Instability of Superoxide Dismutase 1 of Drosophila in Mutants Deficient for Its Cognate Copper Chaperone

Copper,zinc superoxide dismutase (SOD1) in mammals is activated principally via a copper chaperone (CCS) and to a lesser degree by a CCS-independent pathway of unknown nature. In this study, we have characterized the requirement for CCS in activating SOD1 from Drosophila. A CCS-null mutant (Ccsn29E) of Drosophila was created and found to phenotypically resemble Drosophila SOD1-null mutants in terms of reduced adult life span, hypersensitivity to oxidative stress, and loss of cytosolic aconitase activity. However, the phenotypes of CCS-null flies were less severe, consistent with some CCS-independent activation of Drosophila SOD1 (dSOD1). Yet SOD1 activity was not detectable in Ccsn29E flies, due largely to a striking loss of SOD1 protein. In contrast, human SOD1 expressed in CCS-null flies is robustly active and rescues the deficits in adult life span and sensitivity to oxidative stress. The dependence of dSOD1 on CCS was also observed in a yeast expression system where the dSOD1 polypeptide exhibited unusual instability in CCS-null (ccs1Δ) yeast. The residual dSOD1 polypeptide in ccs1Δ yeast was nevertheless active, consistent with CCS-independent activation. Stability of dSOD1 in ccs1Δ cells was readily restored by expression of either yeast or Drosophila CCS, and this required copper insertion into the enzyme. The yeast expression system also revealed some species specificity for CCS. Yeast SOD1 exhibits preference for yeast CCS over Drosophila CCS, whereas dSOD1 is fully activated with either CCS molecule. Such variation in mechanisms of copper activation of SOD1 could reflect evolutionary responses to unique oxygen and/or copper environments faced by divergent species. Copper,zinc superoxide dismutase (SOD1) in mammals is activated principally via a copper chaperone (CCS) and to a lesser degree by a CCS-independent pathway of unknown nature. In this study, we have characterized the requirement for CCS in activating SOD1 from Drosophila. A CCS-null mutant (Ccsn29E) of Drosophila was created and found to phenotypically resemble Drosophila SOD1-null mutants in terms of reduced adult life span, hypersensitivity to oxidative stress, and loss of cytosolic aconitase activity. However, the phenotypes of CCS-null flies were less severe, consistent with some CCS-independent activation of Drosophila SOD1 (dSOD1). Yet SOD1 activity was not detectable in Ccsn29E flies, due largely to a striking loss of SOD1 protein. In contrast, human SOD1 expressed in CCS-null flies is robustly active and rescues the deficits in adult life span and sensitivity to oxidative stress. The dependence of dSOD1 on CCS was also observed in a yeast expression system where the dSOD1 polypeptide exhibited unusual instability in CCS-null (ccs1Δ) yeast. The residual dSOD1 polypeptide in ccs1Δ yeast was nevertheless active, consistent with CCS-independent activation. Stability of dSOD1 in ccs1Δ cells was readily restored by expression of either yeast or Drosophila CCS, and this required copper insertion into the enzyme. The yeast expression system also revealed some species specificity for CCS. Yeast SOD1 exhibits preference for yeast CCS over Drosophila CCS, whereas dSOD1 is fully activated with either CCS molecule. Such variation in mechanisms of copper activation of SOD1 could reflect evolutionary responses to unique oxygen and/or copper environments faced by divergent species. Dismutation of superoxide O2·¯ by cytosolic superoxide dismutase (SOD1) 3The abbreviations used are:SOD1Cu,Zn superoxide dismutaseSOD2Mn superoxide dismutasecACONcytoplasmic aconitaseCCScopper chaperone for Cu,Zn superoxide dismutasedSOD1Drosophila SOD1dCCSDrosophila CCShSOD1human SOD1mACONmitochondrial aconitaseTEMEDtetramethylethylenediamineyCCSyeast CCS6-HD6-hydroxydopamine is dependent upon the cyclic reduction and oxidation of the prosthetic transition metal, copper. The cell must strictly limit concentrations of free copper, while simultaneously ensuring efficient delivery of copper to the SOD1 apoprotein. Copper is inserted into the SOD1 apoprotein by a specific chaperone, the copper chaperone for SOD1 (CCS). Initially identified in yeast as the protein product of the LYS7 gene (1Culotta V.C. Klomp L.W. Strain J. Casareno R.L. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar), CCS molecules have been identified from a wide range of organisms ranging from fungi to various metazoans (2Field L.S. Luk E. Culotta V.C. J. Bioenerg. Biomembr. 2002; 34: 373-379Crossref PubMed Scopus (102) Google Scholar). Cu,Zn superoxide dismutase Mn superoxide dismutase cytoplasmic aconitase copper chaperone for Cu,Zn superoxide dismutase Drosophila SOD1 Drosophila CCS human SOD1 mitochondrial aconitase tetramethylethylenediamine yeast CCS 6-hydroxydopamine CCS is composed of three separate protein domains that function in concert to activate SOD1 with copper (see Fig. 1B). At the N terminus of CCS, domain I resembles the ATX1 family of copper chaperones that harbor the well conserved CXXC Cu(I)-binding motif. The central domain II of CCS has significant homology to SOD1 and is important for forming a CCS-SOD1 heterodimer-docked complex as a prerequisite to copper transfer. Finally the C-terminal domain III contains a critical CXC copper-binding site that inserts copper and oxidizes the intramolecular disulfide in SOD1 (3Culotta V.C. Yang M. O'Halloran T.V. Biochim. Biophys. Acta. 2006; 1763: 747-758Crossref PubMed Scopus (420) Google Scholar, 4Schmidt P.J. Rae T.D. Pufahl R.A. Hamma T. Strain J. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1999; 274: 23719-23725Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). In many models domain I and domain III cysteines together coordinate a copper ion (5Eisses J.F. Stasser J.P. Ralle M. Kaplan J.H. Blackburn N.J. Biochemistry. 2000; 39: 7337-7342Crossref PubMed Scopus (55) Google Scholar, 6Zhu H. Shipp E. Sanchez R.J. Liba A. Stine J.E. Hart P.J. Gralla E.B. Nersissian A.M. Valentine J.S. Biochemistry. 2000; 39: 5413-5421Crossref PubMed Scopus (53) Google Scholar), although the role of domain I cysteines in SOD1 activation in vivo is uncertain. In addition to CCS, SOD1 can be activated by a so-called CCS-independent pathway that is currently of unknown nature but requires reduced GSH (7Carroll M.C. Girouard J.B. Ulloa J.L. Subramaniam J.R. Wong P.C. Valentine J.S. Culotta V.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5964-5969Crossref PubMed Scopus (156) Google Scholar). The mode of copper activation (CCS versus CCS-independent) can vary among different organisms. For example, the SOD1 of bakers' yeast Saccharomyces cerevisiae is totally dependent on CCS for activation, whereas that of the nematode Caenorhabditis elegans only acquires copper through the CCS-independent pathway (8Jensen L.T. Culotta V.C. J. Biol. Chem. 2005; 280: 41373-41379Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In fact, C. elegans lacks an obvious CCS-encoding gene. Mammals express a CCS that is homologous to that of yeast, but mammalian SOD1 can acquire copper by either pathway (7Carroll M.C. Girouard J.B. Ulloa J.L. Subramaniam J.R. Wong P.C. Valentine J.S. Culotta V.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5964-5969Crossref PubMed Scopus (156) Google Scholar, 9Corson L.B. Strain J.J. Culotta V.C. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6361-6366Crossref PubMed Scopus (143) Google Scholar). CCS-encoding genes have been identified in a wide array of metazoans, yet the biology of CCS from nonmammalian metazoans has not been investigated. The invertebrate, Drosophila melanogaster, has provided an ideal organism in which to explore the role of SOD1 in development, survival, and aging. Mutant Drosophila lacking SOD1 survive well as embryos and larvae but experience high rates of mortality as late pupae and early adults. Surviving adults exhibit a median life span about 10! of normal, severely reduced fertility, hypersensitivity to a variety of oxidative stress conditions (10Phillips J.P. Campbell S.D. Michaud D. Charbonneau M. Hilliker A.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2761-2765Crossref PubMed Scopus (310) Google Scholar), retinal degeneration (11Phillips J.P. Tainer J.A. Getzoff E.D. Boulianne G.L. Kirby K. Hilliker A.