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Title: Functional analysis of ICAD-S protein and the development of a novel technology for site-specific protein cleavage in vertebrate cells
Author: Ageichik, Alexander Valery
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2007
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CAD (Caspase Activated DNase) exists in a complex with its inhibitor ICAD in intact cells. To study the particular role of ICAD splice forms ICAD-S and ICAD-L in vivo I constructed DT40 cell lines in which the ICAD or ICAD plus CAD ORFs had been deleted. ICAD and ICAD/CAD double knockouts were characterized by the absence of DNA fragmentation and apoptotic bodies after the induction of apoptosis. To characterise the functions of ICAD-S, I constructed a model system consisting from human (h) hICAD-S, hICAD-L and hCAD components. hICAD-S was not able to work as a chaperone for hCAD in vivo. However, a modified version of hICAD-S hICAD-S2TEV was able to inhibit hCAD activation upon induction of apoptosis in vivo.  Moreover, such inhibition correlated with level of hICAD-S2TEV expression. The inhibitor function of hICAD-S2TEV was confirmed in vitro, where hCAD was activated after hICAD-S2TEV cleavage with TEV protease. The use of CAD as a drug target could be particularly advantageous since CAD is situated at the end of apoptotic pathway. Thus, once activated CAD may avoid upstream regulatory mechanisms that protect cells against certain apoptotic stimuli. I constructed a model system based on ICAD/CAD double knockout to study the possibility that CAD can cause the cell death. This system included hICAD-L2TEV:hCAD and TEV protease under the control of inducible promoter. However, it was not possible to cleave hICAD-L2TEV protein in vivo with TEV protease. An alternative strategy was based on the use of PreScission protease. The active part of PreScission protease is 3C protease from human rhinovirus origin. A cleaved fragment of hICAD-L2PRE protein was detected in DT40 cells expressing PreScission protease. Preliminary data suggest that CAD contributes to cell death in cells expressing hICAD-L2PRE:hCAD and transfected with PreScission protease.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available