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A chemical probe to modulate human GID4 Pro/N-degron interactions

Nature Chemical Biology (2024)Cite this article

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Abstract

The C-terminal to LisH (CTLH) complex is a ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via its substrate receptor Glucose-Induced Degradation 4 (GID4), but its function and substrates in humans remain unclear. Here, we report PFI-7, a potent, selective and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation and quantitative proteomics enabled the identification of GID4 interactors and GID4-regulated proteins. GID4 interactors are enriched for nucleolar proteins, including the Pro/N-degron-containing RNA helicases DDX21 and DDX50. We also identified a distinct subset of proteins whose cellular levels are regulated by GID4 including HMGCS1, a Pro/N-degron-containing metabolic enzyme. These data reveal human GID4 Pro/N-degron targets regulated through a combination of degradative and nondegradative functions. Going forward, PFI-7 will be a valuable research tool for investigating CTLH complex biology and facilitating development of targeted protein degradation strategies that highjack CTLH E3 ligase activity.

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Fig. 1: PFI-7 binds in the substrate binding pocket of human GID4.
Fig. 2: PFI-7 cellular target engagement and selectivity.
Fig. 3: GID4-BioID2 in the presence and absence of proteasome inhibition.
Fig. 4: GID4-BioID2 in the presence and absence of PFI-7.
Fig. 5: PFI-7 treatment alters the proteome of GID4-BioID2 HeLa cells.
Fig. 6: GID4- and PFI-7-dependent changes in RNA helicases and HMGCS1.

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Data availability

The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD038487. The mass spectrometry data of the chemoproteomics experiment have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD044977. The structure of PFI-7 bound to GID4 was deposited to the PDB with accession number 7SLZ. Source data are provided with this paper.

Code availability

R scripts for analysis of proteomics data are freely available at https://github.com/d0minicO/GID4_analysis.

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Acknowledgements

This work was supported by Mitacs Elevate Postdoctoral Fellowship to D.D.G.O., funding from the Canadian Institutes for Health Research (grant nos. MOP-142414 and PJT-169101 to C.S.-P.; FDN154328 to C.H.A.), Natural Sciences and Engineering Research Council of Canada grant nos. RGPIN-2021-02728 to J.M. and RGPIN-2021-03435 to D.B.L. and a Cancer Research Society grant no. 25418 to D.B.-L. M.P.S. is funded by the Deutsche Forschungsgemeinschaft CRC1430 (Project ID 424228829). V.R. and M.G. have received support from the EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no. 875510). We thank M. Robers and K. Riching from Promega for advising on the NanoBRET and target engagement assays. This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science user facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. Mass spectrometry analyses were performed on equipment funded by a grant from the Canada Foundation for Innovation to G.A.L. The Structural Genomics Consortium is a registered charity (no. 1097737) that receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through the Ontario Genomics Institute (OGI-196), EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no. 875510), Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda.

Author information

Author notes

  1. These authors contributed equally: Dominic D. G. Owens, Matthew E. R. Maitland.

Authors and Affiliations

  1. Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada

    Dominic D. G. Owens, Matthew E. R. Maitland, Aliakbar Khalili Yazdi, Xiaosheng Song, Raquel A. C. Machado, Magdalena M. Szewczyk, Cheng Dong, Aiping Dong, Peter Loppnau, Ella C. Adamson, Matthieu Schapira, Masoud Vedadi, Jinrong Min, Dalia Barsyte-Lovejoy & Cheryl H. Arrowsmith

  2. Robarts Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada

    Matthew E. R. Maitland, Xu Wang & Caroline Schild-Poulter

  3. Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada

    Matthew E. R. Maitland, Gilles A. Lajoie & Caroline Schild-Poulter

  4. Don Rix Protein Identification Facility, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada

    Matthew E. R. Maitland & Gilles A. Lajoie

  5. Institute of Molecular Systems Biology at ETH Zurich, Zurich, Switzerland

    Viviane Reber & Matthias Gstaiger

  6. Institut für Pharmazeutische Chemie, Goethe-University Frankfurt, Biozentrum, Frankfurt am Main, Germany

    Martin P. Schwalm, Nicolas Bauer & Stefan Knapp

  7. Structural Genomics Consortium, Goethe-University Frankfurt, Buchmann Institute for Life Sciences, Frankfurt am Main, Germany

