Abstract
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.
Highlights
Cells are known to secrete extracellular vesicles (EVs) into the extracellular milieu that can be categorized into multivesicular bodies (MVBs)-originated exosomes (30–150 nm), microvesicles (MVs)(100–1000 nm) that shed from the plasma membrane or apoptotic bodies (>1000 nm) from dying cells [1].In particular, exosomes and MVs are known to promote intercellular communication in numerous physiological and pathological settings through the transfer of materials such as proteins, mRNA and miRNA to the recipient cells [2,3,4]
In lysosome-mediated protein degradation, the endosomal sorting complex required for transport (ESCRT) machinery is crucial for the sequestration of ubiquitinated proteins at the endosomal membrane, followed by inward budding of the membrane into intraluminal vesicles for degradation [8]
We identify a possible role of newly synthesized cathepsin D on EVs biogenesis in mHypoA 2/28 hypothalamic cells and these results may provide invaluable insight into the regulation of the EVs-lysosome axis and their possible effect on energy homeostasis
Summary
Cells are known to secrete extracellular vesicles (EVs) into the extracellular milieu that can be categorized into multivesicular bodies (MVBs)-originated exosomes (30–150 nm), microvesicles (MVs). Exosomes and MVs are known to promote intercellular communication in numerous physiological and pathological settings through the transfer of materials such as proteins, mRNA and miRNA to the recipient cells [2,3,4]. In lysosome-mediated protein degradation, the endosomal sorting complex required for transport (ESCRT) machinery is crucial for the sequestration of ubiquitinated proteins at the endosomal membrane, followed by inward budding of the membrane into intraluminal vesicles (iLVs) for degradation [8]. Formation of EVs seems to involve only a subset of the ESCRT machinery [9] and non-ubiquitinated proteins can be sorted into the vesicle as well [10]. Exosome biogenesis can occur independent of the ESCRT machinery through the conversion of sphingomyelin to ceramide by the neutral type
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