title: gamma-secretase cleavage is distinct from endoplasmic reticulum degradation of the transmembrane domain of the amyloid precursor protein.
abstract: One of the critical cleavage events that generates Alzheimer's amyloid Abeta peptide occurs within the transmembrane domain (TMD) of the amyloid precursor protein (APP) and is carried out by a poorly understood enzyme activity known as gamma-secretase. To investigate this processing, a probe molecule, H26-57C, was constructed containing the TMD of APP flanked immediately on each side by unique epitope tags. H26-57C-transfected cells secrete a approximately 2.9-kDa fragment, indicating that the lumenal and cytosolic domains of APP are not required for gamma-secretase processing. Pulse-chase experiments indicate that the probe turns over with a half-life of 8 min. No degradation intermediates are detected during the chase period, indicating that TMD turnover is a highly processive mechanism. The protease inhibitors, ALLN and MG132, cause a dramatic (50-fold) increase in the steady-state amount of the probe. All of the inhibitors that prevent degradation of the probe in the rough endoplasmic reticulum increase the amount of the approximately 2.9-kDa fragment that is secreted into the media and also causes a similar increase the secretion of 4 kDa Abeta from APP-transfected cells. These results indicate that the system responsible for the degradation of the probe in the rough endoplasmic reticulum and the intramembrane cleavage by gamma-secretase that produces soluble, secreted Abeta are distinct and opposing processes.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, California 92697-3900, USA.
authors.forename: W L
authors.initials: WL
authors.lastname: Bunnell
authors.forename: H V
authors.initials: HV
authors.lastname: Pham
authors.forename: C G
authors.initials: CG
authors.lastname: Glabe
mesh_terms: D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004285:Dogs; D010450:Endopeptidases / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008565:Membrane Proteins; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010179:Pancreas / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic
chemical_list: D016564:Amyloid beta-Protein Precursor; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D011480:Protease Inhibitors; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde