小泛素相关修饰物SUMO研究进展

蛋白质翻译后修饰对改变蛋白功能、活性或定位都起着非常重要的作用,泛素及其相似蛋白的修饰是其中一种重要形式。与其他诸如磷酸化、乙酰化、糖基化等不同的是,泛素及其相似蛋白的修饰基团本身即是一个小的多肽,通过异肽键与靶蛋白Lys侧链ε-NH2相连,其中小泛素相关修饰物(small ubiquitin—related modifier,SUMO)与蛋白的共价连接是一种新的广泛存在的翻译后修饰形式。SUMO是广泛存在于真核生物中高度保守的蛋白家族,在脊椎动物中有三个SUMO基因,称为SUMO-1,-2,-3,与泛素在二级结构上极其相似,且催化修饰过程的酶体系也具有很高的同源性。然而,与泛素化介导的蛋白酶降解途径不同,SUMO化修饰发挥着更为广泛的功能,如核质转运、细胞周期调控、信号转导、转录活性调控等。     

Post-translational modification of the RhoGTPase activating protein 21, ARHGAP21, by SUMO2/3

ARHGAP21 is a 217 kDa RhoGAP protein shown to modulate cell migration through the control of Cdc42 and FAK activities. In the present work a 250 kDa-ARHGAP21 was identified by mass spectrometry. This modified form is differentially expressed among cell lines and human primary cells. Co-immunoprecipitations and in vitro SUMOylation confirmed ARHGAP21 specific modification by SUMO2/3 and mapped the SUMOylation site to ARHGAP21 lysine K1443. Immunofluorescence staining revealed that ARHGAP21 co-localizes with SUMO2/3 in the cytoplasm and membrane compartments. Interestingly, our results suggest that ARHGAP21 SUMOylation may be related to cell proliferation. Therefore, SUMOylation of ARHGAP21 may represent a way of guiding its function.     

SUMO2 is essential while SUMO3 is dispensable for mouse embryonic development

Small ubiquitin-like modifier (SUMO1–3) conjugation plays a critical role in embryogenesis. Embryos deficient in the SUMO-conjugating enzyme Ubc9 die at the early postimplantation stage. Sumo1^−/− mice are viable, as SUMO2/3 can compensate for most SUMO1 functions. To uncover the role of SUMO2/3 in embryogenesis, we generated Sumo2- and Sumo3-null mutant mice. Here, we report that Sumo3^−/− mice were viable, while Sumo2^−/− embryos exhibited severe developmental delay and died at approximately embryonic day 10.5 (E10.5). We also provide evidence that SUMO2 is the predominantly expressed SUMO isoform. Furthermore, although Sumo2^+/− and Sumo2^+/−;Sumo3^+/− mice lacked any overt phenotype, only 2 Sumo2^+/−;Sumo3^−/− mice were found at birth in 35 litters after crossing Sumo2^+/−;Sumo3^+/− with Sumo3^−/− mice, and these rare mice were considerably smaller than littermates of the other genotypes. Thus, our findings suggest that expression levels and not functional differences between SUMO2 and SUMO3 are critical for normal embryogenesis.     

Commentary on: Calpain-2 participates in the process of calpain-1 inactivation

Calpain belongs to the calcium-dependent non-lysosomal cysteine protease. Calpain-1 (C1) and calpain-2 (C2) expression are ubiquitous in mammals and an important mediator of the action of calcium. Specific substrate cleavage by C1 and C2 is critical for several calcium-dependent cellular pathways including neuronal function, muscle contraction, signal transduction, cell differentiation, proliferation, and apoptosis. Research suggests that C1 and C2 perform similar functions due to their structurally highly similar isoforms. Increasing evidence suggests that C1 and C2 carry out their specific function in vivo. A recent paper published by Shinkai-Ouchi et al. (Bioscience Reports (2020) 40, DOI: 10.1042/BSR20200552) elucidated the mechanism to differentiate the function of each calpain with respect to the efficiency and longevity for proteolysis after activation. Further, the study represented that C1 and C2 do not synergistically perform their work in vitro. On the other hand, the activity of C1 is reduced in presence of C2. This insight establishes the platform for future studies to examine how C2 regulates the C1 for substrate proteolysis.     

Gene expression and cell growth are modified by silencing SUMO2 and SUMO3 expression

Small ubiquitin-like modifier (SUMO) is a group of proteins binding to lysine residues of target proteins and thereby modifying their stability, activity and subcellular localization. Here we report that blocking SUMO2 and SUMO3 conjugation by silencing their expression markedly modifies gene expression. A microRNA-based RNAi system was used to specifically silence SUMO2 and SUMO3 expression simultaneously and stably transfected neuroblastoma B35 cells expressing dual SUMO2/3 microRNA were created. In cells stably expressing SUMO2/3 microRNA, mRNA levels of 105 and 58 known genes were significantly up- and down-regulated, respectively. About 20% of differentially regulated genes were associated with pathways involved in cell growth and differentiation. Cell division was significantly suppressed in SUMO2/3 miRNA expressing cells. Elucidating what effect the silencing of SUMO2/3 expression has on gene expression will help to identify the impact of SUMO2/3 conjugation on the various cellular pathways.     