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8574-8578Crossref PubMed Scopus (90) Google Scholar), and increased rates of spontaneous somatic and germ line mutation (12Woodruff R.C. Phillips J.P. Hilliker A.J. Genome. 2004; 47: 1029-1035Crossref PubMed Google Scholar). The high rate of early mortality in SOD1-null mutants is related to a general pattern of premature aging as reflected by the precocious onset and rapid progression of patterns of marker gene expression that typify normal senescence (13Rogina B. Helfand S.L. Biogerontology. 2000; 1: 163-169Crossref PubMed Scopus (32) Google Scholar). Clearly, SOD1 plays a critical role in the biology and life history of Drosophila, but the biochemistry of the maturation process for this critical enzyme has not been explored. Here we describe a single CCS-encoding gene from Drosophila and the biological and biochemical consequences of losing the copper chaperone for SOD1. Drosophila Stocks, Culture Conditions—The Sod1n108 and ry+5 strains are described in Ref, 10Phillips J.P. Campbell S.D. Michaud D. Charbonneau M. Hilliker A.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2761-2765Crossref PubMed Scopus (310) Google Scholar. hSOD1 was expressed using lines carrying UAS-hSod1 and the GAL4 driver line, daGAL4G32 as described (14Parkes T.L. Elia A.J. Dickinson D. Hilliker A.J. Phillips J.P. Boulianne G.L. Nat. Genet. 1998; 19: 171-174Crossref PubMed Scopus (696) Google Scholar). Stocks were maintained at 25 °C on standard cornmeal and agar medium unless otherwise stated. The copper chaperone coding region was amplified by PCR (primers CCS5 + CCS7) using DNA extracted from a Drosophila Canton S adult cDNA library (Stratagene catalog number 936603). The 930-bp PCR product was blunt-ended and subcloned into the SmaI site of pBluescriptIISK (Stratagene) to generate the vector pBCC. The transformation vector pUCC was constructed by subcloning the EcoRI-NotI fragment of pBCC into PubMed Google Scholar). Yeast strains used in this were from Gralla E.B. Valentine J.S. Culotta V.C. J. Biol. Chem. Full Text PDF PubMed Google and Culotta V.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and K. L.T. Culotta V.C. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). were at °C either in yeast medium with or in medium in Yeast Scholar). medium was with and to in Yeast expression for dSOD1 and were using cDNA for dSOD1 and pUCC for The coding for dSOD1 and were and in dSOD1 and and in The dSOD1 PCR product was with the and into K. L.T. Culotta V.C. J. Biol. Chem. Full Text Full Text PDF PubMed Google with the yeast SOD1 coding with that of dSOD1 in The PCR product was inserted into of K. L.T. Culotta V.C. J. Biol. Chem. Full Text Full Text PDF PubMed Google with an site to the and an site the the coding with that of dSOD1 and were of S. cerevisiae The of was by DNA The Yeast and have been described K. L.T. Culotta V.C. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). I mutation in was using the (Stratagene) using as a P.J. Rae T.D. Pufahl R.A. Hamma T. Strain J. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1999; 274: 23719-23725Abstract Full Text Full Text PDF PubMed Scopus (163) Google in SOD1 SOD1 activity from Drosophila, were in of of was to a an of was on a 10! The was in for by in for and in as described PubMed Scopus Google Scholar). activity was from yeast in medium to an of Yeast were by in a and of activity by using and with was as described PubMed Scopus Google that was to the SOD1 were in The was extracted with with a for SOD1 activity was by the of 6-hydroxydopamine at at °C in of PubMed Scopus Google Scholar). was by using the protein adult were in of and at were on III was by of the in 25 and J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Kirby K. Hilliker A.J. Phillips J.