Biological relevance of Cu

Cu is an essential micronutrient required by all lifeforms. Cu is a transition metal and hence involved in avariety of biological processes viz., embryonicdevelopment, mitochondrial respiration, regulation ofhemoglobin levels as well as hepatocyte and neuronalfunctions. Being a transition metal, Cu gets biologicallyconverted between different redox states namelyoxidized Cu (II) and reduced Cu (I). This uniqueattribute has made Cu metal to get manifested as animportant catalytic co-factor for a variety of metabolicreactions in biological systems. Several reviews1-5

highlighted the participation of Cu in a myriad cellularactivities and physiological processes such as cellular

respiration, iron metabolism, biosynthesis ofneurotransmitter, and free radical detoxification.Therefore, it is worth recalling that Cu is vital for normalhealthy functioning of organisms (Fig. 1).

Source of copper: Rich amounts of copper along withother essential elements found in the soil are taken upby plants using very elaborate transportation machinery.Plants, thus serve as a direct source of elemental copperfor higher organisms. Additionally, human breast milkhas the highest concentration of Cu (0.25 to 6.0 mg/l).For its effective utilization, the elemental copper derivedfrom these sources needs to be absorbed and transportedto metabolically active sites. This process, termedbioavailability, is possibly regulated by four essential

Copper & biological health

S. Krupanidhi, Arun Sreekumar* & C.B. Sanjeevi**

Department of Biosciences, Sri Sathya Sai University, Prasanthi Nilayam, India; *Michigan Center forTranslational Pathology & Department of Pathology, University of Michigan, Ann Arbor, MI, USA &**Center for Molecular Medicine, Department of Molecular Medicine & Surgery, Karolinska Hospital,Stockholm, Sweden

Received January 28, 2008

Cu being a transition metal is ubiquitously engaged in biological systems to derive electrons throughits participation in several enzymatic reactions. Upon bestowing the significance of Cu in biologicalsystems, an elaborate mechanism is set forth by nature for maintaining Cu homeostasis. As aconsequence, a wide variety of proteins viz., family of Cu bearing proteins, cuproenzymes, Cutransporters and Cu chaperone proteins have been manifested for enabling Cu to show its relevancein biological health. In addition, understanding the role of Cu in hepatic and neuronal functionsand also in angiogenesis keeps progressing with the advent of novel molecular tools. The studies ongenetic defects in Cu metabolism causing abnormalities are providing insights leading to the possibleprognostic cues to alleviate the sufferings.

Key words Ceruloplasmin - Cu carriers - Cu chaperones - Cu chelators - Cu metabolism - Cu transporters - Menkes disease -oxygen binding Cu proteins - Wilson’s disease

Indian J Med Res 128, October 2008, pp 448-461

Review Article

448

attributes as defined by Raul6. These include (i) quantumof intake; (ii) dependent variability; (iii) linearitybetween dose and response; and (iv) slope ratio analysis.Solubility of Cu in water or physiological fluids is agood indicator of bioavailability and digestibility.Additionally, copper complexes with variousbiomolecules, thus, facilitating its utilization. Theseinclude complex of copper with lectins andglycoproteins as seen in grains or with amino acids asnoticed in higher organisms including mammals. In fact,amino acids exert a critical role in uptake of copper bythe intestinal membranes. Further, among the essentialamino acids, methionine in the diet enhances Cuabsorption by at least 2-fold. On the contrary, by the sideof cysteine due to its ability to chelate by the side ofcopper coupled to its ability to potentially reduce copperto a monovalent state7, leads to a reduction in itsbioavailability. On a similar note, tripeptide of glutathionehas significant post-absorptive importance in Cutransport. Glutathione forms an intermediary complexwith Cu in the enterocytes before transferring the metalto other target proteins- viz., superoxide dismutase orceruloplamin (CP), etc., thus facilitating its assimilation.This ability of copper to complex with amino acids ororganic acids is extensively exploited in animal nutritionexperiments. As an example, Cu-lysine complex has beenshown to be effective as a supplement in feed for chicksthan for lambs8. Similarly, formulations of proteins withminerals, also termed proteinates, have been found to behighly effective as feed for growing calves in areas withhigh molybdenum contents in foliage9. Notably,

molybdenum competitively inhibits intestinal Cu uptake.On the contrary, various derivatives of copper such aschlorides, acetates, sulfates and carbonates enhance itsbioavailability in higher organisms.

Plants as bioindicators and hyperaccumulators of Cu:A few plants uniquely accumulate copper from theirhabitats viz., soil or water. They are: Aeolanthusbiformifolius, Athyrium yokoscense, Azolla filiculoides,Bacopa monnieri, Brassica juncea L., CallisneriaAmericana, Eichhornia crassipes, Haumaniustrumrobertii, Helianthus annuus, Larrea tridentate, Lemnaminor, Pistia stratiotes and Thlaspi caerulescens. Themetal molybdenum is also accumulated by Thlaspicaerulescens (Brassica). Sheep feeding on Thlaspicaerulescens possibly face the deficiency of copper asthis specific plant is also a hyperaccumulator ofmolybdenum which inhibits intestinal copper uptake.

Copper transport and utilization

Dietary copper, absorbed in the stomach and upperintestinal tract, reaches liver as a complex with serumproteins viz., albumin or transcuperin or the amino acidhistidine10. Importantly, liver is the major store housefor intracellular copper11. Here, copper is reduced tocupric state and transported across plasma membraneby CTR1 transporters as described later. Importantly,as highlighted later in this review, intracellular copperneeds to be maintained in a complex state so as toprevent the oxidative damage caused by free copper toDNA, proteins and membrane components10. Hence,copper transport and utilization involves a complexinterplay between transporters and binding proteins/chaperones. Additionally, Cu plays a vital role as acatalytic co-factor for a variety of metalloenzymes.Keeping the importance of cupric Cu in biologicalfunction, an elaborate mechanism is set forth by Naturefor maintaining Cu homeostasis, which includes a widearray of proteins namely (i) family of Cu bearingproteins, (ii) cuproenzymes, (iii) Cu transporters and(iv) Cu chaperone proteins. It is not surprising for theredundant machinery that Cu is enjoying out of severalheavy metals for its transport and participation incellular metabolism, which guarantees the survival ofliving organisms as conditioned by the strategies andmechanisms of the evolution of metallic proteins.

The family of Cu bearing proteins plays a significantrole in metal detoxification and keeps the Cu in non ioniccurpric state. They are metallothioneins, prion protein,albumin, transcuperin, CP, phycocyanins of blue greenalgae and haemocyanins of blue blooded organisms.

KRUPANIDHI et al: COPPER & BIOLOGICAL HEALTH 449

Fig. 1. Influence of Cu deficiency or excess on the response oforganisms. (1) physiological efficiency, (2) tolerable levels withinbuilt compensatory mechanisms, (3) therapeutic symptomsleading to fatal diseases.

Blue blooded organisms: An interesting copper bindingprotein found in some of the lower eukaryotes ishemocyanin (Hcy). Fig. 2 shows the UV spectrum ofOxy-Hemocyanin (Oxy-Hcy) with a characteristicabsorbance at 340 nm revealing the presence of copper-oxygen complex. Hcys are found in a majority ofarthropods and mollusks, and they are called “BlueBlooded Organisms” by virtue of the fact that their bloodturns blue in color upon oxygenation. Importantly, inthese organisms, hemocyanin associated with blood (alsocalled hemolymph) serves as primary carrier of oxygen.Hcy turns blue upon binding molecular oxygen, aphenomenon that is readily reversible. Notably, suchbinding occurs at high partial pressure of oxygen whichconverts Hcy to Oxy-Hcy. The latter dissociates to releasemolecular oxygen at the vicinity of tissues that have lowoxygen pressure, thus functioning as a mode for oxygentransport12 (Fig. 3).

