A new functional screening system for identification of regulators for the generation of amyloid beta-protein.
Presenilin (PS) is essential for gamma-cleavage, which is required for the generation of amyloid beta-protein (Abeta) from the beta-amyloid precursor protein. However, it remains to be clarified how gamma-cleavage is regulated. To elucidate the regulation of PS-mediated gamma-cleavage, we developed a new functional screening method for identifying cDNA that enhances gamma-cleavage. This screening system utilizes our own developed cell line, where the expression of cDNA that enhances gamma-cleavage confers puromycin resistance. The cDNA library is retrovirally delivered to the above-mentioned cell line, allowing the identification of our target cDNAs by a combination of puromycin resistance selection and Abeta assay screening. With this screening method, we isolated several cDNAs enhancing gamma-cleavage, including the previously reported Herp. Here we also demonstrate that Rab1A, identified with this screening, can be a regulator of Abeta generation. Thus, our established screening method is a powerful tool for identifying multiple regulators involved in gamma-cleavage in the Abeta generation pathway, including modulators of gamma-secretase activity or the intracellular trafficking of factors necessary for gamma-cleavage.
Komano
Hiroto
H
Department of Dementia Research, National Institute for Longevity Sciences, 36-3 Gengo, Morioka, Obu, Aichi 474-8522, Japan. hkomano@nils.go.jp
Shiraishi
Hirohisa
H
Kawamura
Yuuki
Y
Sai
Xiaorei
X
Suzuki
Ryo
R
Serneels
Lutgarde
L
Kawaichi
Masashi
M
Kitamura
Toshio
T
Yanagisawa
Katsuhiko
K
D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D051792:Basic Helix-Loop-Helix Proteins; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D010450:Endopeptidases / Q000378:metabolism; D015723:Gene Library; D005821:Genetic Techniques; D006624:Hippocampus / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D018345:Mice, Knockout; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D012190:Retroviridae / Q000235:genetics; D000072056:Transcription Factor HES-1; D020761:rab1 GTP-Binding Proteins / Q000378:metabolism
D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides; D016282:Aspartic Acid Endopeptidases; D051792:Basic Helix-Loop-Helix Proteins; D018076:DNA, Complementary; D010450:Endopeptidases; D018398:Homeodomain Proteins; D011500:Protein Synthesis Inhibitors; D011691:Puromycin; D000072056:Transcription Factor HES-1; D020761:rab1 GTP-Binding Proteins; C489734:Hes1 protein, mouse; C078493:HES1 protein, human; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse