Aggregation of α-synuclein protein in neuronal cells has been strongly implicated in the pathogenesis of neurodegenerative disorders (Synucleinopathies) including Parkinson's disease and dementia with Lewy bodies. Current experimental evidence indicates that α-synuclein aggregation affects mitochondrial function and impairs autophagy that eliminates misfolded/unfolded proteins and damaged organelles (1, 2).
Interestingly, α-synuclein undergoes a protein post-translational modification termed SUMOylation. It involves attaching the Small Ubiquitin MOdifier-like protein (SUMO, SUMO-1, or SUMO-2/3) to the protein. Moreover, SUMOylation is known to inhibit α-synuclein aggregation (3), so to promote α-synuclein SUMOylation levels may represent a promising therapeutic strategy against synucleinopathies. Furthermore, SUMOylation is rapidly reversible due to the actions of SUMO proteases (SENP1-3, and 5-7), which detach SUMO from SUMOylated proteins in a process known as deSUMOylation. Recently we, and others, have shown that the SUMO protease SENP3 plays important roles in mitochondria-mediated cell death and various autophagic pathways (4-8). Therefore, we hypothesize that (i) SUMOylation repairs autophagy impairment induced by α-synuclein, ii) SENP3 deSUMOylates α-synuclein, and (iii) depletion of SENP3 enhances autophagy through promoting α-synuclein SUMOylation levels for cell survival.
The proposed work will directly test the hypothesis, and it will involve a combination of techniques, including those in molecular biology (e.g., cloning and tagging and site-directed mutagenesis), protein chemistry (e.g., glutathione S-transferase/histidine pulldowns & co-immunoprecipitations followed by western blotting, protein purification and assays for SUMOylation/deSUMOyation), cell biology (e.g., cultures of clonal cell lines and neuronal cells, and DNA & sh/siRNA transfections/CRISPR-Cas9-mediated knockdown/knockout and replacement), together with quantitative analysis of autophagy/mitophagy and imaging & biochemical examinations of cell viability (e.g., cytochrome c release, caspase activation, and MTT and LDH assays) under different experimental conditions.
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