The Copper Chaperone CCS Directly Interacts with Copper/Zinc Superoxide Dismutase

Abstract

Dominantly inherited mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) result in the fatal motor neuron disease familial amyotrophic lateral sclerosis (FALS). These mutations confer a gain-of-function to SOD1 with neuronal degeneration resulting from enhanced free radical generating activity of the copper present in the mutant enzyme. The delivery of copper to SOD1 is mediated through a soluble factor identified as the copper chaperone for SOD1 (CCS). Amino acid sequence alignment of SOD1 and CCS reveals a striking homology with conservation of the amino acids essential for mediating SOD1 homodimerization. Here we demonstrate that CCS and SOD1 directly interact in vitro and in vivo and that this interaction is mediated via the homologous domains in each protein. Importantly, CCS interacts not only with wild-type SOD1 but also with SOD1 containing the common missense mutations resulting in FALS. Our findings therefore reveal a common mechanism whereby different SOD1 FALS mutants may result in neuronal injury and suggest a novel therapeutic approach in patients affected by this fatal disease. Dominantly inherited mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) result in the fatal motor neuron disease familial amyotrophic lateral sclerosis (FALS). These mutations confer a gain-of-function to SOD1 with neuronal degeneration resulting from enhanced free radical generating activity of the copper present in the mutant enzyme. The delivery of copper to SOD1 is mediated through a soluble factor identified as the copper chaperone for SOD1 (CCS). Amino acid sequence alignment of SOD1 and CCS reveals a striking homology with conservation of the amino acids essential for mediating SOD1 homodimerization. Here we demonstrate that CCS and SOD1 directly interact in vitro and in vivo and that this interaction is mediated via the homologous domains in each protein. Importantly, CCS interacts not only with wild-type SOD1 but also with SOD1 containing the common missense mutations resulting in FALS. Our findings therefore reveal a common mechanism whereby different SOD1 FALS mutants may result in neuronal injury and suggest a novel therapeutic approach in patients affected by this fatal disease. copper/zinc superoxide dismutase familial amyotrophic lateral sclerosis copper chaperone for SOD1 dithiothreitol glutathioneS-transferase polymerase chain reaction phosphate-buffered saline copper(II) sulfate. Copper/zinc superoxide dismutase (SOD1)1 is a homodimeric enzyme that catalyzes the disproportionation of superoxide anions via the cyclic oxidation and reduction of a single bound copper ion per subunit (1McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar, 2Fridovich I. Annu. Rev. Biochem. 1995; 64: 97-112Crossref PubMed Scopus (2673) Google Scholar). Linkage studies have revealed that mutations in SOD1 are responsible for 10–15% of cases of the fatal motor neuron disease familial amyotrophic lateral sclerosis (FALS) (3Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. Deng H.-X. Rahmani Z. Krizus A. McKenna-Yasek D. Cayabyab A. Gaston S.M. Berger R. Tanzi R.E. Halperin J. Herzfeldt B. Van den Vergh R. Hung W.-Y. Bird T. Deng G. Mulder D.W. Smyth C. Laing N.G. Soriano E. Pericak-Vance M.A. Haines J. Rouleau G.A. Gusella J.S. Horvitz H.R. Brown R.H. Nature. 1993; 362: 59-62Crossref PubMed Scopus (5402) Google Scholar, 4Deng H.-X. Hentati A. Tainer J. Iqbal Z. Cayabyab A. Hung W.-Y. Getzoff E.D. Hu P. Herzfeldt B. Roos R.P. Warner C. Deng G. Soriano E. Smyth C. Parge H.E. Ahmed A. Roses A.D. Hallewell R.E. Pericak-Vance M.A. Siddique T. Science. 1993; 261: 1047-1051Crossref PubMed Scopus (1330) Google Scholar). 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Genet. 1996; 13: 43-47Crossref PubMed Scopus (1030) Google Scholar, 9Ho Y.S. Gargano M. Cao J. Bronson R.T. Heimler I. Hutz R.J. J. Biol. Chem. 1998; 273: 7765-7769Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Although the precise mechanisms whereby multiple mutations in SOD1, including several that involve the essential copper binding ligands, result in neuronal injury remain unclear, current evidence implicates enhanced free radical generating activity associated with the copper bound by the mutant SOD1 enzymes (10Wiedau-Pazos M. Goto J.J. Rabizadeh S. Gralla E.B. Roe J.A. Lee M.K. Valentine J.S. Bredesen D.E. Science. 