Characterization of a Novel Amino-Terminal Domain From A Copper Transporting P-Type ATPase Implicated in Human

Genetic Disorders of Copper Metabolism

Michael DiDonato

A thesis submitted in conformity with the requirements for the degree of Doctor o f Philosophy Graduate Department o f Biochemistry

University of Toronto

O Copyright by Michael DiDonato 1999

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and

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ABSTRACT

Characterization of a novel copper transporting P-type ATPase implicated in human genetic disorders of copper metabolism

Degree of Doctor of Philosophy, 1999.

Michael DiDonato

Graduate Department of Biochemistry, University of Toronto

The -70 kDa copper binding domain fiom the Wilson disease copper transporting P-

tvpe ATPase was expressed and purified as a fiision to giutathione-S-transferase (GST).

Following purification, a detailed study of the metal binding characteristics of the domain, as

well as the stmctural consequences of metal binding, was carried out. In order to determine

the rnetal binding specificity of the domain, immobilized metal ion affinity (IMAC) and

blotting analyses were performed. The results of these analyses indicate that the domain is

able to bind a wide range of transition metals with varying affinities. The apparent order of

affinity was found to be: Cu(I)/Cu(II)>Zn(Li)>Ni(II)>Co(II). Copper bound to the domain is

in the + I oxidation state as assessed using the colorimetric copper(1) chelator bathocuproine

disulfonic acid (BCS) and x-ray absorption near edge structure (XANES) spectroscopy.

Neutron activation analysis (NAA) indicated a coppedprotein stoichiornetry of 6.5 copper

atoms / mole of protein. In competitive " ~ n blotting experirnents non-radioactive copper

(Cu(1) or Cu(I1)) was able to inhibit %nc binding to the domain in a sigmoidal marner,

suggesting the prcsence of cooperativity. This sigmoidal inhibition pattern was not observed

for any of the other metaIs tested. Detailed structural studies using circular dichroism (CD)

spectroscopy indicate that significant changes in both secondary and tertiary structures take

iii

place in the domain upon copper binding. Furthemore, the structural changes which occur

upon the addition of zinc are significantly different fiom those which occur with copper. X-

ray absorption spectroscopy (XAS) indicates that the copper atoms are ligated by two sulfur

atoms in a distorted linear arrangement. The XANES spectra did not change significantly

with incremental addition of copper to the domain indicating that al1 six binding sites may be

stnrcturally sirnilar.

No amount of experimentation can prove me right; a single experiment can prove me wrong.

-Albert Einstein

Acknowledgments

I would like to thank al1 the people who, in one way or another, have helped me in my

graduate career over the past five years. First and foremost I would like to thank my

supervisor Dr. Bibudhendra Sarkar for allowing me the freedom to pursue my interests in his

lab and for al1 his help and guidance over the years. 1 would also like to thank al1 the

members of my cornmittee, Dr. David Mademan, Dr. Robert Moms and Dr. Diane Cox for

al1 their suggestions, help and encouragement. I could not have reached this point without

the help and assistance of my Iab-mates both past and present. To Suree, thank you for

always bcing there as a friend and for your constant Stream of suggestions and solutions to

rny problerns, you have been a tnie role model. Many thanks to Tokameh who shared with

me both the pain and exhilaration of research during Our tour of duty in the lab. you were

missed over the last year. 1 would also like to thank Nira and Loretta for al1 their help and

assistance over the years. To al1 the members of the Sarkar Lab (past and present) Suree,

Tokameh, Mike, Negah, Lakshmi, Andrew, Damiano, Xuefeng and Jin-you, thank you for

making the lab a great place to work. 1 wil1 miss those lab lunches and the crosswords.

1 must also take this opportunity to thank the person who has had the most influence

on my life, my wife Grace DeSantis. She has helped in more ways than 1 can say and has

becn the rock which has anchored me in the Storm. She has always been there with her

support and bas never tired of hearing my depressing research stories. Her intelligence and

perseverance has always been an inspiration to me.

1 would also like to thank al1 rny fnends for their help, support and friendship over the

years. Thank you to Nana and Natalie first for teaching me molecular biology and for

making the Pulleyblank lab a great place, but more importantly for your fi-iendship and

support through the "rough" times in grad school. To Voula, thanks for al1 the QC on my

data presentation (it really paid off in Edmonton!), for cirafting me into the BGSU and for

enlightening me on the "purpose" of glycerïn soap. To Dina, thanks for al1 the spanakopita

and dolmathakia and for always having interesting stones to tell me. To Randy, thanks for

getting me involved with Aliquotes and giving me an excuse to put up a web site, it was great

whils it lasted.

To everyone else on the third floor who I've missed, thanks for making rny stay at

Sick Kids interesting and fun, you will al1 be missed. Lastly I would like to thank my famiiy

for always supporting my interests and encouraging me to strive for higher goals. I love you

al1 very much.

vii

Table of Contents

ABSTRACT ACKNO WLEDGEMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ABBREVIATIONS

CHAPTER 1

Introduction

1 . Metals in Biological Systems

1.3 Form and Function of Copper in Bmlogical Systems I -2.1 Types of copper 1 2.2 Important copper-containing enzymes in humans

1.3 Copper Homeostasis in Humans 1.3.1 Copper uptake and circulation 1.3 -2 Membrane transport of copper . 1.3.3 Intracellular copper trafficking 1.3.4 Excretionofcopper

1.4 Cation Transporting ATPases 1.4.1 Types of cation transporting ATPases

1.4.1.1 P-type ATPases 1.4.1.2 V-type and F-type ATPases

1.4.2 Meakes and Wilson Disease Copper Transporting ATPases

1.5 Human Genetic Disorders of Copper Metabolism 1 S. 1 Menkes disease

1 -5. 1.1 Genetics of Menkes disease 1 S. 1.2 Clinical and Biochemical findings 1.5.1.3 Currenttreatments 1 .S. 1 -4 Animal models

viii

Table of Contents (cont'd)

1 -5.2 Wilson disease 1 -5.2.1 Genetics of Menkes disease 1 -5.2.2 Clinical and Biochemical findings 1 -5.2.3 Current treatments 1 -5.2.4 Animal models

1.6 Project Rationale

CHAPTER 2

Materials and Metbods

2.1 DNA Preparation 2.1.1 Ce11 lines 2.1 -2 Preparation of competent BL2 1 (DE3) and DH5a Cd1 2.1.3 Transformation of competent BL2 l(DE3) and DHSa CelL

2.1.3.1 Preparation of glyceroI stocks 2.1.4 Purification of plasmid DNA 2.1.5 DNA agarose gel electrophoresis 2.1.6 Purification of DNA restriction fiagrnents from agarose gels 2.1 -7 DNA Sequencing 2.1.8 Restriction enzyme digests and ligation reactions

2.2 Construction of Expression Vectors 2.2.1 Construction of @EX-WCBD 2.2.2 Construction of pGEX-WCBD(P)

2.3 Expression and Purification of GST-WCBD and GST-WCBD(P) 2.3.1 Buffer and reagent preparation 2.3.2 Expression of GST-WCBD and GST-WCBD(P) 2.3.3 Purification of GST-WCBD and GST-WCBD(P) 2.3 -4 SDS-PAGE 2.3.5 Protein quantitation 2.3.6 N-Terminal Amino Acid Sequencing 2.3.7 Arnino Acid Analysis

2.4 Removal of GST Fom GST-WCBD and GST-WCBD(P) 2.4.1 Thrombin cleavage of GST-WCBQ

Table of Contents (cont'd)

2.4.1.1 Optimization of thrombin cleavage conditions 2.4.2 PreScissionm cleavage of GST-WCBD(P)

2.5 Characterization of the Metal Binding Properties of WCBD 2.5.1 Immobilized Meta1 Ion Afinity Chromatography (MAC) 2.5.2 6 5 ~ n 0 Blotting Assay

2.5 -2.1 Effect of pH on 6 S ~ n ( l ~ Blotting Assay 2.5.2.2 Effect of DTT on 6 5 ~ n ( ~ ~ Blotting Assay

2.5.3 Compehtion "z~(II) Blotting Assay 2.5.4 Stoichiometry of metal binding 2.5.5 Neutron Activation Analysis (NAAI

2.6 Characterization of the Copper Binding Sites 2.6.1 Preparation of apo-protein 2.6.2 Preparation of Samples For XAS and CD analysis

2.6.2.1 Preparation of Dry XAS Samples 2.6.2.2 Preparation of Liquid XAS and EPR Samples

2.6.3 EXAFS and XANES Analysis of Cu-GST-WCBD 2.6.4 Oxidative release of copper fiom GST-WCBD

2.7 Conformational Analysis of GST-WCBD and WCBD 2.7.1 Analysis of protein structural changes upon metal binding 2.7.2 Estimation of secondas. structure content

CHAPTER 3

Meta1 Binding Properties of the N-Terminal Copper Binding Domain from the Wilson Disease Copper Transporting P-Type ATPase (WND)

3. L Introduction 105

3.2 Results 1 07 3.2.1 Expression and purification of GST-WCBD, GST-WCBD(P), and

WCBD 107 3 2 . 2 Metal binding charactenstics of GST-WCBD and WCBD 110

3.2.2.1 Immobilized metal ion affinity chrornatograpby (MAC) 110 3.2.2.2 Neutron activation analysis (NAA) 112 3 -2.2.3 6 5 ~ i n c blotting analysis 112

Table of Contents (cont'd)

3 .M.4 Cornpetition 65~?nc blotting analysis

3.3 Surnmary and Discussion

CHAPTER 4

Structural Characterization of the Copper Binding Domain And Its Meta1 Binding Sites

4.1 Introduction

3.2 Results 4.2.1 X-ray absorption spectroscopy (XAS) analysis of GST-WCBD 4.2.2 Circular dichroism analysis of GST-WCBD and WCBD 4.2 -3 Estimation of secondary structure content 4.2.4 Oxidative release of copper from GST-WCBD

4.3 Summary and Discussion 4.3.1 Hypothetical mode1 for the function of the N-terminal copper

binding domain of ATP7B in vivo

CHAPTER 5

General Discussion and Future Directions

5.1 General Discussion

5.2 Future Directions

5.3 Literature Cited

List of Tables

Table 1.1

Table 1.2

TabIe 1.3

Table 1.4

Table 1.5

Table 1.6

Table 1.7

Table

Table

Table 1-10

Table 1.1 1

Table 2.1

Table 4.1

Table 4.2

Function of Some Biologically Important Metal Ions

Physiological Role of Copper-Containing Enzymes in Humans

Developmentally Important Copper-Containing Enzymes in Humans

Dietary Modifiers of Intestinal Copper Uptake in Humans

meta ai Content of Rat Bile

Metal Transporting Proteins Containing N-terminal HMA Domains

Symptoms of Classical Menkes Disease and Occipital Hom Syndrome (OHS)

Typical Copper Measwements in Normal Adults and Menkes Disease Patients

Features of Menkes Disease in Humans and the Mottled Mouse Mutants

Typical Copper Measurements in Normal Adults and Wilson Disease Patients

Cornparison of Biochemical Findings in Wilson Disease and its Animal Models: Toxic Milk Mouse and Bedlington Terriers

CD Parameters Used in the Conformational Analysis of GST-WCBD and WCBD

Secondary Structure Estimation for Metal Titrated WCBD and GST- WCBD Using SELCON and K2D

CD Transitions of Aromatic S ide Chains 141

xii

List of Figures

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

Figure 1.5

Figure 1.6

Figure 1.7

Fi,oure 1.8

Figure 2.1

Figure 2.2

Figure 3.1

Figure 3.3

Figure 3.3

Figure 3.4

Figure 3.5

Figure 3.6

Figure 3.1

Figure 4.2

Figure 4.3

Figure 4.4

Intestinal and Cellular Copper Uptake Pathways

Transport Mechanism of SERCA and Na-/K- ATPases

Topology of Type 1 and Type U ATPases

Cornparison of Wilson (W), Menkes (MNK) and Rat (rWND) Copper Transporting ATPases

Proposed Structure for Wilson and Menkes Copper ATPases

Phylogenetic Relationship Between HMA Containing Proteins

Genomic Structure of the Disease Gene (ATP7A)

Genomic Structure of the Wilson Disease Gene (ATP7B)

Parental and Moditied GST Fusion Expression Vectors

GST-WCBD(P) Expression Vector with PreScissionTM Cleavage Site

Effect of Copper on BL2 1 (DE3) Growth Following Induction

Summary of Typical Purification of WCBD and GST-WCBD

Immobilized Metal Ion Affinity Chromatography (IMAC) of GST- WCBD

EEect of pH on the Binding of

Effect of Reducing Agents on the Binding of 6 5 ~ n

Cornpetition of 6 5 ~ n Binding to WCBD

Normalized Copper XANES Spectra For GST-WCBD with Various Amounts of Copper

EXAFS Spectra For GST-WCBD with Various Amounts of Copper

CD Spectra of GST-WCBD Titrated with Copper

CD Spectra of GST-WCBD Titrated with Zinc

xiii

List of Figures (cont'd)

Figure 4.5 CD Spectra of WCBD Titrated with Copper

Figure 4.6 CD Spectra of WCBD Titrated with Zinc

Figure 4.7 Oxidative Release of Bound Copper From WCBD

Figure 4.8 Hypothetical Mode1 for the Function of WND in vivo

xiv

Abbreviations

p-Me

BCS

BSA

CAPS

cDNA

DNA

DTT

EDTA

EPR

ERDBD

EXAFS

GST

HMA

IPTG

LB

NAA

OMIM

PAGE

PMSF

RT

RT-PCR

SDS

SOC

TAE

TCA

TCEP

WCBD

XANES

?CAS

fi-Mercaptoethanol

Bathocuproinedisulfoaic acid

Bovine serum albumin

3-(cyc1ohexylamino)- 1 -propanesulfonic acid

Cornplimentary deoxyribonucleic acid

Deoxyribonucleic acid

Dithiothreitol

Ethy lenediaminetetraacetic acid

Eiectron paramagnetic resonance

Estrogen receptor DNA binding domain

Extended X-ray absorption fine structure

Glutathione-S-Transferase

Heavy metal associated domain

Isopropyl b-D-Thiogalactopyranoside

Luria-Bertani Broth

Neutron activation analysis

Online Mendelian Inheritance in Man

Polyacrylamide gel electrophoresis

Phenylmethylsulfonyl fluoride

Roorn temperature

Reverse transcription potymerase chain reaction

Sodium dodecyl sulfate

Save Our Cells (medium)

Tris-Acetate-EDTA Buffer

Tnchloroacetic acid

TrÎs(2-carboxyethy1)phosphine

Wilson Disease Copper Binding Dornain

X-ray absorbance near edge structure

X-ray absorption spectroscopy

CHAPTER 1

Introduction

1.1 Metals in Biologieal Systems

Metals play a vital role in the proper hnction of many biological processes and are

therefore integral to the maintenance of life. It is believed by some, that the origin of life

itself was aided in part by the presence of various metals in the primordial environment of the

early Earth (Ochiai, 1995; Osterberg, 1995). The role o f metal ions in biological systems has

evolvcd to include important roles in transport, signaling, catalysis and structure-promotion.

The bioIogical h c t i o n s of some selected metal ions are surnmarized in table 1.1. There are

many attributes of metal ions which make them especially usefiil in biological systems. Two

of the most important are complex formation and redox properties. When metal ions form

complexes, the properties of the metal as well as the ligand are altered. The simplest

cxampIe of this is the alteration of the pK, of water upon the addition of metal. When

iron(II1) is introduced into an aqueous solution the pK, of the bound water ligands is

drarnatically altered and is substantially lower than that of the b u k water, 3.5 versus 15.6

(Burgess, 1978). This property can also be obsewed with other ligands and is integral to the

hnction of proteins which contain a metal factor.

In these cases the metal ion may promote electrophilic activation, charge

neutralization, and the creation of a more effective nucleophile. Alternatively, the metal ion

rnay help establish the proper substrate geornetry, or activate the substrate by imposing a

distortion or strain (Phipps, 1995). In many cases the binding of a metal ion to a protein can

lead to the stabilization of a particular conformation which may be significantly different

than what would be observed in the absence of metal (Glusker, 1991). The redox properties

of certain metal atoms also play an important part in biological systems since the various

oxidation States o f the metal usually have different properties and provide

Table 1.1 Function of Some Biologically Important Metal Ions

Sodium Potassium

Magnesium Calcium

Charge carrier; osmotic balance Charge carrier; osmotic balance

Structure, hydrolase; isomerase Structure, signaling; charge carrier

Vanadium Nitrogen fixation; oxidase

C hromium Involvement in glucose tolerance and rnetabolism

Molybdenum Nitrogen fixation; oxidase; 0x0 transfer

Tungsten Dehydrogenase

Photosynthesis; oxidase: structure Oxidase; dioxygen transport; electron transfer, nitrogen fixation

Cobalt Oxidase; aikyl group transfer

Nickel Hydrogenase ; hydrolase

Copper Oxidase; dioxygen transport; electron transfer

Zinc Structure; hydrolase Adapted tiom (Lippard and Berg, 1994).

an additional control point for modulation of the proteins' activity. Many metalloenzymes

cany out complex redox reactions which are critical to the maintenance of life. These are

usually two electron reactions which ofien involve group transfer as wefl. Examples of these

types of reactions are the addition and removal of oxygen f?om a substrate.

Although a critical component of many enzymes, most metal ions are only required in

trace amount. An excess of metal ionst especially those with redox properties such as copper

and iron, pose a potentiat threat to the organism. As a result, many organisms have had to

evolve specific and efficient methods to regulate both the uptake and efflux of metal ions

from their cells in order to avoid the toxic effects of metal accumulation and the equally toxic

effects of metal deficiency. The result is a whole host of proteins, and control mechanisms

which are only now becoming well understood.

1.2 Form and Function of Copper in Biological Systems

1.2.1 Types of Copper

Copper is an essential element which is present in the active sites of many enzymes

carrying out a variety of fùnctions (Table 1.2). Most of biological copper exists bound to

proteins and these copper binding sites are divided into 3 main types based on their

spectroscopic properties. Type 1 copper is not normally found in humans and consists of a

single copper atoms ligated by four ligands in a distorted tetrahedral arrangement. This type

of copper is characterized by an intense absorption (E = 3000 - 5000 M-' cm-') at - 625 nm,

imparting a blue color to the protein, and an unusually small hyperfine splitting constant in

its EPR spectra (Solomon et al., 1976). The intense blue color of these proteins has led to

Table 1.2 Physiological Role of Copper-Containing Enzymes in Humans

Physiological Role Protein Radical Scavenging CulZn Superoxide Dismutase

Metal Transport

Electron Transport

Ferroxidase Activity

Adenosine and Homoc ysteine Synthesis

Metallothionein Ceruloplasrnin

Metallothionein Cemioplasmin Albumin ATOX I +

c c s 1 +

cox 17+

Cytochrome oxidase

Ceruloplasrnin Ferroxidase II

Adenosy lhomocysteinase

Blood Coagulation Factor V - Factor VI1

' ~ecen t ly identi fied intracellular copper chaperones.

this type of copper also being referred to as 'blue copper'. The type 1 copper center can

accommodate the geometry of both copper(1) and copper(iI). The second type of copper

centers are referred to as 'normal' copper since their spectroscopic properties are similar to

those of rnany low molecular weight copper complexes. This site is characterized by a

hyperfine coupling constant as detected by EPR, which is considerably larger than for type 1

copper and a weak absorption in the W N i s spectrum. Type 2 copper centers usually consist

of a distorted square planar arrangement of ligands and are practically colorless (Harris,

1995). The usual oxidation state for copper in this site is +2. The third type of copper differs

from the first two in that it is a binuclear site. Type 3 copper sites exist as a Cu([)-Cu(1)

couple to which oxygen is able to bind bridging the two copper atoms. The binding of

oxygen as a stable peroxide species results in the oxidation of the copper pair from the +1 to

the +-2 oxidation state (Solomon, 1988). The Cu(I1)-Cu(I1) form of the protein would be

expecied to have an EPR spectrum since Cu(Ii) is a d9 system; however, the proximity of the

two copper atoms (- 3.5 A (Volbeda and Hol, 1988)) results in spin coupling abolishing the

EPR spectra. The oxygenated type 3 copper site is characterized by a strong absorption at

330 nm (Harris, 1995).

Although the copper sites descnbed above are the most common of those found in

bioiogical systems, another class of copper site which is composed of a type 3 and type 2 site

has also been described. This trinuclear site is found in al1 multicopper oxidases described

thus far and is composed of a triangular arrangement of copper atoms with the binuclear type

3 copper site at the base and a type 2 copper site at the apex (Allendorf et al., 1985; Spira-

Solomon el al., 1986). Lastly, mono copper sites based on copper in the +1 oxidation state

have sometimes been referred to as type 4 copper. However, since copper is in the i l

oxidation state, with a dlo closed-shell system, it is spectroscopically inactive and hence not

easily detected. In order to characterize these types of copper centers, techniques such as

magnetic circular dichrosim and various types of XAS must be employed.

1.2.2 Important Cupper Containing Enzymes in Humans

There are many developmentally important enzymes in humans which require copper

to function properly (Table 1.3). -4 deficiency of copper in the body will lead to a decreased

activity of these enzymes with serious consequences. It is this scenario which is present in

Menkes disease, where many of the observed phenotypes can be traced back to a deficiency

in copper-requiring enzymes. Among the most important copper-containing enzymes in

humans are cytochrome c oxidase, ceruloplasmin, lysyl oxidase, and CdZn superoxide

dismutase (SOD).

Ceruloplasmin is a large, secreted, multicopper serum glycoprotein which carries

approximately 95% of al1 copper circulating in the body (Frieden, 1986). The crystal

structure of holoceniloplasmin bas been solved and shows that the holoprotein contains six

copper atoms, two type 1 sites, type 2 and 3 sites and a type 4 site (Zaitseva er al., 1996).

The type 3 and type 2 sites are arranged in trinuclear cluster which is similar to that found in

laccase and imparts ferroxidase activity on the protein (Rydén, 1988). The protein is

synthesized in hepatocytes and copper incorporation takes place early in the secretory

pathway possibly in the golgi or rough endoplasmic reticulum, with N-glycosylation not

being required for copper loading (Sato and Gitlin, 1991). Although the level or availability

of copper has no effect on the synthesis or secretion of the protein, apoceruloplasmin lacking

oxidase activity is unstable and rapidly degraded once secreted (Gitlin et al., 1992). The

Table 1.3 Developmentally 1a i~ ; tûz t Ccnner-Contakg T c Enzymes in Humans

Enzyme Function Consequence of Deficiency

Cytochrome oxidase (EC 1.9.3.1)

Superoxide dismutase (EC 1.15.1.1)

Tyrosinase (EC 1.14.18.1)

Dopamine-f3-hydroxylase (EC 1.14.17.1)

Lysyl oxidase (EC 1.4.3.13)

Electron ttansport Muscle weakness, neurological chah effects, hypothermia

Free radical Implicated in arnyotrophic scavenger lateral sclerosis (ALS)

Melanin synthesis Loss of pigmentation

Catecholamine Neurologie effects production

Cross-linking of Arterial abnormalities, loose collagen and elastin skin and joints

Ferroxidase, possible Anemia, secondary copper (EC 1.16.3.1) copper transporter de ficiency

precise function of ceruloplasmin in the body is not known, although roles in copper

transport (Campbell et al., 198 1 ; Dameron and Harris, 1987; Hilton et al., 1995; Hsieh and

Frieden, 1975; Marceau and Aspin, 1973) and iron metabolism (Frieden, 1986; Hams,

199Sa; Roeser et al., f 970), due to its ferroxidase activity, have been propose&

The trinuclear copper cluster is responsible for activating oxygen during the catalytic

cycle of the protein and residues in both the arnino and carboxy terminus of the protein

provide essentiai ligands for the trinuclear copper center (Zaitseva et al., 1996). Studies have

shown that ceruloplasmin plays a key role in the mobilization and oxidation of iron stores

and its subsequent incorporation into transferrin (Osaki and Johnson, 1969). This is

supported by the observation that animals deprived of dietary copper have little or no

circulating holoceruloplasmin and are unable to release iron into the plasma from tissue

stores (Roeser et al., 1970). Mutations in the ceruloplasmin geiie have been linked to

aceruloplasminemia, a disorder characterized by progressive neurodegeneration of the retinal

and basal ganglia (Harris et al., 1998). The mutations characterized so far have been s h o w

to abolish amino acids in the carboxy terminus which are critical for the formation of the

trinuclear copper cluster (Daimon et al., 1995; Hams et al., 1996; Hams et al., 1995;

Okamoto et al., 1996; Takahashi et al., 1996; Yoshida et al., 1995). This correlates with the

finding of little or no oxidase activity in the plasma of affected individuals (Harris et al.,

1998).

Lysyl oxidase is an extremely important enzyme, particularly during development,

since it is responsible for the formation and fùnction of comective tissue throughout the body

(Linder, 199 1). Lysyl oxidase is a secreted protein of about 32 kDa in size with one tightly

bound copper atom in the +2 oxidation state, ligated by four ligands (at least three of which

are nitrogen) in a distorted tetrahedral environment (Gacheru et al., 1990). The enzyme

functions by catalyzing the oxidative deamination of lysine and hydroxylysine side chains

and oxidizing the &-amino carbon to an aldehyde to form allysine- The resulting aldehyde

groups serve as the center for a cascade of spontaneous reactions involving the side chains of

non-oxidized lysine residues whicti results in the formation of inter- and intramolecular

crosslinks which stabilized the collagen and elastin fibers (Linder and Hazegh-Azam, 1996).

Like other copper oxidases, lysyl oxidase uses molecular oxygen and 6-hydroxy dopa to

cany out the oxidative deamination with the release of ammonia and peroxide.

Lysyl oxidase is widely expressed and is especially abundant in skin, veins, artenes

and other structures containing large amounts of comective tissue (eg. cartilage). It is often

isolated as four isoforms each with a molecular weight of 32 kDa. The enzymes are

synthesized as a pro-polypeptide of 50 kDa which is post-translationally modified by

proteolysis and glycosylatiou to produce the final mature form of 32 kDa (Romero-Chapman

et al., 199 1; Trackman er ai., 1992; Trackman et al., IWO). Little is known about how the

enzyme is regulated; however, many studies have pointed to copper as an important regulator

of Iysyl oxidase. Early reports on the effects of dietary copper deficiency on Iysyl oxidase

activity indicated that depressed copper levels resulted in decreased lysyl oxidase activity in

both chick aorta and lung (Harris, 1986; Harris et al., 1974). The lower lysyl oxidase levels

resulted in the proportion of salt extractable or non-crosslinked collagen to increase in these

tissues. Furthermore, the authors observed that feeding copper deficient animais trace

amounts of copper or adding it to cultures of aortic tissue resulted in the immediate

restoration of lysyl oxidase activity (Harris, 1976; Rayton and Hams, 1979). More recent

studies in rats have s h o w that even though copper deficiency results in a depression of lysyl

oxidase activity, the steady state levels of lysyl oxidase mRNA are unaffected (Rucker et al.,

1996). These results suggest that copper does not seem to bc involved in the regulation of

lysyl oxidase synthesis even though it is required for activity.

Superoxide dismutase (SOD) is a 32 kDa homodimeric protein which usually

contains one mole of copper and one mole of zinc per monomer. Manganese and iron

variants of the enzyme also occur, especially in bacteria (Linder and Hazegh-Azam, 1996).

The copper binding site in copper-zinc SOD is type 2, and the copper atom is coordinated by

four histidine residues, one of which serves as a bridging ligand between the copper atom and

the zinc atom. The copper and zinc sites are located at the bottom of a long narrow charnel

where the substrate diffùses into the active site. Mutation of one of the histidine residues

s~rrounding the copper to a cysteine results in the creation of a stable copper site closely

resembling that of type 1 copper (Lu et al., 1992). Although the activity of SOD requires the

presence of two essential metals, copper and zinc, its expression in many tissues is only

regulated by copper (Harris, 1992a; Harris, 1992). Copper-zinc SOD is present in the

cytoplasm of eukaryotic ceIls where it is responsible for the dismutation of superoxide (Oz*-)

to hydrogen peroxide (H202) and O2 through the cyclic reduction and oxidation of the bound

copper atom (McCord and Fridovich, 1969). Biochemlcal and immunological studies have

indicated that the enzyme is located in peroxisomes (Dhaunsi et al., 1992; Keller et al.,

1991).

It has also been shown that SOD is a critical enzyme in antioxidant defense. Oxygen

exposure has been shown to increase SOD activity as well as mRNA levels in both human

endothelial cells and yeast (Bermingham-McDonogh et al., 1988; Linder, 199 1). This

correlates well with the finding that transgenic mice with elevated SOD activity have

increased resistance to reperfusion injury after stroke, which in many cases is ascribed to

peroxidative processes (Maitre et al., 1993). Genetic analysis has shown that 10-15% of

familial amyotrophic lateral sclerosis ( F U S ) cases are linked to mutations in the SODl gene

(Deng et al., 1993; Rosen et al., 1993).

Many of the point mutations in the SODl gene Iead to a mutant protein which is still

able to bind copper and has normal superoxide scavenging activity (Corson et al., 1998).

These results suggest that loss of superoxide scavenging activity is not the primary cause of

FALS, indicating that FALS may be a result of a gain-of-fûnction associated with these

mutations (Gurney et al., 1994; Ho et al., 1998; Reaurne et al., 1996; Ripps et al., 1995;

Wong et al., 1995). The most promising theory puts forth the idea that these mutations lead

to a less restrictive active site, allowing access of other substrates which could lead to

aberrant chemistry at the copper atom. These substrates may include hydrogen peroxide

( H 2 0 1 ) and peroxynitrite (ON003 which have been shown to react with copper-zinc SOD in

virro to oxidize substrates and catalyze the nitration of phenolic groups, respectively

(Beckman et al., 1993; Hodgson and Fridovich, 1975; Ischiropoulos et al., 1992; Wiedau-

Pazos et al., 1996). The toxic products produced by these aberrant reactions may be the

cause of FALS; however, further work is required to c o n f m these hypotheses.

1.3 Copper Homeostasis in Humans

To avoid the toxic effects ofcopper excess or deficiency, an efficient method for the

uptake, distribution and excretion of copper is needed. Although many aspects of copper

transport have been studied for over the past 30 years, until recently little was known about

the molecular aspects of copper trûfficking within the body. The recent discovery of the

Wilson and Menkes copper transporting ATPases, together with the identification of copper

chaperones, has given us an unprecrdentltd understanding of the mechanisms behind copper

homeostasis. These discoveries and those to follow will be critical in developing new

treatments for disorders of metal metabolism.

1.3.1 Copper Lrptake and Circulation

Copper uptake in humans is confined mainly to the stomach and srnaIl intestines.

Although the overall mechanism of copper uptake in the intestines is not understood, several

studies have identified several key features of the system. It has been demonstrated in

several studies that copper uptake in the intestines is inversely related to the amount of

copper ingested (Tumlund et al., 1989; Tumlund et al., 1985). Thus with very high copper

diets, the absorption of copper in the intestines can be as low as 12% of maximum- A

theoretical absorptive maximum of 65% of ingested copper has been suggested, but, with

typical diets the actual copper uptake is about 40% (Wapnir, 1998). These observations have

strengthened the view that intestinal copper uptake plays a central role in the regulation of

overall copper absorption. Intestinal copper absorption is influenced by many dietary factors

such as pH, vegetable fibers, carbohydrates, and fats (table 1.4). The effect of pH on copper

absorption has been studied in the rat and it was found that the rate of copper absorption in

the ileum is higher at pH 5.8 or 7.8 thao at pH 6.3 - 7.3 (Wapnir and Stiel, 1987).

Sodium has also been suggested to be involved in the uptake of copper by the

intestinal mucosa. Perfusion studies using rat ileum and jejunum found that the rate of

copper uptake was significantly decreased when sodium was omitted fiom the perfusate

Table 1.4 Dietary Modifiers of Intestinal Copper Uptake in Humans

Modifier Effect Fibre

Hemicellulose &

Carbohydrates Fructose Glucose polymers

Fats Long-chain fatty acids Medium-chain fatty acids

Protein High-protein diet Excess amino acids

Organic acids Ascorbic acid Polybasic amino acids

Divalent cations (Zinc, Iron, Tin, Molybdenum) &

NK = Not Known

(Wapnir and Stiel, 1987). A similar result was also observed when amiloride was used to

inhibit the function of the sodium-proton antiporter and the sodium-calcium exchanger

(Wapnir, 1991). Although these results suggest a link between intestinal copper uptake and

sodium, the nutritional and biological significance of such a link is unknown. The effect of

carbohydrates, especially hc tose , has been studied extensively in rats. The effect of high

fructose diets in rats can lead to copper uptake insufficiency and these effects seem to be both

species- and sex-dependent (Fields et al., 1984; Reiser er al., 1983). This effect has also

been o b s e ~ e d in humans where high hc tose diets can Iead to decreased copper status

(Reiser et al., 1985). The widespread use of fructose-containing sweeteners and the finding

that copper sufficiency is closely linked to cholesterol metabolism makes these studies

especially relevant (Kievay, 1987).

The effect of amino acids on intestinal copper absorption is varied and is directly

linked to their concentration in relation to copper. One study in humans found that subjects

whose diet was supplemented with methionine had a net copper absorption which was nearly

double that of unsupplemented controls (Kies et al., 1989). Conversely, an excess of dietary

amino acids such as histidine and cysteine can have serious consequences on copper uptake.

In perhsion studies using rats, excess dietary histidine has been shown to cause increasing

losses of copper through the urine and to reduce copper uptake significantly (Harvey et al.,

1981). These observations probably stem from the ability of histidine to chelate copper,

thereby reducing its bioavailability, or competing for binding with the intestinal transport

proteins. A similar effect bas also been observed for cysteine. In addition to being an

effective chelator for copper, cysteine is also able to reduce copper from the divalent to

monvalent state, having a deleterious effect on copper uptake (Baker and Czarnecki-

Maulden, 1987). Other divalent cation such as zinc, ferrous iron, and starnous tin c m act in

a cornpetitive marner to decrease intestinal copper uptake (Hall et al., 1979; Pekelharing et

al., 1994; Van Campen and Scaife, 1967; Yu et al., 1994a).

The transport of copper from the intestinal lumen to the intestinal mucosa is a canier-

mediated process involving at least one saturable component (Crampton et al., 1965;

Tumlund et al., 1989). Although the identity of the intestinal copper transport protein is not

known, the Menkes disease copper transporting ATPase is a strong candidate in light of the

Menkes phenotype and the strong expression of the protein in the intestinal mucosa (Chelly

el al., 1993; Mercer et al., 1993; Vulpe et al., 1993). Once copper enters the blood Stream it

becomes bound to a variety of proteins. More than 90% of copper is bound to ceniloplasmin,

a large multicopper senun glycoprotein; however, this copper is tightly bound and considered

non-exchangeable (Linder and Hazegh-Azam, 1996). The remaining 5 - 10% is

exchangeable and is bound to albumin, and amino acids as iow molecular weight complexes.

Several carefiil and detailed studies revealed that the main copper-amino acid complex in the

serum was in the form of a 1 :2 copper-histidine complex (Neumann and Sass-Kortsak, 1967;

Sarkar and Kruck, 1966). Further studies also revealed that histidine could form a temary

complex with albumin and copper (Lau and Sarkar, 1971 ; Sarkar and Wigfield, 1968). The

copper binding site on albumin has been very well characterized (Iyer et al., 1978; Laussac

and Sarkar, 1980; Laussac and Sarkar, 1984). The copper binding site on albumin is at the

amino terminus and involves the first three residues (Asp-Ala-His). Copper is ligated in a

pentacoordinate arrangement using the amino-terminal nitrogen, the carboxyl side chain of

aspartic acid, two intervening peptide nitrogens, and an imidazole nitrogen fiom a histidine

residue in the third position. The four nitrogen ligands are in a square-planar arrangement

with the carboxyl oxygen occupying the axial position.

Copper bas also been observed to bind to another large semm protein found in rats

known as transcuprein (Linder, 199 1; Weiss and Linder, 1985). Since this protein has yet to

be cloned and has not been fully characterized, conclusions cannot be drawn regarding its

biological function (Barrow and T a ~ e r , 1988; Goode et al., 1989; Tsai et al., 1992). It is

clear that albumin is responsible for carrying the majority of newly absorbed copper ions in

the blood stream. Approximately 40 pg of copper are requircd to fil1 al1 the high affinity

albumin copper sites in I ml of human serum, but the amount that actually binds in vivo is far

less (Linder and Hazegh-Azam, 1996). Although albumin is an important transporter of

copper in the blood stream, its role in the uptake of copper by various tissues remains

unknown. When albumin is completely absent, as is the case in analbuminernia, there does

not seem to be any disruption in the distribution of copper (Vargas et al., 1994). Although

albumin levels in the nagase rat are 4000 times lower than normal, intravenously injected

67 Cu entered the liver at least as fast in these animals as in normal animals and its

reappearance in ceruloplasmin seemed to be accelerated. These findings suggest that

aIburnin may be playing a passive role in copper transport, acting as a reservoir for newly

absorbed copper. This view is strengthened by the observation that copper uptake by

hepatocytes is inhibited by albumin and no ce11 surface receptors for albumin have been

found on these cells (Ettinger et of., 1986). It is likely that albumin-bound copper must

exchange with other components, which then facilitate its uptake into cells. Transfer of

copper between albumin and transcuprein has been shown, as has exchange between albumin

and histidine (Lau and Sarkar, 1971; Sarkar and Wigfield, 1968; Tsai et al., 1992; Weiss and

Linder, 1985). The former may be important in rodents while the latter is more important in

humans.

1.3.2 Membrane Transport of Copper

There are at least two pathways by which cells take up copper from the bloodstream

(fig. 1.1). The first pathway involves copper release and uptake from ceruloplasmin.

Ceruloplasmin has been shown to be able to donate copper to cells as well as to intracellular,

copper-containing proteins (Campbell et al., 198 1 ; Dameron and Hams, 1987; Hsieh and

Frieden, 1975; Marceau and Aspin, 1973). The uptake of copper from ceruloplasmin is

belicved to occur via interaction witb a ce11 surface ceruloplasmin receptor, releasing

ceruloplasmin bound copper. The released copper is then reduced by a ce11 surface reductase

and enters the ce11 through an energy-independent transport protein. Although the nature of

the uptake protein has not been established conclusively, hCTRl has been proposed to be the

high affinity copper uptake protein in humans (Zhou and Gitschier, 1997). This protein was

initially discovered by its ability to complement the yeast hi@ affinity copper uptake mutant,

ctrl. The human CTRl protein shares 29% identity with its yeast couterpart; however, it is

much smaller in both the intracellular and extracellular metal binding domains (Zhou and

Gitschier, 1997). A second protein, hCTR2, which is similar to hCTRl was discovered

through database searching and both genes are expressed in al1 tissues. The exact role of

these proteins in the uptake of copper remains to be established.

l

[ Intestine

Figure 1.1 Intestinal and Cellular Copper Upiake Pathways. Copper is absorbed from the gastrointestinal tract and enters the exchangeable pool. During uptake, copper is reduced to Cu(1) by a hypothetical membrane reductase and is absorbed by the ce11 through a passive transporter, possibly hCTRI. In the ceruloplasmin (Cp) pathway, copper is released Erom Cp through interaction with its receptor, followed by reduction and uptake. Once inside the cell, copper becomes bound to glutathione (GSH) and various copper chaperones (ATOXl , Ccs 1, Coxl7). The Wilson disease ATPase is positioned in the bans-golgi membrane where it pumps copper into the excretory pathway for incorporation into cuproproteins.

htative ceruloplasmin receptors have been identified in a variety of tissues and ce11

types; however, those which have been isolated, differ in size and composition (Barnes and

Frieden, 1984; Gordon et al., 1987; Kataoka and Tavassoli, 1 984; Kataoka and Tavassoli,

1985; Omoto and Tavassoli, 1990; Orena er al., 1986; Stem and Fneden, 1993; Stevens et

al., 1984). These differing proteins may represent different components of the ceruloplasrnin

receptor, al1 of which have some affinity for ceruloplasmin. Studies on the uptake of

ceruloplasmin-bound copper by human erythroleukemia celIs revealed that ceruloplasmin

itself was not intemalized when copper was taken up by the ce11 (Percival and Harris, 1990).

