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A new vertebrate SUMO enzyme family reveals insights into SUMO-chain assembly

Abstract

SUMO chains act as stress-induced degradation tags or repair factor–recruiting signals at DNA lesions. Although E1 activating, E2 conjugating and E3 ligating enzymes efficiently assemble SUMO chains, specific chain-elongation mechanisms are unknown. E4 elongases are specialized E3 ligases that extend a chain but are inefficient in the initial conjugation of the modifier. We identified ZNF451, a representative member of a new class of SUMO2 and SUMO3 (SUMO2/3)-specific enzymes that execute catalysis via a tandem SUMO-interaction motif (SIM) region. One SIM positions the donor SUMO while a second SIM binds SUMO on the back side of the E2 enzyme. This tandem-SIM region is sufficient to extend a back side–anchored SUMO chain (E4 elongase activity), whereas efficient chain initiation also requires a zinc-finger region to recruit the initial acceptor SUMO (E3 ligase activity). Finally, we describe four human proteins sharing E4 elongase activities and their function in stress-induced SUMO2/3 conjugation.

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Figure 1: ZNF451 enhances SUMO2/3-chain assembly.
Figure 2: Tandem-SIM and zinc-finger regions are required for ZNF451's activity.
Figure 3: The classical zinc finger and both SIMs are required for ZNF451's activity.
Figure 4: The tandem-SIM region presents the catalytic unit.
Figure 5: The inter-SIM region is essential for ZNF451's enzymatic activity.
Figure 6: Four human proteins share the catalytic unit of ZNF451.
Figure 7: ZNF451 regulates stress-induced global SUMO2/3 conjugation.

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References

  1. Hendriks, I.A. et al. Uncovering global SUMOylation signaling networks in a site-specific manner. Nat. Struct. Mol. Biol. 21, 927–936 (2014).

    Article  CAS  Google Scholar 

  2. Lamoliatte, F. et al. Targeted identification of SUMOylation sites in human proteins using affinity enrichment and paralog-specific reporter ions. Mol. Cell. Proteomics 12, 2536–2550 (2013).

    Article  CAS  Google Scholar 

  3. Tammsalu, T. et al. Proteome-wide identification of SUMO2 modification sites. Sci. Signal. 7, rs2 (2014).

    Article  Google Scholar 

  4. Bernardi, R. & Pandolfi, P.P. Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies. Nat. Rev. Mol. Cell Biol. 8, 1006–1016 (2007).

    Article  CAS  Google Scholar 

  5. Galanty, Y., Belotserkovskaya, R., Coates, J. & Jackson, S.P. RNF4, a SUMO-targeted ubiquitin E3 ligase, promotes DNA double-strand break repair. Genes Dev. 26, 1179–1195 (2012).

    Article  CAS  Google Scholar 

  6. Guzzo, C.M. et al. RNF4-dependent hybrid SUMO-ubiquitin chains are signals for RAP80 and thereby mediate the recruitment of BRCA1 to sites of DNA damage. Sci. Signal. 5, ra88 (2012).

    Article  Google Scholar 

  7. Hirota, K. et al. SUMO-targeted ubiquitin ligase RNF4 plays a critical role in preventing chromosome loss. Genes Cells 19, 743–754 (2014).

    Article  CAS  Google Scholar 

  8. Poulsen, S.L. et al. RNF111/Arkadia is a SUMO-targeted ubiquitin ligase that facilitates the DNA damage response. J. Cell Biol. 201, 797–807 (2013).

    Article  CAS  Google Scholar 

  9. Tatham, M.H. et al. RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation. Nat. Cell Biol. 10, 538–546 (2008).

    Article  CAS  Google Scholar 

  10. Yin, Y. et al. SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage. Genes Dev. 26, 1196–1208 (2012).

    Article  CAS  Google Scholar 

  11. Gibbs-Seymour, I. et al. Ubiquitin-SUMO circuitry controls activated fanconi anemia ID complex dosage in response to DNA damage. Mol. Cell 57, 150–164 (2015).

    Article  CAS  Google Scholar 

  12. Guervilly, J.H. et al. The SLX4 complex is a SUMO E3 ligase that impacts on replication stress outcome and genome stability. Mol. Cell 57, 123–137 (2015).

