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

Abstract

Despite the known propensity of small-molecule electrophiles to react with numerous cysteine-active proteins, biological actions of individual signal inducers have emerged to be chemotype-specific. To pinpoint and quantify the impacts of modifying one target out of the whole proteome, we develop a target-protein-personalized “electrophile toolbox” with which specific intracellular targets can be selectively modified at a precise time by specific reactive signals. This general methodology—T-REX (targetable reactive electrophiles & oxidants)—is established by: (1) constructing a platform that can deliver a range of electronic and sterically different bioactive lipid-derived signaling electrophiles to specific proteins in cells; (2) probing the kinetics of targeted delivery concept which revealed that targeting efficiency in cells is largely driven by initial on-rate of alkylation; and (3) evaluating the consequences of protein-target- and small-molecule-signal-specific modifications on the strength of downstream signaling. These data show that T-REX allows quantitative interrogations into the extent to which the Nrf2 transcription factor-dependent antioxidant response element (ARE) signaling is activated by selective electrophilic modifications on Keap1 protein—one of several redox-sensitive regulators of the Nrf2–ARE axis. The results document Keap1 as a promiscuous electrophile-responsive sensor able to respond with similar efficiencies to discrete electrophilic signals, promoting comparable strength of Nrf2–ARE induction. T-REX is also able to elicit cell activation in cases in which whole-cell electrophile flooding fails to stimulate ARE induction prior to causing cytotoxicity. The platform presents a previously unavailable opportunity to elucidate the functional consequences of small-molecule-signal- and protein-target-specific electrophilic modifications in an otherwise unaffected cellular background.

Modulation of Fluorescent Protein Chromophores To Detect Protein Aggregation with Turn-On Fluorescence

“…To this end, we genetically fused Halo-Tag 17 to the POI and synthesized 4 for bioorthogonal conjugation (Figure 4a, hereafter termed as AggTag method). Using the AggTag method, we expect that the fluorophore remains dark when POI is folded.…”

Precision Electrophile Tagging in Caenorhabditis elegans

“…Indicated transgenic Halo worms following transgene induction were treated with bioinert photocaged precursors ( 1–3 ) to specific bioactive lipid-derived electrophiles (LDEs) (HNE 4 , dHNE 5 , and HDE 6 ). 3 At a user-defined time, photouncaging liberates the respective alkyne-functionalized LDEs ( 4–6 ) and proximity enhancement 21 enables labeling of a quintessential electrophile–sensor protein Keap1 (see also Figure S1A). Halo functionality, probe specificity, and LDE differential modification efficiency on target are assessed by three independent readouts.…”

“…3 These photocaged precursors were produced via Williamson ether synthesis, using anthraquinone and a corresponding Grignard-generated allylic bromide. 3 Using an optimized protocol, worms were treated with 30 μM probe for 6 h in liquid medium with OP50 bacteria (food) and then washed three times. After this point, worms were lysed by being frozen–thawed/vortexed with beads.…”

“…The half-life of HNE release in vivo under these conditions was on par with that previously assessed in cultured cells ( t 1/2 < 1–2 min). 3 …”

“…Bulk HNE exposure and T-REX give different extents of HNEylation on Keap1, suggesting that uptake/metabolism is a more significant variable in living model organisms than in living cells where HNEylation efficiencies are largely found to be comparable between the two conditions. 3 (C) Representative data set for input and elution samples. Actin serves as a loading control.…”