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Ubiquitin-specific proximity labeling for the identification of E3 ligase substrates

Nature Chemical Biology (2024)Cite this article

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Abstract

Protein ubiquitylation controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in disease. Moreover, small-molecule degraders that redirect ubiquitylation activities toward disease targets are an emerging and promising therapeutic class. Over 600 E3 ubiquitin ligases are expressed in humans, but their substrates remain largely elusive, necessitating the development of new methods for their discovery. Here we report the development of E3-substrate tagging by ubiquitin biotinylation (E-STUB), a ubiquitin-specific proximity labeling method that biotinylates ubiquitylated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitylated targets of protein degraders, including collateral targets and ubiquitylation events that do not lead to substrate degradation. It also detects known substrates of E3 ligase CRBN and VHL with high specificity. With the ability to elucidate proximal ubiquitylation events, E-STUB may facilitate the development of proximity-inducing therapeutics and act as a generalizable method for E3-substrate mapping.

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Fig. 1: E-STUB enables biotinylation of E3 substrates in a ubiquitin-specific and proximity-dependent manner.
Fig. 2: E-STUB identifies the expected substrates of various molecular glues and PROTACs and demonstrates high specificity.
Fig. 3: E-STUB identifies the induced targets of multikinase degraders, including those without a degradative outcome.
Fig. 4: Collateral ubiquitylation of corepressor complexes by multi-HDAC degraders.
Fig. 5: Identification of physiological E3 substrates.

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Data availability

Raw data files of all MS studies in this study have been deposited in the PRIDE Archive, including PXD042673, PXD042691, PXD042687, PXD047583, PXD042693, PXD042674, PXD042697, PXD042700, PXD042698, PXD042702, PXD042694, PXD042706, PXD021255, PXD042657 and PXD047455. Processed protein abundance values and results from limma analyses are provided in Supplementary Data 1–4. Details on the experimental conditions, associated figures and corresponding PRIDE accession number can be also found in the ‘metadata’ tab in Supplementary Data 1–4. Source data are provided with this paper.

Code availability

The code necessary to reproduce the statistical analysis for quantitative proteomics can be found at: https://doi.org/10.5281/zenodo.10633173.

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Acknowledgements

This work was supported by the Merkin Institute for Transformative Technologies in Healthcare and the Ludwig Center at Harvard. BI-0319 and BI-3663 were provided by Boehringer Ingelheim via its open innovation platform opnMe (available at https://opnme.com). SK-3-91 and SB1-G-187 were provided by N.S. Gray (Stanford University, CA, USA). DB0646 was provided by T. Sim (Yonsei University College of Medicine, Republic of Korea). WH-10417-099 was provided by S.J. Buhrlage (Dana-Farber Cancer Institute, MA, USA). We thank W.G. Kaelin for helpful comments; M. L. Meyerson (Dana-Farber Cancer Institute, MA, USA) for providing the pRK5-puroR and PX330 constructs; J.A. Cutler, M. Slabicki, H. Yoon and Y.-D. Li for helpful discussions; D. Kesar for advice on bioinformatic analysis; K. L. Shaw for proofreading the paper. W.R.S. was supported by R01CA233626 from the National Institutes of Health (NIH).

Author information

Authors and Affiliations

  1. Broad Institute of Harvard and MIT, Cambridge, MA, USA

    Hai-Tsang Huang, Ryan W. Tsai, Shuyao Su, Xu Zhao, James Chen & William R. Sellers

  2. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    Hai-Tsang Huang & William R. Sellers

  3. Harvard Medical School, Boston, MA, USA

    Hai-Tsang Huang & William R. Sellers

  4. Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA

    Ryan J. Lumpkin, Yuan Xiong, Nada Mageed, Katherine A. Donovan & Eric S. Fischer

  5. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Ryan J. Lumpkin, Yuan Xiong, Katherine A. Donovan & Eric S. Fischer

