Studies on the Reversibility of Protein S-Thiolation in Human Endothelial Cells

Abstract

Exposure of human umbilical vein endothelial cells to oxidants, such as hydrogen peroxide and diamide, has been shown to induce protein-specific S-thiolation of cellular proteins. In this study we have now identified glutathione (reduced form) as the major low molecular-weight thiol that is bound to protein substrates in human umbilical vein endothelial (HUVE) cells during oxidative stress and investigated the dose- and time-response relationship of diamide- and hydrogen peroxide-induced S-thiolation of HUVE cell protein. Intact HUVE cells are able to rapidly reduce S-thiolated proteins with almost quantitative reappearance of reduced glutathione in the cells and protection from acute, lytic cytotoxicity. Additionally, studies were performed with detergent-solubilized cell extracts to determine the nature of the reductants operating in HUVE cells to maintain protein thiol homeostasis. The results clearly show the involvement of NADH- and NADPH-dependent systems. These data suggest that the reversible S-thiolation of proteins in these human endothelial cells may represent a significant cellular antioxidant and regulatory mechanism during oxidative stress.