Role of charged residues of the electrostatic loop of hSOD1 in promotion of aggregation: Implications for the mechanism of ALS-associated mutations under amyloidogenic conditions

Protein misfolding and amyloid formation are hallmarks of numerous diseases, including amyotrophic lateral sclerosis (ALS), in which hSOD1 aggregation is involved in pathogenesis. We used two point mutations in the electrostatic loop, G138E and T137R, to analyze charge distribution under destabilizing circumstances to gain more about how ALS-linked mutations affect SOD1 protein stability or net repulsive charge. We show that protein charge is important in the ALS disease process using bioinformatics and experiments. The MD simulation findings demonstrate that the mutant protein differs significantly from WT SOD1, which is consistent with the experimental evidence. The specific activity of the wild type was 1.61 and 1.48 times higher than that of the G138E and T137R mutants, respectively. Under amyloid induction conditions, the intensity of intrinsic and ANS fluorescence in both mutants reduced. Increasing the content of 8-sheet structures in mutants can be attributed to aggregation propensity, which was confirmed using CD polarimetry and FTIR spectroscopy. Our findings show that two ALS-related mutations promote the formation of amyloid-like aggregates at near physiological pH under destabilizing conditions, which were detected using spectroscopic probes such as Congo red and ThT fluorescence, and also further confirmation of amyloid-like species by TEM. Overall, our results provide evidence supporting the notion that negative charge changes combined with other destabilizing factors play an important role in increasing protein aggregation by reducing repulsive negative charges.

Automated summary

Protein misfolding and amyloid formation are key features in diseases like ALS. By using two point mutations in hSOD1, we observed changes in charge distribution and stability. Our findings suggest that protein charge plays a role in ALS and the mutated protein differs significantly from the wild type. Moreover, the mutants showed an increase in amyloid-like aggregates under destabilizing conditions.