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Contributions

H.-T.H. and W.R.S. conceived the project. H.-T.H., R.W.T. and S.S. performed the E-STUB experiments. K.A.D. and N.M. performed the total proteomics experiments. R.J.L. and K.A.D. acquired the MS data. R.J.L., K.A.D., H.-T.H. and X.Z. performed bioinformatic analysis. H.-T.H, R.T., S.S. and J.C. performed the biological validation and analysis. Y.X. synthesized the HDAC degraders. H.-T.H. prepared the figures for the main text and the extended data. H.-T.H. and W.R.S. wrote the paper with critical reading and feedback from the other coauthors. W.R.S. and E.S.F. supervised the project.

Corresponding author

Correspondence to William R. Sellers.

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Competing interests

W.R.S. and H.-T.H. are inventors on a filed patent describing E-STUB (PCT application publication WO/2023/235313). W.R.S. is a board or scientific advisory board (SAB) member and holds equity in Ideaya Biosciences, Civetta Therapeutics, Red Ridge Bio, Delphia Therapeutics and 2Seventy Bio. W.R.S. has consulted for Array, Astex, Epidarex Capital, Ipsen, PearlRiver Therapeutics, Merck Pharmaceuticals, Sanofi, Servier and Syndax Pharmaceuticals and receives research funding from Pfizer Pharmaceuticals, Merck Pharmaceuticals, Ideaya Biosciences, Calico, Boehringer Ingelheim, Bristol Myers Squibb, Novartis Institutes for Biomedical Research, Bayer Pharmaceuticals and Ridgeline Discovery. E.S.F. is a founder, SAB member and equity holder of Civetta Therapeutics, Lighthorse Therapeutics, Proximity Therapeutics and Neomorph (also board of directors). E.S.F. is an equity holder and SAB member for Avilar Therapeutics and Photys Therapeutics and a consultant to Novartis, Sanofi, EcoR1 Capital, Ajax Therapeutics and Deerfield. The Fischer Lab receives or has received research funding from Deerfield, Novartis, Ajax, Interline and Astellas. K.A.D. is a consultant to Kronos Bio and Neomorph. The other authors declare no competing interests.

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Nature Chemical Biology thanks Wei Qin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Development and optimization of E-STUB.

a, Immunoblotting analysis of Avi-ubiquitylated and biotinylated proteins in 293T cells expressing Avi-ubiquitin, 3F-BirA-mCherry, and treated with biotin (50 μM), where indicated. Images from a single experiment. b, Immunoblotting analysis of GSPT1 abundance after 4-hour CC-885 (1 μM) treatment in 293T, 293TCRBN−/− and 293TCRBN−/− cells expressing CRBN-BirA or BirA-CRBN as indicated. Images from a single experiment. c, 293T cells expressing Avi-ubiquitin, CRBN-BirA and V5-IKZF1 were treated with carfilzomib (0.4 μM, 2 hours), and where indicated, POM (1 μM, 1 hour) and biotin (50 μM, 15 minutes). Right: ectopically expressed proteins detected by immunoblotting. Middle: Avi-ubiquitylation and biotinylation of immunoprecipitated V5-IKZF1 detected by immunoblotting. Left: V5-IKZF1 and biotinylated proteins enriched by streptavidin beads detected by immunoblotting. Representative of two independent measurements. POM, pomalidomide. IP, immunoprecipitation. AP, affinity purification. d, 293T cells expressing the indicated variant of BAP-ubiquitin, V5-IKZF1 and CRBN-BirA were treated with carfilzomib (0.4 μM, 2 hours), and where indicated, POM (1 μM, 1 hour) and biotin (50 μM, 15 minutes). Left: ectopically expressed and biotinylated proteins detected by immunoblotting. Right: BAP-ubiquitylation and biotinylation of immunoprecipitated V5-IKZF1 detected by immunoblotting. Representative of two independent measurements. e, 293TCRBN−/− cells expressing the indicated variant of BAP-ubiquitin and CRBN-BirA were treated with carfilzomib (0.4 μM, 3 hours), biotin (50 μM, 15 minutes), and where indicated, CC-885 (1 μM, 2 hours). GSPT1, ectopically expressed proteins, and biotinylated proteins enriched by streptavidin beads were detected by immunoblotting. Representative of two independent measurements.

