Effects of magnesium ions on recombinant human furin: selective activation of hydrolytic activity upon substrates derived from virus envelope glycoprotein

Here we report a detailed analysis of magnesium (Mg(2+)) ion effects on furin hydrolysis of fluorescent resonance energy transfer decapeptide substrates derived from canonical R-X-K/R-R furin cleavage motifs within certain viral envelope glycoproteins and eukaryotic proproteins. Using virus-derived sequences a selective activation of furin by Mg(2+) ions was observed as a result of cooperativity between furin subsites. Furin hydrolysis of the peptides Abz-SRRHKR down arrow FAGV-Q-EDDnp (from measles virus fusion protein F(o)) and Abz-RERRRKKR down arrow GLFG-Q-EDDnp (from Asian avian influenza A, H5N1) was activated between 60- and 80-fold by MgCl(2). It appears that virus envelope glycoprotein mutations have been selected to increase their susceptibility to furin within cells, a location where Mg(2+) is present in adequate concentrations for activation. Both the pH profile of furin and its intrinsic fluorescence were modified by Mg(2+) ions, which bind to furin with a K(d) value of 1.1 mM.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, BrazilUniv Maryland, Dept Anat & Neurobiol, Baltimore, MD 21201 USAUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

Analysis of secreted protein profile and enzymatic activities from Corynebacterium diphtheriae and Bordetella pertussis on production batch media using peptide quenched fluorescent substrates

Proteases were identified and characterized from the culture supernatant of the C. diphtheriae and B. pertussis bacteria. the proteases were secreted in the media and detected at the end of the exponential growth phase. Activity was detected in some fluorescent substrates, based on selected protein sequences such as insuline beta-chain, bradykinin, and synaptobrevin. the proteases were purified by means of gel filtration chromatography. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis ( SDS- PAGE) analysis of the purified proteins indicated, for the main secreted proteins, an estimated molecular mass of 30 kDa in C. diphtheriae and 69 kDa in B. pertussis culture media. the proteases were stable and presented enzymatic activity at 378 degrees C. These proteases were not related to the main toxic compounds described in these two bacteria, but could represent good markers for the fermentation process when the enzyme activity was measured with the fluorescent substrates.Butantan Inst, Biochem & Biophys Lab, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilButantan Inst, Dept Bacteriol, Sect Aerob Vaccines, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilWeb of Scienc

Enzyme specificity and effects of gyroxin, a serine protease from the venom of the South American rattlesnake Crotalus durissus terrificus, on protease-activated receptors

Gyroxin is a serine protease displaying a thrombin-like activity found in the venom of the South American rattlesnake Crotalus durissus terrificus. Typically, intravenous injection of purified gyroxin induces a barrel rotation syndrome in mice. the serine protease thrombin activates platelets aggregation by cleaving and releasing a tethered N-terminus peptide from the G-protein-coupled receptors, known as protease-activated receptors (PARs). Gyroxin also presents pro-coagulant activity suggested to be dependent of PARs activation. in the present work, the effects of these serine proteases, namely gyroxin and thrombin, on PARs were comparatively studied by characterizing the hydrolytic specificity and kinetics using PARs-mimetic FRET peptides. We show for the first time that the short (sh) and long (lg) peptides mimetizing the PAR-1, -2, -3, and -4 activation sites are all hydrolyzed by gyroxin exclusively after the Arg residues. Thrombin also hydrolyzes PAR-1 and -4 after the Arg residue, but hydrolyzes sh and lg PAR-3 after the Lys residue. the k(cat)/K-M values determined for gyroxin using sh and lg PAR-4 mimetic peptides were at least 2150 and 400 times smaller than those determined for thrombin, respectively. for the sh and lg PAR-2 mimetic peptides the k(cat)/K-M values determined for gyroxin were at least 6500 and 2919 times smaller than those determined for trypsin, respectively. the k(cat)/K-M values for gyroxin using the PAR-1 and -3 mimetic peptides could not be determined due to the extreme low hydrolysis velocity. Moreover, the functional studies of the effects of gyroxin on PARs were conducted in living cells using cultured astrocytes, which express all PARs. Despite the ability to cleavage the PAR-I, -2, -3, and -4 peptides, gyroxin was unable to activate the PARs expressed in astrocytes as determined by evaluating the cytosolic calcium mobilization. On the other hand, we also showed that gyroxin is able to interfere with the activation of PAR-1 by thrombin or by synthetic PAR-1 agonist in cultured astrocytes. Taken together, the data presented here allow us showing that gyroxin cleaves PARs-mimetic peptides slowly and it does not induce activation of PARs in astrocytes. Although gyroxin does not mobilize calcium it was shown to interfere with PARs activation by thrombin and PAR-1 agonist. the determination of gyroxin enzymatic specificity and kinetics on PAR-1, -2, -3, and -4 will potentially help to fill the gap in the knowledge in this field, as the PARs are still believed to have a key role for the gyroxin biological effects. (C) 2013 Elsevier B.V. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Depto Farmacol, São Paulo, BrazilUniversidade Federal de São Paulo, Depto Biofis, São Paulo, BrazilIPEN, Ctr Biotecnol, São Paulo, BrazilInst Butantan, Lab Genet, São Paulo, BrazilUniv Estado Amazonas, Escola Super Ciencias Saude, INCT, Manaus, Amazonas, BrazilUniversidade Federal de São Paulo, Depto Farmacol, São Paulo, BrazilUniversidade Federal de São Paulo, Depto Biofis, São Paulo, BrazilWeb of Scienc

Evaluation of NDEL1 oligopeptidase activity in blood and brain in an animal model of schizophrenia: effects of psychostimulants and antipsychotics

Nuclear distribution element-like 1 (NDEL1) enzyme activity is important for neuritogenesis, neuronal migration, and neurodevelopment. We reported previously lower NDEL1 enzyme activity in blood of treated first episode psychosis and chronic schizophrenia (SCZ) compared to healthy control subjects, with even lower activity in treatment resistant chronic SCZ patients, implicating NDEL1 activity in SCZ. Herein, higher NDEL1 activity was observed in the blood and several brain regions of a validated animal model for SCZ at baseline. In addition, long-term treatment with typical or atypical antipsychotics, under conditions in which SCZ-like phenotypes were reported to be reversed in this animal model for SCZ, showed a significant NDEL1 activity reduction in blood and brain regions which is in line with clinical data. Importantly, these results support measuring NDEL1 enzyme activity in the peripheral blood to predict changes in NDEL1 activity in the CNS. Also, acute administration of psychostimulants, at levels reported to induce SCZ-like phenotype in normal rat strains, increased NDEL1 enzyme activity in blood. Therefore, alterations in NDEL1 activity after treatment with antipsychotics or psychostimulants may suggest a possible modulation of NDEL1 activity secondary to neurotransmission homeostasis and provide new insights into the role of NDEL1 in SCZ pathophysiology

Human tissue kallikreins 3 and 5 can act as plasminogen activator releasing active plasmin

Human tissue kallikreins (KLKs) are a group of serine proteases found in many tissues and biological fluids and are differentially expressed in several specific pathologies. Here, we present evidences of the ability of these enzymes to activate plasminogen. Kallikreins 3 and 5 were able to induce plasmin activity after hydrolyzing plasminogen, and we also verified that plasminogen activation was potentiated in the presence of glycosaminoglycans compared with plasminogen activation by tPA. This finding can shed new light on the plasminogen/plasmin system and its involvement in tumor metastasis, in which kallikreins appear to be upregulated. (C) 2013 Elsevier Inc. All rights reserved

