INTERROGATION OF E3 UBIQUITIN LIGASE CATALYSIS BY DEEP MUTATIONAL SCANNING

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 泛素信号转导是一种重要的机制，几乎是每一个细胞的过程。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。虽然E3酶是一大类被充分研究的蛋白质，但人们对E3如何催化这种转移知之甚少。我们计划询问E3连接酶每一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突变体文库。我们在体外泛素化反应中使用UBE4B噬菌体来检测它们的E3活性。在添加了E1和E2酶的情况下，UBE4B利用标记的泛素催化自体素化。这个程序允许我们通过与抗Flag珠子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脱，结合的噬菌体通过与Flag肽的竞争而洗脱。被洗脱的噬菌体被扩增，并接受更多轮的选择。使用高通量测序来确定噬菌体输入池中的基因类型与选择的噬菌体相比，我们可以跟踪每个突变在选择实验中的表现。这项实验将使我们深入了解UBE4B的U-box结构域中每一个氨基酸的功能，并可能揭示E3催化的功能元件。

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.