A yeast genetic assay for caspase cleavage of the amyloid-beta precursor protein.
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 beta-secretase cleaved C-terminal fragment of APP fused to the Ga14 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.
Gunyuzlu
P L
PL
DuPont Pharmaceuticals, Experimental Station, Wilmington, Delaware 19880-0336, USA.
White
W H
WH
Davis
G L
GL
Hollis
G F
GF
Toyn
J H
JH
D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D004789:Enzyme Activation / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D017930:Genes, Reporter; D005821:Genetic Techniques; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015003:Yeasts / Q000235:genetics*
D016564:Amyloid beta-Protein Precursor; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases