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Vardan T Karamyan - One of the best experts on this subject based on the ideXlab platform.
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Long-Range Changes in Neurolysin Dynamics Upon Inhibitor Binding
Journal of chemical theory and computation, 2017Co-Authors: Arzu Uyar, Vardan T Karamyan, Alex DicksonAbstract:Crystal structures of Neurolysin, a zinc metallopeptidase, do not show a significant conformational change upon the binding of an allosteric inhibitor. Neurolysin has a deep channel where it hydrolyzes a short neuropeptide neurotensin to create inactive fragments and thus controls its level in the tissue. Neurolysin is of interest as a therapeutic target since changes in neurotensin level have been implicated in cardiovascular disorders, neurological disorders, and cancer, and inhibitors of Neurolysin have been developed. An understanding of the dynamical and structural differences between apo and inhibitor-bound Neurolysin will aid in further design of potent inhibitors and activators. For this purpose, we performed several molecular dynamics (MD) simulations for both apo and inhibitor-bound Neurolysin. A machine learning method (Linear Discriminant Analysis) is applied to reveal differences between the apo and inhibitor-bound ensembles in an automated way, and large differences are observed on residues ...
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Distribution of non-AT1, non-AT2 binding of 125i-sarcosine, isoleucine8 angiotensin ii in Neurolysin knockout mouse brains. PLoS One 2014
2016Co-Authors: Robert C Speth, Kira L Santos, Ines Schadock, Michael Bader, Eduardo J Carrera, Andrea Linares, Jamala D Swindle, Luz Gonzalez-reiley, Vardan T KaramyanAbstract:The recent identification of a novel binding site for angiotensin (Ang) II as the peptidase Neurolysin (E.C. 3.4.24.16) has implications for the renin-angiotensin system (RAS). This report describes the distribution of specific binding of 125I-Sarcosine1, Isoleucine8 Ang II (125I-SI Ang II) in Neurolysin knockout mouse brains compared to wild-type mouse brains using quantitative receptor autoradiography. In the presence of p-chloromercuribenzoic acid (PCMB), which unmasks the novel binding site, widespread distribution of specific (3 mM Ang II displaceable) 125I-SI Ang II binding in 32 mouse brain regions was observed. Highest levels of binding.700 fmol/g initial wet weight were seen in hypothalamic, thalamic and septal regions, while the lowest level of binding,300 fmol/g initial wet weight was in the mediolateral medulla. 125I-SI Ang II binding was substantially higher by an average of 85 % in wild-type mouse brains compared to Neurolysin knockout brains, suggesting the presence of an additional non-AT1, non-AT2, non-Neurolysin Ang II binding site in the mouse brain. Binding of 125I-SI Ang II to Neurolysin in the presence of PCMB was highest in hypothalamic and ventral cortical brain regions, but broadly distributed across all regions surveyed. Non-AT1, non-AT2, non-Neurolysin binding was also highest i
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Allosteric Potentiation of Peptidase Neurolysin by Small Molecules
Biophysical Journal, 2016Co-Authors: Srinidhi Jayaraman, Naomi J Wangler, Rui Zhu, Yehia Mechref, Thomas J. Abbruscato, David A. Ostrov, Vardan T KaramyanAbstract:Here, we describe discovery of two small molecules which specifically enhance catalytic efficiency of peptidase Neurolysin. A computational approach was used to explore structure of Neurolysin and identify a druggable surface pocket in its hinge region, followed by docking and ranking of 139,735 molecules from NCI DTP database. Top ranking compounds were subjected to pharmacological evaluation. Two structurally related compounds were identified which enhanced the rate of substrate hydrolysis by recombinant (human and rat) and mouse brain-purified Neurolysins in a concentration-dependent manner. Neither the identified modulators nor dynorphin A(1-13), a competitive inhibitor, affected each other's affinity in modulating activity of Neurolysin, suggesting that the modulators do not bind to the substrate binding site. Both modulators reduced Km and increased Vmax values for hydrolysis of the synthetic substrate by Neurolysin in a concentration-dependent manner. The modulators had negligible effect on catalytic activity of thimet oligopeptidase, neprilysin, angiotensin converting enzyme (ACE) and ACE2, indicating that they are specific to Neurolysin. Both modulators also enhanced hydrolysis of endogenous substrates, suggesting that their effect was not linked to the synthetic substrate. The identified molecules could be developed into research tools for evaluation of the (patho)physiological function of Neurolysin. This study is one of few utilizing a structure-based approach for rational identification of enzyme activators, and by that it demonstrates applicability of this methodology for identification of allosteric modulators of other enzymes.