Characteristically, Hcy is non-cellular and foundfreely dissolved in haemolymph. By virtue of its largemolecular size with multiple epitopes, Hcy is a potentimmunogen as evidenced by the development of discretecrescentic arcs in Ouchterlony double immunodiffusionassay upon antibody challenge (Fig. 4).

As revealed in Figs. 5B and C the Hcy fromhemolymph of fresh water field crab and Indian applesnail Pila, showed positive staining with rubeanic acidstain (a stain to detect copper binding proteins),confirming its ability to bind copper13. Additionally, thedetection of copper granules by the histochemicalstaining of hepato-pancreas in the pulmonate gardensnail, Cryptozona ligulata, potentially reveals theexistence of a copper store, probably complexed withmetallothioneins, that could be possibly recruited forHcy biosynthesis (Fig. 5A)14,15. Structurally, molluscanhaemocyanins are composed of multiple subunits (eight)that result from duplications in the gene encoding forthe protein (Fig. 6). These subunits assemble into aquaternary folded architecture with 160 oxygen bindingsites in the native protein16. This is in contrast to theHcy from arthropods, that have only 3 subunits that arefolded up to generate 48 oxygen binding sites3.Importantly, in all these cases, each of the oxygenbinding sites contains 2-Cu atoms and each of the Cuatoms anchors to 3 histidine residues. Further, the twomolecules of copper are bridged together by 2 moleculesof oxygen resulting in the formation of a dioxygenbridge. Thus on the whole, hcy derived from molluscsand arthropods contain 320 and 96 copper atomsrespectively.

450 INDIAN J MED RES, OCTOBER 2008

Fig. 2. The UV visible spectrum of blue blood of Pila globosacontaining hemocyanin revealing the presence of broad peak at340 nm due to CU-O complex.

Fig. 3. Typical sigmoid oxygen equilibrium curve of hemocyanin.The P50 values indicating the affinity of hcy towards oxygen areinfluenced by the factors shown in the diagram. DPG,diphosphoglycerate.

Fig. 4. Immunocrescentric arcs developed by hemocyanin of Pilaglobosa along with its antibodies harvested in mouse.

Partial pressure of oxygen(mm Hg)

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Phenoloxidase is another such copper bindingprotein that binds to dioxygen with a differentphysiological function viz., browning of fruits andvegetables in plants as well as wound healing, skinpigmentation etc in higher organisms. Also, it has a roleduring sclerotization of new exoskeleton in moltinginsects. This contrasting physiological role forphenoloxidase compared to Hcy could be attributed tothe ability of the former to trigger the catecholaseactivity3.

Transporter proteins: In higher organisms and plants,principal copper binding proteins belonging to thefamily of P-type ATPases serve the function ofintracellular copper transport. Included among these arethe two proteins ATP 7A and ATP 7B. Interestingly, thepresence of such a network for Cu transport seems tobe evolutionarily conserved. Notably, prokaryotespossess metal transporting enzymes also termed heavymetal ATPases that protect them from stress caused byheavy metals found in their natural environment. Theseaccording to Nigel et al17 are encoded by the structuralgenes (cutA, cut-B…cut-F) and the regulatory protein,cutR. Additionally, some mutant forms of E.coli harbora plasmid borne version of copper resistance genes alsocalled pco that confers resistance to approximately fivefold higher concentrations of cupric ions than wild typestrains18. Significantly, bacteria endowed with suchheavy metal transporter proteins are now beingexploited commercially in a process termed“Bioleaching”. The latter is an environmentally friendlyprocess for metal recovery which is a cost-effectiveprocess for treating ores that are remote and difficult toaccess. One of the most exploited microorganisms inbioleaching is Acidithiobacillus ferrooxidans.

As mentioned above, a similar paradigm consistingof metal transporters have been described to be existentin plants, specifically in edible portions such as seeds19.Notably, Cu plays a vital role in the physiology of plantsviz., respiration and photosynthesis. Further,photosynthetically active cells require more Cu thanother cells. Two families of Cu transporter proteins havebeen recognized among plants19. Among these, P-typeATPases (PAA) belong to the family called heavy metalATPases (HMA). In Arabidopsis, they function totransport Cu to the stroma of the chloroplast, wherethey play a critical role in maintaining copperhomeostasis. Importantly, mutation in these ATPasesaffects the photosynthetic electron transport, which canbe reversed by addition of Cu. Additionally, these arealso involved in the transport of Cu in roots and flowers

KRUPANIDHI et al: COPPER & BIOLOGICAL HEALTH 451

Fig. 5. A. Cu granules (possibly prohcy) in the hepatopancreas ofa pulmonate snail, Cryptozona ligulata stained with rubeanic acid.Rubeanic acid stains copper binding proteins B. Native PAGE ofthe proteins of hepatopancreas of fresh water field crab (1) andIndian apple snail, Pila (2) stained with rubeanic acid and coomasieblue. C. Native PAGE of the blood proteins of crab (1) and Pila(2) stained similarly.

Fig. 6. Alkaline dissociation of haemocyanin (H) of Pila globosashowing 8 subunits at 8.5 pH shown on Native PAGE gel performedusing PHAST system. M, molecular weight marker.

of plants. The latter is supported by the detection oftranscripts for HMA in these sites19,20. The second familyof Cu transporters viz., COPT (Cu transporters) are alsoidentified in plants. The homologous transporterproteins of the same have been reported in yeast andmammals20. Notably, Arabidopsis exposed to decreasedlevels of copper for a period of 18 h was shown to turnon a compensatory mechanism that involved increasedsynthesis of COPT mRNA21. In addition, the phenotypicmanifestation of reduced copper levels in these plantsinvolved an increase in root length which could bereversed by the addition of Cu19. Further, the importanceof COPT1 knockdown using an anti-sense strategyresulted in an increased frequency of pollenabnormalities even though the experimental plants weregrown under standard nutrient conditions. The latterphenotype was rescued by exogenous addition of Cuhighlighting the importance of this element for thedeveloping pollen19. In addition to transporters, plantsalso contain a class of molecules termed the metallo-chaperones that bind metals and facilitate their transportto target proteins/sites. The expression of such Cuchaperone mRNA is ubiquitously seen in the tissues ofroot, stem, leaf and inflorescence indicating its role asintercellular Cu delivery and recycling. One such copperchaperone seen in plants is cytochrome oxidase 17(COX17). Defects in COX17 lead to the respiratorydeficiency due to the failure of protein to deliver Cu tomitochondrial cytochrome oxidase complex.Interestingly, the various metal transporter proteins likePAA, HMA, COPT, CCH, COX17, etc., form potentialtargets that could be manipulated to enhance mineraldeposits in plants that could possibly alleviate mineraldeficiency in humans and live stock.

