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Results for “Copper-Transporting ATPases”

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PubMed1996

Helicobacter pylori genes hpcopA and hpcopP constitute a cop operon involved in copper export.

We previously reported that a gene hpcopA isolated from Helicobacter pylori is associated with copper transport. In the present study, the DNA upstream of the hpcopA was cloned and the nucleotide sequence analyzed. An open reading frame coding for 124 amino acids was predicted, which was connected in frame to the hpcopA. The deduced protein sequence exhibits striking homology with known copper-transporting P-type ATPases. Disruption of this ORF renders H. pylori hypersensitive to copper present in growth media, indicating that it encodes the N-terminal amino acid sequence of the hpCopA protein. Measurement of copper content in the wild-type and hpcopA-disrupted cells showed that the copper content was increased in the mutant cells, further supporting that the previous proposal that the gene hpcopA is involved in copper export. In addition, the cop operon consisting of the genes hpcopA and hpcopP was identified by primer extension and reverse transcription-polymerase chain reaction. Our results indicate that the genes for copper import and export are located in separate operons within H. pylori.

Adenosine Triphosphatases↗

PubMed1997

Mutation analysis and expression of the mottled gene in the macular mouse model of Menkes disease.

The gene for Menkes disease, an X-linked disorder of copper transport, has recently been identified and shown to encode a copper-transporting P-type ATPase. The macular mutant mouse has been proposed as an animal model for Menkes disease. In the present study, we report the finding of a missense mutation in the mottled gene of the macular mouse. A single base change, T to C, at nucleotide position 4223, is predicted to result in an amino acid change from serine to proline at residue 1382 in the eighth transmembrane domain. This mutation differs from the 6-bp deletion we find in brindled cDNA. With validation of macular as an animal model of Menkes disease, we compared mottled gene expression in the intestine, kidney, and brain of macular and normal mice. In Northern analyses an 8.3-kb transcript was detected in the intestine, kidney, and brain of both normal and macular mice, with the level of transcript in macular approximately 80% that of normal. In situ hybridization studies revealed that the mottled gene was clearly expressed in intestinal epithelial cells, Paneth cells, and renal proximal tubular cells of both normal and macular mice. In normal brain, mottled gene expression was most intensely observed in the choroid plexus, in Ammon's born and the dentate gyrus in the hippocampus, in Purkinje cells, and the granular layer of the cerebellum. The intensity and localization of the signals in the brain of macular mice were similar to those of the controls. The distribution of expression of mottled is correlated with cells and tissues showing histopathology or abnormal copper sequestration in macular and other mutants.

Adenosine Triphosphatases↗

PubMed2002

Molecular mechanism of copper transport in Wilson disease.

Wilson disease is an autosomal recessive disorder of copper metabolism. The Wilson disease protein is a putative copper-transporting P-type ATPase, ATP7B, whose malfunction results in the toxic accumulation of copper in the liver and brain, causing the hepatic and/or neurological symptoms accompanying this disease. The cytosolic N-terminal domain (approximately 70 kDa) of this ATPase comprises six heavy metal-associated domains, each of which contains the conserved metal-binding motif GMTCXXC. The N-terminal domain (Wilson disease copper-binding domain [WCBD]) has been expressed, purified, and characterized using various techniques. The WCBD binds six atoms of copper in the +1 oxidation state competitively, and with a greater affinity than all other metals. The copper atom is coordinated by two cysteines in a distorted linear geometry. Copper binds the WCBD in a cooperative manner and induces secondary and tertiary conformation changes. Zinc binding to the WCBD has also been characterized by circular dichroism spectroscopy and shown to produce conformational changes that are completely different from those induced by copper. The phosphorylation/nucleotide-binding domain of ATP7B has also been expressed and characterized and shown to be capable of binding ATP but lacking ATPase activity. A peptide corresponding to the sixth transmembrane domain of ATP7B has been constructed and shown to undergo secondary conformational changes upon binding a single atom of copper. Finally, a chimeric protein consisting of the WCBD and truncated ZntA, a zinc-transporting ATPase lacking the N-terminal domain, has been constructed and analyzed for metal ion selectivity. These results suggest that the core determines the metal ion specificity of P-type ATPases, and the N-terminal metal-binding domain may play a regulatory role.

