Copper: the Jekyll and Hyde ElementJulian F.B. Mercer1 and Jim Camakaris2

1Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, VIC 3125 and 2Department of Genetics, University of Melbourne, VIC 3010

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Copper was discovered as an essential nutrient in animals in the 1920s and 1930s but it took several decades after this to establish the importance of copper for human health (1). Copper is required for the activity of a range of cuproenzymes, and the most important clinically are cytochrome c oxidase and lysyl oxidase. The ability of copper to undergo facile interconversion between the two oxidation states, Cu (I) and Cu (II), has been utilised in a range of oxidative enzymes. This useful property can be regarded as the Dr Jekyll side of c o p p e r w h i c h p r o v i d e s f u n c t i o n s n e e d e d f o r maintaining good health.

Unfortunately Dr Jekyll is accompanied by the dangerous Mr Hyde. If copper is not handled properly by cells, the activity of Mr Hyde is unleashed in the form of copper-catalysed free radicals. The most damaging of these is the hydroxyl radical, which is formed by copper-catalysed reaction of hydrogen peroxide in the Fenton reaction. Hydroxyl radicals can cause substantial damage to all cellular constituents, including DNA, proteins and membranes. Thus the problem for all organisms living in an oxidising environment is how to use Dr Jekyll without releasing Mr Hyde. This problem was solved by the development of tightly regulated copper homeostatic mechanisms. The identification of the genes involved in a genetic copper deficiency (Menkes disease; Dr Jekyll is out of action), or copper toxicity (Wilson disease; Mr Hyde has escaped) has identified two key players in copper homeostasis, and studies of yeast mutants have revealed other important members of the cast of molecules that regulate copper absorption, distribution and excretion.

Copper Deficiency DiseasesAnimal studies revealed the symptoms of copper

deficiency, which include osteoporosis, anaemia, arterial rupture, neurological defects and, in sheep, wool abnormalities known as 'steely wool'. Species vary widely in their susceptibility to copper deficiency (and toxicity) and the particular symptoms that predominate (1). Anaemia and neutropenia are found in all cases of severe nutritional copper deficiency of animals, including humans. Severe copper deficiency in humans is usually only reported in premature infants and reflects the high demand for copper in the early period of growth (1). The fact that copper deficiency can induce common conditions such as osteoporosis and anaemia, however, raises the issue of how many such cases have chronic marginal copper deficiency as a contributing factor.

The most severe example of copper deficiency is seen in the genetic disorder Menkes disease. This fatal X-

l i n k e d c o n d i t i o n i s c h a r a c t e r i s e d b y g r o w t h retardation, neurological degeneration, connective tissue abnormalities and peculiar hair (2). Menkes disease was first shown to be a copper deficiency disease by David Danks at the Children's Hospital in Melbourne (3). David's familiarity with the symptoms of copper deficiency in animals explains why this seminal discovery in the copper field was made in Australia. Allelic variants of Menkes disease are known and one of these, occipital horn syndrome, is primarily a connective tissue disorder.

The isolation of the Menkes gene by positional cloning provided an insight into not only the disease i t se l f , but a l so the molecular bas is o f copper homeostasis (4-6). It led directly to the cloning of the gene affected in Wilson disease. The Menkes gene (ATP7A) encodes a transmembrane copper P-type ATPase, ATP7A protein, the first heavy metal P-type ATPase to be described in mammals.

Copper Toxicity DisordersWilson disease is an autosomal recessive copper

toxicosis disease caused by a mutation in a Menkes gene paralogue, ATP7B. It is primarily a disease of copper accumulation in the liver that can lead to liver failure, or neurological disease when copper escapes from the liver and deposits in the central nervous system. The Wilson protein, ATP7B, has a central role in copper homeostasis, as it eliminates excess copper from the body in the bile. It is the failure of this excretory mechanism that results in the slow accumulation of copper in hepatocytes. The excess copper causes the death of the hepatocyte and subsequent liver failure. Wilson disease is readily treated with copper chelators if diagnosed before liver failure.Another copper toxicity disorder is known as copper-

associated childhood cirrhosis. This disease appears to be an autosomal recessive condition that requires exposure to excess copper in early childhood for the symptoms to occur (7). The sporadic occurrence of this disease in several countries has caused public health authorities to review the possible risks to the wider population of copper in drinking water, primarily originating in copper pipes. The cases of childhood deaths suggest that genetic subgroups may be at risk and further work into possible polymorphic variants of copper transporters may identify sensitive subgroups.

Neurological Diseases Involving CopperThe amyloid precursor protein (APP), which is

involved in Alzheimer's disease, may have a role in copper homeostasis in neurons (8). Recent findings demonstrate that mice with a knockout of the APP

gene have increased copper accumulation in cultured neurons (9) and that copper regulates the expression of the APP gene (10). Amyloid plaques contain high levels of copper and zinc; and copper bound to Aβ can directly produce hydrogen peroxide, setting up conditions for Fenton chemistry. Copper chelators have been found to dissolve these plaques and to produce a significant improvement in Alzheimer patients (11).

Prion diseases, such as Kuru and mad cow disease, produce fatal neurodegeneration. The prion protein contains copper-binding motifs and the toxic peptide fragment PrP106-126 generates hydroxyl radicals in the presence of copper, strongly suggesting a role for copper in these diseases (12). It is vital that copper homeostasis in the brain is understood in order to clarify the role of copper in aetiology of neurodegenerative diseases.

Key Molecules in Copper HomeostasisThe cloning of the Menkes and Wilson proteins and

studies of yeast mutants have led to the isolation of a range of molecules important for copper homeostasis (summarised in Fig. 1). Copper homeostasis is achieved by a balance between absorption and excretion, and the rates of each process are modulated by the dietary and tissue copper levels. Copper is absorbed from the small intestine and is passed into the blood via the intestinal enterocytes. The most likely transporter at the apical surface of the cell is hCTR1, a trimeric complex that was first identified in yeast (13). In the cytoplasm a group of small molecules known as copper chaperones receive the copper from hCTR1 (14). In Fig. 1 we show only ATOX1 which delivers copper to the Menkes and

Wilson proteins. In the enterocyte, the Menkes protein (ATP7A) transports copper across the basolateral membrane into the circulation, and this explains the block to copper transport at this level in patients with Menkes disease (3). Copper uptake is regulated by the level of dietary copper, and the mechanism possibly involves the endocytosis of hCTR1 (see below). Copper is distributed to tissues in the blood, bound to a variety of molecules and small peptides and the details of this are still being investigated. The hepatocyte plays a pivotal role in copper homeostasis. Much of the absorbed copper is taken up by the hepatocyte, and excess copper is excreted into the bile by the Wilson protein (ATP7B). This process is regulated by the copper-induced trafficking of ATP7B to vesicles which move to the apical surface of the hepatocyte (see next section). The critical function of copper delivery to the brain across the blood brain barrier requires the Menkes protein and, in a mouse model of Menkes disease, copper is trapped in the astrocytes and endothelial cells that form the blood brain barrier (15). The Menkes protein is also required for efflux of copper out of various tissues, such as kidney proximal tubules, and copper accumulates in the kidneys of patients with M e n k e s d i s e a s e . T h u s m u t a t i o n s i n A T P 7 A paradoxically result in overall copper deficiency despite the accumulation of copper in some tissues. When the cellular efflux molecules, ATP7A and ATP7B, are inactivated by mutations as in Menkes and Wilson diseases, intracellular copper rises and this induces the synthesis of the metal binding metallothioneins (MTs) which sequester the excess copper.

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Fig. 1. Some molecules involved in the physiological regulation of copper.The trimeric protein CTR1 is primarily responsible for copper uptake in a variety of cells. The Menkes protein ATP7A effluxes copper across the basolateral surface of the intestinal enterocyte and is also required for copper entry into the brain. The Wilson protein ATP7B is the molecule that removes excess copper from the liver in the bile. Copper exists as Cu (I) [ I ] inside cells and Cu (II) [ II ] in the extracellular environment.

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Copper: the Jekyll and Hyde Element

Trafficking of Copper Transporters:a Central Homeostatic MechanismIn addition to the efflux of copper across the plasma

membrane, both ATP7A and ATP7B supply copper to secreted copper-dependent enzymes, a process that occurs in the trans Golgi network (TGN). In low copper conditions, the transporters are localised in the TGN. However, as copper levels rise in the cytoplasm, the proteins are induced to traffic out of the TGN. This process was first described in our laboratories in the case of the Menkes protein, which traffics to the plasma membrane in response to copper (16) . Subsequently, ATP7B was found to traffic to large vesicles in response to copper. This trafficking is central to the maintenance of cellular and whole body copper levels. The central role of excretion of excess copper in the bile is achieved by trafficking of ATP7B to vesicles closely associated with the apical surface of the cell and thus the biliary canaliculus (17) (Fig. 1). The basolateral targeting of ATP7A is consistent with its proposed role in copper absorption at the basolateral surface of the intestinal enterocyte (18) (Fig. 2).

Regulation of copper uptake by CTR1 also involves trafficking (19). As shown in Fig. 2, in low copper, the prote in is c losely associated with the plasma membrane, but after a period of time in high copper it is found inside the cell in large vesicular structures. Thus, in response to high copper, the transporters display distinct trafficking responses: CTR1 is removed from the plasma membrane region reducing uptake, and ATP7A/B move to the plasma membrane region to facilitate efflux of excess copper.We have been studying the regulation of the

traff icking of ATP7A and AT7B using in vitro mutagenesis to alter various regions of the proteins.

