Sumoylation: A regulatory protein modification in health and disease

Annual Review of Biochemistry

Volumn 82, Issue , 2013, Pages 357-385

  Flotho, Annette ^a   Melchior, Frauke ^a  

^a UNIVERSITY OF HEIDELBERG   (Germany)

Author keywords

E3 ligase; Isopeptidase; Posttranslational modification; SUMO; Ubiquitin

Indexed keywords

HETEROCHROMATIN PROTEIN 1; HETEROCHROMATIN PROTEIN 1ALPHA; LIGASE; PHOSPHATIDYLINOSITOL 3,4,5 TRISPHOSPHATE 3 PHOSPHATASE; PROTEIN UBC9; PROTEINASE; RAC PROTEIN; SUMO 1 PROTEIN; SUMO 2 PROTEIN; SUMO 3 PROTEIN; SUMO ACTIVATING ENZYME SUBUNIT 1; SUMO ACTIVATING ENZYME SUBUNIT 2; SUMO E3 LIGASE; SUMO PROTEASE; SUMO PROTEIN; UNCLASSIFIED DRUG;

ACTIN FILAMENT; AMINO ACID SEQUENCE; AMINO TERMINAL SEQUENCE; CARBOXY TERMINAL SEQUENCE; CELL MIGRATION; CELL NUCLEUS; CLOSTRIDIUM PERFRINGENS; CONSENSUS SEQUENCE; DNA DAMAGE; ENZYME ACTIVITY; ENZYME INACTIVATION; ENZYME LOCALIZATION; ENZYME STRUCTURE; ENZYME SUBSTRATE COMPLEX; EXCISION REPAIR; LISTERIA MONOCYTOGENES; MITOSIS; NONHUMAN; PRIORITY JOURNAL; PROTEIN AGGREGATION; PROTEIN EXPRESSION; PROTEIN FUNCTION; PROTEIN INTERACTION; PROTEIN MOTIF; PROTEIN PHOSPHORYLATION; PROTEIN PROTEIN INTERACTION; REGULATORY MECHANISM; REVIEW; SEQUENCE HOMOLOGY; STREPTOCOCCUS PNEUMONIAE; SUMOYLATION; UNFOLDED PROTEIN RESPONSE;

ANIMALS; GENETIC PREDISPOSITION TO DISEASE; HUMANS; PROTEINS; SIGNAL TRANSDUCTION; SMALL UBIQUITIN-RELATED MODIFIER PROTEINS; SUMOYLATION;

EUKARYOTA;

EID: 84878944582     PISSN: 00664154     EISSN: 15454509     Source Type: Book Series    
DOI: 10.1146/annurev-biochem-061909-093311     Document Type: Review

References (221)

1. Mahajan R, Delphin C, Guan T, Gerace L, Melchior F. 1997. A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 88:97-107
2. Matunis MJ, Coutavas E, Blobel G. 1996. A novel ubiquitin- likemodificationmodulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J. Cell Biol. 135:1457-70
3. Gareau JR, Lima CD. 2010. The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition. Nat. Rev. Mol. Cell Biol. 11:861-71
4. Geiss-Friedlander R, Melchior F. 2007. Concepts in sumoylation: a decade on. Nat. Rev. Mol. Cell Biol. 8:947-56
5. Hay RT. 2005. SUMO: a history of modification. Mol. Cell 18:1-12
6. Johnson ES. 2004. Protein modification by SUMO. Annu. Rev. Biochem. 73:355-82
7. Miura K, Jin JB, Hasegawa PM. 2007. Sumoylation, a post-translational regulatory process in plants. Curr. Opin. Plant Biol. 10:495-502
8. Wilkinson KA, Henley JM. 2010. Mechanisms, regulation and consequences of protein SUMOylation. Biochem. J. 428:133-45
9. Komander D, Rape M. 2012. The ubiquitin code. Annu. Rev. Biochem. 81:203-29
10. Drag M, Salvesen GS. 2008. DeSUMOylating enzymes-SENPs. IUBMB Life 60:734-42
11. Hickey CM, Wilson NR, Hochstrasser M. 2012. Function and regulation of SUMOproteases. Nat. Rev. Mol. Cell Biol. 13:755-66
12. van der Veen AG, Ploegh HL. 2012. Ubiquitin-like proteins. Annu. Rev. Biochem. 81:323-57
13. Saitoh H, Hinchey J. 2000. Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3. J. Biol. Chem. 275:6252-58
14. Guo D, Li M, Zhang Y, Yang P, Eckenrode S, et al. 2004. A functional variant of SUMO4, a new I Bα modifier, is associated with type 1 diabetes. Nat. Genet. 36:837-41
15. Owerbach D, McKay EM, Yeh ET, Gabbay KH, Bohren KM. 2005. A proline-90 residue unique to SUMO-4 prevents maturation and sumoylation. Biochem. Biophys. Res. Commun. 337:517-20
16. Wei W, Yang P, Pang J, Zhang S, Wang Y, et al. 2008. A stress-dependent SUMO4 sumoylation of its substrate proteins. Biochem. Biophys. Res. Commun. 375:454-9
17. Tanaka K, Nishide J, Okazaki K, Kato H, Niwa O, et al. 1999. Characterization of a fission yeast SUMO- 1 homologue, pmt3p, required for multiple nuclear events, including the control of telomere length and chromosome segregation. Mol. Cell. Biol. 19:8660-72
18. Wong KH, Todd RB, Oakley BR, Oakley CE, Hynes MJ, Davis MA. 2008. Sumoylation in Aspergillus nidulans: sumO inactivation, overexpression and live-cell imaging. Fungal Genet. Biol. 45:728-37
19. Alkuraya FS, Saadi I, Lund JJ, Turbe-Doan A, Morton CC, Maas RL. 2006. SUMO1 haploinsufficiency leads to cleft lip and palate. Science 313:1751
20. Evdokimov E, Sharma P, Lockett SJ, Lualdi M, Kuehn MR. 2008. Loss of SUMO1 in mice affects RanGAP1 localization and formation of PML nuclear bodies, but is not lethal as it can be compensated by SUMO2 or SUMO3. J. Cell Sci. 121:4106-13
21. Wang J, Chen L, Wen S, Zhu H, Yu W, et al. 2011. Defective sumoylation pathway directs congenital heart disease. Birth Defects Res. A 91:468-76
22. Zhang FP, Mikkonen L, Toppari J, Palvimo JJ, Thesleff I, Janne OA. 2008. Sumo-1 function is dispensable in normal mouse development. Mol. Cell. Biol. 28:5381-90
23. Yuan H, Zhou J, Deng M, Liu X, Le Bras M, et al. 2010. Small ubiquitin-related modifier paralogs are indispensable but functionally redundant during early development of zebrafish. Cell Res. 20:185-96
24. Saracco SA, Miller MJ, Kurepa J, Vierstra RD. 2007. Genetic analysis of SUMOylation in Arabidopsis: conjugation of SUMO1 and SUMO2 to nuclear proteins is essential. Plant Physiol. 145:119-34
25. Novatchkova M, Bachmair A, Eisenhaber B, Eisenhaber F. 2005. Proteins with two SUMO-like domains in chromatin-associated complexes: the RENi (Rad60-Esc2-NIP45) family. BMC Bioinformatics 6:22
26. Yang K, Moldovan GL, Vinciguerra P, Murai J, Takeda S, DAndrea AD. 2011. Regulation of the Fanconi anemia pathway by a SUMO-like delivery network. Genes Dev. 25:1847-58
27. Prudden J, Perry JJ, Nie M, Vashisht AA, Arvai AS, et al. 2011. DNA repair and global sumoylation are regulated by distinct Ubc9 noncovalent complexes. Mol. Cell. Biol. 31:2299-310
28. Olsen SK, Capili AD, Lu X, Tan DS, Lima CD. 2010. Active site remodelling accompanies thioester bond formation in the SUMO E1. Nature 463:906-12
29. Dye BT, Schulman BA. 2007. Structural mechanisms underlying posttranslational modification by ubiquitin-like proteins. Annu. Rev. Biophys. Biomol. Struct. 36:131-50
30. Schulman BA, Harper JW. 2009. Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. Nat. Rev. Mol. Cell Biol. 10:319-31
31. Bernier-Villamor V, Sampson DA, Matunis MJ, Lima CD. 2002. Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1. Cell 108:345-56
32. Nacerddine K, Lehembre F, Bhaumik M, Artus J, Cohen-Tannoudji M, et al. 2005. The SUMOpathway is essential for nuclear integrity and chromosome segregation in mice. Dev. Cell 9:769-79
33. Subramaniam S, Mealer RG, Sixt KM, Barrow RK, Usiello A, Snyder SH. 2010. Rhes, a physiologic regulator of sumoylation, enhances cross-sumoylation between the basic sumoylation enzymes E1 and Ubc9. J. Biol. Chem. 285:20428-32
34. Capili AD, Lima CD. 2007. Structure and analysis of a complex between SUMO and Ubc9 illustrates features of a conserved E2-Ubl interaction. J. Mol. Biol. 369:608-18
35. Duda DM, van Waardenburg RC, Borg LA, McGarity S, Nourse A, et al. 2007. Structure of a SUMObinding- 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. 369:619-30
36. Knipscheer P, van Dijk WJ, Olsen JV, Mann M, Sixma TK. 2007. Noncovalent interaction between Ubc9 and SUMO promotes SUMO chain formation. EMBO J. 26:2797-807
37. Knipscheer P, Flotho A, Klug H, Olsen JV, Van Dijk WJ, et al. 2008. Ubc9 sumoylation regulatesSUMO target discrimination. Mol. Cell 31:371-82
38. Su YF, Yang T, Huang H, Liu LF, Hwang J. 2012. Phosphorylation ofUbc9 byCdk1 enhances SUMOylation activity. PLoS ONE 7:e34250
39. Boggio R, Passafaro A, Chiocca S. 2007. Targeting SUMO E1 to ubiquitin ligases: a viral strategy to counteract sumoylation. J. Biol. Chem. 282:15376-82
40. Boggio R, Colombo R, Hay RT, Draetta GF, Chiocca S. 2004. A mechanism for inhibiting the SUMO pathway. Mol. Cell 16:549-61
41. Heaton PR, Deyrieux AF, Bian XL, Wilson VG. 2011. HPV E6 proteins target Ubc9, the SUMO conjugating enzyme. Virus Res. 158:199-208
42. Ribet D, Hamon M, Gouin E, Nahori MA, Impens F, et al. 2010. Listeria monocytogenes impairs SUMOylation for efficient infection. Nature 464:1192-95
43. Hershko A, Ciechanover A. 1998. The ubiquitin system. Annu. Rev. Biochem. 67:425-79
44. Plechanovova A, Jaffray EG, Tatham MH, Naismith JH, Hay RT. 2012. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature 489:115-20
45. Reverter D, Lima CD. 2005. Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9- Nup358 complex. Nature 435:687-92
46. Yunus AA, Lima CD. 2009. Structure of the Siz/PIAS SUMO E3 ligase Siz1 and determinants required for SUMO modification of PCNA. Mol. Cell 35:669-82
47. Pichler A, Knipscheer P, Saitoh H, Sixma TK, Melchior F. 2004. The RanBP2 SUMO E3 ligase is neither HECT- nor RING-type. Nat. Struct. Mol. Biol. 11:984-91
48. Andrews EA, Palecek J, Sergeant J, Taylor E, Lehmann AR, Watts FZ. 2005. Nse2, a component of the Smc5-6 complex, is a SUMOligase required for the response to DNA damage. Mol. Cell. Biol. 25:185-96
49. Kahyo T, Nishida T, Yasuda H. 2001. Involvement of PIAS1 in the sumoylation of tumor suppressor p53. Mol. Cell 8:713-18
50. Potts PR, Yu H. 2005. Human MMS21/NSE2 is a SUMO ligase required for DNA repair. Mol. Cell. Biol. 25:7021-32
51. Sachdev S, Bruhn L, Sieber H, Pichler A, Melchior F, Grosschedl R. 2001. PIASy, a nuclear matrixassociated SUMO E3 ligase, represses LEF1 activity by sequestration into nuclear bodies. Genes Dev. 15:3088-103
52. Sapetschnig A, Rischitor G, Braun H, Doll A, Schergaut M, et al. 2002. Transcription factor Sp3 is silenced through SUMO modification by PIAS1. EMBO J. 21:5206-15
53. Schmidt D, Muller S. 2002. Members of the PIAS family act as SUMO ligases for c-Jun and p53 and repress p53 activity. Proc. Natl. Acad. Sci. USA 99:2872-77
