Formation of extracellular vesicles (EVs) has emerged as a novel paradigm in cell-to-cell communication in health and disease. EVs are notably produced during cell death but it had remained unclear whether different modalities of regulated cell death (RCD) influence the biogenesis and composition of EVs. To this end, we performed a comparative analysis of steady-state (ssEVs) and cell death-associated EVs (cdEVs) following TNF-induced necroptosis (necEVs), anti-Fas-induced apoptosis (apoEVs), and ML162-induced ferroptosis (ferEVs) using the same cell line. For each RCD condition, we determined the biophysical and biochemical characteristics of the cell death-associated EVs (cdEVs), the protein cargo, and the presence of methylated ribosomal RNA. We found that the global protein content of all cdEVs was increased compared to steady-state EVs. Qualitatively, the isolated exosomal ssEVs and cdEVs, contained a largely overlapping protein cargo including some quantitative differences in particular proteins. All cdEVs were enriched for proteins involved in RNA splicing and nuclear export, and showed distinctive rRNA methylation patterns compared to ssEVs. Interestingly, necEVs and apoEVs, but strikingly not ferEVs, showed enrichment of proteins involved in ribosome biogenesis. Altogether, our work documents quantitative and qualitative differences between ssEVs and cdEVs.

中文翻译：

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细胞凋亡、坏死性凋亡和铁死亡细胞产生的外泌体细胞外囊泡的系统成分分析

细胞外囊泡（EV）的形成已成为健康和疾病中细胞间通讯的新范例。EVs 主要在细胞死亡过程中产生，但目前尚不清楚调节性细胞死亡 (RCD) 的不同方式是否影响 EVs 的生物发生和组成。为此，我们对 TNF 诱导的坏死性凋亡 (necEVs)、抗 Fas 诱导的细胞凋亡 (apoEVs) 和 ML162 诱导的铁死亡后的稳态 (ssEVs) 和细胞死亡相关 EVs (cdEVs) 进行了比较分析。 ferEVs）使用相同的细胞系。对于每种 RCD 条件，我们确定了细胞死亡相关 EV (cdEV)、蛋白质货物以及甲基化核糖体 RNA 的存在的生物物理和生化特征。我们发现，与稳态 EV 相比，所有 cdEV 的整体蛋白质含量均有所增加。定性地，分离的外泌体 ssEV 和 cdEV 含有大量重叠的蛋白质货物，包括特定蛋白质的一些数量差异。所有 cdEV 都富含参与 RNA 剪接和核输出的蛋白质，并且与 ssEV 相比，显示出独特的 rRNA 甲基化模式。有趣的是，necEV 和 apoEV，但明显不是 ferEV，显示出参与核糖体生物合成的蛋白质富集。总而言之，我们的工作记录了 ssEV 和 cdEV 之间的定量和定性差异。