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化: 一种重要的体内翻译后蛋白质修饰系统

靶蛋白被小泛素相关修饰物(small ubiquitin-related modifier,SUMO)修饰已经成为真核细胞特有的翻译后蛋白质修饰标志之一.SUMO与靶蛋白之间这种可逆的共价连接,在核质运输、DNA与蛋白质结合活性、蛋白质之间相互作用、转录调控、DNA修复以及维持基因组稳定等方面均发挥着重要的调节作用.在许多人类疾病如癌症和神经退化性疾病中,SUMO化修饰作用对疾病的发生与发展起着极为重要的作用.阐明SUMO化修饰在这些疾病中的功能,将为疾病的治疗开辟一条崭新的思路.     

The implication of SUMO in intrinsic and innate immunity

Since its discovery, SUMOylation has emerged as a key post-translational modification involved in the regulation of host-virus interactions. SUMOylation has been associated with the replication of a large number of viruses, either through the direct modification of viral proteins or through the modulation of cellular proteins implicated in antiviral defense. SUMO can affect protein function via covalent or non-covalent binding. There is growing evidence that SUMO regulates several host proteins involved in intrinsic and innate immunity, thereby contributing to the process governing interferon production during viral infection; as well as the interferon-activated Jak/STAT pathway. Unlike the interferon-mediated innate immune response, intrinsic antiviral resistance is mediated by constitutively expressed antiviral proteins (defined as restriction factors), which confer direct viral resistance through a variety of mechanisms. The aim of this review is to evaluate the role of SUMO in intrinsic and innate immunity; highlighting the involvement of the TRIM family proteins, with a specific focus on the mechanism through which SUMO affects i- interferon production upon viral infection, ii-interferon Jak/STAT signaling and biological responses, iii-the relationship between restriction factors and RNA viruses.     

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.     

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.     

Rod/Zw10 Complex Is Required for PIASy-dependent Centromeric SUMOylation

SUMO conjugation of cellular proteins is essential for proper progression of mitosis. PIASy, a SUMO E3 ligase, is required for mitotic SUMOylation of chromosomal proteins, yet the regulatory mechanism behind the PIASy-dependent SUMOylation during mitosis has not been determined. Using a series of truncated PIASy proteins, we have found that the N terminus of PIASy is not required for SUMO modification in vitro but is essential for mitotic SUMOylation in Xenopus egg extracts. We demonstrate that swapping the N terminus of PIASy protein with the corresponding region of other PIAS family members abolishes chromosomal binding and mitotic SUMOylation. We further show that the N-terminal domain of PIASy is sufficient for centromeric localization. We identified that the N-terminal domain of PIASy interacts with the Rod/Zw10 complex, and immunofluorescence further reveals that PIASy colocalizes with Rod/Zw10 in the centromeric region. We show that the Rod/Zw10 complex interacts with the first 47 residues of PIASy which were particularly important for mitotic SUMOylation. Finally, we show that depletion of Rod compromises the centromeric localization of PIASy and SUMO2/3 in mitosis. Together, we demonstrate a fundamental mechanism of PIASy to localize in the centromeric region of chromosome to execute centromeric SUMOylation during mitosis.     

SUMO在转录中的抑制作用

许多调控基因转录的重要蛋白质能被SUMO (small ubiquitin-related modifier)化修饰,这些蛋白质包括转录因子,转录辅助因子和染色质修饰酶.SUMO化修饰对底物蛋白的活性产生影响,在大多数情况下,与转录活性的抑制有关.最近,对SUMO化调控转录的机制有了新的认识,认为SUMO化的一个重要作用是促进转录因子与转录抑制因子之间的相互作用.另一方面,已经发现转录共抑制因子HDAC (组蛋白去乙酰化酶)可以作为SUMO化的底物、效应因子和调控因子,说明乙酰化和SUMO化之间复杂的相互作用对基因转录调控起着非常重要的作用.     

SUMOylation Is Required for Optimal TRAF3 Signaling Capacity

TNF receptor–associated factors (TRAFs) are multifunctional adaptor proteins involved in temporal and spatial coordination of signals necessary for normal immune function. Here, we report that TRAF3, a TRAF family member with a key role in Toll-like and TNF family receptor signaling and suppressor of lymphomagenesis, is post-translationally modified by the small ubiquitin-related modifier (SUMO). Through yeast two-hybrid and co-immunoprecipitation assays we have identified Ubc9, the SUMO conjugating enzyme, as a novel TRAF3-interacting protein. We show that Ubc9-dependent SUMOylation of TRAF3 modulates optimal association with the CD40 receptor, thereby influencing TRAF3 degradation and non-canonical NF-κB activation upon CD40 triggering. Collectively, our findings describe a novel post-translational modification of a TRAF family member and reveal a link between SUMOylation and TRAF-mediated signal transduction.     

