16 Genetic Susceptibility to Heavy Metals in the Environment Diane W. Cox and Lara M. Cullen University of Alberta, Edmonton, Alberta, Canada John R. Forbes McGill University, Montreal, Quebec, Canada 1. INTRODUCTION Many metal ions are required for essential functions, yet are toxic in excess. Metalloproteins play important structural and functional roles in cell metabolism. Homeostatic mechanisms maintain a critical balance to avoid toxicity. Such mechanisms must be able to respond to changes in metal concentration in our environment due to diet or environmental pollutants. Maintenance of balance requires regulated absorption, transport, and excretion mechanisms, controlled by a number of genes whose products ensure correct transport of metals to spe- cific sites. Each metal is expected to have its own series of transporters. However, as we will show in the following pages, several metals are highly dependent upon the concentration of other metals. When any gene within the balanced system is nonfunctional, the balance can be upset. The specific genes involved in human transport are best known for copper and iron. Both metals are associated with diseases involving metal storage, which Copyright © 2002 Marcel Dekker, Inc. can be affected by excessive metal intake. These two metals have important inter- actions. Zinc and molybdate, in the form of tetrathiomolybdate, each have an effect on the transport of copper. Knowledge of the copper transport pathway has increased dramatically in less than a decade and is the major focus of this chapter. Copper and iron are chosen for discussion also because they demonstrate genetic disorders and show environmental consequences. 2. OVERVIEW OF COPPER TRANSPORT IN HUMANS The average daily copper intake is between 1 and 2 mg, and approximately half of this is absorbed (1). This copper plays an essential role in a number of proteins. Cytochrome oxidase is an inner-membrane mitochondrial protein complex, func- tioning as a key enzyme that catalyzes the reduction of oxygen to water, using the free energy of the reaction to generate a proton gradient required for respira- tion. Other proteins that require copper for function are superoxide dismutase (SOD1), which converts superoxide anion to hydrogen peroxide and protects against cellular free radical damage; lysyl oxidase, required for collagen and elas- tase cross-linking, and dopa beta-monooxygenase, which converts dopamine to norepinephrine. In excess, copper generates free radicals and becomes a potent cellular toxin via the Haber-Weiss reaction (2). The consequences of hydroxy radical production in vivo include lipid peroxidation, DNA strand breakage and base oxidation, mitochondrial damage leading to reduced cytochrome oxidase activity, and protein damage. Copper is widespread in the environment, released particularly through mining operations, incineration, weathering of soil, industrial discharge, and sew- age treatment plants. The concentration of copper can be increased during its distribution. Copper, particularly at low pH, can be leeched from copper pipes and plumbing fixtures into the drinking water. When water sits stagnant in pipes, the copper concentration in the water can be markedly increased. Chronic expo- sure to high doses of copper can cause liver disease in normal individuals, for example, with the use of a high-dose copper supplement 10 or more times the daily requirement (3). The ability to excrete copper through the liver is normally high and copper is unlikely to cause damage except in individuals who have abnormalities of biliary excretion. However, there are genetic situations in which individuals can be susceptible to normal amounts of copper in the diet. Human genetic disorders associated with an imbalance of copper homeostasis include Menkes disease, a disorder resulting in overall copper deficiency, and Wilson disease, a disorder of toxicity due to excess copper storage. Aceruloplasminemia, a complete deficiency of ceruloplasmin, is associated with iron storage. Aceru- loplasminemia demonstrates the interplay between iron and copper. Canine cop- per toxicosis, which occurs particularly in Bedlington terriers, is a different disor- der of copper storage. Copyright © 2002 Marcel Dekker, Inc. F IGURE 1 Overview of key features of copper transport, showing diseases that can result from defects in three parts of the pathway. A simplified overview of the mammalian copper pathway is shown in Fig- ure 