SUMO family Ubiquitin-like Modifiers In Higher Eukaryote

高等真核生物中的 SUMO 家族泛素样修饰剂

• 批准号: 6992985
• 负责人: MARY C. DASSO
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6992985
• 关键词: DNA gyrase; DNA topoisomerases; HeLa cells; Xenopus oocyte; cell cycle; cell growth regulation; chemical conjugate; chimeric proteins; cytogenetics; enzyme activity; eukaryote; guanine nucleotide binding protein; guanosinetriphosphatase activating protein; imaging /visualization /scanning; ligase; photochemistry; protein isoforms; protein structure function; ubiquitin

SUMO proteins are a conserved family of ubiquitin-related proteins that become conjugated to substrates in a manner similar to ubiquitin. Fission and budding yeast each contain a single SUMO family protein. These proteins have been implicated in the regulation of the cell cycle in both organisms. There are three human SUMO paralogues: SUMO-1 is about 45% identical to SUMO-2 and SUMO-3, which are 96% identical to each other. The conjugation pathway for all paralogues is similar to the ubiquitin conjugation pathway: SUMO proteins must be processed to yield a C-terminal di-glycine motif. After processing, the first step in the SUMO conjugation pathway is the ATP-dependent formation of a thioester bond between SUMO proteins and their activating (E1) enzyme. The second step is the formation of a thioester bond between SUMO proteins and their conjugating (E2) enzyme, Ubc9. In the last step, an isopeptide bond is formed between SUMO proteins and substrates through the cooperative action of Ubc9 and protein ligases (E3). An important question about SUMO proteins concerns the roles of individual SUMO paralogues within vertebrate cells. It is currently unclear whether SUMO-1, -2 and -3 function in ways that are unique, redundant or antagonistic. Moreover, all three paralogues share common E1 and E2 enzymes, while the specificity of SUMO ligases and proteases is not well understood. It has been difficult to address this question experimentally in the past, because superphysiological levels of individual SUMO proteins can cause a loss of paralogue specificity, and because the dynamics and localizations of these proteins can only be imprecisely estimated within fixed cells. To address the dynamic properties of SUMO paralogues, we have developed stable HeLa-derived cell lines that express biofluorescent SUMO chimeras at levels comparable to the endogenous proteins. Through live imaging and photobleaching studies, we have found that SUMO-1 differs from SUMO-2 and SUMO-3 in both it localization and its dynamics throughout the cell cycle. Additionally, we found significant differences between SUMO-1 dynamics in different subnuclear compartments. Together, our findings demonstrate that mammalian SUMO paralogues show discrete temporal and spatial patterns of utilization throughout the cell cycle, arguing that they are functionally distinct and specifically regulated in vivo. In addition to experiments examining the role of SUMO-1 conjugation in the regulation of RanGAP1, we have sought to identify conjugation targets whose modification is cell cycle-dependent. We have found that Topoisomerase-II is modified exclusively by SUMO-2/3 during mitosis in Xenopus egg extracts; this modification is maximal in metaphase, followed by rapid deconjugation during anaphase. The differential extraction properties of modified and unmodified Topoisomerase-II suggest that SUMO-2/3 conjugation may mobilize Topoisomerase-II from mitotic chromatin in a manner that is important for chromosome segregation. Together, our findings indicate that SUMO-2/3 conjugation of Topoisomerase-II is important for remodeling of mitotic chromosomes at the metaphase-anaphase transition, and that failure of such remodeling could be expected to cause high levels of chromosome mis-segregation in vivo. We have identified the SUMO ligase that is responsible for the mitotic conjugation of Topoisomerase-II, and we are currently investigating how it is regulated by phosphorylation and association to chromatin. We have also been systematically examining the properties and behavior of SUMO ligases (PIAS/Siz family) and SUMO protease (SENPs). We find that PIAS family members show similar patterns of localization within interphase nuclei, but also show subtle differences in their localization on mitotic chromosomes. We have identified domains within one of the PIAS proteins (PIASy) that are responsible for its targeting to interphase nuclei and to chromosomes. Different SENP family members are localized to nucleoli, nucleoplasm and to the nuclear envelope. We are currently pursuing identification of the interactions responsible for such targeting, and investigating the consequences of mis-targeting these enzymes.

