State-of-the-art of metallothioneins at the beginning of the 21st century

R

S

Ma

b

C

a

ARAA

KMMMME

0d

Coordination Chemistry Reviews 256 (2012) 46– 62

Contents lists available at ScienceDirect

Coordination Chemistry Reviews

journa l h o me page: www.elsev ier .com/ locate /ccr

eview

tate-of-the-art of metallothioneins at the beginning of the 21st century

. Capdevilaa,∗, R. Bofill a, Ò. Palaciosa, S. Atrianb

Departament de Química, Facultat de Ciències, Universitat Autònoma de Barcelona, E-08193 Bellaterra (Cerdanyola del Vallès), Barcelona, SpainDepartament de Genètica, Facultat de Biologia, Universitat de Barcelona & Institut de Biomedicina de la Universitat de Barcelona, Avda. Diagonal 645, E-08028 Barcelona, Spain

ontents

1. Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

2.1. Overview, history and scope of MT research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472.2. Sources and origins of metallothionein proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

3. General features, classification, reactivity and function of MTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503.1. The three proposals of classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503.2. Reactivity of MTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

3.2.1. Metal uptake and release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513.2.2. Metal exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513.2.3. Metal transfer between proteins and other biomolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513.2.4. Redox activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

3.3. Proposed biological functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523.4. Differentiation and evolutionary divergence significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

4. Experimental methodologies to approach MT research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534.1. Purification and quantification approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544.2. Chemical and structural characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

5. Three-dimensional structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566. MTs in biomedicine and biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597. Future directions/goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

r t i c l e i n f o

rticle history:eceived 29 April 2011ccepted 9 July 2011vailable online 19 July 2011

eywords:etallothioneinetal-thiolate clusteretal exchange

a b s t r a c t

Metallothioneins (MTs) are a particular type of small metalloprotein that possess a significant amountof Cys residues, which confer upon them an extraordinary capacity for heavy metal binding. Now, morethan 50 years after their discovery and after the publication of thousands of papers, it is generally agreedthat they are involved in different biological functions depending on the organisms and/or isoformsconsidered.

This review attempts to critically review the state-of-the-art of these unusual metal-binding proteins.Special attention is devoted to their chemical and structural characterization and reactivity, including adetailed overview of the most prominent techniques used for purification and quantification of MTs, as

T reactivityvolutionary divergence

well as a section with a comparative description of all the three-dimensional structures so far known.Equally important are the biological aspects of MTs, which have also been extensively analyzed. Thus,their in vivo origin, localization and induction pathways, as well as their proposed biological significanceis discussed. Finally, the most recent applications of MTs in the Biomedicine and Biotechnology fields andthe future goals that, in our opinion, investigators should aim for in MT research are mentioned in the
final sections.
∗ Corresponding author. Tel.: +34 93 581 3323; fax: +34 93 581 3101.E-mail address: [email protected] (M. Capdevila).

010-8545/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.ccr.2011.07.006

© 2011 Elsevier B.V. All rights reserved.

1. Foreword

At the beginning of 2011, reviewing, all the current knowledgein the field of metallothioneins (MTs) was an easily affordable taskafter the excellent reports of Blindauer on the so-called class II MTs

dx.doi.org/10.1016/j.ccr.2011.07.006

http://www.sciencedirect.com/science/journal/00108545

http://www.elsevier.com/locate/ccr

mailto:[email protected]

dx.doi.org/10.1016/j.ccr.2011.07.006

Chem

[Vafottaprtwti“icFify

2

2

tstsp“

eaei9

Kmiat1mppMwotlCa

cmritdbo

M. Capdevila et al. / Coordination

1], Vallee [2] and Maret [3] on the mammalian MT1 and MT2,asák and Faller on mammalian MT3 [4], Freisinger [5] in the sub-rea of plant MTs, Weser [6] in the field of MTs from yeast andungi, and the comprehensive book edited by the Sigel family [7],r the very recent special issue of the JBIC devoted to MTs [8], ashe vast majority of the information has already been collected byhem. Therefore, the present review just attempts to summarizell the current knowledge by distilling the main ideas and beliefsublished in the literature until now, and providing interestedesearchers with all the necessary links to obtain further informa-ion on each specific topic of MT research. Only in the cases wheree consider a precise issue has not been treated widely enough in

he literature, is intensive information included. Additionally, wentend to offer the reader a critical point of view, which is of courseour point of view”, about the kind of information we already haven our hands, in the sense of how relevant is it to unveil the stillontroversial and elusive [9] function/s of these metalloproteins.inally, the last section intends to be a guide to these aspects that,n our opinion, should be studied in more depth, and which are theuture goals that Science should achieve in this field in the comingears.

. Introduction

.1. Overview, history and scope of MT research

Since the discovery in 1957 [10] of a cadmium-binding pro-ein in horse kidney, there have been four international meetingsolely devoted to MTs [11–14]. There are also excellent books onhe subject [7,15,16], chapters in encyclopedias and in comprehen-ive collections [17,18], special journal issues [8], and thousands ofublications, which include more than 100 reviews with the wordmetallothionein” appearing in their title.

Searching the topic “metallothionein” in several databases cov-ring all types of scientific reports (i.e. articles and also conferencebstracts) yields overwhelming results. There are about 13,850ntries in Scifinder [19], 12,350 in the ISI Web of Knowledge [20]f only articles, books and reviews are considered, as well as about300 in PubMed [21].

When analyzing the 12,350 results obtained from the ISI Web ofnowledge database in detail, some interesting observations can beade. If the number of publications per year is considered (Fig. 1),

t is easy to see that the few publications (less than 10) whichppeared at the end of the 1950s – beginning of the 1970s con-rasts with the more than 300 articles published in the decade of the970s. Afterwards, there is a clear increase in production until theiddle of the 1990s. The 1980s were very intense years when the

roceedings of the second international conference on MTs wereublished [12], and the first three-dimensional structures of metal-T complexes (mammalian MT2 coordinating divalent metal ions)ere solved using both crystal X-ray diffraction and NMR method-

logies (see Section 5 for further information). The beginning ofhe 1990s marks a plateau, with an average of about 450–500 pub-ications per year, that would extend for 15 years (until 2005).urrently, it seems that the interest in MT research has increasedgain reaching 600 articles per year between 2009 and 2010.

Interestingly, a glance at the research fields where these publi-ations can be ascribed reveals how the scientific interest on theseetalloproteins has evolved with time. To show this, the results

etrieved from the ISI Web of Knowledge have been refined by list-ng the 10 top journals that have published the most papers at
he beginning of the mentioned plateau (1990–1995, Fig. 2A) anduring the latest five years (2007–2011, Fig. 2B), and the num-er of articles that contain “metallothionein” either in their titler abstract.
istry Reviews 256 (2012) 46– 62 47

Fig. 2 clearly shows that in the first half of the 1990s almost 50%of the publications fell in the molecular biology and biochemistryfields, colored in blue tones, followed by the articles of the toxi-cological area, in brown. During the last 5 years (2007–2011), thefields of interest of research on MTs have experienced a substantialdrift towards the environmental and toxicological areas. Actually,the toxicologically oriented papers constitute about two thirds ofthe whole reports whereas the number of articles in more biolog-ical areas has decreased to a ca. 20% of the total, equaling those ofthe environmental fields, in green.

Concerning the “size” of the family of metalloproteins clusteredunder the term Metallothionein, it is pleasing to realize that in a lit-tle bit more than 50 years many new members have been added tothe initial mammalian MT1. A fast search in a widely used proteindatabank, UniProtKB/(Swiss-Pro and TrEMBL) databases [22], resultsin about 280 records by using the keyword “metallothionein”. How-ever, a fine tuning of this search, mainly consisting of the removalof the metallothionein-like proteins, the putative proteins and thetranscription factors, gives rise to 202 sequences of “real” MTsbelonging to 109 different organisms, from which about 30% aremammalian MTs. This result already highlights something alsodeducible from literature data: the bulk of work on MTs has beencarried out with mammalian isoforms, while those of other organ-isms have received less attention until recently, probably due to theproblems associated with their purification. These authors com-pletely share Blindauer’s statement [1] that this has provoked theperception that knowledge on mammalian/vertebrate MTs couldbe extended to other MTs although every new member belonging tothe other phyla, and that constitute the majority of family members,reveals unforeseen features about MTs features/properties thatcall into question this transference of knowledge from one MT toanother. A non-comprehensive compilation of known MT primarystructures, their number of Cys residues and experimentally con-firmed metal-to-protein stoichiometries can be found elsewhere[1].

2.2. Sources and origins of metallothionein proteins

Analytical (bio)chemists need to overcome formidable chal-lenges in terms of sample preparation and speciation for propermaterial characterization. This obvious requirement becomes anarduous task when coming to MTs, as during the past 50 years ithas made the study of most native MT proteins difficult, or it haseven precluded it. Such problems can be summarized in four mainpoints: extreme polymorphism in a single organism; a complicatedtissular and ontogenic MT gene expression pattern, regulated bya panoply of inducer agents; a minimal basal synthesis level inthe absence of metal induction; and last but not least, the factthat MTs are usually produced in organisms with variable metalload, in different oxidation states and degrees of polymerization.In practice, polymorphism, derived from gene paralogy, impliesthat several MT proteins, to a greater or lesser extent, are encodedby a same organism. Only prokaryots and some unicellular fungiappear to synthesize only one MT protein. On the other hand, inmany unicellular eukaryotes (yeasts, ciliates and fungi), MT mul-tiplicity is already a patent reality (cf. Table 1) [1,23]. In everyorganism class, each MT isogene is expressed following a differenttissue- and time-specific pattern, and furthermore, each isogenemay respond to different types of stimuli, depending on the variouskinds of physiological functions to which MTs have been associ-ated (cf. Section 3.3). This means that preparation, from tissue ororganism crude homogenates, undoubtedly encounters its first dif-
ficulty when attempting to separate the desired MT isoform fromother isoforms, which commonly share high similarity in biochem-ical features. This review includes a special Section (4.1) devotedto MT purification strategies, but it is worth noting that each

48 M. Capdevila et al. / Coordination Chemistry Reviews 256 (2012) 46– 62

0

100

200

300

400

500

600

700

1970

1971

1972

1973

1974

1975

1976

1977

1978

1979

1980

1981

1982

1983

1984

1985

1986

1987

1988

1989

1990

1991

1992

1993

1994

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

Num

ber

of p

ublic

a�om

s

Years

F ) retri fore 1

pn(

tfadctdfpdmi

Ftt

ig. 1. Representation of the publications per year (only articles, books and reviewsonein” in the title, abstract or keywords. For the sake of clarity, the publications be

articular MT protein may require an individual purification ratio-ale [24], which has been reflected in many bibliography surveyscf. Chapters 25–44 in [16]).

It is not daring to assume that the principal inconvenienceso obtain homogenous MT samples on a preparative scale, deriverom its own nature: the MT apoforms (i.e. free of metal ions)re devoid of any three-dimensional structures, adopting a ran-om coil fold in solution. It is precisely metal ion coordination thatonfers them a definite three-dimensional structure, defined byhe resulting metal-thiolate clusters (Section 5). But the fact thatifferent (mainly divalent vs. monovalent) metal ions require dif-erent coordination geometry requirements means that a given MT
olypeptide generates different final three-dimensional structuresepending on the type of bound metal ion [26]. What is more, aetal-MT complex may also include a variable number of metal
ons, and also a mixture of different types of metal ions (typi-

JOURNAL OFBIOLOGICALCHEMISTRY

METH. ENZYM.

BIOCHEM. J.

BIOL. TRACEELEMENT RES.

MOL.CEL.BIOL.

TOXICOL. APPL.PHARMACOL.

TOXICOLOGY

COMP.BIOCHEM.PHYSIOL. C-PHARMACOL.TOXICOL.

ENDOCRINOL.TOXICOL.LETTERS

PROC. NAT.ACAD.

SCIENCESUSA

1990-1995(A)

ig. 2. Relative percentage representation of the amount of published articles for the tophe 1990–1995 (A) and 2007–2011 (B) periods. Blue tones have been employed for puboxicological area and green colors for other fields.

ieved from the ISI Web of Knowledge database [20] containing the topic “metalloth-970 have not been included.

cally, for example, Zn and Cu, if considering physiological samples).This accounts for the heterogeneity of native preparations reportedin the literature. Most significantly, it accounts for the fact thatif the rate of MT synthesis is induced in an organism by metaltreatment in order to increase the yield of purification, the recov-ered complexes will include the inducer metal, and these speciesmay hold no relationship to the physiologically significant com-plexes. Hence, Cd(II) has been mostly used to induce MT synthesisfor purification purposes, and therefore Cd–MT complexes havebeen the species recovered. If it is the corresponding Zn- or theCu–MT complexes which are the subject of study, obtaining theapoform, and reconstituting the Zn- or Cu-complexes in vitro will
become an unavoidable step, but unfortunately equivalence withnatively-folded forms or isostructurality cannot always be assumedor guaranteed, even between very similar metal ions such as Zn(II)and Cd(II) [27].
BIOL. TRACEELEMENT

RES.BIOMETALS

ECOTOXICOL.ENVIRON.SAFETY

COMP.BIOCHEM.PHYSIOL. C-TOXICOL.

PHARMACOL.

AQUATICTOXICOL.

TOXICOL. APPL.PHARMACOL.

ARCH.ENVIRON.CONTAM.TOXICOL.

ENVIRON.TOXICOL. CHEM.

CHEMOSPHERE

ENVIRON.POLLUTION

2007-2011(B)

10 journals containing the word “metallothionein” in their titles/abstracts duringlications in the field of molecular biology and biochemistry, brown tones for the

M.

Capdevila et

al. /

Coordination Chem

istry R

eviews

256 (2012) 46– 6249

Table 1Some examples of members of the 15 MT families proposed in the classification of Binz and Kägi [25]. The examples chosen were those that best exemplify different aspects related to the polymorphisms in each one of thefamilies, as commented in text. UniProtKB/SwissProt accession codes are included after each sequence. (*) This isoform has been recently identified in the Drosophila melanogaster genome, personal communication Prof. WalterSchaffner, Zurich.

1. Vertebrata/mammaliaM. musculus MT1 MDPN-CSCTTGGSCACAGSCKCKECKCTSCKK-CCSCCPVGCAKCAQGCVCKGSS——EKCRCCA (P02802)M. musculus MT2 MDPN-CSCASDGSCSCAGACKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEAS——DKCSCCA (P02798)M. musculus MT3 MDPETCPCPTGGSCTCSDKCKCKGCKCTNCKS-CCSCCPAGCEKCAKDCVCKGEEGAKAEAEKCSCCQ (P28184)M. musculus MT4 MDPGECTCMSGGICICGDNCKCTTCSCKTCRKSCCPCCPPGCAKCARGCICKGGS——DKCSCCP (P47945)

2. MolluscaBivalvia

M. edulis 10MTIV MPAPCNCIETNVCICDTGCSGEGCRCGDACKCSGADCKCSGCKVVCKCSGSCACEGGCTGPSTCKCAPGCSCK (P80249)M. edulis 20MTI MPGPCNCIETNVCICGTGCSGKCCRCGDACKCASG-CGCSGCKVVCKCSGTCKCGCDCTGPTNCKCESGCSCK (P80251)

GastropodaH. pomatia CdMT MSGKGKGEKCTSACRSEPCQCGSKCQCGEGCTCAACKTCNCTSDGCKCGKECTGPDSCKCGSSCSCK (P33187)H. pomatia Cd/CuMT MSGKGS–NCAGSCNSNPCSCGDDCKCGAGCSCVQCHSCQCNNDTCKCGNQCSASGSCKCG-SCGCK (DILZJ8)H. pomatia CuMT MSGRGK–NCGGACNSNPCSCGNDCKCGAGCNCDRCSSCHCSNDDCKCGSQCTGSGSCKCGSACGCK (P55947)

3. CrustaceaC. sapidus CdMT1 MPGPCCNDKCVCQEGGCKAGCQCTSCRCSPCQKCTSGCKCATKEECSKTCTKPCSCCPK (Q548Y3)C. sapidus CdMT2 MPDPCCNDKCECKEGECKTGCKCKSCRCPPCDKCSSECKCTSKEECSKTCSKPCSCCP (Q548Y2)C. sapidus CuMT MPCGCGTSCKCGSGKCCCGSTCNCTTCPSKQSCSCNDGACGSACQCKTSCCCGADCKCSPCPMK (Q9U620)

4. EchinodermataS. purpuratus SpMTA MPDVKCVCCKEGKECACFGQDCCKTGECCKDGTCC-GICTNAACK-CANGCKCGSGCSCTEGNCAC (P04734)S. purpuratus SpMTB MPDVKCVCCKEGNECACKGQDCCTTGECCKGGTCCTGKCSNAACKTCADGCKCGSGCSCTEGNCPC (Q27287)

5. DipteraD. melanogaster MtnA MPCP-CGSGCKCASQATKGSCNCGSDCKC—GGDKKSACGCSK (P04357)D. melanogaster MtnB MVCKGCGTNCQCSAQKCGDNCACNKDCQCVCKNGPKDQCCSNK (P11956)D. melanogaster MtnC MVCKGCGTNCKCQDTKCGDNCACNQDCKCVCKNGPKDQCCKSK (Q9VDN2)D. melanogaster MtnD MGCKACGTNCQCSATKCGDNCACSQQCQCSCKNGPKDKCCSTKN (Q819B4)D. melanogaster MtnE MPCKGCGNNCQCSAGKCGGNCAGNSQCQCAAKTGA-KCCQAK (*)

6. NematodaC. elegans MT1 MACKCDCKNKQCKC–GDK-CECSGDKCCEKYCCEEASEKKCCPAGCKGDCKCANCHCAEQKQCGDKTHQHQGTAAAH (P17511)C. elegans MT2 MVCKCDCKNQNCSCNTGTKDCDCSDAKCCEQYCCPTASEKKCCKSGCAGGCKCANCECAQ—————AAH (P17512)

7. CiliataT. thermophila MTT1 MDKVNNNCCCGENAKPCCTDPNSGCCCVSETNNCCKSDKKECCTGTGEGCKWTGCKCCQPAKSGCCCGDKAKACCTDPNSG

CCCSSKTNKCCDSTNKTECKTCECCK(Q8T6B3)

T. thermophila MTT2 MDTQTQTKVTVGCSCNPCKCQPLCKCGTTAACNCQPCENCDPCSCNPCKCGVTESCGCNPCKCAECKCGSHTEKTSACKCNPCACNPCNCGSTSNCKCNPCKCAECKC

(Q6V594)

T. thermophila MTT3 MEKINNSCCGENTKICCTDLNRQCNCACKTDNCCKPETNECCTDTLEGCKCVDCKCCKSHVTCCHGVNVKSSCLDPNSGYQCASKTDNCCKSDTKECCTGTQEGCKCTNCQCYKQAQQGCCCGDKAKACCTDPNSGCCCSNNKANKCCDATSKKECQVCQCCK

(Q5XQF5)

T. thermophila MTT4 MDTQTQTKVTVGCSCNPCKCQPLCKCGTTAACNCQPCENCDPCSCNPCKCGVTESCGCNPCKCAECKCGSHTEKTSACKCNPCACNPCKCGSTSNCKCNPCKCAECKC

(Q6DMQ9)

T. thermophila MTT5 MDKISGESTKICSKTEEKWCCCPSETQNCCNSDDKQCCVGSGEGCIYVCCKCCKVQAECKCGPNAKYCCIDPNTGNCCVCKTKFCSKSDSKECCPGGSC (Q5EGE0)8. to 13. Fungi

Family 8: N. crassa MT MGDCGCSGASSCNCGSGCSCSNCGSK (P02807)Family 9: C. glabrata MT1 MANDCKCPNGCSCPNCANGGCQCGDKCECKKQSCHGCGEQCKCGSHGSSCHGSCGCGDKCECK (P15113)Family 10: C. glabrata MT2 MPEQVNCQYDCHCSNCACENTCNCCAKPACACTNSASNECSCQTCKCQTCKC (P15114)Family 11: Y. lipolytica MT3 MEFTTAMLGASLISTTSTQSKHNLVNNCCCSSSTSESSMPASCACTKCGCKTCKC (P41927)Family 12: S. cerevisiae CUP1 MFSELINFQNEGHECQCQCGSCKNNEQCQKSCSCPTGCNSDDKCPCGNKSEETKKSCCSGK (P07215)Family 13: S. cerevisiae CRS5 TVKICDCEGECCKDSCHCGSTCLPSCSGGEKCKCDHSTGSPQCKSCGEKCKCETTCTCEKSKCNCEKC (P41902)

14. ProkariotaSynechococcus SmtA (Zn) MTTVTQMKCACPHCLCIVSLNDAIMVDGKPYCSEVCANGTCKENSGCGHAGCGCGSA (P30331)Mycobacterium MymT (Cu) MRVIRMTNYEAGTLLTCSHEGCGCRVRIEVPCHCAGAGDAYRCTCGDELAPVK (P06128)

15. Plantae (4 types)Type 1: A. thaliana MT1 MADSNCGCGSSCKCGDSCSCEKNYNKECDNCSCGSNCSCGSNCNC (P43392)Type 2: A. thaliana MT2 MSCCGGNCGCGSGCKCGNGCGGCKMYPDLGFSGETTTTETFVLGVAPAMKNQYEASGESNNAENDACKCGSDCKCDPCTCK (P25860)Type 3: A. thaliana MT3 MSSNCGSCDCADKTQCVKKGTSYTFDIVETQESYKEAMIMDVGAEENNANCKCKCGSSCSCVNCTCCPN (O22433)Type 4 (or EC): A. thaliana MT4 MADTGKGSSVAGCNDSCGCPSPCPGGNSCRCRMREASAGDQGHMVCPCGEHCGCNPCNCPKTQTQTSAKGCTCGEGCTCASCAT (P93746)

5 Chem

cftbtprsDHowolgbotiatmohbbofMtrrIeDqiptooioctalaicoaptame

ooctZic

0 M. Capdevila et al. / Coordination

In view of this scenario, some attempts were made to syntheti-ally prepare MT samples, mainly short peptides, such as fungal MTorms or separate MT domains of crustaceans and mammals. Thisechnique also allowed any desired MT sequence modification toe designed. However the difficulties in avoiding thiol group oxida-ion during the chemical synthesis of these extremely cysteine-richeptides, and the fact that the final apopeptide also had to be metal-econstituted in vitro, caused a rapid decline in its use (for a review,ee [28]). Fortunately, by the middle of the 1990s, recombinantNA methodology came to change this unenthusiastic panorama.ence, genetic engineering and recombinant synthesis in heterol-gous hosts (optimally E. coli) led to a revolution in MT research,hose fruits are becoming evident after no more than two decades

f application, in view of the explosion of data for the most miscel-aneous types of MTs. First attempts to recombinantly express MTenes were carried out for mammalian isoforms in E. coli [29,30],ut had limited success, probably because of stability problemsf these peculiar proteins in a prokaryotic environment. Alterna-ively, fusion protein systems were attempted, which immediatelymproved the recovery yield [31–33]. Yeast (S. cerevisiae) was alsossayed as alternative eukaryotic heterologous host, with posi-ive results for Drosophila and mammalian MTs [34,35]. However,

ost of these studies were limited to a purely analytical level,nly demonstrating the feasibility of recombinant expression orow it conferred metal resistance to the MT expressing host cells,ut not exploring its application for preparative purposes. At theeginning of the 1990s, the breakthrough came, with the reportf an expression system able to render MT preparations suitableor spectrophotometric and spectroscopic characterization (fish

T [36], human MT [37], and sea urchin MT [38]). Unfortunately,his expression system depended on the use of Cd(II), so that theecovered preparation also required acid treatment and in vitroeconstitution if Zn- or Cu-complexes needed to be characterized.n 1997, we first reported the use of a new E. coli (pGEX-based)xpression system [39,40], which confirmed that the recombinantNA approach was a most valuable tool for the preparation of highuality and high quantity samples of metal-MT complexes, foldedn a life environment (the cytoplasm of the producing cell), com-letely equivalent to the natively produced, as was validated forhe mammalian MT1 isoform [39,40]. Fifteen years of applicationf this methodology offered us the possibility of synthesis of MTsf the most diverse organisms (reviewed in [27] and [41]), avoid-ng the difficulties of native MT purification from original wholerganism and/or tissues, and allowing the direct recovery of the MTomplexed with any desired metal ion, just by supplementation ofhe corresponding bacterial culture. The pGEX expression system islso used in other research groups, significantly in Prof. Freisinger’saboratory, for plant MT preparation [42]. Other non-negligibledvantages of recombinant synthesis are the homogeneity andntegrity of the recovered MT peptides, and, most important, theapacity of design and expression of any desired amino acid mutantr independent MT domain. This has led to functional and structuralnalysis of the role of different protein sequence features and MTrotein regions in many different MTs, from mammals [35,43–46]o invertebrates [47]. Other recombinant expression MT systemsre currently used by several groups for routine MT preparation,ainly the pET-vector based, applied, as latest examples, for C.

legans [48] or human MT synthesis (MT1, [49]; MT3, [50]).Recombinant synthesis has also unveiled unsuspected aspects

f MT capabilities, and in this aspect, the report of the presencef sulfide anions as additional ligands in some types of metal-MTomplexes is of great importance [51]. Hence, it has been shown
hat the Cd-complexes, and to a lesser extent the correspondingn-complexes, formed by the so-called Cu-thioneins significantlynclude S2− anions and that these tertiary aggregates coexist withanonical, binary complexes. This property has susequently been
istry Reviews 256 (2012) 46– 62

shown to occur also when MTs are natively synthesized [52], andsupposedly, the strictness of purification protocols for native sam-ples, not to mention any kind of acid treatment, has precluded itsdiscovery until recombinant technology was introduced in the field.

3. General features, classification, reactivity and function ofMTs

Nowadays, all scientists involved in MTs research agree that themembers of this big family of metalloproteins have the followinggeneral features in common [1]:

• Present in all eukaryotes and identified in some prokaryotes• Small (<10 kDa), cysteine-rich (ranging from 15% to 30%), and

thermostable proteins• High metal content and natively bound to Zn(II) and/or Cu(I), but

with a high affinity for all non-essential d10 metal ions• High thermodynamic stability combined with kinetic lability• ApoMT forms lack secondary structural elements• Three dimensional structure dictated by the bound metal ions• Containing characteristic metal-thiolate “clusters” that provide

them with the typical spectroscopic features of the M–SCys bonds• Absence or scarcity of aromatic amino acids

However, the diversity of the amino acid sequences of theknown MTs, which obviously implies a differentiation in their metalbinding abilities, three-dimensional structures and, consequently,biological functions, calls for a refinement and a further subdivisionof this ever growing collection of metalloproteins.

3.1. The three proposals of classification

The first attempt at classification of MTs arose from an Inter-national Nomenclature Committee established at the SecondInternational Conference on Metallothioneins in 1985 [12], whichrecommended subdividing the known MTs into three classes: thosehomologous to mammalian MT1 and MT2 (Class I), those highlydistant to them (Class II) and the enzymatically synthesized phy-tochelatins and cadystins (Class III). Almost 15 years later, Binz andKägi [25] proposed a second classification system trying to give aresponse to the rag bag that class II metallothioneins had becomewith the incorporation of all the new MTs discovered since the endof the 1980s. Most of the new members of the family had beenincorporated into this subgroup and the lack of homology betweenthem was amazing [14]. Unfortunately, the new proposal, basedon sequence similarities derived from phylogenetic relationships,devised as many groups as different taxons were known and shedno light on the possible function, reactivity or evolution relation-ships among MTs. Some years ago, our group developed [53] anew criterion based on the stoichiometric, spectroscopic and spec-trometric features of the recombinant complexes yielded by thedistinct MT polypeptides when binding a physiological metal ion(Zn(II) or Cu(I)). This classification differentiated between two cat-egories, considering as Zn-thioneins those that kept Zn(II) boundwhen folding in vivo even in the presence of high copper concen-trations in the culture medium and hence generating Zn, Cu-mixedmetal species; while defining as Cu-thioneins those capable ofyielding homometallic Cu(I)-MT species under the same conditions.More recently we have elaborated a fine tuning of this initial two-group classification by proposing a step gradation between the twoextreme MT types [41]. We have also established four bases thatallow location of each MT studied into the sequence that ranges
from genuine Zn-thioneins (such as the CeMT1 isoform of C. elegans,with a clear role for Zn-homeostasis) and genuine Cu-thioneins(like the paradigmatic copper binding yeast Cup1), leavingsome of them in intermediate positions, which highlight their

Chem

me

3

aTctdf((cisostipvbsomnoitr

3

aeHPbMlpfpna

imssatnom

msih

3

d


ulti-purpose character in the sense that do not show a clear pref-rence either for Zn(II) or for Cu(I) [27].

.2. Reactivity of MTs

To progress in the search for the putative function/s of MTs,n intensive knowledge of their chemical reactivity was required.herefore, many studies carried out with MTs, once their chemi-al and structural features had been characterized, were devotedo looking at their reactivity features in detail. As shown by Blin-auer [1], MTs reactivity can be summarized by considering theollowing cases: (i) metal uptake and release, (ii) metal exchange,iii) metal transfer between proteins and other biomolecules, andiv) redox activity. However, although all of these items providehemical information about the MT system, they do not necessar-ly inform about their physiological reactivity. In this context, thetudies reporting reaction of MTs with alkylating agents [54,55]r on their reaction with non-biological competing ligands [55,56]hould not be disregarded. Finally, it has to be noted that the par-icular case of interaction of MT and ATP has also been mentionedn the literature [57]. Unfortunately, the review of all “reactivityapers” revealed that the vast majority of the physiologically rele-ant studies are devoted to mammalian MTs – which have recentlyeen reviewed in [58] –, this impedes us reaching a comprehen-ive view for the whole MT family. More medically or biologicallyriented studies highlight MT–protein interactions, especially forammalian MT3 and its well know GIF activity although also for

on-mammalian MTs [59–61], that could shed light on the physi-logical function/s of MTs, but unfortunately they do not providenformation on the chemical implications of the interactions, andhus cannot be included in the previously reported four types of MTeactivity.

.2.1. Metal uptake and releaseDue to the polydentate thiolate nature of all MTs, their high

ffinity for most heavy metal ions is well known. Therefore, lit-rature data on MT metal binding studies is available for Cd(II),g(II), Cu(I), Cu(II), Ag(I), Au(I), Bi(III), As(III), Co(II), Fe(II), Pb(II),t(II) and Tc(IV) [2]. However, the relevance of these studies reliesoth on the bioavailability of these metals and on the “state” of theT molecules inside the organism. Here, it is appropriate to high-

ight that since 2001, Maret et al. [62] support the idea that a goodercentage of the intra- and extracellular MT is either in its apo-orm or at least as partially-metalated complexes [63], a fact onlyreviously reported for tumor cells [64]. The putative unmetalatedature of MTs obviously facilitates the metal uptake in these casesnd consequently makes the proteins highly reactive.