Source data

Extended Data Fig. 2 Two-day biotin depletion does not affect cellular health and response.

a,b, Two-day growth curves of 293T (a) or A375 (b) cells grown in normal media versus biotin-depleted media. Individual data points were shown for two biological replicates. c,d, Scatter plot showing fold change (FC) in protein abundance of 293T cells treated with SK-3-91 (1 μM) (c) or XY-07-187 (1 μM) (d) for 5 hours when cultured in biotin-depleted media (2-day depletion) versus normal media. Proteins that are significantly degraded in both conditions (log2 FC < −0.585; p-value < 0.001) are highlighted in yellow; proteins only significantly degraded in the biotin-depleted condition are highlighted red; proteins only significantly degraded in the normal media condition are highlighted blue; significant hits that are detected in one condition but not the other are shown in the respective gray boxes labeled n.d. (not detected). e, FC in protein abundance of 293T cells grown in biotin-depleted media (2-day depletion) vs normal media. Total proteomics experiments were evaluated by label-free quantification (timsTOF Pro2) in biological triplicates. log2(FC) and P values were calculated by two-sided moderated t test implemented by the limma package. Adjusted P values of multiple comparisons are included in Supplementary Data 4.

Extended Data Fig. 3 Unbiased discovery of chemically induced substrates by E-STUB and mass spectrometry.

a, Schematic of E-STUB workflow for substrate identification by mass spectrometry. b. Left: rank-ordered abundance plot highlighting top 50 streptavidin-enriched proteins from an E-STUB experiment in 293TCRBN−/− cells expressing CRBN-BirA and A3-Ub after 2-hour CC-885 (1 μM) treatment. Right: heatmap showing the respective abundances of those top 50 streptavidin-enriched proteins in CC-885-treated versus DMSO-treated conditions. c, Immunoblotting analysis of protein inputs and those enriched by Ni-NTA resins from 293T cells expressing His-tagged ubiquitin, Flag-tagged CRBN and the indicated V5-tagged proteins. The cells were treated with carfilzomib (0.4 μM) and, where indicated, with CC-885 (1 μM) for 2 hours. Images from a single experiment. d,e, E-STUB data showing fold change (FC) in abundance of streptavidin-enriched proteins following 1-hour CC-885 (1 μM) treatment in U2OS cells expressing CRBN-BirA and A3-Ub (d) or 1-hour E7820 (1 μM) treatment in 293T cells expressing DCAF15-BirA and A3-Ub (e). All E-STUB experiments were evaluated by label-free quantification (Exploris 480) in biological triplicates. log2(FC) and P values were calculated by two-sided moderated t test implemented by the limma package. Adjusted P values of multiple comparisons are included in Supplementary Data 2.

Source data

Extended Data Fig. 4 E-STUB characterization of multikinase degrader SK-3-91.