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distribution of non at1 non at2 binding of 125i sarcosine1 isoleucine8 angiotensin ii in Neurolysin knockout mouse brains
PLOS ONE, 2014Co-Authors: Robert C Speth, Kira L Santos, Ines Schadock, Michael Bader, Eduardo J Carrera, Catalina Breton, Andrea Linares, Luz Gonzalezreiley, Jamala D Swindle, Vardan T KaramyanAbstract:The recent identification of a novel binding site for angiotensin (Ang) II as the peptidase Neurolysin (E.C. 3.4.24.16) has implications for the renin-angiotensin system (RAS). This report describes the distribution of specific binding of 125I-Sarcosine1, Isoleucine8 Ang II (125I-SI Ang II) in Neurolysin knockout mouse brains compared to wild-type mouse brains using quantitative receptor autoradiography. In the presence of p-chloromercuribenzoic acid (PCMB), which unmasks the novel binding site, widespread distribution of specific (3 µM Ang II displaceable) 125I-SI Ang II binding in 32 mouse brain regions was observed. Highest levels of binding >700 fmol/g initial wet weight were seen in hypothalamic, thalamic and septal regions, while the lowest level of binding <300 fmol/g initial wet weight was in the mediolateral medulla. 125I-SI Ang II binding was substantially higher by an average of 85% in wild-type mouse brains compared to Neurolysin knockout brains, suggesting the presence of an additional non-AT1, non-AT2, non-Neurolysin Ang II binding site in the mouse brain. Binding of 125I-SI Ang II to Neurolysin in the presence of PCMB was highest in hypothalamic and ventral cortical brain regions, but broadly distributed across all regions surveyed. Non-AT1, non-AT2, non-Neurolysin binding was also highest in the hypothalamus but had a different distribution than Neurolysin. There was a significant reduction in AT2 receptor binding in the Neurolysin knockout brain and a trend towards decreased AT1 receptor binding. In the Neurolysin knockout brains, the size of the lateral ventricles was increased by 56% and the size of the mid forebrain (-2.72 to +1.48 relative to Bregma) was increased by 12%. These results confirm the identity of Neurolysin as a novel Ang II binding site, suggesting that Neurolysin may play a significant role in opposing the pathophysiological actions of the brain RAS and influencing brain morphology.
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Distribution of non-AT1, non-AT2 binding of 125I-sarcosine1, isoleucine8 angiotensin II in Neurolysin knockout mouse brains.
PloS one, 2014Co-Authors: Robert C Speth, Kira L Santos, Ines Schadock, Michael Bader, Eduardo J Carrera, Catalina Breton, Andrea Linares, Jamala D Swindle, Luz Gonzalez-reiley, Vardan T KaramyanAbstract:The recent identification of a novel binding site for angiotensin (Ang) II as the peptidase Neurolysin (E.C. 3.4.24.16) has implications for the renin-angiotensin system (RAS). This report describes the distribution of specific binding of 125I-Sarcosine1, Isoleucine8 Ang II (125I-SI Ang II) in Neurolysin knockout mouse brains compared to wild-type mouse brains using quantitative receptor autoradiography. In the presence of p-chloromercuribenzoic acid (PCMB), which unmasks the novel binding site, widespread distribution of specific (3 µM Ang II displaceable) 125I-SI Ang II binding in 32 mouse brain regions was observed. Highest levels of binding >700 fmol/g initial wet weight were seen in hypothalamic, thalamic and septal regions, while the lowest level of binding
Emer S Ferro - One of the best experts on this subject based on the ideXlab platform.
