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K29-linked ubiquitin signaling regulates proteotoxic stress response and cell cycle

Nature Chemical Biology volume 17, pages 896–905 (2021)Cite this article

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Abstract

Protein ubiquitination shows remarkable topological and functional diversity through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understand the physiology of the cell. However, our understanding of its function is rather limited due to the lack of specific binders as tools to detect K29-linked polyubiquitin. In this study, we screened and characterized a synthetic antigen-binding fragment, termed sAB-K29, that can specifically recognize K29-linked polyubiquitin using chemically synthesized K29-linked diubiquitin. We further determined the crystal structure of this fragment bound to the K29-linked diubiquitin, which revealed the molecular basis of specificity. Using sAB-K29 as a tool, we uncovered that K29-linked ubiquitination is involved in different kinds of cellular proteotoxic stress response as well as cell cycle regulation. In particular, we showed that K29-linked ubiquitination is enriched in the midbody and downregulation of the K29-linked ubiquitination signal arrests cells in G1/S phase.

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Fig. 1: Screening for a K29-linked ubiquitin chain-specific binder and the crystal structure of sAB-K29 in complex with K29-linked diubiquitin.
Fig. 2: In vitro characterization of sAB-K29.
Fig. 3: Pull-down and proteomic analyses of HeLa cells using sAB-K29.
Fig. 4: K29-linked polyubiquitination is involved in protein homeostasis and stress response.
Fig. 5: K29-linked polyubiquitination is involved in cell cycle regulation.
Fig. 6: K29-linked polyubiquitination is enriched around the midbody during mitosis.

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Data availability

The atomic model of K29-linked diUb in complex with sAB-K29 has been deposited in the PDB under the accession code 7KEO. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD024428 and PXD024425 (https://www.ebi.ac.uk/pride/). Source data are provided with this paper.

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Acknowledgements

Funding for this work was, in part, provided by the Catalyst Award from the Chicago Biomedical Consortium. This work was supported by Chicago Biomedical Consortium Catalyst Award no. C-086 to M.Z. This work was supported by National Institutes of Health awards R01GM117372 to A.A.K. We thank the National Key R&D Program of China (No. 2017YFA0505200), NSFC (No. 91753205) and NSFC (No. 21621003) for financial support. This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). The Eiger 16M detector on the 24-ID-E beam line is funded by a NIH-ORIP HEI grant (S10OD021527). This research used resources of the Advanced Photon Source, a U.S. 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. We thank H. Deng, X. Meng and M. Han in the Proteomics Facility at Technology Center for Protein Sciences, Tsinghua University, for help in mass spectrometry analysis. We thank V. Bindokas in the Integrated Light Microscopy Core Facility and D. Leclerc in the Flow Cytometry Core Facility at the University of Chicago for help in fluorescent imaging and flow cytometry.

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Author notes

  1. These authors contributed equally: Yuanyuan Yu, Qingyun Zheng, Satchal K. Erramilli, Man Pan.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA

    Yuanyuan Yu, Satchal K. Erramilli, Man Pan, Seongjin Park, Yuan Xie, Jingxian Li, Jingyi Fei, Anthony A. Kossiakoff & Minglei Zhao

  2. Tsinghua-Peking Center for Life Sciences, Department of Chemistry, Tsinghua University, Beijing, China

    Qingyun Zheng & Lei Liu

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Contributions

Y.Y., M.P., L.L. and M.Z. designed all the experiments and interpreted the results. S.K.E and A.A.K performed the sAB selection and evaluation. Y.Y., Q.Z. and M.P. synthesized all diubiquitin molecules and carried out the related biochemical characterizations. Y.Y., J.L. and M.Z. performed crystal screening and data processing. Q.Z., Y.Y. and M.P. performed and interpreted the LC–MS/MS experiments. Y.Y., Y.X., S.P. and J.F. performed the cell-based imaging experiments. M.Z., M.P. and Y.Y. wrote the paper. M.Z., L.L. and A.A.K supervised the project.

Corresponding authors

Correspondence to Man Pan, Anthony A. Kossiakoff, Lei Liu or Minglei Zhao.

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The authors declare no competing interests.

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Peer review information Nature Chemical Biology thanks 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 Chemical synthesis and characterization of biotinylated K29-linked diubiquitin.

a, The synthetic route of biotinylated K29-linked diubiquitin. b, Liquid chromatography and mass spectrometry (LC-MS) analysis of the synthetic biotinylated K29-linked diubiquitin. c, Circular dichroism (CD) spectra of the synthetic biotinylated K29-linked diubiquitin. Recombinant monoubiquitin was used as a control.

