Anti-trypanosomal activity of non-peptidic nitrile-based cysteine protease inhibitors.

Science.gov (United States)

Burtoloso, Antonio C B; de Albuquerque, Sérgio; Furber, Mark; Gomes, Juliana C; Gonçalez, Cristiana; Kenny, Peter W; Leitão, Andrei; Montanari, Carlos A; Quilles, José Carlos; Ribeiro, Jean F R; Rocha, Josmar R

2017-02-01

The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Anti-trypanosomal activity against the CL Brener strain of T. cruzi was observed in the 0.1 μM to 1 μM range for three nitrile-based cysteine protease inhibitors based on two scaffolds known to be associated with cathepsin K inhibition. The two compounds showing the greatest potency against the trypanosome were characterized by EC50 values (0.12 μM and 0.25 μM) that were an order of magnitude lower than the corresponding Ki values measured against cruzain, a recombinant form of cruzipain, in an enzyme inhibition assay. This implies that the anti-trypanosomal activity of these two compounds may not be explained only by the inhibition of the cruzain enzyme, thereby triggering a putative polypharmacological profile towards cysteine proteases.

Identification of semicarbazones, thiosemicarbazones and triazine nitriles as inhibitors of Leishmania mexicana cysteine protease CPB.

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Jörg Schröder

Full Text Available Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas' disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases.

Cysteine proteases and wheat (Triticum aestivum L) under drought: A still greatly unexplored association.

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Botha, Anna-Maria; Kunert, Karl J; Cullis, Christopher A

2017-09-01

Bread wheat (Triticum aestivum L.) provides about 19% of global dietary energy. Environmental stress, such as drought, affects wheat growth causing premature plant senescence and ultimately plant death. A plant response to drought is an increase in protease-mediated proteolysis with rapid degradation of proteins required for metabolic processes. Among the plant proteases that are increased in their activity following stress, cysteine proteases are the best characterized. Very little is known about particular wheat cysteine protease sequences, their expression and also localization. The current knowledge on wheat cysteine proteases belonging to the five clans (CA, CD, CE, CF and CP) is outlined, in particular their expression and possible function under drought. The first successes in establishing an annotated wheat genome database are further highlighted which has allowed more detailed mining of cysteine proteases. We also share our thoughts on future research directions considering the growing availability of genomic resources of this very important food crop. Finally, we also outline future application of developed knowledge in transgenic wheat plants for environmental stress protection and also as senescence markers to monitor wheat growth under environmental stress conditions. © 2017 John Wiley & Sons Ltd.

2-D zymographic analysis of Broccoli (Brassica oleracea L. var. Italica) florets proteases: follow up of cysteine protease isotypes in the course of post-harvest senescence.

Science.gov (United States)

Rossano, Rocco; Larocca, Marilena; Riccio, Paolo

2011-09-01

Zymographic analysis of Broccoli florets (Brassica oleracea L. var. Italica) revealed the presence of acidic metallo-proteases, serine proteases and cysteine proteases. Under conditions which were denaturing for the other proteases, the study was restricted to cysteine proteases. 2-D zymography, a technique that combines IEF and zymography was used to show the presence of 11 different cysteine protease spots with molecular mass of 44 and 47-48kDa and pIs ranging between 4.1 and 4.7. pI differences could be ascribed to different degrees of phosphorylation that partly disappeared in the presence of alkaline phosphatase. Post-harvest senescence of Broccoli florets was characterized by decrease in protein and chlorophyll contents and increase of protease activity. In particular, as determined by 2-D zymography, the presence of cysteine protease clearly increased during senescence, a finding that may represent a useful tool for the control of the aging process. Copyright © 2011 Elsevier GmbH. All rights reserved.

Allicin and derivates are cysteine protease inhibitors with antiparasitic activity.

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Waag, Thilo; Gelhaus, Christoph; Rath, Jennifer; Stich, August; Leippe, Matthias; Schirmeister, Tanja

2010-09-15

Allicin and derivatives thereof inhibit the CAC1 cysteine proteases falcipain 2, rhodesain, cathepsin B and L in the low micromolar range. The structure-activity relationship revealed that only derivatives with primary carbon atom in vicinity to the thiosulfinate sulfur atom attacked by the active-site Cys residue are active against the target enzymes. Some compounds also show potent antiparasitic activity against Plasmodium falciparum and Trypanosoma brucei brucei. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Cysteine and aspartic proteases cathepsins B and D determine the invasiveness of MCF10A neoT cells

International Nuclear Information System (INIS)

Premzl, J.; Kos, J.

2003-01-01

Background. Lysosomal cathepsins B and D have been reported to play a role in various processes leading to progression of malignant disease. In ras-transformed MCF10A neoT cells both enzymes show similar vesicular distribution in perinuclear and peripheral cytoplasmic regions. Results. The co-localization of cathepsins B and D in some vesicles as defined by confocal microscopy supports their co-ordinate activity in the proteolytic cascade. On the other hand, we showed that stefin A, an endogenous intracellular inhibitor of cysteine proteases, did not co-localize with cathepsin B and is presumably not involved in regulation of its enzymatic activity within the vesicles. Intracellular localization of both enzymes was confined to similar vesicles as the fluorescent degradation products of DQ-collagen IV either in individual cells or cell spheroids. The capability of these two enzymes to degrade collagen and other components of extracellular matrix is further supported by the results of Matrigel invasion assay. We showed that specific intracellular (CA-074 Me) and extracellular (CA-074) inhibitors of cathepsin B and pepstatin A, an inhibitor of cathepsin D, significantly reduced invasion of MCF10A neoT cells. Our results also show that in contrast to some other studies the activation peptide of pro-cathepsin D exhibited no mitogenic effect on MCF10A neoT, MCF-7 or HEK-293 cells. Conclusion. We conclude that lysosomal cysteine proteases cathepsins B and D predominantly participate in degradation of extracellular matrix and facilitate invasion of tumour cells. (author)

Cloning and characterization of a novel cysteine protease gene ...

Indian Academy of Sciences (India)

PRAKASH KUMAR

Cysteine proteases can be found in the animal and plant kingdoms as well as in some viruses and bacteria. They have been implemented in many ..... in developing resistance against pathogens and insects in other crops. Acknowledgments.

Site-specific O-Glycosylation on the MUC2 Mucin Protein Inhibits Cleavage by the Porphyromonas gingivalis Secreted Cysteine Protease (RgpB)

DEFF Research Database (Denmark)

van der Post, Sjoerd; Subramani, Durai B; Bäckström, Malin

2013-01-01

that are protease-resistant and has cysteine-rich N and C termini responsible for polymerization. Culture supernatants of Porphyromonas gingivalis, a bacterium that secretes proteases responsible for periodontitis, cleaved the MUC2 C-terminal region, whereas the N-terminal region was unaffected. The active enzyme...

Effects of cysteine on growth, protease production, and catalase activity of Pseudomonas fluorescens.

OpenAIRE

Himelbloom, B H; Hassan, H M

1986-01-01

Cysteine inhibits growth of and protease production by Pseudomonas fluorescens NC3. Catalase activity in P. fluorescens NC3 was increased by cysteine. The addition of exogenous hydrogen peroxide did not increase catalase activity, thus suggesting a role for the endogenous generation of hydrogen peroxide via the autoxidation of cysteine.

