Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

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

Subjects

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

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.

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.

References

  1. Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012).

    Article  CAS  PubMed  Google Scholar 

  2. Swatek, K. N. & Komander, D. Ubiquitin modifications. Cell Res. 26, 399–422 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Yau, R. & Rape, M. The increasing complexity of the ubiquitin code. Nat. Cell Biol. 18, 579–586 (2016).

    Article  CAS  PubMed  Google Scholar 

  4. Lopez-Mosqueda, J. & Dikic, I. Deciphering functions of branched ubiquitin chains. Cell 157, 767–769 (2014).

    Article  CAS  PubMed  Google Scholar 

  5. Meyer, H. J. & Rape, M. Enhanced protein degradation by branched ubiquitin chains. Cell 157, 910–921 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Haakonsen, D. L. & Rape, M. Branching out: improved signaling by heterotypic ubiquitin chains. Trends Cell Biol. 29, 704–716 (2019).

    Article  CAS  PubMed  Google Scholar 

  7. Swatek, K. N. et al. Insights into ubiquitin chain architecture using Ub-clipping. Nature 572, 533–537 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Gautam, A. K. S. & Matouschek, A. Decoding without the cipher. Nat. Chem. Biol. 15, 210–212 (2019).

    Article  Google Scholar 

  9. Miura, G. Unraveling the chain. Nat. Chem. Biol. 13, 1139 (2017).

    Article  CAS  PubMed  Google Scholar 

  10. de la Pena, A. H., Goodall, E. A., Gates, S. N., Lander, G. C. & Martin, A. Substrate-engaged 26S proteasome structures reveal mechanisms for ATP-hydrolysis-driven translocation. Science 362, 6418 (2018).

    Google Scholar 

  11. Ding, Z. et al. Structural snapshots of 26S proteasome reveal tetraubiquitin-induced conformations. Mol. Cell 73, 1150–1161 (2019).

    Article  CAS  PubMed  Google Scholar 

  12. Dong, Y. et al. Cryo-EM structures and dynamics of substrate-engaged human 26S proteasome. Nature 565, 49–55 (2019).

    Article  CAS  PubMed  Google Scholar 

  13. Wang, G. et al. K63-linked ubiquitination in kinase activation and cancer. Front. Oncol. 2, 5 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ikeda, F. & Dikic, I. Atypical ubiquitin chains: new molecular signals. ‘Protein Modifications: Beyond the Usual Suspects’ review series. EMBO Rep. 9, 536–542 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kulathu, Y. & Komander, D. Atypical ubiquitylation—the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages. Nat. Rev. Mol. Cell Biol. 13, 508–523 (2012).

    Article  CAS  PubMed  Google Scholar 

  16. Keusekotten, K. et al. OTULIN antagonizes LUBAC signaling by specifically hydrolyzing Met1-linked polyubiquitin. Cell 153, 1312–1326 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Aalto, A. L. et al. M1-linked ubiquitination by LUBEL is required for inflammatory responses to oral infection in Drosophila. Cell Death Differ. 26, 860–876 (2019).

    Article  CAS  PubMed  Google Scholar 

  18. Van Wijk, S. J. et al. Linear ubiquitination of cytosolic Salmonella typhimurium activates NF-kappa B and restricts bacterial proliferation. Nat. Microbiol. 2, https://doi.org/10.1038/nmicrobiol.2017.66 (2017).

  19. Durcan, T. M. et al. USP8 regulates mitophagy by removing K6-linked ubiquitin conjugates from parkin. EMBO J. 33, 2473–2491 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Cunningham, C. N. et al. USP30 and parkin homeostatically regulate atypical ubiquitin chains on mitochondria. Nat. Cell Biol. 17, 160–169 (2015).

    Article  CAS  PubMed  Google Scholar 

  21. Gersch, M. et al. Mechanism and regulation of the Lys6-selective deubiquitinase USP30. Nat. Struct. Mol. Biol. 24, 920–930 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Matsumoto, M. L. et al. K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody. Mol. Cell 39, 477–484 (2010).

    Article  CAS  PubMed  Google Scholar 

  23. Xu, P. et al. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137, 133–145 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Liu, J. et al. Rhbdd3 controls autoimmunity by suppressing the production of IL-6 by dendritic cells via K27-linked ubiquitination of the regulator NEMO. Nat. Immunol. 15, 612–622 (2014).

    Article  CAS  PubMed  Google Scholar 

  25. Xue, B. et al. TRIM21 promotes innate immune response to RNA viral infection through Lys27-linked polyubiquitination of MAVS. J. Virol. 92, e00321-18 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Lee, Y. R. et al. Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWPI inhibitory pathway. Science 364, 6441 (2019).

    Article  Google Scholar 

  27. Huang, H. et al. K33-linked polyubiquitination of T cell receptor-zeta regulates proteolysis-independent T cell signaling. Immunity 33, 60–70 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Yuan, W. C. et al. K33-linked polyubiquitination of coronin 7 by Cul3-KLHL20 ubiquitin E3 ligase regulates protein trafficking. Mol. Cell 54, 586–600 (2014).

