The Copper Toxicosis Gene Product Murr1 Directly Interacts with the Wilson Disease Protein

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. 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. Copper is an essential micronutrient that functions as an enzymatic cofactor in diverse metabolic pathways of all aerobic organisms (1Pena M.M. Lee J. Thiele D.J. J. Nutr. 1999; 129: 1251-1260Crossref PubMed Scopus (612) Google Scholar). In vertebrates, the liver plays a critical role in copper homeostasis as both the storage site for this metal and the physiologic route for excretion through the biliary system (2Loudianos G. Gitlin J.D. Semin. Liver Dis. 2000; 20: 353-364Crossref PubMed Scopus (141) Google Scholar). In the liver, hepatocytes are responsible for the uptake and storage of copper, as well as the regulation of excretion of this metal into the bile. The molecular mechanisms determining biliary copper excretion have become clearer following identification of the genetic defect in Wilson disease, an inherited disorder resulting in hepatic copper accumulation. The Wilson disease gene encodes a copper-transporting P-type ATPase localized to the trans-Golgi network of hepatocytes and required for biliary copper excretion (3Lutsenko S. Petris M.J. J. Membr. Biol. 2003; 191: 1-12Crossref PubMed Scopus (176) Google Scholar). With an increase in the hepatocyte cytosolic copper concentration, this protein traffics to a vesicular compartment near the canalicular membrane where copper is accumulated for subsequent excretion into the bile (4Hung I.H. Suzuki M. Yamaguchi Y. Yuan D.S. Klausner R.D. Gitlin J.D. J. Biol. Chem. 1997; 272: 21461-21466Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 5Schaefer M. Hopkins R.G. Failla M.L. Gitlin J.D. Am. J. Physiol. 1999; 276: G639-G646PubMed Google Scholar, 6Roelofsen H. Wolters H. Van Luyn M.J. Miura N. Kuipers F. Vonk R.J. Gastroenterology. 2000; 119: 782-793Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 7Huster D. Hoppert M. Lutsenko S. Zinke J. Lehmann C. Mossner J. Berr F. Caca K. Gastroenterology. 2003; 124: 335-345Abstract Full Text PDF PubMed Scopus (126) Google Scholar). The precise mechanisms involved in subsequent copper movement across the canalicular membrane are unknown. Although Wilson disease is the most common disorder resulting in hepatic copper overload, a distinct form of rapidly progressive cirrhosis associated with a marked increase in hepatic copper content has been described in young infants (8Muller T. van de Sluis B. Muller W. Pearson P. Wijmenga C. Eur. J. Med. Res. 1999; 4: 293-297PubMed Google Scholar). A similar form of copper-associated cirrhosis is also observed as an autosomal recessive disorder in inbred Bedlington terriers, and studies in these animals reveal a severe impairment in biliary copper excretion (9Hultgren B.D. Stevens J.B. Hardy R.M. Am. J. Vet. Res. 1986; 47: 365-377PubMed Google Scholar). Recent molecular genetic analysis in these dogs has revealed an in-frame deletion in MURR1, a gene highly conserved in the vertebrate genome and containing an open reading frame of 190 amino acids with no known protein motifs or homologies (10van De Sluis B. Rothuizen J. Pearson P.L. van Oost B.A. Wijmenga C. Hum. Mol. Genet. 2002; 11: 165-173Crossref PubMed Scopus (310) Google Scholar). We now demonstrate that human Murr1 directly interacts with the Wilson disease protein, providing biochemical evidence in support of the proposed role of the MURR1 gene in hepatic copper toxicosis. Cloning and Protein Expression—The coding region of Murr1 was amplified by polymerase chain reaction (PCR) from human liver cDNA using oligonucleotide primers corresponding to the 5′ and 3′ sequence of the open reading frame of human Murr1 (9Hultgren B.D. Stevens J.B. Hardy R.M. Am. J. Vet. Res. 1986; 47: 365-377PubMed Google Scholar). A hemaggluttinin (HA) 1The abbreviations used are: HA, hemaggluttinin; SOD1, copper/zinc superoxide dismutase; CCS, copper chaperone for superoxide dismutase; PBS, phosphate-buffered saline; GST, glutathione S-transferase; DTT, dithiothreitol. epitope was included in the 3′-oligonucleotide, and the amplified fragment was ligated into pCDNA3.1 (Invitrogen). An HA epitope was inserted at the carboxyl terminus of human ceruloplasmin (11Hellman N.E. Kono S. Mancini G.M. Hoogeboom A.J. De Jong G.J. Gitlin J.D. J. Biol. Chem. 2002; 277: 46632-46638Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) and VDU1 (12Li Z. Wang D. Na X. Schoen S.R. Messing E.M. Wu G. Biochem. Biophys. Res. Commun. 