Mechanisms of E2 Conjugating Enzymes

泛素依赖的蛋白水解途径是细胞中最重要的调节机制之一。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如阿尔茨海默病、帕金森病和肌萎缩性侧索硬化症的标志。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，直到它标记靶向降解的蛋白质的赖氨酸。在该级联的第二步中，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缀合物负责募集RING或HECT E3酶以将Ub转移至底物。最近的蛋白质组学实验已经确定，Ub可以后磷酸化和乙酰化。具体地，Ub在位置T7、T12、T14、S20、S57、Y 59和S65处被磷酸化，并且在位置K6、K11、K27、K33、K48和K63处被乙酰化。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转移到/从中央E2缀合酶的影响。

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.