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

描述（由申请人提供）：哺乳动物细胞周期由一个复杂的暂时振荡调节蛋白网络调节，这些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复合物，通过靶向泛素依赖性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连接酶的错误调节与许多恶性肿瘤有关。虽然其他SCF连接酶的错误调节也可能参与其中，但SCF连接酶的完整功能尚未被表征。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（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，它们是SCF底物识别亚基，这表明细胞可以组装多达69个不同的SCF复合物。目前还缺乏关于fbp如何组装成SCF复合物以及该复合物如何在细胞周期进程中重组的综合分析。我的初步数据确定了许多未表征的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。这表明这些连接酶的功能是调节细胞周期，并且FBP的组装不是由FBP表达协调的，而是由细胞周期相依赖性底物可用性协调的。因此，本课题的目的是确定SCF E3泛素连接酶在整个细胞周期内组装的时间动态，进而研究SCF连接酶的独特生物学功能和调节复合物组装的机制。这一目标将通过三个具体目标得到检验。目的1将确定scfe3泛素连接酶的库

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.