amyloid-beta-peptides and acetylleucyl-leucyl-norleucinal

The detection of cell cycle proteins in Alzheimer's disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Abeta properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid beta-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Abeta40 and Abeta42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Abeta42 to Abeta40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Abeta. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a gamma-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668.

Topics: Acetylcysteine; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Benzazepines; Cell Line; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Cyclin D1; Fibroblasts; Glioma; Hexanones; Humans; Hydrocarbons, Chlorinated; Indoles; Leupeptins; Mice; Peptide Fragments; Proteasome Inhibitors; Purines; Recombinant Fusion Proteins; Roscovitine; Threonine

The calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) has been reported to have complex effects on the production of the beta-amyloid peptide (Abeta). In this study, the effects of ALLN on the processing of the amyloid precursor protein (APP) to Abeta were examined in 293 cells expressing APP or the C-terminal 100 amino acids of APP (C100). In cells expressing APP or low levels of C100, ALLN increased Abeta40 and Abeta42 secretion at low concentrations, decreased Abeta40 and Abeta42 secretion at high concentrations, and increased cellular levels of C100 in a concentration-dependent manner by inhibiting C100 degradation. Low concentrations of ALLN increased Abeta42 secretion more dramatically than Abeta40 secretion. ALLN treatment of cells expressing high levels of C100 did not alter cellular C100 levels and inhibited Abeta40 and Abeta42 secretion with similar IC50 values. These results suggest that C100 can be processed both by gamma-secretase and by a degradation pathway that is inhibited by low concentrations of ALLN. The data are consistent with inhibition of gamma-secretase by high concentrations of ALLN but do not support previous assertions that ALLN is a selective inhibitor of the gamma-secretase producing Abeta40. Rather, Abeta42 secretion may be more dependent on C100 substrate concentration than Abeta40 secretion.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Aspartic Acid Endopeptidases; Calpain; Cell Line; Endopeptidases; Gene Expression Regulation; Humans; Leupeptins; Membrane Proteins; Molecular Sequence Data; Mutation; Peptide Fragments; Presenilin-1; Protease Inhibitors; Transfection

The carboxy-terminal ends of the 40- and 42-amino acids amyloid beta-protein (Abeta) may be generated by the action of at least two different proteases termed gamma(40)- and gamma(42)-secretase, respectively. To examine the cleavage specificity of the two proteases, we treated amyloid precursor protein (APP)-transfected cell cultures with several dipeptidyl aldehydes including N-benzyloxycarbonyl-Leu-leucinal (Z-LL-CHO) and the newly synthesized N-benzyloxycarbonyl-Val-leucinal (Z-VL-CHO). All dipeptidyl aldehydes tested inhibited production of both Abeta1-40 and Abeta1-42. Changes in the P1 and P2 residues of these aldehydes, however, indicated that the amino acids occupying these positions are important for the efficient inhibition of gamma-secretases. Peptidyl aldehydes inhibit both cysteine and serine proteases, suggesting that the two gamma-secretases belong to one of these mechanistic classes. To differentiate between the two classes of proteases, we treated our cultures with the specific cysteine protease inhibitor E-64d. This agent inhibited production of secreted Abeta1-40, with a concomitant accumulation of its cellular precursor indicating that gamma(40)-secretase is a cysteine protease. In contrast, this treatment increased production of secreted Abeta1-42. No inhibition of Abeta production was observed with the potent calpain inhibitor I (acetyl-Leu-Leu-norleucinal), suggesting that calpain is not involved. Together, these results indicate that gamma(40)-secretase is a cysteine protease distinct from calpain, whereas gamma(42)-secretase may be a serine protease. In addition, the two secretases may compete for the same substrate. Dipeptidyl aldehyde treatment of cultures transfected with APP carrying the Swedish mutation resulted in the accumulation of the beta-secretase C-terminal APP fragment and a decrease of the alpha-secretase C-terminal APP fragment, indicating that this mutation shifts APP cleavage from the alpha-secretase site to the beta-secretase site.

Topics: Aldehydes; Alzheimer Disease; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Calpain; CHO Cells; Cricetinae; Cysteine Endopeptidases; Dipeptides; Endopeptidases; Enzyme Inhibitors; Leupeptins; Peptide Fragments; Serine Endopeptidases