Extended Data Fig. 2 Purification, crystallization, and structural comparison of K29-linked diubiquitin in complex with sAB-K29.

The gel in panel a is a single-time experiment; The gel in panel c is representative of two independent experiments; n = 2. a, Purification of K29-linked diubiquitin. A mixture of K29- and K48-linked polyubiquitin mixture was assembled by incubating monoubiquitin, UBE1, UBE2L3, and UBE3C overnight. vOTU, a DUB that does not cleave K29-linked chains, was added to the reaction mixture to cleave K48-linked polyubiquitin chains. The resulting mono-, di-, and triubiquitin molecules were separated by cation exchange chromatography. b, Size exclusion chromatography (SEC) of sAB-K29 and sAB-K29 in complex with K29-linked diubiquitin. c, The SDS-PAGE gel of the complex peak in panel b. d, A crystal of the complex mounted in a cryo-loop. e, The distribution of hydrophobic patches on K29-linked diUb. Only the I36 patch of the distal ubiquitin molecule is involved in the interaction with the heavy chain of sAB-K29. The colour code of the complex is the same as that in Fig. 1b. f, Superimposition of the crystal structure of K29-linked diUb (PDB accession code: 4S22) and K29-linked diUb in the complex structure determined in this study. The distal ubiquitin molecules in the two structures were aligned. g, Superimposition of the crystal structure of K29-linked diUb in complex with NZF1 domain of TRABID (PDB accession code: 4S1Z) and K29-linked diUb in the complex structure determined in this study. The distal ubiquitin molecules in the two structures were aligned. NZF1 domain was coloured in pink. h, Superimposition of the crystal structure of K33-linked diUb (PDB accession code: 4XYZ) and K29-linked diUb in the complex structure determined in this study. The distal ubiquitin molecules in the two structures were aligned. The K29-linked diUb from this study in panels f-h are in the same orientation as in Fig. 1b and the left of panel e.

Extended Data Fig. 3 Characterization of sAB-K29 specificity.

Gel panels in this figure are representative of two independent experiments; n = 2. a, Single-point competitive phage ELISA. Biotinylated K29-linked diubiquitin was immobilized and incubated with phage-displayed sAB-K29 in the presence of excess competitors (monoubiquitin and K29-, K27-, and K33-linked diubiquitin) in solution. A decrease in absorption indicated more specific binding. Error bars represent the standard deviations of triplicate experiments. (n = 3 biological replicates, mean ± SD). b, A point mutation in the heavy chain of sAB-K29 (Y112A) abolished its recognition of K29-linked diUb. Monoubiquitin and diubiquitin (~500 ng) with eight types of linkages were loaded on an SDS-PAGE gel. Western blot was performed using sAB-K29 or sAB-K29 (Y112A) as the primary binder. c, Mutation of the heavy chain of sAB-K29 (Y112A) abolished its ability to detect K29-linked polyubiquitin chains by western blot. K29-linked polyubiquitin chains were assembled by an E1-E2-E3 mixture containing UBA1, UBE2L3, and UBE3C. The reactions were followed for 90 minutes at four time points. d, Polyubiquitin chains were assembled by four ubiquitination systems: UBE3C (K48- and K29-linked chains), UBE2S (K11-linked chains), E2-25K (K48-linked chains), and UBC13/Mms2 (K63-linked chains). Parallel western blot using anti-ubiquitin, K48 and K63 linkage-specific antibodies were performed for comparison. e, sAB-K29 could specifically detect K29-linked polyubiquitin chains in vitro. Polyubiquitin chains were assembled as in panel d.

Source data

Extended Data Fig. 4 Pull-down and proteomic analysis of HeLa cells using sAB-K29.

a, Pull-down assay of HeLa cell lysate using sAB-K29. The bound proteins were separated on an SDS-PAGE gel. Gel slices were subjected to label-free quantitative mass spectrometry to identify the enriched proteins. Three biological replicates were performed. sAB-MBP was used in the control pull-down assay. Western blot analysis of the pull-down eluant using sAB-K29 or anti-ubiquitin antibody as the primary binder confirmed that sAB-MBP is an effective control. b, GO analysis (cellular components) of enriched proteins from pull-down experiments of HeLa cells using sAB-K29. The enriched proteins were identified using label-free quantitative mass spectrometry. Three biological and two technical replicates were performed. Significant hits (compared to pull down using sAB-MBP, FDR < 0.05) were subjected to GO analysis. c, Volcano plots of the quantitative mass spectrometry results. Significantly enriched hits (FDR < 0.05) are colored cyan, with some well-documented proteins involved in protein homeostasis, RNA processing, and transcription regulation highlighted and labeled.

