UHRF2, a Ubiquitin E3 Ligase,Acts as a Small Ubiquitin-like Modifier E3 Ligase for Zinc Finger Protein 131

Small ubiquitin-like modifier (SUMO), a member of the ubiquitin-related protein family, is covalently conjugated to lysine residues of its substrates in a process referred to as SUMOylation. SUMOylation occurs through a series of enzymatic reactions analogous to that of the ubiquitination pathway, resulting in modification of the biochemical and functional properties of substrates. To date, four mammalian SUMO isoforms, a single heterodimeric SUMO-activating E1 enzyme SAE1/SAE2, a single SUMO-conjugating E2 enzyme ubiquitin-conjugating enzyme E2I (UBC9), and a few subgroups of SUMO E3 ligases have been identified. Several SUMO E3 ligases such as topoisomerase I binding, arginine/serine-rich (TOPORS), TNF receptor-associated factor 7 (TRAF7), and tripartite motif containing 27 (TRIM27) have dual functions as ubiquitin E3 ligases. Here, we demonstrate that the ubiquitin E3 ligase UHRF2 also acts as a SUMO E3 ligase. UHRF2 effectively enhances zinc finger protein 131 (ZNF131) SUMOylation but does not enhance ZNF131 ubiquitination. In addition, the SUMO E3 activity of UHRF2 on ZNF131 depends on the presence of SET and RING finger-associated and nuclear localization signal-containing region domains, whereas the critical ubiquitin E3 activity RING domain is dispensable. Our findings suggest that UHRF2 has independent functional domains and regulatory mechanisms for these two distinct enzymatic activities.     

SUMO与乳腺癌

小泛素修饰物（Small ubiquitin-like modifier,SUMO）是结构上与泛素类似的一种修饰蛋白,能与一些特定的靶蛋白共价连接。与泛素介导蛋白质的降解不同,SUMO化修饰调控主要对靶蛋白的功能,如在蛋白质的稳定性、细胞定位、信号转导、基因转录调控等方面均发挥着重要的作用。最近的研究表明：SUMO与乳腺癌的发生发展密切相关,它是通过SUMO化修饰参与并影响雌激素受体信号通路来实现的,本文将就此做一综述。     

SUMOylation and cell signalling

SUMOylation is a highly transient post-translational protein modification. Attachment of SUMO to target proteins occurs via a number of specific activating and ligating enzymes that form the SUMO-substrate complex, and other SUMO-specific proteases that cleave the covalent bond, thus leaving both SUMO and target protein free for the next round of modification. SUMO modification has major effects on numerous aspects of substrate function, including subcellular localisation, regulation of their target genes, and interactions with other molecules. The modified SUMO-protein complex is a very transient state, and it thus facilitates rapid response and actions by the cell, when needed. Like phosphorylation, acetylation and ubiquitination, SUMOylation has been associated with a number of cellular processes. In addition to its nuclear role, important sides of mitochondrial activity, stress response signalling and the decision of cells to undergo senescence or apoptosis, have now been shown to involve the SUMO pathway. With ever increasing numbers of reports linking SUMO to human disease, like neurodegeneration and cancer metastasis, it is highly likely that novel and equally important functions of components of the SUMOylation process in cell signalling pathways will be elucidated in the near future.     

SUMO-1 controls the protein stability and the biological function of phosducin

Phosducin regulates Gbetagamma-stimulated signaling by binding to Gbetagamma subunits of heterotrimeric G-proteins. Control of phosducin activity by phosphorylation is well established. However, little is known about other mechanisms that may control phosducin activity. Here we report that phosducin is regulated at the posttranslational level by modification with the small ubiquitin-related modifier, SUMO. We demonstrate modification with SUMO for phosducin in vitro expressed in cells and for native phosducin purified from retina and the heart. A consensus motif for SUMOylation was identified in phosducin at amino acid positions 32-35. Mutation of the conserved lysine 33 to arginine in this motif abolished SUMOylation of phosducin, indicating that SUMO is attached to lysine 33 of phosducin. In transfected cells the steady-state levels of the K33R mutant protein were much lower compared with wild-type phosducin. The investigation of the stability of wild-type phosducin and of phosducinK33R showed a decreased protein stability of the SUMOylation-deficient mutant. The decreased protein stability correlated with increased ubiquitinylation of the SUMOylation-deficient mutant. These findings indicate that SUMOylation protects phosducin from proteasomal degradation. SUMOylation of phosducin decreased its ability to bind Gbetagamma. PhlP, a closely related member of the phosducin family, was not a target for SUMOylation, but its SUMOylation can be achieved by a single amino acid insertion in the conserved N terminus of PhlP. Together, these findings show that phosducin is a previously unrecognized target of SUMO modification and that SUMOylation controls phosducin stability in cells as well as its functional properties.     

