An siRNA screen for endothelial PAR1‐specific deubiquitinases regulating p38 inflammatory signaling

Abstract

Endothelial dysfunction is a hallmark of vascular disease and induced by various inflammatory mediators which signal through GPCRs pathways that are poorly understood. Thrombin induces endothelial inflammatory responses through the activation of protease‐activated receptor 1 (PAR1), a GPCR. Previously, we showed that thrombin‐activated PAR1 is modified by ubiquitin and drives recruitment of transforming growth factor‐β–activated kinase‐1–binding protein 2 (TAB2), an adaptor protein that binds TAB1 to trigger p38‐dependent endothelial barrier disruption, a hallmark of inflammation. However, the regulatory processes that control PAR1‐deubiquitination and associated p38 inflammatory signaling are not known. To fill this gap in knowledge, we sought to identify PAR1‐specific deubiquitinases (DUBs) that removes K63‐linked ubiquitin by conducting an unbiased comprehensive genome‐wide siRNA library screen targeting all 96 human DUB genes in human cultured endothelial cells using multiple PAR1 ubiquitin‐dependent functional readouts. We hypothesize that a PAR1‐specific DUB is essential for terminating thrombin‐induced p38 inflammatory signaling and might be altered in endothelial dysfunction. In an initial screen, endothelial cells were transfected with siRNA pools targeting specific DUBs and PAR1‐ubiquitin dependent responses were assessed. We specifically examined PAR1 cell surface expression, controlled by basal PAR1 ubiquitination, and thrombin‐stimulated p38 activation and downstream inflammatory signaling, which is dependent on agonist‐triggered PAR1 ubiquitination. In preliminary studies have identified several candidates PAR1‐specific DUBs. Currently, we are now investigating the function of top candidate DUBs in regulating the status of PAR1 ubiquitination, cell surface expression and p38 inflammatory signaling. The molecular mechanism responsible for PAR1 deubiquination and relation to controlling the spatio‐temporal dynamics of pro‐inflammatory signaling will be further investigated. Taken together, we anticipate that these studies using a comprehensive siRNA library screening approach will reveal the identity of several DUBS that have important functions in regulating PAR1‐induced ubiquitin driven p38 MAPK endothelial inflammatory signaling.Support or Funding InformationAuthors acknowledge NIH/NIGMS funding support.