Fig. 1: Proteomic screens and in vitro validation of hits. | Nature Communications

Fig. 1: Proteomic screens and in vitro validation of hits.

From: Disrupting the α-synuclein-ESCRT interaction with a peptide inhibitor mitigates neurodegeneration in preclinical models of Parkinson’s disease

Fig. 1: Proteomic screens and in vitro validation of hits.

A Schematic of the experimental design including proteomic screens, identification and validation of hits, target deconvolution, and evaluation in cell and animal models (created using Servier Medical Art at https://smart.servier.com/). For the cytotoxicity screen, we employed our peptide library to identify peptides that rescue cytotoxicity induced by a-syn overexpression and proteostatic stress (due to MG132 administration). To screen our library for peptides that inhibit a-syn oligomers, we used FACS with a split YFP-a-syn system (cells co-expressing V1S and SV2). B Validation of cell viability effect of peptides in A53T and WT a-syn expressing cells under proteostatic stress as well as controls. MG132 was used at a concentration of 10 μM. Experiments were done in triplicate. Data represent means ± s.d. (unpaired two-tailed t-tests; A53T PDpep1, P = 0.0051; A53T PDpep1.1, P = 0.0002; A53T PDpep1.2, P = 0.0035; A53T PDpep1.3, P < 0.0001; WT a-syn PDpep1.3, P = 0.0008; n = 3). C WT a-syn oligomers as measured by luciferase activity. All four peptides showed significant reduction in a-syn oligomers from cells stably expressing split luciferase-a-syn constructs. Experiments were done in triplicate. Data represent means ± s.d. (unpaired two-tailed t-tests; PDpep1, P = 0.0008; PDpep1.1, PDpep1.2, PDpep1.3, P < 0.0001; n = 3). D Co-immunoprecipitation experiment with Flag-CHMP2B and GFP-peptides (n = 3). PDpep1 and PDpep1.3 peptides interacted with Flag-CHMP2B whereas GFP alone (CTL) did not. E Fluorescence polarization (FP) binding assay of a FITC-labeled PDpep1.3 peptide against CHMP2B. Error bars represent ± s.d. of the fit (n = 5). F Knockdown of CHMP2B or VPS4 in A53T a-syn expressing cells under proteostatic stress. Cell viabilities were measured with cells stably expressing different shRNAs and transfected with Scramble or PDpep1.3 peptide. Experiments were done in triplicate. Data represent means ± s.d. (unpaired two-tailed t-tests; PDpep1.3, P = 0.0005; n = 3). **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are provided as a Source Data file.

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