N-terminal cysteine independent effects of oxidative stress on the stability of RGS4 and 5

Abstract

Oxidative post-translational modifications on the sulfhydryl group of cysteines can occur spontaneously or enzymatically. The enzymatic dioxygenation of N-terminal cysteines has emerged as a new oxygen sensing paradigm, catalysed by 2-aminoethanethiol dioxygenase (ADO) in mammals. Conflicting evidence has been reported in recent years on whether this reaction can occur spontaneously in the absence of ADO. Here we sought to address whether cellular oxidative stress can interfere with ADO-catalysed N-terminal cysteine dioxygenation. Using a system to produce titratable intracellular levels of H2O2, we demonstrate that the stability of RGS4 and 5 is not affected by physiological levels of oxidative stress, whether ADO is present or not. However, cytotoxic levels of oxidative stress induced by tert-butyl hydroperoxide did induce a substantial increase in RGS4/5 protein stability that occurred independently of the Cys N-degron pathway. This effect of tBHP was abolished by Fe2+ chelation, lysosomal pH perturbation, and lipid peroxide scavenging. We conclude that N-terminal cysteine dependent proteolysis of RGS4/5 is not sensitive to physiological oxidative stress, but these proteins can be stabilised during the process of oxidative stress-induced cell death through an N-terminal cysteine independent mechanism.