Z-REX: Shepherding Reactive Electrophiles to Specific Proteins Expressed either Tissue-Specifically or Ubiquitously, and Recording the Resultant Functional Electrophile-Induced Redox Responses in Larval Fish

Abstract

This Protocol Extension describes the adaptation of an existing Nature Protocol detailing the use of T-REX (targetable reactive electrophiles and oxidants)-an on-demand redox targeting toolset in cultured cells. The adaptation described here is for use of REX technologies in live zebrafish embryos (Z-REX). Zebrafish embryos expressing a Halo-tagged protein of interest (POI)-either ubiquitously or tissue-specifically-are treated with a HaloTag-specific small-molecule probe housing a photocaged reactive electrophile (either natural electrophiles or synthetic electrophilic drug-like fragments). The reactive electrophile is then photouncaged at a user-defined time, enabling proximity-assisted electrophile-modification of a POI. Functional and phenotypic ramifications of POI-specific modification can then be monitored, by coupling to standard downstream assays, such as, Click chemistry-based POI-labeling and target-occupancy quantification; immunofluorescence or live imaging; RNA-Seq and qRT-PCR analyses of downstream-transcript modulations. Transient expression of requisite Halo-POI in zebrafish embryos is achieved by mRNA injection. Procedures associated with generation of transgenic zebrafish expressing a tissue-specific Halo-POI are also described. The Z-REX experiments can be completed in <1-week using standard techniques. To successfully execute Z-REX, researchers should have basic skills in fish husbandry, imaging, and pathway analysis. Experience with protein or proteome manipulation is useful. This protocol extension is aimed at helping chemical biologists study precision redox events in a model organism and fish biologists perform redox chemical biology.

NCBP1 electrophilic-stress signaling in the nucleus promotes alternatively-spliced S6K1 that dominantly inhibits global translation

“…2A), where electrophile is only available transiently and in substoichiometric amounts 13,15 . Notably, the percentage LO of NCBP1 (Figure 2D) was on par with the best electrophileresponder proteins we have examined [5][6][7][13][14][15][16][17][21][22][23][24][25] . Live imaging validated that localization of NCBP1-Halo is chiefly nuclear-restricted as anticipated (Figure 2E), consistent with an intrinsic nuclearlocalization signal within NCBP1.…”

“…1D, top-row). We note this workflow is different from some of our previous published protocols [5][6][7][13][14][15][16][17] in which we compared responsivity of nuclear and cytosolic proteins 6 . This difference, and the fact that the cell line used in this instance was different from our previous reports, explains why our previously reported hits were not discovered.…”

“…A) HEK293T cells ectopically expressing NCBP1-Flag-TEV-Halo (wt, or indicated Cys-to-Ala single-mutant), were subjected to T-REX (Fig. 2A) and HNE-ligand occupancy was quantified as prevously reported [13][14][15] , while also taking into account differences in expression levels (if any) as analyzed by anti-Flag western blot. See Supplemental Fig.…”

“…Figure 3. The 19 cysteines within NCBP1 play a compensatory HNE-sensing role; only C436-specific HNEylation functionally suppresses protein translation.A) HEK293T cells ectopically expressing NCBP1-Flag-TEV-Halo (wt, or indicated Cys-to-Ala single-mutant), were subjected to T-REX (Fig.2A) and HNE-ligand occupancy was quantified as prevously reported[13][14][15] , while also taking into account differences in expression levels (if any) as analyzed by anti-Flag western blot. See Supplemental Fig.S11Afor corresponding in-gel fluorescence (Cy5) and anti-Flag and anti-tubulin western blot analyses.…”

Structural Insights into Electrophiles and Electrophilic Metabolites Sensing and Signaling: The Missing Information

“…[1,9,13,15 -20,22 -30] Despite the myriad profiling tools (spearheaded by the innovative activity-based protein profiling (ABPP) method and its derivatives) and emerging functionguided proximity mapping-based REX technologies (T-REX, Z-REX, G-REX, and more recently, Localis-REX), several fundamental questions remain unaddressed, particularly with regard to structure/function relationship principles underlying electrophile-sensor protein regulation (Figure 2). [15,16,[18][19][20] makes a protein a KPS, given that a two units change in thiol pKa can only increase the reaction rate with peroxide by no more than 20-fold and that full cysteine thiol deprotonation only enhances the Michael addition reaction rate with the electrophile acrolein by a maximum of 10-fold, i. e., how is the more functionally relevant kinetic selectivity, such as transition state stabilization, achieved? [1] How does RES modification of a single site affect the sensor protein's activity/function, or how does it modulate the function of a binding partner, the interactome or downstream signal transduction?…”