Development of Protein-based Inhibitors of the Proprotein of Convertase SKI-1/S1P PROCESSING OF SREBP-2, ATF6, AND A VIRAL GLYCOPROTEIN

Abstract

Processing of membrane-bound transcription factors such as sterol regulatory element-binding proteins (SREBPs) and the ER-stress response factor ATF6, and glycoproteins of some hemorrhagic fever viruses are initiated by the proprotein convertase SKI-1/S1P. So far, no cellular protein-based inhibitor of the hydrophobicamino acid specific SKI-1 is known. The prosegment of the basic-amino acid specific convertases (e.g. furin and PC5) or 1-PDX, a variant of 1-antitrypsin ( 1-AT) exhibiting an RIPR358 sequence at the reactive site loop, were shown to potently inhibit these secretory proteinases. Accordingly, we tested the SKI-1-inhibitory potential of various point mutants of either the 198 amino acid preprosegment of SKI-1-(1–198) or 1-AT. Transient transfections data showed that, out of numerous mutants studied, the R134E prosegment mutant or the 1-AT reactive site loop variants RRVL358, RRYL358 and RRIL358 are the best specific cellular inhibitors of SKI-1. The observed inhibition of the processing of endogenous SREBP-2, exogenous ATF6 and a PDGF-A (RRLL86) variant were >55% and reach 80% in stable transfectants. We also show that SKI-1 forms SDS-stable complexes with these 1-AT variants, but not with wild-type 1-AT or 1-PDX. Finally, these inhibitors were also shown to affect the processing and stability of the Crimean-Congo hemorrhagic fever virus glycoprotein