GTP-binding to eIF2B as a novel mechanism for G protein activation in protein synthesis initiation

我们研究细胞如何控制营养物质转化为生命所需的新蛋白质--细胞的生长和分裂。蛋白质是执行几乎所有细胞功能的生物机器。每种蛋白质都是由被称为氨基酸的积木组成的，这些氨基酸以链的形式连接并折叠，形成特定的三维结构，这些结构对每种蛋白质发挥各自的作用非常重要。正确制造每种蛋白质所需的指令是由我们基因组中基因的DNA序列决定的。这被称为“蛋白质合成”，这是基因表达途径的最后一步，对于确保在正确的地点和时间解码正确的基因至关重要。蛋白质合成发生在称为核糖体的相对大型和复杂的分子机器中，核糖体在称为信使RNA(MRNAs)的中间分子中解码从基因组传递的指令。每个人类细胞都含有100多万个核糖体。核糖体对mRNA的解码是通过被称为蛋白质合成因子的辅助因子和被称为转移RNA(TRNAs)的适配器的协同作用来实现的。它们将必要的氨基酸与指令结合在一起，以确保在正确的时间制造正确的蛋白质。这一建议涉及蛋白质合成的起始阶段，在该阶段中有一组专门的因子发挥作用，称为蛋白质合成起始因子。它们引导核糖体和一种特殊的tRNA，这种tRNA以氨基酸蛋氨酸(指定为Met-tRNAi)启动蛋白质，并将其连接到每个mRNA的正确起始处。这对于在每个细胞中制造正确的蛋白质是至关重要的，而且必须准确而快速地完成。起始是蛋白质合成过程中最复杂的阶段，也是在分子水平上最不被理解的阶段。这一提议的关键是被指定为eIF2B、eIF2和eIF5的因素。这三个因子通过控制eIF2因子上的能量电荷来调节Met-tRNAi的核糖体结合。能量状态由称为GTP和GDP的结合核苷酸决定。当结合到GTP时，eIF2打开并可以与Met-tRNAi结合，当与GDP结合时，它不能与Met-tRNAi结合，因此被关闭。我们一直在研究这种切换机制，因为它是控制蛋白质合成的核心。EIF5关闭eIF2，而eIF2B将其打开。我们最近发现eIF2B蛋白也与GTP结合。这一意想不到的发现对eIF2B的工作原理有一定的影响。EIF2B是一种复杂的因子，我们已经确定了GTP与蛋白质的哪一部分结合--称为伽马亚单位。我们知道，伽马亚基对于eIF2B的两个已知功能都是关键的，第一个是它的GDP/GTP交换功能，另一个是我们最近发现的第二个角色，这个功能取代了eIF5，使eIF2可以自由地进行核苷酸交换。我们在本提案中描述的实验旨在了解(I)eIF2B为什么与GTP结合，(Ii)它确实在哪里结合，以及(Iii)它对细胞中的每个eIF2B角色有多重要。我们的想法是，GTP直接从eIF2B传递到eIF2，这将是这种类型蛋白质发挥功能的一种新方式。通过详细了解eIF2B对控制蛋白质合成的贡献，它将帮助科学家更好地了解细胞生长，并进一步了解疾病机制，例如消失的白质疾病，这是一种由eIF2B突变引起的遗传性疾病。这项工作也可能对一些行业感兴趣，例如那些生产特定蛋白质作为药物治疗或商业产品的行业，或者那些通过发酵培养细胞的行业，因为对蛋白质合成机制的更好理解将有助于设计优化的商业蛋白质表达或发酵系统。

We study how cells control the conversion of nutrients into the new proteins that are required for life-the growth and division of cells. Proteins are biological machines that perform nearly all cellular functions. Each protein is made from building blocks called amino acids that are linked in chains and folded to make specific 3-dimensional structures that are important for each protein to fulfil their individual roles. The instructions required to make each protein correctly are determined by the DNA sequences of our genes in the genome. Termed 'protein synthesis' this is the final step in the gene expression pathway, which is critical for ensuring that the correct genes are decoded at the correct place and time. Protein synthesis occurs within relatively large and complex molecular machines called ribosomes that decode instructions relayed from the genome within intermediary molecules called messenger RNAs (mRNAs). Human cells each contain over a million ribosomes. mRNA decoding by ribosomes is made possible by the concerted action of 'helpers' called protein synthesis factors and adapters called transfer RNAs (tRNAs). In concert they bring the necessary amino acids together with the instructions to ensure the correct proteins are made at the right time. This proposal concerns the initiation phase of protein synthesis in which a dedicated set of factors act termed protein synthesis initiation factors. They direct the ribosomes and a specialised tRNA that starts proteins with the amino acid methionine (designated Met-tRNAi) to the correct start place on each mRNA. This is critical to make the right proteins in every cell and must be done both accurately and rapidly. Initiation is the most complex phase of protein synthesis and the least well understood at the molecular level. Key to this proposal are factors designated eIF2B, eIF2 and eIF5. These three factors are critical for regulating ribosome binding of Met-tRNAi by controlling the energy charge on the eIF2 factor. The energy status is determined by binding nucleotides called GTP and GDP. When bound to GTP eIF2 is switched on and can bind to Met-tRNAi and when bound to GDP it cannot bind to Met-tRNAi and so is switched off. We have been studying this switching mechanism, as it is central to the control of protein synthesis. eIF5 switches eIF2 off, while eIF2B switches it on. We have recently found that the eIF2B protein also binds GTP. This unexpected finding has implications for how eIF2B works. eIF2B is complex factor and we have identified approximately which part of the protein GTP binds to - called the gamma subunit. We know that the gamma subunit is critical for both eIF2B known functions, first its GDP/GTP exchange function and also a second role we recently found, a function that displaces eIF5 from eIF2 so that eIF2 is free for nucleotide exchange. The experiments we describe in this proposal are aimed at understanding (i) why eIF2B binds GTP, (ii) where exactly it does bind and (iii) how important it is for each of the eIF2B roles in cells. Our idea is that GTP is directly handed from eIF2B to eIF2, which would be a novel way for this type of protein to function. By providing a detailed understanding of the contribution of eIF2B to the control of protein synthesis it will help scientists understand cell growth better and provide further insight into disease mechanisms, for example vanishing white matter disease, a genetic disorder which is caused by mutations in eIF2B. The work may also be of interest to industries eg those that produce specific proteins as drug therapeutics or commercial products or those that grow cells by fermentation, because improved understanding of protein synthesis mechanisms will assist in the design of optimized commercial protein expression or fermentation systems.