Ceruloplasmin

(DeLano 2002)

Submitted to Dr Margaret Brosnan

Submitted by Ryan Hughes

Student 200802692

Due Date November 14 2014

Introduction

Human Ceruloplasmin (hCP) is a member of the multi-copper oxidase

(MCO) ldquobluerdquo family of enzymes (1) Other members of this family are

ascorbate oxidase laccase Fet3 and the recently discovered zyklopen

Only CP Fet3 and zyklopen are able to oxidize inorganic substrates such

as Fe(II) (1) MCOrsquos use copper to couple substrate oxidation with the four-

electron reduction of dioxygen to water (1) hCP is a serum ferroxidase

and as such plays an important role in iron homeostasis (1) hCP contains

greater than 95 of the copper found in plasma It consists of a single

1046 amino-acid (aa) residue polypeptide with 6 distinct cupredoxin type

domains (CDTDrsquos) It is a 132 kDa glycoprotein consisting of 7-8 carbo-

hydrate by mass hCp needs copper to function However it has no role in

the transport or metabolism of the metal hCP is essential for regulating ef-

flux of copper out of many parenchyma cell types

Genetics

hCP is encoded by 20 exons spanning ~ 65 kB of DNA at chromosomal

position 3q23-q24 An hCP pseudogene encodes the carboxyl-terminal 563

aa residues of the protein Although this pseudogene is not expressed It

must be considered when designing any molecular diagnostic test for

aceruloplasminemia (2) Alternative polyadenylation leads to mRNA of 37

and 42 kDa in hepatic tissue hCP is expressed in smaller amounts in the

spleen lungs testes and brain (3) A glycophosphatidylinositol (GPI)ndashan-

chored protein is also produced via alternative splicing of exons 19 and 20

in astrocytes and Sertoli cells (4)

Interaction of Ceruloplasmin With Copper (Structure)

The 6 CDTDrsquos are arranged in a triangular array There are 6 integral cop-

per ions 3 form a trinuclear cluster at the interface of domains 1 amp 6 3 are

mononuclear ions (coordinated to a cystine and two histidine residues) in

domains 2 4 and 6 At these 3 (type 1) copper sites charge transfer be-

tween the cysteine ligand sulfur and the copper result in a strong absorp-

tion at 600 nm This confers an intense blue colour to the protein One type

II copper is coordinated to four imidazole nitrogens in close proximity to

two antiferromagnetically coupled type III coppers that absorb at 330 nm

The type II and III coppers constitute the trinuclear copper cluster where a

dioxygen species may bind (6) All known isoforms of hCP incorporate 6

copper ions into the structure Copper does not affect the rate of synthesis

or secretion of hCP However failure to incorporate copper ions leads to

very high turnover of the apoenzyme (6) Thus high copper levels lead to a

steady state of hCP and low copper levels lead to a decreased in hCP It

has been shown that all six copper atoms must be present for the enzyme

to fold and therefore function properly (6)

Function of Ceruloplasmin

Ceruloplasmin is a ferroxidase that converts the toxic Fe(II) to Fe(III) Free

Fe(II) can react with hydrogen peroxide and molecular oxygen to produce

free radicals Free radicals may induce lipid peroxidation DNA strand

breaks destruction of biomolecules and lead to cell death (7) Fe(III) is in-

corporated into transferrin which is the ldquostorage poolrdquo for iron until it is in-

corporated into oxygen transport molecules such as hemoglobin

Definitive evidence of the physiologic role of hCP in iron homeostasis

came with the discovery of patients with aceruloplasminemia (discussed

later)

Mechanism by Which Ceruloplasmin Functions

Multicopper oxidases such as hCP use the electronic chemistry of bound

copper ions to couple substrate oxidation with four-electron reduction of

dioxygen Electrons pass from the substrate (ferrus iron) to the type I cop-

per then to the trinu-

clear copper cluster

and then to the oxygen

molecule bound at this

site (8)

Ceruloplasmin Deficiency (Wilson Disease)

Wilson disease is an autosomal recessive disorder with a global incidence

considered to be 1 in 30000 (9) Marked decrease in the concentration of

this hCP in serum samples from patients with Wilson disease provides the

basis of a biochemical test for this disorder that is still in clinical use today

Wilson disease does not result from lack of copper Conversely Wilson dis-

ease is an autosomal recessive disorder resulting in hepatic copper accu-

mulation (9) The primary genetic defect lies in a large 21-exon gene on

chromosome 13 The ATP7B gene encodes a 1456-amino-acid protein with

6 copper-binding domains ATP7B protein is a copper transporting ATPase

which brings copper from the trans-golgi to the biliary canaliculus of hepa-

tocytes From there it may be incorporated into hCP to form the holoen-

zyme The excess copper is excreted into to the bile for elimination (9)

Thus mutations in the Wilson protein decrease the amount of stable

haloenzyme increase the amount of unstable apoenzyme and reduce the

rate at which copper may be excreted into the bile All together this causes

reduced levels of hCP and buildup of cytoplasmic copper This leads to the

symptoms of Wilson disease which are Kayser-Fleischer (KF) rings hepati-

tis Cirrhosis and neurological problems (9) Chelation therapy with D-

penicillamine may help but has serious side affects

Ceruloplasmin Deficiency (Menkes disease)

Most dietary copper is absorbed from the proximal small intestine and is di-

rected into the portal circulation with the help of another copper-transport-

ing protein produced by the ATP7A gene Mutations in the ATP7A gene

lead to a copper-deficiency disease called Menkes disease Both of the

ATP7 gene products are required for ceruloplasmin copper loading There-

fore the symptoms and treatment of Menkes are the same as for Wilsonrsquos

(9)

Ceruloplasmin Deficiency (Aceruloplasminemia)

There has been hCP deficiency attributed to autosomal recessive muta-

tions in the hCP gene (10) Heterozygous individuals usually show normal

iron metabolism However in some cases dominant mutations in the hCP

gene lead to iron overload in various parenchyma These mutations were

found to disrupt copper loading of hCP by ATP7B The lack of ferroxidase

activity of hCP has been shown to lead to internalization and degradation

of ferroportin (11)

Most of the iron used each day for hematopoiesis and other essential

needs is recycled from erythrocytes (heme) in the reticuloendothelial sys-

tem Iron transported in the plasma bound to transferrin must be oxidized

prior to binding to this transport protein Ceruloplasmin plays a critical role

in the iron cycle by establishing a rate of iron oxidation sufficient for iron re-

lease from the reticuloendothelial (RI) system The absence of serum ceru-

loplasmin in patients with aceruloplasminemia leads to an accumulation of

iron in the RI system In addition to the accumulation of iron within the RI

system the absence of serum ceruloplasmin also results in increased fer-

rous iron in the plasma which is rapidly removed from the circulation by the

liver pancreas and other tissues When copper cannot be incorporated into

hCP the apoenzyme is quickly degraded Thus a low serum ceruloplasmin

level along with iron accumulation in the basal gan- glia (detected as a

low-intensity signal on T1- and T2-weighted magnetic resonance imaging)

is sufficient for diagnoses

Ceruloplasmin is an endogenous inhibitor of myeloperoxidase (12)

Myeloperoxidase generates hypochlorous acid and reactive radicle

species leading to oxidative stress when it leaks into the plasma hCP defi-

ciency may lead to increased tissue damage in times of increased oxidative

stress Inflammation from cystic fibrosis chronic obstructive pulmonary dis-

ease rheumatoid arthritis and Alzheimer disease may be worsened if hCP

is not in the plasma to act as an antioxidant (12)

Primary Hemochromotosis (Atransferrinemia)

The mechanism of iron overload in aceruloplasminemia is similar to that

which occurs in patients with primary hemochromotosis (PH)(atransferrine-

mia) PH is caused by mutations in the serum transferrin gene leading to

lack of functional transferrin

Copper Deficiency

The daily recommended copper intake is 09 mg (13) Most people get

more than enough from their food and water intake Pregnant and lactating

women persons with inflamed and diseased bowel and persons who have

had bariatric surgery are at greater risk for copper deficiency due to their

decreased copper absorption potential High zinc content of certain dental

adhesives is leading to an increase in zinc-induced copper deficiency

There are about a dozen mammalian copper dependent enzymes Table 1

gives the names and functions of the post prevalent ones

Prohaska JR (2014) Impact of copper deficiency in humans AnnNYA-cadSci 1314 1-5

Copper deficiency constantly leads to anemia (lower-than-normal hemoglo-

bin concentration) (13) This may be partly explained based on decreased

activity of copper ferroxidases hephaestin and ceruloplasmin

Altered function of ATP7A when copper is limiting may contribute to the

symptoms However anemia may result from a variety of compromised

copper-dependent processes including impaired erythropoiesis (13)

Iron Deficiency (Activation of CP by HIF-1)

Iron deficiency leading to hypoxia potentiates production of the regulatory

protein hypoxia-inducible factor (HIF-1) HIF-1 is a heterodimeric transcrip-

tion factor complex HIF-11113114a is the key regulatory component of the com-

plex because it is absent under normal conditions but is upregulated by the

iron chelator desferrioxamine (14) Upon activation the HIF-1a1113114HIF-11113114b

dimer binds to hypoxia-responsive elements (HREs) in multiple genes in-

cluding several with important functions in iron metabolism such as ery-

thropoietin heme oxygenase-1 transferrin and transferrin receptor HIF-1

has been shown to activate CP transcription via one putative hypoxia-re-

sponsive element (HRE) in the 5rsquo regulatory region of the CP gene (14)

This was shown by cloning the 5rsquo flanking region of the CP gene from a hu-

man cDNA library This promoterenhancer region driving a luciferase re-

porter was transfected into HepG2 cells Hypoxia increased luciferase ac-

tivity 5-10 fold (14)

Reactive Oxygen Species (ROS) Regulate CP

ROS decrease the synthesis of both the secretory form of Cp (in hepatic

cells) and membrane-bound GPI-Cp (in astroglial cells) by a novel post-

transcriptional mechanism involving its 31113114-UTR (15) The Cp 3rsquo1113114-UTR-

binding protein complex is decreased by ROS causing increased decay of

Cp mRNA Therefore Cp expression is down-regulated by ROS This may

explain the iron deposition and related damage by increased ROS gener-

ation in neurodegenerative diseases (15)

Conclusion

Investigations into iron-copper interactions have revealed novel aspects of

mineral homeostasis Careful observation of the influence of copper on

iron homeostasis has shed light on divalent metal dependant pathological

mechanisms of human disease (1725 words + 539 words in References)

References

1 Bielli P Bellenchi GC and Calabrese L (2001) Site-directed

mutagenesis of human ceruloplasmin production of a proteolyti-

cally stable protein and structure-activity relationships of type 1

sites JBiolChem 276 2678-2685

2 Koschinsky ML Chow BK Schwartz J Hamerton JL and

MacGillivray RT (1987) Isolation and characterization of a pro-

cessed gene for human ceruloplasmin Biochemistry 26 7760-

7767

3 Koschinsky ML Funk WD van Oost BA and MacGillivray

RT (1986) Complete cDNA sequence of human

preceruloplasmin ProcNatlAcadSciUSA 83 5086-5090

4 Fortna RR Watson HA and Nyquist SE (1999) Glycosyl

phosphatidylinositol-anchored ceruloplasmin is expressed by rat

Sertoli cells and is concentrated in detergent-insoluble membrane

fractions BiolReprod 61 1042-1049

5 Calabrese L Carbonaro M and Musci G (1989) Presence of

coupled trinuclear copper cluster in mammalian ceruloplasmin is

essential for efficient electron transfer to oxygen JBiolChem

264 6183-6187

6 Hellman NE Kono S Mancini GM Hoogeboom AJ De

Jong GJ and Gitlin JD (2002) Mechanisms of copper incorpora-

tion into human ceruloplasmin JBiolChem 277 46632-46638

7 Halliwell B and Gutteridge JM (1984) Oxygen toxicity oxy-

gen radicals transition metals and disease BiochemJ 219 1-

1413 Jeong SY and David S (2003) Glycosylphosphatidylinosi-

tol-anchored ceruloplasmin is required for iron efflux from cells in

the central nervous system JBiolChem 278 27144-27148

8 Messerschmidt A Rossi A Ladenstein R Huber R Bolog-

nesi M Gatti G Marchesini A Petruzzelli R and Finazzi-Agro

A (1989) X-ray crystal structure of the blue oxidase ascorbate ox-

idase from zucchini Analysis of the polypeptide fold and a model

of the copper sites and ligands JMolBiol 206 513-529

9 European Association for Study of Liver (2012) EASL Clinical

Practice Guidelines Wilsons disease JHepatol 56 671-685

10 di Patti MC Maio N Rizzo G De Francesco G Persichini

T Colasanti M Polticelli F and Musci G (2009) Dominant mu-

tants of ceruloplasmin impair the copper loading machinery in

aceruloplasminemia JBiolChem 284 4545-4554

11 De Domenico I Ward DM di Patti MC Jeong SY David

S Musci G and Kaplan J (2007) Ferroxidase activity is required

for the stability of cell surface ferroportin in cells expressing GPI-

ceruloplasmin EMBO J 26 2823-2831

12 Chapman AL Mocatta TJ Shiva S Seidel A Chen B

Khalilova I Paumann-Page ME Jameson GN Winterbourn

CC and Kettle AJ (2013) Ceruloplasmin is an endogenous in-

hibitor of myeloperoxidase JBiolChem 288 6465-6477

13 Prohaska JR (2014) Impact of copper deficiency in humans

AnnNYAcadSci 1314 1-5

14 Mukhopadhyay CK Mazumder B and Fox PL (2000) Role

of hypoxia-inducible factor-1 in transcriptional activation of ceru-

loplasmin by iron deficiency JBiolChem 275 21048-21054

15 Tapryal N Mukhopadhyay C Das D Fox PL and

Mukhopadhyay CK (2009) Reactive oxygen species regulate

ceruloplasmin by a novel mRNA decay mechanism involving its 3-

untranslated region implications in neurodegenerative diseases

JBiolChem 284 1873-1883

Introduction

Human Ceruloplasmin (hCP) is a member of the multi-copper oxidase

(MCO) ldquobluerdquo family of enzymes (1) Other members of this family are

ascorbate oxidase laccase Fet3 and the recently discovered zyklopen

Only CP Fet3 and zyklopen are able to oxidize inorganic substrates such

as Fe(II) (1) MCOrsquos use copper to couple substrate oxidation with the four-

electron reduction of dioxygen to water (1) hCP is a serum ferroxidase

and as such plays an important role in iron homeostasis (1) hCP contains

greater than 95 of the copper found in plasma It consists of a single

1046 amino-acid (aa) residue polypeptide with 6 distinct cupredoxin type

domains (CDTDrsquos) It is a 132 kDa glycoprotein consisting of 7-8 carbo-

hydrate by mass hCp needs copper to function However it has no role in

the transport or metabolism of the metal hCP is essential for regulating ef-

flux of copper out of many parenchyma cell types

Genetics

hCP is encoded by 20 exons spanning ~ 65 kB of DNA at chromosomal

position 3q23-q24 An hCP pseudogene encodes the carboxyl-terminal 563

aa residues of the protein Although this pseudogene is not expressed It

must be considered when designing any molecular diagnostic test for

aceruloplasminemia (2) Alternative polyadenylation leads to mRNA of 37

and 42 kDa in hepatic tissue hCP is expressed in smaller amounts in the

spleen lungs testes and brain (3) A glycophosphatidylinositol (GPI)ndashan-

chored protein is also produced via alternative splicing of exons 19 and 20

in astrocytes and Sertoli cells (4)

Interaction of Ceruloplasmin With Copper (Structure)

The 6 CDTDrsquos are arranged in a triangular array There are 6 integral cop-

per ions 3 form a trinuclear cluster at the interface of domains 1 amp 6 3 are

mononuclear ions (coordinated to a cystine and two histidine residues) in

domains 2 4 and 6 At these 3 (type 1) copper sites charge transfer be-

tween the cysteine ligand sulfur and the copper result in a strong absorp-

tion at 600 nm This confers an intense blue colour to the protein One type

II copper is coordinated to four imidazole nitrogens in close proximity to

two antiferromagnetically coupled type III coppers that absorb at 330 nm

The type II and III coppers constitute the trinuclear copper cluster where a

dioxygen species may bind (6) All known isoforms of hCP incorporate 6

copper ions into the structure Copper does not affect the rate of synthesis

or secretion of hCP However failure to incorporate copper ions leads to

very high turnover of the apoenzyme (6) Thus high copper levels lead to a

steady state of hCP and low copper levels lead to a decreased in hCP It

has been shown that all six copper atoms must be present for the enzyme

to fold and therefore function properly (6)

Function of Ceruloplasmin

Ceruloplasmin is a ferroxidase that converts the toxic Fe(II) to Fe(III) Free

Fe(II) can react with hydrogen peroxide and molecular oxygen to produce

free radicals Free radicals may induce lipid peroxidation DNA strand

breaks destruction of biomolecules and lead to cell death (7) Fe(III) is in-

corporated into transferrin which is the ldquostorage poolrdquo for iron until it is in-

corporated into oxygen transport molecules such as hemoglobin

Definitive evidence of the physiologic role of hCP in iron homeostasis

came with the discovery of patients with aceruloplasminemia (discussed

later)

Mechanism by Which Ceruloplasmin Functions

Multicopper oxidases such as hCP use the electronic chemistry of bound

copper ions to couple substrate oxidation with four-electron reduction of

dioxygen Electrons pass from the substrate (ferrus iron) to the type I cop-

per then to the trinu-

clear copper cluster

and then to the oxygen

molecule bound at this

site (8)

Ceruloplasmin Deficiency (Wilson Disease)

Wilson disease is an autosomal recessive disorder with a global incidence

considered to be 1 in 30000 (9) Marked decrease in the concentration of

this hCP in serum samples from patients with Wilson disease provides the

basis of a biochemical test for this disorder that is still in clinical use today

Wilson disease does not result from lack of copper Conversely Wilson dis-

ease is an autosomal recessive disorder resulting in hepatic copper accu-

mulation (9) The primary genetic defect lies in a large 21-exon gene on

chromosome 13 The ATP7B gene encodes a 1456-amino-acid protein with

6 copper-binding domains ATP7B protein is a copper transporting ATPase

which brings copper from the trans-golgi to the biliary canaliculus of hepa-

tocytes From there it may be incorporated into hCP to form the holoen-

zyme The excess copper is excreted into to the bile for elimination (9)

Thus mutations in the Wilson protein decrease the amount of stable

haloenzyme increase the amount of unstable apoenzyme and reduce the

rate at which copper may be excreted into the bile All together this causes

reduced levels of hCP and buildup of cytoplasmic copper This leads to the

symptoms of Wilson disease which are Kayser-Fleischer (KF) rings hepati-

tis Cirrhosis and neurological problems (9) Chelation therapy with D-

penicillamine may help but has serious side affects

Ceruloplasmin Deficiency (Menkes disease)

Most dietary copper is absorbed from the proximal small intestine and is di-

rected into the portal circulation with the help of another copper-transport-

ing protein produced by the ATP7A gene Mutations in the ATP7A gene

lead to a copper-deficiency disease called Menkes disease Both of the

ATP7 gene products are required for ceruloplasmin copper loading There-

fore the symptoms and treatment of Menkes are the same as for Wilsonrsquos

(9)

Ceruloplasmin Deficiency (Aceruloplasminemia)

There has been hCP deficiency attributed to autosomal recessive muta-

tions in the hCP gene (10) Heterozygous individuals usually show normal

iron metabolism However in some cases dominant mutations in the hCP

gene lead to iron overload in various parenchyma These mutations were

found to disrupt copper loading of hCP by ATP7B The lack of ferroxidase

activity of hCP has been shown to lead to internalization and degradation

of ferroportin (11)

Most of the iron used each day for hematopoiesis and other essential

needs is recycled from erythrocytes (heme) in the reticuloendothelial sys-

tem Iron transported in the plasma bound to transferrin must be oxidized

prior to binding to this transport protein Ceruloplasmin plays a critical role

in the iron cycle by establishing a rate of iron oxidation sufficient for iron re-

lease from the reticuloendothelial (RI) system The absence of serum ceru-

loplasmin in patients with aceruloplasminemia leads to an accumulation of

iron in the RI system In addition to the accumulation of iron within the RI

system the absence of serum ceruloplasmin also results in increased fer-

rous iron in the plasma which is rapidly removed from the circulation by the

liver pancreas and other tissues When copper cannot be incorporated into

hCP the apoenzyme is quickly degraded Thus a low serum ceruloplasmin

level along with iron accumulation in the basal gan- glia (detected as a

low-intensity signal on T1- and T2-weighted magnetic resonance imaging)

is sufficient for diagnoses

Ceruloplasmin is an endogenous inhibitor of myeloperoxidase (12)

Myeloperoxidase generates hypochlorous acid and reactive radicle

species leading to oxidative stress when it leaks into the plasma hCP defi-

ciency may lead to increased tissue damage in times of increased oxidative

stress Inflammation from cystic fibrosis chronic obstructive pulmonary dis-

ease rheumatoid arthritis and Alzheimer disease may be worsened if hCP

is not in the plasma to act as an antioxidant (12)

Primary Hemochromotosis (Atransferrinemia)

The mechanism of iron overload in aceruloplasminemia is similar to that

which occurs in patients with primary hemochromotosis (PH)(atransferrine-

mia) PH is caused by mutations in the serum transferrin gene leading to

lack of functional transferrin

Copper Deficiency

The daily recommended copper intake is 09 mg (13) Most people get

more than enough from their food and water intake Pregnant and lactating

women persons with inflamed and diseased bowel and persons who have

had bariatric surgery are at greater risk for copper deficiency due to their

decreased copper absorption potential High zinc content of certain dental

adhesives is leading to an increase in zinc-induced copper deficiency

There are about a dozen mammalian copper dependent enzymes Table 1

gives the names and functions of the post prevalent ones

Prohaska JR (2014) Impact of copper deficiency in humans AnnNYA-cadSci 1314 1-5

Copper deficiency constantly leads to anemia (lower-than-normal hemoglo-

bin concentration) (13) This may be partly explained based on decreased

activity of copper ferroxidases hephaestin and ceruloplasmin

Altered function of ATP7A when copper is limiting may contribute to the

symptoms However anemia may result from a variety of compromised

copper-dependent processes including impaired erythropoiesis (13)

Iron Deficiency (Activation of CP by HIF-1)

Iron deficiency leading to hypoxia potentiates production of the regulatory

protein hypoxia-inducible factor (HIF-1) HIF-1 is a heterodimeric transcrip-

tion factor complex HIF-11113114a is the key regulatory component of the com-

plex because it is absent under normal conditions but is upregulated by the

iron chelator desferrioxamine (14) Upon activation the HIF-1a1113114HIF-11113114b

dimer binds to hypoxia-responsive elements (HREs) in multiple genes in-

cluding several with important functions in iron metabolism such as ery-

thropoietin heme oxygenase-1 transferrin and transferrin receptor HIF-1

has been shown to activate CP transcription via one putative hypoxia-re-

sponsive element (HRE) in the 5rsquo regulatory region of the CP gene (14)

This was shown by cloning the 5rsquo flanking region of the CP gene from a hu-

man cDNA library This promoterenhancer region driving a luciferase re-

porter was transfected into HepG2 cells Hypoxia increased luciferase ac-

tivity 5-10 fold (14)

Reactive Oxygen Species (ROS) Regulate CP

ROS decrease the synthesis of both the secretory form of Cp (in hepatic

cells) and membrane-bound GPI-Cp (in astroglial cells) by a novel post-

transcriptional mechanism involving its 31113114-UTR (15) The Cp 3rsquo1113114-UTR-

binding protein complex is decreased by ROS causing increased decay of

Cp mRNA Therefore Cp expression is down-regulated by ROS This may

explain the iron deposition and related damage by increased ROS gener-

ation in neurodegenerative diseases (15)

Conclusion

Investigations into iron-copper interactions have revealed novel aspects of

mineral homeostasis Careful observation of the influence of copper on

iron homeostasis has shed light on divalent metal dependant pathological

mechanisms of human disease (1725 words + 539 words in References)

References

1 Bielli P Bellenchi GC and Calabrese L (2001) Site-directed

mutagenesis of human ceruloplasmin production of a proteolyti-

cally stable protein and structure-activity relationships of type 1

sites JBiolChem 276 2678-2685

2 Koschinsky ML Chow BK Schwartz J Hamerton JL and

MacGillivray RT (1987) Isolation and characterization of a pro-

cessed gene for human ceruloplasmin Biochemistry 26 7760-

7767

3 Koschinsky ML Funk WD van Oost BA and MacGillivray

RT (1986) Complete cDNA sequence of human

preceruloplasmin ProcNatlAcadSciUSA 83 5086-5090

4 Fortna RR Watson HA and Nyquist SE (1999) Glycosyl

phosphatidylinositol-anchored ceruloplasmin is expressed by rat

Sertoli cells and is concentrated in detergent-insoluble membrane

fractions BiolReprod 61 1042-1049

5 Calabrese L Carbonaro M and Musci G (1989) Presence of

coupled trinuclear copper cluster in mammalian ceruloplasmin is

essential for efficient electron transfer to oxygen JBiolChem

264 6183-6187

6 Hellman NE Kono S Mancini GM Hoogeboom AJ De

Jong GJ and Gitlin JD (2002) Mechanisms of copper incorpora-

tion into human ceruloplasmin JBiolChem 277 46632-46638

7 Halliwell B and Gutteridge JM (1984) Oxygen toxicity oxy-

gen radicals transition metals and disease BiochemJ 219 1-

1413 Jeong SY and David S (2003) Glycosylphosphatidylinosi-

tol-anchored ceruloplasmin is required for iron efflux from cells in

the central nervous system JBiolChem 278 27144-27148

8 Messerschmidt A Rossi A Ladenstein R Huber R Bolog-

nesi M Gatti G Marchesini A Petruzzelli R and Finazzi-Agro

A (1989) X-ray crystal structure of the blue oxidase ascorbate ox-

idase from zucchini Analysis of the polypeptide fold and a model

of the copper sites and ligands JMolBiol 206 513-529

9 European Association for Study of Liver (2012) EASL Clinical

Practice Guidelines Wilsons disease JHepatol 56 671-685

10 di Patti MC Maio N Rizzo G De Francesco G Persichini

T Colasanti M Polticelli F and Musci G (2009) Dominant mu-

tants of ceruloplasmin impair the copper loading machinery in

aceruloplasminemia JBiolChem 284 4545-4554

11 De Domenico I Ward DM di Patti MC Jeong SY David

S Musci G and Kaplan J (2007) Ferroxidase activity is required

for the stability of cell surface ferroportin in cells expressing GPI-

ceruloplasmin EMBO J 26 2823-2831

12 Chapman AL Mocatta TJ Shiva S Seidel A Chen B

Khalilova I Paumann-Page ME Jameson GN Winterbourn

CC and Kettle AJ (2013) Ceruloplasmin is an endogenous in-

hibitor of myeloperoxidase JBiolChem 288 6465-6477

13 Prohaska JR (2014) Impact of copper deficiency in humans

AnnNYAcadSci 1314 1-5

14 Mukhopadhyay CK Mazumder B and Fox PL (2000) Role

of hypoxia-inducible factor-1 in transcriptional activation of ceru-

loplasmin by iron deficiency JBiolChem 275 21048-21054

15 Tapryal N Mukhopadhyay C Das D Fox PL and

Mukhopadhyay CK (2009) Reactive oxygen species regulate

ceruloplasmin by a novel mRNA decay mechanism involving its 3-

untranslated region implications in neurodegenerative diseases

JBiolChem 284 1873-1883

aa residues of the protein Although this pseudogene is not expressed It

must be considered when designing any molecular diagnostic test for

aceruloplasminemia (2) Alternative polyadenylation leads to mRNA of 37

and 42 kDa in hepatic tissue hCP is expressed in smaller amounts in the

spleen lungs testes and brain (3) A glycophosphatidylinositol (GPI)ndashan-

chored protein is also produced via alternative splicing of exons 19 and 20

in astrocytes and Sertoli cells (4)

Interaction of Ceruloplasmin With Copper (Structure)

The 6 CDTDrsquos are arranged in a triangular array There are 6 integral cop-

per ions 3 form a trinuclear cluster at the interface of domains 1 amp 6 3 are

mononuclear ions (coordinated to a cystine and two histidine residues) in

domains 2 4 and 6 At these 3 (type 1) copper sites charge transfer be-

tween the cysteine ligand sulfur and the copper result in a strong absorp-

tion at 600 nm This confers an intense blue colour to the protein One type

II copper is coordinated to four imidazole nitrogens in close proximity to

two antiferromagnetically coupled type III coppers that absorb at 330 nm

The type II and III coppers constitute the trinuclear copper cluster where a

dioxygen species may bind (6) All known isoforms of hCP incorporate 6

copper ions into the structure Copper does not affect the rate of synthesis

or secretion of hCP However failure to incorporate copper ions leads to

very high turnover of the apoenzyme (6) Thus high copper levels lead to a

steady state of hCP and low copper levels lead to a decreased in hCP It

has been shown that all six copper atoms must be present for the enzyme

to fold and therefore function properly (6)

Function of Ceruloplasmin

Ceruloplasmin is a ferroxidase that converts the toxic Fe(II) to Fe(III) Free

Fe(II) can react with hydrogen peroxide and molecular oxygen to produce

free radicals Free radicals may induce lipid peroxidation DNA strand

breaks destruction of biomolecules and lead to cell death (7) Fe(III) is in-

corporated into transferrin which is the ldquostorage poolrdquo for iron until it is in-

corporated into oxygen transport molecules such as hemoglobin

Definitive evidence of the physiologic role of hCP in iron homeostasis

came with the discovery of patients with aceruloplasminemia (discussed

later)

Mechanism by Which Ceruloplasmin Functions

Multicopper oxidases such as hCP use the electronic chemistry of bound

copper ions to couple substrate oxidation with four-electron reduction of

dioxygen Electrons pass from the substrate (ferrus iron) to the type I cop-

per then to the trinu-

clear copper cluster

and then to the oxygen

molecule bound at this

site (8)

Ceruloplasmin Deficiency (Wilson Disease)

Wilson disease is an autosomal recessive disorder with a global incidence

considered to be 1 in 30000 (9) Marked decrease in the concentration of

this hCP in serum samples from patients with Wilson disease provides the

basis of a biochemical test for this disorder that is still in clinical use today

Wilson disease does not result from lack of copper Conversely Wilson dis-

ease is an autosomal recessive disorder resulting in hepatic copper accu-

mulation (9) The primary genetic defect lies in a large 21-exon gene on

chromosome 13 The ATP7B gene encodes a 1456-amino-acid protein with

6 copper-binding domains ATP7B protein is a copper transporting ATPase

which brings copper from the trans-golgi to the biliary canaliculus of hepa-

tocytes From there it may be incorporated into hCP to form the holoen-

zyme The excess copper is excreted into to the bile for elimination (9)

Thus mutations in the Wilson protein decrease the amount of stable

haloenzyme increase the amount of unstable apoenzyme and reduce the

rate at which copper may be excreted into the bile All together this causes

reduced levels of hCP and buildup of cytoplasmic copper This leads to the

symptoms of Wilson disease which are Kayser-Fleischer (KF) rings hepati-

tis Cirrhosis and neurological problems (9) Chelation therapy with D-

penicillamine may help but has serious side affects

Ceruloplasmin Deficiency (Menkes disease)

Most dietary copper is absorbed from the proximal small intestine and is di-

rected into the portal circulation with the help of another copper-transport-

ing protein produced by the ATP7A gene Mutations in the ATP7A gene

lead to a copper-deficiency disease called Menkes disease Both of the

ATP7 gene products are required for ceruloplasmin copper loading There-

fore the symptoms and treatment of Menkes are the same as for Wilsonrsquos

(9)

Ceruloplasmin Deficiency (Aceruloplasminemia)

There has been hCP deficiency attributed to autosomal recessive muta-

tions in the hCP gene (10) Heterozygous individuals usually show normal

iron metabolism However in some cases dominant mutations in the hCP

gene lead to iron overload in various parenchyma These mutations were

found to disrupt copper loading of hCP by ATP7B The lack of ferroxidase

activity of hCP has been shown to lead to internalization and degradation

of ferroportin (11)

Most of the iron used each day for hematopoiesis and other essential

needs is recycled from erythrocytes (heme) in the reticuloendothelial sys-

tem Iron transported in the plasma bound to transferrin must be oxidized

prior to binding to this transport protein Ceruloplasmin plays a critical role

in the iron cycle by establishing a rate of iron oxidation sufficient for iron re-

lease from the reticuloendothelial (RI) system The absence of serum ceru-

loplasmin in patients with aceruloplasminemia leads to an accumulation of

iron in the RI system In addition to the accumulation of iron within the RI

system the absence of serum ceruloplasmin also results in increased fer-

rous iron in the plasma which is rapidly removed from the circulation by the

liver pancreas and other tissues When copper cannot be incorporated into

hCP the apoenzyme is quickly degraded Thus a low serum ceruloplasmin

level along with iron accumulation in the basal gan- glia (detected as a

low-intensity signal on T1- and T2-weighted magnetic resonance imaging)

is sufficient for diagnoses

Ceruloplasmin is an endogenous inhibitor of myeloperoxidase (12)

Myeloperoxidase generates hypochlorous acid and reactive radicle

species leading to oxidative stress when it leaks into the plasma hCP defi-

ciency may lead to increased tissue damage in times of increased oxidative

stress Inflammation from cystic fibrosis chronic obstructive pulmonary dis-

ease rheumatoid arthritis and Alzheimer disease may be worsened if hCP

is not in the plasma to act as an antioxidant (12)

Primary Hemochromotosis (Atransferrinemia)

The mechanism of iron overload in aceruloplasminemia is similar to that

which occurs in patients with primary hemochromotosis (PH)(atransferrine-

mia) PH is caused by mutations in the serum transferrin gene leading to

lack of functional transferrin

Copper Deficiency

The daily recommended copper intake is 09 mg (13) Most people get

more than enough from their food and water intake Pregnant and lactating

women persons with inflamed and diseased bowel and persons who have

had bariatric surgery are at greater risk for copper deficiency due to their

decreased copper absorption potential High zinc content of certain dental

adhesives is leading to an increase in zinc-induced copper deficiency

There are about a dozen mammalian copper dependent enzymes Table 1

gives the names and functions of the post prevalent ones

Prohaska JR (2014) Impact of copper deficiency in humans AnnNYA-cadSci 1314 1-5

Copper deficiency constantly leads to anemia (lower-than-normal hemoglo-

bin concentration) (13) This may be partly explained based on decreased

activity of copper ferroxidases hephaestin and ceruloplasmin

Altered function of ATP7A when copper is limiting may contribute to the

symptoms However anemia may result from a variety of compromised

copper-dependent processes including impaired erythropoiesis (13)

Iron Deficiency (Activation of CP by HIF-1)

Iron deficiency leading to hypoxia potentiates production of the regulatory

protein hypoxia-inducible factor (HIF-1) HIF-1 is a heterodimeric transcrip-

tion factor complex HIF-11113114a is the key regulatory component of the com-

plex because it is absent under normal conditions but is upregulated by the

iron chelator desferrioxamine (14) Upon activation the HIF-1a1113114HIF-11113114b

dimer binds to hypoxia-responsive elements (HREs) in multiple genes in-

cluding several with important functions in iron metabolism such as ery-

thropoietin heme oxygenase-1 transferrin and transferrin receptor HIF-1

has been shown to activate CP transcription via one putative hypoxia-re-

sponsive element (HRE) in the 5rsquo regulatory region of the CP gene (14)

This was shown by cloning the 5rsquo flanking region of the CP gene from a hu-

man cDNA library This promoterenhancer region driving a luciferase re-

porter was transfected into HepG2 cells Hypoxia increased luciferase ac-

tivity 5-10 fold (14)

Reactive Oxygen Species (ROS) Regulate CP

ROS decrease the synthesis of both the secretory form of Cp (in hepatic

cells) and membrane-bound GPI-Cp (in astroglial cells) by a novel post-

transcriptional mechanism involving its 31113114-UTR (15) The Cp 3rsquo1113114-UTR-

binding protein complex is decreased by ROS causing increased decay of

Cp mRNA Therefore Cp expression is down-regulated by ROS This may

explain the iron deposition and related damage by increased ROS gener-

ation in neurodegenerative diseases (15)

Conclusion

Investigations into iron-copper interactions have revealed novel aspects of

mineral homeostasis Careful observation of the influence of copper on

iron homeostasis has shed light on divalent metal dependant pathological

mechanisms of human disease (1725 words + 539 words in References)

References

1 Bielli P Bellenchi GC and Calabrese L (2001) Site-directed

mutagenesis of human ceruloplasmin production of a proteolyti-

cally stable protein and structure-activity relationships of type 1

sites JBiolChem 276 2678-2685

2 Koschinsky ML Chow BK Schwartz J Hamerton JL and

MacGillivray RT (1987) Isolation and characterization of a pro-

cessed gene for human ceruloplasmin Biochemistry 26 7760-

7767

3 Koschinsky ML Funk WD van Oost BA and MacGillivray

RT (1986) Complete cDNA sequence of human

preceruloplasmin ProcNatlAcadSciUSA 83 5086-5090

4 Fortna RR Watson HA and Nyquist SE (1999) Glycosyl

phosphatidylinositol-anchored ceruloplasmin is expressed by rat

Sertoli cells and is concentrated in detergent-insoluble membrane

fractions BiolReprod 61 1042-1049

5 Calabrese L Carbonaro M and Musci G (1989) Presence of

coupled trinuclear copper cluster in mammalian ceruloplasmin is

essential for efficient electron transfer to oxygen JBiolChem

264 6183-6187

6 Hellman NE Kono S Mancini GM Hoogeboom AJ De

Jong GJ and Gitlin JD (2002) Mechanisms of copper incorpora-

tion into human ceruloplasmin JBiolChem 277 46632-46638

7 Halliwell B and Gutteridge JM (1984) Oxygen toxicity oxy-

gen radicals transition metals and disease BiochemJ 219 1-

1413 Jeong SY and David S (2003) Glycosylphosphatidylinosi-

tol-anchored ceruloplasmin is required for iron efflux from cells in

the central nervous system JBiolChem 278 27144-27148

8 Messerschmidt A Rossi A Ladenstein R Huber R Bolog-

nesi M Gatti G Marchesini A Petruzzelli R and Finazzi-Agro

A (1989) X-ray crystal structure of the blue oxidase ascorbate ox-

idase from zucchini Analysis of the polypeptide fold and a model

of the copper sites and ligands JMolBiol 206 513-529

9 European Association for Study of Liver (2012) EASL Clinical

Practice Guidelines Wilsons disease JHepatol 56 671-685

10 di Patti MC Maio N Rizzo G De Francesco G Persichini

T Colasanti M Polticelli F and Musci G (2009) Dominant mu-

tants of ceruloplasmin impair the copper loading machinery in

aceruloplasminemia JBiolChem 284 4545-4554

11 De Domenico I Ward DM di Patti MC Jeong SY David

S Musci G and Kaplan J (2007) Ferroxidase activity is required

for the stability of cell surface ferroportin in cells expressing GPI-

ceruloplasmin EMBO J 26 2823-2831

12 Chapman AL Mocatta TJ Shiva S Seidel A Chen B

Khalilova I Paumann-Page ME Jameson GN Winterbourn

CC and Kettle AJ (2013) Ceruloplasmin is an endogenous in-

hibitor of myeloperoxidase JBiolChem 288 6465-6477

13 Prohaska JR (2014) Impact of copper deficiency in humans

AnnNYAcadSci 1314 1-5

14 Mukhopadhyay CK Mazumder B and Fox PL (2000) Role

of hypoxia-inducible factor-1 in transcriptional activation of ceru-

loplasmin by iron deficiency JBiolChem 275 21048-21054

15 Tapryal N Mukhopadhyay C Das D Fox PL and

Mukhopadhyay CK (2009) Reactive oxygen species regulate

ceruloplasmin by a novel mRNA decay mechanism involving its 3-

untranslated region implications in neurodegenerative diseases

JBiolChem 284 1873-1883

copper ions into the structure Copper does not affect the rate of synthesis

or secretion of hCP However failure to incorporate copper ions leads to

very high turnover of the apoenzyme (6) Thus high copper levels lead to a

steady state of hCP and low copper levels lead to a decreased in hCP It

has been shown that all six copper atoms must be present for the enzyme

to fold and therefore function properly (6)

Function of Ceruloplasmin

Ceruloplasmin is a ferroxidase that converts the toxic Fe(II) to Fe(III) Free

Fe(II) can react with hydrogen peroxide and molecular oxygen to produce

free radicals Free radicals may induce lipid peroxidation DNA strand

breaks destruction of biomolecules and lead to cell death (7) Fe(III) is in-

corporated into transferrin which is the ldquostorage poolrdquo for iron until it is in-

corporated into oxygen transport molecules such as hemoglobin

Definitive evidence of the physiologic role of hCP in iron homeostasis

came with the discovery of patients with aceruloplasminemia (discussed

later)

Mechanism by Which Ceruloplasmin Functions

Multicopper oxidases such as hCP use the electronic chemistry of bound

copper ions to couple substrate oxidation with four-electron reduction of

dioxygen Electrons pass from the substrate (ferrus iron) to the type I cop-

per then to the trinu-

clear copper cluster

and then to the oxygen

molecule bound at this

site (8)

Ceruloplasmin Deficiency (Wilson Disease)

Wilson disease is an autosomal recessive disorder with a global incidence

considered to be 1 in 30000 (9) Marked decrease in the concentration of

this hCP in serum samples from patients with Wilson disease provides the

basis of a biochemical test for this disorder that is still in clinical use today

Wilson disease does not result from lack of copper Conversely Wilson dis-

ease is an autosomal recessive disorder resulting in hepatic copper accu-

mulation (9) The primary genetic defect lies in a large 21-exon gene on

chromosome 13 The ATP7B gene encodes a 1456-amino-acid protein with

6 copper-binding domains ATP7B protein is a copper transporting ATPase

which brings copper from the trans-golgi to the biliary canaliculus of hepa-

tocytes From there it may be incorporated into hCP to form the holoen-

zyme The excess copper is excreted into to the bile for elimination (9)

Thus mutations in the Wilson protein decrease the amount of stable

haloenzyme increase the amount of unstable apoenzyme and reduce the

rate at which copper may be excreted into the bile All together this causes

reduced levels of hCP and buildup of cytoplasmic copper This leads to the

symptoms of Wilson disease which are Kayser-Fleischer (KF) rings hepati-

tis Cirrhosis and neurological problems (9) Chelation therapy with D-

penicillamine may help but has serious side affects

Ceruloplasmin Deficiency (Menkes disease)

Most dietary copper is absorbed from the proximal small intestine and is di-

rected into the portal circulation with the help of another copper-transport-

ing protein produced by the ATP7A gene Mutations in the ATP7A gene

lead to a copper-deficiency disease called Menkes disease Both of the

ATP7 gene products are required for ceruloplasmin copper loading There-

fore the symptoms and treatment of Menkes are the same as for Wilsonrsquos

(9)

Ceruloplasmin Deficiency (Aceruloplasminemia)

There has been hCP deficiency attributed to autosomal recessive muta-

tions in the hCP gene (10) Heterozygous individuals usually show normal

iron metabolism However in some cases dominant mutations in the hCP

gene lead to iron overload in various parenchyma These mutations were

found to disrupt copper loading of hCP by ATP7B The lack of ferroxidase

activity of hCP has been shown to lead to internalization and degradation

of ferroportin (11)

Most of the iron used each day for hematopoiesis and other essential

needs is recycled from erythrocytes (heme) in the reticuloendothelial sys-

tem Iron transported in the plasma bound to transferrin must be oxidized

prior to binding to this transport protein Ceruloplasmin plays a critical role

in the iron cycle by establishing a rate of iron oxidation sufficient for iron re-

lease from the reticuloendothelial (RI) system The absence of serum ceru-

loplasmin in patients with aceruloplasminemia leads to an accumulation of

iron in the RI system In addition to the accumulation of iron within the RI

system the absence of serum ceruloplasmin also results in increased fer-

rous iron in the plasma which is rapidly removed from the circulation by the

liver pancreas and other tissues When copper cannot be incorporated into

hCP the apoenzyme is quickly degraded Thus a low serum ceruloplasmin

level along with iron accumulation in the basal gan- glia (detected as a

low-intensity signal on T1- and T2-weighted magnetic resonance imaging)

is sufficient for diagnoses

Ceruloplasmin is an endogenous inhibitor of myeloperoxidase (12)