    Article  CAS  Google Scholar 

  13. Ouyang, J. et al. Noncovalent interactions with SUMO and ubiquitin orchestrate distinct functions of the SLX4 complex in genome maintenance. Mol. Cell 57, 108–122 (2015).

    Article  CAS  Google Scholar 

  14. Droescher, M., Chaugule, V.K. & Pichler, A. SUMO rules: regulatory concepts and their implication in neurologic functions. Neuromolecular Med. 15, 639–660 (2013).

    Article  CAS  Google Scholar 

  15. Flotho, A. & Melchior, F. Sumoylation: a regulatory protein modification in health and disease. Annu. Rev. Biochem. 82, 357–385 (2013).

    Article  CAS  Google Scholar 

  16. Dou, H., Buetow, L., Sibbet, G.J., Cameron, K. & Huang, D.T. BIRC7–E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer. Nat. Struct. Mol. Biol. 19, 876–883 (2012).

    Article  CAS  Google Scholar 

  17. Klug, H. et al. Ubc9 sumoylation controls SUMO chain formation and meiotic synapsis in Saccharomyces cerevisiae. Mol. Cell 50, 625–636 (2013).

    Article  CAS  Google Scholar 

  18. Plechanovová, A., Jaffray, E.G., Tatham, M.H., Naismith, J.H. & Hay, R.T. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature 489, 115–120 (2012).

    Article  Google Scholar 

  19. Reverter, D. & Lima, C.D. Insights into E3 ligase activity revealed by a SUMO–RanGAP1–Ubc9–Nup358 complex. Nature 435, 687–692 (2005).

    Article  CAS  Google Scholar 

  20. Wickliffe, K.E., Lorenz, S., Wemmer, D.E., Kuriyan, J. & Rape, M. The mechanism of linkage-specific ubiquitin chain elongation by a single-subunit E2. Cell 144, 769–781 (2011).

    Article  CAS  Google Scholar 

  21. Rytinki, M.M., Kaikkonen, S., Pehkonen, P., Jaaskelainen, T. & Palvimo, J.J. PIAS proteins: pleiotropic interactors associated with SUMO. Cell. Mol. Life Sci. 66, 3029–3041 (2009).

    Article  CAS  Google Scholar 

  22. Hoppe, T. Multiubiquitylation by E4 enzymes: 'one size' doesn't fit all. Trends Biochem. Sci. 30, 183–187 (2005).

    Article  CAS  Google Scholar 

  23. Saitoh, H. & Hinchey, J. Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3. J. Biol. Chem. 275, 6252–6258 (2000).

    Article  CAS  Google Scholar 

  24. Wang, L. et al. SUMO2 is essential while SUMO3 is dispensable for mouse embryonic development. EMBO Rep. 15, 878–885 (2014).

    Article  CAS  Google Scholar 

  25. Hay, R.T. Decoding the SUMO signal. Biochem. Soc. Trans. 41, 463–473 (2013).

    Article  CAS  Google Scholar 

  26. Karvonen, U., Jaaskelainen, T., Rytinki, M., Kaikkonen, S. & Palvimo, J.J. ZNF451 is a novel PML body- and SUMO-associated transcriptional coregulator. J. Mol. Biol. 382, 585–600 (2008).

    Article  CAS  Google Scholar 

  27. Pichler, A., Knipscheer, P., Saitoh, H., Sixma, T.K. & Melchior, F. The RanBP2 SUMO E3 ligase is neither HECT- nor RING-type. Nat. Struct. Mol. Biol. 11, 984–991 (2004).

    Article  CAS  Google Scholar 

  28. Pichler, A., Gast, A., Seeler, J.S., Dejean, A. & Melchior, F. The nucleoporin RanBP2 has SUMO1 E3 ligase activity. Cell 108, 109–120 (2002).

    Article  CAS  Google Scholar 

  29. Krishna, S.S., Majumdar, I. & Grishin, N.V. Structural classification of zinc fingers: survey and summary. Nucleic Acids Res. 31, 532–550 (2003).

    Article  CAS  Google Scholar 

  30. Vogt, B. & Hofmann, K. Bioinformatical detection of recognition factors for ubiquitin and SUMO. Methods Mol. Biol. 832, 249–261 (2012).

