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Title: Characterisation of small protease inhibitors as potential therapeutics
Author: McCrudden, Maeliosa Theresa Christine
ISNI:       0000 0001 3623 4982
Awarding Body: Queen's University of Belfast
Current Institution: Queen's University Belfast
Date of Award: 2008
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Neutrophil-derived serine proteases provide one of the first lines of defense against infection in innate immunity. However, in some disease states, the activity of these proteases becomes excessive and prolonged and so novel lead molecules with the potential to inhibit the excessive activity of these proteases in disease states are constantly being sought. In the current study, three proteinaceous protease inhibitors were chosen for characterisation. The specificity of the Escherichia coli trypsin inhibitor (ecotin) was modulated by site-directed mutagenesis studies; the elastase and cathepsin G-like inhibitor (eglin C) from Hirudo medicinalis was used as a positive control recombinant protein and the functional characteristics of the epididymal protease inhibitor (eppin) were examined. Eppin's anti-bacterial activity had been partially characterised but beyond this, very little functional information was available for the protein. Composed of the Whey acidic protein (WAP) and Kunitz inhibitor motifs, eppin was postulated to have anti-protease and immunomodulatory properties. Expression vectors were constructed for eppin and each of its consensus inhibitor domains and recombinant proteins were subsequently expressed in and purified from E. coli cells. The proteins all had elements of a-helix and p-sheet in their secondary structure, deduced by circular dichrosim (CD) analysis. Eppin and its domains were found to kill E. coli cells via a mechanism of permeabilisation of bacterial membranes resulting in uncoupling of respiratory electron transport but the two domains were required to achieve maximal effect. The Kunitz domain inhibited human neutrophil elastase to a similar extent as the intact protein but the.WAP domain exhibited no such activity. In a model of lipopolysaccharide (LPS)-induced inflammation in a monocytic cell line, eppin was shown 'to inhibit the expression of three pro-inflammatory cytokines. The mechanism of inhibition, in the case of one of the cytokines, was via down-regulation of transcription ofthe cytokine.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available