Myeloperoxidase generates hypochlorous acid and reactive radicle

species leading to oxidative stress when it leaks into the plasma hCP defi-

ciency may lead to increased tissue damage in times of increased oxidative

stress Inflammation from cystic fibrosis chronic obstructive pulmonary dis-

ease rheumatoid arthritis and Alzheimer disease may be worsened if hCP

is not in the plasma to act as an antioxidant (12)

Primary Hemochromotosis (Atransferrinemia)

The mechanism of iron overload in aceruloplasminemia is similar to that

which occurs in patients with primary hemochromotosis (PH)(atransferrine-

mia) PH is caused by mutations in the serum transferrin gene leading to

lack of functional transferrin

Copper Deficiency

The daily recommended copper intake is 09 mg (13) Most people get

more than enough from their food and water intake Pregnant and lactating

women persons with inflamed and diseased bowel and persons who have

had bariatric surgery are at greater risk for copper deficiency due to their

decreased copper absorption potential High zinc content of certain dental

adhesives is leading to an increase in zinc-induced copper deficiency

There are about a dozen mammalian copper dependent enzymes Table 1

gives the names and functions of the post prevalent ones

Prohaska JR (2014) Impact of copper deficiency in humans AnnNYA-cadSci 1314 1-5

Copper deficiency constantly leads to anemia (lower-than-normal hemoglo-

bin concentration) (13) This may be partly explained based on decreased

activity of copper ferroxidases hephaestin and ceruloplasmin

Altered function of ATP7A when copper is limiting may contribute to the

symptoms However anemia may result from a variety of compromised

copper-dependent processes including impaired erythropoiesis (13)

Iron Deficiency (Activation of CP by HIF-1)

Iron deficiency leading to hypoxia potentiates production of the regulatory

protein hypoxia-inducible factor (HIF-1) HIF-1 is a heterodimeric transcrip-

tion factor complex HIF-11113114a is the key regulatory component of the com-

plex because it is absent under normal conditions but is upregulated by the

iron chelator desferrioxamine (14) Upon activation the HIF-1a1113114HIF-11113114b

dimer binds to hypoxia-responsive elements (HREs) in multiple genes in-

cluding several with important functions in iron metabolism such as ery-

thropoietin heme oxygenase-1 transferrin and transferrin receptor HIF-1

has been shown to activate CP transcription via one putative hypoxia-re-

sponsive element (HRE) in the 5rsquo regulatory region of the CP gene (14)

This was shown by cloning the 5rsquo flanking region of the CP gene from a hu-

man cDNA library This promoterenhancer region driving a luciferase re-

porter was transfected into HepG2 cells Hypoxia increased luciferase ac-

tivity 5-10 fold (14)

Reactive Oxygen Species (ROS) Regulate CP

ROS decrease the synthesis of both the secretory form of Cp (in hepatic

cells) and membrane-bound GPI-Cp (in astroglial cells) by a novel post-

transcriptional mechanism involving its 31113114-UTR (15) The Cp 3rsquo1113114-UTR-

binding protein complex is decreased by ROS causing increased decay of

Cp mRNA Therefore Cp expression is down-regulated by ROS This may

explain the iron deposition and related damage by increased ROS gener-

ation in neurodegenerative diseases (15)

Conclusion

Investigations into iron-copper interactions have revealed novel aspects of

mineral homeostasis Careful observation of the influence of copper on

iron homeostasis has shed light on divalent metal dependant pathological

mechanisms of human disease (1725 words + 539 words in References)

References

1 Bielli P Bellenchi GC and Calabrese L (2001) Site-directed

mutagenesis of human ceruloplasmin production of a proteolyti-

cally stable protein and structure-activity relationships of type 1

sites JBiolChem 276 2678-2685

2 Koschinsky ML Chow BK Schwartz J Hamerton JL and

MacGillivray RT (1987) Isolation and characterization of a pro-

cessed gene for human ceruloplasmin Biochemistry 26 7760-

7767

3 Koschinsky ML Funk WD van Oost BA and MacGillivray

RT (1986) Complete cDNA sequence of human

preceruloplasmin ProcNatlAcadSciUSA 83 5086-5090

4 Fortna RR Watson HA and Nyquist SE (1999) Glycosyl

phosphatidylinositol-anchored ceruloplasmin is expressed by rat

Sertoli cells and is concentrated in detergent-insoluble membrane

fractions BiolReprod 61 1042-1049

5 Calabrese L Carbonaro M and Musci G (1989) Presence of

coupled trinuclear copper cluster in mammalian ceruloplasmin is

essential for efficient electron transfer to oxygen JBiolChem

264 6183-6187

6 Hellman NE Kono S Mancini GM Hoogeboom AJ De

Jong GJ and Gitlin JD (2002) Mechanisms of copper incorpora-

tion into human ceruloplasmin JBiolChem 277 46632-46638

7 Halliwell B and Gutteridge JM (1984) Oxygen toxicity oxy-

gen radicals transition metals and disease BiochemJ 219 1-

1413 Jeong SY and David S (2003) Glycosylphosphatidylinosi-

tol-anchored ceruloplasmin is required for iron efflux from cells in

the central nervous system JBiolChem 278 27144-27148

8 Messerschmidt A Rossi A Ladenstein R Huber R Bolog-

nesi M Gatti G Marchesini A Petruzzelli R and Finazzi-Agro

A (1989) X-ray crystal structure of the blue oxidase ascorbate ox-

idase from zucchini Analysis of the polypeptide fold and a model

of the copper sites and ligands JMolBiol 206 513-529

9 European Association for Study of Liver (2012) EASL Clinical

Practice Guidelines Wilsons disease JHepatol 56 671-685

10 di Patti MC Maio N Rizzo G De Francesco G Persichini

T Colasanti M Polticelli F and Musci G (2009) Dominant mu-

tants of ceruloplasmin impair the copper loading machinery in

aceruloplasminemia JBiolChem 284 4545-4554

11 De Domenico I Ward DM di Patti MC Jeong SY David

S Musci G and Kaplan J (2007) Ferroxidase activity is required

for the stability of cell surface ferroportin in cells expressing GPI-

ceruloplasmin EMBO J 26 2823-2831

12 Chapman AL Mocatta TJ Shiva S Seidel A Chen B

Khalilova I Paumann-Page ME Jameson GN Winterbourn

CC and Kettle AJ (2013) Ceruloplasmin is an endogenous in-

hibitor of myeloperoxidase JBiolChem 288 6465-6477

13 Prohaska JR (2014) Impact of copper deficiency in humans

AnnNYAcadSci 1314 1-5

14 Mukhopadhyay CK Mazumder B and Fox PL (2000) Role

of hypoxia-inducible factor-1 in transcriptional activation of ceru-

loplasmin by iron deficiency JBiolChem 275 21048-21054

15 Tapryal N Mukhopadhyay C Das D Fox PL and

Mukhopadhyay CK (2009) Reactive oxygen species regulate

ceruloplasmin by a novel mRNA decay mechanism involving its 3-

untranslated region implications in neurodegenerative diseases

JBiolChem 284 1873-1883

Multicopper oxidases such as hCP use the electronic chemistry of bound

copper ions to couple substrate oxidation with four-electron reduction of

dioxygen Electrons pass from the substrate (ferrus iron) to the type I cop-

per then to the trinu-

clear copper cluster

and then to the oxygen

molecule bound at this

site (8)

Ceruloplasmin Deficiency (Wilson Disease)

Wilson disease is an autosomal recessive disorder with a global incidence

considered to be 1 in 30000 (9) Marked decrease in the concentration of

this hCP in serum samples from patients with Wilson disease provides the

basis of a biochemical test for this disorder that is still in clinical use today

Wilson disease does not result from lack of copper Conversely Wilson dis-

ease is an autosomal recessive disorder resulting in hepatic copper accu-

mulation (9) The primary genetic defect lies in a large 21-exon gene on

chromosome 13 The ATP7B gene encodes a 1456-amino-acid protein with

6 copper-binding domains ATP7B protein is a copper transporting ATPase

which brings copper from the trans-golgi to the biliary canaliculus of hepa-

tocytes From there it may be incorporated into hCP to form the holoen-

zyme The excess copper is excreted into to the bile for elimination (9)

Thus mutations in the Wilson protein decrease the amount of stable

haloenzyme increase the amount of unstable apoenzyme and reduce the

rate at which copper may be excreted into the bile All together this causes

reduced levels of hCP and buildup of cytoplasmic copper This leads to the

symptoms of Wilson disease which are Kayser-Fleischer (KF) rings hepati-

tis Cirrhosis and neurological problems (9) Chelation therapy with D-

penicillamine may help but has serious side affects

Ceruloplasmin Deficiency (Menkes disease)

Most dietary copper is absorbed from the proximal small intestine and is di-

rected into the portal circulation with the help of another copper-transport-

ing protein produced by the ATP7A gene Mutations in the ATP7A gene

lead to a copper-deficiency disease called Menkes disease Both of the

ATP7 gene products are required for ceruloplasmin copper loading There-

fore the symptoms and treatment of Menkes are the same as for Wilsonrsquos

(9)

Ceruloplasmin Deficiency (Aceruloplasminemia)

There has been hCP deficiency attributed to autosomal recessive muta-

tions in the hCP gene (10) Heterozygous individuals usually show normal

iron metabolism However in some cases dominant mutations in the hCP

gene lead to iron overload in various parenchyma These mutations were

found to disrupt copper loading of hCP by ATP7B The lack of ferroxidase

activity of hCP has been shown to lead to internalization and degradation

of ferroportin (11)

Most of the iron used each day for hematopoiesis and other essential

needs is recycled from erythrocytes (heme) in the reticuloendothelial sys-

tem Iron transported in the plasma bound to transferrin must be oxidized

prior to binding to this transport protein Ceruloplasmin plays a critical role

in the iron cycle by establishing a rate of iron oxidation sufficient for iron re-

lease from the reticuloendothelial (RI) system The absence of serum ceru-

loplasmin in patients with aceruloplasminemia leads to an accumulation of

iron in the RI system In addition to the accumulation of iron within the RI

system the absence of serum ceruloplasmin also results in increased fer-

rous iron in the plasma which is rapidly removed from the circulation by the

liver pancreas and other tissues When copper cannot be incorporated into

hCP the apoenzyme is quickly degraded Thus a low serum ceruloplasmin

level along with iron accumulation in the basal gan- glia (detected as a

low-intensity signal on T1- and T2-weighted magnetic resonance imaging)

is sufficient for diagnoses

Ceruloplasmin is an endogenous inhibitor of myeloperoxidase (12)

Myeloperoxidase generates hypochlorous acid and reactive radicle

species leading to oxidative stress when it leaks into the plasma hCP defi-

ciency may lead to increased tissue damage in times of increased oxidative

stress Inflammation from cystic fibrosis chronic obstructive pulmonary dis-

ease rheumatoid arthritis and Alzheimer disease may be worsened if hCP

is not in the plasma to act as an antioxidant (12)

Primary Hemochromotosis (Atransferrinemia)

The mechanism of iron overload in aceruloplasminemia is similar to that

which occurs in patients with primary hemochromotosis (PH)(atransferrine-

mia) PH is caused by mutations in the serum transferrin gene leading to

lack of functional transferrin

Copper Deficiency

The daily recommended copper intake is 09 mg (13) Most people get

more than enough from their food and water intake Pregnant and lactating

women persons with inflamed and diseased bowel and persons who have

had bariatric surgery are at greater risk for copper deficiency due to their

decreased copper absorption potential High zinc content of certain dental

adhesives is leading to an increase in zinc-induced copper deficiency

There are about a dozen mammalian copper dependent enzymes Table 1

gives the names and functions of the post prevalent ones

Prohaska JR (2014) Impact of copper deficiency in humans AnnNYA-cadSci 1314 1-5

Copper deficiency constantly leads to anemia (lower-than-normal hemoglo-

bin concentration) (13) This may be partly explained based on decreased

activity of copper ferroxidases hephaestin and ceruloplasmin

Altered function of ATP7A when copper is limiting may contribute to the

symptoms However anemia may result from a variety of compromised

copper-dependent processes including impaired erythropoiesis (13)

Iron Deficiency (Activation of CP by HIF-1)

Iron deficiency leading to hypoxia potentiates production of the regulatory

protein hypoxia-inducible factor (HIF-1) HIF-1 is a heterodimeric transcrip-

tion factor complex HIF-11113114a is the key regulatory component of the com-

plex because it is absent under normal conditions but is upregulated by the

iron chelator desferrioxamine (14) Upon activation the HIF-1a1113114HIF-11113114b

dimer binds to hypoxia-responsive elements (HREs) in multiple genes in-

cluding several with important functions in iron metabolism such as ery-

thropoietin heme oxygenase-1 transferrin and transferrin receptor HIF-1

has been shown to activate CP transcription via one putative hypoxia-re-

sponsive element (HRE) in the 5rsquo regulatory region of the CP gene (14)

This was shown by cloning the 5rsquo flanking region of the CP gene from a hu-

man cDNA library This promoterenhancer region driving a luciferase re-

porter was transfected into HepG2 cells Hypoxia increased luciferase ac-

tivity 5-10 fold (14)

Reactive Oxygen Species (ROS) Regulate CP

ROS decrease the synthesis of both the secretory form of Cp (in hepatic

cells) and membrane-bound GPI-Cp (in astroglial cells) by a novel post-

transcriptional mechanism involving its 31113114-UTR (15) The Cp 3rsquo1113114-UTR-

binding protein complex is decreased by ROS causing increased decay of

Cp mRNA Therefore Cp expression is down-regulated by ROS This may

explain the iron deposition and related damage by increased ROS gener-

ation in neurodegenerative diseases (15)

Conclusion

Investigations into iron-copper interactions have revealed novel aspects of

mineral homeostasis Careful observation of the influence of copper on

iron homeostasis has shed light on divalent metal dependant pathological

mechanisms of human disease (1725 words + 539 words in References)

References

1 Bielli P Bellenchi GC and Calabrese L (2001) Site-directed

mutagenesis of human ceruloplasmin production of a proteolyti-

cally stable protein and structure-activity relationships of type 1

sites JBiolChem 276 2678-2685

2 Koschinsky ML Chow BK Schwartz J Hamerton JL and

MacGillivray RT (1987) Isolation and characterization of a pro-

cessed gene for human ceruloplasmin Biochemistry 26 7760-

7767

3 Koschinsky ML Funk WD van Oost BA and MacGillivray

RT (1986) Complete cDNA sequence of human

preceruloplasmin ProcNatlAcadSciUSA 83 5086-5090

4 Fortna RR Watson HA and Nyquist SE (1999) Glycosyl

phosphatidylinositol-anchored ceruloplasmin is expressed by rat

Sertoli cells and is concentrated in detergent-insoluble membrane

fractions BiolReprod 61 1042-1049

5 Calabrese L Carbonaro M and Musci G (1989) Presence of

coupled trinuclear copper cluster in mammalian ceruloplasmin is

essential for efficient electron transfer to oxygen JBiolChem

264 6183-6187

6 Hellman NE Kono S Mancini GM Hoogeboom AJ De

Jong GJ and Gitlin JD (2002) Mechanisms of copper incorpora-

tion into human ceruloplasmin JBiolChem 277 46632-46638

7 Halliwell B and Gutteridge JM (1984) Oxygen toxicity oxy-

gen radicals transition metals and disease BiochemJ 219 1-

1413 Jeong SY and David S (2003) Glycosylphosphatidylinosi-

tol-anchored ceruloplasmin is required for iron efflux from cells in

the central nervous system JBiolChem 278 27144-27148

8 Messerschmidt A Rossi A Ladenstein R Huber R Bolog-

nesi M Gatti G Marchesini A Petruzzelli R and Finazzi-Agro

A (1989) X-ray crystal structure of the blue oxidase ascorbate ox-

idase from zucchini Analysis of the polypeptide fold and a model

of the copper sites and ligands JMolBiol 206 513-529

9 European Association for Study of Liver (2012) EASL Clinical

Practice Guidelines Wilsons disease JHepatol 56 671-685

10 di Patti MC Maio N Rizzo G De Francesco G Persichini

T Colasanti M Polticelli F and Musci G (2009) Dominant mu-

tants of ceruloplasmin impair the copper loading machinery in

aceruloplasminemia JBiolChem 284 4545-4554

11 De Domenico I Ward DM di Patti MC Jeong SY David

S Musci G and Kaplan J (2007) Ferroxidase activity is required

for the stability of cell surface ferroportin in cells expressing GPI-

ceruloplasmin EMBO J 26 2823-2831

12 Chapman AL Mocatta TJ Shiva S Seidel A Chen B

Khalilova I Paumann-Page ME Jameson GN Winterbourn

CC and Kettle AJ (2013) Ceruloplasmin is an endogenous in-

hibitor of myeloperoxidase JBiolChem 288 6465-6477

13 Prohaska JR (2014) Impact of copper deficiency in humans

AnnNYAcadSci 1314 1-5

14 Mukhopadhyay CK Mazumder B and Fox PL (2000) Role

of hypoxia-inducible factor-1 in transcriptional activation of ceru-

loplasmin by iron deficiency JBiolChem 275 21048-21054

15 Tapryal N Mukhopadhyay C Das D Fox PL and

Mukhopadhyay CK (2009) Reactive oxygen species regulate

ceruloplasmin by a novel mRNA decay mechanism involving its 3-

untranslated region implications in neurodegenerative diseases

JBiolChem 284 1873-1883

basis of a biochemical test for this disorder that is still in clinical use today

Wilson disease does not result from lack of copper Conversely Wilson dis-

ease is an autosomal recessive disorder resulting in hepatic copper accu-

mulation (9) The primary genetic defect lies in a large 21-exon gene on

chromosome 13 The ATP7B gene encodes a 1456-amino-acid protein with

6 copper-binding domains ATP7B protein is a copper transporting ATPase

which brings copper from the trans-golgi to the biliary canaliculus of hepa-

tocytes From there it may be incorporated into hCP to form the holoen-

zyme The excess copper is excreted into to the bile for elimination (9)

Thus mutations in the Wilson protein decrease the amount of stable

haloenzyme increase the amount of unstable apoenzyme and reduce the

rate at which copper may be excreted into the bile All together this causes

reduced levels of hCP and buildup of cytoplasmic copper This leads to the

symptoms of Wilson disease which are Kayser-Fleischer (KF) rings hepati-

tis Cirrhosis and neurological problems (9) Chelation therapy with D-

penicillamine may help but has serious side affects

Ceruloplasmin Deficiency (Menkes disease)

Most dietary copper is absorbed from the proximal small intestine and is di-

rected into the portal circulation with the help of another copper-transport-

ing protein produced by the ATP7A gene Mutations in the ATP7A gene

lead to a copper-deficiency disease called Menkes disease Both of the

ATP7 gene products are required for ceruloplasmin copper loading There-

fore the symptoms and treatment of Menkes are the same as for Wilsonrsquos

(9)

Ceruloplasmin Deficiency (Aceruloplasminemia)

There has been hCP deficiency attributed to autosomal recessive muta-

tions in the hCP gene (10) Heterozygous individuals usually show normal

iron metabolism However in some cases dominant mutations in the hCP

gene lead to iron overload in various parenchyma These mutations were

found to disrupt copper loading of hCP by ATP7B The lack of ferroxidase

activity of hCP has been shown to lead to internalization and degradation

of ferroportin (11)

Most of the iron used each day for hematopoiesis and other essential

needs is recycled from erythrocytes (heme) in the reticuloendothelial sys-

tem Iron transported in the plasma bound to transferrin must be oxidized

prior to binding to this transport protein Ceruloplasmin plays a critical role

in the iron cycle by establishing a rate of iron oxidation sufficient for iron re-

lease from the reticuloendothelial (RI) system The absence of serum ceru-

loplasmin in patients with aceruloplasminemia leads to an accumulation of

iron in the RI system In addition to the accumulation of iron within the RI

system the absence of serum ceruloplasmin also results in increased fer-

rous iron in the plasma which is rapidly removed from the circulation by the

liver pancreas and other tissues When copper cannot be incorporated into

hCP the apoenzyme is quickly degraded Thus a low serum ceruloplasmin

level along with iron accumulation in the basal gan- glia (detected as a

low-intensity signal on T1- and T2-weighted magnetic resonance imaging)

is sufficient for diagnoses

Ceruloplasmin is an endogenous inhibitor of myeloperoxidase (12)

Myeloperoxidase generates hypochlorous acid and reactive radicle

species leading to oxidative stress when it leaks into the plasma hCP defi-

ciency may lead to increased tissue damage in times of increased oxidative

stress Inflammation from cystic fibrosis chronic obstructive pulmonary dis-

ease rheumatoid arthritis and Alzheimer disease may be worsened if hCP

is not in the plasma to act as an antioxidant (12)

Primary Hemochromotosis (Atransferrinemia)

The mechanism of iron overload in aceruloplasminemia is similar to that

which occurs in patients with primary hemochromotosis (PH)(atransferrine-

mia) PH is caused by mutations in the serum transferrin gene leading to

lack of functional transferrin

Copper Deficiency

The daily recommended copper intake is 09 mg (13) Most people get

more than enough from their food and water intake Pregnant and lactating

women persons with inflamed and diseased bowel and persons who have

had bariatric surgery are at greater risk for copper deficiency due to their

decreased copper absorption potential High zinc content of certain dental

adhesives is leading to an increase in zinc-induced copper deficiency

There are about a dozen mammalian copper dependent enzymes Table 1

gives the names and functions of the post prevalent ones

Prohaska JR (2014) Impact of copper deficiency in humans AnnNYA-cadSci 1314 1-5

Copper deficiency constantly leads to anemia (lower-than-normal hemoglo-

bin concentration) (13) This may be partly explained based on decreased

activity of copper ferroxidases hephaestin and ceruloplasmin

Altered function of ATP7A when copper is limiting may contribute to the

symptoms However anemia may result from a variety of compromised

copper-dependent processes including impaired erythropoiesis (13)

Iron Deficiency (Activation of CP by HIF-1)

Iron deficiency leading to hypoxia potentiates production of the regulatory

protein hypoxia-inducible factor (HIF-1) HIF-1 is a heterodimeric transcrip-

tion factor complex HIF-11113114a is the key regulatory component of the com-

plex because it is absent under normal conditions but is upregulated by the

iron chelator desferrioxamine (14) Upon activation the HIF-1a1113114HIF-11113114b

dimer binds to hypoxia-responsive elements (HREs) in multiple genes in-

cluding several with important functions in iron metabolism such as ery-

thropoietin heme oxygenase-1 transferrin and transferrin receptor HIF-1

has been shown to activate CP transcription via one putative hypoxia-re-

sponsive element (HRE) in the 5rsquo regulatory region of the CP gene (14)

This was shown by cloning the 5rsquo flanking region of the CP gene from a hu-

man cDNA library This promoterenhancer region driving a luciferase re-

porter was transfected into HepG2 cells Hypoxia increased luciferase ac-

tivity 5-10 fold (14)

Reactive Oxygen Species (ROS) Regulate CP

ROS decrease the synthesis of both the secretory form of Cp (in hepatic

cells) and membrane-bound GPI-Cp (in astroglial cells) by a novel post-

transcriptional mechanism involving its 31113114-UTR (15) The Cp 3rsquo1113114-UTR-

binding protein complex is decreased by ROS causing increased decay of

Cp mRNA Therefore Cp expression is down-regulated by ROS This may

explain the iron deposition and related damage by increased ROS gener-

ation in neurodegenerative diseases (15)

Conclusion

Investigations into iron-copper interactions have revealed novel aspects of

mineral homeostasis Careful observation of the influence of copper on

iron homeostasis has shed light on divalent metal dependant pathological

mechanisms of human disease (1725 words + 539 words in References)

References

1 Bielli P Bellenchi GC and Calabrese L (2001) Site-directed

mutagenesis of human ceruloplasmin production of a proteolyti-

cally stable protein and structure-activity relationships of type 1

sites JBiolChem 276 2678-2685

2 Koschinsky ML Chow BK Schwartz J Hamerton JL and

MacGillivray RT (1987) Isolation and characterization of a pro-

cessed gene for human ceruloplasmin Biochemistry 26 7760-

7767

3 Koschinsky ML Funk WD van Oost BA and MacGillivray

RT (1986) Complete cDNA sequence of human

preceruloplasmin ProcNatlAcadSciUSA 83 5086-5090

4 Fortna RR Watson HA and Nyquist SE (1999) Glycosyl

phosphatidylinositol-anchored ceruloplasmin is expressed by rat

Sertoli cells and is concentrated in detergent-insoluble membrane

fractions BiolReprod 61 1042-1049

5 Calabrese L Carbonaro M and Musci G (1989) Presence of

coupled trinuclear copper cluster in mammalian ceruloplasmin is

essential for efficient electron transfer to oxygen JBiolChem

264 6183-6187

6 Hellman NE Kono S Mancini GM Hoogeboom AJ De

Jong GJ and Gitlin JD (2002) Mechanisms of copper incorpora-

tion into human ceruloplasmin JBiolChem 277 46632-46638

7 Halliwell B and Gutteridge JM (1984) Oxygen toxicity oxy-

gen radicals transition metals and disease BiochemJ 219 1-

1413 Jeong SY and David S (2003) Glycosylphosphatidylinosi-

tol-anchored ceruloplasmin is required for iron efflux from cells in

the central nervous system JBiolChem 278 27144-27148

8 Messerschmidt A Rossi A Ladenstein R Huber R Bolog-

nesi M Gatti G Marchesini A Petruzzelli R and Finazzi-Agro

A (1989) X-ray crystal structure of the blue oxidase ascorbate ox-

idase from zucchini Analysis of the polypeptide fold and a model

of the copper sites and ligands JMolBiol 206 513-529

9 European Association for Study of Liver (2012) EASL Clinical

Practice Guidelines Wilsons disease JHepatol 56 671-685

10 di Patti MC Maio N Rizzo G De Francesco G Persichini

T Colasanti M Polticelli F and Musci G (2009) Dominant mu-

tants of ceruloplasmin impair the copper loading machinery in

aceruloplasminemia JBiolChem 284 4545-4554

11 De Domenico I Ward DM di Patti MC Jeong SY David

S Musci G and Kaplan J (2007) Ferroxidase activity is required

for the stability of cell surface ferroportin in cells expressing GPI-

ceruloplasmin EMBO J 26 2823-2831

12 Chapman AL Mocatta TJ Shiva S Seidel A Chen B

Khalilova I Paumann-Page ME Jameson GN Winterbourn

CC and Kettle AJ (2013) Ceruloplasmin is an endogenous in-

hibitor of myeloperoxidase JBiolChem 288 6465-6477

13 Prohaska JR (2014) Impact of copper deficiency in humans

AnnNYAcadSci 1314 1-5

14 Mukhopadhyay CK Mazumder B and Fox PL (2000) Role

of hypoxia-inducible factor-1 in transcriptional activation of ceru-

loplasmin by iron deficiency JBiolChem 275 21048-21054

15 Tapryal N Mukhopadhyay C Das D Fox PL and

Mukhopadhyay CK (2009) Reactive oxygen species regulate

ceruloplasmin by a novel mRNA decay mechanism involving its 3-

untranslated region implications in neurodegenerative diseases

JBiolChem 284 1873-1883

Ceruloplasmin Deficiency (Menkes disease)

Most dietary copper is absorbed from the proximal small intestine and is di-

rected into the portal circulation with the help of another copper-transport-

ing protein produced by the ATP7A gene Mutations in the ATP7A gene

lead to a copper-deficiency disease called Menkes disease Both of the

ATP7 gene products are required for ceruloplasmin copper loading There-

fore the symptoms and treatment of Menkes are the same as for Wilsonrsquos

(9)

Ceruloplasmin Deficiency (Aceruloplasminemia)

There has been hCP deficiency attributed to autosomal recessive muta-

tions in the hCP gene (10) Heterozygous individuals usually show normal

iron metabolism However in some cases dominant mutations in the hCP

gene lead to iron overload in various parenchyma These mutations were

found to disrupt copper loading of hCP by ATP7B The lack of ferroxidase

activity of hCP has been shown to lead to internalization and degradation

of ferroportin (11)

Most of the iron used each day for hematopoiesis and other essential

needs is recycled from erythrocytes (heme) in the reticuloendothelial sys-

tem Iron transported in the plasma bound to transferrin must be oxidized

prior to binding to this transport protein Ceruloplasmin plays a critical role

in the iron cycle by establishing a rate of iron oxidation sufficient for iron re-

lease from the reticuloendothelial (RI) system The absence of serum ceru-

loplasmin in patients with aceruloplasminemia leads to an accumulation of

iron in the RI system In addition to the accumulation of iron within the RI

system the absence of serum ceruloplasmin also results in increased fer-

rous iron in the plasma which is rapidly removed from the circulation by the

liver pancreas and other tissues When copper cannot be incorporated into

hCP the apoenzyme is quickly degraded Thus a low serum ceruloplasmin

level along with iron accumulation in the basal gan- glia (detected as a

low-intensity signal on T1- and T2-weighted magnetic resonance imaging)

is sufficient for diagnoses

Ceruloplasmin is an endogenous inhibitor of myeloperoxidase (12)

