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Zinc-Dependent Hydrolases: Structure-Function Study of Glutamate Carboxypeptidase II and Histone Deacetylase 6
Škultétyová, Ľubica
Zinc-binding proteins represent approximately one tenth of the proteome and a good portion of them are zinc-dependent hydrolases. This thesis focuses on biochemical and structural characterization of glutamate carboxypeptidase II (GCPII) and histone deacetylase 6 (HDAC6), two members of the zinc-dependent metallohydrolase superfamily. We describe here their interactions with natural substrates and inhibitors. GCPII is a homodimeric membrane protease catalyzing hydrolytic cleavage of glutamate from the neurotransmitter N-acetylaspartylglutamate (NAAG) and dietary folates in the central and peripheral nervous systems and small intestine, respectively. This enzyme is associated with several neurological disorders and also presents an ideal target for imaging and treatment of prostate cancer. GCPII inhibitors typically consist of a zinc-binding group (ZBG) linked to an S1' docking moiety (a glutamate moiety or its isostere). As such, these compounds are highly hydrophilic molecules therefore unable to cross the blood-brain barrier and this hampers targeting GCPII to the central nervous system. Different approaches are adopted to alter the S1' docking moiety of the existing inhibitors. As a part of this thesis, we present different strategies relying on replacement of the canonical P1' glutamate residue...
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Antibody derivatives for the detection of human glutamatecarboxypeptidase II
Bělousová, Nikola ; Bařinka, Cyril (advisor) ; Pavlíček, Jiří (referee)
Prostate cancer is one of the most common human malignancies and, consequently it is critical to develop appropriate diagnostic and therapeutic tools. Glutamate carboxypeptidase II (GCPII) is currently being considered one of the most important prostate cancer markers due to its tissue- specific expression. Whereas in healthy prostatic tissue the expression levels of GCPII are low, the transformation into the tumor is associated with the substantial increase of GCPII expression, with the highest levels observed in androgen-independent metastatic tumors. GCPII is thus considered a promising marker for early phase as well as advanced metastatic stages of prostate cancer. Current research is focused on the development of highly sensitive and specific reagents that allow detection of small amounts of GCPII, for example in early stages of cancer. Antibody derivatives are promising molecules for this purpose because they have high affinity and specificity and minimum negative side effects. Protein engineering is a prefered approach for preparation of various antibody molecules that differ in size, binding properties, stability, solubility, and production means. Different types of derivatives are being developed for medical needs such as in vitro diagnosis, therapy, and in vivo imagingSmall molecular...
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Zinc-Dependent Hydrolases: Structure-Function Study of Glutamate Carboxypeptidase II and Histone Deacetylase 6
Škultétyová, Ľubica ; Bařinka, Cyril (advisor) ; Obšil, Tomáš (referee) ; Novák, Petr (referee)
Zinc-binding proteins represent approximately one tenth of the proteome and a good portion of them are zinc-dependent hydrolases. This thesis focuses on biochemical and structural characterization of glutamate carboxypeptidase II (GCPII) and histone deacetylase 6 (HDAC6), two members of the zinc-dependent metallohydrolase superfamily. We describe here their interactions with natural substrates and inhibitors. GCPII is a homodimeric membrane protease catalyzing hydrolytic cleavage of glutamate from the neurotransmitter N-acetylaspartylglutamate (NAAG) and dietary folates in the central and peripheral nervous systems and small intestine, respectively. This enzyme is associated with several neurological disorders and also presents an ideal target for imaging and treatment of prostate cancer. GCPII inhibitors typically consist of a zinc-binding group (ZBG) linked to an S1' docking moiety (a glutamate moiety or its isostere). As such, these compounds are highly hydrophilic molecules therefore unable to cross the blood-brain barrier and this hampers targeting GCPII to the central nervous system. Different approaches are adopted to alter the S1' docking moiety of the existing inhibitors. As a part of this thesis, we present different strategies relying on replacement of the canonical P1' glutamate residue...
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Glutamate Carboxypeptidase II as a Drug Target and a Molecular Address for Cancer Treatment
Knedlík, Tomáš ; Konvalinka, Jan (advisor) ; Stiborová, Marie (referee) ; Souček, Pavel (referee)
Glutamate carboxypeptidase II (GCPII), also known as prostate-specific membrane antigen (PSMA), is a membrane metallopeptidase overexpressed on most prostate cancer cells. Additionally, GCPII also attracted neurologists' attention because it cleaves neurotransmitter N-acetyl-L-aspartyl-L-glutamate (NAAG). Since NAAG exhibits neuroprotective effects, GCPII may participate in a number of brain disorders, which were shown to be ameliorated by GCPII selective inhibitors. Therefore, GCPII has become a promising target for imaging and prostate cancer targeted therapy as well as therapy of neuronal disorders. Globally, prostate cancer represents the second most prevalent cancer in men. With the age, most men will develop prostate cancer. However, prostate tumors are life threatening only if they escape from the prostate itself and start to spread to other tissues. Therefore, considerable efforts have been made to discover tumors earlier at more curable stages as well as to target aggressive metastatic cancers that have already invaded other tissues and become resistant to the standard treatment. Since patients undergoing a conventional therapy (a combination of chemotherapy and surgery) suffer from severe side effects, more effective ways of treatment are being searched for. Novel approaches include selective...
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The effect of Ca2+ ion on the enzymatic activity of human glutamate carboxypeptidase II
Nedvědová, Jana ; Bařinka, Cyril (advisor) ; Hlouchová, Klára (referee)
Human glutamate carboxypeptidase II (GCPII, EC 3.4.17.24) is a homodimeric membrane glycoprotein. GCPII has been studied as a marker of prostate carcinoma and a therapeutic target of neurodegenerative disorders. The extracellular region of the protein is composed of three domains, apical, protease and C-terminal. There are two zinc ions in the active site that are essential for the enzymatic activity. A calcium ion is located between apical and protease domains near the dimeric interface approximately 20 Å away from the active site. Consequently, the Ca2+ ion in unlikely to participate in substrate hydrolysis. The aim of this thesis is to elucidate the function of Ca2+ in GCPII using a combination of molecular-biological, biochemical and biophysical approaches. To this end we prepared series of GCPII variants with mutations in calcium-coordinating amino acids. The mutant constructs were expressed in insect S2 cells and purified by combination of affinity and size exclusion chromatography. Enzymatic activity and thermostability of the mutants were decreased significantly. Furthermore, mutated proteins were aggregation prone and formed a monomeric GCPII species. Our results thus show that Ca2+ ion plays an essential role in proper GCPII folding as well as the formation of a homodimer molecule that is...
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Evaluation of the properties of polymer conjugates which specifically bind proteins and can be used in molecular biology
Parolek, Jan ; Konvalinka, Jan (advisor) ; Liberda, Jiří (referee)
During last three decades, a great effort was invested to the development of polymer conjugates of low molecular drugs with the aim to improve the specific targeting of drugs to diseased tissues, cells and organs. The main reason for this effort was the fact that high molecular weight copolymers have a favourite distribution profile in tissues and organisms. A linker between a polymer backbone and drug has very important role: it is possible to synthesize a biodegradable linker, which can be enzymatically hydrolyzed. Conversely, there is a possibility to synthesize an inert linker, resistant to the hydrolysis. Proper choice of the suitable precursor- polymer is also essential, hence it has to accomplish all of the stringent demands for biocompatibility. Macromolecular polymer-drug conjugates tend to accumulate in solid tumors because of the so called enhanced permeability and retention (EPR) effect. There is a whole range of possible applications of high molecular polymer-drug conjugates. In the introduction part of this thesis, I summarize potential use of drugs based on poly(N-(2-hydroxypropyl)methacrylamide) (HPMA) copolymers. Moreover, I introduce some therapeutically important proteins used in experimental drug discovery. In our laboratory, we have developed a concept of HPMA copolymers...
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Mass Spectrometry-Based Identification of a Potential Binding Partner of Glutamate Carboxypetidase II
Tužil, Jan ; Konvalinka, Jan (advisor) ; Novák, Petr (referee)
English Abstract The incoming paradigm of the network (or systems) biology calls for a new high throughput tool for a wide scale study of protein-protein interactions. Mass spectrometry-based proteomics have experienced a great progress in recent years and have become an indispensable technology of elementary as well as clinical research. Glutamate carboxypeptidase II (GCPII; EC 3.5.17.21) is a transmembrane protein with two known enzymatic activities. Its expression is highly upregulated in some solid tumors and also in tumor-associated neovasculature in general. Nevertheless, none of the two enzymatic activities were shown to be physiologically relevant to these cells. Some facts point at a possible receptor function of GCPII, however, no specific binding partner has been found yet. In the search for potential binding partners and/or ligands of GCPII, a series of methods have been employed, including pull-down experiment, immunoprecipitation and mass spectrometry. Sample preparation and mass spectrometry data processing methodology was specifically developed in order to identify potential binding partners. As one of the outcome of that methodology, the interaction of β-subunit of F1 ATP synthase was selected for further detailed analysis as a putative ligand of GCPII.
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Příprava a charakterisace rekombinantního dermcidinu jako potenciálního proteinového partnera glutamátkarboxypeptidasy II
Tužil, Jan ; Konvalinka, Jan (advisor) ; Pavlíček, Jiří (referee)
A process of forming new blood vessels is necessary for tumour viability and expansion. Without vasculature, tumour stops growing at a size of millimeters. Some tumours, however, undergo an angiogenic switch and start to build up their own vascular architecture. The rate of apoptosis then decreases and the tumour becomes invasive. There are many factors that control the process of physiological angiogenesis. These might or might not relate to tumour tissue as well. Glutamate carboxypeptidase II (GCPII; EC 3.4.17.21) is a type II transmembrane glycoprotein with two known enzymatic activities. GCPII expression is upregulated in prostate cancer and also highly expressed in tumour-associated neovasculature even though none of these enzymatic functions was observed on the endothelium. Although numerous researches suggested that GCPII might serve as a receptor, no natural ligand has been identified yet. Preliminary experiments performed in our laboratory indicated some proteins to be possible natural ligands of GCPII. Therefore, we chose one of them- dermcidin, cloned and expressed this protein in mammalian cells. We investigated its possible interaction with GCPII introducing new detection system utilizing FLAG-tag however, we were not able to approve neither disapprove its interaction in vitro.
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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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