In higher organisms, the absorbed dietary Cu entersliver through entero-hepatic circulation and is furthertransported as a complex with CP or excreted into bile,a process facilitated by a number of Cu chaperones andtransporting proteins (Fig. 7). ATP7A (MND) andATP7B (WND) are also the principal Cu transportersin higher eukaryotes. These transporters contain 8-transmembrane domains in addition to six Cu-bindingmotifs at the N-terminus (MXCXXC; M=methionine,C=Cysteine, X=any amino acid). These Cu bindingmotifs of ATPases reveal that Cu ions are typicallybound to sulphur containing amino acids. They functionlike cation exchangers and use energy from ATPhydrolysis to translocate metal cations across lipidbilayers. Both ATP7A and ATP7B are predominantlylocalized in the transgolgi-network (TGN) and involvedin the delivery of Cu into nascent cuproproteins. ATP7A

transcripts are seen at high levels in muscle, kidney,lung and brain and low levels in placenta and pancreas,while liver contains only trace amounts of thistransporter22-24. ATP7A regulates Cu- efflux when thelevels of the latter become high in epithelial cells. Incontrast, ATP7B expression is higher in the liver whereit regulates the release of copper into bile. Importantly,the transcript levels of both these transporters arepositively regulated by intracellular levels of copper.Further, Cu ATPases also appear in the placenta andlactating breast tissue for transporting Cu to fetus andthrough milk to neo-nates respectively. ATP7A havebeen reported to be present within syncytiotrophoblasts,cytotrophoblasts and fetal vascular endothelial cells.This is consistent with their role in the transport of Cufrom these tissues into the fetal circulation25. In contrastATP7B has been shown to facilitate the export of Cufrom the placenta to the maternal tissues, a mechanismthat protects excessive copper from reaching thedeveloping fetus. Additionally, ATP7A is also reportedto be expressed in luminal epithelial cells of alveoliand ducts of breast tissue, with its expression levelsbeing positively regulated by lactation26. Further, CuATPase activity is also seen in the central nervoussystem where both ATP7A and ATP7B regulate neuronalCu homeostasis. Also, both these are expressed withinretinal pigment epithelium where they regulate therelease of CP that in turn maintains iron homeostasis.

An alternate class of copper transporters found inyeast are, Ctr1 that regulate the influx of copper intothe cytoplasm. These transporters contain threetransmembrane domains with methionine richextracellular motifs at the N-terminal that bind copperand enable its import27. Importantly, dietary Cu (CuII)needs to be converted to its reduced form (CuI) prior toits transport by the Ctrls. The process of biochemical

452 INDIAN J MED RES, OCTOBER 2008

Fig. 7. Enterohepatic uptake, distribution and excretion of Cu.

reduction is carried out by various plasma-associatedreductases.

The mammalian homologue of yeast Ctr1 is calledMURR1; while it’s human orthologue is named CTR1/2. MURR1 is a recently discovered protein chaperonewhose absence has been shown to cause Cu toxicosis,potentially due to hepatic Cu overload (Fig. 8). The latterhas been documented in Bedlington terriers withMURR1 gene mutation that is characterized by deletionof exon 2, resulting in complete absence of thefunctional protein product in liver of affected animals28.Importantly, these terriers exhibit elevated levels oflysosomal Cu content and pronounced reduction inbilary Cu excretion29. This is suggestive of cooperativitybetween MURR1 and ATP7B, to mediate excretion ofexcess copper into bile. Further, imported intracellularcopper has been shown to bind Atox1, which thentransfers the metal to its docking partners in thesecretory pathway (Fig. 8). This reveals a potential roleof Atox1 in the ATP7B-mediated bilary excretion ofexcess Cu. Atox1 has also been implicated in mediatingcopper transfer to CP and tyrosinase. The latter whichis a critical step in melanin generation is supported bythe observation wherein Atox1-null mice have beenshown to exhibit hypo-pigmentation30.

An alternate mode of copper entry involvingendocytosis is facilitated by a class of proteins termedprion protein (PrPC). These are glycoproteins that areexpressed on the plasma membrane. By virtue of itsexpression in the central nervous system as well asperipheral tissues, mutation in PrPC lead to a number of

neurodegenerative disorders that includes Creutzfeld-Jakob disease. In these disorders, the ability of themutant prion to transport copper is significantlyimpaired making neuronal cells susceptible to oxidativestress24. Also, tripeptide glutathione (GSH) binds copperand enables its transport across the blood-brain barrier.

Importantly, in serum, most of the copper istransported by CP. It is synthesized by both hepatocytesand activated macrophages. It is a 132 KDa monomer.In addition to serving as a prime transporter of copperin serum, CP also plays a major role in intestinalabsorption of iron. Significantly, in its role as a carrierof copper in serum, each molecule of CP can bind toseven molecules of copper. Notably, elevated plasmaCP has been shown to have adverse effects oncardiovascular system.

Transcriptional regulation of proteins involved inCu translocation

Prokaryotic Cu homeostatic system has been wellcharacterized in Enterococcus hirae31. Four genes(copY, copZ, copA and copB) are reported to bearranged in the cop operon of E.hirae. CopA and copBencodes for Cu transporting P-type ATPases which arehighly conserved, stabilized and possibly extended intoeukaryotes. CopY encodes for Cu responsive repressorand copZ encodes for a chaperone protein. The copoperon allows growth of E. hirae in Cu-limitingconditions (up to 8 mM Cu). CopA ATPases take Cuwhile it is limiting and copB ATPases bale out excessCu. CopY regulates the expression of cop operon andcopZ translocates Cu intracellularly32,33. CopY is a Zncontaining homodimeric repressor that binds to thepromoter region of the cop operon, thereby regulatingthe synthesis of ATPases and chaperones. It is reportedthat copY is dimeric and belongs to winged-helix typerepressor34. Thus, initially, the package of molecularmachinery for the regulation of heavy metal ions gainedrelevance in the survival of bacteria and hence it wouldnot be a surprise for the eukaryotes to adopt them. Theexpression of the cop operon is low in standard growthmedia whereas induced by 50 fold upon exposure ofbacteria to extracellular Cu35. CopY repressor binds tothe consensus binding site TACANNTGTA, called ‘copbox’36. Experimentally induced mutation in cop-boxprevented its interaction with the repressor. The kineticsof the interaction between the repressor and promoterof cop operon in E. hirae are elaborated by DavidMagnani and Marc Solioz35. The induction of copoperon is facilitated by excess Cu which makes therepressor (CopY) to dissociate from the cop box. This

KRUPANIDHI et al: COPPER & BIOLOGICAL HEALTH 453

Fig. 8. Pictorial illustration of copper transport in liver. Cu chaperones(violet), storing site (MT), distribution (CCO, CuSOD, TGN),secretion of CP and excretion of copper from a hepatocyte are shown.Ctr1 and ATP7B are Cu transporters. The abbreviations shown inthe figure are cited in the text.

E. hirae model has yielded an insight into possibleexistence of a similar molecular architecture ineukaryotes.

Copper-complexes…. A necessity for cellularfunction

In addition to being transported, intracellular copperhas to be sustained in a complexed configuration inorder to prevent its deleterious effects. The latter,possibly are due to the generation of hydroxyl freeradicals by chemical reaction of monomeric copper withhydrogen peroxide. Thus, elemental copper that istrafficked into cells is kept in bound state by a group ofcopper binding proteins or chaperon proteins (Fig. 8).These include Atox1 (antioxidant protein), CCS (Cuchaperone for SOD), COX17, MT1, MT2(metallothionein) and APP (amyloid precursor protein).

In order to understand the biological processesregulated by copper binding proteins, we adopted anenrichment strategy. Firstly, all proteins having either acopper binding domain/functional site were culled fromthe InterPro database (http://www.ebi.ac.uk/interpro).This resulted in a total of 36 proteins that were distributedacross 7 groups based on function/domains/functionalsites (Table). Each group included 3-12 proteins. Proteinsfrom all groups were then used for enrichment analysesusing a bioinformatics tool called Oncomine ConceptMaps (OCM) (www.oncomine.org), developed by DanielRhodes and colleagues37,38. OCM, is an enrichment tool,that allows to systematically linking groups of protein/genes that have a common biological nuance to variousmolecular concepts thus generating novel hypothesis.Notably, we believe that such an enrichment analysis ofcopper binding proteins could potentially reveal variouscellular processes that could be initiated by their action.The various molecular concepts that were used in thisenrichment analyses were derived from both gene andprotein annotations from external databases, andcomputationally-derived regulatory networks. Theexternal annotation included chromosomal locations,protein domains and families, molecular functions,cellular localizations, biological processes, signaling andmetabolic pathways, protein-protein interactionnetworks, protein complexes, and gene expressionsignatures. The regulatory networks were derived byscanning human promoters for known transcription factormotifs and by comparative genomics analyses thatidentified conserved promoter and 3’UTR elements. AP-value cutoff of 5X10-2 was used to cull significantconcepts. In total, data from 12 databases and 335 high-through put datasets were collected and analyzed.

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Table. Classes of copper binding proteins defined by InterPro (http://www.ebi.ac.uk/interpro)

Group 1: Copper type II, ascorbate-dependent monooxygenase1. DBH, Dopamine beta-hydroxylase (dopamine beta-

monooxygenase)2. MOXD1, Monooxygenase, dbh-like 13. PAM, Peptidylglycine alpha-amidating monooxygenase

Group 2: Di-copper centre-containing1. DCT, Dopachrome tautomerase2. TYR, Tyrosinase3. TYRP1, Tyrosinase-related protein 1

Group 3: Copper amine oxidase1. ABP1, Amiloride binding protein 1 [amine oxidase

(copper containing)]2. AOC2, Amine oxidase, copper containing 2 (retina

specific)3. RHBDF1, Rhomboid 5 homolog 1 (drosophila)4. AOC3, Amine oxidase, copper containing 3 (vascular

adhesion protein 1)Group 4: Multicopper oxidase, type 1

1. CP, Ceruloplasmin (ferroxidase)2. F5, Coagulation factor V (proaccelerin, labile factor)3. F8, Coagulation factor Viii, procoagulant component

(haemophilia a)4. HEPH, Hephaestin

Group 5: Copper/Zinc superoxide dismutase1. CR1, Complement component (3b/4b) receptor 1 (knops

blood group)2. PSORS1C1, Psoriasis susceptibility candidate 13. KIAA0467, Kiaa04674. SOD1, Superoxide dismutase 1, soluble (amyotrophic

lateral sclerosis 1)5. SOD3, Superoxide dismutase 3, extracellular6. CCS, Copper chaperone for superoxide dismutase

Group 6: Blue (type 1) copper domain1. NR1H3, Nuclear receptor subfamily 12. LYST, Lysosomal trafficking regulator3. FLJ25006, hypothetical protein flj250064. APR-2, Apoptosis related protein5. SLC35B2, Solute carrier family 35, member b26. IGHG4, Immunoglobulin heavy constant gamma 4 (g4m

marker)7. SIRT7, Sirtuin (silent mating type information regulation

2 homolog) 7 (S. cerevisiae)8. CCDC14, Coiled-coil domain containing 14

Group 7: Multicopper oxidase, copper-binding site1. SLC15A4, Solute carrier family 15, member 42. CP, Ceruloplasmin (ferroxidase)3. F5, Coagulation factor V (proaccelerin, labile factor)4. HYAL4, Hyaluronoglucosamindase 45. SNAI3, Snail homolog 3 (drosophila)6. HEPHL1, Hephaestin-like 17. ITIH3, Inter-alpha (globulin) inhibitor h38. CCDC73, Coiled-coil domain containing 739. SLC14A1, Solute carrier family 14 (urea transporter),

member 110. SLC14A2, Solute carrier family 14 (urea transporter),

member 211. CCIN, Calicin12. HEPH, Hephaestin

Interestingly, as shown in Fig. 9, the coppercontaining proteins play an active role in 3 major cellularprocesses. These include tyrosine metabolism andmelanin biosynthesis (red bridges), amino acidmetabolism (blue bridges) and coagulation cascade (blackbridges). Further, included in the concept that portrayed“tyrosine metabolism and melanin biosynthesis” weremultiple protein-protein complexes involving the proteinsDopachrome tautomerase, Tyrosinase and Tyrosinase-related protein 1, all of which are copper binding proteinsand play a critical role in the above bioprocess. Similarlycopper binding proteins, potentate amino acidmetabolism, by having a functional role in two biologicalprocesses, namely amine oxidase and oxidoreductaseactivity. Additionally, copper binding proteins regulatethe coagulation cascade by forming protein complex withthe PROC protein (inactivator of coagulation factors Vaand VIIIa). Also the proteins that bind copper wereintimately involved in superoxide metabolism.

Among the proteins involved in superoxidemetabolism, CCS plays a key role in the transmission ofCu to pro-form of superoxide dismutase (apo-SOD). CCS

possesses three functional domains. Domain I containsCu-binding site, domain II is homologous to SOD anddomain III contains cysteines essential in the transfer ofCu to apo-SOD. CCS deletion has been documented tomarkedly reduce SOD activity in mice39,40. Third classof chaperone includes COX17, which delivers Cu tocytochrome C oxidase (CCO). CCO is a large proteinfound in the cytoplasm and mitochondrial innermembrane. It has two subunits I and II, each containingCu binding sites. Fourth class of copper chaperoneincludes metallothioneins (MT). These are cysteine richproteins (30%) composed of 61 amino acids. Due to theirhigh redox potential, MT’s regulate intracellular levelsof Zn and Cu in addition to serving as potent mediatorsof toxic metal detoxification. As a part of the former,MT levels tightly regulate copper homeostasis in liver.Interestingly, the pool of MT-Cu complex progressivelydecreases with age in mammals41,42. A fifth class of copperchaperone comprises of the membrane protein β amyloidprecursor protein (APP) that regulates import of the metalinto brain. This is supported by the observation whereincopper levels in the brain of APP null mice are highercompared to their wild type counterparts43.

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Fig. 9. Oncomine concept map analysis of copper binding proteins. Network view of the molecular concept analysis for the copper bindingproteins derived from InterPro (orange node). Each node represents a molecular concept or a set of biologically related genes. The node size isproportional to the number of genes in the concept. Each edge represents a statistically significant enrichment (P<5 x 10-2).

In addition to the transporters, there are a numberof enzymes that require copper as cofactors for theircatalytic activity. Some of the members of this class ofcuproenzymes include Cu/Zn SOD (antioxidantdefense), cytochrome C oxidase (mitochondrialrespiration), CP (iron metabolism), tyrosinase(pigmentation), lysyl oxidase (collagen maturation inconnective tissue), Hephaestin (intestinal iron efflux),dopamine β-hydroxylase (catecholamine synthesis),Peptidylglycine α -amidating monooxygenase (peptide-hormone processing), amine oxidase (removal ofhormones), ascorbate oxidase and catalase oxidase(oxidation of primary alcohols to aldehydes)44.

Interestingly, as would be discussed later, the OCMalso portrayed an enrichment of copper binding proteinsin invasive tumors especially those associated with softtissue (melanoma) and liver.

Prognosis through copper metabolism: Cu is found inall living organisms in trace quantities with an uptakerange in humans being 0.9 to 10 mg/day. The metal, Cuis found as a prosthetic group in metalloenzymesbinding to sulphur residues. Several physiologicalreactions such as electron transfer, detoxification ofreactive oxygen species, connective tissue development,oxygen transport, oxygenation reactions are beingmediated by Cu containing metalloenzymes. When Curegulation fails, a variety of biochemical disturbancesdevelop. The failure in Cu elimination and its effluxleads to Wilson’s and Menkes diseases respectively.Another intriguing role of Cu is reported in thepromotion of angiogenesis for facilitating tumor toprogress. Therefore, by examining the distinguishingfeatures of symptoms due to copper imbalance and itsmetabolism, the possible prophylactic andchemotherapeutic agents could be designed.

Disease symptoms due to Cu deficiency and overload

The disturbance in the levels of Cu is primarily dueto genetic defects. The most prominent among theseare Menkes and Wilson’s diseases.

