Role of Disulfide Cross-Linking of Mutant SOD1 in the Formation of Inclusion-Body-Like Structures

Abstract

BackgroundPathologic aggregates of superoxide dismutase 1 (SOD1) harboring mutations linked to familial amyotrophic lateral sclerosis (fALS) have been shown to contain aberrant intermolecular disulfide cross-links. In prior studies, we observed that intermolecular bonding was not necessary in the formation of detergent- insoluble SOD1 complexes by mutant SOD1, but we were unable to assess whether this type of bonding may be important for pathologic inclusion formation. In the present study, we visually assess the formation of large inclusions by fusing mutant SOD1 to yellow fluorescent protein (YFP).Methodology/Principal FindingsExperimental constructs possessing mutations at all cysteine residues in SOD1 (sites 6, 57, 111, and 146 to F,S,Y,R or G,S,Y,R, respectively) were shown to maintain a high propensity of inclusion formation despite the inability to form disulfide cross-links. Interestingly, although aggregates form when all cysteines were mutated, double mutants of the ALS mutation C6G with an experimental mutation C111S exhibited low aggregation propensity.Conclusions/SignificanceOverall, this study is an extension of previous work demonstrating that cysteine residues in mutant SOD1 play a role in modulating aggregation and that intermolecular disulfide bonds are not required to produce large intracellular inclusion-like structures.

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

“…9, which also shows how the Trx and GSH-Grx systems participate in this process. Mutation in hSOD1 dimers exposes its conserved Cys 57 -Cys 146 disulfide on the protein surface in either one or both subunits (28,29), where it is easy to be reduced by the Trx and Grx systems into dithiol form, followed by the dimer dissociation. We can also find non-conserved disulfide bond forms, as Cys 6 -Cys 111 and Cys 146 -Cys 111 in SOD1 dimers, that are in a balance because of the Trx-GSH/ Grx redox systems.…”

Altered Thiol Chemistry in Human Amyotrophic Lateral Sclerosis-linked Mutants of Superoxide Dismutase 1

“…We hypothesize that in hSOD1 G93A, Cys-111 becomes more sensitive to redox modifications, probably because of increased solvent accessibility. This increased exposure to the solvent may lead to intermolecular thiol/disulfide exchange reactions and aggregation (15,26,38). It is also possible that structural perturbations caused by the mutation result in decreased reactivity of Cys-111 (54) (53).…”

“…Formation of insoluble aggregates containing SOD1 is a common pathophysiological characteristic of ALS (6,11). Non-native disulfide formation contributes to form and/or stabilize SOD1 aggregates that accompany progression of the disease (12)(13)(14)(15). Indeed, it has been suggested that scrambling of the disulfide and the free cysteines in SOD1 results in formation of insoluble aggregates (16).…”

“…Thiol/disulfide exchange reactions involving disulfide 57-146 and the free cysteines of hSOD1 have been proposed to contribute to aggregation and misfolding in ALS (15,16,26,(37)(38)(39). However, description of these reactions at the molecular level is still missing.…”

A structural modeling approach for the understanding of initiation and elongation of ALS-linked superoxide dismutase fibrils

“…The final interface is extended for about 1900 Å 2 , composed of 114 residues participating in the contact through 432 atoms and, as observed for the dimer of dimers, is stabilized by a large number of hydrophobic contacts in the inner region, by 16 long-time persistent hydrogen bonds and by eight salt bridges. The final model, submitted to PDBePISA [39], received the maximum CSS score of 1.0, suggesting that also the hybrid dimer of dimers could have a role in the aggregation [42].…”