Adenosine Triphosphatases↗

PubMed2000

Copper-transporting P-type adenosine triphosphatase (ATP7B) is associated with cisplatin resistance.

The accumulation of cisplatin is decreased in many cisplatin-resistant cell lines, and an active efflux pump for cisplatin exists in some of them, but it has not yet been identified. In this study, we transfected the copper-transporting P-type ATPase cDNA (ATP7B) into human epidermoid carcinoma KB-3-1 cells. The transfectant, KB/WD cell line, which overexpressed the P-type ATPase, ATP7B, was resistant to both cisplatin (8.9-fold) and copper (2.0-fold). The accumulation of cisplatin in KB/WD cells was lower than in mock-transfected KB/CV cells, and the efflux of cisplatin from KB/WD cells was enhanced compared with KB/CV cells. KB/WD cells were sensitive to other heavy metals, such as antimony, arsenate, arsenite, cadmium, and cobalt. ATP7B was overexpressed in cisplatin-resistant prostate carcinoma PC-5 cells but not in the parental PC-3 cells and the revertant PC-5R cells. ATP7B may be involved in cisplatin resistance in some tumors.

Adenosine Triphosphatases↗

PubMed2004

Intracellular trafficking of the human Wilson protein: the role of the six N-terminal metal-binding sites.

The Wilson protein (ATP7B) is a copper-transporting CPx-type ATPase defective in the copper toxicity disorder Wilson disease. In hepatocytes, ATP7B delivers copper to apo-ceruloplasmin and mediates the excretion of excess copper into bile. These distinct functions require the protein to localize at two different subcellular compartments. At the trans-Golgi network, ATP7B transports copper for incorporation into apo-ceruloplasmin. When intracellular copper levels are increased, ATP7B traffics to post-Golgi vesicles in close proximity to the canalicular membrane to facilitate biliary copper excretion. In the present study, we investigated the role of the six N-terminal MBSs (metal-binding sites) in the trafficking process. Using site-directed mutagenesis, we mutated or deleted various combinations of the MBSs and assessed the effect of these changes on the localization and trafficking of ATP7B. Results show that the MBSs required for trafficking are the same as those previously found essential for the copper transport function. Either MBS 5 or MBS 6 alone was sufficient to support the redistribution of ATP7B to vesicular compartments. The first three N-terminal motifs were not required for copper-dependent intracellular trafficking and could not functionally replace sites 4-6 when placed in the same sequence position. Furthermore, the N-terminal region encompassing MBSs 1-5 (amino acids 64-540) was not essential for trafficking, with only one MBS close to the membrane channel, necessary and sufficient to support trafficking. Our findings were similar to those obtained for the closely related ATP7A protein, suggesting similar mechanisms for trafficking between copper-transporting CPx-type ATPases.

Adenosine Triphosphatases↗

PubMed2002

Deletion of the copper transporter CaCCC2 reveals two distinct pathways for iron acquisition in Candida albicans.

Efficient iron acquisition is an essential requirement for growth of pathogenic organisms in the iron-poor host environment. In Saccharomyces cerevisiae, high-affinity iron import depends on the multicopper ferroxidase ScFet3. ScFet3 biogenesis in the trans-Golgi compartment requires a copper-transporting P-type ATPase, ScCcc2. Here, we describe the isolation by functional complementation of a Ccc2 homologue from the pathogenic yeast Candida albicans. CaCcc2 is functionally distinct from a previously described C. albicans copper-transporting P-type ATPase, CaCrp1, which appears to be specifically involved in copper detoxification. Regulation of CaCCC2 and the phenotype of the homozygous CaCCC2 deletion indicate that it is required for high-affinity iron import, making it the bona fide CCC2 homologue of C. albicans. Remarkably, in a mouse model of systemic infection, the Caccc2Delta strain displayed robust proliferation and no significant reduction in pathogenicity, suggesting the existence of alternative mechanisms of iron uptake from host tissues. We identify haemin and haemoglobin as potential iron sources that can be used by C. albicans in a CaCcc2-independent manner.