KSHV latent protein LANA2 inhibits sumo2 modification of p53

Tumor suppressor p53 plays a crucial antiviral role and targeting of p53 by viral proteins is a common mechanism involved in virus oncogenesis. The activity of p53 is tightly regulated at the post-translational levels through a myriad of modifications. Among them, modification of p53 by SUMO has been associated with the onset of cellular senescence. Kaposi´s sarcoma-associated herpesvirus (KSHV) expresses several proteins targeting p53, including the latent protein LANA2 that regulates polyubiquitylation and phosphorylation of p53. Here we show that LANA2 also inhibits the modification of p53 by SUMO2. Furthermore, we show that the reduction of p53-SUMO2 conjugation by LANA2, as well as the p53-LANA2 interaction, both require the SUMOylation of the viral protein and its interaction with SUMO or SUMOylated proteins in a non-covalent manner. Finally, we show that the control of p53-SUMO2 conjugation by LANA2 correlates with its ability to inhibit SUMO2- and type I interferon-induced senescence. These results highlight the importance of p53 SUMOylation in the control of virus infection and suggest that viral oncoproteins could contribute to viral infection and cell transformation by abrogating p53 SUMOylation.     

KSHV latent protein LANA2 inhibits sumo2 modification of p53

Tumor suppressor p53 plays a crucial antiviral role and targeting of p53 by viral proteins is a common mechanism involved in virus oncogenesis. The activity of p53 is tightly regulated at the post-translational levels through a myriad of modifications. Among them, modification of p53 by SUMO has been associated with the onset of cellular senescence. Kaposi´s sarcoma-associated herpesvirus (KSHV) expresses several proteins targeting p53, including the latent protein LANA2 that regulates polyubiquitylation and phosphorylation of p53. Here we show that LANA2 also inhibits the modification of p53 by SUMO2. Furthermore, we show that the reduction of p53-SUMO2 conjugation by LANA2, as well as the p53-LANA2 interaction, both require the SUMOylation of the viral protein and its interaction with SUMO or SUMOylated proteins in a non-covalent manner. Finally, we show that the control of p53-SUMO2 conjugation by LANA2 correlates with its ability to inhibit SUMO2- and type I interferon-induced senescence. These results highlight the importance of p53 SUMOylation in the control of virus infection and suggest that viral oncoproteins could contribute to viral infection and cell transformation by abrogating p53 SUMOylation.     

MAPL is a new mitochondrial SUMO E3 ligase that regulates mitochondrial fission

The modification of proteins by the small ubiquitin‐like modifier (SUMO) is known to regulate an increasing array of cellular processes. SUMOylation of the mitochondrial fission GTPase dynamin‐related protein 1 (DRP1) stimulates mitochondrial fission, suggesting that SUMOylation has an important function in mitochondrial dynamics. The conjugation of SUMO to its substrates requires a regulatory SUMO E3 ligase; however, so far, none has been functionally associated with the mitochondria. By using biochemical assays, overexpression and RNA interference experiments, we characterized the mitochondrial‐anchored protein ligase (MAPL) as the first mitochondrial‐anchored SUMO E3 ligase. Furthermore, we show that DRP1 is a substrate for MAPL, providing a direct link between MAPL and the fission machinery. Importantly, the large number of unidentified mitochondrial SUMO targets suggests a global role for SUMOylation in mitochondrial function, placing MAPL as a crucial component in the regulation of multiple conjugation events.     

Enhanced protein expression in mammalian cells using engineered SUMO fusions: secreted phospholipase A2

SUMOylation, the covalent attachment of SUMO (small ubiquitin-like modifier), is a eukaryotic post-translational event that has been demonstrated to play a critical role in several biological processes. When used as an N-terminal tag or fusion partner, SUMO has been shown to enhance functional protein production significantly by improving folding, solubility, and stability. We have engineered several SUMOs and, through their fusion, developed a system for enhancing the expression and secretion of complex proteins. To demonstrate the fidelity of this fusion technology, secreted phospholipase A(2) proteins (sPLA(2)) were produced using HEK-293T and CHO-K1 cells. Five mouse sPLA(2) homologs were expressed and secreted in mammalian cell cultures using SUMO or SUMO-derived, N-terminal fusion partners. Mean and median increases of 43- and 18-fold, respectively, were obtained using novel SUMO mutants that are resistant to digestion by endogenous deSUMOylases.