Identification of Cleavage Sites Recognized by the 3C-Like Cysteine Protease within the Two Polyproteins of Strawberry Mottle Virus

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Hélène Sanfaçon

2017-04-01

Full Text Available Strawberry mottle virus (SMoV, family Secoviridae, order Picornavirales is one of several viruses found in association with strawberry decline disease in Eastern Canada. The SMoV genome consists of two positive-sense single-stranded RNAs, each encoding one large polyprotein. The RNA1 polyprotein (P1 includes the domains for a putative helicase, a VPg, a 3C-like cysteine protease and an RNA-dependent RNA polymerase at its C-terminus, and one or two protein domains at its N-terminus. The RNA2 polyprotein (P2 is predicted to contain the domains for a movement protein (MP and one or several coat proteins at its N-terminus, and one or more additional domains for proteins of unknown function at its C-terminus. The RNA1-encoded 3C-like protease is presumed to cleave the two polyproteins in cis (P1 and in trans (P2. Using in vitro processing assays, we systematically scanned the two polyproteins for cleavage sites recognized by this protease. We identified five cis-cleavage sites in P1, with cleavage between the putative helicase and VPg domains being the most efficient. The presence of six protein domains in the SMoV P1, including two upstream of the putative helicase domain, is a feature shared with nepoviruses but not with comoviruses. Results from trans-cleavage assays indicate that the RNA1-encoded 3C-like protease recognized a single cleavage site, which was between the predicted MP and coat protein domains in the P2 polyprotein. The cleavage site consensus sequence for the SMoV 3C-like protease is AxE (E or Q/(G or S.

Iron-Binding Protein Degradation by Cysteine Proteases of Naegleria fowleri

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Moisés Martínez-Castillo

2015-01-01

Full Text Available Naegleria fowleri causes acute and fulminant primary amoebic meningoencephalitis. This microorganism invades its host by penetrating the olfactory mucosa and then traveling up the mesaxonal spaces and crossing the cribriform plate; finally, the trophozoites invade the olfactory bulbs. During its invasion, the protozoan obtains nutrients such as proteins, lipids, carbohydrates, and cationic ions (e.g., iron, calcium, and sodium from the host. However, the mechanism by which these ions are obtained, particularly iron, is poorly understood. In the present study, we evaluated the ability of N. fowleri to degrade iron-binding proteins, including hololactoferrin, transferrin, ferritin, and hemoglobin. Zymography assays were performed for each substrate under physiological conditions (pH 7 at 37°C employing conditioned medium (CM and total crude extracts (TCEs of N. fowleri. Different degradation patterns with CM were observed for hololactoferrin, transferrin, and hemoglobin; however, CM did not cause ferritin degradation. In contrast, the TCEs degraded only hololactoferrin and transferrin. Inhibition assays revealed that cysteine proteases were involved in this process. Based on these results, we suggest that CM and TCEs of N. fowleri degrade iron-binding proteins by employing cysteine proteases, which enables the parasite to obtain iron to survive while invading the central nervous system.

Biochemical properties of a novel cysteine protease of Plasmodium vivax, vivapain-4.

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Byoung-Kuk Na

2010-10-01

Full Text Available Multiple cysteine proteases of malaria parasites are required for maintenance of parasite metabolic homeostasis and egress from the host erythrocyte. In Plasmodium falciparum these proteases appear to mediate the processing of hemoglobin and aspartic proteases (plasmepsins in the acidic food vacuole and the hydrolysis of erythrocyte structural proteins at neutral pH. Two cysteine proteases, vivapain (VX-2 and VX-3 have been characterized in P. vivax, but comprehensive studies of P. vivax cysteine proteases remain elusive.We characterized a novel cysteine protease of P. vivax, VX-4, of which orthologs appears to have evolved differentially in primate plasmodia with strong cladistic affinity toward those of rodent Plasmodium. Recombinant VX-4 demonstrated dual substrate specificity depending on the surrounding micro-environmental pH. Its hydrolyzing activity against benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA and Z-Phe-Arg-MCA was highest at acidic pH (5.5, whereas that against Z-Arg-Arg-MCA was maximal at neutral pH (6.5-7.5. VX-4 preferred positively charged amino acids and Gln at the P1 position, with less strict specificity at P3 and P4. P2 preferences depended on pH (Leu at pH 5.5 and Arg at pH 7.5. Three amino acids that delineate the S2 pocket were substituted in VX-4 compared to VX-2 and VX-3 (Ala90, Gly157 and Glu180. Replacement of Glu180 abolished activity against Z-Arg-Arg-MCA at neutral pH, indicating the importance of this amino acid in the pH-dependent substrate preference. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. VX-4 showed maximal activity against actin at neutral pH, and that against P. vivax plasmepsin 4 and hemoglobin was detected at neutral/acidic and acidic pH, respectively.VX-4 demonstrates pH-dependent substrate switching, which might offer an efficient mechanism for the specific cleavage of different substrates in different intracellular

Identification and characterization of MOR-CP, a cysteine protease induced by ozone and developmental senescence in maize (Zea mays L.) leaves.

Science.gov (United States)

Ahmad, Rafiq; Zuily-Fodil, Yasmine; Passaquet, Chantal; Bethenod, Olivier; Roche, Romain; Repellin, Anne

2014-08-01

Among the different classes of endoproteases, cysteine proteases are consistently associated with senescence, defense signaling pathways and cellular responses to abiotic stresses. The objectives of this work were to study the effects of various concentrations of ozone on gene expression and enzymatic activity for papain-like cysteine proteases (PLCPs), in the leaves of maize plants grown under field conditions. Leaves from ranks 12 and 10 (cob leaf) were harvested regularly over a long-term artificial ozone fumigation experiment (50 d). Tissues were tested for transcriptional and activity changes concerning cysteine proteases, using qRT-PCR for the newly identified ozone-responsive PLCP gene (Mor-CP) and synthetic oligopeptide Boc-Val-Leu-Lys-AMC as a PLCP-specific substrate, respectively. Results showed that developmental senescence induced a significant and progressive rise in CP activity, only in the older leaves 10 and had no effect on Mor-CP gene expression levels. On the other hand, ozone dramatically enhanced Mor-CP mRNA levels and global PLCP enzymatic activity in leaves 12 and 10, particularly toward the end of the treatment. Ozone impact was more pronounced in the older leaves 10. Together, these observations concurred to conclude that ozone stress enhances natural senescence processes, such as those related to proteolysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Retaining in-gel zymographic activity of cysteine proteases via a cysteine-supplemented running buffer.

Science.gov (United States)

Vootukuri Reddy, Sreekanth; Philpott, Mike P; Trigiante, Giuseppe

2016-10-01

Zymography is a powerful technique to separate and identify different enzymatic activities on a standard acrylamide gel. For oxidation prone enzymes such as cysteine proteases however, the oxidizing species generated by electrolysis of the gel running buffer may result in partial or complete inactivation, thus compromising the final readout. This can be only partially remedied by subsequent treatment of the gel with reducing agents. We demonstrate the generation of reactive oxidizing species during electrophoresis and discovered that supplementation of the gel running buffer with a minimum of 5 mM cysteine prevents enzyme inactivation and allows retention of proteolytic activity as measured by zymography on model substrate N α-benzoyl-l-arginine p-nitroanilide, without at the same time altering the mobilities of the gel proteins. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hemoglobin cleavage site-specificity of the Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3.

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Shoba Subramanian

Full Text Available The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P(1 - P(4 amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P(2 position. Second, with overlapping peptides spanning alpha and beta globin and proteolysis-dependent (18O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P(2 Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents.

