T-REX on-demand redox targeting in live cells

Abstract

This protocol describes targetable reactive electrophiles and oxidants (T-REX)—a live-cell-based tool designed to (i) interrogate the consequences of specific and time-resolved redox events, and (ii) screen for bona fide redox-sensor targets. A small-molecule toolset comprising photocaged precursors to specific reactive redox signals is constructed such that these inert precursors specifically and irreversibly tag any HaloTag-fused protein of interest (POI) in mammalian and Escherichia coli cells. Syntheses of the alkyne-functionalized endogenous reactive signal 4-hydroxynonenal (HNE (alkyne)) and the HaloTag-targetable photocaged precursor to HNE (alkyne) (also known as Ht-PreHNE or HtPHA) are described. Low-energy light prompts photo-uncaging (t1/2 <1–2 min) and target-specific modification. The targeted modification of the POI enables precisely timed and spatially controlled redox events with no off-target modification. Two independent pathways are described, along with a simple setup to functionally validate known targets or discover novel sensors. T-REX sidesteps mixed responses caused by uncontrolled whole-cell swamping with reactive signals. Modification and downstream response can be analyzed by in-gel fluorescence, proteomics, qRT-PCR, immunofluorescence, fluorescence resonance energy transfer (FRET)-based and dual-luciferase reporters, or flow cytometry assays. T-REX targeting takes 4 h from initial probe treatment. Analysis of targeted redox responses takes an additional 4–24 h, depending on the nature of the pathway and the type of readouts used.

The Die Is Cast: Precision Electrophilic Modifications Contribute to Cellular Decision Making

“…Consistent with prior data suggesting that alkylation of this enzyme leads to loss of protein function, T-REX-enabled selective HNEylation of PTEN in cells resulted in accumulation of endogenous PIP3 phosphoinositide. 33 These data reinforce that inhibitory electrophilic modification on a single target is a functionally relevant event that can intercept canonical currency transfer processes such as phosphosignaling pathways (Figure 4b). Together with the Keap1 example above, these systems provide direct evidence supporting the single-hit model (Figure 3).…”

“…Under T-REX-enabled targeted LDEylation of Keap1 in low stoichiometry (20–60% modification efficiency depending on cell lines/context, RES chemotype, etc. ), 33−36 with no background labeling detected, selective Nrf2 stabilization and gain-of-function Nrf2-driven AR upregulation were observed. No AR activation was observed under otherwise identical conditions when Keap1 was not specifically targeted.…”

“…33−36 T-REX takes advantage of proximity-assisted targeting to enable controlled delivery of a reactive chemical signal to a specific protein of interest upon photoactivation (Figure 4, inset) (Class II proximity enhancement concept 37 ). The nuts and bolts of the platform are detailed elsewhere.…”

“…The nuts and bolts of the platform are detailed elsewhere. 33 Briefly, a protein of interest (POI) is expressed as a genetic fusion to HaloTag (an engineered protein that reacts stoichiometrically and irreversibly with chloroalkanes). Cells expressing the HaloTag-POI construct are treated with a specific chloroalkane-decorated inert probe capable of releasing a single HNE molecule in the presence of light.…”

Precision Electrophile Tagging in Caenorhabditis elegans

“…Built-in controls account for off-target effects and/or artifactual modifications/responses. 4,6 We recently adapted this platform to transient expression in larval zebrafish, 6 a popular vertebrate research model, and uncovered conserved signaling nodes that interchange RES-based non-enzyme-mediated PTM and conventional enzyme-regulated PTMs at a single-POI-precision scale. 10,15 This model provided new insights into redox signaling performed by specific Akt-kinase isoforms in developing fish but is currently incompatible with adult fish, restricting studies on aging.…”