Glutathionylation at Cys-111 Induces Dissociation of Wild Type and FALS Mutant SOD1 Dimers

Abstract

Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) is hypothesized to cause familial amyotrophic lateral sclerosis (FALS) through structural destabilization leading to misfolding and aggregation. Considering the late onset of symptoms as well as the phenotypic variability among patients with identical SOD1 mutations, it is clear that nongenetic factor(s) impact ALS etiology and disease progression. Here we examine the effect of Cys-111 glutathionylation, a physiologically prevalent post-translational oxidative modification, on the stabilities of wild type SOD1 and two phenotypically diverse FALS mutants, A4V and I112T. Glutathionylation results in profound destabilization of SOD1WT dimers, increasing the equilibrium dissociation constant Kd to ~10−20 μM, comparable to that of the aggressive A4V mutant. SOD1A4V is further destabilized by glutathionylation, experiencing an ~30-fold increase in Kd. Dissociation kinetics of glutathionylated SOD1WT and SOD1A4V are unchanged, as measured by surface plasmon resonance, indicating that glutathionylation destabilizes these variants by decreasing association rate. In contrast, SOD1I112T has a modestly increased dissociation rate but no change in Kd when glutathionylated. Using computational structural modeling, we show that the distinct effects of glutathionylation on different SOD1 variants correspond to changes in composition of the dimer interface. Our experimental and computational results show that Cys-111 glutathionylation induces structural rearrangements that modulate stability of both wild type and FALS mutant SOD1. The distinct sensitivities of SOD1 variants to glutathionylation, a modification that acts in part as a coping mechanism for oxidative stress, suggest a novel mode by which redox regulation and aggregation propensity interact in ALS.

Cellular Redox Systems Impact the Aggregation of Cu,Zn Superoxide Dismutase Linked to Familial Amyotrophic Lateral Sclerosis

“…Because of its location in the surface of the protein, Cys 111 may interact with Cys 57 or Cys 146 and reorganize the intramolecular disulfide bond (20) and would reorient the Cu 2ϩ . As it has been described, glutathionylation of Cys 111 increases the proportion of highly fibrillogenic hSOD1 monomers (46,47), interrupting the dimer contact at the interface stereochemically and causing the dissociation. Indeed, the point mutation of Cys 111 in A4V and G93A mutant SOD1 proteins reduces the amount of aggregates (Fig.…”

Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes

“…Our hypothesis, thus, also encompasses aspects of the oxidative damage hypothesis, which purports that reactive oxygen species within highly metabolic neurons put SOD at risk for oxidative damage; it was observed for hydrogen peroxidemediated oxidative damage to active site histidine ligands, leading to copper ion release (102). Furthermore, slightly destabilized mutant proteins may be more sensitive to the effects of oxidative modification, such as glutathionylation (103), leading to their dissociation and misfolding. The destabilization hypothesis also predicts that loss of Zn (39) and ALS mutations introduced into covalently linked SOD dimers, such as those previously developed to increase serum half-life of SOD (104), would increase disease in animal models of ALS.…”

Disulfide Bond As a Switch for Copper-Zinc Superoxide Dismutase Activity in Asthma

“…C6 is well buried in the b-barrel motif and is usually inaccessible to the solvent unless the SOD b-barrel core structure is severely disrupted (42). In contrast, C111 is exposed on the protein surface near the dimer interface and may be oxidized or modified (33). The sulfur atom of cysteine can undergo different types of oxidation: (i) reversible to disulfide (S-S) or sulfenic acid (-SOH) or (ii) irreversible oxidation to sulfinic acid (-SO 2 H) or sulfonic acid (-SO 3 H) (34).…”