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Production of cysteine cathepsins and structural characterization of their interaction with peptidomimetic inhibitors
Wichterle, Filip ; Mareš, Michael (advisor) ; Knejzlík, Zdeněk (referee)
Cysteine cathepsins participate in many pathological processes such as cancer and neurodegen- erative, cardiovascular, or autoimmune diseases. This work is focused on cathepsins B, L, and V (CatB, CatL, CatV), which represent attractive targets for the development of inhibitors as potential chemotherapeutics and diagnostic tools. The aim of this study was to prepare these cathepsins and structurally characterize their complexes with selected synthetic peptidomimetic inhibitors. Recombinant CatB and CatL were prepared in the yeast Pichia pastoris, and the expression conditions were optimized for the production of cathepsin zymogens. A chromato- graphic purification protocol was designed for CatB and CatL, while CatV was purified using a previously developed procedure. The obtained enzymes were used to prepare complexes with six peptidomimetic inhibitors equipped with a carbamate, vinyl sulfone, or azanitrile warhead, which selectively target CatB, CatL, and CatV, respectively. Their inhibition parameters were determined in a kinetic assay and initial crystallization conditions were identified. After opti- mizing the crystallization conditions for CatB with three carbamate inhibitors, crystals suitable for X-ray crystallography were obtained. Based on the crystal structures of these complexes, the...
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Interaction of nucleic acids with RNA polymerase
Janoušková, Martina ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee) ; Knejzlík, Zdeněk (referee)
Regulation of gene expression by RNA polymerase (RNAP) is an essential ability of living organisms, required for their adaption to a changing environment and ultimately enabling their survival. Interaction of RNAP with ribonucleic acids (DNA or RNA) is crucial for transcription and its regulation. This Doctoral Thesis contains two projects addressing interactions of RNAP with nucleic acids: (i) Transcription of modified DNA templates and (ii) Ms1, a small RNA (sRNA) from M. smegmatis. (i) We investigated the influence of modifications in the major groove of DNA on bacterial transcription in vitro. We found out that transcription of modified DNA templates is influenced on the transcription initiation level and that the promoter sequence is important for the effect of the modifications. Furthermore, we successfully performed transcription switch ON and OFF in vitro by bioorthogonal reactions. This regulation of transcription by artificial DNA modifications has a future in biotechnologies and/or medical therapy. (ii) Regulators of transcription are also small non-coding RNAs. These molecules have an important role in gene expression regulation among prokaryotes and eukaryotes. Ms1 is an sRNA found in mycobacteria. It makes a complex with the RNAP core and it is abundant in stationary phase (in amounts...
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