TY - CHAP
T1 - Advanced Yeast Models of Familial Alzheimer Disease Expressing FAD-Linked Presenilin to Screen Mutations and γ-Secretase Modulators
AU - Futai, Eugene
N1 - Funding Information:
We thank Dr. Takeshi Iwatsubo and Dr. Taisuke Tomita (University of Tokyo) for PS1 antisera and Pen2 and Aph-1 clones, Dr. Raphael Kopan (Washington University) for the mNotch1 clone, and Dr. Philip James (University of Wisconsin) for the PJ-69-4A yeast strain. We thank Dr. Taisuke Tomita for helpful discussions and technical suggestions. We also thank the members of our laboratory for encouragement and critical comments.
Publisher Copyright:
© Springer Science+Business Media, LLC, part of Springer Nature 2019.
PY - 2019
Y1 - 2019
N2 - γ-Secretase is a multisubunit membrane protein complex containing catalytic presenilin (PS1 or PS2) and cofactors such as nicastrin, Aph-1, and Pen2. γ-Secretase hydrolyzes the transmembrane domains of type-I membrane proteins, which include the amyloid precursor protein (APP). APP is cleaved by γ-secretase to produce amyloid β peptide (Aβ), which is deposited in the brains of Alzheimer disease patients. However, the mechanism of this unusual proteolytic process within the lipid bilayer remains unknown. We have established a yeast transcriptional activator Gal4p system with artificial γ-secretase substrates containing APP or Notch fragments to examine the enzymatic properties of γ-secretase. The γ-secretase activities were evaluated by transcriptional activation of reporter genes upon Gal4 release from the membrane bound substrates as assessed by growth of yeast or β-galactosidase assay. We also established an in vitro yeast microsome assay system which identified different Aβ species produced by trimming. The yeast system allows for the screening of mutations and chemicals that inhibit or modulate γ-secretase activity. Herein we describe the genetic and biochemical methods used to analyze γ-secretase activity using the yeast reconstitution system. By studying the loss-of-function properties of PS1 mutants, it is possible to successfully screen FAD suppressor mutations and identify γ-secretase modulators (GSMs), which are promising Alzheimer disease therapeutic agents.
AB - γ-Secretase is a multisubunit membrane protein complex containing catalytic presenilin (PS1 or PS2) and cofactors such as nicastrin, Aph-1, and Pen2. γ-Secretase hydrolyzes the transmembrane domains of type-I membrane proteins, which include the amyloid precursor protein (APP). APP is cleaved by γ-secretase to produce amyloid β peptide (Aβ), which is deposited in the brains of Alzheimer disease patients. However, the mechanism of this unusual proteolytic process within the lipid bilayer remains unknown. We have established a yeast transcriptional activator Gal4p system with artificial γ-secretase substrates containing APP or Notch fragments to examine the enzymatic properties of γ-secretase. The γ-secretase activities were evaluated by transcriptional activation of reporter genes upon Gal4 release from the membrane bound substrates as assessed by growth of yeast or β-galactosidase assay. We also established an in vitro yeast microsome assay system which identified different Aβ species produced by trimming. The yeast system allows for the screening of mutations and chemicals that inhibit or modulate γ-secretase activity. Herein we describe the genetic and biochemical methods used to analyze γ-secretase activity using the yeast reconstitution system. By studying the loss-of-function properties of PS1 mutants, it is possible to successfully screen FAD suppressor mutations and identify γ-secretase modulators (GSMs), which are promising Alzheimer disease therapeutic agents.
KW - Alzheimer disease
KW - Amyloid β
KW - Budding yeast
KW - Intramembrane proteolysis
KW - Saccharomyces cerevisiae
KW - γ-Secretase
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U2 - 10.1007/978-1-4939-9736-7_23
DO - 10.1007/978-1-4939-9736-7_23
M3 - Chapter
C2 - 31602624
AN - SCOPUS:85073122260
T3 - Methods in Molecular Biology
SP - 403
EP - 417
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -