A yeast genetic assay for caspase cleavage of the amyloid-β precursor protein

Author： Gunyuzlu Paul White W. Davis Gary Hollis Gregory Toyn Jeremy

ISSN： 1073-6085

Source： Molecular Biotechnology, Vol.15, Iss.1, 2000-05, pp. : 29-37

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Abstract

A functional assay for proteolytic processing of the amyloid precursor protein (APP) was set up in yeast. This consisted of a membrane-bound chimeric protein containing the β-secretase cleaved C-terminal fragment of APP fused to the Gal4 transcription factor. Using this chimera in a GAL-reporter yeast strain, an expression library of human cDNAs was screened for clones that could activate the GAL-reporter genes by proteolytic processing of the membrane-bound APP-Gal4. Two human proteases, caspase-3 and caspase-8, were identified and confirmed to act by a mechanism that involved proteolysis at the site in the APP-Gal4 chimera that corresponded to the natural caspase cleavage site in APP, thus linking a readily scorable phenotype to proteolytic processing of APP. The activation of caspase-3 involved a mechanism that was independent of aspartic acid residue 175 at the cleavage site normally required for processing of caspase-3.