ISGylation controls exosome secretion by promoting lysosomal degradation of MVB proteins.

Carolina Villarroya-Beltri,Daniel Torralba,Francesc Baixauli,Olga Moreno-Gonzalo,Susana Guerra,Sara Baldanta,Carlos Enrich,Francisco Sánchez-Madrid,Irene Fernández-Delgado,María Mittelbrunn

doi:10.1038/ncomms13588

Abstract

Exosomes are vesicles secreted to the extracellular environment through fusion with the plasma membrane of specific endosomes called multivesicular bodies (MVB) and mediate cell-to-cell communication in many biological processes. Posttranslational modifications are involved in the sorting of specific proteins into exosomes. Here we identify ISGylation as a ubiquitin-like modification that controls exosome release. ISGylation induction decreases MVB numbers and impairs exosome secretion. Using ISG15-knockout mice and mice expressing the enzymatically inactive form of the de-ISGylase USP18, we demonstrate in vitro and in vivo that ISG15 conjugation regulates exosome secretion. ISG15 conjugation triggers MVB co-localization with lysosomes and promotes the aggregation and degradation of MVB proteins. Accordingly, inhibition of lysosomal function or autophagy restores exosome secretion. Specifically, ISGylation of the MVB protein TSG101 induces its aggregation and degradation, being sufficient to impair exosome secretion. These results identify ISGylation as a novel ubiquitin-like modifier in the control of exosome production.

Highlights

Exosomes are vesicles secreted to the extracellular environment through fusion with the plasma membrane of specific endosomes called multivesicular bodies (MVB) and mediate cell-to-cell communication in many biological processes
We demonstrate that the ISGylation of the MVB protein TSG101 induces its aggregation and degradation, and this is sufficient for impairing exosome secretion
Nanoparticle tracking analysis (NTA) showed a decrease in the number of particles secreted on ISG15WT overexpression in comparison with ISG15MUT-expressing cells (Supplementary Fig. 1C), indicating that ISGylation is not causing a mere decrease in the sorting of exosomal markers in extracellular vesicles (EVs), but an actual decrease in exosome secretion

Summary

Introduction

Exosomes are vesicles secreted to the extracellular environment through fusion with the plasma membrane of specific endosomes called multivesicular bodies (MVB) and mediate cell-to-cell communication in many biological processes. The ESCRT complex is essential for the sorting of proteins such as epidermal growth factor receptor into MVBs that are degraded through fusion with lysosomes[15], but is involved in the regulation of exosome composition and secretion[16,17]. Another ubiquitin-like protein (UBL) that can modify exosomal proteins is SUMO, whose conjugation to hnRNPA2B1 is essential for the sorting of microRNAs into exosomes[18], and enhances the secretion of a-synuclein into extracellular vesicles (EVs) in an ESCRTdependent manner[19]. Free extracellular human ISG15 is important in IFN-g-dependent anti-mycobacterial immunity[21], whereas free intracellular ISG15 is involved in USP18-mediated downregulation of IFN-a/b signalling[35]

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Results

Conclusion