E3-mediated Ubiquitin-like Protein Ligation

E3介导的泛素样蛋白连接

• 批准号: 8697909
• 负责人: BRENDA A SCHULMAN
• 金额: $ 36.31万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8697909
• 关键词: Active Sites; Affect; Affinity; Apoptosis; Architecture; Autophagocytosis; Binding; Biochemical; Biological Process; Bortezomib; C-terminal; Cachexia; Catalysis; Cell Cycle; Cell division; Clinical Trials; Complex; Covalent Interaction; Cullin 1; Cullin Proteins; Cysteine; DNA; Defect; Development; Disease; Enzymes; Family; Generations; Genes; Goals; Half-Life; Heart Diseases; Human; Hypertension; Immune response; Infection; Knowledge; Lead; Ligation; Link; Lysine; Malignant Neoplasms; Mediating; Modification; Molecular Conformation; Multiple Myeloma; N-terminal; Neurodegenerative Disorders; Oranges; Pathway interactions; Polyubiquitination; Post-Translational Protein Processing; Proteasome Inhibitor; Protein Kinase; Proteins; Reaction; Recruitment Activity; Regulation; Research; Resolution; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Source; Specific qualifier value; Specificity; Structural Biochemistry; Structure; Surface; Tail; Therapeutic; Ubiquitin; Ubiquitin Like Proteins; crosslink; developmental disease; flexibility; human disease; interest; protein function; protein protein interaction; public health relevance; structural biology; thioester; trafficking; ubiquitin-protein ligase

E1-E2-E3 enzyme-mediated covalent attachment of ubiquitin (Ub) or ubiquitin-like proteins (Ubls) such NEDD8 is a predominant form of eukaryotic protein regulation. Ubls modify a vast number of proteins and alter their functions in a variety of ways. For example, Ub/Ubl modifications can affect the target protein's half-life, subcellular localization, enzymatic activity, or ability to interact with protein or DNA partners. As a result, Ub/Ubls regulate numerous biological processes, such as the cell cycle, signal transduction, apoptosis, the immune response, autophagy, and development. Defects in Ub/Ubl pathways are widely associated with diseases, including cancers, developmental disorders, high blood pressure, neurodegenerative disorders, and cachexia. We propose to extend our expertise on Ub/Ubl pathways to mechanisms of ligation by the three largest E3 families: HECT (Homologous to E6AP C-Terminus - 28 predicted in humans), RING (Really Interesting New Gene - 570 predicted in humans) and RBR (RING1-IBR-RING2 - 13 predicted in humans). HECT and RBR E3s participate directly in catalysis, with a catalytic cysteine that forms a covalent intermediate through thioester-linkage with Ub's C-terminus via a 2-step reaction. First, the HECT or RBR E3 binds a thioester-linked E2~Ub intermediate, and Ub is transferred from the E2 catalytic cysteine to an E3 catalytic cysteine. Second, Ub is transferred from the E3 Cys to a target lysine. By contrast, RING E3s promote transfer of Ub or a Ubl from from an E2~Ub/Ubl intermediate to an associated substrate. Among the RING E3s, the largest class consists of the modular, multisubunit Cullin-RING (CRL) family. CRLs function sequentially with distinct E2s to modify distinct targets: first the RING domain binds an E2~NEDD8 intermediate, and the cullin subunit is activated by self-modification with NEDD8. Then a NEDD8-modifed CRL binds a Ub-loaded E2, which is the source of Ub to be transferred to a target. Ultimately, for all three classes of E3, repeated cycles through their enzymatic cascades can lead to polyubiquitination, with specific enzymes selectively linking a donor Ub's C-terminus to distinctive lysines on the acceptor Ub's surface. We propose a research plan focused on structural biology and biochemistry to understand mechanisms underlying Ub/Ubl ligation, determining target specificity, and modulating functions of HECT (Aim 1), CRL (Aim 2), and RBR E3s (Aim 3).

E1-E2-E3 酶介导的泛素 (Ub) 或泛素样蛋白 (Ubls) 的共价连接，例如 NEDD8 是真核蛋白质调节的主要形式。 Ubls 修饰大量蛋白质并改变其 以多种方式发挥作用。例如，Ub/Ubl 修饰可以影响靶蛋白的半衰期， 亚细胞定位、酶活性或与蛋白质或 DNA 伴侣相互作用的能力。因此， Ub/Ubls 调节许多生物过程，例如细胞周期、信号转导、细胞凋亡、 免疫反应、自噬和发育。 Ub/Ubl 通路缺陷与以下因素广泛相关： 疾病，包括癌症、发育障碍、高血压、神经退行性疾病和 恶病质。我们建议将我们在 Ub/Ubl 通路方面的专业知识扩展到三个连接机制 最大的 E3 家族：HECT（与 E6AP C 末端同源 - 在人类中预测有 28 个）、RING（真的 有趣的新基因 - 在人类中预测为 570）和 RBR（RING1-IBR-RING2 - 在人类中预测为 13）。 HECT 和 RBR E3 直接参与催化，催化半胱氨酸形成共价中间体 通过两步反应通过硫酯键与 Ub 的 C 末端连接。首先，HECT 或 RBR E3 结合 硫酯连接的E2~Ub中间体，Ub从E2催化半胱氨酸转移到E3催化 半胱氨酸。其次，Ub 从 E3 Cys 转移至目标赖氨酸。相比之下，RING E3 则促进 将Ub或Ubl从E2~Ub/Ubl中间体转移至相关底物。在RING之中 E3 是最大的类别，由模块化、多亚基 ​​Cullin-RING (CRL) 系列组成。 CRL 功能 依次用不同的 E2 来修改不同的目标：首先，RING 结构域结合 E2~NEDD8 中间体，并通过 NEDD8 自我修饰激活 cullin 亚基。然后是 NEDD8 修改的 CRL 结合载有 Ub 的 E2，它是要转移到目标的 Ub 的来源。最终，对于所有三个类别 E3，通过其酶级联的重复循环可以导致多泛素化，与特定的酶 选择性地将供体 Ub 的 C 末端与受体 Ub 表面上的独特赖氨酸连接。我们提出一个 研究计划侧重于结构生物学和生物化学，以了解 Ub/Ubl 的潜在机制 连接、确定目标特异性以及调节 HECT（目标 1）、CRL（目标 2）和 RBR E3 的功能 （目标 3）。