Aspartyl Protease Inhibitor Pepstatin Binds to the Presenilins of Alzheimer's Disease

Abstract

Mutations in the presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease by altering gamma-secretase cleavage of the amyloid precursor protein, the last step in the generation of Abeta peptide. Ablation of presenilin (PS) genes, or mutation of two critical aspartates, abolishes gamma-secretase cleavage, suggesting that PS may be the gamma-secretases. Independently, inhibition experiments indicate that gamma-secretase is an aspartyl protease. To characterize the putative gamma-secretase activity associated with presenilins, lysates from human neuroblastoma SH-SY5Y and human brain homogenates were incubated with biotin derivatives of pepstatin, followed by immunoprecipitation of PS and associated proteins, and biotin detection by Western blotting. Precipitation with PS1 antibodies, directed to either N-terminal or loop regions, yielded the same 43 kDa band, of apparent molecular mass consistent with that of full-length PS1, although it may represent an aspartyl protease complexed with PS1. Incubation of cell lysates with pepstatin-biotin, followed by streptavidin precipitation and PS1 Western blotting, revealed PS1 fragments and full-length protein, indicating that pepstatin-biotin bound to both cleaved and uncleaved PS1. Binding could be competed by gamma-secretase inhibitor L-685,458 and could not be achieved with a PS1 mutant lacking the two transmembrane aspartates. Pepstatin-biotin was also shown to bind to PS2. PS1 was specifically absorbed to pepstatin-agarose, with an optimal pH of 6. Binding of pepstatin-biotin to PS1 from lymphocytes of a heterozygous carrier of pathologic exon 9 deletion was markedly decreased as compared to control lymphocytes, suggesting that this PS1 mutation altered the pepstatin binding site.

Evidence That Nonsteroidal Anti-inflammatory Drugs Decrease Amyloid β42 Production by Direct Modulation of γ-Secretase Activity

“…The interpretation of these findings is that the mutations have induced subtle conformational changes in the PS-containing ␥-secretase complex or its substrate, APP, which alter the inhibitor interaction with the enzymatic machinery or its substrate (50). And third, several transition state and nontransition state inhibitors have been demonstrated by affinity labeling to directly bind to PS (25,36,51,52).…”

Linear Non-competitive Inhibition of Solubilized Human γ-Secretase by Pepstatin A Methylester, L685458, Sulfonamides, and Benzodiazepines

“…Given this structural complexity, it would not be surprising if an inhibitor binds to a non-catalytic site to interrupt subunit interactions, thereby displaying non-competitive inhibition kinetics. Yet to be proven, evidence exists to suggest strongly that ␥-secretase is an aspartyl protease (12,(17)(18)(19)(20)(21)(22)(23)(24). Therefore, the issue with this model of PME or L685458 binding to an allosteric site is the issue of a transition state analog binding exclusively at a non-catalytic site.…”

“…Given these results, it has been hypothesized that, like ␤-secretase, ␥-secretase is an aspartyl protease (17)(18)(19)(20)(21)(22). Recent pepstatin-derived affinity chromatography of PS1 (23), photo affinity labeling of PS1 with transition state analog L685458 (12), and chemical affinity labeling of PS1 with difluoro peptidomimetics (24) further support ␥-secretase as an aspartyl protease and have led to the tentative identification of presenilins as the catalytic components of ␥-secretase.…”

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

“…Synthetic peptides derived from human sequences were conjugated to diphtheria toxoid and rabbit antibodies generated as follows: Ab 98/1, PS1 NT (1-20) [20]; Ab 00/ 1 and Ab 00/2 PS1 loop (301-317) [21]; Ab 00/12, PS2 loop (307-336); Ab 00/19 NCT CT (691-709]) [22]; Ab 00/22 NCT ectodomain (331-346); Ab 02/45 PEN-2 NT (1-15) and Ab 02/41 APH-1b CT (244-257). Ab 00/6 was raised to human BACE CT (485-501) [23].…”