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Copper & biological health.

Cu being a transition metal is ubiquitously engaged in biological systems to derive electrons through its participation in several enzymatic reactions. Upon bestowing the significance of Cu in biological systems, an elaborate mechanism is set forth by nature for maintaining Cu homeostasis. As a consequence, a wide variety of proteins viz., family of Cu bearing proteins, cuproenzymes, Cu transporters and Cu chaperone proteins have been manifested for enabling Cu to show its relevance in biological health. In addition, understanding the role of Cu in hepatic and neuronal functions and also in angiogenesis keeps progressing with the advent of novel molecular tools. The studies on genetic defects in Cu metabolism causing abnormalities are providing insights leading to the possible prognostic 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

Biological relevance of Cu

Cu is an essential micronutrient required by all life forms. Cu is a transition metal and hence involved in a variety of biological processes viz., embryonic development, mitochondrial respiration, regulation of hemoglobin levels as well as hepatocyte and neuronal functions. Being a transition metal, Cu gets biologically converted between different redox states namely oxidized Cu (II) and reduced Cu (I). This unique attribute has made Cu metal to get manifested as an important catalytic co-factor for a variety of metabolic reactions in biological systems. Several reviews (1-5) highlighted the participation of Cu in a myriad cellular activities and physiological processes such as cellular respiration, iron metabolism, biosynthesis of neurotransmitter, and free radical detoxification. Therefore, it is worth recalling that Cu is vital for normal healthy functioning of organisms (Fig. 1).

Source of copper: Rich amounts of copper along with other essential elements found in the soil are taken up by plants using very elaborate transportation machinery. Plants, thus serve as a direct source of elemental copper for higher organisms. Additionally, human breast milk has the highest concentration of Cu (0.25 to 6.0 mg/l). For its effective utilization, the elemental copper derived from these sources needs to be absorbed and transported to metabolically active sites. This process, termed bioavailability, is possibly regulated by four essential attributes as defined by Raul (6). These include (i) quantum of intake; (ii) dependent variability; (iii) linearity between dose and response; and (iv) slope ratio analysis. Solubility of Cu in water or physiological fluids is a good indicator of bioavailability and digestibility. Additionally, copper complexes with various biomolecules, thus, facilitating its utilization. These include complex of copper with lectins and glycoproteins as seen in grains or with amino acids as noticed in higher organisms including mammals. In fact, amino acids exert a critical role in uptake of copper by the intestinal membranes. Further, among the essential amino acids, methionine in the diet enhances Cu absorption by at least 2-fold. On the contrary, by the side of cysteine due to its ability to chelate by the side of copper coupled to its ability to potentially reduce copper to a monovalent state (7), leads to a reduction in its bioavailability. On a similar note, tripeptide of glutathione has significant post-absorptive importance in Cu transport. Glutathione forms an intermediary complex with Cu in the enterocytes before transferring the metal to other target proteins- viz., superoxide dismutase or ceruloplamin (CP), etc., thus facilitating its assimilation. This ability of copper to complex with amino acids or organic acids is extensively exploited in animal nutrition experiments. As an example, Cu-lysine complex has been shown to be effective as a supplement in feed for chicks than for lambs (8). Similarly, formulations of proteins with minerals, also termed proteinates, have been found to be highly effective as feed for growing calves in areas with high molybdenum contents in foliage (9). Notably, molybdenum competitively inhibits intestinal Cu uptake. On the contrary, various derivatives of copper such as chlorides, acetates, sulfates and carbonates enhance its bioavailability in higher organisms.

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Plants as bioindicators and hyperaccumulators of Cu: A few plants uniquely accumulate copper from their habitats viz., soil or water. They are: Aeolanthus biformifolius, Athyrium yokoscense, Azolla filiculoides, Bacopa monnieri, Brassica juncea L., Callisneria Americana, Eichhornia crassipes, Haumaniustrum robertii, Helianthusannuus, Larrea tridentate, Lemna minor, Pistia stratiotes and Thlaspi caerulescens. The metal molybdenum is also accumulated by Thlaspi caerulescens (Brassica). Sheep feeding on Thlaspi caerulescens possibly face the deficiency of copper as this specific plant is also a hyperaccumulator of molybdenum which inhibits intestinal copper uptake.

Copper transport and utilization

Dietary copper, absorbed in the stomach and upper intestinal tract, reaches liver as a complex with serum proteins viz., albumin or transcuperin or the amino acid histidine (10). Importantly, liver is the major store house for intracellular copper (11). Here, copper is reduced to cupric state and transported across plasma membrane by CTR1 transporters as described later. Importantly, as highlighted later in this review, intracellular copper needs to be maintained in a complex state so as to prevent the oxidative damage caused by free copper to DNA, proteins and membrane components (10). Hence, copper transport and utilization involves a complex interplay between transporters and binding proteins/ chaperones. Additionally, Cu plays a vital role as a catalytic co-factor for a variety of metalloenzymes. Keeping the importance of cupric Cu in biological function, an elaborate mechanism is set forth by Nature for maintaining Cu homeostasis, which includes a wide array of proteins namely (i) family of Cu bearing proteins, (ii) cuproenzymes, (iii) Cu transporters and (iv) Cu chaperone proteins. It is not surprising for the redundant machinery that Cu is enjoying out of several heavy metals for its transport and participation in cellular metabolism, which guarantees the survival of living organisms as conditioned by the strategies and mechanisms of the evolution of metallic proteins.

