Understanding extracellular vesicles biology through the study of protein dynamics using pSILAC-based quantitative proteomics

• Main Author: Tan, Chee Fan
• Other Authors: Sze Siu Kwan
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/137126
• Institution: Nanyang Technological University

Small extracellular vesicles (EVs) are shown to play an important role in intercellular communication as they contain an array of biological molecules such as proteins, nuclei acids and lipids that elicit physiological changes in their recipient organ. Research interests on EVs have increased tremendously and advancing the study on EV-mediated intercellular communication and EVs biogenesis is pertinent in understanding EVs biology and their potential function as therapeutic agent. In this thesis, I have applied a system biology approach driven by pSILAC-based quantitative proteomics to study protein dynamics in EVs derived from mHypoA 2/28 adult hypothalamus cell line and examine their role in EV biogenesis and leptin-mediated EV inter-organ communication. Vesicles formed intracellularly can either fused to the lysosome for degradation or transported to the plasma membrane for secretion. Owing to EV secretory nature, proteins essential for EV biogenesis should have increased protein synthesis rate as compared to those required for protein degradation. Analysis of protein synthesis ratio revealed that newly synthesized cathepsin proteins are preferentially sorted into mHypoA 2/28 EVs over the lysosomes. Functional characterization of these cathepsin proteins revealed a role of cathepsin D in EV biogenesis. Chemical inhibition of cathepsin D promoted EVs secretion, which is accompanied by an upregulation of Rab GTPase cellular expression. Tetraspanin protein expression was also altered in cathepsin D-inhibited EVs. Next, the role of leptin-induced mHypoA 2/28 EVs in energy homeostasis was also investigated through pSILAC-based quantitative proteomics. Leptin treatment increased EVs secretion and promoted the sorting of newly-synthesized proteins into the mHypoA 2/28 EVs. Injection of either leptin-induced or basal mHypoA 2/28 EVs into C57bl/6J mice reduced both the blood glucose level and body weight. Gene analysis of the tibialis anterior muscle from mice injected with basal mHypoA 2/28 EVS showed an increase in GLUT4 expression that is independent of leptin action. Nonetheless, this study established a functional role of mHypoA 2/28 EVs in blood glucose homeostasis. In conclusion, the usage of a pSILAC-based quantitative proteomic methodology in this study has offered a new perspective in EV biogenesis. Furthermore, the ability of mHypoA 2/28 EVs to modulate glucose homeostasis could contribute towards the development of an EV-based therapeutic treatment against metabolic diseases such as hyperglycaemia.