    Article  CAS  Google Scholar 

  31. Capili, A.D. & Lima, C.D. Structure and analysis of a complex between SUMO and Ubc9 illustrates features of a conserved E2-Ubl interaction. J. Mol. Biol. 369, 608–618 (2007).

    Article  CAS  Google Scholar 

  32. Duda, D.M. et al. Structure of a SUMO-binding-motif mimic bound to Smt3p-Ubc9p: conservation of a non-covalent ubiquitin-like protein-E2 complex as a platform for selective interactions within a SUMO pathway. J. Mol. Biol. 369, 619–630 (2007).

    Article  CAS  Google Scholar 

  33. Knipscheer, P., van Dijk, W.J., Olsen, J.V., Mann, M. & Sixma, T.K. Noncovalent interaction between Ubc9 and SUMO promotes SUMO chain formation. EMBO J. 26, 2797–2807 (2007).

    Article  CAS  Google Scholar 

  34. Namanja, A.T. et al. Insights into high affinity small ubiquitin-like modifier (SUMO) recognition by SUMO-interacting motifs (SIMs) revealed by a combination of NMR and peptide array analysis. J. Biol. Chem. 287, 3231–3240 (2012).

    Article  CAS  Google Scholar 

  35. Knipscheer, P. et al. Ubc9 sumoylation regulates SUMO target discrimination. Mol. Cell 31, 371–382 (2008).

    Article  CAS  Google Scholar 

  36. Abascal, F., Tress, M. & Valencia, A. Alternative splicing and co-option of transposable elements: the case of TMPO/LAP2alpha and ZNF451 in mammals. Bioinformatics 31, 2257–2261 (2015).

    Article  CAS  Google Scholar 

  37. Tatham, M.H., Matic, I., Mann, M. & Hay, R.T. Comparative proteomic analysis identifies a role for SUMO in protein quality control. Sci. Signal. 4, rs4 (2011).

    Article  CAS  Google Scholar 

  38. Ilves, I., Petojevic, T., Pesavento, J.J. & Botchan, M.R. Activation of the MCM2–7 helicase by association with Cdc45 and GINS proteins. Mol. Cell 37, 247–258 (2010).

    Article  CAS  Google Scholar 

  39. Johnson, E.S. & Gupta, A.A. An E3-like factor that promotes SUMO conjugation to the yeast septins. Cell 106, 735–744 (2001).

    Article  CAS  Google Scholar 

  40. Yunus, A.A. & Lima, C.D. Structure of the Siz/PIAS SUMO E3 ligase Siz1 and determinants required for SUMO modification of PCNA. Mol. Cell 35, 669–682 (2009).

    Article  CAS  Google Scholar 

  41. Mascle, X.H. et al. Identification of a non-covalent ternary complex formed by PIAS1, SUMO1, and UBC9 proteins involved in transcriptional regulation. J. Biol. Chem. 288, 36312–36327 (2013).

    Article  CAS  Google Scholar 

  42. Tatham, M.H. et al. Unique binding interactions among Ubc9, SUMO and RanBP2 reveal a mechanism for SUMO paralog selection. Nat. Struct. Mol. Biol. 12, 67–74 (2005).

    Article  CAS  Google Scholar 

  43. Werner, A., Flotho, A. & Melchior, F. The RanBP2/RanGAP1*SUMO1/Ubc9 complex is a multisubunit SUMO E3 ligase. Mol. Cell 46, 287–298 (2012).

    Article  CAS  Google Scholar 

  44. Cappadocia, L., Pichler, A. & Lima, C.D. Structural basis for catalytic activation by the ZNF451 SUMO E3 ligase. Nat. Struct. Mol. Biol. doi:10.1038/nsmb.3116 (2 November 2015).

  45. Brown, N.G. et al. Mechanism of polyubiquitination by human anaphase-promoting complex: RING repurposing for ubiquitin chain assembly. Mol. Cell 56, 246–260 (2014).

    Article  CAS  Google Scholar 

  46. Kelly, A., Wickliffe, K.E., Song, L., Fedrigo, I. & Rape, M. Ubiquitin chain elongation requires e3-dependent tracking of the emerging conjugate. Mol. Cell 56, 232–245 (2014).