The family of Cu beating proteins plays a significant role in metal detoxification and keeps the Cu in non ionic curpric state. They are metallothioneins, prion protein, albumin, transcuperin, CP, phycocyanins of blue green algae and haemocyanins of blue blooded organisms.

Blue blooded organisms: An interesting copper binding protein found in some of the lower eukaryotes is hemocyanin (Hcy). Fig. 2 shows the UV spectrum of Oxy-Hemocyanin (Oxy-Hcy) with a characteristic absorbance at 340 nm revealing the presence of copper-oxygen complex. Hcys are found in a majority of arthropods and mollusks, and they are called "Blue Blooded Organisms" by virtue of the fact that their blood turns blue in color upon oxygenation. Importantly, in these organisms, hemocyanin associated with blood (also called hemolymph) serves as primary carrier of oxygen. Hcy turns blue upon binding molecular oxygen, a phenomenon that is readily reversible. Notably, such binding occurs at high partial pressure of oxygen which converts Hcy to Oxy-Hcy. The latter dissociates to release molecular oxygen at the vicinity of tissues that have low oxygen pressure, thus functioning as a mode for oxygen transport (12) (Fig. 3).

Characteristically, Hcy is non-cellular and found freely dissolved in haemolymph. By virtue of its large molecular size with multiple epitopes, Hcy is a potent immunogen as evidenced by the development of discrete crescentic arcs in Ouchterlony double immunodiffusion assay upon antibody challenge (Fig. 4).

As revealed in Figs. 5B and C the Hcy from hemolymph of fresh water field crab and Indian apple snail Pila, showed positive staining with rubeanic acid stain (a stain to detect copper binding proteins), confirming its ability to bind copper (13). Additionally, the detection of copper granules by the histochemical staining of hepato-pancreas in the pulmonate garden snail, Cryptozona ligulata, potentially reveals the existence of a copper store, probably complexed with metallothioneins, that could be possibly recruited for Hcy biosynthesis (Fig. 5A) (14,15). Structurally, molluscan haemocyanins are composed of multiple subunits (eight) that result from duplications in the gene encoding for the protein (Fig. 6). These subunits assemble into a quaternary folded architecture with 160 oxygen binding sites in the native protein (16). This is in contrast to the Hcy from arthropods, that have only 3 subunits that are folded up to generate 48 oxygen binding sites (3). Importantly, in all these cases, each of the oxygen binding sites contains 2-Cu atoms and each of the Cu atoms anchors to 3 histidine residues. Further, the two molecules of copper are bridged together by 2 molecules of oxygen resulting in the formation of a dioxygen bridge. Thus on the whole, hcy derived from molluscs and arthropods contain 320 and 96 copper atoms respectively.

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Phenoloxidase is another such copper binding protein that binds to dioxygen with a different physiological function viz., browning of fruits and vegetables in plants as well as wound healing, skin pigmentation etc in higher organisms. Also, it has a role during sclerotization of new exoskeleton in molting insects. This contrasting physiological role for phenoloxidase compared to Hcy could be attributed to the ability of the former to trigger the catecholase activity (3).

Transporter proteins: In higher organisms and plants, principal copper binding proteins belonging to the family of P-type ATPases serve the function of intracellular copper transport. Included among these are the two proteins ATP 7A and ATP 7B. Interestingly, the presence of such a network for Cu transport seems to be evolutionarily conserved. Notably, prokaryotes possess metal transporting enzymes also termed heavy metal ATPases that protect them from stress caused by heavy metals found in their natural environment. These according to Nigel et al (17) are encoded by the structural genes (cutA, cut-B ... cut-F) and the regulatory protein, cutR. Additionally, some mutant forms of E.coli harbor a plasmid borne version of copper resistance genes also called pco that confers resistance to approximately five fold higher concentrations of cupric ions than wild type strains (18). Significantly, bacteria endowed with such heavy metal transporter proteins are now being exploited commercially in a process termed "Bioleaching". The latter is an environmentally friendly process for metal recovery which is a cost-effective process for treating ores that are remote and difficult to access. One of the most exploited microorganisms in bioleaching is Acidithiobacillus ferrooxidans.

