Ubiquitin-like Proteases Research Articles

In the moonlight: non-catalytic functions of ubiquitin and ubiquitin-like proteases.

Proteases that cleave ubiquitin or ubiquitin-like proteins (UBLs) are critical players in maintaining the homeostasis of the organism. Concordantly, their dysregulation has been directly linked to various diseases, including cancer, neurodegeneration, developmental aberrations, cardiac disorders and inflammation. Given their potential as novel therapeutic targets, it is essential to fully understand their mechanisms of action. Traditionally, observed effects resulting from deficiencies in deubiquitinases (DUBs) and UBL proteases have often been attributed to the misregulation of substrate modification by ubiquitin or UBLs. Therefore, much research has focused on understanding the catalytic activities of these proteins. However, this view has overlooked the possibility that DUBs and UBL proteases might also have significant non-catalytic functions, which are more prevalent than previously believed and urgently require further investigation. Moreover, multiple examples have shown that either selective loss of only the protease activity or complete absence of these proteins can have different functional and physiological consequences. Furthermore, DUBs and UBL proteases have been shown to often contain domains or binding motifs that not only modulate their catalytic activity but can also mediate entirely different functions. This review aims to shed light on the non-catalytic, moonlighting functions of DUBs and UBL proteases, which extend beyond the hydrolysis of ubiquitin and UBL chains and are just beginning to emerge.

Beyond reversal: ubiquitin and ubiquitin-like proteases and the orchestration of the DNA double strand break repair response.

The cellular response to genotoxic DNA double strand breaks (DSBs) uses a multitude of post-translational modifications to localise, modulate and ultimately clear DNA repair factors in a timely and accurate manner. Ubiquitination is well established as vital to the DSB response, with a carefully co-ordinated pathway of histone ubiquitination events being a central component of DSB signalling. Other ubiquitin-like modifiers (Ubl) including SUMO and NEDD8 have since been identified as playing important roles in DSB repair. In the last five years ∼20 additional Ub/Ubl proteases have been implicated in the DSB response. The number of proteases identified highlights the complexity of the Ub/Ubl signal present at DSBs. Ub/Ubl proteases regulate turnover, activity and protein–protein interactions of DSB repair factors both catalytically and non-catalytically. This not only ensures efficient repair of breaks but has a role in channelling repair into the correct DSB repair sub-pathways. Ultimately Ub/Ubl proteases have essential roles in maintaining genomic stability. Given that deficiencies in many Ub/Ubl proteases promotes sensitivity to DNA damaging chemotherapies, they could be attractive targets for cancer treatment.

Structure of a pathogen effector reveals the enzymatic mechanism of a novel acetyltransferase family.

Effectors secreted by the type III secretion system are essential for bacterial pathogenesis. Members of the Yersinia outer-protein J (YopJ) family of effectors found in diverse plant and animal pathogens depend on a protease-like catalytic triad to acetylate host proteins and produce virulence. However, the structural basis for this noncanonical acetyltransferase activity remains unknown. Here, we report the crystal structures of the YopJ effector HopZ1a, produced by the phytopathogen Pseudomonas syringae, in complex with the eukaryote-specific cofactor inositol hexakisphosphate (IP6) and/or coenzyme A (CoA). Structural, computational and functional characterizations reveal a catalytic core with a fold resembling that of ubiquitin-like cysteine proteases and an acetyl-CoA-binding pocket formed after IP6-induced structural rearrangements. Modeling-guided mutagenesis further identified key IP6-interacting residues of Salmonella effector AvrA that are required for acetylating its substrate. Our study reveals the structural basis of a novel class of acetyltransferases and the conserved allosteric regulation of YopJ effectors by IP6.

Effect of mild hypothermia on expression of small ubiquitin-like modifier-specific proteases 3 in brain tissues during cerebral ischemia-reperfusion in mice

Objective To investigate the effect of mild hypothermia on the expression of small ubiquitin-like modifier-specific proteases 3(SENP3)in the brain tissues during cerebral ischemia-reperfusion(I/R)in mice. Methods Ninety-six male C57/BL6 mice, aged 10-12 weeks, weighing 22-30 g, were randomly divided into 3 groups(n=32 each)using a random number table: sham operation group(group S), group I/R, and mild hypothermia group(group H). Cerebral I/R was produced by occlusion of bilateral common carotid arteries for 15 min followed by reperfusion.The surface cooling was started immediately after reperfusion, and the rectal temperature was maintained at 32-34 ℃ for 3 h. At 6, 12, 24 and 72 h of reperfusion, 8 mice were selected from each group and sacrificed.The hippocampi were removed for examination of the pathological changes(with light microscope)and for determination of cell apoptosis(using TUNEL)and expression of SENP3(by Western blot). The apoptosis rate was calculated. Results Compared with group S, the apoptosis rate in hippocampal CA1 region was significantly increased, and the expression of SENP3 was significantly up-regulated at each time point of reperfusion in group I/R(P<0.05). Compared with group I/R, the apoptosis rate in hippocampal CA1 region was significantly decreased, and the expression of SENP3 was significantly down-regulated at each time point of reperfusion(P<0.05), and the pathological changes were significantly reduced in group H. Conclusion The mechanism by which mild hypothermia reduces cerebral I/R injury is associated with inhibition of SENP3 expression in the brain tissues of mice. Key words: Hypothermia, induced; Cysteine endopeptidases; Reperfusion injury; Brain

SUMO proteases: uncovering the roles of deSUMOylation in plants.

Plants have evolved to cope with changing environmental conditions. One way plants achieve this is through post-translational modification of target proteins by ubiquitination and SUMOylation. These post-translational modifiers (PMs) can alter stability, protein-protein interactions, and the overall fate of the protein. Both of these systems have remarkable similarities in terms of the process leading to attachment of the PM to its substrate : having to undertake activation, conjugation, and finally ligation to the target. In the ubiquitin system, there are a vast number of ubiquitin ligase enzymes (E3s) that provide specificity for the attachment of ubiquitin. With the SUMO system, only a small number of SUMO E3 ligases have so far been identified in the fully sequenced plant genomes. In Arabidopsis thaliana, there are only two SUMO E3s, compared to over 1400 ubiquitin E3s, a trend also observed in crop species such as Oryza sativa and Zea mays Recent research indicates that removing SUMO from its substrate by the enzymatically active SUMO proteases is a vital part of this system. A class of SUMO proteases called ubiquitin-like proteases (ULPs) are widespread in all eukaryotes; within plants, both monocot and dicot kingdoms have conserved and divergent ULPs and ULP-like proteases. This paper examines the roles ULPs have in stress responses and highlights the 'fine-tuning' of SUMO attachment/removal in balancing growth versus stress.

Stability of small ubiquitin-like modifier (SUMO) proteases OVERLY TOLERANT TO SALT1 and -2 modulates salicylic acid signalling and SUMO1/2 conjugation in Arabidopsis thaliana.

Small ubiquitin-like modifier proteases 1 and 2 (SUMO1/2) have been linked to the regulation of salicylic acid (SA)-mediated defence signalling in Arabidopsis thaliana. In order to define the role of the SUMO proteases OVERLY TOLERANT TO SALT1 and -2 (OTS1/2) in defence and to provide insight into SUMO1/2-mediated regulation of SA signalling, we examined the status of SA-mediated defences in ots1/2 mutants. The ots1 ots2 double mutant displayed enhanced resistance to virulent Pseudomonas syringae and higher levels of SA compared with wild-type (WT) plants. Furthermore, ots1 ots2 mutants exhibited upregulated expression of the SA biosynthesis gene ICS1 in addition to enhanced SA-responsive ICS1 expression beyond that of WT. SA stimulated OTS1/2 degradation and promoted accumulation of SUMO1/2 conjugates. These results indicate that OTS1 and -2 act in a feedback loop in SA signalling and that de novo OTS1/2 synthesis works antagonistically to SA-promoted degradation, adjusting the abundance of OTS1/2 to moderate SA signalling. Accumulation of SUMO1/2 conjugates coincides with SA-promoted OTS degradation and may play a positive role in SA-mediated signalling in addition to its repressive roles reported elsewhere.

Over-expression of small ubiquitin-like modifier proteases 1 predicts chemo-sensitivity and poor survival in non-small cell lung cancer

Background Non-small cell lung cancer (NSCLC) is one of the most common malignant tumors. Despite the advances in therapy over the years, its mortality remains high. The aim of this study was to evaluate the expression of small ubiquitin-like modifier (SUMO) proteases 1 (SENP1) in NSCLC tissues and its role in the regulation of vascular endothelial growth factor (VEGF) expression. We also investigated the association between the expression level of SENP1 and the clinicopathological features and survival of the patients. Methods A SENP1 small interfering RNA (siRNA) was constructed and transfected into the NSCLC cells. VEGF gene expression was analyzed by real-time polymerase chain reaction (RT-PCR). Immunohistochemistry staining was used to assess the expression of SENP1 in 100 NSCLC patients and its association with the clinicopathological features and survival was analyzed. Results VEGF expression was significantly higher in NSCLC tissues than in normal lung tissues. Inhibition of SENP1 by siRNA was associated with decreased VEGF expression. SENP1 was over-expressed in 55 of the 100 NSCLC samples (55%) and was associated with a moderate and low histological tumor grade (3.6%, 38.2%, and 58.2% in high, moderate and low differentiated tumors, respectively, P=0.046), higher T stage (10.9% in T1, and 89.1% in T2 and T3 tumor samples, P &lt;0.001) and TNM stage (10.9% in stage I, and 89.1% in stages II and III tumor samples, P &lt;0.001). The rate of lymph node metastasis was significantly higher in the SENP1 over-expression group (76.4%) than that in the SENP1 low expression group (33.3%, P &lt;0.001). Sixty three patients received postoperative chemotherapy, including 34 with SENP1 over-expression and 29 with SENP1 low expression. Among the 34 patients with SENP1 over-expression, 22 (64.7%) patients developed recurrence or metastasis, significantly higher than those in the low expression group 27.6% (8/29) (P=0.005). Multivariate Cox regression analysis showed that lymph node metastasis (P=0.015), TNM stage (P=0.001), and SENP1 expression level (P=0.002) were independent prognostic factors for the survival of NSCLC patients. Conclusions SENP1 may be a promising predictor of survival, a predictive factor of chemo-sensitivity for NSCLC patients, and potentially a desirable drug target for lung carcinoma target therapy.

