Mechanisms of action of human translation initiation factors and their regulation

人类翻译起始因子的作用机制及其调控

• 批准号: 10250331
• 负责人: ASSEN G Marintchev
• 金额: $ 33万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10250331
• 关键词: Affect; Alzheimer' s Disease; Animal Disease Models; Back; Binding; Biochemical; Biological Assay; Biophysics; C-terminal; Catalysis; Cells; Cellular Stress Response; Complex; Cryoelectron Microscopy; Data; Disease; Drug Design; Enzyme Inhibitor Drugs; Etiology; Eukaryotic Initiation Factors; Experimental Models; Failure; Fluorescence; GTPase-Activating Proteins; Guanine Nucleotide Dissociation Inhibitors; Guanine Nucleotide Exchange Factors; Guanine Nucleotides; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Human; Hydrolysis; In Vitro; Initiator Codon; Italy; Kinetics; Malignant Neoplasms; Mediating; Medical; Messenger RNA; Metabolic Diseases; Modeling; Mutation; N-terminal; Names; Natural regeneration; Neurodegenerative Disorders; Neuroprotective Agents; Nucleotides; Pathway interactions; Peptide Initiation Factors; Phosphorylation; Phosphotransferases; Play; Preparation; Prion Diseases; Process; Protein Biosynthesis; Proteins; Recycling; Regulation; Reporting; Ribosomes; Role; Site-Directed Mutagenesis; Stimulus; Stress; Structure; System; Systems Analysis; Tail; Testing; Thermodynamics; Transfer RNA; Translation Initiation; Translations; Work; Yeasts; base; biological adaptation to stress; cofactor; dalton; design; enzyme substrate complex; eukaryotic initiation factor-5B; in vitro Assay; in vivo; inhibitor/antagonist; interest; neuron loss; protein function; protein protein interaction; reconstitution; small molecule

Translation initiation is the main target for regulation of protein synthesis. It is a multistep process involving ribosomes, mRNAs, tRNAs, and a number of eukaryotic translation initiation factors (eIFs). Translation initiation relies on a dynamic network of interactions within a mega-dalton translation pre-initiation complex (PIC). The guanine nucleotide exchange factor (GEF) eIF2B is the target of multiple pathways regulating protein synthesis and the cellular stress response. The activity of eIF2B is regulated by phosphorylation and by small molecules, such as nucleotides and cofactors. The α-subunit of the substrate eIF2 is phosphorylated by several stress-induced kinases. Phosphorylated eIF2-GDP (eIF2(α-P)-GDP) is an inhibitor of eIF2B, which triggers the integrated stress response (ISR). ISR dysregulation is involved in the etiology of many diseases, including metabolic disorders and cancer. Neuronal cell death in prion disease, Alzheimer's disease, and other neurodegenerative disorders is mediated by dysregulated ISR stimulation. Accordingly, ISR inhibitors have shown promise as neuroprotective agents in animal models of these diseases. However, despite its exceptional scientific and medical importance, the eIF2B function is poorly understood, which impedes rational drug design. We will employ a combination of NMR, site-directed mutagenesis, biophysical and biochemical assays to: 1. Determine how intramolecular interactions in eIF5 coordinate its interactions with other eIFs eIF5 is an integral part of the PIC, but upon start codon selection, it disengages from most of its binding partners and leaves with only eIF2-GDP. We will identify the roles of competing intramolecular interactions within eIF5 in initiation, and specifically in the rearrangement of the eIF5 interaction network. 2. Elucidate the mechanism of regeneration of the eIF2-GTP·Met-tRNAi ternary complex by eIF2B We will test our model, based on the available data about the eIF2B-catalyzed process of eIF2-GTP·Met- tRNAi ternary complex (TC) regeneration, that eIF2 is channeled from the PIC to eIF5 (as eIF2-GDP), to eIF2B, and then back to eIF5 and the PIC (as TC), without being released free at any stage of the cycle. We will use fluorescence and MST assays to build a complete kinetic framework of the eIF2B-catalyzed process. 3. Develop a complete in vitro reconstituted system for analysis of eIF2B activity and regulation We will develop a complete in vitro assay for eIF2B catalysis that faithfully reproduces all aspects of regulation in vivo. which will allow us to identify the mechanisms of action of any regulator of eIF2B and the steps it affects. We will use our assay to test the effects of a set of regulators and mutations in eIF2B, for which the currently used assay has failed to produce meaningful results. The proposed studies will elucidate the mechanisms of eIF2B catalysis and regulation, and allow rational design of ISR inhibitors for treatment of neurodegenerative disorders and other diseases.

翻译起始是调节蛋白质合成的主要靶点。这是一个多步骤的过程，涉及 核糖体、mRNA、tRNA和许多真核翻译起始因子（eIF）。翻译起始 依赖于巨道尔顿翻译前起始复合物（PIC）内的动态相互作用网络。 鸟嘌呤核苷酸交换因子（GEF）eIF 2B是多途径调控蛋白的靶点 合成和细胞应激反应。eIF 2B的活性受磷酸化和小干扰素的调节。 分子，如核苷酸和辅因子。底物eIF 2的α亚基被几种磷酸化酶磷酸化， 应激诱导的激酶磷酸化eIF 2-GDP（eIF 2（α-P）-GDP）是eIF 2B的抑制剂，可触发 综合应激反应（ISR）。ISR失调涉及许多疾病的病因，包括 代谢紊乱和癌症。朊病毒病、阿尔茨海默病和其他疾病中的神经细胞死亡 神经退行性疾病由失调的ISR刺激介导。因此，ISR抑制剂具有 在这些疾病的动物模型中显示出作为神经保护剂的前景。然而，尽管其特殊的 由于eIF 2B在科学和医学上的重要性，人们对eIF 2B的功能知之甚少，这阻碍了合理的药物设计。 我们将采用NMR、定点诱变、生物物理和生物化学测定的组合来： 1.确定eIF 5分子内相互作用如何协调其与其他eIFs的相互作用 eIF 5是PIC的一个组成部分，但在起始密码子选择后，它脱离了大部分结合 只有eIF 2-GDP的合作伙伴和离开。我们将确定竞争性分子内相互作用的作用 eIF 5在启动中，特别是在eIF 5相互作用网络的重排中。 2.阐明eIF 2B再生eIF 2-GTP·Met-tRNAi三元复合物的机制 我们将根据有关eIF 2B催化eIF 2-GTP·Met过程的可用数据来测试我们的模型- tRNAi三元复合物（TC）再生，eIF 2从PIC被引导至eIF 5（作为eIF 2-GDP），至eIF 2B， 然后返回eIF 5和PIC（作为TC），在循环的任何阶段都不会释放。我们将使用 荧光和MST测定以构建eIF 2B催化过程的完整动力学框架。 3.建立一个完整的体外重组系统，用于分析eIF 2B的活性和调控 我们将开发一种完整的eIF 2B催化体外试验，忠实地再现eIF 2B催化的各个方面。 体内调节。这将使我们能够确定eIF 2B的任何调节剂的作用机制和步骤 它影响。我们将使用我们的测定来测试eIF 2B中的一组调节子和突变的影响， 目前使用的分析不能产生有意义的结果。 这些研究将阐明eIF 2B的催化和调节机制，并允许合理的 设计ISR抑制剂用于治疗神经变性病症和其它疾病。