Abstract

Palmitoyl-Proteomics to Identify Protein Signatures For Large and Small Cancer-derived Extracellular Vesicles in Patient Plasma

Dolores Di Vizio, Professor, Cedars Sinai Medical Center

Extracellular vesicles (EVs) are membrane-enclosed nano- and micro-particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Historically called with various names, depending on the size, cargo, function and cellular and subcellular origin, they are currently categorized in large and small EVs. Our group and others have reported a cancer-specific population of EVs known as large oncosomes that is significantly larger than exosomes-like EVs, and is released by cancer cells during active metastatic migration. Protein S-acylation, also known as palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyze the palmitoyl proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells and patient plasma. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbor proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABBC4 as prostate cancer-specific palmitoyl proteins enriched in both EV populations in comparison with the originating cell lines. Importantly, the presence of the above proteins in EVs was significantly reduced upon inhibition of palmitoylation in the producing cells. We also performed additional experiments on a group of patients with metastatic prostate cancer and narrowed down a list of palmitoylated proteins that can be used as biomarkers in plasma. Finally, the palmitoyl-proteome of EVs in patient plasma significantly differed from the palmitoyl-proteome of whole plasma prior to purify EVs. These results suggest that palmitoylation may be involved in the differential sorting of proteins to distinct EV populations and allow for better detection of disease biomarkers.