Copper-Induced Translocation of the Wilson Disease Protein ATP7B Independent of Murr1/COMMD1 and Rab7
2008; Elsevier BV; Volume: 173; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2008.071134
ISSN1525-2191
AutoresKarl Heinz Weiss, Javier Carbajo-Lozoya, Sabine Tuma, Daniel Gotthardt, Jürgen Reichert, Robert Ehehalt, Wolfgang Stremmel, Joachim Füllekrug,
Tópico(s)Iron Metabolism and Disorders
ResumoWilson 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. 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. Copper is a trace element in the diet, but is required as a protein co-factor for basic cellular processes and therefore essential for all living organisms. However, too much intracellular copper is cytotoxic, leading to the formation of reactive oxygen species. In mammals, intestinal copper uptake does not seem to be regulated, and homeostasis is achieved primarily by adjusting biliary excretion of copper.1Wijmenga C Klomp LW Molecular regulation of copper excretion in the liver.Proc Nutr Soc. 2004; 63: 31-39Crossref PubMed Scopus (70) Google Scholar, 2Prohaska JR Gybina AA Intracellular copper transport in mammals.J Nutr. 2004; 134: 1003-1006Crossref PubMed Scopus (237) Google ScholarWilson disease is characterized by gradual accumulation of copper in tissues, manifested by liver disease and/or neurological symptoms.3Gitlin JD Wilson disease.Gastroenterology. 2003; 125: 1868-1877Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 4Ala A Walker AP Ashkan K Dooley JS Schilsky ML Wilson's disease.Lancet. 2007; 369: 397-408Abstract Full Text Full Text PDF PubMed Scopus (831) Google Scholar This autosomal recessive disorder of copper homeostasis in humans is caused by a functional deficiency of ATP7B, the Wilson disease protein (WDP).5Bull PC Thomas GR Rommens JM Forbes JR Cox DW The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene.Nat Genet. 1993; 5: 327-337Crossref PubMed Scopus (1686) Google Scholar, 6Tanzi RE Petrukhin K Chernov I Pellequer JL Wasco W Ross B Romano DM Parano E Pavone L Brzustowicz LM The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene.Nat Genet. 1993; 5: 344-350Crossref PubMed Scopus (1174) Google Scholar Many different mutations distributed along the whole ATP7B gene lead to Wilson disease.7Thomas GR Forbes JR Roberts EA Walshe JM Cox DW The Wilson disease gene: spectrum of mutations and their consequences.Nat Genet. 1995; 9: 210-217Crossref PubMed Scopus (487) Google Scholar, 8Ferenci P Caca K Loudianos G Mieli-Vergani G Tanner S Sternlieb I Schilsky M Cox D Berr F Diagnosis and phenotypic classification of Wilson disease.Liver Int. 2003; 23: 139-142Crossref PubMed Scopus (654) Google Scholar The WDP is critical for biliary excretion of copper9Terada K Aiba N Yang XL Iida M Nakai M Miura N Sugiyama T Biliary excretion of copper in LEC rat after introduction of copper transporting P-type ATPase, ATP7B.FEBS Lett. 1999; 448: 53-56Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar but also supplies copper ions for the ferroxidase ceruloplasmin,10Terada K Nakako T Yang X-L Iida M Aiba N Minamiya Y Nakai M Sakaki T Miura N Sugiyama T Restoration of holoceruloplasmin synthesis in LEC rat after infusion of recombinant adenovirus bearing WND cDNA.J Biol Chem. 1998; 273: 1815-1820Crossref PubMed Scopus (151) Google Scholar which is the main copper containing protein of serum.11Hellman NE Gitlin JD Ceruloplasmin metabolism and function.Annu Rev Nutr. 2002; 22: 439-458Crossref PubMed Scopus (659) Google Scholar Copper transport defects may also lead to systemic copper deficiency when ATP7A, a related intestinal P-type ATPase, is mutated.12Cox DW Moore SD Copper transporting P-type ATPases and human disease.J Bioenerg Biomembr. 