1996; 271: 515-518Crossref PubMed Scopus (657) Google Scholar, 11Yim M.B. Kang J.-H. Yim H.-S. Kwak H.-S. Chock P.B. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5709-5714Crossref PubMed Scopus (427) Google Scholar, 12Beckman J.S. Carson M.S. Smith C.D. Koppenol W.H. Nature. 1993; 364: 584Crossref PubMed Scopus (784) Google Scholar). The delivery of copper to specific proteins within the cell is mediated by distinct intracellular carrier proteins termed chaperones (13Valentine J.S. Gralla E.B. Science. 1997; 278: 817-818Crossref PubMed Scopus (189) Google Scholar). Consistent with this, recent studies have identified a protein termed the copper chaperone for superoxide dismutase (CCS) as the factor responsible for copper incorporation into SOD1 (14Culotta V.C. Klomp L.W.J. Strain J. Casareno R.L.B. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar). In this study, comparison of the amino acid sequences of SOD1 and CCS revealed a striking homology between this enzyme and its putative chaperone, including residues involved in SOD1 homodimerization. This observation suggested a mechanism by which copper may be delivered to SOD1 by CCS through direct protein-protein interaction, and thus further analysis was undertaken to examine this possibility in vitro and in vivo. The results validate this concept and suggest a novel paradigm whereby the multiple FALS-associated SOD1 mutations may result in neurodegenerative disease. The coding region for full-length CCS was amplified by polymerase chain reaction (PCR) and subcloned into the BamHI and EcoRI sites of pGEX 4T-1 (Amersham Pharmacia Biotech). Similarly, the coding region for amino acids 86–274 (domains B/C) of CCS was amplified by PCR and subcloned into this expression plasmid using the same restriction sites. The coding region for amino acids 1–85 of CCS was amplified by PCR using oligonucleotides with NdeI and EcoRI sites and then subcloned into the pET 28a(+) expression plasmid (Novagen). Full-length and truncated CCS constructs subcloned into glutathione S-transferase (GST) or (His)6vectors were expressed in Escherichia coli BL21 or BL21(DE3) cells and purified as described previously (15Smith D.B. Johnson K.S. Gene (Amst.). 1988; 64: 31-40Crossref Scopus (5028) Google Scholar). GST and His tag column binding assays were performed as described (16Hsu H. Shu H.B. Pau M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1719) Google Scholar). For these assays, the CCS fusion proteins were immobilized on glutathione-agarose beads (Sigma) or His-binding resin (Novagen) and allowed to interact with cell lysate (50 μg of protein) or purified human SOD1 (Sigma) in 100 μl of PBS at 4 °C for 1.5 h. Following binding, the beads were washed extensively with PBS, and protein complexes were released by the addition of sample buffer with dithiothreitol (DTT) and heating at 100 °C for 5 min. Samples were then separated by SDS-PAGE followed by electrophoretic transfer to nitrocellulose. Membranes were blocked with 5% non-fat dry milk and following incubation with specific antisera were developed using enhanced chemiluminescence (Amersham Pharmacia Biotech) as described previously (17Yamaguchi Y. Heiny M.E. Suzuki M. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14030-14036Crossref PubMed Scopus (189) Google Scholar). The copper content of the purified CCS and SOD1 was determined utilizing bicinchoninic acid as described (18Brenner A.J. Harris E.D. Anal. Biochem. 1995; 226: 80-84Crossref PubMed Scopus (169) Google Scholar). COS-1 and HepG2 cells were obtained from the American Type Culture Collection and grown to confluence in medium with fetal calf serum as described (17Yamaguchi Y. Heiny M.E. Suzuki M. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14030-14036Crossref PubMed Scopus (189) Google Scholar). COS-1 cells were transiently transfected with pEFBOS vector containing wild-type or FALS-associated mutants as described previously (19Borchelt D.R. Lee M.K. Slunt H.S. Guarnieri M. Xu Z.-S. Wong P.C. Brown Jr., R.H. Price D.L. Sisodia S.S. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8292-8296Crossref PubMed Scopus (522) Google Scholar). Cells were lysed in PBS supplemented with 1 μm pepstatin, 2.5 μg/ml leupeptin, and 0.2 mm phenylmethylsulfonyl fluoride. After one cycle of freeze-thaw, lysates were precleared overnight with normal rabbit serum and protein A-agarose beads (Sigma). CCS antiserum was added to 1 mg of total protein, incubated with rocking at 4 °C for 1 h, and the immunoprecipitated CCS was extracted from solution by the addition of protein A-agarose beads (16Hsu H. Shu H.B. Pau M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1719) Google Scholar). The coimmunoprecipitated complex was released from the beads by the addition of sample buffer with DTT followed by heating to 100 °C for 5 min. Following SDS-PAGE and transfer to nitrocellulose, immunoblot analysis was performed as described (17Yamaguchi Y. Heiny M.E. Suzuki M. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14030-14036Crossref PubMed Scopus (189) Google Scholar). To generate CCS-specific antisera, a cDNA fragment encoding the first 85 amino acids (domain A; Fig. 1) of CCS was subcloned into pET 28a(+), expressed in E. coli BL21(DE3) cells, and the purified protein was subsequently injected into rabbits (Animal Pharm, Healdsburg, CA). Anti-SOD1 polyclonal antiserum was generated in rabbits by injection of purified human SOD1 (Sigma). For immunofluorescence, HepG2 cells were plated on glass coverslips pretreated with poly-l-lysine, 1 mg/ml, and then fixed in 4% paraformaldehyde prior to incubation with primary and secondary antibodies (17Yamaguchi Y. Heiny M.E. Suzuki M. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14030-14036Crossref PubMed Scopus (189) Google Scholar). For double-immunofluorescence studies, either a murine monoclonal anti-human SOD1 antibody (Sigma) or a sheep polyclonal anti-human catalase antibody (Biodesign) was utilized. Amino acid sequence alignment of human CCS and SOD1 revealed a region in CCS from residues 86–234 (domain B), which is 47% identical to SOD1 (Fig. 1). Importantly, the identical residues include all of the known SOD1 copper and zinc ligands except His120 as well as those amino acids at the SOD1 dimer interface (20Tainer J.A. Getzoff E.D. Richardson J.S. Richardson D.C. Nature. 1983; 306: 284-287Crossref PubMed Scopus (789) Google Scholar) and most of the amino acid residues mutated in FALS (21Siddique T. Deng H.X. Hum. Mol. Gen. 1996; 271: 1406-1409Google Scholar). The first 85 amino acid residues of CCS (domain A) contain the copper binding consensus sequence MXCXXC found in the copper transporting ATPases (22Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (414) Google Scholar, 23Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (412) Google Scholar) and the cytosolic copper chaperones ATX1 (24Lin S.J. Culotta V.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3784-3788Crossref PubMed Scopus (232) Google Scholar, 25Pufahl R. Singer P. Peariso K.L. Lin S.-J. Schmidt P. Culotta V.C. Penner-Hahn J.E. O'Halloran T.V. Science. 1997; 238: 853-856Crossref Scopus (583) Google Scholar) and HAH1 (26Klomp L.W.J. Lin S.-J. Yuan D.S. Klausner R.D. Culotta V.C. Gitlin J.D. J. Biol. Chem. 1997; 272: 9221-9226Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 27Hung I. Casareno R.L.B. LaBesse G. Matthews S. Gitlin J.D. J. Biol. Chem. 1998; 273: 1749-1754Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Analysis of the carboxyl-terminal region of CCS (domain C), which includes residues 235–274, revealed no homology to SOD1 or other known proteins but did indicate a putative peroxisomal localization sequence (AHL) at the COOH terminus (Fig. 1) (28Elgermsa Y. Tabak H.F. Biochim. Biophys. Acta. 1996; 1286: 269-283Crossref PubMed Scopus (70) Google Scholar). SOD1 is a homodimer and the striking homology between CCS and SOD1 suggested to us that these two proteins may form a heteromeric complex during copper delivery. To investigate this, column binding assays were performed utilizing a GST-CCS fusion protein. GST-CCS was immobilized on glutathione-agarose beads and mixed with either cell lysates from COS-1 cells transiently transfected with the