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Neurolysin: From Initial Detection to Latest Advances
Neurochemical Research, 2018Co-Authors: Frédéric Checler, Emer S FerroAbstract:The mechanisms by which peptidergic signals are terminated have been the center of multiple studies leading to the discoveries of novel proteolytic activities. When studying the catabolic fate of neurotensin (NT) in brain and gastrointestinal tract, we detected a novel activity belonging to the metallopeptidases class and apparently distinct from previously known enzymes. Purification and cloning confirmed that this NT-degrading neutral metalloendopeptidase activity was indeed original. It was named endopeptidase 3.4.24.16 according to the IUBMB nomenclature and later, referred to as Neurolysin. This review tells the history of Neurolysin from its initial detection to its purification, cloning, design of specific inhibitors as well as in vitro and in vivo pharmacological studies aimed at delineating its role in the control of NT function. Finally, we discuss very recent advances suggesting a potential role of Neurolysin in pathologies.
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Peptidomic analysis of the Neurolysin-knockout mouse brain
Journal of proteomics, 2014Co-Authors: Leandro M. Castro, Vanessa Rioli, Luiz Juliano, Vitor Oliveira, Diogo Manuel Lopes De Paiva Cavalcanti, Christiane B. De Araujo, Marcelo Y. Icimoto, Fabio C. Gozzo, Maria A. Juliano, Emer S FerroAbstract:Abstract A large number of intracellular peptides are constantly produced following protein degradation by the proteasome. A few of these peptides function in cell signaling and regulate protein–protein interactions. Neurolysin (Nln) is a structurally defined and biochemically well-characterized endooligopeptidase, and its subcellular distribution and biological activity in the vertebrate brain have been previously investigated. However, the contribution of Nln to peptide metabolism in vivo is poorly understood. In this study, we used quantitative mass spectrometry to investigate the brain peptidome of Nln-knockout mice. An additional in vitro digestion assay with recombinant Nln was also performed to confirm the identification of the substrates and/or products of Nln. Altogether, the data presented suggest that Nln is a key enzyme in the in vivo degradation of only a few peptides derived from proenkephalin, such as Met-enkephalin and octapeptide. Nln was found to have only a minor contribution to the intracellular peptide metabolism in the entire mouse brain. However, further studies appear necessary to investigate the contribution of Nln to the peptide metabolism in specific areas of the murine brain. Biological significance Neurolysin was first identified in the synaptic membranes of the rat brain in the middle 80's by Frederic Checler and colleagues. Neurolysin was well characterized biochemically, and its brain distribution has been confirmed by immunohistochemical methods. The Neurolysin contribution to the central and peripheral neurotensin-mediated functions in vivo has been delineated through inhibitor-based pharmacological approaches, but its genuine contribution to the physiological inactivation of neuropeptides remains to be firmly established. As a result, the main significance of this work is the first characterization of the brain peptidome of the Neurolysin-knockout mouse. This article is part of a Special Issue entitled: Proteomics, mass spectrometry and peptidomics, Cancun 2013. Guest Editors: Cesar Lopez-Camarillo, Victoria Pando-Robles and Bronwyn Jane Barkla.
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temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases Neurolysin and thimet oligopeptidase
FEBS Journal, 2002Co-Authors: Vitor Oliveira, Vanessa Rioli, Reynaldo M Gatti, Maria Aparecida Juliano, Alberto Spisni, Antonio C M Camargo, Emer S Ferro, Luiz JulianoAbstract:We report the recombinant Neurolysin and thimet oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R–R or at R–X bonds, and TOP hydrolyzed at R–R or L–R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. The kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. The relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.05–0.2 m for NaCl, KCl, NH4Cl and NaI, and 0.025–0.1 m for Na2SO4. Concentration higher than 0.2 N NH4Cl andNaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na2SO4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na2SO4, while NH4Cl and NaI have very modest effect. High positive values of enthalpy (ΔH*) and entropy (ΔS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. The effects of low temperature and high NaCl concentration on the hydrolytic activities of Neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far-UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.
