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Jonathan D. Gitlin - One of the best experts on this subject based on the ideXlab platform.
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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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biochemical characterization and intracellular localization of the Menkes disease Protein
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Yukitoshi Yamaguchi, M E Heiny, Mariko Suzuki, Jonathan D. GitlinAbstract:Menkes disease is a fatal neurodegenerative disorder of childhood due to the absence or dysfunction of a putative copper-transporting P-type ATPase encoded on the X chromosome. To elucidate the biosynthesis and subcellular localization of this Protein, polyclonal antisera were generated against a bacterial fusion Protein encoding the 4th to 6th copper-binding domains in the amino terminus of the human Menkes Protein. RNA blot analysis revealed abundant Menkes gene expression in several cell lines, and immunoblotting studies utilizing this antiserum readily detected a 178-kDa Protein in lysates from these cells. Pulse–chase studies indicate that this Protein is synthesized as a single-chain polypeptide which is modified by N-linked glycosylation to a mature endoglycosidase H-resistant form. Sucrose gradient fractionation of HeLa cell lysates followed by immunoblotting of individual fractions with antibodies to Proteins of known intracellular location identified the Menkes ATPase in fractions similar to those containing the cation-independent mannose-6-phosphate receptor. Consistent with this observation, confocal immunofluorescence studies of these same cells localized this Protein to the trans-Golgi network and a vesicular compartment with no expression in the nucleus or on the plasma membrane. Taken together, these data provide a unique model of copper transport into the secretory pathway of mammalian cells which is compatible with clinical observations in affected patients and with recent data on homologous Proteins identified in prokaryotes and yeast.
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isolation and characterization of a human liver cdna as a candidate gene for wilson disease
Biochemical and Biophysical Research Communications, 1993Co-Authors: Y Yamaguchi, M E Heiny, Jonathan D. GitlinAbstract:Abstract The putative copper and ATP-binding domains of the human Menkes disease gene were used as probes to screen a human liver cDNA library at reduced stringency. Sixty-five clones which remained positive after tertiary screening were subcloned and sequenced. One of these cDNA clones contains an open reading frame with 65% amino acid homology to the Menkes Protein. Southern blot analysis localizes this cDNA to the region of the Wilson disease locus on chromosome 13. This cDNA detects a 7.5 kB transcript which is present in human liver and cell lines devoid of the Menkes transcript and which is absent in liver from a patient with Wilson disease. These data suggest that this cDNA is a candidate gene for Wilson disease and that the Protein encoded at this locus is a member of the P-type ATPase family.
James Camakaris - One of the best experts on this subject based on the ideXlab platform.
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regulation of prion gene expression by transcription factors sp1 and metal transcription factor 1
Journal of Biological Chemistry, 2009Co-Authors: Shayne A Bellingham, Louise A Coleman, Colin L Masters, James Camakaris, Andrew F HillAbstract:Abstract Prion diseases are associated with the conformational conversion of the host-encoded cellular prion Protein into an abnormal pathogenic isoform. Reduction in prion Protein levels has potential as a therapeutic approach in treating these diseases. Key targets for this goal are factors that affect the regulation of the prion Protein gene. Recent in vivo and in vitro studies have suggested a role for prion Protein in copper homeostasis. Copper can also induce prion gene expression in rat neurons. However, the mechanism involved in this regulation remains to be determined. We hypothesized that transcription factors SP1 and metal transcription factor-1 (MTF-1) may be involved in copper-mediated regulation of human prion gene. To test the hypothesis, we utilized human fibroblasts that are deleted or overexpressing the Menkes Protein (MNK), a major mammalian copper efflux Protein. Menkes deletion fibroblasts have high intracellular copper, whereas Menkes overexpressed fibroblasts have severely depleted intracellular copper. We have utilized this system previously to demonstrate copper-dependent regulation of the Alzheimer amyloid precursor Protein. Here we demonstrate that copper depletion in MNK overexpressed fibroblasts decreases cellular prion Protein and PRNP gene levels. Conversely, expression of transcription factors SP1 and/or MTF-1 significantly increases prion Protein levels and up-regulates prion gene expression in copper-replete MNK deletion cells. Furthermore, siRNA “knockdown” of SP1 or MTF-1 in MNK deletion cells decreases prion Protein levels and down-regulates prion gene expression. These data support a novel mechanism whereby SP1 and MTF-1 act as copper-sensing transcriptional activators to regulate human prion gene expression and further support a role for the prion Protein to function in copper homeostasis. Expression of the prion Protein is a vital component for the propagation of prion diseases; thus SP1 and MTF-1 represent new targets in the development of key therapeutics toward modulating the expression of the cellular prion Protein and ultimately the prevention of prion disease.
