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

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.