1. Ingested copper is absorbed via the small intestine (4) and rapidly enters the bloodstream bound to albumin, histidine, and small peptides (1). The absorbed copper is transported to the liver, where it is excreted into the bile. In the hepato- cyte, a small amount is incorporated into the plasma protein, ceruloplasmin, a 130-kDa protein containing six atoms of copper. Most of the copper in plasma is bound to ceruloplasmin. Normal concentrations of copper in the serum are 3– 10 millimolar in adults. Ceruloplasmin concentration is usually 200–400 mg/L, with higher amounts in young children (5). Ceruloplasmin is an acute-phase re- actant and increases in response to inflammation, liver disease, and malignancy. Copper in the plasma is carried to the kidney, where a small amount is resorbed. Copyright © 2002 Marcel Dekker, Inc. If the concentration of copper is very high in the plasma, as indicated by an elevation in ceruloplasmin, adequate resorption may not take place and a slight increase in urinary copper excretion could occur. Discovery of the genes involved in two copper transport diseases has been important in our understanding both of normal copper transport and of the conse- quences of abnormal functioning of either of these genes. One of these genes, involved in biliary copper excretion, influences our capacity to rid the body of excess copper from the environment. The second gene, involved in a copper- deficiency state, is highly related. 3. DISORDERS OF COPPER TRANSPORT IN HUMANS 3.1 Menkes Disease 3.1.1 Clinical and Biochemical Features Menkes disease is an X-linked recessive copper deficiency usually leading to severe disease and death in early childhood (5,6). This disease, while not influ- enced by environment, is important for the understanding of other aspects of response to copper. The features of the disease are those characteristic of severe copper deficiency. There is a pronounced reduction in all copper-containing enzymes. Resulting features include hypothermia and progressive cerebral degeneration (deficiency of dopamine beta-monooxygenase and cytochrome oxidase), arterial aneurysm, bladder diverticuli, and ligament laxity (deficiency of collagen cross-linking mediated by lysyl-oxidase) and hypopigmentation (deficiency of copper-dependent tyrosinase enzyme activity). Another feature of Menkes disease is distinctive brittle hair with a corkscrew-like appearance. Treatment by copper histidine administration can be effective in part, if started sufficiently early (7,8). The problems with connective tissue begin in utero and are not reversed by this treatment (8). A variant of Menkes disease, occipital horn syndrome, is a mild form of Menkes disease characterized by connective- tissue abnormalities, such as hyperelastic skin, skeletal abnormalities, hernias, bladder diverticuli, and aortic aneurysms, all due to reduced lysyl oxidase activity (9,10). Developmental delay, if present, is less severe than in classical Menkes disease. Biochemical features of Menkes disease reflect the copper deficiency (11,12). These include reduction of liver copper, and low plasma copper and ceruloplasmin. However, the copper level in the duodenal mucosa is two- to threefold higher than in normal individuals. Copper absorption in the intestine is normal, suggesting that there is defect in copper transport out of the intestinal epithelium and into the circulation. An additional diagnostic feature of Menkes disease is that patient fibroblasts in culture accumulate high levels of copper due to defective copper efflux compared with that of normal controls (13,14). Copyright © 2002 Marcel Dekker, Inc. The gene for Menkes disease (designated ATP7A) was identified in 1993 by positional cloning, in a female patient with a translocation breakpoint at the Menkes locus (15–17). Sequencing of the cDNA revealed a predicted protein (ATP7A) similar to a P-type ATPase previously found to transport copper in bacteria (18). ATP7A is expressed in all tissues including the intestine, but not in the liver. The protein mediates copper efflux from cells in peripheral tissues, particularly intestine, as a mechanism for copper homeostasis. Lack of ATP7A in intestinal cells results in the defect of intestinal copper transport and the resulting widespread copper deficiency seen in Menkes disease. ATP7A may also be in- volved in copper incorporation into cuproenzymes in peripheral tissues. Numer- ous ATP7A mutations have been identified in Menkes disease patients (19). Dele- tions, some of many kb in length, have been identified in 15–20% of patients with classic Menkes disease (16,17). Approximately 90% of known mutations are predicted to destroy the protein, causing the severe disease seen in most patients (12,19,20). The few missense mutations or splice site mutations observed are typically found in patients with the milder form, occipital horn syndrome (21,22). 