SUMO modification of PCNA is controlled by DNA

Post-translational modification by the ubiquitin-like protein SUMO is often regulated by cellular signals that restrict the modification to appropriate situations. Nevertheless, many SUMO-specific ligases do not exhibit much target specificity, and--compared with the diversity of sumoylation substrates--their number is limited. This raises the question of how SUMO conjugation is controlled in vivo. We report here an unexpected mechanism by which sumoylation of the replication clamp protein, PCNA, from budding yeast is effectively coupled to S phase. We find that loading of PCNA onto DNA is a prerequisite for sumoylation in vivo and greatly stimulates modification in vitro. To our surprise, however, DNA binding by the ligase Siz1, responsible for PCNA sumoylation, is not strictly required. Instead, the stimulatory effect of DNA on conjugation is mainly attributable to DNA binding of PCNA itself. These findings imply a change in the properties of PCNA upon loading that enhances its capacity to be sumoylated.     

Characterization of a novel posttranslational modification in polypyrimidine tract-binding proteins by SUMO1

Polypyrimidine tract-binding protein 1 (PTBP1) and its brainspecific homologue, PTBP2, are associated with pre-mRNAs and influence pre-mRNA processing, as well as mRNA metabolism and transport. They play important roles in neural differentiation and glioma development. In our study, we detected the expression of the two proteins in glioma cells and predicted that they may be sumoylated using SUMOplot analyses. We confirmed that PTBP1 and PTBP2 can be modified by SUMO1 with co-immunoprecipitation experiments using 293ET cells transiently co-expressing SUMO1 and either PTBP1 or PTBP2. We also found that SUMO1 modification of PTBP2 was enhanced by Ubc9 (E2). The mutation of the sumoylation site (Lys137) of PTBP2 markedly inhibited its modification by SUMO1. Interestingly, in T98G glioma cells, the level of sumoylated PTBP2 was reduced compared to that of normal brain cells. Overall, this study shows that PTBP2 is posttranslationally modified by SUMO1. [BMB Reports 2014; 47(4): 233-238]     

NSMCE2 suppresses cancer and aging in mice independently of its SUMO ligase activity

The SMC5/6 complex is the least understood of SMC complexes. In yeast, smc5/6 mutants phenocopy mutations in sgs1, the BLM ortholog that is deficient in Bloom's syndrome (BS). We here show that NSMCE2 (Mms21, in Saccharomyces cerevisiae), an essential SUMO ligase of the SMC5/6 complex, suppresses cancer and aging in mice. Surprisingly, a mutation that compromises NSMCE2‐dependent SUMOylation does not have a detectable impact on murine lifespan. In contrast, NSMCE2 deletion in adult mice leads to pathologies resembling those found in patients of BS. Moreover, and whereas NSMCE2 deletion does not have a detectable impact on DNA replication, NSMCE2‐deficient cells also present the cellular hallmarks of BS such as increased recombination rates and an accumulation of micronuclei. Despite the similarities, NSMCE2 and BLM foci do not colocalize and concomitant deletion of Blm and Nsmce2 in B lymphocytes further increases recombination rates and is synthetic lethal due to severe chromosome mis‐segregation. Our work reveals that SUMO‐ and BLM‐independent activities of NSMCE2 limit recombination and facilitate segregation; functions of the SMC5/6 complex that are necessary to prevent cancer and aging in mice.     

Characterization of endogenous and recombinant human calpain-10

Calpain-10 is a novel ubiquitous calpain family member that has been implicated as a susceptibility gene for type 2 diabetes. One of the major challenges is that the function of calpain-10 is not yet known. To address this problem, we purified human calpain-10 from different sources, including the endogenous and the recombinant calpain-10 from HeLa S3 and 293F cells, respectively. Both endogenous and recombinant calpain-10 were present as two major forms with different origins. Interestingly, radiolabeled calpain-10 was found to be efficiently cleaved at the N-terminal region by calpain-2, but not by other proteases. None of these calpain-10 proteins have putative proteolytic activity under in vitro conditions when examined using different peptide substrates, including more than 70 in vitro translated, radiolabeled oligopeptides. Our results raise the possibility that calpain-10 may require a special intracellular localization or interacting partner(s) to acquire proteolytic activity, or it functions by interacting with other proteins rather than through its proteolytic activity.     

