T-REX on-demand redox targeting in live cells

• Published in: Nature protocols Vol. 11; no. 12; pp. 2328 - 2356
• Main Authors: Parvez, Saba, Long, Marcus J C, Lin, Hong-Yu, Zhao, Yi, Haegele, Joseph A, Pham, Vanha N, Lee, Dustin K, Aye, Yimon
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.12.2016; Nature Publishing Group
• Subjects: 631/1647/1888; 631/45/612; 631/80/86/2365; 639/638/92/96; Aldehydes - metabolism; Amino Acid Sequence; Analytical Chemistry; Biological Techniques; Blotting, Western; Cell Survival; Computational Biology/Bioinformatics; Cytological Techniques - methods; E coli; Energy transfer; Escherichia coli - cytology; Health aspects; HEK293 Cells; Humans; Kinetics; Life Sciences; Microarrays; Organic Chemistry; Oxidants - metabolism; Oxidation-Reduction; Oxidation-reduction reaction; Oxidizing agents; Physiology; Proteins; Proteomics; Protocol; Sensors; Signal Transduction; New York; United States > US
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2016.114

Investigating the ramifications of site-specific protein redox modification in cells is challenging. This protocol uses HaloTagged proteins and a HaloTag-targetable photocaged 4-hydroxynonenal to elicit target-specific modifications and to trace their effects. 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 ( t 1/2 <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.