C-terminal fragment of presenilin is the molecular target of a dipeptidic γ-secretase-specific inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester)

被引:168
作者
Morohashi, Yuichi
Kan, Toshiyuki
Tominari, Yusuke
Fuwa, Haruhiko
Okamura, Yumiko
Watanabe, Naoto
Sato, Chihiro
Natsugari, Hideaki
Fukuyama, Tohru
Iwatsubo, Takeshi
Tomita, Taisuke
机构
[1] Univ Tokyo, Grad Sch Pharmaceut Sci, Dept Neuropathol & Neurosci, Bunkyo Ku, Tokyo 1130033, Japan
[2] Univ Tokyo, Grad Sch Pharmaceut Sci, Dept Synth Nat Prod Chem, Bunkyo Ku, Tokyo 1130033, Japan
[3] Univ Tokyo, Grad Sch Pharmaceut Sci, Dept Rat Med Sci, Bunkyo Ku, Tokyo 1130033, Japan
关键词
D O I
10.1074/jbc.M513012200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
gamma-Secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1 and, Pen-2 that is responsible for the intramembrane proteolysis of various type I transmembrane proteins, including amyloid beta-precursor protein and Notch. The direct labeling of PS polypeptides by transition-state analogue gamma-secretase inhibitors suggested that PS represents the catalytic center of gamma-secretase. Here we show that one of the major gamma-secretase inhibitors of dipeptidic type, N-[N-( 3,5- difluorophenacetyl)-L-alanyl]-S- phenylglycine t-butyl ester (DAPT), targets the C-terminal fragment of PS, especially the transmembrane domain 7 or more C-terminal region, by designing and synthesizing DAP-BpB (N-[N-(3,5- difluorophenacetyl)-L-alanyl]-(S)-phenylglycine4-(4-(8-biotinamido) octylamino)benzoyl)benzyl) methylamide), a photoactivable DAPT derivative. We also found that DAP-BpB selectively binds to the high molecular weight gamma-secretase complex in an activity-dependent manner. Photolabeling of PS by DAP-BpB is completely blocked by DAPT or its structural relatives (e.g. Compound E) as well as by arylsulfonamides. In contrast, transition-state analogue inhibitor L-685,458 or alpha-helical peptidic inhibitor attenuated the photolabeling of PS1 only at higher concentrations. These data illustrate the DAPT binding site as a novel functional domain within the PS C-terminal fragment that is distinct from the catalytic site or the substrate binding site.
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页码:14670 / 14676
页数:7
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