Regulation of Cellular Growth and Division by Ubiquitin-Mediated Proteolysis

泛素介导的蛋白水解作用对细胞生长和分裂的调节

• 批准号: 8041511
• 负责人: Jennifer A Benanti
• 金额: $ 24.9万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8041511
• 关键词: Address; Binding; Cell Cycle; Cell Cycle Progression; Cell Cycle Proteins; Cell Cycle Regulation; Cells; Chimeric Proteins; Complex; Data; Development; Environment; Event; F Box Domain; F-Box Proteins; Genes; Genetic Transcription; Growth; Human; Libraries; Ligase; Malignant Neoplasms; Mediating; Metabolic; Metabolism; Methods; Microscopy; Nutrient; Phase; Phosphorylation; Phosphotransferases; Process; Proteins; Regulation; Saccharomycetales; Substrate Interaction; Time; Ubiquitin-Protein Ligase Complexes; Ubiquitin-mediated Proteolysis Pathway; Ubiquitination; Yeasts; anaphase-promoting complex; cancer cell; cell growth; design; extracellular; genetic regulatory protein; multicatalytic endopeptidase complex; protein complex; protein degradation; research study; transcription factor; ubiquitin ligase; ubiquitin-protein ligase

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 (APC) 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, we developed a method that utilizes a library of strains expressing GFP-fusion proteins and highthroughput microscopy to identify ubiquitin ligase targets in yeast, and are using this approach to identify the ubiquitin ligases that target 73 unstable cell cycle regulators for destruction. In the experiments proposed here, we will determine the mechanism of turnover of these cell cycle proteins by specific E3s, and examine the importance of these turnover events for nonnal cell cycle progression. The SCF(Gn'l) ubiquitin ligase is known to target both cell cycle and metabolic regulators for destruction. We previously identified the transcription factor Tye7 as a SCF(Grrl) target. Tye7 regulates transcription of metabolic genes and is itself transcriptionally regulated throughout the cell cycle. We will detennine Tye7 is targeted for degradation and analyze the consequence of blocking SCF-mediated turnover. Finally, we will investigate how the cell cycle and metabolic factors influence SCF ligase activity. SCF ligases recognize 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. We will analyze SCF complex composition in different cell cycle and growth states to address these questions.

细胞分裂周期是由定时合成和随后的泛素介导的蛋白水解来协调的 关键的调节蛋白。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（APC）和Skp 1-Cul 1-F-box（SCF）复合物。然而，有许多 细胞周期调节因子，其周转机制仍然未知，以及大量的泛素 其靶标尚未鉴定的连接酶。为了更好地理解泛素连接酶如何控制细胞 循环，我们开发了一种方法，利用表达GFP融合蛋白的菌株库和高通量 显微镜，以确定泛素连接酶的目标，在酵母，并使用这种方法来确定 泛素连接酶靶向73个不稳定的细胞周期调节因子进行破坏。在实验中提出 在这里，我们将确定这些细胞周期蛋白的周转机制，通过特定的E3，并检查 这些周转事件对正常细胞周期进程的重要性。SCF（Gn '1）泛素连接酶是 已知其靶向细胞周期和代谢调节剂以进行破坏。我们之前发现 转录因子Tye 7作为SCF（Grrl）靶标。Tye 7调节代谢基因的转录，其本身是 在整个细胞周期中受到转录调控。我们将确定Tye 7是降解的目标， 分析阻断SCF介导的周转的后果。最后，我们将研究细胞周期如何 代谢因素影响SCF连接酶活性。SCF连接酶通过多种途径之一识别靶标 称为F盒蛋白的模块化衔接子亚基。目前尚不清楚有多少F-box蛋白与 SCF在任何一个时间，以及这些复合物是否在整个细胞周期中变化。我们将分析SCF 在不同的细胞周期和生长状态复杂的组成，以解决这些问题。