Mechanisms of E2 Conjugating Enzymes

E2结合酶的机制

• 批准号: RGPIN-2017-05590
• 负责人: Shaw, Gary
• 金额: $ 3.64万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=664920
• 关键词: Mechanisms; E2; Conjugating; Enzymes

The ubiquitin-dependent proteolysis pathway is one of the most important regulatory mechanisms in the cell. It is responsible for the turnover of damaged or misfolded proteins that are the hallmark of neurodegenerative diseases such as Alzheimer's, Parkinson's and Amyotrophic Lateral Sclerosis diseases. The process of ubiquitination involves the transfer of ubiquitin (Ub) between a series of proteins (E1, E2, E3) until it labels a lysine of a protein targeted for degradation. In the second step of this cascade the Ub molecule is transferred to the catalytic cysteine of one of ~60 possible E2 enzymes forming a covalent but highly unstable E2~Ub thiolester intermediate. The E2~Ub conjugate is responsible for recruiting either a RING or HECT E3 enzyme in order to transfer the Ub to a substrate. Recently proteomic experiments have identified that Ub can be post-translationally phosphorylated and acetylated. Specifically, Ub is phosphorylated at positions T7, T12, T14, S20, S57, Y59 and S65 and acetylated at positions K6, K11, K27, K33, K48 and K63. Our goal is to mechanistically understand the consequences of phosphorylation and acetylation on the structure, stability and folding of Ub and the impact of these modifications on the transfer of Ub to/from the central E2 conjugating enzyme.******The following questions will be addressed;******(1) How does phosphorylation and acetylation alter the conformation and stability of Ub,***(2) How do phosphorylation and acetylation impact the transfer of Ub to/from an E2 enzyme,***(3) How are the conformations and structures of different E2~Ub conjugates modified by phosphorylation and acetylation.******The aims and objectives to answer these questions are,******(1) Synthesize and express selected phosphorylated and acetylated Ub proteins using orthogonal translation methods,***(2) Use chemical and thermal denaturation methods to determine the stabilities and folding of post-translationally modified Ub proteins,***(3) Use NMR spectroscopy to examine the conformations of Ub, phosphorylated at S7, S20 and S57, or acetylated at K6, K11, K48 and K63. Determine structures of modified Ub that indicate the largest apparent changes to uncover the atomic level impact of the post-translational modification,***(4) Use kinetic experiments to determine the rates of loading and unloading of post-translationally modified Ubs with selected E2 enzymes,***(5) Assemble stable E2~Ub conjugate proteins with phosphorylated or acetylated Ub proteins and different E2 enzymes. Determine how post-translationally modified Ub alters the conformations of E2~Ub conjugates.******The results of this work will identify how post-translational modifications of Ub alter the processing of this protein by the E2 conjugating enzyme and ultimately how these modifications affect the entire ubiquitination pathway.

泛素依赖的蛋白分解途径是细胞内最重要的调控机制之一。它负责受损或错误折叠的蛋白质的周转，这些蛋白质是阿尔茨海默氏症、帕金森氏症和肌萎缩侧索硬化症等神经退行性疾病的标志。泛素化的过程涉及泛素(Ub)在一系列蛋白质(E1、E2、E3)之间的转移，直到它标记出目标为降解的蛋白质的赖氨酸。在这个级联的第二步，Ub分子被转移到~60种可能的E2酶之一的催化半胱氨酸上，形成共价但高度不稳定的E2~Ub硫酯中间体。E2~Ub结合物负责募集环状或Hect E3酶，以便将Ub转移到底物上。最近的蛋白质组学实验证明，Ub可以被翻译后磷酸化和乙酰化。具体地说，Ub在T7、T12、T14、S20、S57、Y59和S65位被磷酸化，在K6、K11、K27、K33、K48和K63位被乙酰化。我们的目标是从机制上了解磷酸化和乙酰化对Ub的结构、稳定性和折叠的影响，以及这些修饰对Ub进出中心E2结合酶的影响。*将解决以下问题；*(1)磷酸化和乙酰化如何改变Ub的构象和稳定性，*(2)磷酸化和乙酰化如何影响Ub在E2酶中的转移，*(3)不同的E2~Ub结合物的构象和结构是如何被磷酸化和乙酰化修饰的。*回答这些问题的目的和目标是，*(1)用正交翻译方法合成和表达选定的磷酸化和乙酰化的Ub蛋白，*(2)使用化学和热变性方法来确定翻译后修饰的Ub蛋白的稳定性和折叠，*(3)使用核磁共振波谱来研究在S7、S20和S57处磷酸化或在K6、K11、K48和K63处乙酰化的Ub的构象。确定修饰的Ub的结构表明最大的明显变化以揭示翻译后修饰的原子水平影响，*(4)使用动力学实验来确定翻译后修饰的Ub与选定的E2酶的加载和卸载速率，*(5)与磷酸化或乙酰化的Ub蛋白和不同的E2酶组装稳定的E2~Ub结合蛋白。确定翻译后修饰的Ub如何改变E2~Ub结合物的构象。*这项工作的结果将确定Ub的翻译后修饰如何改变E2结合酶对该蛋白质的加工，并最终如何影响整个泛素化途径。