Role of copper transporters in copper homeostasis

Copper is a redox active metal that is essential for biological function. Copper is potentially toxic; thus, its homeostasis is carefully regulated through a system of protein transporters. Copper is taken up across the lumen surface of the small intestinal microvilli as cuprous ion by Ctr1. Cupric ion may also be taken up, but those processes are less well understood. Within the cell, intestinal as well as others, copper is escorted to specific compartments by metallochaperones. One, CCS, donates copper to superoxide dismutase. Another, COX17, delivers copper to additional chaperones within the mitochondria for synthesis of cytochrome c oxidase. A third chaperone, Atox1, delivers copper to the secretory pathway by docking with 2 P-type ATPases. One, ATP7A, is the protein nonfunctional in Menkes disease. This protein is required for cuproenzyme biosynthesis, and in the enterocyte it is required for copper efflux to portal blood. The second, ATP7B, predominantly expressed in liver, is required for copper metallation of ceruloplasmin and biliary copper excretion. Mutations in ATP7B lead to Wilson disease. Additional intracellular hepatic copper-binding proteins COMMD1 (copper metabolism MURR1 domain) and XIAP (X-linked inhibitor of apoptosis protein) may also be required for excretion. Other proteins involved in copper homeostasis may include metallothionein and amyloid precursor protein. Plasma protein transport of copper from the intestine to liver and in systemic circulation probably includes both albumin and alpha2-macroglobulin. Changes in the expression of copper "transporters" may be useful to monitor copper status of humans, provided a suitable cell type can be sampled.     

Wilson disease

Wilson disease is an autosomal, recessive inherited disorder of copper metabolism, characterized by the accumulation of copper in the body due to defective biliary copper excretion of hepatocytes. Recently, novel components involved in copper metabolism, Wilson disease protein (ATP7B) and copper chaperones, have been identified. It has been demonstrated that ATP7B functions in copper secretion into the plasma, coupled with coeruloplasmin synthesis and biliary copper excretion. Genetic testing may help early diagnosis and with the beginning of therapy the development of symptoms can be prevented. Various mutations of ATP7B have been identified, the most common is in Hungary, the H1069Q mutation. Genetic screening should only be advised if there is a predominant mutation characteristic for the geographic area. The authors discuss the modern diagnostic and therapeutic possibilities of Wilson disease.     

Liver as a key organ in the supply, storage, and excretion of copper

The liver plays an important role in the disposition of copper. Most dietary copper passes through the liver where it can be used for protein and energy production or excreted through the biliary route. Because copper is a prooxidant, its intracellular handling is tightly managed. In Wilson disease, in which synthesis of ceruloplasmin and biliary excretion of copper are defective, copper accumulates in the liver and leads to progressive liver damage. The features of hepatic Wilson disease are highly variable. The spectrum of liver disease includes mild inflammation, fatty liver, an autoimmune disorder, and cirrhosis. Wilson disease thus resembles drug hepatotoxicity, and indeed it can be regarded as a prototypic example of endogenous hepatotoxicity. Biomarkers developed for detecting drug hepatotoxicity may be relevant to Wilson disease. Biomarkers developed through metalloproteomics, which for copper seeks to define a set of proteins that have copper-binding capacity, or through genomic studies may also be relevant to Wilson disease and other disorders of copper handling, whether copper is deficient or overloaded.     

Advances in the understanding of mammalian copper transporters

Copper (Cu) is an essential micronutrient. Its ability to exist in 2 oxidation states (Cu(1+) and Cu(2+)) allows it to function as an enzymatic cofactor in hydrolytic, electron transfer, and oxygen utilization reactions. Cu transporters CTR1, ATP7A, and ATP7B play key roles in ensuring that adequate Cu is available for Cu-requiring processes and the prevention of excess Cu accumulation within cells. Two diseases of Cu metabolism, Menkes disease and Wilson disease, which are caused by mutations in ATP7A and ATP7B, respectively, exemplify the critical importance of regulating Cu balance in humans. Herein, we review recent studies of the biochemical and cell biological characteristics of CTR1, ATP7A, and ATP7B, as well as emerging roles for Cu in new areas of physiology.     