    Article  CAS  Google Scholar 

  47. Lallemand-Breitenbach, V. et al. Arsenic degrades PML or PML–RARα through a SUMO-triggered RNF4/ubiquitin-mediated pathway. Nat. Cell Biol. 10, 547–555 (2008).

    Article  CAS  Google Scholar 

  48. Hofmann, S. et al. A genome-wide association study reveals evidence of association with sarcoidosis at 6p12.1. Eur. Respir. J. 38, 1127–1135 (2011).

    Article  CAS  Google Scholar 

  49. van den Ent, F. & Löwe, J. RF cloning: a restriction-free method for inserting target genes into plasmids. J. Biochem. Biophys. Methods 67, 67–74 (2006).

    Article  CAS  Google Scholar 

  50. Mahajan, R., Delphin, C., Guan, T., Gerace, L. & Melchior, F. A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 88, 97–107 (1997).

    Article  CAS  Google Scholar 

  51. Becker, J. et al. Detecting endogenous SUMO targets in mammalian cells and tissues. Nat. Struct. Mol. Biol. 20, 525–531 (2013).

    Article  CAS  Google Scholar 

  52. Guex, N. & Peitsch, M.C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723 (1997).

    Article  CAS  Google Scholar 

  53. Pettersen, E.F. et al. UCSF Chimera: a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

  54. Knuckles, P. et al. Drosha regulates neurogenesis by controlling neurogenin 2 expression independent of microRNAs. Nat. Neurosci. 15, 962–969 (2012).

    Article  CAS  Google Scholar 

  55. Zheng, Q. et al. Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells. Biotechniques 57, 115–124 (2014).

    Article  CAS  Google Scholar 

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Acknowledgements

Our special thanks go to the members of A.P.'s laboratory for discussions and sharing reagents, and C. Lima, F. Melchior, P. Nielsen, K. Tibbles and R. Sawarkar for discussions and suggestions on the manuscript. We kindly acknowledge R.T. Hay (University of Dundee), H. Walden (University of Dundee) and J. Winter (University Medical Center Mainz) for sharing reagents. This work was supported by the Max Planck Society (to A.P.) and grants from the Deutsche Forschungsgemeinschaft (DFG-SPP1365 PI 917/2-1 to A.P. and DFG-SPP1365 to K.H.), the Academy of Finland (251133 to J.J.P.) and the Sigrid Jusélius Foundation (to J.J.P.). This article is based on work from European Cooperation in Science and Technology (COST) Action (PROTEOSTASIS BM1307 to A.P.), supported by COST.

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Authors and Affiliations

Authors

Contributions

V.K.C., N.E., S.K., M.D. and A.P. designed the experiments; V.K.C. and N.E. purified diverse proteins; V.K.C. established the fluorescence in vitro SUMOylation assay; M.D., N.E. and V.K.C. performed the in vitro assays; N.E. performed pulldown experiments; E.D. and J.R. performed initial experiments, E.D. purified SUMO2ΔSIM, cloned some ZNF451 constructs and provided the purified anti-ZNF451 and anti-RNF4 antibodies; J.R. provided the SUMO(2)ylated Ubc9; J.R. and S.K. designed and S.K. performed ZNF451 CRISPR-Cas9 knockout; S.K., M.D. and N.E. performed all cell-culture experiments; P.S. performed the 3×FLAG-ZNF451 immunoprecipitation; S.Y.I. identified MCM4 by mass spectrometry; K.H. performed the bioinformatics analysis and identified SIM I; J.J.P. conceived the initial idea and provided reagents; N.E., V.K.C., M.D. and A.P. prepared figures, figure legends and methods; and A.P. wrote the manuscript.

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Correspondence to Andrea Pichler.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 SUMO-paralog specificity of E3 ligases.

(a) Immunoblot of a multi-turn-over assay (60 nM Aos1-Uba2, 100 nM Ubc9) to measure SUMO-chain formation activity with increasing ZNF-N concentrations (0, 20, 60 and 180 nM) and indicated SUMO-paralogs (2 μM) monitored after 30 min at 30 °C.

(b) Fluorescent gel of a multi-turn-over assay (60 nM Aos1-Uba2, 100 nM Ubc9, 2 μM DyLight800-labelled SUMO2) to measure SUMO-chain-formation ability with increasing concentrations of ZNF-N, untagged ZNF-N and MBP (0, 20, 60 and 180 nM) monitored after 60 min at 30 °C.