The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein from Clostridium difficile

Energy Technology Data Exchange (ETDEWEB)

Bradshaw, William J. [University of Bath, Claverton Down, Bath BA2 7AY (United Kingdom); Public Health England, Porton Down, Salisbury SP4 0JG (United Kingdom); Kirby, Jonathan M. [Public Health England, Porton Down, Salisbury SP4 0JG (United Kingdom); Thiyagarajan, Nethaji [University of Bath, Claverton Down, Bath BA2 7AY (United Kingdom); Chambers, Christopher J.; Davies, Abigail H. [University of Bath, Claverton Down, Bath BA2 7AY (United Kingdom); Public Health England, Porton Down, Salisbury SP4 0JG (United Kingdom); Roberts, April K.; Shone, Clifford C. [Public Health England, Porton Down, Salisbury SP4 0JG (United Kingdom); Acharya, K. Ravi, E-mail: bsskra@bath.ac.uk [University of Bath, Claverton Down, Bath BA2 7AY (United Kingdom)

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) 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.

The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein from Clostridium difficile

International Nuclear Information System (INIS)

Bradshaw, William J.; Kirby, Jonathan M.; Thiyagarajan, Nethaji; Chambers, Christopher J.; Davies, Abigail H.; Roberts, April K.; Shone, Clifford C.; Acharya, K. Ravi

2014-01-01

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) 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

Thrombin like activity of Asclepias curassavica L. latex: action of cysteine proteases.

Science.gov (United States)

Shivaprasad, H V; Rajesh, R; Nanda, B L; Dharmappa, K K; Vishwanath, B S

2009-05-04

To validate the scientific basis of plant latex to stop bleeding on fresh cuts. Cysteine protease(s) from Asclepias curassavica (Asclepiadaceae) plant latex was assessed for pro-coagulant and thrombin like activities. A waxy material from the latex of Asclepias curassavica latex was removed by freezing and thawing. The resulted latex enzyme fraction was assayed for proteolytic activity using denatured casein as substrate. Its coagulant activity and thrombin like activity were determined using citrated plasma and pure fibrinogen, respectively. Inhibition studies were performed using specific protease inhibitors to know the type of protease. The latex enzyme fraction exhibited strong proteolytic activity when compared to trypsin and exerted pro-coagulant action by reducing plasma clotting time from 195 to 58 s whereas trypsin reduced clotting time marginally from 195 to 155 s. The pro-coagulant activity of this enzyme fraction was exerted by selectively hydrolyzing A alpha and B beta subunits of fibrinogen to form fibrin clot when pure fibrinogen was used as substrate as assessed by fibrinogen-agarose plate method and fibrinogen polymerization assay. Trypsin failed to induce any fibrin clot under similar conditions. The electrophoretic pattern of latex enzyme fraction-induced fibrin clot was very much similar to that of thrombin-induced fibrin clot and mimic thrombin like action. The proteolytic activity including thrombin like activity of Asclepias curassavica latex enzyme fraction was completely inhibited by iodoaceticacid (IAA). Cysteine proteases from Asclepias curassavica latex exhibited strong pro-coagulant action and were found to be specific in its action (Thrombin like). This could be the basis for the use of plant latex in pharmacological applications that justify their use as folk medicine.

A role in immunity for Arabidopsis cysteine protease RD21, the ortholog of the tomato immune protease C14.

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Takayuki Shindo

Full Text Available Secreted papain-like Cys proteases are important players in plant immunity. We previously reported that the C14 protease of tomato is targeted by cystatin-like EPIC proteins that are secreted by the oomycete pathogen Phytophthora infestans (Pinf during infection. C14 has been under diversifying selection in wild potato species coevolving with Pinf and reduced C14 levels result in enhanced susceptibility for Pinf. Here, we investigated the role C14-EPIC-like interactions in the natural pathosystem of Arabidopsis with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa. In contrast to the Pinf-solanaceae pathosystem, the C14 orthologous protease of Arabidopsis, RD21, does not evolve under diversifying selection in Arabidopsis, and rd21 null mutants do not show phenotypes upon compatible and incompatible Hpa interactions, despite the evident lack of a major leaf protease. Hpa isolates express highly conserved EPIC-like proteins during infections, but it is unknown if these HpaEPICs can inhibit RD21 and one of these HpaEPICs even lacks the canonical cystatin motifs. The rd21 mutants are unaffected in compatible and incompatible interactions with Pseudomonas syringae pv. tomato, but are significantly more susceptible for the necrotrophic fungal pathogen Botrytis cinerea, demonstrating that RD21 provides immunity to a necrotrophic pathogen.

Expression, characterization, and cellular localization of knowpains, papain-like cysteine proteases of the Plasmodium knowlesi malaria parasite.

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Rajesh Prasad

Full Text Available Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4. Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains. All three knowpains were present in the food vacuole, active in acidic pH, and capable of degrading haemoglobin at the food vacuolar pH (≈5.5, suggesting roles in haemoglobin degradation. The proteases showed absolute (KP2 and KP3 to moderate (KP4 preference for peptide substrates containing leucine at the P2 position; KP4 preferred arginine at the P2 position. While the three knowpains appear to have redundant roles in haemoglobin degradation, KP4 may also have a role in degradation of erythrocyte cytoskeleton during merozoite egress, as it displayed broad substrate specificity and was primarily localized at the parasite periphery. Importantly, E64 blocked erythrocytic development of P. knowlesi, with enlargement of food vacuoles, indicating inhibition of haemoglobin hydrolysis and supporting the potential for inhibition of knowpains as a strategy for the treatment of malaria. Functional expression and characterization of knowpains should enable simultaneous screening of available cysteine protease

In vivo inhibition of cysteine proteases provides evidence for the involvement of 'senescence-associated vacuoles' in chloroplast protein degradation during dark-induced senescence of tobacco leaves.

Science.gov (United States)

Carrión, Cristian A; Costa, María Lorenza; Martínez, Dana E; Mohr, Christina; Humbeck, Klaus; Guiamet, Juan J

2013-11-01

Breakdown of leaf proteins, particularly chloroplast proteins, is a massive process in senescing leaves. In spite of its importance in internal N recycling, the mechanism(s) and the enzymes involved are largely unknown. Senescence-associated vacuoles (SAVs) are small, acidic vacuoles with high cysteine peptidase activity. Chloroplast-targeted proteins re-localize to SAVs during senescence, suggesting that SAVs might be involved in chloroplast protein degradation. SAVs were undetectable in mature, non-senescent tobacco leaves. Their abundance, visualized either with the acidotropic marker Lysotracker Red or by green fluorescent protein (GFP) fluorescence in a line expressing the senescence-associated cysteine protease SAG12 fused to GFP, increased during senescence induction in darkness, and peaked after 2-4 d, when chloroplast dismantling was most intense. Increased abundance of SAVs correlated with higher levels of SAG12 mRNA. Activity labelling with a biotinylated derivative of the cysteine protease inhibitor E-64 was used to detect active cysteine proteases. The two apparently most abundant cysteine proteases of senescing leaves, of 40kDa and 33kDa were detected in isolated SAVs. Rubisco degradation in isolated SAVs was completely blocked by E-64. Treatment of leaf disks with E-64 in vivo substantially reduced degradation of Rubisco and leaf proteins. Overall, these results indicate that SAVs contain most of the cysteine protease activity of senescing cells, and that SAV cysteine proteases are at least partly responsible for the degradation of stromal proteins of the chloroplast.