    Martin P. Schwalm, Nicolas Bauer & Stefan Knapp

  8. Development and Medical, Pfizer Worldwide Research, Groton, CT, USA

    Matthew F. Calabrese, Matthew S. Dowling, Jisun Lee, Justin I. Montgomery, Thomas N. O’Connell, Chakrapani Subramanyam, Feng Wang & Dafydd R. Owen

  9. Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada

    Matthieu Schapira, Masoud Vedadi & Dalia Barsyte-Lovejoy

  10. Department of Physiology, University of Toronto, Toronto, Ontario, Canada

    Jinrong Min

  11. Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan, China

    Jinrong Min

  12. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

    Cheryl H. Arrowsmith

  13. Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada

    Cheryl H. Arrowsmith

Authors
  1. Dominic D. G. Owens
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  2. Matthew E. R. Maitland
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  3. Aliakbar Khalili Yazdi
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Contributions

M.E.R.M., D.D.G.O., X.W., E.C.A., M.M.S., R.A.C.M., P.L., D.B.-L. and C.S.-P. designed and performed cellular experiments. M.E.R.M. and G.A.L. designed and performed proteomic experiments. D.D.G.O. conducted proteomic data analysis. V.R. and M.G. designed, performed and analyzed the chemoproteomic experiments. N.B., M.P.S. and S.K. designed and performed GID4-tracer NanoBRET assay and generated NB716. A.K.Y. and M.V. designed and performed biophysical experiments. M.F.C., M.S.D., J.L., J.I.M., T.N.O., D.R.O., C.S. and F.W. designed and synthesized compounds. X.S., C.D., J.M. and A.D. performed crystallography studies and solved structures. C.H.A., M.V., J.M., D.B.-L., M.S., G.A.L. and C.S.-P. supervised research. C.H.A., D.B.-L., C.S.-P. and J.M. provided funding. C.H.A., D.B.-L., M.E.R.M. and D.D.G.O. wrote the manuscript.

Corresponding author

Correspondence to Cheryl H. Arrowsmith.

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Competing interests

M.F.C., M.S.D., J.L., J.I.M., T.N.O., C.S., F.W. and D.R.O. are or were employees of Pfizer and some of the authors are shareholders in Pfizer Inc. After submission, D.D.G.O. became an employee of Amphista Therapeutics, a company that is developing targeted protein degradation therapeutic platforms. The remaining authors declare no competing interests.

Peer review

Peer review information

Nature Chemical Biology thanks Nico Dissmeyer, Chao Xu 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 Enrichment profiling of NB716.

The volcano plot shows the proteins enriched by streptavidin affinity purification in the presence of NB716. GID4 is among the most highly enriched proteins. Many known interaction partners shown in blue are also highly enriched. The differential abundance was calculated and significance was determined using a two-tailed moderated t-test with multiple testing correction using the method of Benjamini-Hochberg. Data are from three independent experiments (n = 3).

Extended Data Fig. 2 Cytotoxic profile of PFI-7.

Cell growth curves for HCT116, HEK293T and U2OS cells treated with increasing concentrations of PFI-7 or PFI-E3H1 are shown over three days. Data are from three independent experiments (n = 3).

Extended Data Fig. 3 Characterization of GID4-BioID2 cell line.

a) Upper, schematic and Alphafold2 predicted structure of GID4-BioID2 fusion protein. Lower, partial CTLH complex structure (PDB 7NSC) showing GID4 C-terminal anchor interacting with ARMC8α (Ref. 31 – Sherpa et al, 2021). b) Immunoprecipitation experiments performed in HeLa cells. Left, HA-pull down of BioID2-GID4 fusion protein with immunoblotting to detect CTLH complex members. Right, immunoprecipitation of RanBP9 with immunoblotting to detect BioID2-GID4 (HA) and other CTLH complex members, *=heavy chain. Data are from one experiment (n = 1). c) Immunoblotting to detect BioID2-GID4 or BioID2 alone after doxycycline induction (top). Bottom, streptavidin-based detection of biotinylated proteins. Representative blot shown from one of five independent experiments (n = 5). d) Immunofluorescence imaging of HA in BioID2 and BioID2-GID4-expressing HeLa cells. HA signal is shown in red, DAPI is shown in blue. Representative images are shown from one of three independent experiments (n = 3). Scale bar: 20 µm. e) Nuclear and cytoplasmic fractionation of HeLa cells expressing BioID2-GID4 or HA-GID4 alone. Immunoblotting of Vinculin, SAP62, RanBP9, and HA is shown. Representative blot shown from one of two independent experiments (n = 23). f) Uniform Manifold Approximation and Projection (UMAP) analysis of proximity-dependent biotinylation samples. BioID2 and BioID2-GID4 cells are shown in gray and blue, respectively. MG132-treated samples are indicated as triangles, and samples treated with vehicle (DMSO) are shown as circles. UMAP was done on 196 high confidence GID4 interacting proteins (MG132 SP > 0.9). Data are from three independent experiments (n = 3, BioID2 + MG132) or four independent experiments (n = 4, BioID2 +DMSO, BioID2-GID4 + DMSO, BioID2-GID4 + MG132). g) Spectral counts of CTLH complex members detected in BioID2-GID4. Bar height represents mean and error bars indicate standard deviation. Data are from three independent experiments (n = 3, BioID2 + MG132) or four independent experiments (n = 4, BioID2 +DMSO, BioID2-GID4 + DMSO, BioID2-GID4 + MG132). h) Overlap between high confidence GID4 interactors and interactors of the CTLH complex present in the BioGRID database.

