Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0022716 (Menkes)
 1,057 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Functional analysis of the N-terminal CXXC metal-binding motifs in the human Menkes copper-transporting P-type ATPase expressed in cultured mammalian cells. 1041 25

Cation-transporting P-type ATPases comprise a major membrane protein family, the members of which are found in eukaryotes, eubacteria, and archaea. A phylogenetically old branch of the P-type ATPase family is involved in the transport of heavy-metal ions such as copper, silver, cadmium, and zinc. In humans, two homologous P-type ATPases transport copper. Mutations in the human proteins cause disorders of copper metabolism known as Wilson and Menkes diseases. E. coli possesses two genes for heavy-metal translocating P-type ATPases. We have constructed an expression system for one of them, ZntA, which encodes a 732 amino acid residue protein capable of transporting Zn(2+). A vanadate-sensitive, Zn(2+)-dependent ATPase activity is present in the membrane fraction of our expression strain. In addition to Zn(2+), the heavy-metal ions Cd(2+), Pb(2+), and Ag(+) activate the ATPase. Incubation of membranes from the expression strain with [gamma-(33)P]ATP in the presence of Zn(2+), Cd(2+), or Pb(2+) brings about phosphorylation of two membrane proteins with molecular masses of approximately 90 and 190 kDa, most likely representing the ZntA monomer and dimer, respectively. Although Cu(2+) can stimulate phosphorylation by [gamma-(33)P]ATP, it does not activate the ATPase. Cu(2+) also prevents the Zn(2+) activation of the ATPase when present in 2-fold excess over Zn(2+). Ag(+) and Cu(+) appear not to promote phosphorylation of the enzyme. To study the effects of Wilson disease mutations, we have constructed two site-directed mutants of ZntA, His475Gln and Glu470Ala, the human counterparts of which cause Wilson disease. Both mutants show a reduced metal ion stimulated ATPase activity (about 30-40% of the wild-type activity) and are phosphorylated much less efficiently by [gamma-(33)P]ATP than the wild type. In comparison to the wild type, the Glu470Ala mutant is phosphorylated more strongly by [(33)P]P(i), whereas the His475Gln mutant is phosphorylated more weakly. These results suggest that the mutation His475Gln affects the reaction with ATP and P(i) and stabilizes the enzyme in a dephosphorylated state. The Glu470Ala mutant seems to favor the E2 state. We conclude that His475 and Glu470 play important roles in the transport cycles of both the Wilson disease ATPase and ZntA.
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PMID:Expression and mutagenesis of ZntA, a zinc-transporting P-type ATPase from Escherichia coli. 1052 59

Pmr1, a novel member of the family of P-type ATPases, localizes to the Golgi compartment in yeast where it provides Ca(2+) and Mn(2+) for a variety of normal secretory processes. We have previously characterized Ca(2+) transport in isolated Golgi vesicles, and described an expression system for the analysis of Pmr1 mutants in a yeast strain devoid of background Ca(2+) pump activity [Sorin, A., Rosas, G., and Rao, R. (1997) J. Biol. Chem. 272, 9895-9901]. Here we show, using recombinant bacterial fusions, that an N-terminal EF hand-like motif in Pmr1 binds Ca(2+). Increasing disruptions of this motif led to progressive loss of pump function; thus, the single point mutations D51A and D53A retained pump activity but with drastic reductions in the affinity for Ca(2+) transport, while the double mutant was largely unable to exit the endoplasmic reticulum. In-frame deletions of the Ca(2+)-binding motif resulted in complete loss of function. Interestingly, the single point mutations conferred differential affinities for transport of Ca(2+) and Mn(2+) ions. Further, the proteolytic stability of the catalytic ATP-binding domain is altered by the N-terminal mutations, suggesting an interaction between these two regions of polypeptide. These studies implicate the N-terminal domain of Pmr1 in the modulation of ion transport, and may help elucidate the role of N-terminal metal-binding sites of Cu(2+)-ATPases, defective in Wilson and Menkes disease.
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PMID:An N-terminal EF hand-like motif modulates ion transport by Pmr1, the yeast Golgi Ca(2+)/Mn(2+)-ATPase. 1054 75

The copA gene product, a putative copper-translocating P-type ATPase, has been shown to be involved in copper resistance in Escherichia coli. The copA gene was disrupted by insertion of a kanamycin gene through homologous recombination. The mutant strain was more sensitive to copper salts but not to salts of other metals, suggesting a role in copper homeostasis. The copper-sensitive phenotype could be rescued by complementation by a plasmid carrying copA from E. coli or copB from Enterococcus hirae. Expression of copA was induced by salts of copper or silver but not zinc or cobalt. Everted membrane vesicles from cells expressing copA exhibited ATP-coupled accumulation of copper, presumably as Cu(I). The results indicate that CopA is a Cu(I)-translocating efflux pump that is similar to the copper pumps related to Menkes and Wilson diseases and provides a useful prokaryotic model for these human diseases.
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PMID:CopA: An Escherichia coli Cu(I)-translocating P-type ATPase. 1063 34

The transport and cellular metabolism of Cu depends on a series of membrane proteins and smaller soluble peptides that comprise a functionally integrated system for maintaining cellular Cu homeostasis. Inward transport across the plasma membrane appears to be a function of integral membrane proteins that form the channels that select Cu ions for passage. Two membrane-bound Cu-transporting ATPase enzymes, ATP7A and ATP7B, the products of the Menkes and Wilson disease genes, respectively, catalyze an ATP-dependent transfer of Cu to intracellular compartments or expel Cu from the cell. ATP7A and ATP7B work in concert with a series of smaller peptides, the copper chaperones, that exchange Cu at the ATPase sites or incorporate the Cu directly into the structure of Cu-dependent enzymes such as cytochrome c oxidase and Cu, Zn superoxide dismutase. These mechanisms come into play in response to a high influx of Cu or during the course of normal Cu metabolism.
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PMID:Cellular copper transport and metabolism. 1094 Mar 36

The Enterococcus hirae ATPase CopA is a member of the recently discovered heavy metal ATPases and shares 43% sequence identity with the human Menkes and Wilson copper ATPases. To study CopA biochemically, it was overexpressed in E. coli with an N-terminal histidine tag and purified to homogeneity by nickel affinity chromatography. The purified CopA catalyzed ATP hydrolysis with a V(max) of 0.15 micromol/min/mg and a K(m) for ATP of 0.2 mM and had an optimum pH of 6.25. The activity was 3- to 4-fold stimulated by reconstitution into proteoliposomes. The enzyme formed an acylphosphate intermediate. Its kinetics of formation and the effects of inhibitors and metal ions upon it support a function of CopA in copper transport. Purification and functional reconstitution of CopA provides the basis to study copper transport in vitro.
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PMID:Purification and functional analysis of the copper ATPase CopA of Enterococcus hirae. 1116 79

Copper plays a key role in brain development, function and survival. Alteration of its homeostasis is suggested to be an aetiological factor in several neurodegenerative diseases. However, the molecular mechanisms relating copper to neurodegeneration are still unknown. In the present report, using morphological analyses of brain sections of mottled/brindled mutant (Mo(br/y)) mice, the animal model of the human genetic copper deficiency associated with neurodegeneration (Menkes' disease), we demonstrated that a high degree of apoptotic cells is present in the neocortex and in the hippocampus. Biochemical characterisation revealed decreased levels of copper content and of the activity of the mitochondrial copper-dependent enzyme cytochrome c oxidase. Copper, zinc-superoxide dismutase activity also shows a slight decrease, while no change was observed for glutathione content. Lower levels of ATP were also found, indicative of a copper-dependent impairment of energy metabolism. Changes appear to be specific for the brain, since no alterations in the activity of liver enzymes were found, although the level of copper was strongly decreased. We also tested biochemical factors involved in cell commitment to apoptosis. The expression of the anti-apoptotic protein Bcl-2, which plays a fundamental role in brain development and morphogenesis, was dramatically decreased and the levels of cytochrome c released from mitochondria into the cytosol were significantly increased. On the basis of these findings, we propose that down-regulation of Bcl-2 can cause neurodegeneration triggered by mitochondrial damage due to copper depletion during brain development in Mo(br/y) mice.
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PMID:Neurodegeneration in the animal model of Menkes' disease involves Bcl-2-linked apoptosis. 1131 99

