Protocol | Published:

T-REX on-demand redox targeting in live cells

Nature Protocols volume 11, pages 23282356 (2016) | Download Citation

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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.

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Change history

  • 10 November 2016

    In the version of this article initially published online, there were three errors. (1) In the Reagent Setup, the instructions for preparing LB-ampicillin-chloramphenicol medium gave an incorrect concentration for ampicillin; it should read "100 μg/ml ampicillin." (2) The text for Step 11B(vi) incorrectly described the bromination procedure; it should read: "Brominate 0.35 g of the resulting alcohol 6 (1.4 mmol) in 20 ml of distilled DCM at 4 °C by adding 0.5 g of CBr4 (1.54 mmol) and 0.44 g of PPh3 (1.68 mmol). Stir the mixture for 15 min" (instead of "...by adding 0.5 g of CBr4 (1.54 mmol) and 0.44 g of PPh3 (1.68 mmol) to 20 ml of distilled DCM at 0 °C"). (3) In Figure 10, under the arrow between compounds 12 and 13, the label "(ii) Me2S" should be omitted. These errors have been corrected for all versions of the article.

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Acknowledgements

We thank the laboratories of T. Evans (Weill Cornell Medicine, New York) and J. Zhang (University of California, San Diego) for plasmids encoding zebrafish hspb7 and the lnPAkt (PIP3-reporter) construct, respectively. We acknowledge all of the Aye Laboratory members who have contributed to T-REX redox targeting protocols, particularly J. Li, X. Fang and Y. Fu, as well as Q. Lin of the State University of New York at Albany for assistance with the LC–MS/MS analysis of modifications on the protein pulled down from cells. Funding was provided by the NIH Director's New Innovator award (1DP2GM114850), the National Science Foundation (NSF) CAREER award (CHE-1351400), the Beckman Young Investigator award and the Sloan Research Fellowship (to Y.A.) and the Burroughs Wellcome Funds CTRG (to principal investigator (PI) Y.A. and host T. Evans). S.P. is a Howard Hughes Medical Institute international predoctoral fellow (59108350). J.A.H. acknowledges the CBI training grant (T32GM008500, PI – H. Lin). V.N.P. thanks the Douglas family for an undergraduate research fellowship. D.K.L. thanks the Cornell University P3 scholars program. Imaging and flow cytometry data were acquired at the Cornell University Biotechnology Resource Center (NIH 1S10RR025502) and the Cornell University cytometry core (supported in part by the Empire State Stem Cell Fund), respectively. We acknowledge the NSF (NSF MRI: CHE-1531632 to Y.A. (PI)) for NMR instrumentation support at Cornell University.

Author information

Author notes

    • Saba Parvez
    •  & Marcus J C Long

    These authors contributed equally to this work.

Affiliations

  1. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.

    • Saba Parvez
    • , Marcus J C Long
    • , Hong-Yu Lin
    • , Yi Zhao
    • , Joseph A Haegele
    • , Vanha N Pham
    • , Dustin K Lee
    •  & Yimon Aye
  2. Department of Biochemistry, Weill Cornell Medicine, New York, New York, USA.

    • Yimon Aye

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Contributions

H.-Y.L. and Y.Z. were joint second authors of this work. S.P., M.J.C.L., H.-Y.L., Y.Z., J.A.H., V.N.P., D.K.L. and Y.A. developed protocols. S.P. and V.N.P. contributed to the data associated with T-REX in E. coli cells. M.J.C.L., Y.Z., J.A.H. and D.K.L. obtained the data associated with T-REX in cultured human cells. H.-Y.L. collected LC-MS/MS data. H.-Y.L. and Y.Z. contributed to chemical synthesis. S.P., M.J.C.L. and H.-Y.L. wrote the protocols. Y.A. wrote the manuscript with proofreading/editing contributions from S.P., M.J.C.L., Y.Z. and J.A.H.

Competing interests

Licensing application on this work is being pursued.

Corresponding author

Correspondence to Yimon Aye.

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–4 and Supplementary Tables 1 and 2

Videos

  1. 1.

    Setting up T-REX in cultured mammalian cells:

    Caption text: video shows a proper setup for the light illumination of cultured mammalian cells in a 48-well plate.

  2. 2.

    Setting up T-REX in E. coli

    Caption text: video shows a proper setup for the light illumination step.

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https://doi.org/10.1038/nprot.2016.114

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