Intracellular copper transport in mammals

Copper is an essential cofactor for approximately a dozen cuproenzymes in which copper is bound to specific amino acid residues in an active site. However, free cuprous ions react readily with hydrogen peroxide to yield the deleterious hydroxyl radical. Therefore, copper homeostasis is regulated very tightly, and unbound copper is extremely low in concentration. Copper imported by the plasma membrane transport protein Ctr1 rapidly binds to intracellular copper chaperone proteins. Atox1 delivers copper to the secretory pathway and docks with either copper-transporting ATPase ATP7B in the liver or ATP7A in other cells. ATP7B directs copper to plasma ceruloplasmin or to biliary excretion in concert with a newly discovered chaperone, Murr1, the protein missing in canine copper toxicosis. ATP7A directs copper within the transgolgi network to the proteins dopamine beta-monooxgenase, peptidylglycine alpha-amidating monooxygenase, lysyl oxidase, and tyrosinase, depending on the cell type. CCS is the copper chaperone for Cu,Zn-superoxide dismutase; it delivers copper in the cytoplasm and intermitochondrial space. Cox17 delivers copper to mitochondria to cytochrome c oxidase via the chaperones Cox11, Sco1, and Sco2. Other copper chaperones may exist and might include metallothionein and amyloid precursor protein (APP). Genetic and nutritional studies have illustrated the essential nature of these copper-binding proteins; alterations in their levels are associated with severe pathology.     

Rapid identification of Wilson's disease carriers by denaturing high-performance liquid chromatography

BACKGROUND: Wilson's disease is an autosomal recessive disorder characterized by decreased biliary copper excretion and reduced copper incorporation into ceruloplasmin. The disease gene ATP7B maps to chromosome 13q14.3, contains 21 exons, and encodes a copper-transporting P-type ATPase. ATP7B mutations are scattered over the entire gene, and scanning methods to detect mutation carriers are in demand. We have tested the usefulness of denaturing high-performance liquid chromatography for mutation detection in Wilson's disease. METHODS: Genomic DNA from five Sardinian Wilson's disease families (32 individuals, 8 patients) was subjected to polymerase chain reactions for ATP7B exons 2-21 and the 5' untranslated region. PCR products were analyzed by chromatography and by direct sequencing. RESULTS: Three disease-causing mutations and seven sequence variants were detected by chromatography. Five patients were homozygotes for -441/-427del, and three were compound heterozygotes for V1146M plus 1512-13insT (N505X) and for -441/-427del plus V1146M, respectively. Eighteen unaffected individuals were mutation carriers. Sequence variants comprised V290V, A406S, L456V, R832K, A1140V, the novel K952R, and T991T. The novel intronic IVS18+6c>t change escaped detection by chromatography. CONCLUSIONS: Denaturing high-performance liquid chromatography is a dependable tool for ATP7B screening that is superior to traditional haplotyping. This method allows for fast, sensitive, and specific mutation detection and identification of carriers in Wilson's disease families.     

From gene to disease; Wilson disease: copper storage due to mutations in ATP7B

Wilson disease is an autosomal recessive disorder of copper metabolism. The gene defective in Wilson disease encodes a copper transporting P-type ATPase expressed in the liver. The disturbed export of copper into bile results in accumulation of copper in liver and secondarily in other organs such as the brain. These patients generally present with either hepatic or neurological symptoms.     

Copper exposure induces trafficking of the menkes protein in intestinal epithelium of ATP7A transgenic mice

The final steps in the absorption and excretion of copper at the molecular level are accomplished by 2 closely related proteins that catalyze the ATP-dependent transport of copper across the plasma membrane. These proteins, ATP7A and ATP7B, are encoded by the genes affected in human genetic copper-transport disorders, namely, Menkes and Wilson diseases. We studied the effect of copper perfusion of an isolated segment of the jejunum of ATP7A transgenic mice on the intracellular distribution of ATP7A by immunofluorescence of frozen sections. Our results indicate that ATP7A is retained in the trans-Golgi network under copper-limiting conditions, but relocalized to a vesicular compartment adjacent to the basolateral membrane in intestines perfused with copper. The findings support the hypothesis that the basolateral transport of copper from the enterocyte into the portal blood may involve ATP7A pumping copper into a vesicular compartment followed by exocytosis to release the copper, rather than direct pumping of copper across the basolateral membrane.     

