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)], 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 untested. 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.

A Generalizable Platform for Interrogating Target- and Signal-Specific Consequences of Electrophilic Modifications in Redox-Dependent Cell Signaling

“…On the other hand, T-REX-targeted HNEylation with Ht-Pre-HNE 17 under identical conditions yielded 2 Cys residues HNEylated—C226 and C368 (IS, 51 and 61, respectively). 13 These data further corroborated the hypothesis of a lower number of Cys residues available for CHE 9 over HNE 1 .…”

“…Substituting HNE 1 in place of CHE 9 and replication of the experiment revealed 6 HNEylated residues instead—C77, C151, C226, C273, C319 and C368—with IS’s: 60, 74, 54, 61, 40 and 95, respectively. 13 The increased promiscuity of HNE relative to CHE thus most likely accounts for the differences in overall alkylation rate seen in the progress curves above (Figure 5b). In addition, C151 was identified as an overlap between the two sets of Cys residues characterized to be modified during CHE- as well as HNE-adduction events.…”

“…12 We subsequently extended this method to interrogate whether specific HNEylation of Keap1 in low stoichiometry could elicit an ARE response, or whether subsidiary factors were required. 13 These pilot studies unambiguously demonstrated that Keap1 is a key redox sensor along the Nrf2–ARE cascade—specifically, HNEylation of Keap1 is alone biologically sufficient to elicit an ARE response of magnitude similar to that observed under whole-cell HNE flooding. 13 Thus, T-REX allows quantitation of the relative strength of downstream signaling selectively induced by Keap1-alone HNEylation—information not easily obtainable by whole-cell LDE treatment approaches.…”

“…13 These pilot studies unambiguously demonstrated that Keap1 is a key redox sensor along the Nrf2–ARE cascade—specifically, HNEylation of Keap1 is alone biologically sufficient to elicit an ARE response of magnitude similar to that observed under whole-cell HNE flooding. 13 Thus, T-REX allows quantitation of the relative strength of downstream signaling selectively induced by Keap1-alone HNEylation—information not easily obtainable by whole-cell LDE treatment approaches. 1,3-7 Notably however, widely different biologic responses are reportedly elicited by whole-cell stimulation with structurally different LDEs.…”

“…13 We here examined the extent to which modest chemical perturbations across the various LDE signals delivered selectively to Keap1 in cells modulate the relative levels of Nrf2–ARE activation. We accomplished this goal using two independent quantitative readouts: ( 1 ) western blot analysis probing the upregulation of Nrf2 steady-state levels, and ( 2 ) reporter gene assay quantitating the fold increment of ARE-driven firefly luciferase signal, normalized over constitutively expressed Renilla luciferase (Figure 8).…”