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Study of the cleavage kinetics of Gag polyprotein from HIV-1 virus by the viral proteinase
Krištofičová, Ivica ; Ingr, Marek (advisor) ; Martínek, Václav (referee)
Gag polyprotein is the precursor of HIV-1 structural proteins, required for correct assembly, budding and maturation of viral particle within HIV-1 life cycle. The process of maturation into an infectious virion is dependent on Gag and GagPol cleavage at nine predefined sites by HIV-1 proteinase. Its disruption is one of the main targets of HIV treatment. HIV-1, however, develops resistance to the proteinase inhibitors by creating mutations in both the proteinase and the substrate. The Gag processing by HIV-1 proteinase is a highly sequential process, that happens in specific order and rate. Previous biochemical studies determined the kinetic data of these processes using oligopeptides representing naturally occuring cleavage sites. This thesis describes the cleavage of the Gag polyprotein itself, which is the natural substrate of HIV-1 proteinase. For this purpose, the full-length Gag polyprotein was recombinantly prepared in bacterial expression system. The cleavage was carried out and its products were analyzed via SDS-PAGE and Western blotting. The substrate specificity of the wild-type and mutant HIV-1 proteinase with respect to the full-length wild-type Gag polyprotein was compared. Substantial differences were observed between the rates of individual steps of cleavage by the wild-type and mutant...
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Enzymatic and inhibiting activity in boar epididymal fluid
Davidová, Nina ; Ren, Š. ; Liberda, J. ; Jonáková, Věra ; Maňásková-Postlerová, Pavla
Sperm maturation, represents a key step in the reproduction process. Spermatozoa, particularly the plasma membrane, are exposed to epididymal fluid (EF) components representing the natural environment essential for their post-testicular maturation. Changes in the sperm membrane proteins are influenced by proteolytic and glycosidic enzymes present in the EF. Accordingly, the occurrence of inhibitors in this reproductive organ is very important for the regulation of sperm membrane protein processing. In present study, we monitored protease and glycosidase activities, and inhibitors of metallo- and serine proteinases in boar EF. Additionally, we studied acrosin inhibitor in fluid, spermatozoa and tissue along the epididymis. We chromatographically separated boar EF into several fractions. These fractions were subjected to SDS-electrophoresis and the separated proteins were either studied by zymographic methods or transferred to nitrocellulose membranes for detection of metallo- and serine proteinases and their inhibitors, and acrosin inhibitor by specific antibody, respectively. Acrosin inhibitor was monitored also in the sperm and tissue of the boar epididymis. In boar epididymal fluid, several metallo- and serine proteinases with different molecular masses, and inhibitors of metalloproteinase MMP-9 and acrosin were found. We measured strong activity of mannosidase in this fluid. Using specific antibody, we registered the increasing signal of acrosin inhibitor from caput to cauda epididymis in the spermatozoa, fluid and also tissue. Proteinases and their inhibitors in reproductive fluids may play a significant role in reproduction processes. Especially, acrosin inhibitor in the reproductive tract inactivates prematurely released sperm acrosin and protects spermatozoa and reproductive epithelium against proteolytic degradation. High mannosidase activity in boar EF suggests evident role of mannose structures in the sperm interaction during reproductive events.
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