title: Cell-free generation of the notch1 intracellular domain (NICD) and APP-CTfgamma: evidence for distinct intramembranous "gamma-secretase" activities.
abstract: PSEN1 and PSEN2 encode polytopic membrane proteins, termed presenilin 1 (PS1) and presenilin 2 (PS2) that play an essential role in intramembranous ("gamma-secretase") proteolysis of selected type I membrane proteins, that include Notch1 and beta-amyloid precursor protein (APP). In order to gain insights into biochemical mechanisms underlying gamma-secretase processing of Notch1 and APP, we have developed a novel in vitro assay in which gamma-secretase-mediated generation of S3/NICD and APP-CTFgamma can be readily detected in isolated membrane fractions derived from immortalized PS1+/- mouse embryonic fibroblasts; production of the APP and Notch1 derivatives are inhibited by a highly selective and potent gamma-secretase inhibitor, L-685,458, with a IC50 of approximately 50 pM. In membranes prepared from PS1-deficient fibroblasts, we detected APP-CTFgamma, albeit at low levels. Unexpectedly, and despite the presence of endogenous PS2 in membranes prepared from PS1-deficient fibroblasts, production of the Notch derivatives, S3/NICD, was nearly undetectable in these reactions. Moreover, S3/NICD production is neither detected in detergent-solubilized membrane preparations from PS1-deficient cells, nor in reactions containing PS1-containing membranes that were co-solubilized with membranes from PS -/- cells expressing a chimeric Notch 1 species. These findings strongly suggest that the factors responsible for intramembranous, gamma-secretase proteolysis of APP and Notch1 are neither equivalent, nor exchangeable.
authors.affiliation: Department of Neurobiology, Pharmacology and Physiology, The University of Chicago, IL 60637, USA.
authors.forename: Takesh
authors.initials: T
authors.lastname: Ikeuchi
authors.forename: Sangram S
authors.initials: SS
authors.lastname: Sisodia
mesh_terms: D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D003433:Crosses, Genetic; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts; D008565:Membrane Proteins / Q000737:chemistry / Q000172:deficiency / Q000235:genetics; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics*; D053764:Presenilin-1; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000378:metabolism; D014157:Transcription Factors; D014162:Transfection
chemical_list: D016564:Amyloid beta-Protein Precursor; D008565:Membrane Proteins; C497762:Notch1 protein, mouse; D010446:Peptide Fragments; D053764:Presenilin-1; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D014157:Transcription Factors; C441641:amyloid precursor protein carboxyl-terminal fragment gamma; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509282:Bace1 protein, mouse