Spatial Mechano-Signaling Regulation of GTPases Through Non-Degradative Ubiquitination

Blood flow produces shear stress exerted on the endothelial layer of the vessels. Spatial characterization of the endothelial proteome is required to uncover the mechanisms of endothelial activation by shear stress, as blood flow varies in the vasculature. An integrative ubiquitinome and proteome analysis of shear-stressed endothelial cells demonstrated that the non-degradative ubiquitination of several GTPases is regulated by mechano-signaling. Spatial analysis reveals increased ubiquitination of the small GTPase RAP1 in the descending aorta, a region exposed to laminar shear stress. The ubiquitin ligase WWP2 is identified as a novel regulator of RAP1 ubiquitination during shear stress response. Non-degradative ubiquitination fine-tunes the function of GTPases by modifying their interacting network. Specifically, WWP2-mediated RAP1 ubiquitination at lysine 31 switches the balance from the RAP1/ Talin 1 (TLN1) toward RAP1/ Afadin (AFDN) or RAP1/ RAS Interacting Protein 1 (RASIP1) complex formation, which is essential to suppress shear stress-induced reactive oxygen species (ROS) production and maintain endothelial barrier integrity. Increased ROS production in endothelial cells in the descending aorta of endothelial-specific Wwp2-knockout mice leads to increased levels of oxidized lipids and inflammation. These results highlight the importance of the spatially regulated non-degradative ubiquitination of GTPases in endothelial mechano-activation.