As mentioned above, a similar paradigm consisting of metal transporters have been described to be existent in plants, specifically in edible portions such as seeds (19). Notably, Cu plays a vital role in the physiology of plants viz., respiration and photosynthesis. Further, photosynthetically active cells require more Cu than other cells. Two families of Cu transporter proteins have been recognized among plants (19). Among these, P-type ATPases (PAA) belong to the family called heavy metal ATPases (HMA). In Arabidopsis, they function to transport Cu to the stroma of the chloroplast, where they play a critical role in maintaining copper homeostasis. Importantly, mutation in these ATPases affects the photosynthetic electron transport, which can be reversed by addition of Cu. Additionally, these are also involved in the transport of Cu in roots and flowers of plants. The latter is supported by the detection of transcripts for HMA in these sites (19,20). The second family of Cu transporters viz., COPT (Cu transporters) are also identified in plants. The homologous transporter proteins of the same have been reported in yeast and mammals (20). Notably, Arabidopsis exposed to decreased levels of copper for a period of 18 h was shown to turn on a compensatory mechanism that involved increased synthesis of COPT mRNA (21). In addition, the phenotypic manifestation of reduced copper levels in these plants involved an increase in root length which could be reversed by the addition of Cu (19). Further, the importance of COPT1 knockdown using an anti-sense strategy resulted in an increased frequency of pollen abnormalities even though the experimental plants were grown under standard nutrient conditions. The latter phenotype was rescued by exogenous addition of Cu highlighting the importance of this element for the developing pollen (19). In addition to transporters, plants also contain a class of molecules termed the metallochaperones that bind metals and facilitate their transport to target proteins/sites. The expression of such Cu chaperone mRNA is ubiquitously seen in the tissues of root, stem, leaf and inflorescence indicating its role as intercellular Cu delivery and recycling. One such copper chaperone seen in plants is cytochrome oxidase 17 (COX17). Defects in COX17 lead to the respiratory deficiency due to the failure of protein to deliver Cu to mitochondrial cytochrome oxidase complex. Interestingly, the various metal transporter proteins like PAA, HMA, COPT, CCH, COX17, etc., form potential targets that could be manipulated to enhance mineral deposits in plants that could possibly alleviate mineral deficiency in humans and live stock.

In higher organisms, the absorbed dietary Cu enters liver through entero-hepatic circulation and is further transported as a complex with CP or excreted into bile, a process facilitated by a number of Cu chaperones and transporting proteins (Fig. 7). ATP7A (MND) and ATP7B (WND) are also the principal Cu transporters in higher eukaryotes. These transporters contain 8-transmembrane domains in addition to six Cu-binding motifs at the N-terminus (MXCXXC; M=methionine, C=Cysteine, X=any amino acid). These Cu binding motifs of ATPases reveal that Cu ions are typically bound to sulphur containing amino acids. They function like cation exchangers and use energy from ATP hydrolysis to translocate metal cations across lipid bilayers. Both ATP7A and ATP7B are predominantly localized in the transgolgi-network (TGN) and involved in 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 this transporter (22-24). ATP7A regulates Cu- efflux when the levels of the latter become high in epithelial cells. In contrast, ATP7B expression is higher in the liver where it regulates the release of copper into bile. Importantly, the transcript levels of both these transporters are positively regulated by intracellular levels of copper. Further, Cu ATPases also appear in the placenta and lactating breast tissue for transporting Cu to fetus and through milk to neo-nates respectively. ATP7A have been reported to be present within syncytiotrophoblasts, cytotrophoblasts and fetal vascular endothelial cells. This is consistent with their role in the transport of Cu from these tissues into the fetal circulation (25). In contrast ATP7B has been shown to facilitate the export of Cu from the placenta to the maternal tissues, a mechanism that protects excessive copper from reaching the developing fetus. Additionally, ATP7A is also reported to be expressed in luminal epithelial cells of alveoli and ducts of breast tissue, with its expression levels being positively regulated by lactation (26). Further, Cu ATPase activity is also seen in the central nervous system where both ATP7A and ATP7B regulate neuronal Cu homeostasis. Also, both these are expressed within retinal pigment epithelium where they regulate the release of CP that in turn maintains iron homeostasis.

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An alternate class of copper transporters found in yeast are, Ctrl that regulate the influx of copper into the cytoplasm. These transporters contain three transmembrane domains with methionine rich extracellular motifs at the N-terminal that bind copper and enable its import (27). Importantly, dietary Cu (CuII) needs to be converted to its reduced form (CuI) prior to its transport by the Ctrls. The process of biochemical reduction is carried out by various plasma-associated reductases.

The mammalian homologue of yeast Ctrl is called MURR1; while it's human orthologue is named CTR1/ 2. MURR1 is a recently discovered protein chaperone whose absence has been shown to cause Cu toxicosis, potentially due to hepatic Cu overload (Fig. 8). The latter has been documented in Bedlington terriers with MURR1 gene mutation that is characterized by deletion of exon 2, resulting in complete absence of the functional protein product in liver of affected animals (28). Importantly, these terriers exhibit elevated levels of lysosomal Cu content and pronounced reduction in bilary Cu excretion (29). This is suggestive of cooperativity between MURR1 and ATP7B, to mediate excretion of excess copper into bile. Further, imported intracellular copper has been shown to bind Atox1, which then transfers the metal to its docking partners in the secretory pathway (Fig. 8). This reveals a potential role of Atox1 in the ATP7B-mediated bilary excretion of excess Cu. Atox1 has also been implicated in mediating copper transfer to CP and tyrosinase. The latter which is a critical step in melanin generation is supported by the observation wherein Atox1-null mice have been shown to exhibit hypo-pigmentation (30).