类泛素化修饰蛋白SUMO1的表达纯化及抗体制备

SUMO是近年发现的类泛素化修饰蛋白,可通过异肽键共价连接到靶蛋白上,影响靶蛋白的细胞内定位、稳定性及与其它生物大分子的相互作用. 为研究蛋白质的SUMO化修饰,本文表达并利用亲和层析的方法纯化了重组的人SUMO1,制备了兔抗hSUMO1的多克隆抗体. 经ELISA和免疫印迹检测,获得了灵敏度高、特异性好的抗体,可用于SUMO化修饰靶蛋白的鉴定及SUMO化修饰的生物学功能研究.     

Interferon,restriction factors and SUMO pathways

SUMOylation is a reversible post-translational modification that regulates several cellular processes including protein stability, subcellular localization, protein–protein interactions and plays a key role in the interferon (IFN) pathway and antiviral defense. In human, three ubiquitously expressed SUMO paralogs (SUMO1, 2 and 3) have been described for their implication in both intrinsic and innate immunity. Differential effects between SUMO paralogs are emerging such as their distinctive regulations of IFN synthesis, of IFN signaling and of the expression and function of IFN-stimulated gene (ISG) products. Several restriction factors are conjugated to SUMO and their modifications are further enhanced in response to IFN. Also, IFN itself was shown to increase global cellular SUMOylation and requires the presence of the E3 SUMO ligase PML that coordinates the assembly of PML nuclear bodies. This review focuses on differential effects of SUMO paralogs on IFN signaling and the stabilization/destabilization of ISG products, highlighting the crosstalk between SUMOylation and other post-translational modifications such as ubiquitination and ISGylation.     

Rotavirus Viroplasm Proteins Interact with the Cellular SUMOylation System: Implications for Viroplasm-Like Structure Formation

Posttranslational modification by SUMO provides functional flexibility to target proteins. Viruses interact extensively with the cellular SUMO modification system in order to improve their replication, and there are numerous examples of viral proteins that are SUMOylated. However, thus far the relevance of SUMOylation for rotavirus replication remains unexplored. In this study, we report that SUMOylation positively regulates rotavirus replication and viral protein production. We show that SUMO can be covalently conjugated to the viroplasm proteins VP1, VP2, NSP2, VP6, and NSP5. In addition, VP1, VP2, and NSP2 can also interact with SUMO in a noncovalent manner. We observed that an NSP5 SUMOylation mutant protein retains most of its activities, such as its interaction with VP1 and NSP2, the formation of viroplasm-like structures after the coexpression with NSP2, and the ability to complement in trans the lack of NSP5 in infected cells. However, this mutant is characterized by a high degree of phosphorylation and is impaired in the formation of viroplasm-like structures when coexpressed with VP2. These results reveal for the first time a positive role for SUMO modification in rotavirus replication, describe the SUMOylation of several viroplasm resident rotavirus proteins, and demonstrate a requirement for NSP5 SUMOylation in the production of viroplasm-like structures.     

