Profiling of aggregation-prone motifs in alpha-synuclein and implication for targeted therapeutic development

Abstract

Background: Lewy bodies and neurites formed due to excessive accumulation of α-synuclein are the hallmarks of Parkinson’s disease. Along with PD, α-synuclein aggregates have been prominently observed in Lewy Body Dementia (LBD), Multiple System Atrophy (MSA), and in a subset of Alzheimer’s disease (AD) patients. The structures of the fibrillar region across different synucleinopathies suggest the modification in each conformation is governed by the pathological condition. The structural heterogeneity between oligomers and fibrillar forms of aggregates derived from patient samples is still elusive. The cryo-EM structures of postmortem brain-derived samples of PD, LBS, and MSA patients have revealed the polymorphic nature of well-arranged fibrillar forms with overlapping stretches. The discordance in main aggregation forming segments remains unsolved even with numerous studies employing computational tools and experimental investigations of selected short peptides Methods: We have synthesized a series of offsetting 15-mer peptides covering the entire stretches from recently reported cryo-EM structures of fibrils isolated from patients with all major synucleinopathies. The congregation patterns were experimentally validated by in-vitro assays such as Thioflavin T assay and Congo red binding assay as well as microscopic methods such as CLSM, SEM, and AFM. Results: Our study aims to identify the sticky peptide regions which can act as nucleation points. By utilizing a ThT-based aggregation assay, we have thoroughly profiled aggregation propensity and zeroed in on three aggregation hot spots, which could be the nucleation centres. We have also discovered that the aggregation propensities are highly modulated by the flanking regions and post-translational modifications particularly focusing on acetylation and carbamylation that render the lysine residue chargeless. Conclusions: We are further utilized the offsetting peptide library as a target detection tool for potential therapeutic development. The mildly aggregation-prone sequences appear to be of high interest as they can be utilized as standalone inhibitors or a potential target site for degrader development.