Short Polybasic Peptide Sequences Are Potent Inhibitors of PC5/6 and PC7: Use of Positional Scanning-Synthetic Peptide Combinatorial Libraries as a Tool for the Optimization of Inhibitory Sequences

Abstract

Positional scanning-synthetic peptide combinatorial libraries (PS-SPCLs) are powerful molecular tools to identify enzyme substrate and potent inhibitory sequences and also to provide crucial information about active site determinants. PS-SPCLs have been surveyed for furin, proprotein convertase (PC)2, PC1/3, and PACE4 and proven efficient to identify potent peptidyl inhibitors in the low nanomolar range for furin and PC1/3. We report herein the screenings of nonamidated and acetylated hexapeptide PS-SPCLs for PC5/6A and PC7. The L-configuration library surveys distinctively revealed that L-Arg, L-Lys, and sometimes L-His in all six positions would generate the most potent inhibitors for both enzymes. Based on this clear polybasic preference, L-poly-Arg peptides ranging from four to nine residues were assayed. Inhibitory potency of these polybasic peptides increased with chain length, making nona-L-Arg a potent nanomolar inhibitor of PC5/6A and PC7 (K i of 150 and 120 nM). PC5/6 and PC7 inhibition by nona-L-arginine was equivalent to that of furin (K i of 114 nM) (J Biol Chem 275: [36741][36742][36743][36744][36745][36746][36747][36748][36749] 2000). Nona-D-arginine was a more potent inhibitor of PC5/6 and PC7 than its levorotatory version (K i of 19 and 81 nM), reminiscent of furin (K i of 1.3 nM) (J Biol Chem 279: 36788 -36794, 2004). Our data indicate that certain polyarginine peptides represent potent inhibitors targeting PCs of the constitutive secretory pathway (furin, PC5/6, and PC7). We conclude that basic residues within PC peptide inhibitors might be responsible for targeting PCs in general and for inhibitory potency, but that select amino acid changes will be necessary to acquire true specificity toward a single PC.

Post-Endoplasmic Reticulum Rescue of Unstable MHC Class I Requires Proprotein Convertase PC7

“…To further corroborate this finding, we pharmacologically inhibited pPCs in all above cell lines using the cell-permeable peptide HDA, which efficiently blocks both furin and PC7 with an inhibition constant (K i ) of 106 and 1875 nM, respectively (24). In our previous studies we observed that HDA treatment specifically impaired MHC I surface expression in T2(1-2DN), but not in T2 (TAPwt) cells, consistent with a requirement for post-ER stabilization of MHC I only in the former cell line (Fig.…”

“…2D) underscores the exclusive involvement of this particular pPC, whereas a practically complete furin knockdown is totally neutral in this sense. Of note, HDA has a broad inhibitory capacity against most pPCs (including PC7), blocks furin activity efficiently in vivo, and actually has a .10-fold higher potency toward furin than toward PC7 (24). Further corroborating the selective requirement for PC7, but not furin, more than half of the peptide peaks that are specifically detected in the T2(1-2DN)-and T2(1-2DN/FUR↓)-derived peptide spectra disappear from the respective spectrum of T2(1-2DN/PC7↓) (Fig.…”

Proprotein Convertase PC7 Enhances the Activation of the EGF Receptor Pathway through Processing of the EGF Precursor

“…Although less efficient than Furin, PC7 specifically cleaves overexpressed substrates at Arg2 residues both in vitro (22)(23)(24)(25)(26)(27)(28)(29) and in cell lines (30 -39). Northern blot analyses revealed a wide expression of PC7 mRNA in all rat tissues and cell lines analyzed (40), suggesting that it may have multiple physiological functions.…”

Mammalian Peptide Hormones: Biosynthesis and Inhibition

“…Apart from peptides derived from endogenous inhibitors (see Section 13.4.3.1), positional scanning of synthetic peptide combinatorial libraries has been used to identify potential inhibitory peptides for PC1 [222,223] and PC2 [222] as well as PC5A [224], and to get information about active-site determinants. Accordingly, S 5 , S 4 , and S 3 subsites of PC1 appear to resemble those of PC2, while the S 6 subsite of PC1 appears to be more selective than that of PC2 [222].…”