Adenosine Triphosphatases↗

PubMed1998

Identification of three novel mutations and a high frequency of the Arg778Leu mutation in Korean patients with Wilson disease.

Four mutations--R778L, A874V, L1083F, and 2304delC--in the copper-transporting enzyme, P-type ATPase (ATP7B), were identified in Korean Patients with Wilson disease. Arg778Leu, the most frequently reported mutation of this enzyme, was found in six of eight unrelated patients studied, an allele frequency of 37.5%, which is considerably higher than those in other Asian populations. The novel single nucleotide deletion, 2304delC, was found in one patient. Since a mutation at cDNA nucleotide 2302 (2302insC) had been previously described, this region of the ATP7B gene may be susceptible to gene rearrangements causing Wilson disease.

Adenosine Triphosphatases↗

PubMed2000

Molecular analysis of Wilson disease in Taiwan: identification of one novel mutation and evidence of haplotype-mutation association.

Wilson disease (WND) is caused by a deficiency of the copper-transporting enzyme, P-type ATPase (ATP7B). Twelve different mutations have previously been identified in Taiwan Chinese with Wilson disease. We, herein, report another 4 missense mutations, 1 of which is novel. We did haplotype analysis of Taiwanese WND chromosomes, using three well characterized short tandem repeat markers (haplotype was assigned in the order of D13S314-D13S301-D13S316). Association correlation was found between the mutations and their respective haplotypes. Haplotype-deduced pedigree analysis was shown to be helpful in the mutation analysis of WND chromosomes and in the molecular assessment of both pre-symptomatic WND patients and carriers. Given the complexity and heterogeneity of the mutation spectrum of ATP7B, we suggest that haplotype analysis should be performed before full-scale mutation analysis.

Adenosine Triphosphatases↗

PubMed1998

Functional analysis of copper homeostasis in cell culture models: a new perspective on internal copper transport.

The movement of copper ions across membrane barriers of vital organs and tissues is a priority topic in nutrition and one for which there continues to be little understanding of the mechanism. Reports of membrane-bound, copper-transporting adenosine triphosphatases (Cu-ATPases) selective for copper ions have brought new focus to the problem and prompted fresh ideas. Using a cell culture model approach, we attempted to learn whether transport into and out of cells depends on a Cu-ATPase. Measurement of transport kinetics in fibroblasts, brain glial cells, neuroblastoma cells, and placental cells showed differences in the rates of copper uptake and response to sulfhydryl reagents. BeWo cells, a human choriocarcinoma placental cell line, behaved as did Menkes fibroblasts by avidly absorbing copper but not releasing copper to the immediate environment. Further tests showed that BeWo cells did not express the transcript for the membrane-bound Cu-ATPase that has been identified with Menkes syndrome. Transcript induction, however, was achieved by growing BeWo cells on porous filters that allowed apical and basolateral surfaces to form. With transcript expression, the cells showed a capacity to release copper into the medium. BeWo cells also synthesized a form of ceruloplasmin whose structure differed from that of the plasma protein and hence may be a product of a different gene. BeWo cells may also express the gene for Wilson disease, thus linking Menkes and Wilson proteins to maternal delivery of copper. We constructed a model in which both ATPases work in concert in a vesicle-based transport mechanism. The vesicle model may help us understand the transport of copper across the placenta and all cells in general.

Adenosine Triphosphatases↗

PubMed1995

A Synechococcus gene encoding a putative pore-forming intrinsic membrane protein.

A cyanobacterium, Synechococcus species PCC7942, has a gene encoding a copper-transporting P-type ATPase, which is located in the thylakoid membrane. At the 5'-upstream of this ATPase gene, we identified another gene, which was supposed to be implicated in a copper-transport process. This novel gene was found to encode a putative pore-forming membrane protein that belongs to a growing family of homologous intrinsic membrane proteins (the MIP family of proteins), which include the major intrinsic protein (MIP) from animal lens fibre junction membranes, the tonoplast intrinsic protein (TIP) from vacuolar membranes of higher plants, and the Escherichia coli glycerol facilitator (GlpF) in the cytoplasmic membrane. The deduced product, named SmpX (Synechococcus membrane protein), is highly homologous throughout its entire sequence to these intrinsic membrane proteins which were postulated to be pore-forming proteins involved in a variety of transport processes. The primary amino acid sequence of SmpX shares all properties characteristic for members of the MIP family. SmpX is more similar to the eukaryotic members (e.g., nodulin-26 from soybean) than to the prokaryotic ones.

Amino Acid Sequence↗

PubMedDate not supplied

Metabolic and molecular bases of Menkes disease and occipital horn syndrome.

Menkes disease and occipital horn syndrome (OHS) are related disorders of copper transport that involve abnormal neurodevelopment, connective tissue problems, and often premature death. Location of the gene responsible for these conditions on the X chromosome was indicated by pedigree analysis from the time of these syndromes' earliest descriptions. Characterization of an affected female with an X-autosomal translocation was used to identify the Menkes/OHS gene, which encodes a highly evolutionarily conserved, copper-transporting P-type ATPase. The gene normally is expressed in nearly all human tissues, and it localizes to the trans-Golgi network of cells. However, in over 70% of Menkes and OHS patients studied, expression of this gene has been demonstrated to be abnormal. Major gene deletions detectable by Southern blotting account for 15-20% of patients, and an interesting spectrum of other mutations is evident among 58 families whose precise molecular defects have been reported as of this writing. The center region of the gene seems particularly prone to mutation, and those that influence mRNA processing and splicing appear to be relatively common. Further advances in understanding the molecular and cell biological mechanisms involved in normal copper transport may ultimately yield new and better approaches to the management of these disorders.

Copper↗

PubMed1999

RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson disease-related copper transporter, is required for ethylene signaling in Arabidopsis.

Ethylene is an important regulator of plant growth. We identified an Arabidopsis mutant, responsive-to-antagonist1 (ran1), that shows ethylene phenotypes in response to treatment with trans-cyclooctene, a potent receptor antagonist. Genetic epistasis studies revealed an early requirement for RAN1 in the ethylene pathway. RAN1 was cloned and found to encode a protein with similarity to copper-transporting P-type ATPases, including the human Menkes/Wilson proteins and yeast Ccc2p. Expression of RAN1 complemented the defects of a ccc2delta mutant, demonstrating its function as a copper transporter. Transgenic CaMV 35S::RAN1 plants showed constitutive expression of ethylene responses, due to cosuppression of RAN1. These results provide an in planta demonstration that ethylene signaling requires copper and reveal that RAN1 acts by delivering copper to create functional hormone receptors.

Alkenes↗

PubMed2000

The high copper tolerance of Candida albicans is mediated by a P-type ATPase.

The pathogenic yeast Candida albicans has higher resistance than the baker's yeast Saccharomyces cerevisiae to elevated concentrations of copper. To understand the basis of this differential resistance, we performed a functional screen for C. albicans genes involved in copper detoxification. Here, we report the isolation of two such genes: a metallothionein, CaCUP1, and a copper-transporting P-type ATPase, CaCRP1. Both genes are induced by extracellular copper. Gene disruptions indicated that the copper extrusion pump is responsible for the unusual resistance of C. albicans to copper, whereas the metallothionein is responsible for the residual copper resistance of the Cacrp1Delta mutant. We show further that under acidic and anaerobic conditions, such as prevail in the natural niche of C. albicans, the digestive tract of animals, CaCRP1 function becomes essential for survival in the presence of even very low copper concentrations. These observations suggest that copper in the gastrointestinal tract may present a toxic challenge to which enteric organisms had to adapt.

