The Small Ubiquitin-like Modifiers: Established and emerging roles in diseases Mike Tatham

1. The Small Ubiquitin-like Modifiers: Established and emerging roles in diseases Mike Tatham Ron Hay lab Wellcome Trust Centre for Gene Regulation and Expression University of Dundee ELRIG/SLAS Drug Discovery Manchester 2012

2. Phylogenetic relationship in the ubiquitin like modifier superfamily • SUMO system is only found in Eukaryotes • Yeasts, flies and worms only express a single SUMO • Vertebrates express three paralogues SUMO-1, SUMO-2, SUMO-3 ELRIG/SLAS Drug Discovery Manchester 2012

3. The SUMO conjugation system • SUMO system is only found in Eukaryotes • Yeasts, flies and worms only express a single SUMO • Vertebrates express three paralogues SUMO-1, SUMO-2, SUMO-3 • Like ubiquitin SUMOs are conjugated to protein substrates in a three step mechanism • Most SUMO conjugation occurs within a consensus motif yKXE/D • SUMO conjugation can occur independent of E3s • SUMO-2 and SUMO-3 contain consensus motifs and can modify themselves to form polySUMO chains • Deletion of yeast SUMO is lethal • Deletion of Ubc9 in mice is lethal Ubiquitination Ubiquitin E1 (2) Ubiquitin E2 (~20) Ubiquitin E3 (hundreds) Substrate U U U U Ubiquitin Protease (~100) Substrate Substrate SUMOylation S S S SUMO E1 (1 – SAE1/2) SUMO E2 (1 – Ubc9) [SUMO E3 (10-20?)] S S yKXE yKXD Substrate SUMO Protease (8) ELRIG/SLAS Drug Discovery Manchester 2012

4. Structural overview • SUMO system is only found in Eukaryotes • Yeasts, flies and worms only express a single SUMO • Vertebrates express three paralogues SUMO-1, SUMO-2, SUMO-3 • Like ubiquitin SUMOs are conjugated to protein substrates in a three step mechanism • Most SUMO conjugation occurs within a consensus motif yKXE/D • SUMO conjugation can occur independent of E3s • SUMO-2 and SUMO-3 contain consensus motifs and can modify themselves to form polySUMO chains • Deletion of yeast SUMO is lethal • Deletion of Ubc9 in mice is lethal • SUMOs have low sequence homology to ubiquitin but high 3D structural similarity

5. Cellular characteristics +MG132 Time (h) 0 1 2 3 5 7 • SUMO system is only found in Eukaryotes • Yeasts, flies and worms only express a single SUMO • Vertebrates express three paralogues SUMO-1, SUMO-2, SUMO-3 • Like ubiquitin SUMOs are conjugated to protein substrates in a three step mechanism • Most SUMO conjugation occurs within a consensus motif yKXE/D • SUMO conjugation can occur independent of E3s • SUMO-2 and SUMO-3 contain consensus motifs and can modify themselves to form polySUMO chains • Deletion of yeast SUMO is lethal • Deletion of Ubc9 in mice is lethal • SUMOs have low sequence homology to ubiquitin but high 3D structural similarity • SUMOs are predominantly nuclear proteins • SUMO-1 and SUMO-2/-3 have largely overlapping protein targets with some distinctions U U U U U IB Ubiquitin Substrate S S S S Substrate IB SUMO-2/3 S S S Substrate IB SUMO-1 Total extracts ELRIG/SLAS Drug Discovery Manchester 2012

6. Molecular functions of SUMO SUMOylation • SUMO system is only found in Eukaryotes • Yeasts, flies and worms only express a single SUMO • Vertebrates express three paralogues SUMO-1, SUMO-2, SUMO-3 • Like ubiquitin SUMOs are conjugated to protein substrates in a three step mechanism • Most SUMO conjugation occurs within a consensus motif yKXE/D • SUMO conjugation can occur independent of E3s • SUMO-2 and SUMO-3 contain consensus motifs and can modify themselves to form polySUMO chains • Deletion of yeast SUMO is lethal • Deletion of Ubc9 in mice is lethal • SUMOs have low sequence homology to ubiquitin but high 3D structural similarity • SUMOs are predominantly nuclear proteins • SUMO-1 and SUMO-2/-3 have largely overlapping protein targets with some distinctions • SUMO conjugation does not have a common effect on proteins, but has myriad of protein-specific consequences mediated by SUMO Interaction Motifs (SIMS) SUMO E1 (1 – SAE1/2) SUMO E2 (1 – Ubc9) [SUMO E3 (10-20?)] S Substrate SUMO Protease (8) Altered function Subcellularlocalisation Enzymatic activity Complex formation Further modification Block modifications Substrate SUMO BP SUMO BP S Substrate S S S Substrate S polySUMO BP S S S S S Substrate S Substrate ELRIG/SLAS Drug Discovery Manchester 2012