3.1.2 Animal Models of Menkes Disease The mottled mouse has biochemical and phenotypic signs like those seen in Menkes disease, and the orthologous gene (Atp7a) is defective (22–24). More than 20 mutations are identified in Atp7a that lead to the mottled (Mo) phenotype. Different alleles of the mottled locus lead to mice with a wide range of disease severity, ranging from prenatal lethality to connective tissue abnormalities as in cutis laxa (23,25,26). For example, the brindled mouse (Mo br ) has a 6-bp gene deletion and a phenotype of prenatal lethality, consistent with severe classical Menkes disease (25,27). Although ATP7A protein is expressed in Mo br fibro- blasts, the protein is probably inactive, and does not traffic within the cell, as is required for copper export (discussed below) (28). The blotchy mouse (Mo bl ) has a splice site mutation that interferes with normal splicing, causing markedly re- duced mRNA levels, and features similar to occipital horn syndrome (29,30). 3.2 Wilson Disease 3.2.1 Clinical and Biochemical Features of Wilson Disease Wilson disease (hepatolenticular degeneration) is an autosomal recessive disorder of hepatic copper transport and storage. The disease locus maps to chromosome 13q14.3. This is a disorder in which excess copper in the environment may play a role. Wilson disease affects approximately 1 in 30,000 individuals in most popu- lations, possibly up to 1 in 10,000 in certain populations such as in China, Japan, and Sardinia (5,31). The characteristic defects observed in Wilson disease are reduced excretion of copper into the bile, resulting in a toxic accumulation of copper in the liver and an increase of copper excretion in the urine. The second Copyright © 2002 Marcel Dekker, Inc. defect is a reduced incorporation of copper into ceruloplasmin. Although the plasma ceruloplasmin concentration may lie within the normal range, the incorpo- ration of radioactive or stable isotopes of copper always indicates a defect in the incorporation of copper into ceruloplasmin (32,33). The cloning of the gene in 1993 (34,35) has helped explain the biochemical and clinical changes observed. As a result of mutations in the copper transporter gene, ATP7B, copper accumulates in the liver, first inducing the production of metallothionein, which can apparently maintain copper in a relatively harmless state. The accumulation of copper causes damage to mitochondria. Copper is deposited in renal tubules, and kidney damage occurs to varying degrees. Copper also accumulates in the basal ganglia of the brain causing neurological disease. It is not yet clear whether the neurological damage is due to expression of the gene in the basal ganglia, or to an effect of high levels of circulating plasma, or to the combined effects of both. In Wilson disease, clinical presentation is highly variable (5,31), with age of onset from less than 5 years to greater than 50 years, and clinical manifestations presenting as hepatic or neurological disease. Patients may have chronic or fulmi- nant liver disease, neurological disorder with or without liver involvement, purely psychiatric illness, or isolated acute hemolysis. Renal damage may also occur. Neurological forms manifest as a movement disorder with poor coordination, tremors, and loss of motor control, or with dystonia, with rigidity and gait distur- bance (5,31). Patients with neurological Wilson disease have copper accumula- tion in the liver and reduced plasma ceruloplasmin levels, but may not show clinical evidence of liver damage. Psychiatric disorders can occur in as many as 20% of patients, (5,31). A distinctive feature of patients with Wilson disease is the presence of Kayser-Fleischer rings in the cornea, due to copper deposition in Descemet’s membrane at the outer rim of the cornea. This is occasionally easily visible, but usually is observed only by careful slit lamp examination. Kayser-Fleischer rings are frequently absent in patients with hepatic disease (36). An important biochemical feature of Wilson disease