A study on trypsin, Aspergillus flavus and Bacillus sp. protease inhibitory activity in Cassia tora (L.) syn Senna tora (L.) Roxb. seed extract.

PubMed

Tripathi, Vinayak R; Kumar, Shailendra; Garg, Satyendra K

2011-07-12

Proteases play an important role in virulence of many human, plant and insect pathogens. The proteinaceous protease inhibitors of plant origin have been reported widely from many plant species. The inhibitors may potentially be used for multiple therapeutic applications in viral, bacterial, fungal diseases and physiological disorders. In traditional Indian medicine system, Cassia tora (Senna tora) is reportedly effective in treatment of skin and gastrointestinal disorders. The present study explores the protease inhibitory activity of the above plant seeds against trypsin, Aspergillus flavus and Bacillus sp. proteases. The crushed seeds of Cassia tora were washed thoroughly with acetone and hexane for depigmentation and defatting. The proteins were fractionated by ammonium sulphate (0-30, 30-60, 60-90%) followed by dialysis and size exclusion chromatography (SEC). The inhibitory potential of crude seed extract and most active dialyzed fraction against trypsin and proteases was established by spot test using unprocessed x-ray film and casein digestion methods, respectively. Electrophoretic analysis of most active fraction (30-60%) and SEC elutes were carried employing Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Gelatin SDS-PAGE. Inhibition of fungal spore germination was studied in the presence of dialyzed active inhibitor fraction. Standard deviation (SD) and ANOVA were employed as statistical tools. The crude seeds' extract displayed strong antitryptic, bacterial and fungal protease inhibitory activity on x-ray film. The seed protein fraction 30-60% was found most active for trypsin inhibition in caseinolytic assay (P < 0.001). The inhibition of caseinolytic activity of the proteases increased with increasing ratio of seed extract. The residual activity of trypsin, Aspergillus flavus and Bacillus sp. proteases remained only 4, 7 and 3.1%, respectively when proteases were incubated with 3 mg ml-1 seed protein extract for 60 min. The

pH regulation of recombinant glucoamylase production in Fusarium venenatum JeRS 325, a transformant with a Fusarium oxysporum alkaline (trypsin-like) protease promoter.

PubMed

Wiebe, M G; Robson, G D; Shuster, J R; Trinci, A P

1999-08-05

Fusarium venenatum (formerly Fusarium graminearum) JeRS 325 produces heterologous glucoamylase (GAM) under the regulation of a Fusarium oxysporum alkaline (trypsin-like) protease promoter. The glucoamylase gene was used as a reporter gene to study the effects of ammonium and pH on GAM production under the control of the alkaline protease promoter. Between pH 4.0 and 5.8, GAM production in glucose-limited chemostat cultures of JeRS 325 grown at a dilution rate of 0.10 h-1 (doubling time, 6.9 h) on (NH4)2SO4 medium increased in a linear manner with increase in pH. However, at pH 4.0 and below GAM production was almost completely repressed in glucose-limited chemostat cultures grown on (NH4)2SO4 or NaNO3 medium. Thus GAM production in JeRS 325 is regulated by culture pH, not by the nature of the nitrogen source in the medium. The difficulty of using unbuffered medium when investigating putative ammonium repression is also shown. The study demonstrates the potential for use of the alkaline protease promoter in F. graminearum for the production of recombinant proteins in a pH dependent man ner. Copyright 1999 John Wiley & Sons, Inc.

MOFzyme: Intrinsic protease-like activity of Cu-MOF.

PubMed

Li, Bin; Chen, Daomei; Wang, Jiaqiang; Yan, Zhiying; Jiang, Liang; Deliang Duan; He, Jiao; Luo, Zhongrui; Zhang, Jinping; Yuan, Fagui

2014-10-24

The construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins is challenging due to the high stability of peptide bonds and the importance of proteases in biology and industry. Metal-organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for protease mimic which has remained unknown. Herein, we report that Cu₂(C₉H₃O₆)₄/₃ MOF (which is well known as HKUST-1 and denoted as Cu-MOF here), possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin to bovine serum albumin (BSA) and casein. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin indicating a much higher affinity of BSA for Cu-MOF surface. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA.

MOFzyme: Intrinsic protease-like activity of Cu-MOF

NASA Astrophysics Data System (ADS)

Li, Bin; Chen, Daomei; Wang, Jiaqiang; Yan, Zhiying; Jiang, Liang; Deliang Duan; He, Jiao; Luo, Zhongrui; Zhang, Jinping; Yuan, Fagui

2014-10-01

The construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins is challenging due to the high stability of peptide bonds and the importance of proteases in biology and industry. Metal-organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for protease mimic which has remained unknown. Herein, we report that Cu2(C9H3O6)4/3 MOF (which is well known as HKUST-1 and denoted as Cu-MOF here), possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin to bovine serum albumin (BSA) and casein. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin indicating a much higher affinity of BSA for Cu-MOF surface. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA.

Expression of protease activated receptor-2 (PAR-2) in central airways of smokers and non-smokers

PubMed Central

Miotto, D; Hollenberg, M; Bunnett, N; Papi, A; Braccioni, F; Boschetto, P; Rea, F; Zuin, A; Geppetti, P; Saetta, M; Maestrelli, P; Fabbri, L; Mapp, C

2002-01-01

Background: Protease activated receptor-2 (PAR-2) is a transmembrane G protein coupled receptor preferentially activated by trypsin and tryptase. The protease activated receptors play an important role in most components of injury responses including cell proliferation, migration, matrix remodelling, and inflammation. Cigarette smoking causes an inflammatory process in the central airways, peripheral airways, lung parenchyma, and adventitia of pulmonary arteries. Methods: To quantify the expression of PAR-2 in the central airways of smokers and non-smokers, surgical specimens obtained from 30 subjects undergoing lung resection for localised pulmonary lesions (24 with a history of cigarette smoking and six non-smoking control subjects) were examined. Central airways were immunostained with an antiserum specific for PAR-2 and PAR-2 expression was quantified using light microscopy and image analysis. Results: PAR-2 expression was found in bronchial smooth muscle, epithelium, glands, and in the endothelium and smooth muscle of bronchial vessels. PAR-2 expression was similar in the central airways of smokers and non-smokers. When smokers were divided according to the presence of symptoms of chronic bronchitis and chronic airflow limitation, PAR-2 expression was increased in smooth muscle (median 3.8 (interquartile range 2.9–5.8) and 1.4 (1.07–3.4) respectively); glands (33.3 (18.2–43.8) and 16.2 (11.5–22.2), respectively); and bronchial vessels (54.2 (48.7–56.8) and 40.0 (36–40.4), respectively) of smokers with symptoms of chronic bronchitis with normal lung function compared with smokers with chronic airflow limitation (COPD), but the increase was statistically significant (p<0.005) only for bronchial vessels. Conclusions: PAR-2 is present in bronchial smooth muscle, glands, and bronchial vessels of both smokers and non-smokers. An increased expression of PAR-2 was found in bronchial vessels of patients with bronchitis compared with those with COPD. PMID

Protease-activated receptor 2 activation of myeloid dendritic cells regulates allergic airway inflammation

PubMed Central

2011-01-01

Background A common characteristic of allergens is that they contain proteases that can activate protease-activated receptor (PAR-2); however the mechanism by which PAR-2 regulates allergic airway inflammation is unclear. Methods Mice (wild type and PAR-2-deficient) were sensitized using German cockroach (GC) feces (frass), the isolated protease from GC frass, or through adoptive transfer of GC frass-treated bone marrow-derived dendritic cells (BMDC) and measurements of airway inflammation (cellular infiltration, cytokine expression, and mucin production), serum IgE levels and airway hyperresponsiveness (AHR) were assessed. BMDC were cultured, treated with GC frass and assessed for cytokine production. PAR-2 expression on pulmonary mDCs was determined by flow cytometry. Results Exposure to GC frass induced AHR and airway inflammation in wild type mice; however PAR-2-deficient mice had significantly attenuated responses. To directly investigate the role of the protease, we isolated the protease from GC frass and administered the endotoxin-free protease into the airways of mice in the presence of OVA. GC frass proteases were sufficient to promote the development of AHR, serum IgE, and Th2 cytokine production. PAR-2 expression on mDC was upregulated following GC frass exposure, but the presence of a functional PAR-2 did not alter antigen uptake. To determine if PAR-2 activation led to differential cytokine production, we cultured BMDC in the presence of GM-CSF and treated these cells ex vivo with GC frass. PAR-2-deficient BMDC released significantly less IL-6, IL-23 and TNFα compared to BMDC from wild type mice, suggesting PAR-2 activation was important in Th2/Th17 skewing cytokine production. To determine the role for PAR-2 on mDCs on the initiation of allergic airway inflammation, BMDCs from wild type and PAR-2-deficient mice were treated in the presence or absence of GC frass and then adoptively transferred into the airway of wild type mice. Importantly, GC frass

Peptide affinity labels for thrombin and other trypsin-like proteases

DOEpatents

Shaw, E.N.; Kettner, C.A.

1982-03-09

A peptide affinity label is disclosed of the formula (I): as given in the patent wherein X is a radical capable of acting as a leaving group in a nucleophilic substitution reaction; A is an aromatic amino acid residue; B is H, or a C[sub 1]--C[sub 4] alkyl group, or aryl; Y is selected from the group consisting of hydrogen, aroyl, C[sub 1]--C[sub 6] acyl, and Q--(A)--[sub n], wherein Q = hydrogen, aroyl, or C[sub 1]--C[sub 6] acyl, n = 1--10, A is an amino acid residue selected from the aliphatic, hydroxy-containing, carboxylic acid group, and amide-thereofcontaining, aromatic, sulfur-containing and imino-containing amino acids; and wherein J is selected from the group consisting of --CH[sub 2]--, --CH[sub 2]--CH[sub 2]--, --CH[sub 2]--CH[sub 2]--CH[sub 2]--, --CH[double bond]CH-- and --CH(OH)--CH[sub 2]. The affinity label is useful for irreversibly inactivating thrombin and trypsin-like enzymes and may be used as a potential anticlotting agent. 2 figs.

Peptide affinity labels for thrombin and other trypsin-like proteases

DOEpatents

Shaw, Elliott N.; Kettner, Charles A.

1982-03-09

A peptide affinity label of the formula (I): ##STR1## wherein X is a radical capable of acting as a leaving group in a nucleophilic substitution reaction; A is an aromatic amino acid residue; B is H, or a C.sub.1 -C.sub.4 alkyl group, or aryl; Y is selected from the group consisting of hydrogen, aroyl, C.sub.1 -C.sub.6 acyl, and Q--(A)--.sub.n, wherein Q=hydrogen, aroyl, or C.sub.1 -C.sub.6 acyl, n=1-10, A is an amino acid residue selected from the aliphatic, hydroxy-containing, carboxylic acid group, and amide-thereof-containing, aromatic, sulfur-containing and imino-containing amino acids; and wherein J is selected from the group consisting of --CH.sub.2 --, --CH.sub.2 --CH.sub.2 --,--CH.sub.2 --CH.sub.2 --CH.sub.2 --, --CH.dbd.CH-- and --CH(OH)--CH.sub.2. The affinity label is useful for irreversibly inactivating thrombin and trypsin-like enzymes and may be used as a potential anticlotting agent.

MOFzyme: Intrinsic protease-like activity of Cu-MOF

PubMed Central

Li, Bin; Chen, Daomei; Wang, Jiaqiang; Yan, Zhiying; Jiang, Liang; Deliang Duan; He, Jiao; Luo, Zhongrui; Zhang, Jinping; Yuan, Fagui

2014-01-01

The construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins is challenging due to the high stability of peptide bonds and the importance of proteases in biology and industry. Metal-organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for protease mimic which has remained unknown. Herein, we report that Cu2(C9H3O6)4/3 MOF (which is well known as HKUST-1 and denoted as Cu-MOF here), possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin to bovine serum albumin (BSA) and casein. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin indicating a much higher affinity of BSA for Cu-MOF surface. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA. PMID:25342169

Synthesis of Trypsin-Resistant Variants of the Listeria-Active Bacteriocin Salivaricin P▿

PubMed Central

O'Shea, Eileen F.; O'Connor, Paula M.; Cotter, Paul D.; Ross, R. Paul; Hill, Colin

2010-01-01

Two-component salivaricin P-like bacteriocins have demonstrated potential as antimicrobials capable of controlling infections in the gastrointestinal tract (GIT). The anti-Listeria activity of salivaricin P is optimal when the individual peptides Sln1 and Sln2 are added in succession at a 1:1 ratio. However, as degradation by digestive proteases may compromise the functionality of these peptides within the GIT, we investigated the potential to create salivaricin variants with enhanced resistance to the intestinal protease trypsin. A total of 11 variants of the salivaricin P components, in which conservative modifications at the trypsin-specific cleavage sites were explored in order to protect the peptides from trypsin degradation while maintaining their potent antimicrobial activity, were generated. Analysis of these variants revealed that eight were resistant to trypsin digestion while retaining antimicrobial activity. Combining the complementary trypsin-resistant variants Sln1-5 and Sln2-3 resulted in a MIC50 of 300 nM against Listeria monocytogenes, a 3.75-fold reduction in activity compared to the level for wild-type salivaricin P. This study demonstrates the potential of engineering bacteriocin variants which are resistant to specific protease action but which retain significant antimicrobial activity. PMID:20581174

Inhibition of Protease-Epithelial Sodium Channel Signaling Improves Mucociliary Function in Cystic Fibrosis Airways.

PubMed

Reihill, James A; Walker, Brian; Hamilton, Robert A; Ferguson, Timothy E G; Elborn, J Stuart; Stutts, M Jackson; Harvey, Brian J; Saint-Criq, Vinciane; Hendrick, Siobhan M; Martin, S Lorraine

2016-09-15

In cystic fibrosis (CF) a reduction in airway surface liquid (ASL) height compromises mucociliary clearance, favoring mucus plugging and chronic bacterial infection. Inhibitors of the epithelial sodium channel (ENaC) have therapeutic potential in CF airways to reduce hyperstimulated sodium and fluid absorption to levels that can restore airway hydration. To determine whether a novel compound (QUB-TL1) designed to inhibit protease/ENaC signaling in CF airways restores ASL volume and mucociliary function. Protease activity was measured using fluorogenic activity assays. Differentiated primary airway epithelial cell cultures (F508del homozygotes) were used to determined ENaC activity (Ussing chamber recordings), ASL height (confocal microscopy), and mucociliary function (by tracking the surface flow of apically applied microbeads). Cell toxicity was measured using a lactate dehydrogenase assay. QUB-TL1 inhibits extracellularly located channel activating proteases (CAPs), including prostasin, matriptase, and furin, the activities of which are observed at excessive levels at the apical surface of CF airway epithelial cells. QUB-TL1-mediated CAP inhibition results in diminished ENaC-mediated Na(+) absorption in CF airway epithelial cells caused by internalization of a prominent pool of cleaved (active) ENaCγ from the cell surface. Importantly, diminished ENaC activity correlates with improved airway hydration status and mucociliary clearance. We further demonstrate QUB-TL1-mediated furin inhibition, which is in contrast to other serine protease inhibitors (camostat mesylate and aprotinin), affords protection against neutrophil elastase-mediated ENaC activation and Pseudomonas aeruginosa exotoxin A-induced cell death. QUB-TL1 corrects aberrant CAP activities, providing a mechanism to delay or prevent the development of CF lung disease in a manner independent of CF transmembrane conductance regulator mutation.

Engineering Protein Allostery: 1.05 Å Resolution Structure and Enzymatic Properties of a Na[superscript +]-activated Trypsin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Page, Michael J.; Carrell, Christopher J.; Di Cera, Enrico

2008-05-28

Some trypsin-like proteases are endowed with Na{sup +}-dependent allosteric enhancement of catalytic activity, but this important mechanism has been difficult to engineer in other members of the family. Replacement of 19 amino acids in Streptomyces griseus trypsin targeting the active site and the Na{sup +}-binding site were found necessary to generate efficient Na{sup +} activation. Remarkably, this property was linked to the acquisition of a new substrate selectivity profile similar to that of factor Xa, a Na{sup -} activated protease involved in blood coagulation. The X-ray crystal structure of the mutant trypsin solved to 1.05 {angstrom} resolution defines the engineeredmore » Na{sup +} site and active site loops in unprecedented detail. The results demonstrate that trypsin can be engineered into an efficient allosteric protease, and that Na+ activation is interwoven with substrate selectivity in the trypsin scaffold.« less

Role of Proteases in Extra-Oral Digestion of a Predatory Bug, Andrallus spinidens

PubMed Central

Zibaee, Arash; Hoda, Hassan; Mahmoud, Fazeli-Dinan

2012-01-01

Roles of salivary proteases in the extra-oral digestion of the predatory bug, Andrallus spinidens Fabricius (Hemiptera: Pentatomidae) were studied by using 2% azocasein as a general substrate and specific protease substrates, as well as synthetic and endogenous inhibitors. It was found that salivary glands of A. spinidens have two anterior, two lateral, and two posterior lobes. Azocasein was used to measure the activity of general proteases in the salivary glands using different buffer solutions. The enzyme had the highest activity at pH 8. General protease activity was highest at 40 °C and was stable for 6–16 hours. The use of specific substrates showed that trypsin-like, chymotrypsin-like, aminopeptidase, and carboxypeptidase are the active proteases present in salivary glands, by the maximum activity of trypsin-like protease in addition to their optimal pH between 8–9. Ca2+ and Mg2+ increased proteolytic activity about 216%, while other ions decreased it. Specific inhibitors including SBTI, PMSF, TLCK, and TPCK significantly decreased enzyme activity, as well as the specific inhibitors of methalloproteases including phenanthroline, EGTA, and TTHA. Extracted endogenous trypsin inhibitors extracted from potential prey, Chilo suppressalis, Naranga aenescens, Pieris brassicae, Hyphantria cunea, and Ephestia kuhniella, had different effects on trypsin-like protease activity of A. spinidens salivary glands. With the exception of C. suppressalis, the endogenous inhibitors significantly decreased enzyme activity in A. spinidens. PMID:22954419

Activation of Influenza A Viruses by Host Proteases from Swine Airway Epithelium

PubMed Central

Peitsch, Catharina; Klenk, Hans-Dieter; Garten, Wolfgang

2014-01-01

Pigs are important natural hosts of influenza A viruses, and due to their susceptibility to swine, avian, and human viruses, they may serve as intermediate hosts supporting adaptation and genetic reassortment. Cleavage of the influenza virus surface glycoprotein hemagglutinin (HA) by host cell proteases is essential for viral infectivity. Most influenza viruses, including human and swine viruses, are activated at a monobasic HA cleavage site, and we previously identified TMPRSS2 and HAT to be relevant proteases present in human airways. We investigated the proteolytic activation of influenza viruses in primary porcine tracheal and bronchial epithelial cells (PTEC and PBEC, respectively). Human H1N1 and H3N2 viruses replicated efficiently in PTECs and PBECs, and viruses containing cleaved HA were released from infected cells. Moreover, the cells supported the proteolytic activation of HA at the stage of entry. We found that swine proteases homologous to TMPRSS2 and HAT, designated swTMPRSS2 and swAT, respectively, were expressed in several parts of the porcine respiratory tract. Both proteases cloned from primary PBECs were shown to activate HA with a monobasic cleavage site upon coexpression and support multicycle replication of influenza viruses. swAT was predominantly localized at the plasma membrane, where it was present as an active protease that mediated activation of incoming virus. In contrast, swTMPRSS2 accumulated in the trans-Golgi network, suggesting that it cleaves HA in this compartment. In conclusion, our data show that HA activation in porcine airways may occur by similar proteases and at similar stages of the viral life cycle as in human airways. PMID:24155384

Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation.

PubMed

Edgington-Mitchell, Laura E; Barlow, Nicholas; Aurelio, Luigi; Samha, Aminath; Szabo, Monika; Graham, Bim; Bunnett, Nigel

2017-01-15

Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Aspergillus fumigatus proteases, Asp f 5 and Asp f 13, are essential for airway inflammation and remodelling in a murine inhalation model.

PubMed

Namvar, S; Warn, P; Farnell, E; Bromley, M; Fraczek, M; Bowyer, P; Herrick, S

2015-05-01

In susceptible individuals, exposure to Aspergillus fumigatus can lead to the development of atopic lung diseases such as allergic bronchopulmonary aspergillosis (ABPA) and severe asthma with fungal sensitization (SAFS). Protease allergens including Asp f 5 and Asp f 13 from Aspergillus fumigatus are thought to be important for initiation and progression of allergic asthma. To assess the importance of secreted protease allergens Asp f 5 (matrix metalloprotease) and Asp f 13 (serine protease) in Aspergillus fumigatus-induced inflammation, airway hyperactivity, atopy and airway wall remodelling in a murine model following chronic exposure to secreted allergens. BALB/c mice were repeatedly intranasally dosed over the course of 5 weeks with culture filtrate from wild-type (WT), Asp f 5 null (∆5) or Asp f 13 null (∆13) strains of Aspergillus fumigatus. Airway hyper-reactivity was measured by non-invasive whole-body plethysmography, Th2 response and airway inflammation by ELISA and cell counts, whilst airway remodelling was assessed by histological analysis. Parent WT and ∆5 culture filtrates showed high protease activity, whilst protease activity in ∆13 culture filtrate was low. Chronic intranasal exposure to the three different filtrates led to comparable airway hyper-reactivity and Th2 response. However, protease allergen deleted strains, in particular ∆13 culture filtrate, induced significantly less airway inflammation and remodelling compared to WT culture filtrate. Aspergillus fumigatus-secreted allergen proteases, Asp f 5 and Asp f 13, are important for recruitment of inflammatory cells and remodelling of the airways in this murine model. However, deletion of a single allergen protease fails to alleviate airway hyper-reactivity and allergic immune response. Targeting protease activity of Aspergillus fumigatus in conditions such as SAFS or ABPA may have beneficial effects in preventing key aspects of airway pathology. © 2014 John Wiley & Sons Ltd.

Crystallization and preliminary X-ray diffraction studies of trypsin-like proteases from the gastric fluid of the marine crab Cancer pagurus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hehemann, Jan-Hendrik; Redecke, Lars; Perbandt, Markus

2007-03-01

Two trypsins from the gastric fluid of the marine crab C. pagurus were purified and crystallized and X-ray data were collected to 0.97 and 3.2 Å resolution. The digestive fluid of the marine crab Cancer pagurus (Decapoda, Brachyura) contains highly stable proteases which display enhanced activity in aqueous mixtures of organic solvents. Three trypsins were isolated from the gastric fluid and two of them, C.p.TryII and C.p.TryIII, were purified to homogeneity by anion-exchange chromatography and crystallized by hanging-drop vapour diffusion. Diffraction data were collected at a synchrotron to 0.97 and 3.2 Å resolution, respectively. The crystal of C.p.TryII belongs tomore » the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 52.06, b = 62.00, c = 71.66 Å. Based on the Matthews coefficient, one protein molecule per asymmetric unit is suggested. In contrast, crystals of C.p.TryIII, which belong to the cubic space group P2{sub 1}3 with unit-cell parameters a = b = c = 215.4 Å, are assumed to contain 12 molecules per asymmetric unit.« less

Characterization of the Mamestra configurata (Lepidoptera: Noctuidae) larval midgut protease complement and adaptation to feeding on artificial diet, Brassica species, and protease inhibitor.

PubMed

Erlandson, Martin A; Hegedus, Dwayne D; Baldwin, Douglas; Noakes, Amy; Toprak, Umut

2010-10-01

The midgut protease profiles from 5th instar Mamestra configurata larvae fed various diets (standard artificial diet, low protein diet, low protein diet with soybean trypsin inhibitor [SBTI], or Brassica napus) were characterized by one-dimensional enzymography in gelatin gels. The gut protease profile of larvae fed B. napus possessed protease activities of molecular masses of approximately 33 and 55 kDa, which were not present in the guts of larvae fed artificial diet. Similarly, larvae fed artificial diet had protease activities of molecular masses of approximately 21, 30, and 100 kDa that were absent in larvae fed B. napus. Protease profiles changed within 12 to 24 h after switching larvae from artificial diet to plant diet and vice versa. The gut protease profiles from larvae fed various other brassicaceous species and lines having different secondary metabolite profiles did not differ despite significant differences in larval growth rates on the different host plants. Genes encoding putative digestive proteolytic enzymes, including four carboxypeptidases, five aminopeptidases, and 48 serine proteases, were identified in cDNA libraries from 4th instar M. configurata midgut tissue. Many of the protease-encoding genes were expressed at similar levels on all diets; however, three chymoptrypsin-like genes (McSP23, McSP27, and McSP37) were expressed at much higher levels on standard artificial diet and diet containing SBTI as was the trypsin-like gene McSP34. The expression of the trypsin-like gene McSP50 was highest on B. napus. The adaptation of M. configurata digestive biochemistry to different diets is discussed in the context of the flexibility of polyphagous insects to changing diet sources.

TNF is required for TLR ligand-mediated but not protease-mediated allergic airway inflammation.

PubMed

Whitehead, Gregory S; Thomas, Seddon Y; Shalaby, Karim H; Nakano, Keiko; Moran, Timothy P; Ward, James M; Flake, Gordon P; Nakano, Hideki; Cook, Donald N

2017-09-01

Asthma is associated with exposure to a wide variety of allergens and adjuvants. The extent to which overlap exists between the cellular and molecular mechanisms triggered by these various agents is poorly understood, but it might explain the differential responsiveness of patients to specific therapies. In particular, it is unclear why some, but not all, patients benefit from blockade of TNF. Here, we characterized signaling pathways triggered by distinct types of adjuvants during allergic sensitization. Mice sensitized to an innocuous protein using TLR ligands or house dust extracts as adjuvants developed mixed eosinophilic and neutrophilic airway inflammation and airway hyperresponsiveness (AHR) following allergen challenge, whereas mice sensitized using proteases as adjuvants developed predominantly eosinophilic inflammation and AHR. TLR ligands, but not proteases, induced TNF during allergic sensitization. TNF signaled through airway epithelial cells to reprogram them and promote Th2, but not Th17, development in lymph nodes. TNF was also required during the allergen challenge phase for neutrophilic and eosinophilic inflammation. In contrast, TNF was dispensable for allergic airway disease in a protease-mediated model of asthma. These findings might help to explain why TNF blockade improves lung function in only some patients with asthma.

TNF is required for TLR ligand–mediated but not protease-mediated allergic airway inflammation

PubMed Central

Whitehead, Gregory S.; Thomas, Seddon Y.; Shalaby, Karim H.; Nakano, Keiko; Moran, Timothy P.; Ward, James M.; Flake, Gordon P.; Cook, Donald N.

2017-01-01

Asthma is associated with exposure to a wide variety of allergens and adjuvants. The extent to which overlap exists between the cellular and molecular mechanisms triggered by these various agents is poorly understood, but it might explain the differential responsiveness of patients to specific therapies. In particular, it is unclear why some, but not all, patients benefit from blockade of TNF. Here, we characterized signaling pathways triggered by distinct types of adjuvants during allergic sensitization. Mice sensitized to an innocuous protein using TLR ligands or house dust extracts as adjuvants developed mixed eosinophilic and neutrophilic airway inflammation and airway hyperresponsiveness (AHR) following allergen challenge, whereas mice sensitized using proteases as adjuvants developed predominantly eosinophilic inflammation and AHR. TLR ligands, but not proteases, induced TNF during allergic sensitization. TNF signaled through airway epithelial cells to reprogram them and promote Th2, but not Th17, development in lymph nodes. TNF was also required during the allergen challenge phase for neutrophilic and eosinophilic inflammation. In contrast, TNF was dispensable for allergic airway disease in a protease-mediated model of asthma. These findings might help to explain why TNF blockade improves lung function in only some patients with asthma. PMID:28758900

Identification of a new soybean Kunitz trypsin inhibitor mutation and its effect on Bowman-Birk protease inhibitor content in soybean seed

USDA-ARS?s Scientific Manuscript database

Soybean seeds possess anti-nutritional compounds which inactivate digestive proteases, principally corresponding to two families: Kunitz Trypsin Inhibitors (KTi) and Bowman-Birk Inhibitors (BBI). High levels of raw soybeans/soybean meal in feed mixtures can cause poor weight gain and pancreatic abno...

Differences in PAR-2 activating potential by king crab (Paralithodes camtschaticus), salmon (Salmo salar), and bovine (Bos taurus) trypsin.

PubMed

Larsen, Anett K; Kristiansen, Kurt; Sylte, Ingebrigt; Seternes, Ole-Morten; Bang, Berit E

2013-07-20

Salmon trypsin is shown to increase secretion of the pro-inflammatory cytokine interleukin (IL)-8 from human airway epithelial cells through activation of PAR-2. Secretion of IL-8 induced by king crab trypsin is observed in a different concentration range compared to salmon trypsin, and seems to be only partially related to PAR-2 activation. This report aim to identify differences in the molecular structure of king crab trypsin (Paralithodes camtschaticus) compared to salmon (Salmo salar) and bovine trypsin (Bos taurus) that might influence the ability to activate protease-activated receptor-2 (PAR-2). During purification king crab trypsin displayed stronger binding capacity to the anionic column used in fast protein liquid chromatography compared to fish trypsins, and was identified as a slightly bigger molecule. Measurements of enzymatic activity yielded no obvious differences between the trypsins tested. Molecular modelling showed that king crab trypsin has a large area with strong negative electrostatic potential compared to the smaller negative areas in bovine and salmon trypsins. Bovine and salmon trypsins also displayed areas with strong positive electrostatic potential, a feature lacking in the king crab trypsin. Furthermore we have identified 3 divergent positions (Asp196, Arg244, and Tyr247) located near the substrate binding pocket of king crab trypsin that might affect the binding and cleavage of PAR-2. These preliminary results indicate that electrostatic interactions could be of importance in binding, cleavage and subsequent activation of PAR-2.

Mesotrypsin has evolved four unique residues to cleave trypsin inhibitors as substrates [Mesotrypsin has evolved to cleave trypsin inhibitors as substrates using four unique residues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alloy, Alexandre P.; Kayode, Olumide; Wang, Ruiying

Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursormore » protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. As a result, these findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.« less

Mesotrypsin has evolved four unique residues to cleave trypsin inhibitors as substrates [Mesotrypsin has evolved to cleave trypsin inhibitors as substrates using four unique residues

DOE PAGES

Alloy, Alexandre P.; Kayode, Olumide; Wang, Ruiying; ...

2015-07-14

Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursormore » protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. As a result, these findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.« less

MOLECULAR IDENTIFICATION OF CYSTEINE AND TRYPSIN PROTEASE, EFFECT OF DIFFERENT HOSTS ON PROTEASE EXPRESSION, AND RNAI MEDIATED SILENCING OF CYSTEINE PROTEASE GENE IN THE SUNN PEST.

PubMed

Amiri, Azam; Bandani, Ali Reza; Alizadeh, Houshang

2016-04-01

Sunn pest, Eurygaster integriceps, is a serious pest of cereals in the wide area of the globe from Near and Middle East to East and South Europe and North Africa. This study described for the first time, identification of E. integriceps trypsin serine protease and cathepsin-L cysteine, transcripts involved in digestion, which might serve as targets for pest control management. A total of 478 and 500 base pair long putative trypsin and cysteine gene sequences were characterized and named Tryp and Cys, respectively. In addition, the tissue-specific relative gene expression levels of these genes as well as gluten hydrolase (Gl) were determined under different host kernels feeding conditions. Result showed that mRNA expression of Cys, Tryp, and Gl was significantly affected after feeding on various host plant species. Transcript levels of these genes were most abundant in the wheat-fed E. integriceps larvae compared to other hosts. The Cys transcript was detected exclusively in the gut, whereas the Gl and Tryp transcripts were detectable in both salivary glands and gut. Also possibility of Sunn pest gene silencing was studied by topical application of cysteine double-stranded RNA (dsRNA). The results indicated that topically applied dsRNA on fifth nymphal stage can penetrate the cuticle of the insect and induce RNA interference. The Cys gene mRNA transcript in the gut was reduced to 83.8% 2 days posttreatment. Also, it was found that dsRNA of Cys gene affected fifth nymphal stage development suggesting the involvement of this protease in the insect growth, development, and molting. © 2015 Wiley Periodicals, Inc.

Effects of Mucuna pruriens protease inhibitors on Echis carinatus venom.

PubMed

Hope-Onyekwere, Nnadozie Stanley; Ogueli, Godwin Ifeanyi; Cortelazzo, Alessio; Cerutti, Helena; Cito, Annarita; Aguiyi, John C; Guerranti, Roberto

2012-12-01

The medicinal plant Mucuna pruriens, with reputed anti-snake venom properties has been reported to contain a kunitz-type trypsin inhibitor. This study was undertaken to further evaluate the protease inhibitory potential of gpMuc, a multiform glycoprotein, and other protein fractions from M. pruriens seeds against trypsin, chymotrypsin, Echis carinatus snake venom, ecarin and thrombin. The results showed that gpMuc inhibited both trypsin and chymotrypsin activities and was thermally stable, maintaining its trypsin inhibitory activity at temperatures of up to 50°C. Its structural conformation was also maintained at pH ranges of 4-7. Immunoreactivity study confirms that it contains protease-recognizing epitope on one of its isoforms. The whole protein extract of M. pruriens seeds inhibited prothrombin activation by ecarin and whole E. carinatus venom, and also thrombin-like activity using chromogenic assay. Copyright © 2012 John Wiley & Sons, Ltd.

Characterization of the molecular features and expression patterns of two serine proteases in Hermetia illucens (Diptera: Stratiomyidae) larvae.

PubMed

Kim, Wontae; Bae, Sungwoo; Kim, Ayoung; Park, Kwanho; Lee, Sangbeom; Choi, Youngcheol; Han, Sangmi; Park, Younghan; Koh, Youngho

2011-06-01

To investigate the molecular scavenging capabilities of the larvae of Hermetia illucens, two serine proteases (SPs) were cloned and characterized. Multiple sequence alignments and phylogenetic tree analysis of the deduced amino acid sequences of Hi-SP1 and Hi-SP2 were suggested that Hi-SP1 may be a chymotrypsin- and Hi-SP2 may be a trypsin-like protease. Hi-SP1 and Hi-SP2 3-D homology models revealed that a catalytic triad, three disulfide bonds, and a substrate-binding pocket were highly conserved, as would be expected of a SP. E. coli expressed Hi-SP1 and Hi-SP2 showed chymotrypsin or trypsin activities, respectively. Hi-SP2 mRNAs were consistently expressed during larval development. In contrast, the expression of Hi-SP1 mRNA fluctuated between feeding and molting stages and disappeared at the pupal stages. These expression pattern differences suggest that Hi-SP1 may be a larval specific chymotrypsin-like protease involved with food digestion, while Hi-SP2 may be a trypsin-like protease with diverse functions at different stages.

Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes.

PubMed

Szul, Tomasz; Bratcher, Preston E; Fraser, Kyle B; Kong, Michele; Tirouvanziam, Rabindra; Ingersoll, Sarah; Sztul, Elizabeth; Rangarajan, Sunil; Blalock, J Edwin; Xu, Xin; Gaggar, Amit

2016-03-01

Proteases are important regulators of pulmonary remodeling and airway inflammation. Recently, we have characterized the enzyme prolyl endopeptidase (PE), a serine peptidase, as a critical protease in the generation of the neutrophil chemoattractant tripeptide Pro-Gly-Pro (PGP) from collagen. However, PE has been characterized as a cytosolic enzyme, and the mechanism mediating PE release extracellularly remains unknown. We examined the role of exosomes derived from airway epithelia as a mechanism for PE release and the potential extracellular signals that regulate the release of these exosomes. We demonstrate a specific regulatory pathway of exosome release from airway epithelia and identify PE as novel exosome cargo. LPS stimulation of airway epithelial cells induces release of PE-containing exosomes, which is significantly attenuated by small interfering RNA depletion of Toll-like receptor 4 (TLR4). These differences were recapitulated upon intratracheal LPS administration in mice competent versus deficient for TLR4 signaling. Finally, sputum samples from subjects with cystic fibrosis colonized with Pseudomonas aeruginosa demonstrate elevated exosome content and increased PE levels. This TLR4-based mechanism highlights the first report of nonstochastic release of exosomes in the lung and couples TLR4 activation with matrikine generation. The increased quantity of these proteolytic exosomes in the airways of subjects with chronic lung disease highlights a new mechanism of injury and inflammation in the pathogenesis of pulmonary disorders.

Dual origin of gut proteases in Formosan subterranean termites (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae).

PubMed

Sethi, Amit; Xue, Qing-Gang; La Peyre, Jerome F; Delatte, Jennifer; Husseneder, Claudia

2011-07-01

Cellulose digestion in lower termites, mediated by carbohydrases originating from both termite and endosymbionts, is well characterized. In contrast, limited information exists on gut proteases of lower termites, their origins and roles in termite nutrition. The objective of this study was to characterize gut proteases of the Formosan subterranean termite (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae). The protease activity of extracts from gut tissues (fore-, mid- and hindgut) and protozoa isolated from hindguts of termite workers was quantified using hide powder azure as a substrate and further characterized by zymography with gelatin SDS-PAGE. Midgut extracts showed the highest protease activity followed by the protozoa extracts. High level of protease activity was also detected in protozoa culture supernatants after 24 h incubation. Incubation of gut and protozoa extracts with class-specific protease inhibitors revealed that most of the proteases were serine proteases. All proteolytic bands identified after gelatin SDS-PAGE were also inhibited by serine protease inhibitors. Finally, incubation with chromogenic substrates indicated that extracts from fore- and hindgut tissues possessed proteases with almost exclusively trypsin-like activity while both midgut and protozoa extracts possessed proteases with trypsin-like and subtilisin/chymotrypsin-like activities. However, protozoa proteases were distinct from midgut proteases (with different molecular mass). Our results suggest that the Formosan subterranean termite not only produces endogenous proteases in its gut tissues, but also possesses proteases originating from its protozoan symbionts. Copyright © 2011 Elsevier Inc. All rights reserved.

Expanding proteome coverage with orthogonal-specificity α-Lytic proteases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyer, Jesse G.; Kim, Sangtae; Maltby, David A.

2014-03-01

Bottom-up proteomics studies traditionally involve proteome digestion with a single protease, trypsin. However, trypsin alone does not generate peptides that encompass the entire proteome. Alternative proteases have been explored, but most have specificity for charged amino acid side chains. Therefore, additional proteases that improve proteome coverage by cleavage at sequences complimentary to trypsin may increase proteome coverage. We demonstrate the novel application of two proteases for bottom-up proteomics: wild type alpha-lytic protease (WaLP), and an active site mutant of WaLP, M190A alpha-lytic protease (MaLP). We assess several relevant factors including MS/MS fragmentation, peptide length, peptide yield, and protease specificity. Bymore » combining data from separate digestions with trypsin, LysC, WaLP, and MaLP, proteome coverage was increased 101% compared to trypsin digestion alone. To demonstrate how the gained sequence coverage can access additional PTM information, we show identification of a number of novel phosphorylation sites in the S. pombe proteome and include an illustrative example from the protein MPD2, wherein two novel sites are identified, one in a tryptic peptide too short to identify and the other in a sequence devoid of tryptic sites. The specificity of WaLP and MaLP for aliphatic amino acid side chains was particularly valuable for coverage of membrane protein sequences, which increased 350% when the data from trypsin, LysC, WaLP, and MaLP were combined.« less

Anti-fibrinolytic and anti-microbial activities of a serine protease inhibitor from honeybee (Apis cerana) venom.

PubMed

Yang, Jie; Lee, Kwang Sik; Kim, Bo Yeon; Choi, Yong Soo; Yoon, Hyung Joo; Jia, Jingming; Jin, Byung Rae

2017-10-01

Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putative low-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identified from honeybee (Apis mellifera) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated. In this study, we identified an Asiatic honeybee (A. cerana) venom serine protease inhibitor (AcVSPI) that was shown to act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-like domain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putative low-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide. Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin, but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however, it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPI inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. These findings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor. Copyright © 2017 Elsevier Inc. All rights reserved.

Trypsin activation pathway of rotavirus infectivity.

PubMed Central

Arias, C F; Romero, P; Alvarez, V; López, S

1996-01-01

The infectivity of rotaviruses is increased by and most probably is dependent on trypsin treatment of the virus. This proteolytic treatment specifically cleaves VP4, the protein that forms the spikes on the surface of the virions, to polypeptides VP5 and VP8. This cleavage has been reported to occur in rotavirus SA114fM at two conserved, closely spaced arginine residues located at VP4 amino acids 241 and 247. In this work, we have characterized the VP4 cleavage products of rotavirus SA114S generated by in vitro treatment of the virus with increasing concentrations of trypsin and with proteases AspN and alpha-chymotrypsin. The VP8 and VP5 polypeptides were analyzed by gel electrophoresis and by Western blotting (immunoblotting) with antibodies raised to synthetic peptides that mimic the terminal regions of VP4 generated by the trypsin cleavage. It was shown that in addition to arginine residues 241 and 247, VP4 is cleaved at arginine residue 231. These three sites were found to have different susceptibilities to trypsin, Arg-241 > Arg-231 > Arg-247, with the enhancement of infectivity correlating with cleavage at Arg-247 rather than at Arg-231 or Arg-241. Proteases AspN and alpha-chymotrypsin cleaved VP4 at Asp-242 and Tyr-246, respectively, with no significant enhancement of infectivity, although this enhancement could be achieved by further treatment of the virus with trypsin. The VP4 end products of trypsin treatment were a homogeneous VP8 polypeptide comprising VP4 amino acids 1 to 231 and a heterogeneous VP5, which is formed by two polypeptide species (present at a ratio of approximately 1:5) as a result of cleavage at either Arg-241 or Arg-247. A pathway for the trypsin activation of rotavirus infectivity is proposed. PMID:8709201

Molecular Genetic Analysis of Midgut Serine Proteases in Aedes aegypti Mosquitoes

PubMed Central

Isoe, Jun; Rascón, Alberto A.; Kunz, Susan; Miesfeld, Roger L.

2009-01-01

Digestion of blood meal proteins by midgut proteases provides anautogenous mosquitoes with the nutrients required to complete the gonotrophic cycle. Inhibition of protein digestion in the midgut of blood feeding mosquitoes could therefore provide a strategy for population control. Based on recent reports indicating that the mechanism and regulation of protein digestion in blood fed female Aedes aegypti mosquitoes is more complex than previously thought, we used a robust RNAi knockdown method to investigate the role of four highly expressed midgut serine proteases in blood meal metabolism. We show by Western blotting that the early phase trypsin protein (AaET) is maximally expressed at 3 h post blood meal (PBM), and that AaET is not required for the protein expression of three late phase serine proteases, AaLT (late trypsin), AaSPVI (5G1), and AaSPVII. Using the trypsin substrate analog BApNA to analyze in vitro enzyme activity in midgut extracts from single mosquitoes, we found that knockdown of AaSPVI expression caused a 77.6% decrease in late phase trypsin-like activity, whereas, knockdown of AaLT and AaSPVII expression had no significant effect on BApNA activity. In contrast, injection of AaLT, AaSPVI, and AaSPVII dsRNA inhibited degradation of endogenous serum albumin protein using an in vivo protease assay, as well as, significantly decreased egg production in both the first and second gonotrophic cycles (p<0.001). These results demonstrate that AaLT, AaSPVI, and AaSPVII all contribute to blood protein digestion and oocyte maturation, even though AaSPVI is the only abundant midgut late phase serine protease that appears to function as a classic trypsin enzyme. PMID:19883761

Mesotrypsin Has Evolved Four Unique Residues to Cleave Trypsin Inhibitors as Substrates.

PubMed

Alloy, Alexandre P; Kayode, Olumide; Wang, Ruiying; Hockla, Alexandra; Soares, Alexei S; Radisky, Evette S

2015-08-28

Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. These findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The role of protease activation of inflammation in allergic respiratory diseases.

PubMed

Reed, Charles E; Kita, Hirohito

2004-11-01

Extracellular endogenous proteases, as well as exogenous proteases from mites and molds, react with cell-surface receptors in the airways to generate leukocyte infiltration and to amplify the response to allergens. Stimulation leads to increased intracellular Ca ++ and gene transcription. The most thoroughly investigated receptors, protease-activated receptors (PARs), are 7-transmembrane proteins coupled to G proteins. PARs are widely distributed on the cells of the airways, where they contribute to the inflammation characteristic of allergic diseases. PAR stimulation of epithelial cells opens tight junctions, causes desquamation, and produces cytokines, chemokines, and growth factors. They degranulate eosinophils and mast cells. Proteases contract bronchial smooth muscle and cause it to proliferate. PARs also promote maturation, proliferation, and collagen production of fibroblast precursors and mature fibroblasts. PAR-2, apparently the most important of the 4 PARs that have been characterized, is increased on the epithelium of patients with asthma. Trypsin, a product of injured epithelial cells, and mast cell tryptase are potent activators of PAR-2. Mast cell chymase activates PAR-1. Proteases from mites and molds appear to act through similar receptors. They amplify IgE production to allergens, degranulate eosinophils, and can generate inflammation, even in the absence of IgE. Proteases produced by Aspergillus species to support its growth are presumably responsible for the exuberant IgE, IgG, and granulomatous response of allergic bronchopulmonary aspergillosis. Similar proteases from molds germinating on the respiratory mucosa have been recently been implicated in the pathogenesis of chronic hyperplastic rhinitis and polyps and, by extension, of intrinsic asthma. Finally, proteases from mites and fungi growing in damp, water-damaged buildings might be the basis for the increased prevalence in these buildings of rhinitis, asthma, and other respiratory diseases

Allium sativum Protease Inhibitor: A Novel Kunitz Trypsin Inhibitor from Garlic Is a New Comrade of the Serpin Family.

PubMed

Shamsi, Tooba Naz; Parveen, Romana; Amir, Mohd; Baig, Mohd Affan; Qureshi, M Irfan; Ali, Sher; Fatima, Sadaf

2016-01-01

This study was aimed to purify and characterize the Protease inhibitor (PI) from a plant Allium sativum (garlic) with strong medicinal properties and to explore its phytodrug potentials. Allium sativum Protease Inhibitor (ASPI) was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD) spectroscopy. ASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max)" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2-12 showing a decline in the activity around pH 4-5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10-80°C) but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (μM/min) and Km value of 0.12 μM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM). The Far UV CD depicted 2.0% α -helices and 51% β -sheets at native pH. To conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids present in the reactive sites. Therefore, ASPI

Allium sativum Protease Inhibitor: A Novel Kunitz Trypsin Inhibitor from Garlic Is a New Comrade of the Serpin Family

PubMed Central

Shamsi, Tooba Naz; Parveen, Romana; Amir, Mohd.; Baig, Mohd. Affan; Qureshi, M. Irfan; Ali, Sher; Fatima, Sadaf

2016-01-01

Purpose This study was aimed to purify and characterize the Protease inhibitor (PI) from a plant Allium sativum (garlic) with strong medicinal properties and to explore its phytodrug potentials. Methods Allium sativum Protease Inhibitor (ASPI) was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD) spectroscopy. Results ASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max)" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2–12 showing a decline in the activity around pH 4–5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10–80°C) but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (μM/min) and Km value of 0.12 μM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM). The Far UV CD depicted 2.0% α -helices and 51% β -sheets at native pH. Conclusions To conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids

Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lini.

PubMed

Barata, Ricardo Andrade; Andrade, Milton Hercules Guerra; Rodrigues, Roberta Dias; Castro, Ieso Miranda

2002-01-01

An alkaline serineprotease, capable of hydrolyzing Nalpha-benzoyl- dl arginine p-nitroanilide, was secreted by Fusarium oxysporum var. lini grown in the presence of gelatin as the sole nitrogen and carbon source. The protease was purified 65-fold to electrophoretic homogenity from the culture supernatant in a three-step procedure comprising QSepharose chromatography, affinity chromatography, and FPLC on a MonoQ column. SDS-PAGE analysis of the purified protein indicated an estimated molecular mass of 41 kDa. The protease had optimum activity at a reaction temperature of 45 degrees C and showed a rapid decrease of activity at 48 degrees C. The optimum pH was around 8.0. Characterization of the protease showed that Ca2+ and Mg2+ cations increased the activity, which was not inhibited by EDTA or 1,10-phenanthroline. The enzyme activity on Nalpha-benzoyl-DL arginine p-nitroanilide was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, p-aminobenzamidine dihydrochloride, aprotinin, 3-4 dichloroisocoumarin, and N-tosyl-L-lysine chloromethyl ketone. The enzyme is also inhibited by substrate concentrations higher than 2.5 x 10(-4)M. The protease had a Michaelis-Menten constant of 0.16 mM and a V(max) of 0.60 mumol released product.min(-1).mg(-1) enzyme when assayed in a non-inhibiting substrate concentration. The activity on Nalpha-benzoyl- dl arginine p-nitroanilide was competitively inhibited by p-aminobenzamidine dihydrochoride. A K(i) value of 0.04 mM was obtained.

Pancreatic and Pancreatic-Like Microbial Proteases Accelerate Gut Maturation in Neonatal Rats

PubMed Central

Prykhodko, Olena; Pierzynowski, Stefan G.; Nikpey, Elham; Arevalo Sureda, Ester; Fedkiv, Olexandr; Weström, Björn R.

2015-01-01

Objectives Postnatal gut maturation in neonatal mammals, either at natural weaning or after precocious inducement, is coinciding with enhanced enzymes production by exocrine pancreas. Since the involvement of enzymes in gut functional maturation was overlooked, the present study aimed to investigate the role of enzymes in gut functional maturation using neonatal rats. Methods Suckling rats (Rattus norvegicus) were instagastrically gavaged with porcine pancreatic enzymes (Creon), microbial-derived amylase, protease, lipase and mixture thereof, while controls received α-lactalbumin or water once per day during 14–16 d of age. At 17 d of age the animals were euthanized and visceral organs were dissected, weighed and analyzed for structural and functional properties. For some of the rats, gavage with the macromolecular markers such as bovine serum albumin and bovine IgG was performed 3 hours prior to blood collection to assess the intestinal permeability. Results Gavage with the pancreatic or pancreatic-like enzymes resulted in stimulated gut growth, increased gastric acid secretion and switched intestinal disaccharidases, with decreased lactase and increased maltase and sucrase activities. The fetal-type vacuolated enterocytes were replaced by the adult-type in the distal intestine, and macromolecular transfer to the blood was declined. Enzyme exposure also promoted pancreas growth with increased amylase and trypsin production. These effects were confined to the proteases in a dose-dependent manner. Conclusion Feeding exogenous enzymes, containing proteases, induced precocious gut maturation in suckling rats. This suggests that luminal exposure to proteases by oral loading or, possibly, via enhanced pancreatic secretion involves in the gut maturation of young mammals. PMID:25658606

Pancreatic and pancreatic-like microbial proteases accelerate gut maturation in neonatal rats.

PubMed

Prykhodko, Olena; Pierzynowski, Stefan G; Nikpey, Elham; Arevalo Sureda, Ester; Fedkiv, Olexandr; Weström, Björn R

2015-01-01

Postnatal gut maturation in neonatal mammals, either at natural weaning or after precocious inducement, is coinciding with enhanced enzymes production by exocrine pancreas. Since the involvement of enzymes in gut functional maturation was overlooked, the present study aimed to investigate the role of enzymes in gut functional maturation using neonatal rats. Suckling rats (Rattus norvegicus) were instagastrically gavaged with porcine pancreatic enzymes (Creon), microbial-derived amylase, protease, lipase and mixture thereof, while controls received α-lactalbumin or water once per day during 14-16 d of age. At 17 d of age the animals were euthanized and visceral organs were dissected, weighed and analyzed for structural and functional properties. For some of the rats, gavage with the macromolecular markers such as bovine serum albumin and bovine IgG was performed 3 hours prior to blood collection to assess the intestinal permeability. Gavage with the pancreatic or pancreatic-like enzymes resulted in stimulated gut growth, increased gastric acid secretion and switched intestinal disaccharidases, with decreased lactase and increased maltase and sucrase activities. The fetal-type vacuolated enterocytes were replaced by the adult-type in the distal intestine, and macromolecular transfer to the blood was declined. Enzyme exposure also promoted pancreas growth with increased amylase and trypsin production. These effects were confined to the proteases in a dose-dependent manner. Feeding exogenous enzymes, containing proteases, induced precocious gut maturation in suckling rats. This suggests that luminal exposure to proteases by oral loading or, possibly, via enhanced pancreatic secretion involves in the gut maturation of young mammals.

A trypsin inhibitor from Sapindus saponaria L. seeds: purification, characterization, and activity towards pest insect digestive enzyme.

PubMed

Macedo, Maria Lígia R; Diz Filho, Eduardo B S; Freire, Mariadas Graças M; Oliva, Maria Luiza V; Sumikawa, Joana T; Toyama, Marcos H; Marangoni, Sérgio

2011-01-01

The present paper describes the purification, characterization and determination of the partial primary structure of the first trypsin inhibitor isolated from the family Sapindaceae. A highly stable, potent trypsin inhibitor (SSTI) was purified to homogeneity. SDS-PAGE analysis revealed that the protein consists of a two-polypeptide chain with molecular masses of approximately 15 and 3 kDa. The purified inhibitor inhibited bovine trypsin at a 1:1 M ratio. Kinetic analysis revealed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 10⁻⁹ M for trypsin. The partial NH₂- terminal sequence of 36 amino acids in SSTI indicates homology with other members of the trypsin-inhibitor family from different sources. This inhibitor is highly stable in the presence of denaturing agents. SSTI showed significant inhibitory activity against trypsin-like proteases present in the larval midgut on Anagasta kuehniella, Corcyra cephalonica, Diatreae saccharalis and Anticarsia gemmatalis.

Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Werb, Z.; Aggeler, J.

1978-04-01

We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2 to 24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or ..cap alpha..-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16 to 48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10/sup 6/ cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continuedmore » presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, ..cap alpha../sub 1/-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.« less

Molecular characterization of genes encoding trypsin-like enzymes from Aedes aegypti larvae and identification of digestive enzymes.

PubMed

Soares, Tatiane S; Watanabe, Renata M O; Lemos, Francisco J A; Tanaka, Aparecida S

2011-12-10

Trypsin-like enzymes play an important role in the Aedes aegypti digestive process. The trypsin-like enzymes present in adults were characterized previously, but little is known about trypsins in larvae. In the present work, we identified one of the trypsin enzymes from Ae. aegypti larval midgut using a library of trypsin gene fragments, which was the sequence known as AAEL005607 from the Ae. aegypti genome. Quantitative PCR analysis showed that AAEL005607 was transcribed in all larval instars, but it was not present in adult midgut. In order to confirm transcription data, the trypsin-like enzymes from 4th instar larvae of Ae. aegypti midgut were purified and sequenced. Purified trypsin showed identity with the amino-terminal sequence of AAEL005607, AAEL005609 and AAEL005614. These three trypsins have high amino acids identity, and could all be used as a template for the design of inhibitors. In conclusion, for the first time, digestive enzymes of 4th larval instar of Ae. aegypti were purified and characterized. The knowledge of digestive enzymes present in Ae. aegypti larvae may be helpful in the development of a larvicide. Copyright © 2011 Elsevier B.V. All rights reserved.

Detailed two-dimensional gel proteomic mapping of the feces of the house dust mite Dermatophagoides pteronyssinus and comparison with D. farinae: Reduced trypsin protease content in D. pteronyssinus and different isoforms.

PubMed

Erban, Tomas; Harant, Karel; Hubert, Jan

2017-06-06

Major domestic mite allergens are present in feces. We present a detailed 2D-E-MS/MS proteomic analysis of the Dermatophagoides pteronyssinus feces. Precise cultivation yielded a pure fecal extract. We detected differences in fecal allergens/digestive enzymes between D. pteronyssinus and D. farinae using 2D-E fingerprinting, including unique information on species-specific protease isoforms. Proteomic analysis was performed by 2D-E coupled with MALDI-TOF/TOF identification. The species-specific differences in the fecal extracts of the mites were attributed to trypsin-like proteases known as group 3 allergens. In D. farinae, Der f 3 exhibited high abundance with a pI similar (acidic) to that of the cysteine protease Der f 1 and the chymotrypsin protease Der f 6, whereas in D. pteronyssinus, Der p 3 was rarely detected and exhibited low abundance only at basic pI. Moreover, Der p 9 was detected at a pI of ~ 10, in contrast to Der p 1 and Der p 6, suggesting different compartmentalization in the body. Overall, in D. pteronyssinus feces, allergens of groups 1, 2, 6, and 15 were quantitatively similar to those of D. farinae with the exception of the group 3 and 9 allergens. This work provides novel insights into mite-defecated proteins/digestive enzymes, which are important allergens. Millions of people are affected by allergy and asthma, and their number is growing. In homes, the major triggers of allergy and asthma are the house dust mites Dermatophagoides farinae and D. pteronyssinus, and a clear understanding of the development of diseases caused by these mites is needed. The major sources of mite allergens are their feces, which are deposited in the environment and are easily inhaled as part of aeroplankton. However, descriptions of and comparisons between the major fecal allergens of these two mites are lacking. This study shows that similar group 1 (cysteine protease), 2 (NPC2 family), 6 (chymotrypsin) and 15 (chitinase-like) allergens are present in the feces of

Trypsin from the pyloric caeca of bluefish (Pomatomus saltatrix).

PubMed

Klomklao, Sappasith; Benjakul, Soottawat; Visessanguan, Wonnop; Kishimura, Hideki; Simpson, Benjamin K

2007-12-01

Trypsin was purified from the pyloric caeca of bluefish (Pomatomus saltatrix) by ammonium sulfate precipitation, acetone precipitation and soybean trypsin inhibitor-Sepharose 4B affinity chromatography. Bluefish trypsin migrated as a single band using both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE and had a molecular mass of 28 kDa. The optima pH and temperature for the hydrolysis of benzoyl-dl-arginine-p-nitroanilide (BAPNA) were 9.5 and 55 degrees C, respectively. The enzyme was stable over a broad pH range (7 to 12), but was unstable at acidic pH, and at temperatures greater than 40 degrees C. The enzyme was inhibited by specific trypsin inhibitors: soybean trypsin inhibitor (SBTI), N-p-tosyl-l-lysine chloromethyl ketone (TLCK) and the serine protease inhibitor phenylmethyl sulfonylfluoride (PMSF). CaCl2 partially protected trypsin against activity loss at 40 degrees C, but NaCl (0 to 30%) decreased the activity in a concentration dependent manner. The N-terminal amino acid sequence of trypsin was determined as IVGGYECKPKSAPVQVSLNL and was highly homologous to other known vertebrate trypsins.

The involvement of the cysteine proteases of Clonorchis sinensis metacercariae in excystment.

PubMed

Li, Shunyu; Chung, Young-Bae; Chung, Byung-Suk; Choi, Min-Ho; Yu, Jae-Ran; Hong, Sung-Tae

2004-05-01

The effects of trypsin, bile, trypsin-bile, pepsin, dithiothreitol (DTT) and metacercarial excretory-secretory product (ESP) on the in vitro excystment of Clonorchis sinensis metacercariae were investigated. The majority of metacercariae excysted immediately in trypsin-bile in PBS solution, a process which was complete after 30 min of incubation. When incubated in metacercarial ESP in PBS, excystment was potentiated in the presence of 5 mM DTT, but was inhibited dose-dependently by a cysteine protease inhibitor, iodoacetic acid. Two active protease bands of 28 and 40 kDa were identified in the ESP of metacercariae by gelatin substrate SDS-PAGE. Scanning electron microscopy demonstrated that the larvae in solutions of DTT and ESP migrated through a small hole on the metacercarial wall, whereas larvae were liberated by entire wall disruption in trypsin solution. These results suggest that trypsin is a major extrinsic factor of the rapid excystment of C. sinensis metacercariae, and that endogenous cysteine proteases are also involved in metacercarial excystment. Copyright 2004 Springer-Verlag

Identification of a serine protease as a major allergen (Per a 10) of Periplaneta americana.

PubMed

Sudha, V T; Arora, N; Gaur, S N; Pasha, S; Singh, B P

2008-06-01

Cockroach allergens are associated with the development of asthma, but none of these has been characterized for proteolytic activity. This study was undertaken to isolate and characterize a protease from Periplaneta americana and determine its allergenicity. A serine protease was isolated from P. americana extract using benzamidine sepharose column and characterized by immunobiochemical methods. Allergenicity of the protease was assessed by enzyme-linked immunosorbent assay, immunoblot, intradermal testing, histamine release and peripheral blood mononuclear cells (PBMCs) proliferation. Affinity purified protein of approximately 28 kDa (Per a 10) showed a single band of activity in gelatin zymogram and agarose plate assay. N-terminal sequence (IVGGRPAQI) revealed similarity with mite serine protease allergens and insect trypsins. It demonstrated proteolytic activity with azocollagen > gelatin > defatted-milk > casein including serine protease specific substrate, N-benzoyl-arginine-ethyl-ester-hydrochloride. It was inhibited by serine protease inhibitors, namely aprotinin > pefabloc > AEBSF > PMSF > benzamidine > antipain > leupeptin and trypsin-specific inhibitor (tosyl-lysyl-chloromethyl-ketone) suggesting it to be a trypsin-like serine protease. Per a 10 was recognized as a major allergen, showing IgE reactivity with >80% of cockroach sensitized patients by skin tests and immunoblot. It could induce significant histamine release (P < 0.05) in blood and secretion of interleukin-4 (IL-4) (P < 0.05) and IL-5 (P < 0.05) in culture supernatant of PBMCs from cockroach hypersensitive patients, suggesting a strong allergenic potency. A serine protease isolated from P. americana was demonstrated to be a major allergen (Per a 10). It has a potential for component-based diagnosis of allergy and will be useful in elucidating the mechanism of allergy.

Peptide code-on-a-microplate for protease activity analysis via MALDI-TOF mass spectrometric quantitation.

PubMed

Hu, Junjie; Liu, Fei; Ju, Huangxian

2015-04-21

A peptide-encoded microplate was proposed for MALDI-TOF mass spectrometric (MS) analysis of protease activity. The peptide codes were designed to contain a coding region and the substrate of protease for enzymatic cleavage, respectively, and an internal standard method was proposed for the MS quantitation of the cleavage products of these peptide codes. Upon the cleavage reaction in the presence of target proteases, the coding regions were released from the microplate, which were directly quantitated by using corresponding peptides with one-amino acid difference as the internal standards. The coding region could be used as the unique "Protease ID" for the identification of corresponding protease, and the amount of the cleavage product was used for protease activity analysis. Using trypsin and chymotrypsin as the model proteases to verify the multiplex protease assay, the designed "Trypsin ID" and "Chymotrypsin ID" occurred at m/z 761.6 and 711.6. The logarithm value of the intensity ratio of "Protease ID" to internal standard was proportional to trypsin and chymotrypsin concentration in a range from 5.0 to 500 and 10 to 500 nM, respectively. The detection limits for trypsin and chymotrypsin were 2.3 and 5.2 nM, respectively. The peptide-encoded microplate showed good selectivity. This proposed method provided a powerful tool for convenient identification and activity analysis of multiplex proteases.