Menkes disease is a rare X-linked (Xq13) fataldisorder affecting one out of 200,000 newborn infants,resulting from a mutation in the gene encoding ATP7A.The mutant protein is no longer able to regulate theflux of copper resulting in a systemic deficiency ofcopper45. Specifically, most of the Cu accumulates inintestinal epithelium and kidney while suboptimal levelsof the metal are found in other tissues such as liver andbrain. Menkes disease is a fatal disorder, whereinlethality is preceded by neuronal (cerebral and

cerebellar) degeneration and connective tissueabnormalities during the first 2-4 yr of infancy46. Similarcondition has been reported to occur in sheep wherethe disorder is termed as Kinky Hair Disease47. Theprimary mode of diagnosis involves the use of geneticscreens. Early diagnosis coupled with supplementationof copper (as Cu-histidine complex) could avoidneurodegeneration and lead to reinstatement of normaldevelopment46. Interestingly, cells derived from patientswith Menkes disease exhibit copper accumulation whencultured in vitro48.

Wilson’s disease is a rare autosomal recessive traitmanifested in the chromosome, 13q14.3. The frequencyof occurrence of Wilson’s disease is about 1/30,000 to1/50,000 with a carrier frequency of 1 per cent andheterozygote frequency of 0.86 per cent, (confined towestern world). Notably, this defect is caused due tothe mutation in the gene coding for ATP7B, whoseoriginal function is to regulate the bilary excretion ofexcess copper. ATP7B is encoded by the WND gene.Among several mutations that have been reported forthis gene (>200), the most well studied one is a pointmutation involving replacement of the amino acidhistidine by glutamine at position 1069. The mutatedprotein thus loses the ability to orient ATP in its catalyticsite, thus impairing its normal function49. The resultantis the accumulation of Cu in liver leading to cirrhosisand hemolysis. Advanced stages of the disorder arecharacterized by deposition of excess Cu in brain andeyes in the form of Kayaer-Fleischer ring, which servesas diagnostic marker for Wilson’s disease50. Thetherapeutic measures for this disorder revolve aroundchelating the excess copper using chelating agents suchas tetrathiomolybdate, trientine and penicillamine.

Contrary to copper accumulation, its deficiencycan lead to hypocupremic state. Zatta and Frank44

reported that there was an incidence of 11.3 millionclinically identifiable Cu deficiency cases in 1970,which has since been on the rise. Copper deficiencycould be a result of either inadequate dietary intake(also termed primary copper deficiency) or due toimpairment in its uptake (secondary copperdeficiency). The latter could be caused by the presenceof additional heavy metals in the diet that couldcompetitively diminish copper uptake in the lining ofgastrointestinal tract. Among these, molybdenum isthe most common competitor of copper absorption.Importantly, the relative ratio of dietary Cu: MO havebeen defined to be 4 and 8 51 respectively to achieveoptimal control in nutritional balance and hence copperhomeostasis in ruminants.

456 INDIAN J MED RES, OCTOBER 2008

Additional disorders are caused by mutations invarious cuproenzymes as reported by Prohaska52. Theseinclude, (i) Albinism, wherein an impairment of anenzyme tyrosinase which is a critical intermediate inmelanin biosynthesis, (ii) Over gene dose effect of Cu-Zn SOD noticed in Down Syndrome (trisomy 21) dueto the presence of this gene on the chromosome 21,(iii) X-linked Cutis laxa (or an analogous disorder inmouse termed blotchy mouse), which are characterizedby defects in cross-linking of collagen due to decreasedlysyl oxidase activity, (iv) Mottled mice, an X-linkeddisorder analogous to Menkes disease wherein Cumetabolism is affected. These mice have a mottledappearance due to decreased melanin pigmentationresulting from a reduction in tyrosinase activity, and(v) Toxic milk mutant mouse, a homozygous trait causedby Cu accumulation in liver. This results in a decreasedcopper content in milk of lactating mothers which istoxic to the suckling offspring. By virtue of its similarityto Wilson’s disease in accumulating copper in liver, thetoxic milk mutant mouse could serve as a paradigm forunderstanding the mechanism that underlies thedevelopment of Wilson’s disease.

Role of copper in tumor development and progression:Copper metabolism is a critical component of tumorprogression. Concentration of copper in serum has beenfound to correlate well with tumor development, size,progression as well as recurrence53. Elevated levels ofcirculating copper in serum have been documented incancers of lung, breast, gastrointestinal tract, brain aswell as gynecological cancers54,55. Importantly, copperlevels are higher in metastatic disease compared tolocalized tumors54. This increase in serum copper levelsduring neoplastic progression is reflected inconcomitant increase in the levels of CP, the primarycarrier of copper in serum53. Interestingly, CP has beennominated as potential marker for diagnosis of advancedsolid tumors56. Additional evidence for the role ofcopper in tumor development is derived fromexperiments that show existence of Cu salts in tumorextracts that could stimulate the migration of endothelialcells in vitro57.

The role of copper in tumor progression is bestunderstood in the light of the knowledge that developingtumors require an ample supply of oxygen and nutrientsthat necessitates the development of a well definedvasculature. The process termed angiogenesis is criticalfor tumor proliferation and metastatic spread. Among thevarious factors that lead to initiation of the angiogenicprocess, tumor associated hypoxia seems to play a majorrole. Importantly, copper also plays a major role in the

induction of tumor angiogenesis53. This is supported byexperiments conducted by Parke et al58, wherein dose-dependent neovascularisation (angiogenesis) is noticedupon implanting Cu pellet into rabbit cornea.

Notably, copper exerts its effect on angiogenesis byinducing endothelial cell proliferation and migration bythe way of activation of various angiogenic factors. Thelatter include vascular endothelial growth factor (VEGF),basic fibroblast growth factor (bFGF), tumor necrosisfactor α (TNFα) and Interleukin 1 (IL-1)53. Theseangiogenic factors in turn activate resting endothelial cells(which are otherwise in G0 phase of the cell cycle) andinitiate their proliferation by transitioning them to theG1 phase of the cell cycle. This process of endothelialactivation by copper can be reversed using chelatingagents like penicillamine59, a property that is widelyexploited in designing therapeutic regimens (see sectionbelow). Additionally copper has been thought to exertits effect by binding to proteins like heparin, CP, etc.,making them angiogenic60. The angiogenic property ofthe latter is evident in the observation wherein CP hasbeen reported to induce the formation of capillaries inthe cornea of rabbits58.

To understand the effect of copper in cancer, we usedthe data from an interesting study aimed at predictingthe chemosensitivity of human cancer cell lines61. In thisstudy chemosensitivity predictions were based ontranscriptomic profiling done upon treatment with variouscompounds on a panel of 60 cancer cell lines (NCI-60panel)61. A set of 50 genes were found to be differentiallyregulated between copper sensitive and resistant cell linesupon treatment with 0.0001M copper sulfate. These setof 50 genes were used for enrichment analyses tounderstand the role of copper in tumor progression. Theenrichment analyses was done using OCM as describedabove37. Interestingly, the differentially expressed genesbetween copper sensitive and resistant cell lines mappedto multiple gene expression signatures (red nodes)derived from tumors that included sarcoma, lungcarcinoma, colorectal cancer, etc. (Fig. 10). Furthermore,copper induced genes also mapped to a subset of genesthat are activated upon Src over expression (red node)(Fig.10). This is important in the context of earlier studiesthat have described a critical role for Src in tumordevelopment and progression. These observationsprovide evidence at the molecular level for the role ofcopper in tumor progression.

Brain disorders due to Cu deficiency and/or excess: Thebrain is an organ of bewildering complexity withmultifaceted serendipitous effects. In such a resilient

KRUPANIDHI et al: COPPER & BIOLOGICAL HEALTH 457

situation, the maintenance of adequate Cu levels is vital.Both brain and liver are metabolically active organsystems. Dysfunction of Cu homeostasis due to itsexcess or deficiency as reflected in Wilson’s andMenkes diseases respectively causes severe ailmentsin these two organ systems.

