A chemical genetic approach to the analysis of peroxisome biogenesis

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.

The proposed research is a collaborative project between chemists and plant scientists at the University of Leeds and will employ chemical genetics to study fundamental processes of plant cell biology. Chemical genetics is a powerful, complementary technique to conventional genetics that can provide tools for the study of complex biological phenomena. It is particularly useful in the study of processes where genetic redundancy and/or lethality make it difficult to obtain informative mutants. This is frequently the case with protein trafficking in multicellular organisms. Preliminary screens have identified small molecules which cause novel phenotypes in Arabidopsis seedlings. The molecules disrupt import of proteins into the peroxisome, alter peroxisomal positioning/movement or prevent lipid breakdown. During the research, chemical and biochemical techniques will be used to understand how these small molecules cause the observed phenotypic effects giving insight into interactions which underpin the peroxisomal function. The precise relationships between chemical structure and activity will be determined by screening an expanded range of compounds and computational modelling. In parallel, detailed studies will fully characterise the phenotypes; dissecting the effect of active compounds on individual components known to be part of the peroxisomal import machinery, on different peroxisomal trafficking pathways and on the interaction of peroxisomes with the cytoskeleton. We will then use chemical synthesis to prepare affinity tagged versions of each of the small molecules to identify their precise biomolecular target. Biomolceular approaches will also be used to probe the interaction of the small molecules with likely targets. Identifying how these small molecules can alter protein trafficking pathways will give new insight into the mechanism and regulation of protein trafficking pathways and provide tools to probe these pathways in the living cel