SUSP1 antagonizes formation of highly SUMO2/3-conjugated species

Small ubiquitin-related modifier (SUMO) processing and deconjugation are mediated by sentrin-specific proteases/ubiquitin-like proteases (SENP/Ulps). We show that SUMO-specific protease 1 (SUSP1), a mammalian SENP/Ulp, localizes within the nucleoplasm. SUSP1 depletion within cell lines expressing enhanced green fluorescent protein (EGFP) fusions to individual SUMO paralogues caused redistribution of EGFP-SUMO2 and -SUMO3, particularly into promyelocytic leukemia (PML) bodies. Further analysis suggested that this change resulted primarily from a deficit of SUMO2/3-deconjugation activity. Under these circumstances, PML bodies became enlarged and increased in number. We did not observe a comparable redistribution of EGFP-SUMO1. We have investigated the specificity of SUSP1 using vinyl sulfone inhibitors and model substrates. We found that SUSP1 has a strong paralogue bias toward SUMO2/3 and that it acts preferentially on substrates containing three or more SUMO2/3 moieties. Together, our findings argue that SUSP1 may play a specialized role in dismantling highly conjugated SUMO2 and -3 species that is critical for PML body maintenance.     

A new 30-kDa ubiquitin-related SUMO-1 hydrolase from bovine brain.

SUMO-1 is a ubiquitin-like protein functioning as an important reversible protein modifier. To date there is no report on a SUMO-1 hydrolase/isopeptidase catalyzing the release of SUMO-1 from its precursor or SUMO-1-ligated proteins in mammalian tissues. Here we found multiple activities that cleave the SUMO-1 moiety from two model substrates, (125)I-SUMO-1-alphaNH-HSTVGSMHISPPEPESEEEEEHYC and/or GST-SUMO-1-(35)S-RanGAP1 conjugate, in bovine brain extracts. Of them, a major SUMO-1 C-terminal hydrolase had been partially purified by successive chromatographic operations. The enzyme had the ability to cleave SUMO-1 not only from its precursor but also from a SUMO-1-ligated RanGAP1 but did not exhibit any significant cleavage of the ubiquitin- and NEDD8-precursor. The activity of SUMO-1 hydrolase was almost completely inhibited by N-ethylmaleimide, but not by phenylmethanesulfonyl fluoride, EDTA, and ubiquitin-aldehyde known as a potent inhibitor of deubiquitinylating enzymes. Intriguingly, the apparent molecular mass of the isolated SUMO-1 hydrolase was approximately 30 kDa, which is significantly smaller than the recently identified yeast Smt3/SUMO-1 specific protease Ulp1. These results indicate that there are multiple SUMO-1 hydrolase/isopeptidases in mammalian cells and that the 30-kDa small SUMO-1 hydrolase plays a central role in processing of the SUMO-1-precursor.     

Covalent modification of human immunodeficiency virus type 1 p6 by SUMO-1

The p6 domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein mediates virion budding from infected cells via protein-protein contacts with the class E vacuolar protein sorting factors, Tsg101 and AIP1/ALIX. Interaction with Tsg101 is strengthened by covalent attachment of monovalent ubiquitin to HIV-1 p6. To identify additional host factors that bind to HIV-1 p6, a human cDNA library was screened in the yeast two-hybrid system. HIV-1 p6 was found to interact with small ubiquitin-like modifier 1 (SUMO-1) as well as the E2 SUMO-1 transfer enzyme, Ubc9. Interaction with p6 was also detected with Daxx, a cellular protein to which SUMO-1 is sometimes covalently attached. SUMO-1 was incorporated into HIV-1 virions where it was protected within the virion membrane from digestion by exogenous protease. Of the two lysine residues in p6, lysine 27 uniquely served as a site of covalent SUMO-1 attachment. As previously reported, though, HIV-1 bearing the p6-K27R mutation replicated just like the wild type. Overproduction of SUMO-1 in HIV-1 producer cells had no apparent effect on virion release or on virion protein or RNA content. Infectivity of the resulting virions, though, was decreased, with the defect occurring after membrane fusion, at the time of viral cDNA synthesis. HIV-1 bearing the p6-K27R mutation was insensitive to SUMO-1 overexpression, suggesting that covalent attachment of SUMO-1 to p6 is detrimental to HIV-1 replication.     

