Deubiquitinases as Regulators of Thrombin‐induced p38 Inflammatory Response

Abstract

Vascular endothelial barrier integrity is tightly regulated, while inflammatory mediators may induce endothelial dysfunction, leading to serious conditions such as cardiovascular disease. Among various inflammatory mediators, thrombin has been characterized to activate protease-activated receptor-1 (PAR1), a G-protein coupled receptors (GPCR) which is capable of triggering many downstream signals that ultimately increase vascular leakage. It has been reported that thrombin cleavage upon PAR1 can activate p38 signaling via a non-canonical pathway. Depending on PAR1 ubiquitination, transforming growth factor-β–activated kinase-1–binding proteins (TAB) are recruited to the endosomal cohort of PAR1 and induce p38 self-phosphorylation, eventually leads to endothelial barrier disruption. While such ubiquitin-dependent pro-inflammatory PAR1 signaling pathway has been identified, the understanding of the regulatory mechanism is lacking, especially around the observed PAR1 deubiquitination. We hypothesize that such enzyme is a key to terminate thrombin-induced p38 inflammatory signaling and to mitigate endothelial dysfunction. In order to identify the deubiquitinases (DUB) that are involved in the PAR1-p38 signaling axis, either through regulating PAR1 surface expression or TAB engagement, we have utilized a siRNA library screen targeting all 96 human DUB genes in endothelial cells. Taking multiple functional readouts together, we have been focusing on 4 DUB candidates that was identified to alter p38 activation and PAR1 surface expression regulation. To delineate the 4 candidates’ role in thrombin-induced p38 inflammatory signaling pathway, we are examining the interaction between such DUB and PAR1, as well as other components in the pathway. We expect that these DUB are critically involved in the regulation of the PAR1-specific, ubiquitin-dependent p38 signaling thus elevating their status in developing treatments tuning endothelial inflammatory responses.