Regulation and function of PIASy mediated mitotic SUMOylation in vertebrates

描述(申请人提供)：在细胞分裂过程中，全套染色体需要在后期准确分离，以保持每个子细胞的完整基因组信息。后期染色体分离的失败会导致子代细胞基因组信息的偏差，从而导致发育缺陷和肿瘤的进展。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，染色体分离受多种翻译后蛋白质修饰系统的调节。遗传学和生物化学研究都表明，小泛素样修饰物(SUMO)修饰途径对蛋白质翻译后修饰在有丝分裂的正常进行中起着重要作用。我发现，有丝分裂特异的SUMO-2修饰对于非洲爪哇卵子提取液检测系统中后期染色体的忠实分离起着至关重要的作用。这种有丝分裂的SUMO-2修饰特别需要PIASy蛋白的活性，PIASy蛋白是E3保守的PIAS家族的成员。PIASy介导对多个有丝分裂染色体蛋白的SUMO-2修饰。在有丝分裂中加入突变形式的Ubc9、SUMO E2或抗PIASy中和抗体来抑制SUMO化会导致后期染色体分离受损。在有丝分裂过程中，PIASy介导的SUMO-2底物主要是DNA拓扑异构酶II(Topoll)，它在有丝分裂染色体的组织中具有重要的功能。抑制糖基化改变了Topoll的染色体结合状态。我推测，PIASy介导的有丝分裂SUMO化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关键作用。本研究拟以非洲爪哇卵提取物为模型系统，通过研究PIASy特异性糖基化途径(Aim1)的功能和鉴定新的PIASy底物(Aim3)来研究PIASy特异性糖基化途径(Aim1)的调控机制和有丝分裂糖基化的结果。这项研究将证明SUMO-2修饰在脊椎动物有丝分裂染色体组织中的作用。PIASy介导的SUMO化调控机制的确定将为理解影响多种细胞生理功能的SUMO化途径提供宝贵的信息。

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.