Mechanism of cinnamic acid-induced trypsin inhibition: A multi-technique approach

NASA Astrophysics Data System (ADS)

Zhang, Hongmei; Zhou, Qiuhua; Cao, Jian; Wang, Yanqing

2013-12-01

In order to investigate the association of the protease trypsin with cinnamic acid, the interaction was characterized by using fluorescence, UV-vis absorption spectroscopy, molecular modeling and an enzymatic inhibition assay. The binding process may be outlined as follows: cinnamic acid can interact with trypsin with one binding site to form cinnamic acid-trypsin complex, resulting in inhibition of trypsin activity; the spectroscopic data show that the interaction is a spontaneous process with the estimated enthalpy and entropy changes being -8.95 kJ mol-1 and 50.70 J mol-1 K-1, respectively. Noncovalent interactions make the main contribution to stabilize the trypsin-cinnamic acid complex; cinnamic acid can enter into the primary substrate-binding pocket and alter the environment around Trp and Tyr residues.

Conformational flexibility in the catalytic triad revealed by the high-resolution crystal structure of Streptomyces erythraeus trypsin in an unliganded state

PubMed Central

Blankenship, Elise; Vukoti, Krishna; Miyagi, Masaru; Lodowski, David T.

2014-01-01

With more than 500 crystal structures determined, serine proteases make up greater than one-third of all proteases structurally examined to date, making them among the best biochemically and structurally characterized enzymes. Despite the numerous crystallographic and biochemical studies of trypsin and related serine proteases, there are still considerable shortcomings in the understanding of their catalytic mechanism. Streptomyces erythraeus trypsin (SET) does not exhibit autolysis and crystallizes readily at physiological pH; hence, it is well suited for structural studies aimed at extending the understanding of the catalytic mechanism of serine proteases. While X-ray crystallographic structures of this enzyme have been reported, no coordinates have ever been made available in the Protein Data Bank. Based on this, and observations on the extreme stability and unique properties of this particular trypsin, it was decided to crystallize it and determine its structure. Here, the first sub-angstrom resolution structure of an unmodified, unliganded trypsin crystallized at physiological pH is reported. Detailed structural analysis reveals the geometry and structural rigidity of the catalytic triad in the unoccupied active site and comparison to related serine proteases provides a context for interpretation of biochemical studies of catalytic mechanism and activity. PMID:24598752

Conformational flexibility in the catalytic triad revealed by the high-resolution crystal structure of Streptomyces erythraeus trypsin in an unliganded state.

PubMed

Blankenship, Elise; Vukoti, Krishna; Miyagi, Masaru; Lodowski, David T

2014-03-01

With more than 500 crystal structures determined, serine proteases make up greater than one-third of all proteases structurally examined to date, making them among the best biochemically and structurally characterized enzymes. Despite the numerous crystallographic and biochemical studies of trypsin and related serine proteases, there are still considerable shortcomings in the understanding of their catalytic mechanism. Streptomyces erythraeus trypsin (SET) does not exhibit autolysis and crystallizes readily at physiological pH; hence, it is well suited for structural studies aimed at extending the understanding of the catalytic mechanism of serine proteases. While X-ray crystallographic structures of this enzyme have been reported, no coordinates have ever been made available in the Protein Data Bank. Based on this, and observations on the extreme stability and unique properties of this particular trypsin, it was decided to crystallize it and determine its structure. Here, the first sub-angstrom resolution structure of an unmodified, unliganded trypsin crystallized at physiological pH is reported. Detailed structural analysis reveals the geometry and structural rigidity of the catalytic triad in the unoccupied active site and comparison to related serine proteases provides a context for interpretation of biochemical studies of catalytic mechanism and activity.

Protease-Activated Receptor 2, Dipeptidyl Peptidase I, and Proteases Mediate Clostridium difficile Toxin A Enteritis

PubMed Central

COTTRELL, GRAEME S.; AMADESI, SILVIA; PIKIOS, STELLA; CAMERER, ERIC; WILLARDSEN, J. ADAM; MURPHY, BRETT R.; CAUGHEY, GEORGE H.; WOLTERS, PAUL J.; COUGHLIN, SHAUN R.; PETERSON, ANDERS; KNECHT, WOLFGANG; POTHOULAKIS, CHARALABOS; BUNNETT, NIGEL W.; GRADY, EILEEN F.

2008-01-01

Background & Aims We studied the role of protease-activated receptor 2 (PAR2) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. Methods We injected TxA into ileal loops in PAR2 or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR2 and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333. Results TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR2 deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%−28% and tissue and fluid myeloperoxidase by 31%−71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR2 and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca2+ responses to PAR2 AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. Conclusions PAR2 and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR2 and up-regulates PAR2 and activating proteases, and PAR2 causes inflammation by neurogenic mechanisms. PMID:17570216

Airway uric acid is a sensor of inhaled protease allergens and initiates type 2 immune responses in respiratory mucosa1

PubMed Central

Hara, Kenichiro; Iijima, Koji; Elias, Martha K.; Seno, Satoshi; Tojima, Ichiro; Kobayashi, Takao; Kephart, Gail M.; Kurabayashi, Masahiko; Kita, Hirohito

2014-01-01

While type 2 immune responses to environmental antigens are thought to play pivotal roles in asthma and allergic airway diseases, the immunological mechanisms that initiate the responses are largely unknown. Many allergens have biologic activities, including enzymatic activities and abilities to engage innate pattern-recognition receptors such as TLR4. Here we report that IL-33 and thymic stromal lymphopoietin (TSLP) were produced quickly in the lungs of naïve mice exposed to cysteine proteases, such as bromelain and papain, as a model for allergens. IL-33 and TSLP sensitized naïve animals to an innocuous airway antigen OVA, which resulted in production of type 2 cytokines and IgE antibody and eosinophilic airway inflammation when mice were challenged with the same antigen. Importantly, upon exposure to proteases, uric acid (UA) was rapidly released into the airway lumen, and removal of this endogenous UA by uricase prevented type 2 immune responses. UA promoted secretion of IL-33 by airway epithelial cells in vitro, and administration of UA into the airways of naïve animals induced extracellular release of IL-33, followed by both innate and adaptive type 2 immune responses in vivo. Finally, a potent UA synthesis inhibitor, febuxostat, mitigated asthma phenotypes that were caused by repeated exposure to natural airborne allergens. These findings provide mechanistic insights into the development of type 2 immunity to airborne allergens and recognize airway UA as a key player that regulates the process in respiratory mucosa. PMID:24663677

The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise.

PubMed

Reuther, C; Ganjam, G K; Dolga, A M; Culmsee, C

2014-11-01

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.

Serine protease activity in m-1 cortical collecting duct cells.

PubMed

Liu, Lian; Hering-Smith, Kathleen S; Schiro, Faith R; Hamm, L Lee

2002-04-01

An apical serine protease, channel-activating protease 1 (CAP1), augments sodium transport in A6 cells. Prostasin, a novel serine protease originally purified from seminal fluid, has been proposed to be the mammalian ortholog of CAP1. We have recently found functional evidence for a similar protease activity in the M-1 cortical collecting duct cell line. The purposes of the present studies were to determine whether prostasin (or CAP1) is present in collecting duct cells by use of mouse M-1 cells, to sequence mouse prostasin, and to further characterize the identity of the serine protease activity and additional functional features in M-1 cells. Using mouse expressed sequence tag sequences that are highly homologous to the published human prostasin sequence as templates, reverse transcription-polymerase chain reaction and RACE (rapid amplification of cDNA ends) were used to sequence mouse prostasin mRNA, which shows 99% identical to published mouse CAP1 sequence. A single 1800-bp transcript was found by Northern analysis, and this was not altered by aldosterone. Equivalent short-circuit current (I(eq)), which represents sodium transport in these cells, dropped to 59+/-3% of control value within 1 hour of incubation with aprotinin, a serine protease inhibitor. Trypsin increased the I(eq) in aprotinin-treated cells to the value of the control group within 5 minutes. Application of aprotinin not only inhibited amiloride sensitive I(eq) but also reduced transepithelial resistance (R(te)) to 43+/-2%, an effect not expected with simple inhibition of sodium channels. Trypsin partially reversed the effect of aprotinin on R(te). Another serine protease inhibitor, soybean trypsin inhibitor (STI), decreased I(eq) in M-1 cells. STI inhibited I(eq) gradually over 6 hours, and the inhibition of I(eq) by 2 inhibitors was additive. STI decreased transepithelial resistance much less than did aprotinin. Neither aldosterone nor dexamethasone significantly augmented protease activity

Cockroach protease allergen induces allergic airway inflammation via epithelial cell activation

PubMed Central

Kale, Sagar L.; Agrawal, Komal; Gaur, Shailendra Nath; Arora, Naveen

2017-01-01

Protease allergens are known to enhance allergic inflammation but their exact role in initiation of allergic reactions at mucosal surfaces still remains elusive. This study was aimed at deciphering the role of serine protease activity of Per a 10, a major cockroach allergen in initiation of allergic inflammation at mucosal surfaces. We demonstrate that Per a 10 increases epithelial permeability by disruption of tight junction proteins, ZO-1 and occludin, and enhances the migration of Monocyte derived dendritic cell precursors towards epithelial layer as exhibited by trans-well studies. Per a 10 exposure also leads to secretion of IL-33, TSLP and intracellular Ca2+ dependent increase in ATP levels. Further, in vivo experiments revealed that Per a 10 administration in mice elevated allergic inflammatory parameters along with high levels of IL-33, TSLP, IL-1α and uric acid in the mice lungs. We next demonstrated that Per a 10 cleaves CD23 (low affinity IgE receptor) from the surface of PBMCs and purified B cells and CD25 (IL-2 receptor) from the surface of PBMCs and purified T cells in an activity dependent manner, which might favour Th2 responses. In conclusion, protease activity of Per a 10 plays a significant role in initiation of allergic airway inflammation at the mucosal surfaces. PMID:28198394

Protease Inhibitors from Marine Venomous Animals and Their Counterparts in Terrestrial Venomous Animals

PubMed Central

Mourão, Caroline B.F.; Schwartz, Elisabeth F.

2013-01-01

The Kunitz-type protease inhibitors are the best-characterized family of serine protease inhibitors, probably due to their abundance in several organisms. These inhibitors consist of a chain of ~60 amino acid residues stabilized by three disulfide bridges, and was first observed in the bovine pancreatic trypsin inhibitor (BPTI)-like protease inhibitors, which strongly inhibit trypsin and chymotrypsin. In this review we present the protease inhibitors (PIs) described to date from marine venomous animals, such as from sea anemone extracts and Conus venom, as well as their counterparts in terrestrial venomous animals, such as snakes, scorpions, spiders, Anurans, and Hymenopterans. More emphasis was given to the Kunitz-type inhibitors, once they are found in all these organisms. Their biological sources, specificity against different proteases, and other molecular blanks (being also K+ channel blockers) are presented, followed by their molecular diversity. Whereas sea anemone, snakes and other venomous animals present mainly Kunitz-type inhibitors, PIs from Anurans present the major variety in structure length and number of Cys residues, with at least six distinguishable classes. A representative alignment of PIs from these venomous animals shows that, despite eventual differences in Cys assignment, the key-residues for the protease inhibitory activity in all of them occupy similar positions in primary sequence. The key-residues for the K+ channel blocking activity was also compared. PMID:23771044

Mechanism of cinnamic acid-induced trypsin inhibition: a multi-technique approach.

PubMed

Zhang, Hongmei; Zhou, Qiuhua; Cao, Jian; Wang, Yanqing

2013-12-01

In order to investigate the association of the protease trypsin with cinnamic acid, the interaction was characterized by using fluorescence, UV-vis absorption spectroscopy, molecular modeling and an enzymatic inhibition assay. The binding process may be outlined as follows: cinnamic acid can interact with trypsin with one binding site to form cinnamic acid-trypsin complex, resulting in inhibition of trypsin activity; the spectroscopic data show that the interaction is a spontaneous process with the estimated enthalpy and entropy changes being -8.95 kJ mol(-1) and 50.70 J mol(-1) K(-1), respectively. Noncovalent interactions make the main contribution to stabilize the trypsin-cinnamic acid complex; cinnamic acid can enter into the primary substrate-binding pocket and alter the environment around Trp and Tyr residues. Copyright © 2013 Elsevier B.V. All rights reserved.

Modification of host erythrocyte membranes by trypsin and chymotrypsin treatments and effects on the in vitro growth of bovine and equine Babesia parasites.

PubMed

Okamura, Masashi; Yokoyama, Naoaki; Takabatake, Noriyuki; Okubo, Kazuhiro; Ikehara, Yuzuru; Igarashi, Ikuo

2007-02-01

In the present study, we investigated the effects of protease pretreatments of host erythrocytes (RBC) on the in vitro growth of bovine Babesia parasites (Babesia bovis and B. bigemina) and equine Babesia parasites (B. equi and B. caballi). The selected proteases, trypsin and chymotrypsin, clearly modified several membrane proteins of both bovine and equine RBC, as demonstrated by SDS-PAGE analysis; however, the protease treatments also modified the sialic acid content exclusively in bovine RBC, as demonstrated by lectin blot analysis. An in vitro growth assay using the protease-treated RBC showed that the trypsin-treated bovine RBC, but not the chymotrypsin-treated ones, significantly reduced the growth of B. bovis and B. bigemina as compared to the control. In contrast, the growth of B. equi and B. caballi was not affected by any of these proteases. Thus, the bovine, but not the equine, Babesia parasites require the trypsin-sensitive membrane (sialoglyco) proteins to infect the RBC.

[Fish ovarian fluid contains protease inhibitors].

PubMed

Minin, A A; Ozerova, S G

2015-01-01

Studies of the conditions under which fish egg is activated spontaneously without the sperm showed that the egg retains the ability for fertilization in the ovarian (coelomic) fluid, which surrounds it in the gonad cavity after ovulation. Earlier, we showed that, in artificial media, the spontaneous activation is suppressed by protease inhibitors. In this study, we investigated the presence of natural protease inhibitors in the ovarian fluid and showed that the ovarian fluid of zebrafish and loach contains protease inhibitors, in particular, type I serpin a, a protein inhibitor of trypsin proteases.

Combinatorial Enzyme Design Probes Allostery and Cooperativity in the Trypsin Fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Page, Michael J.; Di Cera, Enrico; St. Louis-MED)

2010-06-14

Converting one enzyme into another is challenging due to the uneven distribution of important amino acids for function in both protein sequence and structure. We report a strategy for protein engineering allowing an organized mixing and matching of genetic material that leverages lower throughput with increased quality of screens. Our approach successfully tested the contribution of each surface-exposed loop in the trypsin fold alone and the cooperativity of their combinations towards building the substrate selectivity and Na{sup +}-dependent allosteric activation of the protease domain of human coagulation factor Xa into a bacterial trypsin. As the created proteases lack additional proteinmore » domains and protein co-factor activation mechanism requisite for the complexity of blood coagulation, they are stepping-stones towards further understanding and engineering of artificial clotting factors.« less

Dysregulation of Protease and Protease Inhibitors in a Mouse Model of Human Pelvic Organ Prolapse

PubMed Central

Budatha, Madhusudhan; Silva, Simone; Montoya, Teodoro Ignacio; Suzuki, Ayako; Shah-Simpson, Sheena; Wieslander, Cecilia Karin; Yanagisawa, Masashi; Word, Ruth Ann; Yanagisawa, Hiromi

2013-01-01

Mice deficient for the fibulin-5 gene (Fbln5−/−) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5−/− mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5−/− mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5−/− and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin) and Elafin] was dysregulated in Fbln5−/− epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice. PMID:23437119

Proteases and caspase-like activity in the yeast Saccharomyces cerevisiae.

PubMed

Wilkinson, Derek; Ramsdale, Mark

2011-10-01

A variety of proteases have been implicated in yeast PCD (programmed cell death) including the metacaspase Mca1 and the separase Esp1, the HtrA-like serine protease Nma111, the cathepsin-like serine carboxypeptideases and a range of vacuolar proteases. Proteasomal activity is also shown to have an important role in determining cell fate, with both pro- and anti-apoptotic roles. Caspase 3-, 6- and 8-like activities are detected upon stimulation of yeast PCD, but not all of this activity is associated with Mca1, implicating other proteases with caspase-like activity in the yeast cell death response. Global proteolytic events that accompany PCD are discussed alongside a consideration of the conservation of the death-related degradome (both at the level of substrate choice and cleavage site). The importance of both gain-of-function changes in the degradome as well as loss-of-function changes are highlighted. Better understanding of both death-related proteases and their substrates may facilitate the design of future antifungal drugs or the manipulation of industrial yeasts for commercial exploitation.

Molecular characterization of two serine proteases expressed in gut tissue of the African trypanosome vector, Glossina morsitans morsitans.

PubMed

Yan, J; Cheng, Q; Li, C B; Aksoy, S

2001-02-01

Serine proteases are major insect gut enzymes involved in digestion of dietary proteins, and in addition they have been implicated in the process of pathogen establishment in several vector insects. The medically important vector, tsetse fly (Diptera:Glossinidiae), is involved in the transmission of African trypanosomes, which cause devastating diseases in animals and humans. Both the male and female tsetse can transmit trypanosomes and both are strict bloodfeeders throughout all stages of their development. Here, we describe the characterization of two putative serine protease-encoding genes, Glossina serine protease-1 (Gsp1) and Glossina serine protease-2 (Gsp2) from gut tissue. Both putative cDNA products represent prepro peptides with hydrophobic signal peptide sequences associated with their 5'-end terminus. The Gsp1 cDNA encodes a putative mature protein of 245 amino acids with a molecular mass of 26 428 Da, while the predicted size of the 228 amino acid mature peptide encoded by Gsp2 cDNA is 24 573 Da. Both deduced peptides contain the Asp/His/Ser catalytic triad and the conserved residues surrounding it which are characteristic of serine proteases. In addition, both proteins have the six-conserved cysteine residues to form the three-cysteine bonds typically present in invertebrate serine proteases. Based on the presence of substrate specific residues, the Gsp1 gene encodes a chymotrypsin-like protease while Gsp2 gene encodes for a protein with trypsin-like activity. Both proteins are encoded by few loci in tsetse genome, being present in one or two copies only. The mRNA expression levels for the genes do not vary extensively throughout the digestive cycle, and high levels of mRNAs can be readily detected in the gut tissue of newly emerged flies. The levels of trypsin and chymotrypsin activities in the gut lumen increase following blood feeding and change significantly in the gut cells throughout the digestion cycle. Hence, the regulation of expression for

Trypsin-sensitive modulation of intestinal epithelial MD-2 as mechanism of lipopolysaccharide tolerance.

PubMed

Cario, Elke; Golenbock, Douglas T; Visintin, Alberto; Rünzi, Michael; Gerken, Guido; Podolsky, Daniel K

2006-04-01

Intestinal epithelial cells (IEC) are constantly exposed to both high concentrations of the bacterial ligand LPS and the serine protease trypsin. MD-2, which contains multiple trypsin cleavage sites, is an essential accessory glycoprotein required for LPS recognition and signaling through TLR4. The aim of this study was to characterize the expression and subcellular distribution of intestinal epithelial MD-2 and to delineate potential functional interactions with trypsin and then alteration in inflammatory bowel disease (IBD). Although MD-2 protein expression was minimal in primary IEC of normal colonic or ileal mucosa, expression was significantly increased in IEC from patients with active IBD colitis, but not in ileal areas from patients with severe Crohn's disease. Endogenous MD-2 was predominantly retained in the calnexin-calreticulin cycle of the endoplasmic reticulum; only a small fraction was exported to the Golgi. MD-2 expression correlated inversely with trypsin activity. Biochemical evidence and in vitro experiments demonstrated that trypsin exposure resulted in extensive proteolysis of endogenous and soluble MD-2 protein, but not of TLR4 in IEC, and was associated with desensitization of IEC to LPS. In conclusion, the present study suggests that endoplasmic reticulum-associated MD-2 expression in IBD may be altered by ileal protease in inflammation, leading to impaired LPS recognition and hyporesponsiveness through MD-2 proteolysis in IEC, thus implying a physiologic mechanism that helps maintain LPS tolerance in the intestine.

Nematode sperm maturation triggered by protease involves sperm-secreted serine protease inhibitor (Serpin)

PubMed Central

Zhao, Yanmei; Sun, Wei; Zhang, Pan; Chi, Hao; Zhang, Mei-Jun; Song, Chun-Qing; Ma, Xuan; Shang, Yunlong; Wang, Bin; Hu, Youqiao; Hao, Zhiqi; Hühmer, Andreas F.; Meng, Fanxia; L'Hernault, Steven W.; He, Si-Min; Dong, Meng-Qiu; Miao, Long

2012-01-01

Spermiogenesis is a series of poorly understood morphological, physiological and biochemical processes that occur during the transition of immotile spermatids into motile, fertilization-competent spermatozoa. Here, we identified a Serpin (serine protease inhibitor) family protein (As_SRP-1) that is secreted from spermatids during nematode Ascaris suum spermiogenesis (also called sperm activation) and we showed that As_SRP-1 has two major functions. First, As_SRP-1 functions in cis to support major sperm protein (MSP)-based cytoskeletal assembly in the spermatid that releases it, thereby facilitating sperm motility acquisition. Second, As_SRP-1 released from an activated sperm inhibits, in trans, the activation of surrounding spermatids by inhibiting vas deferens-derived As_TRY-5, a trypsin-like serine protease necessary for sperm activation. Because vesicular exocytosis is necessary to create fertilization-competent sperm in many animal species, components released during this process might be more important modulators of the physiology and behavior of surrounding sperm than was previously appreciated. PMID:22307610

High feline trypsin-like immunoreactivity in a cat with pancreatitis and hepatic lipidosis.

PubMed

Bruner, J M; Steiner, J M; Williams, D A; Van Alstine, W G; Blevins, W

1997-06-15

A 1.5-year-old domestic shorthair cat was examined because of vomiting and icterus. Clinicopathologic abnormalities included high alanine transaminase, alkaline phosphatase, and gamma-glutamyltransferase activities and high total bilirubin concentration. During abdominal ultrasonography, the left limb and body of the pancreas appeared hypoechoic, and a small quantity of peritoneal effusion was seen. The liver was diffusely hyperechoic, with echogenicity similar to that of the spleen, indicating hepatic lipidosis. Feline trypsin-like immunoreactivity was high, suggesting that the cat also had pancreatitis. The cat was treated with crystalloid fluids and was fed a protein-restricted diet via a percutaneous endoscopically placed gastrostomy tube. The cat's condition continued to deteriorate despite medical treatment, and it was euthanatized. Necropsy confirmed the clinical suspicion of acute pancreatitis and hepatic lipidosis. This case suggests that measurement of trypsin-like immunoreactivity may be useful in cats suspected of having pancreatitis.

Synthesis and biochemical evaluation of triazole/tetrazole-containing sulfonamides against thrombin and related serine proteases

PubMed Central

Siles, Rogelio; Kawasaki, Yuko; Ross, Patrick; Freire, Ernesto

2011-01-01

A small library of 25 triazole/tetrazole-based sulfonamides have been synthesized and further evaluated for their inhibitory activity against thrombin, trypsin, tryptase and chymase. In general, the triazole-based sulfonamides inhibited thrombin more efficiently than the tetrazole counterparts. Particularly, compound 26 showed strong thrombin inhibition (Ki =880 nM) and significant selectivity against other human related serine proteases like trypsin (Ki =729 µM). Thrombin binding affinity of the same compound was determined by ITC and demonstrated that the binding of this new triazole-based scaffold is enthalpically driven, making it a good candidate for further development. PMID:21807511

Serpin-9 and -13 regulate hemolymph proteases during immune responses of Manduca sexta.

PubMed

He, Yan; Wang, Yang; Zhao, Picheng; Rayaprolu, Subrahmanyam; Wang, Xiuhong; Cao, Xiaolong; Jiang, Haobo

2017-11-01

Serpins are a superfamily of proteins, most of which inhibit cognate serine proteases by forming inactive acyl-enzyme complexes. In the tobacco hornworm Manduca sexta, serpin-1, -3 through -7 negatively regulate a hemolymph serine protease system that activates precursors of the serine protease homologs (SPHs), phenoloxidases (POs), Spätzles, and other cytokines. Here we report the cloning and characterization of M. sexta serpin-9 and -13. Serpin-9, a 402-residue protein most similar to Drosophila Spn77Ba, has R 366 at the P1 position right before the cleavage site; Serpin-13, a 444-residue ortholog of Drosophila Spn28Dc, is longer than the other seven serpins and has R 410 as the P1 residue. Both serpins are mainly produced in fat body and secreted into plasma to function. While their mRNA and protein levels were not up-regulated upon immune challenge, they blocked protease activities and affected proPO activation in hemolymph. Serpin-9 inhibited human neutrophil elastase, cathepsin G, trypsin, and chymotrypsin to different extents; serpin-13 reduced trypsin activity to approximately 10% at a molar ratio of 4:1 (serpin: enzyme). Serpin-9 was cleaved at Arg 366 by the enzymes with different specificity, but serpin-13 had four P1 sites (Arg 410 for trypsin-like proteases, Gly 406 and Ala 409 for the elastase and Thr 404 for cathepsin G). Supplementation of induced cell-free hemolymph (IP, P for plasma) with recombinant serpin-9 did not noticeably affect proPO activation, but slightly reduced the PO activity increase after 0-50% ammonium sulfate fraction of the IP had been elicited by bacteria. In comparison, addition of recombinant serpin-13 significantly inhibited proPO activation in IP and the suppression was stronger in the fraction of IP. Serpin-9- and -13-containing protein complexes were isolated from IP using their antibodies. Hemolymph protease-1 precursor (proHP1), HP6 and HP8 were found to be associated with serpin-9, whereas proHP1, HP2 and HP6 were

TsAg5, a Taenia solium cysticercus protein with a marginal trypsin-like activity in the diagnosis of human neurocysticercosis.

PubMed

Rueda, Analiz; Sifuentes, Cecilia; Gilman, Robert H; Gutiérrez, Andrés H; Piña, Ruby; Chile, Nancy; Carrasco, Sebastián; Larson, Sandra; Mayta, Holger; Verástegui, Manuela; Rodriguez, Silvia; Gutiérrez-Correa, Marcel; García, Héctor H; Sheen, Patricia; Zimic, Mirko

2011-12-01

Neurocysticercosis is an endemic parasitic disease caused by Taenia solium larva. Although the mechanism of infection is not completely understood, it is likely driven by proteolytic activity that degrades the intestinal wall to facilitate oncosphere penetration and further infection. We analyzed the publicly available T. solium EST/DNA library and identified two contigs comprising a full-length cDNA fragment very similar to Echinococcus granulosus Ag5 protein. The T. solium cDNA sequence included a proteolytic trypsin-like-domain in the C-terminal region, and a thrombospondin type-1 adherence-domain in the N-terminal region. Both the trypsin-like and adherence domains were expressed independently as recombinant proteins in bacterial systems. TsAg5 showed marginal trypsin-like activity and high sequence similarity to Ag5. The purified antigens were tested in a Western immunoblot assay to diagnose human neurocysticercosis. The sensitivity of the trypsin-like-domain was 96.36% in patients infected with extraparenchymal cysts, 75.44% in patients infected with multiple cysts, and 39.62% in patients with a single cyst. Specificity was 76.70%. The thrombospondin type-1 adherence-domain was not specific for neurocysticercosis. Copyright © 2011 Elsevier B.V. All rights reserved.

Proteolytic Activation of the Protease-activated Receptor (PAR)-2 by the Glycosylphosphatidylinositol-anchored Serine Protease Testisin*

PubMed Central

Driesbaugh, Kathryn H.; Buzza, Marguerite S.; Martin, Erik W.; Conway, Gregory D.; Kao, Joseph P. Y.; Antalis, Toni M.

2015-01-01

Protease-activated receptors (PARs) are a family of seven-transmembrane, G-protein-coupled receptors that are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR-activating proteases described to date are soluble proteases that are active during injury, coagulation, and inflammation. Less investigation, however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI) anchor. Here, we show that the N-terminal domain of PAR-2 is a substrate for testisin and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways, including intracellular Ca2+ mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of the intracellular serum-response element and NFκB signaling pathways and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface. PMID:25519908

Kunitz-type protease inhibitors group B from Solanum palustre.

PubMed

Speransky, Anna S; Cimaglia, Fabio; Krinitsina, Anastasya A; Poltronieri, Palmiro; Fasano, Pasqua; Bogacheva, Anna M; Valueva, Tatiana A; Halterman, Dennis; Shevelev, Alexei B; Santino, Angelo

2007-11-01

Five Kunitz protease inhibitor group B genes were isolated from the genome of the diploid non-tuber-forming potato species Solanum palustre. Three of five new genes share 99% identity to the published KPI-B genes from various cultivated potato accessions, while others exhibit 96% identity. Spls-KPI-B2 and Spls-KPI-B4 proteins contain unique substitutions of the most conserved residues usually involved to trypsin and chymotrypsin-specific binding sites of Kunitz-type protease inhibitor (KPI)-B, respectively. To test the inhibition of trypsin and chymotrypsin by Spls-KPI proteins, five of them were produced in E. coli purified using a Ni-sepharose resin and ion-exchange chromatography. All recombinant Spls-KPI-B inhibited trypsin; K(i) values ranged from 84.8 (Spls-KPI-B4), 345.5 (Spls-KPI-B1), and 1310.6 nM (Spls-KPI-B2) to 3883.5 (Spls-KPI-B5) and 8370 nM (Spls-KPI-B3). In addition, Spls-KPI-B1 and Spls-KPI-B4 inhibited chymotrypsin. These data suggest that regardless of substitutions of key active-center residues both Spls-KPI-B4 and Spls-KPI-B1 are functional trypsin-chymotrypsin inhibitors.

Lectin Activation in Giardia lamblia by Host Protease: A Novel Host-Parasite Interaction

NASA Astrophysics Data System (ADS)

Lev, Boaz; Ward, Honorine; Keusch, Gerald T.; Pereira, Miercio E. A.

1986-04-01

A lectin in Giardia lamblia was activated by secretions from the human duodenum, the environment where the parasite lives. Incubation of the secretions with trypsin inhibitors prevented the appearance of lectin activity, implicating proteases as the activating agent. Accordingly, lectin activation was also produced by crystalline trypsin and Pronase; other proteases tested were ineffective. When activated, the lectin agglutinated intestinal cells to which the parasite adheres in vivo. The lectin was most specific to mannose-6-phosphate and apparently was bound to the plasma membrane. Activation of a parasite lectin by a host protease represents a novel mechanism of hostparasite interaction and may contribute to the affinity of Giardia lamblia to the infection site.

Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion.

PubMed

Pan, Yanbo; Cheng, Kai; Mao, Jiawei; Liu, Fangjie; Liu, Jing; Ye, Mingliang; Zou, Hanfa

2014-10-01

Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins.

Studies on trypsin-like enzymes in sperm and early embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Penn, A.

1975-12-09

Results are reported from a study of acrosomal proteinase, a trypsin-like enzyme (TLE), found in the acrosome of all eutherian mammals studied to date. It has been implicated in the dissolution of a passage for the sperm through the zona pellucida of the egg, a step necessary for in vivo fertilization. A cytochemical procedure employing autoradiographic film as a gelatin substrate is described for in situ detection and localization of acrosomal proteolytic activity. A role for TLE in the early development of embryos is suggested. (CH)

Potentiometric determination of trypsin using a polymeric membrane polycation-sensitive electrode based on current-controlled reagent delivery.

PubMed

Chen, Yan; Ding, Jiawang; Qin, Wei

2012-12-01

A potentiometric biosensor for the determination of trypsin is described based on current-controlled reagent delivery. A polymeric membrane protamine-sensitive electrode with dinonylnaphthalene sulfonate as cation exchanger is used for in situ generation of protamine. Diffusion of protamine across the polymeric membrane can be controlled precisely by applying an external current. The hydrolysis catalyzed with trypsin in sample solution decreases the concentration of free protamine released at the sample-membrane interface and facilitates the stripping of protamine out of the membrane surface via the ion-exchange process with sodium ions from the sample solution, thus decreasing the membrane potential, by which the protease can be sensed potentiometrically. The influences of anodic current amplitude, current pulse duration and protamine concentration in the inner filling solution on the membrane potential response have been studied. Under optimum conditions, the proposed protamine-sensitive electrode is useful for continuous and reversible detection of trypsin over the concentration range of 0.5-5UmL(-1) with a detection limit of 0.3UmL(-1). The proposed detection strategy provides a rapid and reagentless way for the detection of protease activities and offers great potential in the homogeneous immunoassays using proteases as labels. Copyright © 2012 Elsevier B.V. All rights reserved.

Effect of pancreatic proteases on plasma cholecystokinin, secretin, and pancreatic exocrine secretion in response to sodium oleate.

PubMed

Li, P; Lee, K Y; Ren, X S; Chang, T M; Chey, W Y

1990-06-01

The effect of pancreatic proteases or juice on the sodium oleate-stimulated pancreatic secretion and plasma concentrations of secretin and cholecystokinin in anesthetized rats was investigated. Each rat received sodium oleate in a dose of 0.12 mmol.h-1 via a duodenal tube. Sodium oleate infusion significantly increased pancreatic secretion (volume and protein output) compared with the saline given the control group. The increase in pancreatic secretion paralleled significant elevations of plasma concentrations of secretin and cholecystokinin. To determine a possible role of pancreatic proteases on the responses induced by sodium oleate, saline, chymotrypsin, and trypsin, a combination of chymotrypsin and trypsin or pancreatic juice was infused into the duodenum. The pancreatic secretion was significantly reduced by pancreatic proteases or pancreatic juice, and the reduction paralleled the decreases in plasma concentrations of the two hormones. These agents suppressed both pancreatic secretion and plasma hormone levels in the following order of magnitude: (pancreatic juice or chymotrypsin + trypsin) greater than (trypsin) greater than (chymotrypsin). The reduction of pancreatic secretion by pancreatic proteases was reversed by intravenous administration of secretin and cholecystokinin in physiological doses. It is concluded that negative-feedback regulation of pancreatic secretion is operative in the intestinal phase in rats and that both secretin and cholecystokinin are involved in the regulation.

Engineering of Yersinia Phytases to Improve Pepsin and Trypsin Resistance and Thermostability and Application Potential in the Food and Feed Industry.

PubMed

Niu, Canfang; Yang, Peilong; Luo, Huiying; Huang, Huoqing; Wang, Yaru; Yao, Bin

2017-08-30

Susceptibility to proteases usually limits the application of phytase. We sought to improve the pepsin and trypsin resistance of YeAPPA from Yersinia enterocolitica and YkAPPA from Y. kristensenii by optimizing amino acid polarity and charge. The predicted pepsin/trypsin cleavage sites F89/K226 in pepsin/trypsin-sensitive YeAPPA and the corresponding sites (F89/E226) in pepsin-sensitive but trypsin-resistant YkAPPA were substituted with S and H, respectively. Six variants were produced in Pichia pastoris for catalytic and biochemical characterization. F89S, E226H, and F89S/E226H elevated pepsin resistance and thermostability and K226H and F89S/K226H improved pepsin and trypsin resistance and stability at 60 °C and low pH. All the variants increased the ability of the proteins to hydrolyze phytate in corn meal by 2.6-14.9-fold in the presence of pepsin at 37 °C and low pH. This study developed a genetic manipulation strategy specific for pepsin/trypsin-sensitive phytases that can improve enzyme tolerance against proteases and heat and benefit the food and feed industry in a cost-effective way.

Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin.

PubMed

Driesbaugh, Kathryn H; Buzza, Marguerite S; Martin, Erik W; Conway, Gregory D; Kao, Joseph P Y; Antalis, Toni M

2015-02-06

Protease-activated receptors (PARs) are a family of seven-transmembrane, G-protein-coupled receptors that are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR-activating proteases described to date are soluble proteases that are active during injury, coagulation, and inflammation. Less investigation, however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI) anchor. Here, we show that the N-terminal domain of PAR-2 is a substrate for testisin and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways, including intracellular Ca(2+) mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of the intracellular serum-response element and NFκB signaling pathways and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Trypsin from unicorn leatherjacket (Aluterus monoceros) pyloric caeca: purification and its use for preparation of fish protein hydrolysate with antioxidative activity.

PubMed

Zamani, Abbas; Benjakul, Soottawat

2016-02-01

Fish proteases, especially trypsin, could be used to prepare fish protein hydrolysates with antioxidative activities. In this study, trypsin from the pyloric caeca of unicorn leatherjacket was purified by ammonium sulfate precipitation and soybean trypsin inhibitor (SBTI)-Sepharose 4B affinity chromatography. Hydrolysate from Indian mackerel protein isolate with different degrees of hydrolysis (20, 30 and 40% DH) was prepared using the purified trypsin, and antioxidative activities (1,1-diphenyl-2-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging activities, ferric-reducing antioxidant power and ferrous-chelating activity) of the hydrolysate were determined. Trypsin was purified 26.43-fold with a yield of 13.43%. The purified trypsin had a molecular weight (MW) of 23.5 kDa and optimal activity at pH 8.0 and 55 °C. It displayed high stability in the pH range of 6.0-11.0 and was thermally stable up to 50 °C. Both SBTI (0.05 mmol L(-1)) and N-p-tosyl-L-lysine-chloromethylketone (5 mmol L(-1)) completely inhibited trypsin activity. Antioxidative activities of the hydrolysate from Indian mackerel protein isolate increased with increasing DH up to 40% (P < 0.05). Based on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the hydrolysate with 40% DH had a MW lower than 6.5 kDa. The purified protease from unicorn leatherjacket pyloric caeca was identified as trypsin based on its ability to hydrolyze a specific synthetic substrate and the response to specific trypsin inhibitors. The purified trypsin could hydrolyze Indian mackerel protein isolate, and the resulting hydrolysate exhibited antioxidative activity depending on its DH. © 2015 Society of Chemical Industry.

Trypsin-like Proteins of the Fungi as Possible Markers of Phytopathogenicity

USDA-ARS?s Scientific Manuscript database

Sequences of peptidases with conserved motifs around the active site residues that are characteristic of trypsins (similar to trypsin peptidases, STP) were obtained from publicly available fungal genomes and related databases. Among the 74 fungal genomes, 30 species of parasitic Ascomycota contained...

Proteases in doping control analysis.

PubMed

Thevis, M; Maurer, J; Kohler, M; Geyer, H; Schänzer, W

2007-07-01

Urine manipulation in sports drug testing has become a serious problem for doping control laboratories, and recent scandals in elite endurance sports have revealed the problem of urine manipulation presumably using proteases, which will impede the detection of drugs such as erythropoietin (EPO) or other peptide hormones. Using commonly accepted analytical strategies, a protocol was developed enabling the determination of elevated protease activities in doping control specimens followed by the visualization of protein degradation and identification of proteases such as chymotrypsin, trypsin and papain. Therefore, protease detection kits based on fluorescein isothiocyanate-labeled casein were employed, and protease concentrations greater than 15 microg/mL of urine entailed subsequent 1-dimensional gel electrophoretic visualization of urinary proteins. The presence of 20 microg of proteases per mL of urine caused a complete degradation of proteins usually observed in urinary matrices ("trace of burning"), while respective proteases were still detected in spiked urine samples after 10 days of storage at + 4 and - 20 degrees C. Identification of target proteases at respective molecular weights was accomplished using bottom-up sequencing approaches based on in-gel digestion of separated enzymes followed by capillary liquid chromatography--Orbitrap tandem mass spectrometry.

Purification of a 6.5 kDa protease inhibitor from Amazon Inga umbratica seeds effective against serine proteases of the boll weevil Anthonomus grandis.

PubMed

Calderon, L A; Teles, R C L; Leite, J R S A; Franco, O L; Grossi-de-Sá, M F; Medrano, F J; Bloch, C; Freitas, S M

2005-08-01

A 6.5 kDa serine protease inhibitor was purified by anion-exchange chromatography from the crude extract of the Inga umbratica seeds, containing inhibitor isoforms ranging from 6.3 to 6.7 kDa and protease inhibitors of approximately 19 kDa. The purified protein was characterized as a potent inhibitor against trypsin and chymotrypsin and it was named I. umbratica trypsin and chymotrypsin inhibitor (IUTCI). MALDI-TOF spectra of the IUTCI, in the presence of DTT, showed six disulfide bonds content, suggesting that this inhibitor belongs to Bowman-Birk family. The circular dichroism spectroscopy indicates that IUTCI is predominantly formed by unordered and beta-sheet secondary structure. It was also characterized, by fluorescence spectroscopy, as a stable protein at range of pH from 5.0 to 7.0. Moreover, this inhibitor at concentration of 75 microM presented a remarkable inhibitory activity (60%) against digestive serine proteases from boll weevil Anthonomus grandis, an important economical cotton pest.

Aggregation of trypsin and trypsin inhibitor by Al cation.

PubMed

Chanphai, P; Kreplak, L; Tajmir-Riahi, H A

2017-04-01

Al cation may trigger protein structural changes such as aggregation and fibrillation, causing neurodegenerative diseases. We report the effect of Al cation on the solution structures of trypsin (try) and trypsin inhibitor (tryi), using thermodynamic analysis, UV-Visible, Fourier transform infrared (FTIR) spectroscopic methods and atomic force microscopy (AFM). Thermodynamic parameters showed Al-protein bindings occur via H-bonding and van der Waals contacts for trypsin and trypsin inhibitor. AFM showed that Al cations are able to force trypsin into larger or more robust aggregates than trypsin inhibitor, with trypsin 5±1 SE (n=52) proteins per aggregate and for trypsin inhibitor 8.3±0.7 SE (n=118). Thioflavin T test showed no major protein fibrillation in the presence of Al cation. Al complexation induced more alterations of trypsin inhibitor conformation than trypsin. Copyright © 2017 Elsevier B.V. All rights reserved.

Can breathing-like pressure oscillations reverse or prevent narrowing of small intact airways?

PubMed

Harvey, Brian C; Parameswaran, Harikrishnan; Lutchen, Kenneth R

2015-07-01

Periodic length fluctuations of airway smooth muscle during breathing are thought to modulate airway responsiveness in vivo. Recent animal and human intact airway studies have shown that pressure fluctuations simulating breathing can only marginally reverse airway narrowing and are ineffective at protecting against future narrowing. However, these previous studies were performed on relatively large (>5 mm diameter) airways, which are inherently stiffer than smaller airways for which a preponderance of airway constriction in asthma likely occurs. The goal of this study was to determine the effectiveness of breathing-like transmural pressure oscillations to reverse induced narrowing and/or protect against future narrowing of smaller, more compliant intact airways. We constricted smaller (luminal diameter = 2.92 ± 0.29 mm) intact airway segments twice with ACh (10(-6) M), once while applying tidal-like pressure oscillations (5-15 cmH2O) before, during, and after inducing constriction (Pre + Post) and again while only imposing the tidal-like pressure oscillation after induced constriction (Post Only). Smaller airways were 128% more compliant than previously studied larger airways. This increased compliance translated into 196% more strain and 76% greater recovery (41 vs. 23%) because of tidal-like pressure oscillations. Larger pressure oscillations (5-25 cmH2O) caused more recovery (77.5 ± 16.5%). However, pressure oscillations applied before and during constriction resulted in the same steady-state diameter as when pressure oscillations were only applied after constriction. These data show that reduced straining of the airways before a challenge likely does not contribute to the emergence of airway hyperreactivity observed in asthma but may serve to sustain a given level of constriction. Copyright © 2015 the American Physiological Society.

Protease-dependent hemagglutinin cleavage contributes to alteration in chicken hemagglutination by the H3N2 influenza A virus.

PubMed

Yamaoka, Masaoki; Makino, Akiko; Sasahara, Kenji; Nastri, Aldise Mareta; Krisna, Luh Ade Wilan; Purhito, Edith Frederika; Poetranto, Emmanuel Djoko; Wulandari, Laksmi; Yudhawati, Resti; Setiawati, Landia; Setyoningrum, Retno Asih; Shinya, Kyoko

2013-01-01

The human influenza A virus (H3N2) has been the predominant influenza strain since 1992, and one property of this virus is non-agglutination of chicken erythrocytes [Ch(-) virus]. The Ch(-) virus in our study was able to acquire chicken hemagglutination [Ch(+)] by trypsin passage but not by chymotrypsin passage. Moreover, the trypsin-passaged Ch(+) viruses reacquired the Ch(-) property after a further chymotrypsin passage. In particular, genetic analysis showed no evidence of mutations in the hemagglutinin (HA) gene during either trypsin or chymotrypsin passages: the only differences found were in the HA cleavage sites between the trypsin-passaged virus and the chymotrypsin-passaged virus as determined by the N-terminal amino acid sequence. These results suggested that protease-dependent differences at the viral HA cleavage site, rather than genetic mutations, are likely to have a significant effect on the viral ability to produce chicken hemagglutination.

Fully human antibodies against the Protease-Activated Receptor-2 (PAR-2) with anti-inflammatory activity.

PubMed

Giblin, Patricia; Boxhammer, Rainer; Desai, Sudha; Kroe-Barrett, Rachel; Hansen, Gale; Ksiazek, John; Panzenbeck, Maret; Ralph, Kerry; Schwartz, Racheline; Zimmitti, Clare; Pracht, Catrin; Miller, Sandra; Magram, Jeanne; Litzenburger, Tobias

2011-01-01

PAR-2 belongs to a family of G-protein coupled Protease-Activated Receptors (PAR) which are activated by specific proteolytic cleavage in the extracellular N-terminal region. PAR-2 is activated by proteases such as trypsin, tryptase, proteinase 3, factor VIIa, factor Xa and is thought to be a mediator of inflammation and tissue injury, where elevated levels of proteases are found. Utilizing the HuCAL GOLD® phage display library we generated fully human antibodies specifically blocking the protease cleavage site in the N-terminal domain. In vitro affinity optimization resulted in antibodies with up to 1000-fold improved affinities relative to the original parental antibodies with dissociation constants as low as 100 pM. Corresponding increases in potency were observed in a mechanistic protease cleavage assay. The antibodies effectively inhibited PAR-2 mediated intracellular calcium release and cytokine secretion in various cell types stimulated with trypsin. In addition, the antibodies demonstrated potent inhibition of trypsin induced relaxation of isolated rat aortic rings ex vivo. In a short term mouse model of inflammation, the trans vivo DTH model, anti-PAR-2 antibodies showed inhibition of the inflammatory swelling response. In summary, potent inhibitors of PAR-2 were generated which allow further assessment of the role of this receptor in inflammation and evaluation of their potential as therapeutic agents.

Murine Coronavirus Ubiquitin-Like Domain Is Important for Papain-Like Protease Stability and Viral Pathogenesis

PubMed Central

Mielech, Anna M.; Deng, Xufang; Chen, Yafang; Kindler, Eveline; Wheeler, Dorthea L.; Mesecar, Andrew D.; Thiel, Volker; Perlman, Stanley

2015-01-01

ABSTRACT Ubiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation. IMPORTANCE Introducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered

Structural Insights into the Role of the Cyclic Backbone in a Squash Trypsin Inhibitor*

PubMed Central

Daly, Norelle L.; Thorstholm, Louise; Greenwood, Kathryn P.; King, Gordon J.; Rosengren, K. Johan; Heras, Begoña; Martin, Jennifer L.; Craik, David J.

2013-01-01

MCoTI-II is a head-to-tail cyclic peptide with potent trypsin inhibitory activity and, on the basis of its exceptional proteolytic stability, is a valuable template for the design of novel drug leads. Insights into inhibitor dynamics and interactions with biological targets are critical for drug design studies, particularly for protease targets. Here, we show that the cyclization and active site loops of MCoTI-II are flexible in solution, but when bound to trypsin, the active site loop converges to a single well defined conformation. This finding of reduced flexibility on binding is in contrast to a recent study on the homologous peptide MCoTI-I, which suggested that regions of the peptide are more flexible upon binding to trypsin. We provide a possible explanation for this discrepancy based on degradation of the complex over time. Our study also unexpectedly shows that the cyclization loop, not present in acyclic homologues, facilitates potent trypsin inhibitory activity by engaging in direct binding interactions with trypsin. PMID:24169696

Gelatin hydrolysates from farmed Giant catfish skin using alkaline proteases and its antioxidative function of simulated gastro-intestinal digestion.

PubMed

Ketnawa, Sunantha; Martínez-Alvarez, Oscar; Benjakul, Soottawat; Rawdkuen, Saroat

2016-02-01

This work aims to evaluate the ability of different alkaline proteases to prepare active gelatin hydrolysates. Fish skin gelatin was hydrolysed by visceral alkaline-proteases from Giant catfish, commercial trypsin, and Izyme AL®. All antioxidant activity indices of the hydrolysates increased with increasing degree of hydrolysis (P<0.05). The hydrolysates obtained with Izyme AL® and visceral alkaline-proteases showed the highest and lowest radical scavenging capacity, while prepared with commercial trypsin was the most effective in reducing ferric ions and showed the best metal chelating properties. The hydrolysate obtained with Izyme AL® showed the lowest iron reducing ability, but provided the highest average molecular weight (⩾ 7 kDa), followed by commercial trypsin (2.2 kDa) and visceral alkaline-proteases (1.75 kDa). After in vitro gastrointestinal digestion, the hydrolysates showed significant higher radical scavenging, reducing ferric ions and chelating activities. Gelatin hydrolysates, from fish skin, could serve as a potential source of functional food ingredients for health promotion. Copyright © 2015 Elsevier Ltd. All rights reserved.

Inhibition of proteases activity in intestine needs a sustainable acidic environment rather than a transient.

PubMed

Xing, Chang; Xing, Jin-Feng; Ge, Zhi-Qiang

2017-10-01

α-Chymotrypsin (α-CT) and trypsin are important components of the enzymatic barrier. They could degrade the therapeutic proteins and peptides, inhibit their activity consequently, and thereby reduce their oral bioavailability. Acidic agents, as one type of indirect protease inhibitors, have shown proof of concept in clinical trials. We report here the inactivated proteases due to acid influence can be reactivated immediately by environmental pH recovery regardless of how long the inactivation last. To keep the inactivation time of proteases for 4-5 h, we designed and prepared a sustained-release tablet containing citric acid (CA) which can effectively reduce the pH below 5.0 and maintain it for 5 h in the dissolution-reaction medium. The activity of α-CT and trypsin was quantified by analyzing the residual amount of their respective substrates BTEE and TAME. More than 80% of the substrates were survived in 5.0 h of incubation, whereas the common tablet inhibited the proteases activity for only two hours in the same experimental medium. It indicates that the sustained-release tablet loaded with CA can efficiently inhibit the α-CT and trypsin activity longer than the common tablet. The results will be beneficial for designing and formulating the peroral administration of peptide and protein drugs.

Protease inhibitor in scorpion (Mesobuthus eupeus) venom prolongs the biological activities of the crude venom.

PubMed

Ma, Hakim; Xiao-Peng, Tang; Yang, Shi-Long; Lu, Qiu-Min; Lai, Ren

2016-08-01

It is hypothesized that protease inhibitors play an essential role in survival of venomous animals through protecting peptide/protein toxins from degradation by proteases in their prey or predators. However, the biological function of protease inhibitors in scorpion venoms remains unknown. In the present study, a trypsin inhibitor was purified and characterized from the venom of scorpion Mesobuthus eupeus, which enhanced the biological activities of crude venom components in mice when injected in combination with crude venom. This protease inhibitor, named MeKTT-1, belonged to Kunitz-type toxins subfamily. Native MeKTT-1 selectively inhibited trypsin with a Kivalue of 130 nmol·L(-1). Furthermore, MeKTT-1 was shown to be a thermo-stable peptide. In animal behavioral tests, MeKTT-1 prolonged the pain behavior induced by scorpion crude venom, suggesting that protease inhibitors in scorpion venom inhibited proteases and protect the functionally important peptide/protein toxins from degradation, consequently keeping them active longer. In conclusion, this was the first experimental evidence about the natural existence of serine protease inhibitor in the venom of scorpion Mesobuthus eupeus, which preserved the activity of venom components, suggests that scorpions may use protease inhibitors for survival. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers.

PubMed

Yuan, Lin; Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

2017-01-01

Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers

PubMed Central

Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

2017-01-01

Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased

Kinetic Dissection of the Pre-existing Conformational Equilibrium in the Trypsin Fold*

PubMed Central

Vogt, Austin D.; Chakraborty, Pradipta; Di Cera, Enrico

2015-01-01

Structural biology has recently documented the conformational plasticity of the trypsin fold for both the protease and zymogen in terms of a pre-existing equilibrium between closed (E*) and open (E) forms of the active site region. How such plasticity is manifested in solution and affects ligand recognition by the protease and zymogen is poorly understood in quantitative terms. Here we dissect the E*-E equilibrium with stopped-flow kinetics in the presence of excess ligand or macromolecule. Using the clotting protease thrombin and its zymogen precursor prethrombin-2 as relevant models we resolve the relative distribution of the E* and E forms and the underlying kinetic rates for their interconversion. In the case of thrombin, the E* and E forms are distributed in a 1:4 ratio and interconvert on a time scale of 45 ms. In the case of prethrombin-2, the equilibrium is shifted strongly (10:1 ratio) in favor of the closed E* form and unfolds over a faster time scale of 4.5 ms. The distribution of E* and E forms observed for thrombin and prethrombin-2 indicates that zymogen activation is linked to a significant shift in the pre-existing equilibrium between closed and open conformations that facilitates ligand binding to the active site. These findings broaden our mechanistic understanding of how conformational transitions control ligand recognition by thrombin and its zymogen precursor prethrombin-2 and have direct relevance to other members of the trypsin fold. PMID:26216877

Molecular cloning of trypsin cDNAs and trypsin gene expression in the salmon louse Lepeophtheirus salmonis (Copepoda: Caligidae).

PubMed

Johnson, S C; Ewart, K V; Osborne, J A; Delage, D; Ross, N W; Murray, H M

2002-09-01

The salmon louse, Lepeophtheirus salmonis, is a marine ectoparasitic copepod that infects salmonid fishes. We are studying the interactions between this parasite and its salmonid hosts, as it is a common cause of disease in both wild and farmed stocks of salmon. In this paper, we report on the cloning and sequencing of seven trypsin-like enzymes from a cDNA library prepared from whole body preadult female and male L. salmonis. The predicted trypsin activation peptides are 23 or 24 residues in length, considerably longer than previously reported activation peptides of other animals. Differences in the putative signal and activation peptide sequences of the trypsin isoforms suggest that these forms differ in their regulation and function. The calculated molecular weights of the trypsins range from 23.6 to 23.7 kDa. There are eight cysteine residues, which suggest the presence of four disulfide bridges. These trypsins are very similar (>or=46% aa identity) to other crustacean trypsins and insect hypodermins. Using in situ hybridization techniques trypsinogen expression could be identified in all three cell types of the midgut.

Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis

PubMed Central

Bel, Yolanda; Banyuls, Núria; Chakroun, Maissa; Escriche, Baltasar; Ferré, Juan

2017-01-01

Vip3 proteins are secretable proteins from Bacillus thuringiensis whose mode of action is still poorly understood. In this study, the activation process for Vip3 proteins was closely examined in order to better understand the Vip3Aa protein stability and to shed light on its structure. The Vip3Aa protoxin (of 89 kDa) was treated with trypsin at concentrations from 1:100 to 120:100 (trypsin:Vip3A, w:w). If the action of trypsin was not properly neutralized, the results of SDS-PAGE analysis (as well as those with Agrotis ipsilon midgut juice) equivocally indicated that the protoxin could be completely processed. However, when the proteolytic reaction was efficiently stopped, it was revealed that the protoxin was only cleaved at a primary cleavage site, regardless of the amount of trypsin used. The 66 kDa and the 19 kDa peptides generated by the proteases co-eluted after gel filtration chromatography, indicating that they remain together after cleavage. The 66 kDa fragment was found to be extremely resistant to proteases. The trypsin treatment of the protoxin in the presence of SDS revealed the presence of secondary cleavage sites at S-509, and presumably at T-466 and V-372, rendering C-terminal fragments of approximately 29, 32, and 42 kDa, respectively. The fact that the predicted secondary structure of the Vip3Aa protein shows a cluster of beta sheets in the C-terminal region of the protein might be the reason behind the higher stability to proteases compared to the rest of the protein, which is mainly composed of alpha helices. PMID:28387713

Functional analysis of the interactions between reovirus particles and various proteases in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sargent, M.D.; Long, D.G.; Borsa, J.

1977-01-01

The digestion of purified reovirus particles by various proteases including chymotrypsin, trypsin, pronase, papain, bromelain, proteinase K, and fibrinolysin has been examined as it relates to virion transcriptase activation and alteration of infectivity. In every case uncoating to the level of active transcriptase proceeds via two mechanistically distinct steps. All the proteases tested serve to mediate only the first of the two steps, converting intact virions to intermediate subviral particles (ISVP) in which the transcriptase is retained in a latent state. The second step of the uncoating process is mediated by a K/sup +/ ion-triggered, endogenous mechanism and results inmore » conversion of ISVP to cores, concomitant with transcriptase activation and loss of infectivity. All of the tested enzymes, except trypsin, reversibly block the second step of uncoating. These results indicate the generality, with respect to protease employed, of the two-step process for reovirus uncoating and transcriptase activation demonstrated previously with chymotrypsin.« less

From proteases to proteomics.

PubMed

Neurath, H

2001-04-01

This personal and professional autobiography covers the 50-yr period of 1950-2000 and includes the following topics: History of the University of Washington School of Medicine and its Department of Biochemistry (Mount Rainier and the University of Washington, recruiting faculty, biology, research programs); scientific editing (publication, Biochemistry, Protein Science, electronic publication); Europe revisited (Heidelberg, approaching retirement, the German Research Center, reunion in Vienna); and 50 yr of research on proteolytic enzymes (trypsin, carboxypeptidases, mast cell proteases, future developments).

Protease inhibitors from several classes work synergistically against Callosobruchus maculatus.

PubMed

Amirhusin, Bahagiawati; Shade, Richard E; Koiwa, Hisashi; Hasegawa, Paul M; Bressan, Ray A; Murdock, Larry L; Zhu-Salzman, Keyan

2007-07-01

Targeting multiple digestive proteases may be more effective in insect pest control than inhibition of a single enzyme class. We therefore explored possible interactions of three antimetabolic protease inhibitors fed to cowpea bruchids in artificial diets, using a recombinant soybean cysteine protease inhibitor scN, an aspartic protease inhibitor pepstatin A, and soybean Kunitz trypsin inhibitor KI. scN and pepstatin, inhibiting major digestive cysteine and aspartic proteases, respectively, significantly prolonged the developmental time of cowpea bruchids individually. When combined, the anti-insect effect was synergistic, i.e., the toxicity of the mixture was markedly greater than that of scN or pepstatin alone. KI alone did not impact insect development even at relatively high concentrations, but its anti-insect properties became apparent when acting jointly with scN or scN plus pepstatin. Incubating KI with bruchid midgut extract showed that it was partially degraded. This instability may explain its lack of anti-insect activity. However, this proteolytic degradation was inhibited by scN and/or pepstatin. Protection of KI from proteolysis in the insect digestive tract thus could be the basis for the synergistic effect. These observations support the concept that cowpea bruchid gut proteases play a dual role; digesting protein for nutrient needs and protecting insects by inactivating dietary proteins that may otherwise be toxic. Our results also suggest that transgenic resistance strategies that involve multigene products are likely to have enhanced efficacy and durability.

Bauhinia variegata var. variegata trypsin inhibitor: From isolation to potential medicinal applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Evandro Fei; Wong, Jack Ho; Bah, Clara Shui Fern

Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel's foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (K{sub i}, 0.1 x 10{sup -9} M) of BvvTI ranks the highest among them. Besides anti-HIV-1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies.more » These results unveil potential medicinal applications of BvvTI.« less

Bauhinia variegata var. variegata trypsin inhibitor: from isolation to potential medicinal applications.

PubMed

Fang, Evandro Fei; Wong, Jack Ho; Bah, Clara Shui Fern; Lin, Peng; Tsao, Sai Wah; Ng, Tzi Bun

2010-06-11

Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel's foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (K(i), 0.1 x 10(-9)M) of BvvTI ranks the highest among them. Besides anti-HIV-1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies. These results unveil potential medicinal applications of BvvTI. (c) 2010 Elsevier Inc. All rights reserved.

Methylene blue not ferrocene: Optimal reporters for electrochemical detection of protease activity.

PubMed

González-Fernández, Eva; Avlonitis, Nicolaos; Murray, Alan F; Mount, Andrew R; Bradley, Mark

2016-10-15

Electrochemical peptide-based biosensors are attracting significant attention for the detection and analysis of proteins. Here we report the optimisation and evaluation of an electrochemical biosensor for the detection of protease activity using self-assembled monolayers (SAMs) on gold surfaces, using trypsin as a model protease. The principle of detection was the specific proteolytic cleavage of redox-tagged peptides by trypsin, which causes the release of the redox reporter, resulting in a decrease of the peak current as measured by square wave voltammetry. A systematic enhancement of detection was achieved through optimisation of the properties of the redox-tagged peptide; this included for the first time a side-by-side study of the applicability of two of the most commonly applied redox reporters used for developing electrochemical biosensors, ferrocene and methylene blue, along with the effect of changing both the nature of the spacer and the composition of the SAM. Methylene blue-tagged peptides combined with a polyethylene-glycol (PEG) based spacer were shown to be the best platform for trypsin detection, leading to the highest fidelity signals (characterised by the highest sensitivity (signal gain) and a much more stable background than that registered when using ferrocene as a reporter). A ternary SAM (T-SAM) configuration, which included a PEG-based dithiol, minimised the non-specific adsorption of other proteins and was sensitive towards trypsin in the clinically relevant range, with a Limit of Detection (LoD) of 250pM. Kinetic analysis of the electrochemical response with time showed a good fit to a Michaelis-Menten surface cleavage model, enabling the extraction of values for kcat and KM. Fitting to this model enabled quantitative determination of the solution concentration of trypsin across the entire measurement range. Studies using an enzyme inhibitor and a range of real world possible interferents demonstrated a selective response to trypsin

Kallikrein-related peptidase 4 (KLK4) initiates intracellular signaling via protease-activated receptors (PARs). KLK4 and PAR-2 are co-expressed during prostate cancer progression.

