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Rosana Puccia - One of the best experts on this subject based on the ideXlab platform.
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C-Npys (S-3-nitro-2-pyridinesulfenyl) and peptide derivatives can inhibit a serine-Thiol Proteinase activity from Paracoccidioides brasiliensis.
Biochemical and biophysical research communications, 2007Co-Authors: Alisson L. Matsuo, Adriana K. Carmona, Luiz S. Silva, Carlos E. Cunha, Ernesto S. Nakayasu, Igor C. Almeida, Maria A. Juliano, Rosana PucciaAbstract:Abstract The inhibitory capacity of C-Npys (S-[3-nitro-2-pyridinesulfenyl]) derivatives over Thiol-containing serine proteases has never been tested. In the present work we used an extracellular serine-Thiol Proteinase activity from the fungal pathogen Paracoccidioides brasiliensis (PbST) to describe a potent inhibitory capacity of Bzl-C(Npys)KRLTL-NH2 and Bzl-MKRLTLC(Npys)-NH2. The assays were performed with PbST enriched upon affinity chromatography in a p-aminobenzamidine (pABA)-Sepharose column. Although PbST can cleave the fluorescence resonance energy transfer peptide Abz-MKRLTL-EDDnp between L–T, the C(Npys) derivatives were not substrates nor were they toxic in a cell detachment assay, allowing therapeutic use. The best inhibitor was Bzl-C(Npys)KRLTL-NH2 (Ki = 16 nM), suggesting that the peptide sequence promoted a favorable interaction, especially when C(Npys) was placed at a further position from the L–T bond, at the N-terminus. Inhibition was completely reverted with dithioerythritol, indicating that it was due to the reactivity of the C(Npys) moiety with a free SH– group.
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Modulation of the exocellular serine-Thiol Proteinase activity of Paracoccidioides brasiliensis by neutral polysaccharides
Microbes and infection, 2005Co-Authors: Alisson L. Matsuo, Ivarne I.l. Tersariol, Silvia Iovine Kobata, Luiz R. Travassos, Adriana K. Carmona, Rosana PucciaAbstract:Abstract Our group characterized an exocellular serine-Thiol Proteinase activity in the yeast phase of Paracoccidioides brasiliensis (PbST), a dimorphic human pathogen. The fungal Proteinase is able to cleave in vitro, at pH 7.4, proteins associated with the basal membrane, such as human laminin and fibronectin, type IV collagen and proteoglycans. In the present study, we investigated the influence of glycosaminoglycans (GAGs) and neutral polysaccharides upon the serine-Thiol Proteinase activity by means of kinetic analysis monitored with fluorescence resonance energy transfer (FRET) peptides using the substrate Abz-MKALTLQ-EDDnp (Abz = ortho-aminobenzoic acid; EDDnp = ethylenediaminedinitrophenyl). Only neutral polysaccharides exhibited patterns of interaction with the Proteinase, while sulfated GAGs had no effect. Incubation with neutral polysaccharides resulted in a powerful modulation of the enzyme activity, intensely changing the enzyme kinetic parameters of catalysis and affinity for the substrate. Commercial dextran at the highest concentration of 20 μM increased 6.8-fold the enzyme affinity for the substrate. In the presence of 8 μM of purified baker's yeast mannan, the apparent KM of the enzyme increased about 5.5-fold, reflecting a significant inhibition in binding to the peptide substrate. When an exocellular galactomannan (GalMan) complex isolated from P. brasiliensis was added to the reaction mixture at 400 nM, the apparent KM and VMAX decreased about threefold. Moreover, GalMan was able to protect the enzymatic activity at high temperatures, but it caused no effect on the optimum cleavage pH. Our results show a novel modulation mechanism in P. brasiliensis, where a fungal polysaccharide-rich component can stabilize a serine-Thiol proteolytic activity, which is possibly involved in fungal dissemination.
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Exocellular proteolytic activity of Paracoccidioides brasiliensis: cleavage of components associated with the basement membrane.
Medical mycology, 1998Co-Authors: Rosana Puccia, Adriana K. Carmona, J L Gesztesi, L. Juliano, L. R. TravassosAbstract:We have previously characterized an exocellular serine-Thiol Proteinase activity in Paracoccidioides brasiliensis, using as substrates peptides analogous of the internally quenched fluorogenic peptide Abz-MKRLTL-EDDnp. In this communication, detection of maximal Proteinase activity in the culture supernatant fluids followed the abrupt increase in the medium pH, owing to the accumulation of ammonia generated by urease activity. Culture supernatant fluids collected at the peak of Proteinase activity against Abz-MRKLTL-EDDnp were able to cleave components of the basal membrane of the extracellular matrix (EM), including laminin, fibronectin, collagen type IV and proteoglycans, and the proteolytic activity was selectively inhibited both by PMSF and p-HMB (sodium 7-hydroxymercuribenzoate), which are also specific inhibitors of the serine-Thiol Proteinase. Human collagen I, bovine fibrinogen, human immunoglobulin G, BSA or P. brasiliensis gp43 were resistant to proteolysis. The kinetics of appearance of the Proteinase activity against EM substrates coincided with that of proteolysis of Abz-MKRLTL-EDDnp. Moreover, chromatographic fractions of culture supernatants containing the serine-Thiol Proteinase at high specific activity were also active against EM substrates. These data suggest the involvement of this enzyme activity in the degradation of the basement membrane, which is the first step for fungal tissue invasion.
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characterization of an exocellular serine Thiol Proteinase activity in paracoccidioides brasiliensis
Biochemical Journal, 1995Co-Authors: Adriana K. Carmona, Rosana Puccia, L. Juliano, M C F Oliveira, Elaine G Rodrigues, L. R. TravassosAbstract:An exocellular Proteinase activity has been characterized in Paracoccidioides brasiliensis culture filtrates. Chromatographic analysis showed that the activity was eluted from an anion-exchange Resource Q column at 0.08-0.1 M NaCl, and by gel filtration near ovalbumin elution, in a single peak. Purification of the Proteinase, however, was hampered by the low protein yield, in contrast to the high peptidase activity. Numerous chromogenic peptidyl p-nitroanilide derivatives and internally quenched fluorescent peptides, flanked by Abz (O-aminobenzoyl) and EDDnp (ethylenediaminedinitrophenyl), were tested as substrates. Cleavage was observed with Abz-MKRLTL-EDDnp, Abz-FRLVR-EDDnp, and Abz-PLGLLGR-EDDnp at Leu-Thr, Leu-Val and Leu-Leu/Leu-Gly bonds respectively as determined by isolation of the corresponding fragments by HPLC. Leucine at P1 seemed to be restrictive for the activity of the exocellular enzyme, but threonine (P'1) and leucine (P'2) in Abz-MKRLTL-EDDnp apparently were not essential. Also, a pair of alanines could substitute for lysine (P3) and arginine (P2) in this substrate, with a decrease in the Km values. The exocellular peptidase activity of P. brasiliensis had an optimum pH of > 9.0 and was irreversibly inhibited by PMSF, mercuric acetate and p-hydroxymercuribenzoate. Inhibition of the mercuriate compounds could be partially reversed by Cys/EDTA. E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanido)butene] was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. These results suggest that P. brasiliensis exocellular enzyme belongs to the subfamily of SH-containing serine Proteinases.
Adriana K. Carmona - One of the best experts on this subject based on the ideXlab platform.
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C-Npys (S-3-nitro-2-pyridinesulfenyl) and peptide derivatives can inhibit a serine-Thiol Proteinase activity from Paracoccidioides brasiliensis.