Metal release reactions can be easily achieved in vitro, fornstance by acidification or by adding competing ligands to the

etal-MT samples. These studies have afforded distinct apparenttability (thermodynamic studies) and/or rate (kinetic studies) con-tants for various metal-MT systems, which however can differmong themselves by several orders of magnitude depending onhe experimental conditions [1]. In vivo, and with no other part-er to receive the metal ions released, this can only occur in somerganelles such us the so-called “cadmosomes” [65] where theetal-MT species are destroyed.Another idea presented in the literature is the oscillation, by

etal uptake and release, of the metal-MT complexes betweenpecies of different degrees of metalation [66]. This dynamic behav-or of MTs clearly supports one of their proposed biological roles:omeostasis of essential metal ions.

.2.2. Metal exchangeMetal exchange in MTs is a kind of reactivity that clearly

iffers from that mentioned above in the sense that it starts

istry Reviews 256 (2012) 46– 62 51

with a metal-loaded MT (with either Zn(II) or Cu(I)), whichexchanges, totally or partially, its initially bound metal ionsfor others. This possibility relies on the well-known seriesof affinity order of heavy metal ions for the thiolate ligands:Fe(II) ≈ Zn(II) ≈ Co(II) < Pb(II) < Cd(II) < Cu(I) < Au(I) ≈ Ag(I) < Hg(II)< Bi(III) [67], and consequently makes Zn-loaded MTs more reactivethan Cu-loaded MTs.

The overwhelming amount of information in the literature onthe metal displacement reactions that can take place on metal-loaded MTs is impossible to summarize and the reader shouldaddress the specific reviews on each family of MTs in the latest com-prehensive books and review articles mentioned at the beginning[1–8]. All this information, together with that gathered from themetal binding studies of apoMTs, has yielded very valuable in vitroinformation: preferred number and nature of the bound metal ionsof each entire MT and the distinct fragments of the bidominial MTs,geometry, affinity constants and rate constants for the binding sites,interactions between domains, interdominial metal transfer andcooperative mechanisms of metal binding. Again, the occurrenceof the metal ions studied in nature, the experimental conditionsassayed, such as the protein concentration, the pH or ionic strength,and the closeness to in vivo findable situations give more or lesssignificance to the results obtained to interpret the physiologicalfunctions of MTs. In any case, what it is clear is that the mostcommonly found xenobiotic metal ions (Cd(II), Pb(II), Hg(II)) showhigher affinity for thiolate ligands than Zn(II) or Cu(I) and thus, incase of intoxication, MTs can obviously work as detoxifying agents.In fact, MTs are acting as detoxifying agents when intercepting thepathways of antitumor Pt complexes to DNA [68,69].

3.2.3. Metal transfer between proteins and other biomoleculesAs proposed by Maret [70], this encompasses metal transfer

between MTs [18] and also between MTs and other intracellularand/or extracellular biomolecules. Concerning this later issue, mostof the work has been solely attained for the Zn-loaded or apoformsof mammalian MTs, and thus by considering the distinct MT formsas Zn(II) donors/acceptors, thus merely limiting the role of MTs tometal homeostasis. In this context, Zn exchange has been nicelyreviewed by Vallee and Bell [2] for mammalian MT1 and MT2,where the authors collect glutathione, Gal4, alkaline phosphatase,carboxypeptidase A, sorbitol dehydrogenase, glycerol phosphatedehydrogenase, mitochondrial aconitase and the TFIIIA and SP1transcription factors as putative receptors/donors of the Zn(II) ini-tially bound to the proteins. Also, other recent papers highlightmetal exchange processes in which mammalian MT3 is involved[4,71] or in which Zn(II) ions present in MTs can activate matrixmetalloproteinase 9 for collagen cleavage [72]. However, in manyof the previously cited cases, redox activity of the diverse mam-malian MT isoforms is implicit and therefore the reader shoulddirect his/her interest to the next paragraph.

3.2.4. Redox activityIn accordance with Maret [3,73] and Kang [74], Zn–MTs are

redox-active systems that can be oxidized by mild cellular oxidants,such as glutathione disulfide (GSSG) or selenium compounds, withthe concomitant release of Zn(II). Then, the reversibility of thesereactions place the GSH/GSSG and MT/thionein redox pairs into thecontrol of the cellular redox state and, consequently, of the energymetabolism. Moreover, under loaded Zn–MT species can offer freecysteine thiols that can be easily oxidized. Consequently, MTs couldneither be fully Zn(II)-loaded nor fully reduced. In short, redox reac-tivity of Zn–MTs would depend on their Zn(II) load, which in turn
is related to the availability of other Zn(II)-binding proteins. Obvi-ously, if Zn(II) is needed and transferred to other proteins, the thiolreactivity of MTs increases. If, however, there is a low demandfor Zn(II), because sufficient Zn(II) is available, their reactivity is

5 Chem

qa

hmt

rsTtditoiqir

wltoeh

3

bewoiooobaotmcpifitntcbff

tomfttdmMr


uenched, thus supporting the role of MTs in cellular homeostasisnd distribution of Zn(II).

The studies of Vasák’s group [71,75] should also be mentionedere, which showed that Cu(II) acts as the oxidant of the Zn-MT3olecules which incorporate Cu(I), thus protecting the brain from

he deleterious effects of free copper(II).Other types of studies have been devoted to the analysis of the

edox behavior of MTs towards strong oxidants and radical speciesuch as H2O2, NO, OH• and O2

•−, i.e. under oxidative stress [3,74,76].hese very reactive species provoke either the oxidation of cysteineo cystine, with the concomitant release of the bound metal ions, oresulfurization reactions [77], which also release the bound metal

ons but preclude any possibility of metal-MT complex regenera-ion, a process which could be always envisaged if MTs were simplyxidized. However, it is not clear whether the release of these metalons is the main purpose of the presence of MTs or just a conse-uence of the oxidative stress. In any case, the obvious conclusion

s that MTs protect against the deleterious effects of oxidants andeactive radical species.

Sadly, to our knowledge there are no data in the literature abouthat would be the redox reactivity of physiologically relevant MTs

oaded with metal ions other than Zn(II), i.e. CuI-loaded MTs (Cu-hioneins). However, this is not the case for FeII-loaded MTs, wherene-electron reduction process of met-myoglobin and the multi-lectron reduction of some azobenzene derivatives by rat �MT2ave been recently reported [78].

.3. Proposed biological functions

More than ten years ago, the publication of the short reviewy Palmiter [9], entitled “The elusive function of metallothioneins”,xposed the crucial problem of assigning a biological role to theseidely studied proteins. Paradoxically, difficulties do not consist in

bservation a function, but in the fact that MTs have been impliedn tens of physiological processes, usually varying from one type ofrganism to another and even among different isoforms of a samerganism [79]. Therefore it is wise to accept that it makes nonsensef looking for a unique function for the whole set of MT proteins,ut instead different precise functions will be gradually associ-ted with distinct MTs from different species, isoforms or eventher type of variants (e.g. post-translational modifications). Whenackling this question, it will be useful to differentiate betweenolecular function and biological or physiological function. Two pre-

ise molecular functions can be attributed to MTs, related to theireculiar amino acid composition: metal binding and redox activ-

ty. In fact, the reactivity of the sulfur atoms of cysteines accountor these two capabilities, which have been largely characterizedn in vitro experimental approaches (cf. Section 3.2). The trouble ishat these two chemical abilities appear to be involved in a largeumber of different biological processes, which depend on the par-icular physiological needs of each kind of organism. Not only theonservation of MT genes along all the branches of the tree of life,ut also the extraordinary multiplication and diversification of iso-orms, may be related to the ability of MTs to adapt and be usefulor a great diversity of functions.

MTs were first reported as cadmium detoxifying agents [10], sohat heavy metal detoxification was their first, and long assumednly biological function [80]. However, its ability to chelate cad-ium should be basically considered to result from its capacity

or Zn coordination, in view of the equivalent coordination geome-ry requirements for these two metal ions, and taking into accounthe common trace presence of cadmium in the earth geological
eposits of zinc, which may have played a leading role in early lifeolecule development [23,81]. The most likely hypothesis relatesTs to basic metal homeostasis; this is the maintenance of cor-
ect levels of the two main physiological metals, zinc and copper

istry Reviews 256 (2012) 46– 62

[23]. In this context, they can perform several additional spe-cific tasks, such as metal ion reservoirs, metal transport, and/ormetal delivery to target metalloproteins (i.e. a metal-chaperonerole) [9,82–88]. The transfer of zinc from MT to the apoformsof zinc proteins has deserved special attention in view of thecrucial role of zinc proteomes in life systems [89]. Just to men-tion some examples, the four mammalian MTs (MT1 to MT4)have been assigned roles in metal homeostasis, metal transferand redox potential, antioxidant actions, antiapoptotic pathways,immunoregulation and anti-inflammatory processes (MT1, MT2);plus neuronal growth control for MT3; and plus epidermal pro-liferation involvement for MT4, a comprehensive recent reviewbeing available in [58]. In distant organism groups, those har-boring haemocyanin as respiratory oxygen copper transporter,a specific MT isoform is devoted to this metal ion homeostasisin haemocyanin synthesizing cells [90], while another isoformis exclusively synthesized for cadmium detoxification [91]. Withregards to plants, types 1, 2 and 3 MTs have been directly related tocopper metabolism special demands and to oxidative stress defense[5], although it is worth noting that the most well studied plantMT type (type 4 or Ec) is a clear Zn-thionein, isolated from wheatembryos [92]. More extensive information on MT biological impli-cations in mammals and humans is detailed in Section 6, BiomedicalMT applications.

On the other hand, the capacity of the thiol groups to be oxidizedby mild oxidizing agents would facilitate their role as a first defenseagainst oxidative stress [93,94]. Hence, MTs are capable of ROS(reactive oxygen species) and RNS (reactive nitrogen species) scav-enging [95], this protecting the most vulnerable cell components,such as DNA, proteins, and lipid membrane structures [96–99].The presence of apoMT in cells, which is more reactive towardsoxidants, cannot be discarded (see [100] for a recent revision).Noteworthy, metal binding and oxidative stress buffering are notcompletely unrelated functions, either for redox active (CuI/CuII)or for non-redox active metal ions (ZnII). First, because copper ionbinding by MT is supposed to prevent the Fenton effects associ-ated with their reactivity [98]. And second, because a redox cyclebetween Zn-MT and the apothionein resulting from Zn(II) releasehas been extensively demonstrated and discussed by Maret andco-workers [101,102], which could be further coupled to otherwell-known cell redox mechanism, such as the GSH/GSSG equi-librium [74].

The reported data on the implication of MTs in biological pro-cesses were classically obtained from two types of studies: thebiochemical features and abilities of the purified proteins, and thetranscriptional response of the corresponding genes to a certaininducer agent or physiological situation. All the above-mentionedbiological functions fully satisfy these two criteria, on the basisof the synthesis of a given protein being induced by an agenttowards which the protein is biologically active. Hence, MT genepromoters normally include a considerable number of MRE (metalresponsive elements) as well as regulatory sequences respondingto oxidative stress (cf. [103] for a recent revision). However, MTgene transcription also responds to other types of stresses for whichMT involvement is less studied, but has therefore to be assumed,such as heat and cold shock, pH changes, starvation, hormones andcytokines, on a broad variety of drugs [79].

Two modern molecular biology approaches have lately beenapplied to the detection of previously unsuspected MT functions:the analysis of the phenotypes exhibited by genetically engineeredMT-devoid (MT-nulls, knock-outs or KOs), and the proteomic anal-yses of protein–protein interactions. MT KOs have been constructed
for different organisms, from unicellular eukaryotes (yeast) [104],plants [105], invertebrates (Drosophila [106], C. elegans [107]) tomammals (mice [85]). Common traits are that MT absence is notlethal, nor greatly deleterious in the absence of metal overload.

Chem

Bat[weg(Mco[

pnMscesicpDttaginrmwcSdnsaaslm

3

itclufgpamAtBfgd

pr


esides the expected phenotype of reduced metal tolerance, also higher sensitivity to oxidative stress was observed, as well as aendency to worsen support inflammatory and infection processes85]. Results with MT-KOs were somehow disappointing, but it isorth remembering that the construction of complete MT-KOs is

xceptionally troublesome, due to the high redundancy of the MTenes, especially in mammalian organisms. Double mouse MT-KOsMT1,2 null) have been obtained, but these animals still keep theT3 and MT4 isogenes, so that it cannot be ruled out that their

ompensatory overexpression accounts for the almost null effectbserved in normal life conditions – only a tendency to gain weight108].

Finally, the identification of MT-interacting proteins should alsorovide information about physiological functionality. Unfortu-ately, very few reports of MT–protein interaction are available.ost of them refer to the brain specific mammalian isoform, MT3,

howing that it is involved in the regulation of Zn exocytosis onertain types of hippocampus neurons, in protecting neurons fromnvironments of changing oxygen concentration, or in neuronalecretion processes [59,109,110]. Additionally, mammalian MT1/2soforms interact with megalin for internalization in kidney tubuleells [111]. In a different scenario, our research group investigatedrotein interactions in a completely different model organism,rosophila [60]. The most significant observation was the interac-

ion between the two major Drosophila MTs (MtnA and MtnB) withhe Jafrac-1 peroxiredoxin, a thiol-specific antioxidant enzyme, andn equivalent interaction between the respective yeast homolo-ous proteins (Cup1 and Tsa1). These results pointed to a new wayn which MTs could be related with redox equilibrium mainte-ance in organisms, and of particular importance, revealed that theecognition between the MT and the peroxiredoxin molecules wasetal-dependent. Precisely, such interaction was only detectedhen MTs were partially or totally loaded with Zn(II), while the

orresponding Cu-complexes exhibited no interaction at all [60].ince it is well established that the same MT polypeptide adoptsifferent three-dimensional structures depending on the coordi-ated metal ion, and protein–protein interaction is essentially ateric process, these results should not be completely unexpected,lthough as far as we know it is the only case where there has been

search for MT partners by using different metal complexes of theame peptide. Finally, it is obvious that this discovery also high-ights how the physiological meaning of MT–protein interactions

ay be related to the specific metabolism of a given metal ion.