PubMed

Ramsay, Andrew J; Dong, Ying; Hunt, Melanie L; Linn, MayLa; Samaratunga, Hemamali; Clements, Judith A; Hooper, John D

2008-05-02

Kallikrein-related peptidase 4 (KLK4) is one of the 15 members of the human KLK family and a trypsin-like, prostate cancer-associated serine protease. Signaling initiated by trypsin-like serine proteases are transduced across the plasma membrane primarily by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. Here we show, using Ca(2+) flux assays, that KLK4 signals via both PAR-1 and PAR-2 but not via PAR-4. Dose-response analysis over the enzyme concentration range 0.1-1000 nM indicated that KLK4-induced Ca(2+) mobilization via PAR-1 is more potent than via PAR-2, whereas KLK4 displayed greater efficacy via the latter PAR. We confirmed the specificity of KLK4 signaling via PAR-2 using in vitro protease cleavage assays and anti-phospho-ERK1/2/total ERK1/2 Western blot analysis of PAR-2-overexpressing and small interfering RNA-mediated receptor knockdown cell lines. Consistently, confocal microscopy analyses indicated that KLK4 initiates loss of PAR-2 from the cell surface and receptor internalization. Immunohistochemical analysis indicated the co-expression of agonist and PAR-2 in primary prostate cancer and bone metastases, suggesting that KLK4 signaling via this receptor will have pathological relevance. These data provide insight into KLK4-mediated cell signaling and suggest that signals induced by this enzyme via PARs may be important in prostate cancer.

Improvement of porcine islet isolation by inhibition of trypsin activity during pancreas preservation and digestion using α1-antitrypsin.

PubMed

Shimoda, Masayuki; Noguchi, Hirofumi; Fujita, Yasutaka; Takita, Morihito; Ikemoto, Tetsuya; Chujo, Daisuke; Naziruddin, Bashoo; Levy, Marlon F; Kobayashi, Naoya; Grayburn, Paul A; Matsumoto, Shinichi

2012-01-01

Porcine islets are considered to be a promising resource for xenotransplantation. However, it is difficult to isolate porcine islets because of the marked fragility and rapid dissociation. Endogenous trypsin is one of the main factors to damage islets during the isolation procedure. Recent studies have suggested that trypsin inhibitors during the preservation of pancreas or the collagenase digestion can improve the result of islet isolation. In this study, we examined whether α1-antitrypsin (Aralast™), which inhibits several endogenous proteases and has immunomodulatory properties, can protect islets from the proteases and improve the results of porcine islet isolation. Twelve porcine pancreata were divided into three groups: without Aralast group (standard, n = 5), preserved with Aralast using the ductal injection (DI) method (DI, n = 3), and with Aralast using the DI method and in the collagenase solution (DI+C, n = 4). Efficacy of islet isolation was assessed by islet yields, purity, and viability. The trypsin activity of the preservation and the digestion solution during the isolation procedure was measured. During islet isolation, the trypsin activity in DI+C group was significantly inhibited compared to the standard group, whereas DI group showed less effect than DI+C group. The average of postpurification islet equivalents (IEQ) per pancreas weight in the DI+C group was significantly higher than the standard group (standard: 3516 ± 497 IEQ/g, DI: 4607 ± 1090 IEQ/g, DI+C: 7097 ± 995 IEQ/g; p = 0.017 between standard and DI+C). In the DI+C group, stimulation index was higher than in other groups, although there was no significant difference. The presence of Aralast in both DI solution and collagenase solution markedly inhibited trypsin activity during pancreas digestion procedure and improved the porcine islet isolation. Inhibition of trypsin activity by Aralast could improve porcine islet isolation.

Partial trypsin digestion as an indicator of mis-folding of mutant alanine:glyoxylate aminotransferase and chaperone effects of specific ligands. Study of a spectrum of missense mutants.

PubMed

Coulter-Mackie, M B; Lian, Q

2008-07-01

Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme whose deficiency results in primary hyperoxaluria type 1 (PH1). More than 75 PH1 mutations are now documented in the AGT gene (AGXT), of which about 50% are missense. We have previously demonstrated that many such mutants expressed by transcription/translation are subject to generalized degradation by the proteasome and a specific limited trimming by an endogenous ATP-independent protease activity. Here, we report the results of partial digestion using trypsin as a mimic for the endogenous non-proteasomal protease and the use of N-terminal protein sequencing to determine the sensitive site. Partial trypsin digestion also provided an indicator of proper folding of the mutant enzyme. For selected mutations the sensitivity to trypsin could be ameliorated by addition of pyridoxal phosphate or aminooxy acetic acid as specific pharmacological chaperones.

From proteases to proteomics

PubMed Central

Neurath, Hans

2001-01-01

This personal and professional autobiography covers the 50-yr period of 1950–2000 and includes the following topics: History of the University of Washington School of Medicine and its Department of Biochemistry (Mount Rainier and the University of Washington, recruiting faculty, biology, research programs); scientific editing (publication, Biochemistry, Protein Science, electronic publication); Europe revisited (Heidelberg, approaching retirement, the German Research Center, reunion in Vienna); and 50 yr of research on proteolytic enzymes (trypsin, carboxypeptidases, mast cell proteases, future developments). PMID:11274481

Maternally derived trypsin may have multiple functions in the early development of turbot (Scopthalmus maximus).

PubMed

Chi, Liang; Liu, Qinghua; Xu, Shihong; Xiao, Zhizhong; Ma, Daoyuan; Li, Jun

2015-10-01

Trypsin is an important serine protease that is considered to be involved in digestion of protein in teleost fish. Nevertheless, studies on trypsin/trypsinogen in fish embryos are very limited. In this study, the trypsinogen of turbot (Scophthalmus maximus) (tTG) was identified and the expression patterns and activity of trypsinogen/trypsin were investigated. The results showed that the tTG mRNA was evenly distributed in the oocytes and was also expressed along the yolk periphery in early embryos. At later embryo stages and 1 days after hatching (dph), the tTG mRNA concentrated at the alimentary tract and head. Quantitative expression analysis showed that the tTG transcripts decreased after fertilization until the gastrula stage, then increased with the embryo and larvae development. This result was also confirmed by the specific activity analysis of trypsin and in-situ-hybridization (ISH). All of the results indicated that tTG in early embryo stages was maternally derived and expressed by itself after gastrula stages. Additionally, location of tTG mRNA in embryos and larvae was investigated; we considered that trypsin may have multiple functions during the embryo development process. Based on our results regarding trypsinogen in embryos and early development, we concluded that the trypsin/trypsinogen in turbot embryos was inherited from a maternal source and we suggested that trypsin in early development has multiple functions in the process of development. Copyright © 2015 Elsevier Inc. All rights reserved.

Loss of hippocampal serine protease BSP1/neuropsin predisposes to global seizure activity.

PubMed

Davies, B; Kearns, I R; Ure, J; Davies, C H; Lathe, R

2001-09-15

Serine proteases in the adult CNS contribute both to activity-dependent structural changes accompanying learning and to the regulation of excitotoxic cell death. Brain serine protease 1 (BSP1)/neuropsin is a trypsin-like serine protease exclusively expressed, within the CNS, in the hippocampus and associated limbic structures. To explore the role of this enzyme, we have used gene targeting to disrupt this gene in mice. Mutant mice were viable and overtly normal; they displayed normal hippocampal long-term synaptic potentiation (LTP) and exhibited no deficits in spatial navigation (water maze). Nevertheless, electrophysiological studies revealed that the hippocampus of mice lacking this specifically expressed protease possessed an increased susceptibility for hyperexcitability (polyspiking) in response to repetitive afferent stimulation. Furthermore, seizure activity on kainic acid administration was markedly increased in mutant mice and was accompanied by heightened immediate early gene (c-fos) expression throughout the brain. In view of the regional selectivity of BSP1/neuropsin brain expression, the observed phenotype may selectively reflect limbic function, further implicating the hippocampus and amygdala in controlling cortical activation. Within the hippocampus, our data suggest that BSP1/neuropsin, unlike other serine proteases, has little effect on physiological synaptic remodeling and instead plays a role in limiting neuronal hyperexcitability induced by epileptogenic insult.

Cuticle-degrading proteases produced by the entomopathogenic fungus Beauveria bassiana in the presence of coffee berry borer cuticle

PubMed Central

Dias, B.A.; Neves, P.M.O.J.; Furlaneto-Maia, L.; Furlaneto, M.C.

2008-01-01

A Brazilian isolate of Beauveria bassiana (CG425) that shows high virulence against the coffee berry borer (CBB) was examined for the production of subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases. Fungal growth was either in nitrate-medium or in CBB cuticle-containing medium under both buffered and unbuffered conditions. In unbuffered medium supplemented with cuticle, the pH of cultures dropped and Pr1 and Pr2 activities were detected in high amounts only at a pH of 5.5 or higher. In buffered cultures, Pr1 and Pr2 activities were higher in medium supplemented with cuticle compared to activities with nitrate-medium. The Pr1 and Pr2 activities detected were mostly in the culture supernatant. These data suggest that Pr1 and Pr2 proteases produced by strain CG425 are induced by components of CBB cuticle, and that the culture pH influences the expression of these proteases, indicating the occurrence of an efficient mechanism of protein secretion in this fungus. The results obtained in this study extend the knowledge about protease production in B. bassiana CG425, opening new avenues for studying the role of secreted proteases in virulence against the coffee berry borer during the infection process. PMID:24031220

Nicotinamide Inhibits the Lysosomal Cathepsin b-like Protease and Kills African Trypanosomes*

PubMed Central

Unciti-Broceta, Juan D.; Maceira, José; Morales, Sonia; García-Pérez, Angélica; Muñóz-Torres, Manuel E.; Garcia-Salcedo, Jose A.

2013-01-01

Nicotinamide, a soluble compound of the vitamin B3 group, has antimicrobial activity against several microorganisms ranging from viruses to parasite protozoans. However, the mode of action of this antimicrobial activity is unknown. Here, we investigate the trypanocidal activity of nicotinamide on Trypanosoma brucei, the causative agent of African trypanosomiasis. Incubation of trypanosomes with nicotinamide causes deleterious defects in endocytic traffic, disruption of the lysosome, failure of cytokinesis, and, ultimately, cell death. At the same concentrations there was no effect on a cultured mammalian cell line. The effects on endocytosis and vesicle traffic were visible within 3 h and can be attributed to inhibition of lysosomal cathepsin b-like protease activity. The inhibitory effect of nicotinamide was confirmed by a direct activity assay of recombinant cathepsin b-like protein. Taken together, these data demonstrate that inhibition of the lysosomal protease cathepsin b-like blocks endocytosis, causing cell death. In addition, these results demonstrate for the first time the inhibitory effect of nicotinamide on a protease. PMID:23443665

Potent Inhibition of Feline Coronaviruses with Peptidyl Compounds Targeting Coronavirus 3C-like Protease

PubMed Central

Kim, Yunjeong; Mandadapu, Sivakoteswara Rao; Groutas, William C.; Chang, Kyeong-Ok

2012-01-01

Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against feline coronaviruses in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC50 in a nanomolar range) and, furthermore, the combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in cell culture systems. PMID:23219425

Characterization of cDNAs encoding serine proteases and their transcriptional responses to Cry1Ab protoxin in the gut of Ostrinia nubilalis larvae

USDA-ARS?s Scientific Manuscript database

Serine proteases, such as trypsin and chymotrypsin, are the primary digestive enzymes in lepidopteran larvae, and are also involved in Bacillus thuringiensis (Bt) protoxin activation and protoxin/toxin degradation. We isolated and sequenced 34 cDNAs putatively encoding trypsins, chymotrypsins and th...

Allergenic proteases cleave the chemokine CX3CL1 directly from the surface of airway epithelium and augment the effect of rhinovirus.

PubMed

Loxham, M; Smart, D E; Bedke, N J; Smithers, N P; Filippi, I; Blume, C; Swindle, E J; Tariq, K; Howarth, P H; Holgate, S T; Davies, D E

2018-03-01

CX3CL1 has been implicated in allergen-induced airway CD4 + T-lymphocyte recruitment in asthma. As epidemiological evidence supports a viral infection-allergen synergy in asthma exacerbations, we postulated that rhinovirus (RV) infection in the presence of allergen augments epithelial CX3CL1 release. Fully differentiated primary bronchial epithelial cultures were pretreated apically with house dust mite (HDM) extract and infected with rhinovirus-16 (RV16). CX3CL1 was measured by enzyme-linked immunosorbent assay and western blotting, and shedding mechanisms assessed using inhibitors, protease-activated receptor-2 (PAR-2) agonist, and recombinant CX3CL1-expressing HEK293T cells. Basolateral CX3CL1 release was unaffected by HDM but stimulated by RV16; inhibition by fluticasone or GM6001 implicated nuclear factor-κB and ADAM (A Disintegrin and Metalloproteinase) sheddases. Conversely, apical CX3CL1 shedding was stimulated by HDM and augmented by RV16. Although fluticasone or GM6001 reduced RV16+HDM-induced apical CX3CL1 release, heat inactivation or cysteine protease inhibition completely blocked CX3CL1 shedding. The HDM effect was via enzymatic cleavage of CX3CL1, not PAR-2 activation, yielding a product mitogenic for smooth muscle cells. Extracts of Alternaria fungus caused similar CX3CL1 shedding. We have identified a novel mechanism whereby allergenic proteases cleave CX3CL1 from the apical epithelial surface to yield a biologically active product. RV16 infection augmented HDM-induced CX3CL1 shedding-this may contribute to synergy between allergen exposure and RV infection in triggering asthma exacerbations and airway remodeling.

Targeting the membrane-anchored serine protease testisin with a novel engineered anthrax toxin prodrug to kill tumor cells and reduce tumor burden

PubMed Central

Martin, Erik W.; Buzza, Marguerite S.; Driesbaugh, Kathryn H.; Liu, Shihui; Fortenberry, Yolanda M.; Leppla, Stephen H.; Antalis, Toni M.

2015-01-01

The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent. PMID:26392335

Autocrine Extra-Pancreatic Trypsin 3 Secretion Promotes Cell Proliferation and Survival in Esophageal Adenocarcinoma

PubMed Central

Han, Song; Lee, Constance W.; Trevino, Jose G.; Hughes, Steven J.; Sarosi, George A.

2013-01-01

Trypsin or Tumor associated trypsin (TAT) activation of Protease-activated receptor 2 (PAR-2) promotes tumor cell proliferation in gastrointestinal cancers. The role of the trypsin/PAR-2 network in esophageal adenocarcinoma (EA) development has not yet been investigated. The aim of this study is to investigate the role of trypsin/PAR-2 activation in EA tumorogenesis and therapy. We found that esophageal adenocarcinoma cells (EACs) and Barrett’s Metaplasia (BART) expressed high levels of type 3 extra-pancreatic trypsinogen (PRSS3), a novel type of TAT. Activity of secreted trypsin was detected in cultured media from EA OE19 and OE33 cultures but not from BART culture. Surface PAR-2 expression in BART and EACs was confirmed by both flow cytometry and immunofluorescence. Trypsin induced cell proliferation (∼ 2 fold; P<0.01) in all tested cell lines at a concentration of 10 nM. Inhibition of PAR-2 activity in EACs via the PAR-2 antagonist ENMD (500 µM), anti-PAR2 antibody SAM-11 (2 µg/ml), or siRNA PAR-2 knockdown, reduced cell proliferation and increased apoptosis by up to 4 fold (P<0.01). Trypsin stimulation led to phosphorylation of ERK1/2, suggesting involvement of MAPK pathway in PAR-2 signal transduction. Inhibition of PAR-2 activation or siRNA PAR-2 knockdown in EACs prior to treatment with 5 FU reduced cell viability of EACs by an additional 30% (P<0.01) compared to chemotherapy alone. Our data suggest that extra-pancreatic trypsinogen 3 is produced by EACs and activates PAR-2 in an autocrine manner. PAR-2 activation increases cancer cell proliferation, and promotes cancer cell survival. Targeting the trypsin activated PAR-2 pathway in conjunction with current chemotherapeutic agents may be a viable therapeutic strategy in EA. PMID:24146905

A giant protease with potential to substitute for some functions of the proteasome.

PubMed

Geier, E; Pfeifer, G; Wilm, M; Lucchiari-Hartz, M; Baumeister, W; Eichmann, K; Niedermann, G

1999-02-12

An alanyl-alanyl-phenylalanyl-7-amino-4-methylcoumarin-hydrolyzing protease particle copurifying with 26S proteasomes was isolated and identified as tripeptidyl peptidase II (TPPII), a cytosolic subtilisin-like peptidase of unknown function. The particle is larger than the 26S proteasome and has a rod-shaped, dynamic supramolecular structure. TPPII exhibits enhanced activity in proteasome inhibitor-adapted cells and degrades polypeptides by exo- as well as predominantly trypsin-like endoproteolytic cleavage. TPPII may thus participate in extralysosomal polypeptide degradation and may in part account for nonproteasomal epitope generation as postulated for certain major histocompatibility complex class I alleles. In addition, TPPII may be able to substitute for some metabolic functions of the proteasome.

Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

PubMed

Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique

2016-08-01

Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop. Copyright © 2016. Published by Elsevier Inc.

Midgut serine proteases and alternative host plant utilization in Pieris brassicae L.

PubMed Central

Kumar, Rakesh; Bhardwaj, Usha; Kumar, Pawan; Mazumdar-Leighton, Sudeshna

2015-01-01

Pieris brassicae L. is a serious pest of cultivated crucifers in several parts of the world. Larvae of P. brassicae also feed prolifically on garden nasturtium (Tropaeolum majus L., of the family Tropaeolaceae). Proteolytic digestion was studied in larvae feeding on multiple hosts. Fourth instars were collected from cauliflower fields before transfer onto detached, aerial tissues of selected host plants in the lab. Variable levels of midgut proteases were detected in larvae fed on different hosts using protein substrates (casein and recombinant RBCL cloned from cauliflower) and diagnostic, synthetic substrates. Qualitative changes in midgut trypsin activities and quantitative changes in midgut chymotrypsin activities were implicated in physiological adaptation of larvae transferred to T. majus. Midgut proteolytic activities were inhibited to different extents by serine protease inhibitors, including putative trypsin inhibitors isolated from herbivore-attacked and herbivore-free leaves of cauliflower (CfTI) and T. majus (TpTI). Transfer of larvae to T. majus significantly influenced feeding parameters but not necessarily when transferred to different tissues of the same host. Results obtained are relevant for devising sustainable pest management strategies, including transgenic approaches using genes encoding plant protease inhibitors. PMID:25873901

Gold nanoclusters-based chemiluminescence resonance energy transfer method for sensitive and label-free detection of trypsin.

PubMed

You, Xiaoying; Li, Yinhuan; Li, Baoping; Ma, Jie

2016-01-15

A chemiluminescence resonance energy transfer (CRET) platform was developed for sensitive and label-free detection of protease by using trypsin as a model analyte. In this CRET platform, bis(2,4,6-trichlorophenyl)oxalate-hydrogen peroxide chemiluminescence (CL) reaction was utilized as an energy donor and bovine serum albumin (BSA)-stabilized gold nanoclusters (Au NCs) as an energy acceptor. The BSA-stabilized Au NCs triggered the CRET phenomenon by accepting the energy from TCPO-H2O2 CL reaction, thus producing intense CL. In the presence of trypsin, the protein template of BSA-stabilized Au NCs was digested, which frustrated the energy transfer efficiency between the CL donor and the BSA-stabilized Au NCs, leading to a significant decrease in the CL signal. The decreased CL signal was proportional to the logarithm of trypsin concentration in the range of 0.01-50.0µg mL(-1). The detection limit for trypsin was 9ng mL(-)(1) and the relative standard deviations were lesser than 3% (n=11). This Au NCs-based CRET platform was successfully applied to the determination of trypsin in human urine samples, demonstrating its potential application in clinical diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Structural Evidence for Regulation and Specificity of Flaviviral Proteases and Evolution of the Flaviviridae Fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aleshin,A.; Shiryaev, S.; Strongin, A.

2007-01-01

Pathogenic members of the flavivirus family, including West Nile Virus (WNV) and Dengue Virus (DV), are growing global threats for which there are no specific treatments. The two-component flaviviral enzyme NS2B-NS3 cleaves the viral polyprotein precursor within the host cell, a process that is required for viral replication. Here, we report the crystal structure of WNV NS2B-NS3pro both in a substrate-free form and in complex with the trypsin inhibitor aprotinin/BPTI. We show that aprotinin binds in a substrate-mimetic fashion in which the productive conformation of the protease is fully formed, providing evidence for an 'induced fit' mechanism of catalysis andmore » allowing us to rationalize the distinct substrate specificities of WNV and DV proteases. We also show that the NS2B cofactor of WNV can adopt two very distinct conformations and that this is likely to be a general feature of flaviviral proteases, providing further opportunities for regulation. Finally, by comparing the flaviviral proteases with the more distantly related Hepatitis C virus, we provide insights into the evolution of the Flaviviridae fold. Our work should expedite the design of protease inhibitors to treat a range of flaviviral infections.« less

Robust Trypsin Coating on Electrospun Polymer Nanofibers in Rigorous Conditions and Its Uses for Protein Digestion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahn, Hye-Kyung; Kim, Byoung Chan; Jun, Seung-Hyun

2010-12-15

An efficient protein digestion in proteomic analysis requires the stabilization of proteases such as trypsin. In the present work, trypsin was stabilized in the form of enzyme coating on electrospun polymer nanofibers (EC-TR), which crosslinks additional trypsin molecules onto covalently-attached trypsin (CA-TR). EC-TR showed better stability than CA-TR in rigorous conditions, such as at high temperatures of 40 °C and 50 °C, in the presence of organic co-solvents, and at various pH's. For example, the half-lives of CA-TR and EC-TR were 0.24 and 163.20 hours at 40 ºC, respectively. The improved stability of EC-TR can be explained by covalent-linkages onmore » the surface of trypsin molecules, which effectively inhibits the denaturation, autolysis, and leaching of trypsin. The protein digestion was performed at 40 °C by using both CA-TR and EC-TR in digesting a model protein, enolase. EC-TR showed better performance and stability than CA-TR by maintaining good performance of enolase digestion under recycled uses for a period of one week. In the same condition, CA-TR showed poor performance from the beginning, and could not be used for digestion at all after a few usages. The enzyme coating approach is anticipated to be successfully employed not only for protein digestion in proteomic analysis, but also for various other fields where the poor enzyme stability presently hampers the practical applications of enzymes.« less

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens

PubMed Central

Stella, Nicholas A.; Hunt, Kristin M.; Brothers, Kimberly M.; Zhang, Liang; Thibodeau, Patrick H.

2015-01-01

The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes ocular infections in healthy individuals. Secreted protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of protease-positive strains was observed among keratitis isolates than among conjunctivitis isolates. A positive correlation between protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined. Deletion of prtS in clinical keratitis isolate K904 reduced, but did not eliminate, cytotoxicity and secreted protease production. This indicated that PrtS is necessary for full cytotoxicity to ocular cells and implied the existence of another secreted protease(s) and cytotoxic factors. Bioinformatic analysis of the S. marcescens Db11 genome revealed three additional open reading frames predicted to code for serralysin-like proteases noted here as slpB, slpC, and slpD. Induced expression of prtS and slpB, but not slpC and slpD, in strain PIC3611 rendered the strain cytotoxic to a lung carcinoma cell line; however, only prtS induction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of both prtS and slpB genes was defective in secreted protease activity and cytotoxicity to human cell lines. PAGE analysis suggests that SlpB is produced at lower levels than PrtS. Purified SlpB demonstrated calcium-dependent and AprI-inhibited protease activity and cytotoxicity to airway and ocular cell lines in vitro. Lastly, genetic analysis indicated that the type I secretion system gene, lipD, is required for SlpB secretion. These genetic data introduce SlpB as a new cytotoxic protease from S. marcescens. PMID:25939509

Protease-Activated Receptor-2 Activation Contributes to House Dust Mite-Induced IgE Responses in Mice

PubMed Central

Post, Sijranke; Heijink, Irene H.; Petersen, Arjen H.; de Bruin, Harold G.; van Oosterhout, Antoon J. M.; Nawijn, Martijn C.

2014-01-01

Aeroallergens such as house dust mite (HDM), cockroach, and grass or tree pollen are innocuous substances that can induce allergic sensitization upon inhalation. The serine proteases present in these allergens are thought to activate the protease-activated receptor (PAR)-2, on the airway epithelium, thereby potentially inducing allergic sensitization at the expense of inhalation tolerance. We hypothesized that the proteolytic activity of allergens may play an important factor in the allergenicity to house dust mite and is essential to overcome airway tolerance. Here, we aimed to investigate the role of PAR-2 activation in allergic sensitization and HDM-induced allergic airway inflammation. In our study, Par-2 deficient mice were treated with two different HDM extracts containing high and low serine protease activities twice a week for a period of 5 weeks. We determined airway inflammation through quantification of percentages of mononuclear cells, eosinophils and neutrophils in the bronchial alveolar lavage fluid and measured total IgE and HDM-specific IgE and IgG1 levels in serum. Furthermore, Th2 and pro-inflammatory cytokines including IL-5, IL-13, Eotaxin-1, IL-17, KC, Chemokine (C-C motif) ligand 17 (CCL17) and thymic stromal lymphopoietin (TSLP), were measured in lung tissue homogenates. We observed that independent of the serine protease content, HDM was able to induce elevated levels of eosinophils and neutrophils in the airways of both wild-type (WT) and Par-2 deficient mice. Furthermore, we show that induction of pro-inflammatory cytokines by HDM exposure is independent of Par-2 activation. In contrast, serine protease activity of HDM does contribute to enhanced levels of total IgE, but not HDM-specific IgE. We conclude that, while Par-2 activation contributes to the development of IgE responses, it is largely dispensable for the HDM-induced induction of pro-inflammatory cytokines and airway inflammation in an experimental mouse model of HDM

Purification and characterization of a newly serine protease inhibitor from Rhamnus frangula with potential for use as therapeutic drug.

PubMed

Bacha, Abir Ben; Jemel, Ikram; Moubayed, Nadine M S; Abdelmalek, Imen Ben

2017-06-01

Protease inhibitors from plants are well known to be potent inhibitors of the growth of bacteria, fungi, and even certain viruses which make them excellent candidates for use as the lead compounds for the development of novel antimicrobial agents for applications in medicine. In this study, Rhamnus frangula was selected as a protease inhibitor source. The maximum recovery of the protease inhibitor against trypsin was recorded in the crude extract made in 0.1 M phosphate buffer (pH 7.0) and isolated from the mature leaves. Then, the protease inhibitor designated as RfIP1 was purified to homogeneity by Sephadex G50 with an apparent molecular mass of 22.5 kDa and its N-terminal sequence exhibited a high degree of homology with known serine protease inhibitor sequences. The RfIP1 displayed maximal activity at pH 7 and 37 °C. It maintained almost 80% of its maximal activity through a large pH range. The thermo-stability of RfIP1 was markedly enhanced by BSA, CaCl 2, and sorbitol, whereas the addition of Mg 2+ , Zn 2+ , NaTDC, SDS, DTT, and β-ME significantly promoted inhibitory activity. The protease inhibitor displayed high inhibitory activity toward some known proteases (cathepsin B, chymotrypsin, collagenase, thrombin, and trypsin) that have more importance in pharmaceutical industry and it acted as potent inhibitor of some commercially proteases from Aspergillus oryzae, Bacillus sp, and Bacillus licheniformis. The protease inhibitor also possessed an appreciable antibacterial effect against both Gram-positive and Gram-negative bacteria.

Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination.

PubMed

Lu, Haibin; Chandrasekar, Balakumaran; Oeljeklaus, Julian; Misas-Villamil, Johana C; Wang, Zheming; Shindo, Takayuki; Bogyo, Matthew; Kaiser, Markus; van der Hoorn, Renier A L

2015-08-01

Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins. © 2015 American Society of Plant Biologists. All Rights Reserved.

Feces Derived Allergens of Tyrophagus putrescentiae Reared on Dried Dog Food and Evidence of the Strong Nutritional Interaction between the Mite and Bacillus cereus Producing Protease Bacillolysins and Exo-chitinases

PubMed Central

Erban, Tomas; Rybanska, Dagmar; Harant, Karel; Hortova, Bronislava; Hubert, Jan

2016-01-01

Tyrophagus putrescentiae (Schrank, 1781) is an emerging source of allergens in stored products and homes. Feces proteases are the major allergens of astigmatid mites (Acari: Acaridida). In addition, the mites are carriers of microorganisms and microbial adjuvant compounds that stimulate innate signaling pathways. We sought to analyze the mite feces proteome, proteolytic activities, and mite-bacterial interaction in dry dog food (DDF). Proteomic methods comprising enzymatic and zymographic analysis of proteases and 2D-E-MS/MS were performed. The highest protease activity was assigned to trypsin-like proteases; lower activity was assigned to chymotrypsin-like proteases, and the cysteine protease cathepsin B-like had very low activity. The 2D-E-MS/MS proteomic analysis identified mite trypsin allergen Tyr p3, fatty acid-binding protein Tyr p13 and putative mite allergens ferritin (Grp 30) and (poly)ubiquitins. Tyr p3 was detected at different positions of the 2D-E. It indicates presence of zymogen at basic pI, and mature-enzyme form and enzyme fragment at acidic pI. Bacillolysins (neutral and alkaline proteases) of Bacillus cereus symbiont can contribute to the protease activity of the mite extract. The bacterial exo-chitinases likely contribute to degradation of mite exuviae, mite bodies or food boluses consisting of chitin, including the peritrophic membrane. Thus, the chitinases disrupt the feces and facilitate release of the allergens. B. cereus was isolated and identified based on amplification and sequencing of 16S rRNA and motB genes. B. cereus was added into high-fat, high-protein (DDF) and low-fat, low-protein (flour) diets to 1 and 5% (w/w), and the diets palatability was evaluated in 21-day population growth test. The supplementation of diet with B. cereus significantly suppressed population growth and the suppressive effect was higher in the high-fat, high-protein diet than in the low-fat, low-protein food. Thus, B. cereus has to coexist with the mite in

Interspecific differences between D. pulex and D. magna in tolerance to cyanobacteria with protease inhibitors.

PubMed

Kuster, Christian J; Von Elert, Eric

2013-01-01

It is known that cyanobacteria negatively affect herbivores due to their production of toxins such as protease inhibitors. In the present study we investigated potential interspecific differences between two major herbivores, Daphnia magna and Daphnia pulex, in terms of their tolerance to cyanobacteria with protease inhibitors. Seven clones each of D. magna and of D. pulex were isolated from different habitats in Europe and North America. To test for interspecific differences in the daphnids' tolerance to cyanobacteria, their somatic and population growth rates were determined for each D. magna and D. pulex clone after exposure to varying concentrations of two Microcystis aeruginosa strains. The M. aeruginosa strains NIVA and PCC(-) contained either chymotrypsin or trypsin inhibitors, but no microcystins. Mean somatic and population growth rates on a diet with 20% NIVA were significantly more reduced in D. pulex than in D. magna. On a diet with 10% PCC(-), the population growth of D. pulex was significantly more reduced than that of D. magna. This indicates that D. magna is more tolerant to cyanobacteria with protease inhibitors than D. pulex. The reduction of growth rates was possibly caused by an interference of cyanobacterial inhibitors with proteases in the gut of Daphnia, as many other conceivable factors, which might have been able to explain the reduced growth, could be excluded as causal factors. Protease assays revealed that the sensitivities of chymotrypsins and trypsins to cyanobacterial protease inhibitors did not differ between D. magna and D. pulex. However, D. magna exhibited a 2.3-fold higher specific chymotrypsin activity than D. pulex, which explains the observed higher tolerance to cyanobacterial protease inhibitors of D. magna. The present study suggests that D. magna may control the development of cyanobacterial blooms more efficiently than D. pulex due to differences in their tolerance to cyanobacteria with protease inhibitors.

The protease-activated receptor-2 upregulates keratinocyte phagocytosis.

PubMed

Sharlow, E R; Paine, C S; Babiarz, L; Eisinger, M; Shapiro, S; Seiberg, M

2000-09-01

The protease-activated receptor-2 (PAR-2) belongs to the family of seven transmembrane domain receptors, which are activated by the specific enzymatic cleavage of their extracellular amino termini. Synthetic peptides corresponding to the tethered ligand domain (SLIGRL in mouse, SLIGKV in human) can activate PAR-2 without the need for receptor cleavage. PAR-2 activation is involved in cell growth, differentiation and inflammatory processes, and was shown to affect melanin and melanosome ingestion by human keratinocytes. Data presented here suggest that PAR-2 activation may regulate human keratinocyte phagocytosis. PAR-2 activation by trypsin, SLIGRL or SLIGKV increased the ability of keratinocytes to ingest fluorescently labeled microspheres or E. coli K-12 bioparticles. This PAR-2 mediated increase in keratinocyte phagocytic capability correlated with an increase in actin polymerization and *-actinin reorganization, cell surface morphological changes and increased soluble protease activity. Moreover, addition of serine protease inhibitors downmodulated both the constitutive and the PAR-2 mediated increases in phagocytosis, suggesting that serine proteases mediate this functional activity in keratinocytes. PAR-2 involvement in keratinocyte phagocytosis is a novel function for this receptor.

Improving volatile fatty acids production by exploiting the residual substrates in post-fermented sludge: Protease catalysis of refractory protein.

PubMed

Yin, Bo; Liu, Hongbo; Wang, Yuanyuan; Bai, Jie; Liu, He; Fu, Bo

2016-03-01

The real cause to the low yield of volatile fatty acids (VFAs), from inhibition or low biodegradation, is uncertain in sludge anaerobic fermentation. In this study, poor biodegradability of proteins and fast decrease of the indigenous hydrolase activity in the residual post-fermented sludge were found to be the major reasons. With the addition of trypsin or alkaline protease in residual post-fermented sludge after primary alkaline fermentation, degradation efficiency of refractory protein increased by 33.6% and 34.8%, respectively. Accordingly, the VFAs yields were improved by 69.7% and 106.1%, respectively. Furthermore, the activities of added trypsin and alkaline protease could maintain at 13.52 U/mL and 19.11 U/mL in the alkaline fermentation process. This study demonstrated that exploiting the refractory proteins in residual post-fermented sludge by protease addition seems to be a very promising way for improving VFAs yield of conventional alkaline fermentations with waste activated sludge. Copyright © 2015 Elsevier Ltd. All rights reserved.

Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2

PubMed Central

Reinke, Lennart Michel; Hartleib, Anika; Nehlmeier, Inga; Gierer, Stefanie; Hoffmann, Markus; Hofmann-Winkler, Heike; Winkler, Michael

2017-01-01

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated. PMID:28636671

Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2.

PubMed

Reinke, Lennart Michel; Spiegel, Martin; Plegge, Teresa; Hartleib, Anika; Nehlmeier, Inga; Gierer, Stefanie; Hoffmann, Markus; Hofmann-Winkler, Heike; Winkler, Michael; Pöhlmann, Stefan

2017-01-01

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated.

Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamilton, Brian S.; Chung, Changik; Cyphers, Soreen Y.

Highlights: • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza HA cleavage activation. • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza virus infection. • Comparative analysis of HAI-2 for vesicular stomatitis virus and human parainfluenza virus type-1. • Analysis of the activity of HAI-2 in a mouse model of influenza. - Abstract: Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant tomore » the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza.« less

A novel serine protease predominately expressed in macrophages.

PubMed Central

Chen, Cailin; Darrow, Andrew L; Qi, Jian-Shen; D'Andrea, Michael R; Andrade-Gordon, Patricia

2003-01-01

We have identified a novel serine protease designated EOS by sequence identity searches. The deduced protein contains 284 amino acids with an active form containing 248 amino acids starting from an Ile-Val-Gly-Gly motif. The active form comprises a catalytic triad of conserved amino acids: His77, Asp126 and Ser231. It shares 44% identity with beta-tryptase and belongs to the S1 trypsin-like serine-protease family. Interestingly, this gene also maps to human chromosome 16p13.3. The purified protease showed amidolytic activity, cleaving its substrates before arginine residues. Tissue distribution by immunohistochemistry analysis demonstrated that EOS is highly expressed in spleen and moderately expressed in intestine, colon, lung and brain. We confirmed this expression pattern at the mRNA level by performing in situ hybridization. The results from both immunohistochemistry and in situ hybridization indicate that EOS is associated with macrophages. We corroborated this observation by double immunofluorescence using the anti-EOS antibody and an anti-CD68 antibody, a macrophage specific marker. Furthermore, we have detected a dramatic increase in immune staining of EOS in cultured U937 cells treated with PMA, which represent activated macrophages. This up-regulation is also reflected by elevated EOS mRNA in the PMA-treated U937 cells detected by Northern blotting. Since macrophages have important roles in various pathological conditions, such as wound healing, atherosclerosis and numerous inflammatory diseases, the localization of this novel serine protease to active macrophages may help to further the elucidation of the roles of this gene product in modulating these disorders. PMID:12795636

Three Pairs of Protease-Serpin Complexes Cooperatively Regulate the Insect Innate Immune Responses*

PubMed Central

Jiang, Rui; Kim, Eun-Hye; Gong, Ji-Hee; Kwon, Hyun-Mi; Kim, Chan-Hee; Ryu, Kyoung-Hwa; Park, Ji-Won; Kurokawa, Kenji; Zhang, Jinghai; Gubb, David; Lee, Bok-Luel

2009-01-01

Serpins are known to be necessary for the regulation of several serine protease cascades. However, the mechanisms of how serpins regulate the innate immune responses of invertebrates are not well understood due to the uncertainty of the identity of the serine proteases targeted by the serpins. We recently reported the molecular activation mechanisms of three serine protease-mediated Toll and melanin synthesis cascades in a large beetle, Tenebrio molitor. Here, we purified three novel serpins (SPN40, SPN55, and SPN48) from the hemolymph of T. molitor. These serpins made specific serpin-serine protease pairs with three Toll cascade-activating serine proteases, such as modular serine protease, Spätzle-processing enzyme-activating enzyme, and Spätzle-processing enzyme and cooperatively blocked the Toll signaling cascade and β-1,3-glucan-mediated melanin biosynthesis. Also, the levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of a Toll ligand, processed Spätzle, into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors. Unexpectedly, SPN55 and SPN48 were cleaved at Tyr and Glu residues in reactive center loops, respectively, despite being targeted by trypsin-like Spätzle-processing enzyme-activating enzyme and Spätzle-processing enzyme. These cleavage patterns are also highly similar to those of unusual mammalian serpins involved in blood coagulation and blood pressure regulation, and they may contribute to highly specific and timely inactivation of detrimental serine proteases during innate immune responses. Taken together, these results demonstrate the specific regulatory evidences of innate immune responses by three novel serpins. PMID:19858208

Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis).

PubMed

Samac, D; Storey, R

1981-12-01

Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination.

In Vitro Microfluidic Models of Mucus-Like Obstructions in Small Airways

NASA Astrophysics Data System (ADS)

Mulligan, Molly K.; Grotberg, James B.; Sznitman, Josué

2012-11-01

Liquid plugs can form in the lungs as a result of a host of different diseases, including cystic fibrosis and chronic obstructive pulmonary disease. The existence of such fluid obstructions have been found as far down in the bronchiole tree as the sixteenth generation, where bronchiole openings have diameters on the order of a hundred to a few hundred microns. Understanding the propagation of liquid plugs within the bifurcating branches of bronchiole airways is important because their presence in the lungs, and their rupture and break-up, can cause injury to the epithelial cells lining the airway walls as a result of high wall shear stresses. In particular, liquid plug rupture and break-up frequently occurs at airway bifurcations. Until present, however, experimental studies of liquid plugs have generally been restricted to Newtonian fluids that do not reflect the actual pseudoplastic properties of lung mucus. The present work attempts to uncover the propagation, rupture and break-up of mucus-like liquid plugs in the lower generations of the airway tree using microfluidic models. Our approach allows the dynamics of mucus-like plug break-up to be studied in real-time, in a one-to-one in vitro model, as a function of mucus rheology and bronchial tree geometry.

Trypsin coatings on electrospun and alcohol-dispersed polymer nanofibers for trypsin digestion column

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jun, Seung-Hyun; Chang, Mun Seock; Kim, Byoung Chan

2010-09-15

The construction of a trypsin reactor in a chromatography column for rapid and efficient protein digestion in proteomics is described. Electrospun and alcohol-dispersed polymer nanofibers were used for the fabrication of highly stable trypsin coating, which was prepared by a two-step process of covalent attachment and enzyme crosslinking. In a comparative study with the trypsin coatings on asspun and non-dispersed nanofibers, it has been observed that a simple step of alcohol dispersion improved not only the enzyme loading but also the performance of protein digestion. In-column digestion of enolase was successfully performed in less than twenty minutes. By applying themore » alcohol dispersion of polymer nanofibers, the bypass of samples was reduced by filling up the column with well-dispersed nanofibers, and subsequently, interactions between the protein and the enzymes were improved yielding more complete and reproducible digestions. Regardless of alcohol-dispersion or not, trypsin coating showed better digestion performance and improved performance stability under recycled uses than covalently-attached trypsin. The combination of highly stable trypsin coating and alcoholdispersion of polymer nanofibers has opened up a new potential to develop a trypsin column for on-line and automated protein digestion.« less

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens.

PubMed

Shanks, Robert M Q; Stella, Nicholas A; Hunt, Kristin M; Brothers, Kimberly M; Zhang, Liang; Thibodeau, Patrick H

2015-07-01

The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes ocular infections in healthy individuals. Secreted protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of protease-positive strains was observed among keratitis isolates than among conjunctivitis isolates. A positive correlation between protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined. Deletion of prtS in clinical keratitis isolate K904 reduced, but did not eliminate, cytotoxicity and secreted protease production. This indicated that PrtS is necessary for full cytotoxicity to ocular cells and implied the existence of another secreted protease(s) and cytotoxic factors. Bioinformatic analysis of the S. marcescens Db11 genome revealed three additional open reading frames predicted to code for serralysin-like proteases noted here as slpB, slpC, and slpD. Induced expression of prtS and slpB, but not slpC and slpD, in strain PIC3611 rendered the strain cytotoxic to a lung carcinoma cell line; however, only prtS induction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of both prtS and slpB genes was defective in secreted protease activity and cytotoxicity to human cell lines. PAGE analysis suggests that SlpB is produced at lower levels than PrtS. Purified SlpB demonstrated calcium-dependent and AprI-inhibited protease activity and cytotoxicity to airway and ocular cell lines in vitro. Lastly, genetic analysis indicated that the type I secretion system gene, lipD, is required for SlpB secretion. These genetic data introduce SlpB as a new cytotoxic protease from S. marcescens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Highly potent fibrinolytic serine protease from Streptomyces.

PubMed

Uesugi, Yoshiko; Usuki, Hirokazu; Iwabuchi, Masaki; Hatanaka, Tadashi

2011-01-05

We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis. Copyright © 2010 Elsevier Inc. All rights reserved.

Modifications in trypsin digestion protocol for increasing the efficiency and coverage.

PubMed

Syal, Kirtimaan; Tadala, Raghu

2015-01-01

Standard trypsin digestion protocol of proteins followed by MALDI-MS analysis has been realized as an important tool for the identification and characterization of proteins. In this article, we proposed the elimination of the step of 'staining/de-staining of gel pieces' in in-gel digestion protocol in order to improve the efficiency of trypsin digestion. Coomassie dye is known to interfere with digestion of proteins by trypsin and the procedure of staining-de-staining could result in loss of photoaffinity probe, post translational modifications and catalytic activities of enzymes. Further, we studied parameters like hydrophobicity and isoelectric point, and attempted to quantitatively relate it to the efficiency of trypsin digestion. We suggest that properties of proteins should be considered and trypsin digestion protocol should be appropriately modified as per sequence and other information.

Midgut Protease Activity During Larval Development of Anastrepha obliqua (Diptera: Tephritidae) Fed With Natural and Artificial Diet

PubMed Central

Rivera-Ciprian, José Pedro; Aceituno-Medina, Marysol; Guillen, Karina

2017-01-01

Abstract In this study, we examined the activity of two serine proteases (chymotrypsin and trypsin) and two metalloproteases (carboxypeptidases A and B) during larval development in Anastrepha obliqua fed natural (mango fruit) and artificial (formulation used in mass-rearing) diets. Proteolytic activity of chymotrypsin, trypsin, carboxypeptidase A, and carboxypeptidase B was detected in the midgut of different instars of A. obliqua and was strongly affected by the pH and diet type. The protein content of the natural and artificial diets was similar. Enzymatic activity was higher in the midgut of the larvae fed the natural diet than in larvae fed the artificial diet. The activity of the endopeptidases (chymotrypsin and trypsin) was lower than those of the exopeptidases (carboxypeptidases A and B). The pH of the midgut varied from acidic to neutral. The results indicate that in the midgut of the larvae reared on both types of diet, the level of carboxypeptidase activity was approximately 100-fold greater than the level of chymotrypsin activity and 10,000-fold greater than the level of trypsin. In conclusion, carboxypeptidase A and B are the main proteases involved in the digestion of proteins in the larvae of A. obliqua. The natural diet showed a high bioaccessibility. A clear tendency to express high activities of chymotrypsin and trypsin was observed by the third instar. Our research contributes to the planning and development of novel bioaccessibility assays to understand the nutrition processing of A. obliqua larvae under mass-rearing conditions for sterile insect technique.

A New Subtilase-Like Protease Deriving from Fusarium equiseti with High Potential for Industrial Applications.

PubMed

Juntunen, Kari; Mäkinen, Susanna; Isoniemi, Sari; Valtakari, Leena; Pelzer, Alexander; Jänis, Janne; Paloheimo, Marja

2015-09-01

A gene encoding a novel extracellular subtilisin-like protease was cloned from the ascomycete Fusarium equiseti and expressed in Trichoderma reesei. The F. equiseti protease (Fe protease) showed excellent performance in stain removal and good compatibility with several commercial laundry detergent formulations, suggesting that it has high potential for use in various industrial applications. The recombinant enzyme was purified and characterized. The temperature optimum of the Fe protease was 60 °C and it showed high activity in the pH range of 6-10, with a sharp decline in activity at pH above 10. The amino acid specificity of the Fe protease was studied using casein, cytochrome c, and ubiquitin as substrates. The Fe protease had broad substrate specificity: almost all amino acid residues were accepted at position P1, even though it showed some preference for cleavage at the C-terminal side of asparagine and histidine residues. The S4 subsite of Fe protease favors aspartic acid and threonine. The other well-characterized proteases from filamentous fungi, Proteinase K from Engyodontium album, Thermomycolin from Malbranchea sulfurea, and alkaline subtilisins from Bacillus species prefer hydrophobic amino acids in both the S1 and S4 subsites. Due to its different specificity compared to the members of the S8 family of clan SB of proteases, we consider that the Fe protease is a new protease. It does not belong to any previously defined IUBMB groups of proteases.

Pre-equilibrium competitive library screening for tuning inhibitor association rate and specificity toward serine proteases.

PubMed

Cohen, Itay; Naftaly, Si; Ben-Zeev, Efrat; Hockla, Alexandra; Radisky, Evette S; Papo, Niv

2018-04-16

High structural and sequence similarity within protein families can pose significant challenges to the development of selective inhibitors, especially toward proteolytic enzymes. Such enzymes usually belong to large families of closely similar proteases and may also hydrolyze, with different rates, protein- or peptide-based inhibitors. To address this challenge, we employed a combinatorial yeast surface display library approach complemented with a novel pre-equilibrium, competitive screening strategy for facile assessment of the effects of multiple mutations on inhibitor association rates and binding specificity. As a proof of principle for this combined approach, we utilized this strategy to alter inhibitor/protease association rates and to tailor the selectivity of the amyloid β-protein precursor Kunitz protease inhibitor domain (APPI) for inhibition of the oncogenic protease mesotrypsin, in the presence of three competing serine proteases, anionic trypsin, cationic trypsin and kallikrein-6. We generated a variant, designated APPI P13W/M17G/I18F/F34V , with up to 30-fold greater specificity relative to the parental APPI M17G/I18F/F34V protein, and 6500- to 230 000-fold improved specificity relative to the wild-type APPI protein in the presence of the other proteases tested. A series of molecular docking simulations suggested a mechanism of interaction that supported the biochemical results. These simulations predicted that the selectivity and specificity are affected by the interaction of the mutated APPI residues with nonconserved enzyme residues located in or near the binding site. Our strategy will facilitate a better understanding of the binding landscape of multispecific proteins and will pave the way for design of new drugs and diagnostic tools targeting proteases and other proteins. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Plant proteases for bioactive peptides release: A review.

PubMed

Mazorra-Manzano, M A; Ramírez-Suarez, J C; Yada, R Y

2017-04-10

Proteins are a potential source of health-promoting biomolecules with medical, nutraceutical, and food applications. Nowadays, bioactive peptides production, its isolation, characterization, and strategies for its delivery to target sites are a matter of intensive research. In vitro and in vivo studies regarding the bioactivity of peptides has generated strong evidence of their health benefits. Dairy proteins are considered the richest source of bioactive peptides, however proteins from animal and vegetable origin also have been shown to be important sources. Enzymatic hydrolysis has been the process most commonly used for bioactive peptide production. Most commercial enzymatic preparations frequently used are from animal (e.g., trypsin and pepsin) and microbial (e.g., Alcalase® and Neutrase®) sources. Although the use of plant proteases is still relatively limited to papain and bromelain from papaya and pineapple, respectively, the application of new plant proteases is increasing. This review presents the latest knowledge in the use and diversity of plant proteases for bioactive peptides release from food proteins including both available commercial plant proteases as well as new potential plant sources. Furthermore, the properties of peptides released by plant proteases and health benefits associated in the control of disorders such as hypertension, diabetes, obesity, and cancer are reviewed.

Activity of trypsin-like enzymes and gelatinases in rats with doxorubicin cardiomyopathy.

PubMed

Gordiienko, Iu A; Babets, Ya V; Kulinich, A O; Shevtsova, A I; Ushakova, G O

2014-01-01

Activity of trypsin-like enzymes (ATLE) and gelatinases A and B were studied in the blood plasma and extracts from cardiac muscle, cerebral cortex and cerebellum of rats with cardiomyopathy caused by anthracycline antibiotic doxorubicin against the background of preventive application of corvitin and α-ketoglutarate. ATLE significantly increased in blood plasma and extracts from cerebral cortex but decreased in extracts from cardiac muscle and cerebellum in doxorubicin cardiomyopathy (DCMP). In addition, a significant increase of activity of both gelatinases in plasma and tissue extracts was observed. Preventive administration of corvitin and α-ketoglutarate resulted in differently directed changes of activity of the above mentioned enzymes in heart and brain tissues. Obtained data confirm the hypothesis about activation of proteolysis under the influence of anthracycline antibiotics and testify to selective effect of corvitin and α-ketoglutarate on ATLE and gelatinases.

Influenza HA subtypes demonstrate divergent phenotypes for cleavage activation and pH of fusion: implications for host range and adaptation.

PubMed

Galloway, Summer E; Reed, Mark L; Russell, Charles J; Steinhauer, David A

2013-02-01

The influenza A virus (IAV) HA protein must be activated by host cells proteases in order to prime the molecule for fusion. Consequently, the availability of activating proteases and the susceptibility of HA to protease activity represents key factors in facilitating virus infection. As such, understanding the intricacies of HA cleavage by various proteases is necessary to derive insights into the emergence of pandemic viruses. To examine these properties, we generated a panel of HAs that are representative of the 16 HA subtypes that circulate in aquatic birds, as well as HAs representative of the subtypes that have infected the human population over the last century. We examined the susceptibility of the panel of HA proteins to trypsin, as well as human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2). Additionally, we examined the pH at which these HAs mediated membrane fusion, as this property is related to the stability of the HA molecule and influences the capacity of influenza viruses to remain infectious in natural environments. Our results show that cleavage efficiency can vary significantly for individual HAs, depending on the protease, and that some HA subtypes display stringent selectivity for specific proteases as activators of fusion function. Additionally, we found that the pH of fusion varies by 0.7 pH units among the subtypes, and notably, we observed that the pH of fusion for most HAs from human isolates was lower than that observed from avian isolates of the same subtype. Overall, these data provide the first broad-spectrum analysis of cleavage-activation and membrane fusion characteristics for all of the IAV HA subtypes, and also show that there are substantial differences between the subtypes that may influence transmission among hosts and establishment in new species.

Influenza HA Subtypes Demonstrate Divergent Phenotypes for Cleavage Activation and pH of Fusion: Implications for Host Range and Adaptation

PubMed Central

Galloway, Summer E.; Reed, Mark L.; Russell, Charles J.; Steinhauer, David A.

2013-01-01

The influenza A virus (IAV) HA protein must be activated by host cells proteases in order to prime the molecule for fusion. Consequently, the availability of activating proteases and the susceptibility of HA to protease activity represents key factors in facilitating virus infection. As such, understanding the intricacies of HA cleavage by various proteases is necessary to derive insights into the emergence of pandemic viruses. To examine these properties, we generated a panel of HAs that are representative of the 16 HA subtypes that circulate in aquatic birds, as well as HAs representative of the subtypes that have infected the human population over the last century. We examined the susceptibility of the panel of HA proteins to trypsin, as well as human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2). Additionally, we examined the pH at which these HAs mediated membrane fusion, as this property is related to the stability of the HA molecule and influences the capacity of influenza viruses to remain infectious in natural environments. Our results show that cleavage efficiency can vary significantly for individual HAs, depending on the protease, and that some HA subtypes display stringent selectivity for specific proteases as activators of fusion function. Additionally, we found that the pH of fusion varies by 0.7 pH units among the subtypes, and notably, we observed that the pH of fusion for most HAs from human isolates was lower than that observed from avian isolates of the same subtype. Overall, these data provide the first broad-spectrum analysis of cleavage-activation and membrane fusion characteristics for all of the IAV HA subtypes, and also show that there are substantial differences between the subtypes that may influence transmission among hosts and establishment in new species. PMID:23459660

Bovine Pancreatic Trypsin Inhibitor-Trypsin Complex as a Detection System for Recombinant Proteins

NASA Astrophysics Data System (ADS)

Borjigin, Jimo; Nathans, Jeremy

1993-01-01

Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10-14 and 1.1 x 10-13 M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.

Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis*

PubMed Central

Sendler, Matthias; Maertin, Sandrina; John, Daniel; Persike, Maria; Weiss, F. Ulrich; Krüger, Burkhard; Wartmann, Thomas; Wagh, Preshit; Halangk, Walter; Schaschke, Norbert; Mayerle, Julia; Lerch, Markus M.

2016-01-01

Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific. PMID:27226576

Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis.

PubMed

Sendler, Matthias; Maertin, Sandrina; John, Daniel; Persike, Maria; Weiss, F Ulrich; Krüger, Burkhard; Wartmann, Thomas; Wagh, Preshit; Halangk, Walter; Schaschke, Norbert; Mayerle, Julia; Lerch, Markus M

2016-07-08

Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis) 1

PubMed Central

Samac, Deborah; Storey, Richard

1981-01-01

Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling. Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination. PMID:16662104

Comprehensive Analysis of a Vibrio parahaemolyticus Strain Extracellular Serine Protease VpSP37

PubMed Central

Bennici, Carmelo; Quatrini, Paola; Catania, Valentina; Mazzola, Salvatore; Ghersi, Giulio; Cuttitta, Angela

2015-01-01

Proteases play an important role in the field of tissue dissociation combined with regenerative medicine. During the years new sources of proteolytic enzymes have been studied including proteases from different marine organisms both eukaryotic and prokaryotic. Herein we have purified a secreted component of an isolate of Vibrio parahaemolyticus, with electrophoretic mobilities corresponding to 36 kDa, belonging to the serine proteases family. Sequencing of the N-terminus enabled the in silico identification of the whole primary structure consisting of 345 amino acid residues with a calculated molecular mass of 37.4 KDa. The purified enzyme, named VpSP37, contains a Serine protease domain between residues 35 and 276 and a canonical Trypsin/Chimotrypsin 3D structure. Functional assays were performed to evaluate protease activity of purified enzyme. Additionally the performance of VpSP37 was evaluated in tissue dissociations experiments and the use of such enzyme as a component of enzyme blend for tissue dissociation procedures is strongly recommended. PMID:26162075

The SARS-Coronavirus papain-like protease: Structure, function and inhibition by designed antiviral compounds

PubMed Central

Baez-Santos, Yahira M.; St. John, Sarah E.; Mesecar, Andrew D.

2018-01-01

Over ten years have passed since the deadly human coronavirus that causes severe acute respiratory syndrome (SARS-CoV) emerged from the Guangdong Province of China. Despite the fact that the SARS-CoV pandemic infected over 8,500 individuals, claimed over 800 lives and cost billions of dollars in economic loss worldwide, there still are no clinically approved antiviral drugs, vaccines or monoclonal antibody therapies to treat SARS-CoV infections. The recent emergence of the deadly human coronavirus that causes Middle East respiratory syndrome (MERS-CoV) is a sobering reminder that new and deadly coronaviruses can emerge at any time with the potential to become pandemics. Therefore, the continued development of therapeutic and prophylactic countermeasures to potentially deadly coronaviruses is warranted. The coronaviral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), are attractive antiviral drug targets because they are essential for coronaviral replication. Although the primary function of PLpro and 3CLpro are to process the viral polyprotein in a coordinated manner, PLpro has the additional function of stripping ubiquitin and ISG15 from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. Therefore, targeting PLpro with antiviral drugs may have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that may lead to cell death in surrounding, uninfected cells. This review provides an up-to-date discussion on the SARS-CoV papain-like protease including a brief overview of the SARS-CoV genome and replication followed by a more in-depth discussion on the structure and catalytic mechanism of SARS-CoV PLpro, the multiple cellular functions of SARS-CoV PLpro, the inhibition of SARS-CoV PLpro by small molecule inhibitors, and the prospect of inhibiting papain-like protease from other coronaviruses. This paper forms part of a series

Characterization of a neutral protease from lysosomes of rabbit polymorphonuclear leucocytes

PubMed Central

Davies, Philip; Rita, Giuseppe A.; Krakauer, Kathrin; Weissmann, Gerald

1971-01-01

1. The subcellular distribution has been investigated of a protease from rabbit polymorphonuclear leucocytes, obtained from peritoneal exudates. The enzyme, optimally active between pH7.0 and 7.5, hydrolyses histone but not haemoglobin, sediments almost exclusively with a granule fraction rich in other lysosomal enzymes, and is latent until the granules are disrupted by various means. 2. Enzymic analysis of specific and azurophilic granules separated by zonal centrifugation showed that neutral protease activity was confined to fractions rich in enzymes characteristic of azurophile granules. 3. Recovery of neutral protease activity from subcellular fractions was several times greater than that found in whole cells. This finding was explained by the presence of a potent inhibitor of the enzyme activity in the cytoplasm. 4. The effect of the inhibitor was reversed by increasing ionic strength (up to 2.5m-potassium chloride) and by polyanions such as heparin and dextran sulphate, but not by an uncharged polymer, dextran. 5. The enzyme was also inhibited, to a lesser extent, by 1-chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one, soya-bean trypsin inhibitor and ∈-aminohexanoate (∈-aminocaproate). 6. The granule fractions failed to hydrolyse artificial substrates for trypsin and chymotrypsin. 7. Partial separation of the enzyme was achieved by Sephadex gel filtration at high ionic strength and by isoelectric focusing. The partially separated, activated enzyme showed an approximately 300-fold increase in specific activity over that in whole cells. PMID:5126908

Serine Proteases-Like Genes in the Asian Rice Gall Midge Show Differential Expression in Compatible and Incompatible Interactions with Rice

PubMed Central

Sinha, Deepak Kumar; Lakshmi, Mulagondla; Anuradha, Ghanta; Rahman, Shaik J.; Siddiq, Ebrahimali A.; Bentur, Jagadish S.; Nair, Suresh

2011-01-01

The Asian rice gall midge, Orseolia oryzae (Wood-Mason), is a serious pest of rice. Investigations into the gall midge-rice interaction will unveil the underlying molecular mechanisms which, in turn, can be used as a tool to assist in developing suitable integrated pest management strategies. The insect gut is known to be involved in various physiological and biological processes including digestion, detoxification and interaction with the host. We have cloned and identified two genes, OoprotI and OoprotII, homologous to serine proteases with the conserved His87, Asp136 and Ser241 residues. OoProtI shared 52.26% identity with mosquito-type trypsin from Hessian fly whereas OoProtII showed 52.49% identity to complement component activated C1s from the Hessian fly. Quantitative real time PCR analysis revealed that both the genes were significantly upregulated in larvae feeding on resistant cultivar than in those feeding on susceptible cultivar. These results provide an opportunity to understand the gut physiology of the insect under compatible or incompatible interactions with the host. Phylogenetic analysis grouped these genes in the clade containing proteases of phytophagous insects away from hematophagous insects. PMID:21686154

Plant cysteine proteases that evoke itch activate protease-activated receptors

PubMed Central

Reddy, V.B.; Lerner, E.A.

2013-01-01

Background Bromelain, ficin and papain are cysteine proteases from plants that produce itch upon injection into skin. Their mechanism of action has not been considered previously. Objectives To determine the mechanism by which these proteases function. Methods The ability of these proteases to activate protease-activated receptors was determined by ratiometric calcium imaging. Results We show here that bromelain, ficin and papain activate protease-activated receptors 2 and 4. Conclusions Bromelain, ficin and papain function as signalling molecules and activate protease-activated receptors. Activation of these receptors is the likely mechanism by which these proteases evoke itch. PMID:20491769

Cloning, expression and characterisation of an HtrA-like serine protease produced in vivo by Mycobacterium leprae.

PubMed

Ribeiro-Guimarães, Michelle Lopes; Marengo, Eliana Blini; Tempone, Antonio Jorge; Amaral, Julio Jablonski; Klitzke, Clécio F; Silveira, Erika K Xavier da; Portaro, Fernanda Calheta Vieira; Pessolani, Maria Cristina Vidal

2009-12-01

Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40 degrees C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.

Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates.

PubMed

Askin, Samuel P; Morin, Isabelle; Schaeffer, Patrick M

2011-08-15

Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus-GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus-GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader. Copyright © 2011 Elsevier Inc. All rights reserved.

Using Trypsin & Soybean Trypsin Inhibitor to Teach Principles of Enzyme Kinetics

ERIC Educational Resources Information Center

Howard, David R.; Herr, Julie; Hollister, Rhiannon

2006-01-01

Trypsin and soybean trypsin inhibitor (Kunitz inhibitor) can be used in a relatively simple and inexpensive student exercise to demonstrate the usefulness of enzyme kinetics. The study of enzyme kinetics is essential to biology because enzymes play such a crucial role in the biochemical pathways of all living organisms. The data from enzyme…

Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom.

PubMed

He, Ying-Ying; Liu, Shu-Bai; Lee, Wen-Hui; Qian, Jin-Qiao; Zhang, Yun

2008-10-01

Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid residues with a molecular mass of 6339Da. Successful expression of OH-TCI was performed as the maltose-binding fusion protein in E. coli DH5alpha. Much different from Oh11-1, the purified native and recombinant OH-TCI both had strong inhibitory activities against trypsin and chymotrypsin although the sequence identity (74.1%) between them is very high. The inhibitor constants (K(i)) of recombinant OH-TCI were 3.91 x 10(-7) and 8.46 x10(-8)M for trypsin and chymotrypsin, respectively. To our knowledge, it was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom that had equivalent trypsin and chymotrypsin inhibitory activities.

Two variants of the major serine protease inhibitor from the sea anemone Stichodactyla helianthus, expressed in Pichia pastoris.

