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 hydrophobic-amino acid specific SKI-1 is known. The prosegment of the basic-amino acid specific convertases (e.g. furin and PC5) or alpha(1)-PDX, a variant of alpha(1)-antitrypsin (alpha(1)-AT) exhibiting an RIPR(358) 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 alpha(1)-AT. Transient transfections data showed that, out of numerous mutants studied, the R134E prosegment mutant or the alpha(1)-AT reactive site loop variants RRVL(358), RRYL(358) and RRIL(358) 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 (RRLL(86)) variant were >55% and reach approximately 80% in stable transfectants. We also show that SKI-1 forms SDS-stable complexes with these alpha(1)-AT variants, but not with wild-type alpha(1)-AT or alpha(1)-PDX. Finally, these inhibitors were also shown to affect the processing and stability of the Crimean-Congo hemorrhagic fever virus glycoprotein.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Activating Transcription Factor 6
-
Amino Acid Motifs
-
Amino Acid Sequence
-
Animals
-
Binding Sites
-
Blotting, Western
-
CHO Cells
-
Cell Line
-
Cricetinae
-
DNA, Complementary / metabolism
-
DNA-Binding Proteins / chemistry*
-
DNA-Binding Proteins / metabolism
-
Endoplasmic Reticulum / metabolism*
-
Glycoproteins / chemistry
-
Hemorrhagic Fever Virus, Crimean-Congo / metabolism
-
Humans
-
Molecular Sequence Data
-
Mutagenesis, Site-Directed
-
Mutation
-
Oligonucleotides / chemistry
-
Platelet-Derived Growth Factor / metabolism
-
Point Mutation
-
Proprotein Convertases / antagonists & inhibitors*
-
Proprotein Convertases / chemistry*
-
Protein Binding
-
Protein Precursors / chemistry
-
Protein Structure, Tertiary
-
Sequence Homology, Amino Acid
-
Serine Endopeptidases / chemistry*
-
Serpins / chemistry
-
Sodium Dodecyl Sulfate / chemistry
-
Sterol Regulatory Element Binding Protein 2
-
Transcription Factors / chemistry*
-
Transcription Factors / metabolism
-
Transfection
-
Viral Proteins / chemistry*
-
alpha 1-Antitrypsin / chemistry
Substances
-
ATF6 protein, human
-
Activating Transcription Factor 6
-
DNA, Complementary
-
DNA-Binding Proteins
-
Glycoproteins
-
Oligonucleotides
-
Platelet-Derived Growth Factor
-
Protein Precursors
-
SREBF2 protein, human
-
Serpins
-
Sterol Regulatory Element Binding Protein 2
-
Transcription Factors
-
Viral Proteins
-
alpha 1-Antitrypsin
-
alpha 1-antitrypsin Portland
-
platelet-derived growth factor A
-
Sodium Dodecyl Sulfate
-
Proprotein Convertases
-
Serine Endopeptidases
-
membrane-bound transcription factor peptidase, site 1