Mapping the dynamic assembly of SCF E3 ubiquitin ligases during the cell cycle

描述（由申请人提供）：哺乳动物细胞周期受时间振荡调节蛋白的复杂网络调节，所述时间振荡调节蛋白促进或停止细胞生长和分裂。细胞周期控制失调是癌症的一个基本方面。SCF E3泛素连接酶是一种动态蛋白质复合物，通过靶向振荡调节蛋白进行泛素依赖性降解来管理细胞周期。控制细胞周期进程的几种SCF连接酶的失调与许多恶性肿瘤有关。虽然也可能涉及其他SCF连接酶的误调节，但SCF连接酶的完整库仍有待表征。因此，彻底了解SCF连接酶如何调节细胞周期对于确定最佳治疗靶点至关重要。SCF连接酶复合物包含3个不变亚基（Skp 1、Cul 1和Rbx 1）和1个不变底物识别亚基（F-box蛋白（FBP））。人类基因组编码69个FBP，它们是SCF底物识别亚基，表明细胞可以组装多达69个不同的SCF复合物。缺乏对哪些FBPs组装成SCF复合物以及该复合物如何响应细胞周期进程进行重组的综合分析。我的初步数据确定了许多未表征的FBPs，它们在细胞周期中不断表达，但以细胞周期依赖性方式组装成SCF连接酶。这表明，这些连接酶的功能，以调节细胞周期和FBP组装是不协调的FBP表达，而是通过细胞周期阶段依赖性底物的可用性。因此，本研究的目的是确定SCF E3泛素连接酶在整个细胞周期中组装的时间动力学，然后研究SCF连接酶独特的生物学功能和调控复杂组装的机制。将通过三个具体目标来检验这一目标。目的1将鉴定SCF E3泛素连接酶的库， 在不同的细胞周期阶段组装。这一目标将通过利用多模式的方法相结合的多重选择反应监测（SRM）质谱和流式细胞术来解决，以量化的FBPs，不变的SCF亚基，和调节蛋白结合到Cul 1与未结合在不同的细胞周期阶段的丰度。目的2寻求（1）确定以细胞周期依赖性方式组装的SCF连接酶如何通过评估消耗振荡FBPs的细胞中的细胞周期进程来调节细胞周期进程，以及（2）使用质谱法来鉴定影响细胞周期的那些FBPs的底物。目的3旨在确定是否基板的可用性驱动的时间组装成SCF复合物的FBPs在细胞周期。这一目标将通过操纵FBP-底物相互作用和测量对同源SCF组装的影响来测试。这项研究的总体结果不仅将提高我们对细胞增殖和SCF生物学的理解，而且还为理解FBPs的失调如何促进肿瘤发生提供了基础。

DESCRIPTION (provided by applicant): The mammalian cell cycle is regulated by a complex network of temporally oscillating regulatory proteins that either promote or halt cell growth and division. Misregulated cell cycle control is a fundamental aspect of cancer. The SCF E3 ubiquitin ligase is a dynamic protein complex that manages the cell cycle by targeting the oscillating regulatory proteins for ubiquitin-dependent degradation. Misregulation of several SCF ligases that control cell cycle progression is implicated in numerous malignancies. While the misregulation of additional SCF ligases may also be involved, the complete repertoire of SCF ligases is yet to be characterized. Therefore, a thorough understanding of how SCF ligases regulate the cell cycle is critical for identifying optimal therapeutic targets. The SCF ligase complex contains 3 invariant subunits (Skp1, Cul1, and Rbx1) and 1 invariant substrate recognition subunit (F-box protein (FBP)). The human genome encodes for 69 FBPs, which are the SCF substrate recognition subunits, suggesting that cells may assemble up to 69 distinct SCF complexes. Comprehensive analysis of which FBPs assemble into an SCF complex and how the complex reorganizes in response to cell cycle progression is lacking. My preliminary data identified numerous uncharacterized FBPs that are expressed constantly during the cell cycle but assemble into SCF ligases in a cell cycle dependent manner. This suggests that these ligases function to regulate the cell cycle and that FBP assembly is not coordinated by FBP expression but rather by cell cycle phase-dependent substrate availability. Therefore, the objective of this proposal is to determine the temporal dynamics of SCF E3 ubiquitin ligase assembly throughout the cell cycle and to then study the unique biological functions of SCF ligase and the mechanisms regulating complex assembly. This objective will be tested through three specific aims. Aim 1 will identify the repertoire of SCF E3 ubiquitin ligases that temporally assemble in different cell cycle phases. This aim will be addressed by utilizing a multi-modal approach combining multiplex-selected reaction monitoring (SRM) mass spectrometry and flow cytometry to quantify the abundance of FBPs, invariant SCF subunits, and regulatory proteins bound to Cul1 versus unbound in different cell cycle phases. Aim 2 seeks (1) to determine how SCF ligases that assemble in a cell cycle dependent manner regulate cell cycle progression by evaluating cell cycle progression in cells depleted of oscillating FBPs and (2) to use mass spectrometry to identify substrates for those FBPs that influence the cell cycle. Aim 3 seeks to determine if substrate availability drives the temporal assembly of FBPs into SCF complexes during the cell cycle. This aim will be tested by manipulating FBP-substrate interactions and measuring the impact on assembly of the cognate SCF. The overall results of this study will not only improve our understanding of cellular proliferation and SCF biology but also provide a foundation for understanding how misregulation of FBPs contributes to tumorigenesis.