E3-mediated Ubiquitin-like Protein Ligation

E3介导的泛素样蛋白连接

• 批准号: 9088465
• 负责人: BRENDA A SCHULMAN
• 金额: $ 46.57万
• 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/9088465
• 关键词: Active Sites; Affect; Affinity; Apoptosis; Architecture; Autophagocytosis; Binding; Biochemical; Biological Process; Bortezomib; C-terminal; CUL1 gene; Cachexia; Catalysis; Cell Cycle; Cell division; Clinical Trials; Complex; Covalent Interaction; Cullin Proteins; Cysteine; DNA; Defect; Development; Disease; Enzymes; Family; Generations; Genes; Goals; Half-Life; Health; 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; structural biology; targeted agent; thioester; trafficking; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): 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).

描述（由申请人提供）：E1-E2-E3酶介导的泛素（Ub）或泛素样蛋白（Ubls）的共价连接，如NEDD 8是真核蛋白调节的主要形式。Ubls修饰大量蛋白质并改变其在多种蛋白质中的功能 的方式例如，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，在本发明中， Ub/Ubls调节许多生物学过程，如细胞周期、信号转导、凋亡、免疫应答、自噬和发育。Ub/Ubl途径的缺陷与疾病广泛相关，包括癌症、发育障碍、高血压、神经退行性疾病和恶病质。我们建议将我们对Ub/Ubl途径的专业知识扩展到三个最大的E3家族的连接机制：HECT（人类预测的E6 AP C-末端-28同源），RING（人类预测的真正有趣的新基因-570）和RBR（人类预测的RING 1-IBR-RING 2 - 13）。HECT和RBR E3直接参与催化，催化半胱氨酸通过硫酯键与Ub的C-末端通过2步反应形成共价中间体。首先，HECT或RBR E3结合硫酯连接的E2-Ub中间体，并且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转移到靶赖氨酸。相比之下，RING E3促进Ub或Ubl从E2-Ub/Ubl中间体转移到相关底物。在RING E3中，最大的一类由模块化的多亚基Cullin-RING（CRL）家族组成。CRL与不同的E2顺序地起作用以修饰不同的靶标：首先，RING结构域结合E2-NEDD 8中间体，并且cullin亚基通过用NEDD 8进行自我修饰而被激活。然后，NEDD 8修改的CRL绑定Ub加载的E2，它是要传输到目标的Ub的源。最终，对于所有三种类型的E3，通过其酶级联反应的重复循环可以导致聚遍在蛋白化，特定的酶选择性地将供体Ub的C末端连接到受体Ub表面上的独特赖氨酸。我们提出了一个研究计划，重点是结构生物学和生物化学，以了解Ub/Ubl连接的机制，确定目标的特异性，以及HECT（目标1），CRL（目标2）和RBR E3（目标3）的调节功能。