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Splice variants of the gene coding for GCPII and their role in cancer development
Jindrová, Helena ; Konvalinka, Jan (advisor) ; Liberda, Jiří (referee)
Alternative splicing is a mechanism of generating distinct proteins that are encoded by the same gene. These proteins differ in amino acid sequence, overall structure and function. Splicing dysregulations have been shown to be implicated in several pathologic processes including cancer. For example, non-physiological splicing of osteopontin was proved to play a key role in cell progression of breast cancer. Glutamate carboxypeptidase II (also called prostate specific membrane antigen, PSMA) is present in both normal prostate and prostate cancer. Several splice variants of PSMA have been described and it has been suggested that the overexpression of some of them could be involved in the progression of prostate cancer. Nevertheless, more detailed investigation of each of the PSMA splice variant in terms of their occurrence in prostate cancer cells remains to be performed. This thesis focuses on the exploration of the expression of PSMA splice variants with deleted exons 6 and 18 in samples of a cell line derived from human prostate cancer, benign prostate hyperplasia and prostate cancer. For this purpose, RT-PCR was utilized to determine the ratio of deletions of exons 6 and 18 in cDNA of the prostate specific membrane antigen. Furthermore, the ratio of deletions of exon 6 and 18 was determined in...
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Physiological and pathophysiological role of GCPII in the body
Sedlák, František ; Konvalinka, Jan (advisor) ; Klener, Pavel (referee) ; Smetana, Karel (referee)
Glutamate carboxypeptidase II (GCPII) is a metalloprotease responsible for cleaving the neurotransmitter N-acetyl-aspartyl-glutamate in the central nervous system to N-acetyl aspartate and glutamate. At the same time, in the human small intestine, it facilitates folate absorption by cleaving γ-linked glutamate from folyl-poly-γ-glutamate. In humans, GCPII is also expressed in a number of other organs (e.g., kidney and prostate) and tumors, where its physiological function is unknown. In an attempt to characterize the physiological function of the enzyme, we first characterized the commercially available monoclonal antibodies against GCPII. Further, we developed a fully synthetic replacement based on a hydrophilic polymer with bound GCPII inhibitors. We evaluated the suitability of using a murine biomodel to study GCPII function in vivo. We found the difference in GCPII expression profile in mouse and human. We did not observe GCPII in either the mouse prostate or small intestine. To assess physiological and pathophysiological functions of the enzyme we analyzed a GCPII-deficient mouse model. Apart from the observation of enlarged seminal vesicles in older males, we did not detect any other obvious phenotype. Similarly, we confirmed that GCPII cannot cleave amyloid peptides (Aβ1-40 and Aβ1-42)....
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Protein engineering as a tool for the production of antibody derivatives
Šulc, Josef ; Bařinka, Cyril (advisor) ; Mikulecký, Pavel (referee)
This thesis deals with production and properties of disulfide-stabilized single-chain variable fragments of the 5D3 antibody (dsscFv), which specifically recognizes and binds to glutamate carboxypeptidase II (GCPII), an antigen closely related to the prostate carcinoma processes and other tumor diseases. Small antibody fragments are in current focus of development of diagnostic and therapeutic reagents. However, compromised stability of antibody derivatives often results in low production yield or loss of function. Introduction of structural changes by protein engineering is often used to solve the issue. The aim of the study was based on enhancement of protein stability by the introduction of interdomain disulfide bond into the structure of single-chain variable fragment. The effect of modification was evaluated by estimation of production yield and affinity of studied protein. The aforementioned antibody derivative was produced using an Escherichia coli expression system, using specific signal sequences leading the production to the bacterial periplasm. The attempted stabilization was carried out by introducing mutations at LV-G44 and HV-G100 positions, replacing glycines with cysteines. The binding affinity of the derivative for human GCPII was determined using ELISA. This thesis also shows a solved 3D...
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Generation and Characterization of Glutamate Carboxypeptidase II (GCPII)-Deficient Mice
Vorlová, Barbora ; Šácha, Pavel (advisor) ; Eckschlager, Tomáš (referee) ; Bařinka, Cyril (referee)
Glutamate carboxypeptidase II (GCPII) is a transmembrane glycoprotein, which consists of short intracellular and transmembrane domains, and a large extracellular domain possessing carboxypeptidase activity. In the human body, GCPII fulfils a neuromodulatory function in the brain and facilitates folate absorption in the small intestine. In addition to the brain and small intestine, high level of GCPII is also present in the prostate and kidney. However, GCPII function in these tissues has not been determined yet. To study the role of GCPII in detail, several research groups attempted to inactivate GCPII encoding gene Folh1 in mice. Surprisingly, the experiments led to rather conflicting results ranging from embryonic lethality to generation of viable GCPII-deficient mice without any obvious phenotype. This dissertation project aimed to dissect the discrepancy using alternative strategy for gene modification. For this purpose, we designed TALENs that specifically targeted exon 11 of Folh1 gene and manipulated mouse zygotes of C57BL/6NCrl genetic background. We analysed all genetically modified mice of F0 generation for presence of TALEN-mediated mutations and established 5 different GCPII-mutant mouse colonies from founder mice that altogether carried 2 frame-shift mutations and 3 small in-frame...
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Development of analytical tools for quantification and screening for inhibitors of glutamate carboxypeptidases II and III
Navrátil, Václav ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee) ; Šedo, Aleksi (referee)
Glutamate carboxypeptidase II (GCPII) usually called prostate specific membrane antigen (PSMA) is membrane bound metallopeptidase expressed mainly in prostate carcinoma (PCa). Agents targeting GCPII suitable for both imaging and treatment of PCa are in development and they show promising results in advanced clinical trials. Some studies showed that GCPII may serve also as PCa blood serum marker, but this has not been validated due to the lack of methods suitable for accurate detection of GCPII in human blood. Moreover, GCPII is also expressed in brain, where it cleaves inhibitory N-acetyl-α-L- aspartyl-L-glutamate (NAAG) to release excitatory L-glutamate and GCPII inhibition has been shown to be neuroprotective in animal models of several neuropathies. Tight binding inhibitors of GCPII have been identified by rational design, but all have poor bioavailability and thus cannot be used in clinics. Identifying new scaffolds by 'brute force' screening methods is thus essential; however, no such method for GCPII has been developed so far. Glutamate carboxypeptidase III (GCPIII) is also expressed in brain and cleaves NAAG. It is thus an important protein for understanding of GCPII function as well as GCPII targeting in medicine. Here, we focused on development of novel methods for quantification of both...
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Chemically modified Murine Polyomavirus-like particles and their interaction with Prostate-Specific Membrane Antigen (PSMA)
Blažková, Kristýna ; Konvalinka, Jan (advisor) ; Horníková, Lenka (referee)
Prostate cancer is one of the most abundant types of cancer among men and the demand for a specific treatment is very high. In this thesis, I have focused on using Glutamate Carboxypepti- dase II (GCPII), as a target for a proof-of-principle delivery system. GCPII is a transmembrane protein that internalizes after a binding of a ligand and is overexpressed in prostate cancer. Virus-like particles from Murine polyomavirus (VLPs) are a suitable nanocarrier for the delivery of imaging agents and drugs. Here I describe modifying these VLPs with inhibitors of GCPII and fluorescent dyes and characterize their binding to GCPII on surface plasmon resonance and to cells expressing GCPII on confocal microscopy. VLPs carrying a GCPII inhibitor show specific binding to GCPII on surface plasmon reso- nance, however they bind non-specifically to cells that don't express GCPII. Several approaches have been tried to avoid that. The substitution of BC loop on the exterior surface of VLPs that is partially responsible for the binding of sialic acid did not seem to affect specificity on cells. Another approach tested was coating of the wild-type VLPs with large polymer carrying a flu- orescent label and a GCPII inhibitor. After the conjugation of the polymer to the VLP, specific binding and internalization in GCPII-positive...
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Splice variants of the gene coding for GCPII and their role in cancer development
Jindrová, Helena ; Konvalinka, Jan (advisor) ; Liberda, Jiří (referee)
Alternative splicing is a mechanism of generating distinct proteins that are encoded by the same gene. These proteins differ in amino acid sequence, overall structure and function. Splicing dysregulations have been shown to be implicated in several pathologic processes including cancer. For example, non-physiological splicing of osteopontin was proved to play a key role in cell progression of breast cancer. Glutamate carboxypeptidase II (also called prostate specific membrane antigen, PSMA) is present in both normal prostate and prostate cancer. Several splice variants of PSMA have been described and it has been suggested that the overexpression of some of them could be involved in the progression of prostate cancer. Nevertheless, more detailed investigation of each of the PSMA splice variant in terms of their occurrence in prostate cancer cells remains to be performed. This thesis focuses on the exploration of the expression of PSMA splice variants with deleted exons 6 and 18 in samples of a cell line derived from human prostate cancer, benign prostate hyperplasia and prostate cancer. For this purpose, RT-PCR was utilized to determine the ratio of deletions of exons 6 and 18 in cDNA of the prostate specific membrane antigen. Furthermore, the ratio of deletions of exon 6 and 18 was determined in...
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Targetting prostate tumor cells by polyomavirus virus-like particles
Suchanová, Jiřina ; Španielová, Hana (advisor) ; Němečková, Šárka (referee)
The aim of this thesis is to investigate the targeting potential of mouse polyomavirus (MPyV) based virus-like particles (VLPs) as vectors for directed cell delivery of therapeutic or diagnostic compounds. Major capsid protein VP1 of MPyV is able to selfassemble into the noninfectious VLPs. Our main goal is to retarget these VLPs from its native receptor to the prostatic cancer cells by changing the receptor binding site in the surface-exposed loop of VP1. We introduced a peptide ligand CTITSKRTC, which binds prostate-specific membrane antigen (PSMA), by insertion or substitution into BC loop of VP1. These modifications did not change the stability of the particles and genetic substitution prevented the native receptor binding. PSMA-specific binding of modified VLPs was tested by pull-down assay and surface plasmon resonance. In order to further utilize these VLPs, we tested several approaches for preparation of VLPs as vehicles for compounds delivery into eukaryotic cells. Although the method for encapsidation of the DNA into the VLPs in cellular nuclear extracts, which mimic the in vivo conditions, did not enabled us to produce pseudocapsids, we successfully optimized procedure for dissassembly and reassembly of purified particles. This method will be use for encapsidation of molecules into the...
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