Presenilin 1 is linked with γ-secretase activity in the detergent solubilized state

Abstract

␥-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two A␤ peptide isoforms, A␤40 and A␤42. Here we report the detergent solubilization and partial characterization of ␥-secretase. The activity of solubilized ␥-secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the ␤-secretase cleavage site. Cleavage of C100Flag by ␥-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the A␤40 or A␤42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the A␤40 and A␤42 termini. Recovery of catalytically competent, soluble ␥-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable. Solubilized ␥-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of A␤40 and A␤42 in mammalian cells. Upon gel exclusion chromatography, solubilized ␥-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 ؋ 10 6 . Anti-PS1 antibody immunoprecipitates ␥-secretase activity from the solubilized ␥-secretase preparation. These data suggest that ␥-secretase activity is catalyzed by a PS1-containing macromolecular complex.

Baculoviruses expressing the human familial Alzheimer's disease presenilin 1 mutation lacking exon 9 increase levels of an amyloid beta-like protein in Sf9 cells

“…Evidence to date has established that PS1 facilitates g-secretase activity. 9,11,[31][32][33] However, it remains to be elucidated whether PS1 is a protease or is instead a cofactor controlling substrate trafficking and/or presentation. To address this question, PS1 and its recently identified partner, nicastrin, were expressed in a baculovirus-insect cell expression system, along with the g-secretase substrate APP-C99.…”

Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations

“…It is ideal for analysis and partial isolation of membrane Carbonate washed SY5Y membranes (500 lg) were run on BN/PAGE, proteins electroeluted from the PS complex region, treated with reducing sample buffer and analysed on 10% Tris/tricine gels using mini Biorad apparatus. PS1 NTF and full-length PS1 were detected with PS1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] antibody, PS1 loop antibody detected PS1 CTF, NCT was detected at 140 kDa, PEN-2 at 10 kDa and PS2 CTF detected with PS2 CTF antibody. Parallel poly(vinylidene difluoride) strips were also probed with APH 1a and 1b antibodies but APH-1 was not detected.…”

“…The variety of identified substrates indicates a critical role for PS in cell metabolism involving controlled cleavage of protein transmembrane domains and signal transduction. The role of PS in c-secretase activity is compelling and includes lack of Ab amyloid peptide generation and Notch signalling in PS double knockout cells, cofractionation of activity with PS, abolition of activity with mutation of conserved aspartates in transmembrane domains six and seven, binding of c-secretase aspartyl protease inhibitors to PS, as well as identification of homologues with protease-associated domains [2][3][4][5][6]. Following a primary cleavage event that causes ectodomain shedding, PS-dependent Ôc-secretaseÕ proteolysis results in generation of a membrane spanning stub (such as Ab peptide) and a cytoplasmic fragment such as the APP intracellular domain or the Notch intracellular domain which translocates to the nucleus for regulation of gene expression [1].…”

Association of γ-Secretase with Lipid Rafts in Post-Golgi and Endosome Membranes

“…Hence, we first evaluated partitioning of PS1 and nicastrin into DIMs in detergents that are routinely used to isolate lipid rafts from cultured cells: non-ionic detergents Brij-96 (0.5%), Triton X-100 (1%), and Lubrol WX (0.5%). We also used CHAPSO (1%), a zwitterionic detergent commonly used to examine association between ␥-secretase components (55,56), and DIM association of PS1 (38).…”