is hepatic copper accu- mulation due to impaired biliary copper efflux (5). Normal adults typically have 20–50 µg copper/g dry liver, whereas Wilson disease patients have greater that 250 µg/g. Copper also accumulates in the kidney, brain, and cornea. Serum holoc- eruloplasmin (enzymatically active) levels, and consequently serum copper lev- els, are usually below normal, although they may be normal in the presence of liver disease (36). Nonceruloplasmin copper (mostly albumin bound) is increased. Urinary copper excretion is greatly elevated. Apoceruloplasmin, encoded by a gene on chromosome 3, has normal biosynthesis in Wilson disease patients, but copper incorporation into the protein during biosynthesis is impaired. Wilson disease can be effectively treated if diagnosis occurs sufficiently early. Chelating agents are effective for removing excess copper from blood and/ or tissues. Penicillamine, the sulfhydryl-containing amino acid cysteine substi- Copyright © 2002 Marcel Dekker, Inc. tuted with two methyl groups, was introduced by Walshe in 1956, and has rescued many hundreds of patients from this potentially fatal disorder (37). Penicillamine removes copper from plasma, preventing further accumulation by greatly increas- ing urinary excretion of copper. Penicillamine is not particularly effective at removing liver copper stores. Studies in the LEC rat model indicate that peni- cillamine inhibits the accumulation of copper in lysosomes, macrophages, and Kupffer cells, and makes copper soluble, for mobilization from these cellular components (38). Hepatic metallothionein is induced and copper is stored in the liver in a relatively nontoxic form. Trientine, or trien, has also been used exten- sively for chelation (39–41). The dramatic effect of a high oral ingestion of zinc led to the use of oral zinc sulfate as a treatment for Wilson disease, particularly in Europe, where it has been used since 1979 or earlier (42). The use of zinc, often as zinc acetate, is now becoming more widespread worldwide. Oral zinc has a different mode of action from the chelating agents. The mechanism of action is through induction of metallothionein in enterocytes (43). Metallothionein preferentially binds cop- per because of its higher affinity. Copper is eliminated through shedding of the enterocytes during normal turnover. This approach offers a cheap alternative treatment. Although the long-term effectiveness and side effects of zinc therapy have not been as well evaluated as for penicillamine, maintenance zinc treatment in follow-up studies of up to 10 years suggest that zinc is effective as maintenance therapy (44). The interaction of copper and zinc is discussed further below. Ammonium tetrathiomolybdate, one of the newer chelating agents to be used, may be particularly useful for patients with neurological disease, as it does not seem to lead to initial neurological degeneration, as is sometimes observed with penicillamine (45). This reagent is particularly effective at removing copper from the liver, in contrast to trientine or penicillamine. Because of its effective- ness, continuous use could cause copper deficiency. Reversible bone marrow suppression has been noted as an adverse side effect (46). With this agent, as with other chelators, studies must be undertaken to ensure that copper is not mobilized to other tissues. Such studies will be facilitated with the use of proven animal models. Cloning of the Wilson disease gene (ATP7B) was accomplished by conven- tional linkage analysis (47), by physical mapping of the relevant region of chro- mosome 13q14, and finally by its high homology with the Menkes disease gene (34,35). ATP7B encodes a predicted protein characteristic of copper-transporting P-type ATPases, with 57% identity to ATP7A. ATP7A and ATP7B have different tissue expression profiles, leading to the distinct phenotypes of Menkes and Wil- son disease. ATP7B is expressed primarily in liver and kidney; and less in the brain and placenta. Translation of the nucleotide sequence predicted six putative heavy-metal- binding domains, and a mean amino acid identity of 65% between each of the Copyright © 2002 Marcel Dekker, Inc. six domains found at the 5′ end of ATP7A. Both ATP7A and ATP7B also contain highly conserved domains characteristic of the P-type family of cation trans- porting ATPases. The functionally important regions of the predicted protein, similar in the genes for the two disorders, were predicted to be: 1) a transduction domain, containing a Thr-Gly-Glu (TGE) motif, and 2) cation channel and phos- phorylation domains, containing a highly conserved Asp-Lys-Thr-Gly-Thr (DKTGT) motif. The aspartate residue forms a phosphorylated intermediate dur- ing the cation transport cycle. An invariant proline residue in a cysteine-proline- cysteine cluster, located 43 residues N-terminal to this aspartate, is within the predicted cation channel. 