eIF2A in translational control

翻译控制中的 eIF2A

• 批准号: 10348143
• 负责人: Anton A. Komar
• 金额: $ 29.2万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-15 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10348143
• 关键词: 3T3-L1 Cells; Adipocytes; Affect; Age-Months; Antigen Presentation; Apoptosis; Binding; Biochemical; Cell Cycle Progression; Cell Line; Cell model; Cells; Characteristics; Codon Nucleotides; Complex; Cryoelectron Microscopy; Development; Diabetes Mellitus; Disease; Drug Targeting; Escherichia coli; Etiology; Eukaryota; Eukaryotic Initiation Factors; Event; Fatty Liver; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Guanosine Triphosphate; High Fat Diet; Homeostasis; Homologous Gene; In Vitro; Knock-out; Knockout Mice; Link; Lipids; Lymphoma; Major Histocompatibility Complex; Malignant Neoplasms; Mammals; Mediating; Messenger RNA; Metabolic; Metabolic Diseases; Metabolic syndrome; Minor; Modeling; Molecular; Mouse Strains; Mus; Obesity; Outcome; Pathway interactions; Peptide Initiation Factors; Phenotype; Phosphorylation; Phosphotransferases; Physiological; Play; Predisposition; Process; Production; Property; Protein Biosynthesis; Proteins; Reaction; Recombinant Proteins; Recombinants; Regulation; Reporting; Resistance; Ribosomes; Role; Serum; Stimulus; Stress; Structure; System; TP53 gene; Testing; Time; Tissues; Transfer RNA; Translations; Validation; Viral Proteins; biological adaptation to stress; cell growth; glucose tolerance; human disease; in vivo; innovation; interest; knockout animal; mouse model; polypeptide; preference; reconstitution; recruit; response; ribosome profiling; transcriptome; transcriptome sequencing; translation assay; translatome; tumor; tumor initiation; tumor progression

Initiation of protein synthesis in eukaryotes is a complex process requiring more than 12 different initiation factors, comprising over 30 polypeptide chains. The functions of many of these factors have been established in great detail; however, the precise role of some of them and their mechanism of action still remain not well understood. eIF2A is a single chain 65 kDa protein that was initially believed to serve as the functional homologue of prokaryotic IF2, since eIF2A and IF2 catalyze biochemically similar reactions, i.e. they stimulate initiator methionyl-tRNA (Met-tRNAMeti) binding to the small ribosomal subunit. However, subsequent identification of a heterotrimeric 126 kDa factor, eIF2() showed that this factor and not eIF2A is primarily responsible for the binding of Met-tRNAMeti to 40S ribosomal subunits in eukaryotes. In mammals, four stress- activated kinases reduce the level of active eIF2 by phosphorylating the eIF2subunit and, consequently, reducing the global level of translation. However, translation of many cellular and viral proteins appeared to be resistant to eIF2α phosphorylation despite requiring Met-tRNAMeti. It was found that a subset of factors, including eIF2A, can promote efficient recruitment of Met-tRNAMeti to 40S/mRNA complexes under conditions of inhibition of eIF2 activity, or its absence. Recently, eIF2A was also reported to be involved in non-AUG dependent initiation in higher eukaryotes and the control of antigen presentation by major histocompatibility complex (MHC) class I molecules, the integrated stress response and tumor initiation and progression. All of these events were affected by eIF2A silencing in cellular models. Yet, the precise role of eIF2A in vivo, as well as the precise mechanism of its action still remain largely enigmatic. There is a fundamental gap in our understanding of how eIF2A functions in mammalian systems in vivo and ex vivo. To fill in this gap above and to continue the physical and functional characterization of a eukaryotic/mammalian eIF2A, we have created a viable homozygous eIF2A-total knockout mouse strain and obtained recombinant eIF2A expressed in E. coli cells. The ultimate goal of this proposal is to understand the function of eIF2A in vivo and in vitro. This goal will be achieved by a combination of in vitro, ex vivo and in vivo (mouse model) approaches. The outcome of this proposal will be important for understanding the basic mechanisms of the translational control of gene expression in higher eukaryotes, especially as part of the stress response.

真核生物中蛋白质合成的启动是一个复杂的过程，需要超过 12 种不同的启动 因子，包含 30 多个多肽链 其中许多因子的功能已被确定。 然而，其中一些的确切作用及其作用机制仍然不清楚。 eIF2A 是一种单链 65 kDa 蛋白质，最初被认为具有功能性。 原核生物 IF2 的同源物，因为 eIF2A 和 IF2 催化生化相似的反应，即它们刺激 然而，起始子甲硫氨酰-tRNA (Met-tRNAMeti) 与小核糖体亚基结合。 异源三聚体 126 kDa 因子 eIF2() 的鉴定表明，该因子而不是 eIF2A 主要是 在真核生物中，负责 Met-tRNAMeti 与 40S 核糖体亚基的结合，有四种应激-。 激活的激酶通过磷酸化 eIF2α 亚基来降低活性 eIF2 的水平，从而 然而，许多细胞和病毒蛋白的翻译似乎受到影响。 尽管需要 Met-tRNAMeti，但仍能抵抗 eIF2α 磷酸化。 包括 eIF2A，在一定条件下可以促进 Met-tRNAMeti 有效招募至 40S/mRNA 复合物 最近，eIF2A 也被报道与非 AUG 相关。 高等真核生物中的依赖性起始和主要组织相容性对抗原呈递的控制 复合体 (MHC) I 类分子，综合应激反应和肿瘤的发生和进展。 这些事件在细胞模型中受到 eIF2A 沉默的影响，但 eIF2A 在体内的确切作用也是如此。 因为其作用的确切机制在很大程度上仍然是个谜。我们的研究存在根本性的差距。 了解 eIF2A 在体内和离体哺乳动物系统中的功能 填补上述空白。 为了继续真核/哺乳动物 eIF2A 的物理和功能表征，我们创建了 活纯合eIF2A全敲除小鼠品系并获得在大肠杆菌中表达的重组eIF2A 该提案的最终目标是了解 eIF2A 在体内和体外的功能。 通过结合体外、离体和体内（小鼠模型）方法来实现这一结果。 该提案对于理解基因翻译控制的基本机制非常重要 在高等真核生物中表达，特别是作为应激反应的一部分。