An alternate mode of copper entry involving endocytosis is facilitated by a class of proteins termed prion protein ([PrP.sup.C]). These are glycoproteins that are expressed on the plasma membrane. By virtue of its expression in the central nervous system as well as peripheral tissues, mutation in [PrP.sup.C] lead to a number of neurodegenerative disorders that includes Creutzfeld-Jakob disease. In these disorders, the ability of the mutant prion to transport copper is significantly impaired making neuronal cells susceptible to oxidative stress (24). Also, tripeptide glutathione (GSH) binds copper and enables its transport across the blood-brain barrier.

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Importantly, in serum, most of the copper is transported by CR It is synthesized by both hepatocytes and activated macrophages. It is a 132 KDa monomer. In addition to serving as a prime transporter of copper in serum, CP also plays a major role in intestinal absorption of iron. Significantly, in its role as a carrier of copper in serum, each molecule of CP can bind to seven molecules of copper. Notably, elevated plasma CP has been shown to have adverse effects on cardiovascular system.

Transcriptional regulation of proteins involved in Cu translocation

Prokaryotic Cu homeostatic system has been well characterized in Enterococcus hirae (31). Four genes (copY, copZ, copA and copB) are reported to be arranged in the cop operon of E.hirae. CopA and copB encodes for Cu transporting P-type ATPases which are highly conserved, stabilized and possibly extended into eukaryotes. CopY encodes for Cu responsive repressor and copZ encodes for a chaperone protein. The cop operon allows growth of E. hirae in Cu-limiting conditions (up to 8 mM Cu). CopA ATPases take Cu while it is limiting and copB ATPases bale out excess Cu. CopY regulates the expression of cop operon and copZ translocates Cu intracellularly (32,33). CopY is a Zn containing homodimeric repressor that binds to the promoter region of the cop operon, thereby regulating the synthesis of ATPases and chaperones. It is reported that copY is dimeric and belongs to winged-helix type repressor (34). Thus, initially, the package of molecular machinery for the regulation of heavy metal ions gained relevance in the survival of bacteria and hence it would not be a surprise for the eukaryotes to adopt them. The expression of the cop operon is low in standard growth media whereas induced by 50 fold upon exposure of bacteria to extracellular Cu (35). CopY repressor binds to the consensus binding site TACANNTGTA, called 'cop box' (36). Experimentally induced mutation in cop-box prevented its interaction with the repressor. The kinetics of the interaction between the repressor and promoter of cop operon in E. hirae are elaborated by David Magnani and Marc Solioz (35). The induction of cop operon is facilitated by excess Cu which makes the repressor (CopY) to dissociate from the cop box. This E. hirae model has yielded an insight into possible existence of a similar molecular architecture in eukaryotes.

Copper-complexes.... A necessity for cellular function

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

In order to understand the biological processes regulated by copper binding proteins, we adopted an enrichment strategy. Firstly, all proteins having either a copper binding domain/functional site were culled from the InterPro database (http://www.ebi.ac.uk/interpro). This resulted in a total of 36 proteins that were distributed across 7 groups based on function/domains/functional sites (Table). Each group included 3-12 proteins. Proteins from all groups were then used for enrichment analyses using a bioinformatics tool called Oncomine Concept Maps (OCM) (www.oncomine.org), developed by Daniel Rhodes and colleagues (37,38). OCM, is an enrichment tool, that allows to systematically linking groups of protein/ genes that have a common biological nuance to various molecular concepts thus generating novel hypothesis. Notably, we believe that such an enrichment analysis of copper binding proteins could potentially reveal various cellular processes that could be initiated by their action. The various molecular concepts that were used in this enrichment analyses were derived from both gene and protein annotations from external databases, and computationally-derived regulatory networks. The external annotation included chromosomal locations, protein domains and families, molecular functions, cellular localizations, biological processes, signaling and metabolic pathways, protein-protein interaction networks, protein complexes, and gene expression signatures. The regulatory networks were derived by scanning human promoters for known transcription factor motifs and by comparative genomics analyses that identified conserved promoter and 3'UTR elements. A P-value cutoff of 5X[10.sup.-2] was used to cull significant concepts. In total, data from 12 databases and 335 high-through put datasets were collected and analyzed.

Interestingly, as shown in Fig. 9, the copper containing proteins play an active role in 3 major cellular processes. These include tyrosine metabolism and melanin biosynthesis (red bridges), amino acid metabolism (blue bridges) and coagulation cascade (black bridges). Further, included in the concept that portrayed "tyrosine metabolism and melanin biosynthesis" were multiple protein-protein complexes involving the proteins Dopachrome tautomerase, Tyrosinase and Tyrosinase-related protein 1, all of which are copper binding proteins and play a critical role in the above bioprocess. Similarly copper binding proteins, potentate amino acid metabolism, by having a functional role in two biological processes, namely amine oxidase and oxidoreductase activity. Additionally, copper binding proteins regulate the coagulation cascade by forming protein complex with the PROC protein (inactivator of coagulation factors Va and VIIIa). Also the proteins that bind copper were intimately involved in superoxide metabolism.