3) An ATP-binding domain (residues 1240–1291) is a highly conserved region situated at the C-terminal end of a large cytoplasmic ATP-binding domain, including a Gly-Asp-Gly (GDG) motif. Eight hydrophobic regions are predicted to span the cell membrane. In addition to the potential membrane-spanning regions, a small hydrophobic domain is predicted (residues 362–386) including part of copper-binding domain 4. This may affect the tertiary structure of the copper-binding region, forming a pocket. Over 200 mutations have been found in the ATP7B gene of Wilson disease patients (for reference see the Wilson disease database at http:// www.medgen.med.ualberta.ca/database.html). The spectrum of known mutations is different from that of ATP7A (20,48). The majority of known mutations (51%) in ATP7B, as indicated in the database, are single-base-pair missense mutations, which are infrequent (2%) in ATP7A (19). The remaining ATP7B mutations in- clude nonsense, splice site, and small insertion/deletion mutations, sometimes resulting in frame shifts. No gross gene deletions (common in ATP7A)ofATP7B have been observed. Differences in the observed mutation spectrum of ATP7A and ATP7B may be biased since Wilson disease mRNA and genomic structure are not routinely analyzed in patients with Wilson disease. Most mutations are very rare in the population; consequently most patients are compound heterozy- gotes. One mutation, His1069Glu, is found in up to 30% of patients of European origin, up to 65 or 70% in eastern Europe. Homozygotes in several studies have had an onset of about 20 years of age, in the neurological form (49–52). The Arg778Leu mutation is commonly found in Asian populations and is associated with severe early-onset hepatic disease in homozygotes (50,53). The extreme phenotypic variation among Wilson disease patients may be explained in part by allelic heterogeneity of the ATP7B gene, but other genetic and environmental factors must also be involved. 3.2.2 Possible Susceptibility of Heterozygotes to Copper in the Environment There is no evidence reported that heterozygotes, who carry only one mutated gene for Wilson disease, ever develop clinical symptoms. However, they are relatively frequent in the population, on average 1 in 90. Possibly these individu- Copyright © 2002 Marcel Dekker, Inc. als are more susceptible than normal individuals to an increased concentration of copper, from drinking water or other environmental sources. 3.2.3 Copper and Oxidative Damage Excess copper in tissues leads to the production of damaging free radicals and to DNA cleavage (54). Copper overload particularly affects mitochondrial respi- ration and causes a decrease in cytochrome C activity (55). Damage to mitochon- dria is an early pathological effect in the liver. Damage to the liver has been shown to cause increased lipid peroxidation and abnormal mitochondrial respira- tion, both in copper overloaded dogs and in patients with Wilson disease (55). The generation of free radicals by the presence of copper could be particularly accelerated in patients with Wilson disease who lack the antioxidant effects of ceruloplasmin. Wilson disease patients have been shown to have low levels, in the liver, of antioxidants, including ascorbate and urate (56) and α-tocopherol (56,57). These observations suggest that antioxidants may be important adjuncts for saving tissue from damage in patients with Wilson disease. Further studies are needed in this area. 3.2.4 Animal Models of Wilson Disease There are two rodent models of Wilson disease: the LEC rat and the toxic milk (tx) mouse (58,59). Both rodents exhibit hepatic copper accumulation due to reduced biliary copper excretion, and reduced copper incorporation into cerulo- plasmin. The LEC rat has a large deletion removing 25% of the Atp7b coding region (58). Unlike patients with Wilson disease, the LEC rat develops liver tu- mors. Copper and iron may both participate in the induction of DNA damage and malignancy in the rat (60). This rat model is being