CNBP调控细胞分裂的机制研究

The spindle assembly checkpoint (SAC) is a regulatory mechanism required for faithful segregation of chromosomes during cell division. Inaccurate chromosome segregation can result in aneuploidy (the loss or gain whole chromosomes), and might facilitate tumor initiation and progression. The mitotic arrest-deficient 1 (Mad1) is a positive regulator of the SAC. More recently, we identified a novel Mad1-interacting protein, Cellular Nucleic acid Binding Protein (CNBP) using immunoprecipitation (IP)-mass spectrometry. CNBP is a small and strikingly conserved transcript factor. However, CNBP is still an enigmatic protein, either its biological functions or mechanisms have not been comprehensively examined since CNBP was discovered two decades ago. The serendipitous finding of CNBP association with Mad1, indicates its novel function in cell division. We first demonstrated that loss of CNBP results in aneupolidy, defective SAC and abnormal chromosal segeration, indicating of its critical role in the maintaining genome stability and the etiologic linkage to carcinogenesis. Remarkably, we observed that CNBP displays dynamic localization during mitosis. The localization pattern of CNBP is analogous to chromosome passenger complex, which is essential for multiple processes during cell division. CNBP co-localizes with INCENP, implicating that CNBP functions in cell division as a putative chromosome passenger. Therefore, in this proposed project, we will explore the novel role of CNBP in regulating multiple mitotic events to maintain genome stability， thus providing the mechanism for tumorigenesis and therapeutic targets for treatment of tumors.

纺锤体检验点(spindle assembly checkpoint, SAC) 是细胞有丝分裂时染色体精准的遗传到子代细胞的关键机制，从而维持基因组稳定性。染色体分裂异常会导致非整倍体，促进肿瘤的发生与发展。Mad1是参与SAC调控的重要蛋白，我们通过免疫共沉淀-质谱的方法鉴定了一个与Mad1相互作用的新蛋白，CNBP。CNBP是一个转录调控因子，其生物学功能研究的报道非常少。我们首次发现CNBP参与SAC调控，敲除CNBP后染色体分裂异常，导致非整倍体，提示其在维持基因组稳定及肿瘤发生发展中的关键作用。我们还发现CNBP在有丝分裂各期动态定位模式与染色体移动复合物（chromosome passenger complex，CPC）一致，提示其作为新的CPC组分调控细胞有丝分裂。基于这些新发现，此课题旨在研究CNBP调控细胞有丝分裂的分子机制，为肿瘤的发生发展及治疗提供新靶点和思路。

纺锤体检查点(spindle assembly checkpoint, SAC)是细胞有丝分裂时染色体精准的遗传到子代细胞的关键机制，从而维持基因组稳定性。染色体分裂异常会导致非整倍体，促进肿瘤的发生与发展。Mad1是参与SAC调控的重要蛋白，我们通过免疫共沉淀-质谱的方法鉴定了一个与Mad1相互作用的新蛋白，CNBP。CNBP是一个转录调控因子，其生物学功能研究的报道非常少。我们首次发现CNBP参与SAC调控，敲除CNBP后染色体分裂异常，导致非整倍体，提示其在维持基因组稳定及肿瘤发生发展中的关键作用。我们还发现CNBP在有丝分裂各期动态定位模式与染色体移动复合物（chromosome passenger complex，CPC）一致，提示其作为新的CPC组分调控细胞有丝分裂。我们从各方面用先进的分子实验证明了CNBP是CPC的一个新成员，并发现其调控Mad1通路从而参与有丝分裂。CNBP不但调控Mad1的表达，而且还促进Aurora-B的蛋白激酶活性，进而调控细胞周期。这些新的研究成果表明CNBP是一个新的参与调控细胞有丝分裂的分子，为肿瘤的发生发展及治疗提供新靶点和思路。.ATM是DNA损伤反应（DDR）中的关键调节因子，也通过调节Mad1二聚化和SAC在有丝分裂中发挥关键作用。我们进一步证明ATM对Mad2的磷酸化有负调节作用，Mad2是SAC的另一个关键成分，也参与DDR。在机制上，我们发现Mad2的磷酸化在ATM缺陷细胞中异常增加。点突变分析进一步显示，丝氨酸195在ATM消融时主要由Mad2磷酸化介导。从功能上讲，Mad2的磷酸化导致DNA损伤修复能力下降，并与癌细胞放射治疗的耐药性有关。揭示了Mad2磷酸化在ATM缺陷细胞的检查点缺陷和DNA损伤修复中的关键调节作用。

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