INTERROGATION OF E3 UBIQUITIN LIGASE CATALYSIS BY DEEP MUTATIONAL SCANNING

通过深度突变扫描研究 E3 泛素连接酶催化作用

• 批准号: 8365800
• 负责人: STANLEY FIELDS
• 金额: $ 2.18万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365800
• 关键词: Amino Acids; Bacteriophage T7; Bacteriophages; Binding; Biology; Boxing; Catalysis; Cell physiology; Elements; Enzymes; Funding; Fungal Genome; Genotype; Grant; In Vitro; Libraries; Mutation; National Center for Research Resources; Peptides; Principal Investigator; Procedures; Proteins; Reaction; Research; Research Infrastructure; Resources; Scanning; Signal Transduction; Source; Specific qualifier value; Testing; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; United States National Institutes of Health; cost; insight; mutant; research study; ubiquitin-protein ligase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Ubiquitin signaling is an important mechanism is nearly every cellular process. E3 ubiquitin ligases specify the substrate and catalyze the transfer from an E2 ubiquitin conjugating enzyme to that protein. Though E3 enzymes are a large and well-studied class of proteins, little is known about how the E3 catalyzes this transfer. We plan to interrogate the functional consequences of mutation at every amino acid of an E3 ligase. To this end, we have chosen to study the U-box domain of UBE4B and constructed a library of 1 million UBE4B mutants displayed on the coat of T7 phage. We use the UBE4B phage in in vitro ubiquitination reactions to test their E3 activity. With the addition of E1 and E2 enzymes, the UBE4B catalyzes autoubiquitination using Flag-tagged ubiquitin. This procedure allows us to select for enzymatically active UBE4B-phage by incubation with anti-Flag beads. Nonspecifically bound phage are washed away and bound phage are eluted by competition with Flag peptide. The eluted phage are amplified and subjected to more rounds of selection. Using high throughput sequencing to determine genotypes in the input pool of phage versus selected phages, we can track how each mutation performs during the selection experiments. This experiment will give us insight into the function of every amino acid in the U-box domain of UBE4B and may reveal the functional elements of E3 catalysis.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 泛素信号传导是几乎每个细胞过程的重要机制。 E3 泛素连接酶指定底物并催化从 E2 泛素结合酶到该蛋白质的转移。 尽管 E3 酶是一类经过充分研究的大型蛋白质，但人们对 E3 如何催化这种转移知之甚少。 我们计划探究 E3 连接酶每个氨基酸突变的功能后果。 为此，我们选择研究UBE4B的U-box结构域，并构建了展示在T7噬菌体外壳上的100万个UBE4B突变体的文库。 我们在体外泛素化反应中使用 UBE4B 噬菌体来测试其 E3 活性。 添加 E1 和 E2 酶后，UBE4B 使用带有 Flag 标记的泛素催化自身泛素化。 该程序使我们能够通过与抗 Flag 珠子一起孵育来选择具有酶活性的 UBE4B 噬菌体。 非特异性结合的噬菌体被洗掉，结合的噬菌体通过与 Flag 肽竞争而被洗脱。 洗脱的噬菌体被扩增并进行更多轮的选择。 使用高通量测序来确定噬菌体输入池与选定噬菌体的基因型，我们可以跟踪每个突变在选择实验期间的表现。 该实验将使我们深入了解UBE4B U-box结构域中每个氨基酸的功能，并可能揭示E3催化的功能元件。