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Title: Engineering and design of aspartic proteinase inhibitors
Author: Winterburn, Timothy John
ISNI:       0000 0004 2747 4898
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2005
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Aspartic proteinases are of considerable interest to the pharmaceutical, agrochemical and food industries. Many small molecule inhibitors have been synthesised chemically to block the action of individual members of this proteinase family. In contrast, only a handful of naturally-occurring, gene-encoded inhibitors of aspartic proteinases are known. One such inhibitor is IA3, an intrinsically unstructured 68-residue cytosolic protein from Saccharomyces cerevisiae, which operates through an unprecedented mode of action. On contacting the target enzyme, residues 2-32 form an amphipathic a-helix that occludes the active site cleft. The potency and specificity of this inhibitor were investigated by producing >80 variants in the form of recombinant proteins in Escherichia coli or as synthetic peptides preliminary attempts were also made to examine the potential of directed evolution by phage display for the generation of further mutants. The inhibitory activity of these variant polypeptides at different pH values was measured against the natural target proteinase from S. cerevisiae and against vacuolar aspartic proteinases from Pichia pastoris and Aspergillus fumigatus, as well as the human lysosomal enzyme, cathepsin D. New IA3-like sequences were also identified in five closely-related yeasts. One, from Saccharomyces castellii, differed substantially from S. cerevisiae IA3 and so was investigated in parallel. Key elements of the inhibitory sequence (i.e. residues 2-34) in the S. castellii IA3 were identified in relation to their counterparts in S. cerevisiae IA3 and the different effects of their respective C-terminal regions (i.e. residues 34 to C-terminus) were unravelled through the production of chimaeric polypeptides. In this way, potent IA3-derived inhibitors of the P. pastoris and A. fumigatus proteinases were produced, and an effective inhibitor of cathepsin D was identified. IA3 is shown to be remarkably adaptable as an inhibitor, and its potency may be re-directed to inhibit more distant enzymes of commercial interest.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available