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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.
Hypoxia-induced vascular endothelial dysfunction (VED) is a significant contributor to several severe human diseases, including heart disease, stroke, dementia, and cancer. However, current treatment options for VED are limited due to the lack of understanding of the underlying disease mechanisms and therapeutic leads. We recently discovered a heat-stable microprotein in ginseng, called ginsentide TP1, that has been shown to reduce vascular dysfunction in cardiovascular disease models. In this study, we use a combination of functional assays and quantitative pulsed SILAC proteomics to identify new proteins synthesized in hypoxia and to show that ginsentide TP1 provides protection for human endothelial cells against hypoxia and ER stress. Consistent with the reported findings, we also found that hypoxia activates various pathways related to endothelium activation and monocyte adhesion, which in turn, impairs nitric oxide (NO) synthase activity, reduces the bioavailability of NO, and increases the production of reactive oxygen species that contribute to VED. Additionally, hypoxia triggers endoplasmic reticulum stress and initiates apoptotic signaling pathways associated with cardiovascular pathology. Treatment with ginsentide TP1 reduced surface adhesion molecule expression, prevented activation of the endothelium and leukocyte adhesion, restored protein hemostasis, and reduced ER stress to protect against hypoxia-induced cell death. Ginsentide TP1 also restored NO signaling and bioavailability, reduced oxidative stress, and protected endothelial cells from endothelium dysfunction. In conclusion, this study shows that the molecular pathogenesis of VED induced by hypoxia can be mitigated by treatment with ginsentide TP1, which could be one of the key bioactive compounds responsible for the "cure-all" effect of ginseng. This research may lead to the development of new therapies for cardiovascular disorders.
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