Cwp84, a Clostridium difficile cysteine protease, exhibits conformational flexibility in the absence of its propeptide

International Nuclear Information System (INIS)

Bradshaw, William J.; Roberts, April K.; Shone, Clifford C.; Acharya, K. Ravi

2015-01-01

Two structures of Cwp84, a cysteine protease from the S-layer of C. difficile, are presented after propeptide cleavage. They reveal the movement of three loops, two in the active-site groove and one on the surface of the lectin-like domain, exposing a hydrophobic pocket. In recent decades, the global healthcare problems caused by Clostridium difficile have increased at an alarming rate. A greater understanding of this antibiotic-resistant bacterium, particularly with respect to how it interacts with the host, is required for the development of novel strategies for fighting C. difficile infections. The surface layer (S-layer) of C. difficile is likely to be of significant importance to host–pathogen interactions. The mature S-layer is formed by a proteinaceous array consisting of multiple copies of a high-molecular-weight and a low-molecular-weight S-layer protein. These components result from the cleavage of SlpA by Cwp84, a cysteine protease. The structure of a truncated Cwp84 active-site mutant has recently been reported and the key features have been identified, providing the first structural insights into the role of Cwp84 in the formation of the S-layer. Here, two structures of Cwp84 after propeptide cleavage are presented and the three conformational changes that are observed are discussed. These changes result in a reconfiguration of the active site and exposure of the hydrophobic pocket

Detection of cysteine protease in Taenia solium-induced brain granulomas in naturally infected pigs

DEFF Research Database (Denmark)

Mkupasi, Ernatus Martin; Sikasunge, Chummy Sikalizyo; Ngowi, Helena Aminiel

2013-01-01

In order to further characterize the immune response around the viable or degenerating Taenia solium cysts in the pig brain, the involvement of cysteine protease in the immune evasion was assessed. Brain tissues from 30 adult pigs naturally infected with T. solium cysticercosis were subjected...... protease may play a role in inducing immune evasion through apoptosis around viable T. solium cysts....

HvPap-1 C1A Protease Participates Differentially in the Barley Response to a Pathogen and an Herbivore

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Mercedes Diaz-Mendoza

2017-09-01

Full Text Available Co-evolutionary processes in plant–pathogen/herbivore systems indicate that protease inhibitors have a particular value in biotic interactions. However, little is known about the defensive role of their targets, the plant proteases. C1A cysteine proteases are the most abundant enzymes responsible for the proteolytic activity during different processes like germination, development and senescence in plants. To identify and characterize C1A cysteine proteases of barley with a potential role in defense, mRNA and protein expression patterns were analyzed in response to biotics stresses. A barley cysteine protease, HvPap-1, previously related to abiotic stresses and grain germination, was particularly induced by flagellin or chitosan elicitation, and biotic stresses such as the phytopathogenic fungus Magnaporthe oryzae or the phytophagous mite Tetranychus urticae. To elucidate the in vivo participation of this enzyme in defense, transformed barley plants overexpressing or silencing HvPap-1 encoding gene were subjected to M. oryzae infection or T. urticae infestation. Whereas overexpressing plants were less susceptible to the fungus than silencing plants, the opposite behavior occurred to the mite. This unexpected result highlights the complexity of the regulatory events leading to the response to a particular biotic stress.

Targeting cysteine residues of human immunodeficiency virus type 1 protease by reactive free radical species.

Science.gov (United States)

Basu, A; Sehajpal, P K; Ogiste, J S; Lander, H M

1999-01-01

Nitric oxide (NO) is a naturally occurring free radical with many functions. The oxidized form of NO, the nitrosonium ion, reacts with the thiol group of cysteine residues resulting in their modification to S-nitrosothiols. The human immunodeficiency virus type 1 (HIV-1) protease (HIV-PR) has two cysteine residues that are conserved amongst different viral isolates found in patients with acquired immunodeficiency syndrome (AIDS). In an active dimer, these residues are located near the surface of the protease. We have found that treatment of HIV-PR with different NO congeners results in loss of its proteolytic activity and simultaneous formation of S-nitrosothiols. Sodium nitroprusside inhibited HIV-PR up to 70% and S-nitroso-N-acetylpenicillamine completely inhibited the protease within 5 min of treatment. The pattern of inhibition by NO donors is comparable to its inhibition by N-acetyl pepstatin. Using electrospray ionization-mass spectrometry, we identified the modification of HIV-PR by NO as that of S-nitrosation. Our findings point towards a possible role of NO in mediating resistance to HIV-1 infection.

Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination1[W

Science.gov (United States)

Martinez, Manuel; Cambra, Ines; Carrillo, Laura; Diaz-Mendoza, Mercedes; Diaz, Isabel

2009-01-01

Plant cystatins are inhibitors of cysteine-proteases of the papain C1A and legumain C13 families. Cystatin data from multiple plant species have suggested that these inhibitors act as defense proteins against pests and pathogens and as regulators of protein turnover. In this study, we characterize the entire cystatin gene family from barley (Hordeum vulgare), which contain 13 nonredundant genes, and identify and characterize their target enzymes, the barley cathepsin L-like proteases. Cystatins and proteases were expressed and purified from Escherichia coli cultures. Each cystatin was found to have different inhibitory capability against barley cysteine-proteases in in vitro inhibitory assays using specific substrates. Real-time reverse transcription-polymerase chain reaction revealed that inhibitors and enzymes present a wide variation in their messenger RNA expression patterns. Their transcripts were mainly detected in developing and germinating seeds, and some of them were also expressed in leaves and roots. Subcellular localization of cystatins and cathepsin L-like proteases fused to green fluorescent protein demonstrated the presence of both protein families throughout the endoplasmic reticulum and the Golgi complex. Proteases and cystatins not only colocalized but also interacted in vivo in the plant cell, as revealed by bimolecular fluorescence complementation. The functional relationship between cystatins and cathepsin L-like proteases was inferred from their common implication as counterparts of mobilization of storage proteins upon barley seed germination. The opposite pattern of transcription expression in gibberellin-treated aleurones presented by inhibitors and enzymes allowed proteases to specifically degrade B, C, and D hordeins stored in the endosperm of barley seeds. PMID:19759340

Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion

International Nuclear Information System (INIS)

Nishikado, Hideto; Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko; Ogawa, Hideoki; Okumura, Ko; Takai, Toshiro

2015-01-01

Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils. - Highlights: • We identified the murine IL3R as a novel target of papain-family cysteine proteases. • Papain-family cysteine proteases cleaved IL3Rα/CD123 on murine basophils. • Papain suppressed IL3- but not TSLP-induced expansion of murine basophils. • The inactivation of IL3R might be a strategy for pathogens to suppress host immunity

Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion

Energy Technology Data Exchange (ETDEWEB)

Nishikado, Hideto [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko [Laboratory of Proteomics and Biomolecular Science, BioMedical Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Ogawa, Hideoki; Okumura, Ko [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Takai, Toshiro, E-mail: t-takai@juntendo.ac.jp [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan)

2015-05-01

Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils. - Highlights: • We identified the murine IL3R as a novel target of papain-family cysteine proteases. • Papain-family cysteine proteases cleaved IL3Rα/CD123 on murine basophils. • Papain suppressed IL3- but not TSLP-induced expansion of murine basophils. • The inactivation of IL3R might be a strategy for pathogens to suppress host immunity.

Heparin modulates the endopeptidase activity of Leishmania mexicana cysteine protease cathepsin L-Like rCPB2.8.

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Wagner A S Judice

Full Text Available Cysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. Several microorganism pathogens bind to the heparin-like glycosaminoglycans chains of proteoglycans at host-cell surface to promote their attachment and internalization. Here, we have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity.THE DATA ANALYSIS REVEALED THAT THE PRESENCE OF HEPARIN AFFECTS ALL STEPS OF THE ENZYME REACTION: (i it decreases 3.5-fold the k 1 and 4.0-fold the k -1, (ii it affects the acyl-enzyme accumulation with pronounced decrease in k 2 (2.7-fold, and also decrease in k 3 (3.5-fold. The large values of ΔG  =  12 kJ/mol for the association and dissociation steps indicate substantial structural strains linked to the formation/dissociation of the ES complex in the presence of heparin, which underscore a conformational change that prevents the diffusion of substrate in the rCPB2.8 active site. Binding to heparin also significantly decreases the α-helix content of the rCPB2.8 and perturbs the intrinsic fluorescence emission of the enzyme. The data strongly suggest that heparin is altering the ionization of catalytic (Cys(25-S(-/(His(163-Im(+ H ion pair of the rCPB2.8. Moreover, the interaction of heparin with the N-terminal pro-region of rCPB2.8 significantly decreased its inhibitory activity against the mature enzyme.Taken together, depending on their concentration, heparin-like glycosaminoglycans can either stimulate or antagonize the activity of cysteine protease B enzymes during parasite infection, suggesting that this glycoconjugate can anchor parasite cysteine protease at host cell surface.

