Inhibitory specificity and potency of proSAAS-derived peptides toward proprotein convertase 1

Prohormone convertase I (PCI), mediating the proteolytic processing of neural and endocrine precursors, is thought to be regulated by the neuroendocrine protein proSAAS. The PCI inhibitory sequence is mostly confined within a 10-12-amino acid segment near the C terminus of the conserved human proSAAS and contains the critical KR244 dibasic motif. Our results show that the decapeptide proSAAS-(235-244) (235)VLGALLRVKR(244) is the most potent reversible competitive PC1-inhibitor (K-i similar to9 nM). The C-terminally extended proSAAS-(235-246) exhibits a 5-6-fold higher K-i (similar to 51 nM). The additional LE sequence at P1'-P2', resulted in a competitive substrate cleaved by PCI at KR244 down arrow LE246. Systematic alanine scanning and in some cases lysine scanning tested the contribution of each residue within proSAAS(235-246) toward the PCI-inhibition's specificity and potency. The amino acids P1 Arg, P2 Lys, and P4 Arg are all critical for inhibition. Moreover, the aliphatic P3 Val and P5, P6, and P1' Leu significantly affect the degree of enzyme inactivation and PC1 specificity. Interestingly, a much longer N- and C-terminally extended endogenous rat proSAAS-(221-254) called little PenLen, was found to be a 3-fold less potent PCI inhibitor with reduced selectivity but a much better substrate than proSAAS-(235-246). Molecular modeling studies and circular dichroism analysis indicate an extended and poly-L-proline II type structural conformation for proSAAS-(235-244), the most potent PC1 inhibitor, a feature not present in poor PC1 inhibitors.