.4. Differentiation and evolutionary divergence significance

As stated previously, the extreme MT sequence heterogene-ty yields few hints to speculate on MT origin and diversificationhrough evolution, so that any homology-driven structural, bio-hemical or functional inference using mammalian data makesittle sense when considering MTs other than those belonging to thenquestionable homology family of vertebrate MTs. Invertebrate,ungal and plant MTs particularly exhibit high sequence hetero-eneity, both among themselves and in relation to the vertebrateeptides (Table 1). Since no definite physiological roles have beenssigned to these proteins, functional constraints or adaptive trendsodulating their evolutionary history are also difficult to envisage.

major mistake has probably been to assume that these condi-ioning forces have been the same for all type of living beings.ut today, new, more informative data are being gathered when

ocusing physiological and evolutionary studies on each separatedroup of organisms, as was discussed in a recent overview on MT
ifferentiation and evolution [23].
The 20th century belief that MTs were essentially eukaryoticroteins was drastically disproved in our new century, with theeport of MTs, already with extraordinary heterogeneity, in bac-

istry Reviews 256 (2012) 46– 62 53

teria. Hence, the two basic kinds of MTs defined by their metalbinding preference (Zn- and Cu-thioneins, cf. Section 3.1.) arepresent in prokaryotes, respectively represented by the SmtA pep-tide of the cyanobacterium Synechococcus sp. [112] and the MymTpeptide of Mycobacterium tuberculosis [113]. The two MTs exhibitdifferent metal preferences, and have subsequently been associ-ated with different metal ion detoxification capacities. This dualpanorama is exactly the same as that repeatedly encountered inalmost all groups of eukaryotic organisms. It is generally acceptedthat evolution under constant conditions yields a divergent treepattern, while very changing or specific conditions appearing overa previously diversified scenario lead to evolutionary conver-gence events. The complicated scenario of MT evolution seemsto have its explanation in a combination of both phenomena.Hence, primitive eukaryotes shared their habitat with emergingaerobic bacteria, and the proliferation of apparently unrelatedsequences performing either Zn- or Cu-binding functions is fre-quent. Besides, in metazoans (pluricellular eukaryotes), both theproliferation of MT sequences and the coexistence in organismsof Zn-, Cu–MTs and intermediate forms, reaches paroxysmal lev-els. A possible physiological explanation for this could be thatmetazoans can be considered mosaics of very different cell typesand tissues, so each of them can be assimilated to “unicellularenvironments” in terms of metal requirements and the concor-dant evolution of the contained metal-interacting proteins. Theanalysis of the diverse eukaryotic MT sequences (Table 1) showsthat the most probable hypothesis is the existence in a giventaxon of one, or even more, primeval metal-binding that byduplication of events, and subsequent sequence differentiation,generated several MT family members, specialized to a greateror lesser degree in either Zn(II) or Cu(I) coordination. Suppos-edly, this may have been achieved by selection of the proteinsequence features-number and position of the coordinating Cysresidues, presence of other putative coordinative amino acids,the relative distance between them, nature of the intercalatedresidues, total length of the peptide, etc. that fulfil the best coor-dination environment requirements for each particular metal ion.Therefore, it is reasonable to assume that the combination ofprotein sequence features determining optimum divalent or mono-valent metal ion binding, acting on different substrate sequences,was the driving force behind the iterative convergence eventsobserved in MT evolution. As mentioned above, the back and forthgradation from Zn-thioneins to Cu-thioneins converges in a groupof MTs with intermediate properties, such as the yeast Crs5 [114]and the mammalian MT4 isoform [115], exhibiting highly ambiva-lent metal binding preferences. Significantly, these MT isoformsappear in the root of phylogenetic trees, so that it is logical to con-clude that they may reflect primeval, polyvalent forms, where theprogressive duplication yielded new forms that could evolve andbe fixed as metal-specialized isoforms for specific, taxon-related,physiological functions. It is also worth noting that in this case,parallel evolution of the corresponding gene regulatory elementsled to matching expression patterns, i.e. genes are induced by thecognate preferred metal ions and promoters ensure that they areactivated in suitable tissues and conditions. Model examples whereMTs of extremely opposite nature (Zn-thioneins and Cu-thioneins)have been reported, and are known to be synthesized under clearlydivergent induction conditions, can be found in the MT systems ofthe following organisms: snails [116], crustaceans [53,117] and theciliate Tetrahymena [118].

4. Experimental methodologies to approach MT research

Nowadays there is a high number and diversity of spectroscopicanalytical techniques and methodologies that can be applied for

5 Chem

ttismpFtsiopmta

4

soghlaF

dptsepfri(bothhnwesc

ms((Untiaptmiatqtp


he separation, quantification and chemical characterization of pro-eins. However their applicability to the study of metallothioneinss drastically hampered because of the special features of MTs. Theirmall size, preference for binding “spectroscopically silent” d10

etal ions, and the practical absence of aromatic residues in theirrimary structures limit the available/useful scientific approaches.urthermore, researchers need to be aware not only of these par-icular properties of MTs but also of the possibility of coexistingpecies in a sample in order to take into account the effect thatts treatment (pH, buffers, presence of competing ligands, oxidantsr reductants, adventitious metal ions, . . .) can exert in the com-osition and structures of the metal-MT species. Even so, someethodological approaches have been, and currently are, applied to

he study of MTs, although not all of them provide precise, univocalnd valuable information.

.1. Purification and quantification approaches

Since their discovery and until the middle of the 1990s, the mainource of MTs was the purification of native proteins from tissuesf the original organisms. Accordingly, the purification methodolo-ies used were based on techniques such as protein precipitation,eat treatment, conventional gel filtration, and HPLC [15]. Cova-

ent chromatography, polarography, and acidic gel filtration havelso been used for the separation and purification of several MTs.urther developments in this field will be treated below.

Since the very beginning, the methodologies based on theetection of the heavy metals bound to MTs have been the mostopular procedures to quantify MT samples. Therefore, owingo their trait of element specific techniques, atomic absorptionpectroscopy (AAS) [119,120], inductively coupled plasma atomicmission spectroscopy (ICP-AES) [120] or inductively coupledlasma mass spectrometry (ICP-MS) [119] have been widely usedor the quantification of MTs. Following this approach, metal satu-ation procedures [15] have also been developed to quantify MTsn several tissues. Here, treatment of the sample with metal ionssuch as Hg(II), Cd(II) and Ag(I)), with higher affinity for the MTinding sites than those ions initially bound, allow, after removalf the excess metal added, the determination of the MT concen-ration by AAS, ICP-AES or ICP-MS. The metal saturation techniqueas resulted especially useful in those cases of organisms producingomometallic MT isoforms, of well-established stoichiometry andot liable to degradation during the purification process, amonghich gastropod molluscs offer an excellent example [121]. How-

ver, it must be stressed the inadequacy of this methodology inystems of undetermined metal-to-protein ratios under saturationonditions.

Other quantification methodologies have been based on theeasurement of a specific property of the protein itself. Mea-

urement of the specific absorptions, either of the Cd-SCys bonds254 nm) or of the thiolate groups modified by the Ellman’s reagentDTNB) or other related reagents [55] have been routinely used.nfortunately, their low accuracy and the large amount of sampleeeded made them limited and less used than the direct quan-ification methodologies. Radioimmunoassay (RIA) and enzymemmunoassay (ELISA), based on the use of specific antibodies, havelso been applied for detection of specific MTs without the need ofrevious separation procedures, but again their restricted applica-ion have made them of narrow use [122,123]. Special attention

ay be drawn into the quantification of MTs by electrochem-cal methods, based on the reactivity of the -SH group, whichllow quantification at very low concentration levels [124]. Unfor-
unately, only a couple of groups are applying this method foruantification purposes. In this sense, we must pay special atten-ion to previous studies by Dabrio and Rodríguez [125,126] asioneers on these kinds of methodologies to analyze and quantify
istry Reviews 256 (2012) 46– 62

MTs, and to the current developments and applications of Kizek’sgroup [127]. Additionally, the construction of the first isoform-selective biosensor [128] capable of selectively quantifying themammalian MT1 isoform in relatively low purity samples has beenrecently reported.

The important development of the hyphenated techniques forthe analysis of metalloproteins that took place at the end of the1990s, mainly due to the progress made on the coupling of highlyselective separation techniques to element-specific and molecule-specific detectors, offered a new horizon in the quantification ofMTs [129,130]. Therefore, the coupling of capillary electrophore-sis (CE) or high-performance liquid chromatography (HPLC) withmass detectors, such as ICP-MS or ESI-MS, instead of the traditionalatomic detectors based on the use of photons (AAS, flame photoion-ization detector (FPD), ICP-AES, atomic fluorescence spectroscopy(AFS)), has now become the main methodology to be applied forthe analysis of all kind of MTs.

Currently, the exploration of MT levels and of their metalcontent mainly employs size exclusion HPLC, microbore reversed-phase (RP-HPLC) or capillary zone electrophoresis (CZE), intwo-dimensional or multidimensional separation approaches, cou-pled with ICP-MS (in its diverse configurations, such as ICP-Q-MS orICP-TOF-MS) in hyphenated techniques, such as HPLC-ICP-MS, andCZE-ICP-MS [130–132], which allow the full determination, at verylow concentration levels, of the distribution of the desired metalions. In fact, the coupling of a high resolution separation technique,which can offer the possibility of separating species of slightly dif-ferent characteristics, with a highly sensitive detection technique,which normally needs low amounts of sample and, as in the caseof ICP-MS, offers the possibility of analyzing several isotopes of thesame or different elements at once, allows a fast, quantitative andreproducible analysis of the individual components of a sample.These approaches have been used to evaluate the distribution ofcopper and zinc in MTs in chronic liver diseases [133], to detectzinc-containing MTs and the characteristic changes of zinc distri-bution in the serum of healthy and non-healthy patients [134], aswell as to study the accumulation of copper in MTs when comparingAlzheimer’s disease and control brains, demonstrating a signifi-cant difference of metalation in MT1, MT2 and MT3 [135], as nicelyreviewed by Maret [3].

The recent application of other methodologies, such as real-timedetection PCR, laser ablation, and imaging techniques, continues toadvance and to expand the applications of the hyphenated tech-niques for the study of metalloproteins.

4.2. Chemical and structural characterization

Since the discovery of MTs, practically all types of molecularspectroscopies and analytical techniques have been applied for theelucidation of their chemical properties and structure [15]. Conse-quently, it is an almost unaffordable task to make an exhaustive listof all data recorded on MTs over the last 50 years. On the other hand,some comprehensive reviews focusing on this topic have alreadybeen published [136–138]. Hence, the following section will try tospotlight the main advantages and drawbacks of each techniquefrom an historical viewpoint.

Because MTs mainly prefer to bind “spectroscopically silent” d10

metal ions, many of the traditional “bioinorganic” techniques arenot applicable to MTs. However, MTs show ligand-to-metal charge-transfer (LMCT) absorption bands detectable in the UV–vis region.For Zn(II) the LMCT bands fall around 220–240 nm, relatively closeto the absorbance maximum for peptide bonds (215 nm), which
can be problematic in terms of sensitivity and data interpretation,but fortunately most of the absorptions for the other metal ionsfall at higher wavelengths. Therefore, electronic spectroscopy hasbeen the most widely used technique ever since the discovery of

Chem

Mtesdaiatt

ucbpabbtbaldvgtncaqbsussae

tmtfwTid

instavwm

thCawtAtdi


Ts. This technique mainly provides information on the metal-hiolate chromophore absorption; however, the interpretation oflectronic spectra is largely dependent on prior knowledge of thetructure. In addition, the bands are normally broad, with minimaletailed resolution, that is to say the bands only provide an over-ll picture. Nevertheless, this drawback can be partially overcomef mathematically subtracting consecutive UV–vis spectra during

titration, thus generating the so-called difference UV–vis spec-ra, each of them giving information on the contribution of the lastitrating agent addition.

Circular dichroism (CD) spectroscopy has also been widelysed for the study of MTs since the appearance of affordableommercial spectropolarimeters in the late 1970s, and thereforeoth UV–vis and CD spectroscopies have been frequently used inarallel because of their complementarity [139,140]. Since the met-ls bound to MTs receive the chirality from the protein, LMCTands between the cysteine thiolates and the metal ions cane observed in CD spectroscopy and are extremely sensitive tohe degree of compactness of the metal-thiolate clusters that areeing formed. Furthermore, for metals that are not in groups 11nd 12, MLCT bands have also been observed at higher wave-engths [141]. Also, intra- and intermetallic transitions have beenescribed for Cu-MTs [142,143]. In general, CD spectroscopy pro-ides clues about metal-ligand stoichiometries and coordinationeometries, and provides information on how a metal substitu-ion process (e.g. Zn(II) by Cu(I) or Zn(II) by Cd(II)) takes place –umber of stages, presence or not of cooperative processes, globalhange in structure [144]. However, no specific bonds between

metal ion and its ligand/s can be directly assigned, and conse-uently confirmation of structural information should be acquiredy other more powerful techniques. Additionally, it is not pos-ible to assign a particular CD fingerprint to a particular speciesnless a single species is present in solution. Nevertheless, masspectrometric measurements frequently reveal the coexistence ofeveral species in solution, which will be discussed below. CD haslso been widely used as a monitoring technique in demetallationxperiments [145].

Magnetic circular dichroism (MCD) gives information aboutransition metal ion oxidation and spin states and allows the deter-

ination of the metal coordination geometry through prediction ofhe expected MCD bands. In short, MCD provides key informationor unique identification of the symmetry of the chromophores,hich cannot be derived either from UV–vis or CD spectroscopies.

his was used for the study of MTs in the presence of distinct metalons during the 1980s and the 1990s [137], but has since fallen intoisuse.

Stopped-flow (SF) techniques have been widely used in biolog-cal kinetic studies involving relatively rapid reactions, but it wasot until 2002 that Petering’s group used SF spectrophotometry totudy the folding pathway of rabbit liver apoMT [146]. However,he folding of mammalian MT domains in the presence of Zn(II)nd Cd(II) at neutral pH was still too fast to be followed by con-entional SF spectrophotometry, as the folding mostly took placeithin 4 ms, which is approximately the so-called “dead-time” ofixing.Since the discovery of orange luminescence at room tempera-

ure for Cu(I)-MTs by Beltramini and Lerch [147], several groupsave analyzed the emission properties of MTs usually containingu(I), but also with Ag(I), Au(I) or Pt(II) [137,148,149] at both roomnd/or cryogenic temperatures. In titrations of apo- and Zn–MTith Cu(I), the emission intensity profile has been interpreted in

erms of site and/or domain occupancy by the incoming Cu(I) ions.
dditionally, because the excited states are quenched by exposure
o the solvent or molecular oxygen [150], the emission intensityepends on the degree of efficiency in the exclusion of the quench-

ng agents during the copper loading process, which in turn can

istry Reviews 256 (2012) 46– 62 55

be related to the degree of folding of the Cu(I)–MT species beingformed.

Electron paramagnetic resonance (EPR) spectroscopy has alsobeen used for the study of the reaction of MTs with Co(II) [151,152].This technique provides information on the spin state of the para-magnetic center and, by comparison with appropriate inorganiccomplexes, the geometry around the paramagnetic ion can bededuced.

Infrared (IR) and resonance Raman spectroscopies [153–155]have also been used for the characterization of MTs, which haveproved to be useful tools for providing information about the stateof the Cys sulfur atoms in either their metal coordinated or oxidizedform, and the participation of other residues such as His in metalcoordination. However, these techniques show inherent difficultiesdue to congestion of bands in complex systems and to the fact thatstructural interpretations are often indicative but not unique.

Synchrotron radiation X-ray absorption techniques (XANES,XAFS and EXAFS) have been also used for the analysis of the metalbinding in MTs [137]. XANES allows the characterization of theaverage oxidation state of redox active metals and the metal-ligandcoordination geometry/symmetry, whereas XAFS, and particu-larly EXAFS, give information on metal-ligand bond distances,coordinating numbers and types of coordinating atoms. Detailedstructural information has been inferred from EXAFS measure-ments of MTs containing Zn(II), Cd(II), Cu(I) or Hg(II) by comparisonwith inorganic model compounds, using both the metal or sulfuras the absorbing atom. However, the main drawback associatedwith these techniques is that the scattering signal is an aver-age of all the sites of the same element. Therefore, if differentsites exist with different coordination environments, only an aver-age picture of the sites can be obtained. Moreover, fitting datais not always an easy task, and often more than one fitting ispossible.

Finally, two other spectroscopies using high energy radiationhave been used for the study of MTs. X-ray photoelectron spec-troscopy (XPS), which measures the elemental composition andthe chemical and electronic state of the elements that exist withina sample, has been used to characterize the oxidation state ofthe bound copper in chicken and rat liver MT [137], and timedifferential perturbed angular correlation spectroscopy (TDPAC),which uses �-rays emitted from radioisotopes and gives informa-tion about site coordination number and geometry, has been usedin the study of Hg(II) binding to mammalian MT [156].

Nowadays, mass spectrometry (MS) is at the vanguard of thetechniques for characterizing biomolecules. Among the molecu-lar MS techniques, electrospray mass spectrometry (ESI-MS) hasbeen chosen since the beginning of the 1990s to analyze MTs, beingmainly used for the identification of soluble metal-MT species. ESI-MS provides information about intact, folded proteins and in thecase of metalloproteins, allows identification of both the holo- andthe apo species. One of the main advantages of using ESI-MS isthat it provides information on each of the individual species of amixture, as each species displays its own mass. This makes infor-mation available in a short period of time on the number of solublespecies, their stoichiometry or metal content, and, in certain con-ditions, gives an idea about their relative abundances. However,ESI-MS also shows limitations, especially concerning quantificationand determination of stability constants, as well as in the analysis ofspecies stabilized by non-covalent interactions. Therefore, the useof ESI-MS is particularly attractive in the case of MTs because theirfold is induced by the covalent binding of metal ions. As a matterof fact, we should appreciate the research carried out by Fenselau’s
team [157], who initiated the study of MTs by ESI-MS. Since then,the technique has been gradually adopted by other groups withvery distinct research interests on MTs [158]. As in the case of ICP-MS, the popularity of ESI-MS for the analysis of metalloproteins

5 Chem

ea[jMfiZastt[oPttap

la[tt

nriiutmsi

cptciwuHw

edl1

woMrm

swbst

5

d


xperienced an increase at the end of the 1990s due to theppearance of the above mentioned hyphenated techniques120,130,159], thus providing a direct identification of each speciesust after the separation by HPLC or CZE. Nowadays, the use of ESI-

S as a routine methodology is a reality and its application in theeld of MTs affords a high variety of types of analysis: study ofn/Cd and Zn/Cu replacement processes, metal transfer reactions,nd identification of the in vivo metal-MT species formed for MTs ofeveral organisms [47,116,158,160] or individual isoforms (cf. forhe brain-specific mammalian MT3 isoform, the work by Palumaa’seam [161,162]), interaction of antitumor Pt complexes with MTs69,163], study of the oxidation of MTs by H2O2 [164], or even studyf the binding to MTs of non-cognate metals such as Hg(II) [165],b(II) [166], As(III) [167] and Bi(III) [168]. As a particularity, evenhe rate constants of As(III) metallation of specific MT-like pep-ides have been calculated by means of ESI-MS [169] in spite of thelready highlighted limitations of the use of ESI-MS for quantitativeurposes [170].

The future of the application of ESI-MS to the study of metal-oproteins lies in the use of highly sensitive and high resolutionnalyzers (time-of-flight (TOF) [170], linear and orbital ion traps171]), the development of reproducible nano-spray interfaces, andhe improvement in fragmentation methodologies, still scarce inhe analysis of MTs.

MALDI-TOF MS, the other molecular mass spectrometric tech-ique widely used to analyze biomolecules [172], has been moreestrictively applied for the study of MTs. This is mainly due to somenherent limitations of the technique, such as the impossibility ofts coupling with a conventional separation technique, and partic-larly the difficulty in analyzing metalated species. This is probablyhe reason why MALDI-TOF has been only used in a couple of studies

ainly devoted to the identification of isoforms and subisoforms ofeveral organisms in their apoform [173,174], thus bypassing thenformation related with their metal content.

Calorimetric methods have been used to determine equilibriumonstants since 1937, but its application to the study of metallo-roteins is much more recent [175]. Since the end of the 1980s,he use of calorimetric techniques, especially isothermal titrationalorimetry (ITC), has become very useful for calculating the bind-ng constants and other thermodynamic parameters associated

ith protein interactions [176], and it has proven to be particularlyseful when there are a limited number of binding sites [176,177].owever, in more complex systems, such as that of MTs, only oneork has been reported so far [178].

Finally, NMR (nuclear magnetic resonance) has been anxtremely powerful tool for providing information about theynamic structure of MTs, specially in the presence of Cd(II). Excel-

ent reviews on the state-of-the-art and applications of 111Cd or13Cd NMR to the dynamics of Cd–MTs have been published else-here [1,179]. The next section will be mainly centered on the use

f NMR in solving three-dimensional structures of MTs. However,T aggregation problems associated with the high concentrations

equired should not be disregarded when carrying out this type ofeasurement.MTs have also been investigated at the single molecule level by

canning tunneling microscopy (STM) [180]. In contrast to NMR,hich cannot provide information on the relative orientation of

oth domains, STM can reveal the dumbbell shape of some MTs inolution and has also allowed the metal ions buried in the proteino be visualized.

. Three-dimensional structures

From the very first attempts, the elucidation of the three-imensional structure of MTs, or more precisely metal-MT

istry Reviews 256 (2012) 46– 62

complexes, has emerged as a particularly troublesome task. Mam-malian Zn, Cd-MT2 complexes were the first to be solved using the1980s commonly accepted methodology of X-ray diffraction [181].However, simultaneously metal-MT2 complexes were among thefirst protein structures being solved by NMR [182–185]. In fact,after a discrepancy between the structures resulting from the twomethodological approaches, the initially proposed crystal three-dimensional structure of rat MT2 had to be refined, so that a secondcrystal model, essentially coincident with the NMR-solved struc-ture, was thereafter published [186]. Therefore, in some way, MTsallowed recognition of the then emerging NMR technique as a validand reliable strategy for protein structure resolution [187], some-thing that the elapsed time has more than confirmed.

Nowadays, NMR spectroscopy has become the major tool forMT structure determination, and all but 2 of the 32 MT structures(Table 2) currently available in the Protein Data Bank (PDB) havebeen obtained by this technique. Among the main advantages thatNMR offers, is the possibility of obtaining spatially resolved high-resolution information on structure and dynamics across the entiremolecule, as well as the observation of the structure dependenceon pH, ligand and/or temperature conditions. However, in order toobtain good spectra, some conditions must be accomplished, suchas high sample purity, an optimal concentration above 1 mM andthe frequent need of isotopic labeling. The reader should keep inmind that neither the conditions concerning NMR measurements,nor those required for crystallizing proteins, necessarily satisfythose observed at physiological conditions, this casting doubts onthe representativity of the achieved results in terms of their use-fulness to draw a realistic picture of the structural features of MTsin nature, as already highlighted [1].

Nevertheless, and in spite of the potentiality that NMR offersfor structure resolution, the case of MTs has been a tough chal-lenge for several reasons. First, some MTs have a good amount ofGly, a residue that provides very little structural information. Sec-ond, the low variability of the residues in their primary sequencemakes conclusive sequential assignment difficult. And finally, con-ventional NMR spectroscopy (1H, 13C, 15N) normally is not powerfulenough to locate all Cys side chains, and therefore an unambiguousassignment of the metal-Cys connectivities is not possible. All thesephenomena stated above explain the amazing divergence betweenthe considerable number of the known MTs nowadays and the lim-ited number of MT structures that have been determined until now(only 32 PDB entries that encompass 18 structures of 14 distinctMTs, Table 2).

Due to NMR limitations, connectivity patterns have to bedetermined by non-standard experiments, which require thereplacement of the native metal ion bound to MT by an NMR activenucleus – e.g. Cu(I) is regularly replaced by 109Ag(I), and Zn(II) by111Cd(II) or 113Cd(II). However, these metal-substitutions can leadto non-isostructural species because the coordination chemistryand ionic size of these NMR-active nuclei do not necessarily matchthose of the original metal ions [52,188].

Table 2 shows the 18 three-dimensional structures currentlyavailable for 14 MTs, with the exception of mussel Cd7-MT10 [189]and that very recently proposed for the � domain (Zn2Cys6) ofwheat Zn6-Ec-1 [42]. A quick glance at the Table already revealsthat most of them correspond to divalent metal ion-loaded forms(mainly Cd(II)), and that only in the case of yeast and fungi, Cu(I)-and/or Ag(I)-loaded forms have been solved. Among those withZn(II) and/or Cd(II), only five MTs do not belong to the vertebratefamily (crab, lobster, sea urchin, bacteria and wheat – only onedomain –) highlighting again the predominance of vertebrate MT
(rat, rabbit, human, mouse and fish) structures.
Since the first bidominial MT structure was solved in 1986[181] until the publication of the monodominial Cu7- and Ag7-Cup1 structures in 1996 [208], all the information available only

M.

Capdevila et

al. /

Coordination Chem

istry R

eviews

256 (2012) 46– 6257

Table 2Structurally characterized MTs and corresponding amino acid sequences.

Method Metallothionein PDB code Publication years Sequence

X-ray diffraction Rat 4MT2 1983–1991 XMDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCACd5Zn2-MT2 [181,186,190]S. cerevisiae 1RJU 2005 HECQCQCGSCKNNEQCQKSCSCPTGCNSDDKCPCGNCu8-Cup1 [191]

Solution NMR Rat 1MRT (�) 1987–1988 MDPNCSCATDGSCSCAGSCKCKQCKCTSCK(�) KSCCSCCPVGCAKCSQGCICKEASDKCSCCA(�)Cd7-MT2 [182–184] 2MRT (�)Rabbit 1MRB (�) 1985–1988 MDPNCSCAAAGDSCTCANSCTCKACKCTSCK (�) KSCCSCCPPGCAKCAQGCICKGASDKCSCCA (�)Cd7-MT2A [192–197] 2MRB (�)Human 1MHU (�) 1990–1992 MDPNCSCAAGDSCTCAGSCKCKECKCTSCK (�) KSCCSCCPVGCAKCAQGCICKGASDKCSCCA (�)Cd7-MT2 [198,199] 2MHU(�)Human 2F5H, 2FJ4, 2FJ5 (�) 2006 KSCCSCCPAECEKCAKDCVCKGGEAAEAEAEKCSCCQCd7-MT3 [200]Mouse 1DFS (�) 1DFT(�) 1999 MDPNCSCSTGGSCTCTSSCACKNCKCTSCK (�) KSCCSCCPVGCSKCAQGCVCKGAADKCTCCA (�)Cd7-MT1 [201]Mouse 1JI9 (�) 2001 KSCCSCCPAGCEKCAKDCVCKGEEGAKAEAEKCSCCQCd7-MT3 [202]N. coriiceps 1M0J (�) 2003 DPCECSKSGTCNCGGSCTCTNCSCKSCK (�) SCCPCCPSGCTKCASGCVCKGKTCDTSCCQ (�)Cd7-MT [203] 1M0G (�)S. purpuratus 1QJK (�) 1999 PDVKCVCCTEGKECACFGQDCCVTGECCKDGTCCGI (�) ICTNAACKCANGCKCGSGCSCTEGNCAC (�)Cd7-MTA [204] 1QJL (�)C. sapidus 1DME, 1DMF (�N) 1993–1995 MPGPCCNDKCVCQEGGCKAGCQCTSCRCS (�N) SPCQKCTSGCKCATKEECSKTCTKPCSCCPK (�C)Cd6-MT1 [205,206] 1DMC, 1DMD (�C)H. americanus 1J5M (�N) 2002 PGPCCKDKCECAEGGCKTGCKCTSCRCA (�N) PCEKCTSGCKCPSKDECAKTCSKPCKCCP (�C)Cd6-MT1 [207] 1J5L (�C)S. cerevisiae 1AQR, 1AQS, 1FMY 1996, 2000 QNEGHECQCQCGSCKNNEQCQKSCSCPTGCNSDDKCPCGNKSEETKKSCCSGKCu7-Cup1 [208,209]S. cerevisiae 1AOO, 1AQQ 1996 QNEGHECQCQCGSCKNNEQCQKSCSCPTGCNSDDKCPCGNKSEETKKSCCSGKAg7-Cup1 [208]N. crassa 1T2Y 2004 GDCGCSGASSCNCGSGCSCSNCGSKCu6-NcMT [210]S. elongatus PCC 7942 1JJD 2001 MTSTTLVKCACEPCLCNVDPSKAIDRNGLYYCSEACADGHTGGSKGCGHTGCNCHGZn4-SmtA [211]T. aestivum 2KAK 2009 GEHTTCGCGEHCGCNPCACGREGTPSGRANRRANCSCGAACNCASCGSATAPGZn4-�E-EC-1 [212]

58 M. Capdevila et al. / Coordination Chemistry Reviews 256 (2012) 46– 62

F Cd3(Sa ticks a

csvr2a

vzamr

�tccmtadueilao�aebmatCra

tfdicfnog

that this is a Zn4Cys9His2 cluster encompassing 2 ZnCys4 and 2ZnCys3His sites (Fig. 4). Remarkably, SmtA is currently the MT with

ig. 3. 3D structure of the human Cd7-MT2 showing (A) the �domain enclosing thend 1mhu, respectively). The Cd(II) ions are shown as spheres, the Cys residues as s

oncerned mammalian �МT and �МT domains or closely relatedtructures such as that of C. sapidus [205]. Bacterial SmtA pro-ided in 2001 new structural features when it was shown that Hisesidues participated in metal-binding [211] and it was not until009 that new topologies for the metal-MT clusters were reported,s in the case of wheat MT [42,212].

The analysis of the topologies of the metal-thiolate clusters ofertebrate, echinoderm and crustacean MTs containing divalentinc or cadmium reveals only 2 types of clusters (Fig. 3), M3(SCys)9nd M4(SCys)11, located in globular domains that give rise to bido-inial MTs. Both types of metal clusters are structurally related, as

ecently reported by Blindauer et al. [1].On the one hand, the M3(SCys)9 cluster, placed in the so-called

domains (Fig. 3A), has 3 divalent metal ions and 3 bridging cys-eine thiolates forming a six-membered ring in a distorted chaironformation, while the other six Cys act as terminal ligands thusompleting the tetrahedral coordination environment about eachetal ion. These M3(SCys)9 clusters are the unique components of

he crustacean MTs (C. sapidus [206] and H. americanus [207], whichre composed of two � domains)) and are found in one of the twoomains of mammalian [182,186,192,198,201], fish [203], and searchin [204] MTs. As regards mammalian MT3, the exchange broad-ning of the NMR signals of its � domain has, until now, hinderedts structural determination, although homology modeling calcu-ations [202] suggest an identical fold to that observed for �MT1nd �MT2, this latest domain being the most rigid fragment [201]f the three. The bidominial MTs with a fragment denominated

domain, also contain a M4(SCys)11 cluster. It can be describeds being formed by 2 fused six-membered rings, which can adoptither a distorted boat or a chair conformation, sharing 2MII-SCysonds (Fig. 3B). Thus, this cluster contains 5 bridging and 6 ter-inal SCys-donor atoms that satisfy the tetrahedral coordination

bout the four divalent metal ions. A variation on the topology ofhis cluster has been proposed for the mollusc M. galloprovincialisd7-MT10 [189], which encompasses 12 Cys residues instead of theepresentative 11 Cys of all other � domains, to give a response to

new Cd4(SCys)12 stoichiometry.Although mammalian MTs are the best known group of struc-

urally resolved MTs, the flexible hinge region of MT1 and MT2ormed by the conserved Lys–Lys segment (K30-K31) in the mid-le of the sequence leads to a discontinuity in the NMR structure,

mpairing the determination of the mutual orientation of bothonstitutive domains. A similar situation has been encountered
or invertebrate MTs. Consequently, NMR structure calculationseeded to be performed separately for both domains and thenly whole structure available is that provided by crystallo-raphic determination [186]. Interestingly, identical metal-thiolate
Cys)9 cluster and (B) the �domain with the Cd4(SCys)11 cluster (PDB entries 2mhund the S–Cd bonds as solid lines.

clusters, and closely comparable polypeptide folds, have beenreported [185] for rat MII-MT2, with independence of their deter-mination by X-ray diffraction on Zn2Cd5-MT2 [186] or by NMRspectroscopy on Cd7-MT2 [182].

