Sodium arsenite enhances copper accumulation in human lung adenocarcinoma cells.
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In this report, we found that arsenite-resistant human lung adenocarcinoma cells, CL3R15, were more susceptible to CuCl2 than the parental CL3 cells. With the aid of atomic absorption spectrophotometry, we observed that CL3R15 cells accumulated more copper than CL3 cells. We further demonstrated that sodium arsenite treatment resulted in a dose-dependent increase of copper accumulation in the parental CL3 cells. In contrast, copper did not alter the levels of intracellular arsenite in CL3 cells treated in combination with sodium arsenite and CuCl2. Pretreatment of CL3 cells with sodium arsenite resulted in a significant increase of copper accumulation and cytotoxicity. These results indicate that intracellular copper accumulation is enhanced by arsenite. However, arsenite-enhanced copper accumulation was not observed in two fibroblastic cells, GM00220 and GM03700, derived from Menkes patients. The Menkes gene encodes a membrane pump responsible for copper exportation. Our results suggest that Menkes protein is a potential target of arsenite. (+info)
Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane.
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The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus. (+info)
Intracellular localization and loss of copper responsiveness of Mnk, the murine homologue of the Menkes protein, in cells from blotchy (Mo blo) and brindled (Mo br) mouse mutants.
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Menkes disease is an X-linked copper deficiency disorder that results from mutations in the ATP7A ( MNK ) gene. A wide range of disease-causing mutations within ATP7A have been described, which lead to a diversity of phenotypes exhibited by Menkes patients. The mottled locus ( Mo, Atp7a, Mnk ) represents the murine homologue of the ATP7A gene, and the mottled mutants exhibit a diversity of phenotypes similar to that observed among Menkes patients. Therefore, these mutants are valuable models for studying Menkes disease. Two of the mottled mutants are brindled and blotchy and their phenotypes resemble classical Menkes disease and occipital horn syndrome (OHS) in humans, respectively. That is, the brindled mutant and patients with classical Menkes disease are severely copper deficient and have profound neurological problems, while OHS patients and the blotchy mouse have a much milder phenotype with predominantly connective tissue defects. In this study, in an attempt to understand the basis for the brindled and blotchy phenotypes, the copper transport characteristics and intracellular distribution of the Mnk protein were assessed in cultured cells from these mutants. The results demonstrated that the abnormal copper metabolism of brindled and blotchy cells may be related to a number of factors, which include the amount of Mnk protein, the intracellular location of the protein and the ability of Mnk to redistribute in elevated copper. The data also provide evidence for a relationship between the copper transport function and copper-dependent trafficking of Mnk. (+info)
A delicate balance: homeostatic control of copper uptake and distribution.
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The cellular uptake and intracellular distribution of the essential but highly toxic nutrient, copper, is a precisely orchestrated process. Copper homeostasis is coordinated by several proteins to ensure that it is delivered to specific subcellular compartments and copper-requiring proteins without releasing free copper ions that will cause damage to cellular components. Genetic studies in prokaryotic organisms and yeast have identified membrane-associated proteins that mediate the uptake or export of copper from cells. Within cells, small cytosolic proteins, called copper chaperones, have been identified that bind copper ions and deliver them to specific compartments and copper-requiring proteins. The identification of mammalian homologues of these proteins reveal a remarkable structural and functional conservation of copper metabolism between bacteria, yeast and humans. Furthermore, studies on the function and localization of the products of the Menkes and Wilson's disease genes, which are defective in patients afflicted with these diseases, have provided valuable insight into the mechanisms of copper balance and their role in maintaining appropriate copper distribution in mammals. (+info)
Characterization of the Menkes protein copper-binding domains and their role in copper-induced protein relocalization.
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Menkes disease is a fatal X-linked disorder of copper metabolism. The gene defective in Menkes disease (ATP7A) encodes a copper transporting P-type ATPase (MNK or ATP7A) with six copper-binding domains at its N-terminus. MNK is normally localized to the trans -Golgi network in cultured cells, but relocates to the plasma membrane in the presence of elevated extracellular copper. In this study, the role of the six copper-binding domains on copper-induced redistribution is investigated. In a recombinant clone, when all the wild-type copper-binding motifs are mutated from GMXCXXC to GMXSXXS and the cells grown in medium containing elevated copper, relocalization of the recombinant protein to the plasma membrane was not observed. Using the same assay with any one of the six copper-binding domains intact, MNK moves to the plasma membrane in a way indistinguishable from the wild-type protein. Therefore, the copper-binding domains are vital for MNK trafficking and only a single domain is sufficient for this redistribution to occur. (+info)
Defective copper-induced trafficking and localization of the Menkes protein in patients with mild and copper-treated classical Menkes disease.
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Menkes disease is an X-linked disorder of copper metabolism. An overall copper deficiency reduces the activity of copper-dependent enzymes accounting for the clinical presentation of affected individuals. The Menkes gene product (MNK) is a P-type ATPase and is considered to be the main copper efflux protein in most cells. The protein is located primarily at the trans -Golgi network (TGN), but relocalizes to the plasma membrane in elevated copper conditions to expel the excess copper from the cell. Here we report the first missense mutation which causes mild Menkes disease, a mutation in a successfully copper-treated classical Menkes patient and the effect of each mutation on the localization of MNK within the cell. Using western blot analysis, MNK was detectable in cells from both patients, but appeared to be mislocalized in the treated case. In the mild Menkes patient, the protein appeared to be located in the TGN but failed to redistribute towards the cell periphery in response to copper. This is the first description of a mutation in a Menkes patient which affects the trafficking of MNK, and the loss of this process is consistent with the clinical phenotype. (+info)
Functional analysis of the N-terminal CXXC metal-binding motifs in the human Menkes copper-transporting P-type ATPase expressed in cultured mammalian cells.
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The Menkes protein (MNK) is a copper-transporting P-type ATPase, which has six highly conserved metal-binding sites, GMTCXXC, at the N terminus. The metal-binding sites may be involved in MNK trafficking and/or copper-translocating activity. In this study, we report the detailed functional analysis in mammalian cells of recombinant human MNK and its mutants with various metal-binding sites altered by site-directed mutagenesis. The results of the study, both in vitro and in vivo, provide evidence that the metal-binding sites of MNK are not essential for the ATP-dependent copper-translocating activity of MNK. Moreover, metal-binding site mutations, which resulted in a loss of ability of MNK to traffick to the plasma membrane, produced a copper hyperaccumulating phenotype. Using an in vitro vesicle assay, we demonstrated that the apparent K(m) and V(max) values for the wild type MNK and its mutants were not significantly different. The results of this study suggest that copper-translocating activity of MNK and its copper-induced relocalization to the plasma membrane represent a well coordinated copper homeostasis system. It is proposed that mutations in MNK which alter either its catalytic activity or/and ability to traffick can be the cause of Menkes disease. (+info)
The Menkes protein (ATP7A; MNK) cycles via the plasma membrane both in basal and elevated extracellular copper using a C-terminal di-leucine endocytic signal.
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Menkes disease is an X-linked recessive copper deficiency disorder caused by mutations in the ATP7A ( MNK ) gene which encodes a copper transporting P-type ATPase (MNK). MNK is normally localized pre- dominantly in the trans -Golgi network (TGN); however, when cells are exposed to excessive copper it is rapidly relocalized to the plasma membrane where it functions in copper efflux. In this study, the c-myc epitope was introduced within the loop connecting the first and second transmembrane regions of MNK. This myc epitope allowed detection of the protein at the surface of living cells and provided the first experimental evidence supporting the common topological model. In cells stably expressing the tagged MNK protein (MNK-tag), extracellular antibodies were internalized to the perinuclear region, indicating that MNK-tag at the TGN constitutively cycles via the plasma membrane in basal copper conditions. Under elevated copper conditions, MNK-tag was recruited to the plasma membrane; however, internalization of MNK-tag was not inhibited and the protein continued to recycle through cyto- plasmic membrane compartments. These findings suggest that copper stimulates exocytic movement of MNK to the plasma membrane rather than reducing MNK retrieval and indicate that MNK may remove copper from the cytoplasm by transporting copper into the vesicles through which it cycles. Newly internalized MNK-tag and transferrin were found to co-localize, suggesting that MNK-tag follows a clathrin-coated pit/endosomal pathway into cells. Mutation of the di-leucine, L1487 L1488, prevented uptake of anti-myc antibodies in both basal and elevated copper conditions, thereby identifying this sequence as an endocytic signal for MNK. Analysis of the effects of the di-leucine mutation in elevated copper provided further support for copper-stimulated exocytic movement of MNK from the TGN to the plasma membrane. (+info)