This study also found that the addition of ascorbate strongly stimulated uptake of copper

while the addition of cuprous chelators inhibited it. In addition, sulfhydryl-modifying

reagents were also found to inhibit uptake, implicating cysteine residues in the copper upbke

process. Taken together these results suggest that copper is taken up in the monovalent

rather than the divalent form (Harris, 199 1; Percival and Harris, 1989).

The second pathway involves uptake f?om non-ceruloplasmin-bound copper such as

that bound to histidine, albumin, and other low molecular weight complexes (Hams and

Sass-Kortsak, 1967; Sass-Kortsak et al., 1967). As is the case for the ceruloplasmin

pathway, uptake of copper does not involve intemalization o f its ligands (Ettinger et al.,

1986; van den Berg and van den Hamer, 1984). Studies on the kinetics of copper uptake by

cultured hepatocytes support a system which is carrier-mediated, energy-independent and

saturable (Ettinger et al., 1986; McArdle et al., 1988; Schmitt et al., 1983; Stockert et al.,

1986; Weiner and Cousins, 1980). Uptake via this pathway can also be inhibited by other

metals such as zinc or cadmium, reflecting the similar coordination preferences of these

rnetals, or suggesting that the protein involved is a general metal carrier (Ettinger et al., 1986;

Schmitt et al., 1983). As has been mentioned previously, histidine and albumin have

different effects on the uptake of copper. Ln the absence of albumin, at ratios less than 1 : 1

witb copper, histidine has no effect on copper uptake. At higher concentrations, histidine

acts as a weak cornpetitive inhibitor of copper uptake in hepatocytes, but, in the presence of

albumin, stimulates the uptake of copper (Dmvish et al., 1984). Studies on the uptake of

copper by placenta1 cells shows that histidine stimulates copper uptake in the presence of

serum while albumin inbibits it (Mas and Sarkar, 1992). These findings and the observation

that analbuminemia in humans and animals does not result in a disturbance of copper uptake

strongly suggest that histidine plays a central role in the uptake of copper from the

bloodstream.

1.3.3 Intracellular Copper Tra ffrcking

Once copper is taken up by the cell, it enters a series of complex pathways which are

only now becoming understood (fig 1.2). Much of the absorbed copper is immediately

excreted from the ce11 via pathways which wil1 be discussed in the following section. Botb

copper and copper proteins are distributed throughout the ce11 and are present in al1 cellular

organelles including lysosomes, mitochondria, endoplasmic reticulum, nucleus and the

cytosol (Vulpe and Packman, 1995). Examples include metallothionein (MT) in the nucleus,

cytosol, and lysosomes (Janssens et al., l984a; Janssens et al., 1 984), cytochrome c oxidase

in the rnitochondria (Kodama et al., 1989), Iysyl oxidase in the golgi and secretory organelles

(Kuivaniemi et al., 1986), and superoxide dismutase (SOD) in the cytosol and possibly

peroxisomes (Crapo et al., 1992). For many years the mechanism by which copper was

delivered to various proteins and cellular compartments was not understood. Recently a

family of small cytosolic proteins has been identified which are responsible for the delivery

of copper to specific cellular targets. These proteins have corne to be known as copper

chaperones. Al1 of these proteins were initially identified in yeast and their human

orthologues were identified by complementation studies.

The copper chaperone family currently includes three members: ATXlhuman

ATOX 1 (formerly known as HAH1) (Hung er al., 1998; Klomp et al., 1997; Lin et al.,

1997), Lys7/human Ccsl (Culotta et al., 1997; Gamonet and Lauquin, 1998), and

Cox 17/human Cox 17 (Amaravadi et al., 1997; Beers et al., 1997; Glerum et al., 1996). Each

of these proteins delivers copper to a different copper containing protein. ATXl delivers

copper to CCC2 (the yeast homologue of WND), lys7 delivers copper to superoxide

dismutase and Coxl7 delivers copper to the mitochondria for incorporation into cytochrome

c oxidase. A direct interaction between these chaperones and their target proteins has only

been demonstrated in two cases. ATXl and the amino-terminal domain of CCC2 have been

shown to interact using yeast two-hybrid analysis (Pu fahl et al., 1997). In the case of Ccs 1

an in vitro interaction with SOD was demonstrated using fusion proteins and an in vivo

interaction was demonstrated by CO-irnmunoprecipitation (Casareno et al., 1998). Although

it is known that Cox17 is responsible for delivering copper to the mitochondria, no specific

target has yet been identified. A possible target of Coxl7 that has been proposed is Sc01

(Glerum et al., 1996). Sc01 is a mitocbondrial protein which has been shown to be critical

for the proper assembly of cytochrome c oxidase and has been localized to the inner

mitochondnal membrane (Buchwald et al., 199 1 ; Krurnmeck and Rodel, 1990; Petnizzella et

al., 1 998; Schulze and Rodel, 1 988). Despite these suggestive results, a direct interaction

between Coxl7 and Sco 1 has not been demonstrated in vivo or in vitro

In most cases the structure of the chaperone is very similar to its target. The primary

structure of Atxl is very similar to the amino-terminal metal binding repeats in CCC2

(Pufahl et al., 1997). Atxl also contains one copy of the metal binding motif, Met-Thr-Cys-

Xaa-Xaa-Cys, which is most likely involved in metal ligation and is present in two copies at

the amino terminus of CCC2 (Yuan et al., 19951. Similarly, the primary structure of Ccsl

bears a striking similarity to the carboxy-terminus of SOD. Siace SOD is known to

homodimerize, this similarity has Iead to the idea that the chaperone dimerizes with SOD to

facilitate the transfer of copper between the two proteins.(Casareno et al., 1998; Culotta et

al., 1997). Since the cellular target of Coxl7 is unknown, no cornparisons can be made

regarding similarity of structure. The copper binding site of Coxl7 has been characterized

spectroscopically. The protein was found to contain two copper atoms in a binuclear

cuprous-thiolate complex which is substantially more labile than similar clusters in other

proteins such as metallothionein (Srhivasan er al., 1998).

Glutathione is also another major chelator of copper in the cytoplasm; however, its

function may be more important in the case of copper overload than under normal conditions.

Studies on copper-loaded hepatoma cells have indicated that over 60% of cytosolic copper

exists as the copper-glutathione complex (Freedman et a/., 1989). The copper-glutathione

complex can also donate copper to various proteins. In viro studies have shown that the

copper-glutathione complex can donate copper to apo-SOD, apo-metallothionein, and apo-

ceruloplasmin (Cino10 et al., 1990; Ferreira er al., 1993; Musci et al., 1996).

Metallothioneins are small molecular weight (6.5 D a ) metal binding proteins which

have several distinguishing features. They have a high metal binding capacity (7 - IO g

atoms/mol); 30% of the amino acids are cysteine yet the native protein has no disulfide

bonds, are devoid of aromatic residues, and exhibit and substantial amount of polyrnorphism

(Cousins, 1985). Metallothionein was originally isolated as a cadmium and zinc binding

protein, but is usually found naturally as a zinc binding protein (Kagi and Vallee, 1960).

Indeed many of the biological fùnctions of metallothionein seem to revolve around zinc

metabolisrn (Cousins, 1985). It has been suggested that metallotbioneins acts as a reservoir

for zinc, regulating its intracellular availability. In support of this notion, the reversible

exchange of zinc between rnetallothionein and the estrogen receptor (2 zinc finger protein)

has been demonstrated, as has the removal of zinc fiom zinc finger-containing transcription

factors such as Sp 1 and TFIILA (Cano-Gauci and Sarkar, 1996; Zeng et al., 199 1 a; Zeng et

al., 1991).

Metallothionein synthesis can be induced by a variety of factors including heavy

metals, physical stress, glucocorticoids, and heat shock. Metallothionein is able to bind other

heavy rnetals such as copper, cadmium, iron, and mercury (Kagi and Schaffer, 1988).

Although the level of metallothionein induction by zinc is greater than that by copper,

metallothionein has a higher binding affinity for copper than zinc (Lipsky and Gollan, 1987).

It is unlikely that rnetallothionein plays a central role in the intracelluIar trafficking of

copper, and its primary role in relation to copper may be one of detoxification and storage.

Many studies have shown that copper-metallothionein accumulates in the lysosomes of

copper-loaded mimals, probably as a protective rneasure of the hepatotoxic effects of excess

copper (Gooneratne et al., 1980; Johnson et al., 198 1 ; Verity et al., 1967). The copper stored

in metallothionein can be made available to other proteins either following metallothionein

degradation in lysosome (Mehra and Bremner, 1983) or by exchange via complexation with

glutathione (Freedman et al., 1989). Yeast strains which have had their rnetallothionein-like

gene knocked out are able to thrive on normal media and maintain normal levels of copper

enzymes, but are intolerant to copper stress. Expression of mammalian metallothionein in

these strains restores their ability to grow in high copper media, supporting the notion that

metallothioneins play a major role in copper detoxification and are not centrai to normal

intracellalar copper metabolism (Thiele et al., 1986). It is likely that a similar scenario takes

place in mammalian cells.

1.3.4 Excretion of Copper

The efficient excretion of excess copper is an integral component of proper copper

metabolism, and the liver plays an important part in this process. Upon being absorbed by the

liver, copper may be retumed to circulation by incorporation into ceruloplasmin,

incorporated into copper containing enzymes, stored within metallothionein, or excreted into

the bile. Urinary copper excretion is negligible under normal physiologic conditions since

most of the circulating copper is either bound to ceruloplasmin or confiaed within

erythrocytes, and little or no copper penneates the glomemlar capillaries (Owen Jr, 1964).

The smaIl amount of ingested copper that does appear in the urine most likely originates

frorn the amino acid-bound fraction of plasma copper and the dissociation of the copper-

albumin cornplex. Ln the overall scherne of copper homeostasis, urinary excretion does not

appear to play an important role. However, cupmria does occur in the later stages of

pathological conditions which affect primary copper homeostatic mec hanisms such as biliary

copper excretion, copper storage and cemloplasmin synthesis.

Biliary excretion is an important mechanism for the excretion of many trace metals

(table 1.5) and accounts for -80% of the copper leaving the liver (Evans, 1973). The

Table 1.5 Metal Content of Rat Bile

-

Metal Concentration (PM) Manganese 5.4 k 0.8 Iron Copper Zinc Molybdenum Vanadium Chromium Strontium Lead 2.8 = 0.7

Adapted fiom @ijkstra et ai., 1 996).

exact mechanism of copper excretion into the bile is not known; however, three potential

routes can be distinguished: i) lysosomal exocytosis, ii) a glutathione-dependent route (via

glutathione conjugate transporter), and iii) secretion by a copper transporting ATPase. The

idea that lysosomes are the source of copper which is excreted into the bile stems from a

large number of studies which have s h o w that large amounts of copper are stored within

hepatic lysosomes under certain physiologie conditions (Mohan et al., 1995; Sternlieb,

1989). In a kinetic study where 64copper was administered orally to a single Wilson disease

patient the specific activity of hepatic lysosomes was similar to that found in the bile

(Sternlieb et al., 1973). This finding, together with the observation that lysosomes could

normaIly release their contents into the bile by exocytosis (De Duve and Wattiaux, 1966;

LaRusso and Fowler, 1979), strengthened the idea that lysosomes are the primary source of

biliary copper. Studies in copper-loaded rats have also shown an association betweeo biliary

copper output and the activity of lysosomal enzymes in bile (Gross et al., 1989) and the

authors suggest that exocytosis of lysosomal contents into bile represents a major mechanisrn

for biliary copper excretion under these conditions. Much of the copper stored in lysosomes

has been found to exist as a complex with a polymerized form of metallothionein (Gross et

al., 1989).

Glutathione (GSH) has been implicated in the biliary excretion of copper since the

early eighties when it was shown that administration of diethylmaleate (DEM) to rats lead to

a strong reduction in the biliary excretion of copper, concorninent with a decrease in hepatic

GSH levels (Alexander and Aaseth, 1980). The inhibitory effect of DEM is also seen with

intravenously administered copper following DEM treatment. DEM is able to forrn

conjugates with GSH, leading to a lowering of available hepatic and biliary GSH. Analysis

of bile fluids fkom rats after copper administration revealed that copper in the bile is

conjugated to GSH (Alexander and Aaseth, 1980). In studies with normal rats it was noticed

that intravenously administered copper was eliminated in a biphasic manner composed of a

rapid phase immediately after injection, followed by a slow gradua1 phase (Nederbragt,

1989). in normal rats the rapid phase is inhibitable with DEM, but the slow phase is not,

suggesting different mechanisms for each process.(Houwen et al., 1990). This has been

confirmed in the mutant GY rat which, despite doubled hepatic GSH levets, is unable to

secrete GSH into the bile. In these animals, the rapid phase is absent; however, the slow

phase as well as secretion of endogenous copper do not seem to be affected (Dijkstra et al.,

1990; Houwen et al., 1990).

These studies strongly suggest that an adequate supply of intracellular GSH is

required for the proper excretion of both endogenous copper and intravcnously administered

copper into bile. However, the rapid phase of biliary copper excretion is only present when

GSH is able to be secreted into the bile. In light of these findings, it seems probable that the

rapid phase involves transport of copper-GSH complexes into bile (fig. 1.2) and this would

most likely occur through the canalicular multispecific organic anion transporter (cMOAT)

(Lee et al., 1997). This finding has been fùrther elaborated by studies of dietary copper

overload in mutant GY rats. In these studies, it was shown that GY rats fed hi@ copper diets

accumulated copper in their livers to the same extent as control animals and had the same

rate of biiiary copper excretion (Dijkstra et al., 1990; Houwen et al., 1990). This implies that

GSH and cMOAT are not involved in a i s process, leading the authors to suggest that there is

another saturable copper transport system in the rat liver.

The Wilson disease copper transporthg ATPase (WND) has been implicated as this

saturable component in the rat Iiver. Although some have demonstrated ATP dependent

transport in isolated rat liver plasma membranes (Dijkstra et al., 1999, localization studies

have not identified the presence of the Wilson disease ATPase on the canalicular membrane.

However, WND may still play an important part in biliary copper excretion. Localization

studies have shown that WND resides in the trans-golgi under steady-state conditions, but

undergoes a reversible, copper-specific translocation to a cytosolic vesicular cornpartment

under elevated copper concentrations (Hung er al., 1997). These vesicles could be

lysosomes; however, ATP dependent copper transport in pure lysosomal membranes has not

been evaluated. The ATPase would pump copper into this vesicle, which could then fuse

with the canalicular membrane and dump its contents into the bile. In this case, GSH has

been postulated to reduce copper(1I) to copper(I), leading to the formation of copper(1)-GSH,

which could donate copper(1) to WND. The copper chaperone, ATOXI, would also be

involved in the donation of copper to WND, in addition to the copper(1)-GSH pathway. This

step may be critical in the efficient transport of copper by this ATPase, as has been

demonstrated for bac terial copper transporting ATPases (Solioz and Odermatt, 1995). In

either case, it is clear that GSH plays a central role in the biliary excretion of copper.

1.4 Cation Transporting ATPases

Cation transporting ATPases (also known as ion motive ATPases) are responsible for

the translocation of ions across biological membranes and are critical for the maintenance of

ion gradients, proper intracellular ion composition and ion resistance. These membrane

bound purnps are found in bacteria, plants, hngi and animals and function by using the

energy obtained Erom the hydrolysis of ATP to move the ion across the membrane. The

known ATPases have been classified into three major groups; P-type, V-type, and F-type

(FIFo-type) ATPases. P-type ATPases are distinguished fiom the other two types by the

formation of a phosphorylated intennediate during the transport cycle. V- and F-type

ATPases are stnicturally and genetically related; however, they have different mechanisms of

action. F-type ATPases are normally run in reverse using an ion gradient to generate ATP.

An example of this is the ATP synthase, Iocated in the imer mitochondrial membrane, which

uses the H- gradient generated from oxidative respiration to drive the synthesis of ATP fiom

ADP (Fillingame, 1997). In contrast to FrFo ATPases, V-type or vacuolar ATPases function

exclusiveiy as proton pumps using the hydrolysis of ATP to drive the reaction. V-type

ATPases are involved in energizing intracellular compartments of the vacuolar system

providing an electrochemical gradient for the transport of solutes and acidifLing several of

these compartrnents (Gluck, 1992).

Recently, two genes which are defective in two human genetic disorders of copper

metabolism (Menkes and Wilson disesise) have been s h o w to encode copper transporting P-

type ATPases (MNK and WND respectively). These were the first heavy metal transporting

ATPases identified in humans. They are part of a larger family of heavy metal transporters

found in bacteria and yeast, responsible for the transport of a diverse set of metals, including

copper, cadmium, zinc, mercury and arsenic (Siiver, 1996). Although similar in many

respects to other P-type ATPases, the heavy metal ATPases have some unique features, the

hnctions of which are beginning to be elucidated. The following sections will provide a

brief outline of the structural features of each type of ATPase noting similarities and

di fferences.

1.4.1 Types of Cation Transporting ATPases

1.4.1.1 P-TypeATPmes

Characteristic features of P-type ATPases which distinguish h e m fiom the other two

type (V and F) are the formation of a phosphoenzyme intermediate, cycling between two

distinct forms (El and Eî) and inhibition by vanadate (Moller et al., 1996). The mechanisrn

of action of two extensively studied P-type ATPases the Na-IK--ATPase (Glynn and Karlish,

1990) and the sarco(endo)plasmic reticulum c ~ ' - - A T P ~ s ~ (SERCA) (MacLennan et al..

1997a) are depicted in figure 1.2. Generally, this mechanism can be represented by a four

step reaction El -. EIP -. EzP -. Ez -. El and is referred to as the Post-Albers scheme (Post

et al., 1972). in the first step, a phosphorylated intermediate (ElP), with bound cations, is

forrned by the reaction of the enzyme with MgATP. This is a high energy intermediate

which can be dephosphorylated with ADP reforming ATP. Following phosphorylation of the

protein, structural changes occur which lead to the translocation of the bound ions across the

membrane. This in turn resuhs in the conversion of the phosphorylated enzyme to the low-

energy E2P form which is unreactive with ADP. In this state, the afinity of the enzyme for

its respective ions is low. The final steps in the reaction invoive removal of the phosphate

group by hydrolysis and reformation of the El state. in many cases reforming the El state

involves the transfer of other ions, K' for the Na'/K'-ATPase and H for SERCA (Levy et

al., 1990; Yu et ai., 1993). The vanadate ion ( ~ 0 4 ~ 3 is a potent inhibitor of P-type ATPases.

It is a transition state analogue of phosphoryl transfer reactions because it readily adopts a

pentacovalent bipyramidal geomeûy. Its inhibitory action most likely stems from its ability

to form a stable transition state complex with the ATPase (Cantley et al., 1978).

a

1 ATP ADP

2ca2+ - fi-

Figure 1.2 Transport Mechanisrn SERCA and N~'/K' ATPases. A SERCA mechanism. Calcium and ATP bind in the cytoplasm resulting in phosphorylation of the El enzyme (1) which undergoes a conformational change to the EL state (2) releasing calcium into the lumen (3), the enzyme is then dephosphorylated and the El state is reformed (4). B ~a' /J f mechanism. Sodium and ATP bind in the cytoplasm resulting in phosphorylation of the EI enzyme (1) which rapidly changes conformation to E2 (2). A series of reactions then occur, which rcsults in the release of sodium and binding of potassium outside the cell, followed by dephosphorylation of the EI enzyme (3). The dephosphorylated El enzyme undergoes another conformational change back to El, with the release of potassium into the cytoplasm.

The P-type ATPases can be divided into two main groups based on the type of ion

transported, either heavy-metal (Type I) or non-heavy-metal (Type Il) (figure L -3). Ali heavy

metal transporting ATPases have similar hydropathy profiles which are significantly different

from those of non-heavy metal transporting ATPases. Members of the type 1 group include

bacterial copper and cadmium ATPases (Nucifora et al., 1989; Odennatt et al., 1993), the

nitrogen fixation protein fixI (Kahn et ai., 1989), the human copper transporting ATPases

(MNK and WND) (Bull er al., 1993; Tanzi et al., 1993; Vulpe et al., 1993) and rnany others.

Al1 members of this family have between 6 and 8 transmembrane segments with only one

pair of transmembrane segments on the carboxy-terminal side o f the cytoplasmic ATP

binding domain. They also contain a highly hydrophobie region, immediately preceding the

TGES/A conserved motif, which probably gives rise to two additional transmembrane

segments in this region. This is in contrast to members of the type II family which contain

only one pair of transmembrane segments in this region (Lutsenko and Kaplan, 1995).

However, the most striking and interesting feature of the type 1 ATPases is the presence of a

large amino-terminal cytosolic domain which contains between one and six copies of the

metal binding repeat GMTCXXC or M/HXXMDHS/GXM (Bull and Cox, 1994; Petnikhin et

al., 1994; Silver et al., 1993). The variable number of these repeats leads to a great diversity

of molecular weights among members of the type 1 family of ATPases.

In contrast to type 1 ATPases, the type II, non-heavy metal transporting ATPases only

have one pair of transmernbrane segments before the TGES energy transduction motif.

Members of this family have a catalytic subunit which consists of 8 to 10 transmembrane

segments and are approximately 100 kDa in size (Lutsenko and Kaplan, 1995). The type II

ATPases can be further subdivided into two groups IIA and IIB. Type IIA

Figure 1.3 Topology of Type I und Type II P-Type ATPuses. Type 1 (heavy metal) and Type II (non-heavy metal) P- type ATPases. White blocks represent core structure, gray blocks represent putative or additional trammembrane segments. Cooserved motifs are indicated, TGES (energy tranduction), DKTG (phosphorylation site), GDGXDN (hinge reg ion), SEHPL (unknown fÙnc tion, type 1 only), GMTCXXC or M/HXXMDHS/GXM (metal binding, n = 1-6).

ATPases have a heterodimeric structure consisting of a catalytic a-subunit and a

glycosylated f3-subunit. Examples of type IIA ATPases include the Na-/K- and H-/K-

ATPases which exchange intracehlar Na' or HT for extracellular K- (Glynn and Karlish,

1990; Sachs er al., 1989). Type IIB ATPases fimction as monosubunit proteins transporting

divalent cations into intracellular compartments or into rhe extracellular milieu. Members of

this family include the calcium ATPases of the plasma membrane and sarco(endo)plasmic

reticulum and the H*-ATPases of fûngi and plants (Moller et al., 1996). The bacterial

inducible, high affinity K' uptake ATPase (KdpK*-ATPase) is somewhat diffèrent kom both

type 1 and type II ATPases. Like the type II ATPases, this protein has only one pair of

transmembrane segments before the TGES motif and bas no metal binding sites at the amino-

terminus. However, it is also missing two transmembrane segments at the carboxy-terminus

which make it shorter than other type II proteins and, in this respect, it resembles the type 1

proteins. Unlike the heavy metal transporters which fiinction as monomers (Nucifora er al.,

1989; Tsai and Linet, 1993), the KdpK--ATPase is active as a multisubunit complex. The

complex consists of three subunits, A, B, and C, which contain 557, 682, and 189 amino

acids, respectively (Hesse et al., 1984). The phosphorylation site is locatsd in subunit B,

while subunits A and C corne together to form the transmembrane charnel (Altendorf et al.,

1 994; Buurrnan et al., 1995)

The core structure of P-type ATPases contain many conserved motifs. Among these

are the TGES, DKTG, and GDGXDN motifs. The TGES motif is located in the cytoplasmic

Ioop between transmembrane segments 2 and 3, which is sometimes referred to as region B

(Moller et al., 1996). This region bas been implicated in energy transduction based on site

directed mutagenesis of the TGES residues and proteolytic cleavage experiments at residues

close to this motif in the SERCA ATPase (Clarke et al., 1990; Imamura and Kawakita, 1989;

Juul et al., 1995; le Maire et al., 1990). These mutagenesis and cleavage experiments

resulted in proteins which retained caIcium binding and phosphorylation capacity, but were

not able to transport calcium. Based on these results, it has been hypothesized that

modifications in this region prevent the conformational change fiom the EiP fonn to the E-P

forrn, leading to an accumulation of EiP with bound calcium (Moller et al., 1996).

The DKTG motif is located in the large cytoplasmic loop between transmembrane

segments 4 and 5 and is referred to as domain C. The DKTG motif is critical to the function

of the ATPase, since it contains the conserved aspartate residue which is phosphorylated in

the transport cycle of the protein. Mutation analysis in the SERCA ATPase has shown that

both the aspartate and neighboring lysine residue are needed for phosphorylation to take

place and cannot be replaced by any other residues (Maruyama and MacLennan, 1988).

Furthemore, mutation of these residues or transposition of the aspartate and lysine rssidues

(DK -. KD) results in loss of phosphorylation and calcium transport (Maruyama and

MacLennan, 1988). These results suggest that the lysine residue may be part of a

stereospecific, acid-base catalyzed transfer of phosphate fiom ATP to the aspartate residue.

The GDGXDN 'hinge' motif is located at the end of domain C just before it enters the

membrane again with trammembrane segment 5 and is referred to as rcgion J. This region is

predicted to be highly alpha-helical and has been predicted to form part of the 'stalk' of the

ATPase (Moller et al., 1996).

The hinge motif also contains two aspartate residues which seem to be required for

phosphorylation, as indicated by chemical modification and mutagenesis studies on both the

N~*/K+ and SERCA ATPases (Lane et al., 1993; Ohta et al., 1986; Vilsen er al., 199 1).

Modification studies in the N a X ' ATPase also indicate that this region may hteract closely

with the ATP binding region of the protein (Ohta et ai., 1986). The SHEPL motif is only

present in heavy metal transporting P-type ATPases in dornain C, just after the

phosphorylation site. The histidine in this motif is cntical for proper h c t i o n of the protein

since mutation of this residue to serine in the Wilson disease copper ATPase leads to the

Wilson disease phenotype (Petrukhin et al., 1993). Although no specific function has yet

been ascribed to this motif in the metal ATPases, the analogous region in SERCA and plasma

membrane Ca-ATPases is a site of regulation through interaction with phophotamban (James

el al., 1989; Toyofiiku et al., 1993) and calmodulin (Falchetto et al., 199 1 ) respectively.

1.4.1.2 Y-gpe and F-type A TPases

V-type (or vacuolar) ATPases are stnicturally related to F-type ATPases and hence

will be considered together in this section. V- and F-type IT ATPases differ £iom the P-type

ATPases in two important ways. First, V- and F-type ATPases are large multisubunit

complexes, in contrast to P-type ATPases which usually function as monosubunit proteins.

Second, although they utilize the energy of ATP hydrolysis, V- and F-type ATPase do not

utilize a phosphoenzyme intermediate (Nelson and Taiz, 1989). V-type ATPases pump

protons from the cytoplasm to the lumen of vacuoles and other cellular organelles such as

lysosomes, endoplasmic reticulum, golgi bodies and secretory granules. As such, they are

closely involved in the maintenance of cellular pH and energizing intracellular compartments

(Finbow and Harrison, 1997). Since the V-type ATPase does not use a counterion, its

activity is electrogenic, creating an electrical potential difference (AY) across the membrane.

This membrane potential is then used to drive solutes and other ions across the organelle

membrane, as occurs in the accumulation of neurotransmitters in synaptic vesicles and amino

acids in the vacuole lumen (Nelson, 1994). The acid pH conditions created by the pumping

of protons into vacuoles is used for othet functions as well (Mellman, 1992). In lysosomes,

low pH is needed to activate acid hydrolases and the dissociation of receptor-ligand

complexes (eg. Fe(iI1) fiom transferrin) (Brown et al., 1983). V-type ATPases are also

closely involved in the trafficking within the endosomal / lysosomal compartment.

Bafiiomycin Al, a specific inhibitor of V-type ATPases, causes a decrease in the rate of

receptor cycling (Johnson et al., 1993). It has since been found that acidification is a

necessary step in the formation of endosome camer vesicles, which are intermediates

between early and late endosornes (Clague et al., 1994).

F-type ATPases (also known as FIFo-ATP synthases) fùnction in reverse when

compared to V-type ATPases. Whereas V-type ATPases use the hydrolysis of ATP to drive

the creation of a membrane potential, F-type ATPases use a membrane potential to dnve the

synthesis of ATP from ADP. In animal cells, the enzyme is located in the inner

mitochondriai membrane where it uses the proton gradient established by oxidative

respiration to drive the synthesis of ATP. Closely related F-type ATPases are found in the

thylakoid membrane of plant chloroplasts, and in the plasma membrane of bacteria such as E.

coli. In plants, the enzyme functions in electron transport-driven photophosphorylation,

while in bacteria the pump can operate reversibly depending on the nutritional status of the

ce11 (Fillingarne, 1997). Under aerobic growth conditions, a proton gradient is created across

the plasma membrane by electron transport systems by pumping protons from the cytoplasm

to the outside of cell. The proton gradient is then used to generate ATP and is also utilized in

proton-coupled nutrient transport and flagellar rotation (Fillingame, 1997). Under anaerobic

conditions, the proton gradient is generated by the hydrolysis of glycolytically derived ATP

by the FiFo-ATPase, pumphg protons fÎom the cytoplasm to the outside of the cell. Al1 three

FI Fo-ATPases (mitochondrial, chloroplast, and bacterial) are stnicturally and mechanistically

related, however they are regulated differently (Harris, 1995; Mills et al., 1995; Walker,

1994).

The F-type ATPases are composed of two structurally and functionally distinct

sectors terrned FI and Fo. The catalytic sector FI extends Erom the surface of the membrane

as a knob-like projection. The Fi sector c m be removed from the membrane as a water

soIuble protein capable of hydrolyzing ATP (Fillingame, 1997). The F i sector is connected

to the membrane bound Fo sector via a narrow stalk. Wben the Fi sector is not present, the Fo

sector can mediate the passive transport of protons across the membrane. Once properly

assembled, the complex mediates the reversible coupling of ATP hydrolysis to proton

transport. The bacterial FIFo-ATPases are cornposed of eight types of polypeptides in the

following stoichiometry: a 3 B 3 y t k (FL) and ai6,c12 (Fo) (Fillingame, 1997). The a3P3y portion

of FI from beef heart mitochondria has been crystallized and structural analysis shows a

symmetncal hexameric arrangement of alternating a and f3 subunits (55 kDa each) around a

central core (Abrahams et al., 1994). The ATP binding sites are Iocated within the subunit

at the af3 subunit interface. The y subunit (30 kDa) is located in the center of the hexamer

and protrudes partially fiom the bottom of the complex. The three dimensional arrangement

of the subunits in the FO domain is unknown.

The V-type ATPases, like their F-type counterparts, are cornposed of a catalytic VI

sector and a membrane bound Vo sector. The VI sector c m be removed using a variety of

methods, including chaotropic agents, alkaline carbonate, or nitrate and MgATP (Kane et al.,

1989; Lai et al., 1988; Xie and Stone, 1988). Unlike the Fo sector, there is no evidence to

suggest that the Vo sector can passively transport protons in the absence of VI (Finbow and

Harrison, 1 997). The subunits in VI are designated A - G and range in size between 1 1 6 kDa

and 13 kDa (Finbow and Harrison, 1997). Three of these, A, B, and C, have sequence

similarity with a, f!, and y fkom the FiFo-ATPase the others having no obvious relationship.

The stoichiometry of the VI sector is similar to that of the Fi sector, possessing a pseudo-6-

fold symmetry a i s . The Vo sector is composed of three different subunits, two of which are

transmembrane (c and a large glycoprotein) and one which is hydrophilic, but tightly bound

to the integral membrane components (Finbow and Hamson, 1997). Electron microscopy

has shown that the are 6 c subunits in the Vo sector, which are arranged symmetrically

(Holzenburg et al., 1993). The stoichiometry o f the other major subunits is the sarne as in the

FI Fo-ATPase (Nelson, 1992).

The mechanism by which V-type ATPases fùnction is poorly understood at the

moment; however, the recent crystal structure of the FI sector has ailowed a putative

mechanism to be proposed for the F-type-ATPases. Based on their structural similanties,

one would predict the mechanism of action to be quite simiîar for V-type ATPases. The

proposed mechanism is based on a rotating or alternate binding change mechanism. Several

biochemical studies have revealed that ADP + Pi is converted into ATP at two or al1 three of

the highly cooperative binding sites without major input of energy (Boyer, 1993; Boyer et

al., 1973). However, the newly formed ATP molecule remains tightly bound and substrate

binding at another site is coupled to product release at the currently active site. This latter

process requires a major input of energy, which is derived from conformational changes

linked to proton flux through the Fo subunit. A series of elegant experiments have shown

that hydrolysis of ATP at the three catalytic sites in the a3P3 hexamer drives the y subunit

around in three steps (Duncan et al., 1995; Noji et al., 1997; Sabbert et ai., 1997; Zhou el al.,

1996).

In ATP synthesis, this process would operate in reverse, where confonnational

changes in the Fo subunit resulting fiom proton flux would be translated to the y subunit. The

confonnational changes generated by proton binding at the c subunits in the Fo sector are

transmitted to the y subunit in the FI sector via the E subunit. Crosslinking studies have

shown that the E subunit extends the entire length of the s tak region (Aggeler and Capaldi,

1996; Zhang and Fillingame, 1995). The translocation of 4 protons leads to a 120' rotation in

the y subunit, releasing one ATP molecule. III this way, the torque generated in the Fo subunit

by the binding of a protons is transmitted to the y subunit, forcing it to rotate in the center of

the a3 f3; hexamer, releasing new ly synthesized ATP.

1.4.2 Menkes and Wfson Disease Copper Transporiing A TPases

The Menkes (MNK) and Wilson (WND) disease proteins are copper transporting P-

type ATPases which have recently been identified in humans (Bull et al., 1993; Chelly et al.,

1 993; Mercer et ai., 1993; Tanzi et al., 1993; Vulpe et al., 1993). Sequence analysis of these

proteins indicate that they belong to a subfamily of eukaryotic cation transporting P-type

(Type 1) ATPases (see section 1.4.1.1) which is closely related to bacterial heavy metal

ATPases (Bull et al., 1993; Chelly et al., 1993). The Menkes and Wilson proteins share a

high degree of homology with each other. The overall sequence identity between the two

proteins is 57%- however; this figure rises to 79% and higher in the phosphatase,

transduction-phosphorylation, ATP and metal binding domains (Bull et al., 1993). Despite

their similarities, the MNK and WND proteins do have some distinct differences. The

greatest of these is a 78 amino acid deletion in WND between the first and second metal

binding domains (fig. 1.4). The significance of this deletion in the bc t ion of the proteins is

not yet known. interestingly, the rat orthologue of the WND gene is missing metal binding

region 4, but the overall spacing and size of tne amino-terminal metal binding domain is

intact (fige 1.4) (Wu et al., 1994).

The MNK and WND ATPases are type 1, P-type ATPases having several features

which distinguish them fiom other members of the P-type ATPase family. Sorne of these

were discussed in section 1 -4.1.1, including an additional pair of transmembrane segments

immediately before the TGES motif and the absence of four transmembrane segments at the

carboxy-terminus, in cornparison wiîh type II P-type ATPases (see fig. 1.3). In addition to

these differences, the MNK and WND ATPases have a larger amino-terminal domain and

several unique amino acid motifs which are conserved in bacterial heavy metal transporters

(fig 1.5) (Bull et al., 1993; Silver et al., 1993; Tanzi et al., 1993; Vulpe et al., 1993). The

large amino-terminal domain in the MNK and WND protein contains six copies of a metal

binding sequence. The repeat is referred to as the heavy metal-associated (HMA) domain

and has the folIowing consensus sequence: FrVNJ-x(2)-[LIVMFAI-x-C-x-[STAGCDNHI-

C-x(3)-[LIVFG]-x(3)-~JV]-x(9,ll)-~A]-x-[LVFYS]. The highlighted cysteine residues

are strictly conserved in al1 proteins which contain the repeat and they are most likely

involved in the ligation of the metal atom.

The HMA has been found at the amino-terminus of a wide variety of heavy metal

ATPases from various organism involved in the transport of copper, zinc, cadmium and

mercury (table 1.6). The phylogenetic relationship between the proteins listed in table 1.6 is

I Metal Binding

f 1

1 3 ~ 5 6 Td C W h ATP

,- Metai Binding 1 1 2 3 4 5 6 Td C M h ATP

1-' Metal Bindin 1 1 2 3

œ - œ 5 6

m - Td 9

Ch/Ph M

ATP 9

: ,' y: - ç ' .a< J!, y$+'*< y ?x?3 *% < :-:. ;- ,. ;:&!i~ei::~*.:7-k~yf=.-.- \ --,,- +,.. + P. 7- -,-.J$$& . . -... --" -&-,. -Cc?e:Içd.*.n

Figure 1.4 Cornparison of Wilson (WND), Menkes (IWVK) and Rat (rWND) Copper Transporting ATPases. WND contains a large deletion (-78 amino acids) between metal binding repeats 1 and 2. rWND is missing the fourth metal binding repeat but overall spacing is intact. Vertical bars represent transmembrane segments, Td, transduction domain, Ch/Ph, channef phosphorylation domain, ATP, ATP binding domain.

ATX1 ATOXl CopA MerP CadA

ILGMTCQSCVKSIEDRISNLKGIISMKVSL VEGMTCQSCVSSIEGKVRKLQGVVRVKVSL IDGMHCKSCVLNIEENIGQLLGvQS IQVSL IAGMTCASCVHSIEGMISQLEGVQQISVSL IKGMTCASWSNIERNLQKEAGVLSELVAL ITGMTCASCVHNIESKLTRTNGITYASVAL

W-MTCSGCSGAVNKVLTKLEPDVSKIDIS VD-MTCGGCAEAVSRVLNKL- -GGVKYDID ITGMTCANCSARIEKELNEQPGVMSATVNL ITGMTCDSCAVHVKDALEwPGVQsADvCT VQGFTCANCAGKFEKNVKKIPGVQDAKVNF

Figure 1.5 Proposed Structure for Wilson and Menkes Copper A TPuses. A Proposed structure for the Menkes (MNK) and Wilson (WND) disease copper ATPases based on sequence comparison with other cation transporting ATPases. B Aligoment of metal binding domains from WND with other heavy metal transporters. ATXl copper chaperone from S. cerevisiae, ATOX 1 human homologue of ATX 1, CopA copper ATPase from E. hirae, MerP mercury transport protein fiom P. Aeruginosa, CadA cadmium ATPase from S. Atweus. Conserved cysteine residues are bolded. CPC and SEHPL motifs are unique heavy metal transporting ATPases.

Table 1.6 Metai Transporting Proteins Containing Amino-

Terminal HMA Domains

Protein Organism Function EMA Swiss-Prot

YBAR MNK

WND

CCC2

SYNA

CopA

CopP

ATOX 1

ATXI

CUA- 1

ZntA

ZiAA

CadA

Fixi

PacS

CQA CtpB MerA

MerP

E . Coli

Human C. Elegam Rat Mouse

Human Rat Mouse Sheep

S. cerevisiae

S y n e c o c o c ~ ~ ~ 7942

E. Hirae

H . Pylor-i

Human S . cerevisiae

C. Elegans

E . Coli

Synechocystis Sp. PCC 6803

B. Firmus S . A ureus

R. hleliloti

Synechocystis Sp. PCC 6803

M. leprae

M. leprue

P . A emginosa

-

AC # Cu-Transporthg ~TPase ' 1 P78245

Cu-Transporting ATPase Cu-Transporting ATPase Cu-Transporthg ATPase Cu-Transporting ATPase

Cu-Transporting ATPase Cu-Transporting ATPase Cu-Transporting ATPase Cu-Transporting ATPase

Cu-Transporting ATPase

Cu-Transporting A T P ~ S ~ '

Cu-Transporting ATPase

Cu Ion Binding Protein

Copper Chaperone

Copper Chaperone

Cu-Transporting ATPase

Zn-Transporting ATPase

Zn-Transporting ATPase

Cd-Transporting A T P ~ S ~ ' Cd-Transpcrting A T P ~ S ~ '

Nitrogen Fixation

Cation Transp. ATPase

Cation Transp. ATPase

Cation Transp. ATPase Hg@) Reductase

P . Aeruginosa Hg;@) Transport Protein 1 PO413 1 ' ~ h e fourth HMA domains in these proteins are non-hinctional. 'probable function based on primary sequence malysis.

s h o w in figure 1.6. When the HMA dornaia in the CopA protein (E. hirae copper ATPase)

is compared to each of the HMA domains in the WND protein, the degree of similarity rises

from 11% identity for the amino-terminal most HMA domain to 40% identity for the

carboxy-terminal most HMA domain (Petmkhin et al., 1994). The identity between the

carboxy-terminal most HMA domains of MNK and WND is 67%. These observation

suggest that the evolution of the large amino-terminal domain in the MNK and WND

proteins proceeded by gene duplication involving the 'splicing' in of each metal binding

domain to the ATPase core, with the carboxy-terminal most domains being the latest

additions (Petnikhin et al., 1994).

The MNK and WND proteins, as well as al1 bacterial heavy metal transporters,

contain two unique amino acid sequences which are not present in non-heavy metal

transporters, these being the CPC motif in the sixth trammembrane segment and the SEHPL

motif following the phosphorylation site. Particularly interesting is the SEHPL motif which

is absolutely conserved in MNK, WND and bacterial copper ATPase CopA (Odematt el al.,

1993; Petrukhin et al., 1993; Tanzi et al., 1993). This strict evolutionary conservation

suggests that this region is of functional importance, a notion which is strengthened by the

observation that mutation of the histidine residue in this motif to a s e ~ e leads to the Wilson

disease phenotype (Thomas et al., 1995).

Although no specific fùnction has been anributed to this motif in the heavy metal

ATPases, the corresponding region in the sarco(endo)plasmic reticulum Ca-ATPase

(SERCA) is the site of interaction witb phospholamban (James et al., 1989; Toyofiiku et al.,

1993). Phosphotamban is an endogenous, membrane bound inhibitor of SERCA which

interacts with the calcium fkee form of the ATPase and lowers its apparent affhity for

WND (Sheep)

WND (Human)

WND (Rat)

WND (Mouse)

CopA

CadA (B. Fimius)

CadA (S. Aureus)

a

CopP

MerP

Mer A

CCc2

MNK (C. Elaguns)

CUA-1

MNK (Human) m

p SYNA

MNK (Rat)

MNK (Mouse)

L

i

I' YBAR cip.

PacS

Figure 1.6 Phylogenetic Relationship Between HU4 Containing Proteins. Protein sequences obtained from the Swiss-Prot database were aligned using ClustalX with the Blossum 30 amino acid weighting matrix. The phylogenetic tree was calculated using ClustalX. See table 1.6 for Swiss-Prot accession numbers for each protein.

calcium. As calcium concentrations rise and more of the enzyme is converted to the calcium

bound form, phospholamban is released fkom the ATPase. Phospholamban could also be

released from the ATPase through phosphory lation of p hospholamban by c AMP-dependent

protein kinase A (Toyofuku et al., 1993). In the plasma membrane calcium ATPases, the

analagous region was shown to interact with a synthetic calmodulin binding domain

(FaIchetto et al., 1991). The interaction of calmodulin with the plasma membrane calcium

ATPase has been shown to resuit in an approximate 20-fold increase in the affinity for

calcium (Larsen et al., 1981; Larsen and Vincenzi, 1979). In Na-/K- ATPases this region is

susceptible to proteolytic digestion in the presence of potassium, but resistant to digestion

when sodium is present (Jorgensen and Farley, 1988). These fmdings suggest that, in these

ATPases, this region undergoes conformational changes during the transport cycle of the

protein. The amino-terminal metal binding domains could play a regulatory role in addition

to binding copper. In the absence of copper, the metal binding domain may interact with the

SEHPL containing segment; once copper binds, conformational changes would release this

interaction, activating phosphorylation of the enzyme and starting the transport cycle.

As compared to non heavy-metal transporting P-type ATPases, the WND and MNK

proteins have two less transmembranç segments at the carboxy-terminus. In both the

SERCA and Na-/KT ATPases the carboxy-terminal transmembrane segments are intimately

involved in the cation translocation process (Clarke et al., 1989; Karlish et al., 1990). In

addition it is interesting to note that the KdpK7-ATPase in E. coli also lacks these carboxy-

terminal transmembrane segments and requires other subunits to form a fùlly functional

complex (see section 1.4.1.1). These observations suggest the possibility that the WND and

MNK proteins require a second, unidentified subunit. This type of arrangement is seen in the

cadmium efflux systern of S. aureus in which the components of the cadmium resistance

mechanism (cadA and cadB) reside on two separate plasrnids (Nucifora et al., 1989). The

CPC motif present in the sixth trammembrane segment is also strictly conserved between the

MNK, WND and bacterial heavy metal transporting AïTases. The proline residue in this

motif is conserved in ail P-type ATPases and has been shown to be critical in the formation

of high affinity calcium binding sites in the SERCA ATPase (Vilsen et al., 1989). It is likely

that the conserved cysteines in this motif are involved in binding the copper atom transiently

as it moves through the membrane.

The WND and MNK proteins also have some important structural differences when

compared to closely related bacterial heavy metal transporting ATPases. First, the MNK and

WND proteins contain six copies of the HMA domain at their amino-terminus, while only

one or two copies are present in the analogous prokaryotic proteins (Bull et al., 1993. Tanzi

et al., 1993). Second, the WND and MNK proteins contain a long carboxy-terminal

hydrophilic fragment which is similar to that found in the plasma membrane calcium ATPase

and the H- ATPase in plants (Pardo and Serrano, 1989). Lastly, the length of the sequence

between the phosphorylation site to the hinge domain is 70 - 80 amino acids longer than in

the analogous bacterial ATPases, but substantially shorter than in the corresponding region of

other eukaryotic ATPases, As discussed above, this region in eukaryotic caIcium ATPases is

the site of interaction with phospholamban and calmodulin, two proteins which regulate the

function of these ATPases. In contrast, the malarial calcium ATPase is lacking this

sequence and, in its place, has an additional 100 - 200 amino acids which are not found in

vertebrates, suggesting that this ATPase has its own regulatory protein (Kimura er al., 1993).

By anaiogy, it has been suggested that the additional 70 - 80 amino acid residues carboxy-

terminal to the SEHPL motif in WND and MNK may reflect the unique regdatory

requirements of these proteins (Petrukhin et al., 1994).

1.5 Human Genetic Disorders of Copper MetaboUsm

Although trace arnounts of many transition metal elements are needed to sustain life,

arnounts in excess of what is needed are usually detrimental. Therefore, precise control over

the free concentrations of these trace metals is essential to maintain good health. This is

especially important for redox active rnetals such as iron and copper which, if present in

excess, can enhance free radical geaeration and lead to cellular damage (Britten, 1996). In

humans, the two major genetic disorders of copper metabolism are Wilson and Menkes

disease which are characterized by an excess and a deficiency of copper respectively. Recent

advances in the cloning of the genes responsible for these disorders has led to several major

insights into copper transport and intracellular trafficking.

1.5.1 Menkes disease

1 .S. 1.1 Genetics of Menkes Disease

Menkes disease (OMIM 309400) is a genetic disorder of copper metabolism

characterized by an impairment in the absorption of dietary copper and a severe disturbance

in the intracellular transport and trafficking of copper (Danks et al., 1973; Herd et al., 1987).

Menkes and coworkers noted in their original study that the disorder seemed to be confined

to males and was inherited in a sex-linked recessive marner (Menkes et al., 1962). The

incidence of Menkes disease is approximately 1/300,000 live births (Tümer and Hom,

1997b). Early work by a number of researchers mapped the disease locus to band Xq13.3

(Tümer er al., l992b; Tümer et al., 1992a; Verga et al., 1991). Using this early genetic work

as a guide, the Menkes disease gene (ATP7A) was isolated independently by three groups

using positional cloning techniques (Chelly et al., 1993; Mercer et al., 1993; Vulpe et al.,

1993). The gene was found to contain 23 exons which span approximately 150 kb of

genomic DNA (fig. 1.7) (Dierick et al., 1995; Tümer et al., 1995). The exons range in size

from 77 to 726 bp, with introns ranging fiom 60 to 196 kb (Dierick et al,, 1995). Al1 splice

sites were found to conform to the GT-AG rule, except for the donor site of intron 9, which

precedes an exon which is altematively spliced (Dierick et al., t 995). The Menkes gene

(ATP7A) shares some similarity with Wilson disease gene (ATP7B). Following the exon

coding for the fifth metal binding domain (exon 5 in ATP7A and exon 3 in ATP7B), the

coding region of both the Menkes and Wilson proteins are virtually identical (Tiimer er al.,

1995). However, significant differences do occur at the 5' end; where the first four metal

binding domains in the Wilson disease gene are contained in one exon, those of the Meakes

disease gene extend over three exons (Dierick et al., 1995).

The Menkes gene encodes a 1500 amino acid protein which is predicted to be a

copper transporting P-type ATPase. The predicted protein has a high degree of similarity to

that encoded by the Wilson disease gene (see section 1.4.2). The main difference is the

presence of a 78 amino acid insertion between metal binding domains 1 and 2 in the Menkes

protein which is not present in the Wilson disease protein. Northem blot analysis has shown

that the Menkes disease protein is expressed in al1 tissues except the liver. which correlates

welI with the observed clinical and biochemical features of the classical Menkes phenotype

(Cheiiy et al., 1993; Vulpe et al., 1993). Analysis of numerous Menkes cases has revealed

that approximately 15-20% of the mutations found in the Menkes disease gene consist of

- Transmembrane segments

CU - Copper binding repeats

PD - Phosphatase dornain

C - Conserved CPC motif

D - Pbosphorylation domain

A - ATP binding domain

Figure 1.7 Genomic Structure of the Diseuse Gene (ATP7A). Vertical lines indicate the positions o f the introns, the numben indicate the positions of the exons. Adapted fiom (Tümer and Hom, 1997b).

CU CU CU CU CU CU #Ru #

I I I I I I l I I I / I l I I I I I I I I I I I

7 8 9 1 2 1 0 1 11192021 22 23 5 6 3 4

insertions or deletions of varying sizes (Chelly et al., 1993; Mercer et al., 1993; Vulpe et al.,

1993). In a study of 41 unrelated patients presentiag with the ciassical form of Menkes

disease, 1 9 out of 4 1 mutations were due to insertion or deletions (Tiimer et al., 1997a). The

two Iargest of these deletions are a 14 bp deletion in exon 10 and a 10 bp deletion in exon 22.

These deletions and others are predicted to alter the reading h m e of the gene, leading to the

introduction of a stop codon and premature truncations of the protein. Alternatively, these

mutations may lead to the skipping of subsequent mutation-bearing exons, resulting in an

internally deleted protein which may retain some function as has been reported for a 2 bp

deletion in exon 7 @as er al., 1994).

The snidy by Tümer el al. (Tümer et al., 1997a) also discovered that approximately

half of the mutations they identified were Iocated in exons 7-10 and half of these mutations

affected exon 8 alone. Furthemore, five of the twelve point mutations identified in another

series also affect exon 8 (Das et al., 1994). Exon 8 encodes a region of the protein between

metal binding domain 6 and the first transmembrane segment of the protein. Although no

specific function has been attributed to this region in the protein, it may play a role in the

precise localization of the metal binding domain relative to the ATPase core. The

importance of this region of the protein is demonstrated by the finding that individuals

carrying mutations in exon 8 prescnt the classical severe form of Menkes disease (Tümer et

al., 1997a). In contrast, only two mutations have been found in the corresponding region of

the Wilson disease gene (Figus et al., 1995). The vast difference in the fiequency of exon 8

mutations between the two genes could indicate that, despite their sirnilarities, these proteins

may have significant differences in their mechanism of action.

1 .S. 1.2 Clinical and Biochemical findings

Menkes disease was originally described by John Menkes and coworkers in 1962

during their study of five patients from the same family who al1 died before uiree years of

age (Menkes et al., 1962). Patients affected by the classical severe form of the disease

originally described by Menkes and coworkers are unable to absorb copper and hence suffer

from the effects of copper deficiency, As observed by Menkes, Menkes disease has a very

early age of onset with untreated patients usually dying before the age of three. The patients

in the original study were al1 delivered nonnally and had normal birthweights. However, in

the weeks following birth, several phenotypes were observed wbich have now become the

hallmarks of Menkes disease. It was noted that al1 patients gained very little weight despite a

normal diet, and al1 had hair abnormalities either from birth or developing soon thereafter.

These abnormalities consisted of hair that was coarse and brinle and was white in color due

to depigmentation. Microscopic examination of individual hair fibers revealed several gross

abnormalities, they were either twisted (pili torti), Eractured at various intervals (trichorrexis

nodosa), or of varying caliber (monilethrix). However, despite these abnormalities, the level

of sulfhydrys in the hair were found to be normal (Menkes et al., 1962). The presence of

these haïr abnormalities in Menkes patients Ied to the disorder also being referred to as

kinky-haïr syndrome.

Other case studies in the years following Menkes' initial description of the disorder

expanded the list of clinical symptoms (Table 1.7) and have identified a moderate fonn, a

late onset form, and a mild allelic form (occipital hom syndrome, OHS) of the disorder

Table 1.7 Symptoms of Classical Menkes Disease and

Occipital Horn Syndrome (OHS)

Classical Menkes OHS Connective Tissue Symptoms

Tortuous vessels + + Skeletal changes + ++ B ladder diverticulae t ++ Loose skin + -I- Loose joints + +

Neurologie Symptoms Mental retardation Convulsions Hypo thennia Feeding dificulries Muscle tone changes

O ther Symptoms Facial dysmorphism Abnonnal hair Hypopigmentation

Laboratory Findings Serum copper 4 4 Semm ceruloplasmin 4 4 Intracellular copper accumulation +

Life Expectancy <3 years NK' Adapted from (Tümer and Hom, 1997b). ' ~ i f e expectancy for OHS patients is not known. AI1 known OHS patients are still alive with the oldest being 53. N D = Not Detennined, NK = Not Known.

(Danks, 1988; Danks et al., 1972; Hom et al., 1995; Peltonen et al., 1983; Procopis et al.,

1981). Although most patients with OHS have only mild deficiencies, several cases with

more severe symptorns such as psychomotor retardation and seizures have been described

(Mentzel et al., 1999; Wakai et al., 1993). The patients were also found to have extremely

low levels of copper in the senun, brain, and liver, and extremely low ceruloplasmin levels.

Typicai copper measurements fiom Menkes patients are summarized in table 1.8. Attempts

to correct the problem by oral administration of copper failed; however, intravenous injection

of copper was able to nonnatize ceruloplasmin levels and copper clearance fiom the serurn

was also norrnalized (Danks et al*, 1972). This and other evidenced strengthened the idea

that the basic biochemical defect was a severely compromised ability to absorb intestinal

copper, compounded by disturbances in the intracellufar transport and traficking of copper

(Danks et al.. 1973; Herd et al., 1987). Cultured fibroblasts fkom Menkes patients have been

found to accumulate copper in the f o m of copper metallothionein and metallothionein

induction in cultured muscle cells from these patients is elevated relative to normal ceIls

(Beratis et al., 1978; Chan et al., 1978; Goka et al., 1976; Herzberg et al., 1990; Onishi et al.,

1 980; Riordan and Jolicoeur-Paquet, 1 982).

The clinical symptoms of classical Menkes disease can be attributed to a deficiency

of developmentally important cuproenzymes such as lysyl oxidase, tyrosinase, cytochrome c

oxidase, dopamine fi hydroxylase, CdZn superoxide dismutase and amine oxidase (table 1.3)

(Danks, 1995; Menkes, 1988). Lysyl oxidase is an essential enzyme needed for the

crosslinking of connective tissue; a deficiency in this enzyme would lead to weakened

comective tissue, which could lead to the comective tissue defects, such as arterial rupture,

Table 1.8 Typical Copper Measurernents in Normal Adults and

Menkes Disease Patients

Menkes Patient Normal Infant (3-12 Months) (3-12 Months)

Serurn ceruloplasmin OD unitdm1 ~0.08 >0.25 mgk <50 >200

Semm copper (pM) <6 >12

Liver copper (pg/g dry weight) 10 - 20 70 - 140

Duodenal copper (pg/g dry weight) 50 - 80 7 -29 Adapted fkom (Danks, 1995).

observed in Menkes patients (Danks, 1995; Kuivaniemi et al., 1982; Peltonen et al., 1983).

The absence of tyrosinase could lead to the hair depigmentation obsewed in Menkes patients

and low levels of cytochrome c oxidase can result in temperature instability and seizures

(Bankier, 1995). Examination of the cuproenzyme status in hepatocytes fiom murine models

of Menkes disease reveals that they have normal activity, although the level of copper in the

liver is severely depressed (Phillips et al., 1986). This suggests that intracellular copper

transport in the liver is normal in Menkes patients.

The biochemical effects of Menkes disease suggest that the body is in a condition of

copper deficiency. Therefore, the Menkes ATPase would be expected to be present in the

intestinat mucosa where it would function to transfer copper fiom the intestine into the blood

Stream. In Menkes disease, the ATPase is non-functional, resulting in an accumulation of

copper in the intestines in the f o m of copper metallothionein, which is lost fiom the body

when these cells are sloughed off leading to a global copper deficiency. Recent studies using

immunofluorescence techniques have localized the Menkes ATPase to the golgi apparatus

under normal copper concentrations (Petris et al., 1996; Yamaguchi et al., 1996). It has also

been shown that under elevated copper conditions, the ATPase undergoes a reversible,

copper dependent relocalization corn the golgi to the plasma membrane (Petris et al., 1996).

Therefore, under normal copper conditions, the ATPase functions to pump copper into the

golgi for incorporation into cuproenzymes or to pump copper into the body from the

intestinal rnucosa. When elevated copper conditions are encountered, the ATPase is

translocated to the plasma membrane where it functions to expel copper fiom the cell. These

observations explain why fibroblasts fiom Menkes patients have nomai copper uptake

activity, but are not able to expel copper to avoid copper accumulation.

1 S. 1 -3 Current Treatmunts

Since the main problem in Menkes patients is a global deficiency of copper,

treatments have concentrated on restoring normal copper levels in the body by the

administration of copper. However, since intestinal absorption of copper is extremely low in

Menkes patients, the copper complexes must be administered parenterally. Some early

complexes which were evaluated for treatment purposes included copper chloride, copper

sulfate, copper EDTA, and copper albumin. However, it was found that these complexes are

unable to produce significant clinical improvement especially in regards to

neurodegeneration (Garnica, 1984). The only copper complex which has been used

successfully to treat Menkes disease is copper-histidine (Sarkar, 1980). Copper-histidine is a

nonnal component of human serum and has been shown to play an important rote in the

transport and uptake of copper (Mas and Sarkar, 1992; Sarkar, 198 1 ; Sarkar and Kruck,

1966). Detailed copper uptake studies conducted in vitro have suggested that copper uptake

by brain tissue is mediated by complexation to histidine and have also suggested that the

copper-histidine complex is abte to cross the blood-brain barrier (Bamea et al., 1990; Barnea

and Katz, 1990; Hartter and Barnea, 1988; Katz and Bamea, 1990).

Patients treated with subcutaneous injections of copper-histidine have shown

significant clinical and biochemical improvement (L-euder et al., 1993; Sarkar et al., 1993;

Sherwood et al., 1989). The best outcomes have occurred in patients in which treatment was

started as early as possible. These case studies have s h o w a normalization of serum copper,

ceruloplasmin, dopamine and norepinephrine lrvels after three months of treatment (Kreuder

et al., 1993). The norrnalization of dopamine and norepinephrine levels indicate that nonnal

dopamine fLhydroxylase levels may have been restored. The severity of the mutation in the

two longest lived Menkes patients who have responded very well to copper-histidine

treatrnent bas been confirmeci by DNA sequence analysis. It has been discovered that these

two patients harbor mutations in the Menkes disease gene which totally abolish the

production of a functional ATPase. In the first patient a mutation in exon 4 introduces a

premature stop codon in the third metal-domain, resulting in the production of a polypeptide

without the ATPase 'core'. In the second patient the mutation occurs in exon 12, again

leading to the introduction of a stop codon upstream of the phosphatase domain. This

mutation produces a polypeptide which contains the first four traasmembrane segments, but

Iacks the remainder of the ATPase 'core' (Tiimer et al., 1996).

Despite significant clinical and biochemical improvements in these patients,

comective tissue disorders continue to persist, indicating that lysyl oxidase levels are not

being restored by copper-histidine injections. Altematively, the persistence of these

disorders rnay reflect the slow turnover of comective tissues. It is also likely that copper

presented in the copper-histidine complex is not available for incorporation into lysyl

oxidase. Studies camed out in brindled mice, a murine mode1 for Menkes disease, have

shown that subcutaneous injections of copper(i) in an alkyl polyether / sebacic acid solution

are able to increase dramatically the level of lysyl oxidase in these animals (Royce et al.,

1982). These findings suggest that the oxidation state of copper may influence its ability to

penetrate certain intracellular compartments. Hence the treatment of Menkes patients could

be enhanced by providing copper both in the +1 and +2 oxidation state, possibly using

copper-histidine in conjunction with another agent.

1 .S. 1.4 Animal Modeis of Menkes Disease

Animal models are indispensable tools for the study of hurnan disorders since they

permit studies which would otherwise be impossible in humans. For Menkes disease the

main animal models are the mottled mutations in mice (Danks, 1977; Danks, 1986; Danks

and Camakaris, 1983; Hunt, 1974; Xu et al., 1994). A cornparison of the symptoms found in

these animals with Menkes patients is shown in table 1.9 The series of mutants, mottled

(Mo), dappled (MO~P), tortoiseshell (Mo'"), brindled (Mobr), viable brindled ( ~ 0 ~ ~ 3 , blotchy

MO^"), macular ( ~ 0 ~ ' ) and pewter (MoPe') are usually considered allelic and display

extensive phenotypic variability. These series of mutations were identified in the 1950s as

X-tinked mutations which led to patchy coat color in females and were lethal in males. In

hemizygous males the displayed phenotypes include connective tissue disorders in blotchy

and viable brindled, prenatal lethality in dappled and tortoiseshell, severe neurologie disease

in brindled and macular, and isolated coat color defects in pewter (Levinson et al., 1994).

Early studies on the brains h m brindled males revealed that copper levels were

significantly decreased (Hunt, 1974). This led to the proposa1 that a deficiency of copper

was the primary defect in these animals and the diverse phenotypes arose fkom secondary

deficiencies in copper-requiring enzymes (Darwish et al., 1983). This idea was further

strengthened by the finding that cultured cells k o m many organs, except the liver, had

defects in copper export, which resulted in the cytosolic accumulation of copper (Dawish et

al., 1983; Packman, 1987; Sayed et al., 198 1). Defective export and accumulation of copper

are also found in cultured fibroblasts from Menkes patients (Beratis et al., 1978; Chan et al.,

1978; Goka et al., 1976; Onishi et al., 1980). The mouse orthologue of the human Menkes

Table 1.9 Features of Menkes Disease in Humans and the

Mottled Mouse Mutants

Menkes Brindled Blotchy Disease Mouse Mouse

Hair deforrnity +t + +

Hair keratin disulfide deficiency + + 4-

Arterial abnonnalities f+ + / - -+

Lysyl oxidase deficiency + + +

Li fe expectancy 1-2 years 13- 15 days 6- 18 months

Liver copper J. J. J.

Kidney copper 'r + It‘

Intestinal copper 'r 'r 'r Adapted fiom (Danks, 1995).

gene has been cloned, and resides on a region of the mouse X-chromosome which is syntenic

to the region where the human Menkes gene is located (Cecchi and Avner, 1996; Levinson et

al., 1994; Mercer et al., 1994).

1 S.2 Wilson disease

1 S.2.l Genetics of Wilson Disease

Wilson disease (OMIM 277900) is an autosomal recessive disorder of copper

transport which was first described by Kimear Wilson in 19 12 (Wilson, 1912). Wilson

disease is characterized by the toxic accumulation of copper in various tissues but primarily

the liver, kidney, and brain. The fiequency of the disease is approximately 1/100,000 but

seems to be more prevalent in Japan, Sardinia, and China (Reilly er al., 1993; Sternlieb,

1990; Türner and Horn, 199%). Early genetic studies assigned the Wilson disease gene to a

single locus by linkage to the esterase D gene (Bull et al., 1993; Frydman et al., 1985) while

Iater studies placed the gene close to a cluster of polymorphic markers in the q14.3 band of

chromosome 13 (Bowcock et al., 1987; Bowcock et al., 1988; Yuzbasiyan-Gurkan er al.,

1 988). Following these studies, the Wilson disease gene (ATP7B) was cloned independently

by two groups (Bull et al., 1993; Tanzi et al., 1993). The Wilson disease gene consists of 22

exons (fig. 1 .a), with exon 2 1 being expressed in the liver and exon 22 being expressed in the

kidney (Cox, 1995; Petrukhin et al., 1994; Thomas et al., 1995). The exons range in size

from 77 bp to 2355 bp (Petrukhin et al., 1994). The ATP7B transcript is expressed at a high

level in the liver and kidney, with a lower level being detected in the lung and placenta (Bull

el al., 1993). The pattern of expression correlates well with the observed clinical and

biochemical features of the disease (see section 1.5.2.2). The gene is predicted to encode a

- Transmernbrane segments

Cu - Copper binding repeats

PD - Phosphatase domain

C - Conserved CPC motif

O - Phosphorylation domain

A - ATP binding domain

1

Figure 1.8 Genornic Structure of the Wilson Disease Gene (ATP7B). Vertical lines indicate the positions of the introns, the numbers indicate the positions of the exons.

I I I I I I I I I I I I I I I I I I I I I

2

Cu Cu Cu Cu Cu Cu 1 I 3 5 6 7 4 8 910111213141

copper transporting P-type ATPase which has many features in common with other cation

transporting P-type ATPases as well as several bactenal heavy metal transporters (see section

1.4.2).

In contrast to the Menkes gene, most of the mutations in the Wilson disease gene do

not involve large insertions or deletions. Missense mutations in the Wilson disease gene

have been found in al1 exons, with the exception of exons 1-5, which encode the six amino-

tcrminal metal binding motifs, and exon 21, which encodes the carboxy-terminal tail of the

protein (Shah et al., 1997; Thomas et ai., 1995). Many other mutations in the Wilson disease

gene, including small insertioddeletions, nonsense, hmeshifi, and splice site mutations have

been found and are summarized elsewhere (Chuang et ai., 1996; Loudianos et ai., 1996;

Nanji et al., 1997; Shimizu et al., 1995; Thomas et al., 1995). From these studies, it is clear

that the most common missense mutation in many populations is His1069Gln which is

present in about 38% of homozygous Wilson disease patients. The mutation occurs in a

conserved Ioop motif (SEHPL), which is adjacent to the conserved phosphorylation motif

DKTG. No specific function has yet been attributed to this motif, but it seems critical for the

correct fiinction of the heavy metal ATPases, as it is absolutely conserved in the Wilson,

Menkes, and CopA (bacterial copper ATPase) proteins (Odermatt et al., 1993; Petrukhin et

al., 1993; Tanzi et al., 1993).

The high fiequency of the His 1069Gin mutation has permitted a genotypelphenotype

correlation for this mutation. In one study, patients hornozygous for the His1069Gln

mutation were found to have a later average age of onset as compared to the heterozygous

patients, 20 years versus 15 years (Shah et al., 1997). In addition, al1 homozygotes were

found to have an equal incidence of bepatic and neurologic symptoms. The His1069Gln

mutation was also found at high fiequency in a study conducted on Mediterranean

populations (Figus et al., 1995). This study also found that the Hisl069Gin is always

associated with the same haplotype suggesting that it most likely arose from an ancient

mutational event (Figus et al., 1995). The molecular basis of the adverse effects of the

His1069Gln mutation has recently been investigated. Recent studies have s h o w that

transfection of the Wilson cDNA containhg the His 1069Gh mutation into mouse fibroblasts

gives rise to a protein with a 5-fold shorter half-life, which is mislocalized to the endoplasmic

reticulum (Payne et al., 1998a). The authors also noted that the mislocalization was

temperature-sensitive and suggest that misfolding followed by degradation constitutes the

molecular basis of the defect in these Wilson patients.

1 .S. 2.2 Ciinical and Biochemicaljïndings

Wilson disease results fiom the accumulation of toxic levels of copper in various

organs due to a severe deficiency in the excretion of copper fiom the body. Wilson disease

displays extensive clinical heterogeneity and has a variable age of onset which can range

from the age of 8-9 years to the mid-50's. The progression of the disease can be roughly

divided into three distinct clinical phases. in the first phase, copper begins to accumulate in

the cytoplasm of hepatocytes due to impaired biliary excretion of copper (Strickland et al.,

1973). In the second phase, as more copper is absorbed, the cytosolic concentration of

copper rises resulting in the induction of large amounts of metallothionein and to the

chelation and storage of excess copper (Hunziker and Stemlieb, 1991). The high levels of

cytosolic copper in hepatocytes eventually leads to necrosis, resulting in the release of large

amounts of copper into the bloodstream. In the final stage of the disease, increasing arnounts

of copper begin to accumulate in other organs sucb as the brain, kidney and comea. The

sudden influx of large amounts of copper into the bloodstream results in oxidative damage to

erythocyte membranes, leading to hemolytic anemia @obyns er al., 1979; Mowat, 1994).

Wilson disease patients can also be divided into one of three groups, depending on what

combination of symptoms are present. Patients may either display hepatic symptoms,

neurologic symptoms, or both.

The deposition of copper in the comea gives rise to duil-yellow Kayser-Fleischer (K-

F) rings which are a diagnostic hallrnark of Wilson disease (Scheinberg and Stemlieb, 1984).

The presence of K-F rings has been found in 95% of a11 Wilson disease patients and in al1

patients which display neurologic disorders (Danks, 1995). As a result of its prevalence in

the majority of patients, K-F rings have become a diagnostic hallrnark of the disease in the

latter stages. The deposition of copper in the brain during the latter stages of the disease

correlates with the finding that patients with an early age of onset usually display

predominantly hepatic symptoms, while those with a later stage of onset display

predominantly neurologic symptoms (S trickland et al., 1 973 ; Vulpe and Packman, 1995).

The list of symptoms relating to hepatic dysfunction is extensive and varied. These include

jaundice, asyrnptomatic cirrhosis, subacute hepatitis (non-A or B) and chronic aggressive

hepatitis (Mowat, 1994). The neurologic syrnptoms in Wilson disease patients c m include

deteriorating coordination, tremors, dysartbria, drooling, personality changes, dementia,

slurring of speech, ngidity and behavioral problems (Mowat, 1994; Strickland and Leu,

1975). The neurologic symptoms in Wilson disease can be attributed to cerebellar or

extrapyramidal involvement or a combination of both (Dobyns er al., 1979).

The biochemical findings in Wilson disease patients are consistent with a disturbance

in the excretion of copper and, pnmarily, a defect in the biliary excretion of copper

(Strickland et al., 1973). Typical copper measurements in Wilson disease patients are

summarized in table 1.10, along with those in normal adults. Urinary copper levels in

Wilson disease patients are elevated due to the accumulation of copper in the kidneys, which

leads to renal dysfunction in the form of albuminuria and renal rickets (Walshe. 1989). Since

incorporation of copper into cerulopIasmin is also impaired in Wilson disease, there is a

greater proportion of copper bound to albumin and low molecular weight complexes in the

serurn; however, the overall serum concentration of copper is lowered (Barrow and Tanner,

1988). Recent immunohistochemical studies have showm that the Wilson disease ATPase is

Iocalized to the tram-golgi network under steady-state conditions (Hung et al., 1997; Nagano

et ai., 1998). Furthemore, as also observed for the Menkes disease ATPase, these studies

have shown that the ATPase undergoes a rapid, copper dependent relocalization fiom the

golgi to a cytosolic vesicular cornpartment, as well as to the plasma membrane.

The function of the ATPase in the trans-golgi is most likely to transport copper fiom

the cytoplasm into this cornpartment for incorporation in secreted, copper-containing proteins

such as ceruloplasmin. This idea correlates with the finding of low ceruloplasmin levels in

Wilson disease patients. The relocalization of the ATPase to a vesicuku compartment or the

plasma membrane in response to elevated copper levels would lead to efflux of copper fiom

the ceI1, either via the bile (through the fusion of vesicles with the canalicular membrane) or

across the plasma membrane (Dijkstra et al., 1996; Hung et al., 1997). There is also one

recent report which has localized the Wilson disease ATPase to the mitochondria, where it

Table 1.10 TypicaI Copper Measurements in Normal Aduits and

Wilson Disease Patients

Wilson Disease Normal Adult Serum ceruloplasmin OD unitdm1 O - 0.25 0.25 - 0.49 mg/L O - 200 200 - 400

Serum copper (CIM) 3 - 10 1 1 -24

Urinary copper (&24 hours) 100 - 1000 40

Liver copper (pg/g dry weight) 200 - 3000 20 - 50 Adapted fiom (Danks, 1995).

has been suggested to provide copper for incorporation into cytochrome oxidase; however,

this bas yet to be coa fmed by others (Lutsenko and Cooper, 1998).

1.5.2.3 Current Treatments

Treatments for Wilson disease patients have concentrated on the use of chelators to

mobiIize copper fkom the centers of accumulation and promote its excretion from the body.

The first chelator used widely for the treatment of Wilson disease was BAL (2,3-

dimercaptopropanol) penny-Brown and Porter, 195 1). in this initial study of five patients,

the authors observed a significant reversa1 of tremors and rigidity following treatment with

BAL. However, these improvements were only observed afier longterm use and were less

significant in patients with more acute cases of the disease (Cartwright et al., 1954; Matthews

et al., 1952). Furthermore, patients had to deal with painful intramuscular injections and

often side effects. These concems led to the development of D-penicillamine which

improved upon BAL in every respect (Walshe, 1956). It could be administered orally, is less

toxic and more effective than BAL. The standard dose of D-penicillamine for adults is 500

mg given twice daily, with close monitoring of urinary copper. On this dose, urinary copper

excretion can increase to between 4 and 5 mg per day (Sass-Kortsak, 1975).

Treatment with D-peaicillamine usually lasts for a lifetime and produces significant

clinical and biological improvements in a large number of cases. In some patients,

neurologie syrnptoms may disappear, liver fùnction may retum to normal, and Kayser-

Fleischer rings usually fade and then disappear (Sass-Kortsak, 1974). However, despite its

vast irnprovement over BAL, some severe side effects have been reported for D-

penicillarnine as weli. The most common side effects in the early stages of treatrnent include

fever, anorexia, pyridoxine deficiency, and a mild depression of leukocyte and platelet counts

(Mowat, 1994; Sass-Kortsak, 1975; Scheinberg, 1968). In very few cases, serious toxic

effects may include systemic lupus erythematosus, and Goodpasture's syndrome (Mowat,

1994; Sass-Kortsak, 1975). D-penicillamine has also been linked to changes in the

crosslinking of collagen and elastin, leading to serious skin and c o ~ e c t i v e tissue disorders

(Iozurni et al., 1997; Narron et al., 1992; Pasquaii-Ronchetti et al., 1989). There are also at

least w o cases on record of asymptomatic Wilson disease patients experiencing severe

newological deterioration soon after initiation of D-penicillamine therapy (Brewer et al.,

1994; Glass et ai., 1990). There is also a danger in intemipting D-penicillamine treatment

once started. There are several cases on record in which patients have stopped taking their

tablets and irreversible liver damage has developed within two years (Scheinberg et al.,

1987; Walshe and Dixon, 1986).

A number of other chelating agents have been developed and used successfully in

cases where patients develop toxic reactions to D-penicillamine or treatment is intermpted.

These inctude trientine (triethylenetetramine dihydrochloride) (Scheinberg et al., 1987;

Walshe, 1 982), tetrathiomolybdate (Brewer et al., 1994a; Walshe, 1984), and zinc therapy

( Alexiou et al., 1 985; Hoogenraad, 1998; Hoogenraad et al., 1979; Hoogenraad et al., 1978;

Van Caillie-Bertrand et al., t 985). Trientine may be a viable alternative to D-penicillamine

in some cases; no toxic side effects have been observed in 20 patients using the drug for up to

13 years (Scheinberg et al., 1987; Walshe, 1982). Trientine has also been employed

successfully in one female patient who had previously been on D-penicillarnine. Trientine

was administered during pregnancy without adverse affects on the fetus (Devesa et al.,

1995). Trientine may be an effective treatment when D-penicillamine has been withdrawn;

however, commercial availability of the compound is still a problem. in addition, trientine

and D-penicillamine seem to act on different pools of copper. D-penicillamine is able to

mobilize copper from the liver, but cornpetes relatively poorly for copper bound to albumin

(Sarkar et al., 1977). In contrast, trientine cannot mobilize copper €rom the liver, but is

effective in competing for copper bound to albumin (Satkar et al., 1977). Both dnigs are

able to mobilize copper fiom the kidneys, promoting its removal through urinaq excretion.

Tetrathiomolybdate is another chelator with great potential which has been used to

treat Wilson disease. It is an extremely potent chelator of copper which can cause severe

copper deficiency in normal rats within 1 week and is, therefore, not suited for long-tenn

treatment of Wilson disease (Brewer et al., 1991; Mason et al., 1989; Walshe, 1989).

However, tetrathiomolybdate can be used for initial decoppering followed by maintenance

with zinc. At the very least, tetrathiomolybdate is a possible alternative in cases where

toxicity to D-penicillamine develops. Unlike other chelating agents which promote the

mobilization and excretion of copper through the urine, zinc therapy promotes fecal copper

excretion. Oral zinc therapy is thought to promote copper excretion by inducing the

synthesis of metallothionein in the intestine, thereby blocking the absorption of copper

(Hoogenraad, 1998; Hoogenraad et al., 1979; Hoogenraad et al., 1978). Although zinc has a

greater ability to induce metallothionein synthesis than does copper, metallothionein has a

higher affinity for copper than zinc (Lipsky and Gollan, 1987). As a result the newly

synthesized metallothionein sequesters copper in the upper intestine, preventing its transport

into the portal circulation. The sloughing off of intestinal cells into the lumen results in the

elevated copper levels observed in the feces of zinc-treated Wilson patients (Brewer et al.,

1983; Lipsky and Gollan, 1987). Patients with acute liver failure and encephalopathy are

usually beyond the help of chelation therapy. Ln these cases liver transplantation may

provide the only hope for survival. The results of 57 Wilson disease patients who underwent

liver transplantation showed chat there was a 72% survival rate after the first year, with 4

patients requiring retransplantation (Mowat, 1994; Schilslq et al., 1994).

