Inner Centromere Targeting of the Chromosome Passenger Complex

描述（由申请方提供）：有丝分裂在全球和地方层面均受到监管。虽然全局调节驱动有丝分裂进程，但局部调节通过在特定时间和位置开启和关闭各种活动来微调有丝分裂。染色体乘客复合体（CPC）是在局部水平上发挥作用的最具特征的有丝分裂调节因子[1，2]。CPC的定位在整个有丝分裂中是动态的，这对应于其各种有丝分裂活动[1，2]。在前中期和中期，CPC定位于内部着丝粒，在那里它纠正微管和动粒的错误附着[3-10]并产生纺锤体检查点信号[11，12]。关于CPC如何以及在何处被靶向到内部着丝粒，知之甚少。我建议通过HPLC纯化从用MNase消化的有丝分裂染色质中纯化CPC，该方法用于成功鉴定新的动粒蛋白和着丝粒蛋白[13-15]，结合质谱和深度测序来解决这个问题。对所鉴定的蛋白质和DNA的功能分析将为CPC的靶向作用和内着丝粒组装的研究奠定基础。同时，已知TD-60是CPC的着丝粒靶向所需的，其机制未知[16]。为了确定TD-60如何调节CPC靶向，我建议通过功能和结构分析来剖析TD-60的哪些结构域和哪些生化功能参与CPC调节。这项研究的长期目标是了解CPC运动如何与有丝分裂进程相结合，以及这种过程的失调如何有助于肿瘤发生。已发现CPC的核心Aurora-B激酶在各种类型的肿瘤中过表达[17- 27]。此外，靶向Aurora激酶的一类新的小分子已在早期临床试验中被证明是有前途的抗癌药物[28]。然而，细胞毒性是一个不可避免的问题，因为这些药物抑制激酶的活性，具有多种有丝分裂功能。如果我们能找到一种抑制Aurora-B特定功能的方法，这个问题可能会得到解决。了解产品总分类的目标可能是实现这一目标的一个有效和必要的途径。

DESCRIPTION (provided by applicant): Mitosis is regulated at both global levels and local level. While global regulation drives mitotic progression, the localized regulation fine tunes mitosis by turning on and off various activities at specific times and locations. The chromosome passenger complex (CPC) is the best characterized mitotic regulator that operates at the local level [1, 2]. The localization of the CPC is dynamic throughout mitosis, which corresponds to its various mitotic activities [1, 2]. During premetaphase and metaphase the CPC localizes at inner centromere where it corrects misattachment of microtubule and kinetochore [3-10] and generates spindle checkpoint signals [11,12]. Little is known about how and where the CPC is targeted to the inner centromere. I propose to purify CPC from mitotic chromatin digested with MNase by LAP purification, an approach used to successfully identify novel kinetochore proteins and centromere proteins [13-15], combined with mass spectrometry and deep sequencing to address this question. Analysis of the function of the identified proteins and DNAs will lay a strong foundation to study CPC targeting as well as inner centromere assembly. Meanwhile, it is known TD-60 is required for centromere targeting of the CPC with an unknown mechanism [16]. To determine how TD-60 regualtes CPC targeting, I propose to dissect which structural domain and which biochemical function of TD-60 is involved in CPC regulation through function and structure analysis. The long-term goals of this proposal are to understand how CPC movement is coupled to mitotic progression and how deregulation of such a process might contribute to tumorigenesis. Aurora-B kinase, the core of the CPC, has been found to be overexpressed in various types of tumors [17- 27]. In addition, a new class small molecules targeting to Aurora kinases have been proved to be promising anti-cancer drugs in early stage of clinical trials [28]. However, cytotoxicity is an inevitable issue since these drugs inhibit the kinase activity which has a variety of mitotic functions. This issue might be overcome if we can find a way to inhibit a specific function of Aurora-B. Knowledge about CPC targeting might be an efficient and essential way to achieve such a goal.