2002; 294: 700-709Crossref PubMed Scopus (105) Google Scholar) using these same methods. The pEFBOS vector containing human copper/zinc superoxide dismutase (SOD1) was from David Borchelt. For experiments utilizing glutathione S-transferase (GST) this fragment, absent in the HA epitope, was ligated into pGEX-1 (Amersham Bioscieces). pET-32a vector (Novagen) containing amino acids 1–650 corresponding to the copper-binding amino terminus of the human Wilson disease protein as well as recombinant protein produced from this vector were from Amy Rosenzweig. Plasmids used for column binding assays including GST-Atox1, GST-CCS, and pET-32a-CCS and methods for purification of fusion proteins have been described elsewhere (13Hamza I. Schaefer M. Klomp L.W. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13363-13368Crossref PubMed Scopus (241) Google Scholar, 14Casareno R.L. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Wilson disease protein deletion constructs were amplified by PCR from the full-length cDNA and ligated into pCDNA3.1. The fidelity of all amplified fragments was confirmed by dideoxy nucleotide sequencing. Pull-down Assays—COS-7 and HepG2 cells were lysed in PBS, 0.5% Triton X-100, 0.5% Nonidet P-40, 2 mm phenylmethylsulfonyl fluoride. GST and pET fusion proteins were immobilized on glutathione-agarose beads (Sigma) or His·Bind resin (Novagen) and allowed to interact with COS cell lysate (125 μg of protein), HepG2 cell lysate (2 mg), purified recombinant amino-terminal human Wilson disease protein (7 μg), or 35S-labeled in vitro translated protein in 100 μl of PBS at 4 °C for 1.5 h as described previously (13Hamza I. Schaefer M. Klomp L.W. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13363-13368Crossref PubMed Scopus (241) Google Scholar, 14Casareno R.L. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Following binding, the beads were extensively washed in PBS, and protein complexes were released by the addition of sample buffer with dithiothreitol (DTT) and heating at 100 °C for 10 min, separated by SDS-PAGE, and analyzed by immunoblotting with specific antisera as described previously (13Hamza I. Schaefer M. Klomp L.W. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13363-13368Crossref PubMed Scopus (241) Google Scholar, 14Casareno R.L. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). In vitro translation was performed using a reticulocyte lysate transcription/translation system (Promega) in the presence of [35S]methionine and [35S]cysteine. Before interaction studies, 120 of the total reaction was analyzed by SDS-PAGE for quantitation using a PhosphorImager (Amersham Biosciences). Equivalent amounts of each protein were then utilized for interaction studies as described previoualy (13Hamza I. Schaefer M. Klomp L.W. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13363-13368Crossref PubMed Scopus (241) Google Scholar). Transfection, Coimmunoprecipitation, and Immunoblot Analysis— COS-7, HeLa, and HepG2 cells were obtained from the American Type Culture Collection and grown to confluence in medium with fetal calf serum as described (4Hung I.H. Suzuki M. Yamaguchi Y. Yuan D.S. Klausner R.D. Gitlin J.D. J. Biol. Chem. 1997; 272: 21461-21466Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 13Hamza I. Schaefer M. Klomp L.W. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13363-13368Crossref PubMed Scopus (241) Google Scholar). Cells were transfected with pCDNA3.1 containing Murr1, ceruloplasmin, or VDU1 utilizing LipofectAMINE 2000 (Invitrogen) and lysed in 50 mm Hepes, pH 7.5, 0.5% Nonidet P-40, 0.5% Triton X-100, 50 mm NaCl, 10% glycerol, 1 mm DTT, 1 mm EDTA supplemented with protease inhibitors, followed by centrifugation for 15 min at 25,000 × g at 4 °C (14Casareno R.L. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Lysates were precleared overnight with normal rabbit serum and protein A beads (Repligen) followed by immunoprecipitation with affinity-purified rabbit polyclonal antisera to HA (Zymed Laboratories Inc.), human Wilson disease protein, or Menkes disease protein (15Yamaguchi Y. Heiny M.E. Suzuki M. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14030-14035Crossref PubMed Scopus (193) Google Scholar). The coimmunoprecipitated complex was released by addition of sample buffer with DTT followed by heating to 100 °C for 5 min, separated by SDS-PAGE, and analyzed by immunoblotting with specific antisera as described previously (4Hung I.H. Suzuki M. Yamaguchi Y. Yuan D.S. Klausner R.D. Gitlin J.D. J. Biol. Chem. 1997; 272: 21461-21466Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 13Hamza I. Schaefer M. Klomp L.W. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13363-13368Crossref PubMed Scopus (241) Google Scholar, 14Casareno R.L. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 15Yamaguchi Y. Heiny M.E. Suzuki M. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14030-14035Crossref PubMed Scopus (193) Google Scholar). To generate Murr1-specific antisera, a peptide corresponding to amino acids 162–181 of human Murr1 was synthesized, conjugated to keyhole limpet hemocyanin, and injected into rabbits (Covance). The Wilson disease P-type ATPase transports copper into the secretory pathway of hepatocytes for subsequent excretion of this metal into bile (2Loudianos G. Gitlin J.D. Semin. Liver Dis. 2000; 20: 353-364Crossref PubMed Scopus (141) Google Scholar, 3Lutsenko S. Petris M.J. J. Membr. Biol. 2003; 191: 1-12Crossref PubMed Scopus (176) Google Scholar). As an inherited deletion in the MURR1 gene results in impaired biliary copper excretion and hepatic copper toxicosis in Bedlington terriers, we sought to determine whether Murr1 interacts with the Wilson disease protein. Pull-down assays were performed utilizing GST-Murr1, GST-CCS, and GST-Atox1 immobilized on glutathione-agarose beads and mixed with lysates from COS-7 cells transiently transfected with a plasmid containing the human Wilson disease cDNA. The inclusion of the copper chaperones CCS and Atox1 served as useful controls as we have previously reported specific interaction of the Wilson disease protein with Atox1 but not CCS using these same methods (13Hamza I. Schaefer M. Klomp L.W. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13363-13368Crossref PubMed Scopus (241) Google Scholar). Following incubation, eluates were subjected to SDS-PAGE and analyzed by immunoblotting with a polyclonal antisera specific for the human Wilson disease protein. As can be seen in Fig. 1A, the Wilson disease protein was readily detected following incubation of GST-Murr1 with this COS cell lysate. The specificity of this interaction was confirmed by detection of the Wilson disease protein following incubation with GST-Atox1 (Fig. 1A, lane 4) but not with GST or GST-CCS (Fig. 1A, lanes 1 and 3). To determine whether a similar interaction occurs in cells endogenously expressing the Wilson disease protein, these pull-down assays were repeated using HepG2 cells, a human hepatoblastoma-derived cell line known to express the Wilson disease protein (4Hung I.H. Suzuki M. Yamaguchi Y. Yuan D.S. Klausner R.D. Gitlin J.D. J. Biol. Chem. 1997; 272: 21461-21466Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). As observed with transfected COS cell lysates (Fig. 1B, lane 4), interaction of HepG2 lysate with GST-Murr1 resulted in readily detectable Wilson disease protein following analysis of the eluates by immunoblotting (Fig. 1B, lane 6). This interaction between Murr1 and the Wilson disease protein was not dependent upon proteins specific to these cell lysates, as a similar interaction was detected when the pull-down assay was repeated using in vitro translated Wilson disease protein (Fig. 1C, lane 4). The specificity of this interaction was demonstrated by confirming the interaction between Atox1 and the Wilson disease protein (Fig. 1C, lane 2) (13Hamza I. Schaefer M. Klomp L.W. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13363-13368Crossref PubMed Scopus (241) Google Scholar) and CCS and SOD1 (Fig. 1C, lane 7) (14Casareno R.L. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) under these same conditions. To examine this interaction between Murr1 and the Wilson disease protein in vivo, we performed coimmunoprecipitation of transfected COS-7 cells utilizing a polyclonal rabbit antisera specific for an HA epitope. Following immunoprecipitation and washing under gentle conditions, immunoprecipitates were analyzed by SDS-PAGE and the presence of Wilson disease protein confirmed by immunoblotting. Immunoprecipitation of Murr1 from lysates of cotransfected COS cells revealed abundant Wilson disease protein (Fig. 2A, lane 6). This was specific and not the result of the lysis or washing conditions as immunoprecipitation of HA-VDU1 or ceruloplasmin failed to result in detectable Wilson disease protein (Fig. 2A, lanes 3 and 4) even though immunoblotting with antisera to HA (Fig. 2A, lanes 7–12) or the Wilson disease protein (data not shown) reveals equivalent expression of these proteins in the cell lysates. The specificity of this interaction was next examined by repeating these studies in COS cells transfected with the Menkes disease P-type ATPase. This ATPase is highly homologous in structure and function to the Wilson disease protein and functions to deliver copper into the secretory pathway of cells in the gastrointestinal tract, placenta, and blood-brain barrier (3Lutsenko S. Petris M.J. J. Membr. Biol. 2003; 191: 1-12Crossref PubMed Scopus (176) Google Scholar). Although the Menkes disease protein was readily detected in transfected cell lysates (Fig. 2B, lane 6), none was detected following immunoprecipitation of Murr1 (Fig. 2B, lane 3) despite abundant coimmunoprecipitation of the Wilson disease protein (Fig. 2D, lane 2) under these same conditions. In all cases, equivalent expression of Murr1 was observed in each of the transfected cell lysates (Fig. 2C, lanes 1–3). The faint signal detecting Menkes disease protein with the Wilson antisera (Fig. 2D, lane 6) is due to cross-reactivity from overexpression of the Menkes disease protein. The failure to detect interaction between Murr1 and the Menkes disease protein in these experiments supports the concept of a specific functional interaction between Murr1 and the Wilson protein in hepatocytes and is consistent with the liver-specific phenotype of impaired copper homeostasis observed in affected Bedlington terriers (9Hultgren B.D. Stevens J.B. Hardy R.M. Am. J. Vet. Res. 1986; 47: 365-377PubMed Google Scholar, 10van De Sluis B. Rothuizen J. Pearson P.L. van Oost B.A. Wijmenga C. Hum. Mol. Genet. 2002; 11: 165-173Crossref PubMed Scopus (310) Google Scholar). The carboxyl terminus of the Wilson disease protein is predicted to reside in the and motifs that play a role in the of this protein critical to the of biliary copper excretion (3Lutsenko S. Petris M.J. J. Membr. Biol. 2003; 191: 1-12Crossref PubMed Scopus (176) Google Scholar, I. J. Protein Chem. 2002; PubMed Scopus Google Scholar). As the homologous in the Menkes disease protein has been shown to be required for the of this protein M.J. J. M. S. Hum. Mol. Genet. 1998; PubMed Scopus Google Scholar, M.J. Hum. Mol. Genet. 1999; PubMed Scopus Google we next sought to determine whether Murr1 interacts with this region of the Wilson disease protein. Wilson disease protein or a with a deletion of the were into COS cells with Murr1 followed by coimmunoprecipitation and immunoblotting as As can be seen in Fig. equivalent interaction was detected between Murr1 and both of these constructs (Fig. lanes 2 and 3) that the of the Wilson protein is not required for interaction with with antisera to HA (Fig. lanes revealed equivalent expression of Murr1 in these cell lysates. studies (13Hamza I. Schaefer M. Klomp L.W. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13363-13368Crossref PubMed Scopus (241) Google Scholar, D. C. K. B. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Lutsenko S. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, I. A. J. J. P. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, I. J. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar, D. A. G. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) have demonstrated that interaction between the cytosolic copper chaperone Atox1 and the amino-terminal region of the Wilson and Menkes disease proteins is essential for copper to the secretory As studies that Murr1 is localized to the (10van De Sluis B. Rothuizen J. Pearson P.L. van Oost B.A. Wijmenga C. Hum. Mol. Genet. 2002; 11: 165-173Crossref PubMed Scopus (310) Google we sought to determine whether this protein also interacts with Pull-down assays were performed utilizing in vitro translated Murr1 and Wilson disease protein. these conditions, no interaction was detected between Murr1 and Atox1 (Fig. lane 3) despite readily detectable Wilson disease protein in eluates from the GST-Atox1 beads (Fig. lane 4). in vitro translation revealed the interaction between Murr1 and the Wilson disease protein observed in and this was next utilized to the region of the Wilson disease protein with As full-length in vitro translated Wilson disease protein interacts with Murr1 in this pull-down assay (Fig. lane and the specificity of this interaction was confirmed by detection of Wilson disease protein following incubation with GST-Atox1 (Fig. lane 4) but not GST or GST-CCS (Fig. lanes 1 and 3). this assay was repeated utilizing in vitro translated Wilson disease protein to or both of the containing the the critical site and the ATPase (3Lutsenko S. Petris M.J. J. Membr. Biol. 2003; 191: 1-12Crossref PubMed Scopus (176) Google Scholar, I. J. Protein Chem. 2002; PubMed Scopus Google interaction with Murr1 was detected (Fig. lanes and As the specificity of this interaction was confirmed by detection of the Wilson disease proteins the amino-terminal following incubation with GST-Atox1 (Fig. lanes and but not with GST or GST-CCS (Fig. lanes and The deletion studies and suggest that Murr1 interact with the amino terminus of the Wilson disease protein. To directly examine this pull-down assays were repeated utilizing a recombinant protein containing all of the copper-binding motifs in the amino terminus of the human Wilson disease protein. of the eluates by immunoblotting demonstrated interaction with GST-Murr1 (Fig. lane As this recombinant protein was also readily detected in eluates from beads containing GST-Atox1 (Fig. lane 4) but not GST or GST-CCS (Fig. lanes 1 and 3). As was observed previously (Fig. A and and Fig. Wilson protein with Atox1 Murr1 (Fig. lanes 2 and 4). This most for Atox1 as the of Wilson protein is by the of GST-Murr1 (data not To examine this interaction with Murr1, an antisera to human Murr1 was utilized to detect this protein in cell lysates. As can be seen in Fig. Murr1 was detected as a single of in each of the cell of COS cells transfected with Murr1 revealed a corresponding to the transfected protein (Fig. lane 4). lysates from HepG2 cells (Fig. lane were utilized in a pull-down assay with a fusion protein containing the amino-terminal of the human Wilson disease protein, Murr1 was detected (Fig. lane confirming the interaction between Murr1 and the Wilson disease protein amino terminus seen This interaction was specific as no Murr1 was detected in eluates from beads containing the vector or (Fig. lanes and in the gene the human Wilson disease ATPase reveal a critical role for this protein in hepatic copper Gitlin J.D. 2003; PubMed Scopus Google Scholar). The Wilson disease protein is localized to the trans-Golgi network of hepatocytes and with copper traffics to a vesicular compartment that near the membrane M. Hopkins R.G. Failla M.L. Gitlin J.D. Am. J. Physiol. 1999; 276: G639-G646PubMed Google Scholar, 6Roelofsen H. Wolters H. Van Luyn M.J. Miura N. Kuipers F. Vonk R.J. Gastroenterology. 2000; 119: 782-793Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). the involved in this of the Wilson disease protein as well as the and of these at the membrane are not well the data in this Murr1 directly in this known biochemical pathway for biliary copper Recent studies on the molecular mechanisms of the and of have cytosolic proteins with no in are essential for the of and M. Semin. Biol. 2002; PubMed Scopus Google Scholar). As Murr1 has similar biochemical (10van De Sluis B. Rothuizen J. Pearson P.L. van Oost B.A. Wijmenga C. Hum. Mol. Genet. 2002; 11: 165-173Crossref PubMed Scopus (310) Google is to that this protein function in an to biliary copper excretion In support of a data that Murr1 form with proteins in addition to the Wilson disease protein. Y. and J. D. the biochemical findings in this a role for Murr1 in human biliary copper excretion, consistent with the metabolic in Bedlington copper toxicosis as well as the genetic analysis a deletion in the MURR1 gene in affected animals (9Hultgren B.D. Stevens J.B. Hardy R.M. Am. J. Vet. Res. 1986; 47: 365-377PubMed Google Scholar, 10van De Sluis B. Rothuizen J. Pearson P.L. van Oost B.A. Wijmenga C. Hum. Mol. Genet. 2002; 11: 165-173Crossref PubMed Scopus (310) Google Scholar). We David Amy and Amy for of and and for critical of the