The Translation Post Vol.1 Issue 2

Review Article ™ SUMO: The Guardian of the Proteome

By John Mountzouris, Ph.D., Director Product Support

Introduction

Sumoylation is a novel post-translational modification system that has been the object of intense interest in recent years (1-6).  Analogous to ubiquitin, reversible covalent attachment of SUMO (small ubiquitin-like modifier) to lysine residues in substrate proteins alters the properties of the proteins to which SUMO conjugates.  However, in contrast to ubiquitin, SUMO conjugation does not typically lead to degradation of the substrate; rather, sumoylation orchestrates a diverse array of effects on many different biological processes, including protein localization and stability, transcriptional activities, nucleocytoplasmic signaling and transport, and genomic replication, as well as the regulation of gene expression and viral reproduction. 

More than 100 SUMO-1 target proteins have been reported, many whose dysregulation is associated with specific disease states (4, 7, 8).  Protein targets for modification include tumor suppressors p53 (plus regulatory proteins), PML, transcription factors such as receptors for androgen, progesterone and gluticorticoids, c-Jun, c-Myb, and the C/EBP proteins.  DNA repair and replication targets include topoisomerase I and II, PCNA, and histone deacetylase, with other modified proteins including heat shock proteins and signal transduction molecules. 

SUMO Isoforms

In vertebrates, four SUMO isoforms, SUMO-1, SUMO-2, SUMO-3, and SUMO-4, have been identified in vertebrates with distinct functions (9-11).  The SUMO-2 and SUMO-3 proteins share 95% amino acid sequence identity, but only approximately 47% identity with SUMO-1 (Figure 1).

Tissue-specific SUMO-4, identified in human kidney, bears homology to SUMO-2/3, suggesting that some SUMO proteins could have tissue-dependent functions (12).

Figure 1

SUMO-1 conjugates to proteins as a monomer, while SUMO-2 and SUMO-3 conjugate to proteins as higher molecular weight polymers with SUMO-1 terminating further SUMO addition.   The in vivo functions of SUMO-2/3 modifications appear to be distinct from those of SUMO-1. All SUMO isoforms are attached to substrate proteins through a biochemical pathway similar to that of ubiquitination (10).

The Molecular Role of SUMO Modification

SUMO modification is thought to provide a new binding site for interactions with other proteins, thereby acting as a recruiting factor for multiprotein complexes.  For example, sumoylation of RanGAP1 facilitates interaction with the nuclear pore protein RanBP2/Nup358 (13, 14). SUMO modification of transcription factors P300 and Elk-1, permits their recruitment of histone deacetylase 6 (HDAC6) (15) and HDAC2 (16).  SUMO modification sites are often located in variable loop structures, or at flexible termini, (17, 18), suggesting the SUMO modification may not exert its influence through altering conformation. The physical model of how SUMO performs its regulatory function has eluded precise definition.

SUMO modification of target proteins

For SUMO modification, a consensus sequence ψ-K-x-D/E (where lysine is the modified amino acid, ψ is a hydrophobic residue and x is any amino acid residue) is required for SUMO modification both in vivo and in vitro using (Figure 2 and Table 1) (4,19). Conversely to SUMO modification, ubiquitination site selection appears to require merely the lysine side chain, and in many cases mutagenesis studies have failed to reveal preferred ubiquitination sites.

Figure 2

Table 1

HQWF

PML (2)

The mechanism involved in maturation and transfer of SUMO to target substrates parallels that for ubiquitination  (4, 20), comprising four steps: maturation, activation, conjugation, and ligation. In the first step the SUMO protein is cleaved by SUMO-specific carboxyl-terminal hydrolase to produce a carboxyl- terminal diglycine. SUMO modification then proceeds by a three-step enzyme shuttle, analogous to ubiquitin addition (21), a process composed of an E1-E2-E3 enzyme catalyzed cascade of activation (E1), conjugation to E2 (UBC9, Figure 3), which is homologous to the ubiquitin E2s, and finally to the terminal amino group of a lysine side chain in target proteins, which may require E3 (ligase). E1 and E2 appear to be conserved for the SUMO proteins, while several different SUMO ligases (E3) have been identified in higher eukaryotes.  Despite the sequence differences among SUMO paralogues, the activation enzyme E1 and conjugation enzyme E2 do not discriminate among the SUMO molecules (13).  SUMO covalent linkage to the substrate protein is reversible, and SUMO-deconjugating enzymes add an additional layer to the regulation of the sumoylation process (4,19).

