Abstract
Prohormone convertase 1 (PC1), mediating the proteolytic processing of neural and endocrine precursors, is thought to be regulated by the neuroendocrine protein proSAAS. The PC1 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 KR(244) dibasic motif. Our results show that the decapeptide proSAAS-(235-244)( 235)VLGALLRVKR(244) is the most potent reversible competitive PC1-inhibitor (K(i) approximately 9 nm). The C-terminally extended proSAAS-(235-246) exhibits a 5-6-fold higher K(i) ( approximately 51 nm). The additional LE sequence at P1'-P2', resulted in a competitive substrate cleaved by PC1 at KR(244) downward arrowLE(246). Systematic alanine scanning and in some cases lysine scanning tested the contribution of each residue within proSAAS-(235-246) toward the PC1-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 PC1 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.
Highlights
These data agree with those predicted from modeling studies by Lipkind et al (28) and Siezen et al (29), whereby specific Glu and Asp residues in the S1, S2, and S4 subsites of Prohormone convertase 1 (PC1) are thought to intimately contact these basic residues
Generally agreeing with the results of positional scanning synthetic peptide combinatorial 6-mer peptide library studies (18), our data differ with respect to the critical importance of P6 Leu
The P9 Leu was found to be the most critical since its substitution by Ala led to a ϳ15-fold decrease in PC1 inhibition
Summary
Peptide Synthesis—All Fmoc protected amino acids (L-configuration), the coupling reagents, and the solvents were purchased from PerkinElmer Life Sciences, Calbiochem-Novabiochem, and Chem Impex International, (Wood Dale, IL). Determination of Inhibition Constant, Ki, and IC50—For Ki determination, various peptide concentrations (0.12 nM to 700 M) were incubated at 37 °C with respective enzyme (5 l) in the above described buffer (100 l) in the presence of at least two different concentrations of fluorogenic substrate, pERTKR-MCA (100, 50, 25 or 12.5 M) (7, 15). Comparative Analysis of PC Inhibition by proSAAS Peptides—Each proSAAS peptide (750 nM or 25 M) was preincubated with enzyme (5 l) for 15 min in buffer (100 l) before the addition of fluorogenic substrate, pERTKR-MCA (100 M). Effect of Preincubation Time on Enzyme Inhibition—For this study, a representative peptide (proSAAS-(235–244), Table I) (20 M) was preincubated with PC1 (5 l) for 0, 5, 10, 15, and 30 min in buffer (100 l) before the addition of substrate pERTKR-MCA (100 M). Molecular Modeling Study—Three-dimensional theoretical structures of proSAAS peptides and their mutants were generated by computer software hyperchem (version 5.0; Hypercube) with Robek-Polard energy minimization carried out at ambient temperature
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