Signal peptide peptidase dependent cleavage of type II transmembrane substrates releases intracellular and extracellular signals

Kumlesh K Dev; Sandipan Chatterjee; Maribel Osinde; Daniela Stauffer; Hannah Morgan; Monika Kobialko; Uwe Dengler; Heinrich Rueeger; Bruno Martoglio; Giorgio Rovelli

doi:10.1016/j.ejphar.2006.04.011

Signal peptide peptidase dependent cleavage of type II transmembrane substrates releases intracellular and extracellular signals

Eur J Pharmacol. 2006 Jul 1;540(1-3):10-7. doi: 10.1016/j.ejphar.2006.04.011. Epub 2006 Apr 15.

Authors

Kumlesh K Dev¹, Sandipan Chatterjee, Maribel Osinde, Daniela Stauffer, Hannah Morgan, Monika Kobialko, Uwe Dengler, Heinrich Rueeger, Bruno Martoglio, Giorgio Rovelli

Affiliation

¹ Novartis Institutes for BioMedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland. kumlesh_k.dev@novartis.com

PMID: 16697367
DOI: 10.1016/j.ejphar.2006.04.011

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Animals
Aspartic Acid Endopeptidases / antagonists & inhibitors
Aspartic Acid Endopeptidases / genetics
Aspartic Acid Endopeptidases / metabolism*
Binding Sites / genetics
CHO Cells
Cell Line
Cricetinae
Cricetulus
Dipeptides / pharmacology
Endoplasmic Reticulum / metabolism
Extracellular Space / drug effects
Extracellular Space / metabolism
Hepacivirus / genetics
Hepacivirus / metabolism
Humans
Intracellular Space / drug effects
Intracellular Space / metabolism
Luciferases / genetics
Luciferases / metabolism
Molecular Sequence Data
Mutation / genetics
Proteasome Endopeptidase Complex / metabolism
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism*
Sequence Homology, Amino Acid
Signal Transduction*
Substrate Specificity
Transfection
Viral Proteins / genetics
Viral Proteins / metabolism

Substances

1, 3-di-(N-carboxybenzoyl-leucyl-leucyl)amino acetone
Dipeptides
Recombinant Fusion Proteins
Viral Proteins
Luciferases
Aspartic Acid Endopeptidases
signal peptide peptidase
Proteasome Endopeptidase Complex