The Experts below are selected from a list of 1842 Experts worldwide ranked by ideXlab platform
Svetlana Lutsenko - One of the best experts on this subject based on the ideXlab platform.
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human copper transporter atp7b Wilson Disease Protein forms stable dimers in vitro and in cells
Journal of Biological Chemistry, 2017Co-Authors: Samuel Jayakanthan, Lelita T Braiterman, Nesrin M. Hasan, Vinzenz M Unger, Svetlana LutsenkoAbstract:Abstract ATP7B is a copper-transporting P1B-type ATPase (Cu-ATPase) with an essential role in human physiology. Mutations in ATP7B cause the potentially fatal Wilson Disease, and changes in ATP7B expression are observed in several cancers. Despite its physiologic importance, the biochemical information about ATP7B remains limited because of a complex multidomain organization of the Protein. By analogy with the better characterized prokaryotic Cu-ATPases, ATP7B is assumed to be a single-chain monomer. We show that in eukaryotic cells, human ATP7B forms dimers that can be purified following solubilization. Deletion of the four N-terminal metal-binding domains, characteristic for human ATP7B, does not disrupt dimerization, i.e. the dimer interface is formed by the domains that are conserved among Cu-ATPases. Unlike the full-length ATP7B, which is targeted to the trans-Golgi network, 1–4ΔMBD-7B is targeted primarily to vesicles. This result and the analysis of differentially tagged ATP7B variants indicate that the dimeric structure is retained during ATP7B trafficking between the intracellular compartments. Purified dimeric species of 1–4ΔMBD-7B were characterized by a negative stain electron microscopy in the presence of ADP/MgCl2. Single-particle analysis yielded a low-resolution 3D model that provides the first insight into an overall architecture of a human Cu-ATPase, positions of the main domains, and a dimer interface.
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Genetic, metabolic and cellular factors influencing intracellular localization of the Wilson Disease Protein, ATP7B
Molecular Cytogenetics, 2014Co-Authors: Arnab Gupta, Lelita T Braiterman, Ashima Bhattacharjee, Nesrin M. Hasan, Svetlana Lutsenko, Ann Louise HubbardAbstract:Background Wilson Disease (WD) is a disorder of copper accumulation in liver and brain caused by mutations in the coppertransporting ATPase ATP7B that affects 1 in 5000 live births. Under basal conditions, ATP7B Protein localizes to the trans-Golgi network (TGN) but traffics to vesicles in response to high copper. The purpose of the study is to identify genetic, metabolic and regulatory factors that regulate ATP7B function and localization to maintain normal copper homeostasis in cells. The study is divided into three parts, (a) Role of copper in normal Protein folding and its ER exit, (b) Role of regulatory phosphorylation of ATP7B in its trafficking from TGN to vesicles (c) Interaction of ATP7B with regulatory Proteins in its trafficking route.
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cellular copper levels determine the phenotype of the arg875 variant of atp7b Wilson Disease Protein
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Arnab Gupta, Lelita T Braiterman, Sergiy Nokhrin, Oleg Y. Dmitriev, Ashima Bhattacharjee, Ann Louise Hubbard, Svetlana LutsenkoAbstract:In human disorders, the genotype-phenotype relationships are often complex and influenced by genetic and/or environmental factors. Wilson Disease (WD) is a monogenic disorder caused by mutations in the copper-transporting P-type ATPase ATP7B. WD shows significant phenotypic diversity even in patients carrying identical mutations; the basis for such diverse manifestations is unknown. We demonstrate that the 2623A/G polymorphism (producing the Gly875→Arg substitution in the A-domain of ATP7B) drastically alters the intracellular properties of ATP7B, whereas copper reverses the effects. Under basal conditions, the common Gly875 variant of ATP7B is targeted to the trans-Golgi network (TGN) and transports copper into the TGN lumen. In contrast, the Arg875 variant is located in the endoplasmic reticulum (ER) and does not deliver copper to the TGN. Elevated copper corrects the ATP7B-Arg875 phenotype. Addition of only 0.5–5 μM copper triggers the exit of ATP7B-Arg875 from the ER and restores copper delivery to the TGN. Analysis of the recombinant A-domains by NMR suggests that the ER retention of ATP7B-Arg875 is attributable to increased unfolding of the Arg875-containing A-domain. Copper is not required for the folding of ATP7B-Arg875 during biosynthesis, but it stabilizes Protein and stimulates its activity. A chemotherapeutical drug, cisplatin, that mimics a copper-bound state of ATP7B also corrects the “Disease-like” phenotype of ATP7B-Arg875 and promotes its TGN targeting and transport function. We conclude that in populations harboring the Arg875 polymorphism, the levels of bioavailable copper may play a vital role in the manifestations of WD.
