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 蛋白是泛素相关蛋白的保守家族，其以类似于泛素的方式与底物缀合。裂殖酵母和芽殖酵母均含有一种 SUMO 家族蛋白。这些蛋白质与两种生物体细胞周期的调节有关。人类 SUMO 旁系同源物共有 3 种：SUMO-1 与 SUMO-2 和 SUMO-3 的同一性约为 45%，而 SUMO-2 和 SUMO-3 的同一性为 96%。所有旁系同源物的缀合途径与泛素缀合途径相似：SUMO 蛋白必须经过加工才能产生 C 末端二甘氨酸基序。处理后，SUMO 缀合途径的第一步是 SUMO 蛋白与其激活 (E1) 酶之间依赖 ATP 形成硫酯键。第二步是在 SUMO 蛋白与其结合 (E2) 酶 Ubc9 之间形成硫酯键。最后一步，通过 Ubc9 和蛋白连接酶 (E3) 的协同作用，SUMO 蛋白和底物之间形成异肽键。 关于 SUMO 蛋白的一个重要问题涉及脊椎动物细胞内各个 SUMO 旁系同源物的作用。目前尚不清楚SUMO-1、-2和-3是否以独特、冗余或拮抗的方式发挥作用。此外，所有三个旁系同源物都具有共同的 E1 和 E2 酶，而 SUMO 连接酶和蛋白酶的特异性尚不清楚。过去很难通过实验解决这个问题，因为单个 SUMO 蛋白的超生理水平可能导致旁系同源蛋白特异性的丧失，并且因为这些蛋白的动态和定位只能在固定细胞内不精确地估计。为了解决 SUMO 旁系同源物的动态特性，我们开发了稳定的 HeLa 衍生细胞系，其表达生物荧光 SUMO 嵌合体的水平与内源蛋白相当。通过实时成像和光漂白研究，我们发现 SUMO-1 在其定位及其整个细胞周期的动态方面与 SUMO-2 和 SUMO-3 不同。此外，我们发现不同亚核区室中 SUMO-1 动力学之间存在显着差异。总之，我们的研究结果表明，哺乳动物 SUMO 旁系同源物在整个细胞周期中表现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，并且在体内受到特殊调节。 除了检验 SUMO-1 缀合在 RanGAP1 调节中的作用的实验外，我们还试图确定其修饰具有细胞周期依赖性的缀合靶标。我们发现，在非洲爪蟾卵提取物的有丝分裂过程中，拓扑异构酶-II 仅被 SUMO-2/3 修饰；这种修饰在中期最大，随后在后期快速解离。修饰和未修饰拓扑异构酶-II 的差异提取特性表明，SUMO-2/3 接合可能以对染色体分离很重要的方式从有丝分裂染色质中调动拓扑异构酶-II。总之，我们的研究结果表明，拓扑异构酶-II 的 SUMO-2/3 缀合对于有丝分裂染色体在中期-后期转变时的重塑非常重要，并且这种重塑的失败可能会导致体内高水平的染色体错误分离。我们已经鉴定出负责拓扑异构酶-II 有丝分裂缀合的 SUMO 连接酶，目前正在研究它如何通过磷酸化和与染色质的关联进行调节。 我们还系统地研究了 SUMO 连接酶（PIAS/Siz 家族）和 SUMO 蛋白酶（SENP）的特性和行为。我们发现 PIAS 家族成员在间期核内表现出相似的定位模式，但在有丝分裂染色体上的定位也表现出细微的差异。我们已经确定了 PIAS 蛋白 (PIASy) 之一内的结构域，这些结构域负责其靶向间期核和染色体。不同的 SENP 家族成员定位于核仁、核质和核膜。我们目前正在寻求鉴定导致这种靶向的相互作用，并调查错误靶向这些酶的后果。