Functional in vivo and in vitro analysis of the archaeal chaperonin complex

古菌伴侣蛋白复合物的功能体内和体外分析

• 批准号: BB/F003099/1
• 负责人: James Chong
• 金额: $ 8.74万
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF003099%2F1
• 关键词: Functional; vivo; vitro; analysis; archaeal

Proteins have numerous roles inside living organisms. They may catalyse reactions, they may be important parts of cellular structures, they may enable cells to respond to external signals, they may turn genes on or off, and so on. Proteins are made as long chains of amino-acids, but before they can do their job inside the cell, they have to fold into a particular shape. Each shape is unique to each protein. Many problems arise when proteins fail to fold correctly. These may be health problems (for example, diseases such as BSE are associated with proteins failing to fold to their functional shape). In addition, proteins are widely produced in industry, and misfolding of these proteins is a major problem in some cases. A significant finding in recent years is that many proteins have to interact with other proteins called molecular chaperones before they reach their folded state. Molecular chaperones only interact briefly with proteins as they fold, but without this interaction many proteins fail to fold properly. There are various different classes of molecular chaperone. We are particularly interested in the class referred to as 'chaperonins'. Chaperonins are of great interest for two reasons. First, they are essential to all cells, whereas many other types of molecular chaperones can be dispensed with. Second, they have a striking structure, in that all form large complexes with many sub-units that can form cages that other proteins can fold inside. Chaperonins fall into two groups: group I and group II. Group I chaperonins are found in all bacteria and also in mitochondria and chloroplasts, and are moderately well understood. Group II chaperonins are found in the cytosol of eukaryotes (like humans), and are much less well understood. They are also found in the archaea, a group of simple organisms that look like bacteria but are more closely related to eukaryotes. Group II chaperonins are known to be important: in eukaryotic cells, they fold the key proteins actin and tubulin, which together form the internal framework of the cell (the cytoskeleton). They also help fold a protein that can suppress tumour formation, and can help to block the formation of aggregated proteins that cause diseases such as Huntington's chorea. The eukaryotic chaperonins contain eight different types of sub-unit and are hard to study; we do not know the fine details of their structure, for example. The archaeal chaperonins often function with only a single type of sub-unit, and we have an excellent knowledge of their structure. Recently in our group we have developed new ways of studying archaeal chaperonins in cells, and the current proposal aims to use these to learn a lot more about these proteins. We want to find out which parts of the chaperonin are needed for them to work, by changing different amino-acids in the proteins and then looking to see how these altered chaperonins function in cells (in vivo). Remarkably, we have shown that the archaeal chaperonins can also work in bacteria, and we want to study this unexpected finding by looking for mutated proteins that can work even better in bacteria. We will then purify some of these altered proteins and look at their properties using biochemical assays (in vitro). This will let us relate the ability of the proteins to function in vivo with particular properties that they have in vitro. We will also use some of the mutant chaperonins to try to identify other proteins with which they interact. These two approaches (genetic and biochemical) will teach us a lot about the archaeal chaperonins in particular and about chaperonins in general, and will help us to understand the eukaryotic chaperonins in more detail. This understanding has important implications for human and animal health and for biotechnological processes. The work will involve a collaborations between three research teams with highly complementary expertise in this area.

