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• W2073982436 abstract "Pyroglutamyl peptidase II (PPII), a highly specific membrane-bound omegapeptidase, removes N-terminal pyroglutamyl from thyrotropin-releasing hormone (<Glu-His-Pro-NH2), inactivating the peptide in the extracellular space. PPII and enzymes with distinct specificities such as neutral aminopeptidase (APN), belong to the M1 metallopeptidase family. M1 aminopeptidases recognize the N-terminal amino group of substrates or inhibitors through hydrogen-bonding to two conserved residues (Gln-213 and exopeptidase motif Glu-355 in human APN), whereas interactions involved in recognition of pyroglutamyl residue by PPII are unknown. In rat PPII, the conserved exopeptidase residue is Glu-408, whereas the other one is Ser-269. Given that variations in M1 peptidase specificity are likely due to changes in the catalytic region, we constructed three-dimensional models for the catalytic domains of PPII and APN. The models showed a salt bridge interaction between PPII-Glu-408 and PPII-Lys-463, whereas the equivalent APN-Glu-355 did not participate in a salt bridge. Docking of thyrotropin-releasing hormone in PPII model suggested that the pyroglutamyl residue interacted with PPII-Ser-269. According to our models, PPII-S269Q and -K463N mutations should leave Glu-408 in a physicochemical context similar to that found in M1 aminopeptidases; alternatively, PPII-S269E replacement might be sufficient to transform PPII into an aminopeptidase. These hypotheses were supported by site-directed mutagenesis; the mutants lost omegapeptidase but displayed alanyl-aminopeptidase activity. In conclusion, recognition of a substrate without an N-terminal charge requires neutralization of the aminopeptidase anionic binding site; furthermore, shortening of side chain at PPII-269 position is required for adjustment to the pyroglutamyl residue. Pyroglutamyl peptidase II (PPII), a highly specific membrane-bound omegapeptidase, removes N-terminal pyroglutamyl from thyrotropin-releasing hormone (<Glu-His-Pro-NH2), inactivating the peptide in the extracellular space. PPII and enzymes with distinct specificities such as neutral aminopeptidase (APN), belong to the M1 metallopeptidase family. M1 aminopeptidases recognize the N-terminal amino group of substrates or inhibitors through hydrogen-bonding to two conserved residues (Gln-213 and exopeptidase motif Glu-355 in human APN), whereas interactions involved in recognition of pyroglutamyl residue by PPII are unknown. In rat PPII, the conserved exopeptidase residue is Glu-408, whereas the other one is Ser-269. Given that variations in M1 peptidase specificity are likely due to changes in the catalytic region, we constructed three-dimensional models for the catalytic domains of PPII and APN. The models showed a salt bridge interaction between PPII-Glu-408 and PPII-Lys-463, whereas the equivalent APN-Glu-355 did not participate in a salt bridge. Docking of thyrotropin-releasing hormone in PPII model suggested that the pyroglutamyl residue interacted with PPII-Ser-269. According to our models, PPII-S269Q and -K463N mutations should leave Glu-408 in a physicochemical context similar to that found in M1 aminopeptidases; alternatively, PPII-S269E replacement might be sufficient to transform PPII into an aminopeptidase. These hypotheses were supported by site-directed mutagenesis; the mutants lost omegapeptidase but displayed alanyl-aminopeptidase activity. In conclusion, recognition of a substrate without an N-terminal charge requires neutralization of the aminopeptidase anionic binding site; furthermore, shortening of side chain at PPII-269 position is required for adjustment to the pyroglutamyl residue. Amino- or omegapeptidases hydrolyze the peptide bond linking the N-terminal amino acids of peptides, selecting either a free terminal amino group or a <Glu 2The abbreviations used are: <Glu, pyroglutamic acid; TRH, thyrotropin-releasing hormone; PPII, pyroglutamyl peptidase II; rPPII, rat PPII; AP, aminopeptidase; APN, aminopeptidase N; APA, aminopeptidase A; LTA4H, leukotriene A4 hydrolase; L-RAP, leukocyte-derived arginine aminopeptidase; F3, tricorn interacting factor 3; EGFP, enhanced green fluorescent protein; βNA, β-naphthylamide; SCR, structurally conserved regions; PA, pepstatin A. 2The abbreviations used are: <Glu, pyroglutamic acid; TRH, thyrotropin-releasing hormone; PPII, pyroglutamyl peptidase II; rPPII, rat PPII; AP, aminopeptidase; APN, aminopeptidase N; APA, aminopeptidase A; LTA4H, leukotriene A4 hydrolase; L-RAP, leukocyte-derived arginine aminopeptidase; F3, tricorn interacting factor 3; EGFP, enhanced green fluorescent protein; βNA, β-naphthylamide; SCR, structurally conserved regions; PA, pepstatin A. residue at the P1 substrate position. They have been grouped in families according to their catalytic mechanisms and amino acid sequences. The M1 family clusters zinc metallopeptidases widely distributed in nature, with homologues found in bacteria, fungi, plants, invertebrates, amphibians, birds, and mammals (1Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. 1998; (pp. , Academic Press, Inc., San Diego, CA): 994-1032Google Scholar, 2Rawlings N.D. Morton F.R. Barrett A.J. Nucleic Acids Res. 2006; 34: 270-272Crossref PubMed Scopus (463) Google Scholar). They are broadly expressed in animal tissues, where they play critical roles in maturation, activation, and degradation of peptides and, therefore, in a variety of physiological processes. Their abnormal expression has been linked to cancer and other pathological conditions such as cardiovascular disorders (3Nanus D.M. Clin. Cancer Res. 2003; 9: 6307-6309PubMed Google Scholar, 4Sato Y. Biol. Pharm. Bull. 2004; 27: 772-776Crossref PubMed Scopus (92) Google Scholar, 5Albiston A.L. Ye S. Chai S.Y. Protein Pept. Lett. 2004; 11: 491-500Crossref PubMed Scopus (63) Google Scholar). The M1 family contains aminopeptidases (AP) with varied specificities, defined in part by their preference for the type of N-terminal residue; among them are the neutral AP (APN; EC 3.4.11.2), the acidic AP (APA; EC 3.4.11.7) and the basic AP (EC 3.4.11.6), or the leukocyte-derived arginine AP (L-RAP; EC 3.4.). Each of these aminopeptidases has multiple tissue specific roles in vivo. For example, in brain APN controls vasopressin release through the hydrolysis of angiotensin III (6Zini S. Fournie-Zaluski M.C. Chauvel E. Roques B.P. Corvol P. Llorens-Cortes C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11968-11973Crossref PubMed Scopus (286) Google Scholar); peptidases belonging to the oxytocinase subfamily play important roles in maintenance of normal pregnancy, memory retention, blood pressure regulation, and antigen presentation (7Tsujimoto M. Hattori A. Biochim. Biophys. Acta. 2005; 1751: 9-18Crossref PubMed Scopus (134) Google Scholar). All members of the M1 family are aminopeptidases, except for pyroglutamyl peptidase II (PPII, EC 3.4.19.6), a membrane-bound omegapeptidase that catalyzes the inactivation of the neuropeptide thyrotropin-releasing hormone (TRH, <Glu-His-Pro-NH2) in the extracellular space (8Charli J.L. Cruz C. Vargas M.A. Joseph-Bravo P. Neurochem. Int. 1988; 13: 237-242Crossref PubMed Scopus (42) Google Scholar, 9Charli J.L. Méndez M. Vargas M.A. Cisneros M. Assai M. Joseph-Bravo P. Wilk S. Neuropeptides. 1989; 14: 191-196Crossref PubMed Scopus (36) Google Scholar, 10O'Cuinn G. O'Connor B. Elmore M. J. Neurochem. 