SUMO-specific protease SUSP4 positively regulates p53 by promoting Mdm2 self-ubiquitination

The p53 tumour suppressor has a key role in the control of cell growth and differentiation, and in the maintenance of genome integrity. p53 is kept labile under normal conditions, but in response to stresses, such as DNA damage, it accumulates in the nucleus for induction of cell-cycle arrest, DNA repair or apoptosis. Mdm2 is an ubiquitin ligase that promotes p53 ubiquitination and degradation. Mdm2 is also self-ubiquitinated and degraded. Here, we identified a novel cascade for the increase in p53 level in response to DNA damage. A new SUMO-specific protease, SUSP4, removed SUMO-1 from Mdm2 and this desumoylation led to promotion of Mdm2 self-ubiquitination, resulting in p53 stabilization. Moreover, SUSP4 competed with p53 for binding to Mdm2, also resulting in p53 stabilization. Overexpression of SUSP4 inhibited cell growth, whereas knockdown of susp4 by RNA interference (RNAi) promoted of cell growth. UV damage induced SUSP4 expression, leading to an increase in p53 levels in parallel with a decrease in Mdm2 levels. These findings establish a new mechanism for the elevation of cellular p53 levels in response to UV damage.     

Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3

Post-translational modification marked by the covalent attachment of the ubiquitin-like protein SUMO-1/SMT3C has been implicated in a wide variety of cellular processes. Recently, two cDNAs encoding proteins related to SUMO-1 have been identified in human and mouse. The functions and regulation of these proteins, known as SUMO-2/SMT3A and SUMO-3/SMT3B, remain largely uncharacterized. We describe herein quantitative and qualitative distinctions between SUMO-1 and SUMO-2/3 in vertebrate cells. Much of this was accomplished through the application of an antibody that recognizes SUMO-2 and -3, but not SUMO-1. This antibody detected multiple SUMO-2/3-modified proteins and revealed that, together, SUMO-2 and -3 constitute a greater percentage of total cellular protein modification than does SUMO-1. Intriguingly, we found that there was a large pool of free, non-conjugated SUMO-2/3 and that the conjugation of SUMO-2/3 to high molecular mass proteins was induced when the cells were subjected to protein-damaging stimuli such as acute temperature fluctuation. In addition, we demonstrated that SUMO-2/3 conjugated poorly, if at all, to a major SUMO-1 substrate, the Ran GTPase-activating protein RanGAP1. Together, these results support the concept of important distinctions between the SUMO-2/3 and SUMO-1 conjugation pathways and suggest a role for SUMO-2/3 in the cellular responses to environmental stress.     

A novel mammalian Smt3-specific isopeptidase 1 (SMT3IP1) localized in the nucleolus at interphase.

A novel Smt3-specific isopeptidase, SMT3IP1, was cloned using a yeast two-hybrid screen with Smt3b as bait. The clone, named SMT3IP1 (Smt3-specific isopeptidase 1), which bound to Smt3b but not SUMO-1 in the two-hybrid system, was distantly related to budding yeast Saccharomyces cerevisiae Ulp1, human SENP1 or human SUSP1. The catalytic domains in the C-terminal region were very similar, but the N-terminal region was quite different to other enzymes. The cysteine, histidine and asparatic acid residues in the catalytic domains were conserved. SMT3IP1 expressed by the baculovirus-expression system had the ability to cleave SUMO-1 or Smt3b from SUMO-1/RanGAP1 or Smt3b/RanGAP1 conjugates, respectively, and the activity was a little stronger towards the Smt3b conjugate than towards the SUMO-1 conjugate. Furthermore, the enzyme bound more strongly to Smt3a and Smt3b than to SUMO-1 in vitro. The enzyme did not cleave Nedd8 from Nedd8/cullin-1. Nor did it cleave ubiquitin from ubiquitinated p53. SMT3IP1 was localized almost exclusively at the nucleolus during interphase. The N-terminal sequence was responsible for the nucleolar localization of this enzyme. Whether SMT3IP1 functions in the nucleolus or just stays there before it functions in the nucleus, as shown in the case of CDC14 phosphatase, remains to be elucidated.     

African swine fever virus protease, a new viral member of the SUMO-1-specific protease family

African swine fever virus (ASFV) is a complex DNA virus that employs polyprotein processing at Gly-Gly-Xaa sites as a strategy to produce several major core components of the viral particle. The virus gene S273R encodes a 31-kDa protein that contains a "core domain" with the conserved catalytic residues characteristic of SUMO-1-specific proteases and the adenovirus protease. Using a COS cell expression system, it was found that protein pS273R is capable of cleaving the viral polyproteins pp62 and pp220 in a specific way giving rise to the same intermediates and mature products as those produced in ASFV-infected cells. Furthermore, protein pS273R, like adenovirus protease and SUMO-1-specific enzymes, is a cysteine protease, because its activity is abolished by mutation of the predicted catalytic histidine and cysteine residues and is inhibited by sulfhydryl-blocking reagents. Protein pS273R is expressed late after infection and is localized in the cytoplasmic viral factories, where it is found associated with virus precursors and mature virions. In the virions, the protein is present in the core shell, a domain where the products of the viral polyproteins are also located. The identification of the ASFV protease will allow a better understanding of the role of polyprotein processing in virus assembly and may contribute to our knowledge of the emerging family of SUMO-1-specific proteases.     

Characterization of the localization and proteolytic activity of the SUMO-specific protease, SENP1

Modification of proteins by small ubiquitin-like modifier (SUMO) plays an important role in the function, compartmentalization, and stability of target proteins, contributing to the regulation of diverse processes. SUMO-1 modification can be regulated not only at the level of conjugation; it may also be reversed by a class of proteases known as the SUMO-specific proteases. However, current understanding of the regulation, specificity, and function of these proteases remains limited. In this study, we characterize aspects of the compartmentalization and proteolytic activity of the mammalian SUMO-specific protease, SENP1, providing insight into its function and regulation. We demonstrate the presence of a single nonconsensus nuclear localization signal within the N terminus of the protein, the mutation of which results in pronounced cytoplasmic accumulation in contrast to the nuclear accumulation of the parental protein. In addition, we observe that the N terminus of the protein may be essential for the correct regulation of the protease, since expression of the core domain alone results in limited expression and loss of SUMO-1, indicative of constitutive catalytic activity. Consistent with the prediction that the protease is a member of the cysteine family of proteases, we mutated a key cysteine residue and observed that expression of this catalytic mutant had a dominant negative phenotype, resulting in the accumulation of high molecular weight SUMO-1 conjugates. Furthermore, we demonstrate that SENP1 may itself be a target for SUMO-1 modification occurring at a nonconsensus site. Finally, we demonstrate that SENP1 localization is influenced by expression and localization of SUMO-1-conjugated target proteins within the cell.     

SUMO-1共价修饰ataxin-3

为了探讨ataxin-3的正常生理功能以及脊髓小脑型共济失调Ⅲ型/马查多-约瑟夫病的发病机理,采用酵母双杂交技术,选择polyQ扩展突变型ataxin-3全长构建诱饵质粒,筛选成人脑cDNA文库,寻找与之相互作用的蛋白质,筛选到互作蛋白smallubiquitin-likemodifier1(SUMO-1).进一步运用免疫共沉淀技术证实,SUMO-1在哺乳动物细胞中共价修饰野生型和polyQ扩展突变型ataxin-3.免疫荧光共定位实验发现,polyQ扩展突变型ataxin-3形成的核内蛋白聚合体与SUMO-1共定位.研究提示,ataxin-3的正常生理功能可能受SUMO-1的调节,SUMO-1可能参与了脊髓小脑型共济失调Ⅲ型/马查多-约瑟夫病的发病机制.     

Association of the human SUMO-1 protease SENP2 with the nuclear pore

SUMO-1 is a small ubiquitin-like protein that can be covalently conjugated to other proteins. A family of proteases catalyzes deconjugation of SUMO-1-containing species. Members of this family also process newly synthesized SUMO-1 into its conjugatable form. To understand these enzymes better, we have examined the localization and behavior of the human SUMO-1 protease SENP2. Here we have shown that SENP2 associates with the nuclear face of nuclear pores and that this association requires protein sequences near the N terminus of SENP2. We have also shown that SENP2 binds to Nup153, a nucleoporin that is localized to the nucleoplasmic face of the pore. Nup153 binding requires the same domain of SENP2 that mediates its targeting in vivo. Removal of the Nup153-interacting region of SENP2 results in a significant change in the spectrum of SUMO-1 conjugates within the cell. Our results suggest that association with the pore plays an important negative role in the regulation of SENP2, perhaps by restricting its activity to a subset of the conjugated proteins within the nucleus.     