In spite of the differences in the primary structure amongall these bidominial MTs, only minor effects on the global fold-ing and the cluster connectivities to the protein backbone havebeen observed. Finally, it is important to note that the similaritiesbetween both types of metal-thiolate aggregates of MTs and somemodel synthetic inorganic complexes have already been exten-sively discussed in a previous review [18].

Two further cases are also considered important in the descrip-tion of the structures of MTs containing divalent metal ions:bacterial SmtA and wheat Ec-1.

Zn4-SmtA structure was solved in 2001 [211] and clearly illus-trated the previously reported [213] His participation in metalcoordination. Its metal cluster strongly resembles that of theZn4Cys11 aggregates described above with the difference being

Fig. 4. 3D structure of S. elongatus Zn4–SmtA, showing the Zn4(SCys)9(NHis)2 cluster(PDB entry 1jjd). The Zn(II) ions are shown as spheres, the Cys and His residues assticks and the S–Zn and N–Zn bonds as solid lines.

M. Capdevila et al. / Coordination Chem

FZH

t5

fiafisCrttZt

chfifiitbcwCadsCac

FeS

ig. 5. 3D structure of T. aestivum Zn4-�E-Ec-1, showing the Zn(SCys)2(NHis)2 andn3(SCys)9 sites (PDB entry 2kak). The Zn(II) ions are shown as spheres, the Cys andis residues as sticks and the S–Zn and N–Zn bonds as solid lines.

he highest content of secondary structure, where 20 out of the total5 amino acids are part of a “treble-clef” zinc finger fold [214].

Wheat Ec-1, which belongs to type 4 plant MTs, constitutes therst example of this family of known three-dimensional structurend its determination has provided unprecedented results in theeld of MTs [212]. The Zn6-Ec-1 contains an isolated ZnCys2His2ite -close to those reported in some zinc finger domains- in itst �E domain (Fig. 5) that encompasses two Cys-rich regions sepa-ated by a spacer, together with a Zn3Cys9 cluster, which resembleshose described above. The remaining two Zn(II) are located inhe Nt domain, denominated � domain, which contains a dynamicn2Cys6 core compatible with two different cluster connectivitieshat have just been presented in the literature [42].

The unique full three-dimensional structure of a Cu-loaded MTurrently available corresponds to that of S. cerevisiae Cup1, whichas been the object of multiple efforts for a long time. In fact, therst proposal for this high-nuclearity metal-thiolate cluster thatnds no parallel examples in the inorganic world [18] was attained

n 1996 by 109Ag(I) substitution on the Cu-Cup1 form. This revealedhat 10 out of the 12 Cys – 2 Cys did not participate in metalinding-bound 7M(I) ions (M = Cu or Ag) in a single M7(SCys)10luster, where digonal and trigonal coordination of the metal ionsas proposed [208]. In 2000, a NMR reinvestigation of the Cu7-up1 structure was carried out [209] and the results suggested

variety of possible cluster structures fully compatible with theetermined constraints. X-ray diffraction data obtained in 2005,
howed a Cu8(SCys)10 cluster (Fig. 6) with a different structure andys topology than the previous proposals [191], in which 6 Cu(I)re trigonally-planar coordinated while the other 2 are linearlyoordinated to the cysteinic thiolates.
ig. 6. 3D structure of S. cerevisiae Cu8-Cup1, showing the Cu8(SCys)10 cluster (PDBntry 1rju). The Cu(I) ions are shown as spheres, the Cys residues as sticks and the–Cu bonds as solid lines.

istry Reviews 256 (2012) 46– 62 59

The other Cu-binding MT on which the structure has beeninvestigated is that of Neurospora crassa (NcMT). Unfortunately,NMR measurements have only been able to determine thethree-dimensional structure of the polypeptide backbone of theCu6-NcMT cluster, which is consistent with the presence of asingle protein domain representing a new monodominial MTfold [210].

6. MTs in biomedicine and biotechnology

Our time is dominated by criteria of science applicability, thusmany researchers have devoted their efforts to design applicationschemes for MTs. The results are a broad spectrum of possi-bilities, in view of the huge amount of biological processes inwhich MTs have been implicated (biomedical applications) andthe in vitro reactions that their peculiar composition and reactiv-ity may facilitate (biotechnological applications). There is no doubtthat the perspective of gathering further knowledge on MTs in theincoming years will still exponentially increase MT applicabilitypossibilities.

The first biomedical aspects of MTs to be investigated weremostly related to carcinogenesis and the use of metal-basedchemotherapeutic drugs, such as cisplatin. Administration ofcisplatin causes immediate MT synthesis induction, which altersits efficiency as antineoplastic drug [215], but is also beneficialin protecting organs not affected by tumors from the cytotoxiceffects of this drug [216]. MT has also been related to other non-desirable side-effects of cisplatin treatment, such as nephrotoxicity,for which the administration of genetically engineered MT vari-ants has been proposed [111]. Nowadays, it has been shown thataltered MT isogene expression is detectable in nearly all types ofhuman tumors, so this may be used as a potential tumor prog-nosis and progression biomarker [217]. There is a huge amountof data on MT functionality in mammalian central nervous sys-tem (CNS), and the possible implication of both MT1/MT2 andMT3 systems in neurodegenerative diseases (Alzheimer’s disease(AD) and amyotrophic lateral sclerosis (ALS) or multiple sclerosis(MS) among the best characterized) and multiple stress-inducedinflammatory processes (cf. [58] and [218] for excellent revisions).Although the precise basis of MT involvement in these disordersremains to be established, it has been shown that intraperitonealadministration of Zn–MT complexes was a very effective treat-ment to delay the onset and progression of the symptoms of amurine model of ALS, the so-called experimental autoimmuneencephalomyelitis (EAE) [219–221], all data suggesting a putativetherapeutical use of MTs for the treatment of neurodegenera-tive diseases. Mammalian MTs are also neuroprotective agents forcerebral ischemia [222,223], seizure accidents [224], and injuryand burn scar formation enhancers [225]. Therefore, it is not toofar-fetched to imagine future pharmaceutical applications of MTfor all these types of disorders, at least at a symptomatologicallevel.

Biotechnological applications of MTs traditionally refer to heavymetal environmental pollution, caused by human-driven activ-ities, both in bioremediation – the use of living organisms orbiomolecules to alleviate contamination events in natural ecosys-tems – and in the development of suitable biosensor systems to beable to monitor these events and to readily evaluate metal accu-mulation consequences in the affected organisms [226]. The mostsuccessful attempts in constructing recombinant bacterial strainscapable of heavy metal removal from the surrounding environ-
ment were based on MT targeting to bacterial periplasmic space[227], specially to the periplasmic space or to outer membranecompartments of E. coli [32,228–233]. An E. coli strain express-ing an MT-outer membrane maltose fusion protein (LamB) showed

6 Chem

atoEtOiattmshmcgersrNaao

7

rDtmpititWAotat

A

fpCR“1im

R


n almost 20-fold rise in Cd(II) binding, as compared to its wild-ype counterpart [229]. A similar Cd(II)-binding performance wasbtained through the expression of mouse MT on the surface of. coli, Ralstonia metallidurans and Pseudomonas putida as a fusiono the L domain of the IgA protease autotransporter [232,233].ther fusions of MT to bacterial membrane proteins have resulted

n only a minor enhancement of bioaccumulation potential, prob-bly due to protein instability or the inappropriate location ofhe fusion protein [228,230]. For the evaluation of metal con-amination events, it is generally accepted that the amount of

etal-loaded MT in an organism directly correlates with its expo-ure to heavy metals, and thus to the level of contamination of itsabitat. Therefore, the development of accurate MT quantificationethods, in order that MT concentration in indicator organisms

an be adopted as a reliable biomarker parameter, is still a mainoal in analytical biochemistry research, considering the differ-nt difficulties encountered (cf. information in Section 4.1 of thiseview and [126].) MTs have also been largely used for biosen-or construction, both capacitance sensors [234–236] and moreecently, potentiometric electrodes by our own group [128,237].ew developments will include even more sensitive devices [238],nd of easy direct application to the samples to be analyzedfter miniaturization, recycling and conservation requirements areptimized.

. Future directions/goals

As stated at the beginning of this review, Pubmed [21] hasetrieved ca. 9300 references for MT at the middle of March 2011.espite all this work, critical questions remain unanswered for

he vast majority of organisms. For example: Why are there soany isoforms? Do they have different properties? With which

roteins and receptors does MT interact? Do these interactionsnvolve redox reactions and/or metal transfer? What is the func-ional significance of such interactions? Does MT have a functionn the metabolism of metal ions other than zinc? Are there struc-urally defined intermediates in the folding and oxidation of MTs?

hat is the significance of polymorphic forms of MT in diseases?nd beyond human MTs: What are the functions of MTs in otherrganisms? Does their structural variability suggest different func-ions? Hyphenated mass spectrometric techniques with element-nd molecule-specific detection will have a critical role in observa-ion answers to any or all of these questions.

cknowledgments

We wish to thank the Spanish Ministerio de Ciencia e Innovaciónor its continued financial support, and particularly for the currentrojects BIO2009-12513-C02-01 to S. Atrian, and BIO2009-12513-02-02 to M. Capdevila and the COST Action (EU) CM0603 on Freeadicals in Chemical Biology. All the authors are members of theGrup de Recerca de la Generalitat de Catalunya” ref. 2009SGR-457. We also thank Dr. Jean-Didier Maréchal for his assistance

n generating the figures of the three-dimensional structures ofetallothioneins.

eferences

[1] C.A. Blindauer, O.I. Leszczyszyn, Nat. Prod. Rep. 27 (2010) 720.[2] S.G. Bell, B.L. Vallee, ChemBioChem 10 (2009) 55.[3] Y. Lia, W. Maret, J. Anal. At. Spectrom. 23 (2008) 1055.[4] P. Faller, FEBS.J. 277 (2010) 2921 (and references therein).[5] E. Freisinger, Dalton Trans. 47 (2008) 6663.
[6] B. Dolderer, H.J. Hartmann, U. Weser, in: A. Sigel, H. Sigel, R.K.O. Sigel (Eds.),
Metal Ions in Life Sciences Metallothioneins and Related Chelators, vol. 5, RSCPublishing, Cambridge, 2009, p. 83.

[7] A. Sigel, H. Sigel, R.K.O. Sigel, Metal Ions in Life Sciences Metallothioneins andRelated Chelators, vol. 5, RSC Publishing, Cambridge, 2009.

istry Reviews 256 (2012) 46– 62

[8] Special issue of JBIC devoted to MTs. Reference to be included on the galleyproofs.

[9] R.D. Palmiter, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 8428.[10] M. Margoshes, B.L. Vallee, J. Am. Chem. Soc. 79 (1957) 4813.[11] J.H.R. Kägi, M. Nordberg, Experientia Supplementum Metallothionein I, vol.

34, Birkhäuser Verlag, Basel, 1979.[12] J.H.R. Kägi, Y. Kojima, Experientia Supplementum Metallothionein II, vol. 52,

Birkhäuser Verlag, Basel, 1987.[13] K.T. Suzuki, N. Imura, M. Kimura, Metallothionein III, Biological Roles and

Medical Implications , Birkhäuser Verlag, Basel, 1993.[14] C.D. Klaasen, Metallothionein IV , Birkhäuser Verlag, Basel, 1999.[15] M.J. Stillman, C.F. Shaw III, K.T. Suzuki, Metallothioneins. Synthesis, Struc-

ture and Properties of Metallothioneins Phytochelatins and Metal-ThiolateComplexes , VCH Publishers Inc, New York, 1992.

[16] J.F. Riordan, B.L. Vallee, Methods in Enzymology Metallobiochemistry Part BMetallothionein and Related Molecules, vol. 205, Academic Press, San Diego,1991.

[17] M. Vasák, J.H.R. Kägi, in: R.B. King (Ed.), Encyclopedia of Inorganic Chemistry,vol. 4, John Wiley and Sons, Chichester, 1994, p. 2229.

[18] P. González-Duarte, in: J.A. McCleverty, T.J. Meyer (Eds.), ComprehensiveCoordination Chemistry, vol. 8, Elsevier, Amsterdam, 2003, p. 213.

[19] https://scifinder.cas.org/, 2011 (accessed 15.03.11).[20] http://www.isiknowledge.com, 2011 (accessed 15.03.11).[21] Database of the National Center of Biotechnology Information of the

US National Library of Medicine, Bethesda, MD, devoted to medicine-related publications. http://www.ncbi.nlm.nih.gov/pubmed, 2011 (accessed15.03.11).

[22] http://www.uniprot.org/, 2011 (accessed 15.03.11).[23] M. Capdevila, S. Atrian, J. Biol. Inorg. Chem. (2011), doi:10.1007/s00775-011-

0798-3.[24] D.R. Winge, M. Brouwer, Environm. Health Perspect. 65 (1986) 211.[25] http://www.bioc.unizh.ch/mtpage/classif.html, 2011 (accessed 22.03.11).[26] K.B. Nielson, C.L. Atkin, D.R. Winge, J. Biol. Chem. 260 (1985) 5342.[27] Ò. Palacios, S. Atrian, M. Capdevila, JBIC (2011), doi:10.1007/s00775-011-

0827-2.[28] Y. Okada, K. Tanaka, J. Sawada, Y. Kikuchi, in: M.J. Stillman, C.F. Shaw III, K.T.

Suzuki (Eds.), Metallothioneins Synthesis, Structure and Properties of Met-allothioneins, Phytochelatins and Metal-Thiolate Complexes, VCH PublishersInc, New York, 1992, p. 195.

[29] Y. Muruoka, T. Nagaoka, Appl. Environ. Microb. 53 (1987) 204.[30] Y. Hou, R. Kim, S. Kim, Biochim. Biophys. Acta 951 (1988) 230.[31] F.M. Romeyer, F.A. Jacobs, L. Masson, Z. Hanna, R. Brousseau, J. Biotechnol. 8

(1988) 207.[32] F.A. Jacobs, F.M. Romeyer, M. Beauchemin, R. Brousseau, Gene 83 (1989) 95.[33] S. Yamazaki, M. Nakanishi, T. Hamamoto, H. Hirata, A. Ebihara, A. Tokue, Y.

Kagawa, Biochem. Intern. 28 (1992) 451.[34] P. Silar, M. Wegnez, FEBS Lett. 269 (1990) 273.[35] M.J. Cismowski, P.C. Huang, Biochemistry 30 (1991) 6626.[36] P. Kille, P. Stephens, A. Cryer, J. Kay, Biochim. Biophys. Acta 1048 (1990) 178.[37] P. Kille, W.E. Lees, B.M. Darke, R.D. Winge, C.T. Dameron, P. Stephens, J. Kay,

J. Biol. Chem. 267 (1992) 8042.[38] Y.J. Wang, E.A. Mackay, M. Kurasaki, J.H.R. Kägi, Eur. J. Biochem. 225 (1994)

449.[39] M. Capdevila, N. Cols, N. Romero-Isart, R. González-Duarte, S. Atrian, P.

González-Duarte, Cell. Mol. Life Sci. 53 (1997) 681.[40] N. Cols, N. Romero-Isart, M. Capdevila, B. Oliva, P. González-Duarte, R.

González-Duarte, S. Atrian, J. Inorg. Biochem. 68 (1997) 157.[41] R. Bofill, M. Capdevila, S. Atrian, Metallomics 1 (2009) 229.[42] J. Loebus, E.A. Peroza, N. Blüthgen, T. Fox, W. Meyer-Klauke, O. Zerbe, E.