PubMed

García-Fernández, Rossana; Ziegelmüller, Patrick; González, Lidice; Mansur, Manuel; Machado, Yoan; Redecke, Lars; Hahn, Ulrich; Betzel, Christian; Chávez, María de Los Ángeles

2016-07-01

The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of bisphenol S on the structures and activities of trypsin and pepsin.

PubMed

Wang, Yan-Qing; Zhang, Hong-Mei

2014-11-19

The effects of bisphenol S on the structures and activities of trypsin and pepsin were investigated by various methods like UV-visible absorbance, fluorescence, circular dichroism, and molecular docking. The secondary and tertiary structures of trypsin and pepsin were altered by bisphenol S binding, which resulted in the loosening of the skeletons of trypsin and pepsin. In addition, bisphenol S induced microenvironmental changes around tyrosine and tryptophan residues of trypsin and pepsin. The activity experimental results showed that the activity of pepsin decreases obviously with the increasing concentration of BPS, while the activity of trypsin does not change remarkably. The binding and thermodynamic parameters obtained by molecular docking and fluorescence spectroscopy showed that the bindings of bisphenol S to trypsin and pepsin were spontaneous processes and hydrogen bonding and hydrophobic interactions played a vital role in stabilizing the bisphenol S-trypsin and bisphenol S-pepsin complexes. The binding constants (K(A)) of bisphenol S with trypsin were 7.42 × 10(4) (298 K) and 5.91 × 10(4) L/mol (310 K), and those of pepsin were 5.78 × 10(4) (298 K) and 4.44 × 10(4) L/mol (310 K). Moreover, there was one main kind of binding site for bisphenol S on trypsin or pepsin.

Ethanol enhances carbachol-induced protease activation and accelerates Ca2+ waves in isolated rat pancreatic acini.

PubMed

Orabi, Abrahim I; Shah, Ahsan U; Muili, Kamaldeen; Luo, Yuhuan; Mahmood, Syeda Maham; Ahmad, Asim; Reed, Anamika; Husain, Sohail Z

2011-04-22

Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute cases and 70-90% of chronic cases, yet the mechanisms leading to alcohol-associated pancreatic injury are unclear. An early and critical feature of pancreatitis is the aberrant signaling of Ca(2+) within the pancreatic acinar cell. An important conductor of this Ca(2+) is the basolaterally localized, intracellular Ca(2+) channel ryanodine receptor (RYR). In this study, we examined the effect of ethanol on mediating both pathologic intra-acinar protease activation, a precursor to pancreatitis, as well as RYR Ca(2+) signals. We hypothesized that ethanol sensitizes the acinar cell to protease activation by modulating RYR Ca(2+). Acinar cells were freshly isolated from rat, pretreated with ethanol, and stimulated with the muscarinic agonist carbachol (1 μM). Ethanol caused a doubling in the carbachol-induced activation of the proteases trypsin and chymotrypsin (p < 0.02). The RYR inhibitor dantrolene abrogated the enhancement of trypsin and chymotrypsin activity by ethanol (p < 0.005 for both proteases). Further, ethanol accelerated the speed of the apical to basolateral Ca(2+) wave from 9 to 18 μm/s (p < 0.0005; n = 18-22 cells/group); an increase in Ca(2+) wave speed was also observed with a change from physiologic concentrations of carbachol (1 μM) to a supraphysiologic concentration (1 mM) that leads to protease activation. Dantrolene abrogated the ethanol-induced acceleration of wave speed (p < 0.05; n = 10-16 cells/group). Our results suggest that the enhancement of pathologic protease activation by ethanol is dependent on the RYR and that a novel mechanism for this enhancement may involve RYR-mediated acceleration of Ca(2+) waves.

Ethanol Enhances Carbachol-induced Protease Activation and Accelerates Ca2+ Waves in Isolated Rat Pancreatic Acini*

PubMed Central

Orabi, Abrahim I.; Shah, Ahsan U.; Muili, Kamaldeen; Luo, Yuhuan; Mahmood, Syeda Maham; Ahmad, Asim; Reed, Anamika; Husain, Sohail Z.

2011-01-01

Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute cases and 70–90% of chronic cases, yet the mechanisms leading to alcohol-associated pancreatic injury are unclear. An early and critical feature of pancreatitis is the aberrant signaling of Ca2+ within the pancreatic acinar cell. An important conductor of this Ca2+ is the basolaterally localized, intracellular Ca2+ channel ryanodine receptor (RYR). In this study, we examined the effect of ethanol on mediating both pathologic intra-acinar protease activation, a precursor to pancreatitis, as well as RYR Ca2+ signals. We hypothesized that ethanol sensitizes the acinar cell to protease activation by modulating RYR Ca2+. Acinar cells were freshly isolated from rat, pretreated with ethanol, and stimulated with the muscarinic agonist carbachol (1 μm). Ethanol caused a doubling in the carbachol-induced activation of the proteases trypsin and chymotrypsin (p < 0.02). The RYR inhibitor dantrolene abrogated the enhancement of trypsin and chymotrypsin activity by ethanol (p < 0.005 for both proteases). Further, ethanol accelerated the speed of the apical to basolateral Ca2+ wave from 9 to 18 μm/s (p < 0.0005; n = 18–22 cells/group); an increase in Ca2+ wave speed was also observed with a change from physiologic concentrations of carbachol (1 μm) to a supraphysiologic concentration (1 mm) that leads to protease activation. Dantrolene abrogated the ethanol-induced acceleration of wave speed (p < 0.05; n = 10–16 cells/group). Our results suggest that the enhancement of pathologic protease activation by ethanol is dependent on the RYR and that a novel mechanism for this enhancement may involve RYR-mediated acceleration of Ca2+ waves. PMID:21372126

Application and bioactive properties of CaTI, a trypsin inhibitor from Capsicum annuum seeds: membrane permeabilization, oxidative stress and intracellular target in phytopathogenic fungi cells.

PubMed

Silva, Marciele S; Ribeiro, Suzanna Ff; Taveira, Gabriel B; Rodrigues, Rosana; Fernandes, Katia Vs; Carvalho, André O; Vasconcelos, Ilka Maria; Mello, Erica Oliveira; Gomes, Valdirene M

2017-08-01

During the last few years, a growing number of antimicrobial peptides have been isolated from plants and particularly from seeds. Recent results from our laboratory have shown the purification of a new trypsin inhibitor, named CaTI, from chilli pepper (Capsicum annuum L.) seeds. This study aims to evaluate the antifungal activity and mechanism of action of CaTI on phytopathogenic fungi and detect the presence of protease inhibitors in other species of this genus. Our results show that CaTI can inhibit the growth of the phytopathogenic fungi Colletotrichum gloeosporioides and C. lindemuthianum. CaTI can also permeabilize the membrane of all tested fungi. When testing the inhibitor on its ability to induce reactive oxygen species, an induction of reactive oxygen species (ROS) and nitric oxide (NO) particularly in Fusarium species was observed. Using CaTI coupled to fluorescein isothiocyanate (FITC), it was possible to determine the presence of the inhibitor inside the hyphae of the Fusarium oxysporum fungus. The search for protease inhibitors in other Capsicum species revealed their presence in all tested species. This paper shows the antifungal activity of protease inhibitors such as CaTI against phytopathogenic fungi. Antimicrobial peptides, among which the trypsin protease inhibitor family stands out, are present in different species of the genus Capsicum and are part of the chemical arsenal that plants use to defend themselves against pathogens. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds.

PubMed

Báez-Santos, Yahira M; St John, Sarah E; Mesecar, Andrew D

2015-03-01

Over 10 years have passed since the deadly human coronavirus that causes severe acute respiratory syndrome (SARS-CoV) emerged from the Guangdong Province of China. Despite the fact that the SARS-CoV pandemic infected over 8500 individuals, claimed over 800 lives and cost billions of dollars in economic loss worldwide, there still are no clinically approved antiviral drugs, vaccines or monoclonal antibody therapies to treat SARS-CoV infections. The recent emergence of the deadly human coronavirus that causes Middle East respiratory syndrome (MERS-CoV) is a sobering reminder that new and deadly coronaviruses can emerge at any time with the potential to become pandemics. Therefore, the continued development of therapeutic and prophylactic countermeasures to potentially deadly coronaviruses is warranted. The coronaviral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), are attractive antiviral drug targets because they are essential for coronaviral replication. Although the primary function of PLpro and 3CLpro are to process the viral polyprotein in a coordinated manner, PLpro has the additional function of stripping ubiquitin and ISG15 from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. Therefore, targeting PLpro with antiviral drugs may have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that may lead to cell death in surrounding, uninfected cells. This review provides an up-to-date discussion on the SARS-CoV papain-like protease including a brief overview of the SARS-CoV genome and replication followed by a more in-depth discussion on the structure and catalytic mechanism of SARS-CoV PLpro, the multiple cellular functions of SARS-CoV PLpro, the inhibition of SARS-CoV PLpro by small molecule inhibitors, and the prospect of inhibiting papain-like protease from other coronaviruses. This paper forms part of a series of

Zebra chip disease decreases tuber (Solanum tuberosum L.) protein content by attenuating protease inhibitor levels and increasing protease activities.

PubMed

Kumar, G N Mohan; Knowles, Lisa O; Knowles, N Richard

2015-11-01

Zebra chip disease of potato decreases protease inhibitor levels resulting in enhanced serine-type protease activity, decreased protein content and altered protein profiles of fully mature tubers. Zebra-chip (ZC), caused by Candidatus Liberibacter solanacearum (CLso), is a relatively new disease of potato that negatively affects growth, yield, propagation potential, and fresh and process qualities of tubers. Diseased plants produce tubers with characteristic brown discoloration of vascular tissue accompanied by elevated levels of free amino acids and reducing sugars. Here we demonstrate that ZC disease induces selective protein catabolism in tubers through modulating protease inhibitor levels. Soluble protein content of tubers from CLso-infected plants was 33% lower than from non-infected plants and electrophoretic analyses revealed substantial reductions in major tuber proteins. Patatin (~40 kDa) and ser-, asp- (22 kDa) and cys-type (85 kDa) protease inhibitors were either absent or greatly reduced in ZC-afflicted tubers. In contrast to healthy (non-infected) tubers, the proteolytic activity in CLso infected tubers was high and the ability of extracts from infected tubers to inhibit trypsin (ser-type) and papain (cys-type) proteases greatly attenuated. Moreover, extracts from CLso-infected tubers rapidly catabolized proteins purified from healthy tubers (40 kDa patatin, 22 kDa protease inhibitors, 85 kDa potato multicystatin) when subjected to proteolysis individually. In contrast, crude extracts from non-infected tubers effectively inhibited the proteolytic activity from ZC-afflicted tubers. These results suggest that the altered protein profile of ZC afflicted tubers is largely due to loss of ser- and cys-type protease inhibitors. Further analysis revealed a novel PMSF-sensitive (ser) protease (ca. 80-120 kDa) in CLso infected tubers. PMSF abolished the proteolytic activities responsible for degrading patatin, the 22 kDa protease inhibitor(s) and potato

Characterization of Bactrocera dorsalis Serine Proteases and Evidence for Their Indirect Role in Insecticide Tolerance

PubMed Central

Hou, Ming-Zhe; Shen, Guang-Mao; Wei, Dong; Li, Ya-Li; Dou, Wei; Wang, Jin-Jun

2014-01-01

The oriental fruit fly Bactrocera dorsalis (Hendel) causes devastating losses to agricultural crops world-wide and is considered to be an economically important pest. Little is known about the digestive enzymes such as serine proteases (SPs) in B. dorsalis, which are important both for energy supply and mitigation of fitness cost associated with insecticide tolerance. In this study, we identified five SP genes in the midgut of B. dorsalis, and the alignments of their deduced amino acid sequences revealed the presence of motifs conserved in the SP superfamily. Phylogenetic analyses with known SPs from other insect species suggested that three of them were trypsin-like proteases. Analyses of the expression profiles among the different developmental stages showed that all five genes were most abundant in larvae than in other stages. When larvae were continuously fed on diet containing 0.33 μg/g β-Cypermethrin, expression of all five genes were upregulated in the midgut but the larval development was delayed. Biochemical assays were consistent with the increased protease activity exhibited by SPs in the midgut after treatment with β-Cypermethrin. Taken together, these findings provide evidence for the hypothesis that enhanced SP activity may play an indirect role in relieving the toxicity stress of insecticide in B. dorsalis. PMID:24566149

The modifier effects of chymotrypsin and trypsin enzymes on fluorescence lifetime distribution of "N-(1-pyrenyl)maleimide-bovine serum albumin" complex

NASA Astrophysics Data System (ADS)

Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder

2016-02-01

Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases.

A colostrum trypsin inhibitor gene expressed in the Cape fur seal mammary gland during lactation.

PubMed

Pharo, Elizabeth A; Cane, Kylie N; McCoey, Julia; Buckle, Ashley M; Oosthuizen, W H; Guinet, Christophe; Arnould, John P Y

2016-03-01

The colostrum trypsin inhibitor (CTI) gene and transcript were cloned from the Cape fur seal mammary gland and CTI identified by in silico analysis of the Pacific walrus and polar bear genomes (Order Carnivora), and in marine and terrestrial mammals of the Orders Cetartiodactyla (yak, whales, camel) and Perissodactyla (white rhinoceros). Unexpectedly, Weddell seal CTI was predicted to be a pseudogene. Cape fur seal CTI was expressed in the mammary gland of a pregnant multiparous seal, but not in a seal in its first pregnancy. While bovine CTI is expressed for 24-48 h postpartum (pp) and secreted in colostrum only, Cape fur seal CTI was detected for at least 2-3 months pp while the mother was suckling its young on-shore. Furthermore, CTI was expressed in the mammary gland of only one of the lactating seals that was foraging at-sea. The expression of β-casein (CSN2) and β-lactoglobulin II (LGB2), but not CTI in the second lactating seal foraging at-sea suggested that CTI may be intermittently expressed during lactation. Cape fur seal and walrus CTI encode putative small, secreted, N-glycosylated proteins with a single Kunitz/bovine pancreatic trypsin inhibitor (BPTI) domain indicative of serine protease inhibition. Mature Cape fur seal CTI shares 92% sequence identity with Pacific walrus CTI, but only 35% identity with BPTI. Structural homology modelling of Cape fur seal CTI and Pacific walrus trypsin based on the model of the second Kunitz domain of human tissue factor pathway inhibitor (TFPI) and porcine trypsin (Protein Data Bank: 1TFX) confirmed that CTI inhibits trypsin in a canonical fashion. Therefore, pinniped CTI may be critical for preventing the proteolytic degradation of immunoglobulins that are passively transferred from mother to young via colostrum and milk. Copyright © 2015 Elsevier B.V. All rights reserved.

Quercetin suppresses the chymotrypsin-like activity of proteasome via inhibition of MEK1/ERK1/2 signaling pathway in hepatocellular carcinoma HepG2 cells.

PubMed

Ding, Youming; Chen, Xiaoyan; Wang, Bin; Yu, Bin; Ge, Jianhui; Shi, Xiaokang

2018-05-01

The proteasomal system is a promising target for cancer treatment. Quercetin (Que), a flavonoid compound with antitumor ability, displays the inhibitory effect on proteasome activity. However, the underlying molecular mechanisms are ill defined. The present study found that Que treatment significantly reduced the chymotrypsin-like protease activity of proteasome whereas the trypsin- and caspase-like protease activities remained unchanged in HepG2 cancer cells, along with activation of p38 MAPK and JNK and reduction of ERK1/2 phosphorylation. Que-reduced proteasome activity could not be reverted by inhibition of p38 MAPK and JNK signaling pathway. In addition, MEK1 overexpression or knockdown upregulated or downregulated the chymotrypsin-like protease activity of proteasome, respectively. Both Que and MEK1/ERK1/2 inhibitor attenuated the expression levels of proteasome β subunits. These results indicate that Que-induced suppression of MEK1/ERK1/2 signaling and subsequent reduction of proteasome β subunits is responsible for its inhibitory impacts on proteasome activity.

Cloning of the cDNA encoding Scg-SPRP, an unusual Ser-protease-related protein from vitellogenic female desert locusts (Schistocerca gregaria).

PubMed

Chiou, S J; Vanden Broeck, J; Janssen, I; Borovsky, D; Vandenbussche, F; Simonet, G; De Loof, A

1998-10-01

The cDNA coding for a Ser-protease-related protein (Scg-SPRP) was cloned from desert locust (Schistocerca gregaria) midgut. The derived amino acid sequence consists of 260 residues and shows strong sequence similarity to insect trypsin-like molecules. It is, however, likely that Scg-SPRP is not a proteolytically active enzyme and that it plays another physiologically relevant role, since two out of three residues which are indispensable for catalytic activity of Ser-proteases are replaced. Northern analysis revealed that the Scg-SPRP gene is expressed in midgut tissue and that this expression is strongly induced in adult female locusts. Moreover, the occurrence of the transcript (1.2 kb) fluctuates during the molting cycle and during the female reproductive cycle. Juvenile hormone (JH III) dependence of transcription was investigated by chemical allatectomy (precocene I) of adult females. This resulted in inhibition of vitellogenesis and in disappearance of the Scg-SPRP transcript. Expression of Scg-SPRP in precocene-treated locusts could be reinduced by additional treatment with JH III or with 20-OH-ecdysone.

Human eosinophils constitutively express a unique serine protease, PRSS33.

PubMed

Toyama, Sumika; Okada, Naoko; Matsuda, Akio; Morita, Hideaki; Saito, Hirohisa; Fujisawa, Takao; Nakae, Susumu; Karasuyama, Hajime; Matsumoto, Kenji

2017-07-01

Eosinophils play important roles in asthma, especially airway remodeling, by producing various granule proteins, chemical mediators, cytokines, chemokines and proteases. However, protease production by eosinophils is not fully understood. In the present study, we investigated the production of eosinophil-specific proteases/proteinases by transcriptome analysis. Human eosinophils and other cells were purified from peripheral blood by density gradient sedimentation and negative/positive selections using immunomagnetic beads. Protease/proteinase expression in eosinophils and release into the supernatant were evaluated by microarray analysis, qPCR, ELISA, flow cytometry and immunofluorescence staining before and after stimulation with eosinophil-activating cytokines and secretagogues. mRNAs for extracellular matrix proteins in human normal fibroblasts were measured by qPCR after exposure to recombinant protease serine 33 (PRSS33) protein (rPRSS33), created with a baculovirus system. Human eosinophils expressed relatively high levels of mRNA for metalloproteinase 25 (MMP25), a disintegrin and metalloprotease 8 (ADAM8), ADAM10, ADAM19 and PRSS33. Expression of PRSS33 was the highest and eosinophil-specific. PRSS33 mRNA expression was not affected by eosinophil-activating cytokines. Immunofluorescence staining showed that PRSS33 was co-localized with an eosinophil granule protein. PRSS33 was not detected in the culture supernatant of eosinophils even after stimulation with secretagogues, but its cell surface expression was increased. rPRSS33 stimulation of human fibroblasts increased expression of collagen and fibronectin mRNAs, at least in part via protease-activated receptor-2 activation. Activated eosinophils may induce fibroblast extracellular matrix protein synthesis via cell surface expression of PRSS33, which would at least partly explain eosinophils' role(s) in airway remodeling. Copyright © 2017 Japanese Society of Allergology. Production and hosting by Elsevier

Trypsin from the processing waste of the lane snapper (Lutjanus synagris) and its compatibility with oxidants, surfactants and commercial detergents.

PubMed

Espósito, Talita S; Marcuschi, Marina; Amaral, Ian P G; Carvalho, Luiz B; Bezerra, Ranilson S

2010-05-26

A trypsin from the viscera of the lane snapper (Lutjanus synagris) was purified by heat treatment, fractionation with ammonium sulfate and affinity chromatography. The molecular weight of the enzyme was estimated to be 28.4 kDa (SDS-PAGE). The purified enzyme was capable of hydrolyzing the specific substrate for trypsin benzoyl-arginine-p-nitroanilide (BApNA) and was inhibited by benzamidine and tosyl lysine chloromethyl ketone (TLCK), synthetic trypsin inhibitors and phenylmethylsulfonyl fluoride (PMSF), which is a serine-protease inhibitor. The enzyme exhibited maximal activity at pH 9.0 and 45 degrees C and retained 100% of the activity after incubation at the optimal temperature for 30 min. At a concentration of 10 mM, activity was slightly activated by Ca(2+) and inhibited by the following ions in decreasing order: Cd(2+) > Hg(2+) > Cu(2+) > Zn(2+) > Al(3+). The effects of Ba(2+), K(1+) and Li(1+) proved to be less intensive. Using 1% (w/v) azocasein as substrate, the enzyme revealed high resistance (60% residual activity) when incubated with 10% H(2)O(2) for 75 min. The enzyme retained more than 80% activity after 60 min in the presence of different surfactants (Tween 20, Tween 80 and sodium choleate). The alkaline protease demonstrated compatibility with commercial detergents (7 mg/mL), such as Bem-te-vi, Surf and Ala, retaining more than 50% of initial activity after 60 min at 25 degrees C and 30 min at 40 degrees C. The thermostability and compatibility of this enzyme with commercial detergents suggest a good potentiality for application in the detergent industry.

Molecular and immunological characterization of subtilisin like serine protease, a major allergen of Curvularia lunata.

PubMed

Tripathi, Prabhanshu; Nair, Smitha; Singh, B P; Arora, Naveen

2011-03-01

Serine protease from numerous sources have been identified and characterized as major allergens. The present study aimed to clone, express and characterize a serine protease from Curvularia lunata. cDNA library screening identified partial protease clones. A clone showed significant homology to subtilisin like serine proteases from Aspergillus and Penicillium species. Full length sequence was generated by RACE PCR, subcloned in pET vector, protein expressed in Escherichia coli and purified from inclusion bodies yielding 0.5 mg/L of culture. Bioinformatic analysis identified serine protease motifs of subtilase family, catalytic triad and N-glycosylation sites on the primary sequence. The protein resolved at 54-kDa on SDS-PAGE and was recognized as a major allergen on immunoblot with 13/16 C. lunata sensitive patients' sera in ELISA and immunoblot. Recombinant protein reacted with rabbit polyclonal antibodies against alkaline serine proteases from C. lunata. Recombinant protein required 50-56 ng of same protein for 50% inhibition of IgE binding in competitive ELISA. In addition, 13 of 16 patients' samples showed significant basophil histamine release upon stimulation with purified recombinant protein. In conclusion, a 54 kDa major allergen of C. lunata was cloned, expressed, characterized and showed biological activity. It has potential to be used in molecule based approach for allergy diagnosis and therapy. Copyright © 2010 Elsevier GmbH. All rights reserved.

Differential suppression of upper airway motor activity during carbachol-induced, REM sleep-like atonia.

PubMed

Fenik, V; Davies, R O; Pack, A I; Kubin, L

1998-10-01

Microinjections of carbachol into the pontine tegmentum of decerebrate cats have been used to study the mechanisms underlying the suppression of postural and respiratory motoneuronal activity during the resulting rapid eye movement (REM) sleep-like atonia. During REM sleep, distinct respiratory muscles are differentially affected; e.g., the activity of the diaphragm shows little suppression, whereas the activity of some upper airway muscles is quite strong. To determine the pattern of the carbachol-induced changes in the activity of different groups of upper airway motoneurons, we simultaneously recorded the efferent activity of the recurrent laryngeal nerve (RL), pharyngeal branch of the vagus nerve (Phar), and genioglossal branch of the hypoglossal (XII) and phrenic (Phr) nerves in 12 decerebrate, paralyzed, vagotomized, and artificially ventilated cats. Pontine carbachol caused a stereotyped suppression of the spontaneous activity that was significantly larger in Phar expiratory (to 8.3% of control) and XII inspiratory motoneurons (to 15%) than in Phr inspiratory (to 87%), RL inspiratory (to 79%), or RL expiratory motoneurons (to 72%). The suppression in upper airway motor output was significantly greater than the depression caused by a level of hypocapnia that reduced Phr activity as much as carbachol. We conclude that pontine carbachol evokes a stereotyped pattern of suppression of upper airway motor activity. Because carbachol evokes a state having many neurophysiological characteristics similar to those of REM sleep, it is likely that pontine cholinoceptive neurons have similar effects on the activity of upper airway motoneurons during both states.

The active contribution of Toll-like receptors to allergic airway inflammation.

PubMed

Chen, Keqiang; Xiang, Yi; Yao, Xiaohong; Liu, Ying; Gong, Wanghua; Yoshimura, Teizo; Wang, Ji Ming

2011-10-01

Epithelia lining the respiratory tract represent a major portal of entry for microorganisms and allergens and are equipped with innate and adaptive immune signaling receptors for host protection. These include Toll-like receptors (TLRs) that recognize microbial components and evoke diverse responses in cells of the respiratory system. TLR stimulation by microorganism-derived molecules activates antigen presenting cells, control T helper (Th) 1, Th2, and Th17 immune cell differentiation, cytokine production by mast cells, and activation of eosinophils. It is clear that TLR are involved in the pathophysiology of allergic airway diseases such as asthma. Dendritic cells (DCs), a kind of antigen presenting cells, which play a key role in the induction of allergic airway inflammation, are privileged targets for pathogen associated molecular patterns (PAMPs). During the allergic responses, engagement of TLRs on DCs determines the Th2 polarization of the T cells. TLR signaling in mast cells increases the release of IL-5, and TLR activation of airway epithelial cells forces the generation of proallergic Th2 type of cytokines. Although these responses aim to protect the host, they may also result in inflammatory tissue damage in the airway. Under certain conditions, stimulation of TLRs, in particular, TLR9, may reduce Th2-dependent allergic inflammation by induction of Th1 responses. Therefore, understanding the complex regulatory roles of TLRs in the pathogenesis of allergic airway inflammation should facilitate the development of preventive and therapeutic measures for asthmatic patients. Copyright © 2011 Elsevier B.V. All rights reserved.

Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans.

PubMed

Pannkuk, Evan L; Risch, Thomas S; Savary, Brett J

2015-01-01

White nose syndrome (WNS) is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1) was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE), broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction.

Isolation and Identification of an Extracellular Subtilisin-Like Serine Protease Secreted by the Bat Pathogen Pseudogymnoascus destructans

PubMed Central

Pannkuk, Evan L.; Risch, Thomas S.; Savary, Brett J.

2015-01-01

White nose syndrome (WNS) is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1) was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE), broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction. PMID:25785714

The modifier effects of chymotrypsin and trypsin enzymes on fluorescence lifetime distribution of "N-(1-pyrenyl)maleimide-bovine serum albumin" complex.

PubMed

Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder

2016-02-05

Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases. Copyright © 2015 Elsevier B.V. All rights reserved.

An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis*

PubMed Central

Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F.; Cohen, Itay; Henin, Rachel D.; Hockla, Alexandra; Soares, Alexei S.; Papo, Niv; Caulfield, Thomas R.; Radisky, Evette S.

2016-01-01

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis. PMID:27810896

The expression and activation of protease-activated receptor-2 correlate with skin color.

PubMed

Babiarz-Magee, Laura; Chen, Nannan; Seiberg, Miri; Lin, Connie B

2004-06-01

Skin color results from the production and distribution of melanin in the epidermis. The protease-activated receptor-2 (PAR-2), expressed on keratinocytes but not on melanocytes, is involved in melanosome uptake via phagocytosis, and modulation of PAR-2 activation affects skin color. The pattern of melanosome distribution within the epidermis is skin color-dependent. In vitro, this distribution pattern is regulated by the ethnic origin of the keratinocytes, not the melanocytes. Therefore, we hypothesized that PAR-2 may play a role in the modulation of pigmentation in a skin type-dependent manner. We examined the expression of PAR-2 and its activator, trypsin, in human skins with different pigmentary levels. Here we show that PAR-2 and trypsin are expressed in higher levels, and are differentially localized in highly pigmented, relative to lightly pigmented skins. Moreover, highly pigmented skins exhibit an increase in PAR-2-specific protease cleavage ability. Microsphere phagocytosis was more efficient in keratinocytes from highly pigmented skins, and PAR-2 induced phagocytosis resulted in more efficient microsphere ingestion and more compacted microsphere organization in dark skin-derived keratinocytes. These results demonstrate that PAR-2 expression and activity correlate with skin color, suggesting the involvement of PAR-2 in ethnic skin color phenotypes.

Bio-potency of a 21 kDa Kunitz-type trypsin inhibitor from Tamarindus indica seeds on the developmental physiology of H. armigera.

PubMed

Pandey, Prabhash K; Jamal, Farrukh

2014-11-01

A trypsin inhibitor purified from the seeds of Tamarindus indica by Sephadex G-75, DEAE-Sepharose and Trypsin-Sepharose CL-4B columns was studied for its antifeedant, larvicidal, pupicidal and growth inhibitory activities against Helicoverpa armigera larvae. Tamarindus trypsin inhibitor (TTI) exhibited inhibitory activity towards total gut proteolytic enzymes of H. armigera (~87%) and bovine trypsin (~84%). Lethal doses which caused mortality and weight reduction by 50% were 1% w/w and 0.50% w/w, respectively. IC50 of TTI against Helicoverpa midgut proteases and bovine trypsin were ~2.10 µg/ml and 1.68 µg/ml respectively. In larval feeding studies the 21 kDa Kunitz-type protein was found to retard growth and development, prolonged the larval-pupal development durations along with adversely affecting the fertility and fecundity of H. armigera. In artificial diet at 0.5% w/w TTI, the efficiency of conversion of ingested food as well as of digested food, relative growth rate, growth index declined whereas approximate digestibility, metabolic cost, relative consumption rate, consumption index and total developmental period enhanced for H. armigera larvae. These results suggest that TTI has toxic and adverse effect on the developmental physiology of H. armigera and could be useful in controlling the pest H. armigera. Copyright © 2014 Elsevier Inc. All rights reserved.

Protease inhibitor from Moringa oleifera with potential for use as therapeutic drug and as seafood preservative

PubMed Central

Bijina, B.; Chellappan, Sreeja; Krishna, Jissa G.; Basheer, Soorej M.; Elyas, K.K.; Bahkali, Ali H.; Chandrasekaran, M.

2011-01-01

Protease inhibitors are well known to have several applications in medicine and biotechnology. Several plant sources are known to return potential protease inhibitors. In this study plants belonging to different families of Leguminosae, Malvaceae, Rutaceae, Graminae and Moringaceae were screened for the protease inhibitor. Among them Moringa oleifera, belonging to the family Moringaceae, recorded high level of protease inhibitor activity after ammonium sulfate fractionation. M. oleifera, which grows throughout most of the tropics and having several industrial and medicinal uses, was selected as a source of protease inhibitor since so far no reports were made on isolation of the protease inhibitor. Among the different parts of M. oleifera tested, the crude extract isolated from the mature leaves and seeds showed the highest level of inhibition against trypsin. Among the various extraction media evaluated, the crude extract prepared in phosphate buffer showed maximum recovery of the protease inhibitor. The protease inhibitor recorded high inhibitory activity toward the serine proteases thrombin, elastase, chymotrypsin and the cysteine proteases cathepsin B and papain which have more importance in pharmaceutical industry. The protease inhibitor also showed complete inhibition of activities of the commercially available proteases of Bacillus licheniformis and Aspergillus oryzae. However, inhibitory activities toward subtilisin, esperase, pronase E and proteinase K were negligible. Further, it was found that the protease inhibitor could prevent proteolysis in a commercially valuable shrimp Penaeus monodon during storage indicating the scope for its application as a seafood preservative. This is the first report on isolation of a protease inhibitor from M. oleifera. PMID:23961135

Amino acid sequence of a trypsin inhibitor from a Spirometra (Spirometra erinaceieuropaei).

PubMed

Sanda, A; Uchida, A; Itagaki, T; Kobayashi, H; Inokuchi, N; Koyama, T; Iwama, M; Ohgi, K; Irie, M

2001-12-01

A trypsin inhibitor that is highly homologous with bovine pancreatic trypsin inhibitor (BPTI) was co-purified along with RNase from Spirometra (Spirometra erinaceieuropaei). The amino acid sequence of this inhibitor (SETI) and the nucleotide sequence of the cDNA encoding this protein were determined by protein chemistry and gene technology. SETI contains 68 amino acid residues and has a molecular mass of 7,798 Da. SETI has 31 amino acid residues that are identical with BPTI's sequence, including 6 half-cystine and 5 aromatic amino acid residues. The active site Lys residue in BPTI is replaced by an Arg residue in SETI. SETI is an effective inhibitor of trypsin and moderately inhibits a-chymotrypsin, but less inhibits elastase or subtilisin. SETI was expressed by E. coli containing a PelB vector carrying the SETI encoding cDNA; an expression yield of 0.68 mg/l was obtained. The phylogenetic relationship of SETI and the other BPTI-like trypsin inhibitors was analyzed using most likelihood inference methods.

Glycosylation analysis of recombinant neutral protease I from Aspergillus oryzae expressed in Pichia pastoris.

PubMed

Lei, Da; Xu, Yang; He, Qinghua; Pang, Yifeng; Chen, Bo; Xiong, Liang; Li, Yanping

2013-12-01

Neutral protease I from Aspergillus oryzae 3.042 was expressed in Pichia pastoris and its N-glycosylation properties were analyzed. After purification by nickel-affinity chromatography column, the recombinant neutral protease (rNPI) was confirmed to be N-glycosylated by periodicacid/Schiff's base staining and Endo H digestion. Moreover, the deglycosylated protein's molecular weight decreased to 43.3 kDa from 54.5 kDa analyzed by SDS-PAGE and MALDI-TOF-MS, and the hyperglycosylation extent was 21 %. The N-glycosylation site of rNPI was analyzed by nano LC-MS/MS after digesting by trypsin and Glu-C, and the unique potential site Asn41 of mature peptide was found to be glycosylated. Homology modeling of the 3D structure of rNPI indicated that the attached N-glycans hardly affected neutral protease's activity due to the great distance away from the active site of the enzyme.

Cloning, expression and decoding of the cold adaptation of a new widely represented thermolabile subtilisin-like protease.

PubMed

Acevedo, J P; Rodriguez, V; Saavedra, M; Muñoz, M; Salazar, O; Asenjo, J A; Andrews, B A

2013-02-01

Cloning, expression and characterization of a new cold-adapted protease with potential biotechnological application, isolated from Antarctic bacteria. A subtilisin-like gene was isolated from several Antarctic bacterial genus using CODPEHOP-designed primers and a genome walking method. This gene encodes a precursor protein, which undergoes an autocatalytic cleavage resulting in a 34.6 kDa active cold-adapted protease with a maximum activity at 25-35°C and optimum pH of 8.0-9.0. It showed a higher catalytic efficiency at lower temperatures compared to its mesophilic counterpart. Heat-induced inactivation resulted in a very low melting point. Local packing analysis using the homology model indicated Ala284 as an important cold-adaptation determinant, which was corroborated by the site-directed mutagenesis. A new thermolabile subtilisin-like protease has been successfully cloned and analysed, and an important hot spot in the evolution of the cold adaptation and substrate specificity of this enzyme was identified and tested. This work reports a new cold-adapted protease with a vast representation amongst Antarctic genus, suggesting therefore its evolutionary success in this cold environment. Likewise, important sites for genetic potentiation have been identified, which are extrapolated to other enzymes of the same kind. © 2012 The Society for Applied Microbiology.

Tumour necrosis factor α secretion induces protease activation and acinar cell necrosis in acute experimental pancreatitis in mice.

PubMed

Sendler, Matthias; Dummer, Annegret; Weiss, Frank U; Krüger, Burkhard; Wartmann, Thomas; Scharffetter-Kochanek, Karin; van Rooijen, Nico; Malla, Sudarshan Ravi; Aghdassi, Ali; Halangk, Walter; Lerch, Markus M; Mayerle, Julia

2013-03-01

Acute pancreatitis has long been considered a disorder of pancreatic self-digestion, in which intracellular activation of digestive proteases induces tissue injury. Chemokines, released from damaged pancreatic cells then attract inflammatory cells, whose systemic action ultimately determines the disease severity. In the present work the opposite mechanism is investigated; that is, whether and how inflammatory cells can activate intracellular proteases. Using mice either deficient for the CD18-α subunit of the membrane attack complex-1 (MAC-1) complex or tumour necrosis factor (TNF)α, as well as after depletion of leucocyte subpopulations, pancreatitis was induced by 7-hourly caerulein injections (50 μg/kg, intraperitoneally). Pancreatic acini were coincubated in vitro from wild-type and cathepsin-B-deficient animals with phorbol-12-myristate-13-acetate (PMA)-activated neutrophils and macrophages, caerulein or TNFα, and activities of trypsin, cathepsin-B and caspase-3 were measured, as well as necrosis using fluorogenic substrates. TNFα was inhibited with monospecific antibodies. Deletion of CD18 prevented transmigration of leucocytes into the pancreas during pancreatitis, greatly reduced disease severity and abolished digestive protease activation. Depletion of neutrophils and macrophages equally reduced premature trypsinogen activation and disease severity. In vitro activated neutrophils and macrophages directly induced premature protease activation and cell death in pancreatic acini and stimulation of acini with TNFα induced caspase-3 activation and necrosis via a cathepsin-B and calcium-dependent mechanism. Neutralising antibodies against TNFα and genetic deletion of TNFα prevented leucocyte-induced trypsin activity and necrosis in isolated acini. The soluble inflammatory cell mediator TNFα directly induces premature protease activation and necrosis in pancreatic acinar cells. This activation depends on calcium and cathepsin-B activity. The findings

Genomic and exoproteomic analyses of cold- and alkaline-adapted bacteria reveal an abundance of secreted subtilisin-like proteases.

PubMed

Lylloff, Jeanette E; Hansen, Lea B S; Jepsen, Morten; Sanggaard, Kristian W; Vester, Jan K; Enghild, Jan J; Sørensen, Søren J; Stougaard, Peter; Glaring, Mikkel A

2016-03-01

Proteases active at low temperature or high pH are used in many commercial applications, including the detergent, food and feed industries, and bacteria specifically adapted to these conditions are a potential source of novel proteases. Environments combining these two extremes are very rare, but offer the promise of proteases ideally suited to work at both high pH and low temperature. In this report, bacteria from two cold and alkaline environments, the ikaite columns in Greenland and alkaline ponds in the McMurdo Dry Valley region, Antarctica, were screened for extracellular protease activity. Two isolates, Arsukibacterium ikkense from Greenland and a related strain, Arsukibacterium sp. MJ3, from Antarctica, were further characterized with respect to protease production. Genome sequencing identified a range of potential extracellular proteases including a number of putative secreted subtilisins. An extensive liquid chromatography-tandem mass spectrometry analysis of proteins secreted by A. ikkense identified six subtilisin-like proteases as abundant components of the exoproteome in addition to other peptidases potentially involved in complete degradation of extracellular protein. Screening of Arsukibacterium genome libraries in Escherichia coli identified two orthologous secreted subtilisins active at pH 10 and 20 °C, which were also present in the A. ikkense exoproteome. Recombinant production of both proteases confirmed the observed activity. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Trypsin inhibitors from Capsicum baccatum var. pendulum leaves involved in Pepper yellow mosaic virus resistance.

PubMed

Moulin, M M; Rodrigues, R; Ribeiro, S F F; Gonçalves, L S A; Bento, C S; Sudré, C P; Vasconcelos, I M; Gomes, V M

2014-11-07

Several plant organs contain proteinase inhibitors, which are produced during normal plant development or are induced upon pathogen attack to suppress the enzymatic activity of phytopathogenic microorganisms. In this study, we examined the presence of proteinase inhibitors, specifically trypsin inhibitors, in the leaf extract of Capsicum baccatum var. pendulum inoculated with PepYMV (Pepper yellow mosaic virus). Leaf extract from plants with the accession number UENF 1624, which is resistant to PepYMV, was collected at 7 different times (0, 24, 48, 72, 96, 120, and 144 h). Seedlings inoculated with PepYMV and control seedlings were grown in a growth chamber. Protein extract from leaf samples was partially purified by reversed-phase chromatography using a C2/C18 column. Residual trypsin activity was assayed to detect inhibitors followed by Tricine-SDS-PAGE analysis to determine the N-terminal peptide sequence. Based on trypsin inhibitor assays, trypsin inhibitors are likely constitutively synthesized in C. baccatum var. pendulum leaf tissue. These inhibitors are likely a defense mechanism for the C. baccatum var. pendulum- PepYMV pathosystem.

Trypsin inhibitory activity and gel-enhancing effect of sarcoplasmic proteins from common carp.

PubMed

Siriangkanakun, Siriphon; Yongsawatdigul, Jirawat

2012-10-01

Proteinase inhibitory activity of sarcoplasmic protein (SP) extracted from common carp (Cyprinus carpio) muscle and its gel-improving ability were investigated. SPs displayed 89% and 54% inhibitory activity toward trypsin at 40 and 65 °C, respectively. Protein bands with molecular mass of 69, 50, 44, 41, and 35 kDa appeared on trypsin inhibitory activity staining under nonreducing condition when incubated at 40 °C, while 2 protein bands at 54 and 35 kDa were observed at 65 °C. Addition of SP at 0.18 g protein/100 g increased textural properties of threadfin bream surimi gel. However, when SP was added in combination with various CaCl(2) concentrations (0.1% to 0.5%) it did not further improve textural properties as compared to the addition of SP alone. Retention of myosin heavy chain of threadfin bream surimi was greater with the addition of SP. These results indicated that the gel-enhancing effect of common carp SP was due to the inhibitory activity toward endogenous trypsin-like proteinases in threadfin bream surimi. Sarcoplasmic protein from common carp muscle could be used as a functional protein ingredient that minimizes muscle proteolysis and improves textural properties of surimi containing trypsin-like endogenous proteinases. © 2012 Institute of Food Technologists®

Tissue Kallikrein Inhibitors Based on the Sunflower Trypsin Inhibitor Scaffold – A Potential Therapeutic Intervention for Skin Diseases

PubMed Central

Chen, Wenjie; Kinsler, Veronica A.

2016-01-01

Tissue kallikreins (KLKs), in particular KLK5, 7 and 14 are the major serine proteases in the skin responsible for skin shedding and activation of inflammatory cell signaling. In the normal skin, their activities are controlled by an endogenous protein protease inhibitor encoded by the SPINK5 gene. Loss-of-function mutations in SPINK5 leads to enhanced skin kallikrein activities and cause the skin disease Netherton Syndrome (NS). We have been developing inhibitors based on the Sunflower Trypsin Inhibitor 1 (SFTI-1) scaffold, a 14 amino acids head-to-tail bicyclic peptide with a disulfide bond. To optimize a previously reported SFTI-1 analogue (I10H), we made five analogues with additional substitutions, two of which showed improved inhibition. We then combined those substitutions and discovered a variant (Analogue 6) that displayed dual inhibition of KLK5 (tryptic) and KLK7 (chymotryptic). Analogue 6 attained a tenfold increase in KLK5 inhibition potency with an Isothermal Titration Calorimetry (ITC) Kd of 20nM. Furthermore, it selectively inhibits KLK5 and KLK14 over seven other serine proteases. Its biological function was ascertained by full suppression of KLK5-induced Protease-Activated Receptor 2 (PAR-2) dependent intracellular calcium mobilization and postponement of Interleukin-8 (IL-8) secretion in cell model. Moreover, Analogue 6 permeates through the cornified layer of in vitro organotypic skin equivalent culture and inhibits protease activities therein, providing a potential drug lead for the treatment of NS. PMID:27824929

Defense response in non-genomic model species: methyl jasmonate exposure reveals the passion fruit leaves' ability to assemble a cocktail of functionally diversified Kunitz-type trypsin inhibitors and recruit two of them against papain.

PubMed

Botelho-Júnior, Sylvio; Machado, Olga L T; Fernandes, Kátia V S; Lemos, Francisco J A; Perdizio, Viviane A; Oliveira, Antônia E A; Monteiro, Leandro R; Filho, Mauri L; Jacinto, Tânia

2014-08-01

Multiplicity of protease inhibitors induced by predators may increase the understanding of a plant's intelligent behavior toward environmental challenges. Information about defense mechanisms of non-genomic model plant passion fruit (Passiflora edulis Sims) in response to predator attack is still limited. Here, via biochemical approaches, we showed its flexibility to build-up a broad repertoire of potent Kunitz-type trypsin inhibitors (KTIs) in response to methyl jasmonate. Seven inhibitors (20-25 kDa) were purified from exposed leaves by chromatographic techniques. Interestingly, the KTIs possessed truncated Kunitz motif in their N-terminus and some of them also presented non-consensus residues. Gelatin-Native-PAGE established multiple isoforms for each inhibitor. Significant differences regarding inhibitors' activity toward trypsin and chymotrypsin were observed, indicating functional polymorphism. Despite its rarity, two of them also inhibited papain, and such bifunctionality suggests a recruiting process onto another mechanistic class of target protease (cysteine-type). All inhibitors acted strongly on midgut proteases from sugarcane borer, Diatraea saccharalis (a lepidopteran insect) while in vivo assays supported their insecticide properties. Moreover, the bifunctional inhibitors displayed activity toward midgut proteases from cowpea weevil, Callosobruchus maculatus (a coleopteran insect). Unexpectedly, all inhibitors were highly effective against midgut proteases from Aedes aegypti a dipteran insect (vector of neglected tropical diseases) opening new avenues for plant-derived PIs for vector control-oriented research. Our results reflect the KTIs' complexities in passion fruit which could be wisely exploited by influencing plant defense conditions. Therefore, the potential of passion fruit as source of bioactive compounds with diversified biotechnological application was strengthened.

Plasma trypsin in chronic pancreatitis and pancreatic adenocarcinoma.

PubMed

Adrian, T E; Besterman, H S; Mallinson, C N; Pera, A; Redshaw, M R; Wood, T P; Bloom, S R

1979-10-01

We have used a simple and precise radioimmunoassay to measure trypsin in human plasma. Fasting plasma trypsin concentrations were extremely low in patients with chronic pancreatitis with steatorrhoea (5 +/- 2 ng/ml) when compared to healthy controls (86 +/- 7 ng/ml, p less than 0.001). In patients with chronic pancreatitis but no steatorrhoea basal plasma trypsin levels were similar to those of the normal controls (99 +/- 25 ng/ml). A small but significant postprandial rise in plasma trypsin concentrations was observed in normal subjects (mean increment 15 +/- 4%, p less than 0.005, paired t test) but was absent in patients with chronic pancreatitis with steatorrhoea. In contrast to exocrine deficient chronic pancreatitis, other malabsorptive conditions associated with steatorrhoea (active coeliac disease and acute tropical sprue) demonstrated mean fasting trypsin concentrations similar to controls. Patients with adenocarcinoma of the pancreas had basal trypsin concentrations similar to healthy subjects as did patients with adenocarcinoma of the stomach, colon, rectum, brochus, and breast. In some cases measurement of plasma trypsin may be of help in the differential diagnosis of steatorrhoea.

Broad spectrum activity of a lectin-like bacterial serine protease family on human leukocytes.

PubMed

Ayala-Lujan, Jorge Luis; Vijayakumar, Vidhya; Gong, Mei; Smith, Rachel; Santiago, Araceli E; Ruiz-Perez, Fernando

2014-01-01

The serine protease autotransporter from Enterobacteriaceae (SPATE) family, which number more than 25 proteases with apparent diverse functions, have been phylogenetically divided into two distinct classes, designated 1 and 2. We recently demonstrated that Pic and Tsh, two members of the class-2 SPATE family produced by intestinal and extraintestinal pathogenic E. coli, were able to cleave a number of O-glycosylated proteins on neutrophils and lymphocytes resulting in impaired leukocyte functions. Here we show that most members of the class-2 SPATE family have lectin-like properties and exhibit differential protease activity reliant on glycoprotein type and cell lineage. Protease activity was seen in virtually all tested O-glycosylated proteins including CD34, CD55, CD164, TIM1, TIM3, TIM4 and C1-INH. We also show that although SPATE proteins bound and cleaved glycoproteins more efficiently on granulocytes and monocytes, they also targeted glycoproteins on B, T and natural killer lymphocytes. Finally, we found that the characteristic domain-2 of class-2 SPATEs is not required for glycoprotease activity, but single amino acid mutations in Pic domain-1 to those residues naturally occurring in domain-1 of SepA, were sufficient to hamper Pic glycoprotease activity. This study shows that most class-2 SPATEs have redundant activities and suggest that they may function as immunomodulators at several levels of the immune system.

Broad Spectrum Activity of a Lectin-Like Bacterial Serine Protease Family on Human Leukocytes

PubMed Central

Ayala-Lujan, Jorge Luis; Vijayakumar, Vidhya; Gong, Mei; Smith, Rachel; Santiago, Araceli E.; Ruiz-Perez, Fernando

2014-01-01

The serine protease autotransporter from Enterobacteriaceae (SPATE) family, which number more than 25 proteases with apparent diverse functions, have been phylogenetically divided into two distinct classes, designated 1 and 2. We recently demonstrated that Pic and Tsh, two members of the class-2 SPATE family produced by intestinal and extraintestinal pathogenic E. coli, were able to cleave a number of O-glycosylated proteins on neutrophils and lymphocytes resulting in impaired leukocyte functions. Here we show that most members of the class-2 SPATE family have lectin-like properties and exhibit differential protease activity reliant on glycoprotein type and cell lineage. Protease activity was seen in virtually all tested O-glycosylated proteins including CD34, CD55, CD164, TIM1, TIM3, TIM4 and C1-INH. We also show that although SPATE proteins bound and cleaved glycoproteins more efficiently on granulocytes and monocytes, they also targeted glycoproteins on B, T and natural killer lymphocytes. Finally, we found that the characteristic domain-2 of class-2 SPATEs is not required for glycoprotease activity, but single amino acid mutations in Pic domain-1 to those residues naturally occurring in domain-1 of SepA, were sufficient to hamper Pic glycoprotease activity. This study shows that most class-2 SPATEs have redundant activities and suggest that they may function as immunomodulators at several levels of the immune system. PMID:25251283

21 CFR 184.1914 - Trypsin.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Substances Affirmed as GRAS § 184.1914 Trypsin. (a) Trypsin (CAS Reg. No. 9002-07-7) is an enzyme preparation... characterizing enzyme activity is that of a peptide hydrolase (EC 3.4.21.4). (b) The ingredient meets the general requirements and additional requirements for enzyme preparations in the Food Chemicals Codex, 3d ed. (1981), p...

PSA-selective activation of cytotoxic human serine proteases within the tumor microenvironment as a therapeutic strategy to target prostate cancer.

PubMed

Rogers, Oliver C; Anthony, Lizamma; Rosen, D Marc; Brennen, W Nathaniel; Denmeade, Samuel R

2018-04-27

Prostate cancer is the most diagnosed malignancy and the second leading cause of cancer-related death in American men. While localized therapy is highly curative, treatments for metastatic prostate cancer are largely palliative. Thus, new innovative therapies are needed to target metastatic tumors. Prostate-Specific Antigen (PSA) is a chymotrypsin-like protease with a unique substrate specificity that is secreted by both normal and malignant prostate epithelial cells. Previous studies demonstrated the presence of high levels (μM-mM) of enzymatically active PSA is present in the extracellular fluid of the prostate cancer microenvironment. Because of this, PSA is an attractive target for a protease activated pro-toxin therapeutic strategy. Because prostate cancers typically grow very slowly, a strategy employing a proliferation-independent cytotoxic payload is preferred. Recently, it was shown that the human protease Granzyme B (GZMB), at low micromolar concentrations in the extracellular space, can cleave an array of extracellular matrix (ECM) proteins thus perturbing cell growth, signaling, motility, and integrity. It is also well established that other human proteases such as trypsin can induce similar effects. Because both enzymes require N-terminal proteolytic activation, we propose to convert these proteins into PSA-activated cytotoxins. In this study, we examine the enzymatic and cell targeting parameters of these PSA-activated cytotoxic serine proteases. These pro-enzymes were activated robustly by PSA and induced ECM damage that led to the death of prostate cancer cells in vitro thus supporting the potential use of this strategy as means to target metastatic prostate cancers.

PSA-selective activation of cytotoxic human serine proteases within the tumor microenvironment as a therapeutic strategy to target prostate cancer

PubMed Central

Rogers, Oliver C.; Anthony, Lizamma; Rosen, D. Marc; Brennen, W. Nathaniel; Denmeade, Samuel R.

2018-01-01

Prostate cancer is the most diagnosed malignancy and the second leading cause of cancer-related death in American men. While localized therapy is highly curative, treatments for metastatic prostate cancer are largely palliative. Thus, new innovative therapies are needed to target metastatic tumors. Prostate-Specific Antigen (PSA) is a chymotrypsin-like protease with a unique substrate specificity that is secreted by both normal and malignant prostate epithelial cells. Previous studies demonstrated the presence of high levels (μM-mM) of enzymatically active PSA is present in the extracellular fluid of the prostate cancer microenvironment. Because of this, PSA is an attractive target for a protease activated pro-toxin therapeutic strategy. Because prostate cancers typically grow very slowly, a strategy employing a proliferation-independent cytotoxic payload is preferred. Recently, it was shown that the human protease Granzyme B (GZMB), at low micromolar concentrations in the extracellular space, can cleave an array of extracellular matrix (ECM) proteins thus perturbing cell growth, signaling, motility, and integrity. It is also well established that other human proteases such as trypsin can induce similar effects. Because both enzymes require N-terminal proteolytic activation, we propose to convert these proteins into PSA-activated cytotoxins. In this study, we examine the enzymatic and cell targeting parameters of these PSA-activated cytotoxic serine proteases. These pro-enzymes were activated robustly by PSA and induced ECM damage that led to the death of prostate cancer cells in vitro thus supporting the potential use of this strategy as means to target metastatic prostate cancers. PMID:29854290

Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression.

PubMed

Manira, Maarof; Khairul Anuar, Khairoji; Seet, Wan Tai; Ahmad Irfan, Abd Wahab; Ng, Min Hwei; Chua, Kien Hui; Mohd Heikal, Mohd Yunus; Aminuddin, Bin Saim; Ruszymah, Bt Hj Idrus

2014-03-01

Animal-derivative free reagents are preferred in skin cell culture for clinical applications. The aim of this study was to compare the performance and effects between animal-derived trypsin and recombinant trypsin for skin cells culture and expansion. Full thickness human skin was digested in 0.6 % collagenase for 6 h to liberate the fibroblasts, followed by treatment with either animal-derived trypsin; Trypsin EDTA (TE) or recombinant trypsin; TrypLE Select (TS) to liberate the keratinocytes. Both keratinocytes and fibroblasts were then culture-expanded until passage 2. Trypsinization for both cell types during culture-expansion was performed using either TE or TS. Total cells yield was determined using a haemocytometer. Expression of collagen type I, collagen type III (Col-III), cytokeratin 10, and cytokeratin 14 genes were quantified via RT-PCR and further confirmed with immunocytochemical staining. The results of our study showed that the total cell yield for both keratinocytes and fibroblasts treated with TE or TS were comparable. RT-PCR showed that expression of skin-specific genes except Col-III was higher in the TS treated group compared to that in the TE group. Expression of proteins specific to the two cell types were confirmed by immunocytochemical staining in both TE and TS groups. In conclusion, the performance of the recombinant trypsin is comparable with the well-established animal-derived trypsin for human skin cell culture expansion in terms of cell yield and expression of specific cellular markers.

The Three Streptomyces lividans HtrA-Like Proteases Involved in the Secretion Stress Response Act in a Cooperative Manner

PubMed Central

Vicente, Rebeca L.; Gullón, Sonia; Marín, Silvia; Mellado, Rafael P.

2016-01-01

Overproduction of Sec-proteins in S. lividans accumulates misfolded proteins outside of the cytoplasmic membrane where the accumulated proteins interfere with the correct functioning of the secretion machinery and with the correct cell functionality, triggering the expression in S. lividans of a CssRS two-component system which regulates the degradation of the accumulated protein, the so-called secretion stress response. Optimization of secretory protein production via the Sec route requires the identification and characterisation of quality factors involved in this process. The phosphorylated regulator (CssR) interacts with the regulatory regions of three genes encoding three different HtrA-like proteases. Individual mutations in each of these genes render degradation of the misfolded protein inoperative, and propagation in high copy number of any of the three proteases encoding genes results on indiscriminate alpha-amylase degradation. None of the proteases could complement the other two deficiencies and only propagation of each single copy protease gene can restore its own deficiency. The obtained results strongly suggest that the synthesis of the three HtrA-like proteases needs to be properly balanced to ensure the effective degradation of misfolded overproduced secretory proteins and, at the same time, avoid negative effects in the secreted proteins and the secretion machinery. This is particularly relevant when considering the optimisation of Streptomyces strains for the overproduction of homologous or heterologous secretory proteins of industrial application. PMID:27977736

An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis.

PubMed

Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F; Cohen, Itay; Henin, Rachel D; Hockla, Alexandra; Soares, Alexei S; Papo, Niv; Caulfield, Thomas R; Radisky, Evette S

2016-12-16

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Biopotency of serine protease inhibitors from cowpea (Vigna unguiculata) seeds on digestive proteases and the development of Spodoptera littoralis (Boisduval).

PubMed

Abd El-latif, Ashraf Oukasha

2015-05-01

Serine protease inhibitors (PIs) have been described in many plant species and are universal throughout the plant kingdom, where trypsin inhibitors is the most common type. In the present study, trypsin and chymotrypsin inhibitory activity was detected in the seed flour extracts of 13 selected cultivars/accessions of cowpea. Two cowpea cultivars, Cream7 and Buff, were found to have higher trypsin and chymotrypsin inhibitory potential compared to other tested cultivars for which they have been selected for further purification studies using ammonium sulfate fractionation and DEAE-Sephadex A-25 column. Cream7-purified proteins showed two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) corresponding to molecular mass of 17.10 and 14.90 kDa, while the purified protein from Buff cultivar showed a single band corresponding mass of 16.50 kDa. The purified inhibitors were stable at temperature below 60°C and were active at wide range of pH from 2 to 12. The kinetic analysis revealed noncompetitive type of inhibition for both inhibitors against both enzymes. The inhibitor constant (Ki ) values suggested high affinity between inhibitors and enzymes. Purified inhibitors were found to have deep and negative effects on the mean larval weight, larval mortality, pupation, and mean pupal weight of Spodoptera littoralis, where Buff PI was more effective than Cream7 PI. It may be concluded that cowpea PI gene(s) could be potential insect control protein for future studies in developing insect-resistant transgenic plants. © 2014 Wiley Periodicals, Inc.

Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula

PubMed Central

Lomate, Purushottam R.; Bonning, Bryony C.

2016-01-01

Stink bugs negatively impact numerous plant species of agricultural and horticultural importance. While efforts to develop effective control measures are underway, the unique digestive physiology of these pests presents a significant hurdle for either protein- or nucleotide-based management options. Here we report the comparative biochemical and proteomic characterization of proteases and nucleases from the gut, salivary gland and saliva of the southern green stink bug, Nezara viridula. The pH optimum for protease activity was acidic (5 to 6) in the gut with the primary proteases being cysteine proteases, and alkaline (8 to 9) in the saliva and salivary gland with the primary proteases being serine proteases. The serine proteases in saliva differ biochemically from trypsin and chymotrypsin, and the cathepsins in the gut and saliva showed distinct properties in inhibitor assays. Nuclease activity (DNase, RNase, dsRNase) was concentrated in the salivary gland and saliva with negligible activity in the gut. The most abundant proteins of the gut (530) and salivary gland (631) identified by proteomic analysis included four gut proteases along with eight proteases and one nuclease from the salivary gland. Understanding of N. viridula digestive physiology will facilitate the design of new strategies for management of this significant pest. PMID:27282882

Comparison of Conventional and Microwave Treatment on Soymilk for Inactivation of Trypsin Inhibitors and In Vitro Protein Digestibility

PubMed Central

Vagadia, Brinda Harish; Raghavan, Vijaya

2018-01-01

Soymilk is lower in calories compared to cow’s milk, since it is derived from a plant source (no cholesterol) and is an excellent source of protein. Despite the beneficial factors, soymilk is considered as one of the most controversial foods in the world. It contains serine protease inhibitors which lower its nutritional value and digestibility. Processing techniques for the elimination of trypsin inhibitors and lipoxygenase, which have shorter processing time and lower production costs are required for the large-scale manufacturing of soymilk. In this study, the suitable conditions of time and temperature are optimized during microwave processing to obtain soymilk with maximum digestibility with inactivation of trypsin inhibitors, in comparison to the conventional thermal treatment. The microwave processing conditions at a frequency of 2.45 GHz and temperatures of 70 °C, 85 °C and 100 °C for 2, 5 and 8 min were investigated and were compared to conventional thermal treatments at the same temperature for 10, 20 and 30 min. Response surface methodology is used to design and optimize the experimental conditions. Thermal processing was able to increase digestibility by 7% (microwave) and 11% (conventional) compared to control, while trypsin inhibitor activity reduced to 1% in microwave processing and 3% in conventional thermal treatment when compared to 10% in raw soybean. PMID:29316679

Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries

PubMed Central

Harris, Jennifer L.; Backes, Bradley J.; Leonetti, Francesco; Mahrus, Sami; Ellman, Jonathan A.; Craik, Charles S.

2000-01-01

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors. PMID:10869434

Supplementation of broiler diets with high levels of microbial protease and phytase enables partial replacement of commercial soybean meal with raw, full-fat soybean.

PubMed

Erdaw, M M; Perez-Maldonado, R A; Iji, P A

2018-06-01

A 3 × 3 + 1 factorial, involving three levels of protease (0, 15,000 or 30,000 PROT/kg) and three levels of phytase (1,000, 2,000 or 3,000 FYT/kg), was used to evaluate the effect of replacing commercial soybean meal (SBM) with raw, full-fat soybean (RFSB) at 75 g/kg of diet for broilers. A control diet was used for comparison. Each treatment was replicated six times, with nine birds per replicate. The concentration of trypsin inhibitors (TIs) in the test diets was approximately 10,193.4 TIU/kg. Regardless of enzyme supplementation, feed intake (FI) and body weight gain (BWG) of birds in the control group were superior to those on the test diets. Birds that received the protease-free test diets had reduced FI and BWG, but when supplemented with protease, were similar to the control diet in BWG, FI (except 0-35 days) and feed conversion ratio (FCR). When the test diet was supplemented with elevated levels (extradose) of protease and phytase, the BWG was improved during 0-10 days (p = .05) and 0-24 days (p < .01). Regardless of protease supplementation, the weight of thighs was lower for birds fed the test diets. Birds that received the control diet had smaller weight of pancreas. Increasing the level of phytase supplementation reduced (p < .05) the weight of the pancreas. The apparent ileal digestibility (AID) of CP and AA was higher in birds on the control diets, but this was also improved in test diets by protease supplementation. The activities of trypsin (7%), general proteolytic (11%) and lipase (12%) were slightly increased because of protease supplementation. Mucosal depth and apparent villus surface areas were increased by about 2.9% and 20%, respectively, due to supplementation of elevated level of phytase. It can be concluded that RFSB could partially replace SBM in broiler diets, provided the diets are supplemented with elevated levels of protease and phytase. © 2018 Blackwell Verlag GmbH.

