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Vloga membransko vezanih gvanilatnih kinaz v apoptozi : magistrsko delo
Gregorc, Uroš
A group of proteolytic enzymes, caspases, is activated during apoptosis. They in turn cleave different cellular proteins. Activation of caspases and cleavages of their substrates lead to biochemical ...and morphological changes inapoptotic cells. Membrane associated guanylate kinases (MAGUK), proteins involved in cell-cell connections, are also cleaved by caspases. These cleavages allegedly induce disruption of cell-cell interaction complexes, isolation of apoptotic cells and their death without any consequence for surrounding tissue. For our studies we have choosen four members of MAGUK - two memebers of DLG family (DLG1 and DLG4/PSD-95) and MAGI family (MAGI-2 and -3). Initial studies were performed on in vitro translated, 35S-Met radiolabelled, proteins. We have inserted cDNA for selected MAGUK in mammalianexpression vector pcDNA3 (Invitrogen) and translated them with TNTr T7 Coupled Reticulocyte Lysate System (Promega). We have prepared recombinant caspases-3, -6, -7 and -8, and performed cleavage experiments on selected MAGUK. They are all cleaved by caspase-3. While DLG1, MAGI-2 and -3 are cleaved at physiologically significant concentration of caspase-3, DLG4/PSD-95is cleaved only at high caspase-3 concentrations. No other caspase cleaved any of the investigated MAGUK. We have selected a line of spontaneously transformed human keratinocytes (HaCaT) for in vivo studies. These are epithelial cells and when grown in culture they form monolayers. UV irradiation and staurosporine was used for induction of apoptosis. Progressionof apoptosis was monitored by measuring caspase activity in cellular lysates and by PARP (poly (ADP-ribose) polymerase) cleavage. DLG1 cleavage was assayed using Western blots and detection with specific monoclonal antobodies. Caspase activity increased with time as well as amount of cleaved PARP and DLG1. (Abstract truncated at 2000 characters).
Type of material - master's thesis
Publication and manufacture - Ljubljana : [U.Gregorc], 2002
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