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Curr Protoc Chem Biol. 2018 Sep;10(3):e43. doi: 10.1002/cpch.43. Epub 2018 Aug 7.

Single-Protein-Specific Redox Targeting in Live Mammalian Cells and C. elegans.

Author information

1
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York.
2
Ecole Polytechnique Fédérale de Lausanne, Institute of Chemical Sciences and Engineering, Lausanne, Switzerland.
3
Department of Biochemistry, Weill Cornell Medicine, New York, New York.

Abstract

T-REX (targetable reactive electrophiles and oxidants) enables electrophile targeting in living systems with high spatiotemporal precision and at single-protein-target resolution. T-REX allows functional consequences of individual electrophile signaling events to be directly linked to on-target modifications. T-REX is accomplished by expressing a HaloTagged protein of interest (POI) and introducing a Halo-targetable bioinert photocaged precursor to a reactive electrophilic signal (RES). Light exposure releases the unfettered RES on demand, enabling precision modification of the POI due to proximity. Using alkyne-functionalized 4-hydroxynonenal (HNE) as a representative RES, this protocol delineates optimized strategies to (1) execute T-REX in live human cells and C. elegans, (2) quantitate the POI's RES-sensitivity by either azido-fluorescent-dye conjugation or (3) enrich using biotin-azide/streptavidin pulldown procedure in both model systems, and (4) identify the site of RES-labeling on the POI using proteomics. Built-in T-REX controls that allow users to directly confirm on-target/on-site specificity of RES-sensing are also described.

KEYWORDS:

4-hydroxynonenal; C. elegans; HaloTag; T-REX; electrophile modifications; redox signaling

PMID:
30085412
PMCID:
PMC6125161
[Available on 2019-09-01]
DOI:
10.1002/cpch.43
[Indexed for MEDLINE]

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