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of SOD1 from Drosophila, were in The were at for at and the was to a an of 10! were for and of protein was on a The protein was to and with Cu,Zn superoxide dismutase catalog number and catalog number The was used for of expressed in yeast was with cell as described and a that with from species at a (8Jensen L.T. Culotta V.C. J. Biol. Chem. 2005; 280: 41373-41379Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). an and were using the system Drosophila were 25 and on cornmeal of line were The were at 25 were to Drosophila were and on cornmeal for in were to with of or and The were at 25 °C in the and flies were as described (10Phillips J.P. Campbell S.D. Michaud D. Charbonneau M. Hilliker A.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2761-2765Crossref PubMed Scopus (310) Google Scholar). the Copper of SOD1 of Drosophila of yeast and mammalian was identified by of a from a Drosophila by in and by of a cDNA from a D. cDNA library The of the Drosophila gene a coding by of and The is from the The contains the region of high homology to SOD1 (2Field L.S. Luk E. Culotta V.C. J. Bioenerg. Biomembr. 2002; 34: 373-379Crossref PubMed Scopus (102) Google in the central domain II of the domain III CXC is the domain I CXXC copper-binding is 1B). A to molecules in the revealed that the of a CXXC in Drosophila CCS is CCS from fungi to only D. and the copper-binding site However, CCS molecules the CXC copper site in domain CCS lacks domain loss of function mutation of by an of a in the S. T. Google Scholar), was identified by of a with reduced SOD1 activity and a of phenotypes exhibited by SOD1 mutants (see of by and SOD1 activity that is and in The in Ccsn29E of region and from the of the the and of the are normal in and are by in not of SOD1 in CCS-null the only for CCS function is via role in the activation of we SOD1 activity in of and using different the with reduction and the 6-hydroxydopamine reduction A and we were to significant SOD1 activity in of CCS in to have on the activity of the mitochondrial (see Fig. by that CCS in Drosophila, as in yeast and is required for activating of hSOD1 in CCS-null Drosophila. hSOD1 expressed in Drosophila is active in the of CCS. activity of used of adult hSOD1 was expressed in and flies using the are as The UAS-hSod1 is on carrying or human SOD1 rescues flies in CCS. are as that strains are for and for the of which is to a reduction of adult life span with Fig. are carrying Ccsn29E and that hSOD1 to in the of However, of SOD1 protein revealed that the of SOD1 polypeptide in is reduced to about of normal in the loss of SOD1 activity in from the reduced of SOD1 protein in the of CCS. that the of SOD1 activity in is not of a mutation in the gene. that the of SOD1 activity in the was by a mutation in the gene this by the SOD1 in by to a a mutant that an but active of SOD1. The of the gene in to a active SOD1 apoprotein can be by the of a protein in the The in Fig. the of an active that the of SOD1 activity in is not of a mutation in the gene. are to with that the of SOD1 activity and SOD1 protein in is the of the loss of CCS and not of a mutation in the gene. of CCS the of cytosolic and mitochondrial that are of by superoxide J. Biol. Chem. Full Text PDF PubMed Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of in Drosophila are reduced in the of SOD1 and J. Kirby K. Hilliker A.J. Phillips J.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of and in that activity is by about with detectable on the activity of that at the biochemical the of is to a in SOD1. The important this to organism of CCS a the loss of CCS generate a of phenotypes by the loss of reduced adult life span with or on and hypersensitivity to oxidative stress. of phenotypes are Fig. that the of CCS early onset adult mortality with an reduction in the median adult life In contrast, the hypersensitivity to the exhibited by SOD1-null In to a SOD1 on with of SOD1 J. and of the in Drosophila of Scholar), and of SOD1-null mutants T.L. Kirby K. Phillips J.P. Hilliker A.J. Genome. 1998; PubMed Google Scholar), which that is a of SOD1 life span, the of is consistent with the in of a residual of SOD1 activity although the of by of is to median adult life span the median life span of SOD1-null Such were to have to via CCS-independent activation of (see SOD1 in CCS-null Drosophila and (14Parkes T.L. Elia A.J. Dickinson D. Hilliker A.J. Phillips J.P. Boulianne G.L. Nat. Genet. 1998; 19: 171-174Crossref PubMed Scopus (696) Google Scholar), we described the expression of human SOD1 in Drosophila in a the CCS-independent activation of hSOD1 has been described (7Carroll M.C. Girouard J.B. Ulloa J.L. Subramaniam J.R. Wong P.C. Valentine J.S. Culotta V.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5964-5969Crossref PubMed Scopus (156) Google Scholar, 9Corson L.B. Strain J.J. Culotta V.C. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6361-6366Crossref PubMed Scopus (143) Google Scholar). Here we the activation of hSOD1 in flies in the of be from this SOD1 of CCS activation, hSOD1 is active in CCS-null flies hSOD1 to the early mortality and reduced median life span of CCS-null flies the in Drosophila of a CCS-independent pathway of activation of hSOD1 while only activation of homologous CCS-independent of Drosophila SOD1 in Yeast on dSOD1 is to acquire copper of CCS. with yeast, and C. elegans SOD1 have identified a of the terminus that can CCS-independent activation (7Carroll M.C. Girouard J.B. Ulloa J.L. Subramaniam J.R. Wong P.C. Valentine J.S. Culotta V.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5964-5969Crossref PubMed Scopus (156) Google Scholar, L.T. Culotta V.C. J. Biol. Chem. 2005; 280: 41373-41379Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Yeast SOD1 contains and is of CCS-independent activation, whereas mammalian and C. elegans SOD1 with at can be activated of CCS. Drosophila SOD1 also lacks to that dSOD1 not exhibit dependence on CCS for activity. although from the of we the of a residual of SOD1 the instability of dSOD1 in the of CCS CCS-independent activation of dSOD1 that in the Drosophila to a yeast expression system to Drosophila CCS and SOD1. the of dSOD1 in the of CCS, the coding for yeast, and Drosophila SOD1 were of the S. cerevisiae SOD1 gene and for SOD1 activity and protein in versus ccs1Δ yeast. in CCS-null flies dSOD1 in ccs1Δ yeast and dSOD1 activity was to Yet of cell were the dSOD1 polypeptide was readily and CCS-independent activation of dSOD1 to with human SOD1 dSOD1 have the to be activated by the CCS-independent the less phenotypes of a CCS-null with a SOD1-null of CCS in to loss of the dSOD1 polypeptide in ccs1Δ yeast and in CCS flies is and is not observed with yeast and human SOD1 in and loss in dSOD1 can be by either yeast CCS or Drosophila CCS in the ccs1Δ yeast and of dSOD1 requires CCS and as a of yeast CCS that with SOD1 P.J. C. Culotta V.C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google to dSOD1 are by we a mutant of yeast CCS. The conserved cysteines and in domain III of CCS are for copper and disulfide oxidation in and a mutant of CCS can with SOD1 but activate the enzyme P.J. C. Culotta V.C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J.B. M. J.L. Culotta V.C. Genet. PubMed Scopus Google Scholar). in Fig. to that copper and/or disulfide oxidation are the requirement for copper, yeast cells were for copper by with the P.J. Culotta V.C. Genet. PubMed Scopus Google Scholar). of the dSOD1 polypeptide were in cells CCS from yeast or Drosophila that is and that CCS to dSOD1 by activating the enzyme through copper insertion and/or disulfide N of Drosophila Drosophila CCS lacks the copper-binding that is well conserved in CCS molecules from In fact, an of CCS molecules species that with the of Drosophila and CCS, CCS molecules identified to harbor cysteines we observed that Drosophila CCS is at activating yeast SOD1 with the homologous yeast CCS expressed from the yeast gene and CCS was as as yeast CCS in activating human SOD1 and and Drosophila SOD1 exhibited preference for CCS and activation by CCS molecules and the yeast SOD1 preference for yeast CCS reflected loss of the conserved we the of a in yeast CCS. in Fig. this mutant the to fully activate yeast SOD1. the well conserved nature of domain I CCS, cysteines are not for activating SOD1. also that the CCS and yeast SOD1 is not by the of CXXC In SOD1 molecules from organisms exhibit unique in copper activation CCS in Drosophila is by a single of a of phenotypes that are to from a SOD1 and are consistent with the that CCS to have a single function in Drosophila, which is to the activation of by copper. with the of phenotypes by SOD1 mutation or the CCS-null phenotypes are from a of CCS-independent SOD1 activity that is at or the of in and could only be in from yeast cells the of SOD1-null flies with to adult life span and oxidative stress (10Phillips J.P. Campbell S.D. Michaud D. Charbonneau M. Hilliker A.