Additionally, disruption of copper homeostasiscoupled to oxidative stress and free radical generationplay a significant role in the development of Alzheimer’sdisease, amyotrophic lateral sclerosis and Creutzfeld-Jakob disease62.

Among these, the etiology of Alzheimer’s disease isassociated with accumulation of amyloid ß protein (Aß)63.This is produced either by proteolytic processing orsecretion of its precursor, a transmembrane glycoproteinnamed ß-amyloid precursor protein (APP)64. Theaccumulation of Aß leads to the formation of Aß fibrils,which have been shown to exert neurotoxic effects bothin vitro65, 66 and in vivo66. Importantly, recent studies haverevealed the ability of the Aß peptide to generatehydrogen peroxide by reduction of the bound metal67

which mediates the generation of free radicals that playa causal role in oxidative stress induced neurotoxicity,by inducing lipid peroxidation, protein oxidation,

etc.,67-69. In Alzheimer’s, the proteolytic product (Aß) ismutated resulting in its accumulation which is assumedto trigger free radical mediated injury viz., neuronalinjury70. Notably, under normal conditions, APP is knownto bind copper in its reduced state and facilitates itstransport along the length of the neuron from the cellbody to the axonal surface and to plasma membrane ofdendrites63. However, in Alzheimer’s disease, APPfunction is disrupted leading to oxidation of its boundcopper in presence of H

2O

2. This is accompanied by

fragmentation of APP resulting in Aß peptides. Thesefragments are thought to aggregate and lead to oxygenfree radical injury in Alzheimer’s disease71. Additionally,Cu also binds to extracellular plaques and causesinterference in Cu trafficking devices and in turn depletesintracellular Cu repertoire. This would reduce theactivities of cytochrome oxidase and SOD. Thus,increased oxidative stress coupled with the reduction inkey metabolic and defense mechanism could contributesignificantly to neuronal damage. Oral treatment oftransgenic mouse with clioquinol resulted in halving ofAß levels and significantly increased the levels of Cuand Zn in the brain72.

The other neuronal cellular membrane protein is prion.It is associated with the diseases such as neurodegenerativedisorders that include Kuru, Creutzfeld-Jakob disease inhumans, Scarpie in humans and bovine spongiformencephalopathy (mad cow disease) in cattle73. Here again,the conformational change in the protein affects itsfunction. The structural change involves a transition ofnative α-helical prion into a ß-sheet conformationconferring pathogenic potential to the protein. Usingexperiments employing circular dichroism, this misfoldingcoupled to its aggregation have been shown to be mediatedby copper74. Further support for the involvement of coppercomes from studies wherein use of D-Penicillamine(copper-chelator) has shown to result in a delayed onsetof the disease in Scrapie infected mice, presumablymediated by a reduction in the levels of copper in brain aswell as in the circulation75.

In addition to the above disorders, severalneurological disorders have been reported in newbornsdue to the deficiency of Cu44. One such disorder happensto be neonatal ataxia, a disease found in lambs, causeddue to either low Cu or high molybdenum content intheir feed. The disease is characterized by tremors, in-coordination, paralysis and ultimately death51.Significantly, affected lambs show significantdemyelination which affects brain as well as causesnecrosis of neurons76. Importantly, copper deficiency

458 INDIAN J MED RES, OCTOBER 2008

Fig. 10. Oncomine concept map analysis of genes up regulated inNCl-60 cancer cell lines upon treatment with 0.0001 M coppersulfate (black node). Each node represents a molecular concept ora set of biologically related genes. The node size is proportional tothe number of genes in the concept. Each edge represents astatistically significant enrichment (P<1 x 10-4).

has been shown to be causal in inducing the de-myelination in affected animals77.

Cu chelation therapy: Dietary excess Cu intake is notvery common, although there are genetic disorders asdiscussed in the previous sections. The increasedaccumulation leads to hepatitis and neurologicaldisorders. Human Wilson’s disease and Toxic milkmouse are associated with excess accumulation ofcellular Cu. In the former, the defect is manifested inWilson protein (ATP7B) which in its normal form doesfacilitate to eliminate excess Cu ions into bile.Therapeutic approaches to Cu toxicity include the drugsand formulations such as D-penicillamine or trientineto prevent neurodegenerative disorder78. Similarly,tetrathiomolybdate, as a specific Cu chelator have beenused in Toxic milk mouse model in reducing abnormallyhigh Cu79. Since copper plays an important role in tumordevelopment and progression (as discussed above),strategies employing Cu chelators are also beingpursued for cancer therapy80. In contrast, in conditionslike Menkes disease that results from copper deficiency,an approach to supplement copper complexed withhistidine or albumin are being tested79.

Homeopathic formulations using Cu metal:Homeopathy is based on the argument that the body isa self-healing entity, and that symptoms are theexpression of the body attempting to restore its balance.Homeopathic physicians are trained to match thepatient’s symptoms with the accurate remedy. Theybelieve that the remedies themselves never destroydisease, but stimulate the body’s own healing action toget rid itself of the problem. Minerals in the body canbe used as healing agents for specific health problems.Minerals are used in homeopathic remedies to stimulatecorresponding body cells towards metabolic activity andhealth restoration. A few tinctures with the combinationof copper are: (i) Cuprum aceticum, (ii) CuprumArsenicosum, (iii) Cuprum Metallicum and (iv) CuprumSulphuricum81 .

All life forms exploit naturally available Cu formyriad physiological functions. Bacteria, plants, blueblooded organisms and vertebrates have developed themolecular mechanisms to upkeep the Cu homeostasis.The bioavailability of Cu, by complexing with proteinsor amino acids or organic acids constitutingorganometallic complex, facilitates its ease in uptake anddistribution in ecosystem. Literature review reveals thatthe Cu imbalance could be causal in Menkes disease,Wilson’s disease, Kuru, Creutzfeld- Jakob disease, madcow disease as well as induce tumor development and

progression. By its unique attribute of being a catalyticcofactor, Cu occupies an important niche in biologicalsystems. Cu transporters, chaperone proteins and carrierproteins make Cu available to the intricate network ofbiochemical systems. Developments in the field of plantgenetic engineering have been pivotal in defining meansto combat copper deficiency. In the clinical field,management of disorders caused by impaired copperhomeostasis are being combated either using metalchelators or by supplementing the metal in a complexstate with various carriers.

Acknowledgment

The authors thank to Dr M. Sivakumar, University ofWollongong, Australia, for designing a few of the figures shown inthe text. One of the authors (SKN) acknowledges UGC and DST(India) for providing financial support through SAP DRS and FISTprogrammes respectively.

References

1. Aaseth J, Flaten TP, Andersen O. Hereditary iron and copperdeposition: diagnostics, pathogenesis and therapeutics. ScandJ Gastroenterol 2007; 42 : 673-81.

2. Araya M, Pizarro F, Olivares M, Arredondo M, Gonzalez M,Mendez M. Understanding copper homeostasis in humans andcopper effects on health. Biol Res 2006; 39 : 183-7.

3. Decker H, Terwilliger N. Cops and robbers: putative evolutionof copper oxygen-binding proteins. J Exp Biol 2000; 203 :1777-82.

4. Goodman VL, Brewer GJ, Merajver SD. Copper deficiencyas an anti-cancer strategy. Endocr Relat Cancer 2004; 11 :255-63.

5. Srivastava S, Singh BR, Tripathi VN Application of bacterialbiomass as a potential metal indicator. Curr Sci 2005; 89 :1248-51.

6. Wapnir RA. Copper absorption and bioavailability. Am J ClinNutr 1998; 67 : 1054S-60S.

7. Baker DH, Czarnecki-Maulden GL. Pharmacologic role ofcysteine in ameliorating or exacerbating mineral toxicities.J Nutr 1987; 117 : 1003-10.

