Palmitoylation of vacuole membrane protein 1 promotes small extracellular vesicle secretion via interaction with ALIX and influences intercellular communication

Abstract

Background Small extracellular vesicles (EVs), exemplified by exosomes, mediate intercellular communication by transporting proteins, mRNAs, and miRNAs. Post-translational modifications are involved in controlling small EV secretion process. However, whether palmitoylation regulates small EV secretion, remains largely unexplored. Methods Vacuole Membrane Protein 1 (VMP1) was testified to be S-palmitoylated by Palmitoylation assays. VMP1 mutant plasmids were constructed to screen out the exact palmitoylation sites. Small EVs were isolated, identified and compared between wild-type VMP1 or mutant VMP1 transfected cells. Electron microscope and immunofluorescence were used to detect multivesicular body (MVB) number and morphology change when VMP1 was mutated. Immunoprecipitation and Mass spectrum were adopted to identify the protein that interacted with palmitoylated VMP1, while knock down experiment was used to explore the function of targeted protein ALIX. Taking human Sertoli cells (SCs) and human spermatogonial stem cell like cells (SSCLCs) as a model of intercellular communication, SSCLC maintenance was detected by flow cytometry and qPCR at 12 days of differentiation. In vivo, mouse model was established by intraperitoneal injection with palmitoylation inhibitor, 2-bromopalmitate (2BP) for 3 months. Results VMP1 was identified to be palmitoylated at cysteine 263,278 by ZDHHC3. Specifically, palmitoylation of VMP1 regulated its subcellular location and enhanced the amount of small EV secretion. Mutation of VMP1 palmitoylation sites interfered with the morphology and biogenesis of MVBs through suppressing intraluminal vesicle formation. Furthermore, inhibition of VMP1 palmitoylation impeded small EV secretion by affecting the interaction of VMP1 with ALIX, an accessory protein of the ESCRT machinery. Taking SCs and SSCLCs as a model of intercellular communication, we discovered VMP1 palmitoylation in SCs was vital to the growth status of SSCLCs in a co-culture system. Inhibition of VMP1 palmitoylation caused low self-maintenance, increased apoptosis, and decreased proliferation rate of SSCLCs. In vivo, intraperitoneal injection of 2BP inhibited VMP1 palmitoylation and exosomal marker expression in mouse testes, which were closely associated with the level of spermatogenic cell apoptosis and proliferation. Conclusions Our study revealed a novel mechanism for small EV secretion regulated by VMP1 palmitoylation in Sertoli cells, and demonstrated its pivotal role in intercellular communication and SSC niche.

Decoding the Versatile Landscape of Autophagic Protein VMP1 in Cancer: A Comprehensive Review across Tissue Types and Regulatory Mechanisms

“…Despite these challenges, recent discoveries of two posttranslational modificationsubiquitination [39] and palmitoylation [94]-have shed light on potential avenues for modulating VMP1 function. These modifications appear to regulate VMP1 function, suggesting that modulating these modifications could offer a viable method for tightly regulating VMP1 function in tissues where these modifications are more active, while minimizing adverse effects in other tissues.…”

VMP1: a multifaceted regulator of cellular homeostasis with implications in disease pathology

“…Besides undergoing ubiquitination, recent studies have also reported that VMP1 can be palmitoylated at cysteine residues 263 and 278 by ZDHHC3 ( Qu et al, 2024 ). This modification is crucial for the subcellular localization and functional regulation of VMP1.…”

“…And also, palmitoylation of VMP1 enhanced the secretion of small extracellular vesicles, influencing the self-renewal capacity of small cell lung cancer cells and their apoptotic and proliferative behaviors. Disruption of VMP1’s palmitoylation sites impairs the biogenesis of multivesicular bodies and the formation of intraluminal vesicles ( Qu et al, 2024 ).…”