title: The C-terminal fragment of the Alzheimer's disease amyloid protein precursor is degraded by a proteasome-dependent mechanism distinct from gamma-secretase.
abstract: The beta-amyloid protein (Abeta) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by beta-secretase generates a C-terminal fragment (APP-CTFbeta), which is subsequently cleaved by gamma-secretase to produce Abeta. The aim of this study was to examine the cleavage of APP-CTFbeta by gamma-secretase in primary cortical neurons from transgenic mice engineered to express the human APP-CTFbeta sequence. Neurons were prepared from transgenic mouse cortex and proteins labelled by incubation with [35S]methionine and [35S]cysteine. Labelled APP-CTFbeta and Abeta were then immunoprecipitated with a monoclonal antibody (WO2) specific for the transgene sequences. Approximately 30% of the human APP-CTFbeta (hAPP-CTFbeta) was converted to human Abeta (hAbeta), which was rapidly secreted. The remaining 70% of the hAPP-CTFbeta was degraded by an alternative pathway. The cleavage of hAPP-CTFbeta to produce hAbeta was inhibited by specific gamma-secretase inhibitors. However, treatment with proteasome inhibitors caused an increase in both hAPP-CTFbeta and hAbeta levels, suggesting that the alternative pathway was proteasome-dependent. A preparation of recombinant 20S proteasome was found to cleave a recombinant cytoplasmic domain fragment of APP (APPcyt) directly. The study suggests that in primary cortical neurons, APP-CTFbeta is degraded by two distinct pathways, one involving gamma-secretase, which produces Abeta, and a second major pathway involving direct cleavage of APP-CTFbeta within the cytoplasmic domain by the proteasome. These results raise the possibility that defective proteasome function could lead to an increase in Abeta production in the AD brain.
authors.affiliation: Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia.
authors.forename: J
authors.initials: J
authors.lastname: Nunan
authors.forename: M S
authors.initials: MS
authors.lastname: Shearman
authors.forename: F
authors.initials: F
authors.lastname: Checler
authors.forename: R
authors.initials: R
authors.lastname: Cappai
authors.forename: G
authors.initials: G
authors.lastname: Evin
authors.forename: K
authors.initials: K
authors.lastname: Beyreuther
authors.forename: C L
authors.initials: CL
authors.lastname: Masters
authors.forename: D H
authors.initials: DH
authors.lastname: Small
mesh_terms: D000544:Alzheimer Disease / Q000201:enzymology / Q000378:metabolism*; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D015153:Blotting, Western; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors
chemical_list: D016564:Amyloid beta-Protein Precursor; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse; D046988:Proteasome Endopeptidase Complex