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• W2001045107 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 KR244 dibasic motif. Our results show that the decapeptide proSAAS-(235–244)235VLGALLRVKR244 is the most potent reversible competitive PC1-inhibitor (K i ∼9 nm). The C-terminally extended proSAAS-(235–246) exhibits a 5–6-fold higher K i (∼51 nm). The additional LE sequence at P1′-P2′, resulted in a competitive substrate cleaved by PC1 at KR244↓LE246. 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. 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 KR244 dibasic motif. Our results show that the decapeptide proSAAS-(235–244)235VLGALLRVKR244 is the most potent reversible competitive PC1-inhibitor (K i ∼9 nm). The C-terminally extended proSAAS-(235–246) exhibits a 5–6-fold higher K i (∼51 nm). The additional LE sequence at P1′-P2′, resulted in a competitive substrate cleaved by PC1 at KR244↓LE246. 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. proprotein/prohormone convertase amino acid(s) N-(9-fluorenyl)methoxycarbonyl reverse-phase high performance liquid chromatography matrix-assisted laser desorption ionization time-of-flight before transmembrane domain 2-(N-morpholino)ethanesulfonic acid 7-amino 4-methylcoumarin 4-methyl 7-aminocoumarinamide Proprotein convertases (PCs),1 a family of Ca2+-dependent mammalian subtilases, are known to mediate the proteolytic processing at selected sites of many precursor proteins into their functionally active forms (1Seidah N.G. Chrétien M. Brain Res. 1999; 848: 45-62Crossref PubMed Scopus (692) Google Scholar, 2Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). These sites are generally composed of a pair of basic amino acids within the consensus sequence of R/K/H-X n-R↓, wheren = 0, 2, 4, or 6 and X represents any amino acid except cysteine. Numerous potential substrates have so far been identified for PCs. These include hormonal peptides and growth factors, their receptors, cell surface proteins, bacterial toxins, envelope viral glycoproteins, enzymes, transcription factors, and others (1Seidah N.G. Chrétien M. Brain Res. 1999; 848: 45-62Crossref PubMed Scopus (692) Google Scholar, 2Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). The delicate balance between cleaved functional proteins and their precursors is critical for normal growth, function, metabolism, and development as well as in pathophysiologic conditions (1Seidah N.G. Chrétien M. Brain Res. 1999; 848: 45-62Crossref PubMed Scopus (692) Google Scholar, 2Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 3Chrétien M. Mbikay M Gaspar L. Seidah N.G. Proc. Assoc. Am. Physicians. 1995; 107: 47-66PubMed Google Scholar, 4Chrétien, M., Cromlish, J. A., and Seidah, N. G. (1997)Bull. Can. Soc. Biochem. Cell. Biol. 86–96.Google Scholar, 5Molloy S.S. Anderson E.D. Jean F. Thomas G. Trends Cell Biol. 1999; 9: 28-35Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). All PCs are initially synthesized as inactive zymogens that must be proteolytically activated through the autocatalytic cleavage of their inhibitory N-terminal prosegment. A number of studies have already revealed this unique property of prodomains in the regulation of enzymatic activity (6Leduc R. Molloy S.S. Thorne B.A. Thomas G. J. Biol. Chem. 1992; 267: 14304-14308Abstract Full Text PDF PubMed Google Scholar, 7Zhong M. Munzer J.S. Basak A. Benjannet S. Mowla S.J. Decroly E. Chrétien M. Seidah N.G. J. Biol. Chem. 1999; 274: 33913-33920Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 8Boudreault A. Gauthier D. Lazure C. J. Biol. Chem. 1998; 273: 31574-31580Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 9Muller L. Cameron A. Fortenberry Y. Apletalina E.V. Lindberg I. J. Biol. Chem. 2000; 275: 39213-39222Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). However, at least for the neuroendocrine convertases PC1 and PC2, their cellular activity is controlled by endogenous inhibitors. Thus, co-localization, in situhybridization, and other biochemical studies revealed that the production of enzymatically active PC2 requires the presence of a binding protein 7B2 (10Seidah N.G. Hsi K.L. De Serres G. Rochemont J. Hamelin J. Antakly T. Cantin M. Chrétien M. Arch. Biochem. Biophys. 