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Luiz Juliano - One of the best experts on this subject based on the ideXlab platform.
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characterization of thimet Oligopeptidase and neurolysin activities in b16f10 nex2 tumor cells and their involvement in angiogenesis and tumor growth
Molecular Cancer, 2007Co-Authors: Thaysa Paschoalin, Elaine G Rodrigues, Adriana K Carmona, Hugo P. Monteiro, Maria Aparecida Juliano, Luiz Juliano, Vitor Oliveira, Luiz R. TravassosAbstract:Background: Angiogenesis is a fundamental process that allows tumor growth by providing nutrients and oxygen to the tumor cells. Beyond the oxygen diffusion limit from a capillary blood vessel, tumor cells become apoptotic. Angiogenesis results from a balance of pro- and antiangiogenic stimuli. Endogenous inhibitors regulate enzyme activities that promote angiogenesis. Tumor cells may express pro-angiogenic factors and hydrolytic enzymes but also kinin-degrading Oligopeptidases which have been investigated. Results: Angiogenesis induced by B16F10-Nex2 melanoma cells was studied in a co-culture with HUVEC on Matrigel. A stimulating effect on angiogenesis was observed in the presence of B16F10Nex2 lysate and plasma membrane. In contrast, the B16F10-Nex2 culture supernatant inhibited angiogenesis in a dose-dependent manner. This effect was abolished by the endo-Oligopeptidase inhibitor, JA-2. Thimet Oligopeptidase (TOP) and neurolysin activities were then investigated in B16F10-Nex2 melanoma cells aiming at gene sequencing, enzyme distribution and activity, influence on tumor development, substrate specificity, hydrolytic products and susceptibility to inhibitors. Fluorescence resonance energy transfer (FRET) peptides as well as neurotensin and bradykinin were used as substrates. The hydrolytic activities in B16F10-Nex2 culture supernatant were totally inhibited by o-phenanthrolin, JA-2 and partially by Pro-Ile. Leupeptin, PMSF, E-64, Z-Pro-Prolinal and captopril failed to inhibit these hydrolytic activities. Genes encoding M3A enzymes in melanoma cells were cloned and sequenced being highly similar to mouse genes. A decreased proliferation of B16F10-Nex2 cells was observed in vitro with specific inhibitors of these Oligopeptidases. Active rTOP but not the inactive protein inhibited melanoma cell development in vivo increasing significantly the survival of mice challenged with the tumor cells. On Matrigel, rTOP inhibited the bradykinin – induced angiogenesis. A possible regulation of the homologous tumor
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Characterization of thimet Oligopeptidase and neurolysin activities in B16F10-Nex2 tumor cells and their involvement in angiogenesis and tumor growth
Molecular Cancer, 2007Co-Authors: Thaysa Paschoalin, Elaine G Rodrigues, Adriana K Carmona, Hugo P. Monteiro, Maria Aparecida Juliano, Luiz Juliano, Vitor Oliveira, Luiz R. TravassosAbstract:Background Angiogenesis is a fundamental process that allows tumor growth by providing nutrients and oxygen to the tumor cells. Beyond the oxygen diffusion limit from a capillary blood vessel, tumor cells become apoptotic. Angiogenesis results from a balance of pro- and anti-angiogenic stimuli. Endogenous inhibitors regulate enzyme activities that promote angiogenesis. Tumor cells may express pro-angiogenic factors and hydrolytic enzymes but also kinin-degrading Oligopeptidases which have been investigated. Results Angiogenesis induced by B16F10-Nex2 melanoma cells was studied in a co-culture with HUVEC on Matrigel. A stimulating effect on angiogenesis was observed in the presence of B16F10-Nex2 lysate and plasma membrane. In contrast, the B16F10-Nex2 culture supernatant inhibited angiogenesis in a dose-dependent manner. This effect was abolished by the endo-Oligopeptidase inhibitor, JA-2. Thimet Oligopeptidase (TOP) and neurolysin activities were then investigated in B16F10-Nex2 melanoma cells aiming at gene sequencing, enzyme distribution and activity, influence on tumor development, substrate specificity, hydrolytic products and susceptibility to inhibitors. Fluorescence resonance energy transfer (FRET) peptides as well as neurotensin and bradykinin were used as substrates. The hydrolytic activities in B16F10-Nex2 culture supernatant were totally inhibited by o -phenanthrolin, JA-2 and partially by Pro-Ile. Leupeptin, PMSF, E-64, Z-Pro-Prolinal and captopril failed to inhibit these hydrolytic activities. Genes encoding M3A enzymes in melanoma cells were cloned and sequenced being highly similar to mouse genes. A decreased proliferation of B16F10-Nex2 cells was observed in vitro with specific inhibitors of these Oligopeptidases. Active rTOP but not the inactive protein inhibited melanoma cell development in vivo increasing significantly the survival of mice challenged with the tumor cells. On Matrigel, rTOP inhibited the bradykinin – induced angiogenesis. A possible regulation of the homologous tumor enzyme in the perivascular microenvironment is suggested based on the observed rTOP inhibition by an S-nitrosothiol NO donor. Conclusion Data show that melanoma cells secrete endo-Oligopeptidases which have an important role in tumor proliferation in vitro and in vivo. rTOP inhibited growth of subcutaneously injected B16F10-Nex2 cells in mice. TOP from tumor cells and bradykinin in endothelial cells are two antagonist factors that may control angiogenesis essential for melanoma growth. A regulatory role of NO or S-nitrosothiols is suggested.
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Tropolysin, a new Oligopeptidase from African trypanosomes.
Biochemistry, 2005Co-Authors: Rory E. Morty, Vitor Oliveira, István Vadász, Patrick Bulau, Vincent Dive, Werner Seeger, Luiz JulianoAbstract:Oligopeptidases are emerging as important pathogenic factors and therapeutic targets in trypanosome infections. We describe here the purification, cloning, and biochemical analysis of a new Oligopeptidase from two pathogenic African trypanosomes. This Oligopeptidase, which we have called tropolysin (encoded by the trn gene), represents an evolutionarily distant member of the M3A subfamily of metallopeptidases, ancestral to thimet Oligopeptidase, neurolysin, and saccharolysin. The trn gene was present as a single copy per haploid genome, was expressed in both the mammalian and insect stages of the parasite life cycle, and encoded an 84 kDa protein. Both purified and hyperexpressed tropolysin hydrolyzed bradykinin-derived fluorogenic peptide substrates at restricted sites, with an alkaline pH optimum, and were activated by dithiothreitol and reduced glutathione and by divalent metal cations, in the order Zn(2+) > Co(2+) > Mn(2+). Under oxidizing conditions, tropolysin reversibly formed inactive multimers. Tropolysin exhibited a preference for acidic amino acid side chains in P(4), hydrophobic side chains in P(3), and hydrophobic or large uncharged side chains in P(1), P(1)', and P(3)', while the S(2)' site was unselective. Highly charged residues were not tolerated in P(1)'. Tropolysin was responsible for the bulk of the kinin-degrading activity in trypanosome lysates, potently (k(cat) approximately 119 s(-)(1)) inactivated the vasoactive kinins bradykinin and kallidin, and generated angiotensin(1-7) from angiotensin I. This hydrolysis both abolished the capacity of bradykinin to stimulate the bradykinin B(2) receptor and abrogated bradykinin prohypotensive properties in vivo, raising the possibility that tropolysin may play a role in the dysregulated kinin metabolism observed in the plasma of trypanosome-infected hosts.
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Characterization of thimet- and neurolysin-like activities in Escherichia coli M3A peptidases and description of a specific substrate
Archives of biochemistry and biophysics, 2005Co-Authors: Thaysa Paschoalin, Adriana K Carmona, Luiz Juliano, Vitor Oliveira, Luiz R. TravassosAbstract:Abstract M3A Oligopeptidases from Escherichia coli , with hydrolytic properties similar to Zn-dependent mammalian thimet Oligopeptidase (EP 24.15) and neurolysin (EP 24.16), were studied aiming at identification of comparative enzyme and substrate specificity, hydrolytic products, and susceptibility to inhibitors. Fluorescent peptides, neurotensin (NT) and bradykinin (BK), were used as substrates for bacterial lysates. Bacterial enzymes were totally inhibited by o -phenanthrolin, JA-2 and partially by Pro-Ile, but not by leupeptin, PMSF, E-64, and Z-Pro-Prolinal, using internally quenched Abz-GFSPFRQ-EDDnp as substrate. The molecular mass of the bacterial Oligopeptidase activity (77–78 kDa) was determined by gel filtration, and the effect of inhibitors, including captopril, suggested that it results from a combination of Oligopeptidase A (OpdA) and peptidyl dipeptidase Dcp (77.1 and 77.5 kDa, respectively). Recombinant OpdA cloned from the same E. coli strain entirely reproduced the primary cleavage of fluorescent peptides, NT and BK, by the bacterial lysate. Genes encoding these M3A enzymes were those recognized in E. coli genome, bearing identity at the amino acid level (25–31%) with mammalian Zn-dependent Oligopeptidases. We also describe a substrate, Abz-GFSPFRQ-EDDnp, that differentiates bacterial and mammalian Oligopeptidases.
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subsite specificity s3 s2 s1 s2 and s3 of Oligopeptidase b from trypanosoma cruzi and trypanosoma brucei using fluorescent quenched peptides comparative study and identification of specific carboxypeptidase activity
Biochemical Journal, 2003Co-Authors: Jefferson P Hemerly, Rory E. Morty, Vitor Oliveira, Norma W. Andrews, Elaine Del Nery, Maria A Juliano, Luiz JulianoAbstract:We characterized the extended substrate binding site of recombinant Oligopeptidase B enzymes from Trypanosoma cruzi (Tc-OP) and Trypanosoma brucei (Tb-OP), evaluating the specificity of their S3, S2, S1', S2' and S3' subsites. Five series of internally quenched fluorescent peptides based on the substrate Abz-AGGRGAQ-EDDnp [where Abz is o -aminobenzoic acid and EDDnp is N -(2,4-dinitrophenyl)ethylenediamine] were designed to contain amino acid residues with side chains of a minimum size, and each residue position of this substrate was modified. Synthetic peptides of different lengths derived from the human kininogen sequence were also examined, and peptides of up to 17 amino acids were found to be hydrolysed by Tc-OP and Tb-OP. These two Oligopeptidases were essentially arginyl hydrolases, since for all peptides examined the only cleavage site was the Arg-Xaa bond. We also demonstrated that Tc-OP and Tb-OP have a very specific carboxypeptidase activity for basic amino acids, which depends on the presence of at least of a pair of basic amino acids at the C-terminal end of the substrate. The peptide with triple Arg residues (Abz-AGRRRAQ-EDDnp) was an efficient substrate for Tc-OP and Tb-OP: the Arg-Ala peptide bond was cleaved first and then two C-terminal Arg residues were successively removed. The S1' subsite seems to be an important determinant of the specificity of both enzymes, showing a preference for Tyr, Ser, Thr and Gln as hydrogen donors. The presence of these amino acids at P1' resulted in substrates that were hydrolysed with K (m) values in the sub-micromolar range. Taken together, this work supports the view that Oligopeptidase B is a specialized protein-processing enzyme with a specific carboxypeptidase activity. Excellent substrates were obtained for Tb-OP and Tc-OP (Abz-AMRRTISQ-EDDnp and Abz-AHKRYSHQ-EDDnp respectively), which were hydrolysed with remarkably high k (cat) and low K (m) values.
Vitor Oliveira - One of the best experts on this subject based on the ideXlab platform.
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Molecular Translational Studies for Schizophrenia– Roles of Oligopeptidases and Their Substrate Neuropeptides in Clinics and Animal Models
European Psychiatry, 2015Co-Authors: Mirian Hayashi, Vitor Oliveira, Camila M. Yonamine, Ary Gadelha, Rodrigo A. Bressan, Ana Maria Vendramini, Marcela B. Nering, Vanessa C. AbílioAbstract:Schizophrenia (SCZ) is a complex and severe chronic mental disease presenting deficits in an operational measure of sensorimotor gating and cognitive impairments. The history of delayed developmental milestones and the presence of minor physical abnormalities may represent indirect evidences of abnormal prenatal development. Premorbid cognitive, personality and social functioning deficits in patients severely affected are also reported. Among the several candidate risk genes potentially associated with SCZ via pleiotropic mechanisms and/or other genes specific to susceptibility for SCZ, the Disrupted-in-Schizophrenia 1 gene ( DISC1 ) is the most studied. Interestingly, the main binding partner and effector of DISC1 protein is the Nuclear-distribution gene E homolog like-1 (Ndel1), which is an Oligopeptidase capable to degrade small peptides such as bradykinin (BK) and neurotensin (NT). Both neuropeptides were implicated in SCZ, and NT was also suggested to be an endogenous antipsychotic. DISC1 binding inhibits the Ndel1 enzyme activity competing with the peptide substrates, and the described translocation of DISC1 gene cosegregated with SCZ suggest a DISC1 and Ndel1 complex formation impairment with potential deregulation of the Ndel1 total activity. Other Oligopeptidase, as Angiotensin I-Converting Enzyme (ACE), is also able to cleave these peptides. Therefore, our group is focused on measuring the Oligopeptidase activity not only in clinical samples to allow the comparison between patients and health controls (HCs), but we have also performed the same measurements in plasma and different brain regions of animal models aiming to have insights into what may be occurring in the human brain based on peripheral tissues measurements. A significant lower Ndel1 Oligopeptidase activity (Gadelha et al., J Psych Res 2013) and significant higher ACE activity (Gadelha and Vendramine et al., submitted 2014) in the plasma of SCZ patients compared to HCs were observed. Moreover, a potential association of the ACE Oligopeptidase activity with the cognitive/disorganization symptoms was observed in both SCZ patients and animal models. The evaluation of the activity of these Oligopeptidases in patients and animal models treated with the same antipsychotics are currently ongoing in our laboratory. The results presented here support the potential involvement of Ndel1 and ACE in SCZ, and they may contribute to the discovery of molecular biomarkers for diagnosis and/or treatment follow-up of a severe chronic mental disease as SCZ, aiming to contribute to foster the translation of basic neurobiological and behavioral research to an improved integrative understanding of psychopathology for the development of a new and/or optimized treatments. Financial support by FAPESP, CNPq and CAPES.
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characterization of thimet Oligopeptidase and neurolysin activities in b16f10 nex2 tumor cells and their involvement in angiogenesis and tumor growth
Molecular Cancer, 2007Co-Authors: Thaysa Paschoalin, Elaine G Rodrigues, Adriana K Carmona, Hugo P. Monteiro, Maria Aparecida Juliano, Luiz Juliano, Vitor Oliveira, Luiz R. TravassosAbstract:Background: Angiogenesis is a fundamental process that allows tumor growth by providing nutrients and oxygen to the tumor cells. Beyond the oxygen diffusion limit from a capillary blood vessel, tumor cells become apoptotic. Angiogenesis results from a balance of pro- and antiangiogenic stimuli. Endogenous inhibitors regulate enzyme activities that promote angiogenesis. Tumor cells may express pro-angiogenic factors and hydrolytic enzymes but also kinin-degrading Oligopeptidases which have been investigated. Results: Angiogenesis induced by B16F10-Nex2 melanoma cells was studied in a co-culture with HUVEC on Matrigel. A stimulating effect on angiogenesis was observed in the presence of B16F10Nex2 lysate and plasma membrane. In contrast, the B16F10-Nex2 culture supernatant inhibited angiogenesis in a dose-dependent manner. This effect was abolished by the endo-Oligopeptidase inhibitor, JA-2. Thimet Oligopeptidase (TOP) and neurolysin activities were then investigated in B16F10-Nex2 melanoma cells aiming at gene sequencing, enzyme distribution and activity, influence on tumor development, substrate specificity, hydrolytic products and susceptibility to inhibitors. Fluorescence resonance energy transfer (FRET) peptides as well as neurotensin and bradykinin were used as substrates. The hydrolytic activities in B16F10-Nex2 culture supernatant were totally inhibited by o-phenanthrolin, JA-2 and partially by Pro-Ile. Leupeptin, PMSF, E-64, Z-Pro-Prolinal and captopril failed to inhibit these hydrolytic activities. Genes encoding M3A enzymes in melanoma cells were cloned and sequenced being highly similar to mouse genes. A decreased proliferation of B16F10-Nex2 cells was observed in vitro with specific inhibitors of these Oligopeptidases. Active rTOP but not the inactive protein inhibited melanoma cell development in vivo increasing significantly the survival of mice challenged with the tumor cells. On Matrigel, rTOP inhibited the bradykinin – induced angiogenesis. A possible regulation of the homologous tumor
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Characterization of thimet Oligopeptidase and neurolysin activities in B16F10-Nex2 tumor cells and their involvement in angiogenesis and tumor growth
Molecular Cancer, 2007Co-Authors: Thaysa Paschoalin, Elaine G Rodrigues, Adriana K Carmona, Hugo P. Monteiro, Maria Aparecida Juliano, Luiz Juliano, Vitor Oliveira, Luiz R. TravassosAbstract:Background Angiogenesis is a fundamental process that allows tumor growth by providing nutrients and oxygen to the tumor cells. Beyond the oxygen diffusion limit from a capillary blood vessel, tumor cells become apoptotic. Angiogenesis results from a balance of pro- and anti-angiogenic stimuli. Endogenous inhibitors regulate enzyme activities that promote angiogenesis. Tumor cells may express pro-angiogenic factors and hydrolytic enzymes but also kinin-degrading Oligopeptidases which have been investigated. Results Angiogenesis induced by B16F10-Nex2 melanoma cells was studied in a co-culture with HUVEC on Matrigel. A stimulating effect on angiogenesis was observed in the presence of B16F10-Nex2 lysate and plasma membrane. In contrast, the B16F10-Nex2 culture supernatant inhibited angiogenesis in a dose-dependent manner. This effect was abolished by the endo-Oligopeptidase inhibitor, JA-2. Thimet Oligopeptidase (TOP) and neurolysin activities were then investigated in B16F10-Nex2 melanoma cells aiming at gene sequencing, enzyme distribution and activity, influence on tumor development, substrate specificity, hydrolytic products and susceptibility to inhibitors. Fluorescence resonance energy transfer (FRET) peptides as well as neurotensin and bradykinin were used as substrates. The hydrolytic activities in B16F10-Nex2 culture supernatant were totally inhibited by o -phenanthrolin, JA-2 and partially by Pro-Ile. Leupeptin, PMSF, E-64, Z-Pro-Prolinal and captopril failed to inhibit these hydrolytic activities. Genes encoding M3A enzymes in melanoma cells were cloned and sequenced being highly similar to mouse genes. A decreased proliferation of B16F10-Nex2 cells was observed in vitro with specific inhibitors of these Oligopeptidases. Active rTOP but not the inactive protein inhibited melanoma cell development in vivo increasing significantly the survival of mice challenged with the tumor cells. On Matrigel, rTOP inhibited the bradykinin – induced angiogenesis. A possible regulation of the homologous tumor enzyme in the perivascular microenvironment is suggested based on the observed rTOP inhibition by an S-nitrosothiol NO donor. Conclusion Data show that melanoma cells secrete endo-Oligopeptidases which have an important role in tumor proliferation in vitro and in vivo. rTOP inhibited growth of subcutaneously injected B16F10-Nex2 cells in mice. TOP from tumor cells and bradykinin in endothelial cells are two antagonist factors that may control angiogenesis essential for melanoma growth. A regulatory role of NO or S-nitrosothiols is suggested.
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Tropolysin, a new Oligopeptidase from African trypanosomes.
Biochemistry, 2005Co-Authors: Rory E. Morty, Vitor Oliveira, István Vadász, Patrick Bulau, Vincent Dive, Werner Seeger, Luiz JulianoAbstract:Oligopeptidases are emerging as important pathogenic factors and therapeutic targets in trypanosome infections. We describe here the purification, cloning, and biochemical analysis of a new Oligopeptidase from two pathogenic African trypanosomes. This Oligopeptidase, which we have called tropolysin (encoded by the trn gene), represents an evolutionarily distant member of the M3A subfamily of metallopeptidases, ancestral to thimet Oligopeptidase, neurolysin, and saccharolysin. The trn gene was present as a single copy per haploid genome, was expressed in both the mammalian and insect stages of the parasite life cycle, and encoded an 84 kDa protein. Both purified and hyperexpressed tropolysin hydrolyzed bradykinin-derived fluorogenic peptide substrates at restricted sites, with an alkaline pH optimum, and were activated by dithiothreitol and reduced glutathione and by divalent metal cations, in the order Zn(2+) > Co(2+) > Mn(2+). Under oxidizing conditions, tropolysin reversibly formed inactive multimers. Tropolysin exhibited a preference for acidic amino acid side chains in P(4), hydrophobic side chains in P(3), and hydrophobic or large uncharged side chains in P(1), P(1)', and P(3)', while the S(2)' site was unselective. Highly charged residues were not tolerated in P(1)'. Tropolysin was responsible for the bulk of the kinin-degrading activity in trypanosome lysates, potently (k(cat) approximately 119 s(-)(1)) inactivated the vasoactive kinins bradykinin and kallidin, and generated angiotensin(1-7) from angiotensin I. This hydrolysis both abolished the capacity of bradykinin to stimulate the bradykinin B(2) receptor and abrogated bradykinin prohypotensive properties in vivo, raising the possibility that tropolysin may play a role in the dysregulated kinin metabolism observed in the plasma of trypanosome-infected hosts.
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Characterization of thimet- and neurolysin-like activities in Escherichia coli M3A peptidases and description of a specific substrate
Archives of biochemistry and biophysics, 2005Co-Authors: Thaysa Paschoalin, Adriana K Carmona, Luiz Juliano, Vitor Oliveira, Luiz R. TravassosAbstract:Abstract M3A Oligopeptidases from Escherichia coli , with hydrolytic properties similar to Zn-dependent mammalian thimet Oligopeptidase (EP 24.15) and neurolysin (EP 24.16), were studied aiming at identification of comparative enzyme and substrate specificity, hydrolytic products, and susceptibility to inhibitors. Fluorescent peptides, neurotensin (NT) and bradykinin (BK), were used as substrates for bacterial lysates. Bacterial enzymes were totally inhibited by o -phenanthrolin, JA-2 and partially by Pro-Ile, but not by leupeptin, PMSF, E-64, and Z-Pro-Prolinal, using internally quenched Abz-GFSPFRQ-EDDnp as substrate. The molecular mass of the bacterial Oligopeptidase activity (77–78 kDa) was determined by gel filtration, and the effect of inhibitors, including captopril, suggested that it results from a combination of Oligopeptidase A (OpdA) and peptidyl dipeptidase Dcp (77.1 and 77.5 kDa, respectively). Recombinant OpdA cloned from the same E. coli strain entirely reproduced the primary cleavage of fluorescent peptides, NT and BK, by the bacterial lysate. Genes encoding these M3A enzymes were those recognized in E. coli genome, bearing identity at the amino acid level (25–31%) with mammalian Zn-dependent Oligopeptidases. We also describe a substrate, Abz-GFSPFRQ-EDDnp, that differentiates bacterial and mammalian Oligopeptidases.
Andrey V. Karlyshev - One of the best experts on this subject based on the ideXlab platform.
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YPTB3816 of Yersinia pseudotuberculosis strain IP32953 is a virulence-related metallo-Oligopeptidase.
BMC microbiology, 2016Co-Authors: Ali Atas, Alan M. Seddon, Donna C. Ford, Ian A. Cooper, Brendan W. Wren, Petra C. F. Oyston, Andrey V. KarlyshevAbstract:Background Although bacterial peptidases are known to be produced by various microorganisms, including pathogenic bacteria, their role in bacterial physiology is not fully understood. In particular, Oligopeptidases are thought to be mainly involved in degradation of short peptides e.g. leader peptides released during classical protein secretion pathways. The aim of this study was to investigate effects of inactivation of an Oligopeptidase encoding gene opdA gene of Yersinia pseudotuberculosis on bacterial properties in vivo and in vitro, and to test dependence of the enzymatic activity of the respective purified enzyme on the presence of different divalent cations.
