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The Right to Choose: Multiple Pathways for Activating Copper,Zinc Superoxide Dismutase

2009; Elsevier BV; Volume: 284; Issue: 37 Linguagem: Inglês

10.1074/jbc.r109.040410

1083-351X

Jeffry M. Leitch, Priscilla J. Yick, Valeria Culotta,

Alzheimer's disease research and treatments

Since the discovery of SOD1 in 1969, there have been numerous achievements made in our understanding of the enzyme's biochemical reactivity and its role in oxidative stress protection and as a genetic determinant in amyotrophic lateral sclerosis. Many recent advances have also been made in understanding the "activation" of SOD1, i.e. the process by which an inert polypeptide is converted to a mature active enzyme through post-translational modifications. To date, two such activation pathways have been identified: one requiring the CCS copper chaperone and one that works independently of CCS to insert copper and activate SOD1 through oxidation of an intramolecular disulfide. Depending on an organism's lifestyle and complexity, different eukaryotes have evolved to favor one pathway over the other. Some organisms rely solely on CCS for activating SOD1, and others can only activate SOD1 independently of CCS, whereas the majority of eukaryotes appear to have evolved to use both pathways. In this minireview, we shall highlight recent advances made in understanding the mechanisms by which the CCS-dependent and CCS-independent pathways control the activity, structure, and intracellular localization of copper,zinc superoxide dismutase, with relevance to amyotrophic lateral sclerosis and an emphasis on evolutionary biology. Since the discovery of SOD1 in 1969, there have been numerous achievements made in our understanding of the enzyme's biochemical reactivity and its role in oxidative stress protection and as a genetic determinant in amyotrophic lateral sclerosis. Many recent advances have also been made in understanding the "activation" of SOD1, i.e. the process by which an inert polypeptide is converted to a mature active enzyme through post-translational modifications. To date, two such activation pathways have been identified: one requiring the CCS copper chaperone and one that works independently of CCS to insert copper and activate SOD1 through oxidation of an intramolecular disulfide. Depending on an organism's lifestyle and complexity, different eukaryotes have evolved to favor one pathway over the other. Some organisms rely solely on CCS for activating SOD1, and others can only activate SOD1 independently of CCS, whereas the majority of eukaryotes appear to have evolved to use both pathways. In this minireview, we shall highlight recent advances made in understanding the mechanisms by which the CCS-dependent and CCS-independent pathways control the activity, structure, and intracellular localization of copper,zinc superoxide dismutase, with relevance to amyotrophic lateral sclerosis and an emphasis on evolutionary biology. In a seminal 1969 Journal of Biological Chemistry publication, McCord and Fridovich reported a novel enzymatic activity for erythrocuprein, an abundant copper-containing protein of erythrocytes (1McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar). Specifically, erythrocuprein was found to disproportionate (both oxidize and reduce) superoxide anions to oxygen and hydrogen peroxide, representing the first description of a superoxide dismutase enzyme. Erythrocuprein is now widely known as SOD1. SOD1 is highly conserved across all eukaryotic phyla and is present in all cells and tissues, where it is believed to act as a first line of defense against toxicity of superoxide anion radicals. The enzyme may also participate in cell signaling where reactive oxygen species have been invoked (2Juarez J.C. Manuia M. Burnett M.E. Betancourt O. Boivin B. Shaw D.E. Tonks N.K. Mazar A.P. Doñate F. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 7147-7152Crossref PubMed Scopus (200) Google Scholar). Although largely cytosolic, SOD1 also resides in the mitochondrial IMS, 3The abbreviations used are:IMSintermembrane spaceALSamyotrophic lateral sclerosisCu,Zn-SODcopper,zinc superoxide dismutase.where the enzyme can directly remove superoxide generated from the mitochondrial respiratory chain (3Okado-Matsumoto A. Fridovich I. J. Biol. Chem. 2001; 276: 38388-38393Abstract Full Text Full Text PDF PubMed Scopus (813) Google Scholar, 4Sturtz L.A. Diekert K. Jensen L.T. Lill R. Culotta V.C. J. Biol. Chem. 2001; 276: 38084-38089Abstract Full Text Full Text PDF PubMed Google Scholar). intermembrane space amyotrophic lateral sclerosis copper,zinc superoxide dismutase. In 1993, mutations in human SOD1 were linked to specific familial cases of ALS (5Rosen D.R. Siddique T. Patterson D. Figlewicz D. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J. Deng H.X. Rahmani Z. Krizus A. McKenna-Yased D. Cayabyav A. Gaston S. Berger R. Tanzio R. Haperin J. Herzfeldt B. Van den Bergh R. Hung W. Bird T. Deng G. Mulder D. Smyth C. Laing N. Soriano E. Pericack-Vance M. Haines J. Rouleau G. Gusella J. Horvitz H. Brown R. Nature. 1993; 362: 59-62Crossref PubMed Scopus (5564) Google Scholar, 6Deng H.X. Hentati A. Tainer J.A. Iqbal Z. Cayabyabi A. Hung W.Y. Getzoff E.D. Hu P. Herzfeld B. Roos R.P. Warner C. Deng G. Soriano E. Smyth C. Parge H. Ahmed A. Roses A.D. Hallewell R.A. Pericak-Vance M.A. Siddique T. Science. 1993; 261: 1047-1051Crossref PubMed Scopus (1360) Google Scholar). This motor neuron disease is not due to a loss of SOD enzymatic activity, but instead mutations throughout the SOD1 polypeptide result in a dominant gain of a toxic property (7Gurney M.E. Pu H. Chiu A.U. Canto M.C. Polchow C.Y. Alexander D.D. Caliendo J. Hentati A. Kwon Y. Deng H.S. Ehen W. Zhai P. Sufit R.L. Siddique T. Science. 1994; 264: 1772-1775Crossref PubMed Scopus (3491) Google Scholar). The precise nature of this toxic property is still not completely clear, and numerous reviews have been published on the topic (8Chattopadhyay M. Valentine J.S. Antioxid. Redox Signal. 2009; 11: 1603-1614Crossref PubMed Scopus (125) Google Scholar, 9Cleveland D.W. Rothstein J.D. Nat. Rev. Neurosci. 2001; 2: 806-819Crossref PubMed Scopus (1188) Google Scholar, 10Trumbull K.A. Beckman J.S. Antioxid. Redox Signal. 2009; 11: 1627-1639Crossref PubMed Scopus (58) Google Scholar, 11Rothstein J.D. Ann. Neurol. 2009; 65: S3-S9Crossref PubMed Google Scholar, 12Seetharaman S.V. Prudencio M. Karch C. Holloway S.P. Borchelt D.R. Hart P.J. Exp. Biol. Med. 2009; Google Scholar). Nevertheless, much evidence points to a role for SOD1 misfolding in disease. SOD1 is normally quite stable, due in large part to copper and zinc binding and oxidation of an intramolecular disulfide (13Roe J.A. Butler A. Scholler D.M. Valentine J.S. Marky L. Breslauer K.J. Biochemistry. 1988; 27: 950-958Crossref PubMed Scopus (109) Google Scholar, 14Forman H.J. Fridovich I. J. Biol. Chem. 1973; 248: 2645-2649Abstract Full Text PDF PubMed Google Scholar). Copper serves as the catalyst for superoxide disproportionation, whereas zinc and the disulfide participate in proper protein folding. SOD1 can be regulated at the post-translational level through copper insertion and disulfide bond formation. We refer to these maturation processes as "SOD1 activation." Currently, there are two main pathways for activating SOD1, and these shall be the focus of this minireview. In the mid-1990s, it became evident that certain copper-requiring enzymes needed an ancillary factor to acquire their metal cofactor in vivo. A so-called family of "copper chaperones" was discovered, which act to deliver copper to specific sites in the cell (15O'Halloran T.V. Culotta V.C. J. Biol. Chem. 2000; 275: 25057-25060Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar, 16Valentine J.S. Gralla E.B. Science. 1997; 278: 817-818Crossref PubMed Scopus (190) Google Scholar, 17Pufahl R.A. Singer C.P. Peariso K.L. Lin S.J. Schmidt P.J. Fahrni C.J. Culotta V.C. Penner-Hahn J.E. O'Halloran T.V. Science. 