7. Cellular functions of SUMO Golebiowski et al Sci Signal. 2009;2(72):ra24. Tatham et al Sci. Signal. 2011;4(178):rs4 TAP-SUMO-2 cells TAG SUMO Substrate Number of SUMO substrates • Purify SUMO from cells • Identify and quantify proteins by quantitative mass spectrometry-based proteomics • Identified a total of ~900 SUMO substrates Year ~10% of cellular proteins are modified by SUMO ELRIG/SLAS Drug Discovery Manchester 2012

8. SUMO and human diseases Centromere instability, and facial anomalies syndrome Megakaryoblastic leukemia Autoimmune regulation Breast cancer Atypical myeloproliferative disease Prostate cancer Melanoma Immunological disorders Dermatomyositis Squamous cell carcinoma Cancers Renal cell carcinoma Acute Promyelocyticleukaemia Colon cancer Multiple myeloma Ovarian cancer DNA viruses Protozoa RNA viruses Circulatory Diseases SUMO Multiple System Atrophy Extra and Intra-cellular bacteria Amyotrophic lateral sclerosis Alzheimer's disease Infectious diseases Frontotemporal lobar degeneration Congenital heart disease Frontotemporal dementia Familial dilated cardiomyopathy Spinocerebellar ataxia type 1 Obesity Others Spinal and bulbar muscular atrophy Parkinson's disease Neurological disorders Transient global and focal cerebral ischemia Heart failure Amyotrophic lateral sclerosis Neuronal intranuclear inclusion disease Dementia with Lewy Bodies Huntington's disease Muscular dystrophy Liver damage Cystic fibrosis Rheumatoid arthritis ELRIG/SLAS Drug Discovery Manchester 2012

9. SUMO and human diseases Types of evidence linking SUMO with diseases Disease protein x is modified by SUMO which alters its function. SUMO conjugation is altered in disease cells SUMO is abnormally distributed within disease cells Enzymes of the SUMO modification system are abnormally expressed in disease cells SUMO system modulation alters the disease phenotype in model cells Immunolabelling of NII in the hippocampalsubiculum of patients with NIID. (Takahashi-Fujigasaki et al. Neuropathology and Applied Neurobiology (2006), 32 , 92–100) Immunostaining of aggregates in glioma cell models for multiple system atrophy (MSA). • Alzheimer’s disease (Amyloid beta protein (derived from from APP)) • (tau) • Parkinson’s disease (a-synuclein, DJ-1) • Prion disease (PrP) • Polyglutamine diseases Huntington’s (Huntingtin) • Kennedy’s (Androgen Rec) • Dentatorubro-pallidoluysian atrophy (Atrophin-1) • Spinocerebellar ataxia (ATAXIN1, 7) • Tauopathy (tau) • Familial amyotrophic lateral sclerosis (SOD1) Li et al. PNAS 100 (2003) (D.L. Pountney et al. Neuroscience Letters 381 (2005) 74–79) Dorval & Fraser. JBC 281 (2005) Dorval & Fraser. JBC 281 (2005), Shinbo et al. Cell. Death Diff. (2006) Juanes et al. JBC 284 (2009) Steffan et al. Science (2004) Mukherjee et al. JBC (2009) Terashima et al. Neuroreport. 13 (2002) Riley et al. JBC 280 (2005), Janer et al. Hum. Mol. Gen. 19 (2010) Dorval & Fraser. JBC 281 (2005) Fei et al. BBRC 347 (2006) ELRIG/SLAS Drug Discovery Manchester 2012