54. Beliakoff J, Sun Z. 2006. Zimp7 and Zimp10, two novel PIAS-like proteins, function as androgen receptor coregulators. Nuclear Recept. Signal. 4:e017
55. Cheng CH, Lo YH, Liang SS, Ti SC, Lin FM, et al. 2006. SUMO modifications control assembly of synaptonemal complex and polycomplex in meiosis of Saccharomyces cerevisiae. Genes Dev. 20:2067-81
56. Johnson ES, Gupta AA. 2001. An E3-like factor that promotes SUMO conjugation to the yeast septins. Cell 106:735-44
57. Takahashi Y, Kahyo T, Toh-e A, Yasuda H, Kikuchi Y. 2001. Yeast Ull1/Siz1 is a novel SUMO1/Smt3 ligase for septin components and functions as an adaptor between conjugating enzyme and substrates. J. Biol. Chem. 276:48973-77
58. Zhao X, Blobel G. 2005. A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization. Proc. Natl. Acad. Sci. USA 102:4777-82
59. Miura K, Hasegawa PM. 2010. Sumoylation and other ubiquitin-like post-translational modifications in plants. Trends Cell Biol. 20:223-32
60. Novatchkova M, Tomanov K, Hofmann K, Stuible HP, Bachmair A. 2012. Update on sumoylation: defining core components of the plant SUMO conjugation system by phylogenetic comparison. New Phytol. 195:23-31
61. Rytinki MM, Kaikkonen S, Pehkonen P, Jaaskelainen T, Palvimo JJ. 2009. PIAS proteins: pleiotropic interactors associated with SUMO. Cell Mol. Life Sci. 66:3029-41
62. Liu B, Mink S, Wong KA, Stein N, Getman C, et al. 2004. PIAS1 selectively inhibits interferon-inducible genes and is important in innate immunity. Nat. Immunol. 5:891-98
63. Santti H, Mikkonen L, Anand A, Hirvonen-Santti S, Toppari J, et al. 2005. Disruption of the murine PIASx gene results in reduced testis weight. J. Mol. Endocrinol. 34:645-54
64. Roth W, Sustmann C, Kieslinger M, Gilmozzi A, Irmer D, et al. 2004. PIASy-deficient mice display modest defects in IFN andWnt signaling. J. Immunol. 173:6189-99
65. Wong KA, Kim R, Christofk H, Gao J, Lawson G, Wu H. 2004. Protein inhibitor of activated STAT Y (PIASy) and a splice variant lacking exon 6 enhance sumoylation but are not essential for embryogenesis and adult life. Mol. Cell. Biol. 24:5577-86
66. Bischof O, Schwamborn K, Martin N, Werner A, Sustmann C, et al. 2006. The E3 SUMOligase PIASy is a regulator of cellular senescence and apoptosis. Mol. Cell 22:783-94
67. Pichler A, Gast A, Seeler JS, Dejean A, Melchior F. 2002. The nucleoporin RanBP2 has SUMO1 E3 ligase activity. Cell 108:109-20
68. Tatham MH, Kim S, Jaffray E, Song J, Chen Y, Hay RT. 2005. Unique binding interactions among Ubc9, SUMO and RanBP2 reveal a mechanism for SUMO paralog selection. Nat. Struct. Mol. Biol. 12:67-74
69. Werner A, Flotho A, Melchior F. 2012. The RanBP2/RanGAP1SUMO1/Ubc9 complex is a multisubunit SUMO E3 ligase. Mol. Cell 46:287-98
70. Hamada M, Haeger A, Jeganathan KB, van Ree JH, Malureanu L, et al. 2011. Ran-dependent docking of importin-βto RanBP2/Nup358 filaments is essential for protein import and cell viability. J. Cell Biol. 194:597-612
71. Kagey MH, Melhuish TA, Wotton D. 2003. The polycomb protein Pc2 is a SUMO E3. Cell 113:127-37
72. Ismail IH, Gagne JP, Caron MC, McDonald D, Xu Z, et al. 2012. CBX4-mediated SUMO modification regulates BMI1 recruitment at sites of DNA damage. Nucleic Acids Res. 40:5497-510
73. Rajendra R, Malegaonkar D, Pungaliya P, Marshall H, Rasheed Z, et al. 2004. Topors functions as an E3 ubiquitin ligase with specific E2 enzymes and ubiquitinates p53. J. Biol. Chem. 279:36440-44
74. Weger S, Hammer E, Heilbronn R. 2005. Topors acts as a SUMO-1 E3 ligase for p53 in vitro and in vivo. FEBS Lett. 579:5007-12
75. Tago K, Chiocca S, Sherr CJ. 2005. Sumoylation induced by theArf tumor suppressor: a p53-independent function. Proc. Natl. Acad. Sci. USA 102:7689-94
76. Woods YL, Xirodimas DP, Prescott AR, Sparks A, Lane DP, Saville MK. 2004. p14 Arf promotes small ubiquitin-like modifier conjugation of Werners helicase. J. Biol. Chem. 279:50157-66
77. García-Gutierrez P, Juarez-Vicente F, Gallardo-Chamizo F, Charnay P, García-Dominguez M. 2011. The transcription factor Krox20 is an E3 ligase that sumoylates its Nab coregulators. EMBO Rep. 12:1018-23
78. Pelisch F, Gerez J, Druker J, Schor IE, Munoz MJ, et al. 2010. The serine/arginine-rich protein SF2/ASF regulates protein sumoylation. Proc. Natl. Acad. Sci. USA 107:16119-24
79. Li SJ, Hochstrasser M. 1999. A new protease required for cell-cycle progression in yeast. Nature 398:246-51
80. Hay RT. 2007. SUMO-specific proteases: a twist in the tail. Trends Cell Biol. 17:370-76
81. Mukhopadhyay D, Dasso M. 2007. Modification in reverse: the SUMO proteases. Trends Biochem. Sci. 32:286-95
82. Yeh ET. 2009. SUMOylation and de-SUMOylation: wrestling with lifes processes. J. Biol. Chem. 284:8223-77
83. Shin EJ, Shin HM, Nam E, Kim WS, Kim JH, et al. 2012. DeSUMOylating isopeptidase: a second class of SUMO protease. EMBO Rep. 13:339-46