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Cellular copper levels determine the phenotype of the Arg875 variant of ATP7B/Wilson Disease Protein
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Arnab Gupta, Lelita T Braiterman, Sergiy Nokhrin, Oleg Y. Dmitriev, Ashima Bhattacharjee, Ann Louise Hubbard, Svetlana LutsenkoAbstract:In human disorders, the genotype-phenotype relationships are often complex and influenced by genetic and/or environmental factors. Wilson Disease (WD) is a monogenic disorder caused by mutations in the copper-transporting P-type ATPase ATP7B. WD shows significant phenotypic diversity even in patients carrying identical mutations; the basis for such diverse manifestations is unknown. We demonstrate that the 2623A/G polymorphism (producing the Gly875→Arg substitution in the A-domain of ATP7B) drastically alters the intracellular properties of ATP7B, whereas copper reverses the effects. Under basal conditions, the common Gly875 variant of ATP7B is targeted to the trans-Golgi network (TGN) and transports copper into the TGN lumen. In contrast, the Arg875 variant is located in the endoplasmic reticulum (ER) and does not deliver copper to the TGN. Elevated copper corrects the ATP7B-Arg875 phenotype. Addition of only 0.5–5 μM copper triggers the exit of ATP7B-Arg875 from the ER and restores copper delivery to the TGN. Analysis of the recombinant A-domains by NMR suggests that the ER retention of ATP7B-Arg875 is attributable to increased unfolding of the Arg875-containing A-domain. Copper is not required for the folding of ATP7B-Arg875 during biosynthesis, but it stabilizes Protein and stimulates its activity. A chemotherapeutical drug, cisplatin, that mimics a copper-bound state of ATP7B also corrects the “Disease-like” phenotype of ATP7B-Arg875 and promotes its TGN targeting and transport function. We conclude that in populations harboring the Arg875 polymorphism, the levels of bioavailable copper may play a vital role in the manifestations of WD.
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DELIVERY OF THE Cu-TRANSPORTING ATPase ATP7B TO THE PLASMA MEMBRANE IN XENOPUS OOCYTES
Biochimica et Biophysica Acta, 2008Co-Authors: Éva Lörinczi, Ruslan Tsivkovskii, Svetlana Lutsenko, Winfried Haase, Ernst Bamberg, Thomas FriedrichAbstract:Cu-transporting ATPase ATP7B (Wilson Disease Protein) is essential for the maintenance of intracellular copper concentration. In hepatocytes, ATP7B is required for copper excretion, which is thought to occur via a transient delivery of the ATP7B- and copper-containing vesicles to the apical membrane. The currently available experimental systems do not allow analysis of ATP7B at the cell surface. Using epitope insertion, we identified an extracellular loop into which the HA-epitope can be introduced without inhibiting ATP7B activity. The HA-tagged ATP7B was expressed in Xenopus oocytes and the presence of ATP7B at the plasma membrane was demonstrated by electron microscopy, freeze-fracture experiments, and surface luminescence measurements in intact cells. Neither the deletion of the entire N-terminal copper-binding domain nor the inactivating mutation of catalytic Asp1027 affected delivery to the plasma membrane of oocytes. In contrast, surface targeting was decreased for the ATP7B variants with mutations in the ATP-binding site or the intra-membrane copper-binding site, suggesting that ligand-stabilized conformation(s) are important for ATP7B trafficking. The developed system provides significant advantages for studies that require access to both sides of ATP7B in the membrane.
Jonathan D Gitlin - One of the best experts on this subject based on the ideXlab platform.
