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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Hendriks, I.A. et al. Uncovering global SUMOylation signaling networks in a site-specific manner. Nat. Struct. Mol. Biol. 21, 927–936 (2014).
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).
Tammsalu, T. et al. Proteome-wide identification of SUMO2 modification sites. Sci. Signal. 7, rs2 (2014).
Bernardi, R. & Pandolfi, P.P. Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies. Nat. Rev. Mol. Cell Biol. 8, 1006–1016 (2007).
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).
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).
Hirota, K. et al. SUMO-targeted ubiquitin ligase RNF4 plays a critical role in preventing chromosome loss. Genes Cells 19, 743–754 (2014).
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).
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).
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).
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).
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).
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).
Droescher, M., Chaugule, V.K. & Pichler, A. SUMO rules: regulatory concepts and their implication in neurologic functions. Neuromolecular Med. 15, 639–660 (2013).
Flotho, A. & Melchior, F. Sumoylation: a regulatory protein modification in health and disease. Annu. Rev. Biochem. 82, 357–385 (2013).
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).
Klug, H. et al. Ubc9 sumoylation controls SUMO chain formation and meiotic synapsis in Saccharomyces cerevisiae. Mol. Cell 50, 625–636 (2013).
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).
Reverter, D. & Lima, C.D. Insights into E3 ligase activity revealed by a SUMO–RanGAP1–Ubc9–Nup358 complex. Nature 435, 687–692 (2005).
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).
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).
Hoppe, T. Multiubiquitylation by E4 enzymes: 'one size' doesn't fit all. Trends Biochem. Sci. 30, 183–187 (2005).
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).
Wang, L. et al. SUMO2 is essential while SUMO3 is dispensable for mouse embryonic development. EMBO Rep. 15, 878–885 (2014).
Hay, R.T. Decoding the SUMO signal. Biochem. Soc. Trans. 41, 463–473 (2013).
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).
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).
Pichler, A., Gast, A., Seeler, J.S., Dejean, A. & Melchior, F. The nucleoporin RanBP2 has SUMO1 E3 ligase activity. Cell 108, 109–120 (2002).
Krishna, S.S., Majumdar, I. & Grishin, N.V. Structural classification of zinc fingers: survey and summary. Nucleic Acids Res. 31, 532–550 (2003).
Vogt, B. & Hofmann, K. Bioinformatical detection of recognition factors for ubiquitin and SUMO. Methods Mol. Biol. 832, 249–261 (2012).
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).
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).
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).
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).
Knipscheer, P. et al. Ubc9 sumoylation regulates SUMO target discrimination. Mol. Cell 31, 371–382 (2008).
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).
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).
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).
Johnson, E.S. & Gupta, A.A. An E3-like factor that promotes SUMO conjugation to the yeast septins. Cell 106, 735–744 (2001).
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).
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).
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).
Werner, A., Flotho, A. & Melchior, F. The RanBP2/RanGAP1*SUMO1/Ubc9 complex is a multisubunit SUMO E3 ligase. Mol. Cell 46, 287–298 (2012).
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).
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).
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).
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).
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).
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).
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).
Becker, J. et al. Detecting endogenous SUMO targets in mammalian cells and tissues. Nat. Struct. Mol. Biol. 20, 525–531 (2013).
Guex, N. & Peitsch, M.C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723 (1997).
Pettersen, E.F. et al. UCSF Chimera: a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Knuckles, P. et al. Drosha regulates neurogenesis by controlling neurogenin 2 expression independent of microRNAs. Nat. Neurosci. 15, 962–969 (2012).
Zheng, Q. et al. Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells. Biotechniques 57, 115–124 (2014).
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.
Author information
Authors and Affiliations
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.
Corresponding author
Ethics declarations
Competing interests
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)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.3114
This article is cited by
-
Signalling mechanisms and cellular functions of SUMO
Nature Reviews Molecular Cell Biology (2022)
-
Structural basis for the E3 ligase activity enhancement of yeast Nse2 by SUMO-interacting motifs
Nature Communications (2021)
-
Impact of posttranslational modifications in pancreatic carcinogenesis and treatments
Cancer and Metastasis Reviews (2021)
-
SUMO mediated regulation of transcription factors as a mechanism for transducing environmental cues into cellular signaling in plants
Cellular and Molecular Life Sciences (2021)
-
SUMO proteins in the cardiovascular system: friend or foe?
Journal of Biomedical Science (2020)