(c) Fluorescent gel of a multi-turn-over assay (60 nM Aos1-Uba2, 100 nM Ubc9) with labelled SUMO-paralogs (2 μM) to measure SUMO-chain-formation ability with increasing concentrations of MBP-PIAS1 (0, 20, 60 and 180 nM) monitored after 60 min at 30 °C.

(d) as (c) but with increasing concentrations (0, 5, 20, 80 nM) of GST-RanBP2ΔFG.

(e) Fluorescent gel of a multi-turn-over assay (60 nM Aos1-Uba2, 100 nM Ubc9) to measure SUMO-chain-formation ability with increasing concentrations of ZNF-N (0, 20, 60 and 180 nM) and labelled SUMO2 or SUMOK11R (2 μM) monitored after 60 min at 30 °C.

♦, unspecific band, MW, molecular-weight, kDA, kilo-Dalton, S, SUMO, Sn, polySUMO, MBP, Maltose-binding-protein, *, isopeptide-bond, uncropped gels are shown in Supplementary Data Set 1.

Supplementary Figure 2 ZNF-N shows highest conservation in the tandem-SIM and the classical zinc-finger regions.

Multiple sequence alignment of ZNF-N from human (Homo sapiens, NP_001026794), monkey (Macaca mulatta,NP_001244706), dog (Canis lupus familiaris (XP_532184), cow (Bos Taurus, NP_001179613), rat (Rattus norvegicus (NP_001028877), mouse (Mus musculus, NP_598578), chicken (Gallus gallus (XP_419898), frog (Xenopus laevisl, NP_001085555), fish (Danio rerio, XP_001923170). Full conservation is shown in red, high and low conservation in orange and yellow, respectively.

SIM, SUMO interaction motif.

Supplementary Figure 3 Detection of free SUMO2 is inefficient in immunoblot analysis.

Coomassie gel (left panel) versus immunoblot (right panel) of equal amounts of SUMO2 and Ubc9C93K~SUMO2.

Supplementary Figure 4 SUMO(2)ylated Ubc9 does not regulate ZNF451’s E3 activity.

Fluorescent gel of a multi-turn-over assay (60 nM Aos1-Uba2, 2 μM labelled SUMO) to measure SUMO-chain-formation ability with increasing concentrations of ZNF-N (0, 20, 60 and 180 nM) and 100 nM Ubc9 or sumo(2)ylated Ubc9 (S2*Ubc9) monitored after 60 min at 30 °C. ♦, unspecific band, MW, molecular-weight, kDA, kilo-Dalton, S, SUMO, Sn, polySUMO, *, isopeptide-bond, uncropped gels are shown in Supplementary Data Set 1

Supplementary Figure 5 SUMO1 automodification activity of ZNF451 SIMonly versus the RNF4 SIM region.

Multi-turn-over assay (60 nM Aos1-Uba2, 2 μM SUMO1) with increasing concentrations of Ubc9 (0, 4, 20, 100 and 500 nM) to measure automodification activity of 60 nM ZNF451 SIMonly (upper panel) or the RNF4-SIM-region (lower panel) monitored after 30 min at 30 °C.

Supplementary Figure 6 Sequence alignment of the three human ZNF451 isoforms and KIAA1586 isoform 1.

Multiple sequence alignment of human ZNF451 isoforms (Q9Y4E5_1, Q9Y4E5_2, Q9Y4E5_3), and isoform 1 of human KIAA1586 (Q9HCI6_1). Full conservation is shown in red, high and low conservation in orange and yellow, respectively. SIM, SUMO-interaction motif.

Supplementary Figure 7 Validation of homemade antibodies for Ubc9 and RNF4.

Immunoblots of N2a lysates after knockdown with scrambled-siRNA, Ubc9-siRNA or RNF4-siRNA

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 1374 kb)

Supplementary Data Set 1

Uncropped blots (PDF 9466 kb)

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Eisenhardt, N., Chaugule, V., Koidl, S. et al. A new vertebrate SUMO enzyme family reveals insights into SUMO-chain assembly. Nat Struct Mol Biol 22, 959–967 (2015). https://doi.org/10.1038/nsmb.3114

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