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Cloning, expression and characterization of human serine racemase mutants
Nováková, Ilona ; Brynda, Jiří (referee) ; Konvalinka, Jan (advisor)
AAbbssttrraacctt Human serine racemase (hSR) is a cytosolic pyridoxal-5'-phosphate dependent enzyme localized in the central nervous system. It synthesizes D-serine, which is an endogenous coagonist for the N-methyl-D-aspartate (NMDA) receptors and plays a key role in excitatory neurotransmission in the brain. Thus, human serine racemase is a promising target for the treatment of neurodegenerative diseases connected with NMDA receptors. However, few specific inhibitors have been identified to date and the crystal structure of hSR has become available only very recently. We decided to perform a random mutagenesis to determine the amino acid residues critical for the enzyme activity. Ser 84 was reported as a catalytic residue along with Lys 56. After analysis of a double mutant S84G/P111L which retained its capability to convert L-serine to pyruvate, we prepared and characterized the single mutant S84G in order to exclude potential effect of the P111L mutation.on the activity of the analyzed enzyme. KKeeyy wwoorrddss:: D-serine; Serine racemase; PLP-dependent enzymes; Random mutagenesis; Racemases
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Preparation, expression and characterization of mouse GCPIII
Bäumlová, Adriana ; Šebo, Peter (referee) ; Konvalinka, Jan (advisor)
English abstract Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a type II transmembrane glycoprotein which has been discovered in nervous system as an enzyme responsible for the hydrolysis of neuropeptide N-acetyl-L-aspartyl-L-glutamate to N-acetyl-L-aspartate and L-glutamate and that has been hypothesized to influence glutamatergic signaling processes. Except for brain, GCPII was mainly found in prostate, kidney, and small intestine. In small intestine, GCPII cleaves terminal glutamates from polyglutamylated folates facilitating thus absorption of dietary folates. In prostate, this enzyme is known as prostate-specific membrane antigen and is used as a cancer marker. Mus musculus is an important model for studing GCPII and its homologs as a therapeutic target. While human GCPII and its paralog GCPIII are relatively well characterized, no biochemical study of their mouse orthologs is available. That is why mouse glutamate carboxypeptidase III (mGCPIII) was cloned, prepared by recombinant expression in insect cells and characterized. We show that pure mouse GCPIII possesses α-glutamate carboxypeptidase activity which is effectively inhibited by specific inhibitor GCPII, 2-PMPA. We also analyzed sensitivity and specifity of monoclonal antibodies against mouse GCPIII. Immunoblots demonstrate that...
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Analysis of serine racemase expression in the CNS of epileptic patients
Vorlová, Barbora ; Maloy Řezáčová, Pavlína (referee) ; Konvalinka, Jan (advisor)
Serine racemase is a pyridoxal-5'-phosphate dependent enzyme that converts L-serine to D-serine. D-serine is a recognized physiological co-agonist of N-methyl-D-aspartate type of glutamate receptors - key receptors that participate in the neurotransmission in the mammalian brain. Dysfunction of these receptors has been implicated in several neuropathologies, including schizophrenia, brain ischemia, neurodegenerative disorders and epilepsy. Serine racemase is thus a promising pharmaceutical target in these diseases. In this study, three anti-human serine racemase monoclonal antibodies were characterized and the best one was used for the Western blot detection of the enzyme in resected human epileptic tissues. For better interpretation of the results, accuracy of the tissue processing, the protein concentration determination and the Western blot quantification were verified. Finally, the activity of human serine racemase was determined with the L-serine-O-sulfate, the substrate with the highest-affinity to this enzyme. (Thesis in Czech)
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Recombinant aspartic proteases of blood-feeding parasites
Váchová, Jana ; Entlicher, Gustav (referee) ; Konvalinka, Jan (advisor)
The blood fluke Schistosoma mansoni and the hard tick Ixodes ricinus produce an aspartic protease cathepsin D which initiates degradation of hemoglobin, their key nutrient. First, in the presented work, the protocol for refolding and activation of the zymogen of cathepsin D from I. ricinus (IrCatD) was developed and optimized. In acidic pH the propeptide of IrCatD zymogen was removed by an auto-activation mechanism. Further, a kinetic assay with fluorogenic substrates was employed to study functional properties of IrCatD including pH optimum, substrate and inhibition specificities. Second, two isoforms of cathepsin D from S. mansoni (SmCatD) were produced using recombinant expression in E. coli. These recombinant proteases were isolated from inclusion bodies using affinity chromatography under denaturating conditions, and protocol for their refolding was developed and optimized. The studied aspartic proteases are pharmacological targets: inhibitors of SmCatD represent potential chemotherapeutics for the treatment of schistosomiasis, and IrCatD is a candidate antigen for the development of novel anti-tick vaccines.
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Cloning, expression and biochemical characterisation of mouse glutamate carboxypeptidase II.