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YPTB3816 of Yersinia pseudotuberculosis strain IP32953 is a virulence-related metallo-Oligopeptidase
BMC Microbiology, 2016Co-Authors: Ali Atas, Alan M. Seddon, Donna C. Ford, Ian A. Cooper, Brendan W. Wren, Petra C. F. Oyston, Andrey V. KarlyshevAbstract:Background Although bacterial peptidases are known to be produced by various microorganisms, including pathogenic bacteria, their role in bacterial physiology is not fully understood. In particular, Oligopeptidases are thought to be mainly involved in degradation of short peptides e.g. leader peptides released during classical protein secretion pathways. The aim of this study was to investigate effects of inactivation of an Oligopeptidase encoding gene opdA gene of Yersinia pseudotuberculosis on bacterial properties in vivo and in vitro, and to test dependence of the enzymatic activity of the respective purified enzyme on the presence of different divalent cations. Results In this study we found that Oligopeptidase OpdA of Yersinia pseudotuberculosis is required for bacterial virulence, whilst knocking out the respective gene did not have any effect on bacterial viability or growth rate in vitro. In addition, we studied enzymatic properties of this enzyme after expression and purification from E. coli . Using an enzyme depleted of contaminant divalent cations and different types of fluorescently labelled substrates, we found strong dependence of its activity on the presence of particular cations. Unexpectedly, Zn2+ showed stimulatory activity only at low concentrations, but inhibited the enzyme at higher concentrations. In contrast, Co2+, Ca2+ and Mn2+ stimulated activity at all concentrations tested, whilst Mg2+ revealed no effect on the enzyme activity at all concentrations used. Conclusions The results of this study provide valuable contribution to the investigation of bacterial peptidases in general, and that of metallo-Oligopeptidases in particular. This is the first study demonstrating that opdA in Yersinia pseudotuberculsosis is required for pathogenicity. The data reported are important for better understanding of the role of OpdA-like enzymes in pathogenesis in bacterial infections. Characterisation of this protein may serve as a basis for the development of novel antibacterials based on specific inhibition of this peptidase activity.
Emer S Ferro - One of the best experts on this subject based on the ideXlab platform.
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Substrate Capture Assay Using Inactive Oligopeptidases to Identify Novel Peptides.
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Vanessa Rioli, Emer S FerroAbstract:Researchers are always searching for novel biologically active molecules including peptides. With the improvement of equipment for electrospray mass spectrometry, it is now possible to identify hundreds of novel peptides in a single run. However, after identifying the peptide sequences it is expensive to synthesize all the peptides to perform biological activity assays. Here, we describe a substrate capture assay that uses inactive Oligopeptidases to identify putative biologically active peptides in complexes peptide mixtures. This methodology can use any crude extracts of biological tissues or cells, with the advantage to introduce a filter (i.e., binding to an inactive Oligopeptidase) as a prior step in screening to bioactive peptides.
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substrate specificity characterization of recombinant metallo oligo peptidases thimet Oligopeptidase and neurolysin
Biochemistry, 2001Co-Authors: Vitor Oliveira, And Maria A Juliano, Robson L. Melo, Antonio C M Camargo, Emer S Ferro, Marcelo Campos, Luiz JulianoAbstract:We report a systematic and detailed analysis of recombinant neurolysin (EC 3.4.24.16) specificity in parallel with thimet Oligopeptidase (TOP, EC 3.4.24.15) using Bk sequence and its C- and N-terminal extensions as in human kininogen as motif for synthesis of internally quenched fluorescent substrates. The influence of the substrate size was investigated, and the longest peptide susceptible to TOP and neurolysin contains 17 amino acids. The specificities of both Oligopeptidases to substrate sites P4 to P3‘ were also characterized in great detail using seven series of peptides based on Abz-GFSPFRQ-EDDnp taken as reference substrate. Most of the peptides were hydrolyzed at the bond corresponding to P4−F5 in the reference substrate and some of them were hydrolyzed at this bond or at F2−S3 bond. No restricted specificity was found for P1‘ as found in thermolysin as well for P1 substrate position, however the modifications at this position (P1) showed to have large influence on the catalytic constant and the b...
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Molecular and Immunochemical Evidences Demonstrate That EndoOligopeptidase A Is the Predominant Cytosolic Oligopeptidase of Rabbit Brain
Biochemical and biophysical research communications, 2000Co-Authors: Mirian A F Hayashi, Fernanda Calheta Portaro, Emer S Ferro, Denise V. Tambourgi, Mauro Sucupira, Tetsuo Yamane, Beatriz L. Fernandes, Nancy Amaral Rebouças, Antonio C M CamargoAbstract:Abstract Oligopeptidases are tissue endopeptidases that do not attack proteins and are likely to be involved in the maturation and degradation of peptide hormones and neuropeptides. The rabbit brain endoOligopeptidase A and the rat testes soluble metallopeptidase (EC 3.4.24.15) are thiol-activated Oligopeptidases which are able to generate enkephalin from a number of opiod peptides and to inactivate bradykinin and neurotensin by hydrolyzing the same peptide bonds. A monospecific antibody raised against the purified rabbit brain endoOligopeptidase A allowed the identification of a 2.3 kb cDNA coding for a truncated enzyme of 512 amino acids, displaying the same enzymatic features as endoOligopeptidase A. In spite of all efforts, employing several strategies, the full-length cDNA could not be cloned until now. The analysis of the deduced amino acid sequence showed no similarity to the rat testes metalloendopeptidase sequence, except for the presence of the typical metalloprotease consensus sequence [HEXXH]. The antibody raised against recombinant endoOligopeptidase A specifically inhibited its own activity and reduced the thiol-activated Oligopeptidase activity of rabbit brain cytosol to less than 30%. Analysis of the endoOligopeptidase A tissue distribution indicated that this enzyme is mainly expressed in the CNS, whereas the soluble metallo EC 3.4.24.15 is mainly expressed in peripheral tissues.
