Differential role for rapid proteostasis in Rho GTPase- mediated control of quiescent endothelial integrity

Endothelial monolayer permeability is regulated by actin dy-namics and vesicular traffic. Recently, ubiquitination was also implicated in the integrity of quiescent endothelium, as it differentially controls the localization and stability of adhesion and signaling proteins. However, the more general effect of fast protein turnover on endothelial integrity is not clear. Here, we found that inhibition of E1 ubiquitin ligases induces a rapid, reversible loss of integrity in quiescent, primary human endo-thelial monolayers, accompanied by increased F-actin stress fi- bers and the formation of intercellular gaps. Concomitantly, total protein and activity of the actin-regulating GTPase RhoB, but not its close homolog RhoA, increase 10-fold in 5 to 8 h. We determined that the depletion of RhoB, but not of RhoA, the inhibition of actin contractility, and the inhibition of protein synthesis all significantly rescue the loss of cell-cell contact induced by E1 ligase inhibition. Collectively, our data suggest that in quiescent human endothelial cells, the continuous and fast turnover of short-lived proteins that negatively regulate cell-cell contact is essential to preserve monolayer integrity.

Automated summary

Endothelial monolayer permeability is regulated by actin dynamics and vesicular traffic. Inhibition of E1 ubiquitin ligases leads to a rapid, reversible loss of integrity in quiescent endothelial monolayers with increased F-actin stress fibers and intercellular gaps. Fast protein turnover of negative regulators of cell-cell contact is essential for maintaining monolayer integrity in quiescent human endothelial cells.