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Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases Neurolysin and thimet oligopeptidase
European journal of biochemistry, 2002Co-Authors: Vitor Oliveira, Vanessa Rioli, Reynaldo M Gatti, Alberto Spisni, Antonio C M Camargo, Emer S Ferro, Maria A. Juliano, Luiz JulianoAbstract:We report the recombinant Neurolysin and thimet oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R-R or at R-X bonds, and TOP hydrolyzed at R-R or L-R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. The kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. The relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.05-0.2 m for NaCl, KCl, NH4Cl and NaI, and 0.025-0.1 m for Na2SO4. Concentration higher than 0.2 N NH4Cl and NaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na2SO4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na2SO4, while NH4Cl and NaI have very modest effect. High positive values of enthalpy (DeltaH*) and entropy (DeltaS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. The effects of low temperature and high NaCl concentration on the hydrolytic activities of Neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far-UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.
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selective neurotensin derived internally quenched fluorogenic substrates for Neurolysin ec 3 4 24 16 comparison with thimet oligopeptidase ec 3 4 24 15 and neprilysin ec 3 4 24 11
Analytical Biochemistry, 2001Co-Authors: Vitor Oliveira, Jefferson P Hemerly, Maria Aparecida Juliano, Antonio C M Camargo, Emer S Ferro, Marcelo Campos, Luiz JulianoAbstract:Abstract Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for Neurolysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15 or TOP), and neprilysin (EC 3.4.24.11 or NEP). Abz-LYENKPRRPYILQ-EDDnp (where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine and Abz is ortho-aminobenzoic acid) was derived from neurotensin by the introduction of Q-EDDnp at the C-terminal end of peptide and by the substitution of the pyroglutamic (pE) residue at N-terminus for Abz and a series of shorter peptides was obtained by deletion of amino acids residues from C-terminal, N-terminal, or both sides. Neurolysin and TOP hydrolyzed the substrates at PY or YI or RR bonds depending on the sequence and size of the peptides, while NEP cleaved P-Y or Y-I bonds according to its S′1 specificity. One of these substrates, Abz-NKPRRPQ-EDDnp was a specific and sensitive substrate for Neurolysin (kcat = 7.0 s−1, Km = 1.19 μM and kcat/Km = 5882 mM−1 · s−1), while it was completely resistant to NEP and poorly hydrolyzed by TOP and also by prolyl oligopeptidase (EC 3.4.21.26). Neurolysin concentrations as low as 1 pM were detected using this substrate under our conditions and its analogue Abz-NKPRAPQ-EDDnp was hydrolyzed by Neurolysin with kcat = 14.03 s−1, Km = 0.82 μM, and kcat/Km = 17,110 mM−1 · s−1, being the best substrate so far described for this peptidase.
Luiz Juliano - One of the best experts on this subject based on the ideXlab platform.
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Peptidomic analysis of the Neurolysin-knockout mouse brain
Journal of proteomics, 2014Co-Authors: Leandro M. Castro, Vanessa Rioli, Luiz Juliano, Vitor Oliveira, Diogo Manuel Lopes De Paiva Cavalcanti, Christiane B. De Araujo, Marcelo Y. Icimoto, Fabio C. Gozzo, Maria A. Juliano, Emer S FerroAbstract:Abstract A large number of intracellular peptides are constantly produced following protein degradation by the proteasome. A few of these peptides function in cell signaling and regulate protein–protein interactions. Neurolysin (Nln) is a structurally defined and biochemically well-characterized endooligopeptidase, and its subcellular distribution and biological activity in the vertebrate brain have been previously investigated. However, the contribution of Nln to peptide metabolism in vivo is poorly understood. In this study, we used quantitative mass spectrometry to investigate the brain peptidome of Nln-knockout mice. An additional in vitro digestion assay with recombinant Nln was also performed to confirm the identification of the substrates and/or products of Nln. Altogether, the data presented suggest that Nln is a key enzyme in the in vivo degradation of only a few peptides derived from proenkephalin, such as Met-enkephalin and octapeptide. Nln was found to have only a minor contribution to the intracellular peptide metabolism in the entire mouse brain. However, further studies appear necessary to investigate the contribution of Nln to the peptide metabolism in specific areas of the murine brain. Biological significance Neurolysin was first identified in the synaptic membranes of the rat brain in the middle 80's by Frederic Checler and colleagues. Neurolysin was well characterized biochemically, and its brain distribution has been confirmed by immunohistochemical methods. The Neurolysin contribution to the central and peripheral neurotensin-mediated functions in vivo has been delineated through inhibitor-based pharmacological approaches, but its genuine contribution to the physiological inactivation of neuropeptides remains to be firmly established. As a result, the main significance of this work is the first characterization of the brain peptidome of the Neurolysin-knockout mouse. This article is part of a Special Issue entitled: Proteomics, mass spectrometry and peptidomics, Cancun 2013. Guest Editors: Cesar Lopez-Camarillo, Victoria Pando-Robles and Bronwyn Jane Barkla.
