Abstract
Aggregation of α-synuclein (αSyn) into proteinaceous deposits is a pathological hallmark of a range of neurodegenerative diseases including Parkinson’s disease (PD). Numerous lines of evidence indicate that the accumulation of toxic oligomeric and prefibrillar αSyn species may underpin the cellular toxicity and spread of pathology between cells. Therefore, aggregation of αSyn is considered a priority target for drug development, as aggregation inhibitors are expected to reduce αSyn toxicity and serve as therapeutic agents. Here, we used the budding yeast S. cerevisiae as a platform for the identification of short peptides that inhibit αSyn aggregation and toxicity. A library consisting of approximately one million peptide variants was utilized in two high-throughput screening approaches for isolation of library representatives that reduce αSyn-associated toxicity and aggregation. Seven peptides were isolated that were able to suppress specifically αSyn toxicity and aggregation in living cells. Expression of the peptides in yeast reduced the accumulation of αSyn-induced reactive oxygen species and increased cell viability. Next, the peptides were chemically synthesized and probed for their ability to modulate αSyn aggregation in vitro. Two synthetic peptides, K84s and K102s, of 25 and 19 amino acids, respectively, significantly inhibited αSyn oligomerization and aggregation at sub-stoichiometric molar ratios. Importantly, K84s reduced αSyn aggregation in human cells. These peptides represent promising αSyn aggregation antagonists for the development of future therapeutic interventions.
Highlights
Protein misfolding and aggregation is a hallmark event in a growing number of human diseases, including Parkinson’s disease (PD)
Pathological conditions promote αSyn aggregation, especially in connection with genetic mutations (Conway et al, 1998; Fredenburg et al, 2007), molecular crowding (Shtilerman et al, 2002; Uversky et al, 2002), increased αSyn protein levels (Conway et al, 1998; Breydo et al, 2012), post-translational modifications (Stefanis, 2012; Popova et al, 2015), low pH (Ahmad et al, 2012), or oxidative conditions (Hashimoto et al, 1999). in vitro studies suggest that αSyn exists in various conformations and oligomeric states in a dynamic equilibrium, where the monomer can aggregate into small oligomeric species, stabilized by β-sheet interactions, which slowly convert into higher molecular weight insoluble protofibrils and amyloidogenic fibrils, resembling those found in Lewy bodies (LB) (Conway et al, 1998, 2000; Karpinar et al, 2009; BengoaVergniory et al, 2017)
The peptide library was generated by random insertion of 60 nt random sequences flanked by EcoRI and XhoI restriction sites into the scaffold B1 domain of protein G from Staphylococcus aureus at a site of a loop between two β-sheets (Fritz and Green, 1996)
Summary
Protein misfolding and aggregation is a hallmark event in a growing number of human diseases, including Parkinson’s disease (PD). The αSyn protein is intrinsically disordered and can self-assemble into oligomeric protofibrils that can further mature into different types of fibrils and aggregates (Breydo et al, 2012). Pathological conditions promote αSyn aggregation, especially in connection with genetic mutations (Conway et al, 1998; Fredenburg et al, 2007), molecular crowding (Shtilerman et al, 2002; Uversky et al, 2002), increased αSyn protein levels (Conway et al, 1998; Breydo et al, 2012), post-translational modifications (Stefanis, 2012; Popova et al, 2015), low pH (Ahmad et al, 2012), or oxidative conditions (Hashimoto et al, 1999). Inhibitors that prevent oligomerization and aggregation of the protein are expected to serve as therapeutic medicines that can prevent the propagation of the disease (Dehay et al, 2015)
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