Among the proteins involved in superoxide metabolism, CCS plays a key role in the transmission of Cu to pro-form of superoxide dismutase (apo-SOD). CCS possesses three functional domains. Domain I contains Cu-binding site, domain II is homologous to SOD and domain III contains cysteines essential in the transfer of Cu to apo-SOD. CCS deletion has been documented to markedly reduce SOD activity in mice (39,40). Third class of chaperone includes COX17, which delivers Cu to cytochrome C oxidase (CCO). CCO is a large protein found in the cytoplasm and mitochondrial inner membrane. It has two subunits I and II, each containing Cu binding sites. Fourth class of copper chaperone includes metallothioneins (MT). These are cysteine rich proteins (30%) composed of 61 amino acids. Due to their high redox potential, MT's regulate intracellular levels of Zn and Cu in addition to serving as potent mediators of toxic metal detoxification. As a part of the former, MT levels tightly regulate copper homeostasis in liver. Interestingly, the pool of MT-Cu complex progressively decreases with age in mammals (41,42). A fifth class of copper chaperone comprises of the membrane protein [beta] amyloid precursor protein (APP) that regulates import of the metal into brain. This is supported by the observation wherein copper levels in the brain of APP null mice are higher compared to their wild type counterparts (43).

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In addition to the transporters, there are a number of enzymes that require copper as cofactors for their catalytic activity. Some of the members of this class of cuproenzymes include Cu/Zn SOD (antioxidant defense), cytochrome C oxidase (mitochondrial respiration), CP (iron metabolism), tyrosinase (pigmentation), lysyl oxidase (collagen maturation in connective tissue), Hephaestin (intestinal iron efflux), dopamine [[beta]-hydroxylase (catecholamine synthesis), Peptidylglycine [alpha] -amidating monooxygenase (peptide-hormone processing), amine oxidase (removal of hormones), ascorbate oxidase and catalase oxidase (oxidation of primary alcohols to aldehydes) (44).

Interestingly, as would be discussed later, the OCM also portrayed an enrichment of copper binding proteins in invasive tumors especially those associated with soft tissue (melanoma) and liver.

Prognosis through copper metabolism: Cu is found in all living organisms in trace quantities with an uptake range in humans being 0.9 to 10 mg/day. The metal, Cu is found as a prosthetic group in metalloenzymes binding to sulphur residues. Several physiological reactions such as electron transfer, detoxification of reactive oxygen species, connective tissue development, oxygen transport, oxygenation reactions are being mediated by Cu containing metalloenzymes. When Cu regulation fails, a variety of biochemical disturbances develop. The failure in Cu elimination and its efflux leads to Wilson's and Menkes diseases respectively. Another intriguing role of Cu is reported in the promotion of angiogenesis for facilitating tumor to progress. Therefore, by examining the distinguishing features of symptoms due to copper imbalance and its metabolism, the possible prophylactic and chemotherapeutic agents could be designed.

Disease symptoms due to Cu deficiency and overload

The disturbance in the levels of Cu is primarily due to genetic defects. The most prominent among these are Menkes and Wilson's diseases.

Menkes disease is a rare X-linked (Xq13) fatal disorder 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 the flux of copper resulting in a systemic deficiency of copper (45). Specifically, most of the Cu accumulates in intestinal epithelium and kidney while suboptimal levels of the metal are found in other tissues such as liver and brain. Menkes disease is a fatal disorder, wherein lethality is preceded by neuronal (cerebral and cerebellar) degeneration and connective tissue abnormalities during the first 2-4 yr of infancy (46). Similar condition has been reported to occur in sheep where the disorder is termed as Kinky Hair Disease (47). The primary mode of diagnosis involves the use of genetic screens. Early diagnosis coupled with supplementation of copper (as Cu-histidine complex) could avoid neurodegeneration and lead to reinstatement of normal development (46). Interestingly, cells derived from patients with Menkes disease exhibit copper accumulation when cultured in vitro (48).

Wilson's disease is a rare autosomal recessive trait manifested in the chromosome, 13q14.3. The frequency of occurrence of Wilson's disease is about 1/30,000 to 1/50,000 with a carrier frequency of 1 per cent and heterozygote frequency of 0.86 per cent, (confined to western world). Notably, this defect is caused due to the mutation in the gene coding for ATP7B, whose original function is to regulate the bilary excretion of excess copper. ATP7B is encoded by the WND gene. Among several mutations that have been reported for this gene (>200), the most well studied one is a point mutation involving replacement of the amino acid histidine by glutamine at position 1069. The mutated protein thus loses the ability to orient ATP in its catalytic site, thus impairing its normal function (49). The resultant is the accumulation of Cu in liver leading to cirrhosis and hemolysis. Advanced stages of the disorder are characterized by deposition of excess Cu in brain and eyes in the form of Kayaer-Fleischer ring, which serves as diagnostic marker for Wilson's disease (50). The therapeutic measures for this disorder revolve around chelating the excess copper using chelating agents such as tetrathiomolybdate, trientine and penicillamine.