Extended Data Fig. 5 K29-linked ubiquitination is involved in protein homeostasis and stress response.

Image and gel panels in this figure are representative of two independent experiments; n = 2. a-b, Immunofluorescent staining of HeLa cells at prometaphase, metaphase, and anaphase of mitosis. Costaining for K29-Ub with VCP (panel a) or the proteasome (20S, panel b) is shown. Scale bars correspond to 5 µm. c, K29-linked ubiquitination is enriched in condensates occasionally observed in normal HeLa cells. Costaining for K29-Ub with VCP, EIF3B, the proteasome (20 S), and K48-Ub is shown. Scale bar in the bottom left panel corresponds to 20 µm; Scale bars in the other panels correspond to 5 µm. d, Western blots of total ubiquitin, K63- and K48-linked ubiquitin in HeLa cells treated with CuET (an inhibitor of the VCP cofactor Npl4, 1 μM for 4 h), MG132 (a proteasome inhibitor, 20 μM for 4 h), or sodium arsenite (an oxidative stress inducer, 500 μM for 1 h). e, Immunofluorescent staining of HeLa cells with sAB-MBP and costaining for EIF3B in HeLa cells treated with 500 µM sodium arsenite for 1 h or subjected to heat shock at 45 °C for 30 minutes. Scale bars in the top and bottom panels correspond to 20 and 5 μm, respectively. f-h, Immunofluorescent staining of HeLa cells treated with 0.5 μM CuET for 4 h, 10 μM MG132 for 4 h, or 250 μM sodium arsenite for 4 h. Costaining for K29-Ub with VCP, proteasome (20S), and EIF3B is shown. Scale bars correspond to 20 μm. i, Immunofluorescent staining of HeLa cells treated with 500 μM sodium arsenite for 1 h or subjected to heat shock at 45 °C for 30 minutes. Costaining for K29-Ub with G3BP1 and VCP is shown. The scale bar corresponds to 20 μm. j, Western blot of VCP in HeLa cells treated with CuET (1 μM for 4 h) and MG132 (20 μM for 4 h).

Source data

Extended Data Fig. 6 Reduced level of K29-linked ubiquitination led to G1/S arrest.

Gel panels in this figure are representative of two independent experiments; n = 2. a, A truncated form of the human deubiquitinating enzyme (DUB) TRABID (residues 245-697, named Tra) was used to reduce the level of K29-linked polyubiquitination in HeLa cells. Domain diagrams of full-length TRABID and the Tra construct are shown. b, Western blots of K63-linked ubiquitin and total ubiquitin in HeLa cells transfected with the Tra construct or the empty vector. c, Cell cycle analysis of HeLa cells transfected with either the Tra or the Tra-CIM construct by flow cytometry. cMyc and K29-linked polyubiquitin channels are shown. Representative flow cytometry graphs from three biological replicates are shown. d, Cell cycle analysis of HeLa cells transfected with either the Tra construct or the empty vector by flow cytometry. cMyc and K29-linked polyubiquitin channels are shown. Note that the cMyc tag was also expressed in cells transfected with the empty vector. Representative flow cytometry graphs from two biological replicates are shown. e, Cell cycle analysis of normal HeLa cells by flow cytometry. f, Cell cycle analysis of Hela cells transfected Tra construct for 27 h using flow cytometry. Representative flow cytometry graphs from three biological replicates are shown (a total of 5 injections). Highlighted areas were subjected to cell cycle analysis.

Source data

Extended Data Fig. 7 Flow cytometry analysis of cultured cells with reduced level of K29-linked ubiquitination.

a, Cell cycle analysis of HeLa cells transfected with Tra or an empty vector by flow cytometry. Representative flow cytometry graphs from two biological replicates are shown (a total of 5 injections). Highlighted areas were subjected to cell cycle analysis. b, Quantification of the cell cycle analysis results in panel a. Two biological replicates (a total of 5 injections) were included in this experiment. (n = 5 technical replicates, mean ± SD, two-sided Student’s t-test). c, Cell cycle analysis of A549 cells transfected with either the Tra construct or the empty vector by flow cytometry. d, Quantification of the A549 cell cycle analysis results in panel c. (n = 3 biological replicates, mean ± SD, two-sided Student’s t-test). e, An example of manual gating analysis used to obtain populations of cells in this study.

Extended Data Fig. 8 Western blot analysis of HeLa cells with reduced level of K29-linked ubiquitination.

