Regulation and function of PIASy mediated mitotic SUMOylation in vertebrates

描述（由申请人提供）：在细胞分裂期间，全套染色体需要在后期准确分离，以保持每个子细胞中的完整基因组信息。在后期染色体分离的失败导致子细胞中的偏向性基因组信息，这导致发育缺陷并有助于肿瘤进展。有丝分裂染色体的精确结构组织是维持染色体分离完整性的关键因素，而染色体分离受多种翻译后蛋白质修饰系统的调控。遗传学和生物化学研究表明，SUMO（Small Ubiquitin-like Modifier）修饰途径对蛋白质的翻译后修饰在有丝分裂的正常进行中起着重要作用。在非洲爪蟾卵提取物分析系统中，我发现有丝分裂特异性的SUMO-2修饰对于完成后期染色体的忠实分离具有至关重要的作用。这种有丝分裂SUMO-2修饰特别需要PIASy蛋白的活性，PIASy蛋白是E3的保守皮亚斯家族的成员。PIASy介导多种有丝分裂染色体蛋白上的SUMO-2修饰。通过添加突变形式的Ubc 9、SUMO E2或抗PIASy中和抗体来抑制有丝分裂中的SUMO化，导致后期染色体分离受损。在有丝分裂期间PIASy介导的SUMO-2主要底物是DMA拓扑异构酶II（Topol 1），已知其在组织有丝分裂染色体中具有重要功能。SUMO化的抑制改变Topol 1的染色体缔合状态。我推测，PIASy介导的有丝分裂SUMO化具有重要的功能，通过其底物调节有丝分裂染色体的组织，这在后期染色体分离中起着至关重要的作用。本研究拟以非洲爪蟾卵提取物为模型系统，通过确定Topol 1 SUMO化（Aim 2）的功能和鉴定新的PIASy底物（Aim 3），研究PIASy特异性SUMO化途径（Aim 1）的调控机制和有丝分裂SUMO化的结果。本研究将证明SUMO-2修饰在脊椎动物有丝分裂染色体组织中的功能。PIASy介导的SUMO化的调控机制的确定将为了解SUMO化途径对多种细胞生理功能的影响提供宝贵的信息。

DESCRIPTION (provided by applicant): During cell division, the full set of chromosomes needs to separate accurately at anaphase to maintain complete genomic information in each daughter cell. Failure of proper chromosome segregation at anaphase leads to biased genomic information in daughter cells, which causes developmental defect and contributes to tumor progression. Precise structural organization of mitotic chromosomes is a key element in maintaining integrity of chromosome segregation, which is regulated by multiple posttranslational protein modification systems. Both genetic and biochemical studies indicate that the posttranslational protein modification by Small Ubiquitin-like Modifier (SUMO) modification pathway has an important role in normal progression of mitosis. I have found that mitotic specific SUMO-2 modification has a crucial role for completion of faithful chromosome segregation in anaphase in Xenopus egg extract assay system. This mitotic SUMO-2 modification specifically requires the activity of the PIASy protein that is a member of conserved PIAS family of E3. PIASy mediates SUMO-2 modification on multiple mitotic chromosomal proteins. Inhibition of SUMOylation in mitosis by either addition of mutant form of Ubc9, SUMO E2, or anti-PIASy neutralizing antibodies causes compromised chromosome segregation at anaphase. The major PIASy-mediated SUMO-2 substrate during mitosis is DMA topoisomerase II (Topoll), which is known to have an essential function in organizing mitotic chromosomes. Inhibition of SUMOylation alters the chromosomal association status of Topoll. I hypothesize that the PIASy mediated mitotic SUMOylation has an essential function in regulating the organization of mitotic chromosomes via its substrates, which plays a crucial role in chromosome segregation at anaphase. I propose to investigate a regulatory mechanism of PIASy specific SUMOylation pathway (Aim1) and a consequence of mitotic SUMOylation with determining the function of Topoll SUMOylation (Aim2) and identifying novel PIASy substrates (Aim3) by using Xenopus egg extracts as a model system. This investigation will demonstrate the function of SUMO-2 modification in organization of mitotic chromosomes in vertebrates. Determination of regulatory mechanisms of PIASy mediated SUMOylation will provide an invaluable information for understanding SUMOylation pathway that has an impact on diverse cellular physiological functions.