Figure 3

SUMOplot ™ A tool for prediction of SUMO sites

The existence of the relatively well-defined sumoylation consensus motif permitted the creation of an algorithm that can scan protein primary sequences and identify potential sites.  Considering all the proven SUMO targets and sumoylation sequences therein an online tool for predicting SUMO sites has been constructed (sumoplot). Since the acceptor Lys residue can appear surrounded by different residues a methodology has been developed to score and rate the different sites based on well-characterized hydropathicity rules (e.g. Kyte and Doolittle).  The SUMOplot™ score system is based on two criteria: 1) amino acid appearance in the SUMO consensus site observed and shown to bind Ubc9, and 2) propensity of these amino acids to maintain similar hydrophobicity, known to be the major driving force for protein folding. Below is an example of the output of SUMOplot when tested on PML protein (Figure 4). Potential sites are underlined and colored in the sequence as well as extracted and ordered according to their score together with surrounding amino acid sequences. SUMOplot accurately predicts sumoylation sites for a variety of proteins such as TOP1, PML, p53, AP1, RGP1, HSF1 and2, RANG, SP10, ANDR, MYB, ARNT, Q1AD55.

Figure 4

Biological Functions of SUMO Modification

The number of proteins and pathways affected by SUMO regulation, and the function thereof are growing in numbers, such that they cannot be exhaustively covered in this brief review.  However, highlights of key examples in the areas of protein localization, genetic regulation, transcriptional activities, and viral reproduction will serve to demonstrate the diversity of effects that SUMO modification has upon modulation of cellular functions.

Subcellular Localization

Sumoylation participates in regulating the subcellular partitioning of dozens of substrate proteins known to date. For example, RanGAP1, a cytoplasmic protein facilitating protein transport across the nuclear pore complex, is localized to the nuclear pore complex upon SUMO modification (13,14). Intriguingly, components of the sumoylation machinery such as the E2 conjugation enzyme and the E3 ligase are enriched at the nuclear pore complex (21). Nuclear bodies containing multiprotein complexes have yielded rich insight into the role of SUMO modification in protein recruitment and localization. Sumoylation has been found to be required for the subcellular localization of many proteins found in PML nuclear bodies, while the sumoylation state of other proteins does not control their localization to the PML nuclear bodies, but does effect their ability to recruit binding partners. For example, the sumoylated forms of PML and Sp100 are found exclusively in the nucleus (22); this modification is mandatory for PML localization. On the other hand, the targeting and accumulation of Sp100 in these bodies does not depend upon sumoylation (22,23) as revealed through studies mutating the target lysine.

SUMO and the Chromosome

Partitioning of chromosomes into replicated cells is a highly choreographed and intricate process that is dependent upon the proper scheduling of sister chromatid assembly and separation. Observation of dysregulation of this process in yeast SUMO-1 mutants, resulting in defective mitosis and chromosomal segregation, was one of the first findings in a cascade of experiments describing the role of SUMO modification in maintaining genetic integrity (24,25). Sumoylation has also been implicated in the recruitment and retention of proteins to the kinetochore, the protein complex that forms at the centromere and gathers microtubules for anaphase separation (26-28).

Sumoylation of a number of critical tumor suppressor and repair proteins implicated have additionally pointed to the role of SUMO-1 in preserving genomic stability (26-28). p53 and Mdm2 are both targets of sumoylation, which has functional effects on the activities of these proteins (2-4). Evidence indicates that components of the Wnt signaling cascade (e.g. axin, -catenin, LEF/Tcf-4) are also targets of sumoylation, (2-4). A number of DNA repair enzymes are also subject to regulation by sumoylation.   Sumoylation has also been implicated in the repair of the DNA damage mediated by topoisomerase II (29,30). 

SUMO and Transcription Regulation

Sumoylation of proteins involved in the transcription machinery is involved in regulation of gene expression (2,4,31), primarily through repression, with the noticeable exception of heat shock factors, which are upregulated (32,33).  Transcription factors inhibited by SUMO modification of the substrate lysine include STAT1,  c-Jun, CEBPα, c-Myb,  IRF-1, SREBPs, SRF, Elk, AP1, AP2, androgen receptor (AR), glucocorticoid receptor (GR), and progesterone receptor (PR) huntingtin and several others (2, 4, 34).

