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, frequently called 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 analyse 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 harbour proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABCC4 as prostate cancer-specific palmitoyl-proteins abundant in both EV populations. Importantly, localization of the above proteins in EVs was reduced upon inhibition of palmitoylation in the producing cells. Our results suggest that this post-translational modification may play a role in the sorting of the EV-bound secretome and possibly enable selective detection of disease biomarkers.

摘要

细胞外囊泡（EV）是膜包裹的颗粒，在癌症进展中发挥重要作用，并已成为循环生物标志物的有前途的来源。蛋白质S-酰化，通常称为棕榈酰化，已被提议作为一种翻译后机制，调节 EV 生物发生和蛋白质货物分选的动态。然而，技术挑战限制了电动汽车的整个棕榈酰蛋白的大规模分析。我们成功地采用了一种将低背景酰基生物素交换 (LB-ABE) 与无标记蛋白质组学相结合的新方法来分析前列腺癌的大 EV (L-EV) 和小 EV (S-EV) 的棕榈酰蛋白质组细胞。在这里，我们报告了 EV 的第一个棕榈酰蛋白特征，并证明 L-和 S-EV 含有与不同生物过程和亚细胞起源相关的蛋白质。我们将 STEAP1、STEAP2 和 ABCC4 鉴定为前列腺癌特异性棕榈酰蛋白，在两个 EV 群体中含量丰富。重要的是，在生产细胞中抑制棕榈酰化后，上述蛋白质在 EV 中的定位就会减少。我们的结果表明，这种翻译后修饰可能在 EV 结合分泌蛋白组的分选中发挥作用，并可能实现疾病生物标志物的选择性检测。