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T-REX on-demand redox targeting in live cells
Saba Parvez1, Marcus J C Long1, Hong-Yu Lin1
1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.
Nature Protocols
|November 4, 2016
Summary
This study introduces Targetable Reactive Electrophiles and Oxidants (T-REX), a novel live-cell tool for precise redox event analysis and sensor screening. T-REX enables specific protein modification, avoiding uncontrolled cellular responses for accurate biological insights.
Area of Science:
- Cell biology
- Biochemistry
- Chemical biology
Background:
- Redox signaling is crucial for cellular processes.
- Existing methods for studying redox events lack specificity and temporal control.
- Identifying bona fide redox sensors remains challenging.
Purpose of the Study:
- To develop a live-cell tool for interrogating specific, time-resolved redox events.
- To create a method for screening redox-sensor targets.
- To enable precise, spatially controlled redox modifications in cells.
Main Methods:
- Development of photocaged precursors to reactive redox signals (e.g., 4-hydroxynonenal).
- Utilizing HaloTag-fused proteins for targeted labeling of proteins of interest (POIs).
- Employing low-energy light for rapid photo-uncaging and specific POI modification.
Main Results:
- Successful synthesis of alkyne-functionalized 4-hydroxynonenal and its photocaged precursor.
- Demonstration of rapid (t1/2 <1-2 min) photo-uncaging and target-specific modification.
- Validation of the T-REX system for functional analysis of known targets and discovery of novel sensors without off-target effects.
Conclusions:
- T-REX provides a robust platform for precise redox event analysis in live cells.
- The tool enables specific and time-resolved interrogation of redox signaling pathways.
- T-REX facilitates the discovery and validation of redox-sensor proteins.
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