蛋白质在生物体内发挥着多种作用。它们可以催化反应，它们可以是细胞结构的重要组成部分，它们可以使细胞能够对外部信号做出反应，它们可以打开或关闭基因，等等。蛋白质由长链氨基酸组成，但在它们在细胞内发挥作用之前，它们必须折叠成特定的形状。每种蛋白质的每种形状都是独特的。当蛋白质无法正确折叠时会出现许多问题。这些可能是健康问题（例如，疯牛病等疾病与蛋白质未能折叠成其功能形状有关）。此外，蛋白质在工业中广泛生产，并且这些蛋白质的错误折叠在某些情况下是一个主要问题。近年来的一个重要发现是，许多蛋白质在达到折叠状态之前必须与其他称为分子伴侣的蛋白质相互作用。分子伴侣在蛋白质折叠时仅与蛋白质发生短暂的相互作用，但如果没有这种相互作用，许多蛋白质就无法正确折叠。有各种不同类别的分子伴侣。我们对被称为“伴侣蛋白”的类别特别感兴趣。伴侣蛋白之所以引起人们极大的兴趣有两个原因。首先，它们对所有细胞都是必需的，而许多其他类型的分子伴侣可以省去。其次，它们具有引人注目的结构，因为它们都形成具有许多亚基的大型复合物，这些亚基可以形成其他蛋白质可以在其中折叠的笼子。伴侣蛋白分为两组：I 组和 II 组。 I 类伴侣蛋白存在于所有细菌中，也存在于线粒体和叶绿体中，并且人们对它的了解还算比较深入。第二类伴侣蛋白存在于真核生物（如人类）的细胞质中，但对其了解较少。它们也存在于古细菌中，古细菌是一组看起来像细菌的简单生物体，但与真核生物关系更密切。众所周知，第二类伴侣蛋白很重要：在真核细胞中，它们折叠关键蛋白肌动蛋白和微管蛋白，它们一起形成细胞的内部框架（细胞骨架）。它们还有助于折叠一种可以抑制肿瘤形成的蛋白质，并有助于阻止导致亨廷顿舞蹈病等疾病的聚集蛋白质的形成。真核伴侣蛋白包含八种不同类型的亚基，难以研究；例如，我们不知道它们结构的细节。古细菌伴侣蛋白通常仅与单一类型的亚基一起发挥作用，并且我们对其结构有很好的了解。最近，我们小组开发了研究细胞中古菌伴侣蛋白的新方法，当前的提议旨在利用这些方法来更多地了解这些蛋白质。我们希望通过改变蛋白质中的不同氨基酸，然后观察这些改变的伴侣蛋白如何在细胞（体内）中发挥作用，找出伴侣蛋白的哪些部分需要它们发挥作用。值得注意的是，我们已经证明古菌伴侣蛋白也可以在细菌中发挥作用，并且我们希望通过寻找可以在细菌中发挥更好作用的突变蛋白质来研究这一意外发现。然后，我们将纯化其中一些改变的蛋白质，并使用生化测定（体外）观察它们的特性。这将使我们能够将蛋白质在体内发挥作用的能力与它们在体外具有的特定特性联系起来。我们还将使用一些突变伴侣蛋白来尝试识别与它们相互作用的其他蛋白质。这两种方法（遗传和生物化学）将教会我们很多关于古细菌伴侣蛋白和一般伴侣蛋白的知识，并将帮助我们更详细地了解真核伴侣蛋白。这种理解对于人类和动物健康以及生物技术过程具有重要意义。这项工作将涉及三个在该领域具有高度互补专业知识的研究团队之间的合作。

项目成果

Bacterial and eukaryotic systems collide in the three Rs of Methanococcus.

细菌和真核系统在甲烷球菌的三个 R 中发生碰撞。

• DOI: 10.1042/bst0390111
• 发表时间: 2011
• 期刊: Biochemical Society transactions
• 影响因子: 3.9
• 作者: Parker RP

Mutations in subdomain B of the minichromosome maintenance (MCM) helicase affect DNA binding and modulate conformational transitions.

• DOI: 10.1074/jbc.m806973200
• 发表时间: 2009-02-27
• 期刊: The Journal of biological chemistry
• 作者: Jenkinson ER;Costa A;Leech AP;Patwardhan A;Onesti S;Chong JP
• 通讯作者: Chong JP

Effects of N-glycosylation site removal in archaellins on the assembly and function of archaella in Methanococcus maripaludis.

• DOI: 10.1371/journal.pone.0116402
• 发表时间: 2015
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Ding Y;Uchida K;Aizawa S;Murphy K;Berezuk A;Khursigara CM;Chong JP;Jarrell KF
• 通讯作者: Jarrell KF

A large genomic island allows Neisseria meningitidis to utilize propionic acid, with implications for colonization of the human nasopharynx.

• DOI: 10.1111/mmi.12664
• 发表时间: 2014-07
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Catenazzi MC;Jones H;Wallace I;Clifton J;Chong JP;Jackson MA;Macdonald S;Edwards J;Moir JW
• 通讯作者: Moir JW

Rapid Discrimination of Archaeal Tetraether Lipid Cores by Liquid Chromatography-Tandem Mass Spectrometry

• DOI: 10.1016/j.jasms.2008.09.015
• 发表时间: 2009-01-01
• 期刊: JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
• 影响因子: 3.2
• 作者: Knappy, Christopher S.;Chong, James P. J.;Keely, Brendan J.
• 通讯作者: Keely, Brendan J.