1997; 278: 853-856Crossref PubMed Scopus (595) Google Scholar). The first of these identified, "Atx1," delivers copper destined for enzymes in the secretory pathway (18Lin S.J. Pufahl R.A. Dancis A. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1997; 272: 9215-9220Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar); the second, "Cox17," helps supply mitochondrial cytochrome c oxidase with copper (19Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 14504-14509Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar); and the third, which we have named CCS for copper chaperone for SOD1, specifically inserts copper into SOD1 (20Culotta 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). The first description of yeast and human CCS appeared in the Journal of Biological Chemistry in 1997 (20Culotta 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), and since then, CCS has been found widely distributed throughout eukaryotes, expressed ubiquitously together with its partner protein, SOD1. The mechanism by which CCS activates SOD1 has been the subject of numerous reviews (21Rosenzweig A.C. O'Halloran T.V. Curr. Opin. Chem. Biol. 2000; 4: 140-147Crossref PubMed Scopus (173) Google Scholar, 22Furukawa Y. O'Halloran T.V. Antioxid. Redox Signal. 2006; 8: 847-867Crossref PubMed Scopus (109) Google Scholar, 23Culotta V.C. Yang M. O'Halloran T.V. Biochim. Biophys. Acta. 2006; 1763: 747-758Crossref PubMed Scopus (419) Google Scholar), and just a brief synopsis will be provided here. CCS consists of three protein domains (I, II, and III). The central domain II resembles SOD1 and serves to dock CCS with SOD1 (24Schmidt 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). Once the CCS-SOD1 heterodimer has been formed, copper insertion and disulfide oxidation may proceed via a CXC copper-binding motif at the C-terminal CCS domain III. In the CCS-SOD1 docked complex structure solved by Rosenzweig and co-workers (25Lamb A.L. Torres A.S. O'Halloran T.V. Rosenzweig A.C. Nature Struct. Biol. 2001; 8: 751-755Crossref PubMed Scopus (252) Google Scholar), CCS domain III Cys229 forms an intermolecular disulfide with Cys57 of SOD1, which is believed to represent the intermediate in forming the SOD1 intramolecular disulfide (see Fig. 2). The most enigmatic of the CCS domains is domain I. This domain harbors a single CXXC copper-binding motif and is a member of the well conserved Atx1 family of copper-binding structures also found in P-type copper-transporting ATPases and in numerous Atx1-like copper chaperones (17Pufahl R.A. Singer C.P. Peariso K.L. Lin S.J. Schmidt P.J. Fahrni C.J. Culotta V.C. Penner-Hahn J.E. O'Halloran T.V. Science. 1997; 278: 853-856Crossref PubMed Scopus (595) Google Scholar, 21Rosenzweig A.C. O'Halloran T.V. Curr. Opin. Chem. Biol. 2000; 4: 140-147Crossref PubMed Scopus (173) Google Scholar). The CXXC site was originally proposed to bind copper (24Schmidt 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, 26Zhu 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, 27Eisses J.F. Stasser J.P. Ralle M. Kaplan J.H. Blackburn N.J. Biochemistry. 2000; 39: 7337-7342Crossref PubMed Scopus (55) Google Scholar); however, not all CCS molecules contain this motif, including that of Drosophila (28Kirby K. Jensen L.T. Binnington J. Hilliker A.J. Ulloa J. Culotta V.C. Phillips J.P. J. Biol. Chem. 2008; 283: 35393-35401Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) and Schizosaccharomyces pombe (29Laliberté J. Whitson L.J. Beaudoin J. Holloway S.P. Hart P.J. Labbé S. J. Biol. Chem. 2004; 279: 28744-28755Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Furthermore, mutating the CXXC cysteines did not affect Saccharomyces cerevisiae CCS activity in vivo (28Kirby K. Jensen L.T. Binnington J. Hilliker A.J. Ulloa J. Culotta V.C. Phillips J.P. J. Biol. Chem. 2008; 283: 35393-35401Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Similar findings have been reported for recombinant mammalian CCS in vitro (30Stasser J.P. Siluvai G.S. Barry A.N. Blackburn N.J. Biochemistry. 2007; 46: 11845-11856Crossref PubMed Scopus (44) Google Scholar). Although the CXXC motif is not essential, domain I itself is required for CCS activity (24Schmidt 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, 29Laliberté J. Whitson L.J. Beaudoin J. Holloway S.P. Hart P.J. Labbé S. J. Biol. Chem. 2004; 279: 28744-28755Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Domain I may help CCS dock with an upstream source of copper (see Fig. 2). CCS co-localizes with SOD1 in the mitochondrial IMS and can greatly influence the partitioning of SOD1 between the mitochondria and cytosol (4Sturtz L.A. Diekert K. Jensen L.T. Lill R. Culotta V.C. J. Biol. Chem. 2001; 276: 38084-38089Abstract Full Text Full Text PDF PubMed Google Scholar, 31Field L.S. Furukawa Y. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 2003; 278: 28052-28059Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). If CCS activates SOD1 in the cytosol, SOD1 is prevented from entering mitochondria. Conversely, if CCS activates SOD1 in the mitochondria, the mature enzyme is retained in the IMS (Fig. 1). How can CCS and SOD1 enter mitochondria without a mitochondrial targeting presequence? Very recent studies by Hell and co-workers (33Reddehase S. Grumbt B. Neupert W. Hell K. J. Mol. Biol. 2009; 385: 331-338Crossref PubMed Scopus (72) Google Scholar) and Kawamata and Manfredi (34Kawamata H. Manfredi G. Hum. Mol. Genet. 2008; 17: 3303-3317Crossref PubMed Scopus (113) Google Scholar) have shown that this occurs through a disulfide relay system involving Mia40. The Mia40 system for mitochondrial import uses transient intermolecular disulfide bonds to drive the uptake of cysteine-containing proteins into the mitochondrial IMS (32Mesecke N. Terziyska N. Kozany C. Baumann F. Neupert W. Hell K. Herrmann J.M. Cell. 2005; 121: 1059-1069Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). CCS, but not SOD1, interacts with Mia40 to form transient disulfides that are subsequently relayed to SOD1, driving uptake of SOD1 into the IMS (Fig. 1) (33Reddehase S. Grumbt B. Neupert W. Hell K. J. Mol. Biol. 2009; 385: 331-338Crossref PubMed Scopus (72) Google Scholar). In the case of human SOD1, all four SOD1 cysteines are required for mitochondrial uptake of SOD1 (34Kawamata H. Manfredi G. Hum. Mol. Genet. 2008; 17: 3303-3317Crossref PubMed Scopus (113) Google Scholar). However, the CCS cysteines used in disulfide relay are still unknown and may involve either the domain III CXC or domain I CXXC motif described above. The CCS control of mitochondrial uptake of SOD1 could have important implications with regard to SOD1-linked ALS. In one hypothesis for the disease, the mitochondrial pool of mutant SOD1 causes damage to mitochondrial function and structure (11Rothstein J.D. Ann. Neurol. 2009; 65: S3-S9Crossref PubMed Google Scholar). Mitochondrial uptake of ALS mutant SOD1 is increased upon CCS overexpression (35Son M. Puttaparthi K. Kawamata H. Rajendran B. Boyer P.J. Manfredi G. Elliott J.L. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 6072-6077Crossref PubMed Scopus (126) Google Scholar); this CCS-driven uptake of mutant SOD1 results in mitochondrial respiratory defects (36Son M. Leary S.C. Romain N. Pierrel F. Winge D.R. Haller R.G. Elliott J.L. J. Biol. Chem. 2008; 283: 12267-12275Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and, as described below, an acceleration in SOD1-linked motor neuron disease (35Son M. Puttaparthi K. Kawamata H. Rajendran B. Boyer P.J. Manfredi G. Elliott J.L. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 6072-6077Crossref PubMed Scopus (126) Google Scholar, 37Son M. Fu Q. Puttaparthi K. Matthews C.M. Elliott J.L. Neurobiol. Dis. 2009; 34: 155-162Crossref PubMed Scopus (28) Google Scholar). CCS can influence SOD1 protein folding and stability. In both Drosophila and Arabidopsis, the Cu,Zn-SOD polypeptide is unstable in cells lacking CCS (28Kirby K. Jensen L.T. Binnington J. Hilliker A.J. Ulloa J. Culotta V.C. Phillips J.P. J. Biol. Chem. 2008; 283: 35393-35401Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 38Chu C.C. Lee W.C. Guo W.Y. Pan S.M. Chen L.J. Li H.M. Jinn T.L. Plant Physiol. 