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differential subcellular distribution of neurolysin ec 3 4 24 16 and thimet Oligopeptidase ec 3 4 24 15 in the rat brain
Brain Research, 1999Co-Authors: Eduardo Ernst Massarelli, Cláudio Aparecido Casatti, J. A. Bauer, Shigehisa Hirose, Antonio C M Camargo, Marc J. Glucksman, James L. Roberts, Akira Kato, Emer S FerroAbstract:Immunohistochemistry was used to analyze the rat brain distribution of thimet Oligopeptidase and neurolysin. Both enzymes appear ubiquitously distributed within the entire rat brain. However, neuronal perikarya and processes stained for neurolysin, while intense nuclear labeling was only observed for thimet Oligopeptidase. These data suggest that neurolysin and thimet Oligopeptidase, endopeptidases sharing several functional and structural similarities, are present in distinctive intracellular compartments in neuronal cells.
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thimet Oligopeptidase ec 3 4 24 15 a novel protein on the route of mhc class i antigen presentation
Biochemical and Biophysical Research Communications, 1999Co-Authors: Célio Lopes Silva, Fernanda Calheta Portaro, Vânia Luiza Deperon Bonato, Antonio C M Camargo, Emer S FerroAbstract:Abstract The initial processing of antigens leading to major histocompatibility complex (MHC) class I antigenic peptides is carried out by the proteasome. However, how the final epitopes are generated and protected from degradation by cytosolic peptidases remains unknown. Coincidentally, peptides associated with the MHC class I molecules range from 8 to 13 amino acid residues, similarly to the optimum substrate size required for the cytosolic thimet Oligopeptidase. Here we have investigated the putative intracellular function of thimet Oligopeptidase related to antigen presentation. Using a well-characterized antigen-presenting cell system, we were able to demonstrate either inhibition or stimulation of CD8 T cell proliferation and cytotoxicity, manipulating intracellular thimet Oligopeptidase levels with its specific inhibitor cFP-Ala-Ala-Tyr-pAb or loading the enzyme itself into the antigen-presenting cells. Our results suggest that thimet Oligopeptidase should take an important function in the pathway of antigen presentation via MHC class I through a mechanism yet unknown.
Luiz R. Travassos - One of the best experts on this subject based on the ideXlab platform.
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characterization of thimet Oligopeptidase and neurolysin activities in b16f10 nex2 tumor cells and their involvement in angiogenesis and tumor growth
Molecular Cancer, 2007Co-Authors: Thaysa Paschoalin, Elaine G Rodrigues, Adriana K Carmona, Hugo P. Monteiro, Maria Aparecida Juliano, Luiz Juliano, Vitor Oliveira, Luiz R. TravassosAbstract:Background: Angiogenesis is a fundamental process that allows tumor growth by providing nutrients and oxygen to the tumor cells. Beyond the oxygen diffusion limit from a capillary blood vessel, tumor cells become apoptotic. Angiogenesis results from a balance of pro- and antiangiogenic stimuli. Endogenous inhibitors regulate enzyme activities that promote angiogenesis. Tumor cells may express pro-angiogenic factors and hydrolytic enzymes but also kinin-degrading Oligopeptidases which have been investigated. Results: Angiogenesis induced by B16F10-Nex2 melanoma cells was studied in a co-culture with HUVEC on Matrigel. A stimulating effect on angiogenesis was observed in the presence of B16F10Nex2 lysate and plasma membrane. In contrast, the B16F10-Nex2 culture supernatant inhibited angiogenesis in a dose-dependent manner. This effect was abolished by the endo-Oligopeptidase inhibitor, JA-2. Thimet Oligopeptidase (TOP) and neurolysin activities were then investigated in B16F10-Nex2 melanoma cells aiming at gene sequencing, enzyme distribution and activity, influence on tumor development, substrate specificity, hydrolytic products and susceptibility to inhibitors. Fluorescence resonance energy transfer (FRET) peptides as well as neurotensin and bradykinin were used as substrates. The hydrolytic activities in B16F10-Nex2 culture supernatant were totally inhibited by o-phenanthrolin, JA-2 and partially by Pro-Ile. Leupeptin, PMSF, E-64, Z-Pro-Prolinal and captopril failed to inhibit these hydrolytic activities. Genes encoding M3A enzymes in melanoma cells were cloned and sequenced being highly similar to mouse genes. A decreased proliferation of B16F10-Nex2 cells was observed in vitro with specific inhibitors of these Oligopeptidases. Active rTOP but not the inactive protein inhibited melanoma cell development in vivo increasing significantly the survival of mice challenged with the tumor cells. On Matrigel, rTOP inhibited the bradykinin – induced angiogenesis. A possible regulation of the homologous tumor
