gamma-secretase cleavage is distinct from endoplasmic reticulum degradation of the transmembrane domain of the amyloid precursor protein.
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.
Bunnell
W L
WL
Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, California 92697-3900, USA.
Pham
H V
HV
Glabe
C G
CG
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
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