title: Single amino acid substitution in the PC1/3 propeptide can induce significant modifications of its inhibitory profile toward its cognate enzyme.
abstract: The proprotein convertase PC1/3 is synthesized as a large precursor that undergoes proteolytic processing of the signal peptide, the propeptide and ultimately the COOH-terminal tail, to generate the mature form. The propeptide is essential for protease folding, and, although cleaved by an autocatalytic process, it remains associated with the mature form acting as an auto-inhibitor of PC1/3. To further assess the role of certain residues in its interaction with its cognate enzyme, we performed an alanine scan on two PC1/3 propeptide potential cleavable sites ((50)RRSRR(54) and (61)KR(62)) and an acidic region (65)DDD(67) conserved among species. Upon incubation with PC1/3, the ensuing peptides exhibit equal inhibitory potency, lower potency, or higher potency than the wild-type propeptide. The K(i) values calculated varied between 0.15 and 16.5 nm. All but one mutant exhibited a tight binding behavior. To examine the specificity of mutants, we studied their reactivity toward furin, a closely related convertase. The mutation of certain residues also affects the inhibition behavior toward furin yielding propeptides exhibiting K(i) ranging from 0.2 to 24 nm. Mutant propeptides exhibited against each enzyme either different mode of inhibition, enhanced selectivity in the order of 40-fold for one enzyme, or high potency with no discrimination. Hence, we demonstrate through single amino acid substitution that it is feasible to modify the inhibitory behavior of propeptides toward convertases in such a way as to increase or decrease their potency, modify their inhibitory mechanisms, as well as increase their selectivity.
authors.affiliation: Neuropeptides Structure and Metabolism, Institut de Recherches Cliniques de Montréal, 110 Pine Avenue West, Montréal, Quebec H2W 1R7, Canada.
authors.forename: Nadia
authors.initials: N
authors.lastname: Rabah
authors.forename: Dany
authors.initials: D
authors.lastname: Gauthier
authors.forename: Brian C
authors.initials: BC
authors.lastname: Wilkes
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Gauthier
authors.forename: Claude
authors.initials: C
authors.lastname: Lazure
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D045683:Furin / Q000737:chemistry; D006801:Humans; D020128:Inhibitory Concentration 50; D007313:Insecta; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D045664:Proprotein Convertase 1 / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
chemical_list: D009841:Oligonucleotides; D010455:Peptides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D045683:Furin; D045664:Proprotein Convertase 1