10. The SUMO-SIM interaction Q. How do we take advantage of the SUMO system therapeutically? A. It depends on what you want to do! SIM peptide Substrate SUMO SUMO BP SUMO BP S S S S SUMO E1 (1 – SAE1/2) SUMO E2 (1 – Ubc9) [SUMO E3 (10-20?)] S yKXE yKXD Substrate SUMO Protease (8) ELRIG/SLAS Drug Discovery Manchester 2012

11. To be or not to be specific: What can parasites tell us? Q. How do we take advantage of the SUMO system therapeutically? A. It depends on how specific you want to be! Infectious diseases DNA viruses Protozoa RNA viruses Wimmer P, et al. J Virol. 2012 Jan;86(2):642-54. Extra and Intra-cellular bacteria ELRIG/SLAS Drug Discovery Manchester 2012

12. SUMO and human diseases Centromere instability, and facial anomalies syndrome Megakaryoblastic leukemia Autoimmune regulation Breast cancer Atypical myeloproliferative disease Prostate cancer Melanoma Immunological disorders Dermatomyositis Squamous cell carcinoma Cancers Renal cell carcinoma Acute Promyelocyticleukaemia Colon cancer Multiple myeloma Ovarian cancer DNA viruses Protozoa RNA viruses Circulatory Diseases SUMO Multiple System Atrophy Extra and Intra-cellular bacteria Amyotrophic lateral sclerosis Alzheimer's disease Infectious diseases Frontotemporal lobar degeneration Congenital heart disease Frontotemporal dementia Familial dilated cardiomyopathy Spinocerebellar ataxia type 1 Obesity Others Spinal and bulbar muscular atrophy Parkinson's disease Neurological disorders Transient global and focal cerebral ischemia Heart failure Amyotrophic lateral sclerosis Neuronal intranuclear inclusion disease Dementia with Lewy Bodies Huntington's disease Muscular dystrophy Liver damage Cystic fibrosis Rheumatoid arthritis ELRIG/SLAS Drug Discovery Manchester 2012

13. Example of a successful drug therapy involving SUMO - APL • Acute PromyelocyticLeukaemia (APL) • A rare condition driven by a chromosomal translocation resulting in the fusion of the PML and retinoic acid receptor a proteins (PML-RARa) • Very malignant and charaterised by sudden hemorrhages and accumulation of promyelocytes in blood • Retinoic acid and arsenic trioxide treatment induce differentiation of promyelocytes and clinical remission. • PML-RARa and PML are known to be SUMOylated and degraded in response to arsenic De The et al J. Cell. Biol. 2012. 198 No.1 11-21 Liu et al Curr. Op. Chem. Biol. 2012. 16 92-98 ELRIG/SLAS Drug Discovery Manchester 2012

14. Example of a successful drug therapy involving SUMO - APL Ring Finger protein 4 (RNF4) aka SNURF Tatham et al 2008. Nat. Cell. Biol. 10. 5. 538-546 ELRIG/SLAS Drug Discovery Manchester 2012

15. Example of a successful drug therapy involving SUMO - APL Tatham et al 2008. Nat. Cell. Biol. 10. 5. 538-546 ELRIG/SLAS Drug Discovery Manchester 2012

16. A model for SUMO-dependent disease remission ARSENIC SUMO conjugation Ubiquitin conjugation SUMO deconjugation Ubiquitin deconjugation U U U U U U U U U S S S S S S S S S S PML PML PML RNF4 Tatham et al 2008. Nat. Cell. Biol. 10. 5. 538-546 ELRIG/SLAS Drug Discovery Manchester 2012

17. The SUMO system as a therapeutic target - Summary • SUMO is functionally highly pleiotropic affecting many important cellular pathways • There is a range of evidence linking SUMO to significant human diseases. • The precise role of SUMO in many diseases is not determined and so its potential as a therapeutic target is largely unclear • The best approach to modulating SUMO function for individual disease therapy is unclear • There is an academic and clinical argument for small molecule effectors of the SUMO system to help clarify these issues. ELRIG/SLAS Drug Discovery Manchester 2012

18. Acknowledgements Ron Hay Filip Golebiowski (Glasgow) Ellis Jaffray Marie-Claude Geoffroy (Paris) Ivan Matic Amit Garg Jurgen Cox Matthias Mann ELRIG/SLAS Drug Discovery Manchester 2012