Characterization of endogenous APP processing in a cell-free system.

We have developed a simple in vitro assay using tissue homogenates that allows detection and characterization of several endogenous proteolytic activities which convert Alzheimer's amyloid precursor protein (APP) to the smaller, carboxy-terminal fragments, postulated to be intermediates in the formation of ß-amyloid peptide (Aß). Incubation at 37°C results in the degradation of transmembrane APP and formation of a mixture of carboxy-terminal containing peptides with mass values of 9-12 kDa. Epitope mapping and electrophoretic comparison with a truncated APP standard showed one of these peptides to contain the entire Aß sequence. Analysis of pH dependence shows that formation of this carboxy-terminal product as well as another fragment, that is the likely product of 'secretase' activity, requires acidic pH. This suggests that cleavage of full-length APP to secreted forms may take place in an acidic intracellular compartment.

Evaluation of cathepsins D and G and EC 3.4.24.15 as candidate beta-secretase proteases using peptide and amyloid precursor protein substrates.

No single protease has emerged that possesses all the expected properties for beta-secretase, including brain localization, appropriate peptide cleavage specificity, and the ability to cleave amyloid precursor protein exactly at the amino-terminus of beta-amyloid peptide. We have isolated and purified a brain-derived activity that cleaves the synthetic peptide substrate SEVKMDAEF between methionine and aspartate residues, as required to generate the amino-terminus of beta-amyloid peptide. Its molecular size of 55-60 kDa and inhibitory profile indicate that we have purified the metalloprotease EC 3.4.24.15. We have compared the sequence specificity of EC 3.4.24.15, cathepsin D, and cathepsin G for their ability to cleave the model peptide SEVKMDAEF or related peptides that contain substitutions reported to modulate beta-amyloid peptide production. We have also tested the ability of these enzymes to form carboxyl-terminal fragments from full-length, membrane-embedded amyloid precursor protein substrate or amyloid precursor protein that contains the Swedish KM --> NL mutation. The correct cleavage was tested with an antibody specific for the free amino-terminus of beta-amyloid peptide. Our results exclude EC 3.4.24.15 as a candidate beta-secretase. Although cathepsin G cleaves the model peptide correctly, it displays poor ability to cleave the Swedish KM --> NL peptide and does not generate carboxy-terminal fragments that are immunoreactive with amino-terminal-specific antiserum. Cathepsin D does not cleave the model peptide or show specificity for wild-type amyloid precursor protein; however, it cleaves the Swedish "NL peptide" and "NL precursor" substrates appropriately. Our results suggest that cathepsin D could act as beta-secretase in the Swedish type of familial Alzheimer's disease and demonstrate the importance of using full-length substrate to verify the sequence specificity of candidate proteases.

The release of Alzheimer's disease beta amyloid peptide is reduced by phorbol treatment.

Amyloid precursor protein (APP) is cleaved predominantly within the beta amyloid peptide (BAP) domain to release a non-amyloidogenic amino-terminal PN2 fragment. Treatment of cells with phorbol dibutyrate, an agent which activates protein kinase C, has been shown to increase the release of an amino-terminal fragment. A panel of mutant APP reporter constructs was expressed in which each of the potential phosphorylation sites located within the cytoplasmic domain of APP was replaced with alanine residues. Phorbol response patterns were unchanged for each of these mutants, suggesting that induced cleavage occurs independently of APP substrate phosphorylation. We find that phorbol (a) increases the release of a PN2 fragment that is consistent with the normal secretase activity, (b) decreases the release of a shorter amino-terminal APP fragment that is cleaved near the amino terminus of BAP, and (c) decreases the release of BAP which was identified based on electrophoretic mobility, epitope mapping, and radio-sequencing. These data demonstrate that pharmacological treatment can reduce the formation of BAP and suggests that protein kinase C activators could be developed as therapeutic agents to block BAP formation.

Release of amino-terminal fragments from amyloid precursor protein reporter and mutated derivatives in cultured cells.

Abnormal proteolytic processing of amyloid precursor protein (APP) is thought to be central to the formation and deposition of beta amyloid peptide in Alzheimer's disease. A putative "secretase" activity normally releases an amino-terminal APP fragment by cleaving APP at residues within the beta amyloid peptide thereby precluding amyloidogenesis. In order to better understand the requirements for APP cleavage by secretase, we have expressed a modified cDNA construct representing the 751-amino acid isoform of APP (APP-REP) and mutated APP-REP proteins in cultured cells. Here, we show that: (a) APP-REP is predominantly associated with membranes; (b) intracellular turnover and processing of APP-REP is similar to that reported for the intact APP protein; (c) secretion appears unaltered by introduction of the glutamate to glutamine mutation found in the APP gene of patients suffering from hereditary cerebral hemorrhage with amyloidosis of Dutch origin; (d) a mutation in which the 18 juxtamembranous amino acids encompassing the secretase site are deleted also allows release of an amino-terminal fragment into the conditioned medium; and (e) kinetics of cleavage of APP-REP and its mutated derivatives are similar. These results indicate that the secretory cleavage of the extracellular amino-terminal fragments of APP-REP can occur in the presence of different novel juxtamembranous amino acid sequences.