Identification of Deubiquitinases (DUBs) that modulate PAR1-mediated p38 MAPK inflammatory signaling in endothelial cells.

Protease-activated receptor-1 (PAR1), a GPCR for thrombin, induces endothelial dysfunction via multiple signal transduction pathways including p38 mitogen-activated protein kinase (MAPK). We showed that thrombin-stimulates K63-linked ubiquitination of PAR1 that drives recruitment of transforming growth factor-β-activated kinase-1 binding protein-2 (TAB2), an adaptor protein that binds TAB1, which triggers p38 activation on endosomes and promotes endothelial barrier disruption. However, the regulatory processes that control thrombin-stimulated PAR1 ubiquitin-dependent p38 inflammatory signaling are not known. To address this gap in knowledge, we sought to identify specific deubiquitinases (DUBs) that counter thrombin-induced ubiquitin-mediated p38 signaling by conducting an unbiased genome-wide siRNA library screen targeting all 96 human DUB genes in human cultured endothelial cells. We hypothesize that a PAR1-specific DUB is essential for controlling proper dynamics of thrombin-mediated p38 signaling and might be altered in endothelial dysfunction. To systematically identify DUBs, endothelial cells were transfected with pools of 4 siRNAs targeting each of the 96 DUBs and thrombin-stimulated p38 phosphorylation was assessed by immunoblotting. In parallel, a genome-wide DUB siRNA library screen was conducted in HeLa cells. We identified 8 different DUBs that preferentially cleave K63-linked ubiquitin and regulate thrombin-p38 signaling in both endothelial and HeLa cells. A time-course analysis of thrombin-stimulated p38 signaling confirmed that depletion of each of the 8 DUBs alone caused an increase in the magnitude and/or duration of p38 signaling. Among the candidate DUBs, USP34, USP47 and CYLD exhibited the highest expression in both endothelial and HeLa cells detected by RT-qPCR and were further examined. Using individual siRNAs targeting each of the 3 candidate DUBs, we validated that knockdown of either USP34, USP47 or CYLD alone was sufficient to markedly enhance thrombin-induced p38 signaling in endothelial cells. Currently, I am examining the impact of USP34-mediated modulation of thrombin-p38 signaling on endothelial inflammatory responses. I will present new data examining the role of USP34 on thrombin-induced endothelial barrier disruption and induction of cytokine expression. While ubiquitin-dependent signaling is widespread, our knowledge of the processes that control ubiquitin-mediated GPCR signaling is limited, especially related to the function of GPCR-specific DUBs. Here, we report the first genome-wide DUB siRNA library screen on GPCR signaling and identified a subset of DUBs that specifically regulate PAR1-p38 inflammatory signaling that may serve as important therapeutic targets for vascular inflammation.