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- ItemSomente Metadadados1.70 angstrom X-ray structure of human apo kallikrein 1: Structural changes upon peptide inhibitor/substrate binding(Wiley-Blackwell, 2005-03-01) Laxmikanthan, G.; Blaber, S. I.; Bernett, M. J.; Scarisbrick, I. A.; Juliano, M. A.; Blaber, M.; Florida State Univ; Mayo Clin & Mayo Grad Sch Med; Universidade Federal de São Paulo (UNIFESP)Human kallikreins are serine proteases that comprise a recently identified large and closely related 15-member family. the kallikreins include both regulatory- and degradative-type proteases, impacting a variety of physiological processes including regulation of blood pressure, neuronal health, and the inflammatory response. While the function of the majority of the kallikreins remains to be elucidated, two members are useful biomarkers for prostate cancer and several others are potentially useful biomarkers for breast cancer, Alzheimer's, and Parkinson's disease. Human tissue kallikrein (human K1) is the best functionally characterized member of this family, and is known to play an important role in blood pressure regulation. As part of this function, human K1 exhibits unique dual-substrate specificity in hydrolyzing low molecular weight kininogen between both Arg-Ser and Met-Lys sequences. We report the X-ray crystal structure of mature, active recombinant human apo K1 at 1.70 Angstrom resolution. the active site exhibits structural features intermediate between that of apo and pro forms of known kallikrein structures. the S2 to S2' pockets demonstrate a variety of conformational changes in comparison to the porcine homolog of K1 in complex with peptide inhibitors, including the displacement of an extensive solvent network. These results indicate that the binding of a peptide substrate contributes to a structural rearrangement of the active-site Ser 195 resulting in a catalytically competent juxtaposition with the active-site His 57. the solvent networks within the S1 and S1' pockets suggest how the Arg-Ser and Met-Lys dual substrate specificity of human K1 is accommodated. Proteins (C) 2005 Wiley-Liss, Inc.
- ItemSomente MetadadadosBmSI-7, a novel subtilisin inhibitor from Boophilus microplus, with activity toward Pr1 proteases from the fungus Metarhizium anisopliae(Elsevier B.V., 2008-02-01) Sasaki, Sergio D. [UNIFESP]; Lima, Cdssia A. de [UNIFESP]; Lovato, Diogo V. [UNIFESP]; Juliano, Maria A. [UNIFESP]; Torquato, Ricardo J. S. [UNIFESP]; Tanaka, Aparecida S. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)BmSI-7 and BmSI-6, two Boophilus microplus subtilisin inhibitors (BmSI) were purified and characterized from eggs. the inhibitors isolated by classical purification methods presented molecular masses of 7408 and 7271 Da, respectively, by MALDI-TOF-MS. Both BmSI-7 and BmSI-6 inhibited neutrophil elastase (K-i 0.4 and 0.3 nM) and subtilisin A (K-i 1.4 nM for both inhibitors). They also strongly inhibited Pr1 proteases from the fungus Metarhizium anisopliae; BmSI-7 (K-i 50 nM) and BmSI-6 (K-i 2.2 nM). the BmSI-7 full length cDNA was obtained using amino acid sequence information of BmSI-7 peptides generated by proteolytic digestion. BmSI-7 belongs to trypsin inhibitor like cysteine rich domain family (TIL), and it is transcribed in ovary, fat body, gut, salivary gland and haemocytes. BmSI-7 is the first TIL inhibitor described with inhibitory activity toward subtilisin A and Pr1 proteases of entomopathogenic fungi. (C) 2007 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosDiscriminating between the activities of human cathepsin G and chymase using fluorogenic substrates(Wiley-Blackwell, 2011-08-01) Korkmaz, Brice; Jegot, Gwenhael; Lau, Laurie C.; Thorpe, Michael; Pitois, Elodie; Juliano, Luiz [UNIFESP]; Walls, Andrew F.; Hellman, Lars; Gauthier, Francis; Univ Tours; Southampton Gen Hosp; Uppsala Univ; Universidade Federal de São Paulo (UNIFESP)Cathepsin G (CG) (EC 3.4.21.20) and chymase (EC 3.4.21.39) are two closely-related chymotrypsin-like proteases that are released from cytoplasmic granules of activated mast cells and/or neutrophils. We investigated the potential for their substrate-binding subsites to discriminate between their substrate specificities, aiming to better understand their respective role during the progression of inflammatory diseases. in addition to their preference for large aromatic residues at P1, both preferentially accommodate small hydrophilic residues at the S1' subsite. Despite significant structural differences in the S2' subsite, both prefer an acidic residue at that position. the Ala226/Glu substitution at the bottom of the CG S1 pocket, which allows CG but not chymase to accommodate a Lys residue at P1, is the main structural difference, allowing discrimination between the activities of these two proteases. However, a Lys at P1 is accommodated much less efficiently than a Phe, and the corresponding substrate is cleaved by beta 2-tryptase (EC 3.4.21.59). We optimized a P1 Lys-containing substrate to enhance sensitivity towards CG and prevent cleavage by chymase and beta 2-tryptase. the resulting substrate (ABZ-GIEPKSDPMPEQ-EDDnp) [ where ABZ is O-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] was cleaved by CG but not by chymase and tryptase, with a specificity constant of 190 mM(-1).s(-1). This allows the quantification of active CG in cells or tissue extracts where it may be present together with chymase and tryptase, as we have shown using a HMC-1 cell homogenate and a sputum sample from a patient with severe asthma.