used for many studies of the transport of copper, and for the evaluation of new modes of therapy (61,62). LEC rats offer opportunities for experiments attempted at therapy at the gene level. The tx autosomal recessive mutation first arose in the inbred DL mouse strain (63), producing homozygous dams unable to secrete copper into milk. Con- sequently, litters of homozygous tx dams are severely copper deficient and dis- play poor growth, hypopigmentation, hepatic accumulation of copper, and early death (64). In the original tx mouse, the causative mutation is a missense mutation at position 4066 of Atp7b, resulting in a methionine-to-valine substitution in the eighth transmembrane region (59). In 1987, a new autosomal recessive mutation (tx J ) arose in the C3H/HeJ strain at the Jackson Laboratory, Bar Harbor, and was shown to be allelic with the original tx mutation (65). The mutation differs from that of the original tx mouse and is found in the second transmembrane domain (V. Coronado, M. Nanji, D. W. Cox, unpublished). The toxic mutations apparently destroy activity of the protein, as their phenotypes are identical to that of a recently described Atp7b knockout mouse (66). Copyright © 2002 Marcel Dekker, Inc. 3.3 Other Copper Diseases Indian childhood cirrhosis and Tyrolean infantile cirrhosis appear to have a strong environmental component, producing disease when dietary copper is exception- ally high. These diseases may be similar to canine copper toxicosis. 3.3.1 Indian Childhood Cirrhosis Indian childhood cirrhosis occurs in infants and young children, with a fatal out- come unless copper is removed through chelation therapy. This disease has a strong environmental factor. This disease has been recognized and described in India since the late nineteenth century and much as been written on the subject (see reviews in refs. 67,68). Clinical features are in many respects similar to those seen in Wilson disease, except that the onset is generally earlier. In some of the series of patients reported with Indian childhood cirrhosis, Wilson disease could have been the cause as clinical symptoms can manifest as early as 3 years of age (69). The histological criteria suggested for Indian childhood cirrhosis are necro- sis of hepatocytes with ballooning, pericellular collagen disposition, and in- flammatory infiltrate (70). These features are not exclusive to Indian childhood cirrhosis. An important difference between Indian childhood cirrhosis and Wilson disease is that copper, demonstrated by automatic absorption spectrometry in Wilson disease, is usually not stainable by copper stains such orcein rhodanine or rubeanic (71). However, orcein staining has been found as a consistent feature of Indian childhood cirrhosis (72). A different distribution of the copper is also observed. In Wilson disease, copper is found in the cytoplasm. In Indian child- hood cirrhosis, copper is reported to accumulate in the nuclear fraction of hepato- cytes (73). Another difference between Indian childhood cirrhosis and Wilson disease is that the former can be halted by a brief period of penicillamine therapy (74). Once the copper is removed, the children and infants do not have a recur- rence of the disease. Ingested copper appears to play an important role in this disease, although there is probably an underlying genetic predisposition. Large amounts of copper are ingested because of the use of brass pots. The first-born male in the family has been particularly likely to be affected, because of the tendency to favor feed- ing animal milk, which is boiled in the brass pots (67). The brass pots used for food are generally covered with a thin layer of tin, which is replaced frequently. When this layer wears off, copper becomes accessible from the brass. Experi- ments have shown that cow’s milk, with a copper content of about 19 µg/dl, contained 26 µg/dl after 6 h at room temperature, and about 625 µg/dl after boiling (67). With the alteration of feeding patterns, this disease has almost disap- peared in the areas of India in which it was prevalent (68). There are still sporadic cases of Indian childhood cirrhosis, suggesting that the disease is likely heteroge- Copyright © 2002 Marcel Dekker, Inc. [...]