title: Proprotein convertase PC1/3-related peptides are potent slow tight-binding inhibitors of murine PC1/3 and Hfurin.
abstract: The proprotein convertase PC1/3 belongs to the subtilisin/kexin-like endoprotease family and is synthesized as a preproenzyme. To investigate the function of its propeptide, murine proPC1/3 and preproPC1/3 were isolated from the inclusion bodies of recombinant preproPC1/3 baculovirus-infected insect cells, rendered soluble with 6 M guanidine HCl and 20 mM dithiothreitol, and purified by gel filtration and metal-binding affinity chromatography. Two NH2-terminal fragments containing the complete propeptide 1-84 region were obtained after CNBr cleavage, purified, and chemically characterized. Progress curve kinetic analysis with enzymatically active murine 71-kDa PC1/3 or 50-kDa human furin demonstrated that both fragments were potent slow tight-binding inhibitors of either enzyme with Ki in the low nanomolar range. Additional cleavages at Trp residues yielded fragment9-71, which no longer represents a potent inhibitor. Upon incubation at pH 5.5 in the presence of excess 71-kDa murine PC1/3, NH2-terminal fragment1-98 is cleaved at two sites, as revealed through Western blotting using NH2-terminal-directed PC1/3 antibodies. Finally, murine PC2 is inhibited by the proPC1/31-98 peptide, albeit at a much lesser extent with a micromolar Ki and in a strictly competitive manner. These results suggest that the proregion of PC1/3 is an important feature in regulating its activity.
authors.affiliation: Protein Engineering Network of Centre of Excellence and the Laboratory of Neuropeptides Structure and Metabolism, Institut de Recherches Cliniques de Montréal (affiliated with the University of Montréal), Montréal, Québec H2W 1R7, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Boudreault
authors.forename: D
authors.initials: D
authors.lastname: Gauthier
authors.forename: C
authors.initials: C
authors.lastname: Lazure
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors*; D016367:Baculoviridae / Q000235:genetics; D002846:Chromatography, Affinity; D003488:Cyanogen Bromide / Q000737:chemistry; D003594:Cytoplasmic Granules / Q000737:chemistry*; D004792:Enzyme Precursors / Q000302:isolation & purification*; D045683:Furin; D006801:Humans; D007463:Iodobenzoates / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*; D045664:Proprotein Convertase 1; D043484:Proprotein Convertases; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D018411:Spodoptera / Q000166:cytology; D013381:Subtilisins / Q000037:antagonists & inhibitors*
chemical_list: D004792:Enzyme Precursors; D007463:Iodobenzoates; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D043484:Proprotein Convertases; D013381:Subtilisins; D045683:Furin; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D016282:Aspartic Acid Endopeptidases; D003488:Cyanogen Bromide