1983; 225: 525-534Crossref PubMed Scopus (123) Google Scholar), which also serves as a specific temporal endogenous inhibitor of this enzyme (for reviews, see Refs. 1Seidah N.G. Chrétien M. Brain Res. 1999; 848: 45-62Crossref PubMed Scopus (692) Google Scholar, 2Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, and11Muller L. Lindberg I. Prog. Nucleic Acids Res. Mol. Biol. 1999; 63: 69-108Crossref PubMed Scopus (125) Google Scholar). 2Mbikay, M., Seidah, N. G., and Chrétien, M. (2001) Biochem. J. 357,329–342 Subsequent deletion and alanine-scanning studies identified the inhibitor segment as a 16-aa fragment of the 31-aa C-terminal domain of 7B2 (13Apletalina E.V. Juliano M.A. Juliano L. Lindberg I. Biochem. Biophys. Res. Commun. 2000; 267: 940-942Crossref PubMed Scopus (21) Google Scholar). Recently, a granin-like 26-kDa (258–260-aa) neuroendocrine secretory protein, called proSAAS, was identified as a specific PC1 inhibitor (14Fricker L.D. McKinzie A.A. Sun J. Curran E. Qian Y. Yan L. Patterson S.D. Courchesne P.L. Richards B. Levin N. Mzhavia N. Devi L.A. Douglass J. J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar). It is interesting to note that while proSAAS and 7B2 are not homologous, they are of similar size, with an N-terminal proline-rich region; both contain several pairs of basic amino acids and are broadly expressed in neural and endocrine tissues. Very recently, proSAAS was shown to specifically inhibit PC1 and not furin, PC2, PC5, or PC7 (15Qian Y. Devi L.A. Mzhavia N. Munzer S. Seidah N.G. Fricker L.D. J. Biol. Chem. 2000; 275: 23596-23601Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The processing profile of proSAAS was recently reported and revealed that it is cleaved in a tissue-specific fashion at its C terminus into smaller inhibitory peptides (15Qian Y. Devi L.A. Mzhavia N. Munzer S. Seidah N.G. Fricker L.D. J. Biol. Chem. 2000; 275: 23596-23601Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 16Mzhavia N. Berman Y. Che F.Y. Fricker L.D. Devi L.A. J. Biol. Chem. 2001; 276: 6207-6213Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 17Cameron A. Fortenberry Y. Lindberg I. FEBS Lett. 2000; 473: 135-138Crossref PubMed Scopus (69) Google Scholar). The inhibitory segment was mapped to a short 6–12-aa sequence near its C terminus that contains a critical Arg244 (human proSAAS nomenclature) located at the processing site235VLGALLRVKR↓LE246 (15Qian Y. Devi L.A. Mzhavia N. Munzer S. Seidah N.G. Fricker L.D. J. Biol. Chem. 2000; 275: 23596-23601Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 17Cameron A. Fortenberry Y. Lindberg I. FEBS Lett. 2000; 473: 135-138Crossref PubMed Scopus (69) Google Scholar). Interestingly, the peptide LLRVKR was previously identified from a combinatorial peptide library screen aimed at identifying specific PC1 inhibitors (18Apletalina E. Appel J. Lamango N.S. Houghten R.A. Lindberg I. J. Biol. Chem. 1998; 273: 26589-26595Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The in vitro inhibition of PC1 by these short peptides was at least 17-fold better than with the full-length proSAAS (15Qian Y. Devi L.A. Mzhavia N. Munzer S. Seidah N.G. Fricker L.D. J. Biol. Chem. 2000; 275: 23596-23601Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Furthermore, it was reported that the 66-kDa processed form of PC1 is better inhibited by proSAAS than is its 87-kDa precursor (15Qian Y. Devi L.A. Mzhavia N. Munzer S. Seidah N.G. Fricker L.D. J. Biol. Chem. 2000; 275: 23596-23601Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) (Fig.1). In this article, we present detailed kinetic studies on the specificity and potency of PC1 inhibition by the above 12-mer proSAAS peptide and its mutants. Data obtained for the wild type sequence as well as from alanine and lysine scans allowed the identification of the critical aa within this sequence. The specificity and potency of these peptides was also tested against other PCs such as the mammalian furin, PACE4, PC5, PC7, and yeast kexin. The 12-mer proSAAS peptides were compared with that of rat proSAAS-(221–254), also known as little PenLen, which is one of the major processing forms of proSAAS in AtT20 cells (14Fricker L.D. McKinzie A.A. Sun J. Curran E. Qian Y. Yan L. Patterson S.D. Courchesne P.L. Richards B. Levin N. Mzhavia N. Devi L.A. Douglass J. J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar). Finally, circular dichroism and molecular modeling studies were used to correlate the secondary structure of the inhibitory peptides to their potency of PC1 inhibition. 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). The peptides, prepared with carboxyl terminus in the amide form (CONH2) are listed in Table I. The synthesis was accomplished on a solid phase automated peptide synthesizer instrument (Pioneer; PerkinElmer Life Sciences), following theO-hexafluorophospho-[7-azabenzotriazol-1-yl]-N,N,N′,N′-tetramethyluronium/diisopropyl ethyl amine-mediated Fmoc chemistry (19Basak A. Zhong M. Munzer J.S. Chrétien M. Seidah N.G. Biochem. J. 2001; 353: 537-545Crossref PubMed Scopus (89) Google Scholar). The peptides were purified by RP-HPLC on C18 semipreparative (0.94 × 25 cm) and analytical columns (0.46 × 25 cm, Jupitor; Phenomenex) using conditions previously described (19Basak A. Zhong M. Munzer J.S. Chrétien M. Seidah N.G. Biochem. J. 2001; 353: 537-545Crossref PubMed Scopus (89) Google Scholar). The “little PenLen” peptide, rat proSAAS-(221–254), AVDQDLGPEVPPENVLGALLRVKRLENSSPQAPA, was synthesized for us by Research Genetics Inc.Table IList of various proSAAS-derived and -related peptidesNo.Peptide nameAmino acid sequenceMW (M + H)+1h/rSAAS-(235–246)235VLGALLRVKRLE24613642h/rSAAS-(235–246)P2′AVLGALLRVKRLA13063h/rSAAS-(235–246)P1′AVLGALLRVKRAE13224h/rSAAS-(235–246)P1AVLGALLRVKALE12795h/rSAAS-(235–246)P2AVLGALLRVARLE13076h/rSAAS-(235–246)P3AVLGALLRAKRLE13367h/rSAAS-(235–246)P4AVLGALLAVKRLE12798h/rSAAS-(235–246)P5AVLGALARVKRLE13229h/rSAAS-(235–246)P6AVLGAALRVKRLE132210h/rSAAS-(235–246)P8AVLAALLRVKRLE137811h/rSAAS-(235–246)P9AVAGALLRVKRLE132212h/rSAAS-(235–246)P10AALGALLRVKRLE133613h/rSAAS-(235–246)VLGALLRVKR112114h/r-dSAAS-(235–246)vlgallrvkr*112115h/rSAAS-(235–246)P1KVLGALLRVKKLE133716h/rSAAS-(235–246)P4KVLGALLKVKRLE133717h/rSAAS-(235–246)P1KP4KVLGALLKVKKLE130918h/rSAAS-(233–246)P3AP5AVLGALARAKRLE129419rproSAAS-(221–254)221AVDQDLGPEVPPENVLGALLRVKRLENSSPQAPA2543581(little PenLen)All amino acids are in l-configuration except for peptide 14, where all amino acids are in d-configuration (indicated by the front letter “d” and the lower case letters for each amino acid residue (*)), all mutations of amino acids are indicated in boldface type and underlined. “h” and “r” prefixes represent human and rat, respectively. MW, molecular weight. Open table in a new tab All amino acids are in l-configuration except for peptide 14, where all amino acids are in d-configuration (indicated by the front letter “d” and the lower case letters for each amino acid residue (*)), all mutations of amino acids are indicated in boldface type and underlined. “h” and “r” prefixes represent human and rat, respectively. MW, molecular weight. The identities of all peptides and their digests were confirmed by MALDI-TOF mass spectrometry using a Voyageur DE-Pro instrument (PerkinElmer Life Sciences) with α-cyano-4-hydroxycynnamic acid and 2,3-dihydroxybenzoic acid (Aldrich) as matrices (7Zhong M. Munzer J.S. Basak A. Benjannet S. Mowla S.J. Decroly E. Chrétien M. Seidah N.G. J. Biol. Chem. 1999; 274: 33913-33920Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 19Basak A. Zhong M. Munzer J.S. Chrétien M. Seidah N.G. Biochem. J. 2001; 353: 537-545Crossref PubMed Scopus (89) Google Scholar). All of the recombinant forms of PCs were produced by using the vaccinia virus constructs of soluble human furin-BTMD (before transmembrane domain): human PACE4, mouse PC1, mouse PC5A, rat PC7-BTMD, and yeast kexin-BTMD. The enzymes were recovered from serum-free culture media as reported previously (15Qian Y. Devi L.A. Mzhavia N. Munzer S. Seidah N.G. Fricker L.D. J. Biol. Chem. 2000; 275: 23596-23601Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 19Basak A. Zhong M. Munzer J.S. Chrétien M. Seidah N.G. Biochem. J. 2001; 353: 537-545Crossref PubMed Scopus (89) Google Scholar). The recombinant mPC1 used in the present study is obtained from expression of its full-length cDNA, but it contains mostly the C-terminal truncated and enzymatically more active 66-kDa form and some 74- and 87-kDa form as well. All enzyme assays were performed with the fluorogenic substrate pyroglutamyl-Arg-Thr-Lys-Arg-4-methyl-coumaryl-7-amide (Peptides International, Louisville, KY) at pH of either 7.4 (furin) or 6.5 (for other PCs). The assay buffer in all cases was composed of 25 mm Tris, 25 mm Mes, 2.5 mmCaCl2. The concentration of the substrate was maintained at 100 µm unless otherwise mentioned. The amounts of enzymes used were adjusted so as to give approximately the same hydrolytic activity (3.9–4.5 nmol of AMC released/h of incubation) in an aliquot of 5 µl. The release of fluorescence was monitored for 6 h using a spectrofluorometer (Gemini, Molecular Probes, Inc., Eugene, OR) at excitation and emission wavelengths of 370 and 460 nm, respectively). Enzymatic activities were measured either from raw fluorescence readings in end time assay or from the progress curves. The fluorescence of released AMC was measured on-line with a spectrofluorometer every 60 s up to 60 min, and the slope of each curve was assessed with the computer-generated highest point fit. For K i determination, various peptide concentrations (0.12 nm to 700 µm) were incubated at 37 °C with respective enzyme (5 µl) in the above described buffer (100 µl) in the presence of at least two different concentrations of fluorogenic substrate, pERTKR-MCA (100, 50, 25 or 12.5 µm) (7Zhong M. Munzer J.S. Basak A. Benjannet S. Mowla S.J. Decroly E. Chrétien M. Seidah N.G. J. Biol. Chem. 1999; 274: 33913-33920Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 15Qian Y. Devi L.A. Mzhavia N. Munzer S. Seidah N.G. Fricker L.D. J. Biol. Chem. 2000; 275: 23596-23601Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The precise concentration range was selected so as to produce an inhibition of 20–80% of initial activity. For all kinetic measurements, the peptides were preincubated with enzyme for 15 min prior to the addition of the substrate. All assays were performed in duplicate for two independent experiments on a 96-well microplates (flat bottom, black; Dynatec).K i was estimated from Dixon plots, while for IC50 value, a double reciprocal Lineweaver-Burk plot was used (15Qian Y. Devi L.A. Mzhavia N. Munzer S. Seidah N.G. Fricker L.D. J. Biol. Chem. 2000; 275: 23596-23601Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 17Cameron A. Fortenberry Y. Lindberg I. FEBS Lett. 2000; 473: 135-138Crossref PubMed Scopus (69) Google Scholar, 19Basak A. Zhong M. Munzer J.S. Chrétien M. Seidah N.G. Biochem. J. 2001; 353: 537-545Crossref PubMed Scopus (89) Google Scholar). Both initial rate and end time assays were used, and the values obtained for the measured parameter were averaged. Each proSAAS peptide including the little PenLen (20 µm) was incubated for 6 h or overnight at 37 °C with PC1, furin, PACE4, kexin, PC5, or PC7 (5 µl) in buffer (100 µl) as described. The enzyme digests were separated by RP-HPLC using a C18 analytical column with a diode ray detector (Varian, Prostar, CA), and the peaks were analyzed by MALDI-TOF mass spectrometry. Each proSAAS peptide (750 nm or 25 µm) was preincubated with enzyme (5 µl) for 15 min in buffer (100 µl) before the addition of fluorogenic substrate, pERTKR-MCA (100 µm). Fluorescence readings were measured following overnight incubation and compared with the control experiment run in parallel without the peptide. For this study, a representative peptide (proSAAS-(235–244), Table I) (20 µm) was preincubated with PC1 (5 µl) for 0, 5, 10, 15, and 30 min in buffer (100 µl) before the addition of substrate pERTKR-MCA (100 µm). Control experiments were run in parallel without the peptide, and the fluorescence readings were measured after 6 h of reaction. All CD measurements were carried out with a JASCO J-810 spectropolarimeter instrument (Easton, MD) using a 100-µl solution of each peptide in distilled water (concentration 0.2–0.5 mg/ml) in a quartz cell (1-mm path length) as described (20Seebach D Sifferien T. Mathieu P.A. Hane A.M. Krell C.M. Bierbaum D.J. Abele S. Helv. Chim. Acta. 2000; 83: 2849-2864Crossref Scopus (50) Google Scholar). The final corrected CD spectra were obtained by subtracting the spectrum obtained with control water from those of crude samples. 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. Based on earlier studies (15Qian Y. Devi L.A. Mzhavia N. Munzer S. Seidah N.G. Fricker L.D. J. Biol. Chem. 2000; 275: 23596-23601Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), we selected the 12-mer human/rat proSAAS 235VLGALLRVKRLE246 (same as in proSAAS-(233–242) in mouse sequence) as a model peptide for detailed kinetic analysis and positional scanning. As shown in Fig.1, the N-terminal 10 aa of this inhibitory peptide represent the C terminus of the natural processing product obtained by PC1 cleavages at PRRLRR220 and LLRVKR244 and contain the critical Arg244 (15Qian Y. Devi L.A. Mzhavia N. Munzer S. Seidah N.G. Fricker L.D. J. Biol. Chem. 2000; 275: 23596-23601Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar,17Cameron A. Fortenberry Y. Lindberg I. FEBS Lett. 2000; 473: 135-138Crossref PubMed Scopus (69) Google Scholar). Table I lists all of the 19 peptides synthesized, including little PenLen, and analyzed in this work. These include the wild type sequence and its 11 Ala derivatives; three Lys mutants at P1, P4, and P1 + P4; and a P3 + P5 double Ala mutant. Finally, in order to compare the effects of P′ residues on inhibition, we also synthesized the wild type proSAAS-(235–244)235VLGALLRVKR244 as well as its all-dextro derivative. The measured K i of all of the above proSAAS peptides against PC1 and in some cases against furin, PC5, and PC7 are presented in Table II. The competitive nature of inhibition of PC1 by both wild type proSAAS-(235–246) (Fig.2A) and its mutants (not shown) as well as proSAAS-(235–244) (Fig. 2 B) was demonstrated by Dixon plots conducted at four different concentrations of the fluorogenic substrate pERTKR-MCA. The near linear regression and the presence of a single point of intersection are evident. However, a close examination of the graphs revealed a slight hyperbolic nature. This may perhaps be due to some artifact, to the presence of multiple PC1 forms in the enzyme preparation, or alternatively to the adsorption of the peptide or enzyme to the sample tubes. The competitive nature of this inhibition was further confirmed by an observed linear increase of IC50 with substrate concentration (not shown) (21Williams J.W. Morrison J.F. Methods Enzymol. 1979; 63: 437-467Crossref PubMed Scopus (663) Google Scholar, 22Copeland R.A. Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis. Wiley-VCH, New York1996Google Scholar, 23Beynon R.J. Bond J.S. Beynon R.J. Bond J.S. Proteolytic Enzymes: A Practical Approach. IRL Press, Oxford1989: 83-102Google Scholar).Table IIInhibition constant (Ki) of proSAAS and other peptides against PC1, furin, PC5, and PC7PeptidesK i ± S.D.PC1/furin/PC5/PC7PC1furinPC5PC7nmdSAAS-(235–244)122,000 ± 11,900SAAS-(235–244)9 ± 0.5261 ± 44317 ± 9940 ± 321:29:35:103SAAS-(235–246)51 ± 3.839,400 ± 4300100,000 ± 7,0004,750 ± 951:780:1980:94SAAS-(235–246)P2′A293 ± 124,399 ± 22035,963 ± 1,4381,667 ± 671:15:123:6SAAS-(235–246)P1′A1,024 ± 401,280 ± 120231,000 ± 21,6002,330 ± 3301:1:181:2SAAS-(235–246)P1A509,000 ± 15,270SAAS-(235–246)P2A4,503 ± 30SAAS-(235–246)P3A360 ± 7292,000 ± 8000335,000 ± 40,000484 ± 341:274:931:1SAAS-(235–246)P4A286 ± 34SAAS-(235–246)P5A172 ± 41SAAS-(235–246)P6A58 ± 16SAAS-(235–246)P8A143 ± 24SAAS-(235–246)P9A737 ± 110SAAS-(235–246)P10A177 ± 6.6SAAS-(235–246)P1K1,530 ± 198SAAS-(235–246)P4K177,500 ± 3,020SAAS-(235–246)P1KP4KNI (185 µm)SAAS-(235–246)P3AP5A13,700 ± 1,200127 ± 3027,700 ± 72010,817 ± 45411:1:218:85PenLen (rSAAS-(221–254)119 ± 1.519,300 ± 18001,400 ± 2004,630 ± 6001:162:12:39K i values were determined using the linear regression of Dixon plot and Graphit software as described under “Experimental Procedures.” NI, no inhibition, all data were acquired in duplicate and then averaged. The lowest Ki value is indicated by an underline. Open table in a new tab K i values were determined using the linear regression of Dixon plot and Graphit software as described under “Experimental Procedures.” NI, no inhibition, all data were acquired in duplicate and then averaged. The lowest Ki value is indicated by an underline. Overall, the data indicated that the 10-mer235VLGALLRVKR244 lacking any P′ residues was the most powerful PC1 inhibitor (K i ∼9 nm). This value is 5.7-fold lower than the ∼50.5 nm calculated for the K i of the C-terminally extended 12-mer 235VLGALLRVKRLE246 (Table II). Although both peptides are selective PC1 inhibitors, the C-terminal Leu-Glu extension resulted in an enhancement of selectivity for PC1 inhibition by 27- and 57-fold as compared with furin and PC5, respectively. Interestingly, the ∼100-fold lower inhibitory potency toward PC7 was not affected by the dipeptide insertion. Noticeably, Ala mutation of either the P1′ Leu or the P2′ Glu resulted in a drastic loss of potency and selectivity toward PC1 inhibition. This means that the nature of these residues is critical for the observed high PC1-selective inhibitory property of proSAAS-(235–246). Finally, it is worth mentioning that the Ala mutation of P1′ Leu resulted in a complete loss of PC1 selectivity as compared with furin, whereas the P2′ Glu to Ala mutation had a 14-fold lesser effect (Table II). This is reminiscent of the observed critical importance of P1′ Leu for the processing of prorenin that is cleaved by PC1 but not at all by furin (24Benjannet S. Reudelhuber T. Mercure C. Rondeau N. Chrétien M. Seidah N.G. J. Biol. Chem. 1992; 267: 11417-11423Abstract Full Text PDF PubMed Google Scholar). Finally, we note that the loss of selectivity toward PC1 versus PC5 and PC7 is somewhat similar for either Ala mutants but not as drastic as the P1′ mutation for furin. As indicated in Table II, PenLen or rat proSAAS-(221–254) inhibits PC1 with K i ∼120 nm, nearly 3- and 13-fold less efficiently than proSAAS-(235–246) and proSAAS-(235–244), respectively. It also displayed a reduced level of selectivity toward PC1 inhibition as compared with furin, PC7, and especially PC5 (TableII). In order to extend those data and include other convertases such as human PACE4 and yeast kexin, in Fig. 3we present in a bar graph format the extent of enzyme inhibition