Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles

Abstract

Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. 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. 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. 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.

Clinical significance of STEAP1 extracellular vesicles in prostate cancer

“…Recently STEAP1 was found to be expressed on PCa cell culture-derived EVs and, in-line with our work, abundance was not found to indicate disease aggressiveness [40]. It is however important to note that while we did not find prognostic significance from an evaluation of total STEAP1 EVs there may be sub-populations of STEAP1 EVs with predictive or prognostic capabilities.…”

Isolation and Characterization of Urine Microvesicles from Prostate Cancer Patients: Different Approaches, Different Visions

“…3B). These differences would be related to those herein noticed with regards EVs size distribution, or as suggested by some reports, as consequence of differences in the membrane proteomic content of small and large EVs [40]. Moreover, EVs rupture due to UC high-speed centrifugations [38] or the reported variability on EVs yield depending on the equipment and operator technical variability could have affected the results [41].…”

Protein palmitoylation regulates extracellular vesicle production and function in sepsis

“…Previous studies have shown that palmitoylation targets modified cytoplasmic proteins to the sites of membrane budding, leading to their presence in secreted vesicles (Fang et al, 2007;Shen et al, 2011). Consistently, three-fold enrichment of palmitoylated proteins is found in EV proteome compared to human proteome (Mariscal et al, 2020). The distribution of palmitoylated proteins shows that EVs contain the highest percentage of palmitoylated proteins compared to other subcellular compartments (Romancino et al, 2018).…”

“…Palmitoylated proteins on EVs were isolated using resin‐assisted capture as previously described (Beard et al., 2016; Mariscal et al., 2020; Yang et al., 2021). EV samples were lysed in lysis buffer (100 mM HEPES, 25 mM NaCl, 1 mM EDTA, 10 μM palmostatin B, Roche protease inhibitor tablet, pH 7.4).…”