ATP7A transgenic and nontransgenic mice are resistant to high copper exposure

The protein affected in Menkes disease, ATP7A, is a copper (Cu)-transporting P-type ATPase that plays an important role in Cu homeostasis, but the full extent of this role has not been defined at a systemic level. Transgenic mice that overexpress the human ATP7A from the chicken beta-actin composite promoter (CAG) were used to further investigate the physiological function of ATP7A. Overexpression of ATP7A in the mice caused disturbances in Cu homeostasis, with depletion of Cu in some tissues, especially the heart. To investigate the effect of overexpression of ATP7A when dietary Cu intake was markedly increased, normal and transgenic mice were exposed to drinking water containing 300 mg/L of Cu as Cu acetate for 3 mo. Cu exposure resulted in partial restoration of heart Cu concentrations in male transgenic mice. Despite the extended period of Cu exposure, Cu concentrations in the liver remained relatively unaffected, with a significant increase in male nontransgenic mice. Liver pathology was unremarkable except for small areas of fibrosis that were detected only in livers of the Cu-exposed transgenic mice. Intracellular localization of ATP7A in various tissues was not affected by Cu exposure. Plasma Cu concentration and ceruloplasmin oxidase activity were reduced in both Cu-exposed transgenic and nontransgenic mice. The expression levels of other candidate Cu homeostatic proteins, endogenous Atp7b, ceruloplasmin, Ctr1, and transgenic ATP7A were not altered significantly by Cu exposure. Overall, mice are remarkably resistant to high Cu loads and the overexpression of ATP7A has only moderate effects on the response to Cu exposure.     

From Dietary Cholesterol to Blood Cholesterol,Physiological Lipid Fluxes,and Cholesterol Homeostasis

Dietary cholesterol (C) is a major contributor to the endogenous C pool, and it affects the serum concentration of total C, particularly the low-density lipoprotein cholesterol (LDL-C). A high serum concentration of LDL-C is associated with an increased risk for atherosclerosis and cardiovascular diseases. This concentration is dependent on hepatic C metabolism creating a balance between C input (absorption and synthesis) and C elimination (conversion to bile acids and fecal excretion). The daily C absorption rate is determined by dietary C intake, biliary C secretion, direct trans-intestinal C excretion (TICE), and the fractional C absorption rate. Hepatic C metabolism coordinates C fluxes entering the liver via chylomicron remnants (CMR), LDL, high-density lipoproteins (HDL), hepatic C synthesis, and those leaving the liver via very low-density lipoproteins (VLDL), biliary secretion, and bile acid synthesis. The knowns and the unknowns of this C homeostasis are discussed.     

From gene to disease; familial hemiplegic migraine as a result of mutations in a sodium-potassium pump gene

Familial hemiplegic migraine (FHM) is a rare, autosomal dominant subtype of migraine, associated in half of the families with mutations in the CACNA1A gene located on chromosome 19p13, which encodes the Cav2.1-subunit of brain-specific P/Q-type calcium channels. Recently, mutations in a second gene, ATP1A2 on chromosome 1q23, which encodes a sodium-potassium exchange pump subunit, have been identified. The first functional studies indicate that A TP1A2 FHM mutations result in a loss of function of the pump, leading to an increase in extracellular potassium. This is known to evoke cortical spreading depression, the underlying mechanism of migraine aura.     

From gene to disease; Menkes disease: copper deficiency due to an ATP7A-gene defect

Menkes disease is an X-linked recessive disorder characterized by neurological deterioration, failure to thrive, peculiar hair and death in childhood, secondary to mutations in the ATP7A gene. The ATP7A gene encodes for a copper transporting P-type ATPase (ATP7A), which is ubiquitously expressed. A defect of the ATP7A protein leads to both a reduced transport of copper from the intestine into the circulation and into the central nervous system, as well as reduced transport of copper into the Golgi apparatus for incorporation into various copper-dependent enzymes. This results in a systemic copper deficiency as well as reduced activity of various copper-dependent enzymes. The reduced activity of these copper-dependent enzymes accounts for most of the characteristic features ofMenkes disease patients.     

