The mRNA‐Binding Protein HuR Is a Kinetically‐Privileged Electrophile Sensor

Abstract

Dedicated to career of and contributions by Prof. Dr. Michael Graetzel (ISIC, EPFL) on his 75 th birthdayThe key mRNA-binding proteins HuR and AUF1 are reported stress sensors in mammals. Intrigued by recent reports of sensitivity of these proteins to the electrophilic lipid prostaglandin A2 and other redox signals, we here examined their sensing abilities to a prototypical redox-linked lipid-derived electrophile, 4-hydroxynonenal (HNE). Leveraging our T-REX electrophile delivery platform, we found that only HuR, and not AUF1, is a kinetically-privileged sensor of HNE in HEK293T cells, and sensing functions through a specific cysteine, C13. Cells depleted of HuR, upon treatment with HNE, manifest unique alterations in cell viability and Nrf2transcription-factor-driven antioxidant response (AR), which our recent work shows is regulated by HuR at the Nrf2-mRNA level. Mutagenesis studies showed that C13-specific sensing alone is not sufficient to explain HuRdependent stress responsivities, further highlighting a complex context-dependent layer of Nrf2/AR regulation through HuR. Figure 1. HuR, but not AUF1, is a sensor of HNE in HEK293T cells. A) Structures of the native RES prostaglandin A2 (PGA2) and alkyne-functionalized HNE. B) HEK293T cells were treated with HNE(alkyne) (20 μM, 18 h). Click chemistry was used to biotinylate HNE-modified proteins, which were then enriched with streptavidin resin. Shown is a representative blot from four independent experiments. Inset at right: Quantification (mean � SEM) of western blot data from n = 4 independent replicates. ND: not detected.

A Primer on Harnessing Non-enzymatic Posttranslational Modifications for Drug Design

“…Signaling changes measured are significantly greater than predicted based on measured occupancy. This result demonstrates that dominant signaling modes occur in redox sensing, and as this has now been shown for several different proteins/pathways (NF-κB 43,51 , PI3K/Akt 43,49,52 , DNA-damage 51 , and antioxidant response 30,31,43,[45][46][47]50 signaling), we cautiously postulate that such behaviors are not uncommon. Such results reconcile some indirect data that had shown that the occupancy on postulated electrophile sensors necessary to trigger signaling was relatively small.…”

“…For several HNE-sensitive proteins, T-REX delivery efficiency (DE) between 15 and 50% triggers signaling phenotypes. 30,31,[44][45][46][47][48][49][50] In several instances the phenotypes stimulated are similar in magnitude to those observed under bolus dosing, or when an inhibitor is used to fully shut down POI activity. Thus, specific electrophile labeling of single proteins is sufficient to trigger signaling.…”

“…Such results reconcile some indirect data that had shown that the occupancy on postulated electrophile sensors necessary to trigger signaling was relatively small. 2,44,45,50 Although T-REX-mediated electrophile release is chemospecific, ~10 different electrophiles so far, and likely many more, are compatible with this method. T-REX thus can perform unbiased (i.e.…”

Hitting the Bullseye: Endogenous Electrophiles Show Remarkable Nuance in Signaling Regulation

“…This can be either inhibitory, or stimulatory, or potentially gain of function 72 . We have identified proteins that sense electrophiles through T-REX TM but do not seem to respond functionally to electrophiles 73 . These for the time being appear to be proteins that are only capable of achieving very low occupancy under standard T-REX TM methods.…”

Where Electrophile Signaling and Covalent Ligand–Target Mining Converge

“…This article highlights the Aye laboratory research on electrophile signaling and stress response (left panel), wherein precision chemical biology tools are engineered in house aimed at addressing unmet biological questions of significant pharmaceutical relevance. [4,6,9,16,17,[22][23][24][25][26][27][28][29][30][31][32][33][35][36][37][38][39][40][41][42][43][44] We apply these tools in combination with organic chemistry, biochemistry, and genetic principles in relevant in vivo models illustrated. The figure depicts general concept underlying REX-technologies: Halo-tag-protein covalently bound to photocaged-HNE enables light-driven on-demand delivery of covalent small-molecule such as HNE, Fig.…”