Regulation by post-translation modifications in response to stress

通过翻译后修饰来应对压力的调节

• 批准号: 10098111
• 负责人: David Paul Toczyski
• 金额: $ 2.58万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10098111
• 关键词: Antimitotic Agents; Area; Biological; Biological Process; Biology; CRISPR screen; Cell Cycle; Cells; Chimeric Proteins; Cullin Proteins; Defect; Detection; Deubiquitinating Enzyme; Enzymes; F-Box Proteins; Gene Mutation; Genes; Genetic Screening; Human; Ligase; Methods; Mitotic; Modification; Mutation; Pathway interactions; Proteins; Proteome; Regulation; Role; Spectrometry; Stress; Translations; Ubiquitin; inhibitor/antagonist; member; response; ubiquitin ligase

Project Summary/Abstract Ubiquitin ligases (E3s) represent a diverse and conserved group of enzymes, with over 600 members. These ligases collectively attach the small protein ubiquitin to more than twenty five percent of the proteome, thereby regulating the stability or activity of each target. Despite the importance of this set of enzymes, only a small percentage of ubiquitin ligases have well-characterized biological functions. We have conducted a CRISPR screen examining the sensitivity of mutations of genes encoding human ubiquitin ligases and deubiquitinating enzymes to a panel of inhibitors covering a broad range of biological pathways. From this screen, we identified an F box protein, called FBXO42, whose mutation renders cells sensitive to inhibitors of mitosis and causes the accumulation of cells with mitotic defects. F box proteins are substrate adaptors for the SCF cullin RING ligase. One of the greatest challenges in the study of ubiquitin ligases is identifying their substrates. We developed several methods to accomplish this, using (MS) spectrometry and fluorescent detection. Leah will identify mitotic substrates of FBXO42 using "ligase trap" fusion proteins and through genetic screens. By identifying FBXO42 substrates, we will be able to better understand its role in the cell cycle.

项目总结/摘要 泛素连接酶（E3）代表了一组多样且保守的酶，具有超过1000个氨基酸。 600名成员。这些连接酶共同将小蛋白泛素连接到超过 25%的蛋白质组，从而调节每个蛋白质组的稳定性或活性。 目标尽管这组酶的重要性，只有一小部分的 泛素连接酶具有充分表征的生物学功能。我们已进行 CRISPR筛选检查编码人类基因的突变的敏感性 泛素连接酶和去泛素化酶与一组抑制剂， 一系列生物学途径。从这个筛选中，我们鉴定了一种F盒蛋白，称为 FBXO 42，其突变使细胞对有丝分裂抑制剂敏感，并导致细胞分裂。 有丝分裂缺陷的细胞聚集。F盒蛋白是蛋白质的底物衔接子。 SCF cullin RING连接酶。泛素连接酶研究中最大的挑战之一 是识别它们的基质。我们开发了几种方法来实现这一点，使用 (MS)光谱法和荧光检测。莉亚将鉴定出 FBXO 42使用“连接酶陷阱”融合蛋白并通过遗传筛选。通过识别 FBXO 42底物，我们将能够更好地了解其在细胞周期中的作用。