Extracellular vesicles production and proteomic cargo varies with incubation time and temperature.
Extracellular vesicles production and proteomic cargo varies with incubation time and temperature.
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DOI：
10.1016/j.yexcr.2022.113454
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发表时间：
2022-12
影响因子：
3.7
通讯作者：
Sagar Rayamajhi;S. Sulthana;Colin Ferrel;Tej B. Shrestha;S. Aryal
中科院分区：
文献类型：
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作者：
Sagar Rayamajhi;S. Sulthana;Colin Ferrel;Tej B. Shrestha;S. Aryal
细胞外囊泡（EVs）是由细胞分泌的蛋白质脂质双层囊泡的异质群体，是一个重要的生物过程。电动汽车货物可以反映细胞生长的细胞环境条件。使用无血清条件培养基收获EV会导致应激介导的细胞变化，培养时间更长，并影响EV的产生和功能。本研究旨在探讨培养时间和温度对EV生产和蛋白质组学的影响。为此，开发了一种优化的超滤尺寸排斥色谱技术，该技术可分离130 ~ 220 nm范围内的小型电动汽车。结果表明，与非癌细胞（NIH/3T3）相比，癌细胞（K7M2）的ev产量更高，并且随着培养时间的延长和温度的升高而增加。基于质谱的ev蛋白质组学表征显示了孵育时间和温度依赖的蛋白质组学特征。在营养胁迫（孵育72 h）和热胁迫（孵育温度40℃）条件下分离的EV中发现了一组富集的EV蛋白。通过免疫印迹进一步验证了热应激下分离的ev中Serpinb1a的富集。基因富集分析表明，营养胁迫后的EV蛋白富集与转录负调控、氧化应激反应和蛋白折叠有关。同样，热应激后丰富的EV蛋白参与草酰乙酸和天冬氨酸代谢以及谷氨酸分解代谢过程。营养胁迫下分离的EVs具有促增殖活性，热胁迫下分离的EVs具有抗增殖活性。我们的研究结果表明，孵育时间和温度可以改变EV的产量，其蛋白质组学货物和功能，可用于设计基于需求的标准分离参数，用于可重复性EV研究。
Extracellular vesicles (EVs) are heterogenous populations of proteolipid bi-layered vesicles secreted by cells as an important biological process. EVs cargo can reflect the cellular environmental conditions in which cells grow. The use of serum-free conditioned media to harvest EVs leads to stress-mediated cellular changes with longer incubation time and impacts EV production and functionality. This study aims to explore the role of incubation time and temperature on EV production and proteomic cargo. For this purpose, an optimized ultrafiltration-size exclusion chromatography-based technique is developed, which isolates small EVs ranging from 130 to 220 nm. The result shows higher EVs production in cancerous cells (K7M2) compared to noncancerous cells (NIH/3T3), which increases with longer incubation time and elevated temperature. Mass spectrometry-based proteomic characterization of EVs showed incubation time and temperature-dependent proteomic profile. A set of enriched EV proteins were identified in EVs isolated at nutrient-stress (72 h incubation time) and heat-stress (40 °C incubation temperature) environment. Enrichment of Serpinb1a in EVs isolated in heat stress was further validated via immunoblot. Gene enrichment analysis revealed that enriched EV proteins following nutrient stress were involved in negative regulation of transcription, response to oxidative stress, and protein folding. Likewise, enriched EV proteins following heat stress were involved in oxaloacetate and aspartate metabolism, and glutamate catabolic process. EVs isolated under nutrient stress showed pro-proliferative activity whereas EVs isolated under heat stress showed anti-proliferative activity. Our results show that incubation time and temperature can alter EV production, its proteomic cargo, and functionality, which can be used to design need-based standard isolation parameters for reproducible EV research.