314 - Mechanistic studies on antioxidants that inhibits secosterol aldehyde-induced SOD1 aggregation

Abstract

SOD1 aggregation is associated with both sporadic and familial cases of amyotrophic lateral sclerosis (ALS). Recently, we showed that cholesterol secosterol aldehydes conjugates to SOD1 inducing its aggregation (Dantas et al. 2018, Redox Biol. 19,105). In this study, we discuss the effect of glutathione (GSH), N-acetyl-cysteine (NAC) and ascorbate (ASC) as inhibitors apo-SOD1 aggregation induced by secosterol aldehyde in vitro. Notably, all three antioxidants reduced SOD1 aggregation by more than 70% at a concentration of 4 mM and pH 8.4. To understand the aggregation inhibitory mechanism we analysed protein modifications by nLC-MS/MS after trypsin digestion. Inhibitory effects of GSH and NAC is apparently related to the formation of Cys-S-conjugates: GSH-conjugates were detected all Cys residues (C6, C57, C111 and C146); NAC-conjugates were detected only at C6 and C146. Moreover, NAC and ASC were able to significantly inhibit secosterol-Lys adducts located at K3, K9, K30, K122, K128 and K136. Sulfenic acid detection using dimedone revealed that secosterol aldehydes induce protein sulfenic acids, and as expected, there was a considerable reduction in their detection in the samples containing GSH and NAC. Taken together, we found that ASC, GSH and NAC may act as SOD1 aggregation inhibitors by mechanisms involving thiol-conjugation and inhibition of secosterol-Lys adduction.