eIF2A in translational control

翻译控制中的 eIF2A

基本信息

批准号：
10539309
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
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-15 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10539309
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 EIF-2alpha 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

项目摘要

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个不同的起始因素，完成30多种多肽链。许多因素的功能已经建立非常详细地；但是，其中一些及其行动机制的确切作用仍然不佳理解。 EIF2A是单个链65 kDa蛋白，最初被认为用作功能核酸IF2的同源物，因为EIF2A和IF2催化了生化相似的反应，即它们刺激引发剂甲基-TRNA（Met-trnameti）与小核糖体亚基结合。但是，随后异三聚合物126 kDa因子的识别，eIF2（）表明，该因子而不是EIF2A是主要的负责真核生物中Met-trnameti与40S核糖体亚基的结合。在哺乳动物中，四个应力 - 激活的激酶通过磷酸化EIF2subunit，因此降低了活性EIF2的水平，因此，降低全球翻译水平。但是，许多细胞和病毒蛋白的翻译似乎是对需要Met-trnameti的EIF2α磷酸化目的地有抵抗力。发现一部分因素，包括EIF2A在内，可以在条件下有效地促进Met-trnameti的有效募集到40S/mRNA复合物抑制EIF2活性或缺乏。最近，据报道EIF2A参与了NON-AUG 较高的真核生物的依赖起始和主要组织相容性控制抗原表现复合物（MHC）I类分子，综合应力反应和肿瘤倡议和进展。所有人这些事件受细胞模型中EIF2A沉默的影响。然而，eif2a在体内的确切作用也是作为其行动的精确机制，仍然很大程度上神秘。我们有根本的差距了解EIF2A在体内和体内的哺乳动物系统中的功能。填补上面的这一空白和为了继续真核/哺乳动物EIF2A的身体和功能表征，我们创建了一个可行的纯合EIF2A-Total敲除小鼠应变，并获得了在大肠杆菌中表达的重组EIF2A 细胞。该提案的最终目标是了解EIF2A在体内和体外的功能。这个目标将可以通过体外，离体和体内（小鼠模型）方法的组合来实现。结果的结果提案对于理解基因翻译控制的基本机制至关重要在较高的真核生物中的表达，尤其是作为应力反应的一部分。