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the copper toxicosis gene product murr1 directly interacts with the Wilson Disease Protein
Journal of Biological Chemistry, 2003Co-Authors: Leo W. J. Klomp, Cisca Wijmenga, Jonathan D GitlinAbstract:Abstract Copper toxicosis in Bedlington terriers is an autosomal recessive disorder characterized by excessive hepatic copper accumulation in association with a marked decrease in biliary copper excretion. Recent genetic data have revealed that MURR1, a single copy gene on dog chromosome 10q26, is mutated in this disorder. This gene encodes a 190-amino acid open reading frame of unknown function that is highly conserved in vertebrate species. The Wilson Disease Protein is a copper transporting ATPase shown to play a critical role in biliary copper excretion. Here we demonstrate that the Wilson Disease Protein directly interacts with the human homologue of Murr1 in vitro and in vivo and that this interaction is mediated via the copper binding, amino terminus of this ATPase. Importantly, this interaction is specific for this copper transporter, a finding consistent with the observation that impaired copper homeostasis in affected terriers is confined to the liver. Our findings reveal involvement of Murr1 in the defined pathway of hepatic biliary copper excretion, suggest a potential mechanism for Murr1 function in this process, and provide biochemical evidence in support of the proposed role of the MURR1 gene in hepatic copper toxicosis.
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interaction of the copper chaperone hah1 with the Wilson Disease Protein is essential for copper homeostasis
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Iqbal Hamza, Mark Schaefer, Leo W. J. Klomp, Jonathan D GitlinAbstract:The delivery of copper to specific sites within the cell is mediated by distinct intracellular carrier Proteins termed copper chaperones. Previous studies in Saccharomyces cerevisiae suggested that the human copper chaperone HAH1 may play a role in copper trafficking to the secretory pathway of the cell. In this current study, HAH1 was detected in lysates from multiple human cell lines and tissues as a single-chain Protein distributed throughout the cytoplasm and nucleus. Studies with a glutathione S-transferase-HAH1 fusion Protein demonstrated direct Protein–Protein interaction between HAH1 and the Wilson Disease Protein, which required the cysteine copper ligands in the amino terminus of HAH1. Consistent with these in vitro observations, coimmunoprecipitation experiments revealed that HAH1 interacts with both the Wilson and Menkes Proteins in vivo and that this interaction depends on available copper. When these studies were repeated utilizing three Disease-associated mutations in the amino terminus of the Wilson Protein, a marked diminution in HAH1 interaction was observed, suggesting that impaired copper delivery by HAH1 constitutes the molecular basis of Wilson Disease in patients harboring these mutations. Taken together, these data provide a mechanism for the function of HAH1 as a copper chaperone in mammalian cells and demonstrate that this Protein is essential for copper homeostasis.
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functional expression of the Wilson Disease Protein reveals mislocalization and impaired copper dependent trafficking of the common h1069q mutation
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Aimee Payne, Edward J. Kelly, Jonathan D GitlinAbstract:Wilson Disease is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase. To elucidate the function of the Wilson Protein, wild-type and mutant Wilson cDNAs were expressed in a Menkes copper transporter-deficient mottled fibroblast cell line defective in copper export. Expression of the wild-type cDNA demonstrated trans-Golgi network localization and copper-dependent trafficking of the Wilson Protein identical to previous observations for the endogenously expressed Protein in hepatocytes. Furthermore, expression of the Wilson cDNA rescued the mottled phenotype as evidenced by a reduction in copper accumulation and restoration of cell viability. In contrast, expression of an H1069Q mutant Wilson cDNA did not rescue the mottled phenotype, and immunofluorescence studies showed that this mutant Wilson Protein was localized in the endoplasmic reticulum. Consistent with these findings, pulse–chase analysis demonstrated a 5-fold decrease in the half-life of the H1069Q mutant as compared with the wild-type Protein. Maintenance of these transfected cell lines at 28°C resulted in localization of the H1069Q Protein in the trans-Golgi network, suggesting that a temperature-sensitive defect in Protein folding followed by degradation constitutes the molecular basis of Wilson Disease in patients harboring the H1069Q mutation. Taken together, these studies describe a tractable expression system for elucidating the function and localization of the copper-transporting ATPases in mammalian cells and provide compelling evidence that the Wilson Protein can functionally substitute for the Menkes Protein, supporting the concept that these Proteins use common biochemical mechanisms to effect cellular copper homeostasis.
