C-terminal fragment of presenilin is the molecular target of a dipeptidic gamma-secretase-specific inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester).
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)-phenylglycine-4-(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.
Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Yuichi
Y
Morohashi
Toshiyuki
T
Kan
Yusuke
Y
Tominari
Haruhiko
H
Fuwa
Yumiko
Y
Okamura
Naoto
N
Watanabe
Chihiro
C
Sato
Hideaki
H
Natsugari
Tohru
T
Fukuyama
Takeshi
T
Iwatsubo
Taisuke
T
Tomita
D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; D001665:Binding Sites; D002219:Carbamates / Q000494:pharmacology; D002845:Chromatography; D004151:Dipeptides / Q000494:pharmacology; D010450:Endopeptidases / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D008565:Membrane Proteins / Q000737:chemistry*; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D053764:Presenilin-1; D017434:Protein Structure, Tertiary; D051880:Receptors, Notch / Q000378:metabolism; D014280:Triglycerides / Q000494:pharmacology*; D005680:gamma-Aminobutyric Acid / Q000031:analogs & derivatives* / Q000494:pharmacology
D002219:Carbamates; D004151:Dipeptides; C410009:L 685458; D008565:Membrane Proteins; C508853:PSEN1 protein, human; D010455:Peptides; D053764:Presenilin-1; D051880:Receptors, Notch; D014280:Triglycerides; D005680:gamma-Aminobutyric Acid; C057077:1,2-dilinolenoyl-3-(4-aminobutyryl)propane-1,2,3-triol; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human