T-REX on-demand redox targeting in live cells.
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.
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.
Saba
S
Parvez
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.
Marcus J C
MJ
Long
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.
Hong-Yu
HY
Lin
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.
Yi
Y
Zhao
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.
Joseph A
JA
Haegele
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.
Vanha N
VN
Pham
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.
Dustin K
DK
Lee
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.|Department of Biochemistry, Weill Cornell Medicine, New York, New York, USA.
Yimon
Y
Aye
D000447:Aldehydes / Q000378:metabolism; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002470:Cell Survival; D003584:Cytological Techniques / Q000379:methods*; D004926:Escherichia coli / Q000166:cytology; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D015398:Signal Transduction
D000447:Aldehydes; D016877:Oxidants; C027576:4-hydroxy-2-nonenal