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functional expression of the Wilson Disease Protein reveals mislocalization and impaired copper dependent trafficking of the common h1069q mutation
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Aimee Payne, Edward J. Kelly, Jonathan D GitlinAbstract:Wilson Disease is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase. To elucidate the function of the Wilson Protein, wild-type and mutant Wilson cDNAs were expressed in a Menkes copper transporter-deficient mottled fibroblast cell line defective in copper export. Expression of the wild-type cDNA demonstrated trans-Golgi network localization and copper-dependent trafficking of the Wilson Protein identical to previous observations for the endogenously expressed Protein in hepatocytes. Furthermore, expression of the Wilson cDNA rescued the mottled phenotype as evidenced by a reduction in copper accumulation and restoration of cell viability. In contrast, expression of an H1069Q mutant Wilson cDNA did not rescue the mottled phenotype, and immunofluorescence studies showed that this mutant Wilson Protein was localized in the endoplasmic reticulum. Consistent with these findings, pulse–chase analysis demonstrated a 5-fold decrease in the half-life of the H1069Q mutant as compared with the wild-type Protein. Maintenance of these transfected cell lines at 28°C resulted in localization of the H1069Q Protein in the trans-Golgi network, suggesting that a temperature-sensitive defect in Protein folding followed by degradation constitutes the molecular basis of Wilson Disease in patients harboring the H1069Q mutation. Taken together, these studies describe a tractable expression system for elucidating the function and localization of the copper-transporting ATPases in mammalian cells and provide compelling evidence that the Wilson Protein can functionally substitute for the Menkes Protein, supporting the concept that these Proteins use common biochemical mechanisms to effect cellular copper homeostasis.
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Functional Expression of the Menkes Disease Protein Reveals Common Biochemical Mechanisms Among the Copper-transporting P-type ATPases
Journal of Biological Chemistry, 1998Co-Authors: Aimee S. Payne, Jonathan D GitlinAbstract:Abstract Menkes Disease is a fatal neurodegenerative disorder of childhood caused by the absence or dysfunction of a putative P-type ATPase encoded on the X chromosome. To elucidate the function of the Menkes Disease Protein, a plasmid containing the open reading frame of the human Menkes Disease gene was constructed and used to transform a strain of Saccharomyces cerevisiae deficient in CCC2, the yeast Menkes/Wilson Disease gene homologue.ccc2Δ yeast are deficient in copper transport into the secretory pathway, and expression of a wild type human Menkes cDNA complemented this defect, as evidenced by the restoration of copper incorporation into the multicopper oxidase Fet3p. Site-directed mutagenesis demonstrated the essential role of four specific amino acids in this process, including a conserved histidine, which is the site of the most common Disease mutation in the homologous Wilson Disease Protein. The expression of Menkes cDNAs with successive mutations of the conserved cysteine residues in the six amino-terminal MXCXXC metal binding domains confirmed the essential role of these cysteine residues in copper transport but revealed that each of these domains is not functionally equivalent. These data demonstrate that the Menkes Disease Protein functions to deliver copper into the secretory pathway of the cell and that this process involves biochemical mechanisms common to previously characterized members of this P-type ATPase family.
Pernilla Wittung-stafshede - One of the best experts on this subject based on the ideXlab platform.
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Copper relay path through the N-terminus of Wilson Disease Protein, ATP7B.
Metallomics, 2019Co-Authors: Kumaravel Ponnandai Shanmugavel, Pernilla Wittung-stafshedeAbstract:In human cells, copper (Cu) ions are transported by the cytoplasmic Cu chaperone Atox1 to the Wilson Disease Protein (ATP7B) in the Golgi for loading of Cu-dependent enzymes. ATP7B is a membrane-spanning Protein which, in contrast to non-mammalian homologs, has six cytoplasmic metal-binding domains (MBDs). To address the reason for multiple MBDs, we introduced strategic mutations in which one, two or three MBDs had been blocked for Cu binding via cysteine-to-serine mutations (but all six MBDs are present in all) in a yeast system that probes Cu flow through Atox1 and ATP7B. The results, combined with earlier work, support a mechanistic model in which MBD1-3 forms a regulatory unit of ATP7B Cu transport. Cu delivery via Atox1 to this unit, followed by loading of Cu in MBD3, promotes release of inhibitory interactions. Whereas the Cu site in MBD4 can be mutated without a large effect, an intact Cu site in either MBD5 or MBD6 is required for Cu transport. All MBDs, expressed as single-domain Proteins, can replace Atox1 and deliver Cu to full-length ATP7B. However, only MBD6 can deliver Cu to truncated ATP7B where all six MBDs are removed, suggesting a docking role for this structural unit.
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A Luminal Loop of Wilson Disease Protein Binds Copper and Is Required for Protein Activity
Biophysical Journal, 2018Co-Authors: Birgit Köhn, Kumaravel Ponnandai Shanmugavel, Michael Kovermann, Pernilla Wittung-stafshedeAbstract:The copper-transporting ATPase ATP7B is essential for loading of copper ions to copper-dependent enzymes in the secretory pathway; its inactivation results in Wilson Disease. In contrast to copper-ion uptake by the cytoplasmic domains, ATP7B-mediated copper-ion release in the Golgi has not been explored yet. We demonstrate here that a luminal loop in ATP7B, rich in histidine/methionine residues, binds reduced copper (Cu(I)) ions, and identified copper-binding residues play an essential role in ATP7B-mediated metal ion release. NMR experiments on short-peptide models demonstrate that three methionine and two histidine residues are specifically involved in Cu(I) ion binding; with these residues replaced by alanines, no Cu(I) ion interaction is detected. Although more than one Cu(I) ion can interact with the wild-type peptide, removing either all histidine or all methionine residues reduces the stoichiometry to one Cu(I) ion binding per peptide. Using a yeast complementation assay, we show that for efficient copper transport by full-length ATP7B, the complete set of histidine and methionine residues in the lumen loop are required. The replacement of histidine/methionine residues by alanines does not perturb overall ATP7B structure, as the localization of ATP7B variants in yeast cells matches that of the wild-type Protein. Thus, in similarity to ATP7A, ATP7B also appears to have a luminal "exit" copper ion site.
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Probing functional roles of Wilson Disease Protein (ATP7B) copper-binding domains in yeast.
Metallomics, 2017Co-Authors: Kumaravel Ponnandai Shanmugavel, Dina Petranovic, Pernilla Wittung-stafshedeAbstract:After Ctr1-mediated uptake into human cells, copper (Cu) ions are transported by the cytoplasmic Cu chaperone Atox1 to the Wilson Disease Protein (ATP7B) in the Golgi network. Cu transfer occurs via direct Protein-Protein interactions and leads to incorporation of Cu into Cu-dependent enzymes. ATP7B is a large multi-domain membrane-spanning Protein which, in contrast to homologs, has six cytoplasmic metal-binding domains (MBDs). The reason for multiple MBDs is proposed to be indirect modulation of activity but mechanistic studies of full-length ATP7B are limited. We here developed a system that probes Cu flow through human Atox1 and ATP7B Proteins when expressed in yeast. Using this assay, we assessed the roles of the different MBDs in ATP7B and found that the presence of the most N-terminal MBD increased, whereas the third MBD decreased, overall ATP7B-mediated Cu transport activity. Upon removal of all MBDs in ATP7B, the ability to transport Cu disappeared. The designed system can be expanded to include other yeast viability parameters and will be a useful tool for further mechanistic insights on human Cu transport as well as Diseases involving Cu imbalance.
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Disease-causing point-mutations in metal-binding domains of Wilson Disease Protein decrease stability and increase structural dynamics
BioMetals, 2017Co-Authors: Ranjeet Kumar, Candan Ariöz, Niklas Bosaeus, Yaozong Li, Sandra Rocha, Pernilla Wittung-stafshedeAbstract:After cellular uptake, Copper (Cu) ions are transferred from the chaperone Atox1 to the Wilson Disease Protein (ATP7B) for incorporation into Cu-dependent enzymes in the secretory pathway. Human ATP7B is a large multi-domain membrane-spanning Protein which, in contrast to homologues in other organisms, has six similar cytoplasmic metal-binding domains (MBDs). The reason for multiple MBDs is proposed to be indirect modulation of enzymatic activity and it is thus intriguing that point mutations in MBDs can promote Wilson Disease. We here investigated, in vitro and in silico, the biophysical consequences of clinically-observed Wilson Disease mutations, G85V in MBD1 and G591D in MBD6, incorporated in domain 4. Because G85 and G591 correspond to a conserved Gly found in all MBDs, we introduced the mutations in the well-characterized MBD4. We found the mutations to dramatically reduce the MBD4 thermal stability, shifting the midpoint temperature of unfolding by more than 20 °C. In contrast to wild type MBD4 and MBD4D, MBD4V adopted a misfolded structure with a large β-sheet content at high temperatures. Molecular dynamic simulations demonstrated that the mutations increased backbone fluctuations that extended throughout the domain. Our findings imply that reduced stability and enhanced dynamics of MBD1 or MBD6 is the origin of ATP7B dysfunction in Wilson Disease patients with the G85V or G591D mutation.