Activation of mas-related G-protein-coupled receptors by the house dust mite cysteine protease Der p1 provides a new mechanism linking allergy and inflammation.

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Reddy, Vemuri B; Lerner, Ethan A

2017-10-20

Cysteine and serine proteases function via protease-activated and mas-related G-protein-coupled receptors (Mrgprs) to contribute to allergy and inflammation. Der p1 is a cysteine protease and major allergen from the house dust mite and is associated with allergic rhinitis and allergic asthma. Der p1 activates protease-activated receptor 2 and induces the release of the pro-inflammatory cytokine IL-6 from cells. However, the possibility that Der p1 acts on Mrgprs has not been considered. We report here that ratiometric calcium imaging reveals that Der p1 activates the human receptor MRGPRX1 and the mouse homolog MrgprC11, implicated previously in itch. Der p1 cleavage of N-terminal receptor peptides followed by site-directed mutagenesis of the cleavage sites links receptor activation to specific amino acid residues. Der p1 also induced the release of IL-6 from heterologous cells expressing MRGPRX1. In summary, activation of Mrgprs by the allergen Der p1 may contribute to inflammation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Entamoeba histolytica-secreted cysteine proteases induce IL-8 production in human mast cells via a PAR2-independent mechanism.

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Lee, Young Ah; Nam, Young Hee; Min, Arim; Kim, Kyeong Ah; Nozaki, Tomoyoshi; Saito-Nakano, Yumiko; Mirelman, David; Shin, Myeong Heon

2014-01-01

Entamoeba histolytica is an extracellular tissue parasite causing colitis and occasional liver abscess in humans. E. histolytica-derived secretory products (SPs) contain large amounts of cysteine proteases (CPs), one of the important amoebic virulence factors. Although tissue-residing mast cells play an important role in the mucosal inflammatory response to this pathogen, it is not known whether the SPs induce mast cell activation. In this study, when human mast cells (HMC-1 cells) were stimulated with SPs collected from pathogenic wild-type amoebae, interleukin IL-8 mRNA expression and production were significantly increased compared with cells incubated with medium alone. Inhibition of CP activity in the SPs with heat or the CP inhibitor E64 resulted in significant reduction of IL-8 production. Moreover, SPs obtained from inhibitors of cysteine protease (ICP)-overexpressing amoebae with low CP activity showed weaker stimulatory effects on IL-8 production than the wild-type control. Preincubation of HMC-1 cells with antibodies to human protease-activated receptor 2 (PAR2) did not affect the SP-induced IL-8 production. These results suggest that cysteine proteases in E. histolytica-derived secretory products stimulate mast cells to produce IL-8 via a PAR2-independent mechanism, which contributes to IL-8-mediated tissue inflammatory responses during the early phase of human amoebiasis. © Y.A. Lee et al., published by EDP Sciences, 2014.

Entamoeba histolytica-secreted cysteine proteases induce IL-8 production in human mast cells via a PAR2-independent mechanism

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Lee Young Ah

2014-01-01

Full Text Available Entamoeba histolytica is an extracellular tissue parasite causing colitis and occasional liver abscess in humans. E. histolytica-derived secretory products (SPs contain large amounts of cysteine proteases (CPs, one of the important amoebic virulence factors. Although tissue-residing mast cells play an important role in the mucosal inflammatory response to this pathogen, it is not known whether the SPs induce mast cell activation. In this study, when human mast cells (HMC-1 cells were stimulated with SPs collected from pathogenic wild-type amoebae, interleukin IL-8 mRNA expression and production were significantly increased compared with cells incubated with medium alone. Inhibition of CP activity in the SPs with heat or the CP inhibitor E64 resulted in significant reduction of IL-8 production. Moreover, SPs obtained from inhibitors of cysteine protease (ICP-overexpressing amoebae with low CP activity showed weaker stimulatory effects on IL-8 production than the wild-type control. Preincubation of HMC-1 cells with antibodies to human protease-activated receptor 2 (PAR2 did not affect the SP-induced IL-8 production. These results suggest that cysteine proteases in E. histolytica-derived secretory products stimulate mast cells to produce IL-8 via a PAR2-independent mechanism, which contributes to IL-8-mediated tissue inflammatory responses during the early phase of human amoebiasis.

Expression of a pathogen-induced cysteine protease (AdCP) in tapetum results in male sterility in transgenic tobacco.

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Shukla, Pawan; Singh, Naveen Kumar; Kumar, Dilip; Vijayan, Sambasivam; Ahmed, Israr; Kirti, Pulugurtha Bharadwaja

2014-06-01

Usable male sterility systems have immense potential in developing hybrid varieties in crop plants, which can also be used as a biological safety containment to prevent horizontal transgene flow. Barnase-Barstar system developed earlier was the first approach to engineer male sterility in plants. In an analogous situation, we have evolved a system of inducing pollen abortion and male sterility in transgenic tobacco by expressing a plant gene coding for a protein with known developmental function in contrast to the Barnase-Barstar system, which deploys genes of prokaryotic origin, i.e., from Bacillus amyloliquefaciens. We have used a plant pathogen-induced gene, cysteine protease for inducing male sterility. This gene was identified in the wild peanut, Arachis diogoi differentially expressed when it was challenged with the late leaf spot pathogen, Phaeoisariopsis personata. Arachis diogoi cysteine protease (AdCP) was expressed under the strong tapetum-specific promoter (TA29) and tobacco transformants were generated. Morphological and histological analysis of AdCP transgenic plants showed ablated tapetum and complete pollen abortion in three transgenic lines. Furthermore, transcript analysis displayed the expression of cysteine protease in these male sterile lines and the expression of the protein was identified in western blot analysis using its polyclonal antibody raised in the rabbit system.

Structure of the Mature Streptococcal Cysteine Protease Exotoxin mSpeB in Its Active Dimeric Form

DEFF Research Database (Denmark)

Olsen, Johan G; Dagil, Robert; Niclasen, Louise Meinert

2009-01-01

Invasive infections of Streptococcus pyogenes are dependent on the cysteine protease streptococcal pyrogenic exotoxin B. Previous structures of the enzyme have not disclosed the proper active-site configuration. Here, the crystal structure of the mature enzyme is presented to 1.55 A, disclosing...

Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

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Amol Bhargava

Full Text Available Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1, suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK. Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

A cysteine protease inhibitor of plasmodium berghei is essential for exo-erythrocytic development.

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Christine Lehmann

2014-08-01

Full Text Available Plasmodium parasites express a potent inhibitor of cysteine proteases (ICP throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the Plasmodium berghei ICP (PbICP. Excision of the pbicb gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a pbicp-gfp construct fully reversed these defects. Taken together, in P. berghei this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.