2002; 34: 333-338Crossref PubMed Scopus (101) Google Scholar, 13Mercer JF Llanos RM Molecular and cellular aspects of copper transport in developing mammals.J Nutr. 2003; 133: 1481S-1484SPubMed Google Scholar, 14Lutsenko S Barnes NL Bartee MY Dmitriev OY Function and regulation of human copper-transporting ATPases.Physiol Rev. 2007; 87: 1011-1046Crossref PubMed Scopus (553) Google ScholarThe WDP is a copper-translocating ATPase highly expressed in hepatocytes. ATP7B features eight transmembrane domains forming a channel, and shows unidirectional ATP-dependent transport of cytoplasmic copper ions across lipid bilayers. In hepatocytes, translocated copper is secreted on the apical side into the bile or is transferred to the ferroxidase ceruloplasmin at a late Golgi compartment,3Gitlin JD Wilson disease.Gastroenterology. 2003; 125: 1868-1877Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 10Terada K Nakako T Yang X-L Iida M Aiba N Minamiya Y Nakai M Sakaki T Miura N Sugiyama T Restoration of holoceruloplasmin synthesis in LEC rat after infusion of recombinant adenovirus bearing WND cDNA.J Biol Chem. 1998; 273: 1815-1820Crossref PubMed Scopus (151) Google Scholar and is then secreted to the basolateral side (serum).ATP7B shows striking changes in its subcellular localization when copper concentrations are manipulated. 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280: 22222-22232Crossref PubMed Scopus (204) Google Scholar, 38Maine GN Burstein E COMMD proteins: COMMing to the scene.Cell Mol Life Sci. 2007; 64: 1997-2005Crossref PubMed Scopus (79) Google Scholar and there is evidence that the other members of this family also bind to ATP7B.31de Bie P Burstein E van De Sluis B Berger R Wijmenga C Duckett CS Klomp LWJ Several COMMD proteins interact with ATP7B; possible candidate genes for hepatic copper overload disorders with unknown etiology.Eur J Gastroenterol Hepatol. 2006; 18: A52Crossref Google Scholar Murr1 is involved in NF-κB-mediated regulation of gene transcription,39Ganesh L Burstein E Guha-Niyogi A Louder MK Mascola JR Klomp LW Wijmenga C Duckett CS Nabel GJ The gene product Murr1 restricts HIV-1 replication in resting CD4+ lymphocytes.Nature. 2003; 426: 853-857Crossref PubMed Scopus (189) Google Scholar, 40Maine GN Mao X Komarck CM Burstein E COMMD1 promotes the ubiquitination of NF-kappaB subunits through a cullin-containing ubiquitin ligase.EMBO J. 2007; 26: 436-447Crossref PubMed Scopus (207) Google Scholar which has been recently reviewed together with other possible functions of the COMMD family.38Maine GN Burstein E COMMD proteins: COMMing to the scene.Cell Mol Life Sci. 2007; 64: 1997-2005Crossref PubMed Scopus (79) Google Scholar XIAP is another protein interacting with Murr1, and enhanced degradation of XIAP mediated by copper binding sensitizes hepatocytes for apoptosis,41Mufti AR Burstein E Csomos RA Graf PCF Wilkinson JC Dick RD Challa M Son J-K Bratton SB Su GL XIAP is a copper binding protein deregulated in Wilson's disease and other copper toxicosis disorders.Mol Cell. 2006; 21: 775-785Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar providing an unexpected new angle for copper-induced cell damage.Here, we applied confocal microscopy and RNA interference to investigate the copper-induced translocation of the WDP, testing current models2Prohaska JR Gybina AA Intracellular copper transport in mammals.J Nutr. 2004; 134: 1003-1006Crossref PubMed Scopus (237) Google Scholar, 3Gitlin JD Wilson disease.Gastroenterology. 2003; 125: 1868-1877Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 33de Bie P van de Sluis B Klomp L Wijmenga C The many faces of the copper metabolism protein MURR1/COMMD1.J Hered. 