84. Iyer LM, Koonin EV, Aravind L. 2004. Novel predicted peptidases with a potential role in the ubiquitin signaling pathway. Cell Cycle 3:1440-50
85. Suh HY, Kim JH, Woo JS, Ku B, Shin EJ, et al. 2012. Crystal structure of DeSI-1, a novel deSUMOylase belonging to a putative isopeptidase superfamily. Proteins 80:2099-104
86. Schulz S, Chachami G, Kozaczkiewicz L, Winter U, Stankovic-Valentin N, et al. 2012. Ubiquitinspecific protease-like 1 (USPL1) is a SUMO isopeptidase with essential, non-catalytic functions. EMBO Rep. 13:930-38
87. Amsterdam A, Nissen RM, Sun Z, Swindell EC, Farrington S, Hopkins N. 2004. Identification of 315 genes essential for early zebrafish development. Proc. Natl. Acad. Sci. USA 101:12792-97
88. Mullen JR, Chen CF, Brill SJ. 2010. Wss1 is a SUMO-dependent isopeptidase that interacts genetically with the Slx5-Slx8 SUMO-targeted ubiquitin ligase. Mol. Cell. Biol. 30:3737-48
89. Cheng J, Kang X, Zhang S, Yeh ET. 2007. SUMO-specific protease 1 is essential for stabilization of HIF1αduring hypoxia. Cell 131:584-95
90. Van Nguyen T, Angkasekwinai P, Dou H, Lin FM, Lu LS, et al. 2012. SUMO-specific protease 1 is critical for early lymphoid development through regulation of STAT5 activation. Mol. Cell 45:210-21
91. Lee MH, Mabb AM, Gill GB, Yeh ET, Miyamoto S. 2011. NF-B induction of the SUMO protease SENP2: a negative feedback loop to attenuate cell survival response to genotoxic stress. Mol. Cell 43:180-91
92. Maison C, Romeo K, Bailly D, Dubarry M, Quivy JP, Almouzni G. 2012. The SUMO protease SENP7 is a critical component to ensure HP1 enrichment at pericentric heterochromatin. Nat. Struct. Mol. Biol. 19:458-60
93. Castoralova M, Brezinova D, Sveda M, Lipov J, Ruml T, Knejzlik Z. 2012. SUMO-2/3 conjugates accumulating under heat shock orMG132 treatment result largely from new protein synthesis. Biochim. Biophys. Acta 1823:911-19
94. Tatham MH, Matic I, Mann M, Hay RT. 2011. Comparative proteomic analysis identifies a role for SUMO in protein quality control. Sci. Signal. 4:rs4
95. Kamitani T, Nguyen HP, Kito K, Fukuda-Kamitani T, Yeh ET. 1998. Covalent modification of PML by the sentrin family of ubiquitin-like proteins. J. Biol. Chem. 273:3117-20
96. Müller S, Matunis MJ, Dejean A. 1998. Conjugation with the ubiquitin-related modifier SUMO-1 regulates the partitioning of PML within the nucleus. EMBO J. 17:61-70
97. Sternsdorf T, Jensen K, Reich B, Will H. 1999. The nuclear dot protein sp100, characterization of domains necessary for dimerization, subcellular localization, and modification by small ubiquitin-like modifiers. J. Biol. Chem. 274:12555-66
98. Bergink S, Jentsch S. 2009. Principles of ubiquitin and SUMO modifications in DNA repair. Nature 458:461-67
99. Garcia-Dominguez M, Reyes JC. 2009. SUMO association with repressor complexes, emerging routes for transcriptional control. Biochim. Biophys. Acta 1789:451-59
100. Heun P. 2007. SUMOrganization of the nucleus. Curr. Opin. Cell Biol. 19:350-55
101. Mukhopadhyay D, Dasso M. 2010. The fate of metaphase kinetochores is weighed in the balance of SUMOylation during S phase. Cell Cycle 9:3194-201
102. Nagai S, Davoodi N, Gasser SM. 2011. Nuclear organization in genome stability: SUMO connections. Cell Res. 21:474-85
103. Palancade B, Doye V. 2008. Sumoylating and desumoylating enzymes at nuclear pores: underpinning their unexpected duties? Trends Cell Biol. 18:174-83
104. Rothenbusch U, Sawatzki M, Chang Y, Caesar S, Schlenstedt G. 2012. Sumoylation regulates Kap114- mediated nuclear transport. EMBO J. 31:2461-72
105. Maison C, Bailly D, Roche D, Montes de Oca R, Probst AV, et al. 2011. SUMOylation promotes de novo targeting of HP1αto pericentric heterochromatin. Nat. Genet. 43:220-27
106. Huang J, Yan J, Zhang J, Zhu S, Wang Y, et al. 2012. SUMO1 modification of PTEN regulates tumorigenesis by controlling its association with the plasma membrane. Nat. Commun. 3:911
107. Castillo-Lluva S, Tatham MH, Jones RC, Jaffray EG, Edmondson RD, et al. 2010. SUMOylation of the GTPase Rac1 is required for optimal cell migration. Nat. Cell Biol. 12:1078-85
108. Yu J, Zhang D, Liu J, Li J, Yu Y, et al. 2012. RhoGDI SUMOylation at Lys-138 increases its binding activity to Rho GTPase and its inhibiting cancer cell motility. J. Biol. Chem. 287:13752-60
109. Kaminsky R, Denison C, Bening-Abu-Shach U, Chisholm AD, Gygi SP, Broday L. 2009. SUMO regulates the assembly and function of a cytoplasmic intermediate filament protein in C. elegans. Dev. Cell 17:724-35
110. Droescher M, Begitt A, Marg A, Zacharias M, Vinkemeier U. 2011. Cytokine-induced paracrystals prolong the activity of signal transducers and activators of transcription (STAT) and provide a model for the regulation of protein solubility by small ubiquitin-likemodifier (SUMO). J. Biol. Chem. 286:18731-46
111. Krumova P, Meulmeester E, Garrido M, Tirard M, Hsiao HH, et al. 2011. Sumoylation inhibits α- synuclein aggregation and toxicity. J. Cell Biol. 194:49-60
