title: Inhibitory potency and specificity of subtilase-like pro-protein convertase (SPC) prodomains.
abstract: The SPCs (subtilisin-like pro-protein convertases) are a family of enzymes responsible for the proteolytic processing of numerous precursor proteins of the constitutive and regulated secretory pathways. SPCs are themselves synthesized as inactive zymogens. Activation of SPCs occurs via the intramolecular autocatalytic removal of the prodomain. SPC prodomains have been proposed as templates in the development of potent and specific SPC inhibitors. In this study, we investigated the specificity and potency of complete prodomains and short C-terminal prodomain peptides of each SPC on highly purified, soluble enzyme preparations of human SPC1, SPC6, and SPC7. Progress curve kinetic analysis of prodomain peptides and complete prodomains showed competitive inhibitory profiles in the low nanomolar range. Complete prodomains were 5-100 times more potent than C-terminal prodomain peptides, suggesting that N-terminal determinants are involved in the recognition process. However, complete prodomains and prodomain peptides exhibit only a partial specificity toward their cognate enzyme. Ala-scan structure activity studies indicated the importance of basic residues in the P(4), P(5), and P(6) positions for inhibition of SPC1. In contrast, hydrophobic residues in P(6) and P(7), as well as basic residues in P(4) and P(5), were critical for inhibition of SPC7. Our data demonstrated that the use of prodomains as specific inhibitors acting in trans would be of limited usefulness, unless modified into more specific compounds.
authors.affiliation: Department of Pharmacology, Institut de Pharmacologie de Sherbrooke, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
authors.forename: Martin
authors.initials: M
authors.lastname: Fugère
authors.forename: Polizois C
authors.initials: PC
authors.lastname: Limperis
authors.forename: Véronique
authors.initials: V
authors.lastname: Beaulieu-Audy
authors.forename: Frédéric
authors.initials: F
authors.lastname: Gagnon
authors.forename: Pierre
authors.initials: P
authors.lastname: Lavigne
authors.forename: Klaus
authors.initials: K
authors.lastname: Klarskov
authors.forename: Richard
authors.initials: R
authors.lastname: Leduc
authors.forename: Robert
authors.initials: R
authors.lastname: Day
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D002850:Chromatography, Gel; D002942:Circular Dichroism; D017077:Culture Media, Conditioned / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D045683:Furin; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D045702:Proprotein Convertase 5; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D020860:Subtilisin / Q000737:chemistry*; D013381:Subtilisins / Q000737:chemistry*
chemical_list: D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D010455:Peptides; D011994:Recombinant Proteins; D045702:Proprotein Convertase 5; D013381:Subtilisins; C100635:protease SPC7; D020860:Subtilisin; D045683:Furin