Adenosine Triphosphatases↗

PubMed1999

The role of GMXCXXC metal binding sites in the copper-induced redistribution of the Menkes protein.

The Menkes protein (MNK or ATP7A) is a transmembrane, copper-transporting CPX-type ATPase, a subgroup of the extensive family of P-type ATPases. A striking feature of the protein is the presence of six metal binding sites (MBSs) in the N-terminal region with the highly conserved consensus sequence GMXCXXC. MNK is normally located in the trans-Golgi network (TGN) but has been shown to relocalize to the plasma membrane when cells are cultured in media containing high concentrations of copper. The experiments described in this report test the hypothesis that the six MBSs are required for this copper-induced trafficking of MNK. Site-directed mutagenesis was used to convert both cysteine residues in the conserved MBS motifs to serines. Mutation of MBS 1, MBS 6, and MBSs 1-3 resulted in a molecule that appeared to relocalize normally with copper, but when MBSs 4-6 or MBSs 1-6 were mutated, MNK remained in the TGN, even when cells were exposed to 300 microM copper. Furthermore, the ability of the MNK variants to relocalize corresponded well with their ability to confer copper resistance. To further define the critical motifs, MBS 5 and MBS 6 were mutated, and these changes abolished the response to copper. The region from amino acid 8 to amino acid 485 was deleted, resulting in mutant MNK that lacked 478 amino acids from the N-terminal region, including the first four MBSs. This truncated molecule responded normally to copper. Moreover, when either one of the remaining MBS 5 and MBS 6 was mutated to GMXSXXS, the resulting proteins were localized to the TGN in low copper and relocalized in response to elevated copper. These experiments demonstrated that the deleted N-terminal region from amino acid 8 to amino acid 485 was not essential for copper-induced trafficking and that one MBS close to the membrane channel of MNK was necessary and sufficient for the copper-induced redistribution.

Adenosine Triphosphatases↗

PubMed2004

Role of a Streptococcus gordonii copper-transport operon, copYAZ, in biofilm detachment.

Streptococcus gordonii is a pioneer oral bacterium that is associated with the initiation of dental plaque development. Located downstream of the S. gordonii adc operon, which is involved in competence and biofilm formation, were three open reading frames, designated copY, copA and copZ. These open reading frames were homologous to the copYAZ genes in Streptococcus mutans that are involved in copper homeostasis and biofilm detachment. This study examined whether copYAZ genes play any role in the biofilm formation and detachment of S. gordonii. The copY gene encodes a 143-amino acid protein homologous to the negative transcriptional regulator of a copper-transport operon, copA encodes a 748-amino acid copper-transporting P-type ATPase, and copZ encodes a 69-amino acid putative metallochaperone protein in S. mutans. Each open reading frame in the copYAZ operon in S. gordonii was inactivated by insertional mutation and the growth, biofilm formation and detachment of each mutant were examined. S. gordonii copY::specR, copA::specR, and copZ::specR mutants were able to form biofilms on both polystyrene and glass surfaces. However, inactivation of copZ and to a lesser extent copY resulted in phenotypes that were defective in biofilm detachment, which is consistent with previous observations in S. mutans and suggests that the trace element copper might influence biofilm detachment of bacterial biofilms.

Bacterial Adhesion↗

PubMed2000

Foreword: from classic bile physiology to cloned transporters.

Uptake of drugs and metabolites from the circulation into the liver is facilitated by transporter proteins in the basolateral membrane of the hepatocyte. Among these proteins are the sodium taurocholate cotransporting protein, various multispecific transporters for organic anions and cations, transporters for glucose, amino acids, and prostaglandins. The canalicular membrane contains a number of ATP-dependent transporters belonging to the families of P-glycoproteins and multidrug resistance-associated proteins. Transport across the canalicular membrane represents the rate-determining step in the secretion of compounds from blood to bile. Mutations of genes encoding these canalicular transporters are associated with liver diseases such as progressive familial intrahepatic cholestasis and Dubin-Johnson syndrome. Wilson's disease appears to be due to a defect of a copper-transporting P-type ATPase. Also, bile ductuli contribute to bile formation. Mutations in the CFTR gene, encoding a chloride channel in bile duct epithelial cells, leads to the hepatic component of cystic fibrosis.