by 750 nm proSAAS peptides and their selected mutants. For this purpose, we used a 4-h stop time assay in which the level of each enzyme used was adjusted so as to give similar initial pERTKR-MCA activity. In agreement with the K i data (Table II), the shorter 10-mer is more potent than the 12-mer proSAAS peptide on PC1, furin, PC5, and PC7 (Fig. 3, first row). In contrast, PACE4 is not inhibited by either peptide even at this high concentration, whereas kexin is ∼50% inhibited by the 12-mer peptide and not at all by the 10-mer one. The chirality of inhibition is evident by the fact that the all-dextro derivative of the 10-mer peptide does not significantly affect the activity of any enzyme tested. Although not shown, Ala mutations at P1′ and P2′ follow the expected pattern from Table II (i.e. they are both important for the selective inhibition of PC1). The critical importance of the three basic residues at P1, P2, and P4 is also evident, since their Ala mutants lost most of their inhibitory properties against all enzymes (Table II, Fig. 3, second row). Furthermore, Arg to Lys mutations at either P1, P4, or P1 + P4 resulted in a drastic loss of PC1 inhibition, especially for the P4 or P1 + P4 mutations (Table II). These data attest to the critical importance of Arg at the P1 and P4 positions and a basic residue at P2. In addition, the aliphatic residues Val at P3 and P10, and Leu at P5, P6, and P9 also contribute to the inhibitory potency and selectivity of proSAAS-(235–246) toward PC1, with the P9 Leu and P3 Val positions being the most critical (Table II, Fig. 3, third andfourth rows). We also tested whether double P3 Val and P5 Leu to Ala mutations could further influence the inhibitory selectivity of proSAAS-(235–246) toward PC1 versus other convertases. Interestingly, the double mutant is a better inhibitor of furin (K i ∼127 nm) as compared with PC1, PC5, and PC7 (Table II). 246) and Its Ala Mutants by PCs—Previously, it was demonstrated that radioiodinated Tyr + mouse SAAS219–258 was internally cleaved by PC1 (17Cameron A. Fortenberry Y. Lindberg I. FEBS Lett. 2000; 473: 135-138Crossref PubMed Scopus (69) Google Scholar), suggesting that PC1 is inhibited by proSAAS by a mechanism similar to that of PC2 by 7B2 (13Apletalina E.V. Juliano M.A. Juliano L. Lindberg I. Biochem. Biophys. Res. Commun. 2000; 267: 940-942Crossref PubMed Scopus (21) Google Scholar, 25Lindberg I. Van den Herk W.H. Bui C. Batie C.J. Biochemistry. 1995; 34: 5486-5493Crossref PubMed Scopus (76) Google Scholar). However, the exact cleavage site of the PenLen peptide was not determined in this previous study (17Cameron A. Fortenberry Y. Lindberg I. FEBS Lett. 2000; 473: 135-138Crossref PubMed Scopus (69) Google Scholar). To determine this cleavage site as well as to examine whether human proSAAS-(235–246) behaves as a competitive substrate, it was incubated for 18 h with PC1 in the presence of the fluorogenic substrate pERTKR-MCA. The RP-HPLC of the crude digest and the mass spectral data of the isolated peaks are shown in Fig.4. Unlike the fluorogenic peptide, which was partially cleaved by PC1, the peptide proSAAS-(235–246) (R t = 33.7 min; (M + H)+ = 1,369) was completely digested, giving the expected decapeptide VLGALLRVKR244↓ (R t = 21.7 min; (M + H)+ = 1,123). The C-terminal dipeptide LE product eluted with the injection peak and was not analyzed. The two other peaks atR t = 23.1 and 15.1 min were characterized by mass spectrometry as the unreacted fluorogenic substrate pERTKR-MCA ((M + H)+ = 830) and its N-terminal product pERTKR-OH ((M + H)+ = 668). Using a similar approac" @default.
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• W2001045107 title "Inhibitory Specificity and Potency of proSAAS-derived Peptides toward Proprotein Convertase 1" @default.
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