Molecular docking and in vitro studies of soap nut trypsin inhibitor (SNTI) against phospholipase A2 isoforms in therapeutic intervention of inflammatory diseases.

PubMed

Sirisha, Gandreddi V D; Vijaya Rachel, K; Zaveri, Kunal; Yarla, Nagendra Sastry; Kiranmayi, P; Ganash, Magdah; Alkreathy, Huda Mohammad; Rajeh, Nisreen; Ashraf, Ghulam Md

2018-07-15

Therapeutic value of allelochemicals in inflammatory disorders and the potential drug targets need to be elucidated to alleviate tissue and vascular injury. Natural anti-inflammatory agents are known to cause minimal adverse effects. Presence of different secondary metabolites (allelochemicals), protease inhibitors like soap nut trypsin inhibitor (SNTI) from Sapindus trifoliatus and allied compounds from natural sources cannot be blithely ignored as natural therapeutics. In the present study, SNTI, a prospective protease inhibitor isolated from the seeds of Sapindus trifoliatus were subjected to docking against three isoforms of Phospholipase A 2 (PLA 2 ) molecules of the inflammatory pathways which are localized in the membrane, cytosol and pancreas. Eleven ligand molecules were selected from Sapindus trifoliatus and docked against membrane, cytosolic and pancreatic PLA 2 . Cytosolic PLA 2 showed a strong inhibition by Kampferol, a secondary metabolite from seed endosperm of Sapindus trifoliatus. SNTI showed best interaction with membrane PLA 2 in both in silico as well as in in vitro studies. SNTI showed IC 50 value of 29.02 μM in in vitro assay. Docking interaction profiles and in vitro studies validate selected molecules from Sapindus trifoliatus as immunomodulators and can mollify inflammatory responses. Copyright © 2018 Elsevier B.V. All rights reserved.

Hyaluronan mediates airway hyperresponsiveness in oxidative lung injury

PubMed Central

Lazrak, Ahmed; Creighton, Judy; Yu, Zhihong; Komarova, Svetlana; Doran, Stephen F.; Aggarwal, Saurabh; Emala, Charles W.; Stober, Vandy P.; Trempus, Carol S.; Garantziotis, Stavros

2015-01-01

Chlorine (Cl2) inhalation induces severe oxidative lung injury and airway hyperresponsiveness (AHR) that lead to asthmalike symptoms. When inhaled, Cl2 reacts with epithelial lining fluid, forming by-products that damage hyaluronan, a constituent of the extracellular matrix, causing the release of low-molecular-weight fragments (L-HA, <300 kDa), which initiate a series of proinflammatory events. Cl2 (400 ppm, 30 min) exposure to mice caused an increase of L-HA and its binding partner, inter-α-trypsin-inhibitor (IαI), in the bronchoalveolar lavage fluid. Airway resistance following methacholine challenge was increased 24 h post-Cl2 exposure. Intratracheal administration of high-molecular-weight hyaluronan (H-HA) or an antibody against IαI post-Cl2 exposure decreased AHR. Exposure of human airway smooth muscle (HASM) cells to Cl2 (100 ppm, 10 min) or incubation with Cl2-exposed H-HA (which fragments it to L-HA) increased membrane potential depolarization, intracellular Ca2+, and RhoA activation. Inhibition of RhoA, chelation of intracellular Ca2+, blockade of cation channels, as well as postexposure addition of H-HA, reversed membrane depolarization in HASM cells. We propose a paradigm in which oxidative lung injury generates reactive species and L-HA that activates RhoA and Ca2+ channels of airway smooth muscle cells, increasing their contractility and thus causing AHR. PMID:25747964

Cellular and Molecular Biology of Airway Mucins

PubMed Central

Lillehoj, Erik P.; Kato, Kosuke; Lu, Wenju; Kim, Kwang C.

2017-01-01

Airway mucus constitutes a thin layer of airway surface liquid with component macromolecules that covers the luminal surface of the respiratory tract. The major function of mucus is to protect the lungs through mucociliary clearance of inhaled foreign particles and noxious chemicals. Mucus is comprised of water, ions, mucin glycoproteins, and a variety of other macromolecules, some of which possess anti-microbial, anti-protease, and anti-oxidant activities. Mucins comprise the major protein component of mucus and exist as secreted and cell-associated glycoproteins. Secreted, gel-forming mucins are mainly responsible for the viscoelastic property of mucus, which is crucial for effective mucociliary clearance. Cell-associated mucins shield the epithelial surface from pathogens through their extracellular domains and regulate intracellular signaling through their cytoplasmic regions. However, neither the exact structures of mucin glycoproteins, nor the manner through which their expression is regulated, are completely understood. This chapter reviews what is currently known about the cellular and molecular properties of airway mucins. PMID:23445810

Systematic Design of Trypsin Cleavage Site Mutated Exendin4-Cysteine 1, an Orally Bioavailable Glucagon-Like Peptide-1 Receptor Agonist.

PubMed

Sai, Wenbo; Tian, Hong; Yang, Kangmin; Tang, Daoqi; Bao, Jinxiao; Ge, Yang; Song, Xiaoda; Zhang, Yu; Luo, Cheng; Gao, Xiangdong; Yao, Wenbing

2017-03-08

Exendin-4 is a strong therapeutic candidate for the treatment of metabolic syndrome. Related receptor agonist drugs have been on the market since 2005. However, technical limitations and the pain caused by subcutaneous injection have severely limited patient compliance. The goal of the study is to investigate a biologically active exendin-4 analog could be administered orally. Using intraperitoneal glucose tolerance tests, we discovered that exendin4-cysteine administered by oral gavage had a distinct hypoglycemic effect in C57BL/6J mice. Using Rosetta Design and Amber, we designed and screened a series of exendin4-cysteine analogs to identify those that retained biological activity while resisting trypsin digestion. Trypsin Cleavage Site Mutated Exendin4-cysteine 1 (TSME-1), an analog whose bioactivity was similar to exendin-4 and was almost completely resistant to trypsin, was screened out. In addition, TSME-1 significantly normalized the blood glucose levels and the availability of TSME-1 was significantly higher than that of exendin-4 and exendin4-cysteine. Collectively orally administered TSME-1, a trypsin-resistant exendin-4 analog obtained by the system, is a strong candidate for future treatments of type 2 diabetes.

Serine protease activity of Cur l 1 from Curvularia lunata augments Th2 response in mice.

PubMed

Tripathi, Prabhanshu; Kukreja, Neetu; Singh, B P; Arora, Naveen

2009-05-01

Studies with mite allergens demonstrated that proteolytic activity augments allergic airway inflammation. This knowledge is limited to few enzyme allergens. The objective of this study is to investigate the effect of serine protease Cur l 1 from Curvularia lunata in airway inflammation/hyper-responsiveness. Cur l 1 was purified and inactivated using a serine protease inhibitor. Balb/c mice were sensitized with enzymatically active Cur l 1 or C. lunata extract. Sensitized mice were given booster dose on day 14 with active or inactivated Cur l 1. Intranasal challenge was given on day 28, 29, and 30. Airway hyper-responsiveness was measured by plethysmography. Blood, bronchoalveolar lavage fluid (BALF), spleen, and lungs from mice were analyzed for cellular infiltration, immunoglobulins, and cytokine levels. Mice challenged with enzymatically active Cur l 1 demonstrated significantly higher airway inflammation than inactive Cur l 1 group mice (p < 0.01). There was a significant difference in serum IgE and IgG1 levels among mice immunized with active Cur l 1 and inactive Cur l 1 (p < 0.01). IL-4 and IL-5 were higher in BALF and splenocyte culture supernatant of active Cur l 1 than inactive Cur l 1 mice. Lung histology revealed increased eosinophil infiltration, goblet cell hyperplasia and mucus secretion in active group. Proteolytic activity of Cur l 1 plays an important role in airway inflammation and the inactivated Cur l 1 has potential to be explored for immunotherapy.

Crystal structure of the 3C protease from Southern African Territories type 2 foot-and-mouth disease virus

PubMed Central

Yang, Jingjie; Leen, Eoin N.; Maree, Francois F.

2016-01-01

The replication of foot-and-mouth disease virus (FMDV) is dependent on the virus-encoded 3C protease (3Cpro). As in other picornaviruses, 3Cpro performs most of the proteolytic processing of the polyprotein expressed from the large open reading frame in the RNA genome of the virus. Previous work revealed that the 3Cpro from serotype A—one of the seven serotypes of FMDV—adopts a trypsin-like fold. On the basis of capsid sequence comparisons the FMDV serotypes are grouped into two phylogenetic clusters, with O, A, C, and Asia 1 in one, and the three Southern African Territories serotypes, (SAT-1, SAT-2 and SAT-3) in another, a grouping pattern that is broadly, but not rigidly, reflected in 3Cpro amino acid sequences. We report here the cloning, expression and purification of 3C proteases from four SAT serotype viruses (SAT2/GHA/8/91, SAT1/NIG/5/81, SAT1/UGA/1/97, and SAT2/ZIM/7/83) and the crystal structure at 3.2 Å resolution of 3Cpro from SAT2/GHA/8/91. PMID:27168976

Fish trypsins: potential applications in biomedicine and prospects for production.

PubMed

Jesús-de la Cruz, Kristal; Álvarez-González, Carlos Alfonso; Peña, Emyr; Morales-Contreras, José Antonio; Ávila-Fernández, Ángela

2018-04-01

In fishes, trypsins are adapted to different environmental conditions, and the biochemical and kinetic properties of a broad variety of native isoforms have been studied. Proteolytic enzymes remain in high demand in the detergent, food, and feed industries; however, our analysis of the literature showed that, in the last decade, some fish trypsins have been studied for the synthesis of industrial peptides and for specific biomedical uses as antipathogenic agents against viruses and bacteria, which have been recently patented. In addition, innovative strategies of trypsin administration have been studied to ensure that trypsins retain their properties until they exert their action. Biomedical uses require the production of high-quality enzymes. In this context, the production of recombinant trypsins is an alternative. For this purpose, E. coli -based systems have been tested for the production of fish trypsins; however, P. pastoris -based systems also seem to show great potential in the production of fish trypsins with higher production quality. On the other hand, there is a lack of information regarding the specific structures, biochemical and kinetic properties, and characteristics of trypsins produced using heterologous systems. This review describes the potential uses of fish trypsins in biomedicine and the enzymatic and structural properties of native and recombinant fish trypsins obtained to date, outlining some prospects for their study.

Preparation and evaluation of dual-enzyme microreactor with co-immobilized trypsin and chymotrypsin.

PubMed

Meller, Kinga; Pomastowski, Paweł; Grzywiński, Damian; Szumski, Michał; Buszewski, Bogusław

2016-04-01

The preparation of capillary microfluidic reactor with co-immobilized trypsin and chymotrypsin with the use of a low-cost commercially available enzymatic reagent (containing these proteases) as well as the evaluation of its usefulness in proteomic research were presented. The monolithic copolymer synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) was used as a support. Firstly, the polymerization conditions were optimized and the monolithic bed was synthesized in the fused silica capillary modified with 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS). The polymer containing epoxy groups was then modified with 1,6-diaminohexane, followed by the attachment of glutaraldehyde and immobilization of enzymes. The efficiency of the prepared monolithic Immobilized Enzyme Microreactor (μ-IMER) with regard to trypsin activity was evaluated using the low-molecular mass compound (Nα-benzoyl-l-arginine ethyl ester, BAEE). The activities of both enzymes were investigated using a macromolecular protein (human transferrin, Tf) as a substrate. In the case of BAEE, the reaction product was separated from the substrate using the capillary liquid chromatography and the efficiency of the reaction was determined by the peak area of the substrate. The hydrolysis products of transferrin were analyzed with MALDI-TOF which allows for the verification of the prepared enzymatic system applicability in the field of proteomic research. Copyright © 2016 Elsevier B.V. All rights reserved.

Functional characterization of a serine protease inhibitor modulated in the infection of the Aedes aegypti with dengue virus.

PubMed

Soares, Tatiane Sanches; Rodriguez Gonzalez, Boris Luis; Torquato, Ricardo José Soares; Lemos, Francisco Jose Alves; Costa-da-Silva, André L; Capurro Guimarães, Margareth de Lara; Tanaka, Aparecida Sadae

2018-01-01

During feeding with blood meal, female Aedes aegypti can transmit infectious agents, such as dengue, yellow fever, chikungunya and Zika viruses. Dengue virus causes human mortality in tropical regions of the world, and there is no specific treatment or vaccine with maximum efficiency being used for these infections. In the vector-virus interaction, the production of several molecules is modulated by both mosquitoes and invading agents. However, little information is available about these molecules in the Ae. aegypti mosquito during dengue infection. Inhibitors of the pacifastin family have been described to participate in the immune response of insects and Pac2 is the only gene of this family present in Ae. aegypti being then chosen for investigation. Pac2 was expressed in E. coli, purified and analyzed by mass spectrometry and SDS-PAGE. The Pac2 transcript was detected by qPCR, and its protein levels were assessed by Western blotting. The inhibitory activity of Pac2 was measured using its K i , IC 50 and zymography. Mosquito infections with DENV were introduced with the Brazilian ACS-46 DENV-2 strain propagated in C6/36 cells. In the present work, we showed that it is possibly involved in the interaction of the mosquitoes with the dengue virus. The Pac2 transcript was detected in larvae and in both the salivary gland and midgut of Ae. aegypti females, while the native protein was identified in females 3 h post-blood meal. Pac2 is a strong inhibitor of trypsin-like and thrombin-like proteases, which are present in 4th instar larvae midgut and females 24 h after blood meal. During DENV infection, up regulation of Pac2 expression occurs in the salivary gland and midgut. Pac2 is the first Pacifastin inhibitor member described in mosquitoes. Our results suggest that Pac2 acts on mosquito serine proteases, mainly the trypsin-like type, and is under transcriptional control by virus infection signals to allow its survival in the vector or by the mosquito as a defense

Docking, thermodynamics and molecular dynamics (MD) studies of a non-canonical protease inhibitor, MP-4, from Mucuna pruriens.

PubMed

Kumar, Ashish; Kaur, Harmeet; Jain, Abha; Nair, Deepak T; Salunke, Dinakar M

2018-01-12

Sequence and structural homology suggests that MP-4 protein from Mucuna pruriens belongs to Kunitz-type protease inhibitor family. However, biochemical assays showed that this protein is a poor inhibitor of trypsin. To understand the basis of observed poor inhibition, thermodynamics and molecular dynamics (MD) simulation studies on binding of MP-4 to trypsin were carried out. Molecular dynamics simulations revealed that temperature influences the spectrum of conformations adopted by the loop regions in the MP-4 structure. At an optimal temperature, MP-4 achieves maximal binding while above and below the optimum temperature, its functional activity is hampered due to unfavourable flexibility and relative rigidity, respectively. The low activity at normal temperature is due to the widening of the conformational spectrum of the Reactive Site Loop (RSL) that reduces the probability of formation of stabilizing contacts with trypsin. The unique sequence of the RSL enhances flexibility at ambient temperature and thus reduces its ability to inhibit trypsin. This study shows that temperature influences the function of a protein through modulation in the structure of functional domain of the protein. Modulation of function through appearance of new sequences that are more sensitive to temperature may be a general strategy for evolution of new proteins.

Degradation of airway neuropeptides by human lung tryptase.

PubMed

Tam, E K; Caughey, G H

1990-07-01

Several lines of evidence suggest a possible role for mast cell proteases in modulating the biologic effects of neuropeptides. To explore the potential of such interactions in human airway, we examined the activity of human tryptase, the major secretory protease of human lung mast cells, against several neuropeptides with proposed regulatory functions in human airway. Using highly purified tryptase obtained from extracts of human lung, we determined the sites and rats of hydrolysis of vasoactive intestinal peptide (VIP), peptide histidine-methionine (PHM), calcitonin gene-related peptide (CGRP), and the tachykinins substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). Tryptase hydrolyzes VIP rapidly at several sites (Arg12, Arg14, Lys20, and Lys21) with an overall kcat/Km of 1.5 x 10(5) M-1 s-1 and hydrolyzes PHM primarily at a single site (Lys20) with a kcat/Km of 1.9 x 10(4) M-1 s-1. Tryptase also rapidly hydrolyzes CGRP at two sites (Arg18 and Lys24) with a kcat/Km of 2.7 x 10(5) M-1 s-1. The tachykinins are not hydrolyzed by tryptase. These observations raise the possibility that tryptase-mediated degradation of the bronchodilators VIP and PHM combined with exaggerated mast cell release of tryptase may contribute to the increase in bronchial responsiveness and the decrease in immunoreactive VIP in airway nerves associated with asthma. The favorable rates of hydrolysis of CGRP suggest that tryptase may also terminate the effects of CGRP on bronchial and vascular smooth muscle tone and permeability.

Polymorphism and partial characterization of digestive enzymes in the spiny lobster Panulirus argus.

PubMed

Perera, Erick; Moyano, F J; Díaz, M; Perdomo-Morales, R; Montero-Alejo, V; Alonso, E; Carrillo, O; Galich, G S

2008-07-01

We characterized major digestive enzymes in Panulirus argus using a combination of biochemical assays and substrate-(SDS or native)-PAGE. Protease and amylase activities were found in the gastric juice while esterase and lipase activities were higher in the digestive gland. Trypsin-like activity was higher than chymotrypsin-like activity in the gastric juice and digestive gland. Stability and optimal conditions for digestive enzyme activities were examined under different pHs, temperature and ionic strength. The use of protease inhibitors showed the prevalence of serine proteases and metalloproteases. Results for serine proteases were corroborated by zymograms where several isotrypsins-like (17-21 kDa) and isochymotrypsin-like enzymes (23-38 kDa) were identified. Amylases (38-47 kDa) were detected in zymograms and a complex array of non-specific esterases isoenzymes was found in the digestive gland. Isoenzyme polymorphism was found for trypsin, amylase, and esterase. This study is the first to evidence the biochemical bases of the plasticity in feeding habits of P. argus. Distribution and properties of enzymes provided some indication on how the digestion takes place and constitute baseline data for further studies on the digestion physiology of spiny lobsters.

Structural Insights into the Protease-like Antigen Plasmodium falciparum SERA5 and Its Noncanonical Active-Site Serine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hodder, Anthony N.; Malby, Robyn L.; Clarke, Oliver B.

The sera genes of the malaria-causing parasite Plasmodium encode a family of unique proteins that are maximally expressed at the time of egress of parasites from infected red blood cells. These multi-domain proteins are unique, containing a central papain-like cysteine-protease fragment enclosed between the disulfide-linked N- and C-terminal domains. However, the central fragment of several members of this family, including serine repeat antigen 5 (SERA5), contains a serine (S596) in place of the active-site cysteine. Here we report the crystal structure of the central protease-like domain of Plasmodium falciparum SERA5, revealing a number of anomalies in addition to the putativemore » nucleophilic serine: (1) the structure of the putative active site is not conducive to binding substrate in the canonical cysteine-protease manner; (2) the side chain of D594 restricts access of substrate to the putative active site; and (3) the S{sub 2} specificity pocket is occupied by the side chain of Y735, reducing this site to a small depression on the protein surface. Attempts to determine the structure in complex with known inhibitors were not successful. Thus, despite having revealed its structure, the function of the catalytic domain of SERA5 remains an enigma.« less

A clip domain serine protease involved in moulting in the silkworm, Bombyx mori: cloning, characterization, expression patterns and functional analysis.

PubMed

Liu, H-W; Wang, L-L; Meng, Z; Tang, X; Li, Y-S; Xia, Q-Y; Zhao, P

2017-10-01

Clip domain serine proteases (CLIPs), characterized by one or more conserved clip domains, are essential components of extracellular signalling cascades in various biological processes, especially in innate immunity and the embryonic development of insects. Additionally, CLIPs may have additional non-immune functions in insect development. In the present study, the clip domain serine protease gene Bombyx mori serine protease 95 (BmSP95), which encodes a 527-residue protein, was cloned from the integument of B. mori. Bioinformatics analysis indicated that BmSP95 is a typical CLIP of the subfamily D and possesses a clip domain at the N terminus, a trypsin-like serine protease (tryp_spc) domain at the C terminus and a conserved proline-rich motif between these two domains. At the transcriptional level, BmSP95 is expressed in the integument during moulting and metamorphosis, and the expression pattern is consistent with the fluctuating 20-hydroxyecdysone (20E) titre in B. mori. At the translational level, BmSP95 protein is synthesized in the epidermal cells, secreted as a zymogen and activated in the moulting fluid. Immunofluorescence revealed that BmSP95 is distributed into the old endocuticle in the moulting stage. The expression of BmSP95 was upregulated by 20E. Moreover, expression of BmSP95 was downregulated by pathogen infection. RNA interference-mediated silencing of BmSP95 led to delayed moulting from pupa to moth. These results suggest that BmSP95 is involved in integument remodelling during moulting and metamorphosis. © 2017 The Royal Entomological Society.

Effects of an Anticarcinogenic Bowman-Birk Protease Inhibitor on Purified 20S Proteasome and MCF-7 Breast Cancer Cells

PubMed Central

Souza, Larissa da Costa; Camargo, Ricardo; Demasi, Marilene; Santana, Jaime Martins; de Freitas, Sonia Maria

2014-01-01

Proteasome inhibitors have been described as an important target for cancer therapy due to their potential to regulate the ubiquitin-proteasome system in the degradation pathway of cellular proteins. Here, we reported the effects of a Bowman-Birk-type protease inhibitor, the Black-eyed pea Trypsin/Chymotrypsin Inhibitor (BTCI), on proteasome 20S in MCF-7 breast cancer cells and on catalytic activity of the purified 20S proteasome from horse erythrocytes, as well as the structural analysis of the BTCI-20S proteasome complex. In vitro experiments and confocal microscopy showed that BTCI readily crosses the membrane of the breast cancer cells and co-localizes with the proteasome in cytoplasm and mainly in nucleus. Indeed, as indicated by dynamic light scattering, BTCI and 20S proteasome form a stable complex at temperatures up to 55°C and at neutral and alkaline pHs. In complexed form, BTCI strongly inhibits the proteolytic chymotrypsin-, trypsin- and caspase-like activities of 20S proteasome, indicated by inhibition constants of 10−7 M magnitude order. Besides other mechanisms, this feature can be associated with previously reported cytostatic and cytotoxic effects of BTCI in MCF-7 breast cancer cells by means of apoptosis. PMID:24475156

Studies of Inhibitory Mechanisms of Propeptide-Like Cysteine Protease Inhibitors

PubMed Central

Nga, Bui T. T.; Takeshita, Yuki; Yamamoto, Misa; Yamamoto, Yoshimi

2014-01-01

Mouse cytotoxic T-lymphocyte antigen-2α (CTLA-2α), Drosophila CTLA-2-like protein (crammer), and Bombyx cysteine protease inhibitor (BCPI) belong to a novel family of cysteine protease inhibitors (I29). Their inhibitory mechanisms were studied comparatively. CTLA-2α contains a cysteine residue (C75), which is essential for its inhibitory potency. The CTLA-2α monomer was converted to a disulfide-bonded dimer in vitro and in vivo. The dimer was fully inhibitory, but the monomer, which possessed a free thiol residue, was not. A disulfide-bonded CTLA-2α/cathepsin L complex was isolated, and a cathepsin L subunit with a molecular weight of 24,000 was identified as the interactive enzyme protein. Crammer also contains a cysteine residue (C72). Both dimeric and monomeric forms of crammer were inhibitory. A crammer mutant with Cys72 to alanine (C72A) was fully inhibitory, while the replacement of Gly73 with alanine (G73A) caused a significant loss in inhibitory potency, which suggests a different inhibition mechanism from CTLA-2α. BCPI does not contain cysteine residue. C-terminal region (L77-R80) of BCPI was essential for its inhibitory potency. CTLA-2α was inhibitory in the acidic pH condition but stabilized cathepsin L under neutral pH conditions. The different inhibition mechanisms and functional considerations of these inhibitors are discussed. PMID:25045530

Investigation on potential enzyme toxicity of clenbuterol to trypsin

NASA Astrophysics Data System (ADS)

Chai, Jun; Xu, Qifei; Dai, Jinping; Liu, Rutao

2013-03-01

Clenbuterol (CLB) is a kind of β2-adrenergic agonists which was illegally used as feed additives nowadays. The toxic interaction of CLB with trypsin, an important digestive enzyme, was studied in vitro using multi-spectroscopic methods and molecular modeling methods. CLB was proved to bind with trypsin in S1 pocket, forming a complex driven by the dominant force of H-bond. The binding constant was calculated to be 1.79887 × 105 L mol-1 at 289 K and 0.32584 × 105 L mol-1 at 310 K, respectively. The skeleton of trypsin became loosened and unfolded with the amino residues microenvironment changed. The secondary and tertiary structure of trypsin also varied. Molecular modeling studies illustrated specific display of the binding information and explained most of the experiment phenomena. The binding site of CLB induced the fluorescence quenching as well as inhibition of enzyme activity of trypsin. The study confirmed that CLB had potential toxicity on both the structure and function of trypsin and the effects enhanced with the increasing concentration of CLB.

Toll-like receptor-2 agonist-allergen coupling efficiently redirects Th2 cell responses and inhibits allergic airway eosinophilia.

PubMed

Krishnaswamy, Jayendra Kumar; Jirmo, Adan Chari; Baru, Abdul Mannan; Ebensen, Thomas; Guzmán, Carlos A; Sparwasser, Tim; Behrens, Georg M N

2012-12-01

Toll-like receptor (TLR) agonists beneficially modulate allergic airway inflammation. However, the efficiency of TLR agonists varies considerably, and their exact cellular mechanisms (especially of TLR 2/6 agonists) are incompletely understood. We investigated at a cellular level whether the administration of the pharmacologically improved TLR2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol (BPP) conjugated to antigenic peptide (BPP-OVA) could divert an existing Th2 response and influence airway eosinophilia. The effects of BPP-OVA on airway inflammation were assessed in a classic murine sensitization/challenge model and an adoptive transfer model, which involved the adoptive transfer of in vitro differentiated ovalbumin (OVA)-specific Th2 cells. Functional T-cell stimulation by lung dendritic cells (DCs) was determined both in vitro and in vivo, combined with a cytokine secretion analysis. A single mucosal application of BPP-OVA efficiently delivered antigen, led to TLR2-mediated DC activation, and resulted in OVA-specific T-cell proliferation via lung DCs in vivo. In alternative models of allergic airway disease, a single administration of BPP-OVA before OVA challenge (but not BPP alone) significantly reduced airway eosinophilia, most likely through altered antigen-specific T-cell stimulation via DCs. Analyses of adoptively transferred Th2-biased cells after BPP-OVA administration in vivo suggested that BPP-OVA guides antigen-specific Th2 cells to produce significantly higher amounts of IFN-γ upon allergen challenge. In conclusion, our data show for the first time that a single mucosal administration of a TLR 2/6 agonist-allergen conjugate can provoke IFN-γ responses in Th2-biased cells and alleviate allergic airway inflammation.

Synergistic Activity between Two Antifungal Proteins, the Plant Defensin NaD1 and the Bovine Pancreatic Trypsin Inhibitor

PubMed Central

Dawson, Charlotte S.; McKenna, James A.; Quimbar, Pedro; Hayes, Brigitte M. E.; van der Weerden, Nicole L.

2017-01-01

ABSTRACT Defensins are a large family of small, cationic, cysteine-rich proteins that are part of the defense arsenal that plants use for protection against potentially damaging fungal infections. The plant defensin NaD1 from Nicotiana alata is a potent antifungal protein that inhibits growth and kills a variety of fungal pathogens that affect both plant and animal (human) hosts. Some serine protease inhibitors have also been reported to be antifungal molecules, while others have no inhibitory activity against fungi. Here we describe the synergistic activity of the plant defensin NaD1 with a selection of serine protease inhibitors against the plant pathogens Fusarium graminearum and Colletotrichum graminicola and the animal pathogen Candida albicans. The synergistic activity was not related to the protease inhibitory activity of these molecules but may arise from activation of fungal stress response pathways. The bovine pancreatic trypsin inhibitor (BPTI) displayed the most synergy with NaD1. BPTI also acted synergistically with several other antifungal molecules. The observation that NaD1 acts synergistically with protease inhibitors provides the foundation for the design of transgenic plants with improved resistance to fungal disease. It also supports the possibility of naturally occurring accessory factors that function to enhance the activity of innate immunity peptides in biological systems. IMPORTANCE This work describes the increased activity of a natural antifungal peptide in the presence of another antifungal peptide from a different family. This is termed antifungal synergy. Synergy is important for decreasing the amount of antifungal molecule needed to control the disease. Traditionally, naturally occurring antifungal molecules are assayed in isolation. Identification of synergistic interactions between antifungal peptides means that their activities in a complex biological system are likely to be different from what we observe when examining them

Airway inflammation in cystic fibrosis: molecular mechanisms and clinical implications.

PubMed

Cohen-Cymberknoh, Malena; Kerem, Eitan; Ferkol, Thomas; Elizur, Arnon

2013-12-01

Airway epithelial cells and immune cells participate in the inflammatory process responsible for much of the pathology found in the lung of patients with cystic fibrosis (CF). Intense bronchial neutrophilic inflammation and release of proteases and oxygen radicals perpetuate the vicious cycle and progressively damage the airways. In vitro studies suggest that CF transmembrane conductance regulator (CFTR)-deficient airway epithelial cells display signalling abnormalities and aberrant intracellular processes which lead to transcription of inflammatory mediators. Several transcription factors, especially nuclear factor-κB, are activated. In addition, the accumulation of abnormally processed CFTR in the endoplasmic reticulum results in unfolded protein responses that trigger 'cell stress' and apoptosis leading to dysregulation of the epithelial cells and innate immune function in the lung, resulting in exaggerated and ineffective airway inflammation. Measuring airway inflammation is crucial for initiating treatment and monitoring its effect. No inflammatory biomarker predictive for the clinical course of CF lung disease is currently known, although neutrophil elastase seems to correlate with lung function decline. CF animal models mimicking human lung disease may provide an important insight into the pathogenesis of lung inflammation in CF and identify new therapeutic targets.

Orosomucoid-like 3 (ORMDL3) upregulates airway smooth muscle proliferation, contraction, and Ca2+ oscillations in asthma.

PubMed

Chen, Jun; Miller, Marina; Unno, Hirotoshi; Rosenthal, Peter; Sanderson, Michael J; Broide, David H

2017-09-07

Airway hyperresponsiveness is a major feature of asthma attributed predominantly to an extrinsic immune/inflammatory response increasing airway smooth muscle (ASM) contractility. We investigated whether increased ASM expression of orosomucoid-like 3 (ORMDL3), a gene on chromosome 17q21 highly linked to asthma, induced increased ASM proliferation and contractility in vitro and influenced airway contractility and calcium flux in ASM in precision-cut lung slices (PCLSs) from wild-type and hORMDL3 Zp3-Cre mice (which express increased levels of human ORMDL3 [hORMDL3]). Levels of ASM proliferation and contraction were assessed in ASM cells transfected with ORMDL3 in vitro. In addition, airway contractility and calcium oscillations were quantitated in ASM cells in PCLSs derived from naive wild-type and naive hORMDL3 Zp3-Cre mice, which do not have a blood supply. Increased ASM expression of ORMDL3 in vitro resulted in increased ASM proliferation and contractility. PCLSs derived from naive hORMDL3 Zp3-Cre mice, which do not have airway inflammation, exhibit increased airway contractility with increased calcium oscillations in ASM cells. Increased ASM ORMDL3 expression increases levels of ASM sarcoplasmic reticulum Ca 2+ ATPase 2b (SERCA2b), which increases ASM proliferation and contractility. Overall, these studies provide evidence that an intrinsic increase in ORMDL3 expression in ASM can induce increased ASM proliferation and contractility, which might contribute to increased airway hyperresponsiveness in the absence of airway inflammation in asthmatic patients. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Quorum-sensing inhibition abrogates the deleterious impact of Pseudomonas aeruginosa on airway epithelial repair.

PubMed

Ruffin, Manon; Bilodeau, Claudia; Maillé, Émilie; LaFayette, Shantelle L; McKay, Geoffrey A; Trinh, Nguyen Thu Ngan; Beaudoin, Trevor; Desrosiers, Martin-Yvon; Rousseau, Simon; Nguyen, Dao; Brochiero, Emmanuelle

2016-09-01

Chronic Pseudomonas aeruginosa lung infections are associated with progressive epithelial damage and lung function decline. In addition to its role in tissue injury, the persistent presence of P. aeruginosa-secreted products may also affect epithelial repair ability, raising the need for new antivirulence therapies. The purpose of our study was to better understand the outcomes of P. aeruginosa exoproducts exposure on airway epithelial repair processes to identify a strategy to counteract their deleterious effect. We found that P. aeruginosa exoproducts significantly decreased wound healing, migration, and proliferation rates, and impaired the ability of directional migration of primary non-cystic fibrosis (CF) human airway epithelial cells. Impact of exoproducts was inhibited after mutations in P. aeruginosa genes that encoded for the quorum-sensing (QS) transcriptional regulator, LasR, and the elastase, LasB, whereas impact was restored by LasB induction in ΔlasR mutants. P. aeruginosa purified elastase also induced a significant decrease in non-CF epithelial repair, whereas protease inhibition with phosphoramidon prevented the effect of P. aeruginosa exoproducts. Furthermore, treatment of P. aeruginosa cultures with 4-hydroxy-2,5-dimethyl-3(2H)-furanone, a QS inhibitor, abrogated the negative impact of P. aeruginosa exoproducts on airway epithelial repair. Finally, we confirmed our findings in human airway epithelial cells from patients with CF, a disease featuring P. aeruginosa chronic respiratory infection. These data demonstrate that secreted proteases under the control of the LasR QS system impair airway epithelial repair and that QS inhibitors could be of benefit to counteract the deleterious effect of P. aeruginosa in infected patients.-Ruffin, M., Bilodeau, C., Maillé, É., LaFayette, S. L., McKay, G. A., Trinh, N. T. N., Beaudoin, T., Desrosiers, M.-Y., Rousseau, S., Nguyen, D., Brochiero, E. Quorum-sensing inhibition abrogates the deleterious impact

Protease-activated receptor 2 agonist increases cell proliferation and invasion of human pancreatic cancer cells

PubMed Central

XIE, LIQUN; DUAN, ZEXING; LIU, CAIJU; ZHENG, YANMIN; ZHOU, JING

2015-01-01

The aim of this study was to determine the expression of protease-activated receptor 2 (PAR-2) in the human pancreatic cancer cell line SW1990, and to evaluate its effect on cell proliferation and invasion. The expression of PAR-2 protein and mRNA in SW1990 cells was determined by immunocytochemistry and reverse transcription polymerase chain reaction (PCR), respectively. MTT and cell invasion and migration assays, as well as semi-quantitative PCR and zymography analysis, were additionally performed. PAR-2 mRNA was significantly upregulated in the cells treated with trypsin or the PAR-2 activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) (P<0.01), but not in the Val-Lys-Gly-Ile-Leu-Ser group (P>0.05). Trypsin and SLIGKV significantly promoted SW1990 cell proliferation in a dose- and time-dependent manner (P<0.05). Compared with the control group, trypsin and SLIGKV significantly increased the mRNA expression (P<0.01) and gelatinolytic activity (P<0.01) of matrix metalloproteinase (MMP)-2. In conclusion, PAR-2 is expressed in SW1990 cells. PAR-2 activation may promote the invasion and migration of human pancreatic cancer cells by increasing MMP-2 expression. PMID:25452809

Peanut Seed Cultivars with Contrasting Resistance to Aspergillus parasiticus Colonization Display Differential Temporal Response of Protease Inhibitors.

PubMed

Müller, Virginia; Bonacci, Gustavo; Batthyany, Carlos; Amé, María V; Carrari, Fernando; Gieco, Jorge; Asis, Ramón

2017-04-01

Significant efforts are being made to minimize aflatoxin contamination in peanut seeds and one possible strategy is to understand and exploit the mechanisms of plant defense against fungal infection. In this study we have identified and characterized, at biochemical and molecular levels, plant protease inhibitors (PPIs) produced in peanut seeds of the resistant PI 337394 and the susceptible Forman cultivar during Aspergillus parasiticus colonization. With chromatographic methods and 2D-electrophoresis-mass spectrometry we have isolated and identified four variants of Bowman-Birk trypsin inhibitor (BBTI) and a novel Kunitz-type protease inhibitor (KPI) produced in response to A. parasiticus colonization. KPI was detected only in the resistant cultivar, while BBTI was produced in the resistant cultivar in a higher concentration than susceptible cultivar and with different isoforms. The kinetic expression of KPI and BBTI genes along with trypsin inhibitory activity was analyzed in both cultivars during infection. In the susceptible cultivar an early PPI activity response was associated with BBTI occurrence. Meanwhile, in the resistant cultivar a later response with a larger increase in PPI activity was associated with BBTI and KPI occurrence. The biological significance of PPI in seed defense against fungal infection was analyzed and linked to inhibitory properties on enzymes released by the fungus during infection, and to the antifungal effect of KPI.

Comparison of magnetic carboxymethyl chitosan nanoparticles and cation exchange resin for the efficient purification of lysine-tagged small ubiquitin-like modifier protease.

PubMed

Li, Junhua; Zhang, Yang; Shen, Fei; Yang, Yanjun

2012-10-15

A fusion tag that can be purified by the cheap ion-exchanger based on the ionic binding force may provide a cost-effective scheme over other affinity fusion tags. Small ubiquitin-like modifier (SUMO) protease derived from Saccharomyces cerevisiae was fused with a poly lysine tag containing 10 lysine residues at its C-terminus and then expressed in Escherichia coli. The ionic binding force provided by the ploy lysine tag allowed the selective recovery of the small ubiquitin-like modifier protease from recombinant E. coli cell extracts. A preliminary comparative study of the adsorption and elution of poly lysine tagged SUMO protease on Amberlite Cobalamion and magnetite carboxymethyl chitosan nanoparticles was performed. Amberlite Cobalamion and magnetite nanoparticles had the similar elution profile due to the common functional groups - carboxyl groups. The maximum dynamic adsorption capacity of Amberlite Cobalamion and magnetite nanoparticles reached 36.8 and 211.4 mg/g, respectively. The lysine-tagged protease can be simply purified by magnetite nanoparticles from cell extracts with higher purity than that by Amberlite Cobalamion. The superparamagnetic nanoparticles possess the advantages of highly specific, fast and excellent binding of a larger amount of lysine tagged SUMO modifier protease, and it is also easier to separate from the crude biological process liquors compared with the conventional separation techniques of polycationic amino acids fusion proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

Target validation of highly conserved Amblyomma americanum tick saliva serine protease inhibitor 19

PubMed Central

Kim, Tae K.; Radulovic, Zeljko; Mulenga, Albert

2016-01-01

Amblyomma americanum tick serine protease inhibitor (serpin, AAS) 19, is a highly conserved protein that is characterized by its functional domain being 100% conserved across tick species. We also reported that AAS19 was an immunogenic tick saliva protein with anti-haemostatic functions and an inhibitor of trypsin-like proteases including five of the eight serine protease factors in the blood clotting cascade. In this study the goal was to validate the importance of AAS19 in A. americanum tick physiology, assess immunogenicity and investigate tick vaccine efficacy of yeast-expressed recombinant (r) AAS19. We confirm that AAS19 is important to A. americanum fitness and blood meal feeding. AAS19 mRNA disruption by RNAi silencing caused ticks to obtain blood meals that were 50% smaller than controls, and treated ticks being morphologically deformed with 100% of the deformed ticks dying in incubation. We show that rAAS19 is highly immunogenic in that two 500 µg inoculations mixed with TiterMax Gold adjuvant provoked antibody titers of more than 1:320000 that specifically reacted with native AAS19 in unfed and partially fed tick tissue. Since AAS19 is injected into animals during tick feeding, we challenge infested immunized rabbits twice to test if tick infestations of immunized rabbits could act as booster. While in the first infestation significantly smaller tick blood meals were observed on one of the two immunized rabbits, smaller blood meals were observed on both rabbits, but 60% of ticks that engorged on immunized rabbits in the second infestation failed to lay eggs. It is notable that ticks fed faster on immunized animals despite obtaining smaller blood meals. We conclude that rAAS19 is a potential component of cocktail tick vaccine. PMID:26746129

Tri-domain Bifunctional Inhibitor of Metallocarboxypeptidases A and Serine Proteases Isolated from Marine Annelid Sabellastarte magnifica*

PubMed Central

Alonso-del-Rivero, Maday; Trejo, Sebastian A.; Reytor, Mey L.; Rodriguez-de-la-Vega, Monica; Delfin, Julieta; Diaz, Joaquin; González-González, Yamile; Canals, Francesc; Chavez, Maria Angeles; Aviles, Francesc X.

2012-01-01

This study describes a novel bifunctional metallocarboxypeptidase and serine protease inhibitor (SmCI) isolated from the tentacle crown of the annelid Sabellastarte magnifica. SmCI is a 165-residue glycoprotein with a molecular mass of 19.69 kDa (mass spectrometry) and 18 cysteine residues forming nine disulfide bonds. Its cDNA was cloned and sequenced by RT-PCR and nested PCR using degenerated oligonucleotides. Employing this information along with data derived from automatic Edman degradation of peptide fragments, the SmCI sequence was fully characterized, indicating the presence of three bovine pancreatic trypsin inhibitor/Kunitz domains and its high homology with other Kunitz serine protease inhibitors. Enzyme kinetics and structural analyses revealed SmCI to be an inhibitor of human and bovine pancreatic metallocarboxypeptidases of the A-type (but not B-type), with nanomolar Ki values. SmCI is also capable of inhibiting bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase in varying measures. When the inhibitor and its nonglycosylated form (SmCI N23A mutant) were overproduced recombinantly in a Pichia pastoris system, they displayed the dual inhibitory properties of the natural form. Similarly, two bi-domain forms of the inhibitor (recombinant rSmCI D1-D2 and rSmCI D2-D3) as well as its C-terminal domain (rSmCI-D3) were also overproduced. Of these fragments, only the rSmCI D1-D2 bi-domain retained inhibition of metallocarboxypeptidase A but only partially, indicating that the whole tri-domain structure is required for such capability in full. SmCI is the first proteinaceous inhibitor of metallocarboxypeptidases able to act as well on another mechanistic class of proteases (serine-type) and is the first of this kind identified in nature. PMID:22411994

Central domain of IL-33 is cleaved by mast cell proteases for potent activation of group-2 innate lymphoid cells

PubMed Central

Lefrançais, Emma; Duval, Anais; Mirey, Emilie; Roga, Stéphane; Espinosa, Eric; Cayrol, Corinne; Girard, Jean-Philippe

2014-01-01

Interleukin-33 (IL-33) is an alarmin cytokine from the IL-1 family. IL-33 activates many immune cell types expressing the interleukin 1 receptor-like 1 (IL1RL1) receptor ST2, including group-2 innate lymphoid cells (ILC2s, natural helper cells, nuocytes), the major producers of IL-5 and IL-13 during type-2 innate immune responses and allergic airway inflammation. IL-33 is likely to play a critical role in asthma because the IL33 and ST2/IL1RL1 genes have been reproducibly identified as major susceptibility loci in large-scale genome-wide association studies. A better understanding of the mechanisms regulating IL-33 activity is thus urgently needed. Here, we investigated the role of mast cells, critical effector cells in allergic disorders, known to interact with ILC2s in vivo. We found that serine proteases secreted by activated mast cells (chymase and tryptase) generate mature forms of IL-33 with potent activity on ILC2s. The major forms produced by mast cell proteases, IL-3395–270, IL-33107–270, and IL-33109–270, were 30-fold more potent than full-length human IL-331–270 for activation of ILC2s ex vivo. They induced a strong expansion of ILC2s and eosinophils in vivo, associated with elevated concentrations of IL-5 and IL-13. Murine IL-33 is also cleaved by mast cell tryptase, and a tryptase inhibitor reduced IL-33–dependent allergic airway inflammation in vivo. Our study identifies the central cleavage/activation domain of IL-33 (amino acids 66–111) as an important functional domain of the protein and suggests that interference with IL-33 cleavage and activation by mast cell and other inflammatory proteases could be useful to reduce IL-33–mediated responses in allergic asthma and other inflammatory diseases. PMID:25313073

Contribution of SRF, Elk-1, and myocardin to airway smooth muscle remodeling in heaves, an asthma-like disease of horses.

PubMed

Chevigny, Mylène; Guérin-Montpetit, Karine; Vargas, Amandine; Lefebvre-Lavoie, Josiane; Lavoie, Jean-Pierre

2015-07-01

Myocyte hyperplasia and hypertrophy contribute to the increased mass of airway smooth muscle (ASM) in asthma. Serum-response factor (SRF) is a transcription factor that regulates myocyte differentiation in vitro in vascular and intestinal smooth muscles. When SRF is associated with phosphorylated (p)Elk-1, it promotes ASM proliferation while binding to myocardin (MYOCD) leading to the expression of contractile elements in these tissues. The objective of this study was therefore to characterize the expression of SRF, pElk-1, and MYOCD in ASM cells from central and peripheral airways in heaves, a spontaneously occurring asthma-like disease of horses, and in controls. Six horses with heaves and five aged-matched controls kept in the same environment were studied. Nuclear protein expression of SRF, pElk-1, and MYOCD was evaluated in peripheral airways and endobronchial biopsies obtained during disease remission and after 1 and 30 days of naturally occurring antigenic exposure using immunohistochemistry and immunofluorescence techniques. Nuclear expression of SRF (P = 0.03, remission vs. 30 days) and MYOCD (P = 0.05, controls vs. heaves at 30 days) increased in the peripheral airways of horses with heaves during disease exacerbation, while MYOCD (P = 0.04, remission vs. 30 days) decreased in the central airways of control horses. No changes were observed in the expression of pElk-1 protein in either tissue. In conclusion, SRF and its cofactor MYOCD likely contribute to the hypertrophy of peripheral ASM observed in equine asthmatic airways, while the remodeling of the central airways is more static or involves different transcription factors. Copyright © 2015 the American Physiological Society.

JcTI-I: a novel trypsin inhibitor from Jatropha curcas seed cake with potential for bacterial infection treatment.

PubMed

Costa, Helen P S; Oliveira, Jose T A; Sousa, Daniele O B; Morais, Janne K S; Moreno, Frederico B; Monteiro-Moreira, Ana Cristina O; Viegas, Ricardo A; Vasconcelos, Ilka M

2014-01-01

Jatropha curcas seed cake is a low-value by-product resulting from biodiesel production. The seed cake is highly toxic, but it has great potential for biotechnology applications as it is a repository of biomolecules that could be important in agriculture, medicine, and industry. To explore this potential, a novel trypsin inhibitor called JcTI-I was purified by fractionation of the crude extract with trichloroacetic acid (2.5%, v/v) followed by affinity chromatography (Trypsin-Sepharose 4B) and molecular exclusion (Sephacryl S-200). Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration showed that JcTI-I has approximately 20.0~kDa. Mass spectrometry analysis revealed that the intact molecular mass of JcTI-I is 10.252~kDa. Moreover, JcTI-I is a glycoprotein with 6.4% (m/m) carbohydrates, pI of 6.6, N-terminal sequence similarity around 60% to plant albumins and high stability to heat, pH, and salinity. JcTI-I presented antibacterial activity against the human pathogenic bacteria Salmonella enterica subspecies enterica serovar choleraesuis and Staphylococcus aureus, with minimum inhibitory concentration less than 5~μg/mL. Furthermore, JcTI-I did have inhibitory activity against the serine proteases from the tested bacteria. Otherwise, no hemolytic activity of human erythrocytes and signs of acute toxicity to mice were observed for JcTI-I. The results demonstrate the benefits of J. curcas seed cake as a source of trypsin inhibitor with potential for biotechnological application as a new antimicrobial agent against human pathogenic bacteria.

JcTI-I: a novel trypsin inhibitor from Jatropha curcas seed cake with potential for bacterial infection treatment

PubMed Central

Costa, Helen P. S.; Oliveira, Jose T. A.; Sousa, Daniele O. B.; Morais, Janne K. S.; Moreno, Frederico B.; Monteiro-Moreira, Ana Cristina O.; Viegas, Ricardo A.; Vasconcelos, Ilka M.

2014-01-01

Jatropha curcas seed cake is a low-value by-product resulting from biodiesel production. The seed cake is highly toxic, but it has great potential for biotechnology applications as it is a repository of biomolecules that could be important in agriculture, medicine, and industry. To explore this potential, a novel trypsin inhibitor called JcTI-I was purified by fractionation of the crude extract with trichloroacetic acid (2.5%, v/v) followed by affinity chromatography (Trypsin-Sepharose 4B) and molecular exclusion (Sephacryl S-200). Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration showed that JcTI-I has approximately 20.0~kDa. Mass spectrometry analysis revealed that the intact molecular mass of JcTI-I is 10.252~kDa. Moreover, JcTI-I is a glycoprotein with 6.4% (m/m) carbohydrates, pI of 6.6, N-terminal sequence similarity around 60% to plant albumins and high stability to heat, pH, and salinity. JcTI-I presented antibacterial activity against the human pathogenic bacteria Salmonella enterica subspecies enterica serovar choleraesuis and Staphylococcus aureus, with minimum inhibitory concentration less than 5~μg/mL. Furthermore, JcTI-I did have inhibitory activity against the serine proteases from the tested bacteria. Otherwise, no hemolytic activity of human erythrocytes and signs of acute toxicity to mice were observed for JcTI-I. The results demonstrate the benefits of J. curcas seed cake as a source of trypsin inhibitor with potential for biotechnological application as a new antimicrobial agent against human pathogenic bacteria. PMID:24523715

Comparative structure-function characterization of the saposin-like domains from potato, barley, cardoon and Arabidopsis aspartic proteases.

PubMed

Bryksa, Brian C; Grahame, Douglas A; Yada, Rickey Y

2017-05-01

The present study characterized the aspartic protease saposin-like domains of four plant species, Solanum tuberosum (potato), Hordeum vulgare L. (barley), Cynara cardunculus L. (cardoon; artichoke thistle) and Arabidopsis thaliana, in terms of bilayer disruption and fusion, and structure pH-dependence. Comparison of the recombinant saposin-like domains revealed that each induced leakage of bilayer vesicles composed of a simple phospholipid mixture with relative rates Arabidopsis>barley>cardoon>potato. When compared for leakage of bilayer composed of a vacuole-like phospholipid mixture, leakage was approximately five times higher for potato saposin-like domain compared to the others. In terms of fusogenic activity, distinctions between particle size profiles were noted among the four proteins, particularly for potato saposin-like domain. Bilayer fusion assays in reducing conditions resulted in altered fusion profiles except in the case of cardoon saposin-like domain which was virtually unchanged. Secondary structure profiles were similar across all four proteins under different pH conditions, although cardoon saposin-like domain appeared to have higher overall helix structure. Furthermore, increases in Trp emission upon protein-bilayer interactions suggested that protein structure rearrangements equilibrated with half-times ranging from 52 to 120s, with cardoon saposin-like domain significantly slower than the other three species. Overall, the present findings serve as a foundation for future studies seeking to delineate protein structural features and motifs in protein-bilayer interactions based upon variability in plant aspartic protease saposin-like domain structures. Copyright © 2017 Elsevier B.V. All rights reserved.

Polyphenol fatty acid esters as serine protease inhibitors: a quantum-chemical QSAR analysis.

PubMed

Viskupicova, Jana; Danihelova, Martina; Majekova, Magdalena; Liptaj, Tibor; Sturdik, Ernest

2012-12-01

We investigated the ability of polyphenol fatty acid esters to inhibit the activity of serine proteases trypsin, thrombin, elastase and urokinase. Potent protease inhibition in micromolar range was displayed by rutin and rutin derivatives esterified with medium and long chain, mono- and polyunsaturated fatty acids (1e-m), followed by phloridzin and esculin esters with medium and long fatty acid chain length (2a-d, 3a-d), while unmodified compounds showed only little or no effect. QSAR study of the compounds tested provided the most significant parameters for individual inhibition activities, i.e. number of hydrogen bond donors for urokinase, molecular volume for thrombin, and solvation energy for elastase. According to the statistical analysis, the action of elastase inhibitors is opposed to those of urokinase and thrombin. Cluster analysis showed two groups of compounds: original polyphenols together with rutin esters with short fatty acid chain length and rutin esters with long fatty acid chain length.

Molecular and Biochemical Characterization of a Cathepsin B-Like Protease Family Unique to Trypanosoma congolense▿ †

PubMed Central

Mendoza-Palomares, Carlos; Biteau, Nicolas; Giroud, Christiane; Coustou, Virginie; Coetzer, Theresa; Authié, Edith; Boulangé, Alain; Baltz, Théo

2008-01-01

Cysteine proteases have been shown to be essential virulence factors and drug targets in trypanosomatids and an attractive antidisease vaccine candidate for Trypanosoma congolense. Here, we describe an important amplification of genes encoding cathepsin B-like proteases unique to T. congolense. More than 13 different genes were identified, whereas only one or two highly homologous genes have been identified in other trypanosomatids. These proteases grouped into three evolutionary clusters: TcoCBc1 to TcoCBc5 and TcoCBc6, which possess the classical catalytic triad (Cys, His, and Asn), and TcoCBs7 to TcoCBs13, which contains an unusual catalytic site (Ser, Xaa, and Asn). Expression profiles showed that members of the TcoCBc1 to TcoCBc5 and the TcoCBs7 to TcoCBs13 groups are expressed mainly in bloodstream forms and localize in the lysosomal compartment. The expression of recombinant representatives of each group (TcoCB1, TcoCB6, and TcoCB12) as proenzymes showed that TcoCBc1 and TcoCBc6 are able to autocatalyze their maturation 21 and 31 residues, respectively, upstream of the predicted start of the catalytic domain. Both displayed a carboxydipeptidase function, while only TcoCBc1 behaved as an endopeptidase. TcoCBc1 exhibited biochemical differences regarding inhibitor sensitivity compared to that of other cathepsin B-like proteases. Recombinant pro-TcoCBs12 did not automature in vitro, and the pepsin-matured enzyme was inactive in tests with cathepsin B fluorogenic substrates. In vivo inhibition studies using CA074Me (a cell-permeable cathepsin B-specific inhibitor) demonstrated that TcoCB are involved in lysosomal protein degradation essential for survival in bloodstream form. Furthermore, TcoCBc1 elicited an important immune response in experimentally infected cattle. We propose this family of proteins as a potential therapeutic target and as a plausible antigen for T. congolense diagnosis. PMID:18281598

Isolation and Biochemical Characterization of a New Thrombin-Like Serine Protease from Bothrops pirajai Snake Venom

PubMed Central

Zaqueo, Kayena D.; Kayano, Anderson M.; Simões-Silva, Rodrigo; Moreira-Dill, Leandro S.; Fernandes, Carla F. C.; Fuly, André L.; Maltarollo, Vinícius G.; Honório, Kathia M.; da Silva, Saulo L.; Acosta, Gerardo; Caballol, Maria Antonia O.; de Oliveira, Eliandre; Albericio, Fernando; Calderon, Leonardo A.; Soares, Andreimar M.; Stábeli, Rodrigo G.

2014-01-01

This paper presents a novel serine protease (SP) isolated from Bothrops pirajai, a venomous snake found solely in Brazil that belongs to the Viperidae family. The identified SP, named BpirSP-39, was isolated by three chromatographic steps (size exclusion, bioaffinity, and reverse phase chromatographies). The molecular mass of BpirSP-39 was estimated by SDS-PAGE and confirmed by mass spectrometry (39,408.32 Da). The protein was able to form fibrin networks, which was not observed in the presence of serine protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF). Furthermore, BpirSP-39 presented considerable thermal stability and was apparently able to activate factor XIII of the blood coagulation cascade, unlike most serine proteases. BpirSP-39 was capable of hydrolyzing different chromogenic substrates tested (S-2222, S-2302, and S-2238) while Cu2+ significantly diminished BspirSP-39 activity on the three tested substrates. The enzyme promoted platelet aggregation and also exhibited fibrinogenolytic, fibrinolytic, gelatinolytic, and amidolytic activities. The multiple alignment showed high sequence similarity to other thrombin-like enzymes from snake venoms. These results allow us to conclude that a new SP was isolated from Bothrops pirajai snake venom. PMID:24719874

Understanding serine proteases implications on Leishmania spp lifecycle.

PubMed

Alves, Carlos Roberto; Souza, Raquel Santos de; Charret, Karen Dos Santos; Côrtes, Luzia Monteiro de Castro; Sá-Silva, Matheus Pereira de; Barral-Veloso, Laura; Oliveira, Luiz Filipe Gonçalves; da Silva, Franklin Souza

2018-01-01

Serine proteases have significant functions over a broad range of relevant biological processes to the Leishmania spp lifecycle. Data gathered here present an update on the Leishmania spp serine proteases and the status of these enzymes as part of the parasite degradome. The serine protease genes (n = 26 to 28) in Leishmania spp, which encode proteins with a wide range of molecular masses (35 kDa-115 kDa), are described along with their degrees of chromosomal and allelic synteny. Amid 17 putative Leishmania spp serine proteases, only ∼18% were experimentally demonstrated, as: signal peptidases that remove the signal peptide from secretory pre-proteins, maturases of other proteins and with metacaspase-like activity. These enzymes include those of clans SB, SC and SF. Classical inhibitors of serine proteases are used as tools for the characterization and investigation of Leishmania spp. Endogenous serine protease inhibitors, which are ecotin-like, can act modulating host actions. However, crude or synthetic based-natural serine protease inhibitors, such as potato tuber extract, Stichodactyla helianthus protease inhibitor I, fukugetin and epoxy-α-lapachone act on parasitic serine proteases and are promising leishmanicidal agents. The functional interrelationship between serine proteases and other Leishmania spp proteins demonstrate essential functions of these enzymes in parasite physiology and therefore their value as targets for leishmaniasis treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

Kunitzins: Prototypes of a new class of protease inhibitor from the skin secretions of European and Asian frogs.

PubMed

Chen, Xiaole; Wang, He; Shen, Yue; Wang, Lei; Zhou, Mei; Chen, Tianbao; Shaw, Chris

2016-08-19

Amphibian skin secretions contain biologically-active compounds, such as anti-microbial peptides and trypsin inhibitors, which are used by biomedical researchers as a source of potential novel drug leads or pharmacological agents. Here, we report the application of a recently developed technique within our laboratory to "shotgun" clone the cDNAs encoding two novel but structurally-related peptides from the lyophilised skin secretions of one species of European frog, Rana esculenta and one species of Chinese frog, Odorrana schmackeri. Bioanalysis of the peptides established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17, which is a canonical Kunitz-type protease inhibitor motif (-CKAAFC-). Due to the presence of this structural attribute, these peptides were named kunitzin-RE (AAKIILNPKFRCKAAFC) and kunitzin-OS (AVNIPFKVHLRCKAAFC). Synthetic replicates of these two novel peptides were found to display a potent inhibitory activity against Escherichia coli but were ineffective at inhibiting the growth of Staphylococcus aureus and Candida albicans at concentrations up to 160 μM, and both showed little haemolytic activity at concentrations up to 120 μM. Subsequently, kunitzin-RE and kunitzin-OS were found to be a potent inhibitor of trypsin with a Ki of 5.56 μM and 7.56 μM that represent prototypes of a novel class of highly-attenuated amphibian skin protease inhibitor. Substitution of Lys-13, the predicted residue occupying the P1 position within the inhibitory loop, with Phe (F) resulted in decrease in trypsin inhibitor effectiveness and antimicrobial activity against Esherichia coli, but exhibits a potential inhibition activity against chymotrypsin. Copyright © 2016 Elsevier Inc. All rights reserved.

Genome-wide identification and expression profiling of serine proteases and homologs in the diamondback moth, Plutella xylostella (L.).

PubMed

Lin, Hailan; Xia, Xiaofeng; Yu, Liying; Vasseur, Liette; Gurr, Geoff M; Yao, Fengluan; Yang, Guang; You, Minsheng

2015-12-10

Serine proteases (SPs) are crucial proteolytic enzymes responsible for digestion and other processes including signal transduction and immune responses in insects. Serine protease homologs (SPHs) lack catalytic activity but are involved in innate immunity. This study presents a genome-wide investigation of SPs and SPHs in the diamondback moth, Plutella xylostella (L.), a globally-distributed destructive pest of cruciferous crops. A total of 120 putative SPs and 101 putative SPHs were identified in the P. xylostella genome by bioinformatics analysis. Based on the features of trypsin, 38 SPs were putatively designated as trypsin genes. The distribution, transcription orientation, exon-intron structure and sequence alignments suggested that the majority of trypsin genes evolved from tandem duplications. Among the 221 SP/SPH genes, ten SP and three SPH genes with one or more clip domains were predicted and designated as PxCLIPs. Phylogenetic analysis of CLIPs in P. xylostella, two other Lepidoptera species (Bombyx mori and Manduca sexta), and two more distantly related insects (Drosophila melanogaster and Apis mellifera) showed that seven of the 13 PxCLIPs were clustered with homologs of the Lepidoptera rather than other species. Expression profiling of the P. xylostella SP and SPH genes in different developmental stages and tissues showed diverse expression patterns, suggesting high functional diversity with roles in digestion and development. This is the first genome-wide investigation on the SP and SPH genes in P. xylostella. The characterized features and profiled expression patterns of the P. xylostella SPs and SPHs suggest their involvement in digestion, development and immunity of this species. Our findings provide a foundation for further research on the functions of this gene family in P. xylostella, and a better understanding of its capacity to rapidly adapt to a wide range of environmental variables including host plants and insecticides.

A novel serine protease with human fibrino(geno)lytic activities from Artocarpus heterophyllus latex.

PubMed

Siritapetawee, Jaruwan; Thumanu, Kanjana; Sojikul, Punchapat; Thammasirirak, Sompong

2012-07-01

A protease was isolated and purified from Artocarpus heterophyllus (jackfruit) latex and designated as a 48-kDa antimicrobial protease (AMP48) in a previous publication. In this work, the enzyme was characterized for more biochemical and medicinal properties. Enzyme activity of AMP48 was strongly inhibited by phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, indicating that the enzyme was a plant serine protease. The N-terminal amino acid sequences (A-Q-E-G-G-K-D-D-D-G-G) of AMP48 had no sequence similarity matches with any sequence databases of BLAST search and other plant serine protease. The secondary structure of this enzyme was composed of high α-helix (51%) and low β-sheet (9%). AMP48 had fibrinogenolytic activity with maximal activity between 55 and 60°C at pH 8. The enzyme efficiently hydrolyzed α followed by partially hydrolyzed β and γ subunits of human fibrinogen. In addition, the fibrinolytic activity was observed through the degradation products by SDS-PAGE and emphasized its activity by monitoring the alteration of secondary structure of fibrin clot after enzyme digestion using ATR-FTIR spectroscopy. This study presented the potential role to use AMP48 as antithrombotic for treatment thromboembolic disorders such as strokes, pulmonary emboli and deep vein thrombosis. Copyright © 2012 Elsevier B.V. All rights reserved.