112. Fei E, Jia N, Yan M, Ying Z, Sun Q, et al. 2006. SUMO-1 modification increases human SOD1 stability and aggregation. Biochem. Biophys. Res. Commun. 347:406-12
113. Janer A, Werner A, Takahashi-Fujigasaki J, Daret A, Fujigasaki H, et al. 2010. SUMOylation attenuates the aggregation propensity and cellular toxicity of the polyglutamine expanded ataxin-7. Hum. Mol. Genet. 19:181-95
114. Mukherjee S, Thomas M, Dadgar N, Lieberman AP, Iniguez-Lluhi JA. 2009. Small ubiquitin-like modifier (SUMO) modification of the androgen receptor attenuates polyglutamine-mediated aggregation. J. Biol. Chem. 284:21296-306
115. Pichler A, Knipscheer P, Oberhofer E, van Dijk WJ, Korner R, et al. 2005. SUMO modification of the ubiquitin-conjugating enzyme E2-25K. Nat. Struct. Mol. Biol. 12:264-69
116. Matic I, Schimmel J, Hendriks IA, van Santen MA, van de Rijke F, et al. 2010. Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif. Mol. Cell 39:641-52
117. Chang CC, Naik MT, Huang YS, Jeng JC, Liao PH, et al. 2011. Structural and functional roles of Daxx SIM phosphorylation in SUMOparalog-selective binding and apoptosis modulation. Mol. Cell 42:62-74
118. Meulmeester E, Kunze M, Hsiao HH, Urlaub H, Melchior F. 2008. Mechanism and consequences for paralog-specific sumoylation of ubiquitin-specific protease 25. Mol. Cell 30:610-19
119. Zhu J, Zhu S, Guzzo CM, Ellis NA, Sung KS, et al. 2008. Small ubiquitin-related modifier (SUMO) binding determines substrate recognition and paralog-selective SUMO modification. J. Biol. Chem. 283:29405-15
120. Hoege C, Pfander B, Moldovan GL, Pyrowolakis G, Jentsch S. 2002. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419:135-41
121. Papouli E, Chen S, Davies AA, Huttner D, Krejci L, et al. 2005. Crosstalk betweenSUMOand ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p. Mol. Cell 19:123-33
122. Pfander B, Moldovan GL, Sacher M, Hoege C, Jentsch S. 2005. SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase. Nature 436:428-33
123. Kolli N, Mikolajczyk J, Drag M, Mukhopadhyay D, Moffatt N, et al. 2010. Distribution and paralogue specificity of mammalian deSUMOylating enzymes. Biochem. J. 430:335-44
124. Zhu S, Goeres J, Sixt KM, Bekes M, Zhang XD, et al. 2009. Protection from isopeptidase-mediated deconjugation regulates paralog-selective sumoylation of RanGAP1. Mol. Cell 33:570-80
125. Bylebyl GR, Belichenko I, Johnson ES. 2003. The SUMO isopeptidase Ulp2 prevents accumulation of SUMO chains in yeast. J. Biol. Chem. 278:44113-20
126. Tatham MH, Jaffray E, Vaughan OA, Desterro JM, Botting CH, et al. 2001. Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9. J. Biol. Chem. 276:35368-74
127. Blomster HA, Imanishi SY, Siimes J, Kastu J, Morrice NA, et al. 2010. In vivo identification of sumoylation sites by a signature tag and cysteine-targeted affinity purification. J. Biol. Chem. 285:19324-29
128. Bruderer R, Tatham MH, Plechanovova A, Matic I, Garg AK, Hay RT. 2011. Purification and identification of endogenous polySUMO conjugates. EMBO Rep. 12:142-48
129. Hsiao HH, Meulmeester E, Frank BT, Melchior F, Urlaub H. 2009. ChopNSpice amass spectrometric approach that allows identification of endogenous small ubiquitin-like modifier-conjugated peptides. Mol. Cell Proteomics 8:2664-75
130. Golebiowski F, Matic I, Tatham MH, Cole C, Yin Y, et al. 2009. System-wide changes to SUMO modifications in response to heat shock. Sci. Signal. 2:ra24
131. Lallemand-Breitenbach V, Jeanne M, Benhenda S, Nasr R, Lei M, et al. 2008. Arsenic degrades PML or PML-RARαthrough a SUMO-triggered RNF4/ubiquitin-mediated pathway. Nat. Cell Biol. 10:547-55
132. Tatham MH, Geoffroy MC, Shen L, Plechanovova A, Hattersley N, et al. 2008. RNF4 is a poly-SUMOspecific E3 ubiquitin ligase required for arsenic-induced PML degradation. Nat. Cell Biol. 10:538-46
133. Poukka H, Karvonen U, Janne OA, Palvimo JJ. 2000. Covalent modification of the androgen receptor by small ubiquitin-like modifier 1 (SUMO-1). Proc. Natl. Acad. Sci. USA 97:14145-50
134. Hietakangas V, Anckar J, Blomster HA, Fujimoto M, Palvimo JJ, et al. 2006. PDSM, a motif for phosphorylation-dependent SUMO modification. Proc. Natl. Acad. Sci. USA 103:45-50
135. Yang SH, Galanis A, Witty J, Sharrocks AD. 2006. An extended consensusmotif enhances the specificity of substrate modification by SUMO. EMBO J. 25:5083-93
136. Mohideen F, Capili AD, Bilimoria PM, Yamada T, Bonni A, Lima CD. 2009. A molecular basis for phosphorylation-dependent SUMO conjugation by the E2 UBC9. Nat. Struct. Mol. Biol. 16:945-52
137. Picard N, Caron V, Bilodeau S, Sanchez M, Mascle X, et al. 2012. Identification of estrogen receptor β as a SUMO-1 target reveals a novel phosphorylated sumoylation motif and regulation by glycogen synthase kinase 3β. Mol. Cell. Biol. 32:2709-21