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Characterization of thimet Oligopeptidase and neurolysin activities in B16F10-Nex2 tumor cells and their involvement in angiogenesis and tumor growth
Molecular Cancer, 2007Co-Authors: Thaysa Paschoalin, Elaine G Rodrigues, Adriana K Carmona, Hugo P. Monteiro, Maria Aparecida Juliano, Luiz Juliano, Vitor Oliveira, Luiz R. TravassosAbstract:Background Angiogenesis is a fundamental process that allows tumor growth by providing nutrients and oxygen to the tumor cells. Beyond the oxygen diffusion limit from a capillary blood vessel, tumor cells become apoptotic. Angiogenesis results from a balance of pro- and anti-angiogenic stimuli. Endogenous inhibitors regulate enzyme activities that promote angiogenesis. Tumor cells may express pro-angiogenic factors and hydrolytic enzymes but also kinin-degrading Oligopeptidases which have been investigated. Results Angiogenesis induced by B16F10-Nex2 melanoma cells was studied in a co-culture with HUVEC on Matrigel. A stimulating effect on angiogenesis was observed in the presence of B16F10-Nex2 lysate and plasma membrane. In contrast, the B16F10-Nex2 culture supernatant inhibited angiogenesis in a dose-dependent manner. This effect was abolished by the endo-Oligopeptidase inhibitor, JA-2. Thimet Oligopeptidase (TOP) and neurolysin activities were then investigated in B16F10-Nex2 melanoma cells aiming at gene sequencing, enzyme distribution and activity, influence on tumor development, substrate specificity, hydrolytic products and susceptibility to inhibitors. Fluorescence resonance energy transfer (FRET) peptides as well as neurotensin and bradykinin were used as substrates. The hydrolytic activities in B16F10-Nex2 culture supernatant were totally inhibited by o -phenanthrolin, JA-2 and partially by Pro-Ile. Leupeptin, PMSF, E-64, Z-Pro-Prolinal and captopril failed to inhibit these hydrolytic activities. Genes encoding M3A enzymes in melanoma cells were cloned and sequenced being highly similar to mouse genes. A decreased proliferation of B16F10-Nex2 cells was observed in vitro with specific inhibitors of these Oligopeptidases. Active rTOP but not the inactive protein inhibited melanoma cell development in vivo increasing significantly the survival of mice challenged with the tumor cells. On Matrigel, rTOP inhibited the bradykinin – induced angiogenesis. A possible regulation of the homologous tumor enzyme in the perivascular microenvironment is suggested based on the observed rTOP inhibition by an S-nitrosothiol NO donor. Conclusion Data show that melanoma cells secrete endo-Oligopeptidases which have an important role in tumor proliferation in vitro and in vivo. rTOP inhibited growth of subcutaneously injected B16F10-Nex2 cells in mice. TOP from tumor cells and bradykinin in endothelial cells are two antagonist factors that may control angiogenesis essential for melanoma growth. A regulatory role of NO or S-nitrosothiols is suggested.
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Characterization of thimet- and neurolysin-like activities in Escherichia coli M3A peptidases and description of a specific substrate
Archives of biochemistry and biophysics, 2005Co-Authors: Thaysa Paschoalin, Adriana K Carmona, Luiz Juliano, Vitor Oliveira, Luiz R. TravassosAbstract:Abstract M3A Oligopeptidases from Escherichia coli , with hydrolytic properties similar to Zn-dependent mammalian thimet Oligopeptidase (EP 24.15) and neurolysin (EP 24.16), were studied aiming at identification of comparative enzyme and substrate specificity, hydrolytic products, and susceptibility to inhibitors. Fluorescent peptides, neurotensin (NT) and bradykinin (BK), were used as substrates for bacterial lysates. Bacterial enzymes were totally inhibited by o -phenanthrolin, JA-2 and partially by Pro-Ile, but not by leupeptin, PMSF, E-64, and Z-Pro-Prolinal, using internally quenched Abz-GFSPFRQ-EDDnp as substrate. The molecular mass of the bacterial Oligopeptidase activity (77–78 kDa) was determined by gel filtration, and the effect of inhibitors, including captopril, suggested that it results from a combination of Oligopeptidase A (OpdA) and peptidyl dipeptidase Dcp (77.1 and 77.5 kDa, respectively). Recombinant OpdA cloned from the same E. coli strain entirely reproduced the primary cleavage of fluorescent peptides, NT and BK, by the bacterial lysate. Genes encoding these M3A enzymes were those recognized in E. coli genome, bearing identity at the amino acid level (25–31%) with mammalian Zn-dependent Oligopeptidases. We also describe a substrate, Abz-GFSPFRQ-EDDnp, that differentiates bacterial and mammalian Oligopeptidases.