The p150 subunit of CAF-1 causes association of SUMO2/3 with the DNA replication foci

The small ubiquitin-related modifier 2/3 (SUMO2/3) can be post-translationally conjugated to a wide variety of proteins constituting chromatin, the platform for genetic and epigenetic regulation. Nevertheless, it is unclear how SUMO2/3 and SUMO2/3-modified proteins are delivered to the chromatin fibers. Here we report that the largest subunit of chromatin assembly factor 1 (CAF-1), human p150, interacts directly and preferentially with SUMO2/3. Amino acid residue of 98-105 in p150 is essential and sufficient for SUMO2/3 interaction. p150-SUMO2/3 interaction coincided with regions that replicate chromatin fibers, because accumulation of the proliferating cell nuclear antigen (PCNA), and incorporation of bromodeoxyuridine (BrdU) were detected at foci co-localized with both p150 and SUMO2/3 during the S-phase in a cell line expressing epitope-tagged p150. Although inhibition of SUMO2/3 expression had only a small effect on p150 deposition on the replication sites, depletion of p150 led to delocalization of SUMO2/3 from the replication foci. Furthermore, p150 mutants deficient in SUMO2/3 interaction, caused a major reduction of SUMO2/3 at the replication foci. Thus, our findings suggest an expanded role of p150 as a SUMO2/3-interacting factor, and raise the intriguing possibility that p150 plays a role in promoting delivery of SUMO2/3 or SUMO2/3-modified proteins (or both) on chromatin fibers during replication.     

SUMO化和磷酸化的相互作用与共同修饰

翻译后修饰如磷酸化、乙酰化、甲基化、泛素化和SUMO化调节不同蛋白质的不同功能。磷酸化可能是最常见的修饰之一,蛋白质磷酸化通过一系列的激酶和磷酸酶催化,从而改变蛋白质功能。SUMO修饰是一种类泛素化修饰。SUMO修饰包括活化、结合、连接和解离,涉及多个酶多个步骤的催化过程。SUMO化可调节蛋白质相互作用、亚细胞定位、蛋白质稳定性和转录活性。关于磷酸化和SUMO化的蛋白质翻译后修饰,已有广泛研究报道。但很少关注于磷酸化和SUMO化之间的相互作用,以及它们对蛋白质的共同修饰。本文综述了蛋白质磷酸化和SUMO化之间的相互作用,以及共同修饰对细胞生理和肿瘤的影响。     

SUMO4基因多态性与2型糖尿病的关系

为了探讨北京汉族人群小泛素样修饰蛋白4(Small ubiquitin-like modifier 4,SUMO4)基因多态性与2型糖尿病(Type 2 diabetes mellitus,T2DM)的关系,文章采用病例对照设计,选取404例T2DM患者(T2DM组)以及年龄、性别匹配的500例健康对照者(Control组)作为研究对象,应用聚合酶链反应-高分辨熔解曲线(PCR-HRM)技术结合测序验证法,检测SUMO4基因3个单核苷酸多态性位点(rs237025、rs237024及rs600739)的基因型与等位基因分布情况,比较T2DM组糖化血红蛋白(Hemoglobin A1c,HbA1c)在各基因型间的分布,并进行单倍型分析。结果显示:①rs237025的G等位基因在T2DM组出现的频率更高(0.334 vs.0.282,P=0.017);GA基因型携带者患T2DM的风险是AA基因型携带者的1.563倍(P=0.001;OR,1.563;95%CI,1.189-2.053);在显性模型(GG+GA vs.AA)分析中,G等位基因携带者(GG+GA)患T2DM的风险是AA基因型携带者的1.525倍(P=0.002;OR,1.525;95%CI,1.169-1.989)。而rs237024和rs600739多态性未发现与T2DM的易感性相关(P>0.05)。②在T2DM组,rs237025的G等位基因携带者、rs237024的TT基因型携带者及rs600739的GG基因携带者具有较高的HbA1c水平,但各基因型携带者之间HbA1c水平并无统计学差异(P>0.05)。③单倍型AAC、AGC及GGT与T2DM的易感性正相关(OR>1);而单倍型AAT、GAC与T2DM的易感性负相关(OR<1)。据此得出结论:rs237025多态性与北京汉族人群T2DM的易感性相关,rs237024和rs600739多态性可能与T2DM的易感性不相关。     

Control of nuclear HIPK2 localization and function by a SUMO interaction motif

The serine/threonine kinase HIPK2 regulates gene expression programs controlling differentiation and cell death. HIPK2 localizes in subnuclear speckles, but the structural components allowing this localization are not understood. A point mutation analysis allowed mapping two nuclear localization signals and a SUMO interaction motif (SIM) that also occurs in HIPK1 and HIPK3. The SIM binds all three major isoforms of SUMO (SUMO-1-3), while only SUMO-1 is capable of covalent conjugation to HIPK2. Deletion or mutation of the SIM prevented SUMO binding and precluded localization of HIPK2 in nuclear speckles, thus causing localization of HIPK2 to the entire cell. Functional inactivation of the SIM prohibited recruitment of HIPK2 to PML nuclear bodies and disrupted colocalization with other proteins such as the polycomb protein Pc2 in nuclear speckles. Interaction of HIPK2 with Pc2 or PML in intact cells was largely dependent on a functional SIM in HIPK2, highlighting the relevance of SUMO/SIM interactions as a molecular glue that serves to enhance protein/protein interaction networks. HIPK2 mutants with an inactive SIM showed changed activities, thus revealing that non-covalent binding of SUMO to the kinase is important for the regulation of its function.