eIF2A in translational control

真核生物中蛋白质合成的起始是一个复杂的过程，需要12种以上的起始 因子，包含超过30条多肽链。其中许多因素的功能已经确定 然而，其中一些药物的确切作用及其作用机制仍然不清楚， 明白eIF 2A是一种单链65 kDa蛋白，最初被认为是功能性的 原核IF 2的同源物，因为eIF 2A和IF 2催化生物化学相似的反应，即它们刺激 引发剂甲硫氨酰-tRNA（Met-tRNAMeti）结合小核糖体亚基。然而，随后 异源三聚体126 kDa因子eIF 2（异源三聚体eIF 2A）的鉴定表明，该因子而不是eIF 2A主要 在真核生物中负责Met-tRNAMeti与40 S核糖体亚基的结合。在哺乳动物中，有四种压力- 活化的激酶通过磷酸化eIF 2 β亚基降低活性eIF 2的水平，因此， 降低全球翻译水平。然而，许多细胞和病毒蛋白的翻译似乎是 对eIF 2 α磷酸化具有抗性，尽管需要Met-tRNAMeti。结果发现，一些因素， 包括eIF 2A，可以在一定条件下促进Met-tRNAMeti向40 S/mRNA复合物的有效募集。 抑制eIF 2活性或其不存在。最近，eIF 2A也被报道参与非AUG 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 MHC I类分子、整合的应激反应和肿瘤的发生和发展。所有 这些事件在细胞模型中受到eIF 2A沉默的影响。然而，eIF 2A在体内的确切作用，以及 因为其作用的确切机制仍然是个谜。在我们的社会中， 了解eIF 2A如何在体内和离体哺乳动物系统中发挥作用。为了填补上面的空白， 为了继续真核/哺乳动物eIF 2A的物理和功能表征，我们创建了一个 活的纯合eIF 2A-全敲除小鼠品系，并获得在E.杆菌 细胞本提案的最终目标是了解eIF 2A在体内和体外的功能。这一目标将 可以通过体外、离体和体内（小鼠模型）方法的组合来实现。这场 这对于理解基因翻译调控的基本机制具有重要意义。 在高等真核生物中表达，特别是作为应激反应的一部分。

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.