1990; 54: 1-13Crossref PubMed Scopus (81) Google Scholar, 11Bauer K. Heuer H. Ifflander F. Peters A. Schmitmeier S. Shomburg L. Turwitt S. Wilkins M. Kenny A.J. Boustead C.M. Cell-Surface Peptidases In Health And Disease. 1997: 239-248Google Scholar, 12Charli J.L. Vargas M.A. Cisneros M. de Gortari P. Baeza M.A. Jasso P. Bourdais J. Pérez L. Uribe R.M. Joseph-Bravo P. Neurobiology. 1998; 6: 45-57PubMed Google Scholar). TRH is a neurohormone that regulates adenohypophysial secretions and a neurotransmitter with effects on multiple central nervous system functions, including cognition and locomotion (13O'Leary R.M. O'Connor B. J. Neurochem. 1995; 65: 953-963Crossref PubMed Scopus (54) Google Scholar, 14Morley J.E. Life Sci. 1979; 25: 1539-1550Crossref PubMed Scopus (266) Google Scholar, 15Horita A. Life Sci. 1998; 62: 1443-1448Crossref PubMed Scopus (99) Google Scholar, 16Griffiths E.C. Baris C. Visser T.J. Klootwijk W. Regul. Pept. 1985; 10: 145-155Crossref PubMed Scopus (21) Google Scholar, 17Nillni E.A. Sevarino K.A. Endocr. Rev. 1999; 20: 599-648Crossref PubMed Scopus (170) Google Scholar). Clinical studies have reported beneficial effects of its administration in several diseases, but these improvements are of short duration. The rapid inactivation of TRH by PPII in the central nervous system extracellular space (17Nillni E.A. Sevarino K.A. Endocr. Rev. 1999; 20: 599-648Crossref PubMed Scopus (170) Google Scholar, 18Kelly J.A. Essays Biochem. 1995; 30: 133-149PubMed Google Scholar) represents a significant drawback in its potential use as a therapeutic agent. Rat and human PPII cDNAs encode sequences with a high degree of conservation (19Schauder B. Schomburg L. Köhrle J. Bauer K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9534-9538Crossref PubMed Scopus (76) Google Scholar, 20Schomburg L. Turwitt S. Presche G. Lohmann D. Horsthemke B. Bauer K. Eur. J. Biochem. 1999; 265: 415-422Crossref PubMed Scopus (43) Google Scholar). Purification of the brain enzyme indicates that it is a glycoprotein composed of two identical subunits with a molecular mass of 230 kDa when solubilized with trypsin (21Wilk S. Wilk E. Neurochem. Int. 1989; 15: 81-89Crossref PubMed Scopus (43) Google Scholar, 22Bauer K. Eur. J. Biochem. 1994; 224: 387-396Crossref PubMed Scopus (46) Google Scholar). The primary structure of M1 membrane-anchored metallopeptidases includes a small intracellular N-terminal fragment, one transmembrane segment, and a large extracellular C-terminal region that holds the exopeptidase ((G/A)(A/G)MEN) and catalytic (HEXXHX18E) motifs. Soluble members share sequence homology with the ectodomains of M1 membrane peptidases; this similarity increases remarkably in the region surrounding the conserved motifs (20Schomburg L. Turwitt S. Presche G. Lohmann D. Horsthemke B. Bauer K. Eur. J. Biochem. 1999; 265: 415-422Crossref PubMed Scopus (43) Google Scholar). For PPII as for other glucinzins, it was proposed that the His residues and the C-terminal Glu within the catalytic motif (441HEXXHX18E464) coordinate the Zn2+ atom, whereas PPII-Glu-442 activates a water molecule, and PPII-Tyr-528 stabilizes the transition state (23Papadopoulos T. Kelly J.A. Bauer K. Biochemistry. 2001; 40: 9347-9355Crossref PubMed Scopus (16) Google Scholar). Sequence alignments among M1 peptidases show that rat PPII shares 34% amino acid identity with human APN, 32% with mouse APA, 31% with human L-RAP, or 26% with human leukotriene A4 hydrolase (LTA4H; EC 3.3.2.6) (Fig. 1). In contrast to APN, the most similar counterpart in the family, PPII, is a narrow-specificity enzyme hydrolyzing pyroglutamyl from TRH or very closely related tripeptides or tetrapeptides but not from longer peptides such as luteinizing hormone-releasing hormone, bombesin, or neurotensin (24O'Connor B. O'Cuinn G. Eur. J. Biochem. 