The Molecular Enzymology of Collagen Biosynthesis and Matrix Assembly; a Nematode Model System.

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

Parasitic nematodes infect over one third of the global population, and cause many debilitating and chronic diseases, predominantly in the developing world. Human diseases include river blindness and elephatiasis. Very few drugs for treatment are avaliable. All nematodes undergo 5 developmental moults, where a new exoskeleton called the cuticle is formed. This process allows growth and transformation to human infective stages and represents a vulnerable time during the lifecycle. The cuticle is composed of structural collagen-like proteins and this proposal aims to identify and characterize enzymes involved in the formation of this structure with the view to exploiting these as potential drug targets. The aim is to use a relatively simple experimental model organism, the free living nematode Caenorhabditis elegans, to address this problem. It is also envisaged that these studies will contribute to the general understanding of collagen folding, as collagens are also vital in human developement, representing the major structural proteins. Characterization of collagens will thus provide important insights into human diseases that are caused by over-modification or lack of modification of these important structural proteins in humans, such as Ehlers-Danlos syndrome (EDS).

Technical Summary

The structural integrity of multicellular animals is determined by the extracellular matrix (ECM); a structure that is predominantly composed of triple helical collagens. In humans, these proteins are involved in bone, skin and cartilage formation and mutations in collagens and the enzymes involved in their biosynthesis cause serious human diseases, such as osteogenesis imperfecta.
In nematodes, collagens are the major structural proteins of the exoskeleton (or cuticle), a nematode-specific structure that is rapidly and successively synthesised 5 times during development. Parasitic nematodes are important pathogens of man, especially in the developing world, where infections result in chronic debilitating diseases such as river blindness and lymphatic filariasis, caused by filaral nematodes. Parasites can be difficult to clear; there are no vaccines, few effective drugs and resistance is becoming commonplace. A number of essential enzymes are involved in biosynthesis, post-translation modification, processing and assembly of collagens. I propose to identify and characterize these enzymes in a model of nematode system, with the overall goal to increase understanding of collagen biosynthesis. The genetically tractable nematode Caenorhabditis elegans is an excellent system in which to carry out a post-genomic characterization of such enzymes and evaluate their potential as drug targets for the less-amenable parasitic nematode species.

The aims of this proposal are therfore to understand the function of enzymes in the modification, processing and assembly of nematode cuticular collagens; collagen assembly/modification in general; and evaluate their potential as targets of chemotherapy. This will involve characterizing enzymes already identified and discovering and characterizing new ones. I propose to apply post-genomic, genetic, biochemical and structural biology approaches to assess and characterize the roles that potential collagen-associated enzymes play in cuticle synthesis. A genome-wide reverse genetics approach will be employed using avaliable RNAi-clones representing potential collagen-specific enzymes and chaperones on an adult marker strain. Interesting enzymes will be examined with respect to expression patterns and enzymatic activity, and will be structurally characterised where appropriate. All interesting enzymes will also be characterized in a nematode collagen expression system and the effects of specific inhibitors will be addressed. Collaborators will be pursued with collagen biochemists (Dr. J. Myllyharju, Oulu, Finland) and structural biologists (Prof. M. Walkinshaw, Edinburgh) to most effectively characterize the identified enzymes.

Publications

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Page AP (2007) The cuticle. in WormBook : the online review of C. elegans biology

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Richardson JM (2007) Cloning, expression and characterisation of FKB-6, the sole large TPR-containing immunophilin from C. elegans. in Biochemical and biophysical research communications

 
Description BBSRC funding SCIBS (Chemical mapping of the PPIase interactome in C. elegans - development of a systems biology toolbox)
Amount £181,151 (GBP)
Funding ID BB/D006201/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2006 
End 10/2009
 
Description VIP award
Amount £22,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2008 
End 09/2009
 
Description VIP award
Amount £22,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2008 
End 05/2009
 
Title Transgenic Marker strains 
Description We have developed transgenic nematode lines that can be applied to assess collagen disruption, matrix formation and morphogenesis. 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact The following publications 19406744 17586485 17339317 16500660 16452136