Chemical Biology Approaches to Ubiquitination

泛素依赖性蛋白水解途径中的酶催化去除受损蛋白质，这些蛋白质是许多疾病的标志。泛素化涉及泛素（Ub）在一系列E1-活化酶、E2-缀合酶和E3-连接酶之间的转移，最终在靶底物上形成多聚泛素链，所述多聚泛素链被诸如26 S蛋白酶体的受体识别以控制蛋白质降解。最近的蛋白质组学实验表明，大多数酶在Ub途径的后处理修饰（PTM）在不同的细胞应激。在大多数情况下，特异性修饰泛素化酶的蛋白激酶或赖氨酸乙酰转移酶尚未被鉴定。在Ub级联中发现的PTM有可能改变Ub催化所需的酶之间的蛋白质相互作用，并调节许多细胞事件。在我们的第一个NSERC发现资助中，我们：（1）实施正交翻译方法来有效地合成特异性磷酸化和乙酰化的Ub，（2）开发基于FRET的实时动力学测定来测量E2~Ub缀合物的形成，以及（3）鉴定Ub乙酰化如何改变E2~Ub缀合物的形成。在接下来的5年里，我们将在这些成就的基础上确定E2和E3酶的磷酸化和乙酰化如何调节它们的结构、相互作用、Ub转移的动力学和聚泛素链的形成。 本论文主要研究以下问题：（1）E2酶的磷酸化或乙酰化是如何改变E2~Ub结合物的构象和稳定性的？（2）E2或E3酶的磷酸化或乙酰化如何改变Ub的转移？（3）Ub的乙酰化如何改变多聚泛素链的动力学和结构？我们的宗旨和目标是：（1）利用正交翻译方法鉴定和优化磷酸化和乙酰化的E2和E3蛋白的合成。（2）确定乙酰化或磷酸化如何改变Ub转移所需的特定E2~Ub构象。用结构学的方法来研究乙酰化或磷酸化的E2~Ub结合物的构象，并显示与E3酶的相互作用是如何被改变的。（3）确定乙酰化或磷酸化如何改变Ub从E2转移到E3或底物的速率。动力学FRET实验将测量用RING、HECT和RBR E3酶卸载E2~Ub的速率。（4）确定Ub的赖氨酸乙酰化如何改变polyUb链的排列。使用NMR方法确定乙酰化diUb链的构象和相互作用以及与蛋白酶体亚基的相互作用。这项工作将确定Ub，E2和E3酶的PTM如何影响Ub转移的结构和动力学，并提供对下游细胞事件的见解。HQP将获得专业知识，为他们在私营部门和学术界的职业生涯做好准备。所使用的方法有可能在未来获得开发特定PTM抗体的许可。

Enzymes in the ubiquitin-dependent proteolysis pathway catalyze the removal of damaged proteins that are hallmarks of many diseases. Ubiquitination involves the transfer of ubiquitin (Ub) between a series of E1-activating, E2-conjugating and E3-ligating enzymes, ultimately forming polyubiquitin chains on target substrates that are recognized by receptors such as the 26S proteasome to control protein degradation. Recent proteomic experiments show that most enzymes in the Ub pathway are post-translationally modified (PTM) under different cellular stresses. In most cases, the protein kinases or lysine acetytransferases that specifically modify ubiquitination enzymes have not been identified. PTMs uncovered in the Ub cascade have the potential to alter protein interactions between the enzymes needed for Ub catalysis and modulate many cellular events. In our first NSERC Discovery grant we; (1) implemented orthogonal translation methods to efficiently synthesize specifically phosphorylated and acetylated Ub, (2) developed real-time FRET-based kinetic assays to measure E2~Ub conjugate formation and (3) identified how Ub acetylation alters the formation of E2~Ub conjugates. In the next 5 years we will build on these achievements to identify how phosphorylation and acetylation of E2 and E3 enzymes modulates their structures, interactions, kinetics of Ub transfer and formation of polyubiquitin chains. The following questions will be addressed: (1)How do phosphorylation or acetylation of E2 enzymes modify the conformations and stabilities of E2~Ub conjugates? (2)How does phosphorylation or acetylation of an E2 or E3 enzyme alter the transfer of Ub? (3)How does acetylation of Ub alter the dynamics and structures of polyubiquitin chains? Our aims and objectives are: (1)Identify and optimize the synthesis of phosphorylated and acetylated E2 and E3 proteins using orthogonal translation methods, (2)Determine how acetylation or phosphorylation modifies specific E2~Ub conformations needed for Ub transfer. Use structural methods to examine conformations of acetylated or phosphorylated E2~Ub conjugates and show how interactions with E3 enzymes are modified. (3)Determine how acetylation or phosphorylation alters the rate of Ub transfer from an E2 to an E3 or substrate. Kinetic FRET experiments will measure the rates of E2~Ub unloading with RING, HECT and RBR E3 enzymes, (4)Identify how lysine acetylation of Ub modifies the arrangement of a polyUb chain. Use NMR methods to determine conformations and interactions of acetylated diUb chains and interactions with a proteasomal subunit. This work will identify how PTMs of Ub, E2 and E3 enzymes impact the structures and kinetics of Ub transfer and provide insights into downstream cellular events. HQP will gain expertise that will prepare them for careers in the private-sector and academia. The methods used have the potential for future licensing opportunities to develop antibodies for specific PTMs.