Knedlík, Tomáš ; Vaněk, Ondřej (referee) ; Konvalinka, Jan (advisor)
English Abstract Glutamate carboxypeptidase II (GCPII) is a membrane metallopeptidase expressed in many human tissues, predominantly in prostate, brain and small intestine. In brain it cleaves the most abundant peptide neurotransmitter N-acetyl-L-aspartyl-α-L-glutamate into N-acetyl-L-aspartate and free L-glutamate. Thus, GCPII participates in glutamate excitotoxicity through the release of free glutamate into the synaptic cleft. Inhibition of this activity has been shown to be neuroprotective in rats. In the human jejunal brush border, GCPII cleaves off terminal glutamate moieties from poly-γ-glutamylated folates, which can be then transported across the intestinal mucosa. The function of GCPII in human prostate is unknown but it is overexpressed in prostate cancer. Therefore, GCPII is an important marker of prostate cancer and its progression.Moreover, it could become a perspective target for treatment of prostate cancer as well as neuronal disorders associated with glutamate excitotoxicity. For the development and testing of novel drugs and therapeutics it is necessary to have an appropriate animal model. Mouse (Mus musculus) is such a model and it is widely used by many experimentators. However, no detailed comparison of mouse and human GCPII orthologs regarding their enzymatic activity, inhibition...
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Production of mouse recombinant prion protein and analysis of its properties
Krejčiříková, Anna ; Brynda, Jiří (referee) ; Konvalinka, Jan (advisor)
Title: Production of mouse recombinant prion protein and analysis of its properties Author: Anna Krejčiříková Department: Department of Biochemistry Supervisor: doc. RNDr. Jan Konvalinka, CSc., Supervisor's e-mail: konval@uochb.cas.cz Consultant: dr. Ing. Karel Holada, IIM 1stFM CU Abstract: Although prion diseases represent only a small fraction of all known neurodegenra- tive illnesses, they deserve our attention mainly due to the fact that they are lethal, incurable by today, and having a potential to cause a serious epidemic. Even though this topic has been studied for many years, there are still many unanswered questions concerning both the mechanism, and the spread of prion diseases. The most promising current theory is the protein-only hypothesis, which explains the fundamental of the ill- ness by the conversion of cellular prion protein (PrPC ) into pathological prion protein (PrPSc ). The important tool in proving this theory is also a recombinant prion pro- tein. The presented thesis summarizes existing successes in the proving of protein-only hypothesis. In the experimental part, we prepared the mouse recombinant prion pro- tein (mrPrP) in E. coli bacteria. The structure of the purified and renaturated protein was verified by CD spectroscopy. Next, we focus on the effects of the...
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Digestive aspartic protease of Colorado beetle
Srp, Jaroslav ; Jonáková, Věra (referee) ; Konvalinka, Jan (advisor)
Colorado potato beetle (Leptinotarsa decemlineata) is an economically important herbivorous pest. Cathepsin D-like aspartic peptidase (LdCD) plays an important role during protein degradation in the midgut of Colorado potato beetle. This work describes the preparation of two expression systems, namely in Escherichia coli and Pichia pastoris, for the production of recombinant LdCD. The protocol for refolding of denatured LdCD was designed and optimized. Activation of the inactive LdCD zymogen and cleavage of the propetide (activation peptide) were investigated. This process proceeds autocatalytically at acidic pH or with the assistance of the cysteine peptidase legumain. The proteolytic activity of LdCD was characterized using fluorogenic peptidic substrate and protein substrates, and kinetic parameters and pH optimum were determined. The inhibition specificity of LdCD was analyzed using a panel of peptidase inhibitors. LdCD was significantly inhibited by PDI (potato cathepsin D inhibitor), a protein inhibitor produced in potato leaves. This suggests that PDI is a natural defense protein, which is directed against LdCD in the midgut of Colorado potato beetle in order to block the digestion. The potential application of PDI in the construction of transgenic crops resistant against insects is discussed.
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Cathepsin L from the hard tick Ixodes ricinus
Talacko, Pavel ; Entlicher, Gustav (referee) ; Konvalinka, Jan (advisor)
Ticks are globally important parasites involved in transmission of a wide variety of infectious agents. The most common tick species found in Europe is the hard tick Ixodes ricinus, which transmits bacterium Borrelia burgdorferi (a causative agent of Lyme disease) or tick-borne encephalitis virus. Cathepsin proteases are important in the process of digestion of blood proteins in the tick gut. This work is focused on cathepsin L, an important digestive cysteine protease of ticks. Recombinant I. ricinus cathepsin L was expressed in Pichia pastoris and separated from the culture medium by chromatographic purification. N-terminal protein sequencing and labeling by activity-based probe Green-DCG-04 were used for characterization of purified cathepsin L. Substrate and inhibitor specificity were analyzed using peptide substrates and inhibitors. This analysis showed that Z-FR-AMC is a suitable substrate with pH optimum 3.5, and that Z-FF-DMK is an efficient inhibitor. It was demonstrated that cathepsin L cleaves protein substrates in strongly acidic environment (pH 3.5-4.5). Cathepsin L-like proteolytic activity was demonstrated in salivary gland extract and in saliva of the I. ricinus tick. The presence of a cathepsin protease in tick saliva is reported here for the first time. This finding suggests that...
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