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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=711734
• 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.

泛素依赖性蛋白水解途径是细胞中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，这些蛋白质是神经退行性疾病的标志，如阿尔茨海默氏症、帕金森病和肌萎缩侧索硬化症。泛素化过程涉及到泛素（Ub）在一系列蛋白质（E1, E2, E3）之间的转移，直到它标记一个蛋白质的赖氨酸被降解。在这个级联反应的第二步中，Ub分子被转移到约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缀合物负责招募RING或HECT E3酶，以便将Ub转移到底物上。最近的蛋白质组学实验已经确定Ub可以被翻译后磷酸化和乙酰化。具体来说，Ub在T7、T12、T14、S20、S57、Y59和S65位点磷酸化，在K6、K11、K27、K33、K48和K63位点乙酰化。我们的目标是从机制上理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从中心E2偶联酶转移的影响。