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

GTP 与 eIF2B 结合作为蛋白质合成起始中 G 蛋白激活的新机制

• 批准号: BB/M006565/1
• 负责人: Graham Pavitt
• 金额: $ 45.7万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM006565%2F1
• 关键词: GTP; binding; eIF2B; novel; mechanism

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.

我们研究细胞如何控制将营养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。每种蛋白质都是由氨基酸组成的，氨基酸以链的形式连接并折叠，形成特定的三维结构，这对每种蛋白质发挥各自的作用很重要。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”，是基因表达途径的最后一步，对于确保正确的基因在正确的地点和时间被解码至关重要。蛋白质合成发生在被称为核糖体的相对较大和复杂的分子机器中，核糖体解码来自被称为信使rna （mrna）的中间分子中的基因组传递的指令。每个人体细胞含有超过一百万个核糖体。核糖体对mRNA的解码是通过称为蛋白质合成因子的“助手”和称为转移rna （tRNAs）的“适配器”的协同作用实现的。它们协调一致地将必需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。该建议涉及蛋白质合成的起始阶段，其中一组专门的因子被称为蛋白质合成起始因子。它们将核糖体和一种特殊的tRNA引导到每个mRNA上正确的起始位置，这种tRNA会启动含有氨基酸蛋氨酸的蛋白质（称为Met-tRNAi）。这对于在每个细胞中制造正确的蛋白质至关重要，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，也是分子水平上了解最少的阶段。该提案的关键是eIF2B、eIF2和eIF5。这三个因子是通过控制eIF2因子上的能量电荷来调节Met-tRNAi核糖体结合的关键。能量状态是由称为GTP和GDP的结合核苷酸决定的。当与GTP结合时，eIF2被打开，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开关机制，因为它是控制蛋白质合成的核心。eIF5关闭eIF2， eIF2B打开eIF2。我们最近发现eIF2B蛋白也与GTP结合。这一意想不到的发现暗示了eIF2B是如何工作的。eIF2B是一个复杂的因子，我们已经大致确定了GTP结合的部分-称为γ亚基。我们知道伽马亚基对eIF2B的两个已知功能都是至关重要的，首先是它的GDP/GTP交换功能，还有我们最近发现的第二个作用，它将eIF5从eIF2中取代，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理解(i)为什么eIF2B结合GTP， （ii）它确切地结合在哪里，以及（iii） eIF2B在细胞中的每种作用有多重要。我们的想法是GTP直接从eIF2B传递到eIF2，这将是这种蛋白质发挥作用的一种新方式。通过详细了解eIF2B对蛋白质合成控制的贡献，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，例如白质消失病，这是一种由eIF2B突变引起的遗传疾病。这项工作也可能对那些生产特定蛋白质作为药物治疗或商业产品的行业或那些通过发酵培养细胞的行业感兴趣，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。

项目成果

Translational regulation in response to stress in Saccharomyces cerevisiae.

酿酒酵母的压力的翻译调节。

• DOI: 10.1002/yea.3349
• 发表时间: 2019-01
• 期刊: Yeast (Chichester, England)
• 作者: Crawford RA;Pavitt GD
• 通讯作者: Pavitt GD

GTP binding to translation factor eIF2B stimulates its guanine nucleotide exchange activity.

• DOI: 10.1016/j.isci.2021.103454
• 发表时间: 2021-12-17
• 期刊: iScience
• 影响因子: 5.8
• 作者: Kershaw CJ;Jennings MD;Cortopassi F;Guaita M;Al-Ghafli H;Pavitt GD
• 通讯作者: Pavitt GD

GTP Binding to Translation Factor eIF2B Stimulates Its Guanine Nucleotide Exchange Activity

GTP 与翻译因子 eIF2B 结合刺激其鸟嘌呤核苷酸交换活性

• DOI: 10.2139/ssrn.3919941
• 发表时间: 2021
• 期刊: SSRN Electronic Journal
• 作者: Kershaw C

Dynamic changes in eIF4F-mRNA interactions revealed by global analyses of environmental stress responses.

• DOI: 10.1186/s13059-017-1338-4
• 发表时间: 2017-10-27
• 期刊: Genome biology
• 影响因子: 12.3
• 作者: Costello JL;Kershaw CJ;Castelli LM;Talavera D;Rowe W;Sims PFG;Ashe MP;Grant CM;Hubbard SJ;Pavitt GD
• 通讯作者: Pavitt GD

Mutations in SNORD118 cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts.

• DOI: 10.1038/ng.3661
• 发表时间: 2016-10
• 期刊: NATURE GENETICS
• 影响因子: 30.8
• 作者: Jenkinson, Emma M.;Rodero, Mathieu P.;Kasher, Paul R.;Uggenti, Carolina;Oojageer, Anthony;Goosey, Laurence C.;Rose, Yoann;Kershaw, Christopher J.;Urquhart, Jill E.;Williams, Simon G.;Bhaskar, Sanjeev S.;O'Sullivan, James;Baerlocher, Gabriela M.;Haubitz, Monika;Aubert, Geraldine;Baranano, Kristin W.;Barnicoat, Angela J.;Battini, Roberta;Berger, Andrea;Blair, Edward M.;Brunstrom-Hernandez, Janice E.;Buckard, Johannes A.;Cassiman, David M.;Caumes, Rosaline;Cordelli, Duccio M.;De Waele, Liesbeth M.;Fay, Alexander J.;Ferreira, Patrick;Fletcher, Nicholas A.;Fryer, Alan E.;Goel, Himanshu;Hemingway, Cheryl A.;Henneke, Marco;Hughes, Imelda;Jefferson, Rosalind J.;Kumar, Ram;Lagae, Lieven;Landrieu, Pierre G.;Lourenco, Charles M.;Malpas, Timothy J.;Mehta, Sarju G.;Metz, Imke;Naidu, Sakkubai;Ounap, Katrin;Panzer, Axel;Prabhakar, Prab;Quaghebeur, Gerardine;Schiffmann, Raphael;Sherr, Elliott H.;Sinnathuray, Kanaga R.;Soh, Calvin;Stewart, Helen S.;Stone, John;Van Esch, Hide;Van Mol, Christine E. G.;Vanderver, Adeline;Wakeling, Emma L.;Whitney, Andrea;Pavitt, Graham D.;Griffiths-Jones, Sam;Rice, Gillian I.;Revy, Patrick;van der Knaap, Marjo S.;Livingston, John H.;O'Keefe, Raymond T.;Crow, Yanick J.
• 通讯作者: Crow, Yanick J.