8. Pott EB, Henry PR, Ammerman CB, Merritt AM, MadisonJB, Miles RD. Relative bioavailability of Cu in a Cu-lysinecomplex for chicks and lambs. Anim Feed Sci Technol 1994;45 : 193-203.

9. Kincaid RL, Blauwiekel RM, Cronath JD. Supplementationof Cu sulphate or Cu proteinate for growing calves fed foragescontaining molybdenum. J Diary Sci 1986; 69 : 160-3.

10. Lowndes SA, Harris AL. The role of copper in tumourangiogenesis. J Mammary Gland Biol Neoplasia 2005; 10 :299-310.

11. Gu M, Cooper JM, Butler P, Walker AP, Mistry PK, DooleyJS, et al. Oxidative-phosphorylation defects in liver of patientswith Wilson’s disease. Lancet 2000; 356 : 469-74.

12. Krupanidhi S. Respiratory pigments. Biol Educ (India)1988;4 : 104-14.

KRUPANIDHI et al: COPPER & BIOLOGICAL HEALTH 459

13. Krupanidhi S, Laksmikanth T. Detection of haemocyanin innative PAGE gels. Natl Acad Sci Lett 2005; 28 : 353-5.

14. Krupanidhi S, Venkata Reddy V, Padmanabha Naidu B. Somestudies on copper metabolism in the garden snail,Cryptozonaligulata. Indian J Exp Biol 1978; 16 : 249-50.

15. Krupanidhi S. Copper granules in the hepatopancreas of thesnail, Crytpzona ligulata. Curr Sci 1985; 53 : 431-2.

16. van Holde KE, Miller KI. Hemocyanins. Adv Protein Chem1995; 47 : 1-81.

17. Brown NL, Camakaris J, Lee BT, Williams T, Morby AP,Parkhill J, et al. Bacterial resistances to mercury and copper.Cell Biochem 1991; 46 : 106-14.

18. Rouch DLB, Camakaris J. Metal on homeostasis: Molecularbiology and chemistry. Supplement: UCLA Symposia onMolecular & Cellular Biology 1989; 38 : 439-46.

19. Grotz N, Guerinot ML. Molecular aspects of Cu, Fe and Znhomeostasis in plants. Biochim Biophys Acta 2006; 1763 :595-608.

20. Sancenon V, Puig S, Mira H, Thiele DJ, Penarrubia L.Identification of a copper transporter family in Arabidopsisthaliana. Plant Mol Biol 2003; 51 : 577-87.

21. Petris MJ. The SLC31 (Ctr) copper transporter family. PflugersArch 2004; 447 : 752-5.

22. Chelly J, Tumer Z, Tonnesen T, Petterson A, Ishikawa-BrushY, Tommerup N, et al. Isolation of a candidate gene for Menkesdisease that encodes a potential heavy metal binding protein.Nat Genet 1993; 3 : 14-9.

23. Mercer JF, Livingston J, Hall B, Paynter JA, Begy C,Chandrasekharappa S, et al. Isolation of a partial candidategene for Menkes disease by positional cloning. Nat Genet1993; 3 : 20-5.

24. Mufti AR, Burstein E, Duckett CS. XIAP: cell death regulationmeets copper homeostasis. Arch Biochem Biophys 2007; 463: 168-74.

25. La Fontaine S, Mercer JF. Trafficking of the copper-ATPases,ATP7A and ATP7B: role in copper homeostasis. Arch BiochemBiophys 2007; 463 : 149-67.

26. Ackland ML, Anikijenko P, Michalczyk A, Mercer JF.Expression of menkes copper-transporting ATPase, MNK, inthe lactating human breast: possible role in copper transportinto milk. J Histochem Cytochem 1999; 47 : 1553-62.

27. Guo Y, Smith K, Lee J, Thiele DJ, Petris MJ. Identification ofmethionine-rich clusters that regulate copper-stimulatedendocytosis of the human Ctr1 copper transporter. J Biol Chem2004; 279 : 17428-33.

28. van De Sluis B, Rothuizen J, Pearson PL, van Oost BA,Wijmenga C. Identification of a new copper metabolism geneby positional cloning in a purebred dog population. Hum MolGenet 2002; 11 : 165-73.

29. Klomp AE, van de Sluis B, Klomp LW, Wijmenga C. Theubiquitously expressed MURR1 protein is absent in caninecopper toxicosis. J Hepatol 2003; 39 : 703-9.

30. de Bie P, van de Sluis B, Klomp L, Wijmenga C. The manyfaces of the copper metabolism protein MURR1/COMMD1.J Hered 2005; 96 : 803-11.

31. Wimmer R, Dameron CT, Solioz M. Molecular hardware ofCu homeostasis in Enterococcus hirae. Handbook of Cu

pharamacology and toxicology. Tofowa, NJ: Humana Press;2002. p. 527-43.

32. Odermatt A, Krapf R, Solioz M. Induction of the putativecopper ATPases, CopA and CopB, of Enterococcus hirae byAg+ and Cu2+, and Ag+ extrusion by CopB. Biochem BiophysRes Commun 1994; 202 : 44-8.

33. Wunderli-Ye H, Solioz M. Effects of promoter mutations onthe in vivo regulation of the cop operon of Enterococcus hiraeby copper(I) and copper(II). Biochem Biophys Res Commun1999; 259 : 443-9.

34. Gajiwala KS, Burley SK. Winged helix proteins. Curr OpinStruct Biol 2000; 10 : 110-6.

35. Magnani D, Solioz M. Copper chaperone cycling anddegradation in the regulation of the cop operon ofEnterococcus hirae. Biometals 2005; 18 : 407-12.

36. Portmann R, Magnani D, Stoyanov JV, Schmechel A,Multhaup G, Solioz M. Interaction kinetics of the copper-responsive CopY repressor with the cop promoter ofEnterococcus hirae. J Biol Inorg Chem 2004; 9 : 396-402.

37. Rhodes DR, Kalyana-Sundaram S, Tomlins SA, MahavisnoV, Kasper N, Varambally R, et al. Molecular concepts analysislinks tumors, pathways, mechanisms, and drugs. Neoplasia2007; 9 : 443-54.

38. Tomlins SA, Mehra R, Rhodes DR, Cao X, Wang L,Dhanasekaran SM, et al. Integrative molecular conceptmodeling of prostate cancer progression. Nature Genet 2007;39 : 41-51.

39. Prohaska JR, Gybina AA. Intracellular copper transport inmammals. J Nutr 2004; 134 : 1003-6.

40. Wong PC, Waggoner D, Subramaniam JR, Tessarollo L,Bartnikas TB, Culotta VC, et al. Copper chaperone forsuperoxide dismutase is essential to activate mammalian Cu/Zn superoxide dismutase. Proc Natl Acad Sci USA 2000; 97 :2886-91.

41. Coyle P, Philcox JC, Carey LC, Rofe AM. Metallothionein: themultipurpose protein. Cell Mol Life Sci 2002; 59 : 627-47.

42. Hamza I, Faisst A, Prohaska J, Chen J, Gruss P, Gitlin JD.The metallochaperone Atox1 plays a critical role in perinatalcopper homeostasis. Proc Natl Acad Sci USA 2001; 98 : 6848-52.

43. White AR, Reyes R, Mercer JF, Camakaris J, Zheng H, BushAI, et al. Copper levels are increased in the cerebral cortexand liver of APP and APLP2 knockout mice. Brain Res 1999;842 : 439-44.

44. Zatta P, Frank A. Copper deficiency and neurological disordersin man and animals. Brain Res Rev 2007; 54 : 19-33.

45. Daniel KG, Harbach RH, Guida WC, Dou QP. Copper storagediseases: Menkes, Wilsons, and cancer. Front Biosci 2004; 9: 2652-62.