... within their heavy- metal-binding domains Each Cys-box is likely part of an individual subdomain, which together form the entire copper-binding domain Bacterial CPx-type ATPases typically contain only one or two Cys-box motifs within a metal-binding domain A second type of putative copper-binding motif, designated the His-box, was also identified in bacterial CPx-type copper ATPases (117,119) The feature... heavy- metal-binding domain based on homology with the bacterial mercury binding protein MerP and bacterial cadmium efflux protein CadA (15–17,34,35) Each metal-binding motif contains a conserved Gly-Met-X-Cys-X-X-Cys (GMxCxxC) sequence, predicted to bind metal via the cysteine residues ATP7A and ATP7B each contain six Copyright © 2002 Marcel Dekker, Inc copies of Cys-box copper-binding motifs within... large N-terminal domain, predicted to be a soluble heavy- metal-binding domain This domain is joined to the core ATPase portion of the molecule by two additional transmembrane segments, making a total of four between the Nterminal domain and transduction domain of these proteins There is no equivalent predicted N-terminal structure on non -heavy- metal-transporting ATPases, which begin immediately with the. .. subdomains The structure of the fourth ATP7A copper-binding subdomain, solved by Gitschier et al (122), likely represents the prototypical fold of copper-binding subdomains found in the copper-binding domains of CPx-type ATPases, although there may be subtle differences between this structure, solved with bound silver, and a copper-bound form of the protein The individual Cys-box subdomains must then fold in. .. ATPase from other P-type ATPases is the Ser-Glu-His-ProLeu (SEHPL) motif, C-terminal to the putative phosphorylated aspartate residue (35,119) The most common mutation in Wilson disease, His1069Gln, lies in this motif This motif is not found in non -heavy metal P-type transporters 4.3.3 Structure and Metal-Binding Properties of Copper-Binding Domains Some facets of the structure and copper-binding properties... must then fold in relation to each other to form a complete copper-binding domain Metal binding to the entire copper-binding domains of ATP7B and ATP7A has been studied (125,126) The data suggest that at least six atoms of copper, in the Cu(I) form, can bind to the copper-binding domains of ATP7B and ATP7B, with one atom occupying each of the six copper-binding subdomains Results have suggested selective... copper binding to the copperCopyright © 2002 Marcel Dekker, Inc binding domain of ATP7B (127) Copper binding by the copper-binding domain of ATP7A also appears to be cooperative (128) 4.3.4 Copper Transport by CPx-Type ATPases The first copper-transporting CPx-type ATPase shown to be capable of directly transporting copper was the CopB ATPase of Escherichia hirae, which contains two His-box copper-binding... states IRPs bind to these IREs, preventing translation of the ferritin mRNA and obscuring a degradation signal in the 3′ UTR of the transferrin receptor mRNA, increasing stability of the mRNA and thus increasing the receptor levels If intracellular iron levels are high, iron binds to the IRPs and prevents their binding to IREs, allowing translation of ferritin to proceed Also, transferrin receptor mRNA... Instead, non -heavy metal P-type ATPase transporters typically have four additional C-terminal transmembrane segments added to the ATPase core giving a predicted topology of 10 membrane-spanning helices, as supported by biochemical data (113,120,121) 4.3.2 The Heavy Metal P-Type ATPases A striking feature distinguishing CPx-type ATPases from other P-type ATPases is the large N-terminal domain, originally... properties of the copper-binding domains of ATP7B and ATP7A are known The solution structure of the fourth copper-binding subdomain of ATP7A was solved by nuclear magnetic resonance (NMR), using silver as the metal ion (122) Met12 was hypothesized to be a metal-binding ligand, owing to its sulfur moiety and close proximity to the conserved cysteine residues Metal binding by Met12 was not observed in the NMR . copper-bound form of the protein. The individual Cys-box subdomains must then fold in relation to each other to form a complete copper-binding do- main. Metal binding to the entire copper-binding. directly transporting copper was the CopB ATPase of Escherichia hirae, which contains two His-box copper-binding subdomains in its N-terminal copper-binding do- main. The protein was able to transport copper into. Copper-Binding Domains Some facets of the structure and copper-binding properties of the copper-binding domains of ATP7B and ATP7A are known. The solution structure of the fourth copper-binding