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P4‐207: Chemical biological analysis of gamma‐secretase using arylsulfonamide‐type inhibitors
Author(s) -
Sugimoto Yasuaki,
Fuwa Haruhiko,
Yokoshima Satoshi,
Fukuyama Tohru,
Sasaki Makoto,
Tomita Taisuke,
Iwatsubo Takeshi
Publication year - 2008
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2008.05.2275
Subject(s) - chemistry , moiety , biotinylation , in vitro , photoaffinity labeling , biochemistry , peptide , benzophenone , biological activity , stereochemistry , receptor , photochemistry
mer’s disease A amyloid peptides and is therefore a therapeutic target. Its proteolytic activity is contained within high molecular complexes comprised of presenilin (PS), which represents the catalytic subunit, nicastrin, Aph1 and Pen-2. There exist two presenilin gene homologues, encoding PS1 and PS2, and two aph1 homologues in human, that encode Aph1a and Aph1b. A third aph1c gene is also expressed in the mouse. In addition, alternative splicing of aph1a gene can produce two different transcripts with a short (Aph1aS) or a long (Aph1aL) C-terminus. Combination of NCT and Pen-2 with either one PS homologue and with either one Aph1 isoform, would produce six alternative complexes in human. Posttranslational modifications of NCT and PS may result in even a larger number of different -secretase complexes. Methods: This study investigated and compared endogenous -secretase activities in brain and peripheral tissues with the aim to identify distinct complexes with different substrate specificity. Membranes were prepared from brain, liver, kidney, lung, spleen, muscle and thymus of adult normal mice and solubilized with 1% CHAPSO and analysed by western blot for PS and NCT expression, and for -secretase activity in vitro using a recombinant APP C100 substrate. Results: The highest expression of PS1 was observed in the brain, lung and thymus whereas PS2 expression was highest in the liver, lung, thymus, and kidney. Enzymatic activity correlated with PS expression and was greatest in the brain, lung, and liver. Lung and liver activities were inhibited by pepstatin and MG132, but resistant to inhibitors of brain -secretase such as DAPT and L-685,458. Lung -secretase activity was immunoprecipitated with a PS1 NT antibody. It produced a high ratio of A 42/A 40, compared to brain -secretase, and was inhibited by pepstatin, DFK-167, MG132, and NSAID but not by dibenzazepine. Purification of the lung and liver -secretase activities is in progress. Conclusions: Our results demonstrate that there exist pharmacologically distinct -secretase activities, which produce different ratios of A 42/A 40 and can be targeted selectively with alternative inhibitors. (Supported by the NHMRC).