Bile↗

PubMed2003

[Wilson's disease].

Wilson's disease is an autosomal recessive disorder of copper metabolism. The Wilson disease protein is a copper-transporting P-type ATPase, ATP7B, the malfunction of which results in the toxic accumulation of copper in the liver and brain, causing the hepatic and/or neurological symptoms accompanying this disease. Patients present, generally between the ages of 10 and 40 years, with liver disease, neurological disease of a movement disorder type, or behavioral abnormalities, and often with a combination of these. Because Wilson's disease is effectively treated, it is extremely important for physicians to learn to recognize and diagnose the disease. The laboratory diagnosis of Wilson's disease is confirmed by decreased serum ceruloplasmin, increased urinary copper content, and elevated hepatic copper concentration. Molecular genetic analysis is complex as more than 200 unique mutations have been identified and most individuals are compound heterozygotes. The treatment of Wilson's disease must be life long. Copper chelation with penicillamine is an effective therapy in most patients. Another chelating agent which has been used successfully as the initial therapy is trientine. The search for new anticopper drugs for Wilson's disease is culminating in two excellent new drugs: zinc for maintenance therapy and ammonium tetrathiomolybdate (which is to date still an experimental drug) for initial therapy. Liver transplantation is indicated for the fulminant form and in those patients with severe disease not responding to optimal medical management. This paper reviews the pathogenesis, pathology, clinical presentation and diagnosis of the Wilson's disease as well as the most recent views on the molecular genetics and the treatment of this disease.

Hepatolenticular Degeneration↗

PubMed1995

Wilson's disease: a new gene and an animal model for an old disease.

Wilson's disease is an autosomal recessive, inherited disorder of copper metabolism. In normal individuals, copper homeostasis is controlled by the balance between intestinal absorption of dietary copper and hepatic excretion of excess copper in bile. In Wilson's disease, hepatic copper is neither excreted in bile nor incorporated into ceruloplasmin and copper accumulates to toxic levels. The Wilson's disease gene (WND) encodes a putative copper-transporting protein that is expressed almost exclusively in the liver. The predicted structure of the protein product is that of a P-type ATPase with striking homology to bacterial copper transporters and the gene product of another inherited disorder of copper metabolism, Menkes' disease. A rat model of Wilson's disease has recently been identified. The Long-Evans Cinnamon (LEC) rat manifests elevated hepatic copper, defective incorporation of copper into ceruloplasmin, and reduced biliary excretion of copper. The rat homologue of the WND is abnormal in LEC rats. Clinical manifestations of Wilson's disease arise directly from copper-induced damage to hepatocytes (hepatic presentation) or indirectly after the release of copper from the liver with subsequent damage to the brain (neuropsychiatric presentation) and other organs. Genetic heterogeneity (different mutations in a single gene) may account for some of the variability in Wilsonian presentations. The diagnosis of Wilson's disease depends on the demonstration of disordered copper metabolism, manifested as elevated urinary and hepatic copper and low ceruloplasmin levels. However, none of the abnormal findings in Wilson's disease is pathognomonic. Genetic diagnosis, in the absence of family studies, is likely to be difficult since many different mutations result in the disease. Management of Wilson's disease involves decreasing excess levels of copper accumulated in the liver, brain, and other organs. Copper chelation therapy, to increase urinary excretion of copper, is the mainstay of treatment. In addition, oral zinc therapy may be useful at decreasing absorption of dietary copper and rendering tissue copper nontoxic, by increasing the formation of complexes with copper-binding proteins. Liver transplantation can be necessary for individuals with acute hepatic failure or complications of cirrhosis. Gene therapy may evolve in the future; however, medical management is effective in most patients.

Amino Acid Sequence↗