Myeloperoxidase generates hypochlorous acid and reactive radicle

species leading to oxidative stress when it leaks into the plasma hCP defi-

ciency may lead to increased tissue damage in times of increased oxidative

stress Inflammation from cystic fibrosis chronic obstructive pulmonary dis-

ease rheumatoid arthritis and Alzheimer disease may be worsened if hCP

is not in the plasma to act as an antioxidant (12)

Primary Hemochromotosis (Atransferrinemia)

The mechanism of iron overload in aceruloplasminemia is similar to that

which occurs in patients with primary hemochromotosis (PH)(atransferrine-

mia) PH is caused by mutations in the serum transferrin gene leading to

lack of functional transferrin

Copper Deficiency

The daily recommended copper intake is 09 mg (13) Most people get

more than enough from their food and water intake Pregnant and lactating

women persons with inflamed and diseased bowel and persons who have

had bariatric surgery are at greater risk for copper deficiency due to their

decreased copper absorption potential High zinc content of certain dental

adhesives is leading to an increase in zinc-induced copper deficiency

There are about a dozen mammalian copper dependent enzymes Table 1

gives the names and functions of the post prevalent ones

Prohaska JR (2014) Impact of copper deficiency in humans AnnNYA-cadSci 1314 1-5

Copper deficiency constantly leads to anemia (lower-than-normal hemoglo-

bin concentration) (13) This may be partly explained based on decreased

activity of copper ferroxidases hephaestin and ceruloplasmin

Altered function of ATP7A when copper is limiting may contribute to the

symptoms However anemia may result from a variety of compromised

copper-dependent processes including impaired erythropoiesis (13)

Iron Deficiency (Activation of CP by HIF-1)

Iron deficiency leading to hypoxia potentiates production of the regulatory

protein hypoxia-inducible factor (HIF-1) HIF-1 is a heterodimeric transcrip-

tion factor complex HIF-11113114a is the key regulatory component of the com-

plex because it is absent under normal conditions but is upregulated by the

iron chelator desferrioxamine (14) Upon activation the HIF-1a1113114HIF-11113114b

dimer binds to hypoxia-responsive elements (HREs) in multiple genes in-

cluding several with important functions in iron metabolism such as ery-

thropoietin heme oxygenase-1 transferrin and transferrin receptor HIF-1

has been shown to activate CP transcription via one putative hypoxia-re-

sponsive element (HRE) in the 5rsquo regulatory region of the CP gene (14)

This was shown by cloning the 5rsquo flanking region of the CP gene from a hu-

man cDNA library This promoterenhancer region driving a luciferase re-

porter was transfected into HepG2 cells Hypoxia increased luciferase ac-

tivity 5-10 fold (14)

Reactive Oxygen Species (ROS) Regulate CP

ROS decrease the synthesis of both the secretory form of Cp (in hepatic

cells) and membrane-bound GPI-Cp (in astroglial cells) by a novel post-

transcriptional mechanism involving its 31113114-UTR (15) The Cp 3rsquo1113114-UTR-

binding protein complex is decreased by ROS causing increased decay of

Cp mRNA Therefore Cp expression is down-regulated by ROS This may

explain the iron deposition and related damage by increased ROS gener-

ation in neurodegenerative diseases (15)

Conclusion

Investigations into iron-copper interactions have revealed novel aspects of

mineral homeostasis Careful observation of the influence of copper on

iron homeostasis has shed light on divalent metal dependant pathological

mechanisms of human disease (1725 words + 539 words in References)

References

1 Bielli P Bellenchi GC and Calabrese L (2001) Site-directed

mutagenesis of human ceruloplasmin production of a proteolyti-

cally stable protein and structure-activity relationships of type 1

sites JBiolChem 276 2678-2685

2 Koschinsky ML Chow BK Schwartz J Hamerton JL and

MacGillivray RT (1987) Isolation and characterization of a pro-

cessed gene for human ceruloplasmin Biochemistry 26 7760-

7767

3 Koschinsky ML Funk WD van Oost BA and MacGillivray

RT (1986) Complete cDNA sequence of human

preceruloplasmin ProcNatlAcadSciUSA 83 5086-5090

4 Fortna RR Watson HA and Nyquist SE (1999) Glycosyl

phosphatidylinositol-anchored ceruloplasmin is expressed by rat

Sertoli cells and is concentrated in detergent-insoluble membrane

fractions BiolReprod 61 1042-1049

5 Calabrese L Carbonaro M and Musci G (1989) Presence of

coupled trinuclear copper cluster in mammalian ceruloplasmin is

essential for efficient electron transfer to oxygen JBiolChem

264 6183-6187

6 Hellman NE Kono S Mancini GM Hoogeboom AJ De

Jong GJ and Gitlin JD (2002) Mechanisms of copper incorpora-

tion into human ceruloplasmin JBiolChem 277 46632-46638

7 Halliwell B and Gutteridge JM (1984) Oxygen toxicity oxy-

gen radicals transition metals and disease BiochemJ 219 1-

1413 Jeong SY and David S (2003) Glycosylphosphatidylinosi-

tol-anchored ceruloplasmin is required for iron efflux from cells in

the central nervous system JBiolChem 278 27144-27148

8 Messerschmidt A Rossi A Ladenstein R Huber R Bolog-

nesi M Gatti G Marchesini A Petruzzelli R and Finazzi-Agro

A (1989) X-ray crystal structure of the blue oxidase ascorbate ox-

idase from zucchini Analysis of the polypeptide fold and a model

of the copper sites and ligands JMolBiol 206 513-529

9 European Association for Study of Liver (2012) EASL Clinical

Practice Guidelines Wilsons disease JHepatol 56 671-685

10 di Patti MC Maio N Rizzo G De Francesco G Persichini

T Colasanti M Polticelli F and Musci G (2009) Dominant mu-

tants of ceruloplasmin impair the copper loading machinery in

aceruloplasminemia JBiolChem 284 4545-4554

11 De Domenico I Ward DM di Patti MC Jeong SY David

S Musci G and Kaplan J (2007) Ferroxidase activity is required

for the stability of cell surface ferroportin in cells expressing GPI-

ceruloplasmin EMBO J 26 2823-2831

12 Chapman AL Mocatta TJ Shiva S Seidel A Chen B

Khalilova I Paumann-Page ME Jameson GN Winterbourn

CC and Kettle AJ (2013) Ceruloplasmin is an endogenous in-

hibitor of myeloperoxidase JBiolChem 288 6465-6477

13 Prohaska JR (2014) Impact of copper deficiency in humans

AnnNYAcadSci 1314 1-5

14 Mukhopadhyay CK Mazumder B and Fox PL (2000) Role

of hypoxia-inducible factor-1 in transcriptional activation of ceru-

loplasmin by iron deficiency JBiolChem 275 21048-21054

15 Tapryal N Mukhopadhyay C Das D Fox PL and

Mukhopadhyay CK (2009) Reactive oxygen species regulate

ceruloplasmin by a novel mRNA decay mechanism involving its 3-

untranslated region implications in neurodegenerative diseases

JBiolChem 284 1873-1883

tem Iron transported in the plasma bound to transferrin must be oxidized

prior to binding to this transport protein Ceruloplasmin plays a critical role

in the iron cycle by establishing a rate of iron oxidation sufficient for iron re-

lease from the reticuloendothelial (RI) system The absence of serum ceru-

loplasmin in patients with aceruloplasminemia leads to an accumulation of

iron in the RI system In addition to the accumulation of iron within the RI

system the absence of serum ceruloplasmin also results in increased fer-

rous iron in the plasma which is rapidly removed from the circulation by the

liver pancreas and other tissues When copper cannot be incorporated into

hCP the apoenzyme is quickly degraded Thus a low serum ceruloplasmin

level along with iron accumulation in the basal gan- glia (detected as a

low-intensity signal on T1- and T2-weighted magnetic resonance imaging)

is sufficient for diagnoses

Ceruloplasmin is an endogenous inhibitor of myeloperoxidase (12)

Myeloperoxidase generates hypochlorous acid and reactive radicle

species leading to oxidative stress when it leaks into the plasma hCP defi-

ciency may lead to increased tissue damage in times of increased oxidative

stress Inflammation from cystic fibrosis chronic obstructive pulmonary dis-

ease rheumatoid arthritis and Alzheimer disease may be worsened if hCP

is not in the plasma to act as an antioxidant (12)

Primary Hemochromotosis (Atransferrinemia)

The mechanism of iron overload in aceruloplasminemia is similar to that

which occurs in patients with primary hemochromotosis (PH)(atransferrine-

mia) PH is caused by mutations in the serum transferrin gene leading to

lack of functional transferrin

Copper Deficiency

The daily recommended copper intake is 09 mg (13) Most people get

more than enough from their food and water intake Pregnant and lactating

women persons with inflamed and diseased bowel and persons who have

had bariatric surgery are at greater risk for copper deficiency due to their

decreased copper absorption potential High zinc content of certain dental

adhesives is leading to an increase in zinc-induced copper deficiency

There are about a dozen mammalian copper dependent enzymes Table 1

gives the names and functions of the post prevalent ones

Prohaska JR (2014) Impact of copper deficiency in humans AnnNYA-cadSci 1314 1-5

Copper deficiency constantly leads to anemia (lower-than-normal hemoglo-

bin concentration) (13) This may be partly explained based on decreased

activity of copper ferroxidases hephaestin and ceruloplasmin

Altered function of ATP7A when copper is limiting may contribute to the

symptoms However anemia may result from a variety of compromised

copper-dependent processes including impaired erythropoiesis (13)

Iron Deficiency (Activation of CP by HIF-1)

Iron deficiency leading to hypoxia potentiates production of the regulatory

protein hypoxia-inducible factor (HIF-1) HIF-1 is a heterodimeric transcrip-

tion factor complex HIF-11113114a is the key regulatory component of the com-

plex because it is absent under normal conditions but is upregulated by the

iron chelator desferrioxamine (14) Upon activation the HIF-1a1113114HIF-11113114b

dimer binds to hypoxia-responsive elements (HREs) in multiple genes in-

cluding several with important functions in iron metabolism such as ery-

thropoietin heme oxygenase-1 transferrin and transferrin receptor HIF-1

has been shown to activate CP transcription via one putative hypoxia-re-

sponsive element (HRE) in the 5rsquo regulatory region of the CP gene (14)

This was shown by cloning the 5rsquo flanking region of the CP gene from a hu-

man cDNA library This promoterenhancer region driving a luciferase re-

porter was transfected into HepG2 cells Hypoxia increased luciferase ac-

tivity 5-10 fold (14)

Reactive Oxygen Species (ROS) Regulate CP

ROS decrease the synthesis of both the secretory form of Cp (in hepatic

cells) and membrane-bound GPI-Cp (in astroglial cells) by a novel post-

transcriptional mechanism involving its 31113114-UTR (15) The Cp 3rsquo1113114-UTR-

binding protein complex is decreased by ROS causing increased decay of

Cp mRNA Therefore Cp expression is down-regulated by ROS This may

explain the iron deposition and related damage by increased ROS gener-

ation in neurodegenerative diseases (15)

Conclusion

Investigations into iron-copper interactions have revealed novel aspects of

mineral homeostasis Careful observation of the influence of copper on

iron homeostasis has shed light on divalent metal dependant pathological

mechanisms of human disease (1725 words + 539 words in References)

References

1 Bielli P Bellenchi GC and Calabrese L (2001) Site-directed

mutagenesis of human ceruloplasmin production of a proteolyti-

cally stable protein and structure-activity relationships of type 1

sites JBiolChem 276 2678-2685

2 Koschinsky ML Chow BK Schwartz J Hamerton JL and

MacGillivray RT (1987) Isolation and characterization of a pro-

cessed gene for human ceruloplasmin Biochemistry 26 7760-

7767

3 Koschinsky ML Funk WD van Oost BA and MacGillivray

RT (1986) Complete cDNA sequence of human

preceruloplasmin ProcNatlAcadSciUSA 83 5086-5090

4 Fortna RR Watson HA and Nyquist SE (1999) Glycosyl

phosphatidylinositol-anchored ceruloplasmin is expressed by rat

Sertoli cells and is concentrated in detergent-insoluble membrane

fractions BiolReprod 61 1042-1049

5 Calabrese L Carbonaro M and Musci G (1989) Presence of

coupled trinuclear copper cluster in mammalian ceruloplasmin is

essential for efficient electron transfer to oxygen JBiolChem

264 6183-6187

6 Hellman NE Kono S Mancini GM Hoogeboom AJ De

Jong GJ and Gitlin JD (2002) Mechanisms of copper incorpora-

tion into human ceruloplasmin JBiolChem 277 46632-46638

7 Halliwell B and Gutteridge JM (1984) Oxygen toxicity oxy-

gen radicals transition metals and disease BiochemJ 219 1-

1413 Jeong SY and David S (2003) Glycosylphosphatidylinosi-

tol-anchored ceruloplasmin is required for iron efflux from cells in

the central nervous system JBiolChem 278 27144-27148

8 Messerschmidt A Rossi A Ladenstein R Huber R Bolog-

nesi M Gatti G Marchesini A Petruzzelli R and Finazzi-Agro

A (1989) X-ray crystal structure of the blue oxidase ascorbate ox-

idase from zucchini Analysis of the polypeptide fold and a model

of the copper sites and ligands JMolBiol 206 513-529

9 European Association for Study of Liver (2012) EASL Clinical

Practice Guidelines Wilsons disease JHepatol 56 671-685

10 di Patti MC Maio N Rizzo G De Francesco G Persichini

T Colasanti M Polticelli F and Musci G (2009) Dominant mu-

tants of ceruloplasmin impair the copper loading machinery in

aceruloplasminemia JBiolChem 284 4545-4554

11 De Domenico I Ward DM di Patti MC Jeong SY David

S Musci G and Kaplan J (2007) Ferroxidase activity is required

for the stability of cell surface ferroportin in cells expressing GPI-

ceruloplasmin EMBO J 26 2823-2831

12 Chapman AL Mocatta TJ Shiva S Seidel A Chen B

Khalilova I Paumann-Page ME Jameson GN Winterbourn

CC and Kettle AJ (2013) Ceruloplasmin is an endogenous in-

hibitor of myeloperoxidase JBiolChem 288 6465-6477

13 Prohaska JR (2014) Impact of copper deficiency in humans

AnnNYAcadSci 1314 1-5

14 Mukhopadhyay CK Mazumder B and Fox PL (2000) Role

of hypoxia-inducible factor-1 in transcriptional activation of ceru-

loplasmin by iron deficiency JBiolChem 275 21048-21054

15 Tapryal N Mukhopadhyay C Das D Fox PL and

Mukhopadhyay CK (2009) Reactive oxygen species regulate

ceruloplasmin by a novel mRNA decay mechanism involving its 3-

untranslated region implications in neurodegenerative diseases

JBiolChem 284 1873-1883

Primary Hemochromotosis (Atransferrinemia)