138. Guo Z, Kanjanapangka J, Liu N, Liu S, Liu C, et al. 2012. Sequential posttranslational modifications program FEN1 degradation during cell-cycle progression. Mol. Cell 47:444-56
139. Yang SH, Jaffray E, Hay RT, Sharrocks AD. 2003. Dynamic interplay of the SUMO and ERK pathways in regulating Elk-1 transcriptional activity. Mol. Cell 12:63-74
140. Bossis G, Malnou CE, Farras R, Andermarcher E, Hipskind R, et al. 2005. Down-regulation of c-Fos/c- Jun AP-1 dimer activity by sumoylation. Mol. Cell. Biol. 25:6964-79
141. Camuzeaux B, Diring J, Hamard PJ, Oulad-Abdelghani M, Donzeau M, et al. 2008. p38β2-mediated phosphorylation and sumoylation of ATF7 are mutually exclusive. J. Mol. Biol. 384:980-91
142. Tan JA, Song J, Chen Y, Durrin LK. 2010. Phosphorylation-dependent interaction of SATB1 and PIAS1 directs SUMO-regulated caspase cleavage of SATB1. Mol. Cell. Biol. 30:2823-36
143. Chang CW, Chang GD, Chen H. 2011. A novel cyclic AMP/Epac1/CaMKI signaling cascade promotes GCM1 desumoylation and placental cell fusion. Mol. Cell. Biol. 31:3820-31
144. Zhang LJ, Vogel WK, Liu X, Topark-Ngarm A, Arbogast BL, et al. 2012. Coordinated regulation of transcription factor Bcl11b activity in thymocytes by the mitogen-activated protein kinase (MAPK) pathways and protein sumoylation. J. Biol. Chem. 287:26971-88
145. Psakhye I, Jentsch S. 2012. Protein group modification and synergy in theSUMOpathway as exemplified in DNA repair. Cell 151:807-20
146. Johnson ES, Blobel G. 1999. Cell cycle-regulated attachment of the ubiquitin-related protein SUMO to the yeast septins. J. Cell Biol. 147:981-94
147. Makhnevych T, Ptak C, Lusk CP, Aitchison JD, Wozniak RW. 2007. The role of karyopherins in the regulated sumoylation of septins. J. Cell Biol. 177:39-49
148. Silver HR, Nissley JA, Reed SH, Hou YM, Johnson ES. 2011. A role for SUMO in nucleotide excision repair. DNA Repair 10:1243-51
149. Tempe D, Piechaczyk M, Bossis G. 2008. SUMO under stress. Biochem. Soc. Trans. 36:874-78
150. Bossis G, Melchior F. 2006. Regulation of SUMOylation by reversible oxidation of SUMO conjugating enzymes. Mol. Cell 21:349-57
151. Zhou W, Ryan JJ, Zhou H. 2004. Global analyses of sumoylated proteins in Saccharomyces cerevisiae: induction of protein sumoylation by cellular stresses. J. Biol. Chem. 279:32262-68
152. Kurepa J, Walker JM, Smalle J, Gosink MM, Davis SJ, et al. 2003. The small ubiquitin-like modifier (SUMO) protein modification system in Arabidopsis: Accumulation of SUMO1 and -2 conjugates is increased by stress. J. Biol. Chem. 278:6862-72
153. Lee YJ, Miyake S, Wakita H, McMullen DC, Azuma Y, et al. 2007. Protein SUMOylation ismassively increased in hibernation torpor and is critical for the cytoprotection provided by ischemic preconditioning and hypothermia in SHSY5Y cells. J. Cereb. Blood Flow Metab. 27:950-62
154. Yang W, Sheng H, Warner DS, Paschen W. 2008. Transient global cerebral ischemia induces a massive increase in protein sumoylation. J. Cereb. Blood Flow Metab. 28:269-79
155. Yang W, Thompson JW, Wang Z, Wang L, Sheng H, et al. 2012. Analysis of oxygen/glucosedeprivation- induced changes in SUMO3 conjugation using SILAC-based quantitative proteomics. J. Proteome Res. 11:1108-17
156. Datwyler AL, Lattig-Tunnemann G, Yang W, Paschen W, Lee SL, et al. 2011. SUMO2/3 conjugation is an endogenous neuroprotective mechanism. J. Cereb. Blood Flow Metab. 31:2152-9
157. Lee YJ, Mou Y, Maric D, Klimanis D, Auh S, Hallenbeck JM. 2011. Elevated global SUMOylation in Ubc9 transgenic mice protects their brains against focal cerebral ischemic damage. PLoS ONE 6:e25852
158. Huang C, Han Y, Wang Y, Sun X, Yan S, et al. 2009. SENP3 is responsible for HIF-1 transactivation under mild oxidative stress via p300 de-SUMOylation. EMBO J. 28:2748-62
159. Yan S, Sun X, Xiang B, Cang H, Kang X, et al. 2010. Redox regulation of the stability of the SUMO protease SENP3 via interactions with CHIP and Hsp90. EMBO J. 29:3773-86
160. Xu Z, Lam LS, Lam LH, Chau SF, Ng TB, Au SW. 2008. Molecular basis of the redox regulation of SUMO proteases: a protective mechanism of intermolecular disulfide linkage against irreversible sulfhydryl oxidation. FASEB J. 22:127-37
161. Leitao BB, Jones MC, Brosens JJ. 2011. The SUMO E3-ligase PIAS1 couples reactive oxygen species- dependent JNK activation to oxidative cell death. FASEB J. 25:3416-25
162. de la Vega L, Grishina I, Moreno R, Kruger M, Braun T, Schmitz ML. 2012. A redox-regulated SUMO/acetylation switch ofHIPK2 controls the survival threshold to oxidative stress. Mol. Cell 46:472-83
163. Sydorskyy Y, Srikumar T, Jeram SM, Wheaton S, Vizeacoumar FJ, et al. 2010. A novel mechanism for SUMO system control: regulated Ulp1 nucleolar sequestration. Mol. Cell. Biol. 30:4452-62
164. Li SJ, Hochstrasser M. 2003. The Ulp1 SUMO isopeptidase: distinct domains required for viability, nuclear envelope localization, and substrate specificity. J. Cell Biol. 160:1069-81