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2761-2765Crossref PubMed Scopus (310) Google Scholar), that only a of SOD1 is required for adult in the of oxidative stress, whereas the of SOD1 is in oxidative stress. is by the and of CCS on life span and the of onset and of early adult mortality in is less in whereas the hypersensitivity of to is to that of is also that the residual SOD1 activity in the of active SOD1 in or a number of cell in which the CCS-independent activation pathway is we of in of this the expression of the CCS-independent hSOD1 in which of the SOD1-null adult life span (14Parkes T.L. Elia A.J. Dickinson D. Hilliker A.J. Phillips J.P. Boulianne G.L. Nat. Genet. 1998; 19: 171-174Crossref PubMed Scopus (696) Google Scholar), that an active CCS-independent activation could be cell that could activate dSOD1 in the of CCS. Drosophila life span is to SOD1 activity in the of the CCS-independent activation pathway in could also CCS has a on adult life span to the life span reduction by SOD1 and as are consistent with the that although cells can survive normal conditions with some cells the residual of SOD1 activity in conditions of oxidative stress or increased activity is also that cell variation in copper in cell specificity in CCS-independent activation of SOD1. Drosophila SOD1 can be to the of SOD1 molecules that can copper of CCS. the only to on CCS is the SOD1 of S. is that yeast not a CCS yet the to activate of CCS. A is that the for CCS activation function in the For example, CCS activation or molecules that in general and/or Drosophila SOD1 unique in that the is in the of the copper The instability is not of the Drosophila as human SOD1 expressed in CCS-null flies polypeptide and in the yeast expression ccs1Δ loss of dSOD1 but not yeast or human SOD1 in in the mammalian SOD1 polypeptide is J.R. J. J. Cleveland D.W. Gitlin J.D. Wong P.C. Nat. 2002; PubMed Scopus Google Scholar). loss of dSOD1 in the of CCS is in of has been observed with mutants of human SOD1 with M.C. J.B. Hart P.J. L.T. Culotta J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). CCS inserts copper and oxidizes the SOD1 either or of must be important for the dSOD1 molecule. to SOD1 was to in for rapid enzyme activation by CCS the for SOD1 P.J. Culotta V.C. Genet. PubMed Scopus Google Scholar, O'Halloran T.V. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: PubMed Scopus Google Scholar). Yet Drosophila SOD1 not in the the high of enzyme activity that is required to oxidative SOD1 The of CCS-null which are in SOD1 to a oxidative stress is consistent with this is also that copper is in Drosophila, and be to copper-binding SOD1 is the of CCS molecules from harbor an the N terminus that is a well characterized site R.A. C. K. S. C. Culotta J.E. O'Halloran T.V. 1997; PubMed Scopus Google Scholar). with human CCS that the cysteines be for SOD1 activation in vivo Gitlin J.D. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), with CCS in that only the C-terminal CXC cysteines are to and the J.P. J.F. Kaplan J.H. Blackburn N.J. Biochemistry. 2005; PubMed Scopus Google Scholar, J.P. Blackburn N.J. Biochemistry. PubMed Scopus Google Scholar). observed that the is not required for activation of yeast SOD1 in copper and must via the C-terminal CXC of CCS and the must be for lacking the that in the site critical for SOD1 activity oxygen and/or copper are for CCS. the of a in Drosophila CCS copper of SOD1 of the are However, the biological consequences of in copper be by the of SOD1 with the cell in the Drosophila and for the (Ccsn29E) and strains that provided the for the Drosophila also and for in and Drosophila for with and for many with

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