Many transcription factors are regulated by association with PML nuclear bodies, and assembly of these structures requires sumoylation of the PML protein (3,4). The PML gene, involved in the (35,36) chromosomal translocation of acute promyelocytic leukemia (APL), encodes a protein which localizes to the PML Oncogenic Domains (PODs), subnuclear macromolecular structures (37, 35, 38). The intercession of sumoylation upon transcriptional activity is therefore potentially widely broadcast in terms of effects downstream. In illustration, sumoylation of PML recruits corepressor Daxx to PML nuclear bodies, thereby removing Daxx-mediated repression of these genes. Similarly, sumoylation of PML directs p53 to PML nuclear bodies, which stimulates the transcriptional activity of p53; given the ubiquitous role of P53 in cellular signaling pathways, the ramifications of p53 sumoylation are significant.

The effects of SUMO on transcriptional activity may also also operate indirectly -  discovery of sumoylation of a number of transcription co-activators and corepressors, such as GRIP1, SRC-1, and histone deacetylases (HDAC) hints at the complexity of sumoylation in transcriptional network regulation (1,3,4, 31).

SUMO and the Viral life Cycle

In addition to sumoylation of host proteins, the modification of specific viral proteins by SUMO has been shown to play a role in viral reproduction.  Sumoylation of major-immediate-early proteins for human cytomegalovirus, herpes virus, Epstein-Barr, adenovirus, and papillomavirus have been shown to enhance transactivation of key proteins in viral reproduction, and to effect localization of the viral proteins to the host nucleus (39-44).  In addition, specific patterns of sumoylation of host proteins may provide a cellular environment conducive to or deterring viral infection (39). Work in this area is accelerating, given the potential to understand and thereby impair by novel means the viral infections via targeting the sumoylation pathway. 

Conclusion

The dizzying array of biological functions in which the posttranslational modification sumoylation is involved put this modification among those such as phosphorylation that have far-reaching effects on cellular function. Many questions remain to be answered, yet the growing interest in this small protein with big effects on networks for both homeostatisis and change is bound to fuel new discoveries and novel therapeutic approaches to diseases impact by SUMO protein substrates for this fascinating mechanism of biological control.

1. Poukka, H., Aarnisalo, P., Karvonen, U., Palvimo, J. J. & Janne, O. A. (1999) J. Biol. Chem. 274, 19441-19446.

2. Muller, S., Ledl, A. & Schmidt, D. (2004) Oncogene 23, 1998-2008.

3. Seeler, J. S. & Dejean, A. (2003) Nat. Rev. Mol. Cell. Biol. 4, 690-699.

4. Johnson, E. S. (2004) Annu. Rev. Biochem. 73, 355™382.

5. Kotaja, N., Karvonen, U., Janne, O. A. & Palvimo, J. J. (2002) J. Biol. Chem. 277, 30283-30288.

6. Chauchereau, A., Amazit, L., Quesne, M., Guiochon-Mantel, A. & Milgrom, E. (2003) J. Biol. Chem. 278, 12335-12343.

7. Steffan, J. S., Agrawal, N., Pallos, J., Rockabrand, E., Trotman, L. C., Slepko, N., Illes, K., Lukacsovich, T., Zhu, Y. Z., Cattaneo, E., et al. (2004) Science 304, 100-104.

8. Kakizuka, A., Miller, W. H., Jr., Umesono, K., Warrell, R. P., Jr., Frankel, S. R., Murty, V. V., Dmitrovsky, E. & Evans, R. M. (1991) Cell 66, 663-674.

9. Saitoh, H. & Hinchey, J. (2000) J. Biol. Chem. 275, 6252-6258.

10. Hay, R. T. (2001) Trends Biochem. Sci. 26, 332-333.

11. Tatham, M. H., Kim, S., Yu, B., Jaffray, E., Song, J., Zheng, J., Rodriguez, M. S., Hay, R. T. & Chen, Y. (2003) Biochemistry 42, 9959-9969.