Studies on a Novel Serine Protease of a ΔhapAΔprtV Vibrio cholerae O1 Strain and Its Role in Hemorrhagic Response in the Rabbit Ileal Loop Model

PubMed Central

Syngkon, Aurelia; Elluri, Sridhar; Koley, Hemanta; Rompikuntal, Pramod K.; Saha, Dhira Rani; Chakrabarti, Manoj K.; Bhadra, Rupak K.; Wai, Sun Nyunt; Pal, Amit

2010-01-01

Background Two well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL). Methodology/Principal Findings We partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/−0.3 n = 3), CHA6.8 (FA ratio 1.08+/−0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/−0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/−0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/−0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/−0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure. Conclusion Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model. PMID:20927349

Purification and molecular characterization of subtilisin-like alkaline protease BPP-A from Bacillus pumilus strain MS-1.

PubMed

Miyaji, T; Otta, Y; Nakagawa, T; Watanabe, T; Niimura, Y; Tomizuka, N

2006-03-01

The present study was conducted by screening zein-degrading bacteria in an attempt to obtain zein-degrading protease. Soil bacteria were screened by formation of a clear zone on zein plates. Characterization of a zein-degrading bacterium indicated a taxonomic affiliation to Bacillus pumilus, and was named MS-1 strain. The strain produced two different types of extracellular proteases, BPP-A and BPP-B. In this study, we purified and characterized BPP-A because it exhibited a higher ability to hydrolyze zein than BPP-B. When casein was used as the substrate, the optimal pH for BPP-A was 11.0. In BPP-A, zein was better substrate than casein at pH 13.0, whereas casein was better one than zein at pH 11.0. The bppA gene encoded a 383-amino acid pre-pro form of BPP-A, and mature BPP-A contained 275 amino acid residues. It was concluded that BPP-A belonged to the subtilisin family. A zein-degrading bacterium assigned to B. pumilus produced two different types of extracellular proteases, BPP-A and BPP-B. BPP-A exhibited an ability to hydrolyze zein in an extreme alkaline condition. This is a first report on screening for zein-degrading micro-organisms. The subtilisin-like protease BPP-A is possible to utilize as an industrial enzyme for the production of zein hydrolysates.

Characterization of a backbone cleavage product of BMS-196854 (Oncostatin M), a recombinant anti-inflammatory cytokine.

PubMed

Zhao, F; Stein, D J; Paborji, M; Cash, P W; Root, B J; Wei, Z; Knupp, C J

2001-01-01

BMS-196843 (Oncostatin M) is a therapeutic recombinant protein in development. Scale-up process changes led to unexpected instability of the bulk drug substance solution during storage. A product with an apparent higher MW than the parent protein was observed by the size-exclusion chromatography (SEC). This study was aimed to fully characterize the product and to identify a solution to stabilize the protein. SEC, SDS-PAGE, tryptic mapping, and N-terminal sequencing were performed to characterize the unknown product. The effect of pH, temperature, bulk concentration, and immobilized trypsin inhibitor on the degradation rate was studied to elucidate the mechanism and to identify stabilization strategies. Despite the apparent high MW indicated initially by SEC, the unknown was characterized to be a degradation product resulted from a backbone cleavage between residues Arg145-Gly146. The resulting fragments from the backbone cleavage were, however, still linked through an intramolecular disulfide bond. Thus, the final product had a more open structure with an increased hydrodynamic radius compared to the parent protein, which explains the initial SEC results. The site-specific backbone cleavage was suspected to be catalyzed by trypsin-like protease impurities in the bulk solution. The bulk drug substance solution was subsequently treated with immobilized soybean trypsin inhibitor, and the degradation rate was significantly reduced. Furthermore, increasing the solution pH from 5 to 8 led to an increase in the degradation rate, which was consistent with the expected pH dependency of trypsin activity. In addition, the effect of bulk concentration also supported the involvement of protease impurities rather than a spontaneous peptide bond hydrolysis reaction. Trace trypsin-like protease impurities led to an unusual site-specific backbone cleavage of BMS-196854. The proteolytic degradation can be minimized by treating the bulk solution with immobilized soybean trypsin

Trypsin impaired epithelial barrier function and induced IL-8 secretion through basolateral PAR-2: a lesson from a stratified squamous epithelial model.

PubMed

Shan, Jing; Oshima, Tadayuki; Chen, Xin; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

2012-11-15

Immune-mediated injury by the protease-activated receptor-2-interleukin-8 (PAR-2-IL8) pathway may underlie the development of gastroesophageal reflux disease (GERD). However, the localization of PAR-2 and the mechanism of PAR-2 activation remain unclear. This study aimed to address these questions on an esophageal stratified squamous epithelial model and in the human esophageal mucosa of GERD patients. Normal human esophageal epithelial cells were cultured with the air-liquid interface system to establish the model. SLIGKV-NH2 (PAR-2 synthetic agonist), trypsin (PAR-2 natural activator), and weak acid (pH 4, 5, and 6) were added to either the apical or basolateral compartment to evaluate their effects on transepithelial electrical resistance (TEER) and IL-8 production. PAR-2 localization was examined both in the cell model and biopsies from GERD patients by immunohistochemistry. Apical trypsin stimulation induced IL-8 accompanied by decreased TEER in vitro, whereas the effective concentration from the basolateral side was 10 times lower. SLIGKV-NH2 from basolateral but not apical stimulation induced IL-8 production. Apical weak acid stimulation did not influence TEER or IL-8 production. Immunohistochemistry showed intense reactivity of PAR-2 in the basal and suprabasal layers after stimulation with trypsin. A similar PAR-2 reactivity that was mainly located at the basal and suprabasal layers was detected in GERD patients. In conclusion, the activation of the PAR-2-IL-8 pathway probably occurred at the basal and suprabasal layers, while the esophageal epithelial barrier may influence the activation of PAR-2. Under proton pump inhibitor therapy, refluxed trypsin may remain active and be a potential agent in the pathogenesis of refractory GERD.

The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin‐like protease(s) in a subpopulation of microglia in neonatal rat brain

PubMed Central

Hirata, Hiroshi; Ohbuchi, Kengo; Nishi, Kentaro; Maeda, Akira; Kuniyasu, Akihiko; Yamada, Daisuke; Maeda, Takehiko; Tsuji, Akihiko; Sawada, Makoto

2016-01-01

To differentiate subtypes of microglia (MG), we developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG. The 9F5 showed high selectivity for this cell type in Western blot and immunocytochemical analyses and no cross‐reaction with rat peritoneal macrophages (Mφ). We identified the antigen molecule for 9F5: the 50‐ to 70‐kDa fragments of rat glycoprotein nonmetastatic melanoma protein B (GPNMB)/osteoactivin, which started at Lys170. In addition, 9F5 immunoreactivity with GPNMB depended on the activity of furin‐like protease(s). More important, rat type 1 MG expressed the GPNMB fragments, but type 2 MG and Mφ did not, although all these cells expressed mRNA and the full‐length protein for GPNMB. These results suggest that 9F5 reactivity with MG depends greatly on cleavage of GPNMB and that type 1 MG, in contrast to type 2 MG and Mφ, may have furin‐like protease(s) for GPNMB cleavage. In neonatal rat brain, amoeboid 9F5+ MG were observed in specific brain areas including forebrain subventricular zone, corpus callosum, and retina. Double‐immunοstaining with 9F5 antibody and anti‐Iba1 antibody, which reacts with MG throughout the CNS, revealed that 9F5+ MG were a portion of Iba1+ MG, suggesting that MG subtype(s) exist in vivo. We propose that 9F5 is a useful tool to discriminate between rat type 1 MG and other subtypes of MG/Mφ and to reveal the role of the GPNMB fragments during developing brain. GLIA 2016;64:1938–1961 PMID:27464357

Chitosan nanoparticles-trypsin interactions: Bio-physicochemical and molecular dynamics simulation studies.

PubMed

Salar, Safoura; Mehrnejad, Faramarz; Sajedi, Reza H; Arough, Javad Mohammadnejad

2017-10-01

Herein, we investigated the effect of the chitosan nanoparticles (CsNP) on the structure, dynamics, and activity of trypsin. The enzyme activity in complex with the nanoparticles slightly increased, which represents the interactions between the nanoparticles and the enzyme. The kinetic parameters of the enzyme, K m and k cat , increased after adding the nanoparticles, resulting in a slight increase in the catalytic efficiency (k cat /K m ). However, the effect of the nanoparticles on the kinetic stability of trypsin has not exhibited significant variations. Fluorescence spectroscopy did not show remarkable changes in the trypsin conformation in the presence of the nanoparticles. The circular dichroism (CD) spectroscopy results also revealed the secondary structure of trypsin attached to the nanoparticles slightly changed. Furthermore, we used molecular dynamics (MD) simulation to find more information about the interaction mechanisms between the nanoparticles and trypsin. The root mean square deviation (RMSD) of Cα atoms results have shown that in the presence of the nanoparticles, trypsin was stable. The simulation and the calculation of the binding free energy demonstrate that the nonpolar interactions are the most important forces for the formation of stable nanoparticle-trypsin complex. This study has explicitly elucidated that the nanoparticles have not considerable effect on the trypsin. Copyright © 2017. Published by Elsevier B.V.

Porphyrin Induced Laser Deactivation of Trypsinogen-Trypsin Conversion

NASA Astrophysics Data System (ADS)

Perido, Joanna; Brancaleon, Lorenzo

2015-03-01

Pancreatitis is caused by the inflammation of the pancreas, where the digestive enzyme trypsin is activated from the precursor enzyme trypsinogen while still in the pancreas. The presence of trypsin in the pancreas causes auto-activation of trypsinogen, resulting in greater inflammation and auto-digestion of the pancreas. In severe cases, this cascade effect can lead to organ failure, diabetes, and pancreatic cancer. Our hypothesis is that if trypsinogen is prevented from auto-activating into trypsin, then this cascade can be stopped. We propose to do this by inducing conformational changes in trypsinogen when bound to a photoactive porphyrin dye. Porphyrins are comprised of four linked heterocyclic groups forming a flat ring, and bind well with proteins such as trypsinogen. In this study we used spectroscopic techniques to probe the binding of meso-tetrakis (4-sulfonatephenyl) porphyrin (TSPP) to trypsinogen in vitro, as a preliminary step to then prompt and characterize conformational changes of trypsinogen through irradiation. If conformational changes are detected the trypsinogen will be tested for trypsin inactivation. This investigation may provide promising initial results to the possible use of porphyrins as an inhibitor of the self-activation of trypsinogen into trypsin, and a potential inhibitor of pancreatitis. MARC*U-STAR.

A thermostable trypsin inhibitor with antiproliferative activity from small pinto beans.

PubMed

Chan, Yau Sang; Zhang, Yanbo; Sze, Stephen Cho Wing; Ng, Tzi Bun

2014-08-01

Small pinto bean is a cultivar of Phaseolus vulgaris. It produces a 16-kDa trypsin inhibitor that could be purified using anion exchange and size chromatography. Q-Sepharose, Mono Q and Superdex 75 columns were employed for the isolation process. Small pinto bean trypsin inhibitor demonstrated moderate pH stability (pH 2-10) and marked heat stability, with its trypsin inhibitory activity largely retained after exposure to 100 °C for half an hour. The activity was abolished in the presence of dithiothreitol, in a dose-dependent manner, implying that disulfide bonds in small pinto bean trypsin inhibitor are crucial for the activity. The trypsin inhibitor showed a blocked N-terminus. The trypsin inhibitor only slightly inhibited the viability of breast cancer MCF7 and hepatoma HepG2 cells at 125 μM.

Interaction of sodium benzoate with trypsin by spectroscopic techniques

NASA Astrophysics Data System (ADS)

Mu, Yue; Lin, Jing; Liu, Rutao

2011-12-01

The toxicity of sodium benzoate to trypsin was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, UV-visible absorption spectroscopy and circular dichroism (CD) spectroscopy under mimic physiological conditions. Sodium benzoate could unfold trypsin by decreasing the β-sheet structure, which leads to more exposure of internal amino acid groups and the obvious intrinsic fluorescence quenching with the rising concentration of sodium benzoate. The results of spectroscopic measurements indicated that sodium benzoate changed the internal microenvironment of trypsin and induced the alteration of the whole molecule, which were performed toxic effects on the organism. Trypsin and sodium benzoate interacted with each other to produce a substance by van der Waals forces and hydrogen bond, the model of which was shown by AutoDock software.

Evolution of a mass spectrometry-grade protease with PTM-directed specificity.

PubMed

Tran, Duc T; Cavett, Valerie J; Dang, Vuong Q; Torres, Héctor L; Paegel, Brian M

2016-12-20

Mapping posttranslational modifications (PTMs), which diversely modulate biological functions, represents a significant analytical challenge. The centerpiece technology for PTM site identification, mass spectrometry (MS), requires proteolytic cleavage in the vicinity of a PTM to yield peptides for sequencing. This requirement catalyzed our efforts to evolve MS-grade mutant PTM-directed proteases. Citrulline, a PTM implicated in epigenetic and immunological function, made an ideal first target, because citrullination eliminates arginyl tryptic sites. Bead-displayed trypsin mutant genes were translated in droplets, the mutant proteases were challenged to cleave bead-bound fluorogenic probes of citrulline-dependent proteolysis, and the resultant beads (1.3 million) were screened. The most promising mutant efficiently catalyzed citrulline-dependent peptide bond cleavage (k cat /K M = 6.9 × 10 5 M -1 ⋅s -1 ). The resulting C-terminally citrullinated peptides generated characteristic isotopic patterns in MALDI-TOF MS, and both a fragmentation product y 1 ion corresponding to citrulline (176.1030 m/z) and diagnostic peak pairs in the extracted ion chromatograms of LC-MS/MS analysis. Using these signatures, we identified citrullination sites in protein arginine deiminase 4 (12 sites) and in fibrinogen (25 sites, two previously unknown). The unique mass spectral features of PTM-dependent proteolytic digest products promise a generalized PTM site-mapping strategy based on a toolbox of such mutant proteases, which are now accessible by laboratory evolution.

Protease activation during in vivo pancreatitis is dependent on calcineurin activation.

PubMed

Shah, Ahsan U; Sarwar, Amna; Orabi, Abrahim I; Gautam, Samir; Grant, Wayne M; Park, Alexander J; Shah, Adnan U; Liu, Jun; Mistry, Pramod K; Jain, Dhanpat; Husain, Sohail Z

2009-11-01

The premature activation of digestive proenzymes, specifically proteases, within the pancreatic acinar cell is an early and critical event during acute pancreatitis. Our previous studies demonstrate that this activation requires a distinct pathological rise in cytosolic Ca(2+). Furthermore, we have shown that a target of aberrant Ca(2+) in acinar cells is the Ca(2+)/calmodulin-dependent phosphatase calcineurin (PP2B). In this study, we hypothesized that PP2B mediates in vivo protease activation and pancreatitis severity. To test this, pancreatitis was induced in mice over 8 h by administering hourly intraperitoneal injections of the cholecystokinin analog caerulein (50 microg/kg). Treatment with the PP2B inhibitor FK506 at 1 and 8 h after pancreatitis induction reduced trypsin activities by greater than 50% (P < 0.005). Serum amylase and IL-6 was reduced by 86 and 84% relative to baseline (P < 0.0005) at 8 h, respectively. Histological severity of pancreatitis, graded on the basis of pancreatic edema, acinar cell vacuolization, inflammation, and apoptosis, was reduced early in the course of pancreatitis. Myeloperoxidase activity from both pancreas and lung was reduced by 93 and 83% relative to baseline, respectively (P < 0.05). These data suggest that PP2B is an important target of the aberrant acinar cell Ca(2+) rise associated with pathological protease activation and pancreatitis.

Transient removal of proflavine inhibition of bovine beta-trypsin by the bovine basic pancreatic trypsin inhibitor (Kunitz). A case for "chronosteric effects".

PubMed

Antonini, E; Ascenzi, P; Bolognesi, M; Menegatti, E; Guarneri, M

1983-04-25

The formation of the bovine beta-trypsin-bovine basic pancreatic trypsin inhibitor (Kunitz) (BPTI) complex was monitored, making use of three different signals: proflavine displacement, optical density changes in the ultraviolet region, and the loss of the catalytic activity. The rates of the reactions indicated by the three different signals were similar at neutral pH, but diverged at low pH. At pH 3.50, proflavine displacement precedes the optical density changes in the ultraviolet and the loss of enzyme activity by several orders of magnitude in time (Antonini, E., Ascenzi, P., Menegatti, E., and Guarneri, M. (1983) Biopolymers 22, 363-375). These data indicated that the bovine beta-trypsin-BPTI complex formation is a multistage process and led to the prediction that, at pH 3.50, BPTI addition to the bovine beta-trypsin-proflavine complex would remove proflavine inhibition and the enzyme would recover transiently its catalytic activity before being irreversibly inhibited by completion of BPTI binding. The kinetic evidences, by completion of BPTI binding. The kinetic evidences, here shown, verified this prediction, indicating that during the bovine beta-trypsin-BPTI complex formation one transient intermediate occurs, which is not able to bind proflavine but may bind and hydrolyze the substrate. Thus, the observed peculiar catalytic behavior is in line with the proposed reaction mechanism for the bovine beta-trypsin-BPTI complex formation, which postulates a sequence of distinct polar and apolar interactions at the contact area.

Interaction of sodium benzoate with trypsin by spectroscopic techniques.

PubMed

Mu, Yue; Lin, Jing; Liu, Rutao

2011-12-01

The toxicity of sodium benzoate to trypsin was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, UV-visible absorption spectroscopy and circular dichroism (CD) spectroscopy under mimic physiological conditions. Sodium benzoate could unfold trypsin by decreasing the β-sheet structure, which leads to more exposure of internal amino acid groups and the obvious intrinsic fluorescence quenching with the rising concentration of sodium benzoate. The results of spectroscopic measurements indicated that sodium benzoate changed the internal microenvironment of trypsin and induced the alteration of the whole molecule, which were performed toxic effects on the organism. Trypsin and sodium benzoate interacted with each other to produce a substance by van der Waals forces and hydrogen bond, the model of which was shown by AutoDock software. Copyright © 2011 Elsevier B.V. All rights reserved.

SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme.

PubMed

Békés, Miklós; Rut, Wioletta; Kasperkiewicz, Paulina; Mulder, Monique P C; Ovaa, Huib; Drag, Marcin; Lima, Christopher D; Huang, Tony T

2015-06-01

Ubiquitin (Ub) and the Ub-like (Ubl) modifier interferon-stimulated gene 15 (ISG15) participate in the host defence of viral infections. Viruses, including the severe acute respiratory syndrome human coronavirus (SARS hCoV), have co-opted Ub-ISG15 conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub-ISG15-conjugated host proteins. In the present study, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle East respiratory syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that, similar to SARS PLpro, MERS PLpro is both a deubiquitinating (DUB) and a deISGylating enzyme. Further analysis of the intrinsic DUB activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, whereas SARS PLpro prefers to cleave Lys48-linked polyUb chains. Secondly, MERS PLpro cleaves polyUb chains in a 'mono-distributive' manner (one Ub at a time) and SARS PLpro prefers to cleave Lys48-linked polyUb chains by sensing a di-Ub moiety as a minimal recognition element using a 'di-distributive' cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP (Ub-specific protease)-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help to identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses.

NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease.

PubMed

Su, Xun-Cheng; Ozawa, Kiyoshi; Yagi, Hiromasa; Lim, Siew P; Wen, Daying; Ekonomiuk, Dariusz; Huang, Danzhi; Keller, Thomas H; Sonntag, Sebastian; Caflisch, Amedeo; Vasudevan, Subhash G; Otting, Gottfried

2009-08-01

The two-component NS2B-NS3 protease of West Nile virus is essential for its replication and presents an attractive target for drug development. Here, we describe protocols for the high-yield expression of stable isotope-labelled samples in vivo and in vitro. We also describe the use of NMR spectroscopy to determine the binding mode of new low molecular mass inhibitors of the West Nile virus NS2B-NS3 protease which were discovered using high-throughput in vitro screening. Binding to the substrate-binding sites S1 and S3 is confirmed by intermolecular NOEs and comparison with the binding mode of a previously identified low molecular mass inhibitor. Our results show that all these inhibitors act by occupying the substrate-binding site of the protease rather than by an allosteric mechanism. In addition, the NS2B polypeptide chain was found to be positioned near the substrate-binding site, as observed previously in crystal structures of the protease in complex with peptide inhibitors or bovine pancreatic trypsin inhibitor. This indicates that the new low molecular mass compounds, although inhibiting the protease, also promote the proteolytically active conformation of NS2B, which is very different from the crystal structure of the protein without inhibitor.

[Comparative analysis of sensitivity of proteases (chymotrypsin and trypsin) and cholinesterases of different origin to some organophosphorus inhibitors].

PubMed

Rozengart, E V

2009-01-01

The antichymotrypsin, antitrypsin, and anticholinesterase efficiencies of four homologous series of organophosphorus inhibitors are compared: O-ethyl-S-(n-alkyl)methylthiophosphonates, O-(n-alkyl)-S-(n-butyl)methylthiophosphonates, O-(n-alkyl)-S-beta-(ethylmercaptoethylene)methylthiophosphonates, and their methylsulfomethylates. As sources of a-chymotrypsin and trypsin, commercial compounds of Worthington Biochemical Corporation and Leningrad Myasokombinat were tested. Bimolecular constant of the reaction rate was used as the measure of antienzyme efficiency. In all cases, the antichymotrypsin efficiency was lower, while the antitrypsin--essentially higher than the anticholinesterase activity of the studied inhibitors. These differences were found to much depend both on the inhibitor structure and on nature of the cholinesterase compounds.

The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin-like protease(s) in a subpopulation of microglia in neonatal rat brain.

PubMed

Kawahara, Kohichi; Hirata, Hiroshi; Ohbuchi, Kengo; Nishi, Kentaro; Maeda, Akira; Kuniyasu, Akihiko; Yamada, Daisuke; Maeda, Takehiko; Tsuji, Akihiko; Sawada, Makoto; Nakayama, Hitoshi

2016-11-01

To differentiate subtypes of microglia (MG), we developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG. The 9F5 showed high selectivity for this cell type in Western blot and immunocytochemical analyses and no cross-reaction with rat peritoneal macrophages (Mφ). We identified the antigen molecule for 9F5: the 50- to 70-kDa fragments of rat glycoprotein nonmetastatic melanoma protein B (GPNMB)/osteoactivin, which started at Lys(170) . In addition, 9F5 immunoreactivity with GPNMB depended on the activity of furin-like protease(s). More important, rat type 1 MG expressed the GPNMB fragments, but type 2 MG and Mφ did not, although all these cells expressed mRNA and the full-length protein for GPNMB. These results suggest that 9F5 reactivity with MG depends greatly on cleavage of GPNMB and that type 1 MG, in contrast to type 2 MG and Mφ, may have furin-like protease(s) for GPNMB cleavage. In neonatal rat brain, amoeboid 9F5+ MG were observed in specific brain areas including forebrain subventricular zone, corpus callosum, and retina. Double-immunοstaining with 9F5 antibody and anti-Iba1 antibody, which reacts with MG throughout the CNS, revealed that 9F5+ MG were a portion of Iba1+ MG, suggesting that MG subtype(s) exist in vivo. We propose that 9F5 is a useful tool to discriminate between rat type 1 MG and other subtypes of MG/Mφ and to reveal the role of the GPNMB fragments during developing brain. GLIA 2016;64:1938-1961. © 2016 The Authors. Glia Published by Wiley Periodicals, Inc.

In vitro assessment of phthalate acid esters-trypsin complex formation.

PubMed

Chi, Zhenxing; Zhao, Jing; Li, Weiguo; Araghi, Arash; Tan, Songwen

2017-10-01

In this work, interactions of three phthalate acid esters (PAEs), including dimethyl phthalate (DMP), diethyl phthalate (DEP) and dibutyl phthalate (DBP), with trypsin have been studied in vitro, under simulated physiological conditions using multi-spectroscopic techniques and molecular modeling. The results show that these PAEs can bind to the trypsin, forming trypsin-PAEs complexes, mainly via hydrophobic interactions, with the affinity order of DMP > DEP > DBP. Binding to the PAEs is found to result in molecular deformation of trypsin. The modeling results suggest that only DBP can bind with the amino acid residues of the catalytic triad and S1 binding pocket of trypsin, leading to potential competitive enzyme inhibition. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs

The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. colimore » α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.« less

Interaction of methotrexate with trypsin analyzed by spectroscopic and molecular modeling methods

NASA Astrophysics Data System (ADS)

Wang, Yanqing; Zhang, Hongmei; Cao, Jian; Zhou, Qiuhua

2013-11-01

Trypsin is one of important digestive enzymes that have intimate correlation with human health and illness. In this work, the interaction of trypsin with methotrexate was investigated by spectroscopic and molecular modeling methods. The results revealed that methotrexate could interact with trypsin with about one binding site. Methotrexate molecule could enter into the primary substrate-binding pocket, resulting in inhibition of trypsin activity. Furthermore, the thermodynamic analysis implied that electrostatic force, hydrogen bonding, van der Waals and hydrophobic interactions were the main interactions for stabilizing the trypsin-methotrexate system, which agreed well with the results from the molecular modeling study.

The highly antigenic 53/25 kDa Taenia solium protein fraction with cathepsin-L like activity is present in the oncosphere/cysticercus and induces non-protective IgG antibodies in pigs

PubMed Central

Zimic, Mirko; Pajuelo, Mónica; Gilman, Robert H.; Gutiérrez, Andrés H.; Rueda, Luis D.; Flores, Myra; Chile, Nancy; Verástegui, Manuela; Gonzalez, Armando; García, Héctor H.; Sheen, Patricia

2011-01-01

Cathepsin L-like proteases are secreted by several parasites including Taenia solium. The mechanism used by T. solium oncospheres to degrade and penetrate the intestine and infect the host is incompletely understood. It is assumed that intestinal degradation is driven by the proteolytic activity of enzymes secreted by the oncosphere. Blocking the proteolytic activity by an antibody response would prevent the oncosphere penetration and further infection. Serine and cysteine proteases including chymotrypsin, trypsin, elastase, and cathepsin L, are secreted by T. solium and Taenia saginata oncospheres when cultured in vitro, being potential vaccine candidates. However, the purification of a sufficient quantity of proteases secreted by oncospheres to conduct a vaccine trial is costly and lengthy. A 53/25 kDa cathepsin L-like fraction partially purified from T. solium cyst fluid was described previously as an important antigen for immunodiagnostics. In this study we found that this antigen is present in the T. solium oncosphere and is also secreted by the cysticercus. This protein fraction was tested for its ability to protect pigs against an oral challenge with T. solium oncospheres in a vaccine trial. IgG antibodies against the 53/25 kDa cathepsin L-like protein fraction were elicited in the vaccinated animals but did not confer protection. PMID:22119017

The role of proteinase 3 (PR3) and the protease-activated receptor-2 (PAR-2) pathway in dendritic cell (DC) maturation of human-DC-like monocytes and murine DC.

PubMed

Jiang, Bo; Grage-Griebenow, Evelin; Csernok, Elena; Butherus, Kristine; Ehlers, Stefan; Gross, Wolfgang L; Holle, Julia U

2010-01-01

The aim of the study was to assess PAR-2 expression on dendritic cell (DC) subsets and other immune cells of Wegener's granulomatosis (WG) patients and healthy controls (HC) and to investigate whether Proteinase 3 (PR3, a serine protease which can activate PAR2) induces maturation of human DC-like monocytes and murine Flt-3 ligand- and GM-CSF-generated DC. Human peripheral blood cells including DC subsets and Flt-3l- and GM-CSF-generated mouse DC were analysed for expression of PAR-2 and DC maturation markers by flow cytometry before and after stimulation with PR3, trypsin, PAR-2 agonist or LPS for 24 h. There was no difference of PAR-2 expression on PMNs, monocytes, lymphocytes and DC between all WG samples and HC. However, in inactive WG, expression of PAR-2 was downregulated on the cell surface of PMNs, monocytes, lymphocytes, and CD11c+DC compared to active WG and HC. PR3 and PAR2-agonists did not induce upregulation of PAR-2 or maturation markers of human DC-like monocytes in WG and HC. Likewise, murine PR3 did not induce upregulation of PAR-2 or maturation markers in murine DC. PAR-2 expression is downregulated on human peripheral blood cells including CD11c+ DC in inactive WG compared to active WG and HC, possibly reflecting a non-activated status of these cells in inactive disease. PR3 and PAR-2- agonists did not induce maturation of human ex vivo DC-like monocytes in WG and HC and of murine DC, suggesting this pathway is not singularly involved in the maturation of these cell subsets.

Synthesis, modification and docking studies of 5-sulfonyl isatin derivatives as SARS-CoV 3C-like protease inhibitors.

PubMed

Liu, Wei; Zhu, He-Min; Niu, Guo-Jun; Shi, En-Zhi; Chen, Jie; Sun, Bo; Chen, Wei-Qiang; Zhou, Hong-Gang; Yang, Cheng

2014-01-01

The Severe Acute Respiratory Syndrome (SARS) is a serious life-threatening and strikingly mortal respiratory illness caused by SARS-CoV. SARS-CoV which contains a chymotrypsin-like main protease analogous to that of the main picornavirus protease, 3CL(pro). 3CL(pro) plays a pivotal role in the viral replication cycle and is a potential target for SARS inhibitor development. A series of isatin derivatives as possible SARS-CoV 3CL(pro) inhibitors was designed, synthesized, and evaluated by in vitro protease assay using fluorogenic substrate peptide, in which several showed potent inhibition against the 3CL(pro). Structure-activity relationship was analyzed, and possible binding interaction modes were proposed by molecular docking studies. Among all compounds, 8k₁ showed most potent inhibitory activity against 3CL(pro) (IC₅₀=1.04 μM). These results indicated that these inhibitors could be potentially developed into anti-SARS drugs. Copyright © 2013 Elsevier Ltd. All rights reserved.

Nma111p, the pro-apoptotic HtrA-like nuclear serine protease in Saccharomyces cerevisiae: a short survey.

PubMed

Fahrenkrog, Birthe

2011-10-01

The baker's yeast, Saccharomyces cerevisiae, is also capable of undergoing programmed cell death or apoptosis, for example in response to viral infection as well as during chronological and replicative aging. Intrinsically, programmed cell death in yeast can be induced by, for example, H2O2, acetic acid or the mating-type pheromone. A number of evolutionarily conserved apoptosis-regulatory proteins have been identified in yeast, one of which is the HtrA (high-temperature requirement A)-like serine protease Nma111p (Nma is nuclear mediator of apoptosis). Nma111p is a nuclear serine protease of the HtrA family, which targets Bir1p, the only known inhibitor-of-apoptosis protein in yeast. Nma111p mediates apoptosis in a serine-protease-dependent manner and exhibits its activity exclusively in the nucleus. How the activity of Nma111p is regulated has remained largely elusive, but some evidence points to a control by phosphorylation. Current knowledge of Nma111p's function in apoptosis will be discussed in the present review.

Identification of essential genes of Pseudomonas aeruginosa for its growth in airway mucus.

PubMed

Alrahman, Mohammed Abd; Yoon, Sang Sun

2017-01-01

Pseudomonas aeruginosa has been identified as an important causative agent of airway infection, mainly in cystic fibrosis. This disease is characterized by defective mucociliary clearance induced in part by mucus hyper-production. Mucin is a major component of airway mucus and is heavily O-glycosylated, with a protein backbone. Airway infection is known to be established with bacterial adhesion to mucin. However, the genes involved in mucin degradation or utilization remain elusive. In this study, we sought to provide a genetic basis of P. aeruginosa airway growth by identifying those genes. First, using RNASeq analyses, we compared genome-wide expression profiles of PAO1, a prototype P. aeruginosa laboratory strain, grown in M9-mucin (M9M) and M9-glucose (M9G) media. Additionally, a PAO1 transposon (Tn) insertion mutants library was screened for mutants defective in growth in M9M medium. One mutant with a Tn insertion in the xcpU gene (PA3100) was determined to exhibit faulty growth in M9M medium. This gene contributes to the type II secretion system, suggesting that P. aeruginosa uses this secretion system to produce a number of proteins to break down and assimilate the mucin molecule. Furthermore, we screened the PAO1 genome for genes with protease activity. Of 13 mutants, one with mutation in PA3247 gene exhibited defective growth in M9M, suggesting that the PA3247-encoded protease plays a role in mucin utilization. Further mechanistic dissection of this particular process will reveal new drug targets, the inhibition of which could control recalcitrant P. aeruginosa infections.

Molecular glues for manipulating enzymes: trypsin inhibition by benzamidine-conjugated molecular glues† †Electronic supplementary information (ESI) available: Synthesis of TEG–BA, Gluen–BA, mGluen–BA and Gluen–Ph; 1H NMR, 13C NMR, MALDI-TOF MS, electronic absorption, and CD spectra; zeta potential distributions; SLS plots; DLS histograms; and related experimental procedures. See DOI: 10.1039/c5sc00524h Click here for additional data file.

PubMed Central

Mogaki, Rina

2015-01-01

Water-soluble bioadhesive polymers bearing multiple guanidinium ion (Gu+) pendants at their side-chain termini (Gluen–BA, n = 10 and 29) that were conjugated with benzamidine (BA) as a trypsin inhibitor were developed. The Gluen–BA molecules are supposed to adhere to oxyanionic regions of the trypsin surface, even in buffer, via a multivalent Gu+/oxyanion salt-bridge interaction, such that their BA group properly blocks the substrate-binding site. In fact, Glue10–BA and Glue29–BA exhibited 35- and 200-fold higher affinities for trypsin, respectively, than a BA derivative without the glue moiety (TEG–BA). Most importantly, Glue10–BA inhibited the protease activity of trypsin 13-fold more than TEG–BA. In sharp contrast, mGlue27–BA, which bears 27 Gu+ units along the main chain and has a 5-fold higher affinity than TEG–BA for trypsin, was inferior even to TEG–BA for trypsin inhibition. PMID:28706668

Identification and biochemical characterization of small-molecule inhibitors of west nile virus serine protease by a high-throughput screen.

PubMed

Mueller, Niklaus H; Pattabiraman, Nagarajan; Ansarah-Sobrinho, Camilo; Viswanathan, Prasanth; Pierson, Theodore C; Padmanabhan, R

2008-09-01

West Nile virus and dengue virus are mosquito-borne flaviviruses that cause a large number of human infections each year. No vaccines or chemotherapeutics are currently available. These viruses encode a serine protease that is essential for polyprotein processing, a required step in the viral replication cycle. In this study, a high-throughput screening assay for the West Nile virus protease was employed to screen approximately 32,000 small-molecule compounds for identification of inhibitors. Lead inhibitor compounds with three distinct core chemical structures (1 to 3) were identified. In a secondary screening of selected compounds, two compounds, belonging to the 8-hydroxyquinoline family (compounds A and B) and containing core structure 1, were identified as potent inhibitors of the West Nile virus protease, with K(i) values of 3.2 +/- 0.3 microM and 3.4 +/- 0.6 microM, respectively. These compounds inhibited the dengue virus type 2 protease with K(i) values of 28.6 +/- 5.1 microM and 30.2 +/- 8.6 microM, respectively, showing some selectivity in the inhibition of these viral proteases. However, the compounds show no inhibition of cellular serine proteases, trypsin, or factor Xa. Kinetic analysis and molecular docking of compound B onto the known crystal structure of the West Nile virus protease indicate that the inhibitor binds in the substrate-binding cleft. Furthermore, compound B was capable of inhibiting West Nile virus RNA replication in cultured Vero cells (50% effective concentration, 1.4 +/- 0.4 microM; selectivity index, 100), presumably by inhibition of polyprotein processing.

Biochemical analysis of a papain-like protease isolated from the latex of Asclepias curassavica L.

PubMed

Liggieri, Constanza; Obregon, Walter; Trejo, Sebastian; Priolo, Nora

2009-02-01

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain c-II) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The Nterminal sequence (LPSFVDWRQKGVVFPIRNQGQCGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-alpha-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-alpha-CBZ-L-Gln-p-nitrophenyl ester as substrate: K(m)=0.1634 mM, k(cat)=121.48 s(-1), and k(cat)/K(m)=7.4 x 10(5) s(-1)/mM.

Trypsin Inhibitors from Cajanus cajan and Phaseolus limensis Possess Antioxidant, Anti-Inflammatory, and Antibacterial Activity.

PubMed

Shamsi, Tooba Naz; Parveen, Romana; Afreen, Sumbul; Azam, Mudasser; Sen, Priyankar; Sharma, Yamini; Haque, Qazi Mohd Rizwanul; Fatma, Tasneem; Manzoor, Nikhat; Fatima, Sadaf

2018-01-18

Protease inhibitors are one of the most promising and investigated subjects for their role in pharmacognostic and pharmacological studies. This study aimed to investigate antioxidant, anti-inflammatory, and antimicrobial activities of trypsin inhibitors (TIs) from two plant sources (Cajanus cajan and Phaseolus limensis). TI was purified from C. cajan (PUSA-992) by ammonium sulfate precipitation followed by ion exchange chromatography. TI from Phaseolus limensis (lima bean trypsin inhibitor; LBTI) was procured from Sigma-Aldrich, St. Louis, Missouri, United States. The antioxidant activity was analyzed by ferric ion reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). The anti-inflammatory property of TIs was determined by inhibition of albumin denaturation assay. Ascorbic acid and aspirin were used as standards for antioxidant and anti-inflammatory assays, respectively. These TIs were tested against various bacterial and fungal strains. The TIs showed DPPH radical-scavenging activity in a concentration-dependent manner with IC 50 values comparable to ascorbic acid. The FRAP values were also observed comparable to ascorbic acid and followed the trend of dose-dependent manner. The half maximal inhibitory concentration (IC 50 ) values of CCTI and LBTI in anti-inflammatory test showed that LBTI is more potent than CCTI. The TIs showed potent antibacterial activity, but apparently no action against fungi. This study has reported the biological properties of CCTI and LBTI for the first time. The results show that TIs possess the ability to inhibit diseases caused by oxidative stress, inflammation, and bacterial infestation.

Regulation of Hippocampal Glutamate Receptors: Evidence for the Involvement of a Calcium-Activated Protease

NASA Astrophysics Data System (ADS)

Baudry, Michel; Lynch, Gary

1980-04-01

Specific [3H]glutamate binding to rat hippocampal membranes and the calcium-induced increase in this binding are markedly temperature-sensitive and are inhibited by alkylating or reducing agents as well as by various protease inhibitors. N-Ethylmaleimide, chloromethyl ketone derivatives of lysine and phenylalanine, and tosylarginine methyl ester decrease the maximum number of [3H]glutamate binding sites without changing their affinity for glutamate. Preincubation of the membranes with glutamate does not protect the glutamate ``receptors'' from the suppressive effects of these agents. The proteases trypsin and α -chymotrypsin increase the maximum number of [3H]glutamate binding sites. The effects of calcium on glutamate binding are different across brain regions. Cerebellar membranes are almost insensitive whereas hippocampal and striatal membranes exhibit a strong increase in the number of binding sites after exposure to even low concentrations of calcium. These results suggest that an endogenous membrane-associated thiol protease regulates the number of [3H]glutamate binding sites in hippocampal membranes and that this is the mechanism by which calcium stimulates glutamate binding. The possibility is discussed that the postulated mechanisms participate in synaptic physiology and in particular may be related to the long-term potentiation of transmission found in hippocampus under certain conditions.

Dual core quantum dots for highly quantitative ratiometric detection of trypsin activity in cystic fibrosis patients

NASA Astrophysics Data System (ADS)

Castelló Serrano, Iván; Stoica, Georgiana; Matas Adams, Alba; Palomares, Emilio

2014-10-01

We present herein two colour encoded silica nanospheres (2nanoSi) for the fluorescence quantitative ratiometric determination of trypsin in humans. Current detection methods for cystic fibrosis diagnosis are slow, costly and suffer from false positives. The 2nanoSi proved to be a highly sensitive, fast (minutes), and single-step approach nanosensor for the screening and diagnosis of cystic fibrosis, allowing the quantification of trypsin concentrations in a wide range relevant for clinical applications (25-350 μg L-1). Furthermore, as trypsin is directly related to the development of cystic fibrosis (CF), different human genotypes, i.e. CF homozygotic, CF heterozygotic, and unaffected, respectively, can be determined using our 2nanoSi nanospheres. We anticipate the 2nanoSi system to be a starting point for non-invasive, easy-to-use and cost effective ratiometric fluorescent biomarkers for recessive genetic diseases like human cystic fibrosis. In a screening program in which the goal is to detect disease and also the carrier status, early diagnosis could be of great help.We present herein two colour encoded silica nanospheres (2nanoSi) for the fluorescence quantitative ratiometric determination of trypsin in humans. Current detection methods for cystic fibrosis diagnosis are slow, costly and suffer from false positives. The 2nanoSi proved to be a highly sensitive, fast (minutes), and single-step approach nanosensor for the screening and diagnosis of cystic fibrosis, allowing the quantification of trypsin concentrations in a wide range relevant for clinical applications (25-350 μg L-1). Furthermore, as trypsin is directly related to the development of cystic fibrosis (CF), different human genotypes, i.e. CF homozygotic, CF heterozygotic, and unaffected, respectively, can be determined using our 2nanoSi nanospheres. We anticipate the 2nanoSi system to be a starting point for non-invasive, easy-to-use and cost effective ratiometric fluorescent biomarkers for

Inhibition of trypsin by condensed tannins and wine.

PubMed

Gonçalves, Rui; Soares, Susana; Mateus, Nuno; de Freitas, Victor

2007-09-05

Phenolic compounds are abundant vegetable secondary metabolites in the human diet. The ability of procyanidin oligomers and wine polyphenols to inhibit trypsin activity was studied using a versatile and reliable in vitro method. The hydrolysis of the chromogenic substrate N-benzoyl-d,l-arginine-p-nitroanilide (BApNA) by trypsin was followed by spectrophotometry in the presence and absence of condensed tannins and wine. A clear relationship between the degree of polymerization of procyanidins and enzymatic inhibition was observed. Trypsin activity inhibition was also detected in several types of wine. In general, the inhibition increased with the concentration of phenolic compounds in wines. These results may be relevant when considering these compounds as antinutritional factors, thereby contributing to a reduced absorption of nutrients.

The Activity of Trypsin

ERIC Educational Resources Information Center

Russo, Salvatore F.; Holzman, Tom

1977-01-01

Describes an experiment that illustrates the following points concerning the experimental determination of trypsin activity: (1) there is a difference in basing enzyme concentration on weight, absorbance, or active sites; and (2) the method of expressing enzyme concentration determines the value of specific, molecular, and catalytic center…

Jojoba seed meal proteins associated with proteolytic and protease inhibitory activities.

PubMed

Shrestha, Madan K; Peri, Irena; Smirnoff, Patricia; Birk, Yehudith; Golan-Goldhirsh, Avi

2002-09-25

The jojoba, Simmondsia chinensis, is a characteristic desert plant native to the Sonoran desert. The jojoba meal after oil extraction is rich in protein. The major jojoba proteins were albumins (79%) and globulins (21%), which have similar amino acid compositions and also showed a labile thrombin-inhibitory activity. SDS-PAGE showed two major proteins at 50 kDa and 25 kDa both in the albumins and in the globulins. The 25 kDa protein has trypsin- and chymotrypsin-inhibitory activities. In vitro digestibility of the globulins and albumins resembled that of casein and soybean protein concentrates and was increased after heat treatment. The increased digestibility achieved by boiling may be attributed to inactivation of the protease inhibitors and denaturation of proteins.

Effects of soybean Kunitz trypsin inhibitor on the cotton boll weevil (Anthonomus grandis).

PubMed

Franco, Octávio L; Dias, Simoni C; Magalhães, Claudio P; Monteiro, Ana C S; Bloch, Carlos; Melo, Francislete R; Oliveira-Neto, Osmundo B; Monnerat, Rose G; Grossi-de-Sá, Maria Fátima

2004-01-01

The cotton boll weevil, Anthonomus grandis, is an economically important pest of cotton in tropical and subtropical areas of several countries in the Americas, causing severe losses due to their damage in cotton floral buds. Enzymatic assays using gut extracts from larval and adult boll weevil have demonstrated the presence of digestive serine proteinase-like activities. Furthermore, in vitro assays showed that soybean Kunitz trypsin inhibitor (SKTI) was able to inhibit these enzymes. Previously, in vivo effects of black-eyed pea trypsin chymotrypsin inhibitor (BTCI) have been demonstrated towards the boll weevil pest. Here, when neonate larvae were reared on an artificial diet containing SKTI at three different concentrations, a reduction of larval weight of up to 64% was observed for highest SKTI concentration 500 microM. The presence of SKTI caused an increase in mortality and severe deformities of larvae, pupae and adult insects. This work therefore represents the first observation of a Kunitz trypsin inhibitor active in vivo and in vitro against A. grandis. Bioassays suggested that SKTI could be used as a tool in engineering crop plants, which might exhibit increased resistance against cotton boll weevil.

The sequence and X-ray structure of the trypsin from Fusarium oxysporum.

PubMed

Rypniewski, W R; Hastrup, S; Betzel, C; Dauter, M; Dauter, Z; Papendorf, G; Branner, S; Wilson, K S

1993-06-01

The trypsin from Fusarium oxysporum is equally homologous to trypsins from Streptomyces griseus, Streptomyces erythraeus and to bovine trypsin. A DFP (diisopropylfluorophosphate) inhibited form of the enzyme has been crystallized from 1.4 M Na2SO4, buffered with citrate at pH 5.0-5.5. The crystals belong to space group P2(1) with cell parameters a = 33.43 A, b = 67.65 A, c = 39.85 A and beta = 107.6 degrees. There is one protein molecule in the asymmetric unit. X-ray diffraction data to a resolution of 1.8 A were collected on film using synchrotron radiation. The structure was solved by molecular replacement using models of bovine and S. griseus trypsins and refined to an R-factor of 0.141. The overall fold is similar to other trypsins, with some insertions and deletions. There is no evidence of the divalent cation binding sites seen in other trypsins. The covalently bound inhibitor molecule is clearly visible.

Alternative Enzymes Lead to Improvements in Sequence Coverage and PTM Analysis

PubMed Central

Hooper, Kyle; Rosenblatt, Michael; Urh, Marjeta; Saveliev, Sergei; Hosfield, Chris; Kobs, Gary; Ford, Michael; Jones, Richard; Amunugama, Ravi; Allen, David; Brazas, Robert

2013-01-01

The profiling of proteins using biological mass spectrometry (bottom up proteomics) most commonly requires trypsin. Trypsin is advantageous in that it produces peptides of optimal charge and size. However, for applications in which the proteins under investigation are part of a complex mixture or not isolated at high levels (i.e. low ng from an immunoprecipitation), sequence coverage is rarely complete. In addition, we have found that in several cases, like phosphorylation, acetylation, and methylation, alternative proteases are required to prepare peptides suitable for MS detection. This poster will provide specific examples which demonstrate this observation. For example, the application of a combined Trypsin/ Lys-C mixture reduces the number of missed cleavages by more than 3-fold producing samples with lower CV's (for biological replicates). The mixture is also well-suited for the complete proteolysis of hydrophobic, compact proteins. The addition of chymotrypsin and elastase has been found to be useful for identifying phosphorylation sites on proteins, especially on sequences where the site of phosphorylation inhibits trypsin (i.e. proximal to K or R). Many epigenetic applications have focused on histone modifications, like lysine acetylation and arginine methylation. Alternative proteases like Asp-N, Glu-C, and chymotrypsin have been especially useful given the fact that the modified K and R residues are resistant to c-terminal cleavage by trypsin. Finally, in the case of serum profiling, the addition of the endoglycosidase, PNGase F has been found to improve sequence coverage due to the removal of N-linked glycans.

Dipeptidyl peptidase IV, aminopeptidase N and DPIV/APN-like proteases in cerebral ischemia

PubMed Central

2012-01-01

Background Cerebral inflammation is a hallmark of neuronal degeneration. Dipeptidyl peptidase IV, aminopeptidase N as well as the dipeptidyl peptidases II, 8 and 9 and cytosolic alanyl-aminopeptidase are involved in the regulation of autoimmunity and inflammation. We studied the expression, localisation and activity patterns of these proteases after endothelin-induced occlusion of the middle cerebral artery in rats, a model of transient and unilateral cerebral ischemia. Methods Male Sprague-Dawley rats were used. RT-PCR, immunohistochemistry and protease activity assays were performed at different time points, lasting from 2 h to 7 days after cerebral ischemia. The effect of protease inhibitors on ischemia-dependent infarct volumes was quantified 7 days post middle cerebral artery occlusion. Statistical analysis was conducted using the t-test. Results Qualitative RT-PCR revealed these proteases in ipsilateral and contralateral cortices. Dipeptidyl peptidase II and aminopeptidase N were up-regulated ipsilaterally from 6 h to 7 days post ischemia, whereas dipeptidyl peptidase 9 and cytosolic alanyl-aminopeptidase were transiently down-regulated at day 3. Dipeptidyl peptidase 8 and aminopeptidase N immunoreactivities were detected in cortical neurons of the contralateral hemisphere. At the same time point, dipeptidyl peptidase IV, 8 and aminopeptidase N were identified in activated microglia and macrophages in the ipsilateral cortex. Seven days post artery occlusion, dipeptidyl peptidase IV immunoreactivity was found in the perikarya of surviving cortical neurons of the ipsilateral hemisphere, whereas their nuclei were dipeptidyl peptidase 8- and amino peptidase N-positive. At the same time point, dipeptidyl peptidase IV, 8 and aminopeptidase N were targeted in astroglial cells. Total dipeptidyl peptidase IV, 8 and 9 activities remained constant in both hemispheres until day 3 post experimental ischemia, but were increased (+165%) in the ipsilateral cortex at day 7

Intensity fading MALDI-TOF mass spectrometry and functional proteomics assignments to identify protease inhibitors in marine invertebrates.

PubMed

Covaleda, Giovanni; Trejo, Sebastian A; Salas-Sarduy, Emir; Del Rivero, Maday Alonso; Chavez, Maria Angeles; Aviles, Francesc X

2017-08-08

Proteases and their inhibitors have become molecules of increasing fundamental and applicative value. Here we report an integrated strategy to identify and analyze such inhibitors from Caribbean marine invertebrates extracts by a fast and sensitive functional proteomics-like approach. The strategy works in three steps: i) multiplexed enzymatic inhibition kinetic assays, ii) Intensity Fading MALDI-TOF MS to establish a link between inhibitory molecules and the related MALDI signal(s) detected in the extract(s), and iii) ISD-CID-T 3 MS fragmentation on the parent MALDI signals selected in the previous step, enabling the partial or total top-down sequencing of the molecules. The present study has allowed validation of the whole approach, identification of a substantial number of novel protein protease inhibitors, as well as full or partial sequencing of reference molecular species and of many unknown ones, respectively. Such inhibitors correspond to six protease subfamilies (metallocarboxypeptidases-A and -B, pepsin, papain, trypsin and subtilisin), are small (1-10KDa) disulfide-rich proteins, and have been found at diverse frequencies among the invertebrates (13 to 41%). The overall procedure could be tailored to other enzyme-inhibitor and protein interacting systems, analyzing samples at medium-throughput level and leading to the functional and structural characterization of proteinaceous ligands from complex biological extracts. Invertebrate animals, and marine ones among, display a remarkable diversity of species and contained biomolecules. Many of their proteins-peptides have high biological, biotechnological and biomedical potential interest but, because of the lack of sequenced genomes behind, their structural and functional characterization constitutes a great challenge. Here, looking at the small, disulfide-rich, proteinaceous inhibitors of proteases found in them, it is shown that such problem can be significatively facilitated by integrative multiplexed

The Human Airway Epithelial Basal Cell Transcriptome

PubMed Central

Wang, Rui; Zwick, Rachel K.; Ferris, Barbara; Witover, Bradley; Salit, Jacqueline; Crystal, Ronald G.

2011-01-01

Background The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. Methodology/Principal Findings Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the “human airway basal cell signature” as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. Conclusion/Significance The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem

Less is More: Membrane Protein Digestion Beyond Urea-Trypsin Solution for Next-level Proteomics.

PubMed

Zhang, Xi

2015-09-01

The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea-trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting-not destroying-structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Less is More: Membrane Protein Digestion Beyond Urea–Trypsin Solution for Next-level Proteomics*

PubMed Central

Zhang, Xi

2015-01-01

The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea–trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting—not destroying—structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. PMID:26081834

Fibrin(ogen)olytic activity of bumblebee venom serine protease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu Yuling; Joint Laboratory between Dong-A University and Shenyang Pharmaceutical University, Shenyang Pharmaceutical University, Shenyang; Choo, Young Moo

Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olyticmore » enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis. - Graphical abstract: Display Omitted Highlights: > Bumblebee venom serine protease (Bt-VSP) is a fibrin(ogen)olytic enzyme. > Bt-VSP activates prothrombin. > Bt-VSP directly degrades fibrinogen into fibrin degradation products. > Bt-VSP is a hemostatically active protein that is a potent clinical agent.« less

From nonpeptide toward noncarbon protease inhibitors: Metallacarboranes as specific and potent inhibitors of HIV protease

PubMed Central

Cígler, Petr; Kožíšek, Milan; Řezáčová, Pavlína; Brynda, Jíří; Otwinowski, Zbyszek; Pokorná, Jana; Plešek, Jaromír; Grüner, Bohumír; Dolečková-Marešová, Lucie; Máša, Martin; Sedláček, Juraj; Bodem, Jochen; Kräusslich, Hans-Georg; Král, Vladimír; Konvalinka, Jan

2005-01-01

HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a Ki value of 2.2 nM and a submicromolar EC50 in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 Å resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3′ subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition. PMID:16227435

Stimulatory effects of bombesin on plasma trypsin release and exocrine pancreatic secretion in dogs.

PubMed

Kiriyama, S; Hayakawa, T; Kondo, T; Shibata, T; Kitagawa, M; Sakai, Y; Sobajima, H; Ikei, N; Kodaira, T; Hamaoka, T

1990-01-01

We examined the effect of bombesin on plasma trypsin release and exocrine pancreatic secretion in dogs. Bombesin significantly increased plasma immunoreactive trypsin (IRT). Atropine significantly inhibited the response of plasma IRT to bombesin. Pancreatic trypsin secretion was also increased by bombesin, as well as bicarbonate and protein outputs. Atropine failed to inhibit pancreatic trypsin secretion. In conclusion, bombesin has a stimulatory effect on plasma trypsin release mediated by a cholinergic mechanism and different from pancreatic secretion.

Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease

PubMed Central

1992-01-01

Serum mannose-binding protein (MBP) is a C-type lectin that binds to terminal mannose and N-acetylglucosamine moieties present on surfaces of certain pathogens and activates the classical complement pathway. In the present study, we describe the mechanism underlying the activation triggered by MBP. The human serum MBP fraction was obtained by sequential affinity chromatography on mannan-Sepharose, anti-IgM- Sepharose and anti-MBP-Sepharose in the presence of calcium ions. This fraction contained a C1s-like serine protease as assessed by C4 consumption. The C1s-like serine protease, designated MBP-associated serine protease (MASP), was separated from MBP by rechromatography on anti-MBP-Sepharose in the presence of ethylenediaminetetraacetic acid. MASP exhibited both C4- and C2-consuming activities. The molecular mass of MASP was estimated to be 83 kD with two polypeptides of heavy (66 kD) and light (L) (31 kD) chains linked by disulfide bonds. The serine residue responsible for protease activity is located on the L chain. Reconstitution experiments using MASP and MBP revealed that combination of the two components restores C4- and C2-activating capacity on mannan. Based on analyses of molecular size, antigenicity, and 11 NH2- terminal amino acid sequences of the L chain, we conclude that MASP is a novel protein different from C1r or C1s. Our findings are not in accord with a proposed mechanism by which MBP utilizes the C1r2-C1s2 complex to initiate the classical complement pathway. PMID:1460414

Comparative evaluation of bioactivity of crystalline trypsin for drying by Fourier-transformed infrared spectroscopy.

PubMed

Otsuka, Makoto; Fukui, Yuya; Ozaki, Yukihiro

2009-03-01

The purpose of this study was to evaluate the enzymatic stability of colloidal trypsin powder during heating in a solid-state by using Fourier transform infrared (FT-IR) spectra with chemoinformatics and generalized two-dimensional (2D) correlation spectroscopy. Colloidal crystalline trypsin powders were heated using differential scanning calorimetry. The enzymatic activity of trypsin was assayed by the kinetic degradation method. Spectra of 10 calibration sample sets were recorded three times with a FT-IR spectrometer. The maximum intensity at 1634cm(-1) of FT-IR spectra and enzymatic activity of trypsin decreased as the temperature increased. The FT-IR spectra of trypsin samples were analyzed by a principal component regression analysis (PCR). A plot of the calibration data obtained was made between the actual and predicted trypsin activity based on a two-component model with gamma(2)=0.962. On the other hand, a 2D method was applied to FT-IR spectra of heat-treated trypsin. The result was consistent with that of the chemoinformetrical method. The results for deactivation of colloidal trypsin powder by heat-treatment indicated that nano-structure of crystalline trypsin changed by heating reflecting that the beta-sheet was mainly transformed, since the peak at 1634cm(-1) decreased with dehydration. The FT-IR chemoinformetrical method allows for a solid-state quantitative analysis of the bioactivity of the bulk powder of trypsin during drying.

Cystic Fibrosis Transmembrane Conductance Regulator in Sarcoplasmic Reticulum of Airway Smooth Muscle. Implications for Airway Contractility

PubMed Central

Cook, Daniel P.; Rector, Michael V.; Bouzek, Drake C.; Michalski, Andrew S.; Gansemer, Nicholas D.; Reznikov, Leah R.; Li, Xiaopeng; Stroik, Mallory R.; Ostedgaard, Lynda S.; Abou Alaiwa, Mahmoud H.; Thompson, Michael A.; Prakash, Y. S.; Krishnan, Ramaswamy; Meyerholz, David K.; Seow, Chun Y.

2016-01-01

Rationale: An asthma-like airway phenotype has been described in people with cystic fibrosis (CF). Whether these findings are directly caused by loss of CF transmembrane conductance regulator (CFTR) function or secondary to chronic airway infection and/or inflammation has been difficult to determine. Objectives: Airway contractility is primarily determined by airway smooth muscle. We tested the hypothesis that CFTR is expressed in airway smooth muscle and directly affects airway smooth muscle contractility. Methods: Newborn pigs, both wild type and with CF (before the onset of airway infection and inflammation), were used in this study. High-resolution immunofluorescence was used to identify the subcellular localization of CFTR in airway smooth muscle. Airway smooth muscle function was determined with tissue myography, intracellular calcium measurements, and regulatory myosin light chain phosphorylation status. Precision-cut lung slices were used to investigate the therapeutic potential of CFTR modulation on airway reactivity. Measurements and Main Results: We found that CFTR localizes to the sarcoplasmic reticulum compartment of airway smooth muscle and regulates airway smooth muscle tone. Loss of CFTR function led to delayed calcium reuptake following cholinergic stimulation and increased myosin light chain phosphorylation. CFTR potentiation with ivacaftor decreased airway reactivity in precision-cut lung slices following cholinergic stimulation. Conclusions: Loss of CFTR alters porcine airway smooth muscle function and may contribute to the airflow obstruction phenotype observed in human CF. Airway smooth muscle CFTR may represent a therapeutic target in CF and other diseases of airway narrowing. PMID:26488271

Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Mi Na; Lee, Kyung Eun; Hong, Jung Yeon

Highlights: Black-Right-Pointing-Pointer Hyperoxia induces apoptosis and chitinase 3-like 1 expression in human airway epithelial cells. Black-Right-Pointing-Pointer Presence of chitinase 3-like 1 affects airway epithelial cell death after hyperoxic exposure. Black-Right-Pointing-Pointer Silencing chitinase 3-like 1 manipulate the phosphorylation of ERK, p38 and Akt. -- Abstract: Background: Exposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associatedmore » with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood. Purpose: In this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells. Method: The human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process. Results: Hyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure

Proteases in agricultural dust induce lung inflammation through PAR-1 and PAR-2 activation.

PubMed

Romberger, Debra J; Heires, Art J; Nordgren, Tara M; Souder, Chelsea P; West, William; Liu, Xiang-de; Poole, Jill A; Toews, Myron L; Wyatt, Todd A

2015-08-15

Workers exposed to aerosolized dust present in concentrated animal feeding operations (CAFOs) are susceptible to inflammatory lung diseases, such as chronic obstructive pulmonary disease. Extracts of dust collected from hog CAFOs [hog dust extract (HDE)] are potent stimulators of lung inflammatory responses in several model systems. The observation that HDE contains active proteases prompted the present study, which evaluated the role of CAFO dust proteases in lung inflammatory processes and tested whether protease-activated receptors (PARs) are involved in the signaling pathway for these events. We hypothesized that the damaging proinflammatory effect of HDE is due, in part, to the proteolytic activation of PARs, and inhibiting the proteases in HDE or disrupting PAR activation would attenuate HDE-mediated inflammatory indexes in bronchial epithelial cells (BECs), in mouse lung slices in vitro, and in a murine in vivo exposure model. Human BECs and mouse lung slice cultures stimulated with 5% HDE released significantly more of each of the cytokines measured (IL-6, IL-8, TNF-α, keratinocyte-derived chemokine/CXC chemokine ligand 1, and macrophage inflammatory protein-2/CXC chemokine ligand 2) than controls, and these effects were markedly diminished by protease inhibition. Inhibition of PARs also blunted the HDE-induced cytokine release from BECs. In addition, protease depletion inhibited HDE-induced BEC intracellular PKCα and PKCε activation. C57BL/6J mice administered 12.5% HDE intranasally, either once or daily for 3 wk, exhibited increased total cellular and neutrophil influx, bronchial alveolar fluid inflammatory cytokines, lung histopathology, and inflammatory scores compared with mice receiving protease-depleted HDE. These data suggest that proteases in dust from CAFOs are important mediators of lung inflammation, and these proteases and their receptors may provide novel targets for therapeutic intervention in CAFO dust-induced airways disease.

The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein from Clostridium difficile

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bradshaw, William J.; Public Health England, Porton Down, Salisbury SP4 0JG; Kirby, Jonathan M.

2014-07-01

The crystal structure of Cwp84, an S-layer protein from Clostridium difficile is presented for the first time. The cathepsin L-like fold of cysteine protease domain, a newly observed ‘lectin-like’ domain and several other features are described. Clostridium difficile is a major problem as an aetiological agent for antibiotic-associated diarrhoea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The mechanism of C. difficile surface-layer (S-layer) biogenesis is also largely unknown but involves the post-translational cleavage of a single polypeptide (surface-layer protein A; SlpA)more » into low- and high-molecular-weight subunits by Cwp84, a surface-located cysteine protease. Here, the first crystal structure of the surface protein Cwp84 is described at 1.4 Å resolution and the key structural components are identified. The truncated Cwp84 active-site mutant (amino-acid residues 33–497; C116A) exhibits three regions: a cleavable propeptide and a cysteine protease domain which exhibits a cathepsin L-like fold followed by a newly identified putative carbohydrate-binding domain with a bound calcium ion, which is referred to here as a lectin-like domain. This study thus provides the first structural insights into Cwp84 and a strong base to elucidate its role in the C. difficile S-layer maturation mechanism.« less

Dual functionality of β-tryptase protomers as both proteases and cofactors in the active tetramer.