2005; 139: 425-436Crossref PubMed Scopus (144) Google Scholar). By comparison, mammalian wild-type SOD1 is generally stable without CCS; however, CCS effects become obvious with ALS mutant forms of human SOD1. ALS SOD1 mutants are rapidly degraded when expressed in yeast cells lacking CCS (39Carroll M.C. Outten C.E. Proescher J.B. Rosenfeld L. Watson W.H. Whitson L.J. Hart P.J. Jensen L.T. Culotta V.C. J. Biol. Chem. 2006; 281: 28648-28656Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), and in mammalian cell culture, overexpression of CCS can prevent aggregation of ALS mutant SOD1 (40Proescher J.B. Son M. Elliott J.L. Culotta V.C. Hum. Mol. Genet. 2008; 17: 1728-1737Crossref PubMed Scopus (56) Google Scholar, 41Furukawa Y. Kaneko K. Yamanaka K. O'Halloran T.V. Nukina N. J. Biol. Chem. 2008; 283: 24167-24176Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 42Cozzolino M. Pesaresi M.G. Amori I. Crosio C. Ferri A. Nencini M. Carri M.T. Antioxid. Redox Signal. 2009; 11: 1547-1558Crossref PubMed Scopus (74) Google Scholar). On the basis of these findings, one might predict a protective effect of CCS in SOD1-linked ALS. This prediction was put to the test by Elliott and co-workers when they overexpressed CCS in mouse models for SOD1-linked ALS. The results were dramatic and surprising. Rather than being protective, CCS overexpression caused a drastic acceleration in motor neuron disease (35Son M. Puttaparthi K. Kawamata H. Rajendran B. Boyer P.J. Manfredi G. Elliott J.L. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 6072-6077Crossref PubMed Scopus (126) Google Scholar, 37Son M. Fu Q. Puttaparthi K. Matthews C.M. Elliott J.L. Neurobiol. Dis. 2009; 34: 155-162Crossref PubMed Scopus (28) Google Scholar). CCS overexpression resulted in an increase in disulfide-reduced SOD1 rather than the anticipated increase in disulfide oxidation (37Son M. Fu Q. Puttaparthi K. Matthews C.M. Elliott J.L. Neurobiol. Dis. 2009; 34: 155-162Crossref PubMed Scopus (28) Google Scholar, 40Proescher J.B. Son M. Elliott J.L. Culotta V.C. Hum. Mol. Genet. 2008; 17: 1728-1737Crossref PubMed Scopus (56) Google Scholar). Because disulfide-reduced SOD1 is the substrate for mitochondrial import (31Field L.S. Furukawa Y. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 2003; 278: 28052-28059Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), mitochondrial uptake of mutant SOD1 was enhanced (35Son M. Puttaparthi K. Kawamata H. Rajendran B. Boyer P.J. Manfredi G. Elliott J.L. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 6072-6077Crossref PubMed Scopus (126) Google Scholar). Curiously, there was no obvious SOD1 aggregation in the mice (35Son M. Puttaparthi K. Kawamata H. Rajendran B. Boyer P.J. Manfredi G. Elliott J.L. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 6072-6077Crossref PubMed Scopus (126) Google Scholar, 37Son M. Fu Q. Puttaparthi K. Matthews C.M. Elliott J.L. Neurobiol. Dis. 2009; 34: 155-162Crossref PubMed Scopus (28) Google Scholar, 40Proescher J.B. Son M. Elliott J.L. Culotta V.C. Hum. Mol. Genet. 2008; 17: 1728-1737Crossref PubMed Scopus (56) Google Scholar), but recent cell culture studies have indicated that CCS overexpression can lead to increased aggregation of mitochondrial SOD1 without affecting the majority of SOD1 that resides in the cytosol (42Cozzolino M. Pesaresi M.G. Amori I. Crosio C. Ferri A. Nencini M. Carri M.T. Antioxid. Redox Signal. 2009; 11: 1547-1558Crossref PubMed Scopus (74) Google Scholar). By leading to enhanced mitochondrial uptake and mitochondrial aggregation of mutant SOD1, overexpression of CCS can accelerate motor neuron disease (Fig. 1). Together, these studies underscore the need for an intricate balance between SOD1 and CCS. SOD1 is normally present at a ≥10-fold molar excess over its copper chaperone (22Furukawa Y. O'Halloran T.V. Antioxid. Redox Signal. 2006; 8: 847-867Crossref PubMed Scopus (109) Google Scholar, 43Rothstein J.D. Dykes-Hoberg M. Corson L.B. Becker M. Cleveland D.W. Price D.L. Culotta V.C. Wong P.C. J. Neurochem. 