The mechanism of iron overload in aceruloplasminemia is similar to that

which occurs in patients with primary hemochromotosis (PH)(atransferrine-

mia) PH is caused by mutations in the serum transferrin gene leading to

lack of functional transferrin

Copper Deficiency

The daily recommended copper intake is 09 mg (13) Most people get

more than enough from their food and water intake Pregnant and lactating

women persons with inflamed and diseased bowel and persons who have

had bariatric surgery are at greater risk for copper deficiency due to their

decreased copper absorption potential High zinc content of certain dental

adhesives is leading to an increase in zinc-induced copper deficiency

There are about a dozen mammalian copper dependent enzymes Table 1

gives the names and functions of the post prevalent ones

Prohaska JR (2014) Impact of copper deficiency in humans AnnNYA-cadSci 1314 1-5

Copper deficiency constantly leads to anemia (lower-than-normal hemoglo-

bin concentration) (13) This may be partly explained based on decreased

activity of copper ferroxidases hephaestin and ceruloplasmin

Altered function of ATP7A when copper is limiting may contribute to the

symptoms However anemia may result from a variety of compromised

copper-dependent processes including impaired erythropoiesis (13)

Iron Deficiency (Activation of CP by HIF-1)

Iron deficiency leading to hypoxia potentiates production of the regulatory

protein hypoxia-inducible factor (HIF-1) HIF-1 is a heterodimeric transcrip-

tion factor complex HIF-11113114a is the key regulatory component of the com-

plex because it is absent under normal conditions but is upregulated by the

iron chelator desferrioxamine (14) Upon activation the HIF-1a1113114HIF-11113114b

dimer binds to hypoxia-responsive elements (HREs) in multiple genes in-

cluding several with important functions in iron metabolism such as ery-

thropoietin heme oxygenase-1 transferrin and transferrin receptor HIF-1

has been shown to activate CP transcription via one putative hypoxia-re-

sponsive element (HRE) in the 5rsquo regulatory region of the CP gene (14)

This was shown by cloning the 5rsquo flanking region of the CP gene from a hu-

man cDNA library This promoterenhancer region driving a luciferase re-

porter was transfected into HepG2 cells Hypoxia increased luciferase ac-

tivity 5-10 fold (14)

Reactive Oxygen Species (ROS) Regulate CP

ROS decrease the synthesis of both the secretory form of Cp (in hepatic

cells) and membrane-bound GPI-Cp (in astroglial cells) by a novel post-

transcriptional mechanism involving its 31113114-UTR (15) The Cp 3rsquo1113114-UTR-

binding protein complex is decreased by ROS causing increased decay of

Cp mRNA Therefore Cp expression is down-regulated by ROS This may

explain the iron deposition and related damage by increased ROS gener-

ation in neurodegenerative diseases (15)

Conclusion

Investigations into iron-copper interactions have revealed novel aspects of

mineral homeostasis Careful observation of the influence of copper on

iron homeostasis has shed light on divalent metal dependant pathological

mechanisms of human disease (1725 words + 539 words in References)

References

1 Bielli P Bellenchi GC and Calabrese L (2001) Site-directed

mutagenesis of human ceruloplasmin production of a proteolyti-

cally stable protein and structure-activity relationships of type 1

sites JBiolChem 276 2678-2685

2 Koschinsky ML Chow BK Schwartz J Hamerton JL and

MacGillivray RT (1987) Isolation and characterization of a pro-

cessed gene for human ceruloplasmin Biochemistry 26 7760-

7767

3 Koschinsky ML Funk WD van Oost BA and MacGillivray

RT (1986) Complete cDNA sequence of human

preceruloplasmin ProcNatlAcadSciUSA 83 5086-5090

4 Fortna RR Watson HA and Nyquist SE (1999) Glycosyl

phosphatidylinositol-anchored ceruloplasmin is expressed by rat

Sertoli cells and is concentrated in detergent-insoluble membrane

fractions BiolReprod 61 1042-1049

5 Calabrese L Carbonaro M and Musci G (1989) Presence of

coupled trinuclear copper cluster in mammalian ceruloplasmin is

essential for efficient electron transfer to oxygen JBiolChem

264 6183-6187

6 Hellman NE Kono S Mancini GM Hoogeboom AJ De

Jong GJ and Gitlin JD (2002) Mechanisms of copper incorpora-

tion into human ceruloplasmin JBiolChem 277 46632-46638

7 Halliwell B and Gutteridge JM (1984) Oxygen toxicity oxy-

gen radicals transition metals and disease BiochemJ 219 1-

1413 Jeong SY and David S (2003) Glycosylphosphatidylinosi-

tol-anchored ceruloplasmin is required for iron efflux from cells in

the central nervous system JBiolChem 278 27144-27148

8 Messerschmidt A Rossi A Ladenstein R Huber R Bolog-

nesi M Gatti G Marchesini A Petruzzelli R and Finazzi-Agro

A (1989) X-ray crystal structure of the blue oxidase ascorbate ox-

idase from zucchini Analysis of the polypeptide fold and a model

of the copper sites and ligands JMolBiol 206 513-529

9 European Association for Study of Liver (2012) EASL Clinical

Practice Guidelines Wilsons disease JHepatol 56 671-685

10 di Patti MC Maio N Rizzo G De Francesco G Persichini

T Colasanti M Polticelli F and Musci G (2009) Dominant mu-

tants of ceruloplasmin impair the copper loading machinery in

aceruloplasminemia JBiolChem 284 4545-4554

11 De Domenico I Ward DM di Patti MC Jeong SY David

S Musci G and Kaplan J (2007) Ferroxidase activity is required

for the stability of cell surface ferroportin in cells expressing GPI-

ceruloplasmin EMBO J 26 2823-2831

12 Chapman AL Mocatta TJ Shiva S Seidel A Chen B

Khalilova I Paumann-Page ME Jameson GN Winterbourn

CC and Kettle AJ (2013) Ceruloplasmin is an endogenous in-

hibitor of myeloperoxidase JBiolChem 288 6465-6477

13 Prohaska JR (2014) Impact of copper deficiency in humans

AnnNYAcadSci 1314 1-5

14 Mukhopadhyay CK Mazumder B and Fox PL (2000) Role

of hypoxia-inducible factor-1 in transcriptional activation of ceru-

loplasmin by iron deficiency JBiolChem 275 21048-21054

15 Tapryal N Mukhopadhyay C Das D Fox PL and

Mukhopadhyay CK (2009) Reactive oxygen species regulate

ceruloplasmin by a novel mRNA decay mechanism involving its 3-

untranslated region implications in neurodegenerative diseases

JBiolChem 284 1873-1883

Prohaska JR (2014) Impact of copper deficiency in humans AnnNYA-cadSci 1314 1-5

Copper deficiency constantly leads to anemia (lower-than-normal hemoglo-

bin concentration) (13) This may be partly explained based on decreased

activity of copper ferroxidases hephaestin and ceruloplasmin

Altered function of ATP7A when copper is limiting may contribute to the

symptoms However anemia may result from a variety of compromised

copper-dependent processes including impaired erythropoiesis (13)

Iron Deficiency (Activation of CP by HIF-1)

Iron deficiency leading to hypoxia potentiates production of the regulatory

protein hypoxia-inducible factor (HIF-1) HIF-1 is a heterodimeric transcrip-

tion factor complex HIF-11113114a is the key regulatory component of the com-

plex because it is absent under normal conditions but is upregulated by the

iron chelator desferrioxamine (14) Upon activation the HIF-1a1113114HIF-11113114b

dimer binds to hypoxia-responsive elements (HREs) in multiple genes in-

cluding several with important functions in iron metabolism such as ery-

thropoietin heme oxygenase-1 transferrin and transferrin receptor HIF-1

has been shown to activate CP transcription via one putative hypoxia-re-

sponsive element (HRE) in the 5rsquo regulatory region of the CP gene (14)

This was shown by cloning the 5rsquo flanking region of the CP gene from a hu-

man cDNA library This promoterenhancer region driving a luciferase re-

porter was transfected into HepG2 cells Hypoxia increased luciferase ac-

tivity 5-10 fold (14)

Reactive Oxygen Species (ROS) Regulate CP

ROS decrease the synthesis of both the secretory form of Cp (in hepatic

cells) and membrane-bound GPI-Cp (in astroglial cells) by a novel post-

transcriptional mechanism involving its 31113114-UTR (15) The Cp 3rsquo1113114-UTR-

binding protein complex is decreased by ROS causing increased decay of

Cp mRNA Therefore Cp expression is down-regulated by ROS This may

explain the iron deposition and related damage by increased ROS gener-

ation in neurodegenerative diseases (15)

Conclusion

Investigations into iron-copper interactions have revealed novel aspects of

mineral homeostasis Careful observation of the influence of copper on

iron homeostasis has shed light on divalent metal dependant pathological

mechanisms of human disease (1725 words + 539 words in References)

References

1 Bielli P Bellenchi GC and Calabrese L (2001) Site-directed

mutagenesis of human ceruloplasmin production of a proteolyti-

cally stable protein and structure-activity relationships of type 1

sites JBiolChem 276 2678-2685

2 Koschinsky ML Chow BK Schwartz J Hamerton JL and

MacGillivray RT (1987) Isolation and characterization of a pro-

cessed gene for human ceruloplasmin Biochemistry 26 7760-

7767

3 Koschinsky ML Funk WD van Oost BA and MacGillivray

RT (1986) Complete cDNA sequence of human

preceruloplasmin ProcNatlAcadSciUSA 83 5086-5090

4 Fortna RR Watson HA and Nyquist SE (1999) Glycosyl

phosphatidylinositol-anchored ceruloplasmin is expressed by rat

Sertoli cells and is concentrated in detergent-insoluble membrane

fractions BiolReprod 61 1042-1049

5 Calabrese L Carbonaro M and Musci G (1989) Presence of

coupled trinuclear copper cluster in mammalian ceruloplasmin is

essential for efficient electron transfer to oxygen JBiolChem

264 6183-6187

6 Hellman NE Kono S Mancini GM Hoogeboom AJ De

Jong GJ and Gitlin JD (2002) Mechanisms of copper incorpora-

tion into human ceruloplasmin JBiolChem 277 46632-46638

7 Halliwell B and Gutteridge JM (1984) Oxygen toxicity oxy-

gen radicals transition metals and disease BiochemJ 219 1-

1413 Jeong SY and David S (2003) Glycosylphosphatidylinosi-

tol-anchored ceruloplasmin is required for iron efflux from cells in

the central nervous system JBiolChem 278 27144-27148

8 Messerschmidt A Rossi A Ladenstein R Huber R Bolog-

nesi M Gatti G Marchesini A Petruzzelli R and Finazzi-Agro

A (1989) X-ray crystal structure of the blue oxidase ascorbate ox-

idase from zucchini Analysis of the polypeptide fold and a model

of the copper sites and ligands JMolBiol 206 513-529

9 European Association for Study of Liver (2012) EASL Clinical

Practice Guidelines Wilsons disease JHepatol 56 671-685

10 di Patti MC Maio N Rizzo G De Francesco G Persichini

T Colasanti M Polticelli F and Musci G (2009) Dominant mu-

tants of ceruloplasmin impair the copper loading machinery in

aceruloplasminemia JBiolChem 284 4545-4554

11 De Domenico I Ward DM di Patti MC Jeong SY David

S Musci G and Kaplan J (2007) Ferroxidase activity is required

for the stability of cell surface ferroportin in cells expressing GPI-

ceruloplasmin EMBO J 26 2823-2831

12 Chapman AL Mocatta TJ Shiva S Seidel A Chen B

Khalilova I Paumann-Page ME Jameson GN Winterbourn

CC and Kettle AJ (2013) Ceruloplasmin is an endogenous in-

hibitor of myeloperoxidase JBiolChem 288 6465-6477

13 Prohaska JR (2014) Impact of copper deficiency in humans

AnnNYAcadSci 1314 1-5

14 Mukhopadhyay CK Mazumder B and Fox PL (2000) Role

of hypoxia-inducible factor-1 in transcriptional activation of ceru-

loplasmin by iron deficiency JBiolChem 275 21048-21054

15 Tapryal N Mukhopadhyay C Das D Fox PL and

Mukhopadhyay CK (2009) Reactive oxygen species regulate

ceruloplasmin by a novel mRNA decay mechanism involving its 3-

untranslated region implications in neurodegenerative diseases

JBiolChem 284 1873-1883

Altered function of ATP7A when copper is limiting may contribute to the

symptoms However anemia may result from a variety of compromised

copper-dependent processes including impaired erythropoiesis (13)

Iron Deficiency (Activation of CP by HIF-1)

Iron deficiency leading to hypoxia potentiates production of the regulatory

protein hypoxia-inducible factor (HIF-1) HIF-1 is a heterodimeric transcrip-

tion factor complex HIF-11113114a is the key regulatory component of the com-

plex because it is absent under normal conditions but is upregulated by the

iron chelator desferrioxamine (14) Upon activation the HIF-1a1113114HIF-11113114b

dimer binds to hypoxia-responsive elements (HREs) in multiple genes in-

cluding several with important functions in iron metabolism such as ery-

thropoietin heme oxygenase-1 transferrin and transferrin receptor HIF-1

has been shown to activate CP transcription via one putative hypoxia-re-

sponsive element (HRE) in the 5rsquo regulatory region of the CP gene (14)

This was shown by cloning the 5rsquo flanking region of the CP gene from a hu-

man cDNA library This promoterenhancer region driving a luciferase re-

porter was transfected into HepG2 cells Hypoxia increased luciferase ac-

tivity 5-10 fold (14)

Reactive Oxygen Species (ROS) Regulate CP

ROS decrease the synthesis of both the secretory form of Cp (in hepatic

cells) and membrane-bound GPI-Cp (in astroglial cells) by a novel post-

transcriptional mechanism involving its 31113114-UTR (15) The Cp 3rsquo1113114-UTR-

binding protein complex is decreased by ROS causing increased decay of

Cp mRNA Therefore Cp expression is down-regulated by ROS This may

explain the iron deposition and related damage by increased ROS gener-

ation in neurodegenerative diseases (15)

Conclusion

Investigations into iron-copper interactions have revealed novel aspects of

mineral homeostasis Careful observation of the influence of copper on

iron homeostasis has shed light on divalent metal dependant pathological

mechanisms of human disease (1725 words + 539 words in References)