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Molecular Cancer BioMed Central
2007Co-Authors: Thaysa Paschoalin, Luiz Juliano, Vitor Oliveira, Maria A. Juliano, Adriana K Carmona, Elaine G Rodrigues, Hugo P Monteiro, Luiz R TravassosAbstract:Characterization of thimet oligopeptidase and Neurolysin activities in B16F10-Nex2 tumor cells and their involvement in angiogenesis and tumor growt
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temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases Neurolysin and thimet oligopeptidase
FEBS Journal, 2002Co-Authors: Vitor Oliveira, Vanessa Rioli, Reynaldo M Gatti, Maria Aparecida Juliano, Alberto Spisni, Antonio C M Camargo, Emer S Ferro, Luiz JulianoAbstract:We report the recombinant Neurolysin and thimet oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R–R or at R–X bonds, and TOP hydrolyzed at R–R or L–R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. The kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. The relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.05–0.2 m for NaCl, KCl, NH4Cl and NaI, and 0.025–0.1 m for Na2SO4. Concentration higher than 0.2 N NH4Cl andNaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na2SO4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na2SO4, while NH4Cl and NaI have very modest effect. High positive values of enthalpy (ΔH*) and entropy (ΔS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. The effects of low temperature and high NaCl concentration on the hydrolytic activities of Neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far-UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.
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Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases Neurolysin and thimet oligopeptidase
European journal of biochemistry, 2002Co-Authors: Vitor Oliveira, Vanessa Rioli, Reynaldo M Gatti, Alberto Spisni, Antonio C M Camargo, Emer S Ferro, Maria A. Juliano, Luiz JulianoAbstract:We report the recombinant Neurolysin and thimet oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R-R or at R-X bonds, and TOP hydrolyzed at R-R or L-R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. The kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. The relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.05-0.2 m for NaCl, KCl, NH4Cl and NaI, and 0.025-0.1 m for Na2SO4. Concentration higher than 0.2 N NH4Cl and NaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na2SO4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na2SO4, while NH4Cl and NaI have very modest effect. High positive values of enthalpy (DeltaH*) and entropy (DeltaS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. The effects of low temperature and high NaCl concentration on the hydrolytic activities of Neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far-UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.
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selective neurotensin derived internally quenched fluorogenic substrates for Neurolysin ec 3 4 24 16 comparison with thimet oligopeptidase ec 3 4 24 15 and neprilysin ec 3 4 24 11
Analytical Biochemistry, 2001Co-Authors: Vitor Oliveira, Jefferson P Hemerly, Maria Aparecida Juliano, Antonio C M Camargo, Emer S Ferro, Marcelo Campos, Luiz JulianoAbstract:Abstract Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for Neurolysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15 or TOP), and neprilysin (EC 3.4.24.11 or NEP). Abz-LYENKPRRPYILQ-EDDnp (where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine and Abz is ortho-aminobenzoic acid) was derived from neurotensin by the introduction of Q-EDDnp at the C-terminal end of peptide and by the substitution of the pyroglutamic (pE) residue at N-terminus for Abz and a series of shorter peptides was obtained by deletion of amino acids residues from C-terminal, N-terminal, or both sides. Neurolysin and TOP hydrolyzed the substrates at PY or YI or RR bonds depending on the sequence and size of the peptides, while NEP cleaved P-Y or Y-I bonds according to its S′1 specificity. One of these substrates, Abz-NKPRRPQ-EDDnp was a specific and sensitive substrate for Neurolysin (kcat = 7.0 s−1, Km = 1.19 μM and kcat/Km = 5882 mM−1 · s−1), while it was completely resistant to NEP and poorly hydrolyzed by TOP and also by prolyl oligopeptidase (EC 3.4.21.26). Neurolysin concentrations as low as 1 pM were detected using this substrate under our conditions and its analogue Abz-NKPRAPQ-EDDnp was hydrolyzed by Neurolysin with kcat = 14.03 s−1, Km = 0.82 μM, and kcat/Km = 17,110 mM−1 · s−1, being the best substrate so far described for this peptidase.