2005; 96: 803-811Crossref PubMed Scopus (65) Google Scholar that hypothesize an interaction of Murr1 and ATP7B during this intracellular trafficking step. We found no evidence for a role of Murr1, suggesting that this protein is involved in a different step of copper excretion. Furthermore, we analyzed the reported relationship between ATP7B and Rab7-positive endosomes.42Harada M Kawaguchi T Kumemura H Terada K Ninomiya H Taniguchi E Hanada S Baba S Maeyama M Koga H Ueno T Furuta K Suganuma T Sugiyama T Sata M The Wilson disease protein ATP7B resides in the late endosomes with Rab7 and the Niemann-Pick C1 protein.Am J Pathol. 2005; 166: 499-510Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar Although we could document a partial co-localization between Murr1 and Rab7, the WDP was not observed in Rab7-positive endosomes, even after copper-induced translocation to cytoplasmically dispersed vesicles.Materials and MethodsAntibodiesThe antibody against ATP7B was essentially prepared as described.18Hung IH Suzuki M Yamaguchi Y Yuan DS Klausner RD Gitlin JD Biochemical characterization of the Wilson disease protein and functional expression in the yeast Saccharomyces cerevisiae.J Biol Chem. 1997; 272: 21461-21466Crossref PubMed Scopus (287) Google Scholar Oligonucleotides were designed and used to amplify the region of the Wilson protein encoding amino acids 325 to 635. This region was amplified by polymerase chain reaction (PCR) and subcloned in the pGEX-2T vector (Amersham Pharmacia Biotech, Uppsala, Sweden). Escherichia coli BL21 cells harboring the expression plasmid were grown to an optical density of 1.5 at 600 nm at 31°C and induced with isopropyl 1-thio-β-d-galactopyranoside. Cultures were harvested by centrifugation, resuspended in phosphate-buffered saline (PBS) containing 1% Triton X-100, and lysed using a high-pressure cell crusher. The supernatant was incubated with glutathione-agarose beads. Bound glutathione S-transferase (GST) fusion protein was thrombin-cleaved to obtain the ATP7B-fragment. New Zealand White rabbits were immunized with 4 × 100 mg of this recombinant protein (immunization was performed according to standard procedures by EuroGentec, Seraing, Belgium).Affinity purification of the antiserum was performed using AminoLink Plus columns as recommended by the manufacturer (Pierce, Rockford, IL). Recombinant protein was coupled to the columns using 0.1 mol/L sodium citrate, 0.05 mol/L sodium carbonate, pH 10. Remaining active sites were blocked by sodium cyanoborohydride solution (5 mol/L NaCNBH3 in 0.01 mol/L NaOH). An additional pre-elution step using elution buffer (0.2 mol/L glycine-HCl, pH 2.6) was performed to remove noncovalently bound recombinant protein. After washing the column with PBS (pH 7.2), the antiserum was loaded to the column. After repeated washing, the bound antibodies were eluted from the column using the elution buffer. Protein concentration of the eluate was determined using a mini Bradford assay. The eluted antibody solution was rebuffered in PBS and 5% bovine serum albumin using PD-10 columns. For long-term storage 50% glycerol was added to the rebuffered antibody solution. The monoclonal COMMD1/Murr1 antibody (M01, clone 2A12) was obtained from Abnova Corporation (Jhongli City, Taiwan). The goat anti-Rab7 antibody (Sc6563) was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Monoclonal anti β-actin antibody (clone AC-15) was obtained from Sigma (St. Louis, MO).Cell CultureHeLa [American Type Culture Collection (ATCC) no.: CCL-2], Ptk2 (ATCC no.: CCL-56; derived from normal kidney of Potorous tridactylis), CaCo-2 (ATCC no.: HTB-37), MDCK (ATCC no.: CCL-34) cells were cultured according to the ATCC protocols. The immortalized human keratinocyte cell line, HaCaT, and the human hepatoma cell line, HuH7, were cultured in Dulbecco's modified Eagle's medium (high glucose 4.5 g/L), supplemented with glutamine (2 mmol/L), penicillin (400 U/ml), streptomycin (50 ng/ml), and 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37°C. The phoenix-gp packaging cell line was cultured as previously described.43Schuck S Manninen A Honsho M Fullekrug J Simons K Generation of single and double knockdowns in polarized epithelial cells by retrovirus-mediated RNA interference.Proc Natl Acad Sci USA. 2004; 101: 4912-4917Crossref PubMed Scopus (82) Google ScholarExpression PlasmidsRab7-GFP,44Bucci C Thomsen P Nicoziani P McCarthy J van Deurs B Rab7: a key to lysosome biogenesis.Mol Biol Cell. 2000; 11: 467-480Crossref PubMed Scopus (788) Google Scholar GFP-Rab7-DN(T22N),44Bucci C Thomsen P Nicoziani P McCarthy J van Deurs B Rab7: a key to lysosome biogenesis.Mol Biol Cell. 2000; 11: 467-480Crossref PubMed Scopus (788) Google Scholar Rab11-GFP,45Sönnichsen B De Renzis S Nielsen E Rietdorf J Zerial M Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11.J Cell Biol. 2000; 149: 901-914Crossref PubMed Scopus (797) Google Scholar Rab9-YFP,46Barbero P Bittova L Pfeffer SR Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells.J Cell Biol. 2002; 156: 511-518Crossref PubMed Scopus (232) Google Scholar and caveolin-1-GFP47Pelkmans L Kartenbeck J Helenius A Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER.Nat Cell Biol. 2001; 3: 473-483Crossref PubMed Scopus (1041) Google Scholar were provided by Marino Zerial (MPI Molecular Cell Biology and Genetics, Dresden, Germany) and Lucas Pelkmans (ETH, Zurich, Switzerland), respectively. The T2-GFP plasmid48Storrie B White J Rottger S Stelzer EH Suganuma T Nilsson T Recycling of Golgi-resident glycosyltransferases through the ER reveals a novel pathway and provides an explanation for nocodazole-induced Golgi scattering.J Cell Biol. 1998; 143: 1505-1521Crossref PubMed Scopus (292) Google Scholar was supplied by Jamie White (EMBL, Heidelberg, Germany). The full-length ATP7B-cDNA5Bull PC Thomas GR Rommens JM Forbes JR Cox DW The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene.Nat Genet. 1993; 5: 327-337Crossref PubMed Scopus (1686) Google Scholar was provided by John R. Forbes (Department of Medical Genetics, University of Alberta, Edmonton, Canada) in a yeast vector. ATP7B-cDNA was subcloned using BamHI into pcDNA3 (Invitrogen, Carlsbad, CA), resulting in a mammalian expression vector. Generation of fluorescent protein-MURR1 fusion constructs was performed as follows: the complete coding region of human MURR1 was amplified from the total cDNA of the human CaCo-2 cells by PCR using sense primer (5′-ACGTAAGCTTACCATGGCGGCGGGCGAGCTTG-3′) and antisense primer (5′-CAC TGATCAGCCAGCCTAACGCGGATCCACGT-3′). The sense primer introduced a HindIII site and the consensus Kozak sequence ACC in front of the starter ATG, the antisense primer covered a BamHI restriction site and the stop codon. The HindIII to BamHI fragment of whole-length Murr1 cDNA was cloned upstream of green fluorescent protein (GFP) cDNA in the mammalian expression vector pEGFP-N1 (BD Biosciences Clontech, Mountain View, CA). The correct sequence was confirmed by bidirectional sequencing. Murr1-RFP was derived from the Murr1-GFP plasmid by replacing GFP (BamHI, NotI) with PCR-modified monomeric RFP.49Campbell RE Tour O Palmer AE Steinbach PA Baird GS Zacharias DA Tsien RY A monomeric red fluorescent protein.Proc Natl Acad Sci USA. 2002; 99: 7877-7882Crossref PubMed Scopus (1987) Google Scholar Murr1-GFP and Murr1-mRFP had identical localizations when co-expressed (see Supplemental Figure 1 at http://ajp.amjpathol.org).ImmunoblottingEqual amounts of protein (50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels followed by electrophoretic transfer to nitrocellulose membranes, incubated with the primary antibody and visualized using enhanced chemiluminescence detection. Immunostaining of β-actin served as a further loading control.Transfection, Copper Exposure, and ImmunofluorescenceFor transient transfection cells were grown on coverslips in a six-well plate (10 cm2/well) to a density of ∼80% and transfected using 4 μg of plasmid DNA and 10 μl of Lipofectamine 2000 reagent (Invitrogen) per well according to the manufacturer's protocol. Six hours after transfection, cells were washed in prewarmed PBS (pH 7.2) and normal culture medium was added to the cells. Copper exposure experiments were started 46 hours after transfection. Before copper exposure, cells were washed twice in prewarmed PBS (pH 7.2) to remove culture medium. Cells were then either incubated with prewarmed Dulbecco's modified Eagle's medium containing 100 μmol/L of the copper chelator bathocuproinedisulfonic acid (BCS) or 100 μmol/L copper sulfate (CuSO4) for 2 hours.After repeated washing in PBS, cells grown on coverslips were fixed with methanol (−20°C) for 3 minutes. With this fixation procedure Murr1-GFP and Murr1-RFP showed a clean vesicular distribution. Use of paraformaldehyde emphasized the cytosolic and nuclear localization of Murr1 (see Supplemental Figure 1, a and b, at http://ajp.amjpathol.org). Antibodies were incubated in PBS containing 0.1% saponin (Sigma), 0.5% gelatin (Teleostan gelatin, Sigma), and 5 mg/ml bovine serum albumin. Corresponding secondary antibodies (donkey anti-rabbit/mouse; Dianova, Hamburg, Germany) were used conjugated to Cy3 or fluorescein isothiocyanate. Coverslips were mounted in Prolong Gold antifade mounting medium (Molecular Probes, Leiden, The Netherlands) and confocal images were taken on a TCS SP2 microscope (objective: ×63 magnification, oil immersion, NA 1.32; Leica Microsystems, Wetzlar, Germany). Double-immunofluorescence images were taken sequentially, and parameters were adjusted so that all light intensities were in the recording range. Intensity of the laser beam and photo multiplier levels were adjusted for each slide and each fluorophore (Cy3, fluorescein isothiocyanate, GFP, RFP, YFP, lysotracker); line averaging was set to 4 to optimize signal to noise ratio. Images shown were derived from representative single confocal planes and arranged with Adobe Photoshop and Adobe Illustrator (Adobe, San Jose, CA).RNA InterferenceTarget sequences for human Murr1/COMMD139Ganesh L Burstein E Guha-Niyogi A Louder MK Mascola JR Klomp LW Wijmenga C Duckett CS Nabel GJ The gene product Murr1 restricts HIV-1 replication in resting CD4+ lymphocytes.Nature. 2003; 426: 853-857Crossref PubMed Scopus (189) Google Scholar and Rab750Jäger S Bucci C Tanida I Ueno T Kominami E Saftig P Eskelinen EL Role for Rab7 in maturation of late autophagic vacuoles.J Cell Sci. 2004; 117: 4837-4848Crossref PubMed Scopus (680) Google Scholar were 5′-AAGUCUAUUGCGUCUGCAGAC-3′ and 5′-CGGUUCCAGUCUCUCGGUG-3′, respectively. Oligonucleotides encoding the corresponding shRNAs were designed and cloned into pSuper as described51Brummelkamp TR Bernards R Agami R A system for stable expression of short interfering RNAs in mammalian cells.Science. 2002; 296: 550-553Crossref PubMed Scopus (3942) Google Scholar except that BglII and XhoI were used. For confirmation, plasmids were sequenced in both directions.Cloning of the retroviral plasmids, transfection of the packaging cell line, collection of pseudotyped retrovirus particles, and transduction of HuH7 cells were as published.43Schuck S Manninen A Honsho M Fullekrug J Simons K Generation of single and double knockdowns in polarized epithelial cells by retrovirus-mediated RNA interference.Proc Natl Acad Sci USA. 2004; 101: 4912-4917Crossref PubMed Scopus (82) Google Scholar Briefly, the shRNA expression cassette was transferred by subcloning into the XhoI/EcoRI site of pRVH1-puro. Cells of the human phoenix gag-pol packaging cell line were grown in 10-cm dishes to near confluence. Using Lipofecta
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