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Preparation of human and schistosomal cathepsins L in enzymatically active form
Horáková, Lenka ; Mareš, Michael (advisor) ; Ječmen, Tomáš (referee)
Cathepsin L-like proteases are involved in many pathological processes and their inhibitors are attractive molecules for the development of new drugs. This thesis focuses on human cathepsin L (hCL) and cathepsin L3 from the parasitic blood fluke Schistosoma mansoni (SmCL3). hCL is associated with cancer and Covid-19, while SmCL3 is the digestive enzyme of S. mansoni causing the disease schistosomiasis. The aim of the thesis was to prepare recombinant cathepsins L in an enzymatically active form that is sensitive to inhibitors. In the case of hCL, conditions for the controlled autoactivation of precursor forms to the mature form of the enzyme were identified and this pH- and temperature-dependent process was optimized. In the case of SmCL3, a protocol for purification of the mature form from the culture media of the yeast Komagataella pastoris was developed. The sensitivity of the prepared hCL and SmCL3 to inhibition was demonstrated using a model peptidomimetic vinyl sulfone whose IC50 values were in the sub/nanomolar concentration range. [IN CZECH] Key words: proteolytic enzymes, enzyme activity and inhibition, functional proteomics, recombinant protein expression, protein structure
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Recombinant expression and functional characterization of plant Kunitz inhibitors
Rybáriková, Renata ; Mareš, Michael (advisor) ; Hlouchová, Klára (referee)
PDI ("potato cathepsin D inhibitor ") and NID ("novel inhibitor of cathepsin D ") from potato (Solanum tuberosum) belong to the protein family of Kunitz inhibitors (I3 family, Merops database). These 20 kDa isoinhibitors with the typical β-trefoil architecture inhibit aspartic and serine peptidases. In this thesis, the constructs for recombinant expression of PDI and NID in the yeast Pichia pastoris system were prepared and high-producing colonies were selected. Both proteins were identified in the cultivation media by mass spectrometry and N-terminal sequencing. A purification protocol for PDI with three chromatographic steps was designed. Analogous functional properties were demonstrated for the purified recombinant PDI and the native PDI isolated from a natural source. Analysis of the inhibitory specificity showed that PDI is a potent inhibitor of selected aspartic peptidases from the A1 family and serine peptidases from the S1 family, including a relevant enzyme of insect origin. This finding supports the hypothesis that Kunitz inhibitors are involved in plant defense against herbivorous insects. The inhibitors prepared within the project will be used for analysis of the reactive centers against target peptidases by protein crystallography. (In Czech) Key words: proteolytic enzymes, activity...
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Preparation and biochemical characterization of protease inhibitor equistatin
Polatová, Daniela ; Mareš, Michael (advisor) ; Bořek Dohalská, Lucie (referee)
Equistatin from the sea anemone Actinia equina contains a protein domain Eqd2 which inhibits aspartic peptidases and has not been characterized in detail. Recombinant Eqd2 was produced in the yeast expression system, and a protocol for its chromatographic purification was designed. The inhibitory specificity of Eqd2 was determined using a fluorescence inhibition assay, showing that Eqd2 is a highly selective inhibitor of cathepsin D-like and pepsin-like aspartic peptidases of family A1. Furthermore, size exclusion chromatography was used to analyze the Eqd2-peptidase complex and Eqd2 oligomerization in solution. Initial screening of crystallization conditions for Eqd2 was performed towards its structural analysis. This work provides important new information about Eqd2 as a unique type of natural inhibitors of aspartic peptidases. Its interaction mechanism can be exploited in the development of synthetic mimetics for regulation of medically important peptidases. (In Czech) Key words: peptidase inhibitors, proteolytic enzymes, activity and inhibition of enzymes, recombinant expression, protein purification, protein crystallization, equistatin
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