Regulation of Cellular Growth and Division by Ubiquitin-Mediated Proteolysis

描述(由申请人提供)：细胞分裂周期由关键调控蛋白的定时合成和随后泛素介导的蛋白分解来协调。已知的调节细胞周期的两种泛素连接酶复合体是后期促进复合体(ARC)和Skp1-Cul1-F-box(SCF)复合体。然而，有许多细胞周期调节因子的周转机制尚不清楚，以及大量的泛素连接酶的靶标尚未确定。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融合蛋白的全面文库和高通量显微镜来识别酵母中的泛素连接酶靶标。在此之前，我使用这项技术来识别泛素连接酶SCF(Grr1)的几个靶点。我现在将使用这项技术来鉴定以破坏75个不稳定的细胞周期调节因子为目标的泛素连接酶，并将检查这些周转事件对正常细胞周期进程的重要性。我鉴定了转录因子Tye7是SCF(Grr1)的靶标。Tye7调控代谢基因的转录，并在整个细胞周期中受到转录调控。我将确定Tye7如何成为降级目标，并分析阻止Grr1介导的营业额的后果。SCF通过许多称为F-box蛋白的模块化适配器亚基中的一个识别靶点。目前尚不清楚在同一时间有多少F-box蛋白与SCF形成复合体，以及这些复合体是否在整个细胞周期中发生变化。我将分析干细胞因子复合体在不同细胞周期和生长状态下的组成，以解决这些问题。在奖项的指导阶段，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，我将学习更多的技术，参加职业发展计划，帮助我成为独立的科学家，并获得一个独立的职位。体内的大多数细胞都严格控制着生长和分裂，因此只有在适当的时候才会复制。当这些控制机制失效，细胞在错误的时间分裂时，就会导致癌症的发展。细胞分裂周期受调控分子定时合成和破坏的调节。了解这些分子是如何定时破坏的，将增加我们对癌细胞破坏过程的理解。

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.