Identification and characterisation of extracellular vesicle associated palmitoylated proteins

Abstract

Extracellular vesicles (EVs) are membrane-enclosed particles enveloping a cargo ofbioactive molecules, encompassing proteins, nucleic acids, and lipids, that aresecreted by virtually all cell types and play an essential role in intercellular communication in various physiological and pathological processes. Critically, cellshave evolved the ability to adapt the repertoire of cargo they selectively package intoEVs, suggesting the existence of different mechanisms that control cargo sorting.An array of post-translational modifications (PTMs) have been shown to regulateprotein sorting into EVs. One such PTM, palmitoylation, involves the reversibleattachment of acyl groups such as palmitate on to cysteine amino acids and regulates the trafficking and localisation of thousands of diverse cellular proteins. Because palmitoylation anchors proteins to cellular membranes, it is a proposed mechanism toregulate protein targeting to EVs. The aim of this study is to explore the associationbetween protein palmitoylation and protein sorting to EVs.This work identified of a total of 2515 unique palmitoylated proteins associated withEVs, representing an approximate 4-fold enrichment of palmitoylated proteins in EVs compared to the whole proteome, and strongly supports the argument thatpalmitoylation is a predictor and critical sorting signal for proteins (i) destined as EVcargo, or (ii) involved in EV biogenesis and trafficking.Three identified palmitoyl-EV proteins, the chaperone protein HSP70, an accessoryprotein of the endosomal sorting complex required for transport (ESCRT) ALIX, andthe metabolic serine hydrolase ABHD16A, were analysed. ABHD16A and ALIX wereboth found to be palmitoylated in HEK293T cells and ABHD16A was found to bepalmitoylated in the EVs isolated from conditioned cell media from HEK29T cells.Subsequent analysis identified at least two sites for ABHD16A palmitoylation, Cys7 and Cys 284/285, and determined that palmitoylation at these cysteines has two roles:(i) to protect ABHD16A from proteasomal degradation; and (ii) in ABHD16Adepalmitoylase activity. Future work will determine the role for palmitoylation in ABHD16A trafficking to EVs.

Date of Award	Mar 2024
Original language	English
Awarding Institution	Coventry University
Supervisor	Jennifer Greaves (Supervisor) & James Dayus (Supervisor)