A chemical genetic approach to the analysis of peroxisome biogenesis

所有的生物体都是由细胞组成的。像植物和动物这样的复杂生物有许多不同类型的细胞组成组织和器官，执行不同的功能。例如，植物的叶子可以进行光合作用，人类的心脏可以将血液输送到全身。这些过程和许多其他不同的过程不仅需要细胞之间的相互作用，还需要细胞内化学反应的适当组织。除了最简单的生物外，所有生物的细胞都含有称为细胞器的隔室。这些隔室需要蛋白质才能发挥作用。细胞质内产生的蛋白质通过蛋白质运输途径传递到细胞器。这些蛋白质携带的信号被其他蛋白质识别，这些蛋白质将它们分类并运送到正确的位置。其中一种细胞器是过氧化物酶体。如果过氧化物酶体出了问题，后果是可怕的。先天性过氧化物酶体缺陷的儿童患有严重的残疾，通常死亡。具有完全缺陷的过氧化物酶体的植物不能产生有活力的种子。由于蛋白质运输途径的重要性，科学家们想要详细了解它们是如何工作的。我们已经知道了很多，主要是通过敲除制造这些途径所需蛋白质的基因的功能。但有时敲除基因会导致生物体死亡，这不是很有用，或者没有效果，因为可能还有其他基因可以发挥与缺陷基因相同的功能。可以进入细胞并抑制其中一些蛋白质功能的小化学物质可以提供一种非常有用的替代方法来了解这些过程是如何工作的，因为化学物质可以在特定的时间点添加并在稍后被移除。它们可以以不同的量添加，这样它们就不会产生致命的影响，而且从添加化学物质的那一刻起，就可以监测其影响。如果有不止一种类似的蛋白质执行相同的功能，同一种化学物质可能会影响所有类似的蛋白质，而敲除一个基因只会影响由该基因产生的蛋白质。大自然，在合成化学的帮助下，已经产生了数以百万计的化学结构。理论上，应该有一种小的化学物质干扰每个细胞中每个蛋白质的功能。我们已经确定了一小组化学物质会干扰蛋白质进入过氧化物酶体，另一组化学物质会改变过氧化物酶体在细胞内的移动方式。另一种化合物阻止过氧化物酶体分解脂肪。该项目的目的是确定这些分子与细胞机制的哪些特定部分相互作用，以便我们能够更多地了解蛋白质进入过氧化物酶体的机制和调节的精确细节。首先，我们将了解引起所观察到的效果所必需的小分子的精确分子特征。与此同时，我们将详细描述每种分子的影响，例如，计算出引起每种反应需要多少分子，以及暴露于化学物质是否会改变已知参与过氧化物酶体蛋白质运输的某些蛋白质的水平。然后，我们将使用一系列不同的化学和生化技术来确定小分子的精确生物分子目标。通过准确地了解小分子与哪个生物分子结合，我们将获得对细胞运作的新见解。更好地了解这些过程最终可能会导致新药的开发，或者为了生物技术目的而操纵植物或微生物中的这些途径的能力。

All organisms are made up of cells. Complex organisms like plants and animals have many different types of cells organised into tissues and organs that perform different functions. Examples are a plant leaf, that carries out photosynthesis and the human heart that pumps blood around our body. Each of these and many other different processes require not only the interaction between cells but the proper organisation of chemical reactions within cells. Cells of all but the simplest organisms contain compartments called organelles. These compartments require proteins in order to function. Proteins made within the cytoplasm of the cell are delivered to organelles by protein trafficking pathways. The proteins carry signals that are recognised by other proteins that sort and deliver them to the correct place. One of these organelles is the peroxisome. If peroxisomes go wrong the consequences are dire. Children born with defective peroxisomes suffer very serious disabilities and usually die. Plants with completely defective peroxisomes cannot produce viable seeds. Because of the importance of protein trafficking pathways, scientists want to understand how they work in detail. We already know a lot, mainly from knocking out the function of genes that make proteins that are needed for these pathways. But sometimes knocking out genes causes the organism to die, which isn't very useful, or has no effect, because there may be other genes that can perform the same function as the defective gene. Small chemicals that can enter cells and inhibit the function of some of these proteins can offer a very useful alternative way to understand how these processes work, because chemicals can be added at specific time points and taken away later on. They can be added in different amounts so that they do not have a lethal effect and the effects can be monitored from the moment the chemical is added. If there is more than one similar protein carrying out the same function the same chemical will probably affect all the similar proteins, whereas knocking out a gene will only affect the protein made by that one gene. Nature, augmented by synthetic chemistry, has produced literally millions of chemical structures. In theory there should be a small chemical that interferes with the function of every protein in every cell. We have identified a small group of chemicals which interfere with the import of proteins into peroxisomes and another group which alter how peroxisomes move around the cell. A further compound blocks the breakdown of fats by the peroxisome. The aim of this project is identify which specific parts of the cellular machinery these molecules are interacting with, so that we can understand more about the precise details of how the mechanism and regulation of proteins import into peroxisomes. Firstly we will understand the precise molecular features of the small molecules that are necessary to cause the observed effect. At the same time we will characterise the effects of each molecule in detail, for example working out how much molecule is needed to cause each type of response and also if exposure to the chemical changes the levels of certain proteins known to be involved in peroxisomal protein trafficking. We will then use a range of different chemical and biochemical techniques to identify the precise biomolecular target of the small molecules. By understanding exactly which biomolecule the small molecule binds to we will gain new insights into the operation of the cell. Understanding these processes better could eventually lead to the development of new drugs or the ability to manipulate these pathways in plants or micro-organisms for biotechnological purposes.