Biochemical and biophysical research communications, 2007Co-Authors: Alisson L. Matsuo, Adriana K. Carmona, Luiz S. Silva, Carlos E. Cunha, Ernesto S. Nakayasu, Igor C. Almeida, Maria A. Juliano, Rosana PucciaAbstract:Abstract The inhibitory capacity of C-Npys (S-[3-nitro-2-pyridinesulfenyl]) derivatives over Thiol-containing serine proteases has never been tested. In the present work we used an extracellular serine-Thiol Proteinase activity from the fungal pathogen Paracoccidioides brasiliensis (PbST) to describe a potent inhibitory capacity of Bzl-C(Npys)KRLTL-NH2 and Bzl-MKRLTLC(Npys)-NH2. The assays were performed with PbST enriched upon affinity chromatography in a p-aminobenzamidine (pABA)-Sepharose column. Although PbST can cleave the fluorescence resonance energy transfer peptide Abz-MKRLTL-EDDnp between L–T, the C(Npys) derivatives were not substrates nor were they toxic in a cell detachment assay, allowing therapeutic use. The best inhibitor was Bzl-C(Npys)KRLTL-NH2 (Ki = 16 nM), suggesting that the peptide sequence promoted a favorable interaction, especially when C(Npys) was placed at a further position from the L–T bond, at the N-terminus. Inhibition was completely reverted with dithioerythritol, indicating that it was due to the reactivity of the C(Npys) moiety with a free SH– group.
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Modulation of the exocellular serine-Thiol Proteinase activity of Paracoccidioides brasiliensis by neutral polysaccharides
Microbes and infection, 2005Co-Authors: Alisson L. Matsuo, Ivarne I.l. Tersariol, Silvia Iovine Kobata, Luiz R. Travassos, Adriana K. Carmona, Rosana PucciaAbstract:Abstract Our group characterized an exocellular serine-Thiol Proteinase activity in the yeast phase of Paracoccidioides brasiliensis (PbST), a dimorphic human pathogen. The fungal Proteinase is able to cleave in vitro, at pH 7.4, proteins associated with the basal membrane, such as human laminin and fibronectin, type IV collagen and proteoglycans. In the present study, we investigated the influence of glycosaminoglycans (GAGs) and neutral polysaccharides upon the serine-Thiol Proteinase activity by means of kinetic analysis monitored with fluorescence resonance energy transfer (FRET) peptides using the substrate Abz-MKALTLQ-EDDnp (Abz = ortho-aminobenzoic acid; EDDnp = ethylenediaminedinitrophenyl). Only neutral polysaccharides exhibited patterns of interaction with the Proteinase, while sulfated GAGs had no effect. Incubation with neutral polysaccharides resulted in a powerful modulation of the enzyme activity, intensely changing the enzyme kinetic parameters of catalysis and affinity for the substrate. Commercial dextran at the highest concentration of 20 μM increased 6.8-fold the enzyme affinity for the substrate. In the presence of 8 μM of purified baker's yeast mannan, the apparent KM of the enzyme increased about 5.5-fold, reflecting a significant inhibition in binding to the peptide substrate. When an exocellular galactomannan (GalMan) complex isolated from P. brasiliensis was added to the reaction mixture at 400 nM, the apparent KM and VMAX decreased about threefold. Moreover, GalMan was able to protect the enzymatic activity at high temperatures, but it caused no effect on the optimum cleavage pH. Our results show a novel modulation mechanism in P. brasiliensis, where a fungal polysaccharide-rich component can stabilize a serine-Thiol proteolytic activity, which is possibly involved in fungal dissemination.
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Exocellular proteolytic activity of Paracoccidioides brasiliensis: cleavage of components associated with the basement membrane.
Medical mycology, 1998Co-Authors: Rosana Puccia, Adriana K. Carmona, J L Gesztesi, L. Juliano, L. R. TravassosAbstract:We have previously characterized an exocellular serine-Thiol Proteinase activity in Paracoccidioides brasiliensis, using as substrates peptides analogous of the internally quenched fluorogenic peptide Abz-MKRLTL-EDDnp. In this communication, detection of maximal Proteinase activity in the culture supernatant fluids followed the abrupt increase in the medium pH, owing to the accumulation of ammonia generated by urease activity. Culture supernatant fluids collected at the peak of Proteinase activity against Abz-MRKLTL-EDDnp were able to cleave components of the basal membrane of the extracellular matrix (EM), including laminin, fibronectin, collagen type IV and proteoglycans, and the proteolytic activity was selectively inhibited both by PMSF and p-HMB (sodium 7-hydroxymercuribenzoate), which are also specific inhibitors of the serine-Thiol Proteinase. Human collagen I, bovine fibrinogen, human immunoglobulin G, BSA or P. brasiliensis gp43 were resistant to proteolysis. The kinetics of appearance of the Proteinase activity against EM substrates coincided with that of proteolysis of Abz-MKRLTL-EDDnp. Moreover, chromatographic fractions of culture supernatants containing the serine-Thiol Proteinase at high specific activity were also active against EM substrates. These data suggest the involvement of this enzyme activity in the degradation of the basement membrane, which is the first step for fungal tissue invasion.
