Abstract
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.
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References
De Strooper B, Iwatsubo T, Wolfe MS (2012) Presenilins and γ-secretase: structure, function, and role in Alzheimer disease. Cold Spring Harb Perspect Med 2:a006304
Selkoe DJ (2011) Alzheimer’s disease. Cold Spring Harb Perspect Biol 3:a004457
Takasugi N, Tomita T, Hayashi I, Tsuruoka M, Niimura M, Takahashi Y, Thinakaran G, Iwatsubo T (2003) The role of presenilin cofactors in the γ-secretase complex. Nature 422:438–441
Edbauer D, Winkler E, Regula JT, Pesold B, Steiner H, Haass C (2003) Reconstitution of γ-secretase activity. Nat Cell Biol 5:486–488
Bai X, Yan C, Yang G, Lu P, Ma D, Sun L, Zhou R, Scheres SHW, Shi Y (2015) An atomic structure of human γ-secretase. Nature 525:212–217
Wolfe MS, Xia W, Ostaszewski BL, Diehl TS, Kimberly WT, Selkoe DJ (1999) Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. Nature 398:513–517
Shen J, Kelleher RJ 3rd. (2007) The presenilin hypothesis of Alzheimer’s disease: evidence for a loss-of-function pathogenic mechanism. Proc Natl Acad Sci U S A 104:403–409
Wolfe MS (2007) When loss is gain: reduced presenilin proteolytic function leads to increased Aβ42/Aβ40. EMBO Rep 8:141–146
Tomita T (2014) Molecular mechanism of intramembrane proteolysis by γ-secretase. J Biochem 156:195–201
Sun L, Li X, Shi Y (2016) Structural biology of intramembrane proteases: mechanistic insights from rhomboid and S2P to γ-secretase. Curr Opin Struct Biol 37:97–107
Bruckner A, Polge C, Lentze N, Auerbach D, Schlattner U (2009) Yeast two-hybrid, a powerful tool for systems biology. Int J Mol Sci 10:2763–2788
Futai E, Yagishita S, Ishiura S (2009) Nicastrin is dispensable for gamma-secretase protease activity in the presence of specific mutations. J Biol Chem 19:13013–13022
Yagishita S, Futai E, Ishiura S (2008) In vitro reconstitution of gamma-secretase activity using yeast microsomes. Biochem Biophys Res Commun 377:141–145
Yonemura Y, Futai E, Yagishita S, Suo S, Tomita T, Iwatsubo T, Ishiura S (2011) Comparison of presenilin 1 and presenilin 2 γ-secretase activities using a yeast reconstitution system. J Biol Chem 286:44569–44575
Futai E, Osawa S, Cai T, Fujisawa T, Ishiura S, Tomita T (2016) Suppressor mutations for presenilin 1 familial Alzheimer disease mutants modulate γ-secretase activities. J Biol Chem 291:435–446
James P, Halladay J, Craig EA (1996) Genomic libraries and a host strain designed for high efficient two-hybrid selection in yeast. Genetics 144:1425–1436
Miller CA 3rd, Martinat MA, Hyman LE (1998) Assessment of aryl hydrocarbon receptor complex interactions using pBEVY plasmids: expression vectors with bi-directional promoters for use in Saccharomyces cerevisiae. Nucleic Acids Res 26:3577–3583
Mumberg D, Müller R, Funk M (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156:119–122
Gietz RD, Woods RA (2002) Transformation of yeast by the Liac/SS carrier DNA/PEG method. Mol Enzymol 350:87–96
Tomita T, Takikawa R, Koyama A, Morohashi Y, Takasugi N, Saido TC, Maruyama K, Iwatsubo T (1999) C terminus presenilin is required for overproduction of amyloidgenic Aβ42 through stabilization and endoproteolysis of presenilin. J Neurosci 19:10627–10634
Clontech (2001) Yeast protocols handbook, Publication PT3024–1. Clontech, Mountain View, CA
Wuestehube LJ, Schekman RW (1992) Reconstitution of transport from endoplasmic reticulum to Golgi complex using endoplasmic reticulum-enriched membrane fraction from yeast. Methods Enzymol 219:124–136
Yagishita S, Morishima-Kawashima M, Ishiura S, Ihara Y (2008) Aβ46 is processed to Aβ40 and Aβ43, but not to Aβ42, in the low density membrane domains. J Biol Chem 283:733–738
Qi-Takahara Y, Morishima-Kawashima M, Tanimura Y, Dolios G, Hirotani N, Horikoshi Y, Kametani F, Maeda M, Saido TC, Wang R, Ihara Y (2005) Longer forms of amyloid beta protein: implications for the mechanism of intramembrane cleavage by gamma-secretase. J Neurosci 25:436–445
Ono Y, Torii F, Ojima K, Doi N, Yoshioka K, Kawabata Y, Labeit D, Labeit S, Suzuki K, Abe K, Maeda T, Sorimachi H (2006) Suppressed disassembly of autolyzing p94/CAPN3 by N2A connectin/titin in a genetic reporter system. J Biol Chem 281:18519–18531
Acknowledgments
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.
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Futai, E. (2019). Advanced Yeast Models of Familial Alzheimer Disease Expressing FAD-Linked Presenilin to Screen Mutations and γ-Secretase Modulators. In: Oliver, S.G., Castrillo, J.I. (eds) Yeast Systems Biology. Methods in Molecular Biology, vol 2049. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9736-7_23
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DOI: https://doi.org/10.1007/978-1-4939-9736-7_23
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