Expression of Mutant Epitope-Tagged TACE Constructs in TACE Knockout Fibroblasts and the Importance of TACE Cytoplasmic Domain in PKC-Mediated Phosphorylation

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Authors
Januszewski, Jacob
Issue Date
2005
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en_US
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Alzheimer's disease (AD) is its most common form of dementia affecting the elderly population. It is speculated that one of the essential upstream mediators of AD is the accumulation of amyloid beta (Aβ)-containing plaques in the brain. Ab is derived from the cleavage of the amyloid precursor protein (APP) by β-and γ-secretases. On the contrary, there exists a competing pathway to that of the β-and y-secretases, in which another enzyme called α-secretase cleaves APP within the Aβ region. The result of this cleavage precludes plaque formation in the brain, and produces a non-pathogenic sAPPα fragment. TACE (TNF-α Converting Enzyme) is a well known a-secretase that competes with the AD-causing β-and γ-secretase pathway. Thus, elucidating the mechanism of TACE regulation will potentially play an important role in understanding ways to reduce the amount of Ab produced in disease states. Recent in vitro studies have shown that protein kinase C (PKC) is directly involved in the phosphorylation of the TACE cytoplasmic domain. In order to determine if the cytoplasmic domain of TACE plays a significant role in in vivo PKC-mediated phosphorylation, several mutant versions of the TACE cytoplasmic domain were produced for transfection into TACE knockout fibroblasts. Since this lab has not yet shown if the TACE knockout fibroblasts are transfectable, this became an important factor in the experiment. Here we show that TACE knockout fibroblasts are transfectable, and that cellular detection of equivalent levels of epitope-tagged TACE constructs is possible. Experiments are ongoing to determine if our mutant TACE constructs will produce altered levels of sAPPα. Wild type or mutant TACE constructs will be expressed and then stimulated with PMA to determine if the mutant version of TACE reduces the amount of sAPPα produced.
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Kalamazoo, Mich. : Kalamazoo College.
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