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characterization of an exocellular serine Thiol Proteinase activity in paracoccidioides brasiliensis
Biochemical Journal, 1995Co-Authors: Adriana K. Carmona, Rosana Puccia, L. Juliano, M C F Oliveira, Elaine G Rodrigues, L. R. TravassosAbstract:An exocellular Proteinase activity has been characterized in Paracoccidioides brasiliensis culture filtrates. Chromatographic analysis showed that the activity was eluted from an anion-exchange Resource Q column at 0.08-0.1 M NaCl, and by gel filtration near ovalbumin elution, in a single peak. Purification of the Proteinase, however, was hampered by the low protein yield, in contrast to the high peptidase activity. Numerous chromogenic peptidyl p-nitroanilide derivatives and internally quenched fluorescent peptides, flanked by Abz (O-aminobenzoyl) and EDDnp (ethylenediaminedinitrophenyl), were tested as substrates. Cleavage was observed with Abz-MKRLTL-EDDnp, Abz-FRLVR-EDDnp, and Abz-PLGLLGR-EDDnp at Leu-Thr, Leu-Val and Leu-Leu/Leu-Gly bonds respectively as determined by isolation of the corresponding fragments by HPLC. Leucine at P1 seemed to be restrictive for the activity of the exocellular enzyme, but threonine (P'1) and leucine (P'2) in Abz-MKRLTL-EDDnp apparently were not essential. Also, a pair of alanines could substitute for lysine (P3) and arginine (P2) in this substrate, with a decrease in the Km values. The exocellular peptidase activity of P. brasiliensis had an optimum pH of > 9.0 and was irreversibly inhibited by PMSF, mercuric acetate and p-hydroxymercuribenzoate. Inhibition of the mercuriate compounds could be partially reversed by Cys/EDTA. E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanido)butene] was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. These results suggest that P. brasiliensis exocellular enzyme belongs to the subfamily of SH-containing serine Proteinases.
Bilqees Bano - One of the best experts on this subject based on the ideXlab platform.
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Biochemical, immunological and kinetic characterization and partial sequence analysis of a Thiol Proteinase inhibitor from Bubalus bubalis kidney: An attempt targeting kidney disorders.
International journal of biological macromolecules, 2016Co-Authors: Anas Shamsi, Azaj Ahmed, Bilqees BanoAbstract:In the present study a Thiol Proteinase inhibitor was isolated from buffalo kidney making use of ammonium sulphate precipitation and gel filtration chromatography on Sephacryl S-100HR column. Purified inhibitor is homogeneous as it displayed a single band in gel electrophoresis both under reducing and non-reducing environment and is of 65KDa as revealed by gel filtration and SDS PAGE. Kinetic studies revealed the presence of reversible accompanied with competitive mode of inhibition; showing maximum efficacy against papain (Ki=2.90×10-4). It was maximally active at pH 8.0 and was stable for a period of 30, 60 and 90 days at 37, 4 and -20°C respectively. Immunological studies confirmed its purity of epitopes as a single precipitin line is obtained in immunodiffusion. N-terminal analysis revealed that it shared a good homology with mouse kidney cystatin as well as with Human Cys C and Cys E thereby advocating its use as a model for various human oriented studies which targets how the kidney cystatin level varies in accordance with various drugs that are currently being used as a target for variety of diseases.
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Conformational changes during amyloid fibril formation of pancreatic Thiol Proteinase inhibitor: effect of copper and zinc.
