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/F002483/1
• 负责人: Peter Lund
• 金额: $ 43.94万
• 依托单位: University of Birmingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2007
• 资助国家: 英国
• 起止时间: 2007 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF002483%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.

蛋白质在生物体中有许多作用。它们可能催化反应，它们可能是细胞结构的重要组成部分，它们可能使细胞对外部信号作出反应，它们可能开启或关闭基因，等等。蛋白质是由长链氨基酸构成的，但在它们能在细胞内发挥作用之前，它们必须折叠成特定的形状。每种蛋白质的形状都是独一无二的。当蛋白质不能正确折叠时，就会出现许多问题。这些可能是健康问题（例如，疯牛病等疾病与蛋白质无法折叠成其功能形状有关）。此外，蛋白质在工业中广泛生产，在某些情况下，这些蛋白质的错误折叠是一个主要问题。近年来的一项重大发现是，许多蛋白质在达到折叠状态之前必须与称为分子伴侣的其他蛋白质相互作用。分子伴侣只在蛋白质折叠时与它们短暂地相互作用，但如果没有这种相互作用，许多蛋白质就不能正常折叠。分子伴侣有很多不同的种类。我们特别感兴趣的一类被称为“伴侣”。有两个原因引起了人们的极大兴趣。首先，它们对所有细胞都是必不可少的，而许多其他类型的分子伴侣是可以省略的。其次，它们有一个引人注目的结构，它们都形成有许多亚基的大复合物，这些复合物可以形成笼子，其他蛋白质可以折叠进去。伴侣蛋白分为两组：I组和II组。I族伴侣蛋白存在于所有细菌中，也存在于线粒体和叶绿体中，人们对它的了解还算充分。II族伴侣蛋白存在于真核生物（如人类）的细胞质中，人们对其知之甚少。它们也存在于古细菌中，古细菌是一组看起来像细菌但与真核生物关系更密切的简单生物。II族伴侣蛋白是非常重要的：在真核细胞中，它们折叠关键蛋白肌动蛋白和微管蛋白，这两种蛋白共同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8种不同类型的亚基，研究难度较大；例如，我们不知道它们结构的细节。古细菌伴侣蛋白通常只与单一类型的亚基一起起作用，我们对它们的结构有很好的了解。最近，在我们的小组中，我们开发了研究细胞中古细菌伴侣蛋白的新方法，目前的建议旨在利用这些方法来了解更多关于这些蛋白质的信息。我们想通过改变蛋白质中不同的氨基酸，找出伴侣蛋白的哪些部分需要它们发挥作用，然后观察这些改变后的伴侣蛋白在细胞（体内）中的功能。值得注意的是，我们已经证明了古细菌伴侣蛋白也能在细菌中起作用，我们想通过寻找能在细菌中发挥更好作用的突变蛋白来研究这一意想不到的发现。然后，我们将纯化其中一些改变的蛋白质，并使用生化分析（体外）观察它们的特性。这将使我们把蛋白质在体内发挥作用的能力与它们在体外具有的特殊性质联系起来。我们还将使用一些突变的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（遗传学和生物化学）将使我们对古细菌伴侣蛋白有更多的了解，并有助于我们更详细地了解真核生物伴侣蛋白。这一认识对人类和动物健康以及生物技术进程具有重要意义。这项工作将涉及在该领域具有高度互补性专业知识的三个研究小组之间的合作。

项目成果

Archaea at St Andrews.

• DOI: 10.1186/gb-2008-9-9-321
• 发表时间: 2008
• 期刊: Genome biology
• 影响因子: 12.3
• 作者: Large AT;Lund PA
• 通讯作者: Lund PA

Replacement of GroEL in Escherichia coli by the Group II Chaperonin from the Archaeon Methanococcus maripaludis.

• DOI: 10.1128/jb.00317-16
• 发表时间: 2016-10-01
• 期刊: Journal of bacteriology
• 影响因子: 3.2
• 作者: Shah R;Large AT;Ursinus A;Lin B;Gowrinathan P;Martin J;Lund PA
• 通讯作者: Lund PA