Functional biochemistry and enzymology
Investigation of a hemolymph protein, PAS-110, and its interaction systemPAS-110 is a 110 kDa target protein in the hemolymph of Manduca sexta to PrtA, a serralysin enzyme of an insect pathogen bacterium, Photorhabdus. This protein is still unknown (according to databases) but it supposedly has a role in the innate immunity. Therefore our aim is to clone, express it, and to investigate its function (e.g. with searching for interacting proteins and RNAi technique) and its expression under various conditions (e.g. with antibody and/or RT-PCR after infection with pathogen or apathogen bacterium).
Investigation of an inhibitor of PrtA, a serralysin-type metalloproteaseThe inhibitor of PrtA (PAI) is the first such molecule, of eukaryotic host origin, which inhibits a metzincin, a serralysin-type metalloprotease. PAI is immune inducible, thus participates in the immune response of the insect (Manduca sexta, tobacco horned worm) upon bacterial infection. We are investigating the kinetic parameters and molecular properties of the inhibition and working on the cloning of the inhibitor. Our further goal is to find a supposed precursor of PAI and the enzyme which activates it. These also will be new components of the immune system of the insect. To investigate the specificity of PAI inhibition we will compare its function with that of other inhibitors of M10 metalloproteases, and determine to what degree PAI is able to discriminate M10 metalloproteases: those of bacterial origin (M10B) from those of eukaryotic origin (M10A). To understand the effect of PAI we will study its mechanism and the X-ray structure of its complex with PrtA.
(University of Bath, U. K.)
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- Marokházi, J., Mihala, N., Hudecz, F., Fodor, A., Gráf, L. & Venekei, I. Cleavage site analysis of a serralysin-like protease, PrtA, from an insect pathogen Photorhabdus luminescens and development of a highly sensitive and specific substrate. (2007). Febs J 274, 1946-56.
- Marokházi, J., Lengyel, K., Pekar, S., Felföldi, G., Patthy, A., Gráf, L., Fodor, A. & Venekei, I. Comparison of proteolytic activities produced by entomopathogenic Photorhabdus bacteria: strain- and phase-dependent heterogeneity in composition and activity of four enzymes. (2004)Appl Environ Microbiol 70, 7311-20.
- Marokházi, J., Koczan, G., Hudecz, F., Gráf, L., Fodor, A. & Venekei, I. Enzymic characterization with progress curve analysis of a collagen peptidase from an enthomopathogenic bacterium, Photorhabdus luminescens. (2004)Biochem J 379, 633-40.
- Szabó, E., Venekei, I., Böcskei, Z., Náray-Szabó, G. & Gráf, L. Three dimensional structures of S189D chymotrypsin and D189S trypsin mutants: the effect of polarity at site 189 on a protease-specific stabilization of the substrate-binding site. (2003)J Mol Biol 331, 1121-30.