Molecular biology reports, 2011Co-Authors: Medha Priyadarshini, Bilqees BanoAbstract:Pancreatic Thiol Proteinase inhibitor (PTPI), a variant of cystatin superfamily of cysteine protease inhibitors, has been isolated from pancreas of Capra hircus. In the present study, we examined the effects of acid denaturation and a co-solvent on PTPI with a focus on protein conformational changes and amyloid fibril formation. The results demonstrate that PTPI can form amyloid like fibrils. Acid denaturation as studied by CD and fluorescence spectroscopy showed that PTPI populates three partly unfolded species, a native like state at pH 3.0, a structured molten globule at pH 1.0 and partly unfolded species at pH 2.0, from each of which amyloid like fibrils grow as assessed by Thioflavin T (ThT) spectroscopy. Effect of trifluoroethanol (TFE) on acid induced states of PTPI was analyzed. TFE stabilized each of the three acid-induced intermediates at predenaturational concentrations (10%) and accelerated fibril formation. Morphology of the protein species at the beginning and end of reactions was observed using transmission electron microscopy. Solvent conditions were decisive for final fibril morphology. Biometals, Cu(2+) and Zn(2+) produced a concentration dependent decline in ThT fluorescence suggesting deaggregation of the fibrils. When added prior to amyloid fibril initiation 50 μM Cu(2+) or 10 μM Zn(2+) prevented any amyloid aggregation. Implications for therapeutics in view of Cu(2+) and Zn(2+) as essential micronutrients are suggested.
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Protein unfolding studies of Thiol-Proteinase inhibitor from goat (Capra hircus) muscle in the presence of urea and GdnHCl as denaturants
European Biophysics Journal, 2011Co-Authors: Mohammad Aatif, Safikur Rahman, Bilqees BanoAbstract:In recent years, many advances have been made in the understanding of functional and structural characteristics of protein evolution from denaturant-based studies that subject the protein to a change in the microenvironment. This paper reports the chemical denaturation of purified goat muscle cystatin (GMC) a Thiol-Proteinase inhibitor, using urea and guanidine hydrochloride (GdnHCl). The subtle conformational changes of GMC were monitored by intrinsic fluorescence, extrinsic fluorescence, and CD spectroscopic techniques. Further, the activity of GMC as a function of increasing concentration of denaturants was also studied. It was found that increasing the concentration of GdnHCl significantly enhances the inactivation and unfolding of the inhibitor (GMC). In urea-induced denaturation, the intrinsic and extrinsic fluorescence intensity reveals significant structural changes in the inhibitor. Further, it was found that at low concentrations of urea, up to 0.5–1.0 M , there was quenching of fluorescence intensity compared with the native form and a red shift of 5 nm was observed up to 5–8 M . The results presented in this paper suggest that GdnHCl-induced denaturation of GMC follows a simple two-state rule in which native → denatured state transition occurs in a single step. However denaturation with urea proceeds through an intermediate or non-native state.
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Cystatin like Thiol Proteinase inhibitor from pancreas of Capra hircus: purification and detailed biochemical characterization
Amino Acids, 2010Co-Authors: Medha Priyadarshini, Bilqees BanoAbstract:A Thiol Proteinase inhibitor from Capra hircus (goat) pancreas (PTPI) isolated by ammonium sulphate precipitation (20–80%) and gel filtration chromatography on Sephacryl S-100HR, with 20.4% yield and 500-fold purification, gave molecular mass of 44 kDa determined by its electrophoretic and gel filtration behavior, respectively. The stokes radius, diffusion and sedimentation coefficients of PTPI were 27.3 Ǻ, 7.87 × 10^−7 cm^2 s^−1 and 3.83 s, respectively. It was stable in pH range 3–10 and up to 70°C (critical temperature, E _a = 21 kJ mol^−1). Kinetic analysis revealed reversible and competitive mode of inhibition with PTPI showing the highest inhibitory efficiency against papain ( K _ i = 5.88 nM). The partial amino acid sequence analysis showed that it shared good homology with bovine parotid and skin cystatin C. PTPI possessed 17.18% α helical content assessed by CD spectroscopy. The hydropathy plot of first 24 residues suggested that most amino acids of this stretch might be in the hydrophobic core of the protein.
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Cystatin like Thiol Proteinase inhibitor from pancreas of Capra hircus: purification and detailed biochemical characterization
Amino Acids, 2010Co-Authors: Medha Priyadarshini, Bilqees BanoAbstract:A Thiol Proteinase inhibitor from Capra hircus (goat) pancreas (PTPI) isolated by ammonium sulphate precipitation (20–80%) and gel filtration chromatography on Sephacryl S-100HR, with 20.4% yield and 500-fold purification, gave molecular mass of 44 kDa determined by its electrophoretic and gel filtration behavior, respectively. The stokes radius, diffusion and sedimentation coefficients of PTPI were 27.3 Ǻ, 7.87 × 10^−7 cm^2 s^−1 and 3.83 s, respectively. It was stable in pH range 3–10 and up to 70°C (critical temperature, E _a = 21 kJ mol^−1). Kinetic analysis revealed reversible and competitive mode of inhibition with PTPI showing the highest inhibitory efficiency against papain ( K _ i = 5.88 nM). The partial amino acid sequence analysis showed that it shared good homology with bovine parotid and skin cystatin C. PTPI possessed 17.18% α helical content assessed by CD spectroscopy. The hydropathy plot of first 24 residues suggested that most amino acids of this stretch might be in the hydrophobic core of the protein.
L. R. Travassos - One of the best experts on this subject based on the ideXlab platform.
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Exocellular proteolytic activity of Paracoccidioides brasiliensis: cleavage of components associated with the basement membrane.
Medical mycology, 1998Co-Authors: Rosana Puccia, Adriana K. Carmona, J L Gesztesi, L. Juliano, L. R. TravassosAbstract:We have previously characterized an exocellular serine-Thiol Proteinase activity in Paracoccidioides brasiliensis, using as substrates peptides analogous of the internally quenched fluorogenic peptide Abz-MKRLTL-EDDnp. In this communication, detection of maximal Proteinase activity in the culture supernatant fluids followed the abrupt increase in the medium pH, owing to the accumulation of ammonia generated by urease activity. Culture supernatant fluids collected at the peak of Proteinase activity against Abz-MRKLTL-EDDnp were able to cleave components of the basal membrane of the extracellular matrix (EM), including laminin, fibronectin, collagen type IV and proteoglycans, and the proteolytic activity was selectively inhibited both by PMSF and p-HMB (sodium 7-hydroxymercuribenzoate), which are also specific inhibitors of the serine-Thiol Proteinase. Human collagen I, bovine fibrinogen, human immunoglobulin G, BSA or P. brasiliensis gp43 were resistant to proteolysis. The kinetics of appearance of the Proteinase activity against EM substrates coincided with that of proteolysis of Abz-MKRLTL-EDDnp. Moreover, chromatographic fractions of culture supernatants containing the serine-Thiol Proteinase at high specific activity were also active against EM substrates. These data suggest the involvement of this enzyme activity in the degradation of the basement membrane, which is the first step for fungal tissue invasion.
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characterization of an exocellular serine Thiol Proteinase activity in paracoccidioides brasiliensis
Biochemical Journal, 1995Co-Authors: Adriana K. Carmona, Rosana Puccia, L. Juliano, M C F Oliveira, Elaine G Rodrigues, L. R. TravassosAbstract:An exocellular Proteinase activity has been characterized in Paracoccidioides brasiliensis culture filtrates. Chromatographic analysis showed that the activity was eluted from an anion-exchange Resource Q column at 0.08-0.1 M NaCl, and by gel filtration near ovalbumin elution, in a single peak. Purification of the Proteinase, however, was hampered by the low protein yield, in contrast to the high peptidase activity. Numerous chromogenic peptidyl p-nitroanilide derivatives and internally quenched fluorescent peptides, flanked by Abz (O-aminobenzoyl) and EDDnp (ethylenediaminedinitrophenyl), were tested as substrates. Cleavage was observed with Abz-MKRLTL-EDDnp, Abz-FRLVR-EDDnp, and Abz-PLGLLGR-EDDnp at Leu-Thr, Leu-Val and Leu-Leu/Leu-Gly bonds respectively as determined by isolation of the corresponding fragments by HPLC. Leucine at P1 seemed to be restrictive for the activity of the exocellular enzyme, but threonine (P'1) and leucine (P'2) in Abz-MKRLTL-EDDnp apparently were not essential. Also, a pair of alanines could substitute for lysine (P3) and arginine (P2) in this substrate, with a decrease in the Km values. The exocellular peptidase activity of P. brasiliensis had an optimum pH of > 9.0 and was irreversibly inhibited by PMSF, mercuric acetate and p-hydroxymercuribenzoate. Inhibition of the mercuriate compounds could be partially reversed by Cys/EDTA. E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanido)butene] was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. These results suggest that P. brasiliensis exocellular enzyme belongs to the subfamily of SH-containing serine Proteinases.
