Abstract 2373 Investigating Proteins Associated with the α-Synuclein DNAJC5 Complex

Parkinson's disease (PD) is a neurodegenerative condition often accompanied by the accumulation of misfolded α-synuclein (α-syn) proteins. In the United States, approximately 500,000 people are diagnosed with PD, imposing a significant burden on society. The pathology involves α-syn, a protein that forms fibrillar aggregates. These aggregates constitute a major part of Lewy bodies in dopaminergic neurons. Previously, we identified DNAJC5, a membrane-anchored co-chaperone of HSC70, as responsible for the secretion of α-syn in both non-neuronal and neuronal cells. DNAJC5 has been shown to form a palmitoylated oligomer on endosomal membranes to facilitate the translocation of α-syn into the organelle. Endosome fusion at the plasma membrane is believed to discharge α-syn to the cell exterior. The secretion of α-syn is independent of its unfolding, suggesting a new mode of membrane translocation. Building on our previous work, I aim to explore the mechanism of α-syn secretion by identifying and analyzing potential membrane proteins that interact with DNAJC5 in an α-syn-dependent manner. I utilized TurboID-based proximity labeling to capture interacting membrane proteins of the α-syn-DNAJC5 complex. Split TurboID reconstitutes its biotinylation activity through spatial restriction. I used α-syn and DNAJC5 as paired bait molecules to biotinylate membrane target proteins in transfected HEK293T cells. I assessed the efficiency of TurboID labeling through streptavidin blotting. The labeling experiment revealed various unidentified proteins in proximity to the complex, suggesting that other proteins could be involved in α-syn secretion. My future plans involve mapping the membrane proteins involved in the translocation of α-syn during its unconventional secretion. After identification, I will perform a siRNA knockdown to decrease the expression of gene(s) encoding the protein of interest. If successful, I will then use CRISPR/Cas9 to generate knockout cell line(s) and overexpress the gene(s) to examine the effect on α-synuclein secretion. Fluorescence imaging will be conducted to examine the subcellular localization of candidate proteins. This work will lay the groundwork for understanding the unconventional membrane translocation mechanism of α-syn and suggest a new path to intervene in the pathology spreading in PD. My research is funded by Howard Hughes Medical Institute (HHMI) and the Summer Undergraduate Research Fellowship (SURF).