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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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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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