PubMed

Maun, Henry R; Liu, Peter S; Franke, Yvonne; Eigenbrot, Charles; Forrest, William F; Schwartz, Lawrence B; Lazarus, Robert A

2018-04-16

Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of the allergic inflammatory responses in asthma. During acute hypersensitivity reactions, mast cells degranulate, releasing active tetramer as a complex with proteoglycans. Extensive efforts have focused on developing therapeutic β-tryptase inhibitors, but its unique activation mechanism is less well explored. Tryptase is active only after proteolytic removal of the pro-domain followed by tetramer formation via two distinct symmetry-related interfaces. We show that the cleaved I16G mutant cannot tetramerize, likely due to impaired insertion of its N-terminus into its 'activation pocket', indicating allosteric linkage at multiple sites on each protomer. We engineered cysteines into each of the two distinct interfaces (Y75C for small or I99C for large) to assess the activity of each tetramer and disulfide-locked dimer. Using size-exclusion chromatography and enzymatic assays, we demonstrate that the two large tetramer interfaces regulate enzymatic activity, elucidating the importance of this protein-protein interaction for allosteric regulation. Notably, the I99C large interface dimer is active, even in the absence of heparin. We show that a monomeric β-tryptase mutant (I99C*:Y75A:Y37bA where C* is cysteinylated Cys99) cannot form a dimer or tetramer, yet is active, but only in the presence of heparin. Thus heparin both stabilizes the tetramer and allosterically conditions the active site. We hypothesize that each β-tryptase protomer in the tetramer has two distinct roles, acting both as a protease and as a cofactor for its neighboring protomer, to allosterically regulate enzymatic activity, providing a rationale for direct correlation of tetramer stability with proteolytic activity. Copyright © 2018, The American Society for Biochemistry and Molecular Biology.

Detergent alkaline proteases: enzymatic properties, genes, and crystal structures.

PubMed

Saeki, Katsuhisa; Ozaki, Katsuya; Kobayashi, Tohru; Ito, Susumu

2007-06-01

Subtilisin-like serine proteases from bacilli have been used in various industrial fields worldwide, particularly in the production of laundry and automatic dishwashing detergents. They belong to family A of the subtilase superfamily, which is composed of three clans, namely, true subtilisins, high-alkaline proteases, and intracellular proteases. We succeeded in the large-scale production of a high-alkaline protease (M-protease) from alkaliphilic Bacillus clausii KSM-K16, and the enzyme has been introduced into compact heavy-duty laundry detergents. We have also succeeded in the industrial-scale production of a new alkaline protease, KP-43, which was originally resistant to chemical oxidants and to surfactants, produced by alkaliphilic Bacillus sp. strain KSM-KP43 and have incorporated it into laundry detergents. KP-43 and related proteases form a new clan, oxidatively stable proteases, in subtilase family A. In this review, we describe the enzymatic properties, gene sequences, and crystal structures of M-protease, KP-43, and related enzymes.

Mass spectrometric analysis of electrophoretically separated allergens and proteases in grass pollen diffusates

PubMed Central

Raftery, Mark J; Saldanha, Rohit G; Geczy, Carolyn L; Kumar, Rakesh K

2003-01-01

Background Pollens are important triggers for allergic asthma and seasonal rhinitis, and proteases released by major allergenic pollens can injure airway epithelial cells in vitro. Disruption of mucosal epithelial integrity by proteases released by inhaled pollens could promote allergic sensitisation. Methods Pollen diffusates from Kentucky blue grass (Poa pratensis), rye grass (Lolium perenne) and Bermuda grass (Cynodon dactylon) were assessed for peptidase activity using a fluorogenic substrate, as well as by gelatin zymography. Following one- or two-dimensional gel electrophoresis, Coomassie-stained individual bands/spots were excised, subjected to tryptic digestion and analysed by mass spectrometry, either MALDI reflectron TOF or microcapillary liquid chromatography MS-MS. Database searches were used to identify allergens and other plant proteins in pollen diffusates. Results All pollen diffusates tested exhibited peptidase activity. Gelatin zymography revealed high Mr proteolytic activity at ~ 95,000 in all diffusates and additional proteolytic bands in rye and Bermuda grass diffusates, which appeared to be serine proteases on the basis of inhibition studies. A proteolytic band at Mr ~ 35,000 in Bermuda grass diffusate, which corresponded to an intense band detected by Western blotting using a monoclonal antibody to the timothy grass (Phleum pratense) group 1 allergen Phl p 1, was identified by mass spectrometric analysis as the group 1 allergen Cyn d 1. Two-dimensional analysis similarly demonstrated proteolytic activity corresponding to protein spots identified as Cyn d 1. Conclusion One- and two-dimensional electrophoretic separation, combined with analysis by mass spectrometry, is useful for rapid determination of the identities of pollen proteins. A component of the proteolytic activity in Bermuda grass diffusate is likely to be related to the allergen Cyn d 1. PMID:14577842

Papain-like cysteine proteases in Carica papaya: lineage-specific gene duplication and expansion.

PubMed

Liu, Juan; Sharma, Anupma; Niewiara, Marie Jamille; Singh, Ratnesh; Ming, Ray; Yu, Qingyi

2018-01-06

Papain-like cysteine proteases (PLCPs), a large group of cysteine proteases structurally related to papain, play important roles in plant development, senescence, and defense responses. Papain, the first cysteine protease whose structure was determined by X-ray crystallography, plays a crucial role in protecting papaya from herbivorous insects. Except the four major PLCPs purified and characterized in papaya latex, the rest of the PLCPs in papaya genome are largely unknown. We identified 33 PLCP genes in papaya genome. Phylogenetic analysis clearly separated plant PLCP genes into nine subfamilies. PLCP genes are not equally distributed among the nine subfamilies and the number of PLCPs in each subfamily does not increase or decrease proportionally among the seven selected plant species. Papaya showed clear lineage-specific gene expansion in the subfamily III. Interestingly, all four major PLCPs purified from papaya latex, including papain, chymopapain, glycyl endopeptidase and caricain, were grouped into the lineage-specific expansion branch in the subfamily III. Mapping PLCP genes on chromosomes of five plant species revealed that lineage-specific expansions of PLCP genes were mostly derived from tandem duplications. We estimated divergence time of papaya PLCP genes of subfamily III. The major duplication events leading to lineage-specific expansion of papaya PLCP genes in subfamily III were estimated at 48 MYA, 34 MYA, and 16 MYA. The gene expression patterns of the papaya PLCP genes in different tissues were assessed by transcriptome sequencing and qRT-PCR. Most of the papaya PLCP genes of subfamily III expressed at high levels in leaf and green fruit tissues. Tandem duplications played the dominant role in affecting copy number of PLCPs in plants. Significant variations in size of the PLCP subfamilies among species may reflect genetic adaptation of plant species to different environments. The lineage-specific expansion of papaya PLCPs of subfamily III might

Spiking of serum specimens with exogenous reporter peptides for mass spectrometry based protease profiling as diagnostic tool.

PubMed

Findeisen, Peter; Peccerella, Teresa; Post, Stefan; Wenz, Frederik; Neumaier, Michael

2008-04-01

Serum is a difficult matrix for the identification of biomarkers by mass spectrometry (MS). This is due to high-abundance proteins and their complex processing by a multitude of endogenous proteases making rigorous standardisation difficult. Here, we have investigated the use of defined exogenous reporter peptides as substrates for disease-specific proteases with respect to improved standardisation and disease classification accuracy. A recombinant N-terminal fragment of the Adenomatous Polyposis Coli (APC) protein was digested with trypsin to yield a peptide mixture for subsequent Reporter Peptide Spiking (RPS) of serum. Different preanalytical handling of serum samples was simulated by storage of serum samples for up to 6 h at ambient temperature, followed by RPS, further incubation under standardised conditions and testing for stability of protease-generated MS profiles. To demonstrate the superior classification accuracy achieved by RPS, a pilot profiling experiment was performed using serum specimens from pancreatic cancer patients (n = 50) and healthy controls (n = 50). After RPS six different peak categories could be defined, two of which (categories C and D) are modulated by endogenous proteases. These latter are relevant for improved classification accuracy as shown by enhanced disease-specific classification from 78% to 87% in unspiked and spiked samples, respectively. Peaks of these categories presented with unchanged signal intensities regardless of preanalytical conditions. The use of RPS generally improved the signal intensities of protease-generated peptide peaks. RPS circumvents preanalytical variabilities and improves classification accuracies. Our approach will be helpful to introduce MS-based proteomic profiling into routine laboratory testing.

Alcohol and Airways Function in Health and Disease

PubMed Central

Sisson, Joseph H.

2007-01-01

The volatility of alcohol promotes the movement of alcohol from the bronchial circulation across the airway epithelium and into the conducting airways of the lung. The exposure of the airways through this route likely accounts for many of the biologic effects of alcohol on lung airway functions. The impact of alcohol on lung airway functions is dependent on the concentration, duration and route of exposure. Brief exposure to mild concentrations of alcohol may enhance mucociliary clearance, stimulates bronchodilation and probably attenuates the airway inflammation and injury observed in asthma and COPD. Prolonged and heavy exposure to alcohol impairs mucociliary clearance, may complicate asthma management and likely worsens outcomes including lung function and mortality in COPD patients. Non-alcohol congeners and alcohol metabolites act as triggers for airway disease exacerbations especially in atopic asthmatics and in Asian populations who have a reduced capacity to metabolize alcohol. Research focused on the mechanisms of alcohol-mediated changes in airway functions has identified specific mechanisms that mediate alcohol effects within the lung airways. These include prominent roles for the second messengers calcium and nitric oxide, regulatory kinases including PKG and PKA, alcohol and acetaldehyde-metabolizing enzymes such as aldehyde dehydrogenase type 2 (ALDH2). The role alcohol may play in the pathobiology of airway mucus, bronchial blood flow, airway smooth muscle regulation and the interaction with other airway exposure agents, such as cigarette smoke, represent opportunities for future investigation. PMID:17764883

Alcohol and airways function in health and disease.

PubMed

Sisson, Joseph H

2007-08-01

The volatility of alcohol promotes the movement of alcohol from the bronchial circulation across the airway epithelium and into the conducting airways of the lung. The exposure of the airways through this route likely accounts for many of the biologic effects of alcohol on lung airway functions. The effect of alcohol on lung airway functions is dependent on the concentration, duration, and route of exposure. Brief exposure to mild concentrations of alcohol may enhance mucociliary clearance, stimulates bronchodilation, and probably attenuates the airway inflammation and injury observed in asthma and chronic obstructive pulmonary disease (COPD). Prolonged and heavy exposure to alcohol impairs mucociliary clearance, may complicate asthma management, and likely worsens outcomes including lung function and mortality in COPD patients. Nonalcohol congeners and alcohol metabolites act as triggers for airway disease exacerbations especially in atopic asthmatics and in Asian populations who have a reduced capacity to metabolize alcohol. Research focused on the mechanisms of alcohol-mediated changes in airway functions has identified specific mechanisms that mediate alcohol effects within the lung airways. These include prominent roles for the second messengers calcium and nitric oxide, regulatory kinases including PKG and PKA, alcohol- and acetaldehyde-metabolizing enzymes such as aldehyde dehydrogenase 2. The role alcohol may play in the pathobiology of airway mucus, bronchial blood flow, airway smooth muscle regulation, and the interaction with other airway exposure agents, such as cigarette smoke, represents opportunities for future investigation.

Potential toxicity of phthalic acid esters plasticizer: interaction of dimethyl phthalate with trypsin in vitro.

PubMed

Wang, Yaping; Zhang, Guowen; Wang, Langhong

2015-01-14

Dimethyl phthalate (DMP) is widely used as a plasticizer in industrial processes and has been reported to possess potential toxicity to the human body. In this study, the interaction between DMP and trypsin in vitro was investigated. The results of fluorescence, UV–vis, circular dichroism, and Fourier transform infrared spectra along with cyclic voltammetric measurements indicated that the remarkable fluorescence quenching and conformational changes of trypsin resulted from the formation of a DMP–trypsin complex, which was driven mainly by hydrophobic interactions. The molecular docking and trypsin activity assay showed that DMP primarily interacted with the catalytic triad of trypsin and led to the inhibition of trypsin activity. The dimensions of the individual trypsin molecules were found to become larger after binding with DMP by atomic force microscopy imaging. This study offers a comprehensive picture of DMP–trypsin interaction, which is expected to provide insights into the toxicological effect of DMP.

Protease-Resistant Peptide Ligands from a Knottin Scaffold Library

PubMed Central

Getz, Jennifer A.; Rice, Jeffrey J.; Daugherty, Patrick S.

2011-01-01

Peptides within the knottin family have been shown to possess inherent stability, making them attractive scaffolds for the development of therapeutic and diagnostic agents. Given its remarkable stability to proteases, the cyclic peptide kalata B1 was employed as a scaffold to create a large knottin library displayed on the surface of E. coli. A library exceeding 109 variants was constructed by randomizing seven amino acids within a loop of the kalata B1 scaffold and screened using fluorescence-activated cell sorting to identify peptide ligands specific for the active site of human thrombin. Refolded thrombin binders exhibited high nanomolar affinities in solution, slow dissociation rates, and were able to inhibit thrombin’s enzymatic activity. Importantly, 80% of a knottin-based thrombin inhibitor remained intact after a two hour incubation both with trypsin and with chymotrypsin, demonstrating that modifying the kalata B1 sequence did not compromise its stability properties. In addition, the knottin variant mediated 20-fold enhanced affinity for thrombin, when compared to the same seven residue binding epitope constrained by a single disulfide bond. Our results indicate that peptide libraries derived from the kalata B1 scaffold can yield high affinity protein ligands that retain the remarkable protease resistance associated with the parent scaffold. More generally, this strategy may prove useful in the development of stable peptide ligands suitable for in vivo applications. PMID:21615106

A study of proteases and protease-inhibitor complexes in biological fluids

PubMed Central

Granelli-Piperno, A; Reich, E

1978-01-01

We have (a) screened a variety of cell lines and body fluids for plasminogen activators and (b) studied the activity of proteases bound to α2- macroglobulin after exposing the complexes to partial degradation and/or denaturing procedures to unmask proteolytic activity. The respective results show (a) that the plasminogen activators in urine and cell culture media are generally of lower molecular weight than those in plasma; and (b) that proteases bound to α2-macroglobulin recover the ability to attack macromolecular substrates after exposure to sodium dodecyl sulfate while retaining the electrophoretic mobility of the protease inhibitor complex. This indicates that the protease and inhibitor are probably linked by covalent bonds. In contrast, other complexes formed between proteases and inhibitors of lower molecular weight (such as soybean or Kunitz inhibitors) are fully dissociated by sodium dodecyl sulfate (SDS). The experiments described were based on a new procedure for detecting proteolytic enzyme activity in SDS-polyacrylamide gels. The method relies on solutions of nonionic detergents for extracting SDS, after which the electrophoretic gel is applied to an indicator gel consisting of a fibrin- agar mixture. The method is sensitive, permitting the detection of proteinases in less than 1 μl of fresh plasma, and it is effective for resolving small differences in molecular weight. The procedure can be quantitated and, with minor modifications appropriate to each particular system, it has been applied to a broad spectrum of serine enzymes and proenzymes, including some that function in the pathways of fibrinolysis, coagulation and kinin-generation. Other potential applications appear likely. PMID:78958

Bile-pancreatic juice-independent increases in pancreatic proteases and intestinal cholecystokinin by dietary protein in rats.

PubMed

Hara, H; Ochi, Y; Kasai, T

1998-02-01

Luminal bile-pancreatic juice (BPJ) is involved in the induction of pancreatic proteases in rats fed a high-protein diet. Recently, we have demonstrated that a BPJ-independent mechanism is responsible for enhancement of pancreatic secretion after feeding of a dietary protein in chronic BPJ-diverted rats. The aim of the present study was to explore the existence of a BPJ-independent mechanism during adaptation of the exocrine pancreas to dietary protein. Rats, whose BPJ was diverted into the ileum through a common bile-pancreatic duct catheter for 5 days (PBD rat), were fed a fat-free diet containing 25% or 60% casein for 3 days. Messenger RNA levels for pancreatic enzymes, cholecystokinin, and secretin in the jejunal mucosa were evaluated by northern blotting method. Pancreatic trypsin and chymotrypsin activities and mRNA levels of their zymogens were higher in PBD rats than in rats whose diverted BPJ was returned into the duodenum (PBD returned rat). In the PBD groups, pancreatic protease activities were further increased by 3-day feeding of a high-protein diet without changes in mRNA levels of these proteases. Cholecystokinin mRNA was increased after feeding of a high-protein diet in the PBD rats. These results indicate that pancreatic proteases are induced by feeding a high-protein diet by a mechanism independent of luminal BPJ, which is associated with an increase in intestinal cholecystokinin mRNA level.

Thylakoid membrane protein topography: transmembrane orientation of the chloroplast cytochrome b-559 psbE gene product.

PubMed

Tae, G S; Black, M T; Cramer, W A; Vallon, O; Bogorad, L

1988-12-27

Protease accessibility and antibody to a COOH-terminal peptide were used as probes for the in situ topography of the Mr 10,000 psbE gene product (alpha subunit) of the chloroplast cytochrome b-559. Exposure of thylakoid membranes to trypsin or Staphylococcus aureus V8 protease cleaved the alpha subunit to a slightly smaller polypeptide (delta Mr approximately -1000) as detected on Western blots, without loss of reactivity to COOH-terminal antibody. The disappearance of the parent Mr 10,000 polypeptide from thylakoids in the presence of trypsin correlated with the appearance of the smaller polypeptide with delta Mr = -750, the conversion having a half-time of approximately 15 min. Exposure of inside-out vesicles to trypsin resulted in almost complete loss of reactivity to the antibody, showing that the COOH terminus is exposed on the lumenal side of the membrane. Removal of the extrinsic polypeptides of the oxygen-evolving complex resulted in an increase of the accessibility of the alpha subunit to trypsin. These data establish that the alpha subunit of cytochrome b-559 crosses the membrane once, as predicted from its single, 26-residue, hydrophobic domain. The NH2 terminus of the alpha polypeptide is on the stromal side of the membrane, where it is accessible, most likely at Arg-7 or Glu-6/Asp-11, to trypsin or V8 protease, respectively. As a consequence of this orientation, the single histidine residue in the alpha subunit is located on the stromal side of the hydrophobic domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiated human airway organoids to assess infectivity of emerging influenza virus.

PubMed

Zhou, Jie; Li, Cun; Sachs, Norman; Chiu, Man Chun; Wong, Bosco Ho-Yin; Chu, Hin; Poon, Vincent Kwok-Man; Wang, Dong; Zhao, Xiaoyu; Wen, Lei; Song, Wenjun; Yuan, Shuofeng; Wong, Kenneth Kak-Yuen; Chan, Jasper Fuk-Woo; To, Kelvin Kai-Wang; Chen, Honglin; Clevers, Hans; Yuen, Kwok-Yung

2018-06-26

Novel reassortant avian influenza H7N9 virus and pandemic 2009 H1N1 (H1N1pdm) virus cause human infections, while avian H7N2 and swine H1N1 virus mainly infect birds and pigs, respectively. There is no robust in vitro model for assessing the infectivity of emerging viruses in humans. Based on a recently established method, we generated long-term expanding 3D human airway organoids which accommodate four types of airway epithelial cells: ciliated, goblet, club, and basal cells. We report differentiation conditions which increase ciliated cell numbers to a nearly physiological level with synchronously beating cilia readily discernible in every organoid. In addition, the differentiation conditions induce elevated levels of serine proteases, which are essential for productive infection of human influenza viruses and low-pathogenic avian influenza viruses. We also established improved 2D monolayer culture conditions for the differentiated airway organoids. To demonstrate the ability of differentiated airway organoids to identify human-infective virus, 3D and 2D differentiated airway organoids are applied to evaluate two pairs of viruses with known distinct infectivity in humans, H7N9/Ah versus H7N2 and H1N1pdm versus an H1N1 strain isolated from swine (H1N1sw). The human-infective H7N9/Ah virus replicated more robustly than the poorly human-infective H7N2 virus; the highly human-infective H1N1pdm virus replicated to a higher titer than the counterpart H1N1sw. Collectively, we developed differentiated human airway organoids which can morphologically and functionally simulate human airway epithelium. These differentiated airway organoids can be applied for rapid assessment of the infectivity of emerging respiratory viruses to human. Copyright © 2018 the Author(s). Published by PNAS.

Carrier free immobilization and characterization of trypsin.

PubMed

Menfaatli, Esra; Zihnioglu, Figen

2015-04-01

Pancreatic trypsin was immobilized by cross-linked enzyme aggregates (CLEA) which is a carrier free immobilization method. Ammonium sulfate was chosen for enzyme precipitation which was followed by cross linking of formed aggregates via glutaraldehyde. Concentrations of precipitant and cross linker were respectively optimized as 60% ammonium sulfate and 1% glutaraldehyde. Optimum pH and temperature for CLEA was increased compared to free enzyme. Furthermore, pH, thermal and storage stability were improved. Presence of additives had no effects on enzyme activity. Prepared cross-linked trypsin aggregates are convenient for in situ protein fragmentation and can be used for protein identification.

Subsite specificity of trypanosomal cathepsin L-like cysteine proteases. Probing the S2 pocket with phenylalanine-derived amino acids.

PubMed

Lecaille, F; Authié, E; Moreau, T; Serveau, C; Gauthier, F; Lalmanach, G

2001-05-01

The S2 subsite of mammalian cysteine proteinases of the papain family is essential for specificity. Among natural amino acids, all these enzymes prefer bulky hydrophobic residues such as phenylalanine at P2. This holds true for their trypanosomal counterparts: cruzain from Trypanosoma cruzi and congopain from T. congolense. A detailed analysis of the S2 specificity of parasitic proteases was performed to gain information that might be of interest for the design of more selective pseudopeptidyl inhibitors. Nonproteogenic phenylalanyl analogs (Xaa) have been introduced into position P2 of fluorogenic substrates dansyl-Xaa-Arg-Ala-Pro-Trp, and their kinetic constants (Km, kcat/Km) have been determined with congopain and cruzain, and related host cathepsins B and L. Trypanosomal cysteine proteases are poorly stereoselective towards D/L-Phe, the inversion of chirality modifying the efficiency of the reaction but not the Km. Congopain binds cyclohexylalanine better than aromatic Phe derivatives. Another characteristic feature of congopain compared to cruzain and cathepsins B and L was that it could accomodate a phenylglycyl residue (kcat/Km = 1300 mM-1.s-1), while lengthening of the side chain by a methylene group only slightly impaired the specificity constant towards trypanosomal cysteine proteases. Mono- and di-halogenation or nitration of Phe did not affect Km for cathepsin L-like enzymes, but the presence of constrained Phe derivatives prevented a correct fitting into the S2 subsite. A model of congopain has been built to study the fit of Phe analogs within the S2 pocket. Phe analogs adopted a positioning within the S2 pocket similar to that of the Tyr of the cruzain/Z-Tyr-Ala-fluoromethylketone complex. However, cyclohexylalanine has an energetically favorable chair-like conformation and can penetrate deeper into the subsite. Fitting of modeled Phe analogs were in good agreement with kinetic parameters. Furthermore, a linear relationship could be established with

The cysteine-rich domain regulates ADAM protease function in vivo.

PubMed

Smith, Katherine M; Gaultier, Alban; Cousin, Helene; Alfandari, Dominique; White, Judith M; DeSimone, Douglas W

2002-12-09

ADAMs are membrane-anchored proteases that regulate cell behavior by proteolytically modifying the cell surface and ECM. Like other membrane-anchored proteases, ADAMs contain candidate "adhesive" domains downstream of their metalloprotease domains. The mechanism by which membrane-anchored cell surface proteases utilize these putative adhesive domains to regulate protease function in vivo is not well understood. We address this important question by analyzing the relative contributions of downstream extracellular domains (disintegrin, cysteine rich, and EGF-like repeat) of the ADAM13 metalloprotease during Xenopus laevis development. When expressed in embryos, ADAM13 induces hyperplasia of the cement gland, whereas ADAM10 does not. Using chimeric constructs, we find that the metalloprotease domain of ADAM10 can substitute for that of ADAM13, but that specificity for cement gland expansion requires a downstream extracellular domain of ADAM13. Analysis of finer resolution chimeras indicates an essential role for the cysteine-rich domain and a supporting role for the disintegrin domain. These and other results reveal that the cysteine-rich domain of ADAM13 cooperates intramolecularly with the ADAM13 metalloprotease domain to regulate its function in vivo. Our findings thus provide the first evidence that a downstream extracellular adhesive domain plays an active role in regulating ADAM protease function in vivo. These findings are likely relevant to other membrane-anchored cell surface proteases.

THE INACTIVATION OF DILUTE SOLUTIONS OF CRYSTALLINE TRYPSIN BY X-RADIATION

PubMed Central

McDonald, Margaret R.

1954-01-01

The activity of dilute solutions of crystalline trypsin is destroyed by x-rays. The inactivation is an exponential function of the radiation dose. The reaction yield of inactivation is independent of the intensity at which the radiation is delivered or the quality of the x-rays. The reaction yield increases with increasing concentration of trypsin, varying from 0.06 to 0.7 micromoles per liter per 1000 r for trypsin solutions ranging from 1 x 10–7 to 2 x 10–4 M. PMID:13192318

Investigation of trypsin-CdSe quantum dot interactions via spectroscopic methods and effects on enzymatic activity.

PubMed

Kaur, Gurvir; Tripathi, S K

2015-01-05

The paper presents the interactions between trypsin and water soluble cadmium selenide (CdSe) quantum dots investigated by spectrophotometric methods. CdSe quantum dots have strong ability to quench the intrinsic fluorescence of trypsin by a static quenching mechanism. The quenching has been studied at three different temperatures where the results revealed that electrostatic interactions exist between CdSe quantum dots and trypsin and are responsible to stabilize the complex. The Scatchard plot from quenching revealed 1 binding site for quantum dots by trypsin, the same has been confirmed by making isothermal titrations of quantum dots against trypsin. The distance between donor and acceptor for trypsin-CdSe quantum dot complexes is calculated to be 2.8 nm by energy transfer mechanisms. The intrinsic fluorescence of CdSe quantum dots has also been enhanced by the trypsin, and is linear for concentration of trypsin ranging 1-80 μl. All the observations evidence the formation of trypsin-CdSe quantum dot conjugates, where trypsin retains the enzymatic activity which in turn is temperature and pH dependent. Copyright © 2014 Elsevier B.V. All rights reserved.

Serine proteases in rodent hippocampus.

PubMed

Davies, B J; Pickard, B S; Steel, M; Morris, R G; Lathe, R

1998-09-04

Brain serine proteases are implicated in developmental processes, synaptic plasticity, and in disorders including Alzheimer's disease. The spectrum of the major enzymes expressed in brain has not been established previously. We now present a systematic study of the serine proteases expressed in adult rat and mouse hippocampus. Using a combination of techniques including polymerase chain reaction amplification and Northern blotting we show that tissue-type plasminogen activator (t-PA) is the major species represented. Unexpectedly, the next most abundant species were RNK-Met-1, a lymphocyte protease not reported previously in brain, and two new family members, BSP1 (brain serine protease 1) and BSP2. We report full-length sequences of the two new proteases; homologies indicate that these are of tryptic specificity. Although BSP2 is expressed in several brain regions, BSP1 expression is strikingly restricted to hippocampus. Other enzymes represented, but at lower levels, included elastase IV, proteinase 3, complement C2, chymotrypsin B, chymotrypsin-like protein, and Hageman factor. Although thrombin and urokinase-type plasminogen activator were not detected in the primary screen, low level expression was confirmed using specific polymerase chain reaction primers. In contrast, and despite robust expression of t-PA, the usual t-PA substrate plasminogen was not expressed at detectable levels.

Extraglottic airway devices: technology update.

PubMed

Sharma, Bimla; Sahai, Chand; Sood, Jayashree

2017-01-01

Extraglottic airway devices (EADs) have revolutionized the field of airway management. The invention of the laryngeal mask airway was a game changer, and since then, there have been several innovations to improve the EADs in design, functionality, safety and construction material. These have ranged from changes in the shape of the mask, number of cuffs and material used, like rubber, polyvinylchloride and latex. Phthalates, which were added to the construction material in order to increase device flexibility, were later omitted when this chemical was found to have serious adverse reproductive outcomes. The various designs brought out by numerous companies manufacturing EADs resulted in the addition of several devices to the airway market. These airway devices were put to use, many of them with inadequate or no evidence base regarding their efficacy and safety. To reduce the possibility of compromising the safety of the patient, the Difficult Airway Society (DAS) formed the Airway Device Evaluation Project Team (ADEPT) to strengthen the evidence base for airway equipment and vet the new extraglottic devices. A preuse careful analysis of the design and structure may help in better understanding of the functionality of a particular device. In the meantime, the search for the ideal EAD continues.

The subtilisin-like protease SBT3 contributes to insect resistance in tomato

PubMed Central

Meyer, Michael; Huttenlocher, Franziska; Cedzich, Anna; Procopio, Susanne; Stroeder, Jasper; Pau-Roblot, Corinne; Lequart-Pillon, Michelle; Pelloux, Jérôme; Stintzi, Annick; Schaller, Andreas

2016-01-01

Subtilisin-like proteases (SBTs) constitute a large family of extracellular plant proteases, the function of which is still largely unknown. In tomato plants, the expression of SBT3 was found to be induced in response to wounding and insect attack in injured leaves but not in healthy systemic tissues. The time course of SBT3 induction resembled that of proteinase inhibitor II and other late wound response genes suggesting a role for SBT3 in herbivore defense. Consistent with such a role, larvae of the specialist herbivore Manduca sexta performed better on transgenic plants silenced for SBT3 expression (SBT3-SI). Supporting a contribution of SBT3 to systemic wound signaling, systemic induction of late wound response genes was attenuated in SBT3-SI plants. The partial loss of insect resistance may thus be explained by a reduction in systemic defense gene expression. Alternatively, SBT3 may play a post-ingestive role in plant defense. Similar to other anti-nutritive proteins, SBT3 was found to be stable and active in the insect’s digestive system, where it may act on unidentified proteins of insect or plant origin. Finally, a reduction in the level of pectin methylesterification that was observed in transgenic plants with altered levels of SBT3 expression suggested an involvement of SBT3 in the regulation of pectin methylesterases (PMEs). While such a role has been described in other systems, PME activity and the degree of pectin methylesterification did not correlate with the level of insect resistance in SBT3-SI and SBT3 overexpressing plants and are thus unrelated to the observed resistance phenotype. PMID:27259555

Medullary thyroid carcinoma: ectopic production of peptides with ACTH-like, corticotrophin releasing factor-like and prolactin production-stimulating activities.

PubMed

Birkenhäger, J C; Upton, G V; Seldenrath, H J; Krieger, D T; Tashjian, A H

1976-10-01

A 45-year-old women had medullary tyroid carcinoma associated with Cushing's syndrome and galactorrhoea. Elevated plasma immunoreactive ACTH and cortisol were partially suppressed by intravenous dexamethasone, appreciably raised by lysine vasopressin, and urinary excretion of 17-oxogenic steroids slightly elevated by metyrapone. A large arterio-venous increase in plasma corticotrophin releasing factor-like activity across the thyroid gland was observed and tumour tissue contained corticotrophin releasing factor-like activity. Biologically active ACTH was not detected in tumour extracts before incubation with trypsin, but after trypsinization a value of 3.2 mU per gram was obtained. Arterial plasma contained biologically active ACTH (1.5 mU/100 ml) prior to trypsinization. Venous effluent from the thyroid gland contained biologically active (9.6 mU/100 ml) and immunoreactive ACTH (970 pg/ml) before trypsinization. Tumour extracts also contained prolactin production-stimulating activity. These findings can explain the Cushing's syndrome and the galactorrhoea both of which disappeared completely after thyroidectomy.

Comparative analysis of procoagulant and fibrinogenolytic activity of crude protease fractions of turmeric species.

PubMed

Shivalingu, B R; Vivek, H K; Nafeesa, Zohara; Priya, B S; Swamy, S Nanjunda

2015-08-22

Turmeric rhizome is a traditional herbal medicine, which has been widely used as a remedy to stop bleeding on fresh cuts and for wound healing by the rural and tribal population of India. To validate scientific and therapeutic application of turmeric rhizomes to stop bleeding on fresh cuts and its role in wound healing process. The water extracts of thoroughly scrubbed and washed turmeric rhizomes viz., Curcuma aromatica Salisb., Curcuma longa L., Curcuma caesia Roxb., Curcuma amada Roxb. and Curcuma zedoria (Christm.) Roscoe. were subjected to salting out and dialysis. The dialyzed crude enzyme fractions (CEFs) were assessed for proteolytic activity using casein as substrate and were also confirmed by caseinolytic zymography. Its coagulant activity and fibrinogenolytic activity were assessed using human citrated plasma and fibrinogen, respectively. The type of protease(s) in CEFs was confirmed by inhibition studies using specific protease inhibitors. The CEFs of C. aromatica, C. longa and C. caesia showed 1.89, 1.21 and 1.07 folds higher proteolytic activity, respectively, compared to papain. In contrast to these, C. amada and C. zedoria exhibited moderate proteolytic activity. CEFs showed low proteolytic activities compared to trypsin. The proteolytic activities of CEFs were confirmed by caseinolytic zymography. The CEFs of C. aromatica, C. longa and C. caesia showed complete hydrolysis of Aα, Bβ and γ subunits of human fibrinogen, while C. amada and C. zedoria showed partial hydrolysis. The CEFs viz., C. aromatica, C. longa, C. caesia, C. amada and C. zedoria exhibited strong procoagulant activity by reducing the human plasma clotting time from 172s (Control) to 66s, 84s 88s, 78s and 90s, respectively. The proteolytic activity of C. aromatica, C. longa, C. caesia and C. amada was inhibited (>82%) by PMSF, suggesting the possible presence of a serine protease(s). However, C. zedoria showed significant inhibition (60%) against IAA and moderate inhibition (30

Long-Acting Beta Agonists Enhance Allergic Airway Disease.

PubMed

Knight, John M; Mak, Garbo; Shaw, Joanne; Porter, Paul; McDermott, Catherine; Roberts, Luz; You, Ran; Yuan, Xiaoyi; Millien, Valentine O; Qian, Yuping; Song, Li-Zhen; Frazier, Vincent; Kim, Choel; Kim, Jeong Joo; Bond, Richard A; Milner, Joshua D; Zhang, Yuan; Mandal, Pijus K; Luong, Amber; Kheradmand, Farrah; McMurray, John S; Corry, David B

2015-01-01

Asthma is one of the most common of medical illnesses and is treated in part by drugs that activate the beta-2-adrenoceptor (β2-AR) to dilate obstructed airways. Such drugs include long acting beta agonists (LABAs) that are paradoxically linked to excess asthma-related mortality. Here we show that LABAs such as salmeterol and structurally related β2-AR drugs such as formoterol and carvedilol, but not short-acting agonists (SABAs) such as albuterol, promote exaggerated asthma-like allergic airway disease and enhanced airway constriction in mice. We demonstrate that salmeterol aberrantly promotes activation of the allergic disease-related transcription factor signal transducer and activator of transcription 6 (STAT6) in multiple mouse and human cells. A novel inhibitor of STAT6, PM-242H, inhibited initiation of allergic disease induced by airway fungal challenge, reversed established allergic airway disease in mice, and blocked salmeterol-dependent enhanced allergic airway disease. Thus, structurally related β2-AR ligands aberrantly activate STAT6 and promote allergic airway disease. This untoward pharmacological property likely explains adverse outcomes observed with LABAs, which may be overcome by agents that antagonize STAT6.

SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme

PubMed Central

Békés, Miklós; Rut, Wioletta; Kasperkiewicz, Paulina; Mulder, Monique P. C.; Ovaa, Huib; Drag, Marcin; Lima, Christopher D.; Huang, Tony T.

2015-01-01

Ubiquitin (Ub) and the ubiquitin-like modifier interferon stimulated gene 15 (ISG15) participate in the host defense of viral infections. Viruses, including the Severe Acute Respiratory Syndrome human coronavirus (SARS hCoV), have co-opted Ub/ISG15-conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub/ISG15-conjugated host proteins. Here, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle Eastern Respiratory Syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that similar to SARS PLpro, MERS PLpro is both a deubiquitinating and a deISGylating enzyme. Further analysis of the intrinsic deubiquitinating enzyme (DUB) activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, while SARS PLpro prefers to cleave Lys48-linked polyUb chains. Second, MERS PLpro cleaves polyUb chains in a “mono-distributive” manner (one Ub at a time), and SARS PLpro prefers to cleave K48-linked poly-Ub chains by sensing a di-Ub moiety as a minimal recognition element using a “di-distributive” cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses. PMID:25764917

Structural Basis for the Ubiquitin-Linkage Specificity and deISGylating Activity of SARS-CoV Papain-Like Protease

PubMed Central

Ratia, Kiira; Kilianski, Andrew; Baez-Santos, Yahira M.; Baker, Susan C.; Mesecar, Andrew

2014-01-01

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a papain-like protease (PLpro) with both deubiquitinating (DUB) and deISGylating activities that are proposed to counteract the post-translational modification of signaling molecules that activate the innate immune response. Here we examine the structural basis for PLpro's ubiquitin chain and interferon stimulated gene 15 (ISG15) specificity. We present the X-ray crystal structure of PLpro in complex with ubiquitin-aldehyde and model the interaction of PLpro with other ubiquitin-chain and ISG15 substrates. We show that PLpro greatly prefers K48- to K63-linked ubiquitin chains, and ISG15-based substrates to those that are mono-ubiquitinated. We propose that PLpro's higher affinity for K48-linked ubiquitin chains and ISG15 stems from a bivalent mechanism of binding, where two ubiquitin-like domains prefer to bind in the palm domain of PLpro with the most distal ubiquitin domain interacting with a “ridge” region of the thumb domain. Mutagenesis of residues within this ridge region revealed that these mutants retain viral protease activity and the ability to catalyze hydrolysis of mono-ubiquitin. However, a select number of these mutants have a significantly reduced ability to hydrolyze the substrate ISG15-AMC, or be inhibited by K48-linked diubuiquitin. For these latter residues, we found that PLpro antagonism of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) signaling pathway is abrogated. This identification of key and unique sites in PLpro required for recognition and processing of diubiquitin and ISG15 versus mono-ubiquitin and protease activity provides new insight into ubiquitin-chain and ISG15 recognition and highlights a role for PLpro DUB and deISGylase activity in antagonism of the innate immune response. PMID:24854014

Matrix metalloproteinase-2: A key regulator in coagulation proteases mediated human breast cancer progression through autocrine signaling.

PubMed

Das, Kaushik; Prasad, Ramesh; Ansari, Shabbir Ahmed; Roy, Abhishek; Mukherjee, Ashis; Sen, Prosenjit

2018-06-02

Cell invasion is attributed to the synthesis and secretion of proteolytically active matrix-metalloproteinases (MMPs) by tumor cells to degrade extracellular matrix (ECM) and promote metastasis. The role of protease-activated receptor 2 (PAR2) in human breast cancer migration/invasion via MMP-2 up-regulation remains ill-defined; hence we investigated whether TF-FVIIa/trypsin-mediated PAR2 activation induces MMP-2 expression in human breast cancer. MMP-2 expression and the signaling mechanisms were analyzed by western blotting and RT-PCR. MMP-2 activity was measured by gelatin zymography. Cell invasion was analyzed by transwell invasion assay whereas; wound healing assay was performed to understand the cell migratory potential. Here, we highlight that TF-FVIIa/trypsin-mediated PAR2 activation leads to enhanced MMP-2 expression in human breast cancer cells contributing to tumor progression. Knock-down of PAR2 abrogated TF-FVIIa/trypsin-induced up-regulation of MMP-2. Again, genetic manipulation of AKT or inhibition of NF-ĸB suggested that PAR2-mediated enhanced MMP-2 expression is dependent on the PI3K-AKT-NF-ĸB pathway. We also reveal that TF, PAR2, and MMP-2 are over-expressed in invasive breast carcinoma tissues as compared to normal. Knock-down of MMP-2 significantly impeded TF-FVIIa/trypsin-induced cell invasion. Further, we report that MMP-2 activates p38 MAPK-MK2-HSP27 signaling axis that leads to actin polymerization and induces cell migration. Pharmacological inhibition of p38 MAPK or MK2 attenuates MMP-2-induced cell migration. The study delineates a novel signaling pathway by which PAR2-induced MMP-2 expression regulates human breast cancer cell migration/invasion. Understanding these mechanistic details will certainly help to identify crucial targets for therapeutic interventions in breast cancer metastasis. Copyright © 2018. Published by Elsevier Masson SAS.

Mechanism of curcumin-induced trypsin inhibition: Computational and experimental studies

NASA Astrophysics Data System (ADS)

Wang, Yan-Qing; Zhang, Hong-Mei; Kang, Yi-Jun; Gu, Yun-Lan; Cao, Jian

2016-03-01

In the present study, the experimental and theoretical methods were used to analyze the binding interaction of food dye, curcumin with trypsin. The results of fluorescence spectroscopic measurements indicated that curcumin binding resulted in the obviously intrinsic fluorescence quenching with the increase concentration of curcumin. This binding interaction is a spontaneous process with the estimated enthalpy and entropy changes being -15.70 kJ mol-1 and 40.25 J mol-1 K-1, respectively. Hydrogen bonds and hydrophobic forces played an important role in the complex formation between curcumin and trypsin. Moreover, curcumin could enter into the primary substrate-binding pocket and makes the activity of trypsin decrease remarkably with the increasing concentration of curcumin.

21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test... measure by immunochemical techniques the inter-alpha trypsin inhibitor (a protein) in serum and other body fluids. Measurement of inter-alpha trypsin inhibitor may aid in the diagnosis of acute bacterial...

21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test... measure by immunochemical techniques the inter-alpha trypsin inhibitor (a protein) in serum and other body fluids. Measurement of inter-alpha trypsin inhibitor may aid in the diagnosis of acute bacterial...

21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test... measure by immunochemical techniques the inter-alpha trypsin inhibitor (a protein) in serum and other body fluids. Measurement of inter-alpha trypsin inhibitor may aid in the diagnosis of acute bacterial...

21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test... measure by immunochemical techniques the inter-alpha trypsin inhibitor (a protein) in serum and other body fluids. Measurement of inter-alpha trypsin inhibitor may aid in the diagnosis of acute bacterial...

Fish skin gelatin hydrolysates produced by visceral peptidase and bovine trypsin: Bioactivity and stability.

PubMed

Ketnawa, Sunantha; Benjakul, Soottawat; Martínez-Alvarez, Oscar; Rawdkuen, Saroat

2017-01-15

The peptidase from the viscera of farmed giant catfish was used for producing gelatin hydrolysates (HG) and compared with those produced from commercial bovine trypsin (HB). The degree of hydrolysis (DH) observed suggests that proteolytic cleavage rapidly occurred within the first 120min of incubation, and there was higher DH in HG than in HB. HG demonstrated the highest ACE-inhibitory activity, DPPH, ABTS radical scavenging activity, and FRAP. HB showed the highest FRAP activity. The DPPH radical scavenging activity of HG was quite stable over the pH range of 1-11, but it increased slightly when the heating duration time reached 240min at 100°C. The ACE-inhibitory activity of HG showed the highest stability at a pH of 7, and it remained very stable at 100°C for over 15-240min. The visceral peptidase from farmed giant catfish could be an alternative protease for generating protein hydrolysates with desirable bioactivities. The resulting hydrolysates showed good stability, making them potential functional ingredients for food formulations. Copyright © 2016 Elsevier Ltd. All rights reserved.

The intracellular carboxyl tail of the PAR-2 receptor controls intracellular signaling and cell death.

PubMed

Zhu, Zhihui; Stricker, Rolf; Li, Rong yu; Zündorf, Gregor; Reiser, Georg

2015-03-01

The protease-activated receptors are a group of unique G protein-coupled receptors, including PAR-1, PAR-2, PAR-3 and PAR-4. PAR-2 is activated by multiple trypsin-like serine proteases, including trypsin, tryptase and coagulation proteases. The clusters of phosphorylation sites in the PAR-2 carboxyl tail are suggested to be important for the binding of adaptor proteins to initiate intracellular signaling to Ca(2+) and mitogen-activated protein kinases. To explore the functional role of PAR-2 carboxyl tail in controlling intracellular Ca(2+), ERK and AKT signaling, a series of truncated mutants containing different clusters of serines/threonines were generated and expressed in HEK293 cells. Firstly, we observed that lack of the complete C-terminus of PAR-2 in a mutated receptor gave a relatively low level of localization on the cell plasma membrane. Secondly, the shortened carboxyl tail containing 13 amino acids was sufficient for receptor internalization. Thirdly, the cells expressing truncation mutants showed deficits in their capacity to couple to intracellular Ca(2+) and ERK and AKT signaling upon trypsin challenge. In addition, HEK293 cells carrying different PAR-2 truncation mutants displayed decreased levels of cell survival after long-lasting trypsin stimulation. In summary, the PAR-2 carboxyl tail was found to control the receptor localization, internalization, intracellular Ca(2+) responses and signaling to ERK and AKT. The latter can be considered to be important for cell death control.

A cathepsin L-like protease from Strongylus vulgaris: an orthologue of Caenorhabditis elegans CPL-1.

PubMed

Ultaigh, Sinéad Nic An; Carolan, James C; Britton, Collette; Murray, Linda; Ryan, Michael F

2009-04-01

Cathespin L-like proteases (CPLs), characterized from a wide range of helminths, are significant in helminth biology. For example, in Caenorhabditis elegans CPL is essential for embryogenesis. Here, we report a cathepsin L-like gene from three species of strongyles that parasitize the horse, and describe the isolation of a cpl gene (Sv-cpl-1) from Strongylus vulgaris, the first such from equine strongyles. It encodes a protein of 354 amino acids with high similarity to other parasitic Strongylida (90-91%), and C.elegans CPL-1 (87%), a member of the same Clade. As S.vulgaris cpl-1 rescued the embryonic lethal phenotype of the C.elegans cpl-1 mutant, these genes may be orthologues, sharing the same function in each species. Targeting Sv-CPL-1 might enable novel control strategies by decreasing parasite development and transmission.

An AC-5 cathepsin B-like protease purified from Haemonchus contortus excretory secretory products shows protective antigen potential for lambs

PubMed Central

De Vries, Erik; Bakker, Nicole; Krijgsveld, Jeroen; Knox, Dave P.; Heck, Albert J.R.; Yatsuda, Ana Patricia

2009-01-01

The immunogenic properties of cysteine proteases obtained from excretory/secretory products (ES) of Haemonchus contortus were investigated with a fraction purified with a recombinant H. contortus cystatin affinity column. The enrichment of H. contortus ES for cysteine protease was confirmed with substrate SDS-PAGE gels since the cystatin-binding fraction activity was three times higher than total ES, despite representing only 3% of total ES. This activity was inhibited by a specific cysteine protease inhibitor (E64) and by recombinant cystatin. The one-dimensional profile of the cystatin-binding fraction displayed a single band with a molecular mass of 43 kDa. Mass spectrometry showed this to be AC-5, a cathepsin B-like cysteine protease which had not been identified in ES products of H. contortus before. The cystatin binding fraction was tested as an immunogen in lambs which were vaccinated three times (week 0, 2.5 and 5), challenged with 10 000 L3 H. contortus (week 6) before necropsy and compared to unvaccinated challenge controls and another group given total ES (n = 10 per group). The group vaccinated with cystatin-binding proteins showed 36% and 32% mean worm burden and eggs per gram of faeces (EPG) reductions, respectively, compared to the controls but total ES was almost without effect. After challenge the cystatin-binding proteins induced significantly higher local and systemic ES specific IgA and IgG responses. PMID:19401141

21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5890 Inter-alpha trypsin inhibitor immunological test system. (a) Identification. An inter... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Inter-alpha trypsin inhibitor immunological test...

Effect of bombesin and cholecystokinin on plasma immunoreactive trypsin in humans.

PubMed

de Jong, A J; Klamer, M; Jansen, J B; Hopman, W P; Lamers, C B

1987-01-01

Since bombesin is a potent stimulus of the release of cholecystokinin (CCK), it has been suggested that the stimulatory effect of bombesin on pancreatic enzyme secretion is mediated by CCK. The present study was undertaken to determine the role of CCK in the bombesin-induced stimulation of plasma immunoreactive trypsin. Plasma CCK was measured by radioimmunoassay using the antibody T204, which binds to all biologically active sulfated COOH-terminal CCK-peptides. Plasma trypsin was also measured by radioimmunoassay. Infusion of 5 ng/kg/min bombesin in 6 healthy volunteers increased plasma CCK from 1.2 +/- 0.2-8.9 +/- 0.7 pM (p less than 0.0001). The peak increment in plasma CCK during bombesin (9.3 +/- 0.6 pM) was accompanied by a significant rise in plasma trypsin from 206 +/- 21-334 +/- 44 ng/ml (p less than 0.01). However, when similar increases in plasma CCK were achieved by infusion of 0.018 CU/kg/min CCK-33 (9.9 +/- 0.8 pM) or by intraduodenal instillation of 250 ml 20% Intralipid (9.7 +/- 1.9 pM), no significant changes in plasma trypsin were observed. It is therefore concluded that the stimulatory effect of bombesin on plasma immunoreactive trypsin is not mediated by CCK.

Purification and characterization of a trypsin inhibitor from the seeds of Artocarpus heterophyllus Lam.

PubMed

Lyu, Junchen; Liu, Yuan; An, Tianchen; Liu, Yujun; Wang, Manchuriga; Song, Yanting; Zheng, Feifei; Wu, Dan; Zhang, Yingxia; Deng, Shiming

2015-05-01

A proteinaceous inhibitor against trypsin was isolated from the seeds of Artocarpus heterophyllus Lam. by successive ammonium sulfate precipitation, ion-exchange, and gel-filtration chromatography. The trypsin inhibitor, named as AHLTI (A. heterophyllus Lam. trypsin inhibitor), consisted of a single polypeptide chain with a molecular weight of 28.5 kDa, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel-filtration chromatography. The N-terminal sequence of AHLTI was DEPPSELDAS, which showed no similarity to other known trypsin inhibitor sequence. AHLTI completely inhibited bovine trypsin at a molar ratio of 1:2 (AHLTI:trypsin) analyzed by native polyacrylamide gel electrophoresis, inhibition activity assay, and gel-filtration chromatography. Moreover, kinetic enzymatic studies were carried out to understand the inhibition mechanism of AHLTI against trypsin. Results showed that AHLTI was a competitive inhibitor with an equilibrium dissociation constant (Ki) of 3.7 × 10(-8) M. However, AHLTI showed weak inhibitory activity toward chymotrypsin and elastase. AHLTI was stable over a broad range of pH 4-8 and temperature 20-80°C. The reduction agent, dithiothreitol, had no obvious effect on AHLTI. The trypsin inhibition assays of AHLTI toward digestive enzymes from insect pest guts in vitro demonstrated that AHLTI was effective against enzymes from Locusta migratoria manilensis (Meyen). These results suggested that AHLTI might be a novel trypsin inhibitor from A. heterophyllus Lam. belonging to Kunitz family, and play an important role in protecting from insect pest. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Searching for discrimination rules in protease proteolytic cleavage activity using genetic programming with a min-max scoring function.

PubMed

Yang, Zheng Rong; Thomson, Rebecca; Hodgman, T Charles; Dry, Jonathan; Doyle, Austin K; Narayanan, Ajit; Wu, XiKun

2003-11-01

This paper presents an algorithm which is able to extract discriminant rules from oligopeptides for protease proteolytic cleavage activity prediction. The algorithm is developed using genetic programming. Three important components in the algorithm are a min-max scoring function, the reverse Polish notation (RPN) and the use of minimum description length. The min-max scoring function is developed using amino acid similarity matrices for measuring the similarity between an oligopeptide and a rule, which is a complex algebraic equation of amino acids rather than a simple pattern sequence. The Fisher ratio is then calculated on the scoring values using the class label associated with the oligopeptides. The discriminant ability of each rule can therefore be evaluated. The use of RPN makes the evolutionary operations simpler and therefore reduces the computational cost. To prevent overfitting, the concept of minimum description length is used to penalize over-complicated rules. A fitness function is therefore composed of the Fisher ratio and the use of minimum description length for an efficient evolutionary process. In the application to four protease datasets (Trypsin, Factor Xa, Hepatitis C Virus and HIV protease cleavage site prediction), our algorithm is superior to C5, a conventional method for deriving decision trees.

Human and bovine spinal disc mechanics subsequent to trypsin injection.

PubMed

Alsup, Jeremy; Bishop, Timothy; Eggett, Dennis; Bowden, Anton E

2017-10-01

To investigate the biomechanical effects of injections of a protease on the characteristics of bovine coccygeal and human lumbar disc motion segments. Mechanics of treated tissues were measured immediately after injection and 3 h after injection. Motion segments underwent axial rotation and flexion-extension loading. Stiffness and neutral zone parameters experienced significant changes over time, with bovine tissues more strongly affected than human cadaver tissues. This was true in both axial rotation and flexion-extension. The treatment type significantly affected the neutral zone measurements in axial rotation. Hysteresis parameters were impacted by control injections. The extrapolation of bovine coccygeal motion testing results to human lumbar disc mechanics is not yet practical. The injected treatment may have a smaller impact on disc mechanics than time in testing. Viscoelasticity of human lumbar discs may be impacted by any damage to the annulus fibrosis induced by needlestick. Preclinical testing of novel spinal devices is essential to the design validation and regulatory processes, but current testing techniques rely on cadaveric testing of primarily older spines with essentially random amounts of disc degeneration. The present work investigates the viability of using trypsin injections to create a more uniform preclinical model of disc degeneration from a mechanics perspective, for the purpose of testing spinal devices. Such a model would facilitate translation of new spinal technologies to clinical practice.

THE INACTIVATION OF DILUTE SOLUTIONS OF CRYSTALLINE TRYPSIN BY X-RADIATION

PubMed Central

McDonald, Margaret R.

1955-01-01

The proteolytic activity of dilute solutions of clystalline trypsin is destroyed by x-rays, the amount of inactivation being an exponential function of the radiation dose. The reaction yield increases steadily with increasing concentration of trypsin, varying, as the concentration of enzyme is increased from 1 to 300 µM, from 0.068 to 0.958 micromole of trypsin per liter inactivated per 1000 r with 0.005 N hydrochloric acid as the solvent, from 0.273 to 0.866 with 0.005 N sulfuric acid as the solvent, and from 0.343 to 0.844 with 0.005 N nitric acid as the solvent. When the reaction yields are plotted as a function of the initial concentration of trypsin, they fall on a curve given by the expression Y α XK, in which Y is the reaction yield, X is the concentration of trypsin, and K is a constant equal to 0.46, 0.20, and 0.16, respectively, with 0.005 N hydrochloric, sulfuric, and nitric acids as solvents. The differences between the reaction yields found with chloride and sulfate ions in I to 10 µM trypsin solutions are significant only in the pH range from 2 to 4. The amount of inactivation obtained with a given dose of x-rays depends on the pH of the solution being irradiated and the nature of the solvent. The reaction yield-pH curve is a symmetrical one, with minimum yields at about pH 7. Buffers such as acetate, citrate, borate and barbiturate, and other organic molecules such as ethanol and glucose, in concentrations as low as 20 µM, inhibit the inactivation of trypsin by x-radiation. Sigmoid inactivation-dose curves instead of exponential ones are obtained in the presence of ethanol. The reaction yields for the inactivation of trypsin solutions by x-rays are approximately 1.5 times greater when the irradiation is done at 26°C. than when it is done at 5°C., when 0.005 N hydrochloric acid is the solvent. The dependence on temperature is less when 0.005 N sulfuric acid is used, and is negligible with 0.005 N nitric acid. The difficulties involved in

Indispensable Role of Proteases in Plant Innate Immunity.

PubMed

Balakireva, Anastasia V; Zamyatnin, Andrey A

2018-02-23

Plant defense is achieved mainly through the induction of microbe-associated molecular patterns (MAMP)-triggered immunity (MTI), effector-triggered immunity (ETI), systemic acquired resistance (SAR), induced systemic resistance (ISR), and RNA silencing. Plant immunity is a highly complex phenomenon with its own unique features that have emerged as a result of the arms race between plants and pathogens. However, the regulation of these processes is the same for all living organisms, including plants, and is controlled by proteases. Different families of plant proteases are involved in every type of immunity: some of the proteases that are covered in this review participate in MTI, affecting stomatal closure and callose deposition. A large number of proteases act in the apoplast, contributing to ETI by managing extracellular defense. A vast majority of the endogenous proteases discussed in this review are associated with the programmed cell death (PCD) of the infected cells and exhibit caspase-like activities. The synthesis of signal molecules, such as salicylic acid, jasmonic acid, and ethylene, and their signaling pathways, are regulated by endogenous proteases that affect the induction of pathogenesis-related genes and SAR or ISR establishment. A number of proteases are associated with herbivore defense. In this review, we summarize the data concerning identified plant endogenous proteases, their effect on plant-pathogen interactions, their subcellular localization, and their functional properties, if available, and we attribute a role in the different types and stages of innate immunity for each of the proteases covered.

Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target.

PubMed

Withers-Martinez, Chrislaine; Suarez, Catherine; Fulle, Simone; Kher, Samir; Penzo, Maria; Ebejer, Jean-Paul; Koussis, Kostas; Hackett, Fiona; Jirgensons, Aigars; Finn, Paul; Blackman, Michael J

2012-05-15

Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

How anaesthesiologists understand difficult airway guidelines-an interview study.

PubMed

Knudsen, Kati; Pöder, Ulrika; Nilsson, Ulrica; Högman, Marieann; Larsson, Anders; Larsson, Jan

2017-11-01

In the practice of anaesthesia, clinical guidelines that aim to improve the safety of airway procedures have been developed. The aim of this study was to explore how anaesthesiologists understand or conceive of difficult airway management algorithms. A qualitative phenomenographic design was chosen to explore anaesthesiologists' views on airway algorithms. Anaesthesiologists working in three hospitals were included. Individual face-to-face interviews were conducted. Four different ways of understanding were identified, describing airway algorithms as: (A) a law-like rule for how to act in difficult airway situations; (B) a cognitive aid, an action plan for difficult airway situations; (C) a basis for developing flexible, personal action plans for the difficult airway; and (D) the experts' consensus, a set of scientifically based guidelines for handling the difficult airway. The interviewed anaesthesiologists understood difficult airway management guidelines/algorithms very differently.

Effect of atropine and somatostatin on bombesin-stimulated plasma immunoreactive trypsin release in man.

PubMed

de Jong, A J; Klamer, M; Lamers, C B

1987-01-01

This study was undertaken to determine the effect of atropine and somatostatin, two inhibitors of intraduodenal pancreatic enzyme secretion, on bombesin-stimulated release of plasma immunoreactive trypsin in 6 healthy volunteers. Infusion of 5 ng/kg.min bombesin during 30 min induced significant increases in plasma trypsin from 206 +/- 20 to 334 +/- 44 ng/ml (p less than 0.01). Atropine (15 ng/kg as i.v. bolus followed by 5 ng/kg.h) had no influence on the bombesin-stimulated increase in plasma immunoreactive trypsin (207 +/- 20 to 326 +/- 54 ng/ml). Somatostatin (125 micrograms as i.v. bolus followed by 125 micrograms/h) also failed to inhibit the plasma trypsin response to bombesin (207 +/- 18 to 663 +/- 166 ng/ml). These results point to major differences in the regulation of plasma and intraduodenal trypsin secretion.

The protease-activated receptor 2 regulates pigmentation via keratinocyte-melanocyte interactions.

PubMed

Seiberg, M; Paine, C; Sharlow, E; Andrade-Gordon, P; Costanzo, M; Eisinger, M; Shapiro, S S

2000-01-10

Close association exists between melanocytes, the pigment melanin-producing cells in the body, and their neighboring keratinocytes. Keratinocytes are the pigment recipients and skin pigmentation is the result of this interaction. While the chemical basis of melanin production (melanogenesis) is well documented, the molecular mechanism of melanosome transfer needs to be elucidated. We are now providing first evidence that the protease-activated receptor 2 (PAR-2) expressed on keratinocytes, but not on melanocytes, is involved in melanosome transfer and therefore may regulate pigmentation. Activation of PAR-2 with trypsin or with the peptide agonist SLIGRL induced pigmentation in both two- and three-dimensional cocultures of keratinocytes and melanocytes, but not in cocultures that were spatially separated, indicating the need for intimate cell-cell contact. Topical application of SLIGRL on human skin transplanted on SCID mice resulted in a visible skin darkening. Histological examination revealed increased deposits of melanin in the keratinocytes. Inhibition of PAR-2 activation by RWJ-50353, a serine protease inhibitor, resulted in depigmentation and changes in expression of melanogenic-specific genes. Keratinocyte-melanocyte contact was essential for this depigmenting effect. Topical application of this inhibitor induced lightening of the dark skin Yucatan swine, which was confirmed by histochemical analysis. The results presented here suggest a novel mechanism for the regulation of pigmentation, mediated by the activation or inhibition of the keratinocyte receptor PAR-2. Copyright 2000 Academic Press.

Reduced Sodium Transport With Nasal Administration of the Prostasin Inhibitor Camostat in Subjects With Cystic Fibrosis

PubMed Central

Reeves, Ginger; Hathorne, Heather; Solomon, G. Martin; Abbi, Smita; Renard, Didier; Lock, Ruth; Zhou, Ping; Danahay, Henry; Clancy, John P.; Waltz, David A.

