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

描述(申请人提供)：哺乳动物的细胞周期是由一个复杂的网络调节的时间振荡调节蛋白，或促进或停止细胞的生长和分裂。失调的细胞周期控制是癌症的一个基本方面。SCF E3泛素连接酶是一种动态的蛋白质复合体，通过针对依赖泛素的降解的振荡调节蛋白来管理细胞周期。控制细胞周期进程的几种SCF连接酶调控不当与许多恶性肿瘤有关。虽然也可能涉及其他SCF连接酶的错误调节，但SCF连接酶的完整谱系尚未确定。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连接酶复合体含有3个不变亚基(Skp1、Cul1和Rbx1)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，这是SCF底物识别亚单位，这表明细胞可能组装多达69个不同的SCF复合体。缺乏对哪些FBP组装成SCF复合体以及该复合体如何响应细胞周期进程进行重组的全面分析。我的初步数据确定了许多未定性的FBP，它们在细胞周期中不断表达，但以细胞周期依赖的方式组装成SCF连接酶。这表明这些连接酶起着调节细胞周期的作用，FBP的组装不是通过FBP的表达来协调的，而是通过细胞周期相依赖的底物可用性来协调的。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态，进而研究SCF连接酶独特的生物学功能和调控复合体组装的机制。这一目标将通过三个具体目标来检验。目标1将确定SCF E3泛素连接酶谱系，这些泛素连接酶暂时 聚集在不同的细胞周期阶段。这一目标将通过结合多重选择反应监测(SRM)质谱仪和流式细胞术的多模式方法来实现，以定量不同细胞周期阶段与Cul1结合的FBPs、不变的SCF亚单位和调节蛋白的丰度。目的2寻求(1)通过评估缺失振荡的FBPs的细胞周期进程，确定以细胞周期依赖的方式组装的SCF连接酶如何调节细胞周期进程；(2)使用质谱仪来确定那些影响细胞周期的FBPs的底物。目的3试图确定在细胞周期中，底物的可用性是否驱动FBP在时间上组装成SCF复合体。这一目标将通过操纵FBP与底物的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，而且还将为理解FBPs的错误调控如何促进肿瘤的发生提供基础。

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.