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copper depletion down regulates expression of the alzheimer s disease amyloid β precursor Protein gene
Journal of Biological Chemistry, 2004Co-Authors: Shayne A Bellingham, Gerd Multhaup, Debomoy K. Lahiri, Bryan Maloney, Sharon La Fontaine, James CamakarisAbstract:Alzheimer's disease is characterized by the accumulation of amyloid-β peptide, which is cleaved from the amyloid-β precursor Protein (APP). Reduction in levels of the potentially toxic amyloid-β has emerged as one of the most important therapeutic goals in Alzheimer's disease. Key targets for this goal are factors that affect the regulation of the APP gene. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for APP and amyloid-β in copper homeostasis. We hypothesized that metals and in particular copper might alter APP gene expression. To test the hypothesis, we utilized human fibroblasts overexpressing the Menkes Protein (MNK), a major mammalian copper efflux Protein. MNK deletion fibroblasts have high intracellular copper, whereas MNK overexpressing fibroblasts have severely depleted intracellular copper. We demonstrate that copper depletion significantly reduced APP Protein levels and down-regulated APP gene expression. Furthermore, APP promoter deletion constructs identified the copper-regulatory region between -490 and +104 of the APP gene promoter in both basal MNK overexpressing cells and in copper-chelated MNK deletion cells. Overall these data support the hypothesis that copper can regulate APP expression and further support a role for APP to function in copper homeostasis. Copper-regulated APP expression may also provide a potential therapeutic target in Alzheimer's disease.
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copper regulated trafficking of the Menkes disease copper atpase is associated with formation of a phosphorylated catalytic intermediate
Journal of Biological Chemistry, 2002Co-Authors: Michael J Petris, Daniel Strausak, James Camakaris, Ilia Voskoboinik, Roxana M Llanos, Michael A Cater, Kathryn Smith, Byungeun Kim, Julian F. B. MercerAbstract:The Menkes Protein (MNK; ATP7A) is a copper-transporting P-type ATPase that is defective in the copper deficiency disorder, Menkes disease. MNK is localized in the trans-Golgi network and transports copper to enzymes synthesized within secretory compartments. However, in cells exposed to excessive copper, MNK traffics to the plasma membrane where it functions in copper efflux. A conserved feature of all P-type ATPases is the formation of an acyl-phosphate intermediate, which occurs as part of the catalytic cycle during cation transport. In this study we investigated the effect of mutations within conserved catalytic regions of MNK on intracellular localization and trafficking from the trans-Golgi network (TGN). Our findings suggest that mutations that block formation of the phosphorylated catalytic intermediate also prevent copper-induced relocalization of MNK from the TGN. Furthermore, mutations in the phosphatase domain, which resulted in hyperphosphorylation of MNK, caused constitutive trafficking from the TGN to the plasma membrane. A similar effect on trafficking was observed with a phosphatase mutation in the closely related copper ATPase, ATP7B, affected in Wilson disease. These findings suggest that the copper-induced trafficking of the Menkes and Wilson disease copper ATPases is associated with the phosphorylated intermediate that is formed during the catalysis of these pumps. Our findings describe a novel mechanism for regulating the subcellular location of a transport Protein involving the recognition of intermediate conformations during catalysis.
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Menkes copper translocating p type atpase atp7a biochemical and cell biology properties and role in Menkes disease
Journal of Bioenergetics and Biomembranes, 2002Co-Authors: Ilia Voskoboinik, James CamakarisAbstract:The Menkes copper-translocating P-type ATPase (ATP7A; MNK) is a ubiquitous Protein that regulates the absorption of copper in the gastrointestinal tract. Inside cells the Protein has a dual function: it delivers copper to cuproenzymes in the Golgi compartment and effluxes excess copper. The latter property is achieved through copper-dependent vesicular trafficking of the Menkes Protein to the plasma membrane of the cell. The trafficking mechanism and catalytic activity combine to facilitate absorption and intercellular transport of copper. The mechanism of catalysis and copper-dependent trafficking of the Menkes Protein are the subjects of this review. Menkes disease, a systemic copper deficiency disorder, is caused by mutations in the gene encoding the Menkes Protein. The effect of these mutations on the catalytic cycle and the cell biology of the Menkes Protein, as well as predictions of the effect of particular mutant MNKs on observed Menkes disease symptoms will also be discussed.
Julian F. B. Mercer - One of the best experts on this subject based on the ideXlab platform.
