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

蛋白质在活着的有机体中有许多作用。它们可能催化反应，它们可能是细胞结构的重要部分，它们可能使细胞对外部信号做出反应，它们可能开启或关闭基因，等等。蛋白质是由长链的氨基酸构成的，但在它们能够在细胞内发挥作用之前，它们必须折叠成特定的形状。每种形状对每种蛋白质都是独一无二的。当蛋白质不能正确折叠时，许多问题就会出现。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，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。分子伴侣在折叠时只与蛋白质短暂地相互作用，但如果没有这种相互作用，许多蛋白质就无法正确折叠。分子伴侣有各种不同的类别。我们对被称为“监护人”的课程特别感兴趣。伴侣之所以引起人们的极大兴趣，有两个原因。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可以省去。其次，它们具有惊人的结构，因为它们都形成了带有许多亚单位的大型复合体，这些亚单位可以形成笼子，其他蛋白质可以在其中折叠。伴侣蛋白分为两组：第一组和第二组。第一组伴侣蛋白存在于所有细菌中，也存在于线粒体和叶绿体中，人们对其了解较多。第二类伴侣蛋白存在于真核生物(如人类)的胞浆中，人们对它的了解要少得多。在古生菌中也发现了它们，古生菌是一群看起来像细菌但与真核生物关系更近的简单有机体。已知第二组伴侣蛋白很重要：在真核细胞中，它们折叠关键蛋白肌动蛋白和微管蛋白，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。真核细胞伴侣蛋白包含八种不同类型的亚单位，很难研究；例如，我们不知道它们结构的细节。古生菌的伴侣蛋白通常只与一种类型的亚单位起作用，我们对其结构有很好的了解。最近，在我们的团队中，我们开发了在细胞中研究古细菌伴侣蛋白的新方法，目前的提议旨在利用这些方法来了解更多关于这些蛋白质的信息。我们希望通过改变蛋白质中不同的氨基酸，然后观察这些改变后的伴侣蛋白在细胞中(在体内)的功能，来找出它们工作所需的伴侣蛋白的哪些部分。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，我们想通过寻找可以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。然后我们将提纯这些改变的蛋白质中的一些，并使用生化分析(体外)来观察它们的性质。这将使我们将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(遗传和生化)将教会我们许多关于古生物伴侣蛋白的知识和一般的关于伴侣蛋白的知识，并将帮助我们更详细地了解真核生物的伴侣蛋白。这一理解对人类和动物健康以及生物技术进程具有重要影响。这项工作将涉及三个在这一领域具有高度互补性的专业知识的研究小组之间的合作。

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.