Freisinger, J. Biol. Inorg. Chem. 16 (2011) 683.[43] M.J. Cismowski, S.S. Narula, I.M. Armitage, M.L. Chernaik, P.C. Huang, J. Biol.

Chem. 266 (1991) 24390.[44] C.W. Cody, P.C. Huang, Biochemistry 32 (1993) 5127.[45] C.W. Cody, P.C. Huang, Biochem. Biophys. Res. Commun. 202 (1994) 954.[46] N. Romero-Isart, N. Cols, M. Termansen, J.L. Gelpí, R. González-Duarte, S.

Atrian, M. Capdevila, P. González-Duarte, Eur. J. Biochem. 259 (1999) 519.[47] R. Bofill, R. Orihuela, M. Romagosa, J. Domènech, S. Atrian, M. Capdevila, FEBS

J. 276 (2009) 7040.[48] S. Zeitoun-Ghandour, J. Charnock, M.M.E. Hodson, O.I. Leszczyszyn, C.A. Blin-

dauer, S.R. Stürzenbaum, FEBS J. 277 (2010) 2531.[49] D.E.K. Sutherland, M.J. Willans, M.J. Stillman, Biochemistry 49 (2010) 3593.[50] G. Meloni, M. Vasák, Free Radical Biol. Med. 50 (2011) 1471.[51] M. Capdevila, J. Domènech, A. Pagani, L. Tío, L. Villarreal, S. Atrian, Angew.

Chem. Int. Ed. Engl. 44 (2005) 4618.[52] R. Orihuela, F. Monteiro, A. Pagani, M. Capdevila, S. Atrian, Chem. Eur. J. 16

(2010) 12363.[53] M. Valls, R. Bofill, R. González-Duarte, P. González-Duarte, M. Capdevila, S.

Atrian, J. Biol. Chem. 276 (2001) 32835.[54] J.D. Otvos, D.H. Petering, C.F. Shaw, Comments Inorg. Chem. 9 (1989) 1.[55] C.F. Shaw III, M.M. Savas, D.H. Petering, in: J.F. Riordan, B.L. Vallee (Eds.), Met-

allobiochemistry Part B, Metallothionein and Related Molecules, Methods in
Enzymology, vol. 205, Academic Press, San Diego, 1991, p. 401.
[56] D.H. Petering, S. Krezoski, P. Chen, A. Pattanaik, C.F. Shaw III, in: M.J. Still-man, C.F. Shaw III, K.T. Suzuki (Eds.), Metallothioneins. Synthesis, Structureand Properties of Metallothioneins, Phytochelatins and Metal-Thiolate Com-plexes, VCH Publishers Inc, New York, 1992, p. 164.

https://scifinder.cas.org/

http://www.isiknowledge.com/

http://www.ncbi.nlm.nih.gov/pubmed

http://www.uniprot.org/

http://www.bioc.unizh.ch/mtpage/classif.html

http://dx.doi.org/10.1007/s00775-011-0827-2

Chem
[57] W. Maret, G. Heffron, H.A.O. Hill, D. Djuricic, L.J. Jiang, B.L. Vallee, Biochemistry41 (2002) 1689.

[58] J. Hidalgo, R. Chung, M. Penkowa, M. Vasák, in: A. Sigel, H. Sigel, R.K.O. Sigel(Eds.), Metal Ions in Life Sciences Metallothioneins and Related Chelators, vol.5, RSC Publishing, Cambridge, 2009, p. 279.

[59] I. El Ghazi, B.L. Martin, I.A. Armitage, Int. J. Alzheimers Dis. (2011), doi:10.406/2011/208634.

[60] L. Tío, M.A. Pagani, S. Atrian, Proteomics 9 (2009) 2568.[61] E.A. Ostrakhovich, P.E. Olsson, S. Jiang, M.G. Cherian, FEBS Lett. 580 (2006)

1235.[62] Y. Yang, W. Maret, B.L. Vallee, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 5556.[63] A. Krezel, W. Maret, J. Biol. Inorg. Chem. 13 (2008) 401.[64] A. Pattanaik, C.F. Shaw III, D.H. Pettering, J. Garvey, A.J. Kraker, J. Inorg.

Biochem. 54 (1994) 91.[65] R. Orihuela, J. Domènech, R. Bofill, C. You, E.A. Mackay, J.H.R. Kägi, M. Capdev-

ila, S. Atrian, J. Biol. Inorg. Chem. 13 (2008) 801.[66] A. Krezel, W. Maret, J. Am. Chem. Soc. 129 (2007) 10911.[67] M. Vasák, in: J.F. Riordan, B.L. Vallee (Eds.), Metallobiochemistry Part B,

Metallothionein and Related Molecules, Methods in Enzymology, vol. 205,Academic Press, San Diego, 1991, p. 452.

[68] A.V. Karotki, M. Vasák, Biochemistry 47 (2008) 10961.[69] A.V. Karotki, M. Vasák, J. Biol. Inorg. Chem. 14 (2009) 1129.[70] W. Maret, BioMetals 22 (2009) 149.[71] G. Meloni, V. Sonois, T. Delaine, L. Guilloreau, A. Gillet, J. Teissie, P. Faller, M.

Vasák, Nat. Chem. Biol. 4 (2008) 366.[72] O. Zitka, S. Krizkova, D. Huska, V. Adam, J. Hubalek, T. Eckschlager, R. Kizek,

Electrophoresis 32 (2011) 1.[73] W. Maret, Exp. Gerontol. 43 (2008) 363.[74] Y.J. Kang, Exper. Biol. Med. 231 (2006) 1459.[75] G. Meloni, P. Faller, M. Vasák, J. Biol. Chem. 282 (2007) 16068.[76] C. Montoliu, P. Monfort, J. Carrasco, Ò. Palacios, M. Capdevila, J. Hidalgo, V.

Felipo, J. Neurochem. 75 (2000) 266.[77] A. Torreggiani, J. Domènech, R. Orihuela, C. Ferreri, S. Atrian, M. Capdevila, C.

Chatgilialoglu, Chem. Eur. J. 15 (2009) 6015.[78] Y. Sano, A. Onoda, R. Sakurai, H. Kitagishi, T. Hayashi, J. Inorg. Biochem. 105

(2011) 702.[79] P. Coyle, J.C. Philcox, L.C. Carey, A.M. Rofe, Cell. Mol. Life Sci. 59 (2002)

627.[80] C.D. Klaasen, J. Liu, S. Chouhuri, Annu. Rev. Pharmacol. Toxicol. 39 (1999)

267.[81] D.H. Hamer, Annu. Rev. Biochem. 55 (1986) 913.[82] W. Feng, J. Cai, W.M. Pierce, R.B. Franklin, W. Maret, F.W. Benz, Y.J. Kang,

Biochem. Biophys. Res. Commun. 332 (2005) 853.[83] F. Haq, M. Mahoney, J. Koropatnick, J. Mutat. Res. 533 (2003) 211.[84] K.T. Suzuki, S. Kanno, S. Misawa, Y. Sumi, Res. Commun. Chem. Pathol. Phar-

macol. 82 (1993) 225.[85] B.A. Mastersm, E.J. Kelly, C.J. Quaife, R.L. Brinster, R.D. Palmiter, Proc. Natl.

Acad. Sci. U. S. A. 91 (1994) 584.[86] F. Trinchella, M. Riggio, S. Filosa, M.G. Volpe, E. Parisi, R. Scudiero, Comp.

Biochem. Physiol., Part C: Toxicol. Pharmacol. 144 (2006) 272.[87] J.C. Amiard, C.S. Amiard-Triquet, B.J. Pellerin, P.S. Rainbow, Aquat. Toxicol. 76

(2006) 160.[88] A.O. Udom, F.O. Brady, Biochem. J. 187 (1980) 329.[89] C. Jacob, W. Maret, B.L. Vallee, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 3489.[90] R. Dallinger, M. Chabicovsky, E. Hödl, C. Prem, P. Hunziker, C. Manzl, Am. J.

Physiol. 189 (2005) R1185–R1195.[91] M. Chabicovsky, W. Klepal, R. Dallinger, Environ. Toxicol. Chem. 23 (2004)

648.[92] B.G. Lane, R. Kajioka, T.D. Kennedy, Biochem. Cell Biol. 65 (1987) 1001.[93] P.J. Thornalley, M. Vasák, Biochim. Biophys. Acta 827 (1985) 36.[94] M.V. Kumari, M. Hiramatsu, M. Ebadi, Free Radic. Res. 29 (1998) 93.[95] M. Yoshida, Y. Saegusa, A. Fukuda, Y. Afama, S. Owada, Toxicology 213 (2005)

74.[96] M. Sato, I. Bremner, Free Radic. Biol. Med. 14 (1993) 325.[97] L. Cai, M. Satoh, C. Tohyama, M.G. Cherian, Toxicology 132 (1999) 85.[98] A. Viarengo, B. Burlando, N. Ceratto, I. Panfoli, Cell Mol. Biol. 46 (2000) 407.[99] M. Vasák, J. Trace Elem. Med. Biol. 19 (2005) 13.

[100] D.H. Petering, J. Zhu, S. Krezoski, J. Meeusen, C. Kiekenbush, S. Krull, T. Specher,M. Dughish, Exp. Biol. Med. 231 (2006) 1528.

[101] Y. Chen, W. Maret, Eur. J. Biochem. 268 (2001) 3346.[102] W. Maret, C. Jacob, B.L. Vallee, E.H. Fisher, Proc. Natl. Acad. Sci. U. S. A. 96

(1999) 1936.[103] K. Balamurugan, W. Schaffner, in: A. Sigel, H. Sigel, R.K.O. Sigel (Eds.), Metal

Ions in Life Sciences Metallothioneins and Related Chelators, vol. 5, RSC Pub-lishing, Cambridge, 2009, p. 31.

[104] V.C. Culotta, W.R. Howard, X.F. Liu, J. Biol. Chem. 269 (1994) 25295.[105] W.J. Guo, M. Meetam, P. Goldsbrough, Plant Physiol. 146 (2008) 1697.[106] D. Egli, H. Yepiskoposyan, A. Selvaraj, K. Balamurugan, R. Rajaman, A. Simons,

G. Multhaup, S. Mettler, A. Vardanyan, O. Georgiev, W. Schaffner, Mol. Cell.Biol. 26 (2006) 2686.

[107] S. Hughes, S.R. Stürzenbaum, Environ. Pollut. 145 (2007) 395.
[108] J.H. Beattie, A.M. Wood, A.M. Newman, I. Bremner, K.H.A. Choo, A.E. Michalska,
J.S. Duncan, P. Trayhurn, Proc. Natl. Acad. Sci. U. S. A. 95 (1994) 358.[109] M. Knipp, G. Meloni, B. Roschitzki, M. Vasák, Biochemistry 44 (2005) 3159.[110] D.W. Lahti, J.D. Hoekman, A.M. Tokheim, B.L. Martin, I.M. Armitage, Protein

Sci. 14 (2005) 1151.

istry Reviews 256 (2012) 46– 62 61

[111] R.B. Klassen, K. Crenshaw, R. Kozyraki, P.J. Verroust, L. Tío, S. Atrian,P.L. Allen, T.G. Hammond, Am. J. Physiol. Renal. Physiol. 287 (2004)F393–F403.

[112] R.W. Olafson, W.D. McCubbin, C.M. Kay, Biochem. J. 251 (1988) 691.[113] B. Gold, H. Deng, R. Bryk, D. Vargas, D. Eliezer, J. Roberts, X. Jiang, C. Nathan,

Nat. Chem. Biol. 4 (2008) 609.[114] A. Pagani, L. Villarreal, M. Capdevila, S. Atrian, Mol. Microbiol. 63 (2007) 256.[115] L. Tío, L. Villarreal, S. Atrian, M. Capdevila, J. Biol. Chem. 279 (2004) 24403.[116] Ò. Palacios, A. Pagani, S. Pérez-Rafael, M. Egg, M. Höckner, A. Kloss-

Brandstätter, M. Capdevila, S. Atrian, R. Dallinger, BMC Biol. 9 (4) (2011),doi:10.1186/1741-7007-9-4.

[117] M. Brouwer, R. Syring, T.H. Brouwer, J. Inorg. Biochem. 88 (2002) 228.[118] S. Díaz, F. Amaro, D. Rico, V. Campos, L. Benítez, A. Martín-González, E.P.

Hamilton, E. Orias, J.C. Gutiérrez, PLoS One 3 (2007) e291.[119] K.T. Suzuki, Analusis 26 (1998) M57–M61.[120] R. Lobinski, H. Chassaigne, J. Szpunar, Talanta 46 (1998) 133.[121] R. Dallinger, M. Chabicovsky, B. Lagg, R. Schipflinger, H.G. Weirich, B. Berger,

Environ. Toxicol. Chem. 23 (2004) 902.[122] R.K. Mehra, I. Bremner, Biochem. J. 213 (1983) 459.[123] K. Nakajima, T. Kodaira, M. Kato, K. Nakazato, Y. Tokita, H. Kikuchi, H. Sekine,

K. Suzuki, T. Nagamine, Clin. Chim. Acta 411 (2010) 758.[124] V. Adam, I. Fabrik, V. Kohoutkova, P. Babula, J. Hubalek, R. Vrba, L. Trnkova, R.

Kizek, Int. J. Electrochem. Sci. 5 (2010) 429.[125] M. Dabrio, A.R. Rodríguez, Electroanalysis 12 (2000) 1026.[126] M. Dabrio, A.R. Rodríguez, G. Bordin, M.J. Bebianno, M. De Ley, I. Sestakova,

M. Vasák, M. Nordberg, J. Inorg. Biochem. 88 (2002) 123.[127] V. Adam, I. Fabrik, T. Eckschlager, M. Stiborova, L. Trnkova, R. Kizek, Trends

Anal. Chem. 29 (2010) 409.[128] M. Capdevila, A. González-Bellavista, M. Munoz, S. Atrian, E. Fàbregas, Chem.

Commun. 46 (2010) 2040.[129] J.L. Gómez-Ariza, T. Garcia-Barrera, F. Lorenzo, V. Bernal, M.J. Villegas, V.

Oliveira, Anal. Chim. Acta 524 (2004) 15.[130] A. Prange, D. Schaumloffel, Anal. Bioanal. Chem. 373 (2002) 441.[131] K. Polec, M. Pérez-Calvo, O. Garcia-Arribas, J. Szpunar, B. Ribas-Ozonas, R.

Lobinski, J. Inorg. Biochem. 88 (2002) 197.[132] B. Michalke, Electrophoresis 26 (2005) 1584.[133] S. Kubo, H. Fukuda, M. Ebara, N. Ikota, H. Saisho, H. Nakagawa, T. Ozawa, M.

Yukawa, K. Kato, T. Satoh, T. Watayo, H. Sakurai, Biol. Pharm. Bull. 28 (2005)1137.

[134] P. Bratter, R. Brunetto, H.J. Gramm, S. Recknagel, H. Siemes, J. Trace Elem.Electrolytes Health Dis. 6 (1992) 251.

[135] A. Prange, D. Schaumloffel, P. Bratter, A.N. Richarz, C. Wolf, Fresenius J. Anal.Chem. 371 (2007) 764.

[136] T.T. Ngu, M.J. Stillman, Dalton Trans. 28 (2009) 5425.[137] M.J. Sillman, Coord. Chem. Rev. 144 (1995) 461.[138] T.T. Ngu, M.J. Stillman, IUBMB Life 61 (2009) 438.[139] A.Y.C. Law, M.J. Stillman, Biochem. Biophys. Res. Commun. 102 (1981) 397.[140] W. Bernhard, M. Good, M. Vasák, J.H.R. Kägi, Inorg. Chim. Acta 79 (1983) 154.[141] M.J. Stillman, C.F. Shaw III, K.T. Suzuki (Eds.), Metallothioneins. Synthesis,

Structure and Properties of Metallothioneins, Phytochelatins and Metal-Thiolate Complexes, VCH Publishers Inc, New York, 1992, p. 60.