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Protein Interactions that Enable Safe and Efficient Copper Ion Transport in the Human Cytoplasm
Biophysical Journal, 2016Co-Authors: Pernilla Wittung-stafshedeAbstract:Although copper (Cu) is an essential metal for most living organisms, high levels and free such ions are toxic. In humans, after cellular uptake via the membrane-bound importer Ctr1, Cu is transported to targets by cytoplasmic Cu chaperones: Atox1 delivers Cu to membrane-bound P1B-type ATPases (i.e., ATP7B or Wilson Disease Protein) in the Golgi (secretory path; here, most Cu-dependent enzymes are loaded with Cu) whereas CCS delivers Cu specifically to cytoplasmic superoxide dismutase. In contrast to bacterial andyeast homologs, ATP7B has six similar metal-binding domains protruding into the cytoplasm: possibly, conformational changes among these regulate overall ATP7B activity. To reveal underlying molecular mechanisms as well as thermodynamic and kinetic driving forces for human Cu transport - from the cell membrane to the Golgi - our strategy involves a range of complementary biophysical experiments on purified Proteins, domain constructs and engineered variants. From our studies, we have discovered that (a) the cytoplasmic C-terminus of Ctr1 binds Cu through its HCH motif with a moderate affinity that allows for Cu delivery to Atox1, (b) Atox1 can interact with CCS and exchange Cu implying cross-reactivity between cytoplasmic chaperones, (c) transfer of Cu from Atox1 to metal-binding domains in ATP7B proceeds through Cu-bridged hetero-Protein dimers displaying enthalpy-entropy compensation, (d) conformational changes and domain-domain interactions within ATP7B depend on Cu loading status and minute changes in solvent conditions, and (e), in addition to its cytoplasmic chaperone activity, Atox1 may have functionality in the nucleus as it interacted with several DNA-binding Proteins in a yeast two-hybrid screen.
Joachim Füllekrug - One of the best experts on this subject based on the ideXlab platform.
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copper induced translocation of the Wilson Disease Protein atp7b independent of murr1 commd1 and rab7
American Journal of Pathology, 2008Co-Authors: Karl Heinz Weiss, Daniel Gotthardt, Wolfgang Stremmel, Javier Carbajo Lozoya, S. Tuma, Jürgen Reichert, Robert Ehehalt, Joachim FüllekrugAbstract:Wilson Disease is a genetic disorder of copper metabolism. Impaired biliary excretion results in a gradual accumulation of copper, which leads to severe Disease. The specific gene defect lies in the Wilson Disease Protein, ATP7B, a copper-transporting ATPase that is highly active in hepatocytes. The two major functions of ATP7B in the liver are the copper loading of ceruloplasmin in the Golgi apparatus, and the excretion of excess copper into the bile. In response to elevated copper levels, ATP7B shows a unique intracellular trafficking pattern that is required for copper excretion from the Golgi apparatus into dispersed vesicles. We analyzed the translocation of ATP7B by both confocal microscopy and RNA interference, testing current models that suggest the involvement of Murr1/COMMD1 and Rab7 in this pathway. We found that although the ATP7B translocation is conserved among nonhepatic cell lines, there is no co-localization with Murr1/COMMD1 or the Rab marker Proteins of the endolysosomal system. Consistent with this finding, the translocation of ATP7B was not impaired by the depletion of either Murr1/COMMD1 or Rab7, or by a dominant-negative Rab7 mutant. In conclusion, our data suggest that the translocation of ATP7B takes place independently of Rab7-regulated endosomal traffic events. Murr1/COMMD1 plays a role in a later step of the copper excretion pathway but is not involved in the translocation of the Wilson Disease Protein.
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Copper-induced translocation of the Wilson Disease Protein ATP7B independent of Murr1/COMMD1 and Rab7.
American Journal of Pathology, 2008Co-Authors: Karl Heinz Weiss, Daniel Gotthardt, Wolfgang Stremmel, Javier Carbajo Lozoya, S. Tuma, Jürgen Reichert, Robert Ehehalt, Joachim FüllekrugAbstract:Wilson Disease is a genetic disorder of copper metabolism. Impaired biliary excretion results in a gradual accumulation of copper, which leads to severe Disease. The specific gene defect lies in the Wilson Disease Protein, ATP7B, a copper-transporting ATPase that is highly active in hepatocytes. The two major functions of ATP7B in the liver are the copper loading of ceruloplasmin in the Golgi apparatus, and the excretion of excess copper into the bile. In response to elevated copper levels, ATP7B shows a unique intracellular trafficking pattern that is required for copper excretion from the Golgi apparatus into dispersed vesicles. We analyzed the translocation of ATP7B by both confocal microscopy and RNA interference, testing current models that suggest the involvement of Murr1/COMMD1 and Rab7 in this pathway. We found that although the ATP7B translocation is conserved among nonhepatic cell lines, there is no co-localization with Murr1/COMMD1 or the Rab marker Proteins of the endolysosomal system. Consistent with this finding, the translocation of ATP7B was not impaired by the depletion of either Murr1/COMMD1 or Rab7, or by a dominant-negative Rab7 mutant. In conclusion, our data suggest that the translocation of ATP7B takes place independently of Rab7-regulated endosomal traffic events. Murr1/COMMD1 plays a role in a later step of the copper excretion pathway but is not involved in the translocation of the Wilson Disease Protein.
