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cIAP1-based degraders induce degradation via branched ubiquitin architectures

Abstract

Targeted protein degradation through chemical hijacking of E3 ubiquitin ligases is an emerging concept in precision medicine. The ubiquitin code is a critical determinant of the fate of substrates. Although two E3s, CRL2VHL and CRL4CRBN, frequently assemble with proteolysis-targeting chimeras (PROTACs) to attach lysine-48 (K48)-linked ubiquitin chains, the diversity of the ubiquitin code used for chemically induced degradation is largely unknown. Here we show that the efficacy of cIAP1-targeting degraders depends on the K63-specific E2 enzyme UBE2N. UBE2N promotes degradation of cIAP1 induced by cIAP1 ligands and subsequent cancer cell apoptosis. Mechanistically, UBE2N-catalyzed K63-linked ubiquitin chains facilitate assembly of highly complex K48/K63 and K11/K48 branched ubiquitin chains, thereby recruiting p97/VCP, UCH37 and the proteasome. Degradation of neo-substrates directed by cIAP1-recruiting PROTACs also depends on UBE2N. These results reveal an unexpected role for K63-linked ubiquitin chains and UBE2N in degrader-induced proteasomal degradation and demonstrate the diversity of the ubiquitin code used for chemical hijacking.

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Fig. 1: Degrader-activated cIAP1 assembles branched ubiquitin chains.
Fig. 2: UBE2N promotes degrader-induced cIAP1 degradation and apoptosis.
Fig. 3: K63 linkages serve as the base for K11/K48/K63 heterotypic ubiquitin chains.
Fig. 4: Architecture of branched ubiquitin chains conjugated on cIAP1.
Fig. 5: UBE2N and UBE2D cooperatively assemble branched ubiquitin chains onto cIAP1 in vitro.
Fig. 6: Generality of UBE2N-mediated (neo-)substrate degradation.

Data availability

The raw datasets for large-scale proteomics analyses have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD029945. MS/MS spectra were blasted against the SwissProt-reviewed H. sapiens reference proteome (UniProt version 2017-10-25). Source data for western blots and graphs are presented in the Source Data files. Source data are provided with this paper.

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Acknowledgements

We thank Y. Demizu for preliminary results, H. Tsuchiya for discussions and Y. Kawase and S. Ono for technical assistance. This work was supported, in part, by JSPS KAKENHI (grant nos. JP21H02433, JP18H05498 and JP20K21408 to F.O.; JP18H05498 to Y.S.; JP19H00997 to K.T.; JP18H05504 to A.O.; and JP18H05502 to M.N.), AMED-CREST (grant no. 21458950 to F.O.), the Takeda Science Foundation (to F.O.) and the Naito Foundation (to F.O.).

Author information

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Contributions

F.O. designed the project and analyzed the data. Y.A. and F.O. performed most of the cell-based and in vitro experiments and mass spectrometric analyses. M.M., Y.M. and A.K.-S. assisted with cell-based experiments. A.E. and F.O. performed TMT-based proteomics. M.N., Y.S. and K.T. provided reagents and advice. S.D., M.S., G.H. and A.O. performed chemical synthesis of ubiquitin derivatives. F.O. wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Fumiaki Ohtake.

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Nature Chemical Biology thanks John Harling, Yogesh Kulathu 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 Role of UBE2N in the degradation of cIAP1.

a. Generation of UBE2N/mut HCT116 cell lines using CRISPR/Cas9 genome editing. The amino acid sequences of the alleles in clone #23 are shown. b. Biological triplicates for the experiment presented in Fig. 2a. c. Attenuated UBE2N does not affect global cellular substrate ubiquitylation. Wild-type and UBE2N /mut HCT116 cells were treated with 20 μM MG132 for 1 or 2 h as indicated. Ub (P4D1): anti-Ub antibody.

Source data

Extended Data Fig. 2 Analysis of cIAP1 degradation.

a. Protein levels of UBE2N in wild-type (WT) and UBE2N/mut HCT116 cells (lanes 1-8) and HCT116 cells treated with the indicated siRNAs (lanes 9–17) were analyzed using quantitative immunoblotting. The data represent raw data for Fig. 2d. b. HCT116 cells transfected for 72 h with the indicated siRNAs were treated with 10 nM LCL-161 for the indicated times (h). Scr: scrambled siRNA. Arrows indicate mono- and di-ubiquitylated cIAP1. c. Wild-type (WT) and UBE2N/mut HCT116 cells were treated with the indicated concentrations of LCL-161 for 24 h, and cell lysates were used for western blot analysis.

Source data

Extended Data Fig. 3 Analysis of K48/K63 branched linkages.

a. Enzymatic specificities of OTUB1* and AMSH*. These enzymes specifically cleave their cognate target linkages. b. Schematic representation of the method used to detect and quantify K48/K63 branched linkages. Ubiquitin chains containing the Ub(R54A) mutant were used for in-gel digestion, and the K48/K63 branched linkage–derived signature peptide was quantified. c. Related to Fig. 4a-b, HEK293T cells were transfected with FLAG-cIAP1 (wild-type or the H588A mutant) and Ub (R54A). cIAP1-conjugated ubiquitin chains were purified using an anti-FLAG antibody, and stained with linkage-specific anti-K48Ub or anti-K63Ub antibodies. d. UBE2N is required for LCL-161–induced assembly of K48/K63 branched ubiquitin chains on cIAP1. HEK293T cells transfected with the indicated siRNAs and FLAG-cIAP1 were treated with 100 nM LCL-161 for 50 min. MG132 was added to all the samples for 60 min. The cell lysates were used for immunoprecipitation analysis using an anti-FLAG antibody. The precipitated ubiquitin chains were quantified using Ub-AQUA/PRM. Three biological replicates.

Source data

Extended Data Fig. 4 Chemical synthesis of GG branched Ub.

a. Outline for the synthesis of GG branched Ub(Δ75,76). b. HPLC chromatograms for the desulfurization steps and MALDI-TOF mass spectrometry of the purified products. (a) K48/K63-2xGG, (b) K11/K48-2xGG, and (c) K11/K63-2xGG. c. Mass spectrometry data for the Ub(1–74)-2xGG species. The Ub compounds were used for LC-MS analysis.

Extended Data Fig. 5 Signature MS2 ions of 2xGG-Ub(1-74) species.

a. Schematic representation of the Ub(1–74) species modified with two di-Gly remnants (2xGG Ub) and the corresponding masses of the y ions. Because K11-, K48-, and K63-linkages account for ~95% of the total ubiquitin chains, most of the 2xGG Ubs are modified at either K11/K48, K11/K63, or K48/K63. When the 2xGG Ub peptides are fragmented using higher collision energy dissociation (HCD), the y38 and y12 ions have different masses depending on the sites where the GGs are conjugated. Specifically, the y38 ion containing two GGs represents the signature for K48/K63 branched linkages. Likewise, the y12 ion that contains no GGs represents the signature for K11/K48 branched linkages.

Extended Data Fig. 6 Assembly of branched ubiquitin chains on cIAP1 in vitro.

a. Scheme for the sequential 3-step ubiquitylation assay in fig. 5b–e. b. The band intensities for Ub1, Ub2/Ub3, and poly-Ub bands and smears in the anti-cIAP1 blot (Fig. 5a) were quantified. Two biological replicates. c. 2-step in vitro ubiquitylation of cIAP1. cIAP1 was first incubated with UBE2D3 and, after the resin was washed, secondly with UBE2N-UBE2V1. The ubiquitin chains conjugated on cIAP1 were analyzed by western blotting. d. Heatmap representation of the LCL-161–enriched interactants shown in Fig. 5f. Proteins enriched more than 2-fold after treatment with LCL-161 are shown. Means for two biological replicates. e. Related to Fig. 5f and Extended Data Fig. 6d, heatmap presentation of FLAG-cIAP1 interacting proteins. Two biological replicates. See Supplementary Dataset 1 for details.

Source data

Extended Data Fig. 7 Database analysis of UBE2N.

a-b. Correlation between UBE2N expression levels and poor prognosis using a Kaplan–Meier survival plot. Relationship between UBE2N expression levels and (a) relapse-free survival for breast cancer patients or (b) overall survival for lung cancer patients was analyzed using the KM plotter database. c. Proteins significantly accumulated after the UBE2N knockdown in Fig. 6d were used for gene ontology (GO) analysis. One-sided P values for significantly enriched categories are shown. In DAVID, Fisher’s Exact test is adopted to measure the gene-enrichment in annotation terms.

Extended Data Fig. 8 Schematic model.

cIAP1 is activated by ligands, and the E2 ubiquitin-conjugating enzyme UBE2N mediates the attachment of K63-linked ubiquitin chains to cIAP1. This facilitates further assembly of K11/K48- and K48/K63-linked branched ubiquitin linkages on the distal surfaces of the ubiquitin chains. The highly complex ubiquitin chains associate with the p97–UCH37–proteasome axis, thereby targeting cIAP1 for degradation. Targeted degradation of a neo-substrate induced by a cIAP1-based PROTAC is also dependent on UBE2N and K63-linked ubiquitin chains. These results reveal a mechanism in which a non-canonical ubiquitin code mediates chemically-induced target degradation, which suggests diverse mechanisms are activated to promote the degradation of neo-substrates.

Supplementary information

Supplementary Information

Supplementary Notes

Reporting Summary

Supplementary Data 1

LC–MS analysis of FLAG-cIAP1-interacting proteins, related to Fig. 5f

Supplementary Data 2

LC–MS analysis of siUBE2N-enriched proteins, related to Fig. 6d

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Akizuki, Y., Morita, M., Mori, Y. et al. cIAP1-based degraders induce degradation via branched ubiquitin architectures. Nat Chem Biol (2022). https://doi.org/10.1038/s41589-022-01178-1

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