a, Bubble chart showing fold change (FC) in abundance (represented by size of dots) and statistical significance (represented by saturation of color) of significant biotinylated proteins (log2 FC > 1; P value < 0.001) in an E-STUB experiment where 293TCRBN−/− cells expressing CRBN-BirA and A3-Ub were treated with SK-3-91 (1 μM) for 15 minutes, 1 hour and 4 hours. b, E-STUB data showing FC in abundance of streptavidin-enriched proteins following 1-hour SK-3-91 (1 μM) treatment in 293TCRBN−/− cells expressing CRBN-BirA and A3-Ub. c, FC in abundance (represented by size of dots) and statistical significance (represented by saturation of color) from b overlaid on top of a volcano plot displaying FC in protein abundance of 293T cells treated with SK-3-91 (1 μM) for 5 hours. Proteins identified in total proteomics that are not detected by E-STUB are shown as cross (×). d, Scatter plot showing FC in abundance of streptavidin-enriched proteins described in b versus FC in total protein abundance described in c. Protein that are both significantly biotinylated (log2(FC) > 1; P value < 0.001) and degraded (log2(FC) < −0.585; P value < 0.001) are highlighted in yellow; proteins only significantly biotinylated are highlighted in red; proteins only significantly degraded are highlighted in blue; significant hits that are detected by one assay but not the other are shown in the respective gray boxes labeled n.d. (not detected). The E-STUB experiment shown in a was evaluated by label-free quantification (Exploris 480) in biological triplicates. The E-STUB experiment shown in b–d was evaluated by label-free quantification (timsTOF Pro2) in biological triplicates. Total proteomics experiments were evaluated by TMT quantitative proteomics (Exploris 480) in at least biological duplicates30. log2(FC) and P values were calculated by two-sided moderated t test implemented by the limma package. Adjusted P values of multiple comparisons are included in Supplementary Data 2 and 4.

Extended Data Fig. 5 Targeted engagement does not predict induced ubiquitylation.

a–e, Scatterplots comparing induced bio-ubiquitylation (E-STUB) and kinase engagement (KiNativ) for SK-3-91 (a), SB1-G-187 (b), DB0646 (c), WH-10417-099 (d) and SK-3-91 (e; timsTOF Pro2). KiNativ data were previously published30. E-STUB data correspond to those shown in Fig. 3a (a), Fig. 3d (b), Fig. 3g (c), Fig. 3j (d) and Extended Data Fig. 4b (e). The coloring scheme for each data point also follows the rules specified in Fig. 3c,f,i,l and Extended Data Fig. 4d.

Extended Data Fig. 6 Protein degradation kinetics in response to multi-HDAC degrader XY-07-187.

Immunoblotting analysis of select HDACs, corepressor complex proteins and known short half-life proteins from 293T cells treated with CHX (50 μg/ml), XY-07-187 (1 μM) or both over a 6-hour time course. CHX, cycloheximide. Representative images of two independent measurements.

Source data

Extended Data Fig. 7 Substrate identification utilizing mutant E3 ubiquitin ligases.

a, E-STUB data showing fold change (FC) in abundance of streptavidin-enriched proteins in 293T cells expressing A3-Ub and VHL-BirA versus 293TCRBN−/− cells expressing A3-Ub and CRBN-BirA. b,c, E-STUB data showing FC in abundance of streptavidin-enriched proteins in 293TCRBN−/− cells expressing A3-Ub and CRBN(W386A)-BirA versus CRBN(WT)-BirA (b) or CRBN(D249Y)-BirA versus CRBN(WT)-BirA (c) after 1-hour CC-885 (1 μM) treatment. All E-STUB experiments were evaluated by label-free quantification (Exploris 480) in biological triplicates. log2(FC) and P values were calculated by two-sided moderated t test implemented by the limma package. Adjusted P values of multiple comparisons are included in Supplementary Data 2.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2.

Reporting Summary

Supplementary Data 1

Quantification of streptavidin-enriched proteins of all E-STUB experiments in this study.

Supplementary Data 2

Differential analysis implemented by the limma package of all E-STUB experiments in this study.

Supplementary Data 3

Protein quantification of all total proteomics experiments in this study.

Supplementary Data 4

Differential analysis implemented by the limma package of all total proteomics experiments in this study.

Source data

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Source Data Extended Data Fig. 1

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Huang, HT., Lumpkin, R.J., Tsai, R.W. et al. Ubiquitin-specific proximity labeling for the identification of E3 ligase substrates. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01590-9

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