12. Bohren, K. M., Nadkarni, V., Song, J. H., Gabbay, K. H., and Owerbach, D. (2004) J. Biol. Chem. 279, 27233™27238.

13. Matunis, M. J., Wu, J. & Blobel, G. (1998) J. Cell Biol. 140, 499-509.

14. Mahajan, R., Delphin, C., Guan, T., Gerace, L. & Melchior, F. (1997) Cell 88, 97-107.

15. Girdwood, D., Bumpass, D., Vaughan, O. A., Thain, A., Anderson, L. A., Snowden, A. W., Garcia-Wilson, E., Perkins, N. D. & Hay, R. T. (2003) Mol. Cell 11, 1043-1054.

16. Yang, S. H. & Sharrocks, A. D. (2004) Mol. Cell 13, 611-617.

17. Bernier-Villamor, V., Sampson, D. A., Matunis, M. J. & Lima, C. D. (2002) Cell 108, 345-356.

18. Rodriguez, M. S., Desterro, J. M., Lain, S., Midgley, C. A., Lane, D. P. & Hay, R. T. (1999) EMBO J. 18, 6455-6461.

19. Melchior, F. 2000. SUMO: Nonclassical ubiquitin. Annu. Rev. Cell Dev. Biol.   16: 591-626.

20. Schwartz, D. C., and Hochstrasser, M. (2003) Trends Biochem. Sci. 28, 321™328.

21. Zhang, H., Saitoh, H., and Matunis, M. J. (2002) Mol. Cell. Biol. 22, 6498™6508.

22. Sternsdorf, T., Jensen, K., and Will, H. (1997) J. Cell Biol. 139, 1621™1634.

23. Sternsdorf, T., Jensen, K., Reich, B., and Will, H. (1999) J. Biol. Chem. 274, 12555.

24. Yamamoto, H., Ihara, M., Matsuura, Y., and Kikuchi, A. (2003) EMBO J. 22, 2047™2059.

25. Tanaka, K., Nishide, J., Okazaki, K., Kato, H., Niwa, O., Nakagawa, T., Matsuda, H., Kawamukai, M., and Murakami, Y. (1999) Mol. Cell. Biol. 19, 8660™8672.

26.  Joseph, J., Tan, S. H., Karpova, T. S., McNally, J. G., and Dasso, M. (2002) J. Cell Biol. 156, 595™602.

27. Fukagawa, T., Regnier, V., and Ikemura, T. (2001) Nucleic Acids Res. 29, 3796™3803.

28. Meluh, P. B., and Koshland, D. (1995) Mol. Biol. Cell 6, 793™807.

29. Mao, Y., Sun, M., Desai, S. D., and Liu, L. F. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4046™4051.

30. Mo, Y. Y., Yu, Y., Shen, Z., and Beck, W. T. (2002) J. Biol. Chem. 277, 2958™2964.

31. Verger, A., Perdomo, J., and Crossley, M. (2003) EMBO Rep. 4, 137™142.

32. Hong, Y., Rogers, R., Matunis, M. J., Mayhew, C. N., Goodson, M. L., Park-Sarge, O. K., and Sarge, K. D. (2001) J. Biol. Chem. 276, 40263™40267.

33. Goodson, M. L., Hong, Y., Rogers, R., Matunis, M. J., Park-Sarge, O. K., and Sarge, K. D. (2001) J. Biol. Chem. 276, 18513™18518.

34. Schmidt, D., and Muller, S. (2003) Cell Mol. Life Sci. 60, 2561™2574.

35. Jensen, K., Shiels, C., Freemont, P.S. (2001) Oncogene. 20, 7223™7233.

36. Grimwade, D., Solomon, E. (1997) Curr. Topics Microbiol. Immunol. 220, 81-112.

37. Brasch, K., Ochs, R.L. (1992) Exp Cell Res. 202, 211-23.

38. Hodges, M., Tissot, C., Howe, K., Grimwade, D. and Freemont, P. S. (1998) Am. J. Hum. Genet. 63, 297-304.

39. Wilson VG, Rangasamy D. Virus Res. (2001) 81, 17-27.

40. Rangasamy D, Wilson VG. J Biol Chem. (2000) 275, 30487-95.

41. Nevels M, Brune W, Shenk T. J Virol. (2004) 78, 7803-12.

42. Colombo R, Boggio R, Seiser C, Draetta GF, Chiocca S. EMBO Rep. (2002) 3, 1062-8.

43. Izumiya Y, Ellison TJ, Yeh ET, Jung JU, Luciw PA, Kung HJ., J Virol. (2005) 79, 9912-25.

44. Gurer C, Berthoux L, Luban J. J Virol. (2005) 79, 910-7.