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2006. Alteration of copper physiology in mice overexpressing the human Menkes Protein ATP7A
2016Co-Authors: Roxana M Llanos, Magali Wright, A Deal, Julian F. B. MercerAbstract:tein (ATP7A) is defective in the Cu deficiency disorder Menkes disease and is an important contributor to the maintenance of physi-ological Cu homeostasis. To investigate more fully the role of ATP7A, transgenic mice expressing the human Menkes gene ATP7A from chicken -actin composite promoter (CAG) were produced. The transgenic mice expressed ATP7A in lung, heart, liver, kidney, small intestine, and brain but displayed no overt phenotype resulting from expression of the human Protein. Immunohistochemical analysis re-vealed that ATP7A was found primarily in the cardiac muscle, smooth muscle of the lung, distal tubules of the kidney, intestinal enterocytes, and patches of hepatocytes, as well as in the hippocampus, cerebel-lum, and choroid plexus of the brain. In 60-day- and 300-day-old mice, Cu concentrations were reduced in most tissues, consistent with ATP7A playing a role in Cu efflux. The reduction in Cu was most pronounced in the hearts of older T22#2 females (24%), T22#2 male
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copper metabolism of astrocytes
Frontiers in Aging Neuroscience, 2013Co-Authors: Ralf Dringen, Ivo F Scheiber, Julian F. B. MercerAbstract:This short review will summarize the current knowledge on the uptake, storage and export of copper ions by astrocytes and will address the potential roles of astrocytes in copper homeostasis in the normal and diseased brain. Astrocytes in culture efficiently accumulate copper by processes that include both the copper transporter Ctr1 and Ctr1-independent mechanisms. Exposure of astrocytes to copper induces an increase in cellular glutathione (GSH) content as well as synthesis of metallothioneins, suggesting that excess of copper is stored as complex with GSH and in metallothioneins. Furthermore, exposure of astrocytes to copper accelerates the release of GSH and of glycolytically generated lactate. Astrocytes are able to export copper and express the Menkes Protein ATP7A. This Protein undergoes reversible, copper-dependent trafficking between the trans-Golgi network and vesicular structures. The ability of astrocytes to efficiently take up, store and export copper suggests that astrocytes play a key role in the supply of neurons with copper and that astrocytes should be considered as target for therapeutic inventions that aim to correct disturbances in brain copper homeostasis.
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Kinetic analysis of the interaction of the copper chaperone Atox1 with the metal binding sites of the Menkes Protein.
Journal of Biological Chemistry, 2003Co-Authors: Daniel Strausak, Michelle K. Howie, Stephen D. Firth, Andrea Schlicksupp, Rüdiger Pipkorn, Gerd Multhaup, Julian F. B. MercerAbstract:Abstract Excess copper is effluxed from mammalian cells by the Menkes or Wilson P-type ATPases (MNK and WND, respectively). MNK and WND have six metal binding sites (MBSs) containing a CXXC motif within their N-terminal cytoplasmic region. Evidence suggests that copper is delivered to the ATPases by Atox1, one of three cytoplasmic copper chaperones. Attempts to monitor a direct Atox1-MNK interaction and to determine kinetic parameters have not been successful. Here we investigated interactions of Atox1 with wild-type and mutated pairs of the MBSs of MNK using two different methods: yeast two-hybrid analysis and real-time surface plasmon resonance (SPR). A copper-dependent interaction of Atox1 with the MBSs of MNK was observed by both approaches. Cys to Ser mutations of conserved CXXC motifs affected the binding of Atox1 underlining the essentiality of Cys residues for the copper-induced interaction. Although the yeast two-hybrid assay failed to show an interaction of Atox1 with MBS5/6, SPR analysis clearly demonstrated a copper-dependent binding with all six MBSs highlighting the power and sensitivity of SPR as compared with other, more indirect methods like the yeast two-hybrid system. Binding constants for copper-dependent chaperone-MBS interactions were determined to be 10–5-10–6 m for all the MBSs representing relatively low affinity binding events. The interaction of Atox1 with pairs of the MBSs was non-cooperative. Therefore, a functional difference of the MBSs in the MNK N terminus cannot be attributed to cooperativity effects or varying affinities of the copper chaperone Atox1 with the MBSs.
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copper regulated trafficking of the Menkes disease copper atpase is associated with formation of a phosphorylated catalytic intermediate
Journal of Biological Chemistry, 2002Co-Authors: Michael J Petris, Daniel Strausak, James Camakaris, Ilia Voskoboinik, Roxana M Llanos, Michael A Cater, Kathryn Smith, Byungeun Kim, Julian F. B. MercerAbstract:The Menkes Protein (MNK; ATP7A) is a copper-transporting P-type ATPase that is defective in the copper deficiency disorder, Menkes disease. MNK is localized in the trans-Golgi network and transports copper to enzymes synthesized within secretory compartments. However, in cells exposed to excessive copper, MNK traffics to the plasma membrane where it functions in copper efflux. A conserved feature of all P-type ATPases is the formation of an acyl-phosphate intermediate, which occurs as part of the catalytic cycle during cation transport. In this study we investigated the effect of mutations within conserved catalytic regions of MNK on intracellular localization and trafficking from the trans-Golgi network (TGN). Our findings suggest that mutations that block formation of the phosphorylated catalytic intermediate also prevent copper-induced relocalization of MNK from the TGN. Furthermore, mutations in the phosphatase domain, which resulted in hyperphosphorylation of MNK, caused constitutive trafficking from the TGN to the plasma membrane. A similar effect on trafficking was observed with a phosphatase mutation in the closely related copper ATPase, ATP7B, affected in Wilson disease. These findings suggest that the copper-induced trafficking of the Menkes and Wilson disease copper ATPases is associated with the phosphorylated intermediate that is formed during the catalysis of these pumps. Our findings describe a novel mechanism for regulating the subcellular location of a transport Protein involving the recognition of intermediate conformations during catalysis.
Aimee Payne - One of the best experts on this subject based on the ideXlab platform.
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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.
Edward J Kelly - One of the best experts on this subject based on the ideXlab platform.
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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.