Regulation of Cellular Growth and Division by Ubiquitin-Mediated Proteolysis

描述（由申请人提供）：细胞分裂周期是由定时合成和随后的泛素介导的关键调节蛋白的蛋白质水解来安排的。已知调节细胞周期的两种泛素连接酶复合物是后期促进复合物（ARC）和Skp1-Cul1-F-box （SCF）复合物。然而，有许多细胞周期调节因子的转换机制尚不清楚，还有大量泛素连接酶的靶点尚未确定。为了更好地了解泛素连接酶如何控制细胞周期，我开发了一种方法，利用gfp融合蛋白的综合文库和高通量显微镜来识别酵母中的泛素连接酶靶点。在此之前，我使用该技术鉴定了一种泛素连接酶SCF（Grrl）的几个靶点。我现在将使用这项技术来识别泛素连接酶，其目标是破坏75个不稳定的细胞周期调节因子，我将检查这些周转事件对正常细胞周期进程的重要性。我确定转录因子Tye7是SCF（Grrl）的靶标。Tye7调节代谢基因的转录，并在整个细胞周期中受到转录调节。我将确定如何靶向降解Tye7，并分析阻断grr1介导的周转的后果。SCF通过称为F-box蛋白的许多模块化适配器亚基之一识别靶标。目前尚不清楚在任何时候有多少F-box蛋白与SCF复合物，以及这些复合物是否在整个细胞周期中发生变化。我将分析SCF复合物在不同细胞周期和生长状态下的组成来解决这些问题。在该奖项的指导阶段，我将进行拟议的筛选，这将产生许多研究途径，将在独立阶段进行。此外，我将学习额外的技术，并参加职业发展计划，这将有助于我成为一个独立的科学家，并获得一个独立的职位。身体中的大多数细胞严格控制生长和分裂，因此它们只在适当的时候复制。当这些控制被破坏，细胞在不正确的时间分裂时，就会导致癌症的发展。细胞分裂周期是由调控分子的定时合成和破坏来调节的。了解这些分子的定时破坏是如何发生的，将增加我们对癌细胞中被破坏过程的理解。

DESCRIPTION (provided by applicant): The cell division cycle is orchestrated by the timed synthesis and subsequent ubiquitin-mediated proteolysis of key regulatory proteins. Two ubiquitin ligase complexes that are known to regulate the cell cycle are the Anaphase Promoting Complex (ARC) and Skp1-Cul1-F-box (SCF) complex. However, there are numerous cell cycle regulators for which the mechanism of turnover remains unknown, and a large number of ubiquitin ligases whose targets have not been identified. To better understand how ubiquitin ligases control the cell cycle, I have developed a method that utilizes a comprehensive library of GFP-fusion proteins and high- throughput microscopy to identify ubiquitin ligase targets in yeast. Previously, I used this technique to identify several targets of one ubiquitin ligase, SCF(Grrl). I will now use this technology to identify the ubiquitin ligases that target the destruction of 75 unstable cell cycle regulators and I will examine the importance of these turnover events for normal cell cycle progression. I identified the transcription factor Tye7 as a target of SCF(Grrl). Tye7 regulates transcription of metabolic genes and is itself transcriptionally regulated throughout the cell cycle. I will determine how Tye7 is targeted for degradation and analyze the consequence of blocking Grr1-mediated turnover. SCF recognizes targets through one of many modular adaptor subunits called F-box proteins. It is unknown how many F-box proteins complex with the SCF at any one time, and whether these complexes change throughout the cell cycle. I will analyze SCF complex composition in different cell cycle and growth states to address these questions. During the mentored phase of the award, I will carry out the proposed screen, which will generate many avenues of research that will be pursued during the independent phase. Moreover, I will learn additional techniques and participate in career development programs that will assist me in becoming and independent scientist and obtaining an independent position. Most cells in the body tightly control growth and division so that they only duplicate when appropriate. When these controls break down and cells divide at incorrect times, this leads to the development of cancer. The cell division cycle is regulated by the timed synthesis and destruction of regulatory molecules. Understanding how timed destruction of these molecules occurs will increase our understanding of processes disrupted in cancer cells.