Unconventional Ubiquitin E2 enzymes and their activation by E3 ligases

泛素蛋白的翻译后修饰是真核细胞中一个重要且极其保守的过程，涉及许多生理和疾病途径。泛素E3连接酶，其中环型E3连接酶占绝大多数，传统上被认为是这一过程的特定调节因子。它们通过调节泛素E2结合酶起作用，在修饰过程中，泛素E2与泛素形成硫酯键，并将泛素转移到目标蛋白的赖氨酸上。重要的是，由于RING E3s变构激活E2s的赖氨酸反应活性，因此不参与化学修饰，因此是E2s决定了反应的产物，即在靶蛋白泛素化的哪个残基上发生，以及是单泛素化还是多泛素化。因此，对大约40种不同的E2酶进行彻底的分子理解是至关重要的。近年来，已经发现了能够将泛素转移到赖氨酸氨基功能以外的活性基团的E2s。矛盾的是，这些不寻常的和典型的赖氨酸反应性E2s都使用保守的催化结构域，尽管它的尺寸很小（~17 kDa），但似乎能够容纳令人难以置信的催化多样性。在这里，我建议研究两个基本上未被表征的具有非常规反应性的E2s:(1) Ube2J2，据报道，除了赖氨酸残基外，Ube2J2还能将Ub转化为丝氨酸和苏氨酸；(2)Ube2W，它对靶蛋白n端的n - α氨基具有特异性。我对这两种酶的结构和生化分析将有助于揭示它们的活性位点如何适应它们不寻常的特异性，以及它们是如何被同源的RING E3连接酶机制调节的。此外，我的分子发现的相关性将在体内进行评估。最终，这项工作将导致更全面地了解非赖氨酸泛素化的分子方面和意义。

Posttranslational modification of proteins with Ubiquitin is an essential and extremely conserved process in eukaryotic cells that is involved in numerous physiological and disease pathways. Ubiquitin E3 ligases, of which RING-type E3 ligases represent the vast majority, have traditionally been viewed as specific regulators of this process. They act by regulating Ubiquitin E2 conjugating enzymes, which, during modification, form a thioester bond with Ubiquitin and can transfer it onto a lysine in a target protein. Importantly, since RING E3s allosterically activate lysine reactivity of E2s and thus do not chemically participate in modification, it is the E2s that determine the product of the reaction, i.e. at what residue of the target protein ubiquitylation takes place and whether it is mono- or polyubiquitylation. Thus, a thorough molecular understanding of all the roughly 40 different E2 enzymes is crucial. Recent years have witnessed the identification of E2s that are able to transfer Ubiquitin to reactive groups other than the amino function of lysines. Paradoxically, both, these unusual and the canonical, lysine-reactive E2s, use a conserved catalytic domain, which despite its small size (~17 kDa) appears to be able to accommodate an incredible catalytic diversity. Here, I propose to study two of the largely uncharacterized E2s with unconventional reactivity: (1) Ube2J2, which was reported to transfer Ub to serine and threonine in addition to lysine residues and (2) Ube2W, which is specific with the N-alpha amino group of target protein N-termini. My structural and biochemical analysis of these two enzymes will help to uncover how their active sites can accommodate their unusual specificity and how they are mechanistically regulated by their cognate RING E3 ligases. In addition, the relevance of my molecular findings will be assessed in vivo. Ultimately, this work will lead to a more comprehensive understating of the molecular aspects and significance of the non-lysine ubiquitylation.