Tissue proteinases and their inhibitors: Characterisation, genetic engineering and structure-function relationship

The proposed project deals with biochemical studies of cysteine proteinases, mostly of human origin, with emphasys on cathepsins B, H, L, S and C and cruzipain from T. cruzi. Studies were performed on natural and recombinant proteins. Refolding of recombinant sulphonated procathepsin S from 6M urea by kinetic stopped-flow measurements were studies. pH-induced conformational transitions of the inhibitory propeptide of human cathepsin L contribute to further understanding of the mechanism of zymogen activation at acidic pH. Porcine spleen cathepsin H was isolated, its crystal structure was determined and its aminopeptidase activity explained. Based on known kinetics and structural data the revised definition of substrate binding sites of papain-like cysteine proteinases was proposed. The oligomeric nature of human cathepsin C was investigated. Studies on the role of cathepsins and their inhibitors stefins in prognosis, diagnosis and cancer therapy were continued. The folding of stefin A and B under denaturating conditions were studied. The discovery of invariant chain (Ii) of MHC class-II molecules and equistatin was essential for discovery of thyropins, a new superfamily of protein inhibitors of cysteine proteinases. Colorado potato beetle larve on equistatin-coted potato leaves were strongly retarded in their growth indicating high potential in biotechnology.