A novel regulator of cell cycle and cell death

描述（由申请人提供）：我们的长期目标是阐明一种新型蛋白C53在调节细胞周期进程和DNA损伤反应中的作用，并探索该蛋白作为癌症治疗新方法的潜在开发利用。调控细胞周期和细胞对DNA损伤的反应对动物发育和衰老以及癌症等人类疾病的发病机制至关重要。细胞周期调控和DNA损伤反应的缺陷可能导致基因组不稳定和肿瘤发生。细胞利用所谓的检查点机制来确保在启动下一阶段之前完成特定阶段，从而保持遗传物质的真实传递。其中一个关键检查点被称为G2/M检查点，它调节有丝分裂进入和细胞周期蛋白依赖性激酶1 （Cdk1）的激活，Cdk1是细胞分裂进程的驱动力。我们最近发现Cdk5激活因子p35结合蛋白C53是细胞周期和DNA损伤反应的一种新的调节因子。我们的研究表明，C53缺失导致细胞周期缺陷和对化疗药物诱导的细胞凋亡的抗性。此外，C53过表达消除了G2/M检查点介导的Cdk1失活，从而使癌细胞对各种DNA损伤剂敏感。我们进一步发现C53缺失导致Cdk1失活和延迟有丝分裂进入，而C53过表达可拮抗chk1介导的Cdk1失活。重要的是，我们发现内源性C53蛋白的一部分定位于中心体，中心体是一种新兴的细胞器，它是整合许多重要信号通路的关键多功能平台。基于我们的初步研究，我们假设C53在不受干扰的细胞周期进程中起着至关重要的作用。通过拮抗中心体相关Chk1的活性，C53在中心体上作为细胞周期蛋白B1/Cdk1激酶初始激活的积极启动子。为了验证我们的假设，我们的目标是：1)研究C53与中心体的关联。为此，我们将通过gfp -中心蛋白稳定细胞、免疫荧光和生化分析来研究C53与中心体的关联及其对接机制。此外，我们将制作一个中心体靶向C53，以测试C53中心体定位对其功能是否重要。2)表征C53在G2/M转变过程中Cdk1/cyclin B1初始激活中的作用。为此，我们将使用同步细胞和免疫荧光分析来检测C53对中心体Cdk1/cyclin B1初始激活的影响。此外，我们将测试C53是否通过影响中心体Chk1和其他中心体相关的有丝分裂调节因子的活性来发挥其功能。这项研究将为细胞周期控制和检查点反应提供新的调控机制。这将为开发该蛋白作为新的肿瘤治疗靶点奠定基础。

DESCRIPTION (provided by applicant): Our long-term goal is to elucidate the roles of a novel protein C53 in regulation of cell cycle progression and DNA damage response, and to explore potential exploitation of this protein as a novel therapeutic approach for cancer treatment. Regulation of cell cycle and cellular response to DNA damage is critical for animal development and ageing, and the pathogenesis of human diseases like cancer. Defects in cell cycle regulation and DNA damage response may lead to genome instability and oncogenesis. Cells utilize the so-called checkpoint mechanisms to ensure the completion of a particular phase before the next phase is initiated, thereby maintaining truthful transmission of genetic material. One of the key checkpoints is so-called the G2/M checkpoint that regulates mitotic entry and activation of cyclin-dependent kinase 1 (Cdk1), a driving force for progression of cell division. We have recently identified Cdk5 activator p35 binding protein C53 as a novel regulator of cell cycle and DNA damage response. Our study demonstrated that C53 depletion leads to defective cell cycle and resistance to apoptosis induced by chemotherapeutic agents. Additionally, C53 overexpression abolished the G2/M checkpoint-mediated Cdk1 inactivation, thereby sensitizing cancer cells to various DNA damage agents. We further found that C53 depletion results in inactivation of Cdk1 and delayed mitotic entry, while C53 overexpression antagonizes Chk1-mediated Cdk1 inactivation. Importantly, we found that a fraction of endogenous C53 protein localizes at the centrosome, an organelle that is emerging as a pivotal multi- functional platform that integrates many important signal pathways. Based upon our preliminary study, we hypothesize that C53 plays a crucial role in unperturbed cell cycle progression. By antagonizing the activity of centrosome-associated Chk1, C53 acts as a positive promoter of initial activation of cyclin B1/Cdk1 kinase at the centrosome. To test our hypothesis, we aim: 1) To Investigate the centrosomal association of C53. In this aim, by using GFP-centrin stable cells and immunofluorescence and biochemical analyses, we will investigate the centrosomal association of C53 and its docking mechanism. Moreover, We will make a centrosome-targeted C53 to test whether C53 centrosomal localization is important for its function. 2) To characterize the role of C53 in initial activation of Cdk1/cyclin B1 during the G2/M transition. In this aim, we will use synchronized cells and immunofluorescence assays to examine the effect of C53 on initial activation of Cdk1/cyclin B1 at the centrosome. Furthermore, we will test whether C53 exerts its function through affecting the activity of centrosomal Chk1 and other centrosome-associated mitotic regulators. This study will provide insight on a novel regulatory mechanism for cell cycle control and checkpoint response. It will lay a foundation for exploitation of this protein as a novel therapeutic target in cancer treatment.