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

Abstract

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 (Ccs n29E ) 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 Ccs n29E 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 (ccs1⌬) yeast. The residual dSOD1 polypeptide in ccs1⌬ yeast was nevertheless active, consistent with CCS-independent activation. Stability of dSOD1 in ccs1⌬ 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.

Human superoxide dismutase 1 (hSOD1) maturation through interaction with human copper chaperone for SOD1 (hCCS)

“…These results are particularly interesting considering that some organisms lack the D1 MXCXXC copper-binding motif, including Drosophila melanogaster and Anopheles gambiae (23) and the fission yeast Saccharomyces pombe (21). Because CCS from D. melanogaster is able to activate D. melanogaster SOD1 and ySOD1 fully without exhibiting copper chaperone activity (23), it appears that the primary function of CCS is that of a disulfide isomerase. This then poses the question of how SOD1 is obtaining copper in the absence of a functional D1.…”

“…Also, human CCS-independent SOD1 maturation is known to require glutathione (19). Thus, there are at least three mechanisms for SOD1 maturation: a first mechanism whereby SOD1 is dependent on CCS for both copper transfer (via D1) and disulfide transfer (via D3), such as is observed for yeast under copper-limiting conditions (33); a second mechanism whereby SOD1 acquires copper from another source and then the disulfide from CCS (via D3), such as is likely for D. melanogaster (23); and a third mechanism comprising a completely CCS-independent system, such as is observed for Caenorhabditis elegans, which lacks CCS altogether (24), as well as in organisms in which the chaperone-dependent systems are also present (19). For organisms in which both CCS-dependent and independent mechanisms have been described, such as for humans, CCSdependent maturation is more efficient (22).…”

“…Understanding these mechanisms is of critical importance because immature forms of SOD1 are believed to be linked to the neurodegenerative disease familial amyotrophic lateral sclerosis (14)(15)(16)(17). Although chaperoneindependent maturation of SOD1 has been reported in humans (hSOD1) (18,19) and other organisms (20)(21)(22)(23), the majority of the SOD1 activation within the cell, or 100% of activation in the case of yeast SOD1 (ySOD1), is via the SOD1-specific chaperone protein, copper chaperone for SOD1 (CCS) (24)(25)(26)(27)(28). CCS also likely exhibits disulfide isomerase activity (29,30).…”

Improved functional abilities of the life-extended Drosophila mutant Methuselah are reversed at old age to below control levels

“…It is unclear why antioxidant intervention by mth mutation leads to improved sensorimotor performance during early stages of life when These results are also informative when compared to patterns of functional decline observed for another lifeextended Drosophila line that carries the human gene superoxide dismutase (SOD1), an enzyme whose function is to remove oxidants from the system (Mccord and Fridovic 1969;Parkes et al 1998;Phillips et al 2000). While broad expression of the human SOD1 gene has no effect on Drosophila longevity (Kirby et al 2008) selective expression of this gene in motorneurons extends lifespan by 40 % (Parkes et al 1998). This increased lifespan is coupled with significant improvements to complex functions that include optomotor efficiency in tracking a visual target during flight (Petrosyan et al 2012a), and longer sustained flight during early and middle stages of life (Petrosyan et al 2012b).…”

Action of protoporphyrin-IX (PP-IX) in the lifespan of <i>Drosophila melanogaster</i> deficient in endogenous antioxidants, Sod and Cat

“…In Drosophila, deficiency of cytoplasmic CuZn-Sod (Sod1) in Sod1-null mutants imparts reduced lifespan, neurodegeneration, infertility, and hypersensitivity to further oxidative stress [37][38][39]. The gene encoding the enzyme is located in chromosome 3 [40].…”