165. Panse VG, Kuster B, Gerstberger T, Hurt E. 2003. Unconventional tethering of Ulp1 to the transport channel of the nuclear pore complex by karyopherins. Nat. Cell Biol. 5:21-27
166. Conti L, Price G, ODonnell E, Schwessinger B, Dominy P, Sadanandom A. 2008. Small ubiquitin-like modifier proteases overly tolerant to salt1 and -2 regulate salt stress responses in Arabidopsis. Plant Cell 20:2894-908
167. Pinto MP, Carvalho AF, Grou CP, Rodrguez-Borges JE, Sa-Miranda C, Azevedo JE. 2012. Heat shock induces a massive but differential inactivation of SUMO-specific proteases. Biochim. Biophys. Acta 1823:1958-66
168. Wu SY, Chiang CM. 2009. Crosstalk between sumoylation and acetylation regulates p53-dependent chromatin transcription and DNA binding. EMBO J. 28:1246-59
169. Finkbeiner E, Haindl M, Müller S. 2011. The SUMO system controls nucleolar partitioning of a novel mammalian ribosome biogenesis complex. EMBO J. 30:1067-78
170. Panse VG, Kressler D, Pauli A, Petfalski E, Gnadig M, et al. 2006. Formation and nuclear export of preribosomes are functionally linked to the small-ubiquitin-related modifier pathway. Traffic 7:1311-21
171. Hardeland U, Steinacher R, Jiricny J, Schar P. 2002. Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover. EMBO J. 21:1456-64
172. Danielsen JR, Povlsen LK, Villumsen BH, Streicher W, Nilsson J, et al. 2012. DNA damage-inducible SUMOylation of HERC2 promotes RNF8 binding via a novel SUMO-binding zinc finger. J. Cell Biol. 197:179-87
173. Pilla E, Moller U, Sauer G, Mattiroli F, Melchior F, Geiss-Friedlander R. 2012. A novel SUMO1- specific interacting motif in dipeptidyl peptidase 9 (DPP9) that is important for enzymatic regulation. J. Biol. Chem. 287:44320-29
174. Alegre KO, Reverter D. 2011. Swapping small ubiquitin-like modifier (SUMO) isoform specificity of SUMO proteases SENP6 and SENP7. J. Biol. Chem. 286:36142-51
175. Hecker CM, Rabiller M, Haglund K, Bayer P, Dikic I. 2006. Specification of SUMO1- and SUMO2- interacting motifs. J. Biol. Chem. 281:16117-27
176. Song J, Durrin LK, Wilkinson TA, Krontiris TG, Chen Y. 2004. Identification of a SUMO-binding motif that recognizes SUMO-modified proteins. Proc. Natl. Acad. Sci. USA 101:14373-78
177. Song J, Zhang Z, Hu W, Chen Y. 2005. Small ubiquitin-like modifier (SUMO) recognition of a SUMO binding motif: a reversal of the bound orientation. J. Biol. Chem. 280:40122-29
178. Husnjak K, Dikic I. 2012. Ubiquitin-binding proteins: decoders of ubiquitin-mediated cellular functions. Annu. Rev. Biochem. 81:291-322
179. Seet BT, Dikic I, Zhou MM, Pawson T. 2006. Reading protein modifications with interaction domains. Nat. Rev. Mol. Cell Biol. 7:473-83
180. Kerscher O. 2007. SUMO junction-whats your function? New insights through SUMO-interacting motifs. EMBO Rep. 8:550-55
181. vanWijk SJ, Müller S, Dikic I. 2011. Shared and unique properties of ubiquitin and SUMO interaction networks in DNA repair. Genes Dev. 25:1763-69
182. Lin DY, Huang YS, Jeng JC, Kuo HY, Chang CC, et al. 2006. Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of sumoylated transcription factors. Mol. Cell 24:341-54
183. Armstrong AA, Mohideen F, Lima CD. 2012. Recognition of SUMO-modified PCNA requires tandem receptor motifs in Srs2. Nature 483:59-63
184. Prudden J, Pebernard S, Raffa G, Slavin DA, Perry JJ, et al. 2007. SUMO-targeted ubiquitin ligases in genome stability. EMBO J. 26:4089-101
185. Sun H, Leverson JD, Hunter T. 2007. Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins. EMBO J. 26:4102-12
186. Uzunova K, Gottsche K, Miteva M, Weisshaar SR, Glanemann C, et al. 2007. Ubiquitin-dependent proteolytic control of SUMO conjugates. J. Biol. Chem. 282:34167-75
187. Xie Y, Kerscher O, Kroetz MB, McConchie HF, Sung P, Hochstrasser M. 2007. The yeast Hex3Slx8 heterodimer is a ubiquitin ligase stimulated by substrate sumoylation. J. Biol. Chem. 282:34176-84
188. Zhang XD, Goeres J, Zhang H, Yen TJ, Porter AC, Matunis MJ. 2008. SUMO-2/3 modification and binding regulate the association of CENP-E with kinetochores and progression through mitosis. Mol. Cell 29:729-41
189. Stehmeier P, Müller S. 2009. Phospho-regulatedSUMOinteractionmodules connect the SUMOsystem to CK2 signaling. Mol. Cell 33:400-9
190. Ullmann R, Chien CD, Avantaggiati ML, Müller S. 2012. An acetylation switch regulates SUMOdependent protein interaction networks. Mol. Cell 46:759-70
191. Praefcke GJ, Hofmann K, Dohmen RJ. 2012. SUMO playing tag with ubiquitin. Trends Biochem. Sci. 37:23-31
192. Ulrich HD, Walden H. 2010. Ubiquitin signalling in DNA replication and repair. Nat. Rev. Mol. Cell Biol. 11:479-89
193. Bekker-Jensen S, Mailand N. 2011. The ubiquitin- and SUMO-dependent signaling response to DNA double-strand breaks. FEBS Lett. 585:2914-19