Alisson L. Matsuo - One of the best experts on this subject based on the ideXlab platform.
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C-Npys (S-3-nitro-2-pyridinesulfenyl) and peptide derivatives can inhibit a serine-Thiol Proteinase activity from Paracoccidioides brasiliensis.
Biochemical and biophysical research communications, 2007Co-Authors: Alisson L. Matsuo, Adriana K. Carmona, Luiz S. Silva, Carlos E. Cunha, Ernesto S. Nakayasu, Igor C. Almeida, Maria A. Juliano, Rosana PucciaAbstract:Abstract The inhibitory capacity of C-Npys (S-[3-nitro-2-pyridinesulfenyl]) derivatives over Thiol-containing serine proteases has never been tested. In the present work we used an extracellular serine-Thiol Proteinase activity from the fungal pathogen Paracoccidioides brasiliensis (PbST) to describe a potent inhibitory capacity of Bzl-C(Npys)KRLTL-NH2 and Bzl-MKRLTLC(Npys)-NH2. The assays were performed with PbST enriched upon affinity chromatography in a p-aminobenzamidine (pABA)-Sepharose column. Although PbST can cleave the fluorescence resonance energy transfer peptide Abz-MKRLTL-EDDnp between L–T, the C(Npys) derivatives were not substrates nor were they toxic in a cell detachment assay, allowing therapeutic use. The best inhibitor was Bzl-C(Npys)KRLTL-NH2 (Ki = 16 nM), suggesting that the peptide sequence promoted a favorable interaction, especially when C(Npys) was placed at a further position from the L–T bond, at the N-terminus. Inhibition was completely reverted with dithioerythritol, indicating that it was due to the reactivity of the C(Npys) moiety with a free SH– group.
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Modulation of the exocellular serine-Thiol Proteinase activity of Paracoccidioides brasiliensis by neutral polysaccharides
Microbes and infection, 2005Co-Authors: Alisson L. Matsuo, Ivarne I.l. Tersariol, Silvia Iovine Kobata, Luiz R. Travassos, Adriana K. Carmona, Rosana PucciaAbstract:Abstract Our group characterized an exocellular serine-Thiol Proteinase activity in the yeast phase of Paracoccidioides brasiliensis (PbST), a dimorphic human pathogen. The fungal Proteinase is able to cleave in vitro, at pH 7.4, proteins associated with the basal membrane, such as human laminin and fibronectin, type IV collagen and proteoglycans. In the present study, we investigated the influence of glycosaminoglycans (GAGs) and neutral polysaccharides upon the serine-Thiol Proteinase activity by means of kinetic analysis monitored with fluorescence resonance energy transfer (FRET) peptides using the substrate Abz-MKALTLQ-EDDnp (Abz = ortho-aminobenzoic acid; EDDnp = ethylenediaminedinitrophenyl). Only neutral polysaccharides exhibited patterns of interaction with the Proteinase, while sulfated GAGs had no effect. Incubation with neutral polysaccharides resulted in a powerful modulation of the enzyme activity, intensely changing the enzyme kinetic parameters of catalysis and affinity for the substrate. Commercial dextran at the highest concentration of 20 μM increased 6.8-fold the enzyme affinity for the substrate. In the presence of 8 μM of purified baker's yeast mannan, the apparent KM of the enzyme increased about 5.5-fold, reflecting a significant inhibition in binding to the peptide substrate. When an exocellular galactomannan (GalMan) complex isolated from P. brasiliensis was added to the reaction mixture at 400 nM, the apparent KM and VMAX decreased about threefold. Moreover, GalMan was able to protect the enzymatic activity at high temperatures, but it caused no effect on the optimum cleavage pH. Our results show a novel modulation mechanism in P. brasiliensis, where a fungal polysaccharide-rich component can stabilize a serine-Thiol proteolytic activity, which is possibly involved in fungal dissemination.