Contrary to copper accumulation, its deficiency can lead to hypocupremic state. Zatta and Frank (44) reported that there was an incidence of 11.3 million clinically identifiable Cu deficiency cases in 1970, which has since been on the rise. Copper deficiency could be a result of either inadequate dietary intake (also termed primary copper deficiency) or due to impairment in its uptake (secondary copper deficiency). The latter could be caused by the presence of additional heavy metals in the diet that could competitively diminish copper uptake in the lining of gastrointestinal tract. Among these, molybdenum is the most common competitor of copper absorption. Importantly, the relative ratio of dietary Cu: MO have been defined to be 4 and 8 (51) respectively to achieve optimal control in nutritional balance and hence copper homeostasis in ruminants.

Additional disorders are caused by mutations in various cuproenzymes as reported by Prohaska (32). These include, (i) Albinism, wherein an impairment of an enzyme tyrosinase which is a critical intermediate in melanin biosynthesis, (ii) Over gene dose effect of Cu-Zn SOD noticed in Down Syndrome (trisomy 21) due to the presence of this gene on the chromosome 21, (iii) X-linked Cutis laxa (or an analogous disorder in mouse termed blotchy mouse), which are characterized by defects in cross-linking of collagen due to decreased lysyl oxidase activity, (iv) Mottled mice, an X-linked disorder analogous to Menkes disease wherein Cu metabolism is affected. These mice have a mottled appearance due to decreased melanin pigmentation resulting from a reduction in tyrosinase activity, and (v) Toxic milk mutant mouse, a homozygous trait caused by Cu accumulation in liver. This results in a decreased copper content in milk of lactating mothers which is toxic to the suckling offspring. By virtue of its similarity to Wilson's disease in accumulating copper in liver, the toxic milk mutant mouse could serve as a paradigm for understanding the mechanism that underlies the development of Wilson's disease.

Role of copper in tumor development and progression: Copper metabolism is a critical component of tumor progression. Concentration of copper in serum has been found to correlate well with tumor development, size, progression as well as recurrence (53). Elevated levels of circulating copper in serum have been documented in cancers of lung, breast, gastrointestinal tract, brain as well as gynecological cancers (54,55). Importantly, copper levels are higher in metastatic disease compared to localized tumors (54). This increase in serum copper levels during neoplastic progression is reflected in concomitant increase in the levels of CP, the primary carrier of copper in serum (53). Interestingly, CP has been nominated as potential marker for diagnosis of advanced solid tumors (56). Additional evidence for the role of copper in tumor development is derived from experiments that show existence of Cu salts in tumor extracts that could stimulate the migration of endothelial cells in vitro (57).

The role of copper in tumor progression is best understood in the light of the knowledge that developing tumors require an ample supply of oxygen and nutrients that necessitates the development of a well defined vasculature. The process termed angiogenesis is critical for tumor proliferation and metastatic spread. Among the various factors that lead to initiation of the angiogenic process, tumor associated hypoxia seems to play a major role. Importantly, copper also plays a major role in the induction of tumor angiogenesis (53). This is supported by experiments conducted by Parke et al (58), wherein dosedependent neovascularisation (angiogenesis) is noticed upon implanting Cu pellet into rabbit cornea.

Notably, copper exerts its effect on angiogenesis by inducing endothelial cell proliferation and migration by the way of activation of various angiogenic factors. The latter include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumor necrosis factor [alpha] (TNF[alpha]) and Interleukin 1 (IL-1) (53). These angiogenic factors in turn activate resting endothelial cells (which are otherwise in GO phase of the cell cycle) and initiate their proliferation by transitioning them to the GI phase of the cell cycle. This process of endothelial activation by copper can be reversed using chelating agents like penicillamine (59), a property that is widely exploited in designing therapeutic regimens (see section below). Additionally copper has been thought to exert its effect by binding to proteins like heparin, CP, etc., making them angiogenic (60). The angiogenic property of the latter is evident in the observation wherein CP has been reported to induce the formation of capillaries in the cornea of rabbits (58).

To understand the effect of copper in cancer, we used the data from an interesting study aimed at predicting the chemosensitivity of human cancer cell lines (61). In this study chemosensitivity predictions were based on transcriptomic profiling done upon treatment with various compounds on a panel of 60 cancer cell lines (NCI-60 panel) (61). A set of 50 genes were found to be differentially regulated between copper sensitive and resistant cell lines upon treatment with 0.0001M copper sulfate. These set of 50 genes were used for enrichment analyses to understand the role of copper in tumor progression. The enrichment analyses was done using OCM as described above (37). Interestingly, the differentially expressed genes between copper sensitive and resistant cell lines mapped to multiple gene expression signatures (red nodes) derived from tumors that included sarcoma, lung carcinoma, colorectal cancer, etc. (Fig. 10). Furthermore, copper induced genes also mapped to a subset of genes that are activated upon Src over expression (red node) (Fig. 10). This is important in the context of earlier studies that have described a critical role for Src in tumor development and progression. These observations provide evidence at the molecular level for the role of copper in tumor progression.