pEFBOS vector containing the SOD1 gene or purified human SOD1 with or without the addition of CuSO4 or DTT. Following incubation, eluates were subjected to SDS-PAGE and analyzed by immunoblotting with a polyclonal antisera specific for SOD1. As can be seen in Fig. 2 A, SOD1 was readily detected following incubation of GST-CCS with cell lysates. Although variability in the amount of SOD1 recovered was observed, no consistent effect on this interaction was detected with the addition of copper or DTT. The CCS-SOD1 interaction is direct and not mediated by additional components in the cell lysate, because incubation of GST-CCS with purified SOD1 gave identical results (Fig. 2 A, lanes 6–8). To further evaluate the effect of copper on this interaction, column binding assays were performed utilizing apo- and holo-CCS and apo- and holo-SOD1. The copper status of the purified proteins was verified by bicinchoninic acid assay (18Brenner A.J. Harris E.D. Anal. Biochem. 1995; 226: 80-84Crossref PubMed Scopus (169) Google Scholar). In each case the copper status of the protein had no effect on the observed binding (data not shown), suggesting that structural elements independent of copper binding are essential for the interaction of CCS and SOD1. To further examine the specificity of CCS-SOD1 interaction and to determine which domains of CCS function in this process, column binding assays were repeated using GST or His-tagged constructs containing the indicated CCS domains or the human copper chaperone HAH1 (Fig. 2 B). Cell lysates from COS-1 cells overexpressing human SOD1 were mixed with these proteins complexed to the agarose beads. As anticipated from the homology noted above, a CCS construct containing domains B and C was found to interact with SOD1 in a manner identical to that observed previously with full-length CCS. In contrast, a CCS construct containing only domain A did not interact with SOD1, indicating that the copper-binding MXCXXC motif is not necessary for CCS-SOD1 interaction. The interaction of CCS and SOD1 observed here is highly specific, as no SOD1 was recovered following incubation of cell lysates with GST or GST-HAH1 (Fig. 2 B, lanes 1and 2). An A4V mutation in SOD1 is responsible for almost 50% of SOD1 mutations in FALS cases (21Siddique T. Deng H.X. Hum. Mol. Gen. 1996; 271: 1406-1409Google Scholar). This A4V mutant retains significant superoxide dismutase activity, indicating the presence of copper in the mutant enzyme (19Borchelt D.R. Lee M.K. Slunt H.S. Guarnieri M. Xu Z.-S. Wong P.C. Brown Jr., R.H. Price D.L. Sisodia S.S. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8292-8296Crossref PubMed Scopus (522) Google Scholar). As CCS is responsible for copper delivery to wild-type SOD1, we examined interaction between CCS and SOD1 A4V utilizing the column binding assay and COS-1 cell lysates expressing this mutant protein. These studies revealed interaction of SOD1 A4V with CCS similar to that observed for wild-type SOD1 (Fig. 2 C, lanes 2 and 3). Identical results were obtained using lysates from COS-1 cells expressing several other common SOD1 FALS mutants (19Borchelt D.R. Lee M.K. Slunt H.S. Guarnieri M. Xu Z.-S. Wong P.C. Brown Jr., R.H. Price D.L. Sisodia S.S. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8292-8296Crossref PubMed Scopus (522) Google Scholar) (data not shown). Two FALS-associated SOD1 mutants, H46R (29Carri M.T. Battistoni A. Polizio F. Desideri A. Rotilio G. FEBS Lett. 1994; 356: 314-316Crossref PubMed Scopus (78) Google Scholar) and H48Q, are devoid of copper to of essential copper and mutations result in significant motor neuron disease (21Siddique T. Deng H.X. Hum. Mol. Gen. 1996; 271: 1406-1409Google Scholar). these mutants also a similar interaction with CCS (Fig. 2 C, lanes 4 and to the possibility that this chaperone may interact with and to copper to SOD1, the enzyme readily a a common mechanism for motor neuron disease associated with different FALS mutants the of CCS interaction with SOD1 resulting in of free These in vitro revealed a direct interaction between CCS and SOD1. To examine this in we performed utilizing a rabbit polyclonal antiserum specific for domain A of CCS. with this antiserum revealed CCS as a single protein in human and HepG2 cell lysates (Fig. A, lanes 1 and 2). of lysates from HepG2 cells with this antisera also detected CCS (Fig. A, SOD1 in these same cells by immunoblotting and (Fig. A, lanes and as anticipated from the of homology in the region to generate the CCS antisera (domain A Fig. no SOD1 was detected with the CCS antisera these (Fig. A, lanes and the specificity of the CCS antisera, we performed of HepG2 cell lysates (Fig. B). Following and were analyzed by SDS-PAGE and the presence of CCS by immunoblotting (Fig. B, lanes 1 and 2). After of the CCS from this the presence of SOD1 was examined utilizing antisera (Fig. B, lanes and As can be seen from the of CCS results in the presence of SOD1 in these same This of SOD1 with CCS was specific and not the result of as with antisera for the copper chaperone HAH1 did not result in SOD1 (Fig. C, identical utilizing either SOD1 or CCS antisera in of SOD1 (Fig. C, lanes 2 and 3). In all no specific were detected following with and the to SOD1 was blocked SOD1 antisera was with purified SOD1 prior to immunoblotting (data not shown). As be anticipated the SOD1 antisera in these did with CCS the of similar using this These studies suggested that copper delivery to SOD1 is mediated via a direct interaction with CCS. studies have revealed that SOD1 is a soluble protein in the and of cells J.W. J.D. J. Cell Biol. 1988; PubMed Scopus Google Scholar, J.D. T. C. J.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). that CCS and SOD1 interact in these proteins be in the same intracellular To examine this we performed of HepG2 cells using the CCS antisera and a murine monoclonal antibody that did not with CCS (data not shown). In these CCS was found the and with a in the (Fig. a and revealed that the of CCS in these cells was identical to that of SOD1 (Fig. and prior to antibody in a of the for CCS and SOD1 within the and the consistent with the concept that these are within the cell (Fig. and analysis of CCS revealed a peroxisomal localization sequence in the terminus (Fig. and recent studies have suggested that in SOD1 is in to S. R. J. 1998; Scopus Google no was observed for either SOD1 or CCS studies were performed in these same cells utilizing a sheep anti-human catalase antibody that identified (data not shown). is essential and recent studies have revealed a complex of intracellular copper mediated by chaperones to this to specific proteins from injury (13Valentine J.S. Gralla E.B. Science. 1997; 278: 817-818Crossref PubMed Scopus (189) Google Scholar). Although the mechanism by which mutant SOD1 results in neuronal injury unclear, is that copper is of this process, as of this the of FALS SOD1 mutants (10Wiedau-Pazos M. Goto J.J. Rabizadeh S. Gralla E.B. Roe J.A. Lee M.K. Valentine J.S. Bredesen D.E. Science. 1996; 271: 515-518Crossref PubMed Scopus (657) Google Scholar, M.E. A.D. P. J. 1997; PubMed Scopus Google Scholar). Our that CCS directly interacts with FALS-associated SOD1 mutants is consistent with the results that these same mutants can copper in via this chaperone Strain J.J. Culotta V.C. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). As studies on these SOD1 mutants have revealed binding sites H. Goto J.J. A. Roe J.A. J.A. C. Bredesen D.E. Gralla E.B. Valentine J.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google findings suggest a novel paradigm by which multiple SOD1 FALS mutants may result in neuronal The delivery of copper by CCS to a protein either or to this to a is consistent with the to FALS in transgenic mice SOD1, because no CCS-SOD1 interaction and thus copper transfer A.G. Elliott J.L. Hoffman E.K. Kowall N.W. Ferrante R.J. Siwek D.F. Wilcox H.M. Flood D.G. Beal M.F. Brown Jr., R.H. Scott R.W. Snider W.D. Nat. Genet. 1996; 13: 43-47Crossref PubMed Scopus (1030) Google Scholar, 9Ho Y.S. Gargano M. Cao J. Bronson R.T. Heimler I. Hutz R.J. J. Biol. Chem. 1998; 273: 7765-7769Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). in this findings CCS in a in of neuronal injury in FALS and suggest a novel therapeutic approach this CCS-SOD1 interaction to copper delivery to the mutant enzyme. Culotta for and for Borchelt for the SOD1 wild-type and FALS expression and and for of the