A chemical genetic approach to the analysis of peroxisome biogenesis
Lead Research Organisation:
University of Leeds
Department Name: Ctr for Plant Sciences
Abstract
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.
Technical Summary
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
Publications
Warriner S
(2007)
A Novel Approach to the Solid-Phase Synthesis of Peptides with a Tetrazole at the C-Terminus
in Synlett
Brown LA
(2008)
Shuttles and cycles: transport of proteins into the peroxisome matrix (review).
in Molecular membrane biology
Lanyon-Hogg T
(2010)
Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes.
in Biology of the cell
Baker A
(2010)
Peroxisome biogenesis and positioning.
in Biochemical Society transactions
Brown L
(2011)
A small molecule with differential effects on the PTS1 and PTS2 peroxisome matrix import pathways
in The Plant Journal
Hu J
(2012)
Plant peroxisomes: biogenesis and function.
in The Plant cell
Theodoulou FL
(2013)
Peroxisome membrane proteins: multiple trafficking routes and multiple functions?
in The Biochemical journal
Brown LA
(2013)
An inhibitor of oil body mobilization in Arabidopsis.
in The New phytologist
Lanyon-Hogg T
(2014)
PEX14 binding to Arabidopsis PEX5 has differential effects on PTS1 and PTS2 cargo occupancy of the receptor.
in FEBS letters
Baker A
(2014)
The life of the peroxisome: from birth to death.
in Current opinion in plant biology
Skoulding NS
(2015)
Experimental validation of plant peroxisomal targeting prediction algorithms by systematic comparison of in vivo import efficiency and in vitro PTS1 binding affinity.
in Journal of molecular biology
McLachlan DH
(2016)
The Breakdown of Stored Triacylglycerols Is Required during Light-Induced Stomatal Opening.
in Current biology : CB
Cross LL
(2016)
Peroxisome biogenesis, protein targeting mechanisms and PEX gene functions in plants.
in Biochimica et biophysica acta
Description | We have identified 3 small molecules that interfere with various aspects of peroxisome biology. One is an inhibitor of fatty acid mobilisation in seeds and two affect the localisation of proteins to the peroxisome. One further small molecular affects the movement of membrane proteins around the plant endomembrane system. 3 publications have arisen from these findings. these chemicals will be useful tools to further interrogate tehse aspects of plant biology |
Exploitation Route | These chemical tools are available to others in the scientific community to use. This has led to a recent publication in which the inhibitor of oil mobilisation that we characterised was used to investigate the role of lipid breakdown in stomatal guard cell opening. See associated paper by McLachlan et al., 2016 Current Biology |
Sectors | Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology |
Description | Japan Partnering award |
Amount | £36,000 (GBP) |
Funding ID | 474346 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 04/2008 |
End | 03/2011 |
Description | Research programme grant |
Amount | £225,906 (GBP) |
Funding ID | RPG-2012-516 |
Organisation | The Leverhulme Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 10/2012 |
End | 09/2016 |
Description | Stuart Warriner |
Organisation | University of Leeds |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Biology side of a chemical biology project. Construct production, confocal microscopy, plant physiology transgenic plant production |
Collaborator Contribution | Chemical synthesis, biophysical biochemistry |
Impact | 10.1016/j.febslet.2014.05.038 10.1042/BJ20131136 10.1111/nph.12467 10.1111/j.1365-313X.2010.04473.x 10.1042/BST0380807 10.1042/BC20090159 10.1055/s-2007-986661 multidisciplinary collaboration between chemistry and biology |
Start Year | 2006 |
Description | Chemical Biology Investigations of Peroxisome Protein Import |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Type Of Presentation | Keynote/Invited Speaker |
Geographic Reach | International |
Primary Audience | Participants in your research or patient groups |
Results and Impact | Invited talk at EMBO special interest meeting Vienna no actual impacts realised to date |
Year(s) Of Engagement Activity | 2011 |
Description | Genetic and small molecule-based approaches to the study of peroxisomal beta-oxidation |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Participants in your research or patient groups |
Results and Impact | Research seminar at the Plant Laboratory Michigan State University no actual impacts realised to date |
Year(s) Of Engagement Activity | 2010 |
Description | Inaugural Irene Manton lacture 2015 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Other audiences |
Results and Impact | I was invited by the local Athena Swan organisers to give the inaugural Irene Manton Lecture to celebrate women in science in our Faculty. I gave my lectures on 'The ins and outs of plant cells' on 8th May 2015 |
Year(s) Of Engagement Activity | 2015 |
Description | Modern Cellular Imaging |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Type Of Presentation | Paper Presentation |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Talk to members of the public at the Otley Science cafe no actual impacts realised to date |
Year(s) Of Engagement Activity | 2011 |