Immobilised native plant cysteine proteases: packed-bed reactor for white wine protein stabilisation

OpenAIRE

Benucci, Ilaria; Lombardelli, Claudio; Liburdi, Katia; Acciaro, Giuseppe; Zappino, Matteo; Esti, Marco

2015-01-01

This research presents a feasibility study of using a continuous packed-bed reactor (PBR), containing immobilised native plant cysteine proteases, as a specific and mild alternative technique relative to the usual bentonite fining for white wine protein stabilisation. The operational parameters for a PBR containing immobilised bromelain (PBR-br) or immobilised papain (PBR-pa) were optimised using model wine fortified with synthetic substrate (Bz-Phe-Val-Arg-pNA). The effectiveness of PBR-br, ...

Natural inhibitors of tumor-associated proteases

International Nuclear Information System (INIS)

Magdolen, U.; Krol, J.; Sato, S.; Schmitt, M.; Magdolen, V.; Krueger, A.; Mueller, M.M.; Sperl, S.

2002-01-01

The turnover and remodelling of extracellular matrix (ECM) is an essential part of many normal biological processes including development, morphogenesis, and wound healing. ECM turnover also occurs in severe pathological situations like artherosclerosis, fibrosis, tumor invasion and metastasis. The major proteases involved in this turnover are serine proteases (especially the urokinase-type plasminogen activator/plasmin system), matrix metalloproteases (a family of about 20 zinc-dependent endopeptidases including collagenases, gelatinases, stromelysins, and membrane-type metalloproteases), and cysteine proteases. In vivo, the activity of these proteases is tightly regulated in the extracellular space by zymogen activation and/or controlled inhibition. In the present review, we give an overview on the structure and biochemical properties of important tumor-associated protease inhibitors such as plasminogen activator inhibitor type 1 and type 2 (PAI-1, PAI-2), tissue inhibitors of metalloproteinases (TIMP-1, -2, -3, and -4), and the cysteine protease inhibitor cystatin C. Interestingly, some of these inhibitors of tumor-associated proteases display multiple functions which rather promote than inhibit tumor progression, when the presence of inhibitors in the tumor tissue is not balanced. (author)

Generation of an antibody that recognizes Plasmodium chabaudi cysteine protease (chabaupain-1) in both sexual and asexual parasite life cycle and evaluation of chabaupain-1 vaccine potential.

Science.gov (United States)

Armada, Ana; Gazarini, Marcos L; Gonçalves, Lídia M; Antunes, Sandra; Custódio, Ana; Rodrigues, Armanda; Almeida, António J; Silveira, Henrique; Rosário, Virgílio do; Santos-Gomes, Gabriela; Domingos, Ana

2013-09-01

Malaria cysteine proteases have been shown to be immunogenic and are being exploited as serodiagnostic markers, drug and vaccine targets. Several Plasmodium spp. cysteine proteases have been described and the best characterized of these are the falcipains, a family of papain-family enzymes. Falcipain-2 and falcipain-3 act in concert with other proteases to hydrolyze host erythrocyte hemoglobin in the parasite food vacuole. Falcipain-1 has less similarity to the other falcipains and its physiological role in parasite asexual blood stage still remains uncertain. Immunolocalization studies using an antibody developed against the Plasmodium chabaudi recombinant chabaupain-1, the falcipain-1 ortholog, were performed confirming its cellular localization in both erythrocyte and mosquito ookinete stage. Immunostaining of chabaupain-1 preferentially in apical portion of parasite ookinete suggests that this protease may be related with parasite egression from mosquito midgut. Immune responses to chabaupain-1 were evaluated using two different adjuvants, chitosan nanoparticles and hydroxide aluminum. Mice immunized with the recombinant protein alone or in association with nanoparticles were challenged with P. chabaudi showing that immunization with the recombinant protein confers partial protection to blood stage infection in BALB/c animal model. Copyright © 2013 Elsevier Inc. All rights reserved.

The papain inhibitor (SPI) of Streptomyces mobaraensis inhibits bacterial cysteine proteases and is an antagonist of bacterial growth

NARCIS (Netherlands)

S. Zindel (Stephan); W.E. Kaman (Wendy); S. Fröls (Sabrina); F. Pfeifer (Felicitas); A. Peters (Annette); J.P. Hays (John); H.-L. Fuchsbauer (Hans-Lothar)

2013-01-01

textabstractA novel papain inhibitory protein (SPI) from Streptomyces mobaraensis was studied to measure its inhibitory effect on bacterial cysteine protease activity (Staphylococcus aureus SspB) and culture supernatants (Porphyromonas gingivalis, Bacillus anthracis). Further, growth of Bacillus

Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease

International Nuclear Information System (INIS)

Gershenfeld, H.K.; Hershberger, R.J.; Shows, T.B.; Weissman, I.L.

1988-01-01

A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage λgt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16 + natural killer cells and CD3 + , CD16 - T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. The authors propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells

Inhibitory effects of bromelain, a cysteine protease derived from pineapple stem (Ananas comosus), on intestinal motility in mice.

Science.gov (United States)

Borrelli, F; Capasso, R; Severino, B; Fiorino, F; Aviello, G; De Rosa, G; Mazzella, M; Romano, B; Capasso, F; Fasolino, I; Izzo, A A

2011-08-01

Bromelain (BR) is a cysteine protease with inhibitory effects on intestinal secretion and inflammation. However, its effects on intestinal motility are largely unexplored. Thus, we investigated the effect of this plant-derived compound on intestinal contractility and transit in mice. Contractility in vitro was evaluated by stimulating the mouse isolated ileum, in an organ bath, with acetylcholine, barium chloride, or electrical field stimulation. Motility in vivo was measured by evaluating the distribution of an orally administered fluorescent marker along the small intestine. Transit was also evaluated in pathophysiologic states induced by the pro-inflammatory compound croton oil or by the diabetogenic agent streptozotocin. Bromelain inhibited the contractions induced by different spasmogenic compounds in the mouse ileum with similar potency. The antispasmodic effect was reduced or counteracted by the proteolytic enzyme inhibitor, gabexate (15 × 10(-6)  mol L(-1) ), protease-activated receptor-2 (PAR-2) antagonist, N(1) -3-methylbutyryl-N(4) -6-aminohexanoyl-piperazine (10(-4) mol L(-1) ), phospholipase C (PLC) inhibitor, neomycin (3 × 10(-3) mol L(-1) ), and phosphodiesterase 4 (PDE4) inhibitor, rolipram (10(-6)  mol L(-1) ). In vivo, BR preferentially inhibited motility in pathophysiologic states in a PAR-2-antagonist-sensitive manner. Our data suggest that BR inhibits intestinal motility - preferentially in pathophysiologic conditions - with a mechanism possibly involving membrane PAR-2 and PLC and PDE4 as intracellular signals. Bromelain could be a lead compound for the development of new drugs, able to normalize the intestinal motility in inflammation and diabetes. © 2011 Blackwell Publishing Ltd.

Significance of Cuscutain, a cysteine protease from Cuscuta reflexa, in host-parasite interactions.

Science.gov (United States)

Bleischwitz, Marc; Albert, Markus; Fuchsbauer, Hans-Lothar; Kaldenhoff, Ralf

2010-10-22

Plant infestation with parasitic weeds like Cuscuta reflexa induces morphological as well as biochemical changes in the host and the parasite. These modifications could be caused by a change in protein or gene activity. Using a comparative macroarray approach Cuscuta genes specifically upregulated at the host attachment site were identified. One of the infestation specific Cuscuta genes encodes a cysteine protease. The protein and its intrinsic inhibitory peptide were heterologously expressed, purified and biochemically characterized. The haustoria specific enzyme was named cuscutain in accordance with similar proteins from other plants, e.g. papaya. The role of cuscutain and its inhibitor during the host parasite interaction was studied by external application of an inhibitor suspension, which induced a significant reduction of successful infection events. The study provides new information about molecular events during the parasitic plant--host interaction. Inhibition of cuscutain cysteine proteinase could provide means for antagonizing parasitic plants.