Profiles of 67Cu in blood, bile, urine and faeces from 67Cu-primed lambs: effect of 99Mo-labelled tetrathiomolybdate on the metabolism of recently stored tissue 67Cu

1. The relative importance of excretory routes in the removal of recently stored 67Cu following tetrathiomolybdate (TTM) administration was studied. Lambs fed on either 5 mg Cu/kg dry matter (DM) or 35 mg Cu/kg DM, were primed intravenously (iv) with 67Cu and challenged 27 h later with 99Mo-labelled TTM given either iv or intraduodenally (id). The profiles of 67Cu and 99Mo and of Cu and Mo in blood, bile, urine and faeces were measured. 2. Level of dietary Cu and route of administration of 99Mo-TTM affected the amplitude of blood, bile and urine profiles of 67Cu and stable Cu, but not the pattern of the responses observed. 3. The present study describes for the first time increased excretion of endogenous 67Cu through gastrointestinal secretions other than the bile due to TTM administration. 4. Administration of TTM resulted in the immediate release of 67Cu from storage compartments in the body into the blood circulation. Changes in stable Cu levels in blood, bile, urine and faeces, and gut and systemic effects were evident. Biliary and urinary Cu excretion due to TTM was rapid and maximal within 24 h of injection. 5. Administration of 67Cu iv resulted in the immediate excretion of 67Cu in bile in a pulsatile, constant pattern. A similar pattern of 67Cu excretion into bile in synchrony with that of 99Mo was observed after 99Mo-labelled TTM administration. 6. The similar pattern of biliary 67Cu excretion observed after injection of 67Cu and after injection of 99Mo-labelled TTM 27 h later is discussed in relation to the times required to process the Cu through different hepatic pathways for excretion in bile.     

Altered cytoskeletal organization and secretory response of thrombin-activated platelets from copper-deficient rats

The cytoskeletal organization that occurs following thrombin-induced activation of platelets was investigated in rats consuming either a copper-deficient diet containing less than 1 microgram Cu/g or a copper-adequate diet containing 5.5 micrograms Cu/g. Within 30 s following thrombin activation, the amount of polymerized actin in Triton X-100 extracts of whole platelets from copper-deficient rats was greater than the amount in platelets from copper-adequate rats. Electrophoretic analysis of the cytoskeletal proteins obtained from low speed centrifugation of the Triton X-100 extracts indicated that the myosin content of the cytoskeleton increased with time and reached higher levels following activation in platelets from copper-deficient rats. Actin content of the cytoskeleton also increased with time following activation. However, the difference in cytoskeletal actin content of platelets from copper-deficient and copper-adequate rats was not as great as that observed for myosin. The rate of ATP secretion from thrombin-activated platelets was also increased by copper deficiency. Myosin association with the platelet cytoskeleton may be involved in platelet secretion following thrombin activation. Thus, the increased association of myosin with the cytoskeleton and concomitant increase in ATP secretion suggest that the normal mechanism for stimulus-response coupling is altered in thrombin-activated platelets from copper-deficient rats.     

The first genetically supported case of chronic benign pemphigus (Hailey-Hailey disease in Hungary

Hailey-Hailey disease, or chronic benign pemphigus (MIM# 169600), is a genodermatosis arising in adult age with recurrent vesicles and erosions primarily in the flexural areas. It is an autosomal dominant skin disorder characterized by abnormal keratinocyte adhesion in the suprabasal layers of the epidermis. ATP2C1, encoding the human secretory pathway Ca(2+)-ATPase (hSPCA1), was recently identified as the defective gene in Hailey-Hailey disease. More than 82 different ATP2C1 mutations have been described up to date. In this study, a case of Hailey-Hailey disease is presented where a nucleotide change (1402C > T) in the decoding region of ATP2C1 resulted in a premature stop mutation (R468X). This defect has been reported earlier in a patient of European descent. A brief molecular genetic review of the disorder is also given.     

Age and copper intake do not affect copper absorption,measured with the use of 65Cu as a tracer,in young infants

BACKGROUND: Copper homeostasis involves a high degree of regulation in which changes in absorption and biliary excretion are the main mechanisms. Whether neonates and small infants can make these changes efficiently is unknown. OBJECTIVE: We evaluated the effect of age and copper intake on copper absorption in infants during the first 3 mo of life. DESIGN: Thirty-nine healthy infants (19 infants aged 1 mo and 20 infants aged 3 mo) were selected. One-half of the subjects were randomly assigned to receive oral supplementation of 80 mg Cu (as copper sulfate). kg body wt(-1). d(-1) for 15 d. At the end of the trial, copper absorption was measured by using orally administered (65)Cu as a tracer and fecal monitoring of recovered (65)Cu. RESULTS: Mean (+/- SD) copper absorption at 1 mo of age was 83.6 +/- 5.8% and 74.8 +/- 9.1% for the unsupplemented and supplemented infants, respectively. The corresponding figures at 3 mo of age were 77.6 +/- 15.2% and 77.7 +/- 11.3%. A two-way analysis of variance showed that age, copper supplementation, and the interaction between age and copper supplementation did not have a significant effect on copper absorption. There was an inverse correlation between total fecal copper and the percentage of (65)Cu absorption (r = -0.50, P < 0.003). CONCLUSION: Copper absorption in young infants is high but does not respond to copper intake within the range tested.     

Fatty acids and glucose in high concentration down-regulates ATP synthase β-subunit protein expression in INS-1 cells

Chronic hyperglycemia and hyperlipidemia exert deleterious effects on β-cell function and impair glucose-induced insulin release, referred to as glucotoxicity and lipotoxticity. These abnormalities are associated with decreased glucose-induced ATP production; ATP serves as an important signal for insulin secretion. To investigate the mechanism of the impaired ATP formation, we examined the effects of elevated glucose and fatty acids levels on ATP synthase β-subunit expression, ATP content and insulin secretion in INS-1 insulinoma β-cells. ATP synthase β-subunit expression was measured by western blot, ATP content was monitored by ATP luminescence and insulin secretion detected by radio immunoassay. Our result indicated that chronic exposure to high doses of fatty acids together with high levels glucose produced a marked decrease in ATP synthase β-subunit protein expression. Reduction of ATP synthase β-subunit protein expression occurred with a decreased intracellular ATP concentration and insulin secretion at high fatty acid concentrations. These results indicate that high glucose together with fatty acids impair the production of ATP in β-cells through the suppression of mitochondrial ATP synthesis. We conclude that ATP synthase β-subunit may have an important role in the glucolipotoxicity of islet cells and suggest that ATP synthase β-subunit might be a target of lipotoxicity in β-cells.     

New molecular features of cholestatic diseases of the liver

Hepatic uptake and biliary excretion of bile salts and non-bile salt organic anions is mediated by specific transport proteins located at the basolateral and canalicular membranes of hepatocytes. Several hepatobiliary transport systems have been identified and cloned over the past years. This development has facilitated molecular biological and genetic analyses of these transporters in experimental cholestasis and human cholestatic liver diseases. Evidence now exists that decreased or even absent expression of hepatobiliary transport systems may explain impaired transport function resulting in hyperbilirubinemia and cholestasis. This review summarizes the molecular defects in hepatocellular membrane transporters associated with hereditary and acquired forms of cholestatic liver diseases. The increasing information on the molecular regulation of hepatobiliary transport systems should bring new insights into the pathophysiology and treatment of human cholestatic liver diseases.     

KIF5C基因突变导致神经发育障碍研究进展

KIF5C基因是神经发育障碍如孤独症的相关候选基因。KIF5C基因编码KIF5C驱动蛋白,帮助沿微管长距离顺行运送神经突成熟所需要货物,对神经元发育重要。驱动蛋白包括头部马达结构域、茎部二聚化结构域、及尾部结构域。头部马达结构域外显子区是基因突变常见区域,偶见内含子突变报道;c.709G>A是热点突变位点,造成p.Glu237Lys氨基酸突变。KIF5C基因突变造成儿童常见神经发育障碍包括皮质发育畸形、小头畸形、癫痫、发育迟缓/智力障碍,及孤独症样特征等;KIF5C基因突变造成相关障碍的机制尚未明了,可能通过影响马达结构域水解ATP能力;深入研究致病机制及寻找新的疾病治疗靶标重要;KIF5C基因治疗有待进一步研究。