Studies have shown that the process of extracellular vesicles (EVs) secretion and lysosome status are linked. When the lysosome is under stress, the cells would secrete more EVs to maintain cellular homeostasis. However, the process that governs lysosomal activity and EVs secretion remains poorly defined and we postulated that certain proteins essential for EVs biogenesis are constantly synthesized and preferentially sorted to the EVs rather than the lysosome. A pulsed stable isotope labelling of amino acids in cell culture (pSILAC) based quantitative proteomics methodology was employed to study the preferential localization of the newly synthesized proteins into the EVs over lysosome in mHypoA 2/28 hypothalamic cell line. Through proteomic analysis, we found numerous newly synthesized lysosomal enzymes—such as the cathepsin proteins—that preferentially localize into the EVs over the lysosome. Chemical inhibition against cathepsin D promoted EVs secretion and a change in the EVs protein composition and therefore indicates its involvement in EVs biogenesis. In conclusion, we applied a heavy isotope pulse/trace proteomic approach to study EVs biogenesis in hypothalamic cells. The results demonstrated the regulation of EVs secretion by the cathepsin proteins that may serve as a potential therapeutic target for a range of neurological disorder associated with energy homeostasis.

中文翻译：

---

通过重同位素脉冲/痕迹蛋白质组学方法探索下丘脑细胞的细胞外囊泡生物发生。

研究表明，细胞外囊泡（EVs）的分泌过程与溶酶体状态相关。当溶酶体处于应激状态时，细胞会分泌更多的电动汽车以维持细胞的稳态。但是，控制溶酶体活性和EV分泌的过程仍然定义不清，我们假设某些对于EV生物发生必不可少的蛋白质是不断合成的，并且优先排列在EV而非溶酶体上。基于细胞培养（pSILAC）的定量蛋白质组学方法中的氨基酸脉冲稳定同位素标记，用于研究mHypoA 2/28下丘脑细胞系中新合成的蛋白质相对于溶酶体的优先定位在EV中。通过蛋白质组学分析，我们发现了许多新合成的溶酶体酶（例如组织蛋白酶蛋白），它们比溶酶体优先位于电动汽车中。对组织蛋白酶D的化学抑制作用促进了EV的分泌和EV蛋白质组成的变化，因此表明它参与了EV的生物发生。总之，我们应用重同位素脉冲/痕量蛋白质组学方法研究下丘脑细胞中的电动汽车生物发生。结果表明，组织蛋白酶可以调节EV的分泌，这可能是与能量稳态相关的一系列神经系统疾病的潜在治疗靶标。总之，我们应用重同位素脉冲/痕量蛋白质组学方法研究下丘脑细胞中的电动汽车生物发生。结果表明，组织蛋白酶可以调节EV的分泌，这可能是与能量稳态相关的一系列神经系统疾病的潜在治疗靶标。总之，我们应用重同位素脉冲/痕量蛋白质组学方法研究下丘脑细胞中的电动汽车生物发生。结果表明，组织蛋白酶可以调节EV的分泌，这可能是与能量稳态相关的一系列神经系统疾病的潜在治疗靶标。