2013-01-01

Background: Prostasin, a trypsin-like serine protease, is a channel-activating protease and major regulator of epithelial sodium channel-mediated sodium absorption. Its direct inhibition by camostat represents a potential approach to inhibiting sodium transport in cystic fibrosis (CF). Methods: To determine whether a topical formulation of camostat represents an efficacious and tolerable approach to reducing Na+ transport in the CF airway, we conducted a two-part randomized, double-blind, placebo-controlled, crossover, ascending single-dose study to evaluate the pharmacodynamics, safety, and pharmacokinetics of camostat administered through a nasal spray pump in subjects with CF. Nasal potential difference (PD) was measured before and after treatment, and safety and pharmacokinetics were assessed by a standardized approach. Results: In part 1, nine subjects were enrolled, and six completed crossover dosing at the maximally tolerated dose. The change in maximal (most polarizing) basal PD 2 h following administration of camostat was +13.1 mV (1.6-mg dose group) compared with −8.6 mV following placebo (P < .005). Intrasubject change in Ringer and amiloride-sensitive PDs exhibited similar and consistent responses. Bayesian analysis in an additional six subjects in part 2 estimated a dose of 18 μg/mL to provide 50% of the maximum effect. There was no significant change in chloride transport or total nasal symptom score, nasal examination rating, and laboratory parameters. Conclusions: This study establishes the proof of concept that a reduction in sodium transport in the human CF airway can be achieved through inhibition of prostasin activity, identifying a potential therapeutic target in the disease. Trial registration: ClinicalTrials.gov; No.: NCT00506792; URL: www.clinicaltrials.gov PMID:23412700

Highly Efficient Gluten Degradation by Lactobacilli and Fungal Proteases during Food Processing: New Perspectives for Celiac Disease▿

PubMed Central

Rizzello, Carlo G.; De Angelis, Maria; Di Cagno, Raffaella; Camarca, Alessandra; Silano, Marco; Losito, Ilario; De Vincenzi, Massimo; De Bari, Maria D.; Palmisano, Francesco; Maurano, Francesco; Gianfrani, Carmen; Gobbetti, Marco

2007-01-01

Presently, the only effective treatment for celiac disease is a life-long gluten-free diet. In this work, we used a new mixture of selected sourdough lactobacilli and fungal proteases to eliminate the toxicity of wheat flour during long-time fermentation. Immunological (R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay [ELISA] and R5 antibody-based Western blot), two-dimensional electrophoresis, and mass spectrometry (matrix-assisted laser desorption ionization-time of flight, strong-cation-exchange-liquid chromatography/capillary liquid chromatography-electrospray ionization-quadrupole-time of flight [SCX-LC/CapLC-ESI-Q-TOF], and high-pressure liquid chromatography-electrospray ionization-ion trap mass spectrometry) analyses were used to determine the gluten concentration. Assays based on the proliferation of peripheral blood mononuclear cells (PBMCs) and gamma interferon production by PBMCs and intestinal T-cell lines (iTCLs) from 12 celiac disease patients were used to determine the protein toxicity of the pepsin-trypsin digests from fermented wheat dough (sourdough). As determined by R5-based sandwich and competitive ELISAs, the residual concentration of gluten in sourdough was 12 ppm. Albumins, globulins, and gliadins were completely hydrolyzed, while ca. 20% of glutenins persisted. Low-molecular-weight epitopes were not detectable by SCX-LC/CapLC-ESI-Q-TOF mass spectrometry and R5-based Western blot analyses. The kinetics of the hydrolysis of the 33-mer by lactobacilli were highly efficient. All proteins extracted from sourdough activated PBMCs and induced gamma interferon production at levels comparable to the negative control. None of the iTCLs demonstrated immunoreactivity towards pepsin-trypsin digests. Bread making was standardized to show the suitability of the detoxified wheat flour. Food processing by selected sourdough lactobacilli and fungal proteases may be considered an efficient approach to eliminate gluten toxicity

Amprenavir, new protease inhibitor, approved.

PubMed

James, J S

1999-05-07

A new protease inhibitor, amprenavir (Agenerase), has received FDA marketing approval. The approval was based on two 24-week controlled trials and safety data in more than 1,400 patients under FDA accelerated-approval rules. Amprenavir is approved for patients 4 years of age and older. The drug is taken twice daily, with or without food. Side effects include gastrointestinal disturbances, rashes, and oral paresthesia. Severe or life-threatening rashes have occurred in 1 percent of all patients. Pregnant women should not use the drug unless necessary. The drug was developed by Vertex Pharmaceuticals Inc. and is being marketed by Glaxo Wellcome. Some studies suggest that amprenavir is less likely than other protease inhibitors to be associated with lipid metabolism problems. It may have a resistance profile different from that of other protease inhibitors, and therefore may cause different cross resistance problems. Amprenavir appears to be synergistic with abacavir (Ziagen) in laboratory tests.

Tunable protease-activatable virus nanonodes.

PubMed

Judd, Justin; Ho, Michelle L; Tiwari, Abhinav; Gomez, Eric J; Dempsey, Christopher; Van Vliet, Kim; Igoshin, Oleg A; Silberg, Jonathan J; Agbandje-McKenna, Mavis; Suh, Junghae

2014-05-27

We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus-receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery.

Tunable Protease-Activatable Virus Nanonodes

PubMed Central

2015-01-01

We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus–receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery. PMID:24796495

21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

Code of Federal Regulations, 2013 CFR

2013-04-01

...) Conditions of use. The drug is used as an aid in the treatment of external wounds and assists healing by... delivered to the wound site contains 0.12 milligram of crystalline trypsin, 87.0 milligrams of Peru balsam... gram delivered to the wound site contains 0.1 milligram of crystalline trypsin, 72.5 milligrams of Peru...

21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

Code of Federal Regulations, 2012 CFR

2012-04-01

...) Conditions of use. The drug is used as an aid in the treatment of external wounds and assists healing by... delivered to the wound site contains 0.12 milligram of crystalline trypsin, 87.0 milligrams of Peru balsam... gram delivered to the wound site contains 0.1 milligram of crystalline trypsin, 72.5 milligrams of Peru...

21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

Code of Federal Regulations, 2011 CFR

2011-04-01

... delivered to the wound site contains 0.12 milligram of crystalline trypsin, 87.0 milligrams of Peru balsam... gram delivered to the wound site contains 0.1 milligram of crystalline trypsin, 72.5 milligrams of Peru...) Conditions of use. The drug is used as an aid in the treatment of external wounds and assists healing by...

21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

Code of Federal Regulations, 2010 CFR

2010-04-01

...) Conditions of use. The drug is used as an aid in the treatment of external wounds and assists healing by... delivered to the wound site contains 0.12 milligram of crystalline trypsin, 87.0 milligrams of Peru balsam... gram delivered to the wound site contains 0.1 milligram of crystalline trypsin, 72.5 milligrams of Peru...

Proteases as therapeutics

PubMed Central

Craik, Charles S.; Page, Michael J.; Madison, Edwin L.

2015-01-01

Proteases are an expanding class of drugs that hold great promise. The U.S. FDA (Food and Drug Administration) has approved 12 protease therapies, and a number of next generation or completely new proteases are in clinical development. Although they are a well-recognized class of targets for inhibitors, proteases themselves have not typically been considered as a drug class despite their application in the clinic over the last several decades; initially as plasma fractions and later as purified products. Although the predominant use of proteases has been in treating cardiovascular disease, they are also emerging as useful agents in the treatment of sepsis, digestive disorders, inflammation, cystic fibrosis, retinal disorders, psoriasis and other diseases. In the present review, we outline the history of proteases as therapeutics, provide an overview of their current clinical application, and describe several approaches to improve and expand their clinical application. Undoubtedly, our ability to harness proteolysis for disease treatment will increase with our understanding of protease biology and the molecular mechanisms responsible. New technologies for rationally engineering proteases, as well as improved delivery options, will expand greatly the potential applications of these enzymes. The recognition that proteases are, in fact, an established class of safe and efficacious drugs will stimulate investigation of additional therapeutic applications for these enzymes. Proteases therefore have a bright future as a distinct therapeutic class with diverse clinical applications. PMID:21406063

The chitinase-like protein YKL-40 increases mucin5AC production in human bronchial epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Chunyi; Li, Qi; Zhou, Xiangdong, E-mail: zxd999@263.net

2013-11-01

Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases. However, the regulatory mechanisms that mediate excessive mucin production remain elusive. Recently, the level of YKL-40, a chitinase-like protein, has been found to be significantly increased in chronic inflammatory airway diseases and has been shown to be associated with the severity of these diseases. In this study, we sought to explore the effect of YKL-40 on mucin5AC (MUC5AC) production in chronic inflammatory airway diseases and the potential signaling pathways involved in this process. We found that elevated YKL-40 levels increased the mRNA and protein expression of MUC5ACmore » in a dose- and time-dependent manner, in association with the phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB), reflecting their activation. These responses were significantly suppressed by the knockdown of protease-activating receptor 2 (PAR2) with specific small interfering RNA or the inhibitors of ERK and NF-κB. YKL-40-induced MUC5AC overproduction was also effectively attenuated by the inhibitor of focal adhesion kinase (FAK). Taken together, these results imply that YKL-40 can stimulate excessive MUC5AC production through PAR2- and FAK-mediated mechanisms. - Highlights: • MUC5AC is the major secreted mucin in chronic inflammatory airway diseases. • YKL-40 is a prototype of the chitinase-like protein in mammals. • YKL-40 is an active player in chronic inflammatory airway diseases. • YKL-40 can increase MUC5AC production via PAR2-mediated pathway. • FAK is another candidate to mediate YKL-40-induced MUC5AC overexpression.« less

Magnetic nanoparticles coated with polyaniline to stabilize immobilized trypsin

NASA Astrophysics Data System (ADS)

Maciel, J. C.; D. Mercês, A. A.; Cabrera, M.; Shigeyosi, W. T.; de Souza, S. D.; Olzon-Dionysio, M.; Fabris, J. D.; Cardoso, C. A.; Neri, D. F. M.; C. Silva, M. P.; Carvalho, L. B.

2016-12-01

It is reported the synthesis of magnetic nanoparticles via the chemical co-precipitation of Fe 3+ ions and their preparation by coating them with polyaniline. The electronic micrograph analysis showed that the mean diameter for the nanoparticles is ˜15 nm. FTIR, powder X-ray diffraction and Mössbauer spectroscopy were used to understand the chemical, crystallographic and 57Fe hyperfine structures for the two samples. The nanoparticles, which exhibited magnetic behavior with relatively high spontaneous magnetization at room temperature, were identified as being mainly formed by maghemite ( γFe2O3). The coated magnetic nanoparticles (sample labeled "mPANI") presented a real ability to bind biological molecules such as trypsin, forming the magnetic enzyme derivative (sample "mPANIG-Trypsin"). The amount of protein and specific activity of the immobilized trypsin were found to be 13±5 μg of protein/mg of mPANI (49.3 % of immobilized protein) and 24.1±0.7 U/mg of immobilized protein, respectively. After 48 days of storage at 4 ∘C, the activity of the immobilized trypsin was found to be 89 % of its initial activity. This simple, fast and low-cost procedure was revealed to be a promising way to prepare mPANI nanoparticles if technological applications addressed to covalently link biomolecules are envisaged. This route yields chemically stable derivatives, which can be easily recovered from the reaction mixture with a magnetic field and recyclable reused.

How anaesthesiologists understand difficult airway guidelines—an interview study

PubMed Central

Knudsen, Kati; Nilsson, Ulrica; Larsson, Anders; Larsson, Jan

2017-01-01

Background In the practice of anaesthesia, clinical guidelines that aim to improve the safety of airway procedures have been developed. The aim of this study was to explore how anaesthesiologists understand or conceive of difficult airway management algorithms. Methods A qualitative phenomenographic design was chosen to explore anaesthesiologists’ views on airway algorithms. Anaesthesiologists working in three hospitals were included. Individual face-to-face interviews were conducted. Results Four different ways of understanding were identified, describing airway algorithms as: (A) a law-like rule for how to act in difficult airway situations; (B) a cognitive aid, an action plan for difficult airway situations; (C) a basis for developing flexible, personal action plans for the difficult airway; and (D) the experts’ consensus, a set of scientifically based guidelines for handling the difficult airway. Conclusions The interviewed anaesthesiologists understood difficult airway management guidelines/algorithms very differently. PMID:29299973

Toll-like receptor 2 is upregulated by hog confinement dust in an IL-6 dependent manner in the airway epithelium

PubMed Central

Bailey, KL; Poole, JA; Mathisen, TL; Wyatt, TA; Von Essen, SG; Romberger, DJ

2009-01-01

Hog confinement workers are at high risk to develop chronic bronchitis as a result of their exposure to organic dust. Chronic bronchitis is characterized by inflammatory changes of the airway epithelium. A key mediator in inflammation is Toll-like receptor 2 (TLR2). We investigated the role of TLR2 in pulmonary inflammation induced by hog confinement dust. Normal Human Bronchial Epithelial Cells (NHBE) were grown in culture and exposed to hog confinement dust extract. Hog confinement dust upregulated airway epithelial cell TLR2 mRNA in a concentration and time-dependent manner using real-time PCR. There was a similar increase in TLR2 protein at 48 hours as shown by Western blot. TLR2 was upregulated on the surface of airway epithelial cells as shown by flow cytometry. A similar upregulation of pulmonary TLR2 mRNA and protein was shown in a murine model of hog confinement dust exposure. Hog confinement dust is known to stimulate epithelial cells to produce IL-6. In order to determine whether TLR2 expression was being regulated by IL-6, the production of IL-6 was blocked using an IL-6 neutralizing antibody. This resulted in attenuation of the dust-induced upregulation of TLR2. To further demonstrate the importance of IL-6 in the regulation of TLR2, NHBE were directly stimulated with recombinant human IL-6. IL-6 alone was able to upregulate TLR2 in airway epithelial cells. Hog confinement dust upregulates TLR2 in the airway epithelium through an IL-6 dependent mechanism. PMID:18359883

21 CFR 184.1914 - Trypsin.

Code of Federal Regulations, 2012 CFR

2012-04-01

... defined in § 170.3(o)(9) of this chapter to hydrolyze proteins or polypeptides. (2) The ingredient is used... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Trypsin. 184.1914 Section 184.1914 Food and Drugs... characterizing enzyme activity is that of a peptide hydrolase (EC 3.4.21.4). (b) The ingredient meets the general...

21 CFR 184.1914 - Trypsin.

Code of Federal Regulations, 2014 CFR

2014-04-01

...: (1) The ingredient is used as an enzyme as defined in § 170.3(o)(9) of this chapter to hydrolyze... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Trypsin. 184.1914 Section 184.1914 Food and Drugs... activity is that of a peptide hydrolase (EC 3.4.21.4). (b) The ingredient meets the general requirements...

21 CFR 184.1914 - Trypsin.

Code of Federal Regulations, 2010 CFR

2010-04-01

... defined in § 170.3(o)(9) of this chapter to hydrolyze proteins or polypeptides. (2) The ingredient is used... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Trypsin. 184.1914 Section 184.1914 Food and Drugs... characterizing enzyme activity is that of a peptide hydrolase (EC 3.4.21.4). (b) The ingredient meets the general...

21 CFR 184.1914 - Trypsin.

Code of Federal Regulations, 2013 CFR

2013-04-01

... practice conditions of use: (1) The ingredient is used as an enzyme as defined in § 170.3(o)(9) of this... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Trypsin. 184.1914 Section 184.1914 Food and Drugs... characterizing enzyme activity is that of a peptide hydrolase (EC 3.4.21.4). (b) The ingredient meets the general...

Network Analyses Reveal Pervasive Functional Regulation Between Proteases in the Human Protease Web

PubMed Central

Fortelny, Nikolaus; Cox, Jennifer H.; Kappelhoff, Reinhild; Starr, Amanda E.; Lange, Philipp F.; Pavlidis, Paul; Overall, Christopher M.

2014-01-01

Proteolytic processing is an irreversible posttranslational modification affecting a large portion of the proteome. Protease-cleaved mediators frequently exhibit altered activity, and biological pathways are often regulated by proteolytic processing. Many of these mechanisms have not been appreciated as being protease-dependent, and the potential in unraveling a complex new dimension of biological control is increasingly recognized. Proteases are currently believed to act individually or in isolated cascades. However, conclusive but scattered biochemical evidence indicates broader regulation of proteases by protease and inhibitor interactions. Therefore, to systematically study such interactions, we assembled curated protease cleavage and inhibition data into a global, computational representation, termed the protease web. This revealed that proteases pervasively influence the activity of other proteases directly or by cleaving intermediate proteases or protease inhibitors. The protease web spans four classes of proteases and inhibitors and so links both recently and classically described protease groups and cascades, which can no longer be viewed as operating in isolation in vivo. We demonstrated that this observation, termed reachability, is robust to alterations in the data and will only increase in the future as additional data are added. We further show how subnetworks of the web are operational in 23 different tissues reflecting different phenotypes. We applied our network to develop novel insights into biologically relevant protease interactions using cell-specific proteases of the polymorphonuclear leukocyte as a system. Predictions from the protease web on the activity of matrix metalloproteinase 8 (MMP8) and neutrophil elastase being linked by an inactivating cleavage of serpinA1 by MMP8 were validated and explain perplexing Mmp8 −/− versus wild-type polymorphonuclear chemokine cleavages in vivo. Our findings supply systematically derived and

Action of trypsin on structural changes of collagen fibres from sea cucumber (Stichopus japonicus).

PubMed

Liu, Zi-Qiang; Tuo, Feng-Yan; Song, Liang; Liu, Yu-Xin; Dong, Xiu-Ping; Li, Dong-Mei; Zhou, Da-Yong; Shahidi, Fereidoon

2018-08-01

Trypsin, a representative serine proteinase, was used to hydrolyse the collagen fibres from sea cucumber (Stichopus japonicus) to highlight the role of serine proteinase in the autolysis of sea cucumber. Partial disaggregation of collagen fibres into collagen fibrils upon trypsin treatment occurred. The trypsin treatment also caused a time-dependent release of water-soluble glycosaminoglycans and proteins. Therefore, the degradation of the proteoglycan bridges between collagen fibrils might account for the disaggregation of collagen fibrils. For trypsin-treated collagen fibres (72 h), the collagen fibrils still kept their structural integrity and showed characteristic D-banding pattern, and the dissolution rate of hydroxyproline was just 0.21%. Meanwhile, Fourier transform infrared analysis showed the collagen within trypsin-treated collagen fibres (72 h) still retaining their triple-helical conformation. These results suggested that serine proteinase participated in the autolysis of S. japonicus body wall by damaging the proteoglycan bridges between collagen fibrils and disintegrating the latter. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cloned cytolytic T-effector cells and their malignant variants produce an extracellular matrix degrading trypsin-like serine proteinase.

PubMed Central

Simon, M M; Simon, H G; Fruth, U; Epplen, J; Müller-Hermelink, H K; Kramer, M D

1987-01-01

This report describes the distribution of a trypsin-like proteinase in defined homogeneous cytolytic T-cell lines (CTLL) and their in vitro and in vivo derived malignant T-lymphoma variants. By means of chromogenic peptide substrates, we found the enzyme to attack preferentially at the carboxy terminus of arginine, in particular when non-polar amino acids were present in the amino terminal neighbouring position. The enzyme was identified by means of various inhibitors as a serine type proteinase having a pH optimum around 8 X 5. Affinity chromatography in connection with molecular sieving resulted in a 200-fold purification and indicated a molecular weight (MW) of about 50,000 for the proteinase. The enzyme was found to be highly expressed in antigen-specific CTLL as well as in their tumorigenic variants. Both intact lymphocytes of all CTLL tested and Triton X-100 lysates or enriched proteinase preparations thereof were able to degrade a high molecular weight protein (casein) and to release high molecular weight split products from the sulphated proteoglycans in subendothelial extracellular matrix. The results are discussed with respect to the invasiveness of normal and malignant T lymphocytes and the proteinase is suggested to be crucially involved in the process of cellular migration in vivo. Images Figure 1 PMID:3546101

Airway structure and function in Eisenmenger's syndrome.

PubMed

McKay, K O; Johnson, P R; Black, J L; Glanville, A R; Armour, C L

1998-10-01

The responsiveness of airways from patients with Eisenmenger's syndrome (n = 5) was compared with that in airways from organ donors (n = 10). Enhanced contractile responses to cholinergic stimulation were found in airways from patients with Eisenmenger's syndrome. The maximal responses to acetylcholine, carbachol, and parasympathetic nerve stimulation in airway tissue from these patients were 221%, 139%, and 152%, respectively, of the maximal responses obtained in donor tissue. Further, relaxation responses to isoproterenol and levocromakalim were absent (n = 2) or markedly impaired (n = 3) in airways from patients with Eisenmenger's syndrome. This attenuated relaxation response was nonspecific in that it was also absent after vasoactive intestinal peptide, sodium nitroprusside, papaverine, and electrical field application. These observations can most likely be explained by a decrease in intrinsic smooth muscle tone, as precontraction of airways revealed relaxation responses that were equivalent to those obtained in donor tissues. Morphometric analysis of tissues used for the functional studies revealed no differences in the airway dimensions (internal perimeter) or airway wall components (e.g., smooth muscle, cartilage) or total area to explain these observations. Although the mechanism for this observed decrease in intrinsic airway smooth muscle tone is not certain, it may be due to alteration in the substructure of the airway wall or, alternatively, may result from the continued release of depressant factors in the vicinity of the smooth muscle which permanently alters smooth muscle responsiveness.

Toll-Like Receptor–2/6 and Toll-Like Receptor–9 Agonists Suppress Viral Replication but Not Airway Hyperreactivity in Guinea Pigs

PubMed Central

Evans, Scott E.; Dickey, Burton F.; Fryer, Allison D.; Jacoby, David B.

2013-01-01

Respiratory virus infections cause airway hyperreactivity (AHR). Preventative strategies for virus-induced AHR remain limited. Toll-like receptors (TLRs) have been suggested as a therapeutic target because of their central role in triggering antiviral immune responses. Previous studies showed that concurrent treatment with TLR2/6 and TLR9 agonists reduced lethality and the microbial burden in murine models of bacterial and viral pneumonia. This study investigated the effects of TLR2/6 and TLR9 agonist pretreatment on parainfluenza virus pneumonia and virus-induced AHR in guinea pigs in vivo. Synthetic TLR2/6 lipopeptide agonist Pam2CSK4 and Class C oligodeoxynucleotide TLR9 agonist ODN2395, administered in combination 24 hours before virus infection, significantly reduced viral replication in the lung. Despite a fivefold reduction in viral titers, concurrent TLR2/6 and TLR9 agonist pretreatment did not prevent virus-induced AHR or virus-induced inhibitory M2 muscarinic receptor dysfunction. Interestingly, the TLR agonists independently caused non–M2-dependent AHR. These data confirm the therapeutic antiviral potential of TLR agonists, while suggesting that virus inhibition may be insufficient to prevent virus-induced airway pathophysiology. Furthermore, TLR agonists independently cause AHR, albeit through a distinctly different mechanism from that of parainfluenza virus. PMID:23449736

Dissection of the binding of hydrogen peroxide to trypsin using spectroscopic methods and molecular modeling

NASA Astrophysics Data System (ADS)

Song, Wei; Yu, Zehua; Hu, Xinxin; Liu, Rutao

2015-02-01

Studies on the effects of environmental pollutants to protein in vitro has become a global attention. Hydrogen peroxide (H2O2) is used as an effective food preservative and bleacher in industrial production. The toxicity of H2O2 to trypsin was investigated by multiple spectroscopic techniques and the molecular docking method at the molecular level. The intrinsic fluorescence of trypsin was proved to be quenched in a static process based on the results of fluorescence lifetime experiment. Hydrogen bonds interaction and van der Waals forces were the main force to generate the trypsin-H2O2 complex on account of the negative ΔH0 and ΔS0. The binding of H2O2 changed the conformational structures and internal microenvironment of trypsin illustrated by UV-vis absorption, fluorescence, synchronous fluorescence, three-dimensional (3D) fluorescence and circular dichroism (CD) results. However, the binding site was far away from the active site of trypsin and the trypsin activity was only slightly affected by H2O2, which was further explained by molecular docking investigations.

Efficacy of Trypsin in Treating Coral Snake Envenomation in the Porcine Model.

PubMed

Parker-Cote, Jennifer L; O'Rourke, Dorcas P; Brewer, Kori L; Lertpiriyapong, Kvin; Punja, Mohan; Bush, Sean P; Miller, Susan N; Meggs, William J

2015-12-01

Antivenom is the definitive treatment for venomous snakebites. Alternative treatments warrant investigation because antivenom is sometimes unavailable, expensive, and can have deleterious side effects. This study assesses the efficacy of trypsin to treat coral snake envenomation in an in vivo porcine model. A randomized, blinded study was conducted. Subjects were 13 pigs injected subcutaneously with 1 mL of eastern coral snake venom (10 mg/mL) in the right distal hind limb. After 1 min, subjects were randomized to have the envenomation site injected with either 1 mL of saline or 1 mL of trypsin (100 mg/mL) by a blinded investigator. Clinical endpoint was survival for 72 h or respiratory depression defined as respiratory rate <15 breaths per minute, falling pulse oximetry, or agonal respirations. Fisher's exact t test was used for between group comparisons. Average time to toxicity for the saline control was 263 min (191-305 min). The development of respiratory depression occurred more frequently in control pigs than treated pigs (p = 0.009). Four of the six pigs that received trypsin survived to the end of the 3-day study. No control pigs survived. Two of the trypsin treatment pigs died with times to toxicity of 718 and 971 min. Survival to 12 and 24 h was significantly greater in the trypsin treatment group (p = 0.002, p = 0.009, respectively). Local injection of trypsin, a proteolytic enzyme, at the site of envenomation decreased the toxicity of eastern coral snake venom and increased survival significantly. Further investigation is required before these results can be extended to human snakebites.

Effect of Moringa oleifera flower extract on larval trypsin and acetylcholinesterase activities in Aedes aegypti.

PubMed

Pontual, Emmanuel Viana; Napoleão, Thiago Henrique; Dias de Assis, Caio Rodrigo; de Souza Bezerra, Ranilson; Xavier, Haroudo Satiro; Navarro, Daniela Maria do Amaral Ferraz; Coelho, Luana Cassandra Breitenbach Barroso; Paiva, Patrícia Maria Guedes

2012-03-01

Aedes aegypti control is crucial to reducing dengue fever. Aedes aegypti larvae have developed resistance to organophosporous insecticides and the use of natural larvicides may help manage larval resistance by increasing elements in insecticide rotation programs. Here, we report on larvicidal activity of Moringa oleifera flower extract against A. aegypti L(1), L(2), L(3), and L(4) as well as the effect of flower extract on gut trypsin and whole-larval acetylcholinesterase from L(4.) In addition, the heated flower extract was investigated for larvicidal activity against L(4) and effect on larval gut trypsin. Moringa oleifera flower extract contains a proteinaceous trypsin inhibitor (M. oleifera flower trypsin inhibitor, MoFTI), triterpene (β-amyrin), sterol (β-sitosterol) as well as flavonoids (kaempferol and quercetin). Larvicidal activity was detected against L(2), L(3), and L(4) (LC(50) of 1.72%, 1.67%, and 0.92%, respectively). Flower extract inhibited L(4) gut trypsin (MoFTI K(i) = 0.6 nM) and did not affect acetylcholinesterase activity. In vivo assay showed that gut trypsin activity from L(4) treated with M. oleifera flower extract decreased over time (0-1,440 min) and was strongly inhibited (98.6%) after 310 min incubation; acetylcholinesterase activity was not affected. Thermal treatment resulted in a loss of trypsin inhibitor and larvicidal activities, supporting the hypothesis that flower extract contains a proteinaceous trypsin inhibitor that may be responsible for the deleterious effects on larval mortality. © 2012 Wiley Periodicals, Inc.

The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

PubMed

Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

2003-01-01

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

Mast Cell Proteases as Protective and Inflammatory Mediators

PubMed Central

Caughey, George H.

2014-01-01

Proteases are the most abundant class of proteins produced by mast cells. Many of these are stored in membrane-enclosed intracellular granules until liberated by degranulating stimuli, which include cross-linking of high affinity IgE receptor FcεRI by IgE bound to multivalent allergen. Understanding and separating the functions of the proteases is important because expression differs among mast cells in different tissue locations. Differences between laboratory animals and humans in protease expression also influence the degree of confidence with which results obtained in animal models of mast cell function can be extrapolated to humans. The inflammatory potential of mast cell proteases was the first aspect of their biology to be explored and has received the most attention, in part because some of them—notably tryptases and chymases—are biomarkers of local and systemic mast cell degranulation and anaphylaxis. Although some of the proteases indeed augment allergic inflammation and are potential targets for inhibition to treat asthma and related allergic disorders, they are protective and even anti-inflammatory in some settings. For example, mast cell tryptases may protect from serious bacterial lung infections and may limit the “rubor” component of inflammation caused by vasodilating neuropeptides in the skin. Chymases help to maintain intestinal barrier function and to expel parasitic worms, and may support blood pressure during anaphylaxis by generating angiotensin II. In other life-or-death examples, carboxypeptidase A3 and other mast cell peptidases limit systemic toxicity of endogenous peptides like endothelin and neurotensin during septic peritonitis, and inactivate venom-associated peptides. On the other hand, mast cell peptidase-mediated destruction of protective cytokines, like IL-6, can enhance mortality from sepsis. Peptidases released from mast cells also influence non-mast cell proteases, such as by activating matrix metalloproteinase cascades

Nociceptin effects in the airways.

PubMed

Peiser, C; Undem, B J; Fischer, A

2000-07-01

The opioid-like heptadecapeptide nociceptin (NC) has the following effects in the airways (investigated in isolated tracheae and bronchi from guinea pig or rat): the electric field stimulation (EFS)-induces release of acetylcholine (ACh), the tachykinin substance P (SP) and calcitonin gene-related peptide (CGRP) is reduced after pretreatment with NC, and EFS-induced tachykinergic nonadrenergic-noncholinergic (NANC) bronchoconstriction is inhibited by NC. Both the NC-mediated inhibition of neurotransmission and of smooth muscle contraction occurred in a concentration-dependent manner. Because these effects were naloxone-insensitive, were blocked by the NC receptor antagonist [F/G]NC(1-13)NH(2), and could be mimicked by the NC analogs, NCNH(2) and NC(1-13)NH(2), it is thought that they are distinct from the classic opioid receptors. That these pharmacological actions of NC are of relevance for airway physiology is highly probable given the presence of NC-immunoreactivity in the nerve fibers of the airways and of opioid-like receptor (ORL-1) transcripts in the jugular ganglia, from where the tachykinin-containing afferents arise.

The incomplete anti-Rh antibody agglutination mechanism of trypsinized ORh+ red cells.

PubMed Central

Margni, R A; Leoni, J; Bazzurro, M

1977-01-01

The capacity for binding to trypsinized and non-trypsinized ORh+ red cells, of the IgG incomplete anti-Rh antibody and its F(ab')2 and Fc fragments has been investigated. An analysis has also been made of the capacity of non-specific human IgG, aggregated non-specific human IgG, human IgM (19S) and IgM (7S), and of fragments Fcgamma, Fcmu and Fc5mu to inhibit the agglutination of trypsinized ORh+ red cells by the IgG incomplete anti-Rh antibody. The results obtained indicate that these antibodies behave in a similar manner to that of nonprecipitating antibodies, and that the agglutination of trypsinized red cells seems to be a mixed reaction due to the interaction of an Fab fragment with its Rh antigenic determinant present in the surface of a red cell and the Fc of the same molecule with a receptor for Fc present in adjacent red cells. The trypsin treatment apparently results in the liberation of occult Fc receptors. It has also been demonstrated that in the agglutination of ORh+ red cells by IgG incomplete anti-Rh antibody in the presence of albumin, interaction must occur in some manner between the albumin and the Fc fragment since the F(ab')2 fragment does not give rise to agglutination under such conditions. Images Figure 1 PMID:415968

The role of protease-activated receptor-2 on pulmonary neutrophils in the innate immune response to cockroach allergen

PubMed Central

2012-01-01

Background Serine proteases in German cockroach (GC) have been shown to mediate allergic airway inflammation through the activation of protease activated receptor (PAR)-2. Neutrophils play an important role in regulating the innate immune response, and are recruited into the airways following GC frass exposure. As such, we investigated the role of PAR-2 in airway neutrophil recruitment, activation and cytokine production following allergen exposure. Methods Wild type and PAR-2-deficient mice were administered a single intratracheal instillation of PBS or GC frass and neutrophil recruitment, expression of PAR-2, CD80, CD86, and MHC class II were assessed by flow cytometry and levels of tumor necrosis factor (TNF)α was assessed by ELISA. Uptake of AlexaFluor 405-labeled GC frass by neutrophils was performed by flow cytometry. Results Neutrophil recruitment in the lung and airways following GC frass exposure was significantly decreased in PAR-2-deficient mice compared to wild type mice. GC frass exposure increased the level of PAR-2 on pulmonary neutrophils and increased numbers of PAR-2-positive neutrophils were found in the lungs; however PAR-2 did not play a role in meditating allergen uptake. Comparing wild type and PAR-2-deficient mice, we found that a single exposure to GC frass increased levels of CD80 and CD86 on pulmonary neutrophils, an effect which was independent of PAR-2 expression. Neutrophils isolated from the whole lungs of naïve PAR-2-deficient mice treated ex vivo with GC frass produced significantly less TNFα than in similarly treated wild type neutrophils. Lastly, neutrophils were isolated from the bronchoalveolar lavage fluid of wild type and PAR-2-deficient mice following a single intratracheal exposure to GC frass. Airway neutrophils from PAR-2-deficient mice released substantially decreased levels of TNFα, suggesting a role for PAR-2 in neutrophil-derived cytokine production. Conclusions Together these data suggest PAR-2 expression can be

Eotaxin-3 (CCL26) exerts innate host defense activities that are modulated by mast cell proteases.

PubMed

Gela, A; Kasetty, G; Jovic, S; Ekoff, M; Nilsson, G; Mörgelin, M; Kjellström, S; Pease, J E; Schmidtchen, A; Egesten, A

2015-02-01

During bacterial infections of the airways, a Th1-profiled inflammation promotes the production of several host defense proteins and peptides with antibacterial activities including β-defensins, ELR-negative CXC chemokines, and the cathelicidin LL-37. These are downregulated by Th2 cytokines of the allergic response. Instead, the eosinophil-recruiting chemokines eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26 are expressed. This study set out to investigate whether these chemokines could serve as innate host defense molecules during allergic inflammation. Antibacterial activities of the eotaxins were investigated using viable count assays, electron microscopy, and methods assessing bacterial permeabilization. Fragments generated by mast cell proteases were characterized, and their potential antibacterial, receptor-activating, and lipopolysaccharide-neutralizing activities were investigated. CCL11, CCL24, and CCL26 all showed potent bactericidal activity, mediated through membrane disruption, against the airway pathogens Streptococcus pneumoniae, Staphylococcus aureus, Nontypeable Haemophilus influenzae, and Pseudomonas aeruginosa. CCL26 retained bactericidal activity in the presence of salt at physiologic concentrations, and the region holding the highest bactericidal activity was the cationic and amphipathic COOH-terminus. Proteolysis of CCL26 by chymase and tryptase, respectively, released distinct fragments of the COOH- and NH2 -terminal regions. The COOH-terminal fragment retained antibacterial activity while the NH2 -terminal had potent LPS-neutralizing properties in the order of CCL26 full-length protein. An identical fragment to NH2 -terminal fragment generated by tryptase was obtained after incubation with supernatants from activated mast cells. None of the fragments activated the CCR3-receptor. Taken together, the findings show that the eotaxins can contribute to host defense against common airway pathogens and that their activities are modulated by

The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage

PubMed Central

Pettit, Steven C.; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H.

2003-01-01

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation. PMID:12477841

Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris.

PubMed

Sreenivas, Suma; Krishnaiah, Sateesh M; Govindappa, Nagaraja; Basavaraju, Yogesh; Kanojia, Komal; Mallikarjun, Niveditha; Natarajan, Jayaprakash; Chatterjee, Amarnath; Sastry, Kedarnath N

2015-01-01

Glargine is an analog of Insulin currently being produced by recombinant DNA technology using two different hosts namely Escherichia coli and Pichia pastoris. Production from E. coli involves the steps of extraction of inclusion bodies by cell lysis, refolding, proteolytic cleavage and purification. In P. pastoris, a single-chain precursor with appropriate disulfide bonding is secreted to the medium. Downstream processing currently involves use of trypsin which converts the precursor into two-chain final product. The use of trypsin in the process generates additional impurities due to presence of Lys and Arg residues in the Glargine molecule. In this study, we describe an alternate approach involving over-expression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (Arg-Arg) at the end of B-chain of Glargine. KEX2 gene over-expression in Pichia was accomplished by using promoters of varying strengths to ensure production of greater levels of fully functional two-chain Glargine product, confirmed by HPLC and mass analysis. In conclusion, this new production process involving Kex2 protease over-expression improves the downstream process efficiency, reduces the levels of impurities generated and decreases the use of raw materials.

Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization.

PubMed

Cabrera-Muñoz, Aymara; Rojas, Laritza; Gil, Dayrom F; González-González, Yamile; Mansur, Manuel; Camejo, Ayamey; Pires, José R; Alonso-Del-Rivero Antigua, Maday

2016-10-01

Cenchritis muricatus protease inhibitor II (CmPI-II) is a tight-binding serine protease inhibitor of the Kazal family with an atypical broad specificity, being active against several proteases such as bovine pancreatic trypsin, human neutrophil elastase and subtilisin A. CmPI-II 3D structures are necessary for understanding the molecular basis of its activity. In the present work, we describe an efficient and straightforward recombinant expression strategy, as well as a cost-effective procedure for isotope labeling for NMR structure determination purposes. The vector pCM101 containing the CmPI-II gene, under the control of Pichia pastoris AOX1 promoter was constructed. Methylotrophic Pichia pastoris strain KM71H was then transformed with the plasmid and the recombinant protein (rCmPI-II) was expressed in benchtop fermenter in unlabeled or (15)N-labeled forms using ammonium chloride ((15)N, 99%) as the sole nitrogen source. Protein purification was accomplished by sequential cation exchange chromatography in STREAMLINE DirectHST, anion exchange chromatography on Hitrap Q-Sepharose FF and gel filtration on Superdex 75 10/30, yielding high quantities of pure rCmPI-II and (15)N rCmPI-II. Recombinant proteins displayed similar functional features as compared to the natural inhibitor and NMR spectra indicated folded and homogeneously labeled samples, suitable for further studies of structure and protease-inhibitor interactions. Copyright © 2016 Elsevier Inc. All rights reserved.

HIV-1 protease-substrate coevolution in nelfinavir resistance.

PubMed

Kolli, Madhavi; Ozen, Ayşegül; Kurt-Yilmaz, Nese; Schiffer, Celia A

2014-07-01

Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Trypsin inhibitor from tamarindus indica L. seeds reduces weight gain and food consumption and increases plasmatic cholecystokinin levels

PubMed Central

do Nascimento Campos Ribeiro, Joycellane Alline; Serquiz, Alexandre Coellho; dos Santos Silva, Priscila Fabíola; Barbosa, Patrícia Batista Barra Medeiros; Sampaio, Tarcísio Bruno Montenegro; de Araújo, Raimundo Fernandes; de Oliveira, Adeliana Silva; Machado, Richele Janaina Araújo; Maciel, Bruna Leal Lima; Uchôa, Adriana Ferreira; dos Santos, Elizeu Antunes; de Araújo Morais, Ana Heloneida

2015-01-01

OBJECTIVES: Seeds are excellent sources of proteinase inhibitors, some of which may have satietogenic and slimming actions. We evaluated the effect of a trypsin inhibitor from Tamarindus indica L. seeds on weight gain, food consumption and cholecystokinin levels in Wistar rats. METHODS: A trypsin inhibitor from Tamarindus was isolated using ammonium sulfate (30–60%) following precipitation with acetone and was further isolated with Trypsin-Sepharose affinity chromatography. Analyses were conducted to assess the in vivo digestibility, food intake, body weight evolution and cholecystokinin levels in Wistar rats. Histological analyses of organs and biochemical analyses of sera were performed. RESULTS: The trypsin inhibitor from Tamarindus reduced food consumption, thereby reducing weight gain. The in vivo true digestibility was not significantly different between the control and Tamarindus trypsin inhibitor-treated groups. The trypsin inhibitor from Tamarindus did not cause alterations in biochemical parameters or liver, stomach, intestine or pancreas histology. Rats treated with the trypsin inhibitor showed significantly elevated cholecystokinin levels compared with animals receiving casein or water. CONCLUSION: The results indicate that the isolated trypsin inhibitor from Tamarindus reduces weight gain by reducing food consumption, an effect that may be mediated by increased cholecystokinin. Thus, the potential use of this trypsin inhibitor in obesity prevention and/or treatment should be evaluated. PMID:25789523

Trypsin inhibitor from tamarindus indica L. seeds reduces weight gain and food consumption and increases plasmatic cholecystokinin levels.

PubMed

Ribeiro, Joycellane Alline do Nascimento Campos; Serquiz, Alexandre Coellho; Silva, Priscila Fabíola dos Santos; Barbosa, Patrícia Batista Barra Medeiros; Sampaio, Tarcísio Bruno Montenegro; Araújo Junior, Raimundo Fernandes de; Oliveira, Adeliana Silva de; Machado, Richele Janaina Araújo; Maciel, Bruna Leal Lima; Uchôa, Adriana Ferreira; Santos, Elizeu Antunes dos; Morais, Ana Heloneida de Araújo

2015-02-01

Seeds are excellent sources of proteinase inhibitors, some of which may have satietogenic and slimming actions. We evaluated the effect of a trypsin inhibitor from Tamarindus indica L. seeds on weight gain, food consumption and cholecystokinin levels in Wistar rats. A trypsin inhibitor from Tamarindus was isolated using ammonium sulfate (30-60%) following precipitation with acetone and was further isolated with Trypsin-Sepharose affinity chromatography. Analyses were conducted to assess the in vivo digestibility, food intake, body weight evolution and cholecystokinin levels in Wistar rats. Histological analyses of organs and biochemical analyses of sera were performed. The trypsin inhibitor from Tamarindus reduced food consumption, thereby reducing weight gain. The in vivo true digestibility was not significantly different between the control and Tamarindus trypsin inhibitor-treated groups. The trypsin inhibitor from Tamarindus did not cause alterations in biochemical parameters or liver, stomach, intestine or pancreas histology. Rats treated with the trypsin inhibitor showed significantly elevated cholecystokinin levels compared with animals receiving casein or water. The results indicate that the isolated trypsin inhibitor from Tamarindus reduces weight gain by reducing food consumption, an effect that may be mediated by increased cholecystokinin. Thus, the potential use of this trypsin inhibitor in obesity prevention and/or treatment should be evaluated.

Reproducibility of airway luminal size in asthma measured by HRCT.

PubMed

Brown, Robert H; Henderson, Robert J; Sugar, Elizabeth A; Holbrook, Janet T; Wise, Robert A

2017-10-01

Brown RH, Henderson RJ, Sugar EA, Holbrook JT, Wise RA, on behalf of the American Lung Association Airways Clinical Research Centers. Reproducibility of airway luminal size in asthma measured by HRCT. J Appl Physiol 123: 876-883, 2017. First published July 13, 2017; doi:10.1152/japplphysiol.00307.2017.-High-resolution CT (HRCT) is a well-established imaging technology used to measure lung and airway morphology in vivo. However, there is a surprising lack of studies examining HRCT reproducibility. The CPAP Trial was a multicenter, randomized, three-parallel-arm, sham-controlled 12-wk clinical trial to assess the use of a nocturnal continuous positive airway pressure (CPAP) device on airway reactivity to methacholine. The lack of a treatment effect of CPAP on clinical or HRCT measures provided an opportunity for the current analysis. We assessed the reproducibility of HRCT imaging over 12 wk. Intraclass correlation coefficients (ICCs) were calculated for individual airway segments, individual lung lobes, both lungs, and air trapping. The ICC [95% confidence interval (CI)] for airway luminal size at total lung capacity ranged from 0.95 (0.91, 0.97) to 0.47 (0.27, 0.69). The ICC (95% CI) for airway luminal size at functional residual capacity ranged from 0.91 (0.85, 0.95) to 0.32 (0.11, 0.65). The ICC measurements for airway distensibility index and wall thickness were lower, ranging from poor (0.08) to moderate (0.63) agreement. The ICC for air trapping at functional residual capacity was 0.89 (0.81, 0.94) and varied only modestly by lobe from 0.76 (0.61, 0.87) to 0.95 (0.92, 0.97). In stable well-controlled asthmatic subjects, it is possible to reproducibly image unstimulated airway luminal areas over time, by region, and by size at total lung capacity throughout the lungs. Therefore, any changes in luminal size on repeat CT imaging are more likely due to changes in disease state and less likely due to normal variability. NEW & NOTEWORTHY There is a surprising lack

Co-evolution of insect proteases and plant protease inhibitors.

PubMed

Jongsma, Maarten A; Beekwilder, Jules

2011-08-01

Plants are at the basis of the food chain, but there is no such thing as a "free lunch" for herbivores. To promote reproductive success, plants evolved multi-layered defensive tactics to avoid or discourage herbivory. To the detriment of plants, herbivores, in turn, evolved intricate strategies to find, eat, and successfully digest essential plant parts to raise their own offspring. In this battle the digestive tract is the arena determining final victory or defeat as measured by growth or starvation of the herbivore. Earlier, specific molecular opponents were identified as proteases and inhibitors: digestive proteases of herbivores evolved structural motifs to occlude plant protease inhibitors, or alternatively, the insects evolved proteases capable of specifically degrading the host plant inhibitors. In response plant inhibitors evolved hyper-variable and novel protein folds to remain active against potential herbivores. At the level of protease regulation in herbivorous insects, it was shown that inhibition-insensitive digestive proteases are up-regulated when sensitive proteases are inhibited. The way this regulation operates in mammals is known as negative feedback by gut-luminal factors, so-called 'monitor peptides' that are sensitive to the concentration of active enzymes. We propose that regulation of gut enzymes by endogenous luminal factors has been an open invitation to plants to "hijack" this regulation by evolving receptor antagonists, although yet these plant factors have not been identified. In future research the question of the co-evolution of insect proteases and plant inhibitors should, therefore, be better approached from a systems level keeping in mind that evolution is fundamentally opportunistic and that the plant's fitness is primarily improved by lowering the availability of essential amino acids to an herbivore by any available mechanism.

Investigate the Binding of Catechins to Trypsin Using Docking and Molecular Dynamics Simulation

PubMed Central

Cui, Fengchao; Yang, Kecheng; Li, Yunqi

2015-01-01

To explore the inhibitory mechanism of catechins for digestive enzymes, we investigated the binding mode of catechins to a typical digestive enzyme-trypsin and analyzed the structure-activity relationship of catechins, using an integration of molecular docking, molecular dynamics simulation and binding free energy calculation. We found that catechins with different structures bound to a conservative pocket S1 of trypsin, which is comprised of residues 189–195, 214–220 and 225–228. In the trypsin-catechin complexes, Asp189 by forming strong hydrogen bonding, and Gln192, Trp215 and Gly216 through hydrophobic interactions, all significantly contribute to the binding of catechins. The number and the position of hydroxyl and aromatic groups, the structure of stereoisomers, and the orientation of catechins in the binding pocket S1 of trypsin all affect the binding affinity. The binding affinity is in the order of Epigallocatechin gallate (EGCG) > Epicatechin gallate (ECG) > Epicatechin (EC) > Epigallocatechin (EGC), and 2R-3R EGCG shows the strongest binding affinity out of other stereoisomers. Meanwhile, the synergic conformational changes of residues and catechins were also analyzed. These findings will be helpful in understanding the knowledge of interactions between catechins and trypsin and referable for the design of novel polyphenol based functional food and nutriceutical formulas. PMID:25938485

Biochemical Characterization of An Arginine-Specific Alkaline Trypsin from Bacillus licheniformis.

PubMed

Gong, Jin-Song; Li, Wei; Zhang, Dan-Dan; Xie, Min-Feng; Yang, Biao; Zhang, Rong-Xian; Li, Heng; Lu, Zhen-Ming; Xu, Zheng-Hong; Shi, Jin-Song

2015-12-17

In the present study, we isolated a trypsin-producing strain DMN6 from the leather waste and identified it as Bacillus licheniformis through a two-step screening strategy. The trypsin activity was increased up to 140 from 20 U/mL through culture optimization. The enzyme was purified to electrophoretic homogeneity with a molecular mass of 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the specific activity of purified enzyme is 350 U/mg with Nα-Benzoyl-L-arginine ethylester as the substrate. The optimum temperature and pH for the trypsin are 65 °C and pH 9.0, respectively. Also, the enzyme can be significantly activated by Ba(2+). This enzyme is relatively stable in alkaline environment and displays excellent activity at low temperatures. It could retain over 95% of enzyme activity after 180 min of incubation at 45 °C. The distinguished activity under low temperature and prominent stability enhance its catalytic potential. In the current work, the open reading frame was obtained with a length of 1371 nucleotides that encoded a protein of 456 amino acids. These data would warrant the B. licheniformis trypsin as a promising candidate for catalytic application in collagen preparation and leather bating through further protein engineering.

Chalcones isolated from Angelica keiskei inhibit cysteine proteases of SARS-CoV.

PubMed

Park, Ji-Young; Ko, Jin-A; Kim, Dae Wook; Kim, Young Min; Kwon, Hyung-Jun; Jeong, Hyung Jae; Kim, Cha Young; Park, Ki Hun; Lee, Woo Song; Ryu, Young Bae

2016-01-01

Two viral proteases of severe acute respiratory syndrome coronavirus (SARS-CoV), a chymotrypsin-like protease (3CL(pro)) and a papain-like protease (PL(pro)) are attractive targets for the development of anti-SARS drugs. In this study, nine alkylated chalcones (1-9) and four coumarins (10-13) were isolated from Angelica keiskei, and the inhibitory activities of these constituents against SARS-CoV proteases (3CL(pro) and PL(pro)) were determined (cell-free/based). Of the isolated alkylated chalcones, chalcone 6, containing the perhydroxyl group, exhibited the most potent 3CL(pro) and PL(pro) inhibitory activity with IC50 values of 11.4 and 1.2 µM. Our detailed protein-inhibitor mechanistic analysis of these species indicated that the chalcones exhibited competitive inhibition characteristics to the SARS-CoV 3CL(pro), whereas noncompetitive inhibition was observed with the SARS-CoV PL(pro).

In silico insights into protein-protein interactions and folding dynamics of the saposin-like domain of Solanum tuberosum aspartic protease.

PubMed

De Moura, Dref C; Bryksa, Brian C; Yada, Rickey Y

2014-01-01

The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum) plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L.) plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family.

In Silico Insights into Protein-Protein Interactions and Folding Dynamics of the Saposin-Like Domain of Solanum tuberosum Aspartic Protease

PubMed Central

De Moura, Dref C.; Bryksa, Brian C.; Yada, Rickey Y.

2014-01-01

The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum) plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L.) plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family. PMID:25188221

Protease and Protease-Activated Receptor-2 Signaling in the Pathogenesis of Atopic Dermatitis

PubMed Central

Lee, Sang Eun; Jeong, Se Kyoo

2010-01-01

Proteases in the skin are essential to epidermal permeability barrier homeostasis. In addition to their direct proteolytic effects, certain proteases signal to cells by activating protease-activated receptors (PARs), the G-protein-coupled receptors. The expression of functional PAR-2 on human skin and its role in inflammation, pruritus, and skin barrier homeostasis have been demonstrated. Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by genetic barrier defects and allergic inflammation, which is sustained by gene-environmental interactions. Recent studies have revealed aberrant expression and activation of serine proteases and PAR-2 in the lesional skin of AD patients. The imbalance between proteases and protease inhibitors associated with genetic defects in the protease/protease inhibitor encoding genes, increase in skin surface pH, and exposure to proteolytically active allergens contribute to this aberrant protease/PAR-2 signaling in AD. The increased protease activity in AD leads to abnormal desquamation, degradation of lipid-processing enzymes and antimicrobial peptides, and activation of primary cytokines, thereby leading to permeability barrier dysfunction, inflammation, and defects in the antimicrobial barrier. Moreover, up-regulated proteases stimulate PAR-2 in lesional skin of AD and lead to the production of cytokines and chemokines involved in inflammation and immune responses, itching sensation, and sustained epidermal barrier perturbation with easier allergen penetration. In addition, PAR-2 is an important sensor for exogenous danger molecules, such as exogenous proteases from various allergens, and plays an important role in AD pathogenesis. Together, these findings suggest that protease activity or PAR-2 may be a future target for therapeutic intervention for the treatment of AD. PMID:20879045

Trypsin inhibitor screening in traditional Chinese medicine by using an immobilized enzyme microreactor in capillary and molecular docking study.

PubMed

Cheng, Mengxia; Chen, Zilin

2017-08-01

A trypsin immobilized enzyme microreactor was successfully prepared in capillary for studying enzyme kinetics of trypsin and online screening of trypsin inhibitors from traditional Chinese medicine through capillary electrophoresis. Trypsin was immobilized on the inner wall at the inlet of the capillary treated with polydopamine. The rest of the capillary was used as a separation channel. The parameters including the separation efficiency and the activity of immobilized trypsin were comprehensively evaluated. Under the optimal conditions, online screening of trypsin inhibitors each time can be carried out within 6 min. The Michaelis-Menten constant of immobilized trypsin was calculated to be 0.50 mM, which indicated high affinity of the immobilized trypsin for the substrate. The half-maximal inhibitory concentration of known inhibitor of benzamidine hydrochloride hydrate as a model inhibitor was 13.32 mM. The proposed method was successfully applied to screen trypsin inhibitors from 15 compounds of traditional Chinese medicine. It has been found that baicalin showed inhibitory potency. Molecular docking study well supported the experimental result by exhibiting molecular interaction between enzyme and inhibitors. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A peptide-based approach to evaluate the adaptability of influenza A virus to humans based on its hemagglutinin proteolytic cleavage site

PubMed Central

Straus, Marco R.; Whittaker, Gary R.

2017-01-01

Cleavage activation of the hemagglutinin (HA) protein by host proteases is a crucial step in the infection process of influenza A viruses (IAV). However, IAV exists in eighteen different HA subtypes in nature and their cleavage sites vary considerably. There is uncertainty regarding which specific proteases activate a given HA in the human respiratory tract. Understanding the relationship between different HA subtypes and human-specific proteases will be valuable in assessing the pandemic potential of circulating viruses. Here we utilized fluorogenic peptides mimicking the HA cleavage motif of representative IAV strains causing disease in humans or of zoonotic/pandemic potential and tested them with a range of proteases known to be present in the human respiratory tract. Our results show that peptides from the H1, H2 and H3 subtypes are cleaved efficiently by a wide range of proteases including trypsin, matriptase, human airway tryptase (HAT), kallikrein-related peptidases 5 (KLK5) and 12 (KLK12) and plasmin. Regarding IAVs currently of concern for human adaptation, cleavage site peptides from H10 viruses showed very limited cleavage by respiratory tract proteases. Peptide mimics from H6 viruses showed broader cleavage by respiratory tract proteases, while H5, H7 and H9 subtypes showed variable cleavage; particularly matriptase appeared to be a key protease capable of activating IAVs. We also tested HA substrate specificity of Factor Xa, a protease required for HA cleavage in chicken embryos and relevant for influenza virus production in eggs. Overall our data provide novel tool allowing the assessment of human adaptation of IAV HA subtypes. PMID:28358853

DNA-directed trypsin immobilization on a polyamidoamine dendrimer-modified capillary to form a renewable immobilized enzyme microreactor.

PubMed

Wu, Nan; Wang, Siming; Yang, Ye; Song, Jiayi; Su, Ping; Yang, Yi

2018-07-01

A novel type of trypsin capillary microreactor was developed based on a DNA-directed immobilization (DDI) technique applied to a fused-silica capillary modified with polyamidoamine (PAMAM) dendrimers. Trypsin binding to the inner wall of the capillary was confirmed by confocal laser scanning microscopy. The properties of the trypsin-DNA conjugated, PAMAM-modified capillary microreactor were investigated by monitoring hydrolysis of Nα-benzoyl- L -arginine ethyl ester. Through the hybridization and dehybridization of the DNA, the inner wall of the capillary functionalized with trypsin can be regenerated, thus indicating the renewability of this enzyme microreactor. In addition, these results demonstrated that introduction of PAMAM enabled higher amounts of trypsin to be immobilized, markedly improving the enzymolysis efficiency, compared with traditional modified capillaries. The digestion performance of the trypsin capillary microreactor was further evaluated by digesting cytochrome C, and a peptide numbers of 8, and a sequence coverage of 59% were obtained. This renewable and efficient immobilized trypsin capillary microreactor combines advantages of both DDI technology and PAMAM, and is potentially adaptable to high-throughput enzyme assays in biochemical and clinical research. Copyright © 2018. Published by Elsevier B.V.

sup 1 H assignments and secondary structure determination of the soybean trypsin/chymotrypsin Bowman-Birk inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Werner, M.H.; Wemmer, D.E.

1991-04-09

The {sup 1}H resonance assignments and secondary structure of the trypsin/chymotrypsin Bowman-Birk inhibitor from soybeans were determined by nuclear magnetic resonance spectroscopy (NMR) at 600 MHz in an 18% acetonitrile-d{sub 3}/aqueous cosolvent. Resonances from 69 to 71 amino acids were assigned sequence specifically. Residues Q11-T15 form an antiparallel {beta}-sheet with residues Q21-S25 in the tryptic inhibitory domain and an analogous region of antiparallel sheet forms between residues S38-A42 and Q48-V52 in the chymotryptic inhibitory domain. The inhibitory sites of each fragment (K16-S17 for trypsin, L43-S44 for chymotrypsin) are each part of a type VI like turn at one end ofmore » their respective region of the antiparallel {beta}-sheet. These structural elements are compared to those found in other Bowman-Birk inhibitors.« less

Immobilization of Trypsin in Lignocellulosic Waste Material to Produce Peptides with Bioactive Potential from Whey Protein

PubMed Central

Bassan, Juliana Cristina; de Souza Bezerra, Thaís Milena; Peixoto, Guilherme; da Cruz, Clariana Zanutto Paulino; Galán, Julián Paul Martínez; Vaz, Aline Buda dos Santos; Garrido, Saulo Santesso; Filice, Marco; Monti, Rubens

2016-01-01

In this study, trypsin (Enzyme Comission 3.4.21.4) was immobilized in a low cost, lignocellulosic support (corn cob powder—CCP) with the goal of obtaining peptides with bioactive potential from cheese whey. The pretreated support was activated with glyoxyl groups, glutaraldehyde and IDA-glyoxyl. The immobilization yields of the derivatives were higher than 83%, and the retention of catalytic activity was higher than 74%. The trypsin-glyoxyl-CCP derivative was thermally stable at 65 °C, a value that was 1090-fold higher than that obtained with the free enzyme. The trypsin-IDA-glyoxyl-CCP and trypsin-glutaraldehyde-CCP derivatives had thermal stabilities that were 883- and five-fold higher, respectively, then those obtained with the free enzyme. In the batch experiments, trypsin-IDA-glyoxyl-CCP retained 91% of its activity and had a degree of hydrolysis of 12.49%, while the values for trypsin-glyoxyl-CCP were 87% and 15.46%, respectively. The stabilized derivative trypsin-glyoxyl-CCP was also tested in an upflow packed-bed reactor. The hydrodynamic characterization of this reactor was a plug flow pattern, and the kinetics of this system provided a relative activity of 3.04 ± 0.01 U·g−1 and an average degree of hydrolysis of 23%, which were suitable for the production of potentially bioactive peptides. PMID:28773482

Membrane protease degradomics: proteomic identification and quantification of cell surface protease substrates.

PubMed

Butler, Georgina S; Dean, Richard A; Smith, Derek; Overall, Christopher M

2009-01-01

The modification of cell surface proteins by plasma membrane and soluble proteases is important for physiological and pathological processes. Methods to identify shed and soluble substrates are crucial to further define the substrate repertoire, termed the substrate degradome, of individual proteases. Identifying protease substrates is essential to elucidate protease function and involvement in different homeostatic and disease pathways. This characterisation is also crucial for drug target identification and validation, which would then allow the rational design of specific targeted inhibitors for therapeutic intervention. We describe two methods for identifying and quantifying shed cell surface protease targets in cultured cells utilising Isotope-Coded Affinity Tags (ICAT) and Isobaric Tags for Relative and Absolute Quantification (iTRAQ). As a model system to develop these techniques, we chose a cell-membrane expressed matrix metalloproteinase, MMP-14, but the concepts can be applied to proteases of other classes. By over-expression, or conversely inhibition, of a particular protease with careful selection of control conditions (e.g. vector or inactive protease) and differential labelling, shed proteins can be identified and quantified by mass spectrometry (MS), MS/MS fragmentation and database searching.

Activation of Bt Protoxin Cry1Ac in Resistant and Susceptible Cotton Bollworm.

PubMed

Wei, Jizhen; Liang, Gemei; Wang, Bingjie; Zhong, Feng; Chen, Lin; Khaing, Myint Myint; Zhang, Jie; Guo, Yuyuan; Wu, Kongming; Tabashnik, Bruce E

2016-01-01

Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins.

Activation of Bt Protoxin Cry1Ac in Resistant and Susceptible Cotton Bollworm

PubMed Central

Liang, Gemei; Wang, Bingjie; Zhong, Feng; Chen, Lin; Khaing, Myint Myint; Zhang, Jie; Guo, Yuyuan; Wu, Kongming; Tabashnik, Bruce E.

2016-01-01

Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins. PMID:27257885

Functional analyses of the three simian hemorrhagic fever virus nonstructural protein 1 papain-like proteases.

PubMed

Vatter, Heather A; Di, Han; Donaldson, Eric F; Radu, Gertrud U; Maines, Taronna R; Brinton, Margo A

2014-08-01

The N-terminal region of simian hemorrhagic fever virus (SHFV) nonstructural polyprotein 1a is predicted to encode three papain-like proteases (PLP1α, PLP1β, and PLP1γ). Catalytic residues and cleavage sites for each of the SHFV PLP1s were predicted by alignment of the SHFV PLP1 region sequences with each other as well as with those of other arteriviruses, and the predicted catalytic residues were shown to be proximal by homology modeling of the SHFV nsp1s on porcine respiratory and reproductive syndrome virus (PRRSV) nsp1 crystal structures. The functionality of the predicted catalytic Cys residues and cleavage sites was tested by analysis of the autoproteolytic products generated in in vitro transcription/translation reactions done with wild-type or mutant SHFV nsp1 constructs. Cleavage sites were also analyzed by mass spectroscopy analysis of selected immunoprecipitated cleavage products. The data showed that each of the three SHFV PLP1s is an active protease. Cys63 was identified as the catalytic Cys of SHFV PLP1α and is adjacent to an Ala instead of the canonical Tyr observed in other arterivirus PLP1s. SHFV PLP1γ is able to cleave at both downstream and upstream nsp1 junction sites. Although intermediate precursor polyproteins as well as alternative products generated by each of the SHFV PLP1s cleaving at sites within the N-terminal region of nsp1β were produced in the in vitro reactions, Western blotting of SHFV-infected, MA104 cell lysates with SHFV nsp1 protein-specific antibodies detected only the three mature nsp1 proteins. SHFV is unique among arteriviruses in having three N-terminal papain-like protease 1 (PLP1) domains. Other arteriviruses encode one or two active PLP1s. This is the first functional study of the SHFV PLP1s. Analysis of the products of in vitro autoprocessing of an N-terminal SHFV nonstructural 1a polypeptide fragment showed that each of the three SHFV PLP1s is active, and the predicted catalytic Cys residues and cleavage sites