SUMO Proteases

SUMO proteases (Ulp’s) have peptidase activity (Lois, 2010; Miura, Jin & Hasegawa, 2007) to process precursor SUMO molecules (Conti et al., 2008; Dye & Schulman, 2007; Mukhopadhyay & Dasso, 2007) and isopeptidase activity to recycle SUMO from substrates (Miura, Jin & Hasegawa, 2007). These combined together function as part of the SUMOylation process are illustrated in Figure

6-1.

Figure 6-1 – The SUMOylation cycle is maintained by dual functions of SUMO protease as a peptidase for maturation of the SUMO protein and an isopeptidase for de-SUMOylation. Stages of SUMOylation processing (solid arrows) and recycling (dashed arrows) are shown. SUMO = Small Ubiquitin-Like Modifier, E1 = SUMO activating enzyme and E2 = SUMO conjugating enzyme. Taken from GAM1

and the SUMO pathway (Boggio & Chiocca, 2005).

Only a low number of protein substrates are ordinarily SUMOylated at any set time, suggesting that SUMO proteases have an essential regulatory role during

SUMOylation (Johnson, 2004). The overall function of these proteases is to regulate and maintain the cellular pool of SUMO available for protein modification (Lois, 2010), by controlling the equilibrium between the SUMOylated and deSUMOylated states of any given protein (Conti et al., 2008). This essential function in the SUMO cycle, requiring additional specificity, may be why there are a high number of Ulp’s compared with SUMO E1 and E2 enzymes, suggesting a complex regulation of signalling at the level of deSUMOylation. Requirement for certain protease-substrate interactions or selectivity for different SUMO proteases for a particular SUMO isoform could be necessary (Conti et al., 2008). Significant in-vitro cleavage assays showed SUMO substrate preferences for Arabidopsis SUMO proteases, which in some cases were governed by the N-terminal domain of the protease (Chosed et al., 2006). Studies in the yeast species Saccharomyces cerevisiae show that the N-terminal domain modulates Ulp1 activity, target binding ability and localisation (Mossessova & Lima, 2000; Li & Hochstrasser, 2003). Evidence shows that distinction in the N-terminal domain may define the basis of specificity of particular SUMO proteases for certain signalling pathways in Arabidopsis (Conti et al., 2008).

SUMO proteases have been discovered in a number of eukaryotic species from humans and animals. In humans, SUMOylation is regulated by a family of sentrin/SUMO-specific proteases, termed SENP’s. There are seven SENP’s (SENP 1, 2, 3, 5, 6, 7, 8) identified in human species of which there has been variable characterisation, with SENP1 and SENP2 the best defined family members

modified proteins (Rajan et al., 2005), while SENP3 and SENP5 form a subfamily of SENP’s that control formation of SUMO 2 and SUMO 3 conjugates, and in a lesser extent SUMO 1 modification (Gong & Yeh, 2006). SENP7 is found to be responsible for chromatin relaxation, with deSUMOylation by this protease necessary to uphold an appropriate chromatin environment for DNA repair (Garvin et al., 2013).

Homologous to the SENP’s in humans are a group of SENP’s/Ulp’s in yeast, called Ulp1 and Ulp2 (Smt4) (Li & Hochstrasser, 1999; 2000). Ulp1 is a protein fundamental to yeast that co-localises with nuclear pore proteins. Deletion of

Ulp1 means yeast becomes non-viable. Ulp2 localises primarily to the nucleus and gene deletion of Ulp2 leads to abnormal growth of yeast and hypersensitivity to DNA damage (Taylor et al., 2002).

There are also SUMO proteases found to be important in plant-pathogen interactions. Avirulence factors such as Xanthomonas campertris vesicatoria4 (AvrXV4) from the plant pathogenic bacterial species X.campestris can play a role as a SUMO protease. It appears to reduce SUMO conjugation onto target proteins in order to disrupt defence in host plant cells (Roden et al., 2004).

In Arabidopsis thaliana there have at least eight SUMO proteases identified, and

some of these have been at least partially characterized. AtUlp1a, AtUlp1c, AtUlp1d and AtESD4 are reported to have a variable N-terminal domain and conserved C-terminal domain associated with catalytic activity (Kurepa et al., 2003; Chosed et al., 2006; Colby et al., 2006).

Ulp1a, also known as ESD-Like SUMO Protease 1 (ELS1) is the least well understood, with little information on its localization, phenotype, or any work done with knockout lines (Lois, 2010) until recent work found that although it is homologous to ESD4 in sequence they have very distinct functions. ELS1 is localised to the cytoplasm, potentially in association in endo-membranes, and is only very weakly detected in the nucleolus, but not other parts of the nucleus (Hermkes et al., 2011). This is in contrast to the other plant SUMO proteases, which are all nuclear localised (Colby et al., 2006). The ELS1 protease has also been reported to have an essential role in SUMO maturation (Li & Hochstrasser, 1999; Hermkes et al., 2011).

Early in Short Days 4 (ESD4) is a far better characterised SUMO protease, which links together the role of SUMO metabolism and control of flowering time (Murtas et al., 2003). The mutation esd4 was found to convey an extreme early- flowering phenotype in Arabidopsis, and an accelerated transition from vegetative growth to flowering, especially emphasised in short-day conditions which normally delays flowering (Reeves et al., 2002). The esd4 mutant was restricted in regulation of SUMOylation, due to the absence of a functional SUMO protease and had further phenotypes including broadening of silique, alteration of phyllotaxy and dwarf appearance. It was also reported to be localised to the nuclear envelope of cells (Murtas et al., 2003).

The other two proteases, referred to as Ulp1c and Ulp1d, are also called OTS1 and OTS2, and are SUMO proteases involved in responses to high salinity (Conti et al., 2008). These two SUMO proteases will be focused on in section 6.1.3.

While there are more numerous, compared with other parts of SUMO machinery, the conservation in sequence among these proteases is much lower than other elements of this machinery. Sequence identity amongst SUMO proteases is between 12 and 44% for the four characterised isoforms that have been mentioned, making discovery of any further SUMO proteases in

Arabidopsis challenging (Lois, 2010). These differences are due to a high degree

of divergence in the Ulp N-terminal domain responsible for regulation. Even the two OTS SUMO proteases, which function redundantly, have only 25% N- terminal sequence identity compared with 70% identity in their catalytic domains (Mukhopadhyay & Dasso, 2007).