46. Gu YH, Kodama H, Sato E, Mochizuki D, Yanagawa Y,Takayanagi M, et al. Prenatal diagnosis of Menkes disease bygenetic analysis and copper measurement. Brain Dev 2002;24 : 715-8.

47. Menkes JH. Kinky hair disease: twenty five years later. BrainDev 1988; 10 : 77-9.

48. Horn N. Menkes’ X-linked disease: prenatal diagnosis andcarrier detection. J Inherit Metab Dis 1983; 6 (Suppl 1) : 59-62.

460 INDIAN J MED RES, OCTOBER 2008

49. Shim H, Harris ZL. Genetic defects in copper metabolism.J Nutr 2003; 133 : 1527S-31S.

50. Sarkar B. Treatment of Wilson and menkes diseases. ChemRev 1999; 99 : 2535-44.

51. Underwood EJ, Suttle NF. Cu. The mineral nutrition of livestock.3rd ed. New York: CABI Publsihing Oxon; 2001.p. 283-342.

52. Prohaska JR. Genetic diseases of copper metabolism. ClinPhysiol Biochem 1986; 4 : 87-93.

53. Nasulewicz A, Mazur A, Opolski A. Role of copper in tumourangiogenesis-clinical implications. J Trace Elem Med Biol2004; 18 : 1-8.

54. Zowczak M, Iskra M, Torlinski L, Cofta S. Analysis of serumcopper and zinc concentrations in cancer patients. Biol TraceElem Res 2001; 82 : 1-8.

55. Yoshida D, Ikeda Y, Nakazawa S. Quantitative analysis ofcopper, zinc and copper/zinc ratio in selected human braintumors. J Neurooncol 1993; 16 : 109-15.

56. Senra Varela A, Lopez Saez JJ, Quintela Senra D. Serumceruloplasmin as a diagnostic marker of cancer. Cancer Lett1997; 121 : 139-45.

57. Hu GF. Copper stimulates proliferation of human endothelialcells under culture. J Cell Biochem 1998; 69 : 326-35.

58. Parke A, Bhattacherjee P, Palmer RM, Lazarus NR.Characterization and quantification of copper sulfate-inducedvascularization of the rabbit cornea. Am J Pathol 1988;130 : 173-8.

59. Pan Q, Kleer CG, van Golen KL, Irani J, Bottema KM, BiasC, et al. Copper deficiency induced by tetrathiomolybdatesuppresses tumor growth and angiogenesis. Cancer Res 2002;62 : 4854-9.

60. Ziche M, Jones J, Gullino PM. Role of prostaglandin E1 andcopper in angiogenesis. J Natl Cancer Inst 1982; 69 : 475-82.

61. Staunton JE, Slonim DK, Coller HA, Tamayo P, Angelo MJ,Park J, et al. Chemosensitivity prediction by transcriptionalprofiling. Proc Natl Acad Sci USA 2001; 98 : 10787-92.

62. Miranda S, Opazo C, Larrondo LF, Munoz FJ, Ruiz F,Leighton F, et al. The role of oxidative stress in the toxicityinduced by amyloid beta-peptide in Alzheimer’s disease. ProgNeurobiol 2000; 62 : 633-48.

63. Selkoe DJ. The cell biology of beta-amyloid precursor proteinand presenilin in Alzheimer’s disease. Trends Cell Biol 1998;8 : 447-53.

64. Soto C, Branes MC, Alvarez J, Inestrosa NC. Structuraldeterminants of the Alzheimer’s amyloid beta-peptide.J Neurochem 1994; 63 : 1191-8.

65. Yankner BA. Mechanisms of neuronal degeneration inAlzheimer’s disease. Neuron 1996; 16 : 921-32.

66. Soto C, Sigurdsson EM, Morelli L, Kumar RA, Castano EM,Frangione B. Beta-sheet breaker peptides inhibit

fibrillogenesis in a rat brain model of amyloidosis: implicationsfor Alzheimer’s therapy. Nature Med 1998; 4 : 822-6.

67. Huang X, Atwood CS, Hartshorn MA, Multhaup G, GoldsteinLE, Scarpa RC, et al. The A beta peptide of Alzheimer’s diseasedirectly produces hydrogen peroxide through metal ionreduction. Biochemistry 1999; 38 : 7609-16.

68. Butterfield DA, Hensley K, Cole P, Subramaniam R, AksenovM, Aksenova M, et al. Oxidatively induced structural alterationof glutamine synthetase assessed by analysis of spin labelincorporation kinetics: relevance to Alzheimer’s disease.J Neurochem 1997; 68 : 2451-7.

69. Stadtman ER. Metal ion-catalyzed oxidation of proteins:biochemical mechanism and biological consequences. FreeRadic Boil Med 1990; 9 : 315-25.

70. Wong PC, Rothstein JD, Price DL. The genetic and molecularmechanisms of motor neuron disease. Curr Opin Neurobiol1998; 8 : 791-9.

71. Multhaup G, Ruppert T, Schlicksupp A, Hesse L, Bill E,Pipkorn R, et al. Copper-binding amyloid precursor proteinundergoes a site-specific fragmentation in the reduction ofhydrogen peroxide. Biochemistry 1998; 37 : 7224-30.

72. Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD,McLean CA, et al. Treatment with a copper-zinc chelatormarkedly and rapidly inhibits beta-amyloid accumulation inAlzheimer’s disease transgenic mice. Neuron 2001; 30 : 665-76.

73. Cerpa W, Varela-Nallar L, Reyes AE, Minniti AN, InestrosaNC. Is there a role for copper in neurodegenerative diseases?Mol Aspects Med 2005; 26 : 405-20.

74. Jones CE, Abdelraheim SR, Brown DR, Viles JH. PreferentialCu2+ coordination by His96 and His111 induces beta-sheetformation in the unstructured amyloidogenic region of theprion protein. J Biol Chem 2004; 279 : 32018-27.

75. Sigurdsson EM, Brown DR, Alim MA, Scholtzova H, CarpR, Meeker HC, et al. Copper chelation delays the onset ofprion disease. J Biol Chem 2003; 278 : 46199-202.

76. Picco SJ, De Luca JC, Mattioli G, Dulout FN. DNA damageinduced by copper deficiency in cattle assessed by the Cometassay. Mutat Res 2001; 498 : 1-6.

77. Kumar N, Gross JB, Jr, Ahlskog JE. Copper deficiencymyelopathy produces a clinical picture like subacute combineddegeneration. Neurology 2004; 63 : 33-9.

78. Offen D, Gilgun-Sherki Y, Barhum Y, Benhar M, Grinberg L,Reich R, et al. A low molecular weight copper chelator crossesthe blood-brain barrier and attenuates experimental autoimmuneencephalomyelitis. J Neurochem 2004; 89 : 1241-51.

79. Czachor JD, Cherian MG, Koropatnick J. Reduction of copperand metallothionein in toxic milk mice by tetrathiomolybdate,but not deferiprone. J Inorg Biochem 2002; 88 : 213-22.

80. Cai L, Li XK, Song Y, Cherian MG. Essentiality, toxicologyand chelation therapy of zinc and copper. Curr Med Chem2005; 12 : 2753-63.

81. Clarke J. A dictionary of practical materia medica. B. JainPublishers Pvt. Ltd.; 1990; 1: 633-44.

KRUPANIDHI et al: COPPER & BIOLOGICAL HEALTH 461

Reprint requests: Dr S. Krupanidhi, Department of Biosciences, Sri Sathya Sai UniversityPrasanthi Nilayam 515 134, Indiae-mail: krupanidhi_srirama@yahoo.com