Sumo蛋白是一种保守的泛素相关蛋白家族，它们以类似于泛素的方式与底物结合。裂变和萌芽的酵母各个含有单一相扑族蛋白。这些蛋白质与两种生物的细胞周期的调节有关。有三个人类的SUMO旁系同源物：SUMO-1大约45％与SUMO-2和SUMO-3相同，彼此相同96％。所有副产品的共轭途径与泛素结合途径相似：必须处理SUMO蛋白以产生C末端二甘氨酸基序。经过处理后，共和偶联途径的第一步是Sumo蛋白与其激活（E1）酶之间硫酯键的ATP依赖性形成。第二步是相扑蛋白及其共轭（E2）酶UBC9之间形成硫酯键。在最后一步中，通过UBC9和蛋白蛋白连接酶的合作作用（E3），在Sumo蛋白和底物之间形成了异肽键。 关于相扑蛋白的一个重要问题涉及脊椎动物细胞中单个相对词的作用。目前尚不清楚SUMO -1，-2和-3是否以独特，多余或对抗的方式函数。此外，这三个旁系同源物具有共同的E1和E2酶，而Sumo连接酶和蛋白酶的特异性尚不清楚。过去很难实验地解决这个问题，因为单个相扑蛋白的超生理水平可能会导致副产品特异性的丧失，并且因为这些蛋白质的动力学和局部化只能在固定细胞中进行不准确的估计。为了解决相扑的动态特性，我们开发了稳定的HELA衍生细胞系，这些细胞系以与内源性蛋白相媲美的水平表达生物荧光的Sumo Chimeras。通过实时成像和光漂白研究，我们发现SUMO-1在IT定位及其在整个细胞周期中都与SUMO-2和SUMO-3不同。此外，我们发现不同亚核室中的SUMO-1动力学之间存在显着差异。总之，我们的发现表明，在整个细胞周期中，哺乳动物相o的旁系同源物显示出离散的时间和空间利用模式，认为它们在功能上是不同的，并且在体内特异性调节。 除了研究SUMO-1结合在Rangap1调控中的作用的实验外，我们还试图确定其修饰依赖细胞周期依赖性的共轭靶标。我们发现，在异武肠卵提取物中有丝分裂期间，拓扑异构酶-II仅通过SUMO-2/3进行了修饰。这种修饰在中期中最大，然后在后期过程中快速解偶。修饰和未修饰的拓扑异构酶-II的差异提取特性表明，SUMO-2/3结合可以以一种对染色体隔离很重要的方式从有丝分裂染色质中动员拓扑异构酶-II。总之，我们的发现表明，TOPOISomerase-II的SUMO-2/3共轭对于中期 - 解相体过渡时的有丝分裂染色体的重塑很重要，并且预计这种重塑的失败会导致高水平的染色体染色体误差降低体体。我们已经确定了负责拓扑异构酶-II有丝分裂偶联的相扑酶，并且我们目前正在研究如何通过磷酸化和与染色质的关联来调节它。 我们还一直在系统地检查相扑连接酶（PIAS/SIZ家族）和相扑蛋白酶（SENP）的性质和行为。我们发现，PIAS家族在相间核中显示出相似的定位模式，但也显示出其在有丝分裂染色体上的定位差异。我们已经确定了一个pias蛋白（PIASY）中的结构域，该域负责其靶向相间核和染色体。不同的SENP家族成员位于核核，核质和核包膜上。我们目前正在识别负责此类靶向的相互作用，并调查误解这些酶的后果。