Vitor Oliveira - One of the best experts on this subject based on the ideXlab platform.
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Peptidomic analysis of the Neurolysin-knockout mouse brain
Journal of proteomics, 2014Co-Authors: Leandro M. Castro, Vanessa Rioli, Luiz Juliano, Vitor Oliveira, Diogo Manuel Lopes De Paiva Cavalcanti, Christiane B. De Araujo, Marcelo Y. Icimoto, Fabio C. Gozzo, Maria A. Juliano, Emer S FerroAbstract:Abstract A large number of intracellular peptides are constantly produced following protein degradation by the proteasome. A few of these peptides function in cell signaling and regulate protein–protein interactions. Neurolysin (Nln) is a structurally defined and biochemically well-characterized endooligopeptidase, and its subcellular distribution and biological activity in the vertebrate brain have been previously investigated. However, the contribution of Nln to peptide metabolism in vivo is poorly understood. In this study, we used quantitative mass spectrometry to investigate the brain peptidome of Nln-knockout mice. An additional in vitro digestion assay with recombinant Nln was also performed to confirm the identification of the substrates and/or products of Nln. Altogether, the data presented suggest that Nln is a key enzyme in the in vivo degradation of only a few peptides derived from proenkephalin, such as Met-enkephalin and octapeptide. Nln was found to have only a minor contribution to the intracellular peptide metabolism in the entire mouse brain. However, further studies appear necessary to investigate the contribution of Nln to the peptide metabolism in specific areas of the murine brain. Biological significance Neurolysin was first identified in the synaptic membranes of the rat brain in the middle 80's by Frederic Checler and colleagues. Neurolysin was well characterized biochemically, and its brain distribution has been confirmed by immunohistochemical methods. The Neurolysin contribution to the central and peripheral neurotensin-mediated functions in vivo has been delineated through inhibitor-based pharmacological approaches, but its genuine contribution to the physiological inactivation of neuropeptides remains to be firmly established. As a result, the main significance of this work is the first characterization of the brain peptidome of the Neurolysin-knockout mouse. This article is part of a Special Issue entitled: Proteomics, mass spectrometry and peptidomics, Cancun 2013. Guest Editors: Cesar Lopez-Camarillo, Victoria Pando-Robles and Bronwyn Jane Barkla.
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Molecular Cancer BioMed Central
2007Co-Authors: Thaysa Paschoalin, Luiz Juliano, Vitor Oliveira, Maria A. Juliano, Adriana K Carmona, Elaine G Rodrigues, Hugo P Monteiro, Luiz R TravassosAbstract:Characterization of thimet oligopeptidase and Neurolysin activities in B16F10-Nex2 tumor cells and their involvement in angiogenesis and tumor growt
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temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases Neurolysin and thimet oligopeptidase
FEBS Journal, 2002Co-Authors: Vitor Oliveira, Vanessa Rioli, Reynaldo M Gatti, Maria Aparecida Juliano, Alberto Spisni, Antonio C M Camargo, Emer S Ferro, Luiz JulianoAbstract:We report the recombinant Neurolysin and thimet oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R–R or at R–X bonds, and TOP hydrolyzed at R–R or L–R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. The kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. The relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.05–0.2 m for NaCl, KCl, NH4Cl and NaI, and 0.025–0.1 m for Na2SO4. Concentration higher than 0.2 N NH4Cl andNaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na2SO4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na2SO4, while NH4Cl and NaI have very modest effect. High positive values of enthalpy (ΔH*) and entropy (ΔS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. The effects of low temperature and high NaCl concentration on the hydrolytic activities of Neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far-UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.