1985; 150: 47-52Crossref PubMed Scopus (55) Google Scholar, 25Elmore M.A. Griffiths E.C. O'Connor B. O'Cuinn G. Neuropeptides. 1990; 15: 31-36Crossref PubMed Scopus (33) Google Scholar, 26O'Leary R.M. O'Connor B. Int. J. Biochem. Cell Biol. 1995; 27: 881-890Crossref PubMed Scopus (12) Google Scholar, 27Gallagher S.P. O'Connor B. Int. J. Biochem. Cell Biol. 1998; 30: 115-133Crossref PubMed Scopus (10) Google Scholar). PPII substrates have the general structure <Glu-XY, where X is a moderately bulky and uncharged residue, and Y is Pro, Ala, Trp, Pro-Gly, Pro-NH2 (TRH), Pro-β-naphthylamine (βNA), or Pro-7-amino-4-methyl coumarin (20Schomburg L. Turwitt S. Presche G. Lohmann D. Horsthemke B. Bauer K. Eur. J. Biochem. 1999; 265: 415-422Crossref PubMed Scopus (43) Google Scholar, 25Elmore M.A. Griffiths E.C. O'Connor B. O'Cuinn G. Neuropeptides. 1990; 15: 31-36Crossref PubMed Scopus (33) Google Scholar, 28Friedman T.C. Wilk S. J. Neurochem. 1986; 46: 1231-1239Crossref PubMed Scopus (70) Google Scholar, 29O'Connor B. O'Cuinn G. Eur. J. Biochem. 1984; 144: 271-278Crossref PubMed Scopus (93) Google Scholar, 30Kelly J.A. Slator G.R. Tipton K.F. Williams C.H. Bauer K. J. Biol. Chem. 2000; 275: 16746-16751Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Despite the importance of M1 peptidases in the metabolism of peptides, the structural determinants ensuring their strict exopeptidase action and specificity are poorly explored. LTA4H, a soluble bifunctional enzyme (epoxide hydrolase and aminopeptidase) was the first M1 metallopeptidase with a crystallographic structure solved (31Thunnissen M.M. Nordlund P. Haeggstrom J.Z. Nat. Struct. Biol. 2001; 8: 131-135Crossref PubMed Scopus (254) Google Scholar). Recently, the structure of the tricorn-interacting factor F3 (F3) was also reported (32Kyrieleis O.J.P. Goettig P. Kiefersauer R. Huber R. Brandstetter H. J. Mol. Biol. 2005; 349: 787-800Crossref PubMed Scopus (69) Google Scholar). The LTA4H and F3 structures consist of three and four domains. Domains I, II, and IV of F3 are equivalent to the three domains of the LTA4H structure, whereas the barrel-like β-sheet structure of domain III is a unique feature of F3. Superposition of the single domains of LTA4H and F3 demonstrates a variable degree of similarity; the N-terminal domain forming a saddle-like structure that covers the active site, and the thermolysin-like catalytic domain, including the zinc-binding residues, are very similar in these proteins. In contrast, the C-terminal domains are partially conserved and differ considerably in their relative positions (32Kyrieleis O.J.P. Goettig P. Kiefersauer R. Huber R. Brandstetter H. J. Mol. Biol. 2005; 349: 787-800Crossref PubMed Scopus (69) Google Scholar). The remarkable structural similarity between the LTA4H and F3 catalytic domains, the high sequence conservation around the M1 catalytic domains, and site-directed mutagenesis studies (20Schomburg L. Turwitt S. Presche G. Lohmann D. Horsthemke B. Bauer K. Eur. J. Biochem. 1999; 265: 415-422Crossref PubMed Scopus (43) Google Scholar, 33Rudberg P. Tholander F. Thunnissen M. Haeggström J.Z. J. Biol. Chem. 2002; 277: 1398-1404Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 34Luciani N. Marie-Claire C. Ruffet E. Beaumont A. Roques B.P. Fournié-Zaluski M.C. Biochemistry. 1998; 37: 686-692Crossref PubMed Scopus (100) Google Scholar, 35Vazeux G. Iturrioz X. Corvol P. Llorens-Cortés C. Biochem. J. 1998; 334: 407-413Crossref PubMed Scopus (71) Google Scholar, 36Rozenfeld R. Iturrioz X. Okada M. Maigret B. Llorens-Cortes C. Biochemistry. 2003; 42: 14785-14793Crossref PubMed Scopus (40) Google Scholar) suggest that they use a common catalytic mechanism and that different N-terminal residue preferences (neutral (APN), acidic (APA), arginyl (L-RAP), or PPII) are supported by discrete changes near the active site. In the M1 aminopeptidases, specific recognition of the free N-terminal group of substrates and inhibitors involves hydrogen bonding with two conserved residues. The structure of LTA4H in complex with the competitive inhibitor bestatin shows that Glu-271, located within the exopeptidase motif, and Gln-136 are positioned in the active site; both make hydrogen bonds to the free amine of the inhibitor, which chemically resembles a peptide substrate, suggesting their participation in the binding of the N-terminal group of substrates. Experimental analysis as well as examination of the x-ray structure of LTA4H-E271Q inactive mutant, indicates that Glu-271 carboxylate is not only involved in the N-terminal recognition but also has a critical role in the aminopeptidase activity (33Rudberg P. Tholander F. Thunnissen M. Haeggström J.Z. J. Biol. Chem. 2002; 277: 1398-1404Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). It is proposed that the counterparts of LTA4H-Glu-271, APA-Glu-352, and APN-Glu-355 interact with the free amino group of substrates and inhibitors via a hydrogen bond, with their negative charge stabilizing the transition state (34Luciani N. Marie-Claire C. Ruffet E. Beaumont A. Roques B.P. Fournié-Zaluski M.C. Biochemistry. 1998; 37: 686-692Crossref PubMed Scopus (100) Google Scholar, 35Vazeux G. Iturrioz X. Corvol P. Llorens-Cortés C. Biochem. J. 1998; 334: 407-413Crossref PubMed Scopus (71) Google Scholar). Additionally, APA-Glu-215, counterpart of LTA4H-Gln-136, is involved in the exopeptidase specificity by interacting with the N-terminal amine of the substrate, contributing together with the exopeptidase motif Glu-352 to the correct positioning of substrates and inhibitors in the active site (36Rozenfeld R. Iturrioz X. Okada M. Maigret B. Llorens-Cortes C. Biochemistry. 2003; 42: 14785-14793Crossref PubMed Scopus (40) Google Scholar). In conclusion, recognition of the α-amino group of substrates, or inhibitors, by M1 aminopeptidases implicates hydrogen bond and charge interaction with a glutamate residue within the exopeptidase motif (anionic binding site) as well as another hydrogen bond with the Glu or Gln residue equivalent to APA-Glu-215 or LTA4H-Gln-136. Unlike aminopeptidase substrates, TRH does not have an α-amino group at its N terminus, implying that in PPII substrate recognition differs from the rest of the M1 family members. In PPII, exopeptidase motif Glu (PPII-Glu-408) is conserved, and its replacement by Gln leads to a completely inactive enzyme, whereas the E408D mutant has a very low catalytic activity (due to a decreased Vmax value, whereas Km is not affected) (23Papadopoulos T. Kelly J.A. Bauer K. Biochemistry. 2001; 40: 9347-9355Crossref PubMed Scopus (16) Google Scholar). This suggests that position and negative charge of PPII-Glu-408 carboxylate are critical for catalysis but not for substrate binding. In the absence of structural information to understand the omegapeptidase specificity of PPII and to compare it with that of M1 aminopeptidases, we performed multiple alignments of M1 family members and constructed by homology modeling three-dimensional models for part of rat PPII, human APN, and L-RAP ectodomains; TRH was docked in the PPII model. We predicted that substituting one or two PPII-specific residues for M1 family residues was sufficient to migrate from PPII to alanyl-aminopeptidase specificity. Site-directed mutagenesis experiments supported these theoretical predictions. Materials—Restriction enzymes and DNA-modifying enzymes were obtained from New England Biolabs (Beverly, MA). DNA purifications were done using Qiagen (Valencia, CA) kits: QIAquick PCR purification kit, QIAquick gel extraction kit, and plasmid Mini and Midi kits. TaqDNA polymerase, pcDNA3.1/HisA expression vector, and liposome transfection reagent Lipofectamine 2000 were from Invitrogen. pEGFP-N3 expression vector and anti-enhanced green fluorescent protein (EGFP) antibody (BD Living Colors) were from Clontech (Palo Alto, CA). Monoclonal anti-poly-His/alkaline phosphatase conjugate (clone HIS-1), phenylmethylsulfonyl fluoride, iodoacetamide, pepstatin A (PA), and deoxyribonuclease I (DNase I) were obtained from Sigma. pBluescript II KS and SK were from Stratagene (La Jolla, CA). Synthetic substrates Ala-βNA, (Glu-βNA, <Glu-βNA, and TRH-βNA were from Bachem (Bunderdorf, Switzerland). Actinonin was from Alexis Biochemicals (San Diego, CA). Nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate were from Roche. Immobilon-P membranes were from Millipore Corp. (Bedford, MA). For DNA sequencing, we used a Dye Terminator Cycle Sequencing Ready Reaction kit from Applied Biosystems (Foster City, CA). Multiple alignments were performed with ClustalW. Modeling of Catalytic Domains of PPII, PPII Mutants, APN, and L-RAP (Lys-392)—By homology modeling, three-dimensional models were constructed for the catalytic domains of rat PPII and human APN (residues 244–623 and 190–567) using as template the x-ray crystallographic structure of human LTA4H (31Thunnissen M.M. Nordlund P. Haeggstrom J.Z. Nat. Struct. Biol. 2001; 8: 131-135Crossref PubMed Scopus (254) Google Scholar). PPII and APN models contained only the region most conserved between template and PPII or APN because we were interested in studying the structure-function relationship for the active sites. The method was similar to that used for APA modeling (37Rozenfeld R. Iturrioz X. Maigret B. Llorens-Cortes C. J. Biol. Chem. 2002; 277: 29242-29252Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). InsightII (Accelrys Software Inc., San Diego, CA) was used to construct the models. We aligned the sequences of APN or PPII with that of LTA4H taking into account the multiple alignment between several proteins of the M1 family and the secondary structure of the template as well as experimental information from previous site-directed mutagenesis studies. Aligned regions, in which we assumed that the secondary structure was conserved, were defined as structurally conserved regions (SCRs), and unaligned regions were defined as loops. Initial models were obtained by transfer of coordinates from the template to PPII or APN sequences in the SCRs and completed by adding the missing loops using the loop search tool. The loop length, the anchor regions corresponding to the three residues at either end of the loop, and the position of critical residues were taken into account. Steric conflicts were corrected during model building using the rotamer library. In each case we minimized the initial model using the conjugate gradient method, a cutoff of 20 Å, keeping the backbone fixed (except for the loops) and the side chains free to move. A second round of minimization was repeated without any restriction. We used the Accelrys CFF97 force field. We added a Zn2+ atom based on its position in the template structure. The minimized model was placed in a water box (dimensions: 80, 95, 80 Å), and 5 or 15 water molecules were substituted with sodium ions to achieve neutrality for PPII or APN system. To eliminate crashes between sodium ions and solvent, we deleted water molecules surrounding the ions (radius = 2 Å). The completed systems (protein, Zn2+, counter-ions, and water molecules) were transferred to NAMD using CHARMM27 force field. Once in NAMD, the refinement procedure continued with several energy minimization steps followed by molecular dynamics. We began by fixing the backbone of the protein, whereas side chains, water molecules, and ions were variables; in a subsequent step the whole system was relaxed. Periodic boundary conditions were used with the same cutoff to truncate non-bonded interactions. The dielectric constant was 1. We checked the stability of the models during molecular dynamics (500 ps); backbone atoms did not have residual mean square deviations greater than 1.5 Å. PPII mutants were modeled by substituting the desired side chain in PPII model followed by energy minimization and 300 ps of molecular dynamics. Because of the sequence similarity between L-RAP and LTA4H sequences, the crystal structure of human LTA4H was also chosen as a template for construction of a three-dimensional model of human L-RAP (Lys-392) (residues 118–494); three-dimensional modeling was performed by using CPH model 2.0 homology modeling server (38Lund O. Nielsen M. Lundegaard C. Worning P. Abstract at the CASP5 conference. 2002; : A102Google Scholar). Docking of TRH in PPII Model—To understand the structural basis of TRH recognition by PPII, we performed docking studies. Using InsightII, TRH was manually docked in the PPII catalytic domain model according to bestatin position in the template structure (31Thunnissen M.M. Nordlund P. Haeggstrom J.Z. Nat. Struct. Biol. 2001; 8: 131-135Crossref PubMed Scopus (254) Google Scholar). All water molecules surrounding the peptide in a radius of 2 Å were deleted, and the PPII-TRH system was subjected to minimization steps followed by molecular dynamics (500 ps). Expression Plasmids and Site-directed Mutagenesis—Expression vectors for rat PPII (pN3/rPPII), poly-His-tagged rPPII (pcDNA3.1/HisA-rPPII), and EGFP fused in-frame to the C terminus of rPPII (pEGFPN3/rPPII) were constructed as previously described (39Chávez-Gutiérrez L. Bourdais J. Aranda G. Vargas M.A. Matta-Camacho E. Ducancel F. Segovia L. Joseph-Bravo P. Charli J.L. J. Neurochem. 2005; 92: 807-817Crossref PubMed Scopus (13) Google Scholar). In pcDNA3.1/HisA-rPPII, the rPPII coding sequence was in-frame C-terminal to the poly-His tag. pEGFP-N3 expression vector for EGFP was used as the control vector in transfections. Site-directed mutagenesis was done according to the one-step overlap extension PCR method (40Urban A. Neukirchen S. Jaeger K.E. Nucleic Acids Res. 1997; 25: 2227-2228Crossref PubMed Scopus (181) Google Scholar). The PCR amplifications were performed with 35 cycles of denaturation (92 °C, 30 s), annealing (55 °C, 30 s), and extension (72 °C, 1 min). Fragment XmaI/SacI of rat PPII cDNA was cloned into both pBlueScript II/KS and SK. These vectors served as PCR templates in the presence of one universal primer (T7, 5′-GTAATACGACTCACTCACTATAGGGC-3′) and 2 mutagenic primers, each containing the mutagenic substitution into a 20-nucleotide overlapping region. S269E, 5′-ACTCAGTTTGAACCTACGCATGCCAGGAAG-3′ and 5′-ATGCGTAGGTTCAAACTGAGTAACACCGAG-3′; S259A, 5′-ACTCAGTTTGCACCTACGCATGCCAGGAAG-3′ and 5′-ATGCGTAGGTGCAAACTGAGTAACACCGAG-3′; S269Q, 5′-ACTCAGTTTCAACCTACGCATGCCAGGAAG-3′ and 5′-ATGCGTAGGTTGAAACTGAGTAACACCGAG-3′; K463N, 5′-GTGTGGTTGAACGAAGGCTTTGCTCACTAC-3′ and 5′-AAAGCCTTCGTTCAACCACACATCTTCCCA-3′; K463R, 5′-GTGTGGTTGAGGGAAGGCTTTGCTCACTAC-3′ and 5′-AAAGCCTTCCCTCAACCACACATCTTCCCA-3′; K463Q, 5′-GTGTGGTTGCAGGAAGGCTTTGCTCACTAC-3′ and 5′-AAAGCCTTCCTGCAACCACACATCTTCCCA-3′. PCR products were digested (XmaI/SacI), separated by gel electrophoresis, and purified using the gel extraction kit. Each mutant sequence was subcloned back into the wild type, poly-His, or EGFP-tagged PPII expression vector. Double mutants were constructed sequentially using the same protocol. The presence of the mutation and the absence of nonspecific mutations were confirmed by DNA sequencing. Cell Culture, Transfection, Membrane Preparation, and Fluorescence Microscopy—COS-7 or C6 glioma cells were cultured and transfected as previously described (39Chávez-Gutiérrez L. Bourdais J. Aranda G. Vargas M.A. Matta-Camacho E. Ducancel F. Segovia L. Joseph-Bravo P. Charli J.L. J. Neurochem. 