1 -5.2.4 Animal Models

Several animal models for Wilson disease have been identified and al1 display clinical

and biochemical symptoms which are very close to those found in human Wilson disease

patients. These animal models include the toxic miik (tx) rnouse (Rauch et al., 1986; Rauch

and Wells, 1999, the Long Evans Cimamon (LEC) rat (Li et al., 1991) and bedlington

temers (Su et al., 1982; Twedt et al., 1979). The biochemical features of these animals, as

compared to Wilson disease, are summarized in table 1.1 1. The toxic milk mouse and the

LEC rat display very similar clinical and biochemical features. The toxic milk mutation was

first recognized as an autosomal recessive trait which was lethal to the pups of affected

females due to a severe copper deficiency in their milk (Rauch, 1983). In affected fernales,

copper accumulates in the liver, leading to histological changes and cirrhosis in the absence

of any neurological effects. It has been shown that the excess copper which accumulates in

the toxic mice is in the fonn of copper metallothionein (Koropatnick and Cherian, 1993;

Rauch et al., 1986).

Table 1.1 1 Cornparison of Biochemical Findings in Wilson Disease and its

Animal Models: Toxic Milk Mouse and Bedlington Temers

Wilson Bedlington Toxic Milk Disease Terrier Mouse

Hepatic copper accumulation +t -ii+ ++ dg dry weight) 200-3000 2000-12000 300-400

Reduced biliary copper excretion f.t + NK

Reduced copper incorporation into ceruloplasmin fft f+ $-t.

Liability to hemolytic crisis + ++ +

Chronic Iiver ceIl damage -+-t + / - tt-

Copper overflow to other tissues ++ + / - + / - Adapted From (Danks, 1995).

Analysis of metallothionein mRNA levels in the liver of these anirnals shows that

they are normal in younger tx mice and highly increased in adult tx mice (Mercer et al.,

1992). This finding suggests that metallothionein regulation is normal in these animals and

excess copper is leading to the induction of meta!lothionein. Morphological examination of

livers from adult tx mice has revealed a pattern of damage which is significantly different

from that observed in Wilson patients and bas led to some doubts as to the validity of the t.r

mouse as a model for Wilson disease (Biempica et al., 1988). However, recent studies have

mappcd the tx mutation to chromosome 8 where it has been shown to reside in the rnurine

orthologue of the human Wilson disease gene confirming that the fx mouse is, indeed, a valid

model for Wilson disease (Raucb and Wells, 1995; Reed et al., 1995; Theophilos et al.,

1996). hterestingly, the fourth metal binding domain in the mouse and rat orthologues of the

Wilson disease gene has been altered such that one or both of the conserved cysteine residues

are no longer present (Theophilos et al., 1996; Wu et al., 1994).

The LEC rat is probably the most used and hence best understood model of Wilson

disease. This inbred rat strain was established from a closed colony of normal Long-Evans

parental rats through successive generations of sibmating (Yoshida et al., 1 987). LEC rats

suffer from spontaneous hepatitis, hemolytic anemia and jaundice at approximately 4 months

of age, followed by death in 50% of animals due to fulminant hepatitis (Li et al., 1991;

Yoshida et al., 1987). The surviving 50% suffer fiom chronic hepatitis and usually go on to

develop livcr cancer (Masuda et al., 1988; Powell, 1994; Sawaki et al., 1990; Yoshida er al.,

1987). In contrast to Wilson disease, neurological abnormalities resulting from copper

accumulation in the brain are rarely seen in LEC rats. As in Wilson disease, LEC rats

accumulate copper in the liver, but semm ceruloplasmin activity is severely depressed (Li et

al., 199 1 a; Schilsky et al., I994a). Genetic analysis has shown that the mutation causing the

spontaneous hepatitis in LEC rats resides in a gene on chromosome 16 which is the rat

orthologue of thc Wilson disease gene (Muramatsu et al., 1994; Wu et al., 1994). The

mutation is predicted to give nse to a premature stop codon leading to termination of the

protein prior to the ATPase 'core'. Recently, a novel splice variant of the Wilson disease

gene in rats has been identified (Bo rjigin et a[., 1999). This variant is expressed in the brain

and eye and its activity is apparently regulated by the circadian system. Tbe functional

significance of this variant has yet to be identified.

The bedlington terrier mode1 is probably the least understood and, therefore, the least

used of the Wilson disease animal models. Copper toxicosis (CT) in bedlington temers is an

autosomal recessive disorder characterized by a defect in the biliary excretion of copper,

leading to copper accumulation in the liver and to cirrhosis (van de Sluis et al., 1999).

Despite its similarity to Wilson disease, CT in bedlington temers does have some significant

differences. Bedlington temers with CT do not show neurological abnormalities, Kayser-

Fleisher rings are absent and ceruloplasmin levels are observed to be normal (Hunt et al.,

1986; Twedt et al., 1979). Genetic analysis has failed to locate mutations in the ATP7B gene

and no linkage has been shown to esterase D (ESD) or retinoblastoma (RB), two genes which

are tightly linked to ATP7B in humans and mice (Reed et al., 1995; Scheffer et al., 1992;

Yuzbasiyan-Gurkan et al., 1993). However, acute hepatitis in the LEC rat is also not linked

to RB, suggesting that not al1 mammalian species share a syntenic relationship beween RB

and ATP7B (Sasaki et al., 1993). Taken together these results suggest that CT in bedlington

terriers may involve a gene which is not homologous to the Wilson disease gene in humans.

In support of this possibility, recent mapping studies have determined that the CT locus in

bedlington terriers maps to a region on dog chromosome 10 which is syntenic to a region on

chromosome 2 in humans, thereby elirninating ATP7B as a possible candidate gene (van de

Sluis et al., 1999). In lieu of these observations, the exact cause of CT in bedlington terriers

remains unknown.

1.6 Project Rationale

The discovery of the genes responsible for Menkes and Wilson diseases, and the

copper transporting ATPases they encode, has advanced our understanding of intracellular

copper trafficking. These two ATPases are the first members (in humans) of a rapidly

growing class of heavy metal iransporting ATPases which have recently been discovered in a

variety of organisrns. Although similar to other cation transporting P-type ATPases, the

heavy metal ATPases have several features which distinguish them from non-heavy metal

transporting ATPases (see section 1.4). Among these are the amino acid motifs (CPC and

SEHPL) which are cntical for proper fiinction of the ATPase, but are absent in the non-heavy

metal ATPases. The other unique and probably most interesting feature of the heavy metal

ATPases is the presence of a large amino-terminal domain which contains between 1 and 6

copies of the heavy metal associated (HMA) domain. The MNK and WND proteins both

contain six HMAs at their amino-terminus, while those from bacteria and lower animais

contain between 1 and 2 copies.

Comparison of the amino acid sequence from the HMAs in the MNK and WND

proteins with that from bacterial proteins clearly indicates that the human HMAs are derived

from their bacterial counterparts ( P e t W i n et al., 1994). However, one obvious difference

between the human proteins and their bacterial counterparts is the number of HMAs present.

Therefore, one of the underlying objectives of this work is to determine the significance of

having six HMAs in the human protein when only one or two seem to be sufficient in the

bacterial and yeast proteins. Several biochemical studies have hinted at the possibility that

the HMA domains may have roles in the ATPase additional to binding metals. Mutational

and functional studies have shown that the HMAs in the WND and MNK proteins are not

functionally equivalent and that not al1 are needed for the transport of copper to occur

(Forbes et al., 1999; Hung et al., 1997; Payne and Gitlin, 1998). Furthemore, in vivo

subcellular localization studies using fluorescent antibodies have s h o w that both the MNK

and WND proteins reside in the trans-golgi network and both undergo translocation to the

plasma membrane and a cytosolic vacuolar cornpartment under elevated copper

concentrations (Hung et al., 1997; Nagano et al., 1998; Petris er al., 1996). This unique

ligand-rnediated response has led to the hypothesis that the amino-terminal domain in the

iMNK and WND proteins serves as a 'sensor' for cytoplasmic concentration of copper which,

in tuni, could regulate the activity of the ATPase.

Both the HMAs and the large amino-terminal metal binding domain in the MNK and

WND proteins are novel protein entities which have not been characterized in any other

known protein. A thorough understanding of their fimction in the context of the whole

ATPase can only be gained following the characterization of the metal binding sites as well

as the structural dynamics of the amino-terminal domain as a whole. With this objective in

mind, the WCBD was systematically characterized, starting with the cloning, expression and

purification of the domain. Once purified, the WCBD was analyzed by determining i) its

metal binding characteristics, ii) its relative affinity for copper, iii) the structural effect of

metal binding, and iv) the structural properties of the copper binding sites. The results from

such an analysis may then be combined fiom fùnctional and mutational studies camied out in

systems using the entire ATPase. in the absence of a three dimensional structure, the metal

binding and structural dynamics studies will provide a wealth of information on this novel

and previously uncharactenzed domain. These studies will increase Our knowledge of the

roIe of this domain in the ATPase and could lead to the development of new pharmaceutical

agents for the treatment o f Menkes and Wilson diseases. Furthexmore, these studies will also

point the way to deciphering the structure and function o f other heavy metal hansporting

ATPases by providing a mode1 for the structure and iùnction of the large amino-terminal

metal binding domain.

CHAPTER 2

Materials and Methods

2.1 DNA Preparation

2.1.1 Ceii lines

Two separate ce11 lines were used for the maintenance of plasmid DNA and for the

expression of GST fusion proteins. E. cofi strain DHSa (supE44 AlacU169 (480 facZAM 15)

hsdR 1 7 recA 1 end4 1 w A 9 6 thi-1 relA 1) was used for the maintenance and production of

plasmid DNA for the construction of the GST expression vector constnicts, since they were

found to lead to higher purity plasmid preps (Hanahan, 1983). E. coli strain BLSl(DE3)

(hsd' gai (AcIrs857 indl Sam7 nin5 lacUVS-T7 gene 1)) were used for the expression of

GST fusion proteins as recommended in the GST Gene Fusion System Handbook

(Amersham-Pharmacia Biotech) (Studier and Moffatt, 1986).

2.1.2 Preparation of competent BLZI(DE3) and DHSa Celis

Preparation of competent BL2 l(DE3) and DH5a cells was carried out using the

calcium chloride method (Sambrook et al., 1989). Briefly, cells were grown in LB broth (1%

W/V peptone, 0.5% wlv yeast extract, 1 % w/v NaCl) to an of 0.4-0.6. The cells were

then cooled on ice for 10 minutes and harvested by centrifiigation for 10 minutes at 3000 x g,

4 ' ~ . The pellets were resuspended in ice-cold 0.1 M CaClz. The cells were then centrifûged

as above and resuspended in O. 1 M CaC12, 10% glycerol (2 ml / 50 ml original culture) and

stored at -70'~.

2.1.3 Transformation of competent BLZI(DE3) and DHSa Ce&

Calcium chloride competent cells were transformed by the heat-shock method as

described in the TA Cloning Kit (Invitrogen). Briefly, 50 pl of competent cells were thawed

on ice and mixed with 1 pl DNA @urified plasmid or tiom a ligation reaction), 2 pl 0.5 M f3-

Me and incubated on ice for 30 minutes. Following this incubation, the cells were heat-

shocked for 30 seconds in a 4 2 ' ~ waterbath and returned to ice for 2 minutes. 450 pl of

prewarmed SOC media (2% w/v peptone, 0.5% w/v yeast extract, 0.05% w/v NaCl, 20 mM

glucose, 2.5 mM KC1, 10 rnM MgC12) was added to the cells and left to recover for 1 hour at

3 7 ' ~ before they were selected on LB-ampicillin (100 mg/L) agar plates.

2.1.3.1 Preparation of glycerd stocks

A single positive clone of BL21(DE3) aansfonned with either pGEX-WCBD or

pGEX-WCBD(P) was inoculated into 5 ml LB-ampicillin and grown overnight with shaking

at 3 7 ' ~ . To 1 ml of the ovemight culture was added 0.5 ml of sterile glycerol stock solution

(65% v/v glycerol, 0.1 M MgSOs, 0.025 M Tris-HCI, pH 8.0) was added. The mixture was

split into aliquots (250 pi) and stored at - 7 0 ' ~ until needed for expression. Glycerol stocks

prepared in this manner were stable for >l year if kept at -70'~.

2.1.4 Purijication of plasmid DNA

SmaIl scale preparation of plasmid DNA was carried out using the QIAprep Minprep

Kit (QIAGEN) which uses the modified alkaline lysis method of Bimboim and Doly

(Birnboim and Doly, 1979). Transformed E. Coli were grown ovemight in 5 ml of LB broth

supplemented with ampicillin (100 mg/L). The following day, cells were hawested by

centrifugation (5 minutes, 3000 x g, 4 ' ~ ) and processed as specified in the QIAprep miniprep

handbook. Double stranded plasmid DNA was quantitated using its absorbance at 260 nm

( 1 .O unit = 50 pg/ml double stranded DNA) (Sambrook et al., 1989).

2.1.5 DNA agarose gel electrophoresis

Gel electrophoresis was used to determine the size and purity of purïfied plasmids

and for the separation of different-sized DNA fragments pior to purification. Agarose slab

gels using the TAE buffer system were used (Sambrook et al., 1989). Al1 1% agarose gels

were prepared in 0.5X TAE buffer containing 1 pg/ml ethidium bromide and were run at a

constant voltage (100 V) until adequate separation was achieved. Ethidium bromide stained

nudeic acid bands were visuaiized using ultraviolet iight.

2.1.6 Purijication of DNA restriction fragments from agarose gels

DNA hgments were separated by agarose gel electrophoresis and purified using the

SephaglasTM BandPrep Kit (Amersham-Pharmacia Biotech), which is a modification of the

Na1 method (Vogelstein and Gillespie, 1979). Purified DNA fragments were quantified

using their absorbance at 260 nm and used without further purification in subsequent

procedures.

2.1.7 DNA Sequencing

Double-stranded plasmid DNA was sequenced by the dideoxy sequencing method

(Sanger et al., 1977) using the T7 SequencingTM Kit (Amersham-Pharmacia Biotech)

according to the manufacturer's directions. The purity of the plasmid preparations was

checked prior to sequencing by measuring the OD (260 nm 1 280 nm) ratio and ensuring that

it was at least 1.8.

2.1.8 Restriction envme digests and ligation reactiuns

Restriction and modiQing enzymes used in these reactions were obtained from

Amersham-Pharmacia Biotech and New England Biolabs (NEB). The reactions were carried

out under the conditions specified by the manufacturer, using the buffers supplied. Al1

Iigation reactions were carried out using T4 DNA ligase fiom NEB. The vector and insert

were ligated at a 1 : 1, 1 :3, 1 5 , and 1 : 10 molar ratio starting with 50 ng of vector and the

corresponding amount of insert calculated using equation 1.

(Y bp insert)(SO ng vector) X ng insert =

(size in bp of vector)

The value obtained for X fiom this equation gives the amount of insert for a 1 : 1 molar ratio.

Multiples of X (3X, 5X and 10X) were used for the other ratios. Generally 400 units of

ligase (NEB unit definition) were added to each Iigation reaction, total reaction volume was

20 pl. Ligation reactions behveen fragments of DNA with compatible ends were carried out

ovemight at 16 '~ . Usually, 1 pl of the ligase reaction was used to tmnsform bacteria and the

remainder was stored at -20'~.

2.2 Construction of Expression Vectors

2.2.1 Construction o f p GEX- WCBD

The 2-kilobase cDNA encoding the Wilson disease protein copper binding domain

(WCBD), was produced in two fragments from total human liver RNA using reverse

transcriptase-polymerase chain reaction (RT-PCR). This reaction was performed by J.R.

Forbes in the laboratory of Dr. D.W. Cox. Primers were designed to create a BamHI

restriction site at the 5' end and a Sul 1 site at the 3' end. The two cDNA fragments

overlapped at an Nsil restriction site. The pnmers used for the 5' fragment were (5' side) 5'-

TATCGGATCCATGCCTGAGC-AGGAGAGA-3' (5'-BamHI), and (3' side) 5' -

AACTTTAAAATTCCCAGGTGG-3' (OL- 1); 3' fragment (3' side) 5'-

ACTTGTCGACTCACTGCTTTATTTCCATTTTG-3' (3'-Sa1 1), and (5' side) 5'-

GGAATGCATTGTAAGTCTTGGCG-3' (OL-2). A stop codon (bold) was inserted just

before the San site; restriction sites are underlined. The two fragments were digested with

BarnHI and .Msi 1 or Sul 1 and Nsi 1, respectively, and following purification fkom an agarose

gel, ligated into the BamHi and Sul 1 sites of the GST expression vector pGEX-4-T-2

(Amersham-Pharmacia Biotech, Inc) to create the expression vector pGEX-WCBD (Fig.

2.1). This vector contains a thrombin cleavage site between the GST moiety and the WCBD.

The fidelity of the constmct was confinned by dideoxy sequencing.

2.2.2 Construction of pGEX- WCBD(P)

The WCBD DNA insert was excised from pGEX-WCBD using BamHI and Sali,

purified on a 1% agarose gel and inserted into the BamHI and Sul 1 sites of the GST fusion

expression vector pGEX-6P-2 (Amersham-Pharmacia Biotech, Inc) to create the expression

vector pGEX-WCBD(P) (Fig. 2.2). This vector contains a PreScissionTM (human rhinovirus

3C protease) cleavage site between the GST moiety and the WCBD (Walker et al., 1994).

The fidelity of the construct was confirmed by dideoxy sequencing.

C Leu - Val - Pro - Arg - J - Gly - Ser

Figure 2.1. Parental and Modifxed GST Fusion Expression Vectors. A. parental GST fusion expression vector, B GST-WCBD expression vector containing - 2 Kb WCBD cDNA insert, C thrombin protease recognition site located between GST and WCBD. Cleavage by the protease occws at the Arg - Gly bond. D thrombin 5 and 3 amino acid consensus sequences. P 1 and P2 are hydrophobie amino acids, P3 and P4 are nonacidic amino acids, P 1' or P2' are Gly

B Leu-Glu-Val-Leu-Phe-Gln- J -GIy-Pro

Figure 2.2. GST- WCBD(P) Expression Vecfor with PreScissionTM Cleavage S i t e . A GST-WCBD(P) expression vector with PreScissionm protease ckavage site between GST and WCBD. B PreScissionm protease recognition site. Cleavage occurs at the Gin - Gly peptide bond.

2.3 Expression and Purification of GST-WCBD and GST-WCBD(P)

2.3.1 Bu ffer and reagent preparuîion

Al1 buffers and reagent solutions were prepared using purified distilled water with a

conductivity of at least 18 M. Deionized urea was used in al1 buffers requiring it and was

prepared as follows: approxirnately 100 - 150 g of AG501-X8 (D) mixed bed resin @IO-

RAD) was added to 2 L of 8 M urea in distiIled water and stirred overnight at RT; the resin

was then removed from the solution by vacuum filtration. Al1 buffers not containing

reducing agents (DTT or f3-Me) were argon purged for at least 30 minutes at RT prior to use.

Vinyl, powder free gloves (Baxter Healthcare Corp) and clear plastic pipette tips were used

in al1 manipulations to avoid metal contamination. Residual metal contamination in dialysis

tubing was removed prior to use by boiling the tubing for approximately 1 to 2 hours in 5 to

6 changes of distilled water.

2.3.2 Expression of GST- WCBD and GST- WCBD(P)

An aliquot of a glycerol stock containing BL2 1 (DE3) transformed with either pGEX-

WCBD or pGEX-WCBD(P) was inoculated into 100 ml of LB supplernented with ampicillin

(100 pg/ml) and grown ovemight at 3 7 ' ~ with shaking. Following overnight growth, the

culture was divided in two and diluted into 2 x 1.5 L fiesh LB supplemented with ampicillin

and 100 PM CuS04 (Tris-buffered, pH 7.0) in 2.8 L Fembach flasks. The cultures were

grown with shaking at 3 7 ' ~ until midlog phase was reached (kW of 0.6-0.8), at which point

they were induced with 0.1 mM IPTG for 3-4 hotus. Following the induction period, the

cells were hawested by centrifugation at 4420 x g for 15 minutes, at 4 ' ~ . Tbe supernatant

was poured off and the bacterial pellets were either stored at -20 '~ ovemight or lysed

immediately.

2.3.3 Purification of GST- WCBD and GST- WCBD(P)

The bacterial pellets were resuspended in lysis buffer (20 mM Tris-HCI, pH 8.0, 130

mM NaCl, 1 mM EDTA, 5 mM DTT, 1 0% glycerol, I mM PMSF, 1 % Triton X- 1 00 and 0.1

mg/ml lysozyme) and lyseci by two cycles of fieeze-thaw. The lysate was centrifüged at

118,000 x g for 45 minutes, at 4 ' ~ . The soluble fraction was decanted and stored at 4 ' ~ .

Fusion protein which localized to inclusion bodies was solubilized and refolded, essentially

as described previously (Dombroski et al., 1992). Briefly, inclusion bodies were solubilized

by homogenization in solubilization buffer (50 mM Tris-HCI, pH 8.0,6 M Urea, 8 mM DTT,

1 mM EDTA) and the reviilting suspension was centrifûged at 1 18,000 x g for 45 minutes at

4 ' ~ . Solubilized fusion protein was refolded by extensive dialysis against refolding buffer

(50 mM Tris-HC1, pH 8.0, 20% glycerol, 5 mM DTT, 1 rnM EDTA). Refolded fusion

protein was combined with the soluble fraction. The resulting mixture was made 1% in

Triton X- 100 and applied to a glutathione Sepbarose 4B column previously equilibrated with

TEND-T (20 mM tris-HC1 pH 8.0, 130 mM NaCl, 1 m M EDTA, 5 mM DTT, 1% Triton X-

100). The column was washed extensively with TEND-T followed by TEND (same as

TEND-T but without Triton X-100). The fusion protein was eluted fiom the column with

TEND-U (20 rnM Tris-HCI, pH 8.0, 130 NaCl, 1mM EDTA, 5 mM DTT, 6 M Urea).

Protein elution was monitored by OD2so readings and samples fiom each fraction were

analyzed by SDS-PAGE.

Fractions containing fusion protein were combined and applied to a Q-sepharose fast

flow column (anion exchange), previously equilibrated with TEND-U at a flow rate of 2.2 ml

/ minute, collecting 5 ml fractions. After sample application, the column was washed

extensively with TEND-U and ODzao reading were taken on each fraction to monitor protein

content. When readings retmed to baseline values, the fbsion protein was eluted with

a linear salt gradient (130 - 250 mM NaCl). Peak fractions were analyzed by SDS-PAGE

and those containing fùsion protein were pooled and refolded as described above.

2.3.4 SDS-PA GE

SDS-PAGE was performed on a mini slab gel apparatus (Hoefer Scientific

Instruments) in the buffer system developed by Laemrnli (Laemmli, 1970). To visualize the

fusion protein as well as the WCBD, we prepared 10% separating gels overlaid with 4%

stacking gels. The reduction of disulfide bonds and the incorporation of SDS were achieved

by mixing protein samples with an equal volume of 2X loading buffer containing 0.72 M p-

Me, boiling for 5 minutes, and immediately loading onto the gel. Gels were run at a constant

voltage (175 V) until separation was achieved, as determined fiom the mobility of the dye

front. Following electrophoresis, the gel was stained in a 0.125% w/v solution of Coomassie

Brilliant Blue R-250 dissolved in 40% v/v methanol, 10% v/v acetic acid, 50% distiiled

water, with sbaking for 15 minutes at RT. The gel was destained by incubation in 10% v/v

methanol, 10% v/v acetic acid, 80% distilled water, with shaking overnight. Destained gels

were photographed using the UVP gel documentation system in conjunction with UVP-Grab-

I F " software version 1.57b (DiaMed Lab Supplies). Molecular weights of the proteins were

estimated by comparison with wide range molecutar weight marker run in paralle1 (200 - 6

kDa, NOVEX Mark 12 Standards).

2.3.5 Protein quanriration

Quantitation of the amount of fiision protein or WCBD was camed out using the

BCA Protein Assay Kit (Pierce). The assay was carried out as outlined in the instruction

manual for the standard protocol. The concentration of the protein sample was detennined

by comparison to a standard curve denved tiom BSA (O - 50 pg, run in duplicate). Results

fiom this assay agreed very closely to those obtained by amino acid analysis.

2.3.6 N- Terminal Amino Acid Sequencing

N-terminal amino acid sequencing was carried out on the purified WCBD to confirm

its identity. The samples were run on 10% SDS-PAGE gels and electroblotted ont0 PVDF

membranes (BIORAD). Electroblotting was carried out in a Hoefer electroblot transfer

apparatus (Arnersham-Phannacia Biotech) at a constant current of 0.5 A for 45 minutes using

CAPS transfer buffer (10 mM CAPS pH 1 1 .O, 10% v/v methanol) cooled with circulating

water. Following blotting, the membrane was washed in two changes of transfer buffer and

then stained with O. 1% w/v Coomassie Bnlliant Blue R-250 dissolved in 50% v/v methanol,

50% distilled water for 1-2 minutes. The membrane was then destained with 50% v/v

methanol, 10% v/v acetic acid, 40% distilled water for 5 minutes and then washed with

several changes of distilled water for 5-10 minutes. The membrane was air dned and

submitted for N-terminal sequence analysis. Seven cycles of automated amino acid

sequencing was carried out on a Porton instruments Gas-Phase Micro Sequencer mode1 PI

2090E with online PTH amino acid aoalysis using a Hewlett-Packard HPLC, mode1 1090L.

N-terminal sequencing reactions were carrïed out using Edman degradation (Matsudaira,

1989)

2.3.7 Amino Acid Analysis

Purified fùsion protein was analyzed for total amino acid content to confirm the

identity of the protein and to determine concentration. The fusion protein was hydrolyzed

with hydrochloric acid (6 M) for 48 hours and the amino acids were reacted with

phenylisothiocyanate (PITC) to form phenylthiohydantoin (PTH) amino acid denvatives.

The PTH-amino acid derivatives were analyzed and quantitated by HPLC in cornparison with

a standard mixture of PTH-derivitized amino acids (Smith, 1997). Fusion protein

concentration was determined by dividing the co~centrations of PTH-Leu, PTH-Ala, PTH-Ile

by 79, 66, and 59 respectively (corresponding to the molar amounts of each residue present

in the fusion protein) and calculating the average of the values obtained.

2.4 Removal of GST from GST-WCBD and GST-WCBD(P)

2.4.1 Thrombin cieavage of GST- WCBD

Thrombin cIeavage of GST-WCBD was either carried out on the cotumn or on the

purified hsion protein in solution. For cleavage of the column-bound fusion protein, 10

units of human thrombin (Sigma-Aldrich) / mg of fusion protein was added to 1 bed volume

of TEND (20 m M Tris-HCI, pH 8.0, 130 mM NaCl, 1 mM EDTA, 5 mM DTT) and applied

to the column. The reaction mixture was incubated at RT for 30-45, minutes followed by

elution of pure WCBD in the same buffer. Thrombin was inhibited by the addition of PMSF

to a final concentration of 1 mM. For cleavage of purified fision protein in solution, 10 units

of human thrombin / mg of fusion protein was added to the protein solution and the reaction

was incubated for 30-45 minutes. After the incubation period, thrombin activity was

inhibited by the addition of PMSF (1 rnM final concentration) and the mixture was applied to

a glutathione affinity column. The flow-through which contained the purified WCBD was

collected.

2.4.1.1 Optimizaîion of thrombin cleavage cortditions

Thrombin cleavage conditions for GST-WCBD were optimized in the following

marner to minimize the non-specific degradation of the WCBD. To a 2 ml solution of the

purified fusion protein (0.48 mg / ml) were added 9.5 cleavage units of human thrombin and

the reaction mixture was incubated at RT. Samples (50 pl) were taken out every 30 minutes

for the first 4 hours and then every hour thereafter and mixed with an equal volume of 2X

loading buffer containing 0.5 mM PMSF. The samples were boiled for 5 minutes and

analyzed on 15% SDS-PAGE slab gels.

2.4.2 PreScission- clerrvage of GST- WCBD(P)

Removal of GST fiom GST-WCBD(P) was carried out on the purified fiision protein

in refolding buffer (50 mM Tris-HCI, pH 8.0, 1 mM DTT, 20% glycerol). Briefly, 1 unit of

PreScissionm protease (Amersham-Pharrnacia Biotech) / mg fusion protein was added to the

protein solution and the reaction mixture was incubated at SOC for 16-48 hrs. The progress of

the cleavage reaçtion was monitored by removing aliquots several time intervals and

analyzing them by SDS-PAGE. Once the reaction was judged complete, the mixture was

passed over a glutathione affinity column to remove free GST and the protease (the protease

is supplied as a h i o n with GST). The flow-through contained the purified WCBD.

2.5 Characterization of the Metal Binding Properties of WCBD

2.5.1 Imnrobifized Metal Ion A ffiniw Chromatography (IMA C)

Samples of the purified fusion protein were dialyzed against MAC buffer (20 mM

NaH2P04, pH 7.0, 0.5 M NaCl) with or without 6 M urea and applied to chetating sepharose

fast flow columns (Amersham-Pharmacia Biotech) charged with either Co(ll), Ni(II), Zn(II),

Cu(II), Fe(II) or Fe(III) as per the manufacturer's instructions. Elution of the hsion protein

from the matrix was achieved either by lowering the pH to 6.0 or 4.0 or by the sequential

addition of chelators (EDTA, imidazole, BCS). Samples of each fraction were analyzed on

10% SDS-PAGE gels.

2.5.2 6 5 ~ r 1 ( ~ Blotting Assay

65 Zn(Q blotting analysis was performed essentially as described previously (Schiff et

al., 1988) with the following modifications. Samples of GST-WCBD, WCBD, GST and

estrogen receptor DNA binding domain (ERDBD) were subjected to SDS-PAGE and

electroblotted onto nitrocellulose membranes (BIORAD). Electroblotting was carried out in a

Hoefer electroblot transfer apparatus (Amersham-Pharmacia Biotech) at a constant current of

0.3 A for I .5 hours using CAPS transfer buffer (10 mM CAPS, pH 1 1.0) cooled with

circulating water. Following electroblotting, the membranes were equilibrated in metal

binding buffer (100 rnM Tris-HC1, pH 7.0, 50 mM NaCl, 1 rnM DIT) for 2 hours at RT with

shaking. The membranes were then probed with 10 pCi of ' 'z~cI~ (30 pM final Znfl1)

concentration) in 20 ml metal binding buffer, without DTT unless otherwise noted, for 1 hour

at RT, with shaking. The membranes were then washed twice (15 minutes each) with the

same buffer. The membranes were then wrapped in plastic wrap and exposed to Biomax MR

film (Kodak) for 22 hours at -70'~ with intensifying screen. Following autoradiography, the

film was developed using a M3SA X-OMAT processor (Kodak).

2 S.2.l Eflect of pH on 6 s ~ n ( ; r ~ Bloning Assay

The effect of pH on the binding of zinc to the domain was assessed using the 6S~n(lI)

blotting assay. The assay was c d e d out as described above, except that the pH of the metal

binding buffer was varied from 6.5 to 9.0 in steps of 0.5 pH units (6 separate blots). The

experiment was carried out using GST-WCBD, WCBD, GST and ERDBD.

65 2.5.2.2 Eflect of DTT on Zn(Z1) Blotting Assay

The effect of DTT on the binding of zinc to the domain was assessed using the

65 Zn@) blotting assay. The assay was carried out as described above, at either pH 8.0 or pH

6.5, under the following conditions: in the presence of 1 m . DTT during the addition of

metal, in the complete absence of DTT throughout the assay; and in the presence of 1 mM

DTT only dunng the equilibration step (standard assay conditions). The experiment was

carried out using GST-WCBD, WCBD, GST and ERDBD.

2.5.3 Contpeiition " ~ n f l l ) Blofting Assay

The standard 6 s ~ n ( ~ ~ ) blotting assay was cm-ied out as described above, using

purified WCBD, except that the membranes were probed with 6 S ~ n ( ~ in the presence of a

non-radioactive competitor metal at concentrations ranging fkom 0.01 mM to 1 mM. Blots

probed with 6s~n(tI) in the absence of a competitor metal were used as controls. The sigoals

from the autoradiograms were quantified using the gel-plotting macros in the NIH Image

1.61 software package. The percent zinc bound was calculated by dividing the signal

obtained in the presence of a competitor to that obtained in its absence (taken as 100% zinc

binding). In some instances the strips were stained with O. 1% wlv Amido Black dissolved in

45% vlv rnethanol, 10% v/v acetic acid, 45% distilled water for 1-2 minutes, followed by

destaining in several changes of 45% vlv methanol, 10% vlv acetic acid, 45% distilled water.

Stained membranes were analyzed to confirm that equal amounts of protein were present in

each lane.

2 S.4 Stoichiometry of metal binding

The copper binding capacity of the domain was evaluated by NAA. Briefly, urea-

denatured fusion protein was dialyzed against refolding buffer containing copper (20 mM

Tris-HC1, pH 8.0, 10% glycerol, 0.1 mM DTT, 0.1 mM CuC12). Following dialysis, the

protein concentration was detennined using the BCA protein assay (Pierce), and the samples

were then dialyzed extensively against 1 % fonnic acid (pH 3.0) and Iyophilized over a 4 day

period on a Freezernobile 25XL lyophilizer (Virtis). Approximately 1.225 mg of the

lyophilized fusion protein (0.0129 prnol) was submitted for copper determination by NAA.

2.5.5 Neutron Activation Analysis (i'VA4)

Neutron activation analysis was performed at the Slowpoke reactor facility at the

University of Toronto. In general samples analyzed for copper were irradiated for 2 hours

using a neutron flux of 1 .O x 10" neutrons/cm2/s. Samples were counted after a 3 hour delay

for 600 seconds per sample. The amount of copper is each sample was determined by

comparing the amount of (î+ = 5.1 minutes, 1039 keV) in the sample to an external

standard using the comparator method. Samples analyzed for copper and zinc were

irradiated for 16 hours using a neutron flux of 2.5 x 10" neutrons/cm2/s. Samples were

counted afier a 6 hour delay for 1000 seconds per sample. The amount of copper and zinc in

each sample was determined by comparing the amount of %u and 6 9 m ~ n (L, = 12.8 hours,

5 1 1 keV and t;, = 13.8 hours, 438.7 keV respectively) in the sample to an extemal standard

using the comparator method (Hancock, 1976; Hancock, 1984; Hancock, 1978).

2.6 Characterizatioa of the Copper Binding Sites

2.6.1 Preparation of apo-pro rein

Meta1 removal fiom the fùsion protein as well as the WCBD was achieved as follows:

A solution containing the fusion protein or the WCBD was made 0.5 M in p-Me and

incubated at 4 ' ~ for 6-8 hrs. Following this incubation, the proteins were rapidly

precipitated by the addition of TCA to a final concentration of 10%, followed by

centrifugation at 3000 x g for 15 minutes at 4 ' ~ . The protein pellet was resolubilized in 0.5

M Tris base, 6 M Urea, 0.5 M fi-Me with sbaking at 4 ' ~ ovemight. AAer resolubilization,

the protein was again precipitated with TCA and resolubilized in 0.5 M Tris, 6 M Urea. The

protein was then refolded by dialysis against modified refoiding buffer (50 mM Tris-Acetate,

pH 8.0,20% glycerol) and then refolding buffer \vithout glycerol. The protein concentration

was deterrnined by the BCA protein assay (Pierce) and then analyzed for metal content by

NAA.

2.6.2 Preparation of Sampfes For XAS and CD anaiysis

Fusion protein and WCBD containing V ~ ~ O U S amounts of copper or zinc were

prepared as follows. DTT was added to samples of apo-GST-WCBD or apo-WCBD to a

final concentration of 1 mM and incubated on ice for 30 minutes. The required amount of

copper or zinc (using CuSOa and ZnSOs) was then added to achieve the desired final molar

ratio of metaVprotein and incubated at RT for at least 30 minutes. A 15 fold molar excess of

metal was usually used to achieve full reconstitution of the protein with metal. The unbound

metal and DTT were then removed by extensive dialysis against 25 mM Tris-Acetate pH 8.0

(25 rnM Tris-HCI pH 8.0 for CD samples). The protein concentration was again conf~rmed

by the BCA protein assay and metal content was assessed by neutron activation. Al1 dialysis

buffers were purged with argon before use and diaIysis was performed in sealed containers.

Typical concentrations of apo-fusion protein used in this procedure were between 1 and 5

mg/ml. In some cases an alternative procedure for the addition of copper or zinc to the apo-

protein was used to eliminate the possible interference of exogenous sulfûr-containing

compounds. In this method, the protein sulhydryls were reduced by the addition of a 1:1

molar ratio (with respect to cysteine concentration) of TCEP. Copper was then added to the

apo-protein in the presence of 1 mM ascorbic acid. Ascorbic acid was omitted during the

addition of zinc to the apo-protein.

2.6.2.1 Preparation of & X4S Sampies

Dry XAS samples were prepared as described above (2.6.2), except that the samples

were dialyzed against 25 m M Ammonium Acetate, pH 7.5 afier incubation with the metal.

Following protein concentration and metal content determination, the samples were

lyophilized by fust flash fieezing them using a dry ice / acetone bath and then lyophilizing

them for 4 days on a Freezemobile 25XL lyophilizer (Virtis). After lyophilization, the

samples were shipped to Dr. Que's lab at the University of Minnesota for XAS analysis.

2.6.2.2 Preparation of Liquid XAS and EPR Samp /es

Liquid samples for XAS and EPR were prepared as described above (2.6.2), except

that the apo protein solutions were more concentrated (16 mg/ml) and, after incubation with

metal the protein was dialyzed against 35 mM Tris-Acetate, pH 8.0, 30% glycerol. Empty

EPR tubes and XAS sample holders were obtained fiom Dr. Que's lab and filled with 0.75 ml

and 0.2-0.3 ml of sample, respectively. The XAS sample was fiozen by placing the holder

on top of a piece of dry ice while the EPR sample was flash fkozen using liquid nitrogen.

The filled tubes and sarnple holder were then shipped back to Dr. Que's lab on dry ice for

analysis.

2.6.3 EX4 FS and XANES AnalysrS of Cu-GST- WCBD

EXAFS and XANES analysis were carried out by Hua-Fen Hsu in Dr. Que's Iab, as

follows. X-ray absorption spectra were collected between 8799 eV and 9726 eV at beamline

X9B of the National Synchrotroa Light Source (NSLS) at Brookhaven National Laboratory.

The protein data were collected in fluorescence mode (Aexp (CflCo)) at 10-20 K by using a

flat Si(220) double-crystal monochromator and a 13-element Ge detector. The

monochromator was calibrated using the edge energy of copper foi1 at 8979.0 eV. Data

extraction and analysis was perfonned as previously described (Scarrow et al., 1987; Shu et

al., 1995). To fit the EXAFS contribution from copper scatterers, the empirical amplitude

reduction factor A and the shell-specific edge AE were extracted from the XAS data of

crystallogaphically characterized mode1 complexes, [ C U ( S C ~ H ~ ) ~ ] ~ - and [~u(Im)4]~+.

2.6.4 Oxidutive releese of copper from WCBD

To samples of copper reconstituted fusion protein (1 -8 mg/ml, 18.4 ,A) were added

either hydrogen peroxide (HIOZ) or cent ammonium nitrate (Ce(NH&(N03)6) to a final

concentration of 250 pM and the reaction was incubated at RT for 2 hours. One sample did

not receive any oxidizing agents and was used as the controt. The samples were then

dialyzed extensively against 25 m M Tris-Acetate, pH 8.0, at 4 ' ~ and an aliquot fiom each

sarnple was taken (1 -53 mg protein) and submitted for copper determination by NAA.