For example, both proteins have dileucine motifs close to their C-termini and these motifs are required for the return of the protein from the plasma membrane (or vesicles) to the trans Golgi (20). The six metal binding sites in the N-terminal region of these molecules are also important for the trafficking process, as mutation of this region blocks trafficking (21). The trigger for trafficking appears to be the formation of a high energy aspartyl-phosphate intermediate (Fig. 3) that is characteristic of P-type ATPases. It is likely that the f o r m a t i o n o f t h e a c y l p h o s p h a t e i n d u c e s a conformation that exposes a trafficking signal. Interestingly, if ATP7A has a mutation in the domain that removes the acyl phosphate (the phosphatase domain), the protein is constitutively located on the plasma membrane (22). It also appears likely that kinase phosphorylation of ATP7A and ATP7B is associated with trafficking (23). Recently we have identified a putative PDZ target motif at the C-terminus of ATP7A which appears to be involved in targeting of ATP7A to the basolateral membrane (18).

Functional Effect of Mutations that Cause Menkes Disease and its VariantsMutations of the Menkes gene result in three distinct

clinical phenotypes: classical Menkes disease, mild Menkes disease and occipital horn syndrome. Classical Menkes disease causes death in early childhood, mild Menkes disease is characterised by less severe neurological defects, and occipital horn syndrome is a connective tissue disorder (2, 24). Classical Menkes disease results when there is little, if any, active ATP7A formed from the mutant gene. Mild Menkes disease appears to result from missense mutations that allow some residual Cu transport activity. The protein has lost its ability to traffic in response to copper, but as it is located in the TGN, it supplies sufficient copper to lysyl oxidase, thus explaining why the connective tissue defects are not pronounced. Occipital horn syndrome is caused by splice site mutations that allow a small amount of normal splicing and therefore a small amount of normal ATP7A to be formed. However, this protein is constitutively localised on the plasma membrane and insufficient copper is supplied to lysyl oxidase, thus explaining the pronounced connective tissue defects (24).

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Fig. 2. Trafficking of copper transporters maintains cellular copper homeostasis.CTR1 t ra f f i c s f rom the p lasma membrane to endosomal-like vesicles in response to high copper, thus limiting copper uptake. ATP7A traffics from the trans Golgi network to the basolateral plasma membrane when cytoplasmic copper increases, and ATP7B traffics to subapical vesicles in high copper.E = endosomal-like vesicles, N = nucleus, SC = subapical compartment, TGN = trans Golgi network.

Fig. 3. Proposed reaction cycle of the Menkes copper-translocating P-type ATPase (ATP7A).T h e e n z y m e h a s t w o b a s i c E 1 a n d E 2 conformation states and forms a high energy Cu-dependent acyl-phosphate* intermediate. The cycle results in translocation of Cu to the lumen (e.g. of the TGN) or to the outside of the cell.

Copper Homeostasis in Other Organisms − Model SystemsElegant studies in yeast have identified a number of

cytosolic 'Cu chaperones', which are involved in transporting Cu to various subcellular compartments (14). The yeast copper-translocating P-type ATPase CCC2 functions in delivering Cu to the multicopper ferroxidase, Fet3 (analogous to ATP7B delivering Cu to caeruloplasmin in the case of mammals), in the Golgi compartment.

The bacterium Enterococcus hirae has two copper-translocating P-type ATPases, CopA, which is involved in Cu uptake, and CopB, which is involved in Cu efflux (25). These are regulated at the transcription level. This is in contrast to the mammalian copper-transporting ATPases which do not appear to be regulated at the transcriptional level, but which exhibit Cu-regulated trafficking as described above.

Recently the fruitfly Drosophila melanogaster has been investigated as a model system for understanding copper homeostasis. It provides a potentially powerful metazoan model which is amenable to sophisticated genetic analysis and has a well annotated genome. Using mutants , the Ctr1B Cu t ransporter was characterised as being vital for normal growth and at particular stages of development (26). We have recently demonstrated that cultured Drosophila cells express a number of putat ive or thologues of human Cu homeostasis genes (27). Using double stranded RNA interference, we have characterised genes involved in copper detoxification (27). These approaches should lead to discovery of novel candidate genes involved in Cu homeostasis.

The copper-regulated trafficking of the copper transporters provide a fascinating example of a tightly coordinated homeostatic mechanism that helps keep the Mr Hyde of copper under control while allowing Dr Jekyll to carry out useful functions. Further analysis of these mechanisms and other components will lead to clarification of the importance of copper toxicity and deficiency in common diseases, and perhaps lead to the identification of genetic variants of the transporters that result in sensitivity to copper deficiency or toxicity.

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