Brain disorders due to Cu deficiency and/or excess: The brain is an organ of bewildering complexity with multifaceted serendipitous effects. In such a resilient situation, the maintenance of adequate Cu levels is vital. Both brain and liver are metabolically active organ systems. Dysfunction of Cu homeostasis due to its excess or deficiency as reflected in Wilson's and Menkes diseases respectively causes severe ailments in these two organ systems.

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Additionally. disruption of copper homeostasis coupled to oxidative stress and free radical generation play a significant role in the development of Alzheimer's disease, amyotrophic lateral sclerosis and Creutzfeid-Jakob disease (62).

Among these, the etiology of Alzheimer's disease is associated with accumulation of amyloid [beta] protein (A[beta]) (63). This is produced either by proteolytic processing or secretion of its precursor, a transmembrane glycoprotein named [beta]-amyloid precursor protein (APP) (64). The accumulation of A[beta] leads to the formation of A[beta] fibrils, which have been shown to exert neurotoxic effects both in vitro (65,66) and in vivo (66). Importantly, recent studies have revealed the ability of the A[beta] peptide to generate hydrogen peroxide by reduction of the bound metal (67) which mediates the generation of free radicals that play a causal role in oxidative stress induced neurotoxicity, by inducing lipid peroxidation, protein oxidation, etc., (67-69). In Alzheimer's, the proteolytic product (A[beta]) is mutated resulting in its accumulation which is assumed to trigger free radical mediated injury viz., neuronal injury (70). Notably, under normal conditions, APP is known to bind copper in its reduced state and facilitates its transport along the length of the neuron from the cell body to the axonal surface and to plasma membrane of dendrites (63). However, in Alzheimer's disease, APP function is disrupted leading to oxidation of its bound copper in presence of [H.sub.2[O.sub.2]. This is accompanied by fragmentation of APP resulting in A[beta] peptides. These fragments are thought to aggregate and lead to oxygen free radical injury in Alzheimer's disease (71). Additionally, Cu also binds to extracellular plaques and causes interference in Cu trafficking devices and in turn depletes intracellular Cu repertoire. This would reduce the activities of cytochrome oxidase and SOD. Thus, increased oxidative stress coupled with the reduction in key metabolic and defense mechanism could contribute significantly to neuronal damage. Oral treatment of transgenic mouse with clioquinol resulted in halving of A[beta] levels and significantly increased the levels of Cu and Zn in the brain (72).

The other neuronal cellular membrane protein is prion. It is associated with the diseases such as neurodegenerative disorders that include Kuru, Creutzfeld-Jakob disease in humans, Scarpie in humans and bovine spongiform encephalopathy (mad cow disease) in cattle (73). Here again, the conformational change in the protein affects its function. The structural change involves a transition of native [alpha]-helical prion into a [beta]-sheet conformation conferring pathogenic potential to the protein. Using experiments employing circular dichroism, this misfolding coupled to its aggregation have been shown to be mediated by copper (74). Further support for the involvement of copper comes from studies wherein use of D-Penicillamine (copper-chelator) has shown to result in a delayed onset of the disease in Scrapie infected mice, presumably mediated by a reduction in the levels of copper in brain as well as in the circulation (75).

In addition to the above disorders, several neurological disorders have been reported in newborns due to the deficiency of Cu (44). One such disorder happens to be neonatal ataxia, a disease found in lambs, caused due to either low Cu or high molybdenum content in their feed. The disease is characterized by tremors, in-coordination, paralysis and ultimately death (51). Significantly, affected lambs show significant demyelination which affects brain as well as causes necrosis of neurons (76). Importantly, copper deficiency has been shown to be causal in inducing the demyelination in affected animals (77).

Cu chelation therapy: Dietary excess Cu intake is not very common, although there are genetic disorders as discussed in the previous sections. The increased accumulation leads to hepatitis and neurological disorders. Human Wilson's disease and Toxic milk mouse are associated with excess accumulation of cellular Cu. In the former, the defect is manifested in Wilson protein (ATP7B) which in its normal form does facilitate to eliminate excess Cu ions into bile. Therapeutic approaches to Cu toxicity include the drugs and formulations such as D-penicillamine or trientine to prevent neurodegenerative disorder (78). Similarly, tetrathiomolybdate, as a specific Cu chelator have been used in Toxic milk mouse model in reducing abnormally high Cu (79). Since copper plays an important role in tumor development and progression (as discussed above), strategies employing Cu chelators are also being pursued for cancer therapy (80). In contrast, in conditions like Menkes disease that results from copper deficiency, an approach to supplement copper complexed with histidine or albumin are being tested (79).