2.7 Conformational Analysis of GST-WCBD and WCBD

2.7.1 Anaiysis of protein structural changes upon metaf binding

Samples for CD analysis containhg either copper or zinc were prepared as described

in section 2.6.2 and analyzed on a Jasco 5-720 spectropolarimeter. For analysis of changes in

secondary structure, samples were loaded into a O. 1 cm path length CD ce11 and spectra were

recorded from 300 - 190 nm. For analysis of changes in tertiary structure, samples were

loaded into a 2 cm path length CD ceil and spectra recorded from 400 - 250 m. In botb

cases, spectra were recorded using the parameters listed in table 2.1. The spectra were

Table 2.1 CD Parameters for the ConformationaI

GST-WCBD and WCBD

- .

Resolution 1 0.2 n m l

Band Width Sensitivity

1.0 nm 10 mdeg

1 Accumulation 1 4 1

Response Speed

1 second 50 d s e c o n d

corrected for the contribution of the buffer and noise reduced and the data were converted to

molar ellipticity using the software supplied with the spectrometer. Molar ellipticity values

per residue were calculated by dividing the molar ellipticity by either 867 for GST-WCBD or

649 for the WCBD.

2.7.2 Estimation of secondaty structure content

Secondary structure content estimation of both the WCBD and the fusion protein

reconstituted with various amotmts of copper and zinc was carried out using the SELCON

(self consistent method) and K2D (neural network) programs (Andrade et al., 1993; Bohm et

al., 1992; Merelo et al., 1994; Sreerama and Woody, 1994; Sreerama and Woody, 1993).

For the SELCON program, CD data between 260 - 200 nm and the secondary structure

assignrnents and protein data base of W. C. Johnson Jr., containing 33 proteins, were used

(Johnson, 1990). Parameters for the SELCON programs were as follows:

CONSTl (limit for sum of fiactions /sum fk - 1.01) CONST2 (limit for hctions, fk >)

For the K2D program, CD data between 240 and 200 nm was used. The SELCON program

was used to predict a-helix, fi-sheet, 6-turns, and other conformations, while the K2D

0.15 -0.15

NITR (max number of iterations for self-consistency) CONVRG (convergence criteria for sel-consistency)

program was used to calculate a-helix, fi-sheet and random conformations. The KîD

10 0.005

prograrn was found to give very good estimates of f3 structure and this value was used in

conjunction with the values from SELCON to estimate overall protein secondary structure

content (Greenfield, 1996). These and other programs for the estimation of secondary

structure from CD spectra are available a t the following website

http://www2.urndnj.edulcdrwjweb/index.htm. 1 gratefiilly acknowledge the assistance of Dr.

Norma J. Greenfield in the use and application of these programs.

CHAPTER 3

Metal Binding Properties of the N-Terminal Copper Binding Domain from the Wilson Disease Copper

Transporting P-Type ATPase (ATP7B)

The rnajority of the material presented in chapter 3 has been published in: DiDonato, M.,

Narindrasorasak, S., Forbes, J.R., Cox, D.W., and Sarkar, B. ( 1 997) Expressiori, Purification,

and Meta1 Binding Properties of the N-terminal Domain fiom the Wilson Disease Putative

Copper-transporting ATPase (ATP7B). J. BioZ. Chem. 272(52), 33279-33282.

3.1 Introduction

In many ways the presence of copper in biological systems is a double edged sword.

Although trace amounts of copper are absolutely required to sustain life, an excess or a

deficiency of copper can lead to serious biochemical defects (Danks, 1995; DiDonato and

Sarkar, 1997). Many aspects of copper transport bave been studied in the past, however in

recent years, key discoveries have been made which have allowed us to start dissecting the

detailed mechanism of intracellular copper traficking and copper homeostasis. The greatest

of these discoveries has been the isolation of the gene responsible for both Menkes (Chelly et

al., 1993; Mercer et al., 1993; Vulpe et al., 1993) and Wilson (Bull et al., 1993; Tanzi et al.,

1993) diseases, two major genetic disorders of copper metabolism in humans. The discovery

of these genes and the putative copper transporting P-type ATPases they encode, have

opened up new avenues of research in the copper transport are.1.

Understanding the mechanism of action of these proteins in vivo will be a necessary

first step in understanding the disease mechanism. ïhese ATPases represent a new class of

human heaw metai traasporting ATPases and have several interesting features. Although

they share a great deal of homology with each other as well as with other cation transporting

ATPases, they do have some features which are not present in other ATPases (Bull et al.,

1993; Tanzi et al., 1993; Vulpe et al., 1993). Among these are a conserved Cys-Pro-Cys

motif in transmembrane six, a conserved Ser-Glu-His-Pro-Leu motif in a cytoplasmic loop of

unknown fünction and a large amino-terminal region containing six copies of the heavy

rnetal-associated domain (HMA). The consensus sequence for the HMA, which spans the

complete domain is: &IVN]-x(2)-[LIVMFA]-x-C-x-[STAGCDNH]-CIx(3)-FIVFG]-x(3)-

[LIVI-x(9,11)-DVA]-x-[LVFYS]. The HMA has also been found in the metal binding

domains of various bacterial heavy metal transporting proteins (Odermatt et al., 1994; Phung

et al., 1993). The two cysteine residues (bold) are strictly conserved and are most likely

involved in metal ligation.

The presence of the large amino-terminal copper binding domain in the Menkes and

Wilson proteins raises many questions as to its fùnction. The repeated copper-binding motif

represents a totally new and uncharacterized protein and its copper-binding site. Although it

is likely that the rwo conserved cysteine residues in the motif participate in the ligation of

copper, nothing m e r is known about this motif or its function in vivo. To begin to

characterize this motif and the copper bindiag sites, we have developed a bacterial expression

system for the production of the entire amino-terminal metal binding domain of the Wilson

disease protein (ATP7B) îüsed to glutathione-S-emsferase (GST).

3.2 Results

3.2. f Expression and purljica fion of GST- WCBD, GST- WCBD (P), and WCBD

The 2 kbp cDNA encoding the entire amino-terminal metal binding domain (residues

1-649, -70 kDa in size) of the Wilson disease ATPase was produced, cloned and expressed

as described in sections 2.2 and 2.3. As iilustrated in figure 3.1, E. c d i transformed with the

expression plasmid pGEX-WCBD exhibited slightly better growth following induction in

media supplemented with increasing amounts of copper when compared to E. coli

transformed with the parental expression vector pGEX-4T-2. This indicates that the

expressed fusion protein may be offsetting the damaging effects of excess copper on the

bacteria by binding it and lowering its fiee concentration.

A typical purification of WCBD, GST-WCBD, and GST-WCBD(P) is shown in

figure 3.2. Approximately 70% of the expressed fusion protein was found in inclusion

bodies, while 30% remained soluble. Fusion protein localized to inclusion bodies was

recovered by solubilization and refolding from urea. Using a combination of glutathione

affinity chromatography, and denaturing ion exchange chromatography the fùsion protein

was obtained in a >90% pure form. The identity of the protein was confinned by amino-

terminal sequencing.

Treatment o f the purified fiision protein eluted fiom the glutathione affinity column

with 6 M urea in the absence of reducing agents witb BCS, gave rise to a reddish-orange

color (hm, = 480 nm) indicative of the CU(I)(BCS)~' complex (Chen et al., 1996). This

suggests that the domain binds copper in the +1 oxidation state and that copper is being

incorporated into the protein during its production in bacteria. Absorption spectra of the

purified fusion protein in the visible and UV regions do not show any features which would

O 1 2 3 4 Time (hm)

Figure 3.1 Ejfect of Copper on BLZZ(DE3) Growth Following Induction. Growth of BL2 l(DE3) traasformed with either A pGEX- 4T-2 or B pGEX-WCBD. Tris-buffered copper was added to the media at the indicated finai concentration at time of induction. At the times indicated, O&()() readings were taken on aliquots of cells, diluted 10 fold.

, GST- WCBD

W C B D

Figure 3.2 Sztmmary of Typical Purr@cation of WCBD and GST- WCBD. GST-WCBD was expressed and purified as described in section 2.3. A. purified WCBD eluted fiom glutathione-sepharose 4 8 resin following 45 minutes cleavage of GST-WCBD with thrombin on the colurnn. B. GST-WCBD eluted with urea fiom glutathione affinity resin following solubilization and refolding fkom inclusion bodies. C. purified GST-WCBD eluted from Q-sepharose FF anion exchange resin. Proteins derived from the modified vector pGEX-WCBD(P) were expressed and purified using the sarne procedure except, cleavage was camied out using the PreScission protease.

indicate the presence of copper in the +2 state, M e r suggesting the presence of CuO. The

metal bound to the domain appears to be fairly strong since it is able to withstand denaturing,

refolding and the presence of reducing agents (10 mM glutathione, 5 rnM DTT). Metal

binding under these conditions has also been observed for the estrogen receptor DNA

binding domain (2 zinc finger protein having 4 cysteines in each finger) where a combination

of low pH, urea, DTT and chelating agents are needed to remove the bound metal (Conte et

al., 1996).

3.2.2 Metal binding characteristics of GST-WCBD and WCBD

3.2.2.1 Immübifited metai ion affrniry chromptograpiy (7MA C)

To investigate the metal binding properties of the domain, both immobilized metal

ion affinity chromatography (MAC) and 6 5 ~ i n c binding assays were carried out using the

fusion protein and the cleaved domain. The results of IMAC using the purïfied fusion

protein are presented in figure 3.3. The expenment was run both under non-denaturing and

denaturing conditions in the absence of reducing agents with nearly identical results. GST

alone did not bind to uncharged metai columns and had sorne weak, non-specific interactions

under non-denaturing conditions on metal charged columns. The fusion protein did not bind

to columns charged with either Fe@) or Fe(II1). The other transition metals showed varying

affinities for the domain. Based on the sequential elution conditions used, the apparent order

of affinity for the different transition metals for the domain was:

Cu(Ii)>>Zn(II)>Ni(n)>Co(II).

The variation in affinity may reflect the inability of the protein metal binding sites to

adopt the preferred conformation for that metal atom. Interestingly, the fusion protein could

PH Imidazo le 50 rnM , y EDTA 50rnM BCS

Figure 3.3 Immobilized Metul Ion Affinity Chrornaiography (IMAC) of GST- WCBD. Samples (S) of the fusion protein were applied to chelating sepharose FF columns charged with the indicated metal under non-denaturing conditions. Protein which did not bind to the column came off in the flow through (FT). Bound protein was eluted by sequential treatment with low pH (6.5 - 4.0), increasing amounts of imidazole (LOO - 500 mM), EDTA and BCS. No binding was observed to columns charged with i o n (+2 or +3). Very similar results were obtained using denaturing conditions.

only be released from the Cu(1I)-charged column by the addition of the specific copper

chelator BCS. Also, elution of the fusion protein with BCS was accompanied by the

formation of the reddish-orange CUO(BCS)~- cornplex, indicating that copper was bound to

the domain in the + l oxidation state. Considering that the colwnn was charged with Cu(I1)

in the absence of reducing agents, this observation suggests that Cu@) atoms may be

reduced to Cu(T) upon binding to the domain.

3.2.2.2 Neutron activation anaiysis (nAA)

To determine the stoichiometry of metal binding to the domain, neutron activation

analysis was used to quantitate the amount of copper bound to GST-WCBD following

reconstitution with excess rnetal during refolding. A sarnple (1.225 mg) of GST-WCBD

(-96 kDa, 0.0 129 pmol) was analyzed by NAA and found to contain 0.089 I 0.005 pmol of

copper. This analysis revealed that 6.5 - 7.3 moles of copper are bound per mole of fusion

protein. Similar ratios were obtained if refolded apo-fùsion protein was reconstitued with

copper. The amount of copper bound was found to be the same for both GST-WCBD and

WCBD, indicating that GST does not bind any copper and that the domain is able to bind 6 -

7 copper atoms. These results are consistent with similar observations made during the

characterization of Coxl7, which was also expressed as a fusion to GST (Srinivasan et al.,

1998).

3.2 J.3 6 5 ~ i n e blotting anak'ysis

In order to determine if WCBD is able to bind zinc and to m e r investigate the

rnetal binding properties of WCBD, a 6Stinc blotting assay was ernployed. Preliminary

experiments indicated that both GST-WCBD and WCBD were able to bind zinc under the

experimental conditions used. To determine the effect of pH on zinc binding, the assay was

carried out over a range of pH values fkom 6.5 - 9.0; these results are summarized in figure

3.4. Both GST-WCBD and WCBD are able to bind zinc over the entire pH range evaluated.

Progressive loss of zinc binding activity is only seen at the upper end of the pH range and

may be related to the pK, of the ligatiag atoms. GST alone did not bind appreciable amounts

of zinc, while the ERDBD (positive control) seemed to be much more sensitive to variations

in pH. The effect of reducing agents (DTT) on the binding of zinc was also examined.

These results are summarized in figure 3.5. The 6Szinc binding assay was carried out at pH

6.5 and 8.0 in either the absence or presence of DTT or just equilibrating the blot with DTT

and then rernoving it before addition of 65zinc. In the absence of DTT, zinc binding activity

was not observed at either pH value. However, strong zinc binding was observed when DTT

was used in the equilibration step only or if it was present throughout the assay. M e n

present throughout the assay, DTT was able to decrease the amount of non-specific binding

dramatically, as observed at pH 8.0. This is most likely due to the ability of DTT to chelate

non-specific and weakly bound zinc atoms. The requirement for DTT pretreatment suggests

that cysteine residues are directly involved in metal Iigation and that a fiee sulfhydryl is

required for metal ligation. This supports the notion that the two cysteine residues in the

HMA domain, strictly consewed in each of the metal binding domains of ATP7B, as well as

many bacterial heavy metal transporters, are crucial for metal ligation (O'Halloran, 1993;

Silver er al., 1989).

F C G E F C G E

Figure 3.4 Eflect of p H on the Bhding of "zinc. Samples of GST- WCBD (F), WCBD (C), GST (G), and the estrogen receptor DNA binding domain (E) were subjected to "zinc blotting analysis as described in section 2.5.2. In each case, blotting was perforrned in metal binding buffer at the indicated pH in the presence of 1 mM DTT.

G E C F M

G E C F G E C F M G E C F

Figure 3.5 Effect of Reducing Agents on the Binding of 6S~inc . Samples of GST-WCBD (F), WCBD (C), GST (G), and the estrogen receptor DNA binding domain (E) were subjected to "zinc blotting analysis, as described in section 2.5.2, at two pH values (6.5 and 8.0). M, molecular weight markers. To test the effects of reducing agents, DTT was either omitted fkom the assay (-DTT), included only during the equilibration step (+DTT/Eq), or included throughout the assay (+DIT).

3.2.2.4 Comptition "-'zinc b folomng ana&&

The ability of WCBD to bind copper and other transition metals was investigated

using a competition "zinc blot. The results of this analysis are presented in figure 3.6. AI1

competition 6Szinc blots were camed out using purified WCBD and DTT was present only in

the equilibration step. Several other transition metals were able to compete successfully with

zinc for binding to the domain. In particular, Cd(II), Au@), and H g 0 were found to have

the highest affinities for the domain relative to zinc. Both Mn@) and Ni(II) were not abte to

compete with zinc for binding to the domain when present in concentrations fiom 0.01 to O. 1

mM. Blotting with 63~ i ( I I ) directly instead of 6 S ~ n ( ~ ~ ) did not give rise to a signal on the

autoradiograph indicating Iack of binding to the domain. Co@), C r o , and Fe@) were the

weakest cornpetitors of the transition metals tested. The binding of zinc also seemed to be

specific, since a large excess of both Mg(I1) and Ca(I1) did not compete at al1 for zinc

binding. Results with copper were very dramatic and unique compared to the other metals

studied. At low concentrations, copper was able to decrease zinc binding by about 30%.

However, as the concentration of copper was raised, the afinity of copper seemed to increase

rapidly. This pattern was reproducible and independent of whether copper was presented in

the + l or +2 oxidation state, suggesting that the domain has similar afinities for both Cu(I)

and Cu(I1). The behavior of copper as a cornpetitor for zinc binding was similar to that

observed for the cooperative binding of ligands to proteins, indicating a possible cooperative

binding mechanism for copper which was not observed for the other transition metals tested.

0.00 1 0.0 1 o. 1 Competitor Concentration (mM)

100 90 80 70

60 50 40 30 20 10 O

Figure 3.6 Cornpetition of 6 5 ~ i n c Binding to WCBD Samples of purified WCBD were subjected to corn etition 6Szinc blotting analysis as descnbed in section 2.5.2.

6P In each case the final zinc concentration was 30 p M (-IO pCi). A, competition of 65 zinc binding with various transition metals. Al1 cornpetitor metals were added as the chloride salt at the indicated concentration. Ni(II), Mn@), Mg(lf), and Ca(II) showed no aEnity for the domain relative to zinc. B, competition of zinc binding with C u 0 and Cu(II). C u 0 was presented as CuClz while Cu(1) was presented as tetrakis(acetonitrile)copper(I) hexafluorophosphate. This complex was prepared in an argon-purged soIution of 2% acetonitrile.

- - - - 0 +

- + FetIII) + Cdrr)

- -0- cd(II) - -c cr(rrr)

- 6 Hg(W + Au(1Ii)

- + Ca(IQ - -X- Mg(1T)

0-

0.00 1 0.0 1 o. 1 1

Competitor Concentration (mM)

3.3 Surnmary and Discussion

We have expressed the amino-terminal metal binding domain of ATP7B as a tiision

to GST and studied its metal binding properties. We have shown that the domain binds

approximateiy 6 moles of copper per mole of protein regardless of whether GST is present or

absent. Using MAC, we have been able to detemine that the domain is able to bind a

variety of different transition metals with the apparent order of affinity:

Cu(I)/Cu(II)>Zn(II)>Ni(TI)>Co(II) under both denaturing and native conditions. Speci fic

binding of metalloproteins with intemal metal binding sites to IMAC columns under

denaturing conditions has also been observed for troponin T, which contains four repeated

rnetal binding motifs (Jin and Smiliie, 1994). In this assay, we did not observe binding of the

dornain to columns charged with either Fe(II) or F e o . This could indicate that the domain

had no affinity for iron or that the metal binding sites were unable to ligate iron while it was

bound to the MAC matrix, possibly due to steric effects.

The domain bound very strongly to columns charged with copper, as expected, and

could only be released with the specific copper chelator, BCS. Upon elution fÎom the

colurnn, the presence of Cu(1) in the protein was indicated by the formation of the reddish-

orange Cu(I)(BCS)-- cornplex, a surprising result considering that the column was charged

with Cu(I1) and non-reducing conditions were used. This observation suggests that the

domain is able to reduce copper from the +2 to +l oxidation state upon biading. This could

occur through the oxidation of a pair of cysteines in one domain followed by the binding of

copper to a reduced pair of cysteines in another domain. This gives rise to the possibility that

the domain could bind both C u 0 and Cu@) in vivo pnor to transport. The oxidation state of

the transported copper is not yet known, however there is one report which suggests that it

rnay be in the Cu@) state (Bingharn et al., 1996).

An in depth study of the metal binding properties of the domain using 65zinc blotting

techniques was performed. The results from this analysis have confmed and expanded

upon those obtained in the M A C experiments with two exceptions. Although the fusion

protein bound to [MAC columns charged with Ni(II), Ni(I1) was not able to compete

successfully with zinc for binding to WCBD in competition ''zinc blotting experiments. This

situation could aise if the affinity for zinc was much greater than that for nickel, in which

case nickel would continuously be competed away from the protein by zinc. This was tested

by blotting with 63nickel directly instead of 6Szinc. Binding of 63nickel to WCBD was not

observed, indicating that either the domain is not able to bind nickel or that the affinity for

nickel is extremely low. The converse situation was observed for iron, where binding was

not observed to iron-charged M A C columns, but iron was able to act as a cornpetitor for

zinc binding in competition blotting expenments.

The reasons for these discrepancies are not clear and they may be due to the inherent

differences in the two methods used. In MAC, the metal is immobilized on a matrix and the

protein must bind to the metal while it is bound to the matrix; in the blotting experiments, the

metal must bind to protein which is immobilized on nitrocellulose. in IMAC, the protein is,

in essence, 'sharing' the metal with the malrix, with some ligands coming fkom the matrix and

others fiom the protein in a temary form. Bearing this in mind, the results fiom the blotting

assays may more accurately reflect the binding properties of the domain, since al1 ligands

originate fiom the protein itself. The binding of transition metals other than copper to the

WCBD has been observed by snother laboratory (Lutsenko et al., 1997), and are in

agreement with what has been found in our studies. However, we have used two

independent methods and a wider range of metals then was used in the study by Lutsenko et

al. (1997) to assess the meta1 specificity of the WCBD.

The unique characteristics observed for copper as a cornpetitor may have important

consequences for the function of the domain in vivo. Although the blotting experiments

cannot be used to establish the presence of cooperative copper binding conclusiveIy, they are

very suggestive. The binding of zinc to the domain is not surprising since the Gly - Met -

Thr - Cys - Xaa - Xaa - Cys motif is also present in the bacterial zinc transporting ATPase,

ZntA (Rensing et al., 1997). What is not clear is how the specificity for copper is conferred

on the ATPase. If cooperative copper binding is taking place, it may be the basis of the

specificity. A cooperative binding mode1 for copper would require structural changes to take

place upon successive metal binding, which would bring the ligating residues to the

appropriate geometry for copper binding through tertiary folding of the protein. We have

studied the structural changes which occur in the WCBD using circular dichroism

spectroscopy and these results are presented in the following chapter.

CHAPTER 4

Structural Characterization of the Wilson Disease Copper Binding Domain And Its Meta1 Binding Sites

4.1 Introduction

The HMA domain is found in proteins fiom a wide range of organisms which are

invotved in the transport of a variety of heavy metals, including zinc, copper, cadmium and

mercury. In most lower organisms, one or two copies of the HMA are present; in bigher

organisms five or six copies are comrnon. Although it is common!y known that these

domains are involved in ligating the metal atorn prior to transport, little is known about the

structure of these domains and how the same domain can serve to ligate diverse metal atoms.

Furthemore, information regarding inter-domain interactions in multiple HMA domain

proteins (Menkes and Wilson disease ATPases) is practically nonexistent. The structural

information that is available is derived fiom studies on proteins with oniy one copy of the

HMA or fiom a single HMA excised fiom a protein containing multiple copies (Gitschier et

ai., 1998; Pufahl et al., 1997).

The NMR solution structure of the fourth HMA domain fiom the Menkes disease

protein complexed with silver(1) revealed Iinear coordination of the metal by the conserved

cysteine residues (Gitschier et al., 1998). Silver is known to prefer linear coordination and it

could be possible that the binding of silver to the domain may force linear coordination on

the metal binding site. Therefore, it is not clear if the conformation this domain adopts in the

presence of silver would be similar to that adopted in the presence of copper(I), which could

bind in either a linear, trigonal, or tetrahedral fashion. Additionally, the authors fouad that

the conformation of the apo-protein was essentially the same as the holoprotein. An XAS

study of the yeast copper chaperone ATX1, which contains one copy of the HMA, revealed

that copper iigation could be either Iinear or trigonal with al1 sulhr ligands (Pufahl et al.,

1997). In this system, it was proposed by the authors that both conserved cysteines and a

methionine were involved in Iigating the copper atom. Recently, a report has appeared fiom

an EXAFS study of the full amino-terminal metal-binding domain of the Menkes protein

fûsed to maltose binding protein (MBP). The authors of this report propose a cornpletely

linear ligation mode1 invotving only the conserved cysteine residues and also suggest a

copper - copper interaction (Ralle et al., 1998). However, this report does not give any

details regarding the copper:protein ratio or the purity of the sample, which would have

allowed usefùl conclusions to be draw from the data.

A recent study of the second metal binding domain fiom the Menkes disease protein

has shown that one domain does not undergo structural changes upon metal binding

(Harrison et al., 1999). The study of a single metal binding domain fiom either the Menkes

or Wilson disease protein can help in understanding the sub-structure of the intact domain;

however, these studies cannot reveal any information on how the entire domain rnay function

in vivo. It has been assumed that the conserved cysteine residues in the HMA repeats are the

only residues involved in Iigating the metal atom. However, analysis of the prirnary

sequence of the Wilson disease protein reveals six additional cysteine residues, evenly

distributed among the six HMA domains, which may play a role in metal ligation.

Furthemore, other potential Iigating residues such as histidine are also present in abundance

in the amino-termina1 metal-binding domain. Single domain studies or studies involving

single HMA proteins would not be able to detemine the potential fùnction of these or other

residues with regards to metal ligation. In order to address some of these questions, we have

begun a detailed structural study of the copper binding sites in the WCBD, using XAS (in

coIlaboration with Dr. Lawrence Que), in addition to analyzing the structural changes which

take place upon metal binding to the WCBD, using circular dichroism spectroscopy.

4.2 Results

4.2.1 X-ray absorption spectroscopy (X4S) anaiysis of GST- WCBD

A detailed analysis of the copper binding sites was carrîed out using XAS techniques

in collaboration with Dr. Lawrence Que, Jr. and Hua-Fen Hsu at the University of Minnesota.

Since the WCBD contains six copper binding sites, XAS techniques carried out on the fully

reconstituted domain (6 bound copper atoms) will give structural information which is

averaged over the six sites. To address the question of whether each copper binding site is

the same structurally, the XAS analysis was carried out on the fùsion protein containing

either 2, 3 or 5 copper atoms. The copper x-ray absorption near edge structure (XANES)

spectra for al1 three samples are shown in figure 4.1. Al1 three spectra are very similar and

exhibit a feature at 8983 eV which is consistent with the 1s -. 4p transition of Cu(Q ions

(Kau et al., 1987). The intensity of the transition at 8983 eV is correlated with the geometry

around the copper atom. Tetrathedrally coordinated copper(1) atoms have low intensity

transitionso while linear, two coordinate sites, have we!I resolved, high intensity transitions.

The observed transition for the WCBD is weaker than that of diagonal compounds but

stronger than that for trigonal compounds.

The extended x-ray absorption fine structure (EXAFS) spectra of the three samples

are shown in figure 4.2. The EXAFS spectra for al1 the samples are very similar both in k-

space and r'-space (fig. 4.2 A and B), which is consistent with the results of the XANES

spectra of the same samples. The ru-space spectra of al1 samples exhibit a prominent feature

centered around r' = 1.8 A (r - r' + 0.4 A) fiom a shell of sulfur scatterers. The best fit for the

first coordination sphere was obîained assuming a single shell of two s u l h atoms at 2.17-

2.19 A. A single shell with one or three sulfur atoms or splitting the shell into one sulfùr and

8970 8975 8980 8985 8990 8995 9000

Energy (eV)

Figure 4.1 Normalized Copper XANES Spectra For GST-WCBD with Vurious Amounts of Copper. XANES spectra were taken fiom lyophilized samples of GST-WCBD containing either 2 (2 Cu), 3 (3 Cu), or 5 (5 Cu) atoms. The amount of copper was quantified using neutron activation analysis. Cu(SR)z : [N(C3 H~)~]CU(SCLOH&; Cu(SR)3: [(Cs Hs)4P] [c~(SC6H5)3]-

Figure 4.2 EiAFS Spectra For GST- WCBD with Various Amoun fs of Copper. 2 Cu (solid line), 3 Cu (dotted line), 5 Cu (dash-dotted line). A EXAFS in k-space, B EXAFS in r'-space (r - r' + 0.4 A), C r'-space EXAFS for 5 Cu sample overlaid with that fiom Cu(SR)z (dot) and CU(SR)~ (diamond).

one low Z atom subshell at 2.17 - 2.19 and 1.9 - 2.1 A respectively, resulted in poorer fits.

The r'-space spectra of al1 samples also exhibit a minor peak at r' = 2.3 A which is not present

in the spectra of the mode1 compounds [ C U ( S C ~ ~ H ~ ~ ) ~ ] - and [CU(SP~)~]~- . A good fit for this

feature could not be obtained using either sulfbr or copper atoms.

4.2.2 Circuiar dichroisna anaiysis of GST- WCBD and WCBD

The confonnational dynamics of WCBD upon binding of metal were studied using

circular dichroism spectroscopy. Spectra of samples with increasing amounts of either

copper or zinc were recorded in the secondary structure region (250 - 200 nm) and the

aromatic region (250 - 400 nm). The spectra of the fusion protein and the WCBD alone

reconstituted with either copper or zinc are presented in figures 4.3, 4.4, 4.5 and 4.6. The

results obtained for the fusion protein are very simiiar to those of the WCBD alone, with

some exceptions. Addition of copper to the apo-fusion protein induced an increasc in

secondary structure (fig- 4.3 A). The magnitude of the increase was the greatest on going

from the apo state to the 2: 1 state and fiom the 2: 1 to 4: 1 state. Going fiom 4: 1 to 6: 1 or 8: 1

did not result in any further significant secondary structure changes. Signi ficant changes

were also observed in the aromatic region (fig. 4.3 B). The changes in this region pardleled

those observed in the secondary structure region with the Iargest changes occurring between

apo and 2: 1 and fiom 2: 1 to 4: 1. A large difference is also observed on going fiom 4: 1 to

6: 1 , whic h is not observed in the secondary structure region. No signi ficant changes were

observed on going fi-om 6: 1 to 8: 1 .

The changes observed upon the addition of zinc to the apo-fusion protein are very

different from those obtained with copper (fig. 4.4). Although the addition of zinc to the

apo-protein does cause an initial increase in secondary structure, additional molar equivalents

seems to cause an altemating increase and decrease in secondary structure (fig. 4.4 A). This

pattern is not o b s e ~ e d for copper, suggesting that the binding of zinc induces a structure

which is significantly different fiom that with copper. The differences between copper and

zinc are also observed in the aromatic region (fig. 4.4 B) where the pattern of changes is

significantly different from what is observed with copper. Substantial changes fkom the apo-

protein spectra are only observed on going fiorn apo to 4: 1 and fiom 4: 1 to 8: 1. The spectra

of the apo-fusion protein and the 2: 1 and 6: 1 zinc reconstituted protein are virtually identical.

Tbese observations, like those in the secondary structure spectra, suggest that the binding of

zinc to the dornain leads to a very different structure fiom that obtained with copper. This

finding may have significant implications for the metal selectivity of the pump in vivo.

The titration of the WCBD alone with copper produced results which were similar to

those observed with the fusion protein with some exceptions (fig. 4.5). A general increase in

secondary structure was observed upon progressive addition of copper. The magnitude of the

change was greatest between the apo form and the 2:1-form and to a lesser extent fiom the

4: 1-fom to the 6: 1-form (fig. 4.5 A). In contrast to the firsion protein spectra, addition of

copper equivalents above 6:l resulted in an apparent loss of secondary structure in the

domain. These differences could result fiom the presence and absence of GST, which may

be influencing certain structural changes in the fusion protein. The changes in the aromatic

region are also similar to those observed in the fusion protein, with some differences (fig. 4.5

B). The largest changes are observed on going fiom apo to 2: 1 and fkom 2: 1 to 4: 1 ; however,

in contrast to the fusion protein, only small changes were observed on

Wavelength (nm)

250 260 270 280 290 300 310 320 330 340 350 Wavelength (MI)

Figure 4.3 CD Spectra of GST- WCBD Titrared wirh Copper. The fusion protein was rcconstituted with copper to the indicated ratio and CD spectra recorded. A. Secondary structure region, B. Aromatic region.

Wavelength (MI)

250 260 270 280 290 300 310 320 330 340 350 Wavelength (nm)

Figure 4.4 CD Spectra of GST- WCBD Titrated with Zinc. The h i o n protein was reconstituted with zinc to the indicated ratio and CD spectra recorded. A. Secondary structure region, B. Aromatic region.

Wavelength (nm)

250 260 270 280 290 300 310 320 330 340 350 Wavelength (nm)

Figure 4.5 CD Spectra of WCBD Titrated wirh Copper. The W C B D was reconstituted with copper to the indicated ratio and CD spectra recorded. A. Secondary structure region, B. Aromatic region.

Wavelength (nm)

10 C

250 260 270 280 290 300 310 320 330 340 350

Wavelength (nrn)

Figure 4.6 CD Specfra of WCBD Titrafed with Zinc. The WCBD was reconstituted with zinc to the indicated ratio and CD spectra recorded. A. Secondary structure region, B. Aromatic region.

going tiom 4: 1 to 6: 1. There is also a significant change on going from 6: 1 to 8: 1, which was

not observed in the fbsion protein.

Titration of WCBD alone with zinc produced a pattem of conformational changes

(fig. 4.6) which were different fiom that of copper and resembled those obtained fiom the

addition of zinc to the apo &ion protein. The initial addition of zinc resulted in an apparent

loss of secondary structure. The addition of increasing amounts of zinc produced an

alternating increase and decrease in the amount of secondary structure present (fig. 4.6A).

This pattern was significantly different fiom that obtained with copper and resembled the

pattern found upon the addition of zinc to the fûsion protein. The aromatic region (fig. 4.6B)

also displayed features which were different fiom those obtained with copper. With the

copper titrations, an initial increase at 260 nm was observed fiom the apo to 2: 1 reconstituted

protein. At higher ratios this increase subsided and was replaced with an increase in the 290

nm range. For the zinc samples, there was only a small increase at 260 nm for the addition of

2 equivalents of zinc and at higher ratios no increases in the 290 nm range were obsewed.

These results suggest that the binding of zinc by the WCBD results in a conformation which

is significantly different fiom that with copper.

4.2.3 Estimation of Secondary Structure Content

The secondary structure content of the WCBD with various copper ratios (0,2,4,6, 8

metal atoms / mol protein) and of the fusion protein with zinc and copper (0, 2, 4,6, 8 metal

atoms / mol protein) were estimated as described in section 2.7.2. The results of this analysis

are presented in table 4.1. Each of the different metal ratios represents a separate protein

sample prepared fiom the same apo stock. Meta! concentrations were confirmed by N U ,

and protein concentrations were confirmed using the BCA protein assay.

4.2.4 Oxidutive refease of copper from GST- WCBD

The ability of oxidizing agents to liberate bound copper fiom the fùsion protein was

evaluated and these results are presented in figure 4.7. The results indicated that only

hydrogen peroxide was able to effect the release of approximately 2 copper atoms fiom the

domain after a 2 hour incubation. Addition of the oxidizing agent, ceric ammonium nitrate,

caused the protein to precipitate with no apparent loss of copper.

Table 4.1 Secondary Structure Estimation for Metal Titrated WCBD

and GST-WCBD Using SELCON and K2D

Metal : Protein Ratio Apo 2: 1 4: 1 6: 1 8: 1

CU-WCBD a-Helix (%) p-~heet' (%) p-Tms (%)

Other (%)

Zn-WCBD a-Helix (%) 6-~heet' (%) p-Tunis (%)

Other (%)

CU-GST-WCBD a-Helix (%) p-sheett (%) f3-Turns (%)

Other (%)

Other (%) 25 3 1 25 43 27 'values in brackets indicate f3-Sheet content estimated by the K2D program, al1 others are fiorn SELCON.

Control Hydrogen Ceric Ammonium Peroxide Nitratet

Figure 4.7 Oxidative Release of Bound Copper From WCBD. Copper reconstitued fusion protein was incubated for 2 hours in the presence of the indicated oxidizing agent. Remaining bound copper was quantified by NAA. tThis compound caused the protein to precipitate.

4.3 Summary and Discussion

The XAS studies of the WCBD with sub-stoichiometric amounts of copper have

provided a wealth of detailed structural information. The XANES spectra (fig. 4.1) M e r

confim the previous observation that copper bound to the WCBD is in the +1 oxidation state

by the presence of a feature at 8983 eV. This feature is attributed to a 1s -, 4p transition

which is characteristic of Cu(1) atorns. The intensity of this transition in the spectra of

WCBD is weaker than that for tinear copper thiolate complexes. This observation suggests a

distorted, two coordinate geometry around the copper atom. This distortion may arise fiom

protein conformational effects or the presence of a weak interaction from a third ligand.

There is some evidence of the latter due to the presence of the minor peak at r' = 2.3 A in the

EXAFS spectra. Good fits for this feature could not be obtained with either copper or su1 fur

atoms and, therefore, the identity of this scatterer remains undetermined.

This featwe is not present in the spectra of the mode1 compounds but is observed in

the EXAFS spectra of the Menkes protein (Ralle et al., 1998). The authors of this paper

suggest the possibility that this feature arises from the formation of copper clusters in the

domain. Although this is a definite possibility, the study by Ralle et al. is not detailed

enough to draw this conclusion. The XAS studies presented in this thesis are more detailed

since various ratios of copper were used. The results show that the feature at r' = 2.3 A

increases in intensity as greater amounts of copper are added. This could lend support to the

idea that copper clusters are forrned as more copper becomes bound to the domain; however,

the low intensity of this peak indicates that this is not a major form of the protein in these

samples. in preliminary experimcnts using samples which were reconstituted with copper in

the presence of TCEP and ascorbic acid, this feature is of greater intensity. The intensity of

the peak also rises as increasing amounts of copper are bound to the domain. Interestingly,

this peak is also a major feature in the spectra of non-lyophilized samples prepared in the

presence of DTT. It is possible that the domain can adopt multiple conformations under

different conditions. Further studies will be needed to establish the identity of the scatterer

which gives rise to this feature conclusively.

Fitting of the EXAFS data indicate that the first coordination sphere consists of two

sulfùr atoms with a Cu-S distance of 2.17 - 2.19 A. This distance is much shorter than those

in trigonal Cu(I)-thiolate complexes and is slightly longer than those of linear Cu(i)-thiolate

complexes (Coucouvanis et al., 1980; Fujisawa et al., 1998; Koch et al., 1984). Trigonal

Cu(1)-thiolate complexes have Cu-S bond distances of 2.27 - 2.38 A, whereas linear Cu(&

thiolate complexes have Cu-S bond distances of 2.14 - 2-15 A in structurally characterized

mode1 complexes. The Cu-S bond distances in the WCBD are similar to those found in the

Menkes pro tein (2.1 7 A) but are muc h shorter than those found in the yeast copper c haperone

ATX1 (2.25 A), which has only one HMA domain (Pufahl et al., 1997). This implies that the

copper sites in the WCBD are similar to those in the Menkes disease protein but significantly

different than that in ATXl. These observations indicate that the presence of multiple HMA

domains have an influence on the copper coordination environment- In light of these

findings, studies based on a single domain fkom the WCBD or those without carefully

characterized metal stoichiometries cannot provide an accurate representation of the domain

as a whole.

The structure dynamics of the WCBD (and GST-WCBD) upon titration with either

copper or zinc were studied using circula dichroism spectroscopy. Both the secondary

structure region (200 - 270 nrn) and the aromatic region (250 - 350 nm) were analyzed for

structural changes upon metal binding. The conformational changes which were obsewed

upon metal binding were similar for both the WCBD and GST-WCBD proteins (for the same

metal) with only minor differences. In the secondary structure region of proteins titrated

with copper (figs. 4.3, 4.5, panel A), the general observation is that the ellipticity increases

upon binding of greater amounts of copper. This increase suggests an increase or

stabilization of secondary structures relative to the metal free (apo) protein. This was

observed in both the fiision protein and the WCBD alone. In both cases, the magnitude of

the change is greatest between the apo fonn and the 2:l copper-fonn. However, in the

WCBD, another significant increase takes place on going fiom 4 to 6 copper atoms and this

is not observed in the fiision protein. Additionally, adding more than the stoichiometric

amount of copper to the WCBD resulted in an apparent destabilization of secondary structure

relative to the apo-protein. This phenomenon was not observed in the fùsion protein. These

differences are most likely the result of the presence of GST, which is able to form dimers in

solution. We have observed that, d- ing purification, the fusion protein is substantially more

soluble and Iess proue to aggregation than the WCBD alone. Furthermore, addition of excess

copper to concentrated solutions of the apo-WCBD usually results in the formation of a

precipitate, a phenomenon which is not obsewed with the fusion protein. Although

precipitate formation was not obsewed in the CD samples, soluble aggregates of the WCBD

may have been forrned at the higher copper ratios, leading to the apparent loss of secondary

structure observed in the CD spectra of the WCBD with an 8: 1 copper:protein ratio.