Crystal structure of a novel cysteinless plant Kunitz-type protease inhibitor

International Nuclear Information System (INIS)

Hansen, Daiane; Macedo-Ribeiro, Sandra; Verissimo, Paula; Yoo Im, Sonia; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela

2007-01-01

Bauhinia bauhinioides Cruzipain Inhibitor (BbCI) is a cysteine protease inhibitor highly homologous to plant Kunitz-type inhibitors. However, in contrast to classical Kunitz family inhibitors it lacks cysteine residues and therefore disulfide bridges. BbCI is also distinct in the ability to inactivate enzymes belonging to two different classes, cysteine and serine proteases. Besides inhibiting the cysteine protease cruzipain, BbCI also inhibits cathepsin L and the serine proteases HNE (human neutrophil elastase) and PPE (porcine pancreatic elastase). Monoclinic crystals of the recombinant inhibitor that diffract to 1.7 A resolution were obtained using hanging drop method by vapor diffusion at 18 o C. The refined structure shows the conservative β-trefoil fold features of the Kunitz inhibitors. In BbCI, one of the two characteristic S-S bonds is replaced by the water-mediated interaction between Tyr125 and Gly132. In this work we explore the structural differences between Kunitz-type inhibitors and analyze the essential interactions that maintain the protein structural stability preserving its biological function

Vibrio Type III Effector VPA1380 Is Related to the Cysteine Protease Domain of Large Bacterial Toxins

Science.gov (United States)

Calder, Thomas; Kinch, Lisa N.; Fernandez, Jessie; Salomon, Dor; Grishin, Nick V.; Orth, Kim

2014-01-01

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium and one of the leading causes of food-borne gastroenteritis. Its genome harbors two Type III Secretion Systems (T3SS1 and T3SS2), but only T3SS2 is required for enterotoxicity seen in animal models. Effector proteins secreted from T3SS2 have been previously shown to promote colonization of the intestinal epithelium, invasion of host cells, and destruction of the epithelial monolayer. In this study, we identify VPA1380, a T3SS2 effector protein that is toxic when expressed in yeast. Bioinformatic analyses revealed that VPA1380 is highly similar to the inositol hexakisphosphate (IP6)-inducible cysteine protease domains of several large bacterial toxins. Mutations in conserved catalytic residues and residues in the putative IP6-binding pocket abolished toxicity in yeast. Furthermore, VPA1380 was not toxic in IP6 deficient yeast cells. Therefore, our findings suggest that VPA1380 is a cysteine protease that requires IP6 as an activator. PMID:25099122

Vibrio type III effector VPA1380 is related to the cysteine protease domain of large bacterial toxins.

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Thomas Calder

Full Text Available Vibrio parahaemolyticus is a Gram-negative halophilic bacterium and one of the leading causes of food-borne gastroenteritis. Its genome harbors two Type III Secretion Systems (T3SS1 and T3SS2, but only T3SS2 is required for enterotoxicity seen in animal models. Effector proteins secreted from T3SS2 have been previously shown to promote colonization of the intestinal epithelium, invasion of host cells, and destruction of the epithelial monolayer. In this study, we identify VPA1380, a T3SS2 effector protein that is toxic when expressed in yeast. Bioinformatic analyses revealed that VPA1380 is highly similar to the inositol hexakisphosphate (IP6-inducible cysteine protease domains of several large bacterial toxins. Mutations in conserved catalytic residues and residues in the putative IP6-binding pocket abolished toxicity in yeast. Furthermore, VPA1380 was not toxic in IP6 deficient yeast cells. Therefore, our findings suggest that VPA1380 is a cysteine protease that requires IP6 as an activator.

Dynamic viscoelasticity of protease-treated rice batters for gluten-free rice bread making.

Science.gov (United States)

Honda, Yuji; Inoue, Nanami; Sugimoto, Reina; Matsumoto, Kenji; Koda, Tomonori; Nishioka, Akihiro

2018-03-01

Papain (cysteine protease), subtilisin (Protin SD-AY10, serine protease), and bacillolysin (Protin SD-NY10, metallo protease) increased the specific volume of gluten-free rice breads by 19-63% compared to untreated bread. In contrast, Newlase F (aspartyl protease) did not expand the volume of the rice bread. In a rheological analysis, the viscoelastic properties of the gluten-free rice batters also depended on the protease categories. Principal component analysis (PCA) analysis suggested that the storage and loss moduli (G' and G″, respectively) at 35 °C, and the maximum values of G' and G″, were important factors in the volume expansion. Judging from the PCA of the viscoelastic parameters of the rice batters, papain and Protin SD-AY10 improved the viscoelasticity for gluten-free rice bread making, and Protin SD-NY effectively expanded the gluten-free rice bread. The rheological properties differed between Protin SD-NY and the other protease treatments.

Significance of Cuscutain, a cysteine protease from Cuscuta reflexa, in host-parasite interactions

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Fuchsbauer Hans-Lothar

2010-10-01

Full Text Available Abstract Background Plant infestation with parasitic weeds like Cuscuta reflexa induces morphological as well as biochemical changes in the host and the parasite. These modifications could be caused by a change in protein or gene activity. Using a comparative macroarray approach Cuscuta genes specifically upregulated at the host attachment site were identified. Results One of the infestation specific Cuscuta genes encodes a cysteine protease. The protein and its intrinsic inhibitory peptide were heterologously expressed, purified and biochemically characterized. The haustoria specific enzyme was named cuscutain in accordance with similar proteins from other plants, e.g. papaya. The role of cuscutain and its inhibitor during the host parasite interaction was studied by external application of an inhibitor suspension, which induced a significant reduction of successful infection events. Conclusions The study provides new information about molecular events during the parasitic plant - host interaction. Inhibition of cuscutain cysteine proteinase could provide means for antagonizing parasitic plants.

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

Science.gov (United States)

Park, Eunsook; Woo, Jongchan; Dinesh-Kumar, SP

2014-01-01

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes. PMID:24658121

Relative contribution of matrix metalloprotease and cysteine protease activities to cytokine-stimulated articular cartilage degradation

DEFF Research Database (Denmark)

Sondergaard, B C; Henriksen, K; Wulf, H

2006-01-01

OBJECTIVE: Both matrix metalloprotease (MMP) activity and cathepsin K (CK) activity have been implicated in cartilage turnover. We investigated the relative contribution of MMP activity and CK activity in cartilage degradation using ex vivo and in vivo models. METHODS: Bovine articular cartilage...... explants were stimulated with oncostatin M (OSM) 10 ng/ml and tumor necrosis factor-alpha (TNF-alpha) 20 ng/ml in the presence or absence of the broad-spectrum MMP inhibitor GM6001 and the cysteine protease inhibitor, E64. Cartilage degradation was evaluated in the conditioned medium by glycosaminoglycans...... was measured from CK-deficient mice. RESULTS: OSM and TNF-alpha combined induced significant (Pcartilage degradation products measured by hydroxyproline and CTX-II compared to vehicle control. The cytokines potently induced MMP expression, assessed by zymography, and CK expression...