Dynamins at a glance

The superfamily of dynamins includes classical dynamins and dynamin-related proteins. Classical dynamins are proteins that share sequence similarity with the first described dynamin, which is a large GTPase with five characteristic domains. Dynamin-related proteins differ from classical dynamins in

Chlamydia trachomatis‐derived deubiquitinating enzymes in mammalian cells during infection

Chlamydia trachomatis is an obligate intracellular bacterium that causes a variety of diseases in humans. C. trachomatis has a complex developmental cycle that depends on host cells for replication, during which gene expression is tightly regulated. Here we identify two C. trachomatis proteases that possess deubiquitinating and deneddylating activities. We have designated these proteins ChlaDub1 and ChlaDub2. The genes encoding ChlaDub1 and ChlaDub2 are present in all Chlamydia species except for Chlamydia pneumoniae, and their catalytic domains bear similarity to the catalytic domains of other eukaryotic ubiquitin-like proteases (Ulp). The C. trachomatis DUBs react with activity-based probes and hydrolyse ubiquitinated and neddylated substrates. ChlaDub1 and ChlaDub2 represent the first known bacterial DUBs that possess both deubiquitinating and deneddylating activities.

Chemistry-based functional proteomics: mechanism-based activity-profiling tools for ubiquitin and ubiquitin-like specific proteases.

Determining the biological function of newly discovered gene products requires the development of novel functional approaches. To facilitate this task, recent developments in proteomics include small molecular probes that target proteolytic enzyme families including serine, threonine, and cysteine proteases. For the families of ubiquitin (Ub) and ubiquitin-like (UBL)-specific proteases, such tools were lacking until recently. Here, we review the advances made in the development of protein-based active site-directed probes that target proteases specific for ubiquitin and ubiquitin-like proteins. Such probes were applied successfully to discover and characterize novel Ub/UBL-specific proteases. Ub/UBL processing and deconjugation are performed by a diverse set of proteases belonging to several different enzyme families, including members of the ovarian tumor domain (OTU) protease family. A further definition of this family of enzymes will benefit from a directed chemical proteomics approach. Some of the Ub/UBL-specific proteases react with multiple Ub/UBLs and members of the same protease family can recognize multiple Ub/UBLs, underscoring the need for tools that appropriately address enzyme specificity.

Reply

As an increasing number of novel mechanisms for NF-κB pathway inhibition by bacteria are reported, we become more aware of the diversity of interactions that mediate the host-pathogen relationship and that hold clues to development of effective anti-inflammatory therapies. Our findings on the Salmonella AvrA effector may represent an example of physiological inhibition of NF-κB activation at the level of IκB ubiquitination. Ubiquitination is an enzymatically mediated covalent modification of substrate proteins that generally results in the proteasomal degradation of the modified proteins. The ubiquitination of IκB by the SCF-βTrCP complex and its subsequent degradation is a classical example. Recently, it has become apparent that modification by ubiquitin and the closely related proteins SUMO and NEDD8 are involved in myriad other regulatory roles including signaling and intracellular trafficking. An expanding list of ubiquitin ligases and hydrolases suggests a substantial regulatory network similar to the many known cellular kinases and phosphatases. As Dr. Peek mentioned, subsequent studies will be focused on identifying the target substrate(s) acted upon by S. typhimurium AvrA. AvrA has sequence and predicted structural similarities with several other bacterial and viral proteases that are capable of cleavage of the isopeptide bond formed between ubiquitin (or ubiquitin-like) monomers and the conjugated protein (Curr Opin Microbiol 2002;5:38–43). Regulatory events mediated by ubiquitin and ubiquitin-like proteins have been implicated in at least 4 steps of the NF-κB activation pathway and in other proinflammatory signaling pathways (Nat Immunol 2002;3:20–26). Thus, potential targets of AvrA include cellular proteins required for phospho-IκB ubiquitination that are posttranslationally modified by ubiquitin, SUMO, or NEDD8. The study of ubiquitin-like proteases in bacteria with anti-inflammatory or proapoptotic effects on their host cells should reveal evolutionarily conserved regulatory mechanisms that may be fruitful targets for anti-inflammatory drug discovery. As Dr. Peek pointed out, isogenic inactivation of avrA partially reversed inhibition of IL-8 secretion induced by wild-type nonpathogenic strains, but the effect was incomplete, raising the possibility that there may be other bacterial mediators contributing to the suppression of IκB ubiquitination. The recent publication of the genomic sequences of Salmonella and other enteric pathogens will greatly facilitate the search for candidate bacterial effector proteins with similar function.

Yersinia lead SUMO attack.

Little is known about the mechanism by which Yops, proteins that Yersinia inject into the cytosol of macrophage, cause downregulation of the inflammatory response and diseases such as the plague. Now it appears that Yops are the first bacterial member of a new family of ubiquitin-like proteases.