Regulation and structural organisation of a key regulatory translation factor eIF2B

蛋白质的生产（蛋白质合成）是活细胞的一个基本过程。这是它们成长、分裂和生存所必需的。蛋白质的合成是由激素（如胰岛素）刺激的，当营养缺乏时，蛋白质的合成就会减少。氨基酸（蛋白质的组成部分）的可用性在控制这一过程中尤为重要。申请人的实验室研究蛋白质合成通常是如何控制的，以及为什么这些控制系统中的错误会导致人类疾病。控制蛋白质合成的关键细胞成分之一是一种称为“真核起始因子2B”（eIF2B）的蛋白质。申请人研究eIF2B及其管制已超过15年。eIF2B的活性受到氨基酸和激素的刺激。在蛋白质合成减缓的压力条件下，它被抑制。几年前，申请人的实验室发现了胰岛素和eIF2B控制之间的关键联系，这有助于为更好地理解胰岛素的作用铺平道路。这个实验室现在已经确定了一种氨基酸控制eIF2B的新方法。eIF2B由五个称为亚单位的组分组成，其中一个执行eIF2B的细胞功能。众所周知，这一基因对eIF2B的控制很重要，eIF2B通过添加或去除磷酸基团（称为“磷酸化-去磷酸化”，一种非常常见的调节蛋白质的方式）来起作用。其他四个亚基在控制eIF2B或eIF2B“复合体”的组装中具有辅助功能。最近的研究表明，eIF2B亚基基因的变化（突变）是导致一种遗传性严重脑疾病的原因，这种疾病被称为“白质消失”（简称VWM），主要影响儿童。这强调了一个事实，即适当的行动和控制eIF2B是至关重要的。了解这些突变如何影响eIF2B以及为什么会导致脑部疾病是很重要的。这个项目的主要目的是：1。找出氨基酸如何控制eIF2B的活性：特别是我们希望确定传递有关氨基酸可用性的信息以控制eIF2B活性的链接（所谓的“信号组件”）。这个目标对于理解蛋白质合成是如何被控制的很重要。它还将提供有关营养物质被动物细胞检测并影响其功能的方式的有价值的新信息。2. 以发现控制eIF2B功能的其他途径。通过应用最先进的“蛋白质组学”技术，我们将了解更多关于通过磷酸化-去磷酸化控制eIF2B的信息。我们将运用这项技术来了解eIF2B在影响蛋白质合成的不同条件下的控制方式。这一目标对于更全面地了解动物细胞中的关键调控分子eIF2B的控制非常重要。3. 来研究eIF2B的五个不同亚基是如何共同作用以产生正常功能的eIF2B蛋白的，尽管我们的初步数据已经提供了对此的见解。我们将根据这些信息来确定这种蛋白质是如何工作的。我们将同时使用生化方法和新的生物物理技术。这方面也与理解引起VWM疾病的突变如何影响eIF2B的功能非常相关。4. 运用我们已经建立的方法来探索VWM突变如何影响eIF2B的功能、组装和控制。这项工作将有助于了解导致VWM的疾病过程和eIF2B本身的特性。5. 已知eIF2B控制特定蛋白质的产生。我们将利用在这个项目中获得的信息来扩展我们对它是如何做到这一点的理解，以及对哺乳动物细胞遗传信息表达的影响。

The production of proteins (protein synthesis) is an essential process for living cells. It is needed for them to grow, divide and survive. Protein synthesis is stimulated by hormones, like insulin, and decreased when nutrients are scarce. The availability of amino acids, the building blocks for proteins, is especially important in controlling this process. The applicant's laboratory studies how protein synthesis is normally controlled and why errors in these control systems lead to human disease. One of the key cellular components that control protein synthesis is a protein termed 'eukaryotic initiation factor 2B' (eIF2B). The applicant has studied eIF2B and its control for a more than 15 years. The activity of eIF2B is stimulated by amino acids and by hormones. It is inhibited under stressful conditions where protein synthesis slows down. Several years ago, the applicant's laboratory identified a key link between insulin and the control of eIF2B, which helped to pave the way to a better understanding of the actions of insulin. This laboratory has now identified a new way in which amino acids can control eIF2B. eIF2B is made up of five components, called subunits, one of which carries out the cellular function of eIF2B. This is the one that is known to be important for the control of eIF2B, which operates through the addition or removal of phosphate groups (called 'phosphorylation-dephosphorylation', a very common way of regulating proteins). The other four subunits have ancillary functions in the control of eIF2B or in the assembly of the eIF2B 'complex'. Recent work has shown that changes (mutations) in the genes for the subunits of eIF2B are responsible for an inherited severe brain disease called 'vanishing white matter' (VWM for short) which mainly affects children. This underlines the fact that the proper actions and control of eIF2B are crucial. It is important to understand how these mutations affect eIF2B and why this leads to a brain disease. The principal aims of this project are: 1. to find out how amino acids control the activity of eIF2B: in particular we wish to identify the links (so-called 'signalling components') that relay information about the availability of amino acids to control eIF2B's activity. This goal is important for understanding how protein synthesis is controlled. It will also provide valuable new information about the ways that nutrients are detected by animal cells and affect their functions. 2. to discover other ways in which the function of eIF2B can be controlled. By applying start-of-the-art 'proteomic' techniques, we will learn more about the control of eIF2B by phosphorylation-dephosphorylation. We will apply this technology to learn more about the ways in which eIF2B is controlled under different conditions that affect protein synthesis. This aim is important for achieving a more complete understanding of the control of eIF2B, a key regulatory molecule in animal cells. 3. to investigate how the five different subunits of eIF2B work together to create a properly functioning eIF2B protein, although our initial data have already provided insights into this. We will build on this information to establish how this protein works. We will use both biochemical methods and new biophysical techniques. This aspect is also very relevant for understanding how mutations that cause the disease VWM affect the function of eIF2B. 4. to apply our established methods to explore how VWM mutations affect the function, assembly and control of eIF2B. This work will be valuable in understanding both the disease processes that lead to VWM and the properties of eIF2B itself. 5. eIF2B is known to control the production of specific proteins. We will use information gained in this project to extend our understanding of how it does this, and of the implications for the expression of the genetic information of mammalian cells.