Source data

Extended Data Fig. 4 DDX50 and DDX21 association with GID4 and CTLH complex.

a) Immunoprecipitation of FLAG-GID4 in HEK293T cells expressing FLAG-GID4 treated with 1 µM PFI-7N or PFI-7 for 24 hours. Data are from two experiments (n = 2). b) Immunoprecipitation of RanBP9 in wild type (WT) and ARMC8 knock-out (ARMC8 KO) HeLa cells. Immunoblotting was done for DDX21/50, FLAG, RanBP9, Muskelin and ARMC8α. Data are from one experiment (n = 1). c) Confocal imaging analysis of U2OS cells transduced with a doxycycline inducible lentiviral expression vector coding for N terminally FLAG-tagged GID4. FLAG-GID4 expression was induced for 24 h and is shown in green, DDX21/50 (antibody recognizes both endogenous proteins) is shown in red, and DAPI is shown in blue. Scale bar represents 15 µm. Representative images are shown from four independent experiments. d) Quantification of fluorescence signal intensity over a cross section of cell nuclei. A rolling average with a window size of 8 was used to smooth data and maximum intensities were scaled to 1. One cell from each condition was quantified from one of four independent experiments.

Source data

Extended Data Fig. 5 Proteomic samples used and overlap with other datasets.

a) Proteomics samples with number of replicates and treatment conditions shown. Data are from four independent experiments (n = 4). These replicate numbers apply to all panels of the figure. b) Number of quantified proteins detected in each sample. Bar height represents mean values and error bars indicate standard deviation. N = 4 independent experiments. c) Protein abundances of CTLH complex members. Median-centered log2 intensities are shown, with blue dots indicating DMSO-treated samples and PFI-7-treated samples shown in red. Boxplot midline indicates median values, bounds of the box indicate 25th and 75th percentiles, and maxima and minima indicate the largest point above or below 1.5 * interquartile range. N = 4 independent experiments. d) Overlap between proteins significantly changed after PFI-7 treatment or GID4 overexpression and medium confidence GID4 interactors (SP > 0.6 in any condition), left. Right, PFI-7-dependent and GID4-dependent proteins overlap with Pro/N-degron-containing proteins. SP scores derived from N = 3 independent experiments in BioID2-GID4 data, protein abundance change significance derived from n = 4 independent expression proteomics experiments.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2, Tables 1 and 2 and Note.

Reporting Summary

Supplementary Data 1–6

Proteomics data.

Supplementary Data 7

Unprocessed western blots for Supplementary Fig. 2.

Source data

Source Data Fig. 1

SPR and fluorescence polarization data.

Source Data Fig. 2

NanoBRET quantification of GID4 with Pro/N-degron, GID4-tracer titration and PFI-7 tracer competition.

Source Data Fig. 4

NanoBRET quantification of GID4 interaction with DDX21 and DDX50.

Source Data Fig. 6

Quantification of HMGCS1 levels by western blot after PFI-7 and cycloheximide treatments.

Source Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 3

Unprocessed western blots.

Source Data Extended Data Fig. 4

Unprocessed western blots.

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Owens, D.D.G., Maitland, M.E.R., Khalili Yazdi, A. et al. A chemical probe to modulate human GID4 Pro/N-degron interactions. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01618-0

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