Palmitoylation Regulates Extracellular Vesicle Production and Cargo Contents

Abstract

DHHC21 belongs to a family of palmitoyl acyltransferases (PATs) and catalyzes the addition of palmitic acid to cysteine residue(s) of a protein. This reversible posttranslational modification dynamically controls protein localization and function, thus regulating various cellular processes. Extracellular vesicles (EVs) are membrane-encapsulated particles that serve as a critical component in intercellular communication. Recent studies have shown that palmitoylated proteins are highly enriched in EVs, however, there is limited information regarding the role of PATs in EV production and function. In this study, we investigated whether and how DHHC21 regulates EV formation and cargo content during septic inflammation caused by cecal ligation and puncture (CLP). EVs were isolated from the plasma of wild-type (WT) and DHHC21 functional deficient (Zdhhc21dep/dep) mice via ultracentrifugation followed by size-exclusion chromatography 24 hours after sham or CLP challenge. Nanoparticle tracking analysis showed no significant difference in the plasma level of EVs between WT and Zdhhc21 dep/dep mice under basal conditions. However, DHHC21 loss-of-function greatly reduced sepsis-induced increase in EV production. Next, palmitoylated proteins in EVs were separated using resin-assisted capture and quantitatively analyzed using liquid chromatography with tandem mass spectrometry. A total of 548 palmitoylated proteins were identified. A clustered heatmap revealed that the EV palmitoyl-protein profiles were remarkably different between CLP-challenged and sham-treated WT mice. Gene ontology (GO) PANTHER pathway analysis on sepsis-altered EV palmitoylated proteins showed that those proteins were highly associated with integrin signaling, inflammation, blood coagulation, and plasminogen activation. More importantly, DHHC21 was involved in regulating EV palmitoyl-protein profile during sepsis, as evidenced by a distinctive protein expression pattern of EVs in Zdhhc21dep/dep mice after CLP. These findings suggest that DHHC21 contributes to the regulation of EV production and cargo protein content during sepsis.