A differential requirement for SUMOylation in proliferating and non-proliferating cells during Drosophila development

SUMOylation is a highly conserved post-translational modification shown to modulate target protein activity in a wide variety of cellular processes. Although the requirement for SUMO modification of specific substrates has received significant attention in vivo and in vitro, the developmental requirements for SUMOylation at the cell and tissue level remain poorly understood. Here, we show that in Drosophila melanogaster, both heterodimeric components of the SUMO E1-activating enzyme are zygotically required for mitotic progression but are dispensable for cell viability, homeostasis and DNA synthesis in non-dividing cells. Explaining the lack of more pleiotropic effects following a global block of SUMO conjugation, we further demonstrate that low levels of global substrate SUMOylation are detected in mutants lacking either or both E1 subunits. These results not only suggest that minimal SUMOylation persists in the absence of Aos1/Uba2, but also show that the process of cell division is selectively sensitive to reductions in global SUMOylation. Supporting this view, knockdown of SUMO or its E1 and E2 enzymes robustly disrupts proliferating cells in the developing eye, without any detectable effects on the development or differentiation of neighboring post-mitotic cells.     

The gap junction channel protein connexin 43 is covalently modified and regulated by SUMOylation

SUMOylation is a posttranslational modification in which a member of the small ubiquitin-like modifier (SUMO) family of proteins is conjugated to lysine residues in specific target proteins. Most known SUMOylation target proteins are located in the nucleus, but there is increasing evidence that SUMO may also be a key determinant of many extranuclear processes. Gap junctions consist of arrays of intercellular channels that provide direct transfer of ions and small molecules between adjacent cells. Gap junction channels are formed by integral membrane proteins called connexins, of which the best-studied isoform is connexin 43 (Cx43). Here we show that Cx43 is posttranslationally modified by SUMOylation. The data suggest that the SUMO system regulates the Cx43 protein level and the level of functional Cx43 gap junctions at the plasma membrane. Cx43 was found to be modified by SUMO-1, -2, and -3. Evidence is provided that the membrane-proximal lysines at positions 144 and 237, located in the Cx43 intracellular loop and C-terminal tail, respectively, act as SUMO conjugation sites. Mutations of lysine 144 or lysine 237 resulted in reduced Cx43 SUMOylation and reduced Cx43 protein and gap junction levels. Altogether, these data identify Cx43 as a SUMOylation target protein and represent the first evidence that gap junctions are regulated by the SUMO system.     

Analysis of Human Cytomegalovirus-Encoded SUMO Targets and Temporal Regulation of SUMOylation of the Immediate-Early Proteins IE1 and IE2 during Infection

Post-translational modification of proteins by members of the small ubiquitin-like modifier (SUMO) is involved in diverse cellular functions. Many viral proteins are SUMO targets and also interact with the cellular SUMOylation system. During human cytomegalovirus (HCMV) infection, the immediate-early (IE) proteins IE1 and IE2 are covalently modified by SUMO. IE2 SUMOylation promotes its transactivation activity, whereas the role of IE1 SUMOylation is not clear. We performed in silico, genome-wide analysis to identify possible SUMOylation sites in HCMV-encoded proteins and evaluated their modification using the E. coli SUMOylation system and in vitro assays. We found that only IE1 and IE2 are substantially modified by SUMO in E. coli, although US34A was also identified as a possible SUMO target in vitro. We also found that SUMOylation of IE1 and IE2 is temporally regulated during viral infection. Levels of SUMO-modified form of IE1 were increased during the early phase of infection, but decreased in the late phase when IE2 and its SUMO-modified forms were expressed at high levels. IE2 expression inhibited IE1 SUMOylation in cotransfection assays. As in IE2 SUMOylation, PIAS1, a SUMO E3 ligase, interacted with IE1 and enhanced IE1 SUMOylation. In in vitro assays, an IE2 fragment that lacked covalent and non-covalent SUMO attachment sites, but was sufficient for PIAS1 binding, effectively inhibited PIAS1-mediated SUMOylation of IE1, indicating that IE2 expression negatively regulates IE1 SUMOylation. We also found that the IE2-mediated downregulation of IE1 SUMOylation correlates with the IE1 activity to repress the promoter containing the interferon stimulated response elements. Taken together, our data demonstrate that IE1 and IE2 are the main viral SUMO targets in HCMV infection and that temporal regulation of their SUMOylation may be important in the progression of this infection.