Obesity modulates the immune response to oxidized LDL in hypertensive patients

Obesity and hypertension have been recognized as inflammatory diseases capable of activating the immune system, thus contributing to an increased cardiovascular risk. However, the link between adaptive immunity, obesity, and hypertension is poorly understood. We investigated the relationship of the body mass index (BMI) on the inflammatory, vascular, and immune responses in patients with hypertension na < ve of anti-hypertensive treatment. Hypertensive patients (N = 88) were divided into three groups: normal weight (NW), overweight (OW), and obese (OB) subjects. Anti-oxidized LDL autoantibodies (anti-oxLDL Abs), anti-ApoB-D peptide (anti-ApoB-D) Abs, interleukin (IL)-8 and IL-10, flow-mediated dilation (FMD) of the brachial artery, and 24-h ambulatory blood pressure monitoring (ABPM) were assessed. OB patients presented lower levels of anti-oxLDL Abs and IL-10, higher levels of IL-8, and impaired FMD, when compared to NW and OW (P < 0.05), without differences between groups regarding anti-ApoB-D Abs. After adjusting for age, systolic and diastolic blood pressure, anti-oxLDL Abs were inversely correlated with BMI and waist circumference (r = -0.24, P = 0.02 and r = -0.25, P = 0.02, respectively), whereas ApoB-D correlated with 24-h ABPM (r = 0.22, P = 0.05 for systolic, and r = 0.29, P = 0.01 for diastolic blood pressure). Regression analyses showed inverse associations of anti-oxLDL Abs with BMI (beta = -0.05, P = 0.01) and waist circumference (beta = -0.01, P = 0.02); anti-ApoB-D Abs were associated with systolic and diastolic 24-h ABPM (beta = 0.96, P = 0.04; beta = 1.02, P = 0.005, for systolic and diastolic 24-h ABPM, respectively). Among hypertensive patients, obesity modulates the immune and inflammatory milieu, determining an unfavorable balance of cytokines and reduction in titers of anti-oxLDL Abs. Twenty-four hour ABPM is associated with titers of anti-ApoB-D Abs.National Institute of Complex Fluids, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Med, Div Cardiol, BR-04039030 São Paulo, BrazilNatl Inst Complex Fluids, São Paulo, BrazilUniv São Paulo, Inst Biomed Sci, Dept Immunol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04039030 São Paulo, BrazilNatl Inst Nanomat Integrated Markers, Recife, PE, BrazilUniversidade Federal de São Paulo, Dept Med, Div Cardiol, BR-04039030 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04039030 São Paulo, BrazilWeb of Scienc

Leviserpin: A Serine Peptidase Inhibitor (Serpin) from the Sugarcane Weevil Sphenophorus levis

Serine peptidase inhibitors (serpins) form a superfamily of proteins covering abroad spectrum of different biological functions. Here we describe the inhibitory characterization of leviserpin, the first serpin from the sugar cane weevil Sphenophorus levis. Leviserpin was able to inhibit bovine trypsin by the formation of the covalent complex serpin-peptidase, demonstrated by SDS-PAGE and mass spectroscopy analysis. We also have determined the cleavage site at the reactive center loop, by the analysis of the polypeptides released from de C-terminus of leviserpin. Moreover we investigated the mRNA expression of leviserpin in different stages of S. levis development. Thus the specificity of leviserpin, in addition with its mRNA coding being transcribed through all lifecycle of the insect, can suggest a possible role in defense mechanism by regulating the action of prophenoloxidase (proPO) activating enzyme.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fed Univ ABC, Ctr Nat & Human Sci, Biol Chem Lab, BR-09210170 Santo Andre, SP, BrazilUniv Fed Sao Carlos, Mol Biol Lab, Dept Genet & Evolut, BR-13565905 Sao Carlos, SP, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilFAPESP: 98/14138-2FAPESP: 06/53607-6CNPq: 312701/2009-8Web of Scienc

Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds

The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K-i 14 nM).BuXI and BvTI are highly homologous (70%). the major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition.Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. the catalytic efficiency k(cat)/K-m 4.3 x 10(7) M(-1)sec(-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate.Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P-1') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa.Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.Universidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilEMBRAPA, Campo Grande, MS USAUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc

Characterization of angiotensin I-converting enzyme N-domain selectivity using positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides

Somatic angiotensin I-converting enzyme (ACE) has two homologous active sites (N and C domains) that show differences in various biochemical properties. in a previous study, we described the use of positional-scanning synthetic combinatorial (PS-SC) libraries of fluorescence resonance energy transfer (FRET) peptides to define the ACE C-domain versus N-domain substrate specificity and developed selective substrates for the C-domain (Bersanetti et al., 2004). in the present work, we used the results from the PS-SC libraries to define the N-domain preferences and designed selective substrates for this domain. the peptide Abz-GDDVAK(Dnp)-OH presented the most favorable residues for N-domain selectivity in the P-3 to P-1' positions. the fluorogenic analog Abz-DVAK(Dnp)-OH (Abz=ortho-aminobenzoic acid; Dnp=2,4-dinitrophenyl) showed the highest selectivity for ACE N-domain (k(cat)/K-m = 1.76 mu M-1.s(-1)). Systematic reduction of the peptide length resulted in a tripeptide that was preferentially hydrolyzed by the C-domain. the binding of Abz-DVAK(Dnp)-OH to the active site of ACE N-domain was examined using a combination of conformational analysis and molecular docking. Our results indicated that the binding energies for the N-domain-substrate complexes were lower than those for the C-domain-substrate, suggesting that the former complexes are more stable.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)National Research FoundationUCTUniversidade Federal de São Paulo, Dept Biophys, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Hlth Informat Dept, BR-04023062 São Paulo, BrazilUCT Fac Hlth Sci, Div Med Biochem, Inst Infect Dis & Mol Med, ZA-7925 Cape Town, South AfricaUniv Cape Town, Dept Chem, ZA-7700 Cape Town, South AfricaUniversidade Federal de São Paulo, Dept Biophys, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Hlth Informat Dept, BR-04023062 São Paulo, BrazilWeb of Scienc

A Bowman-Birk protease inhibitor purified, cloned, sequenced and characterized from the seeds of Maclura pomifera (Raf.) Schneid

A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of Maclura pomifera , a dicotyledonous plant belonging to the Moraceae family. In this work, we report a Bowman-Birk inhibitor (BBI) isolated, purified, cloned, and characterized from Maclura pomifera seeds (MpBBI), the first of this type from a species belonging to Moraceae family. MpBBI was purified to homogeneity by RP-HPLC, total RNA was extracted from seeds of M. pomifera, and the 3'RACE-PCR method was applied to obtain the cDNA, which was cloned and sequenced. Peptide mass fingerprinting (PMF) analysis showed correspondence between the in silico-translated protein and MpBBI, confirming that it corresponds to a new plant protease inhibitor. The obtained cDNA encoded a polypeptide of 65 residues and possesses 10 cysteine residues, with molecular mass of 7379.27, pI 6.10, and extinction molar coefficient of 9105 M-1 cm(-1). MpBBI inhibits strongly trypsin with K (i) in the 10(-10) M range and was stable in a wide array of pH and extreme temperatures. MpBBI comparative modeling was applied to gain insight into its 3D structure and highlighted some distinguishing features: (1) two non-identical loops, (2) loop 1 (CEEESRC) is completely different from any known BBI, and (3) the amount of disulphide bonds is also different from any reported BBI from dicot plants.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)CYTEDCONICETPHusisTherapeutics, 3210 Merryfield Row, San Diego, CA 92121 USAUniv Nacl La Plata, Fac Ciencias Exactas, Ctr Invest Proteinas Vegetales, RA-1900 La Plata, Buenos Aires, ArgentinaUniv Fed Sao Paulo, Dept Biofis, BR-04044020 Sao Paulo, BrazilUniv Autonoma Barcelona, IBB, E-08193 Barcelona, SpainUniv Nacl Arturo Jauretche, Inst Ciencias Salud, 1888 Florencio Varela, Buenos Aires, DF, ArgentinaCITEC, Gonnet, B1897, Buenos Aires, DF, ArgentinaInst Multidisciplinar Biol Celular IMBICE, Lab Neurofisiol, B1906APO, Buenos Aires, DF, ArgentinaUniv Fed Sao Paulo, Dept Biofis, BR-04044020 Sao Paulo, BrazilFAPESP: 12/50191-4RCYTED: Red tematica PROMAL 210RT0398CONICET: PIP 0297Web of Scienc