Pellino-1, an adaptor protein of interleukin-1 receptor/toll-like receptor signaling,is sumoylated by Ubc9

Covalent modifications of the Pellino-1 protein are essential for transmitting innate immune response signals downstream, as the phosphorylation and polyubiquitination of Pellino-1 mediated by the IRAK proteins appear to have roles in regulating Pellino-1 function. In this study, we demonstrate that the Pellino-1 protein is post-translationally modified by small-ubiquitin-related modifier-1 (SUMO-1). Sumoylation assays with Pellino-1 and SUMO-1 expression plasmids reveal that the Pellino-1 protein is sumoylated in vitro and in vivo. Treatment of SUMO-1 specific protease 1 (SENP1) inhibited the sumoylation of the Pellino-1 protein and a GST pull-down assay as well as a yeast two hybrid assay showed that Pellino-1 binds to the SUMO-conjugating enzyme, Ubc9. Furthermore, we identified the five lysine residues of the Pellino-1 protein where SUMO-1 covalently attaches. Some of the sumoylated sites overlap with previously identified ubiquitination sites, suggesting competition between sumoylation and ubiquitination, as well as suggesting that the sumoylated Pellino-1 protein may have a cellular function distinct from previously identified functions.     

Effects of PTX1 expression on growth and tumorigenicity of the prostate cancer cell line PC-3

PTX1 is a gene identified by subtractive hybridization on the basis that it is expressed in normal prostate and not in prostate carcinoma. It encodes a nuclear protein that is downregulated in prostate carcinoma. Expression constructs containing PTX1 cDNA in both sense and antisense orientations were transfected into prostate tumor cell line, PC-3 cells. The effects of the expression of PTX1 and antisense PTX1 on PC-3 cells were examined using cell growth, proliferation, soft agar, invasion chamber, senescence-associated beta-galactosidase, and nude mice assays. Cells transfected with PTX1 construct in the sense orientation were growth-arrested. These cells displayed multiple morphological changes consistent with cellular senescence, including the expression of a senescence-associated beta-galactosidase. On the other hand, expression of antisense PTX1 RNA in PC-3 cells resulted in uncontrolled cell growth and increase of invasive potential. In nude mice, cells expressing antisense PTX1 grew sixfold faster than the control. These results suggest that PTX1 may play an important role in the growth and tumorigenicity of PC-3 cells.     

Evidence for Covalent Modification of the Nuclear Dot–associated Proteins PML and Sp100 by PIC1/SUMO-1

PML and Sp100 proteins are associated with nuclear domains, known as nuclear dots (NDs). They were discovered in the context of leukemic transformation and as an autoantigen in primary biliary cirrhosis, respectively. Both proteins are expressed in the form of many COOH-terminally spliced variants, and their expression is enhanced by interferons (IFN). The recent finding that PIC1/SUMO-1, a small ubiquitin-like protein, is covalently linked to the RanGAP1 protein of the nuclear pore complex and also binds PML in yeast cells led us to determine whether PML is covalently modified by PIC1/SUMO-1 and whether the same is true for Sp100. We found an immune reaction of PML and Sp100 proteins with a PIC1/SUMO-1–specific monoclonal antibody by immunoblotting when using cell extracts prepared from stably transfected cells inducibly expressing one isoform of each protein as well as from nontransfected cells. In contrast, both proteins did not react when synthesized in vitro. Immunofluorescence staining showed that PIC1/SUMO-1 colocalized with Sp100 and PML in NDs except in mitotic cells, in which PML and Sp100 are dissociated. Cell fractionation and immunoblotting demonstrated that PIC1/SUMO-1 immunoreactive Sp100 in IFN-treated and untreated cells was exclusively nuclear, whereas nonmodified Sp100 was also found in the cytoplasm. Taken together, these data strongly suggest covalent modification of specific nuclear isoforms of Sp100 and PML by PIC1/SUMO-1. This modification may play a regulatory role in ND structure, composition, and function.