Activation of Cu,Zn-Superoxide Dismutase in the Absence of Oxygen and the Copper Chaperone CCS

Abstract

Eukaryotic Cu,Zn-superoxide dismutases (SOD1s) are generally thought to acquire the essential copper cofactor and intramolecular disulfide bond through the action of the CCS copper chaperone. However, several metazoan SOD1s have been shown to acquire activity in vivo in the absence of CCS, and the Cu,Zn-SOD from Caenorhabditis elegans has evolved complete independence from CCS. To investigate SOD1 activation in the absence of CCS, we compared and contrasted the CCSindependent activation of C. elegans and human SOD1 to the strict CCS-dependent activation of Saccharomyces cerevisiae SOD1. Using a yeast expression system, both pathways were seen to acquire copper derived from cell surface transporters and compete for the same intracellular pool of copper. Like CCS, CCS-independent activation occurs rapidly with a preexisting pool of apo-SOD1 without the need for new protein synthesis. The two pathways, however, strongly diverge when assayed for the SOD1 disulfide. SOD1 molecules that are activated without CCS exhibit disulfide oxidation in vivo without oxygen and under copper-depleted conditions. The strict requirement for copper, oxygen, and CCS in disulfide bond oxidation appears exclusive to yeast SOD1, and we find that a unique proline at position 144 in yeast SOD1 is responsible for this disulfide effect. CCS-dependent and -independent pathways also exhibit differential requirements for molecular oxygen. CCS activation of SOD1 requires oxygen, whereas the CCS-independent pathway is able to activate SOD1s even under anaerobic conditions. In this manner, Cu,Zn-SOD from metazoans may retain activity over a wide range of physiological oxygen tensions.

Copper Chaperone-Dependent and -Independent Activation of Three Copper-Zinc Superoxide Dismutase Homologs Localized in Different Cellular Compartments in Arabidopsis

“…When these residues were replaced by dual Pro residues in ySOD1, CCS-independent activities for both hSOD1 and wSod-1 were inhibited. A recent study further confirmed that the Pro at residue 144 but not 142 restricted ySOD1 disulfide formation in the absence of CCS, which played a key role in blocking CCS-independent activation (Leitch et al, 2009a). The existence of additional essential factors in this pathway remains to be determined.…”

“…However, further investigation revealed significant differences between the two orthologs. The C-terminal amino acid residues 142P/144P of ySOD1 prevented its activation by the alternative pathway (Carroll et al, 2004), and this effect is mediated primarily by 144P (Leitch et al, 2009a); when 144P was mutated to 144L, this ySOD1 variant showed clear activity in the absence of CCS. For CSD2, the amino acid residues corresponding to 142P/144P of ySOD1 are 143G/145L (Fig.…”

“…For CSD2, the amino acid residues corresponding to 142P/144P of ySOD1 are 143G/145L (Fig. 4A), which are typical for a CSD protein capable of being activated by the CCS-independent pathway (Carroll et al, 2004;Leitch et al, 2009a). The CSD2 protein can be activated by this pathway because the DTP-CSD2 (cytoplasmlocalized CSD2) was active in Atccs (Fig.…”

Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

“…Ccs1 is known as the sole cupper provider for Sod1p in yeast [20], but absence of CCS1 gene clearly generates more damaging conditions than absence of SOD1. Consistent with this observation, ccs1D mutants were previously shown to have more oxidative damages than sod1D and sod2D mutants [21].…”

Assessment of chronological lifespan dependent molecular damages in yeast lacking mitochondrial antioxidant genes

“…Therefore, CCS1 seems to be one of the most important mitochondrial antioxidant genes in the chronological aging process. However, Ccs1 is not strictly necessary for Sod1p function since Sod1p can supply its copper in a Ccs1-independent manner [31]. Thus, the importance of CCS1 gene in aging could be because of its roles in providing copper to other proteins such as Cox11 rather than Sod1 [32].…”