The Menkes protein is a transmembrane copper translocating P-type ATPase. Mutations in the Menkes gene that affect the function of the Menkes protein may cause Menkes disease in humans, which is associated with severe systemic copper deficiency. The catalytic mechanism of the Menkes protein, including the formation of transient acylphosphate, is poorly understood. We transfected and overexpressed wild-type and targeted mutant Menkes protein in yeast and investigated its transient acyl phosphorylation. We demonstrated that the Menkes protein is transiently phosphorylated by ATP in a copper-specific and copper-dependent manner and appears to undergo conformational changes in accordance with the classical P-type ATPase model. Our data suggest that the catalytic cycle of the Menkes protein begins with the binding of copper to high affinity binding sites in the transmembrane channel, followed by ATP binding and transient phosphorylation. We propose that putative copper-binding sites at the N-terminal domain of the Menkes protein are important as sensors of low concentrations of copper but are not essential for the overall catalytic activity.
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PMID:The regulation of catalytic activity of the menkes copper-translocating P-type ATPase. Role of high affinity copper-binding sites. 1137 92

The Enterococcus hirae CopB ATPase (EC 3.6.1.3) confers copper resistance to the organism by expelling excess copper. Two related human ATPase genes, ATP7A (EC 3.6.1.36) and ATP7B (EC 3.6.1.36), have been cloned as the loci of mutations causing Menkes and Wilson diseases, diseases of copper metabolism. Many mutations in these genes have been identified in patients. Since it has not yet been possible to purify the human copper ATPases, it has proved difficult to test the impact of mutations on ATPase function. Some mutations occur in highly conserved sequence motifs, suggesting that their effect on function can be tested with a homologous enzyme. Here, we used the E. hirae CopB ATPase to investigate the impact of such mutations on enzyme function in vivo and in vitro. The Menkes disease mutation of Cys-1000-->Arg, changing the conserved Cys-Pro-Cys ('CPC') motif, was mimicked in CopB. The corresponding Cys-396-->Ser CopB ATPase was unable to restore copper resistance in a CopB knock-out mutant in vivo. The purified mutant ATPase still formed an acylphosphate intermediate, but possessed no detectable ATP hydrolytic activity. The most frequent Wilson disease mutation, His-1069-->Gln, was introduced into CopB as His-480-->Gln (H480Q). This mutant CopB also failed to confer copper resistance to a CopB knock-out strain. Purified H480Q CopB formed an acylphosphate intermediate and retained a small, but significant, ATPase activity. Our results reveal that Cys-396 and His-480 of CopB are key residues for ATPase function, and similar roles are suggested for Cys-1000 and His-1069 of Menkes and Wilson ATPases respectively.
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PMID:Structure-function analysis of purified Enterococcus hirae CopB copper ATPase: effect of Menkes/Wilson disease mutation homologues. 1141 52

Escherichia coli CopA is a Cu(I)-translocating P-type ATPase that is involved in copper export and resistance. It is an orthologue of the human Menkes and Wilson disease-related proteins. Each of those two human copper pumps has six N-terminal Cys(X)(2)Cys sequences, but their function in transport is unclear. CopA has two N-terminal Cys(X)(2)Cys sequences, GLSC(14)GHC(17) and GMSC(110)ASC(113). The requirement of these cysteine motifs was investigated by mutagenesis of the codons for all four cysteine residues, singly and in combination. Cells of a copA deletion strain expressing genes for the mutant genes were nearly as resistant to copper as the wild type. In addition, everted membrane vesicles from cells expressing the mutant copA genes exhibited ATP-coupled accumulation of copper similar to that of the wild type. The results indicate that neither of two N-terminal Cys(X)(2)Cys motifs is required for either resistance or transport.
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PMID:Escherichia coli CopA N-terminal Cys(X)(2)Cys motifs are not required for copper resistance or transport. 1150 54


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