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Localization of the Wilson Disease Protein in murine intestine
Journal of Anatomy, 2008Co-Authors: Karl Heinz Weiss, Judith Wurz, Daniel Gotthardt, Uta Merle, Wolfgang Stremmel, Joachim FüllekrugAbstract:Wilson Disease is an inherited disorder of human copper metabolism, characterized by gradual accumulation of copper in tissues, predominantly liver and brain. The gene defect lies in the Wilson Disease Protein ATP7B, a copper transporting ATPase highly active in hepatocytes. In the liver, ATP7B is essential for excretion of excess copper into the bile and for copper loading of ceruloplasmin in the Golgi apparatus. The extrahepatic role of ATP7B is not yet completely understood. We analysed the intestinal expression of ATP7B in mice using RT-PCR, Western blot and indirect immunofluorescence. We found abundant expression of ATP7B in stomach and small intestine, but not in colon. Using confocal microscopy we demonstrate a Golgi localization of ATP7B in enterocytes. In response to elevated copper, the Wilson Disease Protein shows an intracellular trafficking pattern in the intestinal polarized cell line CaCo-2, moving away from the Golgi apparatus to dispersed vesicles. This suggests a role for intestinal ATP7B in sequestration of copper in intracellular vesicles for maintenance of copper homeostasis in the enterocyte. In conclusion, the expression of ATP7B in the small intestine might represent an additional regulatory mechanism to fine-tune intestinal copper absorption.
Oleg Y. Dmitriev - One of the best experts on this subject based on the ideXlab platform.
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cellular copper levels determine the phenotype of the arg875 variant of atp7b Wilson Disease Protein
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Arnab Gupta, Lelita T Braiterman, Sergiy Nokhrin, Oleg Y. Dmitriev, Ashima Bhattacharjee, Ann Louise Hubbard, Svetlana LutsenkoAbstract:In human disorders, the genotype-phenotype relationships are often complex and influenced by genetic and/or environmental factors. Wilson Disease (WD) is a monogenic disorder caused by mutations in the copper-transporting P-type ATPase ATP7B. WD shows significant phenotypic diversity even in patients carrying identical mutations; the basis for such diverse manifestations is unknown. We demonstrate that the 2623A/G polymorphism (producing the Gly875→Arg substitution in the A-domain of ATP7B) drastically alters the intracellular properties of ATP7B, whereas copper reverses the effects. Under basal conditions, the common Gly875 variant of ATP7B is targeted to the trans-Golgi network (TGN) and transports copper into the TGN lumen. In contrast, the Arg875 variant is located in the endoplasmic reticulum (ER) and does not deliver copper to the TGN. Elevated copper corrects the ATP7B-Arg875 phenotype. Addition of only 0.5–5 μM copper triggers the exit of ATP7B-Arg875 from the ER and restores copper delivery to the TGN. Analysis of the recombinant A-domains by NMR suggests that the ER retention of ATP7B-Arg875 is attributable to increased unfolding of the Arg875-containing A-domain. Copper is not required for the folding of ATP7B-Arg875 during biosynthesis, but it stabilizes Protein and stimulates its activity. A chemotherapeutical drug, cisplatin, that mimics a copper-bound state of ATP7B also corrects the “Disease-like” phenotype of ATP7B-Arg875 and promotes its TGN targeting and transport function. We conclude that in populations harboring the Arg875 polymorphism, the levels of bioavailable copper may play a vital role in the manifestations of WD.
