title: Signal peptide peptidase dependent cleavage of type II transmembrane substrates releases intracellular and extracellular signals.
abstract: The intramembrane-cleaving proteases (I-CLiPs) presenilin-1 and -2 (PS1 and PS2), signal peptide peptidase (SPP) and the Site-2 protease (S2P) catalyze critical steps in cell signaling and are implicated in diseases such as Alzheimer's disease, hepatitis C virus (HCV) infection and cholesterol homeostasis. Here we describe the development of a cellular assay based on cleavage of the transmembrane sequence of the HCV core protein precursor, releasing intra- and extra-cellular signals that represent sequential signal peptidase and SPP cleavage, respectively. We find that the SPP inhibitor (Z-LL)2-ketone (IC50 = 1.33 microM) and the gamma-secretase potent inhibitors NVP-AHW700-NX (IC50 = 51 nM) and LY411575 (IC50 = 61 nM) but not DAPT dose dependently inhibited SPP but not signal peptidase cleavage. Our data confirm that type II orientated substrates, like the HCV transmembrane sequence, are sequentially cleaved by signal peptidase then SPP. This dual assay provides a powerful tool to pharmacologically analyze sequential cleavage events of signal peptidase and SPP and their regulation.
authors.affiliation: Novartis Institutes for BioMedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland. kumlesh_k.dev@novartis.com
authors.forename: Kumlesh K
authors.initials: KK
authors.lastname: Dev
authors.forename: Sandipan
authors.initials: S
authors.lastname: Chatterjee
authors.forename: Maribel
authors.initials: M
authors.lastname: Osinde
authors.forename: Daniela
authors.initials: D
authors.lastname: Stauffer
authors.forename: Hannah
authors.initials: H
authors.lastname: Morgan
authors.forename: Monika
authors.initials: M
authors.lastname: Kobialko
authors.forename: Uwe
authors.initials: U
authors.lastname: Dengler
authors.forename: Heinrich
authors.initials: H
authors.lastname: Rueeger
authors.forename: Bruno
authors.initials: B
authors.lastname: Martoglio
authors.forename: Giorgio
authors.initials: G
authors.lastname: Rovelli
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004151:Dipeptides / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013379:Substrate Specificity; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C415363:1, 3-di-(N-carboxybenzoyl-leucyl-leucyl)amino acetone; D004151:Dipeptides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D008156:Luciferases; D016282:Aspartic Acid Endopeptidases; C460085:signal peptide peptidase; D046988:Proteasome Endopeptidase Complex