2005; 92: 807-817Crossref PubMed Scopus (13) Google Scholar). Cells were collected 48 h post-transfection, and total membranes were prepared essentially as described (41Bourdais J. Romero F. Urostegui B. Cisneros M. Joseph-Bravo P. Charli J.L. Neuropeptides. 2000; 34: 83-88Crossref PubMed Scopus (8) Google Scholar). Briefly, cells were homogenized in 50 mm potassium phosphate buffer, pH 7.5 (buffer A), 0.3 μm phenylmethylsulfonyl fluoride, 1 μm iodoacetamide, 1 μm PA, 2.5 mm MgCl2, 0.1 mg/ml DNase I by freezing and thawing on ice (3×). Total membranes were collected by centrifugation (90,000 × g, 45 min), the pellet was washed once with buffer A, 1 m NaCl, and centrifugation was repeated. Finally, the pellet was homogenized in buffer A and stored at –80 °C until use. Protein concentrations were determined by the Bradford assay (42Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). For microscopic analysis, cells were observed under an eclipse TE300 microscope (Nikon, Melville NY) equipped with the cool snap software at 40× magnification. EGFP fluorescence was detected using the EPI-FL filter block (Nikon). SDS-PAGE and Western Blot Analysis—Membrane preparations were submitted to reducing SDS-polyacrylamide gel electrophoresis, proteins were transferred, and poly-His or EGFP-tagged PPII (wild type or mutants) was detected as described (39Chávez-Gutiérrez L. Bourdais J. Aranda G. Vargas M.A. Matta-Camacho E. Ducancel F. Segovia L. Joseph-Bravo P. Charli J.L. J. Neurochem. 2005; 92: 807-817Crossref PubMed Scopus (13) Google Scholar). The protein expression levels were estimated by densitometric scanning with a Fluor-S Multi-Imager (Bio-Rad). Peptidase Activity Determination—PPII activity was determined using 400 μm TRH-βNA as substrate in a coupled assay with excess dipeptidyl aminopeptidase IV (EC 3.4.14.5) essentially as described (41Bourdais J. Romero F. Urostegui B. Cisneros M. Joseph-Bravo P. Charli J.L. Neuropeptides. 2000; 34: 83-88Crossref PubMed Scopus (8) Google Scholar); assay buffer (50 mm Na3PO4, pH 7.5) included 0.2 mm N-ethyl maleimide, an inhibitor of pyroglutamyl peptidase I (EC 3.4.19.3), and 0.2 mm bacitracin, an inhibitor of prolyl endopeptidase (EC 3.4.21.26); both are soluble enzymes able to degrade TRH in vitro. Alanyl- or glutamylaminopeptidases activities were assayed with 400 μm Ala-βNA or 400 μm Glu-βNA in 100 mm Tris-HCl, pH 7.5. To determine pyroglutamyl peptidase activity, assay buffer (50 mm Na3PO4, pH 7.5) included 1 mm N-ethylmaleimide; the enzymatic reaction was initiated by the addition of 400 μm <Glu-βNA. All enzymatic assays were performed at 37 °C under initial velocity conditions in duplicate or triplicate, and their mean was taken as one determination for each independent transfection. Released βNA was determined in a fluorometer (excitation, 335 nm; emission, 410 nm). Activities of wild type PPII and mutants were normalized by total protein or PPII expression levels for non-tagged or poly-His-tagged proteins. Multiple alignments for the M1 family were performed to study the conservation of the residues involved in the recognition of the α-amino group of substrates and inhibitors by M1 aminopeptidases. Exopeptidase motif Glu" @default.
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• W2073982436 date "2006-07-01" @default.
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• W2073982436 title "Homology Modeling and Site-directed Mutagenesis of Pyroglutamyl Peptidase II" @default.
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• W2073982436 doi "https://doi.org/10.1074/jbc.m601392200" @default.
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