- ItemSomente MetadadadosLow molecular weight squash trypsin inhibitors from Sechium edule seeds(Elsevier B.V., 2006-02-01) Laure, H. J.; Faca, V. M.; Izumi, C.; Padovan, J. C.; Greene, L. J.; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP); Rockefeller UnivNine chromatographic components containing trypsin inhibitor activity were isolated from Sechium edule seeds by acetone fractionation, gel filtration, affinity chromatography and RP-HPLC in an overall yield of 46% of activity and 0.05% of protein. the components obtained with highest yield of total activity and highest specific activity were sequenced by Edman degradation and their molecular masses determined by mass spectrometry. the inhibitors contained 31, 32 and 27 residues per molecule and their sequences were: SETI-IIa, EDRKCPKILMRCKRDSDCLAKCTCQESGYCG; SETI-IIb, EEDRKCPKILMRCKRDSDCLAKCTCQESGYCG and SETI-V, CPRILMKCKLDTDCFPTCTCRPSGFCG. SETI-IIa and SETI-IIb, which differed by an amino-terminal E in the IIb form, were not separable under the conditions employed. the sequences are consistent with consensus sequences obtained from 37 other inhibitors: CPriIlmeCk_DSDCla_C_C_G_CG, where capital letters are invariant amino acid residues and lower case letters are the most preserved in this position. SETI-II and SETI-V form complexes with trypsin with a 1: 1 stoichiometry and have dissociation constants of 5.4 x 10(-11) M and 1.1 x 10(-9) M, respectively. (c) 2005 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosMeasurement of free and membrane-bound cathepsin G in human neutrophils using new sensitive fluorogenic substrates(Portland Press, 2002-09-15) Attucci, Sylvie; Korkmaz, Brice; Juliano, Luiz [UNIFESP]; Hazouard, Eric; Girardin, Catherine; Brillard-Bourdet, Michele; Rehault, Sophie; Anthonioz, Phillipe; Gauthier, Francis; Univ Tours; Universidade Federal de São Paulo (UNIFESP)Activated human polymorphonuclear neutrophils at inflammatory sites release the chymotrypsin-like protease cathepsin G, together with elastase and proteinase 3 (myeloblastin), from their azurophil granules. the low activity of cathepsin G on synthetic substrates seriously impairs studies designed to clarify its role in tissue inflammation. We have solved this problem by producing new peptide substrates with intramolecularly quenched fluorescence. These substrates were deduced from the sequence of putative protein targets of cathepsin G, including the reactive loop sequence of serpin inhibitors and the N-terminal domain of the protease-activated receptor of thrombin, PAR-1. Two substrates were selected, Abz-TPFSGQ-EDDnp and Abz-EPFWEDQ-EDDnp, that are cleaved very efficiently by cathepsin G but not by neutrophil elastase or proteinase 3, with specificity constants (k(cat)/K-m) in the 10(5) M-1 (.) s(-1) range. They can be used to measure subnanomolar concentrations of free enzyme in vitro and at the surface of neutrophils purified from fresh human blood. Purified neutrophils express 0.02-0.7 pg of cathepsin G/cell (n = 15) at their surface. This means that about 10(4) purified cells may be enough to record cathepsin G activity within minutes. This may be most important for investigating the role of cathepsin G as an inflammatory agent, especially in bronchoalveolar lavage fluids from patients with pulmonary inflammatory disorders.