References

1 Bielli P Bellenchi GC and Calabrese L (2001) Site-directed

mutagenesis of human ceruloplasmin production of a proteolyti-

cally stable protein and structure-activity relationships of type 1

sites JBiolChem 276 2678-2685

2 Koschinsky ML Chow BK Schwartz J Hamerton JL and

MacGillivray RT (1987) Isolation and characterization of a pro-

cessed gene for human ceruloplasmin Biochemistry 26 7760-

7767

3 Koschinsky ML Funk WD van Oost BA and MacGillivray

RT (1986) Complete cDNA sequence of human

preceruloplasmin ProcNatlAcadSciUSA 83 5086-5090

4 Fortna RR Watson HA and Nyquist SE (1999) Glycosyl

phosphatidylinositol-anchored ceruloplasmin is expressed by rat

Sertoli cells and is concentrated in detergent-insoluble membrane

fractions BiolReprod 61 1042-1049

5 Calabrese L Carbonaro M and Musci G (1989) Presence of

coupled trinuclear copper cluster in mammalian ceruloplasmin is

essential for efficient electron transfer to oxygen JBiolChem

264 6183-6187

6 Hellman NE Kono S Mancini GM Hoogeboom AJ De

Jong GJ and Gitlin JD (2002) Mechanisms of copper incorpora-

tion into human ceruloplasmin JBiolChem 277 46632-46638

7 Halliwell B and Gutteridge JM (1984) Oxygen toxicity oxy-

gen radicals transition metals and disease BiochemJ 219 1-

1413 Jeong SY and David S (2003) Glycosylphosphatidylinosi-

tol-anchored ceruloplasmin is required for iron efflux from cells in

the central nervous system JBiolChem 278 27144-27148

8 Messerschmidt A Rossi A Ladenstein R Huber R Bolog-

nesi M Gatti G Marchesini A Petruzzelli R and Finazzi-Agro

A (1989) X-ray crystal structure of the blue oxidase ascorbate ox-

idase from zucchini Analysis of the polypeptide fold and a model

of the copper sites and ligands JMolBiol 206 513-529

9 European Association for Study of Liver (2012) EASL Clinical

Practice Guidelines Wilsons disease JHepatol 56 671-685

10 di Patti MC Maio N Rizzo G De Francesco G Persichini

T Colasanti M Polticelli F and Musci G (2009) Dominant mu-

tants of ceruloplasmin impair the copper loading machinery in

aceruloplasminemia JBiolChem 284 4545-4554

11 De Domenico I Ward DM di Patti MC Jeong SY David

S Musci G and Kaplan J (2007) Ferroxidase activity is required

for the stability of cell surface ferroportin in cells expressing GPI-

ceruloplasmin EMBO J 26 2823-2831

12 Chapman AL Mocatta TJ Shiva S Seidel A Chen B

Khalilova I Paumann-Page ME Jameson GN Winterbourn

CC and Kettle AJ (2013) Ceruloplasmin is an endogenous in-

hibitor of myeloperoxidase JBiolChem 288 6465-6477

13 Prohaska JR (2014) Impact of copper deficiency in humans

AnnNYAcadSci 1314 1-5

14 Mukhopadhyay CK Mazumder B and Fox PL (2000) Role

of hypoxia-inducible factor-1 in transcriptional activation of ceru-

loplasmin by iron deficiency JBiolChem 275 21048-21054

15 Tapryal N Mukhopadhyay C Das D Fox PL and

Mukhopadhyay CK (2009) Reactive oxygen species regulate

ceruloplasmin by a novel mRNA decay mechanism involving its 3-

untranslated region implications in neurodegenerative diseases

JBiolChem 284 1873-1883

Reactive Oxygen Species (ROS) Regulate CP

ROS decrease the synthesis of both the secretory form of Cp (in hepatic

cells) and membrane-bound GPI-Cp (in astroglial cells) by a novel post-

transcriptional mechanism involving its 31113114-UTR (15) The Cp 3rsquo1113114-UTR-

binding protein complex is decreased by ROS causing increased decay of

Cp mRNA Therefore Cp expression is down-regulated by ROS This may

explain the iron deposition and related damage by increased ROS gener-

ation in neurodegenerative diseases (15)

Conclusion

Investigations into iron-copper interactions have revealed novel aspects of

mineral homeostasis Careful observation of the influence of copper on

iron homeostasis has shed light on divalent metal dependant pathological

mechanisms of human disease (1725 words + 539 words in References)

References

1 Bielli P Bellenchi GC and Calabrese L (2001) Site-directed

mutagenesis of human ceruloplasmin production of a proteolyti-

cally stable protein and structure-activity relationships of type 1

sites JBiolChem 276 2678-2685

2 Koschinsky ML Chow BK Schwartz J Hamerton JL and

MacGillivray RT (1987) Isolation and characterization of a pro-

cessed gene for human ceruloplasmin Biochemistry 26 7760-

7767

3 Koschinsky ML Funk WD van Oost BA and MacGillivray

RT (1986) Complete cDNA sequence of human

preceruloplasmin ProcNatlAcadSciUSA 83 5086-5090

4 Fortna RR Watson HA and Nyquist SE (1999) Glycosyl

phosphatidylinositol-anchored ceruloplasmin is expressed by rat

Sertoli cells and is concentrated in detergent-insoluble membrane

fractions BiolReprod 61 1042-1049

5 Calabrese L Carbonaro M and Musci G (1989) Presence of

coupled trinuclear copper cluster in mammalian ceruloplasmin is

essential for efficient electron transfer to oxygen JBiolChem

264 6183-6187

6 Hellman NE Kono S Mancini GM Hoogeboom AJ De

Jong GJ and Gitlin JD (2002) Mechanisms of copper incorpora-

tion into human ceruloplasmin JBiolChem 277 46632-46638

7 Halliwell B and Gutteridge JM (1984) Oxygen toxicity oxy-

gen radicals transition metals and disease BiochemJ 219 1-

1413 Jeong SY and David S (2003) Glycosylphosphatidylinosi-

tol-anchored ceruloplasmin is required for iron efflux from cells in

the central nervous system JBiolChem 278 27144-27148

8 Messerschmidt A Rossi A Ladenstein R Huber R Bolog-

nesi M Gatti G Marchesini A Petruzzelli R and Finazzi-Agro

A (1989) X-ray crystal structure of the blue oxidase ascorbate ox-

idase from zucchini Analysis of the polypeptide fold and a model

of the copper sites and ligands JMolBiol 206 513-529

9 European Association for Study of Liver (2012) EASL Clinical

Practice Guidelines Wilsons disease JHepatol 56 671-685

10 di Patti MC Maio N Rizzo G De Francesco G Persichini

T Colasanti M Polticelli F and Musci G (2009) Dominant mu-

tants of ceruloplasmin impair the copper loading machinery in

aceruloplasminemia JBiolChem 284 4545-4554

11 De Domenico I Ward DM di Patti MC Jeong SY David

S Musci G and Kaplan J (2007) Ferroxidase activity is required

for the stability of cell surface ferroportin in cells expressing GPI-

ceruloplasmin EMBO J 26 2823-2831

12 Chapman AL Mocatta TJ Shiva S Seidel A Chen B

Khalilova I Paumann-Page ME Jameson GN Winterbourn

CC and Kettle AJ (2013) Ceruloplasmin is an endogenous in-

hibitor of myeloperoxidase JBiolChem 288 6465-6477

13 Prohaska JR (2014) Impact of copper deficiency in humans

AnnNYAcadSci 1314 1-5

14 Mukhopadhyay CK Mazumder B and Fox PL (2000) Role

of hypoxia-inducible factor-1 in transcriptional activation of ceru-

loplasmin by iron deficiency JBiolChem 275 21048-21054

15 Tapryal N Mukhopadhyay C Das D Fox PL and

Mukhopadhyay CK (2009) Reactive oxygen species regulate

ceruloplasmin by a novel mRNA decay mechanism involving its 3-

untranslated region implications in neurodegenerative diseases

JBiolChem 284 1873-1883

cally stable protein and structure-activity relationships of type 1

sites JBiolChem 276 2678-2685

2 Koschinsky ML Chow BK Schwartz J Hamerton JL and

MacGillivray RT (1987) Isolation and characterization of a pro-

cessed gene for human ceruloplasmin Biochemistry 26 7760-

7767

3 Koschinsky ML Funk WD van Oost BA and MacGillivray

RT (1986) Complete cDNA sequence of human

preceruloplasmin ProcNatlAcadSciUSA 83 5086-5090

4 Fortna RR Watson HA and Nyquist SE (1999) Glycosyl

phosphatidylinositol-anchored ceruloplasmin is expressed by rat

Sertoli cells and is concentrated in detergent-insoluble membrane

fractions BiolReprod 61 1042-1049

5 Calabrese L Carbonaro M and Musci G (1989) Presence of

coupled trinuclear copper cluster in mammalian ceruloplasmin is

essential for efficient electron transfer to oxygen JBiolChem

264 6183-6187

6 Hellman NE Kono S Mancini GM Hoogeboom AJ De

Jong GJ and Gitlin JD (2002) Mechanisms of copper incorpora-

tion into human ceruloplasmin JBiolChem 277 46632-46638

7 Halliwell B and Gutteridge JM (1984) Oxygen toxicity oxy-

gen radicals transition metals and disease BiochemJ 219 1-

1413 Jeong SY and David S (2003) Glycosylphosphatidylinosi-

tol-anchored ceruloplasmin is required for iron efflux from cells in

the central nervous system JBiolChem 278 27144-27148

8 Messerschmidt A Rossi A Ladenstein R Huber R Bolog-

nesi M Gatti G Marchesini A Petruzzelli R and Finazzi-Agro

A (1989) X-ray crystal structure of the blue oxidase ascorbate ox-

idase from zucchini Analysis of the polypeptide fold and a model

of the copper sites and ligands JMolBiol 206 513-529

9 European Association for Study of Liver (2012) EASL Clinical

Practice Guidelines Wilsons disease JHepatol 56 671-685

10 di Patti MC Maio N Rizzo G De Francesco G Persichini

T Colasanti M Polticelli F and Musci G (2009) Dominant mu-

tants of ceruloplasmin impair the copper loading machinery in

aceruloplasminemia JBiolChem 284 4545-4554

11 De Domenico I Ward DM di Patti MC Jeong SY David

S Musci G and Kaplan J (2007) Ferroxidase activity is required

for the stability of cell surface ferroportin in cells expressing GPI-

ceruloplasmin EMBO J 26 2823-2831

12 Chapman AL Mocatta TJ Shiva S Seidel A Chen B

Khalilova I Paumann-Page ME Jameson GN Winterbourn

CC and Kettle AJ (2013) Ceruloplasmin is an endogenous in-

hibitor of myeloperoxidase JBiolChem 288 6465-6477

13 Prohaska JR (2014) Impact of copper deficiency in humans

AnnNYAcadSci 1314 1-5

14 Mukhopadhyay CK Mazumder B and Fox PL (2000) Role

of hypoxia-inducible factor-1 in transcriptional activation of ceru-

loplasmin by iron deficiency JBiolChem 275 21048-21054

15 Tapryal N Mukhopadhyay C Das D Fox PL and

Mukhopadhyay CK (2009) Reactive oxygen species regulate

ceruloplasmin by a novel mRNA decay mechanism involving its 3-

untranslated region implications in neurodegenerative diseases

JBiolChem 284 1873-1883

6 Hellman NE Kono S Mancini GM Hoogeboom AJ De

Jong GJ and Gitlin JD (2002) Mechanisms of copper incorpora-

tion into human ceruloplasmin JBiolChem 277 46632-46638

7 Halliwell B and Gutteridge JM (1984) Oxygen toxicity oxy-

gen radicals transition metals and disease BiochemJ 219 1-

1413 Jeong SY and David S (2003) Glycosylphosphatidylinosi-

tol-anchored ceruloplasmin is required for iron efflux from cells in

the central nervous system JBiolChem 278 27144-27148

8 Messerschmidt A Rossi A Ladenstein R Huber R Bolog-

nesi M Gatti G Marchesini A Petruzzelli R and Finazzi-Agro

A (1989) X-ray crystal structure of the blue oxidase ascorbate ox-

idase from zucchini Analysis of the polypeptide fold and a model

of the copper sites and ligands JMolBiol 206 513-529

9 European Association for Study of Liver (2012) EASL Clinical

Practice Guidelines Wilsons disease JHepatol 56 671-685

10 di Patti MC Maio N Rizzo G De Francesco G Persichini

T Colasanti M Polticelli F and Musci G (2009) Dominant mu-

tants of ceruloplasmin impair the copper loading machinery in

aceruloplasminemia JBiolChem 284 4545-4554

11 De Domenico I Ward DM di Patti MC Jeong SY David

S Musci G and Kaplan J (2007) Ferroxidase activity is required

for the stability of cell surface ferroportin in cells expressing GPI-

ceruloplasmin EMBO J 26 2823-2831

12 Chapman AL Mocatta TJ Shiva S Seidel A Chen B

Khalilova I Paumann-Page ME Jameson GN Winterbourn

CC and Kettle AJ (2013) Ceruloplasmin is an endogenous in-

hibitor of myeloperoxidase JBiolChem 288 6465-6477

13 Prohaska JR (2014) Impact of copper deficiency in humans

AnnNYAcadSci 1314 1-5

14 Mukhopadhyay CK Mazumder B and Fox PL (2000) Role

of hypoxia-inducible factor-1 in transcriptional activation of ceru-

loplasmin by iron deficiency JBiolChem 275 21048-21054

15 Tapryal N Mukhopadhyay C Das D Fox PL and

Mukhopadhyay CK (2009) Reactive oxygen species regulate

ceruloplasmin by a novel mRNA decay mechanism involving its 3-

untranslated region implications in neurodegenerative diseases

JBiolChem 284 1873-1883

10 di Patti MC Maio N Rizzo G De Francesco G Persichini

T Colasanti M Polticelli F and Musci G (2009) Dominant mu-

tants of ceruloplasmin impair the copper loading machinery in

aceruloplasminemia JBiolChem 284 4545-4554

11 De Domenico I Ward DM di Patti MC Jeong SY David

S Musci G and Kaplan J (2007) Ferroxidase activity is required

for the stability of cell surface ferroportin in cells expressing GPI-

ceruloplasmin EMBO J 26 2823-2831

12 Chapman AL Mocatta TJ Shiva S Seidel A Chen B

Khalilova I Paumann-Page ME Jameson GN Winterbourn

CC and Kettle AJ (2013) Ceruloplasmin is an endogenous in-

hibitor of myeloperoxidase JBiolChem 288 6465-6477

13 Prohaska JR (2014) Impact of copper deficiency in humans

AnnNYAcadSci 1314 1-5

14 Mukhopadhyay CK Mazumder B and Fox PL (2000) Role

of hypoxia-inducible factor-1 in transcriptional activation of ceru-

loplasmin by iron deficiency JBiolChem 275 21048-21054

15 Tapryal N Mukhopadhyay C Das D Fox PL and

Mukhopadhyay CK (2009) Reactive oxygen species regulate

ceruloplasmin by a novel mRNA decay mechanism involving its 3-

untranslated region implications in neurodegenerative diseases

JBiolChem 284 1873-1883

15 Tapryal N Mukhopadhyay C Das D Fox PL and

Mukhopadhyay CK (2009) Reactive oxygen species regulate

ceruloplasmin by a novel mRNA decay mechanism involving its 3-

untranslated region implications in neurodegenerative diseases

JBiolChem 284 1873-1883