Human beta-secretase activity in yeast detected by a novel cellular growth selection system.
Sequential processing of the transmembrane amyloid precursor protein (APP) by the beta-secretase BACE and by the gamma-secretase causes secretion of Abeta peptides. Extracellular aggregation of these peptides in the brain is a major hallmark of Alzheimer's disease. For therapeutic purposes and the development of specific inhibitors, it is important to characterize these secretases. We have established a cellular growth selection system for functional expression of human BACE in the yeast Saccharomyces cerevisiae. A fragment of APP bearing the beta-site, the transmembrane domain and the cytosolic tail was fused to the C-terminus of the yeast enzyme invertase, which is normally secreted to allow cell growth in the presence of sucrose as the sole carbon source. The resulting invertase-APP fusion protein was expressed as a type-I transmembrane protein in intracellular compartments of yeast cells lacking endogenous invertase. In these cells, co-expression of human BACE restored cell growth on selective plates upon cleavage of the invertase-APP fusion protein. The cellular growth selection system presented here can be generally applied to screen for secretases that specifically cleave membrane-bound substrates. Furthermore, this system provides the basis for a high-throughput screen for identifying secretase inhibitors that are active in eukaryotic cells.
Lüthi
Urs
U
ESBATech AG, Wagistrasse 21, CH-8952 Zurich-Schlieren, Switzerland.
Schaerer-Brodbeck
Claudia
C
Tanner
Stefan
S
Middendorp
Oliver
O
Edler
Karin
K
Barberis
Alcide
A
D053829:Amyloid Precursor Protein Secretases; D001053:Apolipoproteins / Q000737:chemistry; D016282:Aspartic Acid Endopeptidases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000737:chemistry; D002462:Cell Membrane / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D010450:Endopeptidases; D004789:Enzyme Activation; D006026:Glycoside Hydrolases / Q000737:chemistry; D006801:Humans; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development*; D000685:Serum Amyloid A Protein / Q000737:chemistry; D013379:Substrate Specificity; D043324:beta-Fructofuranosidase
D001053:Apolipoproteins; D011993:Recombinant Fusion Proteins; D000685:Serum Amyloid A Protein; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human