194. Galanty Y, Belotserkovskaya R, Coates J, Polo S, Miller KM, Jackson SP. 2009. Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA double-strand breaks. Nature 462:935-39
195. Morris JR, Boutell C, Keppler M, Densham R, Weekes D, et al. 2009. The SUMOmodification pathway is involved in the BRCA1 response to genotoxic stress. Nature 462:886-90
196. Bekker-Jensen S, Rendtlew Danielsen J, Fugger K, Gromova I, Nerstedt A, et al. 2010. HERC2 coordinates ubiquitin-dependent assembly of DNA repair factors on damaged chromosomes. Nat. Cell Biol. 12:80-86; suppl. 1-12
197. Hu X, Paul A, Wang B. 2012. Rap80 protein recruitment to DNA double-strand breaks requires binding to both small ubiquitin-like modifier (SUMO) and ubiquitin conjugates. J. Biol. Chem. 287:25510-19
198. Luo K, Zhang H, Wang L, Yuan J, Lou Z. 2012. Sumoylation of MDC1 is important for proper DNA damage response. EMBO J. 31:3008-19
199. Strauss C, Goldberg M. 2011. Recruitment of proteins to DNA double-strand breaks: MDC1 directly recruits RAP80. Cell Cycle 10:2850-57
200. Strauss C, Halevy T, Macarov M, Argaman L, Goldberg M. 2011. MDC1 is ubiquitylated on its tandem BRCT domain and directly binds RAP80 in a UBC13-dependent manner. DNA Repair 10:806-14
201. Yin Y, Seifert A, Chua JS, Maure JF, Golebiowski F, Hay RT. 2012. SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage. Genes Dev. 26:1196-208
202. Galanty Y, Belotserkovskaya R, Coates J, Jackson SP. 2012. RNF4, a SUMO-targeted ubiquitin E3 ligase, promotes DNA double-strand break repair. Genes Dev. 26:1179-95
203. Demarque MD, Nacerddine K, Neyret-Kahn H, Andrieux A, Danenberg E, et al. 2011. Sumoylation by Ubc9 regulates the stem cell compartment and structure and function of the intestinal epithelium in mice. Gastroenterology 140:286-96
204. Kanakousaki K, Gibson MC. 2012. A differential requirement for SUMOylation in proliferating and non-proliferating cells during Drosophila development. Development 139:2751-62
205. Bettermann K, Benesch M, Weis S, Haybaeck J. 2012. SUMOylation in carcinogenesis. Cancer Lett. 316:113-25
206. Bawa-Khalfe T, Yeh ET. 2010. SUMO losing balance: SUMO proteases disrupt SUMO homeostasis to facilitate cancer development and progression. Genes Cancer 1:748-52
207. Kessler JD, Kahle KT, Sun T, Meerbrey KL, Schlabach MR, et al. 2012. A SUMOylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis. Science 335:348-53
208. Stielow B, Kruger I, Diezko R, Finkernagel F, Gillemans N, et al. 2010. Epigenetic silencing of spermatocyte-specific and neuronal genes by SUMO modification of the transcription factor Sp3. PLoS Genet. 6:e1001203
209. Lee FY, Faivre EJ, Suzawa M, Lontok E, Ebert D, et al. 2011. Eliminating SF-1 (NR5A1) sumoylation in vivo results in ectopic hedgehog signaling and disruption of endocrine development. Dev. Cell 21:315-27
210. Bertolotto C, Lesueur F, Giuliano S, Strub T, de Lichy M, et al. 2011. A SUMOylation-defectiveMITF germline mutation predisposes to melanoma and renal carcinoma. Nature 480:94-98
211. Yokoyama S, Woods SL, Boyle GM, Aoude LG, MacGregor S, et al. 2011. A novel recurrent mutation in MITF predisposes to familial and sporadic melanoma. Nature 480:99-103
212. Wang J, Feng XH, Schwartz RJ. 2004. SUMO-1 modification activated GATA4-dependent cardiogenic gene activity. J. Biol. Chem. 279:49091-98
213. Kim EY, Chen L, Ma Y, Yu W, Chang J, et al. 2011. Expression of sumoylation deficient Nkx2. 5 mutant in Nkx2. 5 haploinsufficient mice leads to congenital heart defects. PLoS ONE 6:e20803
214. Zhang YQ, Sarge KD. 2008. Sumoylation regulates lamin A function and is lost in lamin A mutants associated with familial cardiomyopathies. J. Cell Biol. 182:35-39
215. Kho C, Lee A, Jeong D, Oh JG, Chaanine AH, et al. 2011. SUMO1-dependentmodulation of SERCA2a in heart failure. Nature 477:601-5
216. Sarge KD, Park-Sarge OK. 2009. Sumoylation and human disease pathogenesis. Trends Biochem. Sci. 34:200-5
217. Rodriguez MS, Dargemont C, Hay RT. 2001. SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. J. Biol. Chem. 276:12654-59
218. Yang XJ, Gregoire S. 2006. A recurrent phospho-sumoyl switch in transcriptional repression and beyond. Mol. Cell 23:779-86
219. Stankovic-Valentin N, Deltour S, Seeler J, Pinte S, Vergoten G, et al. 2007. An acetylation/deacetylation- SUMOylation switch through a phylogenetically conservedKXEPmotif in the tumor suppressor HIC1 regulates transcriptional repression activity. Mol. Cell. Biol. 27:2661-75
220. Yang Y, Fu W, Chen J, Olashaw N, Zhang X, et al. 2007. SIRT1 sumoylation regulates its deacetylase activity and cellular response to genotoxic stress. Nat. Cell Biol. 9:1253-62
221. Brandl A, Wagner T, Uhlig KM, Knauer SK, Stauber RH, et al. 2012. Dynamically regulated sumoylation of HDAC2 controls p53 deacetylation and restricts apoptosis following genotoxic stress. J. Mol. Cell Biol. 4:284-93