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Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases Neurolysin and thimet oligopeptidase
European journal of biochemistry, 2002Co-Authors: Vitor Oliveira, Vanessa Rioli, Reynaldo M Gatti, Alberto Spisni, Antonio C M Camargo, Emer S Ferro, Maria A. Juliano, Luiz JulianoAbstract:We report the recombinant Neurolysin and thimet oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R-R or at R-X bonds, and TOP hydrolyzed at R-R or L-R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. The kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. The relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.05-0.2 m for NaCl, KCl, NH4Cl and NaI, and 0.025-0.1 m for Na2SO4. Concentration higher than 0.2 N NH4Cl and NaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na2SO4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na2SO4, while NH4Cl and NaI have very modest effect. High positive values of enthalpy (DeltaH*) and entropy (DeltaS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. The effects of low temperature and high NaCl concentration on the hydrolytic activities of Neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far-UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.
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selective neurotensin derived internally quenched fluorogenic substrates for Neurolysin ec 3 4 24 16 comparison with thimet oligopeptidase ec 3 4 24 15 and neprilysin ec 3 4 24 11
Analytical Biochemistry, 2001Co-Authors: Vitor Oliveira, Jefferson P Hemerly, Maria Aparecida Juliano, Antonio C M Camargo, Emer S Ferro, Marcelo Campos, Luiz JulianoAbstract:Abstract Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for Neurolysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15 or TOP), and neprilysin (EC 3.4.24.11 or NEP). Abz-LYENKPRRPYILQ-EDDnp (where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine and Abz is ortho-aminobenzoic acid) was derived from neurotensin by the introduction of Q-EDDnp at the C-terminal end of peptide and by the substitution of the pyroglutamic (pE) residue at N-terminus for Abz and a series of shorter peptides was obtained by deletion of amino acids residues from C-terminal, N-terminal, or both sides. Neurolysin and TOP hydrolyzed the substrates at PY or YI or RR bonds depending on the sequence and size of the peptides, while NEP cleaved P-Y or Y-I bonds according to its S′1 specificity. One of these substrates, Abz-NKPRRPQ-EDDnp was a specific and sensitive substrate for Neurolysin (kcat = 7.0 s−1, Km = 1.19 μM and kcat/Km = 5882 mM−1 · s−1), while it was completely resistant to NEP and poorly hydrolyzed by TOP and also by prolyl oligopeptidase (EC 3.4.21.26). Neurolysin concentrations as low as 1 pM were detected using this substrate under our conditions and its analogue Abz-NKPRAPQ-EDDnp was hydrolyzed by Neurolysin with kcat = 14.03 s−1, Km = 0.82 μM, and kcat/Km = 17,110 mM−1 · s−1, being the best substrate so far described for this peptidase.
Mitsuyoshi Ueda - One of the best experts on this subject based on the ideXlab platform.
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specificity
2015Co-Authors: Tetsuya Kadonosono, Michiko Kato-murai, Mitsuyoshi UedaAbstract:Alteration of substrate specificity of rat Neurolysin from matrix metalloproteinase-2/9-type to-3-typ
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Novel high-throughput system for production of new medicines-integration and combination with molecular display and combinatorial bioengineering
Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan, 2009Co-Authors: Mitsuyoshi UedaAbstract:To demonstrate the practical use of a novel high-throughput screening system by single cells constructed by the molecular display method, a yeast cell chip microchamber array was developed. As applications, peptides, peptidases, and antibodies were examined. Neurolysin originally recognizes substrates with six-amino-acid-long residues, cleaving a peptide bond in the center position of the substrate amino acid sequence. To alter the recognition of the P2' amino acid of substrates by Neurolysin, six residues of Neurolysin which might be involved in the formation of the Neurolysin S2' subsite were individually and comprehensively substituted by semirational mutagenesis coupled with the yeast molecular display system. The protein libraries of mutant Neurolysins were displayed on the yeast cell surface and screening was carried out using two fluorescence-quenching peptides, the matrix metalloproteinase-2/9- and MMP-3-specific substrates. Among mutant Neurolysins, one mutant Neurolysin with a marked change in substrate specificity was successfully obtained. Furthermore, skillful display of antibodies (H and L chains) on the cell surface of yeast cells suggested the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach. Accordingly, the combination of the molecular display and combinatorial bioengineering would lead to produce novel medicines.
