Substoichiometric Hydroxynonenylation of a Single Protein Recapitulates Whole‐cell‐stimulated Antioxidant Response

Abstract

Lipid-derived electrophiles (LDEs) that can directly modify proteins have emerged as important small-molecule cues in cellular decision-making. However, because these diffusible LDEs can modify many targets [e.g., >700 cysteines are modified by the well-known LDE 4-hydroxynonenal (HNE) (by Cravatt et al., Nature Methods, 2014, 11, 79-85)], establishing the functional consequences of LDE modification on individual targets remains devilishly difficult. Whether LDE modifications on a single protein are biologically sufficient to activate discrete redox signaling response downstream also remains an untested hypothesis. Herein, using T-REX (targetable reactive electrophiles and oxidants)—an approach aimed at selectively flipping a single redox switch in cells at a precise time —we show that a modest level (~34%) of HNEylation on a single target is sufficient to elicit the pharmaceutically-important antioxidant response element (ARE) activation, and the resultant strength of ARE induction recapitulates that observed from whole-cell electrophilic perturbation. These data provide the first evidence that single-target LDE modifications are important individual events in mammalian physiology. Research involving the development and applications of the new T-REX idea is supported by the NIH Director's New Innovator Award (1DP2GM114850), NSF CAREER Award (CHE-1351400), and the Beckman Young Investigator Award (to Y.A.).