Dipeptidyl peptidase IV (DPP-IV) is a serine protease, which executes its proteolytic activity by cleaving X-Pro dipeptides from the N-termini of its substrates. Furthermore, DPP-IV exhibits many biological functions independent of its enzymatic aktivity. Previous studies in our laboratory proved increased expression of DPP-IV in high-grade astrocytic tumours. To evaluate the enzymatic and non-enzymatic functions of DPP-IV in a glioma model, clones of asctrocytic cell line U373MG transfected by enzymatically inactive, mutated DPP-IV (mutDPP-IV) and enzymatically active, wild type DPP-IV (wtDPP-IV), were prepared. Enzymatically inactive mutDPP-IV was prepared using point mutation the active site serine residue. Cells U373MG were transfected using a doxycycline inducible Tet-On® system. For further analysis of the transgenic forms of DPP-IV, methods were used for verification of protein expression, enzymatic activity and subcellular localization. Doxycycline induced U373MG mutDPP-IV and U373MG wtDPP-IV cells, expressing mutated and wild type DPP-IV, respectivelly, exhibited increased expression of transgenic DPP-IV in a concentration and time dependent manner. Doxycycline induced U373MG wtDPP-IV cells exhibited both increased expression and enzymatic activity of DPP-IV. In contrast, DPP-IV enzymatic...

The majority of the mitochondrial proteins is synthetized on the cytosolic ribosomes in the form of the protein precursors bearing mitochondrion-targeting signal presequences. Once the protein precursor has reached the mitochondrial matrix the signal presequence is no longer necessary and is cleaved off by heterodimeric mitochondrial processing peptidase (MPP; α/β). Although the crystal structure of MPP is available, the MPP mechanism of function is still matter of discussion. An all atomic, non-restrained molecular dynamics (MD) simulation in explicit water was used to study in detail the structural features of the highly conserved glycine-rich loop (GRL) of the regulatory α-subunit of the yeast MPP. Wild-type and GRL-deleted MPP structures were studied both in the presence and absence of a substrate in the peptidase active site. Targeted MD simulations were employed to study the mechanism of substrate translocation from the GRL to the peptidase active site. We demonstrate that the natural conformational flexibility of the GRL is crucial for the substrate translocation process from outside the enzyme towards the MPP active site. We show that the α-helical conformation of the substrate is important not only during its initial interaction with MPP (i.e. substrate recognition), but also later, at...

Blood fluke Schistosoma mansoni is one of the most important human parasites. Proteolytic system of schistosoma is crucial for parasite - host interactions. Therefore some of the proteases became potential therapeutic targets. This work is focused on not yet characterized serine protease SmSP2. SmSP2 is newly discovered protease of S. mansoni, whose biological role is unknown. This protease is highly expressed in developmental stages parasitizing humans. SmSP2 was recombinantly expressed in prokaryotic and eukaryotic expression system (E. coli a P. pastoris) and purified using chromatographic methods. Recombinant SmSP2 was used for polyclonal antibody production. Conditions for refolding were optimized. Basic biochemical properties of the protease were detected and substrate amino acid preferences for P1 - P4 sites for single aminoacids were identified using synthetic fluorogenic peptides for positional scanning substrate combinatorial library (PS-SCL). (In Czech)