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Alteration of substrate specificity of rat Neurolysin from matrix metalloproteinase-2/9-type to -3-type specificity by comprehensive mutation
Protein engineering design & selection : PEDS, 2008Co-Authors: Tetsuya Kadonosono, Michiko Kato-murai, Mitsuyoshi UedaAbstract:kyoto-u.ac.jp The substrate specificity of rat brain Neurolysin was rapidly modified by semirational mutagenesis coupled with a yeast molecular display system. Neurolysin mainly recognizes substrates with sequential six residues close to the scissile bond in polypeptides, cleaving a peptide bond in the center position of the six residues. To alter the recognition of the P2 0 amino acid of substrates by Neurolysin, six residues of Neurolysin, Asp467, Arg470, Glu510, Tyr606, Tyr610 and Tyr611, which might be involved in the formation of the Neurolysin S2 0 subsite, were individually and comprehensively substituted. The protein libraries of mutant Neurolysins comprising 120 species were displayed on the yeast cell surface and screening was carried out using two fluorescence-quenching peptides, the matrix metalloproteinase-2/9- (MMPs-2/9-) and MMP-3-specific substrates, which consisted of similar amino acids, except for alanine (for MMPs-2/9) or glutamic acid (for MMP-3) at the P2 0 amino acid position. Among mutant Neurolysins, the Y610L mutant Neurolysin exhibited a marked change in substrate specificity. Steady-state kinetic analysis of the purified Y610L mutant Neurolysin revealed that the binding efficiency toward the MMP-3-specific substrate was about 3-fold higher than that toward the MMP-2/9-specific substrate. These results indicate that Tyr610 of Neurolysin is the important residue to recognize the P2 0 amino acid of
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Substrate specificity of rat brain Neurolysin disclosed by molecular display system and putative substrates in rat tissues
Applied Microbiology and Biotechnology, 2007Co-Authors: Tetsuya Kadonosono, Michiko Kato, Mitsuyoshi UedaAbstract:To search for the substrates, other than neurotensin, of rat brain Neurolysin, a novel method of determining peptidase activity was developed using a yeast molecular display system. This is a useful and convenient method of handling homogenously pure proteins to evaluate the properties of Neurolysin. The Neurolysin gene was ligated to the C-terminal half of the α-agglutinin gene with a FLAG tag sequence and a yeast cell-surface molecular displaying plasmid was constructed. Display of Neurolysin with correct folding and appropriate activity was verified by immunofluorescence staining and activity measurement of a bradykinin-related peptide. The cleavage sites of peptides were determined by high-performance liquid chromatography (HPLC) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results showed the amino acid preferences of hydrophobic, aromatic, and basic residues, which were the same as those of soluble Neurolysin. Moreover, this method clearly showed the presence of two recognition motifs in Neurolysin. By using these motifs, novel substrate candidates of Neurolysin in rat tissues were screened, and several bioactive peptides that regulate feeding were found. We also discussed the ubiquitous distribution of Neurolysin in rat tissues and the functions of substrate candidate peptides.
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Metallopeptidase, Neurolysin, as a novel molecular tool for analysis of properties of cancer-producing matrix metalloproteinases-2 and -9
Applied Microbiology and Biotechnology, 2007Co-Authors: Tetsuya Kadonosono, Michiko Kato, Mitsuyoshi UedaAbstract:To compare the substrate preferences of rat brain Neurolysin and cancer-producing matrix metalloproteinases (MMPs), which have the same architecture in their catalytic domains, the cleavage activity of Neurolysin toward MMP-specific fluorescence-quenching peptides was quantitatively measured. The results show that Neurolysin effectively cleaved MOCAc [(7-methoxy coumarin-4-yl) acetyl]-RPKPYANvaWMK(Dnp[2,4-dinitrophenyl])-NH_2, a specific substrate of MMP-2 and MMP-9, but hardly cleaved MOCAc-RPKPVENvaWRK(Dnp)-NH_2, a specific substrate of MMP-3, suggesting that Neurolysin has a similar substrate preference to MMP-2 and MMP-9. A structural comparison between Neurolysin and MMP-9 showed the similar key amino acid residues for substrate recognition. The possible application of Neurolysin displayed on the yeast cell surface, as a safe protein alternative to MMP-2 and MMP-9 which induce cancer cell growth, invasion, and metastasis, to analysis of properties of the MMPs, including the screening of inhibitors and analysis of inhibition mechanism etc., are also discussed.
