Inner Centromere Targeting of the Chromosome Passenger Complex

描述（由申请人提供）：有丝分裂在全球和地方层面都受到监管。虽然全局调控推动有丝分裂进程，但局部调控通过在特定时间和地点开启和关闭各种活动来精细调节有丝分裂。染色体客运复合体（CPC）是最具特征的有丝分裂调控因子，它在局部水平起作用[1,2]。CPC的定位在整个有丝分裂过程中是动态的，这与其各种有丝分裂活动相对应[1,2]。在中期前和中期，CPC定位于内着丝粒，在那里它纠正微管和着丝粒的失联[3-10]，并产生纺锤体检查点信号[11,12]。对于CPC如何以及在何处靶向内部着丝粒，我们知之甚少。我建议通过LAP纯化，结合质谱和深度测序，从mase消化的有丝分裂染色质中纯化CPC。LAP纯化是一种成功鉴定新型着丝点蛋白和着丝粒蛋白的方法[13-15]。分析鉴定的蛋白质和dna的功能将为研究CPC靶向和内部着丝粒组装奠定坚实的基础。同时，我们也知道有一种机制未知的CPC的着丝粒靶向需要TD-60。为了确定TD-60如何调控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调控。该建议的长期目标是了解CPC运动如何与有丝分裂进展相结合，以及放松这一过程如何有助于肿瘤发生。CPC的核心Aurora-B激酶已被发现在各种类型的肿瘤中过表达[17- 27]。此外，一类新的靶向Aurora激酶的小分子药物已在早期临床试验中被证明是有前景的抗癌药物。然而，由于这些药物抑制具有多种有丝分裂功能的激酶活性，细胞毒性是一个不可避免的问题。如果我们能找到一种方法来抑制极光- b的特定功能，这个问题可能会得到解决。关于CPC目标的知识可能是实现这一目标的有效和必要的方法。

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.