Homeopathic formulations using Cu metal: Homeopathy is based on the argument that the body is a self-healing entity, and that symptoms are the expression of the body attempting to restore its balance. Homeopathic physicians are trained to match the patient's symptoms with the accurate remedy. They believe that the remedies themselves never destroy disease, but stimulate the body's own healing action to get rid itself of the problem. Minerals in the body can be used as healing agents for specific health problems. Minerals are used in homeopathic remedies to stimulate corresponding body cells towards metabolic activity and health restoration. A few tinctures with the combination of copper are: (i) Cuprum aceticum, (ii) Cuprum Arsenicosum, (iii) Cuprum Metallicum and (iv) Cuprum Sulphuricum (81).

All life forms exploit naturally available Cu for myriad physiological functions. Bacteria, plants, blue blooded organisms and vertebrates have developed the molecular mechanisms to upkeep the Cu homeostasis. The bioavailability of Cu, by complexing with proteins or amino acids or organic acids constituting organometallic complex, facilitates its ease in uptake and distribution in ecosystem. Literature review reveals that the Cu imbalance could be causal in Menkes disease, Wilson's disease, Kuru, Creutzfeld- Jakob disease, mad cow disease as well as induce tumor development and progression. By its unique attribute of being a catalytic cofactor, Cu occupies an important niche in biological systems. Cu transporters, chaperone proteins and carrier proteins make Cu available to the intricate network of biochemical systems. Developments in the field of plant genetic engineering have been pivotal in defining means to combat copper deficiency. In the clinical field, management of disorders caused by impaired copper homeostasis are being combated either using metal chelators or by supplementing the metal in a complex state with various carriers.

Acknowledgment

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

Received January 28, 2008

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Reprint requests: Dr S. Krupanidhi, Department of Biosciences, Sri Sathya Sai University Prasanthi Nilayam 515 134, India e-mail: krupanidhi_srirama@yahoo.com

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

Department of Biosciences, Sri Sathya Sai University, Prasanthi Nilayam, India; * Michigan Center for Translational Pathology & Department of Pathology, University of Michigan, Ann Arbor, MI, USA & ** Center for Molecular Medicine, Department of Molecular Medicine & Surgery, Karolinska Hospital, Stockholm, Sweden
Table. Classes of copper binding proteins defined by
InterPro (http://www.uk/interpro)

Group 1: Copper type 11, ascorbate-dependent monooxygenase

 1. DBH, Dopamine beta-hydroxylase (dopamine beta-
 monooxygenase)
 2. MOXD1, Monooxygenase, dbh-like 1
 3. PAM, Peptidylglycine alpha-amidating monooxygenase

Group 2: Di-copper centre-containing

 l. DCT, Dopachrome tautomerase
 2. TYR, Tyrosinase
 3. TYRP1, Tyrosinase-related protein 1

Group 3: Copper amine oxidase

 1. 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 dismutase

 1. CR1, Complement component (36/4b) receptor I (knops
 blood group)
 2. PSORS1C1, Psoriasis susceptibility candidate 1
 3. KIAA0467, Kiaa0467
 4. SOD1, Superoxide dismutase 1, soluble (amyotrophic
 lateral sclerosis 1)
 5. SOD3, Superoxide dismutase 3, extracellular
 6. CCS, Copper chaperone for superoxide dismutase

Group 6: Blue (type 1) copper domain

 1. NR1H3, Nuclear receptor subfamily 1
 2. LYST, Lysosomal trafficking regulator
 3. FLJ25006, hypothetical protein flj25006
 4. APR-2, Apoptosis related protein
 5. SLC35B2, Solute carrier family 35, member b2
 6. IGHG4, Immunoglobulin heavy constant gamma 4 (g4m
 marker)
 7. SIRT7, Sirtuin (silent mating type information regulation
 2 homolog) 7 (S. cerevisiae)
 8. CCDC 14, Coiled-coil domain containing 14

Group 7: Multicopper oxidase, copper-binding site

 1. SLC15A4, Solute carrier family 15, member 4
 2. CP, Ceruloplasmin (ferroxidase)
 3. F5, Coagulation factor V (proaccelerin, labile factor)
 4. HYAL4, Hyaluronoglucosamindase 4
 5. SNAD, Snail homolog 3 (drosophila)
 6. HEPHL1, Hephaestin-like I
 7. ITIH3. Inter-alpha (globulin) inhibitor h3
 8. CCDC73, Coiled-coil domain containing 73
 9. SLC14A1 Solute carrier family 14 (urea transporter),
 member 1
 10. SLC14A2, Solute carrier family 14 (urea transporter),
 member 2
 11. CCIN, Calicin
 12. HEPH, Hephaestin
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Author:Krupanidhi, S.; Sreekumar, Arun; Sanjeevi, C.B.
Publication:Indian Journal of Medical Research
Article Type:Clinical report
Geographic Code:9INDI
Date:Oct 1, 2008
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