    Article  CAS  PubMed  Google Scholar 

  29. Karim, M. et al. Nonproteolytic K29-linked ubiquitination of the PB2 replication protein of influenza A viruses by proviral cullin 4-based E3 ligases. mBio 11, e00305–e00320 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Liu, C., Liu, W. X., Ye, Y. H. & Li, W. Ufd2p synthesizes branched ubiquitin chains to promote the degradation of substrates modified with atypical chains. Nat. Commun. 8, 14274 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Tsuchiya, H. et al. Ub-ProT reveals global length and composition of protein ubiquitylation in cells. Nat. Commun. 9, 524–524 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Kristariyanto, Y. A. et al. K29-selective ubiquitin binding domain reveals structural basis of specificity and heterotypic nature of k29 polyubiquitin. Mol. Cell 58, 83–94 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Michel, M. A., Swatek, K. N., Hospenthal, M. K. & Komander, D. Ubiquitin linkage-specific affimers reveal insights into K6-linked ubiquitin signaling. Mol. Cell 68, 233–246 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Newton, K. et al. Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 134, 668–678 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Matsumoto, M. L. et al. Engineering and structural characterization of a linear polyubiquitin-specific antibody. J. Mol. Biol. 418, 134–144 (2012).

    Article  CAS  PubMed  Google Scholar 

  36. Yau, R. G. et al. Assembly and function of heterotypic ubiquitin chains in cell-cycle and protein quality control. Cell 171, 918–933 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Miersch, S. et al. Scalable high throughput selection from phage-displayed synthetic antibody libraries. J. Vis. Exp. 2015, 51492 (2015).

    Google Scholar 

  38. Pan, M. et al. Quasi-racemic X-ray structures of K27-linked ubiquitin chains prepared by total chemical synthesis. J. Am. Chem. Soc. 138, 7429–7435 (2016).

    Article  CAS  PubMed  Google Scholar 

  39. Pan, M. et al. Chemical synthesis of structurally defined phosphorylated ubiquitins suggests impaired Parkin activation by phosphorylated ubiquitins with a non-phosphorylated distal unit. CCS Chem. 1, 476–489 (2019).

    Article  CAS  Google Scholar 

  40. Miller, K. R. et al. T cell receptor-like recognition of tumor in vivo by synthetic antibody fragment. PLoS ONE 7, e43746 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Paduch, M. et al. Generating conformation-specific synthetic antibodies to trap proteins in selected functional states. Methods 60, 3–14 (2013).

    Article  CAS  PubMed  Google Scholar 

  42. Michel, M. A. et al. Assembly and specific recognition of K29- and K33-linked polyubiquitin. Mol. Cell 58, 95–109 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Li, Y. M. et al. Irreversible site-specific hydrazinolysis of proteins by use of sortase. Angew. Chem. Int. Ed. Engl. 53, 2198–2202 (2014).

    Article  CAS  PubMed  Google Scholar 

  44. Skrott, Z. et al. Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4. Nature 552, 194–199 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Pan, M. et al. Seesaw conformations of Npl4 in the human p97 complex and the inhibitory mechanism of a disulfiram derivative. Nat. Commun. 12, 121 (2021).

  46. Jolly, C., Usson, Y. & Morimoto, R. I. Rapid and reversible relocalization of heat shock factor 1 within seconds to nuclear stress granules. Proc. Natl Acad. Sci. USA 96, 6769–6774 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Tsai, W. C. et al. Arginine demethylation of G3BP1 promotes stress granule assembly. J. Biol. Chem. 291, 22671–22685 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Licchesi, J. D. et al. An ankyrin-repeat ubiquitin-binding domain determines TRABID’s specificity for atypical ubiquitin chains. Nat. Struct. Mol. Biol. 19, 62–71 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Ramadan, K. et al. Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin. Nature 450, 1258–1262 (2007).

    Article  CAS  PubMed  Google Scholar 

  50. Meyer, H., Bug, M. & Bremer, S. Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system. Nat. Cell Biol. 14, 117–123 (2012).

    Article  CAS  PubMed  Google Scholar 

  51. Qu, Q. et al. Highly efficient synthesis of polyubiquitin chains. Adv. Sci. 5, 1800234–1800234 (2018).

    Article  Google Scholar 

  52. Studier, F. W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).

    Article  CAS  PubMed  Google Scholar 

  53. Kabsch, W. XDS. Acta Crystallogr. D. Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Sliwiak, J., Jaskolski, M., Dauter, Z., McCoy, A. J. & Read, R. J. Likelihood-based molecular-replacement solution for a highly pathological crystal with tetartohedral twinning and sevenfold translational noncrystallographic symmetry. Acta Crystallogr. D. Biol. Crystallogr. 70, 471–480 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D. Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  57. Dominik, P. K. et al. Conformational chaperones for structural studies of membrane proteins using antibody phage display with nanodiscs. Structure 24, 300–309 (2016).

    Article  CAS  PubMed  Google Scholar 

  58. Mukherjee, S. et al. Engineered synthetic antibodies as probes to quantify the energetic contributions of ligand binding to conformational changes in proteins. J. Biol. Chem. 293, 2815–2828 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Fei, J. et al. RNA biochemistry. Determination of in vivo target search kinetics of regulatory noncoding RNA. Science 347, 1371–1374 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Park, S. et al. Conducting multiple imaging modes with one fluorescence microscope. J. Vis. Exp. https://doi.org/10.3791/58320 (2018).

Download references

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.

Author information

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

Authors
  1. Yuanyuan Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qingyun Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Satchal K. Erramilli
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Man Pan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Seongjin Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yuan Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jingxian Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jingyi Fei
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Anthony A. Kossiakoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Lei Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Minglei Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Source data

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.

Source data

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.

Source data

Source Data Fig. 2

Unprocessed western blots and/or gels.

Source Data Fig. 3

Unprocessed western blots and/or gels.

Source Data Fig. 4

Unprocessed western blots and/or gels.

Source Data Fig. 5

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 3

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 5

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 6

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 8

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 9

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 10

Unprocessed western blots and/or gels.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-021-00823-5

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing