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T-REX on-demand redox targeting in live cells

This article has been updated

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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Figure 1: Strategies for studying cellular redox responses.
Figure 2: On-target, on-demand redox signaling enabled by T-REX.
Figure 3: T-REX approach allows flexibility while enabling quantification of modification and response at numerous points.
Figure 4: Commercial HaloTag library allows discovery and validation of 'first responders' to a specific LDE using T-REX.
Figure 5: Assessment of N- versus C-terminal HaloTagging on T-REX functionality, exemplified by Keap1 LDE targeting.
Figure 6: T-REX targeting is equally efficient in human (HEK-293) cells and E. coli.
Figure 7: Flow-cytometry-based ARE-GFP reporter assay quantifying T-REX-mediated activation of antioxidant response in a subpopulation of live HEK-293 cells.
Figure 8: Immunofluorescence analysis of endogenous PIP3 phosphoinositide in fixed cells subsequent to PTEN-targeted redox modification enabled by T-REX in live cells.
Figure 9: FRET-based biosensor assay in live cells, reporting the levels of endogenous PIP3 subsequent to PTEN-targeted redox modification enabled by T-REX.
Figure 10

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.

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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.

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Correspondence to Yimon Aye.

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Supplementary Figure 1 Executing T-REX in mammalian cells

HEK-293 cells cultured (a) in 2x 55 cm2 adherent cell culture plates, and (b and c) in a 48-well multi-well adherent cell culture plate. No cover was placed on the plates during photo-uncaging. See Main Text for detailed experimental conditions and equipment specifications. Also see Supplementary Videos 1, 2

Supplementary Figure 2 Evaluation of time-dependent redox signal release in cells in T-REX method and validation that HaloTag does not react with HNE.

(a) Measurements of HNE release efficiency in cells. HEK293T cells expressing HaloTag alone treated under standard T-REX conditions with Ht-PreHNE were exposed to UV light (0.3 mW/cm2, 365 nm) for the indicated time periods at which point the cells were harvested, lysed, and subjected to Click coupling and in-gel fluorescence analysis followed by western blot. Error bars designate SD (N=3). (b) Controls to show that HaloTag does not react with HNE. Purified recombinant HaloTag was treated with either the photocaged precursor Ht-PreHNE (Figure 2, inset, and Figure 10) (2 equiv., lane a, positive control), or directly with reactive electrophile HNE (Figure 10) (0, 2, 4, 8, 16 equiv., lane b, c, d, e, f, respectively). After 20-min incubation, the samples were analyzed by in-gel fluorescence. M, molecular weight ladder.

Supplementary Figure 3 UV light exposure employed in T-REX is non-invasive: representative data for γ-H2AX1 and NF-κB2, markers for DNA damage and inflammatory signaling, respectively.

(a) HEK293T cells were exposed to UV light (0.3 mW/cm2, 340 nm) for the indicated time periods. Mitomycin C (10 μg/ml for 24 h)3 and aphidicolin (10 μg/ml for 36 h)4 serve as positive controls. After 12 hours post the end of UV illumination, cells were fixed and analyzed by standard immunofluorescence imaging method using γ-H2AX antibody (Millipore 05-636 at 1:1000 dilution). Data show mean +/- S.D. N > 50 cells. (b) HEK293T cells stably expressing NRE-inducible firefly luciferase5 were transfected with the respective plasmids encoding indicated transgene (empty vector, HaloTag alone, or Halo-Keap1 and Renilla luciferase) under constitutive CMV promoters. 24 hours post transfection, half of the plates were exposed to UV light (0.3 mW/cm2, 365 nm) over 20 min. Phorbol 12-myristate 13-acetate (PMA) (10 ng/mL, 18 h) was used as a positive control for NRE activation5. NRE activation was measured after 18 hrs. Error bars designate S.D. (N = 8 biological replicates).

Supplementary Figure 4 T-REX screen of Halo ORF clones for the discovery of novel electrophile-sensitive targets and pulldown validation of expressed proteins exemplified by zebrafish HSPB7.

(a) T-REX-enabled gel-based screen for bona fide HNE-sensitive targets using Halo-ORFeome library (Promega). Individual wells in a 48-well plate contained live HEK-293 cells ectopically expressing a unique HaloTagged gene of interest. The cells were subjected to T-REX-HNE(alkyne) targeting on demand. Post cell lysis, all samples were treated with TEV protease and subsequently subjected to Click coupling reaction with Cy5 azide. Probing with Halo antibody allowed evaluation of expression level (and/or solubility under the lysis conditions used). The “hit” bands on Cy5-fluorescent gel were judged against Halo protein level revealed by western blot. For example, RRM1, PRKCD, p53R2, and Keap1 (positive control) had roughly similar expression levels. Only RRM1 and Keap1 were HNE-sensitive although all four targets have been previously identified to be potentially redox/HNE-sensitive6–10. See Main Text for discussion. , a non-specific band. Also see Figure 4 and procedural details in Main Text. (b) Zebrafish HSPB7 expression and protein ID of the band shown in Figure 4a was validated by enrichment from HEK-293 cells ectopically expressing Halo-HSPB7 with the use of HaloTag PEG-Biotin ligand (Promega G8592) and streptavidin sepharose beads (GE Healthcare, cat. no. 17-5113-01), and subsequent on-bead TEV-protease cleavage followed by gel electrophoresis analysis. Theoretical MW of HSPB7 ~ 18 kDa. L, MW ladder.

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Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 1 and 2 (PDF 2343 kb)

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. (MP4 35071 kb)

Setting up T-REX in E. coli

Caption text: video shows a proper setup for the light illumination step. (MP4 3685 kb)

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Parvez, S., Long, M., Lin, HY. et al. T-REX on-demand redox targeting in live cells. Nat Protoc 11, 2328–2356 (2016). https://doi.org/10.1038/nprot.2016.114

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