title: A faster migrating variant masquerades as NICD when performing in vitro gamma-secretase assays with bacterially expressed Notch substrates.
abstract: Intramembrane proteolysis is a new and rapidly growing field. In vitro assays utilizing recombinant substrates for gamma-secretase, an intramembrane-cleaving enzyme, are critically important in order to characterize the biochemical properties of this unusual enzyme. Several recombinant Notch proteins of varying length are commonly used as in vitro substrates for CHAPSO-solubilized gamma-secretase. Here we report that several recombinant Notch constructs undergo limited or no proteolysis in vitro. Instead, upon incubation with or without gamma-secretase, variants of the intact protein migrate during SDS-PAGE at the location expected for the gamma-secretase specific cleavage products. In addition, we show that addition of aspartyl- and gamma-secretase specific protease inhibitors are able to retard the formation of these variants independent of gamma-secretase, which could lead to the erroneous conclusion that Notch cleavage by solubilized gamma-secretase was achieved in vitro even when no proteolysis occurred. In contrast, substrates produced in mammalian or insect cells are cleaved efficiently in vitro. These observations suggest that in vitro studies reliant on recombinant, bacterially produced Notch TMD should be performed with the inclusion of additional controls able to differentiate between actual cleavage and this potential artifact.
authors.affiliation: Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8231, St. Louis, Missouri 63110, USA.
authors.forename: Preston C
authors.initials: PC
authors.lastname: Keller
authors.forename: Taisuke
authors.initials: T
authors.lastname: Tomita
authors.forename: Ikuo
authors.initials: I
authors.lastname: Hayashi
authors.forename: Dilip
authors.initials: D
authors.lastname: Chandu
authors.forename: Jason D
authors.initials: JD
authors.lastname: Weber
authors.forename: David P
authors.initials: DP
authors.lastname: Cistola
authors.forename: Raphael
authors.initials: R
authors.lastname: Kopan
mesh_terms: D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; D002460:Cell Line; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D051880:Receptors, Notch; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human