1999; 72: 422-429Crossref PubMed Scopus (107) Google Scholar), which is normally sufficient to maintain SOD1 activity, yet a drastic change in this ratio, such as that obtained in the CCS-overexpressing mouse, can negatively impact on SOD1, particularly in the case of ALS mutants. It is possible that CCS overexpression precludes alternative methods for activating Cu,Zn-SOD such as that described below. When initially discovered in 1997, CCS was believed to represent the sole means for activating Cu,Zn-SOD in vivo. The bulk of the earlier studies were conducted on bakers' yeast SOD1, where ccs1Δ null strains were seen to express an inactive SOD1 lacking both copper and the intramolecular disulfide (20Culotta 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, 44Furukawa Y. Torres A.S. O'Halloran T.V. EMBO J. 2004; 23: 2872-2881Crossref PubMed Scopus (293) Google Scholar). However, when the CCS−/− null mouse was created by Wong et al. (45Wong P.C. Waggoner D. Subramaniam J.R. Tessarollo L. Bartnikas T.B. Culotta V.C. Price D.L. Rothstein J. Gitlin J.D. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 2886-2891Crossref PubMed Scopus (265) Google Scholar) in 2000, it was noted that a fraction of murine SOD1 retained activity. Moreover, this CCS-independent activation of mammalian SOD1 was mirrored in a yeast expression system (46Carroll 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). It became clear that CCS was not the only way to activate SOD1. Based on the vast homology (54% identity) between yeast and human SOD1, it was surprising that the two enzymes exhibited striking differences in their requirement for CCS. We observed that a single amino acid near the SOD1 C terminus was key. Pro144 in S. cerevisiae SOD1 is a leucine in human SOD1, and a single P144L (or P144S or P144Q) mutation in yeast SOD1 is sufficient to confer CCS independence to the yeast enzyme (47Leitch J.M. Jensen L.T. Bouldin S.D. Outten C.E. Hart P.J. Culotta V.C. J. Biol. Chem. 2009; 284: 21863-21871Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Our most recent studies have shown a role for Pro144 in controlling the disulfide (see below). Using Pro144 as an indicator, we have surveyed the available genome sequences for CCS-dependent SOD1 molecules. Among nearly 270 SOD1 sequences examined across all phyla of plants, animals, fungi, and protists, the vast majority of molecules are seen to contain Leu, Ala, or Val at the corresponding position 144. The occurrence of Pro144 is extremely rare and restricted to Ascomycota fungi. Even so, Pro144 is not inclusive of all Ascomycota fungi, as fission yeast SOD1 lacks Pro144 and indeed shows some CCS-independent activity (29Laliberté J. Whitson L.J. Beaudoin J. Holloway S.P. Hart P.J. Labbé S. J. Biol. Chem. 2004; 279: 28744-28755Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Like humans (46Carroll 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) and fission yeast (29Laliberté J. Whitson L.J. Beaudoin J. Holloway S.P. Hart P.J. Labbé S. J. Biol. Chem. 2004; 279: 28744-28755Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), the Cu,Zn-SOD molecules of the plant Arabidopsis thaliana (38Chu C.C. Lee W.C. Guo W.Y. Pan S.M. Chen L.J. Li H.M. Jinn T.L. Plant Physiol. 2005; 139: 425-436Crossref PubMed Scopus (144) Google Scholar), Drosophila melanogaster (28Kirby K. Jensen L.T. Binnington J. Hilliker A.J. Ulloa J. Culotta V.C. Phillips J.P. J. Biol. Chem. 2008; 283: 35393-35401Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), mice (45Wong P.C. Waggoner D. Subramaniam J.R. Tessarollo L. Bartnikas T.B. Culotta V.C. Price D.L. Rothstein J. Gitlin J.D. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 2886-2891Crossref PubMed Scopus (265) Google Scholar), and avians 4L. T. Jensen, M. Spaulding, and V. C. Culotta, unpublished data.can all be activated with or without CCS, consistent with the absence of Pro144. Even so, in all these cases, maximal SOD1 activity is obtained with CCS. CCS may represent the preferred route of enzyme activation, whereas the CCS-independent pathway acts as a backup under conditions in which CCS is limited. Although CCS is well conserved between organisms as distant as fungi and mammals, CCS is not present in all eukaryotes. Upon completion of the Caenorhabditis elegans genome, it became clear that this organism lacks an obvious CCS-encoding locus. Using a yeast expression model, we found that the two Cu,Zn-SOD molecules of C. elegans are completely CCS-independent, i.e. they can be activated only by the CCS-independent pathway and will not accept copper from CCS (48Jensen L.T. Culotta V.C. J. Biol. Chem. 2005; 280: 41373-41379Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The complete independence from CCS may not be unique to C. elegans. An inspection of available genome sequences revealed a striking pattern. Thus far, there have been no CCS-like loci identified in any organism of the Nematoda phylum or in Platyhelminthes (including pathogenic flatworms) for the 17 species with documented Cu,Zn-SODs (Table 1). Also, none can be identified in mollusks, even though 11 species contain documented Cu,Zn-SOD molecules. In arthropods, CCS is present throughout Insecta, but as of yet, none have been identified in arachnids (Table 1). By comparison, CCS molecules are found widely distributed throughout all chordates. Although it is difficult to establish an absence of CCS in organisms with incomplete genomes, it is unlikely that C. elegans is the only specie that has evolved complete independence from CCS.TABLE 1Number of putative Cu,Zn-SOD- and CCS-encoding loci that can be identified in currently available data bases for the indicated phyla and classSOD1-encoding genesaNumber of non-extracellular Cu,Zn-SOD genes found in the given taxonomic class.CCS-like locibNumber of CCS or CCS-like genes found in the given taxonomic class.NematodaChromadorea110Enoplea10PlatyhelminthesCestoda20Trematoda40MolluscaBivalvia60Gastropoda50ArthropodaArachnida40Insecta4820a Number of non-extracellular Cu,Zn-SOD genes found in the given taxonomic class.b Number of CCS or CCS-like genes found in the given taxonomic class. Open table in a new tab In an attempt to understand the mechanism of SOD1 activation without CCS, we analyzed the activation patterns for Cu,Zn-SOD molecules that are completely CCS-dependent (S. cerevisiae) and compared them with those that solely use the CCS-independent pathway (C. elegans SOD1) and with those that employ both pathways (human SOD1). Using a yeast expression system, we observed no differences in the apparent kinetics of copper activation, and the CCS-dependent and CCS-independent pathways appear to draw upon the same limited pool of copper (47Leitch J.M. Jensen L.T. Bouldin S.D. Outten C.E. Hart P.J. Culotta V.C. J. Biol. Chem. 2009; 284: 21863-21871Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). It is possible that the same upstream copper source is used for the two pathways (Fig. 2A). Although the pathways could not be distinguished at the level of copper, a striking difference was noted with the disulfide. The intramolecular disulfide in Cu,Zn-SOD1 is unusual in that the reducing environment of the cytosol tends to maintain protein thiols in the reduced state. In fact, Cu,Zn-SOD bears the only known stable disulfide in the cytosol. In elegant studies by O'Halloran and co-workers (44Furukawa Y. Torres A.S. O'Halloran T.V. EMBO J. 2004; 23: 2872-2881Crossref PubMed Scopus (293) Google Scholar), the S. cerevisiae SOD1 disulfide was shown to be oxidized by Cu-CCS, yet we recently observed that this is not necessarily true for CCS-independent SOD1 molecules. The disulfide of C. elegans Cu,Zn-SOD is retained in the oxidized state regardless of copper or CCS conditions, and the disulfide of human SOD1 is still ≈50% oxidized in the absence of CCS and copper (47Leitch J.M. Jensen L.T. Bouldin S.D. Outten C.E. Hart P.J. Culotta V.C. J. Biol. Chem. 2009; 284: 21863-21871Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). We conclude from these studies that CCS is required for those Cu,Zn-SOD molecules that have a low propensity for disulfide oxidation in the reducing environment of the cell. Thus, S. cerevisiae SOD1 is an exception rather than a rule in that its disulfide cysteines are uniquely dependent on Cu-CCS for oxidation. The key to this dependence is the aforementioned Pro144 in SOD1. The disulfide cysteines of P144S, P144L, and P144Q derivatives of S. cerevisiae SOD1 are readily oxidized in the absence of CCS and copper, similar to what is seen with human SOD1 (47Leitch J.M. Jensen L.T. Bouldin S.D. Outten C.E. Hart P.J. Culotta V.C. J. Biol. Chem. 2009; 284: 21863-21871Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). This proline lies within 5.5 Å of the disulfide Cys146 (Fig. 2B) and may place a conformational restriction on disulfide formation that is overcome only in the presence of Cu-CCS (Fig. 2A). A second important difference between the CCS-dependent and CCS-independent pathways lies in the requirement for molecular oxygen. O'Halloran and co-workers (49Brown N.M. Torres A.S. Doan P.E. O'Halloran T.V. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 5518-5523Crossref PubMed Scopus (119) Google Scholar) have demonstrated that CCS activation of SOD1 requires molecular oxygen. Surprisingly, however, there is no similar oxygen dependence with CCS-independent activation. We observed CCS-independent activation of human SOD1 even under hypoxic and anoxic conditions (47Leitch J.M. Jensen L.T. Bouldin S.D. Outten C.E. Hart P.J. Culotta V.C. J. Biol. Chem. 2009; 284: 21863-21871Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Hence, CCS-independent activation allows SOD1 activity to be maintained over a range of oxygen conditions. This can be particularly critical in tissues of multicellular organisms where oxygen tensions range from near atmospheric to hypoxic. Why have a vast number of eukaryotes evolved to use dual pathways for activating SOD1 as opposed to just one (e.g. only CCS in bakers' yeast or only the CCS-independent method in nematodes)? The answer may lie in the dual role of SOD1 in oxidative stress protection and cell signaling in higher organisms. At one extreme, the maximal SOD1 activity needed under aerobic conditions and oxidative stress can be achieved through oxygen-regulated CCS. At the opposite extreme, under hypoxic conditions, where oxidative stress is not a concern, the reactive oxygen species products and reactants of SOD can affect cell signaling processes. With low oxygen, SOD1 activity levels are titrated down as needed through a loss of CCS-mediated activation, with the residual SOD1 activity retained through the CCS-independent pathway. Cu,Zn-SOD is just now celebrating its 40th birthday. Over the decades, this enzyme has made so many benchmarks in the field of reactive oxygen biology, from being the first enzyme known to disproportionate superoxide to being a critical genetic determinant in a fatal motor neuron disease. Although originally believed to be highly active in all cells and tissues, we now know that the activity and structure of the enzyme are quite variable and can be controlled by extrinsic factors, such as oxygen and CCS. The evolutionary biology of SOD1 enzyme activation is in itself an intriguing story, where organisms exhibit the "right to choose" the optimal method for activating SOD1 to fit their complexity and lifestyle. Although a great deal is understood about CCS, the factors for CCS-independent activation of SOD1 are still unknown. It is quite likely that CCS-independent and CCS-dependent pathways use the same source of copper, such as glutathione, and that the only difference lies at the level of disulfide bond oxidation (Fig. 2A). In addition, we have only begun to scratch the surface of the role of SOD1 in cell signaling. The enzyme is not just for oxidative stress protection, and studies in the upcoming years are likely to reveal how SOD1 activation pathways work to modulate cell signals involving superoxide and hydrogen peroxide.