[142] B. Roschitzki, M. Vasák, J. Biol. Inorg. Chem. 7 (2002) 611.[143] A. Presta, A.R. Green, A. Zelazowski, M.J. Stillman, Eur. J. Biochem. 227 (1995)

226.[144] W. Lu, A.J. Zelazowski, M.J. Stillman, Inorg. Chem. 32 (1993) 919.[145] M.J. Stillman, W. Cai, A.J. Zelazowski, J. Biol. Chem. 262 (1987) 4538.[146] J. Ejnik, J. Robinson, J.Y. Zhu, H. Försterling, C.F. Shaw III, D.H. Petering, J. Inorg.

Biochem. 88 (2002) 144.[147] M. Beltramini, M. Lerch, FEBS Lett. 127 (1981) 201.[148] R. Bogumil, P. Faller, D.L. Pountney, M. Vasák, Eur. J. Biochem. 238 (1996) 698.[149] M. Brouwer, T.H. Brouwer, Arch. Biochem. Biophys. 351 (1998) 257.[150] A.R. Green, M.J. Stillman, Inorg. Chim. Acta 226 (1994) 275.[151] D.L. Poutney, M. Vasák, Eur. J. Biochem. 209 (1992) 335.[152] J.D. Otvos, H.R. Engeseth, D.G. Nettesheim, C.R. Hilt, in: J.H.R. Kägi, Y. Kojima

(Eds.), Metallothionein II, Birkhäuser, Basel, 1987, p. 171.[153] J. Pande, C. Pande, D. Gilg, M. Vasák, R. Callender, J.H.R. Kägi, Biochemistry 25

(1986) 5526.[154] T. Elgren, D.E. Wilcox, Biochem. Biophys. Res. Commun. 163 (1989) 1093.[155] A. Torreggiani, J. Domènech, S. Atrian, M. Capdevila, A. Tinti, Biopolymers 89

(2008) 1114.[156] À. Leiva-Presa, M. Capdevila, P. González-Duarte, Eur. J. Biochem. 271 (2004)

4872.[157] X.L. Yu, M. Wojciechowski, C. Fenselau, Anal. Chem. 65 (1993) 1355.[158] O.I. Leszczyszyn, C.A. Blindauer, Phys. Chem. Chem. Phys. 12 (2010) 13408.[159] H. Chassaigne, R. Lobinski, Analyst 123 (1998) 2125.[160] C.A. Blindauer, M. Tahir Razi, D.J. Campopiano, P.J. Sadler, J. Biol. Inorg. Chem.

12 (2007) 393.[161] P. Palumaa, E. Eriste, O. Njunkova, L. Pokras, H. Jörnvall, R. Sillard, Biochemistry

41 (2002) 6158.[162] P. Palumaa, E. Eriste, K. Kruusel, L. Kangur, H. Jörnvall, R. Sillard, Cell. Mol. Biol.

49 (2003) 763.
[163] D. Esteban-Fernández, B. Canas, I. Pizarro, M.A. Palacios, M.M. Gómez-Gómez,
J. Anal. At. Spectrom. 22 (2007) 1113.[164] S. Krizkova, V. Adam, R. Kizek, Electrophoresis 30 (2009) 4029.[165] J.C. Shen, Z.Y. Huang, Z.X. Zhuang, X.R. Wang, F.S.C. Lee, Appl. Organomet.

Chem. 19 (2005) 140.

6 Chem

[[

[

[[[

[[[

[

[

[

[

[[

[

[

[

[

[

[

[[

[

[[

[

[

[

[

[

[

[

[

[

[[


166] Ò. Palacios, À. Leiva-Presa, S. Atrian, R. Lobinski, Talanta 72 (2007) 480.167] T.T. Ngu, M.D. Dryden, M.J. Stillman, Biochem. Biophys. Res. Commun. 401

(2010) 69.168] T.T. Ngu, S. Krecisz, M.J. Stillman, Biochem. Biophys. Res. Commun. 396 (2010)

206.169] T.T. Ngu, J.A. Lee, T.B.J. Pinter, M.J. Stillman, J. Inorg. Biochem. 104 (2010) 232.170] S. Pérez-Rafael, S. Atrian, M. Capdevila, Ò. Palacios, Talanta 83 (2011) 1057.171] S. Mounicou, L. Ouerdane, B. L’Azou, I. Passagne, C. Ohayon-Courtes, J. Szpunar,

R. Lobinski, Anal. Chem. 82 (2010) 6947.172] J. Szpunar, Anal. Bioanal. Chem. 378 (2004) 54.173] Y. Ogra, K.T. Suzuki, Res. Commun. Mol. Path. Pharmacol 102 (1998) 149.174] R.Y. Wang, D.A. Sens, A. Albrecht, S. Garrett, S. Somji, M.A. Sens, X.N. Lu, Anal.

Chem. 79 (2007) 4433.175] K.M. Keating, L.R. Ghosaini, D.P. Giedroc, K.R. Williams, J.E. Coleman, J.M.

Sturtevant, Biochemistry 27 (1988) 5240.176] N.E. Grossoehme, A.M. Spuches, D.E. Wilcox, J. Biol. Inorg. Chem. 15 (2010)

1183.177] E. Chekmeneva, R. Prohens, J.M. Díaz-Cruz, C. Arino, M. Esteban, Environ. Sci.

Technol. 42 (2008) 2860.178] D.Y. Chu, Y. Tang, Y. Huan, W.G. He, W. Cao, Thermochim. Acta 352 (2000)

205.179] M. Vasák, Biodegradation 9 (1998) 501.180] J.J. Davis, H.A.O. Hill, A. Kurz, C. Jacob, W. Maret, B.L. Vallee, Phys. Chem.

Commun. 1 (1998) 12.181] W.F. Furey, A.H. Robbins, L.L. Clancy, D.R. Winge, B.C. Wang, C.D. Stout, Science

231 (1986) 704.182] E. Wörgötter, G. Wagner, M. Vasák, J.H.R. Kägi, K. Wüthrich, Eur. J. Biochem.

167 (1987) 457.183] M. Vasák, E. Wörgötter, G. Wagner, J.H.R. Kägi, K. Wüthrich, J. Mol. Biol. 196

(1987) 711.184] P. Schultze, E. Wörgötter, W. Braun, G. Wagner, M. Vásak, J.H.R. Kägi, K.

Wüthrich, J. Mol. Biol. 203 (1988) 251.185] W. Braun, M. Vasák, A.H. Robbins, C.D. Stout, G. Wagner, J.H.R. Kägi, K.

Wüthrich, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 10124.186] A.H. Robbins, D.E. McRee, M. Williamson, S.A. Collett, N.H. Xuong, W.F. Furey,

B.C. Wang, C.D. Stout, J. Mol. Biol. 221 (1991) 1269.187] K. Wüthrich, Nat. Struct. Biol. 8 (2001) 923.188] Ò. Palacios, K. Polec-Pawlak, R. Lobinski, M. Capdevila, P. González-Duarte, J.

Biol. Inorg. Chem. 8 (2003) 831.189] G. Digilio, C. Bracco, L. Vergani, M. Botta, D. Osella, A. Viarengo, J. Biol. Inorg.

Chem. 14 (2009) 167.190] K.A. Melis, D.C. Carter, C.D. Stout, D.R. Winge, J. Biol. Chem. 258 (1983) 6255.191] V. Calderone, B. Dolderer, H.J. Hartmann, H. Echner, C. Luchinat, C. Del Bianco,

S. Mangani, U. Weser, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 51.192] M.H. Frey, G. Wagner, M. Vasák, O.W. Sørensen, D. Neuhaus, E. Wörgötter,

J.H.R. Kägi, R.R. Ernst, K. Wüthrich, J. Am. Chem. Soc. 107 (1985) 6847.193] D. Neuhaus, G. Wagner, M. Vasák, J.H.R. Kägi, K. Wüthrich, Eur. J. Biochem.

151 (1985) 257.194] W. Braun, G. Wagner, E. Wörgötter, M. Vasák, J.H.R. Kägi, K. Wüthrich, J. Mol.

Biol. 187 (1986) 125.195] G. Wagner, D. Neuhaus, E. Wörgötter, M. Vasák, J.H.R. Kägi, K. Wüthrich, J.

Mol. Biol. 187 (1986) 131.196] G. Wagner, D. Neuhaus, E. Wörgötter, M. Vasák, J.H.R. Kägi, K. Wüthrich, Eur.

J. Biochem. 157 (1986) 275.197] A. Arseniev, P. Schultze, E. Wörgötter, W. Braun, G. Wagner, M. Vásak, J.H.R.

Kägi, K. Wüthrich, J. Mol. Biol. 201 (1988) 637.198] B.A. Messerle, A. Schäffer, M. Vasák, J.H.R. Kägi, K. Wüthrich, J. Mol. Biol. 214

(1990) 765.199] B.A. Messerle, A. Schäffer, M. Vásak, J.H.R. Kägi, K. Wüthrich, J. Mol. Biol. 225

(1992) 433.200] H. Wang, Q. Zhang, B. Cai, H.Y. Li, K.H. Sze, Z.X. Huang, H.M. Wu, H.Z. Sun, FEBS

Lett. 580 (2006) 795.201] K. Zangger, G. Öz, J.D. Otvos, I.M. Armitage, Protein Sci. 8 (1999) 2630.202] G. Öz, K. Zangger, I.M. Armitage, Biochemistry 40 (2001) 11433.

istry Reviews 256 (2012) 46– 62

[203] C. Capasso, V. Carginale, O. Crescenzi, D. Di Maro, E. Parisi, R. Spadaccini, P.A.Temussi, Structure 11 (2003) 435.

[204] R. Riek, B. Prêcheur, Y.Y. Wang, E.A. Mackay, G. Wider, P. Güntert, A.Z. Liu,J.H.R. Kägi, K. Wüthrich, J. Mol. Biol. 291 (1999) 417.

[205] S.S. Narula, M. Brouwer, X.Y. Hua, I.M. Armitage, Magn. Reson. Chem. 31(1993) 96.

[206] S.S. Narula, M. Brouwer, X.Y. Hua, I.M. Armitage, Biochemistry 34 (1995) 620.[207] A. Munoz, F.H. Försterling, C.F. Shaw, D.H. Petering, J. Biol. Inorg. Chem. 7

(2002) 713.[208] C.W. Peterson, S.S. Narula, I.M. Armitage, FEBS Lett. 379 (1996) 85.[209] I. Bertini, H.J. Hartmann, T. Klein, G.H. Liu, C. Luchinat, U. Weser, Eur. J.

Biochem. 267 (2000) 1008.[210] P.A. Cobine, R.T. McKay, K. Zangger, C.T. Dameron, I.M. Armitage, Eur. J.

Biochem. 271 (2004) 4213.[211] C.A. Blindauer, M.D. Harrison, J.A. Parkinson, A.K. Robinson, J.S. Cavet, N.J.

Robinson, P.J. Sadler, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 9593.[212] E.A. Peroza, R. Schmucki, P. Güntert, E. Freisinger, O. Zerbe, J. Mol. Biol. 387

(2009) 207.[213] M.J. Daniels, J.S. Turner-Cavet, R. Selkirk, H.Z. Sun, J.A. Parkinson, P.J. Sadler,

N.J. Robinson, J. Biol. Chem. 273 (1998) 22957.[214] C.A. Blindauer, P.J. Sadler, Acc. Chem. Res. 38 (2005) 62.[215] M. Satoh, M.G. Cherian, N. Imura, H. Shimizu, Cancer Res. 54 (1994) 5255.[216] Y. Kondo, E.S. Woo, A.E. Michalska, K.H. Choo, J.S. Lazo, Cancer Res. 55 (1995)

2021.[217] M.G. Cherian, A. Jayasurya, B.H. Bay, Mutat. Res. 533 (2003) 201.[218] M. Vasák, G. Meloni, in: A. Sigel, H. Sigel, R.K.O. Sigel (Eds.), Metal Ions in

Life Sciences Metallothioneins and Related Chelators, vol. 5, RSC Publishing,Cambridge, 2009, p. 319.

[219] M. Penkowa, J. Hidalgo, Glia 32 (2000) 247.[220] M. Penkowa, J. Hidalgo, Exp. Neurol. 170 (2001) 1.[221] M. Penkowa, L. Tío, M. Giralt, A. Quintana, A. Molinero, S. Atrian, M. Vasák, J.

Hidalgo, J. Neurosc. Res. 23 (2006) 974.[222] D.M. Durnam, R.D. Palmiter, Exp. Suppl. 52 (1987) 457.[223] M. Penkowa, M. Giralt, J. Camats, J. Hidalgo, J. Comp. Neurol. 444 (2002) 174.[224] M. Aschner, M.G. Cherian, C.D. Klaassen, R.D. Palmiter, J.C. Erickson, A.I. Bush,

Toxicol. Appl. Pharmacol. 142 (1997) 229.[225] N.M. Morellini, N.L. Giles, S. Rea, K.F. Adcroft, S. Falder, C.E. King, S.A. Dunlop,

L.D. Beazley, A.K. West, F.M. Wood, M.W. Fear, Wound Repair Regen. 16 (2008)682.

[226] J.C. Gutiérrez, F. Amaro, A. Martín-González, BioEssays 31 (2009) 805 (andreferences therein).

[227] M. Pazirandeh, L.A. Chrisey, J.M. Mauro, J.R. Campbell, B.P. Gaber, Appl. Micro-biol. Biotechnol. 43 (1995) 1112.

[228] F.M. Romeyer, F.A. Jacobs, R. Brousseau, Appl. Environ. Microbiol. 56 (1990)2748.

[229] C. Sousa, P. Kotrba, T. Ruml, A. Cebolla, V. de Lorenzo, J. Bacteriol. 180 (1998)2280.

[230] M. Valls, R. González-Duarte, S. Atrian, V. de Lorenzo, Biochimie 80 (1998) 1.[231] P. Kotrba, P. Pospisil, V. de Lorenzo, T. Ruml, J. Recept. Signal Trans. 19 (1999)

703.[232] M. Valls, S. Atrian, V. de Lorenzo, L.A. Fernández, Nat. Biotechnol. 18 (2000)

661.[233] M. Valls, V. de Lorenzo, R. González-Duarte, S. Atrian, J. Inorg. Biochem. 79

(2000) 219.[234] I. Bontidean, J. Ahlqvist, A. Mulchandani, W. Chen, W. Bae, R.K. Mehra, A.

Mortari, E. Csöregi, Biosens. Bioelectron. 18 (2003) 547.[235] I. Bontidean, C. Berggren, G. Johansson, E. Csoregi, B. Mattiasson, J.R. Lloyd,

K.J. Kenneth, J. Jakeman, N.L. Brown, Anal. Chem. 70 (1998) 4162.[236] P. Corbisier, C. Van der Lelie, B. Borremans, A. Provoost, V. de Lorenzo, N.L.

Brown, J.R. Lloyd, J.L. Hobman, E. Csoregi, G. Johansson, B. Mattiasson, Anal.Chim. Acta 387 (1999) 235.

[237] A. González-Bellavista, S. Atrian, M. Munoz, M. Capdevila, E. Fàbregas, Talanta77 (2009) 1528.

[238] V. Adam, J. Baloun, I. Fabrik, L. Trnkova, R. Kizek, Sensors 8 (2008) 2293.

Documents

State-of-the-art of metallothioneins at the beginning of the 21st century