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Cellular copper levels determine the phenotype of the Arg875 variant of ATP7B/Wilson Disease Protein
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Arnab Gupta, Lelita T Braiterman, Sergiy Nokhrin, Oleg Y. Dmitriev, Ashima Bhattacharjee, Ann Louise Hubbard, Svetlana LutsenkoAbstract:In human disorders, the genotype-phenotype relationships are often complex and influenced by genetic and/or environmental factors. Wilson Disease (WD) is a monogenic disorder caused by mutations in the copper-transporting P-type ATPase ATP7B. WD shows significant phenotypic diversity even in patients carrying identical mutations; the basis for such diverse manifestations is unknown. We demonstrate that the 2623A/G polymorphism (producing the Gly875→Arg substitution in the A-domain of ATP7B) drastically alters the intracellular properties of ATP7B, whereas copper reverses the effects. Under basal conditions, the common Gly875 variant of ATP7B is targeted to the trans-Golgi network (TGN) and transports copper into the TGN lumen. In contrast, the Arg875 variant is located in the endoplasmic reticulum (ER) and does not deliver copper to the TGN. Elevated copper corrects the ATP7B-Arg875 phenotype. Addition of only 0.5–5 μM copper triggers the exit of ATP7B-Arg875 from the ER and restores copper delivery to the TGN. Analysis of the recombinant A-domains by NMR suggests that the ER retention of ATP7B-Arg875 is attributable to increased unfolding of the Arg875-containing A-domain. Copper is not required for the folding of ATP7B-Arg875 during biosynthesis, but it stabilizes Protein and stimulates its activity. A chemotherapeutical drug, cisplatin, that mimics a copper-bound state of ATP7B also corrects the “Disease-like” phenotype of ATP7B-Arg875 and promotes its TGN targeting and transport function. We conclude that in populations harboring the Arg875 polymorphism, the levels of bioavailable copper may play a vital role in the manifestations of WD.
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Mechanism of tumor resistance to cisplatin mediated by the copper transporter ATP7BThis paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergon
Biochemistry and Cell Biology, 2011Co-Authors: Oleg Y. DmitrievAbstract:The Wilson Disease Protein (ATP7B) is a copper-transporting ATPase that is responsible for regulating copper homeostasis in human tissues. ATP7B is associated with cancer resistance to cisplatin, one of the most widely used anticancer drugs. This minireview discusses the possible mechanisms of tumor resistance to cisplatin mediated by ATP7B. Cisplatin binds to the N-terminal cytosolic domain of ATP7B, which contains multiple copper-binding sites. Active platinum efflux catalyzed by ATP7B is unlikely to significantly contribute to cisplatin resistance in vivo. Transient platinum sequestration in the metal-binding domain followed by transfer to an acceptor Protein or a low molecular weight compound is proposed as an alternative mechanism of cisplatin detoxification in the cell.
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solution structure of the n domain of Wilson Disease Protein distinct nucleotide binding environment and effects of Disease mutations
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Oleg Y. Dmitriev, Clinton T Morgan, Ruslan Tsivkovskii, Frits Abildgaard, John L Markley, Svetlana LutsenkoAbstract:Wilson Disease Protein (ATP7B) is a copper-transporting P1B-type ATPase that regulates copper homeostasis and biosynthesis of copper-containing enzymes in human tissues. Inactivation of ATP7B or related ATP7A leads to severe neurodegenerative disorders, whereas their overexpression contributes to cancer cell resistance to chemotherapeutics. Copper-transporting ATPases differ from other P-type ATPases in their topology and the sequence of their nucleotide-binding domain (N-domain). To gain insight into the structural basis of ATP7B function, we have solved the structure of the ATP7B N-domain in the presence of ATP by using heteronuclear multidimensional NMR spectroscopy. The N-domain consists of a six-stranded β-sheet with two adjacent α-helical hairpins and, unexpectedly, shows higher similarity to the bacterial K+-transporting ATPase KdpB than to the mammalian Ca2+-ATPase or Na+,K+-ATPase. The common core structure of P-type ATPases is retained in the 3D fold of the N-domain; however, the nucleotide coordination environment of ATP7B within this fold is different. The residues H1069, G1099, G1101, I1102, G1149, and N1150 conserved in the P1B-ATPase subfamily contribute to ATP binding. Analysis of the frequent Disease mutation H1069Q demonstrates that this mutation does not significantly affect the structure of the N-domain but prevents tight binding of ATP. The structure of the N-domain accounts for the disruptive effects of >30 known Wilson Disease mutations. The unique features of the N-domain provide a structural basis for the development of specific inhibitors and regulators of ATP7B.