Gel panels in this figure are representative of two independent experiments; n = 2. a-d, Western blots of CyclinA, CDC 27, STAT3, and Cyclin D1 in HeLa cells transfected with the Tra construct or the empty vector. e, Tra level of transfected HeLa cells treated with puromycin, cycloheximide, or α-amanitin. Western blot was performed using an anti-cMyc antibody. f-g, New protein synthesis was not affected after K29-linked ubiquitination was reduced in HeLa cells. HeLa cells were transfected with the Tra construct and treated with puromycin, cycloheximide, or α-amanitin. Western blot was performed using anti-puromycin antibody (f) or sAB-K29 (g).

Source data

Extended Data Fig. 9 K29-linked ubiquitination is involved in mitosis.

Gel panels and image panels in this figure are representative of two independent experiments; n = 2. a, Western blot analysis of K29-linked ubiquitination during mitosis. HeLa cells were synchronized to prometaphase by treatment with thymidine and nocodazole. Aliquots of cells were taken and lysed at the indicated time points, followed by western blot using sAB-K29 as the primary binder. b, A diagram showing migration of the K29-linked polyubiquitination signal during the telophase of mitosis. Roughly four stages could be distinguished based on localization and morphology. c, 3D STORM images of K29-linked polyubiquitin at telophase 3 and 4. The orthographic projection image of the highlighted area in telophase 4 (bottom cyan box) along the long axis (white arrow) is shown in the upper right inset. Rendering was conducted by Visual Molecular Dynamics (VMD) with GLSL rendering, Orthographic display, and Surf drawing. Scale bars in the left images correspond to 2 μm. Scale bars in the right images correspond to 250 nm. d, Immunofluorescent staining of HeLa cells at different stages of mitosis. Costaining for K29-linked ubiquitin with α-tubulin is shown. Cells were pre-extracted before fixation. e, Immunofluorescent staining of HeLa cells with sAB-MBP. Costaining for α-tubulin is shown. Cells were pre-extracted before fixation. f, Immunofluorescent staining of HeLa cells at the telophase of mitosis. Costaining for K29-linked ubiquitin with either Aurora B or CDC27 (a negative control) is shown. Cells were pre-extracted before fixation. Scale bars in panels d-f correspond to 5 μm.

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Extended Data Fig. 10 Identification of midbody proteins involved in K29-linked polyubiquitination.

Gel panels and image panels in this figure are representative of two independent experiments; n = 2. a, sAB-K29 could be used to pull down K29-linked diUb in vitro under denaturing conditions (up to 2 M urea). b, sAB-K29 could be used to specifically pull down K29-linked diUb under denaturing conditions (2 M urea). c, Volcano plot of the quantitative mass spectrometry results after pull-down experiments in HeLa cells synchronized to the telophase of mitosis using sAB-K29 under denaturing conditions (1 M urea). Significant hits (FDR < 0.05) are colored blue, with those involved in midbody assembly highlighted in red. d, Immunofluorescent staining of HeLa cells at the telophase of mitosis. Costaining for K29-Ub with pTBK, PLK1, INCENP, and MKLP1 is shown. Scale bars correspond to 5 μm. e, Immunoprecipitation of synchronized HeLa cells under denaturing conditions using antibodies against TBK1, PLK1, INCENP, and MKLP1. Western blot was performed using sAB-K29 as the primary binder. Anti-IgG was used in the control experiment. f, Immunoprecipitation of HeLa cells (either synchronized to the telophase of mitosis or unsynchronized) using sAB-K29. Antibodies against TBK1, PLK1, INCENP, and MKLP1 were used for western blot.

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Supplementary information

Supplementary Information

Supplementary Table 1.

Reporting Summary

Supplementary Table 2

Proteins identified from pull-down and proteomic analysis using sAB-K29.

Supplementary Video 1

3D STORM visualization (rotation by a shorter axis) of K29-linked ubiquitin in the midbody of dividing HeLa cells. Rendering was conducted by visual molecular dynamics with GLSL rendering, orthographic display and surf drawing.

Supplementary Video 2

3D STORM visualization (rotation by a longer axis) of K29-linked ubiquitin in the midbody of dividing HeLa cells. Rendering was conducted by visual molecular dynamics with GLSL rendering, orthographic display and surf drawing.

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Yu, Y., Zheng, Q., Erramilli, S.K. et al. K29-linked ubiquitin signaling regulates proteotoxic stress response and cell cycle. Nat Chem Biol 17, 896–905 (2021). https://doi.org/10.1038/s41589-021-00823-5

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