- ItemSomente MetadadadosPurification, biochemical and functional characterization of miliin, a new thiol-dependent serine protease isolated from the latex of Euphorbia milii(Bentham Science Publ Ltd, 2008-07-01) Moro, L. P.; Murakami, M. T.; Cabral, H.; Vidotto, A.; Tajara, E. H.; Arni, R. K.; Juliano, L. [UNIFESP]; Bonilla-Rodriguez, G. O.; UNESP; Ctr Struct Mol Biol; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)Miliin, a new thiol-dependent serine protease purified from the latex of Euphorbia milii possesses a molecular weight of 79 kDa, an isoelectric point of 4.3 and is optimally active at 60 degrees C in the pH range of and 7.5-11.0. Activity tests indicate that milliin is a thiol-dependent serine protease.
- ItemSomente MetadadadosSpecificity of S '(1) and S '(2) subsites of human tissue kallikrein using the reactive-centre loop of kallistatin: the importance of P '(1) and P '(2) positions in design of inhibitors(Portland Press, 2003-05-01) Pimenta, Daniel C. [UNIFESP]; Fogaca, Sandro E.; Melo, Robson L.; Juliano, Luiz [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)We have demonstrated that the S-1' and S-2', subsites of human tissue kallikrein (hK1) play determinant roles in the recognition and hydrolysis of substrates. the presence of serine at position P-1' and arginine at P-2' resulted in the best substrate, Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, which was derived from the kallistatin reactive-centre loop sequence and quencher groups o-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp). Serine and arginine are also the residues at positions P-1' and P-2' in human kininogen, from which hK1 releases Lys-bradykinin. Several peptide analogues of Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, in which the Ser and Arg residues were substituted with various other amino acids, were synthesized and tested as substrates. Most of them were hydrolysed slowly, although they showed significant binding to hK1, as demonstrated by their competitive inhibition constants (K-i). Using this information, six peptides were designed, synthesized and assayed as inhibitors of hK1. Abz-Lys-Phe-Phe-Pro-Arg-Gln-EDDnp, Abz-Lys-Phe-Arg-Pro-Arg-Gln-EDDnp and acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH2 inhibited hK1 in the range 20-30 nM (letters in italics denote the D-form of the amino acid). the peptide acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH2 was a weak inhibitor for other serine proteases, as indicated by the higher K-i values compared with hK1, but this peptide was a potent inhibitor of human plasma kallikrein, which has a Ki value of 8 nM. This result was surprising, since this enzyme is known to be a restricted arginyl-hydrolase. in conclusion, acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH2 can be used as a leader compound to design specific inhibitors for hK1, plasma kallikrein, or for both at same time, if the inhibition of kinin release is the main goal.
- ItemSomente MetadadadosSynthesis, biological evaluation, and docking studies of PAR2-AP-derived pseudopeptides as inhibitors of kallikrein 5 and 6(Walter de Gruyter Gmbh, 2015-01-01) Severino, Beatrice; Fiorino, Ferdinando; Corvino, Angela; Caliendo, Giuseppe; Santagada, Vincenzo; Assis, Diego Magno [UNIFESP]; Oliveira, Juliana R. [UNIFESP]; Juliano, Luiz [UNIFESP]; Manganelli, Serena; Benfenati, Emilio; Frecentese, Francesco; Perissutti, Elisa; Juliano, Maria Aparecida [UNIFESP]; Univ Naples Federico II; Universidade Federal de São Paulo (UNIFESP); Ist Ric Farmacol Mario NegriA series of protease activated receptor 2 activating peptide (PAR2-AP) derivatives (1-15) were designed and synthesized. the obtained compounds were tested on a panel of human kallikreins (hKLK1, hKLK2, hKLK5, hKLK6, and hKLK7) and were found completely inactive toward hKLK1, hKLK2, and hKLK7. Aiming to investigate the mode of interaction between the most interesting compounds and the selected hKLKs, docking studies were performed. the described compounds distinguish the different human tissue kallikreins with compounds 1 and 5 as the best hKLK5 and hKLK6 inhibitors, respectively.