The changes in the aromatic region were also very similar between the fusion protein

and the WCBD. The changes which occwred in the aromatic region spectra upon addition of

copper paralleled those in the secondary structure region. The largest changes in spectral line

shape were observed on going fiom the apo-protein to the 2:l copper form and again fiom

the 2: 1 form to the 4: 1 copper fom. It is interesting to note that, in the WCBD, the 2: 1 and

4:1 copper forms seemed very similar in secondary structure but their tertiary structure

spectra were significantly different. A similar phenomena was also observed in the fiision

protein. This would seem to indicate that, although the secondary structure remains

relatively constant, the relative orientation of secondary structure elements within the domain

are changing, depending on how much metal is bound. These tertiary structure changes

could be the ba is of cooperativity in the domain where binding of copper to one site leads to

a modification of binding properties at another site. Cooperativity of copper binding to the

WCBD was suggested by competition zinc blotting experirnents using copper.

The spectra in the aromatic region *se fiom the rc-R' electronic transitions kom the

aromatic side chains of tyrosine, tryptophan, and phenylalanine residues. However, because

these residues contain complete or partial planes of symmetry, the transition a ise entirely

from interaction with neighboring groups (Wingfield and Pain, 1996). A detailed

interpretation of the spectra in this region is extremely difficult, since many of the transitions

overlap (table 4.2). In proteins which contain several of each kind of aromatic amino acid

(each in a different environment), deconvolution of the spectra to isolate contributions fiom

each residue is nearly impossible in the absence of either a three dimensional structure or

detailed mutational studies. Despite these difficulties, we can observe several patterns in

both the fiision protein and the WCBD, which occur upon copper binding. In going from the

apo to 2: 1 copper form a decrease (more positive) in ellipticity occurs in the 250 to 270 o . .

region. When copper is added to a 4: 1 ratio, the ellipticity in this region increases while the

ellipticity in the 270 - 290 nrn region decreases. The former region contains mainly

Table 4.2 CD Transitions of Aromatic Side chahst

--

Amino ~ x d Electronic Peak Transitions Wavelengths (nm)

Pheny lalanine 'Lb 262,268

Tyrosine

Tryptophan

far W 277,283

268,289 (shoulders)

' ~ a t a from (Strickland, 1974). ' ~ a ~ be seen as a low intensity peak or shoulder fiom a Trp in a nonpolar environment. n nu su al peak observed in lysozyme.

phenylalanine and tyrosine contributions, while the latter region contains mainly tryptophan

contributions (WingfieId and Pain, 1996). It is clear that the largest changes in the spectra

(both line shape and intensity) occur between the 2 and 4 copper fonns of the protein. This

indicates that a significant tertiary structure change is taking place afier the first few coppers

are bound, possibly reflecting the cooperative binding of copper aAer the fust few are bound.

This correlates well with the cornpetition zinc blotting studies (see section 3.2.2.4). At lower

copper concentrations zinc binding was reduced in a linear fashion and this was followed by

a sharp transition at hi@ copper concentrations, which is indicative of cooperative binding.

The disulfide bond can also make significant contributions to the ellipticity in CD

spectra because it does not contain an inherent plane of symmetry. The disulfide bond bas a

broad band of ellipticity in the 240 - 350 nm range and can augment or diminish the signal

from tyrosine and tryptophan residues (Wingfield and Pain, 1996). This M e r complicates

the interpretation of the spectra in this region. In cases where the disulfide makes a

significant contribution to the spectra, its presence is confirmed by ellipticity above 320 m.

This feature is present in both the fusion protein and the WCBD when they were titrated with

copper. It is most prominent in the apo form, since without copper and reducing agents the

18 cysteine residues in the domain will readily oxidize to form disulfide bonds. Furthemore,

this feature decreases as the amount of copper increases, since the cysteine residues are now

participating in metal ligation and are unavailable for disulfide formation. When six copper

atoms are bound, the ellipticity above 320 nm is nonexistent, suggesting that no disulfides are

present. The domain contains twelve conserved cysteine residues which are located in six

HMA domains (2 per domain) and are involved in ligating the metal. In addition, another six

cysteine residues are evenly interspersed between the HMA domains. It is not known

whether these form disulfides or are fiee in the native domain. The CD results indicate the

absence of disulfides in the fully copper-reconstituted state, indicating that either these

cysteines remain reduced or that they too are involved in metal ligation. However, at this

point we do not have any evidence to support one view over the other.

In contrast to the spectra obtained with copper, those obtained fiom the titration with

zinc are very different in both the secondaxy structure and aromatic regions (figs. 4.4 and

4.6). The addition of zinc to both the apo hision protein and the apo WCBD resulted in a

strange pattern of atternating increase and decrease of ellipticity which is in stark contrast to

the progressive increase in ellipticity observed for the copper samples. The same was mie

for the aromatic region, where the pattern of changes observed were not as significant and

did not resemble those for the proteins titrated with copper. In particular, the ellipticity

above 320 nm, which is indicative of the presence of disulfide bonds, did not decrease in a

Iinear fashion, as was observed with copper. Although we saw a decrease on going fkom the

apo to the 2 and 4 copper forms, no additional increase was observed on going fiom 4 to 6 or

8 coppers. These observations suggest that although zinc is able to bind to the domain (as

seen in IMAC and zinc blotting) it is not able to induce the same structure in the domain as

copper; this may be an important factor for the function of this dornain in vivo. Differences

in the conformation of the domain with zinc and copper may be due to the different

geometrical preferences of each metal. While zinc predominantly forms tetrahedral

complexes, copper can adopt conformations ranging fiom linear to distorted tetrahedral. This

distinction may play a role in conferring specificity of metal binding to the dornain.

Two computer programs SELCON and K2D were used to estimate the secondary

structure content of the fusion protein and the WCBD with various amounts of copper and

zinc (table 4.1). The SELCON program (Sreerama and Woody, 1994; Sreerama and Woody,

1993) uses CD data between 260 and 200 nm to predict a-helix, 6-structure, and fi-turns in

globular proteins. The program starts by arranging the proteins in the database in order of

increasing RMS difference fiom the CD spectrum to be analyzed and the spectra which are

most dissimilar are deleted. An initial guess of the unknown protein is made and this

conformation is included in the basis set, which is deconvoluted using singular value

decomposition (SVD). The secondary structure of the unknown protein is then caiculated.

The solution replaces the initial guess and the process is repeated until self-consistency is

attained. The K2D is a neural net program (Andrade et al., 1993; Merelo et al., 1994) which

uses a technique called proteinotopic mapping to calculate a-helix and 6-structure using CD

data between 240 and 200 am. It has been found to give the best estimate of f3-sheet content

using this data range and was used in conjunction with the SELCON program (Andrade et

al., 1993; Merelo et al., 1994).

The general trend in the WCBD titrated with copper was an increase in a-helix

content (-6%), a decrease in 6-sheet content (-IO%), no change in the fi-turn content and a

slight decrease in 'other' conformations (-2%). In contrast, titrating the WCBD with zinc

produced a fluctuation in the secondary structure content which followed the patterns seen in

the spectra. Both the trend and magnitude of changes were substantially different from those

produced with copper, further supporting the idea that these metals induce very different

conformations in the domain upon binding. The trends for the fusion proteins with either

copper or zinc were similar to those for the GST fiee domains. The absolute amount of each

secondary structure element was higher than for the WCBD alone due to the contributions of

GST to the spectra. The effect of copper binding seems to be one of stabilizing or increasing

a-helical content at the expense of f3-sbeet content. The binding of copper could rigidiS the

secondary structure around the metal binding site, stabilizing secondary structural elements

which, in the absence of metal, adopt a different conformation. Alternatively, stabilization

could be achieved by interaction of distant parts of the domain which are brought into close

proxirnity by the binding of copper.

These studies have shown that copper is bound fairly strongly to the domain;

however, at some point, the copper must be removed fiom the domain. We have investigated

the possibility that copper may be removed by oxidation. After incubation of copper WCBD

with either hydrogen peroxide or ceric ammonium nitrate, only 2 copper atoms were

liberated fiom the domain with hydrogen peroxide. Ceric ammonium nitrate resulted in the

precipitation of the protein; however, when copper was measured in the dialyzed precipitate,

al1 six copper atoms were still present. These results indicate that, at least with hydrogen

peroxide, it is possible to release some of the bound copper. The other copper atoms rnay be

located in regions inaccessible to the oxidizing agent (buried in the core) or involved in

structures which are resistant to oxidation. These results suggest the possibility that copper

could be transported in the +2 oxidation state. There is at least one study where ATP-

dependent transport of copper(1I) has been detected in the golgi apparatus of rat hepatocytes

(Bingharn et al., 1996). Further research into the form of the transported copper, as well as

the kinetics of copper binding to the amino-teminal domain, is needed.

4.3.1 Hypofhetical Model for the Function ofthe WCBD in vivo

The current work has provided insights into the mechanism by which metal is bound

to the WCBD and the structural changes which take place upon copper binding. Functional

studies, both in cultured cells and yeast mode1 systems have also provided important insights

into the role of the amino-terminal metal-binding repeats in the context of the whole ATPase.

Immunohistochemical studies in HepG2 cells have s h o w that, under normal, steady-state

conditions, the WND protein is Iocalized in the membrane of the tram-golgi (Hung et al.,

1997). However, if the concentration of copper was elevated in the media, a protein

translocation event was observed which resulted in the appearance of the WND protein in the

plasma membrane as well as an unidentified cytoplasrnic vacuolar location (Hung et al.,

1997). Furthemore, this translocation event was reversible and dependent on copper, since

removal of copper fiom the media resulted in the WND protein recycling back to the trans-

golgi. This phenomena has also been observed for the MNK protein and it is likely that these

proteins continuously recycle between the trans-golgi and the plasma membrane under steady

state conditions; elevated copper concentrations may shift the equilibrium toward plasma

membrane localization (Nagano et al., 1998; Petris et al., 1996).

It has also been shown that not al1 of the metal binding repeats are needed to mediate

the transport fûnction of the purnp (Forbes et al., 1999). Using a yeast complementation

assay, Forbes et al. (1999) were able to demonstrate that the mutation of metal repeats 1

through 4 did not affect the transport activity of the pump. Mutation of repeats 1 through 5

resulted in an approximate 5% drop in transport activity, while mutation of al1 six domains

totally abolished transport activity. This is in contrat to what has been found for the MNK

protein. In MNK, sequential mutation of the metal binding repeats does not result in a

significant loss of transport activity until the third motif is mutated (Payne and Gitlin, 1998).

This suggests that the metal binding repeats in the MNK protein are not functionally

equivalent and that motif three may play a central role in the binding and/or transport of the

metal. In the WND protein the data suggest that the metal binding repeats closer to the

membrane are more important than those at the amino-terminus- The metal binding sites in

the WND protein are also not functionally equivalent, since swapping the amino-terminal

three domains with the three domains closer to the membrane does not result in a fùnctional

protein (Forbes et al., 1999).

Taken together, these results can be used to construct a hypothetical model of how the

arnino-terminal metal-binding domain hnctions in the context of the whole ATPase (!Tg.

4.8). In this model, the WND protein would reside in the tram-golgi membrane under

normal physiological conditions where it would finction to pump copper fiom the cytoplasm

into the tram-golgi lumen. This copper could then be incorporated into secreted copper-

containing enzymes such as lysyl oxidase and ceruloplasmin. When unoccupied with copper,

the amino-terminal metal-binding domain interacts with the other cytosolic loops of the

ATPase (possibly the SEHPL motif region), preventing the transport of copper through the

pump. The binding of copper (most likely to the rnetal repeats closer to the trammembrane

segments) triggers a conformational change in the metal binding domain which disrupts or

changes its interaction with the cytosolic loops of the ATPase. This conformational change

could also promote the phosphorylation of the enzyme, initiating the transport cycle and

allow copper to be pumped into the tram-golgi lumen.

When the cytoplasmic concentrations of copper rise (as in copper overload), the

additional metal binding repeats would become progressively occupied with copper leading

to further conformational changes in the domain. These conforrnational changes would

somehow trigger the translocation of the WND (and MNK) proteins fiom the trans-golgi to

both the plasma membrane and a cytoplasmic vacuolar cornpartment. At the plasma

TGN Lumen

CopeOr ) Binding

Cytoplasm

1 A Extracellular 1

1. *[Cul* 2. Translocation

Figure 4.8 Hypothetical Model for the Function of WND in vivo. Under normal physiological conditions, the WND protein is localized in the bans-golgi network (TGN). When not occupied with copper, the metal binding domain interacts with the cytosolic loops of the ATPase. Binding of copper to the metal repeats closest to the transmembrane segments results in a conformational change wbich stimulates the phosphorylation of the ATPase and initiation of copper transport into the TGN lumen. Under conditions of elevated cytoplasmic copper, al1 of the metal binding repeats become occupied with copper resulthg in further conformational changes and the translocation of WND fiom the TGN to the plasma membrane (PM). At the PM, the WND protein would pump copper nom the cytoplasm into the extracellular space, lowering the cytoplasmic concentration of copper. As intracellular copper levels decrease, the metal binding repeats become progressively unoccupied and WND is recycled back to the TGN. A similar mechanism could also take place with the MNK protein.

membrane the WND and MNK proteins would be involved in pumping copper out of the ce11

in an effort to lower the cytoplasmic concentrations of copper. As the intracellular levels of

copper decrease, the metal binding domain would become progressively unoccupied leading

to the recycling of WND back to the tram-golgi membrane. The nature of the cytoplasmic

vacuolar compartment has not been analyzed. A recent study has shown that these

cytoplasmic vesicles are localized towards the hepatocyte canalicular membrane and may

provide a route for the excretion of copper into the bile (Scbaefer ez al., 1999). It is possible

that this compartment could be a lysosome, since copper pumped into lysosomes could then

be deposited into the bile through fùsion with the canalicular membrane. If true, this would

explain why biliary copper excretion in Wilson disease patients is severely compromised.

This point will have to be clarified by fùture studies.

Several details of the mechanism presented in figure 4.8 still remain to be elucidated.

For example, it is not known whether the copper atoms bound to the amino-terminal domain

are actually transported through the ATPase or if copper atorns fkom the cytoplasm are

transported. In either case, it seems clear from several studies of the WND and -MNK

proteins that not al1 the metal binding repeats are needed to allow transport activity (Hung et

al., 1997; Nagano et al., 1998; Petris et al., 1996). If copper bound to the amino-terminal

domain is transported, these observations suggest that copper is onIy transported from a few

of the metal binding sites (those closer to the membrane in WND, and those closer to the

amino-terminus in MNK). In this case, the other repeats could be Eûnctioning in a regulatory

role, acting as 'sensors' of intracellular copper concentration. If the amino-terminal bound

copper atoms are not transported, then the entire domain could be playing a role in regulating

the activity of the protein. The oxidation state of the transported copper atom is also not

known. Although we have shown that copper bound to the amino-terminal domain is in the

+ 1 oxidation state (DiDonato et al., 1997), some evidence suggests that copper is transported

in the +2 oxidation state (Bingham et al., 1996). If transported in the +2 oxidation state, the

amino-terminal bound copper would have to be oxidized prior to transport through the

membrane. This may be mediated by the protein itself or by an extemal oxidizing agent such

as hydrogen peroxide.

CHAPTER 5

General Discussion and Future Directions

5.1 General Discussion

The results presented in this thesis represent the fmt detailed characterization studies

of the copper binding domain fiom the Wilson disease copper transporting ATPase. These

studies have shown that the WCBD can bind a variety of transition metals in addition to

copper with varying affinities. This finding is intriguing since it leads to the question of

whether these other metals are traosported by the WND ATPase as well. This question has

not yet been addressed directly and will be an important topic for friture research. However,

we do know that the primary defect in Wilson disease is one of impaired copper emux Erom

the liver, leading to copper overload, which suggests that the main purpose of the WND

protein is to transport copper. Therefore, in the cytoplasm of the hepatocyte, what

mechanism does the protein employ to ensure specificity for copper? It is possible that the

protein never has an opportunity to interact with other metals in vivo due to their localization

in different cellular compartments. Ln recent years, a series of copper chaperones have been

identified, which are responsible for targetting copper to specific intracellular targets

(Valentine and Gralla, 1997). Similar proteins could exist for other transition metals to

facilitate their delivery to specific cellular compartments.

Altematively, the metal binding domain itself could be responsible for conferring

metal specificity on the protein. Bo& the 6Sz in~ blotting studies and the circula dichroism

studies have shown that the binding of copper to the WCBD may have some unique

properties which could result in specificity for copper as the transported ion. The

cornpetition 65zinc bloning studies have shown that a cooperative binding mode may exist for

copper, but not for the other metals. The structural changes in the domain induced by copper

binding are also very different fiom those induced by the binding of zinc. In the intact

protein, the binding of copper to the amino-terminal domain could trigger specific structural

changes which lead to the initiation of the transport cycle. Zinc would not be able to induce

the same conformation in the amino-terminal domain and, hence, the transport cycle could

not be initiated. XAS studies have shown that copper is ligated by two sulfur atoms in a

distorted linear arrangement. Zinc would not be able to bind to the domain in the same

manner since it prefers tetrahedral coordination and it is likely that the binding of zinc would

force the domain into a non-native conformation, an idea which is supported by our CD zinc

titration results.

The detailed structural data presented in this thesis, together with in vivo and in vitro

work of other groups, has allowed the formulation of a hypothetical mode1 for the function of

the LWD protein in vivo and the role the WCBD plays in this mechanism (fig. 4.8). In this

modei, the translocation of the protein, which is observed at elevated copper concentrations,

is rnediated by conformationai changes resulting from copper binding to the amino-terminal

domain. However, recent studies have indicated that this may not occur in the Menkes

protein. When the cysteine residues in HMA domains 1, 6, or 1-3 were mutated to serine in

the Menkes protein, the protcin was still able to relocate to the plasma membrane in response

to elevated copper (Strausak et al., 1999). However, if HMAs 4-6 or 1-6 were mutated, the

protein did not respond to elevated copper concentrations. Further analysis revealed that the

first four HMAs could be eliminated without loss of copper induced relocation; mutation of

the remaining two HMAs abolished its response to copper. Although the study by Strausak,

et al. (1999) evaluated the effects of these mutations on copper induced relocation, they did

not measure copper transport activity. An earlier report has shown that al1 transport activity

is lost wheo the cysteine residues in the fmt three HMA domains of the Menkes protein are

mutated (Payne and Gitlin, 1998).

These observations are in sharp contrast to similar studies performed on the Wilson

protein, which show that the first five HMA domains can be mutated without loss of copper

transport activity (Forbes et al., 1999). Taken together, these studies illustrate that the HMA

domains are not bctionally equivalent and that the amino-terminal domains of the Menkes

and Wilson proteins do not hnction in similar manners. In light of this, the results of the

study by Strausak, et al. (1999) cannot be extrapolated easily to the Wilson disease. Similar

studies on the Wilson disease protein will be needed to answer these questions.

5.2 Future Directions

The expression and characterization of the amino-teminal copper binding domain

from the WiIson disease copper transporting ATPase has given us invaluable information on

the possible fiuiction of this domain in the context of the entire protein. However, despite the

detailed analysis which has been carried out thusfar, several important questions concerning

the function of this domain in the transport of copper through the ATPase remain to be

addressed. Among these is the mechanism by which copper is delivered to the ATPase and

the subsequent steps which allow it to be transported across the membrane. The discovery of

a family of cytoplasmic copper chaperones has provided new avenues of research in this

area. Much of what is known about the fûnction of the copper chaperones has been derived

from complernentation studies in yeast. The yeast copper chaperone ATXI has been shown

to interact with the amino-terminal domain of ccc2, the yeast orthologue of WND, by yeast

two-hybnd analysis (Pufahl et al., 1997). The association of ATXl and ccc2 was also shown

to be dependent on the presence of copper. Recently, the human orthologue of ATXl has

been isolated and is known as ATOXl (formerly known as HAHI) (Klomp et al., 1997).

Due to the simiiarity between the ATXl and ATOXl proteins, an interaction bctween

ATOXI and the amino-terminal domain of the Wilson disease protein has been assumed to

exist, but has not been demonstrated experimentally .

In order to address the question of how copper is delivered to the domain in vivo, an

association behveen recombinant ATOX 1 and GST-WCBD wilt be demonstrated in vitro and

then characterized to determine the mechanism by which copper is transferred fiom ATOX 1

to the WCBD. The ba i s of the assay would consist of fornihg the complex in viîro and then

isolating it using glutathione affinity chromatography. Truncation and site-directed

mutagenesis studies will then De employed to isolate the region of the proteins which are

critical for cornplex formation and metal transfer. Expenments with the tnuicated protein

will establish if the chaperone is delivering copper to a specific HMA domain or has no

specificity for individual HMA domains. Together, these studies will help to define the

functional significance (if any) for each HMA repeat in the copper binding domain. The KD

values for copper binding to either the WCBD or ATOXl are also unknown. Solution ligand

binding experiments will be used to determine the dissociation constant for copper bound to

either the WCBD or ATOX1.

Another question, which is currently not addressed in the literature, is how copper is

transported across the membrane by the ATPase. Our studies have shown that once copper is

bound to the domain it is fairly resistant to oxidation and remains bound in the presence of

excess reducing agents, denaturants, and chelating agents such as EDTA. These observations

indicate that copper is bound tightly to the WCBD and lead to the question of how copper

can be removed from the WCBD once bound. We have investigated the possibility that

copper may be released by oxidation during the transport cycle. Prolonged exposure of the

the copper loaded WCBD to excess hydrogen peroxide resulted in the release of only two

copper atoms. In the intact protein, interactions between the amino-terminal domain and

other parts of the ATPase could also facilitate its release from the WCBD and its

translocation across the membrane. To M e r analyze this possibility, experiments must be

conducted using the entire ATPase. The Wilson disease ATPase couid be expressed in a

mammalian system and isolated in vesicles in a fùnctional form. Using this preparation,

detailed transport studies could be performed. If inverted vesicles could be prepared, such

that copper is pumped into the vesicte, the oxidation state of the transported copper could be

determined by examining the copper in the vesicles after the assay is complete. The

oxidation state of the copper inside the vesicles could easily be determined by electron

paramagnetic resonance (EPR)

The structural studies presented in this thesis have shown that significant structural

changes take place in the domain upon metal binding. Although both copper and zinc are

able to bind to the domain, the structures they induce upon binding are very different. This

difference could be the basis for the specificity of the ATPase. The HMA domain is found in

many proteins which bind a varie5 of metals (table 1.6), suggesting that it may be a general

metal binding motif. Studies on mtA, a zinc transporting ATPase in bacteria containing one

HMA domain, have shown that this protein c m transport cadmium as well as zinc, two

rnetals with similar ligation geometries (Rensing et al., 1997). Our cornpetition 65zinc

blotting studies have suggested that the binding of copper to the domain may have some

degree of cooperativity and that this property is unique to copper- In light of this

observation, we propose that the cooperativity observed for copper binding may contribute to

the specificity of the ATPase. To m e r examine the role of the metal binding domain and

the possible role it plays in conferring specificity to the ATPase, a chimeric protein could be

created containing the copper binding domain fiom the WND protein and the ATPase "core"

of zntA. The ability or inability of this chimera to transport copper could help confirm the

hypothesis that the WCBD is playing a part in determining the specificity of the transported

metal.

Although these experiments will give a very good understanding of how copper is

transferred and bound to the domain, a complete understanding of the fünction of this domain

wiIl require a detailed three-dimensional structure. For this reason, the three-dimensional

structure of the WCBD will be determined by x-ray crystallography. NMR will also be used

to determine the structure of a truncated fom (-35 kDa) of the domain, which contains only

KMA domains 4, 5, and 6, since biochemical snidies have indicated that these are of greater

importance to the function of the Wilson disease protein than the others (Forbes et al., 1999).

These structural studies, coupled with ongoing XAS experiments using copper and zinc

reconstituted proteins, will allow us to better define the role of this novel dornain in the

overall mechanism of the ATPase.

5.3 Literature Cited

Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994). Structure at 2.8 A resolution of F 1-ATPase from bovine heart rnitochondria. Nature 370,62 1-628.

Aggeler, R., and Capaldi, R. A. (1996). Nucleotide-dependent movement of the epsilon subunit between alpha and beta subunits in the Escherichia coli FlFO-type ATPase. 1. BioI. Chem. 271, 13888-13891.

Alexander, J., and Aaseth, J. (1980). Biliary excretion of copper and zinc in the rat as influenced by diethyhaleate, selenite and diethyldithiocarbamate. Biochem. Pharmacol. 29, 2139-2133.

Alexiou, D., Hatzis, T., and Koutselinis, A. (1985). Maintenance treatment of Wilson's disease with oral zinc. Apropos of a child treated for 4 years. Archives Francaises de Pediatrie 42,44749.

Allendorf, M. D., Spira, D. J., and Solomon, E. 1. (1985). Low-temperature magnetic circular d ic hro ism studies of native laccase: spectroscopie evidence for exogenous ligand bi-idging at a trinuclear copper active site. Proc Nat1 Acad Sci U S A 82,3063-3067.

Altendorf, K., Voelkner, P., and Puppe, W. (1994). The sensor kinase KdpD and the response regulator KdpE control expression of the kdpFABC operon in Escherichia coli. Res. Microbiol. 145,374-38 1.

Amaravadi, R., Glerum, D. M., and Tzagoloff, A. (1997). Isolation of a cDNA encoding the human homolog of COX 17, a yeast gene essential for mitochondrial copper recruitment. Hum. Genet. 99,329-333.

Andrade, M. A., Chacon, P., Merelo, J. J., and Moran, F. (1993). Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng. 6, 383-390.

Baker, D. H., and Czarnecki-Maulden, G. L. (1987). Pharmacologic role of cysteine in ameliorating or exacerbating mineral toxicities. J. Nutr. 11 7, 1003- 10 10.

Bankier, A. (1 995). Menkes disease. J. Med. Genet. 32,2 13-5.

Barnea, A., Hartter, D. E., Cho, G., Bhasker, K. R., Katz, B. M., and Edwards, M. D. (1990). Further characterization of the process of in vitro uptake of radiolabeled copper by the rat brain. J. Inorg. Biochem. 40, 103-10.

Bamea, A., and Katz, B. M. (1990). Uptake of 67copper complexed to 3H-histidine by brain hypothalamic slices: evidence that dissociation of the complex is not the only factor determining 67copper uptake. J. Inorg. Biochem. 40,8 1-93.

Barnes, G., and Frieden, E. (1984). Ceruloplasmin receptors of erythrocytes. Biochem. Biophys. Res. Commun. 125, 157-162.

Barrow, L., and Tanner, M. S. (1988). Copper distribution among serum proteins in paediatric liver disorders and malignancies. Eur. J. Clin. hvest. 18,555-60.

Beckman, J. S., Carson, M., Smith, C. D., and Koppenol, W. H. (1993). ALS, SOD and peroxynitrite. Nature 364, 584.

Beers, J., Glerum, D. M., and Tzagoloff, A. ( 1 997). Puri fication, characterization, and localization of yeast Coxl7p, a rnitochondrial copper shuttle. J. Biol. Chem. 272, 33191- 33196.

Beratis, N. G., Price, P., Labadie, G., and Hirschhom, K. (1978). 64Cu metabolism in Menkes and normal cultured skin fibroblasts. Pediatr. Res. f 2,699-702.

Bermingham-McDonogh, O., Gralla, E. B., and Valentine, J. S. (1988). The copper, zinc- superoxide dismutase gene of Saccharomyces cerevisiae: cloning, sequencing, znd biological activity. Proc Nat1 Acad Sci U S A 85,47894793.

Biernpica, L., Rauch, H., Quintana, N., and Stemlieb, 1. (1988). Morphologie and chemical studies on a murine mutation (toxic miik mice) resulting in hepatic copper toxicosis. Lab. Invest. 59, 500-508.

Bingham, M. J., Ong, T. J., Ingledew, W. J., and McArdle, H. J. (1996). ATP-dependent copper transporter, in the Golgi apparatus of rat hepatocytes, transports C u 0 not Cu@). Am. J. Physiol. 271, G741-6.

Bimboim, H. C., and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1 5 1 3-23.

Bohm, G., Muhr, R., and Jaenicke, R. (1992). Quantitative analysis of protein far W circular dichroism spectra by neural networks. Protein Eng. 5, 19 1 - 195.

Borjigin, J., Payne, A. S., Deng, J., Li, X., Wang, M. M., Ovodenko, B., Gitlin, J. D., and Snyder, S. H. (1999). A novel pineal night-specific ATPase encoded by the wilson disease gene. J. Neurosci. 19, 1018-26.

Bowcock, A. M., Farrer, L. A., Cavalli-Sforza, L. L., Hebert, J. M., Kidd, K. K., Frydman, M., and Bonne-Tamir, B. (1987). Mapping the Wilson disease locus to a cluster of linked polyrnorphic markers on chromosome 13. Am. J. Hum. Genet. 41,27-35.

Bowcock, A. M., Farrer, L. A., Hcbert, J. M., Agger, M., Sternlieb, I., Scheinberg, 1. H., Buys, C. H., Scheffer, H., Frydman, M., Chajek-Saul, T., and et al. (1988). Eight closely linked loci place the Wilson disease locus within 13q14-q21. Am. J. Hum. Genet. 43,664-74.

Boyer, P. D. (1993). The binding change mechanism for ATP synthase-some probabilities and possibilities. Biochim. Biophys. Acta 1140,215-250.

Boyer, P. D., Cross, R. L., and Momsen, W. (1973). A new concept for energy coupling in oxidative phosphorylation based on a molecular explanation of the oxygen exchange reactions. Proc Nat1 Acad Sci U S A 70, 283 7-2839.

Brewer, G. J., Dick, R. D., Johnson, V., Wang, Y., Yuzbasiyan-Gurkan, V., Kluin, K., Fink, 3. K., and Aisen, A. (1994a). Treatment of Wilson's disease with ammonium tetrathiomolybdate. 1. initial therapy in 17 neurologically affected patients. Arch. Neurol. 51, 545-554.

Brewer, G. J., Dick, R. D., Yuzbasiyan-Gurkin, V., Tankanow, R., Young, A. B., and Kluin, K. J. (199 1). Initial therapy of patients with Wilson's disease with tetrathiomolybdate. Arch. Neurol. 48,42-7.

Brewer, G. J., Hill, G. M., Prasad, A. S., Cossack, Z . T., and Rabbani, P. (1983). Oral zinc therapy for Wilson's disease. Am. htem. Med. 99,3 14-9.

Brewer, G. J., Turkay, A., and Yuzbaziyan-Gurkan, V. (1 994). Development of neurologie symptoms in a patient with asymptomatic Wilson's disease treated with penicillamine. Arch. Neurol. 51,304-5.

Britton, R. S. (1996). Metal-induced hepatotoxicity. Semin. Liver Dis. 16, 3-12.

Brown, M. S., Anderson, R. G., and Goldstein, J. L. (1 983). Recycling receptors: the round- trip itinerary of migrant membrane proteins. Cell32,663-667.

Buchwald, P., Krurnrneck, G., and Rodel, G. (1991). Immunological identification of yeast SC01 protein as a component of the imer mitochondrial membrane. Mol. Gen. Genet. 229, 41 3-20.

Bull, P. C., and Cox, D. W. (1994). Wilson disease and Menkes disease: new handles on heavy-metal transport. Trends Genet. 10,246-52.

Bull, P. C., Thomas, G. R., Rommens, J. M., Forbes, J. R., and Cox, D. W. (1993). The Wilson disease gene is a putative copper transporthg P-type ATPase similar to the Menkes genc. Nat. Genet. 5, 327-37.

Burgess, J. (1 978). Meta1 Ions in Solution (Chichester: Ellis Horwood Ltd).

Buurman, E. T., Kim, K. T., and Epstein, W. (1995). Genetic evidence for two sequentially occupied K+ binding sites in the Kdp transport ATPase. J. Biol. Chem. 270,6678-6685.

Campbell, C. H. important source 678,27-38.

, Brown, R., and Linder, M. C. (198 1). Circulating ceruloplasmin is an of copper for normal and malignant animal cells. Biochim. Biophys. Acta

Cano-Gauci, D. F., and Sarkar, B. (1 996). Reversible zinc exchange between metallothionein and the estrogen receptor zinc finger. FEBS Lett. 386, 1 4 .

Cantley, L. C., Jr., Cantley, L. G., and Josephson, L. (1978). A characterization of vanadate interactions with the (Na,K)-ATPase. Mechanistic and regulatory implications. J. Biol. Chem. 253,736 1-7368-

Cartwright, G . E., Hodges, R E., Gubler, C. J., Mahoney, 1. P., Daum, K., Wintrobe, M. M., and Bean, W. B. (1954). Studies on copper metabolism. xiii. Hepatolenticular Degeneration. J. Clin. Invest. 33, 1487.

Casareno, R. L. B., Waggoner, D., and Gitlin, J. D. (1 998). The copper chaperone CCS directly interacts with copperl zinc superoxide dismutase. J. Biol. Chem. 273,23625-23628.

Cecchi. C., and Amer, P. (1 996). Genomic organization of the mottled gene, the mouse homologue of the human menkes disease gene. Gcnomics 37,96- 104.

Chan, W. Y., Gamica, A. D., and Remert, O. M. (1978). CeIl culhue studies of Menkes kinky hair disease. Clin. Chim. Acta 88,495-507.

Chelly, J., Tumer, Z., T a ~ e s e n , T., Petterson, A., Ishikawa-Brush, Y., Tommerup, N., Hom, N.. and Monaco, A. P. (1993). Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein. Nat. Genet. 3, 14-9.

Chen. P., Onana, P., Shaw, C. F. r., and Petering, D. H. (1996). Characterization of calf liver Cu,Zn-metallothionein: naturally variabIe Cu and Zn stoichiometries. Biochem. J. 3 17, 389- 94.

Chang , L. M., Wu, H. P., h g , M. H., Wang, T. R., Sue, W. C., Lin, B. J., Cox, D. W., and Tai, T. Y. (1996). High fiequency of two mutations in codon 778 in exon 8 of the ATP7B gene in Taiwanese families with Wilson disease. J. Med. Genet. 33,52 1-523.

Ciriolo, M. R., Desideri, A., Paci, M., and Rotilio, G. (1990). Reconstitution of Cu,Zn- superoxide dismutase by the CuO.glutathione cornplex. J. Biol. Chem. 265, 11030-4.

Clague, M. J., Urbe, S-, Aniento, F., and Gmenberg, J. (1994). Vacuolar ATPase activity is required for endosomal carrier vesicle formation. 1. Biol. Chem. 269,21-24.

Clarke, D. M., Loo, T. W., Inesi, G., and Mademan, D. H. (1989). Location of high afinity Ca2+-binding sites within the predicted transmembrane domain o f the sarcoplasmic reticulum Cd+-ATPase. Nature 339,476-478.

Clarke, D. M., Loo, T. W., and MacLennan, D. H. (1990). Functional consequences of mutations of conserved amino acids in the beta-strand domain of the Cd(+)-ATPase of sarcoplasmic reticulum. J. Biol. Chem. 265, 14088-14092.

Conte, D., Narindrasorasak, S., and Sarkar, B. (1996). In vivo and in vitro iron-replaced zinc finger generates £kee radicals and causes DNA damage. J. Biol. Chem. 271, 5 125-30.

Corson, L. B., Strain, J. J., Culotta, V. C., and Cleveland, D. W. (1998). Chaperone- facilitated copper binding is a property common to several classes of familial amyotrophie lateral sclerosis-linked superoxide dismutase mutants. Proc Natl Acad Sci U S A 95, 636 1 - 6366.

Coucouvanis, D., Murphy, C. N., and Kanodia, S. K. (1980). Metal-Mercaptide Chemistry. Synthesis and Structural Characterization of the [Cu(SC6H5)3]2- Anion. A Rational Spthesis and the structure of the [Cu4(SC6H5)6]2- Cluster. Iaorg. Chem. 19, 2993-2998.

Cousins, R. J. (1985). Absorption, transport, and hepatic metabolism of copper and zinc: special reference to metallothioneh and ceruloplasmin. Physiol. Rev. 65, 238-309.

Cox, D. W. (1995). Genes of the copper pathway. Am. J. Hum. Genet. 56,828-34.

Crampton, R. F., Mathews, D. M., and Poisner, R. (1965). Observations on the mechanism of absorption of copper by the small intestine. J. Physiol. 178, 1 1 1 - 126.

Crapo, J. D., Oury, T., Rabouille, C., Slot, J. W., and Chang, L. Y. (1992). Copper,zinc superoxide dismutase is primarily a cytosolic protein in human cells. Proc Natl Acad Sci U S A 89. 10405- 10409.

Culotta, V. C., Klomp, L. W. J., Strain, J., Casareno. R. L. B., Krems, B., and Gitlin, J. D. (1 997). The copper chaperone for superoxide dismutase. J. Biol. Chem. 272,23469-23472.

Daimon, M., Kato, T., Kawanami, T., Tominaga, M., Igarashi, M., Yamatani, K., and Sasaki, H. (1995). A nonsense mutation of the ceruloplasmin gene in hereditary ceruloplasmin deficiency with diabetes meIlitus. Biochem. Biophys. Res. Commun. 21 7, 89-95.

Dameron, C. T., and Harris, E. D. (1987). Regulation of aonic CuZn-superoxide dismutase with copper. Caeruloplasmin and albumin re-activate and transfer copper to the enzyme in culture. Biochem. J. 248,669-75.

Danks, D. M. (1977). Copper transport and utilisation in Menkes' syndrome and in mottled mice. Inorg. Perspect. Biol. Med. 1, 73.

Danks, D. M. (1995). Disorders of Copper Transport. In Metabolic Basis of Inherited Disease, C. R. Scriver, A. 1. Beaudet, W. S. Sly and D. Valle, eds. (New York: McGraw- Hill), pp. 22 1 1-2235.

Danks, D. M. (1988). The mild fonn of Menkes disease: progress report on the onginal case. Am. J. Med. Genet. 30,859-64.

Danks, D. M. (1986). Of mice and men, metals and mutations. J. Med. Genet. 23,99-106.

Danks, D. M., and Carnakaris, J. (1 983). Mutations affecting trace elements in humans and animais. A genetic approach to an understanding of trace elements. Adv. Hum. Genet. 13, 149-2 16.

Danks, D. M., Campbell, P. E., Walker-Smith, J., Stevens, B. J., Gillespie, J. M., Blomfield, J., and Turner, B. (1972). Menkes' kinky-hair syndrome. Lancet 1, 1100-2.

Danks, D. M., Cartwright, E., Stevens, B. J-, and Townley, R- R. (1973). Menkes' kinky hair disease: further definition of the defect in copper transport. Science 179, 1 140-2.

Danvish, H. M., Cheney, J. C., Schmitt, R, C., and Ettinger, M. J. (1984). Mobilization of copper(I1) from plasma components and mechanisms of hepatic copper transport. Am. J. Physiol. 246, G72-79.

Danvish, H. M., Hoke, J. E., and Ettinger, M. J. (1983). Kinetics of Cu(I1) transport and accumuIation by hepatocytes fkom copper-deficient mice and the brindled mouse mode1 of Menkes disease. J. Biol. Chem. 258, 13621-6.

Das, S., Levinson, B., Whitney, S., Vulpe, C., Packman, S., and Gitschier, J. (1994). Diverse mutations in patients with Menkes disease often lead to exon skipping. Am. J. Hum. Genet. 55, 883-9.

De Duve, C., and Wattiaux, R. (1966). Functions of lysosomes. Annu. Rev. Physiol. 28,435- 492.

Deng, H. X., Hentati, A., Tainer, J. A., Iqbal, Z., Cayabyab, A., Hung, W. Y., Getzoff, E. D., Hu, P., Herzfeidt, B., Roos, R. P., Wamer, C., Deng, G., Soriano, E., Smyth, C., Parge, H. E., Ahmed, A., Roses, A. D., Hailewell, R. E., Pericak-Vance, M. A., and Siddique, T. (1993). Arnyotrophic Iateral sclerosis and structural defects in Cu,Zn superoxide dismutase. Science 261, 1047-1051.

Demy-Brown, D., and Porter, H. (195 1). The effect of BAL (2,3-dimercaptopropanol) on hepatolenticular degeneration (Wilson's disease). N. Engl. J. Med. 245, 9 17-925.

Devesa, R., Alvarez, A., de las Heras, G., and Ramon de Miguel, J. (1995). Wilson's disease treated with trientine during pregnancy. Journal of Pediatric Gastroenterology & Nutrition 20, 102-3.

Dhaunsi, G. S., Gulati, S., Singh, A. K., Orak, 3. K., Asayama, K., and Singh, 1. (1992). Demonstration of Cu-Zn superoxide dismutase in rat liver peroxisomes. Biochemical and immunochemical evidence. J. Biol. Chem. 267,6870-6873.

DiDonato, M., Narindrasorasak, S., Forbes, J. R., Cox, D. W., and Sarkar, B. (1997). Expression, purification, and metal binding properties of the N-terminal domain fiom the Wilson disease putative copper-transporting ATPase (ATP7B). J. Biol. Chem. 2 72,33279.

DiDonato, M., and Sarkar, B. (1997). Copper transport and its alterations in Menkes and Wilson diseases. Bba Mol Basis Dis 1360, 3-16.

Dierick, H. A., Ambrosini, L., Spencer, J., Glover, T. W., and Mercer, J. F. (1995). Molecular structure of the Menkes disease gene (ATP7A). Genomics 28,462-9.

Dijkstra, M., In 't Veld, G., van den Berg, G. J., Muller, M., Kuipers, F., and Vonk, R J. (1995). Adenosine triphosphate-dependent copper transport in isolated rat liver plasma membranes. J. Clin. Invest. 95,4 12-4 16.

Dijkstra, M., Kuipers, F., Havinga, R., Smit, E. P., and Vonk, R. J. (1990). Bile secretion of trace elements in rats with a congenital defect in hepatobiliary transport of glutathione. Pediatr. Rcs. 28, 339-343.

Dijkstra, M., Vonk, R. J., and Kuipers, F. (1996). How does copper get into bile? New insights into the mechanism(s) of hepatobiliary copper transport. J. Hepatol. 24, 109- 120.

Dobyns, W. B., Goldstein, N. P., and Gordon, H. (1979). Clinical spectmm of Wilson's disease (hepatolenticular degeneration). Mayo Clin. Proc. 54,3542.

Dombroski, A. J., Walter, W. A., Record, M. T.. Jr., Siegele, D. A., and Gross, C. A. (1992). Polypeptides containing highly consewed regions of transcription initiation factor sigma 70 exhibit specificity of binding to promoter DNA. Ce11 70, 501 -1 2.

Duncan, T. M., Bulygin, V. V., Zhou, Y., Hutcheon, M. L., and Cross, R. L. (1995). Rotation of subunits during catalysis by Escherichia coli FI-ATPase. Proc Nat1 Acad Sci U S A 92, 10964- 10968.

Ettinger, M. J., Darwish, H. M., and Schmitt, R. C. (1986). Mechanism of copper transport f?om plasma to hepatocytes. Federation Proceedings 45,2800-2804.

Evans, G. W. (1973). Copper homeostasis in the mammalian system. Physiol. Rev. 53, 535- 570.

Falchetto, R., Vorherr, T., Bninner, J., and Carafoli, E. (1991). The plasma membrane C d + pump contains a site that interacts with its calmodulin-binding domain. J. Biol. Chem. 266, 2930-2936.

Ferreira, A. M., Ciriolo, M. R., Marcocci, L., and Rotilio, G. (1993). Copper(l) transfer into metallothionein mediated by glutathione. Biochem. J. 292,673-6.

Fields, M., Ferretti, R. J., Reiser, S., and Smith, J. C., Jr. (1984). The severity of copper deficiency in rats is determined by the type of dietary carbohydrate. Proc. Soc. Exp. Biol. Med. 175, 530-537.

Figus, A., Angius, A., Loudianos, G., Bertini, C., Dessi, V., Loi, A., Deiana, M., Lovicu, M., Olla, N., Sole, G., Devirgiliis, S., Lilliu, F., Farci, A. M. G., Nurchi, A., Giacchino, R., Barabino, A., Marazzi, M., Zancan, L., Greggio, N. A., Marcellini, M., Solinas, A., Deplano, A., Barbera, C., Devoto, M., Ozsoylu, S., Kocak, N., Akar, N., Karayalcin, S., Mokini. V., Cullufi, P., Balestrieri, A., Cao, A., and Pirastu, M. (1995). Molecular pathology and haplotype analysis of Wilson disease in Mediterranean populations. Am. I. Hum. Genet. 57, 13 1 8- 1324.

Fillingame, R. H. (1 997). Coupling H+ banspon and ATP synthesis in F 1 FO-ATP synthases: glimpses of interacting parts in a dynamic molecular machine. J. Exp. Biol. 2OO,2 17-224.

Finbow, M. E., and Harrison, M. A. (1997). The vacuolar H+-ATPase: a universal proton purnp of eukaryotes. Biochem. J. 324,697-712.

Forbes, J. R., Hsi, G., and Cox, D. W. (1999). Role of the Copper-binding Domain in the Coppcr Transport Fuaction of ATPïB, the P-type ATPase Defective in Wilson Disease. J. Biol. Chern. 274, 12408- 12413.

Freedman, J. H., Ciriolo, M. R., and Peisach, 1. (1989). The role of glutathione in copper metabolism and toxicity. J. Biol. Chem. 264, 5598-5605.

Frieden, E. (1986). Perspectives on copper biochemistry. Clinical Physiology & Biochemistry 4, 1 1-9.

Frydman, M., Bonne-Tamir, B., Farrer, L. A., Comeally, P. M., Magazanik, A., Ashbel, S., and Goldwitch, 2. (1985). Assignment of the gene for Wilson disease to chromosome 13: linkage to the esterase D locus. Proc. Natl. Acad. Sci. USA 82, 18 19-2 1.

Fujisawa, K., Imai, S., Kitajima, M., and Moro-oka, Y. (1998). Preparation, Spectroscopie Characterization, and Molecular Stnicture of CopperO Aliphatic Thiolate Complexes. horg. Chem. 37, 168-169.

Gacheru, S. N., Trackman, P. C., Shah, M. A., O'Gara, C. Y., Spacciapoli, P., Greenaway, F. T., and Kagan, H. M. (1 990). Structural and catalytic properties of copper in lysyl oxidase. J. Biol. Chem. 265, 19022-19027.

Gamonet, F., and Lauquin, G. J. M. (1998). The Saccharomyces cerevisiae LYS7 gene is involved in oxidative stress protection. Eur. J. Biochern. 251, 7 16-723.

Garnica, A. D. (1984). The failure of parenteral copper therapy in Menkes Kinky Hair syndrome. Eur. J. Pediatr. 142,98-102.

Gitlin, J. D., Schroeder, J. J., Lee-Ambrose, L. M., and Cousins, R. J. (1992). Mechanisms of caeruloplasmin biosynthesis in normal and copper-deficient rats. Biochem. J. 282,835-839.

Gitschier. J., Moffat, B., Reilly, D., Wood, W. I., and Fairbrother, W. J. (1998). Solution structure of the fourth metal-binding domain from the Menkes copper-transporting ATpase. Nature Struct Biology 5,47-54.

Glass, J. D., Reich, S. G., and DeLong, M. R. (1990). Wilson's disease. Development of neurological disease after beginning penicillamine therapy. Arch. Neurol. 47,595-6.

Glerum, D. M., Shtanko, A., and Tzagoloff, A. (1996). Characterization of COX17, a yeast gene involved in copper metabolism and assembly o f cytochrome oxidase, J. Biol. Chem. 2 71, 14504- 14509.

Glerum, D. M., Shtanko, A., and Tzagoloff, A. (1996). SC01 and SC02 act as high copy suppressors of a mitochondrial copper recruitment defect in Saccharomyces cerevisiae. 1. Biol. Chem. 271,2053 1-20535.

Gluck, S. (1992). V-ATPases of the plasma membrane. J. Exp. Biol. 172,29-37.

Glusker, J. P. (1991). Advances in Protein Chemistry, Volume 42, C. B. Anfinsen, J. T. Edsall, F. M. Richards and D. S. Eisenberg, eds. (San Diego: Academic Press).

Glym, 1. M., and Karlish, S. J. (1990). OccIuded cations in active transport. Annu. Rev. B ioc hem. 59, 1 7 1 -205.

Goka, T. J., Stevenson, R. E., Hefferan, P. M., and Howell, R. R. (1976). Menkes disease: a biochemical abnonnality in cultured human fibroblasts- Proc. Natl. Acad. Sci. USA 73, 604- 6.

Goode, C. A., Dinh, C. T., and Linder, M. C. (1989). Mechanism of copper transport and delivery in mammals: review and recent findings. Adv. Exp. Med. Biol. 258, 13 1-144.

Gooneratne, S. R., Howell, J. M., and Cook, R. D. (1980). An ultrastructural and morphometric study of the liver of normal and copper-poisoned sheep. Am. J. Pathol. 99, 429-449.

Gordon, D. T., Leinart, A. S., and Cousins, R. J. (1987). Portal copper transport in rats by albumin. Am. J. Physiol. 252, E327-333.

Greenfield, N. J. (1 996). Methods to estimate the conformation of proteins and polypeptides fkom circular dichroism data. Anal. Biochem. 235, 1- 10.

Gross, J. B., Jr., Myers, B. M., Kost, L. J., Kuntz, S. M., and LaRusso, N. F. (1989). Biliary copper excretion by hepatocyte lysosomes in the rat. Major excretory pathway in experimental copper overload. J. Clin. hvest. 83, 30-39.

Gurney, M. E., Pu, H., Chiu, A. Y., Da1 Canto, M. C., Polchow, C. Y., Alexander, D. D., Caliendo, J., Hentati, A., Kwon, Y. W., Deng, H. X., Chen, W. C., Zhai, P., Sufit, R. L., and Siddique, T. (1994). Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science 264, 1 772- 1775.

Hall, A. C., Young, B. W., and Bremner, 1. (1979). intestinal metallothionein and the mutual antagonism between copper and zinc in the rat. J. inorg. Biochem. 11,57-66.

Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557-80.

Hancock, R. G. V. (1976). Low flux multielement instrumental neutron activation analysis in archaeometry. Analyt. Chem. 48, 1443- 1445.

Hancock, R. G. V. (1984). On the source of clay used for Cologne Roman pottesr- Archaeometry 26,2 10-2 1 7.

Hancock, R. G. V. (1978). Some aspects of the analysis of ancient artifacts by neutron activation analysis. J. Internat. Inst. Conserv. - Canad. Grp. 3,2 1-27.

Hams, D. A. (1995). Protein and peptide inhibitors of the FI-ATPase. Biochem. Soc. Trons. 23, 790-793.

Harris, D. I., and Sass-Kortsak, A. (1967). The influence of amino acids on copper uptake by rat liver slices. J. Clin. Lnvest. 46, 659-667.

Harris, E. D. (1986). Biochemical defect in chick lung resulting from copper deficiency. J. Nutr. 116, 252-258.

Harris, E. D. (1992a). Copper as a cofactor and regulator of copper,zinc superoxide dismutase. J. Nutr. 122,636-640.

Harris, E. D. ( 199 1). Copper transport: an overview. Proc. Soc. Exp. Biol. Med. 196, 130- 140.

Hams, E. D. (1976). Copper-induced activation of aortic lysyl oxidase in vivo. Proc Nat1 Acad Sci U S A 73,371-374.

Hams, E. D. (1995). Individual functional roles of metal ions in vivo. Essential metal ions: copper. in Handbook of Metal Ligand Interactions in Biological Fluids, G. Berthon, ed. (New York: Marcel Dekker), pp. 2 19-23 1.

Harris, E. D. (1 995a). The iron-copper comection: The link to ceruloplasmin grows stronger. Nutr. Rev. 53, 170-1 73.

Harris, E. D. (1992). Regdation of antioxidant enzymes. FASEB J. 6,2675-2683.

Hams, E. D., Gomerman, W. A., Savage, J. E., and O'Dell, B- L. (1974). Connective tissue amine oxidase. II. Purification and partial characterization of Lysyl oxidase fiom chick aorta. Biochim. Biophys. Acta 341,332-344.

Harris, 2. L., Klomp, L. W. J., and Gitlin, J. D. (1998). Acerulopiasminemia: an inherited neurodegenerative disease with impairment of iron homeostasis. Am. J. Clin. Nutr. 67, S972- S977.

Harris, 2. L,, Migas, M. C., Hughes, A. E., Logan, J. I., and Gitlin, J. D. (1996). Familial dementia due to a M e s h i f i mutation in the cemloplasmin geoe. Q. J. bled. 89,355-9.

Harris, 2. L., Talcahashi, Y., Miyajima, H., Serizawa, M., MacGillivray, R. T., and Gitlin, J. D. (1995). Aceruloplasminemia: molecular characterization of this disorder of iron metabolism. Proc. Natl. Acad. Sci. USA 92, 2539-43.

Harrison, M. D., Meier, S., and Dameron, C. T. (1999). Characterisation of copper-binding to the second sub- domain of the Menkes protein ATPase (MNKr2). Bba Mol Basis Dis 1453, 254-260.

Hartter, D. E., and Barnea, A. ( I 988). Brain tissue accumulates 67copper by two ligand- dependent saturable processes. A high affinity, low capacity and a low affinity, high capacity process. J. Biol. Chem. 263,799-805.

Harvey, P. W., Hunsaker, H. A., and Allen, K. G. (1981). Dietary L-histidine-induced hypercholesterolemia and hypocupremia in the rat. J. Nutr. 11 1,639-647.

Herd, S. M., Carnakaris, J., Christofferson, R., Wookey, P., and Danks, D. M. (1987). Uptake and efflux of copper-64 in Menkesf-disease and normal continuous lymphoid ce11 lines. Biochem. J. 247,341-7.

Herzberg, N. H., Wolterman, R. A., van den Berg, G. J., Barth, P. G., and Bolhuis, P. A. ( 1 990). Metallothionein in Menkes' disease: induction in cultured muscle cells. J. Neurol. Sci. 100, 50-6.

Hesse, J. E., Wieczorek, L., Altendorf, K., Reicin, A. S., Dorus, E., and Epstein, W. (1984). Sequence homology between two membrane transport ATPases, the Kdp- ATPase of Escherichia coli and the Cd+-ATPase of sarcoplasmic reticulum. Proc Natl Acad Sci U S A 81,4746-4750.

Hilton, M., Spenser, D. C., Ross, P., Ramsey, A., and Mcardle, H. J. (1995). Characterisation of the copper uptake mechanism and isolation of the cemloplasmin receptor/copper transporter in human placental vesicles. Biochimica et Biophysica Acta - General Subjects 1245, 153-160.

Ho, Y. S., Gargano, M., Cao, J., Bronson, R. T., Heimler, I., and Hutz, R. J. (1998). Reduced fertility in female mice lacking copper-zinc superoxide dismutase. J. Biol. Chem. 273, 7765- 7769.

Hodgson, E. K., and Fridovich, 1. (1975). The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: inactivation of the enzyme. Biochemistry 14,5294-5299.

Holzenhwg, A., Jones, P. C., Franklin, T., Pali, T., Heimburg, T., Marsh, D., Findlay, J. B., and Finbow, M. E. (1993). Evidence for a common structure for a class of membrane channels. Eur. J. Biochem. 213, 21-30.

Hoogenraad, T. U. ( 1998). Zinc treatment of Wilson's disease. J. Lab. Clin. Med. 132, 240- 24 1.

Hoogenraad, T. U., Koevoet, R., and de Ruyter Korver, E. G. (1979). Oral zinc sulphate as long-term treatment in Wilson's disease (hepatolenticular degeneration). Eur. Neurol. 18, 205- 1 1.

Hoogenraad, T. U., Van den Hamer, C. J., Koevoet, R., and Kower, E. G. (1978). Oral zinc in Wilson's disease. Lancet 2, 1262.

Hom, N., Tonnesen, T., and Tumer, 2. (1995). Clinical variability of Menkes disease. In Metals and Genetics, B. Sarkar, ed. (New York: Marcel and Dekker), pp. 285-303.

Houwen, R., Dijkstra, M., Kuipers, F., Smit, E. P., Havinga, R., and Vonk, R. J. (1990). Two pathways for biliary copper excretion in the rat. The role of glutathione. Biochem. Pharmacol. 39, 1039- 1044.

Hsieh, H. S., and Frieden, E. (1975). Evidence for ceruloplasmin as a copper transport protein. Biochemical & Biophysical Research Communications 67, 1326-3 1.

Hung, 1. H., Casareno, R. L. B., Labesse, G., Mathews, F. S., and Gitlin, J. D. (1998). HAHI is a copper-binding protein with distinct amino acid residues mediating copper homeostasis and antioxidant defense. J. Biol. Chem. 273, 1749- t 754.

Hung, 1. H., Suzuki, M., Yamaguchi, Y., Yuan, D. S., Klausner, R. D., and Gitlin, J. D. (1 997). Biochernical characterization of the Wilson disease protein and functional expression in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 272,2 146 1-2 1466.

Hunt, D. (1974). Primary defect in copper transport underlies mottled mutants in the rnouse. Nature 249, 852-854.

Hunt, D. M., Wake, S. A., Mercer, J. F., and Danks, D. M. (1986). A study of the role of metallothionein in the inherited copper toxicosis of dogs. Biochem. J. 236,409-4 15.

Hunziker, P. E., and Sternlieb, 1. (1991). Copper metallothioneh in patients with hepatic copper overload. Eur. J. Clin. Invest. 2 1,466-7 1.

Imamura, Y., and Kawakita, M. (1989). Purification of limited tryptic fragments of Ca2+,Mg2+-adenosine triphosphatase of the sarcoplasmic reticulum and identification of conformation-sensitive cleavage sites. J Biochem (Tokyo) 105, 775-78 1.

Iozumi, K., Nakagawa, H., and Tamaki, K- (1997). Penicillamine-induced degenerative dermatoses: report of a case and brief review of such dermatoses. J. Dennatol. 24,458-65.

Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M., Martin, J. C.? Smith. C. D., and Bechan , J. S. ( I 992). Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase- Arc h. Biochem. Biophys. 298,43 1-437.

Iyer, K. S., Lau, S. J., Laurie, S. H., and Sarkar, B. (1978). Synthesis of the native copper(I1)- transport site of human serum albumin and its copper(II)-binding properties. Biochem. J. 169, 61-69.

James, P., Inui, M., Tada, M., Chiesi, M., and Carafoli, E. (1989). Nature and site of phospholamban regulation of the C d + pump of sarcoplasmic reticulum. Nature 342,90-2.

Janssens, A. R., Bosman, F. T., Ruiter, D. J., and Van den Hamer, C. J. (1984a). Immunohistochemical demonstration of the cytoplasmic copper-associated protein in the liver in primary biliary cirrhosis: its identification as metallothionein. Liver 4, 139-47.

Janssens, A. R., Van Noord, M. J., Van Hoek, C. J., Ruiter, D. J., Mauw, B. J., and Van den Hamer, C. J. (1984). The lysosomal copper concentration in the liver in primary biliary cirrhosis. Liver 4, 396-40 1.

Jin, J. P., and Smillie, L. B. (1994). An unusual metal-bindhg cluster found exclusively in the avian breast muscle troponin T of Galliformes and Craciformes. FEBS Lett. 341, 135-40.

Johnson, G. F., Morell, A. G., Stockert, R. J., and Stemlieb, 1. (1981). Hepatic lysosomal copper protein in dogs with an inherited copper toxicosis. Hepatology 1,243-248.

Johnson, L. S., Dunn, K. W., Pytowski, B., and McGraw, T. E. (1993). Endosome acidification and receptor trafficking: bafilomycin Al slows receptor extemalization by a mechanism involving the receptor's internalization motif. Mol. Biol. Ce11 4, 125 1-1 266.

Johnson, W. C., Jr. (1 990). Protein secondary structure and circular dichroism: a practical guide. Proteins 7, 205-2 14.

Jorgensen, P. L., and Farley, R. A. (1988). Proteolytic cleavage as a tool for studying structure and conformation of pure membrane-bound Na+, K+-ATPase. Methods Enzymol. 156, 29 1-30 1.

Juul, B., Turc, H., Durand, M. L-, Gomez de Gracia, A., Denoroy, L., Moller, J. V., Champeil, P., and le Maire, M. (1995). Do transmembrane segments in proteolyzed sarcoplasmic reticulurn Ca(2+)- ATPase retain their functional C d + binding properties afier removal of cytoplasmic fragments by proteinase K? J. Biol. Chem. 270,20123-20134.

Kagi, J. H., and Schaffer, A. (1988). Biochemistry of metallothionein. Biochemistry 27, 8509-85 15.

Kagi, J. H. R., and Vallee, B. L. (1960). Metallothionein: a cadmium and zinc containing protein fiom equine renal cortex. J. Biol. Chem. 235, 3460-3465.

Kahn, D., David, M., Domergue, O., Daveran, M. L., Ghai, J., Hirsch, P. R., and Batut, J. (1989). Rhizobium meliloti fixGHI sequence predicts involvement of a specific cation purnp in syrnbiotic nitrogen fixation. J. Bacteriol. 171,929-39-

Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989). Biochemical characterization of the yeast vacuolar H(+)-ATPase. J. Biol. Chem. 264, 19236- 19244.

Karlish, S. J., Goldshlegcr, R., and Stein, W. D. (1990). A 19-kDa C-terminal tryptic fragment of the alpha chain of NaK-ATPase is essential for occlusion and transport of cations. Proc Nat1 Acad Sci U S A 87,4566-4570.

Katao ka, M., and Tz-.vassoli, M. (1 984). Ceruloplasmin receptors in liver ce11 suspensions are limited to the endothelium. Exp. Ce11 Res. 155,232-240.

Kataoka, M., and Tavassoli, M. (1985). Identification of ceruloplasmin receptors on the surface of human blood monocytes, granulocytes, and lymphocytes. Exp. Hematol. 13, 806- 810.

Katz, B. M., and Barnea, A. (1990). The ligand specificity for uptake of complexed copper- 67 by brain hypothalamic tissue is a iünction of copper concentration and copper:ligand molar ratio. J. Biol. Chem. 265, 2017-21.

Kau, L.-S., Spira-Solomon, D. J., Pemer-Hahn, J. E., Hodgson, K. O., and Solomon, E. 1. (1987). X-ray Absorption Edge Detennination of the Oxidation State and Coordination Number of Copper: Application to the Type 3 Site in Rhus vernicijieru Laccase and Its Reaction with Oxygen. J. Am. Chem. Soc. 109,6433-6442.

Keller, G. A., Warner, T. G., Steimer, K. S., and Hallewell, R. A. (1991). Cu,Zn superoxide dismutase is a peroxisomal enzyme in human fibroblasts a d hepatoma cells. Proc Nat1 Acad Sci U S A 88, 738 1-7385.

Kies, C., Chuang, J. H., and Fox, H. M. (1989). Copper utilization in humans as affected by amino acid supplements. FASEB J. 3, A360.

Kimura, M., Yamaguchi, Y., Takada, S., and Tanabe, K. (1993). Cloning of a Ca(2+)- ATPase gene of Plasmodium falciparum and cornparison with vertebrate Ca(2+)-ATPases. J. Cell Sci. 104, 1 129- 1 136.

Klevay, L. M. (1987). Dietary copper: a powerfbl determinant of cholesterolemia. Med. Hypotheses 24, 1 1 1 - 1 19.

Klomp, L. W. J., Lin, S. J., Yuan, D. S., Klausner, R. D., Culotta, V. C., and Gitlin, J. D. (1997). Identification and functional expression of HAHI, a novel human gene involved in copper homeostasis. J. Biol. Chem. 272,922 1-9226.

Koch, S. A,, MiIlart M., and O'Sitllivan, T. (1984). Stenc Control of Metal-Thiolate Coordination: Synthesis and Structure of Monomeric Cobalt(n) and Copper(1) Thiolate Complexes. Inorg. Chem. 23, 122- 124.

Kodama, H., Okabe, I., Yanagisawa, M., and Kodama, Y. (1989). Copper deficiency in the mitochondria of cultured skin fibroblasts fiom patients with Menkes syndrome. J. Meri t . Metab. Dis. 12, 386-389.

Koropatnick, J., and Cherian, M. G. (1993). A mutant mouse (tx) with increased hepatic metallothionein stability and accumulation. Biochem. J. 296,443-449.

Kreuder, J., Otten, A., Fuder, H., Turner, Z., Teunesen, T., Horn, N., and Dralle, D- (1993). Clinical and biochemical consequences of copper-histidine therapy in Menkes disease. Eur. I. Pediatr. 152, 828-32.

Krummeck, G., and Rodel, G. (1990). Yeast S C 0 1 protein is required for a post-translational step in the accumulation of mitochondrial cytochrome c oxidase subunits 1 and II. Curr. Genet. 18, 13-5.

Kuivaniemi, H., Ala-Kokko, L., and Kivirikko, K. 1. (1986). Secretion of lysyl oxidase by cultured human skin fibroblasts and effects o f monensin, nigericin, tunicamycin and colchicine. Biochim. Biophys. Acta 883,326-334.

Kuivaniemi, H., Peltonen, L., Palotie, A., Kaitila, I., and Kivirikko, K. 1. (1982). Abnormal copper metabolism and deficient lysyl oxidase activity in a heritable connective tissue disorder. J. Clin. Invest. 69, 730-3.

Laemmli, U. K. (1970). Clcavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680-5.

Lai, S. P., Randall, S. K., and Sze, H. (1988). Peripheral and integral subunits of the tonoplast H+-ATPase from oat roots. J. Biol. Chem. 263, 16731-16737.

Lane, L. K., Feldmann, J. M., Flarsheim, C. E., and Rybczynski, C. L. (1993). Expression of rat alpha 1 Na,K-ATPase containing substitutions of "essential" amino acids in the catalytic center. J. Biol. Chem. 268, 1793047934.

Larsen, F. L., Katz, S., and Roufogalis, B. D. (1981). Calmodulin regdation of Ca2+ transport in human erythrocytes. Biochem. i. 200, 185-9 1.

Larsen, F. L., and Vincenzi, F. F. (1979). Calcium transport across the plasma membrane: stimulation by calmodulin. Science 204, 306-9.

LaRusso, N. F., and Fowler, S. (1979). Coordinate secretion of acid hydrolases in rat bile. J. Clin. Invest, 64,948-954.

Lau, S. J., and Sarkar, B. (1971). Ternary coordination complex between human serum alburnin, copper (II), and L-histidine. J. Biol. Chem. 246,5938-5943.

Laussac, J. P., and Sarkar, B. ( 1980). 13Carbon-nuclear magnetic resonance investigation of the Cu(II)-binding to the native sequence peptide representing the Cu(1I)-transport site of human albumin. Evidence for the involvement of the beta-carboxyl side chain of aspartyl residuc. J. Biol. Chem. 255, 7563-7568.

Laussac, J. P., and Sarkar, B. (1984). Characterization of the copper(I1)- and nickel(I1)- transport site of human serum albumin. Studies of copperi11) and nickel(1I) binding to peptide 1-24 of human s e m albumin by 13C and 1 H NMR spectroscopy. Biochemistry 23, 2832-2838.

le Maire, M., Lund, S., Viel, A., ChampeiIt P., and Moller, J. V. (1990). Ca2(+)-induced conformational changes and location of C d + transport sites in sarcoplasmic reticulum Cd(+)-ATPase as dctected by the use of proteolytic e&e (Va). J. ~ i o 1 . - ~ h e m . 265, 1 1 1 1- 1123.

Lee, T. K., Li, L., and Ballatori, N. (1997). Hepatic glutathione and glutathione S-conjugate transport mechanisrns. Yale J. Biol. Med. 70, 287-300.

Levinson, B., Vulpe, C., Elder, B., Martin, C., Verley, F-, Packman, S., and Gitschier, J. (1991). The rnottled gene is the mouse homologue of the Menkes disease gene. Nat. Genet. 6, 369-73.

Levy, D., Seigneuret, M., Bluzat, A., and Rigaud, J. L. (1990). Evidence for proton countertransport by the sarcoplasmic reticulum Ca2(+)-ATPase during calcium transport in reconstituted proteoliposomes with low ionic penneability. J. Biol. Chem. 265, 1952449534.

Li, Y., Togashi, Y., Sato, S., Emoto, T., Kang, J. H., Takeichi, N., Kobayashi, H., Kojima, Y., Une, Y., and Uchino, J. (1991a). Abnormal copper accumulation in non-cancerous and cancerous liver tissues of LEC rats developing hereditary hepatitis and spontaneous hepatoma. Jpn. J. Cancer Res. 82,490-492.

Li, Y., Togashi, Y., Sato, S., Emoto, T., Kang, J. H., Takeichi, N., Kobayashi, H., Kojima, Y., Une, Y.. and Uchino, J. (1991). Spontaneous hepatic copper accumulation in Long-Evans Cimamon rats with hereditary hepatitis. A mode1 of Wilson's disease. J. Clin. hvest. 87, 1858-61.

Lin, S. J., Pufahl, R. A., Dancis, A., Ohalloran, T. V., and Culotta, V. C. (1997). A role for the Saccharomyces cerevisiae ATXl gene in copper traficking and iron transport. J. Biol. Chem. 272,92 15-9220.

Linder, M. C. (1991). Biochemistry of Copper, E. Frieden, ed. (New York: Plenum), pp. 163- 239.

Linder, M. C., and Hazegh-Azam, M. (1996). Copper biochemistry and molecular biology. Am. J. Clin. Nutr. 63. 7973-8 1 1 S.

Lippard, S. J., and Berg, J. M. (1994). Principles of Bioinorganic Chernistry (Mill Valley, California: University Science Books).

Lipsky, M. A., and Gollan, J. L. (1 987). Treatment of Wilson's disease: in D-penicillarnine we trust--what about zinc? Hepatology 7, 593-595.

Loudianos, G., Dessi, V., Angius, A., Lovicu, M., Loi, A., Deiana, M., Akar, N., Vajro, P., Figus, A., Cao, A., and Pirastu, M. (1996). Wilson disease mutations associated with uncornmon haplotypes in Mediterranean patients. Hum. Genet. 98, 640-2.

Lu, Y., Gralla, E. B., Roe, J. A., and Vaientine, J. S. (1992). Redesign of a Type 2 into a Type 1 Copper Protein: Construction and Characterization of Yeast Copper-Zinc Superoxide Dismutase Mutants. J. Amer. Chern. Soc. 114, 3560.

Lutsenko, S., and Cooper, M. J. (1998). Localization of the Wilson's disease protein product to mitochondria. Proc. Natl. Acad. Sci. USA 95, 6004-6009.

Lutsenko, S., and Kaplan, J. H. (1995). Organization of P-type ATPases: Significance of structural diversity. Biochemistry 34, 15607-1 56 13.

Lutsenko, S., Petmichin, K., Cooper, M. J., Gilliam, C. T., and Kaplan, J. H. (1997). N- terminal domains of human copper-transporting adenosine triphosphatases (the Wilson's and Menkes disease proteins) bind copper selectively in vivo and in vitro with stoichiometry of one copper per metal-binding repeat. J. Biol. Chem. 272, 18939-1 8944.

MacLeman, D. H., Rice, W. J., and Green, N. M. (1997a). The mechanism of Cd+ transport by sarco(endo)plasmic reticulurn Cd+- ATPases. J. Biol. Chem. 272, 288 15-8.

Maitre, B., Jornot, L., and Junod, A. F. (1993). Effects of inhibition of catalase and superoxide dismutase activity on antioxidant enzyme mRNA levels. Am. J. Physiol. 265, L636-643.

Marceau, N., and Aspin, N. (1973). The intracellular distribution of the radiocopper derived fiom ceruloplasmin and fiom albumin. Biochim. Biophys. Acta 328,338-50.

Maruyama, K., and MacLennan, D. H. (1 988). Mutation of aspartic acid-35 1, lysine-352, and lysine-5 15 alters the Ca2+ transport activity of the Cd-+-ATPase expressed in COS- 1 cells. Proc Nat1 Acad Sci U S A 85,33 14-33 18.

Mas, A., and Sarkar, B. (1992). Uptake of 67Cu by isoiated human trophoblast cells. Biochim. Biophys. Acta 1 f 35, 123-8.

Mason, J., McQuaid, A., and Pheifîèr, H. (1989). Can Wilson's disease patients be decoppered? Lancet 1, 1455.

Masuda, R., Yoshida, M. C., Sasaki, M., Dempo, K., and Mori, M. (1988). High susceptibili ty to hepatocellular carc inoma development in LEC rats with heredi tary hepatitis. Jpn. J. Cancer Res. 79, 828-835.

Matsudaira, P. T. (1989). A Practical Guide to Protein and Peptide Purification for Microsequencing (San Diego: Academic Press).

Matthews, W. B., Miline, M. D., and Bell, M. (1952). The metabolic disorder in hepatolenticular degeneration. Quart- J- ~Med. 21,245.

McArdle, H. J., Gross, S. M., and Danks, D. M. (1988). Uptake of copper by mouse hepatocytes. J. Cell. Physiol. 136,373-378.

McCord, J. M., and Fridovich, 1. ( 1 969). Superoxide dismutase. An enzymic fiinction for erythrocuprein (hemocuprein). J. Biol. Chem. 244,6049-6055.

Mehra, R. K., and Bremner, 1. (1983). The degradation of copper-metallothionein-1 and II in rat liver as influenced by the intracellular distribtution of the proteins. Biochem. Soc. Trans. 11, 774-775.

Mellrnan, 1. (1992). The Importance of Being Acid: the Role of Acidification in Intracellular Membrane Traffic. J. Exp. Biol. 172, 39-45-

Menkes, J. H. (1988). Kinky hair disease: twenty five years later. Brain & Development 10, 77-9,

Menkes, J. H., Alter, M., Steigleder, G. K., Weakley, D. R., and Sung, J. H. (1962). A sex- linked recessive disorder with retardation of growth, peculiar hair, and focal cerebral and cerebellar degeneration. Pediatricts 29, 764-779.

Mentzel, H. J., Seidel, J., Vogt, S., Vogt, L., and Kaiser, W. A. (1999). Vascular complications (splenic and hepatic artery aneurysms) in the occipital horn syndrome: report of a patient and review of the literature. Pediatr. Radiol. 29, 19-22.

Mercer, J. F., Grimes, A., Ambrosini, L., Lockhart, P., Paynter, J. A., Dierick, H., and Glover, T. W. (1994). Mutations in the murine homologue of the Menkes gene in dappled and blotchy mice. Nat. Genet. 6,374-8.

Mercer, J. F., Grimes, A., and Rauch, H. (1992). Hepatic metallothionein gene expression in toxic milk mice. J. Nutr. 122, 1254- 1259.

Mercer, J. F., Livingston. J., Hall, B., Paynter, J. A., Begy, C., Chandrasekharappa, S., Lockhart, P., Grimes, A., Bhave, M., Siemieniak. D., and et al. (1993). Isolation of a partial candidate gene for Menkes disease by positional cloning. Nat. Genet. 3,20-5.

Merelo, J. J., Andrade, M. A., Pneto, A., and Moran, F. (1994). Proteinotopic feature maps. Neurocomputing 6, 1 - 1 2.

Mills. J. D., Austin, P. A., and Turton, J. S. (1995). Thioredoxin and control of FOFI: function and distribution. Biochem. Soc. Trans. 23, 775-780.

Mohan, P., Failla, M., Bremner, I., Arthur-Smith, A., and Kerzner, B. (1995). Biliary copper excretion in the neonatal rat: role of glutathione and metaliothionein. Hepatology 21, 105 1 - 1017.

Moller, J. V., Juul, B., and le Maire, M. (1996). Structural organization, ion transport, and energy transduction of P-type ATPases. Biochim. Biophys. Acta 1286, 1-5 1.

Mowat, A. P. (1994). Liver Disorders in Childhood, 3rd ed. Edition (Cambridge: Butterworth-Heinemann).

Muramatsu, Y., Yamada, T., Miura, M., Sakai, T., Suzuki, Y., Serikawa, T., Tanzi, R. E., and Matsumoto, K. (1 994). Wilson's disease gene is homologous to hts causing abnormal copper transport in Long-Evans cinnarnon rats. Gastroenterology 107, 1 189-92.

Musci, G., Di Marco, S., Bellenchi, G. C., and Caiabrese, L. (1996). Reconstitution of cemloplasrnin by the Cu(I)-glutathione complex. Evidence for a role of Mg2+ and ATP. J. Biol. Chern. 271, 1972-8.

Nagano, K., Nakumura, K., Urakami, K. I., Umeyama, K., Uchiyama, H., Koiwai, K., Hattori, S., Yamamoto, T., Matsuda, I., and Endo, F. (1998). Intracellular distribution of the Wilson's disease gene product (ATPase7B) after in vitro and in vivo exogenous expression in hepatocytes fiom the LEC rat, an animal mode1 of Wilson's disease. Hepatology 27, 799-807.

Nanji, M. S., Nguyen, V. T. T., Kawasoe, J. H., Inui, K., Endo, F., Nakajima, T., Anezaki, T., and Cox, D. W. (1997). Haplotype and mutation analysis in Japanese patients with Wilson disease. Am. J. Hum. Genet. 60, 1423-1429.

Narron, G. H., Zec, N., Neves, R. I., Manders, E. K., and Sexton, F. M., Jr. (1992). Penicillamine-induced pseudoxanthoma eIasticum-like skin changes requiring rhytidectomy. Ann. Plast. Surg. 29, 367-70.

Nederbragt, H. (1 989). Effect of the glutathione-depleting agents diethylmaleate, phorone and buthionine sulfoximine on biliary copper excretion in rats. Biochem. Pharmacol. 38, 3399-3406.

Nelson, N. (1994). Energizing porters by proton-motive force. J. Exp. Biol. 196, 7-13.

Nelson, N. (1 992). Organellar proton-ATPases. Curr. Opin. Ceil Biol. 4,654-660.

Nelson, N., and Taiz, L. (1 989). The evolution of H+-ATPases. Trends Biochem. Sci. 14, 113-1 16.

Neumann, P. Z., and Sass-Kortsak, A. (1967). The state of copper in human serum: evidence for an amino acid-bound fraction. J. Clin. invest. 46,646-658.

Noji, H,, Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1 997). Direct observation of the rotation of F 1 -ATPase. Nature 386, 299-302.

Nucifora, G., Chu, L., Misra, T. K., and Silver, S. (1989). Cadmium resistance fiom Staphylococcus aureus plasmid p1258 cadA gene results fiom a cadmium-efflux ATPase. Proc Natl Acad Sci U S A 86,3544-3548.

O'Halloran, T. V. (1993). Transition metals in cootrol of gene expression. Science 261, 715- 25.

Ochiai, E. 1. (1995). Prebiotic metal ion-ligand interactions and the origin of life. In Handbook of Metal-Ligand Interactions in Biological Fluids, G. Berthon, ed. (New York: Marcel Dekker), pp. 1-9.

Odermatt, A., Krapf, R., and Solioz, M. (1994). Induction of the putative copper ATPases, CopA and CopB, of Enterococcus hirae by Ag+ and Cu2+, and Ag+ extrusion by CopB. Biochemical & Biophysical Rcsearch Communications 202,44-48.

Odermatt, A., Suter, H., Krapf, R., and Solioz, M. (1993). Primary structure of two P-type ATPases involved in copper homeostasis in Enterococcus hirae. J. Biol. Chem. 268, 12775-9.

Ohta, T., Nagano, K., and Yoshida, M. (1986). The active site structure of Na+/K+- transporting ATPase: location of the 5'-(p-fluorosulfonyl)benzoyladenosine binding site and soluble peptides released by irypsin. Proc Natl Acad Sci U S A 83,207 1-2075.

Okamoto, N., Wada, S., Oga, T., Kawabata, Y., Baba, Y., Habu, D., Takeda, Z., and Wada, Y. (1996). Hereditary ceruloplasmin deficiency with hemosiderosis. Hum. Genet. 97, 755- 758.

Omoto, E., and Tavassoli, M. (1990). Purification and partial characterization of ceruloplasmin receptors from rat liver endothelium. Arch. Biochern. Biophys. 282, 34-38.

Onishi, T., Inubushi, H., Tokugawa, S., Muramatsu, M., Nishikawa, K., Suzuki, Y., and Miyao, M. (1980). Abnormal copper metabolism in menkes cultured fibroblasts. Eur. J. Pediatr. 134, 205- 10.

Orena, S. J., Goodc, C. A., and Linder, M. C . (1986). Binding and uptake of copper fiom ceruloplasmin. Biochem. Biophys. Res. Commun. 139,822-829.

Osaki, S., and Johnson, D. A. (1969). Mobilization of liver iron by ferroxidase (ceruloplasmin). J. Biol. Chem. 244, 5757-8.

Osterberg, R. (1995). Ongin of metal ions occurring in living systems. In Handbook of MetaI-Ligand Interactions in Biological Fluids, G- Berthon, ed. (New York: Marcel Dekker), pp. 10-28.

Owen Jr, C. A. (1964). Distribution of copper in the rat. Am. J. Physiol. 207,446-448.

Packman, S. (1 987). Regdation of copper metabolism in the mottled mouse. Arch. Dermatol. 123, 1545- 1547a.

Pardo, J, M., and Serrano, R. (1 989). Structure of a plasma membrane H+-ATPase gene fiom the plant Arabidopsis thaliana. J. Biol. Chem. 264, 8557-8562.

Pasquali-Ronchetti, I., Quaglino, D., Jr., Baccarani-Contri, M., Hayek, J., and Galassi, G. (1 989). Dermal alterations in patients wità Wilson's disease treated with D-penicillamine. J. Submicrosc. Cytol. Pathol. 21, 13 1-9.

Payne, A. S., and Gitlin, J. D. (1998). Functional expression of the Menkes disease protein reveals common biochemical mechanisms among the copper- transporting P-type ATPases. J. Biol. Chem. 273,3765-3770.

Payne, A. S., Kelly, E. J., and Gitlin, J. D. (1998a). Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation. Proc. Natl. Acad. Sci. USA 95, 10854-10859.

Pekelharing, H. L., Lemrnens, A. G., and Beynen, A. C. (1994). Iron, copper and zinc status in rats fed on diets containing various concentrations of tin. Br. J. Nutr. 71, 103-109.

Peltonen, L., Kuivaniemi, H., Palotie, A., Horn, N., Kaitila, I., and Kivirikko, K. 1. (1983). Alterations in copper and collagen metabolism in the Menkes syndrome and a new subtype of the Ehlers-Dados syndrome. Biochemistry 22,6 156-63.

Percival, S. S., and Hams, E. D. (1989). Ascorbate enhances copper transport from ceruloplasmin into human K562 cells. J. Nutr. 119. 779-784.

Percival, S. S., and Harris, E. D. (1990). Copper transport from ceruloplasmin: characterization of the cellular uptake mechanism. Am. J. Physiol. 258, C 140- 146.

Petris, M. J., Mercer, J. F., Culvenor, J. G., Lockhart, P., Gleeson, P. A., and Camakaris, J. ( 1996). Ligand-regulated transport of the Menkes copper P-type ATPase efflwr pump fiom the Golgi apparatus to the plasma membrane: a novel mechanism of regulated traficking. EMBO J. 15,6084-95.

Petrukhin, K., Fischer, S. G., Pirastu, M., Tanzi, R. E., Chernov, I., Devoto, M., Brzustowicz, L. M., Cayanis, E., Vitale, E., Russo, I. J., Matseoane, D., Boukhgalter, B., Wasco, W., Figus, A. L., Loutianos, J., Cao, A., Sternlieb, I., Evgrafov, O., Parano, E., Pavone, L., Warburton, D., Ott, J., Penchaszadeh, G. K., Scheinberg, 1. H., and Gilliam, T. C. (1993). Mapping, cloning and genetic characterization of the region containhg the Wilson disease gene. Nat. Genet. 5, 338-43.

Petrukhin, K., Lutsenko, S., Chemov, L, Ross, B. M., Kaplan, J. H., and Gilliam, T. C. (1994). Characterization of the Wilson disease gene encoding a P-type copper transporting ATPase: genomic organization, alternative spticing, and stnicture/fhction predictions. Hum. Mol. Genet. 3, 1647-1656.

Petmzzella, V., Tiranti, V., Fernandez, P., Ianna, P., Carrozzo, R., and Zeviani, M. (1998). Identification and characterization of human cDNAs specific to BCS 1, PET1 12, SC0 1, COX 1 5, and COXl1, five genes involved in the formation and function of the mitochondrial respiratory chain. Genomics 54,494-504.

Phillips, M., Camakaris, J., and Danks, D. M. (1986). Comparïsons of copper deficiency States in the murine mutants blotchy and brindled. Changes in copper-dependent enzyme activity in 13-day-old mice. Biochem. J. 238, 177-83.

Phipps, D. A. (1995). Mctal ion physiology: role, action, and mechanism. In Handbook of Metal-Ligand Interactions in Biological Fluids, G. Berthon, ed. (New York: MarceI Dekker), pp. 89- 106.

Phung, L. T., Ajlani, G., and HaseLicorn, R. (1994). P-type ATPase fiom the cyanobacterium Synechococcus 7942 related to the human Menkes and Wilson disease gene products. Proc. Natl. Acad. Sci. USA 91,965 1-4.

Post, R. L., Hegyvary, C., and Kume, S. (1972). Activation by adenosine triphosphate in the phosphorylation kinetics of sodium and potassium ion transport adenosine triphosphatase. J. BioI. Chem. 247,6530-6540.

Powell, C. J. (1994). Copper-overload causes cancer? The LEC rat: a mode1 for human hepatitis, Liver cancer, and rnuch more. Human & Experimental Toxicology 13,9 10-2.

Procopis, P., Carnakaris, J., and Danks, D. M. (1981). A mild form of Menkes' syndrome. J. Pediatr. 98, 97-99.

Pufahl, R. A., Singer, C. P., Peariso, K. L., Lin, S. J., Schmidt, P. J., F a h i , C. J., Culotta, V. C., PemerHahn, J. E., and Ohalloran, T. V. (1997). Metal ion chaperone fwiction of the soluble Cu(I) receptor Atx 1. Science 278, 853-856.

Ralle, M., Cooper, M. J., Lutsenko, S., and Blackburn, N. J. (1 998). The Menkes disease protein binds copper via novel 2-coordinate Cu(I)-cysteinates in the N-terminal domain. J. Am. Chem. Soc. 120, 13525-13526.

Rauch, H. (1983). Toxic milk, a new mutation affecting Cooper metabolism in the mouse. J. Hered. 74, 14 1-144.

Rauch, H., Dupuy, D., Stockert, R. J., and Sternlieb, 1. (1986). Hepatic copper and superoxide dismutase activity in toxic milk mutant mice. In Superoxide and Superoxide Dismutase in Chemistry, Biology, and Medicine., G. Rotilio, ed. (Amsterdam: Elsevier Science).

Rauch, H., and Wells, A. J. (1995). The toxic milk mutation, tx, which results in a condition resembling Wilson disease in humans, is linked to mouse chromosome 8. Genomics 29,55 1 - 2.

Rayton, J. K., and Harris, E. D. (1979). Induction of lysyl oxidase with copper. Properties of an in vitro system. J. Biol. Chem. 254,62 1-626.

Reaume. A. G., Elliott, J. L., Hoffman, E. K., Kowall, N. W., Ferrante, R. J., Siwek, D. F., Wilcox, H. M., Flood, D. G., Beal, M. F., Brown, R. H., ir., Scott, R. W., and Snider, W. D. (1 996). Motor neurons in Cw'Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced ce11 death after axonal injury. Nat. Genet. 13,43-7.

Reed, V., Williamson, P., Bull, P. C., Cox, D. W., and Boyd, Y. (1995). Mapping of the mouse homologue of the Wilson disease gene to mouse chromosome 8. Genomics 28, 573- 575.

Reilly, M., Daly, L., and Hutchinson, M. (1993). An epidemiological study of Wilson Disease in the Republic of Ireland. J. Neurol. Neurosurg. Psychiatry 56,298-200.

Reiser, S., Ferretti, R. J., Fields, M., and Smith, J- C., Jr. (1983). Role of dietary h c t o s e in the enhancement of mortality and biochemical changes associated with copper deficiency in rats. Am. J. Clin. Nutr. 38,214-222.

Reiser, S., Smith, J. C., Jr., Mertz, W., Holbrook, J. T., Scholfield, D. J., Powell, A. S., Canfield, W. K., and Canary, J. J. (1985). indices of copper status in humans consurning a typical American diet containing either h c t o s e or starch. Am. 3. Clin. Nutr. 42,242-25 1.

Rensing, C., Mitra, B., and Rosen, B. P. (1997). The mtA gene of Escherichia coli encodes a Zn(I1)- translocating P-type ATPase. Proc. Natl. Acad. Sci. USA 94, 14326-1433 1.

Riordan, J. R., and Jolicoeur-Paquet, L. (1982). Metallothioneh accumulation may account for intracellular copper retention in ~Menkes' disease. J. Biol. Chem. 257,4639-45.

Ripps, M. E., Huntley, G . W., Hof, P. R., Morrison, J. H., and Gordon, J, W. (1995). Transgenic mice expressing an altered murine superoxide dismutase gene provide an animal mode1 of amyotrophic lateral sclerosis. Proc Nat1 Acad Sci U S A 92,689-693.

Roeser, H. P., Lee, G. R., Nacht, S., and Cartwright, G. E. (1970). The role of ceruloplasmin in iron metabolism. J- Clin. invest. 49,2408- 1 7.

Romero-Chapman, N., Lee, J., Tinker, D., Uriu-Hare, J. Y., Keen, C. L., and Rucker, R. R- (1 99 1 ). Purification, properties and influence of dietary copper on accumulation and fùnctional activity of lysyl oxidase in rat skin. Biochem. J. 275,657-662.

Rosen, D. R., Siddique, T., Patterson, D., Figlewicz, D. A-, Sapp, P., Hentati, A., Donaldson, D., Goto, J., O'Regan, J. P., Deng, H. X., Rahmani, Z. , Krizus, A., McKe~a-Yasek, D., Cayabyab, A., Gaston, S. M., Berger, R., Tanzi, R. E., Halperin, J., Herzfeldt, B., Van den Vergh, R., Hung, W.-Y., Bird, T., Deng, G., Mulder, D. W., Smyth, C., Laing, N. G., Soriano, E., Pericak-Vance, M. A., Haines, l., Rouleau, G. A., Gusella, J. S., Horvitz, H. R., and Brown, R. H. (1993). Mutations in CdZn superoxide dismutase gene are associated with familial arnyotrophic lateral sclerosis. Nature 362, 59-62.

Royce, P. M., Carnakaris, J., Mann, J. R., and Danks, D. M. (1982). Copper metabolisrn in mottled mouse mutants. The effect of copper therapy on lysyl oxidase activity in brïndled (Mobr) mice. Biochem. J. 202,369-71.

Rucker, R. B., Romerochapman, N., Wong, T., Lee, J., Steinberg, F. M., Mcgee, C., Clegg, M. S., Reiser, K., Kosonen, T., Uriuhare, J. Y., Murphy, J., and Keen, C. L. (1996). Modulation of lysyl oxidase by dietary copper in rats. J. Nutr. 126, 51-60.

Rydén, L. (1988). Evolution of blue copper proteins. In Oxidases and Related Redox Systems, T. E. King, H. S. Mason and M. Momson, eds. (New York: Alan R. Liss, Inc.), pp. 349-368.

Sabbert, D., Engelbrecht, S., and Juge, W. (1 997). Functional and idling rotatory motion within Fi-ATPase. Proc Natl Acad Sci U S A 94,4401-4405.

Sachs, G., Munson, K., Balaji, V. N., Aures-Fischer, D., Hersey, S. I., and Hall, K. (1989). Functional domains of the gastric HK ATPase. J. Bioenerg. Biomembr. 21, 573-588.

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed. Edition (New York: Cold Spring Harbor Laboratory Press).

Sanger, F., Niclden, S., and Coulson, A. R. (1977). DNA sequencing with chain-terrninating inhibitors. Proc Natl Acad Sci U S A 74,5463-7.

Sarkar, B. (1980). Recent trends in the application of coordination chemistry in biology and medicine. In (IUPAC) Coordination Chemistry, D. Banerjea, ed. (Oxford and New York: Pergamon Press), pp. 19 1-200.

Sarkar, B. (1981). Transport of copper. In Meta1 Ions in Biological Systems, H. Sigel, ed. (New York: Marcel Dekker), pp. 233-28 1.

Sarkar, B., and Knick, T. P. A. (1966). Copper-amino acid complexes in human serum. in The Biochemistry of Copper, J. Peisach, P. Aisen and W. E. Blumberg, eds. (New York: Academic Press), pp. 1 83- 196.

Sarkar, B., Lingertat-Walsh, K., and Clarke, J. T. (1993). Copper-histidine therapy for Menkes disease. J. Pediatr. 123, 828-30.

Sarkar, B., Sass-Kortsak, A., Clarke, R., Laurie, S. H., and Wei, P. (1977). A comparative study of in vitro and in vivo interaction of D- penicillamine and triethylenetetramine with copper. Proc R Soc Med 70, 13-8.

Sarkar, B., and Wigfield, Y. (1968). Evidence for albumin--cu(lI)--amino acid temary complex. Can J Biochem 46,60 1-607.

Sasaki, N.. Hayashizaki, Y., and Kasai, N. (1993). Absence of linkage between the retinoblastoma gene and hts gene in the LEC rat: a mode1 of human Wilson's disease. Jpn. J. Cancer Res. 84, 10 19- 1022.

Sass-Kortsak, A. (1974). Hepatolenticular Degeneration. In Handbuch der inneren Medizin (Berlin: Springer-Verlag), pp. 627-665.

Sass-Kortsak, A. (1975). Wilson's disease. A treatable liver disease in children. Pediatr. Clin. North Am. 22.963-84.

Sass-Kortsak, A., Clarke, R.: Hams, D. 1. M., Neumann, P. Z., and Sarkar, B. (1967). The biological transport of copper. Prog. Neuro. Genet. 175,625-63 1.

Sato, M., and Gitlin, J. D. (1991). Mechanisms of copper incorporation during the biosynthesis of human ceruloplasmin. J. Biol. Chem. 266,s 128-34.

Sawaki, M., Enomoto, K., Takahashi, H., Nakajima, Y., and Mori, M. (1990). Phenotype of preneoplastic and neoplastic liver lesions during spontaneous liver carcinogenesis of LEC rats. Carcinogenesis 11, 1857- 186 1.

Sayed, A. K., Edwards, J. A., and Banneman, R. M. (198 1). Copper metabolism of cultured fibroblasts fiom the brindled mouse (gene symbol Mobr). Proc. Soc. Exp. Biol. Med. 166, 153-6.

Scarrow, R C., Maroney, M. J., Palmer, S. M., Roe, A. L., Que, L., Ir., Salowe, S. P., and Stubbe, J. (1987). EXAFS studies of binuclear iron proteins: Hemerythrin and Ribonucleotide Reductase. J. Amer. Chem. Soc. 109, 7857-7864.

Schaefer, M., Hopkins, R. G., Failla, M. L., and Gitlin, J. D. (1999). Hepatocyte-specific localization and copper-dependent trafficking of the Wilson's disease protein in the liver. Amer J Physiol Gastrointest L 39, G639-(3646.

Scheffer, H., Houwen, R. H., Te Meerman, G. I., Loessner, J., Bachmam, B., Kunert, E., Verlind, E., and Buys, C. H. (1992). Identification of crossovers in WiIson disease families as reference points for a genetic localization of the gene. Hum. Genet. 89, 607-61 1.

Scheinberg, 1. H. (1 968). Toxicity of penicillamine. Postgrad. Med. J., Suppl: 1 1-4.

Scheinberg, 1. H., Jaffe, M. E., and Stemlieb, 1. (1987). The use of trientine in preventing the effects of intempting penicillamine therapy in Wilson's disease. N. Engl. J. Med. 31 7, 209- 13.

Scheinberg, 1. H., and Stemlieb, 1. (1984). Wilson's Disease (Philadelphia: Saunders).

Schiff, L. A., Nibert, M. L., and Fields, B. N. (1988). Characterîzation of a zinc blotting technique: Evidence that a retroviral gag protein binds zinc. Proc. Natl. Acad. Sci. 85, 4195- 4 199.

Schilsky, M. L., Scheinberg, 1. H., and Sternlieb, 1. (1994). Liver transplantation for Wilson's disease: indications and outcome. Hepatology 19, 583-587.

Schilsky, M. L., Stockert, R. J., and Sternlieb, 1. (1 994a). Pleiotropic effect of LEC mutation: a rodent mode1 of Wilson's disease. Am. J. Physiol. 266, G907-13.

Schmitî, R. C., Danvish, H. M., Cheney, J. C., and Ettinger, M. J. (1983). Copper transport kinetics by isolated rat hepatocytes. Am. 1. Physiol. 244, G183- 19 1.

Schulze, M., and Rodel, G. (1988). SC0 1, a yeast nuclear gene essential for accumulation of mitochondrial cytochrorne c oxidase subunit II. Mol. Gen. Genet. 211,492-8.

Shah, A. B., Chernov, I., Zhang, H. T., Ross, B. M., Das, K., Lutsenko, S., Parano, E., Pavone, L., Evgrafov, O., IvanovaSmolenskaya, 1. A., Ameren, G., Westermark, K-, Urrutia, F. H., Penchaszadeh, G. K., Sternlieb, I., Scheinberg, 1. H., Gilliam, T. C., and Petrukhin, K. (1997). Identification and analysis of mutations in the Wilson disease gene (ATP7B): Population frequencies, genotype- phenotype correlation, and functional analyses. Am. J. Hum. Genet. 61,3 17-328.

Sherwood, G., Sarkar, B., and Kortsak, A. S. ( 1989). Copper histidinate therapy in Menkes' disease: prevention of progressive neurodegeneration. J. Inberit. Metab. Dis. 12 Suppl2,393- 6.

Shimizu, N., Kawase, C.. Nakazono, H., Hemmi, H., Shimatake, H., and Aoki, T. (1995). A novel RNA splicing mutation in Japanese patients with Wilson disease. Biochem. Biophys. Res. Commun. 21 7, 16-30.

Shu, L., Chiou, Y. M., Orville, A. M., Miller, M. A., Lipscomb, J. D., and Que, L., Jr. (1995). X-ray absorption spectroscopic studies of the Fe(II) active site of catechol2,3-dioxygenase. Implications for the extradiol cleavage mechanism. Biochemistry 34,6649-59.

Silver, S. ( 1 996). Bacterial resistances to toxic metal ions--a review. Gene 179, 9- 19.

Silver, S., Nucifora, G-. Chu, L., and Misra, T. K. (1989). Bacterial resistance ATPases: primary pumps for exporting toxic cations and anions. Trends Biochem. Sci. 14, 76-80.

Silver, S., Nucifora, G., and Phung, L. T. (1993). Human Menkes X-chromosome disease and the staphylococcal cadmium-resistance ATPase: a remarkable similarity in protein sequences. Mol. Microbiol. 10, 7- 12.

Smith, A. J. (1 997). Amino acid analysis. Methods Enzyrnol. 289,419-26.

Solioz, M., and Odermatt, A. (1995). Copper and silver transport by CopB-ATPase in membrane vesicles of Enterococcus hirae. J. Bioi. Chem. 270,921 7-922 1.

Solomon, E. 1. ( 1 988). CoupIed binuclear copper proteins: catalytic mechanism and structure-reactivity correlations. In Oxidases and Related Redox Systems, T. E. King, H. S. Mason and M. Morrison, eds. (New York: Alan R. Liss, hc.), pp. 309-330.

Solomon, E. I., Hare, J. W., and Gray, H. B. (1976). Spectroscopie studies and a structural mode1 for blue copper centers in proteins. Proc Nat1 Acad Sci U S A 73, 1389-1393.

Spira-Solomon, D. J., Allendorf, M. D., and Solomon, E. 1. (1986). Low-temperature magnetic circular dichroism studies of native laccase: confinnation of a trinuclear copper active site. J. Amer. Chem. Soc. 108, 53 18-5328.

Sreerama, N., and Woody, R. W. (1 994). Protein secondary structure fiom circular dichroism spectroscopy. Combining variable selection principle and cluster analysis with neural network, ridge regression and self-consistent methods. J. Mol. Biol. 242,497-507.

Sreerama, N., and Woody, R. W. (1993). A self-consistent method for the analysis of protein secondary structure fiom circular dichroism. Anal. Biochem. 209, 32-44.

Srinivasan, C., Posewitz, M. C., George, G. N., and Winge, D, R. (1998). Characterization of the copper chaperone Cox17 of Saccharomyces cerevisiae. Biochemistry 37,7572-7577.

Stem, R. V., and Frieden, E. (1993). Partial purification of the rat erythrocyte ceruloplasmin receptor monitored by an electrophoresis mobility shift assay. Anal. Biochem. 212,221-228.

Stemlieb, 1. (1 989). Hepatic copper toxicosis. J. Gastroenterol. Hepatol. 4, 175- 18 1.

SternIieb, 1. ( 1990). Perspectives on Wilson disease. Hepatology 12, 1234- 1239.

Sternlieb, I., Van den Hamer, C. J., Morell, A. G., Alpert, S., Gregoriadis, G., and Scheinberg, 1. H. (1973). Lysosomal defect of hepatic copper excretion in Wilson's disease (hepatolenticular degeneration). Gastroenterology 64, 99- 105.

Stevens, M. D., DiSilvestro, R. A., and Harris, E. D. (1984). Specific receptor for ceruloplasmin in membrane fragments from aortic and heart tissues. Biochemistry 23, 261- 266.

Stockert, R. J., Grushoff, P. S., Morell, A. G., Bentley, G. E., O'Brien, H. A., Scheinberg, 1. H., and Sternlieb, 1. (1986). Transport and intracellular distribution of copper in a human hepatoblastoma ce11 line, HepG2. Hepatology 6,6044.

Strausak, D., Fontaine, S. L., Hill, J., Firth, S. D., Lockhart, P. J., and Mercer, 1. F. (1999). The Role of GMXCXXC Metal Binding Sites in the Copper-induced Redistribution of the Menkes Protein. J. Biol. Chem. 2 74, 1 1 170- 1 1 177.

Strickland, E. H. (1974). Aromatic contributions to circular dicbroism spectra of proteins. CRC Crit. Rev. Biochem. 2, 1 13- 175.

Strickland, G. T., Frommer, D., Leu, M. L., Pollard, R., Sherlock, S., and Cumings, J. N. (1973). Wilson's disease in the United Kingdom and Taiwan. 1. General characteristics of 142 cases and prognosis. II. A genetic analysis of 88 cases. Quarterly Journal of Medicine 42,6 19-38.

Strickland, G. T., and Leu, M. Id. (1975). Wilson's disease. Clinical and laboratory maniestations in 40 patients. Medicine 54, 1 13-37.

Studier, F. W., and Moffatt, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 1 13-30.

Su, L. C., Ravanshad, S., Owen, C. A., Jr., McCalI, J. T., Zollman, P. E., and Hardy, R. M. (1 982). A comparison of copper-loading disease in Bedlington terriers and Wilson's disease iri humans. Am. J. Physiol. 243, G226-30.

Takahashi, Y., Miyajima, H., Shirabe, S., Nagataki, S., Suenaga, A., and Gitlin, J. D. (1996). Characterization of a nonsense mutation in the ceruloplasmin gene resulting in diabetes and neurodegenerative disease. Hum. Mol. Genct. 5,8 1-84.

Tanzi, R. E., Petnikhin, K., Chemov, I., Pcllequer, J. L., Wasco, W., Ross, B., Romano, D. M., Parano, E., Pavone, L., Brzustowicz, L. M., Devoto, M., Peppercom, J., Bush, A. I., Sternlieb. I., Piratsu. M., Gusella, J. F., Evgrafov, O., Penchaszadeh, G. K., Honig, B., Edelman, 1. S., Soares, M. B., Schcinberg, 1. H., and Gilliam, T. C. (1993). The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nat. Genet. 5, 344-50.

Theophilos, M. B-, Cox, D. W., and Mercer, 3. F. B. (1996). The toxic milk mouse is a murine mode1 of Wilson disease. Hum, Mol. Genet. 5, 16 19-1624.

Thiele, D. J., Walling, M. J., and Hamer. D. H. (1986). Mammalian metallothionein is functional in yeast. Science 231, 854-856.

Thomas, G. R., Forbes, J. R., Roberts, E. A., Walshe, J. M., and Cox, D. W. (1995) The Wilson disease gene: spectrum of mutations and their consequences. Nat. Genet. 9,210-7.

Toyofuku, T., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1 993). Identification of regions in the Ca(2t)-ATPase of sarcoplasmic reticulum that affect fünctional association with phospholamban. J. Biol. Chem. 268,2809-28 15.

Trackman, P. C., Bedell-Hogan, D., Tang, J., and Kagan, H. M. (1992). Post-translational glycosylation and proteolytic processing of a lysyl oxidase precursor, J. Biol. Chem. 267, 8666-867 1.

Trackman, P. C., Pratt, A. M., Wolanski, A., Tang, S. S., Offner, G. D., Troxler, R. F., and Kagan, H. M. (1990). Cloning of rat aorta lysyl oxidase cDNA: complete codons and predicted amino acid scquence.

Tsai, K. J., and Linet, A. L. (1993). Formation of a phosphorylated enzyme intermediate by the cadA Cd(2+)- ATPase. Arch. Biochem. Biophys. 305,267-270.

Tsai, M. T., Dinh, C. T., and Linder, M. C. (1992). interactions of transcuprein and albumin in copper transport. FASEB J. 6, A 1095.

Tümer, Z., Chelly, J., Tornmerup, N., Ishikawa-Brush, Y., Tannesen, T., Monaco, A. P., and Hom, N. (1992b). Characterization of a 1.0 Mb YAC contig spaming two chromosome breakpoints related to Menkes disease. Hum. Mol. Genet. 1,483-9.

Tümer, Z., and Hom, N. (1997b). Menkes disease: Recent advances and new aspects. J. Med. Genet. 34,265-274.

Tümer, Z., Horn, N., Tonnesen, T., Christodoulou, J., Clarke, J. T. R., and Sarkar, B. (1996). Early copper-histidine treatment for Menkes disease. Nat. Genet. 12, 1 1 - 13.

Tümer, Z., Lund, C., Tolshave, J., Vural, B., Tonnesen, T., and Hom, N. (1997a). Identification of point mutations in 41 unrelated patients affected with Menkes disease. Am. J. Hum. Genet. 60,63-7 1.

Tümer, Z.? Tommenip, Net Tannesen, T.: Kreuder, J., Craig, 1. W., and Hom, N. (1992a). Mapping of the Menkes locus to Xq13.3 distal to the X-inactivation center by an intrachromosomal insertion of the segment Xq 13 -3-q2 1.2. Hum. Genet. 88,668-72-

Tümer, Z., Vural, B., Tomesen, T., Chelly, J., Monaco, A. P., and Hom, N. (1995). Characterization of the exon structure of the Meakes disease gene using vectorette PCR. Genomics 26,43743.

Tumlund, J. R., Keycs, W. R., Anderson, H. L., and Acord, L. L. (1989). Copper absorption and retention in young men at three levels of dietary copper by use o f the stable isotope 65Cu. Am. J. Clin. Nutr. 49, 870-878.

Turnlund, J. R., King, J. C., Gong, B., Keyes, W, R., and Michel, M. C. (1985). A stable isotope study of copper absorption in young men: effect of phytate and alpha-cellulose. Am. J. Clin. Nutr. 42, 18-23.

Twedt, D. C., Sternlieb, I., and Gilbertson, S. R. (1979). Clinical, morphologie, and chernical studies on copper toxicosis of Bedlington Terriers. J. Am. Vet. Med. Assoc. 175,269-75.

Vakntine, J. S., and Gralla, E. B. (1997). Delivering copper inside yeast and human cells. Science 2 78, 8 1 7-8 1 8.

Van Caillie-Bertrand, M., Degenhart, H. J., Luijendijk, I., Bouquet, J., and Sinaasappel, M. (1985). Wilson's disease: assessrnent of D-penicillamine treatment. Arch. Dis. Child. 60, 652-655.

Van Campen, D. R., and Scaife, P. U. (1967). Zinc interference with copper absorption in rats. J. Nutr. 91,473-476.

van de Sluis, B. J. A., Breen, M., Nanji, M., van Wolferen, M., de Jong, P., Binns, M. M., Pearson, P. L., Kuipers, J., Rothuizen, J., Cox, D. W., Wijmenga, C., and van Oost, B. A. (1999). Genetic mapping of the copper toxicosis locus in Bedlington temers to dog chromosome 10, in a region syntenic to human chromosome region 2p13- p16. Hum. Mol. Genet. 8, 50 1-507.

van den Berg, G. J., and van den Hamer, C. J. (1984). Trace metal uptake in liver cells. 1. influence of albumin in the medium on the uptake of copper by hepatoma cells. J. inorg. Biochem. 22,7344.

Vargas, E. J., Shoho, A. R., and Linder, M. C. (1994). Copper transport in the Nagase analbuminemic rat. Am. J. Physiol. 267, G259-269.

Verga, V., Hall, B. K., Wang, S. R., Johnson, S., Higgins, J. V., and Glover, T. W. (1991). Localization of the translocation breakpoint in a femate with Menkes syndrome to Xq13.2- q13.3 proximal to PGK- 1. Am. J. Hum. Genet. 48, 1 133-8.

Verity, M. A., Garnbell, J. K., Reith, A. R., and Brown, W. J. (1967). Subcellular distribution and enzyme changes fotlowing subacute copper intoxication, Lab. Invest. 16,580-590.

Vilsen, B., Andersen, J. P., Clarke, D. M., and MacLennan, D. H. (1989). Functional consequences of proline mutations in the cytoplasmic and transmembrane sectors of the Cd(+)-ATPase of srirçoplasmic reticulum. J. BioI. Chern. 264,2 1024-2 1030.

Vilsen, B., Andersen, J. P., and MacLeman, D. H. (199 1). Functional consequences of alterations to amino acids located in the hinge domain of the Ca(2+)-ATPase of sarcoplasmic reticulum. J. Biol. Chcm. 266, 16 157-1 6 164.

Vogelstein, B., and Gillespie, D. (1979). Preparative and analytical purification of DNA fiom agarose. Proc Nat1 Acad Sci U S A 76,6 15-9.

Volbeda, A., and Holt W. G. J. ( 1985). Structure of arthropodan hemocyanin. In Oxidases and Related Redox Systems, T. E. King, H. S. Mason and M. Momson, eds- (New York: Alan R. Liss, Inc.), pp. 29 1-308.

Vulpe, C., Levinson, B.. Whitney, S., Packman, S., and Gitschier, J. (1993). Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase. Nat. Genet. 3 ,7 - 13.

Vulpe, C. D., and Packman, S. (1995). Cellular copper transport. Annu. Rev. Nutr. 15, 293- 322.

Wakai, S., Ishikawa, Y., Nagaoka, M., Okabe, M., Minami, R., and Hayakawa, T. (1993). Central nervous system involvement and generalized muscular atrophy in occipital horn syndrome: Ehlers-Danlos type iX. A first Japanese case. 1. Neurol. Sci. 116, 1-5.

Walker, J. E. (1994). The regdation of catalysis in ATP synthase. C m . Opin. Struct. Biol. 4, 912-918.

Walker, P. A., Leong, L. E., Ng, P. W., Tan, S. H., Waller, S., Murphy, D., and Porter, A. G. (1994). Efficient and rapid affinity purification of proteins using recombinant fusion proteases. Biotechnoiogy (N Y) 12,601 -5.

Walshe, J. M. (1984). Copper: its role in the pathogenesis of liver disease. Semin. Liver Dis. 4,252-263.

Walshe, J. M. (1956). Penicillamine, a new oral therapy for Wilson disease. Am. J. Med. 21, 487-495.

Walshe, J. M. (1982). Treatment of Wilson's disease with trientine (triethylene tetrarnine) dihydrochloride. Lancet 1,643-7.

Walshe, J. M. (1 989). Wilson's disease patients can be decoppered. Lancet 2,228.

Walshe, J. M. (1989). Wïlson's disease presenting with features of hepatic dysfunction: a clinical analysis of eighty-seven patients. Quarterly Journal of Medicine 70, 253-63.

Walshe, J. M., and Dixon, A. K. (1986). Dangers of non-cornpliance in Wilson's disease. Lancet 1,845-847.

Wapnir, R. A. (1998). Copper absorption and bioavailability. Am. J. Clin. Nutr. 67, S1054- S 1060.

Wapnir, R. A. (199 1). Copper-sodium linkage during intestinal absorption: inhibition by amiloride. Proc. Soc. Exp. Biol. Med. 196,4 10-4 14.

Wapnir, R. A., and Stiel, L. (1987). Intestinal absorption of copper: effect of sodium. Proc. Soc. Exp. Biol. Med. 185, 277-282.

Weiner, A. L., and Cousins, R. J. (1980). Copper accumulation and metabolism in primary monolayer cultures of rat liver parenchymal cells. Biochim. Biophys. Acta 629, 1 13-125.

Weiss, K. C., and Linder, M. C. (1985). Copper transport in rats involving a new plasma protein. Am. J. Physiol. 249, E77-88.

Wiedau-Pazos, M., Goto, J. J., Rabizadeh, S., Gralla, E. B., Roe, J. A., Lee, M. K., Valentine, J. S., and Bredesen, D. E. (1996). Altered reactivity of superoxide dismutase in familial amyotrophie lateral sclerosis. Science 271, 5 15-5 18.

Wilson, S. A. K. (1 9 12 j. Progressive lenticular degeneration: A familial nervous disease associated with cirrhosis of the liver. Brain 34, 295-508.

Wingfield, P. T., and Pain, R. H. (1996). Determining the CD Spectrum of a Protein. In Current Protocols in Protein Science, J. E. Coligan, B. M. Dunn, H. L. Ploegh, D. W. Speicher and P. T. Wingfield, eds. (New York: John Wiley & Sons, Inc.), pp. 7.6.1-7.6-23.

Wong, P. C., Pardo, C. A., Borchelt, D. R., Lee, M. K., Copeland, N. G., Jenkins, N. A., Sisodia, S. S., Cleveland, D. W., and Price, D. L. (1995). An adverse property of a familial

ALS-linked SODl mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria. Neuron 14, 1 105- 1 1 16.

Wu, J., Forbes, J. R., Chen, H. S., and Cox, D. W. (1994). The LEC rat bas a deletion in the copper transporting ATPase gene homologous to the Wilson disease gene. Nat. Genet. 7, 541-5.

Xie, X. S., and Stone, D. K. (1988). Partial resolution and reconstitution of the subuni9 of the clathrin- coated vesicle proton ATPase responsible for Cd+-activated ATP hydrolysis. J. Biol. Chem. 263,9859-9867.

Xu. G. Q., Yarnano. T.. and Shirnad- M. (1994). Copper distribution in fetus and placenta of the macular mutant rnouse as a modcl of Menkes kinky hair disease. Biol. Neonate 66, 302- 1 o.

Yamaguchi, Y., Heiny, M. E., Suzuki, M., and Gitlin, J. D. (1996). Biochemical characterization and intracellular localization of the Menkes disease protein. Proc. Natl. Acad. Sci. USA 93, 14030-5.

Yoshida, K., Furihata, K., Takeda, S., Nakamura, A., Yamamoto, K., Morita, H., Himuta, S., Ikeda, S ., Shimizu, N., and Yanagisawa, N. ( 1995). A mutation in the ceruloplasmin gene is associated with systemic hemosiderosis in humans. Nat. Genet. 9,267-72.

Yoshida, M. C., Masuda, R., Sasaki, M., Takeichi, N., Kobayashi, H., Dempo, K., and Mori, M. (1987). New mutation causing hereditary hepatitis in the laboratory rat. J. Hered. 78, 36 1- 365.

Yu, S., West, C. E.. and Beynen, A. C. (1994a). Increasing intakes of iron reduce status, absorption and biliary excretion of copper in rats. Br. J. Nutr. 71, 887-895.

Yu, X., Carroll, S . ! Rigaud, 1. L., and Inesi, G. (1993). H+ countertransport and electrogenicity of the sarcoplasmic rcticulum C d + pump in reconstituted proteoliposomes. Biophys. J. 64, 1232- 1242.

Yuan, D. S., Steaman, R., Dancis, A., Dunn, T., Beeler, T., and Klausner, R. D. (1995). The Menkes~Wilson disease gene homologue in yerist provides copper to a cemloplasmin-Iike oxidase required for iron uptake. Proc. Natl. Acad. Sci. USA 92, 2632-6.

Yuzbasiyan-Gurkan, V., Brewer, G. J., Boerwinkle, E., and Venta, P. J. (1988). Linkage of the Wilson disease gene to chrornosomc 13 in North-American pedigrees. Am. J. Hum. Genet. 42, 825-9.

Yuzbasiyan-Gurkan, V., Wagnitz, S., Blanton, S. H., and Brewer, G. J. (1993). Linkage studies of the esterase D and retinoblastorna genes to canine copper toxicosis: a mode1 for Wilson disease. Genomics 15, 86-90.

Zaitseva, I., Zaitsev, V., Card, G., Moshkov, K., Bax, B., Ralph, A., and Lindley, P. (1996). The X-ray structure of human serum ceruloplasmin at 3.1 angstrom: Nature of the copper centres. J Bi01 Inorg Chem 1, 15-23.

Zeng, J., Heuchel, R., Schafier, W., and Kagi, J. H. (1991a). Thionein (apometallothionein) c m modulate DNA binding and transcription activation by zinc finger containing factor Spl. FEBS Lett. 279, 3 10-3 12.

Zeng, J., Vallee, B. L., and Kagi, J. H. (1991). Zinc transfer fiom transcription factor IIIA fingers to thionein clusters. Proc Natl Acad Sci U S A 88,9984-9988.

Zhang, Y., and Fillingame. R- H. (1995). Subunits coupling H+ transport and ATP synthesis in the Escherichia coli ATP synthase. Cys-Cys cross-linking of FI subunit epsilon to the polar loop of FO subunit c. J. Biol. Chem. 270,24609-24614-

Zhou, B., and Gitschier, 1. (1997). hCTR1: a human gene for copper uptake identified by complementation in yeast. Proc Natl Acad Sci U S A 94, 748 1-7486.

Zhou, Y., Duncan, T. M., Bulygin, V. V., Hutcheon, M. L., and Cross, R. L. (1996). ATP hydrolysis by membrane-bound Escherichia coli FOFl causes rotation of the gamma subunit relative to the beta subunits. Biochim. Biophys. Acta 1275, 96-100.