Identification, classification and expression pattern analysis of sugarcane cysteine proteinases

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Gustavo Coelho Correa

2001-12-01

Full Text Available Cysteine proteases are peptidyl hydrolyses dependent on a cysteine residue at the active center. The physical and chemical properties of cysteine proteases have been extensively characterized, but their precise biological functions have not yet been completely understood, although it is known that they are involved in a number of events such as protein turnover, cancer, germination, programmed cell death and senescence. Protein sequences from different cysteine proteinases, classified as members of the E.C.3.4.22 sub-sub-class, were used to perform a T-BLAST-n search on the Brazilian Sugarcane Expressed Sequence Tags project (SUCEST data bank. Sequence homology was found with 76 cluster sequences that corresponded to possible cysteine proteinases. The alignments of these SUCEST clusters with the sequence of cysteine proteinases of known origins provided important information about the classification and possible function of these sugarcane enzymes. Inferences about the expression pattern of each gene were made by direct correlation with the SUCEST cDNA libraries from which each cluster was derived. Since no previous reports of sugarcane cysteine proteinases genes exists, this study represents a first step in the study of new biochemical, physiological and biotechnological aspects of sugarcane cysteine proteases.Proteinases cisteínicas são peptidil-hidrolases dependentes de um resíduo de cisteína em seu sítio ativo. As propriedades físico-químicas destas proteinases têm sido amplamente caracterizadas, entretanto suas funções biológicas ainda não foram completamente elucidadas. Elas estão envolvidas em um grande número de eventos, tais como: processamento e degradação protéica, câncer, germinação, morte celular programada e processos de senescência. Diferentes proteinases cisteínicas, classificadas pelo Comitê de Nomenclatura da União Internacional de Bioquímica e Biologia Molecular (IUBMB como pertencentes à sub

Identification, Characterization and Down-Regulation of Cysteine Protease Genes in Tobacco for Use in Recombinant Protein Production.

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Kishor Duwadi

Full Text Available Plants are an attractive host system for pharmaceutical protein production. Many therapeutic proteins have been produced and scaled up in plants at a low cost compared to the conventional microbial and animal-based systems. The main technical challenge during this process is to produce sufficient levels of recombinant proteins in plants. Low yield is generally caused by proteolytic degradation during expression and downstream processing of recombinant proteins. The yield of human therapeutic interleukin (IL-10 produced in transgenic tobacco leaves was found to be below the critical level, and may be due to degradation by tobacco proteases. Here, we identified a total of 60 putative cysteine protease genes (CysP in tobacco. Based on their predicted expression in leaf tissue, 10 candidate CysPs (CysP1-CysP10 were selected for further characterization. The effect of CysP gene silencing on IL-10 accumulation was examined in tobacco. It was found that the recombinant protein yield in tobacco could be increased by silencing CysP6. Transient expression of CysP6 silencing construct also showed an increase in IL-10 accumulation in comparison to the control. Moreover, CysP6 localizes to the endoplasmic reticulum (ER, suggesting that ER may be the site of IL-10 degradation. Overall results suggest that CysP6 is important in determining the yield of recombinant IL-10 in tobacco leaves.

Cysteine Protease (Capparin from Capsules of Caper (Capparis spinosa

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Yasar Demir

2008-01-01

Full Text Available Proteases are enzymes that perform very important functions in organisms and are used for a variety of objectives in vitro. In recent years, proteases have been used for clinical, pharmaceutical (alimentary digestion, anti-inflammatory, etc. and industrial applications (cheese production, meat tenderizing, leather tanning. In this research, a protease has been purified from capsules of caper (Capparis spinosa and characterized. Caper plants have been used for food and medicine since ancient times. The plant grows abundantly in certain regions of Turkey. Ammonium sulphate fractionation and a CM Sephadex column were used for purification of the enzyme. The purification enzyme has an optimum pH=5.0 and its optimum temperature was 60 °C. The vmax and Km values determined by Lineweaver-Burk graphics were 1.38 μg/(L·min and 0.88 μg/L, respectively. The purification degree and the molecular mass of the enzyme (46 kDa were determined by SDS-PAGE and gel filtration chromatography. It was investigated whether the purified and characterized protease could cause milk to congeal or digest chicken and cow meat. The results show that protease can be used for industrial production.

Activation of ADAM 12 protease by copper

DEFF Research Database (Denmark)

Loechel, F; Wewer, Ulla M.

2001-01-01

Conversion of latent proteases to the active form occurs by various mechanisms characteristic for different protease families. Here we report that the disintegrin metalloprotease ADAM 12-S is activated by Cu(II). Copper activation is distinct from the cysteine switch component of latency: elimina......Conversion of latent proteases to the active form occurs by various mechanisms characteristic for different protease families. Here we report that the disintegrin metalloprotease ADAM 12-S is activated by Cu(II). Copper activation is distinct from the cysteine switch component of latency......: elimination of the ADAM 12 cysteine switch by a point mutation in the propeptide had no effect on copper activation, whereas mutation of an unpaired cysteine residue in the catalytic domain resulted in a mutant form of ADAM 12-S that was insensitive to copper. This suggests a multi-step activation mechanism...... for ADAM 12 involving both furin cleavage and copper binding....

A protein extract and a cysteine protease inhibitor enriched fraction from Jatropha curcas seed cake have in vitro anti-Toxoplasma gondii activity.

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Soares, A M S; Carvalho, L P; Melo, E J T; Costa, H P S; Vasconcelos, I M; Oliveira, J T A

2015-06-01

Toxoplasma gondii is a parasite of great medical and veterinary importance that has worldwide distribution and causes toxoplasmosis. There are few treatments available for toxoplasmosis and the search for plant extracts and compounds with anti-Toxoplasma activity is of utmost importance for the discovery of new active drugs. The objective of this study was to investigate the action of a protein extract and a protease inhibitor enriched fraction from J. curcas seed cake on developing tachyzoites of T. gondii-infected Vero cells. The protein extract (JcCE) was obtained after solubilization of the J. curcas seed cake with 100 mM sodium borate buffer, pH 10, centrifugation and dialysis of the resulting supernatant with the extracting buffer. JcCE was used for the in vitro assays of anti-Toxoplasma activity at 0.01, 0.1, 0.5, 1.5, 3.0 and 5.0 mg/ml concentration for 24 h. The results showed that JcCE reduced the percentage of infection and the number of intracellular parasites, but had no effect on the morphology of Vero cells up to 3.0 mg/mL. The cysteine protease inhibitor enriched fraction, which was obtained after chromatography of JcCE on Sephadex G-75 and presented a unique protein band following SDS-PAGE, reduced both the number of T. gondii infected cells and intracellular parasites. These results suggest that both JcCE and the cysteine protease inhibitor enriched fraction interfere with the intracellular growth of T. gondii. Copyright © 2015 Elsevier Inc. All rights reserved.

The cysteine protease CEP1, a key executor involved in tapetal programmed cell death, regulates pollen development in Arabidopsis.

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Zhang, Dandan; Liu, Di; Lv, Xiaomeng; Wang, Ying; Xun, Zhili; Liu, Zhixiong; Li, Fenglan; Lu, Hai

2014-07-01

Tapetal programmed cell death (PCD) is a prerequisite for pollen grain development in angiosperms, and cysteine proteases are the most ubiquitous hydrolases involved in plant PCD. We identified a papain-like cysteine protease, CEP1, which is involved in tapetal PCD and pollen development in Arabidopsis thaliana. CEP1 is expressed specifically in the tapetum from stages 5 to 11 of anther development. The CEP1 protein first appears as a proenzyme in precursor protease vesicles and is then transported to the vacuole and transformed into the mature enzyme before rupture of the vacuole. cep1 mutants exhibited aborted tapetal PCD and decreased pollen fertility with abnormal pollen exine. A transcriptomic analysis revealed that 872 genes showed significantly altered expression in the cep1 mutants, and most of them are important for tapetal cell wall organization, tapetal secretory structure formation, and pollen development. CEP1 overexpression caused premature tapetal PCD and pollen infertility. ELISA and quantitative RT-PCR analyses confirmed that the CEP1 expression level showed a strong relationship to the degree of tapetal PCD and pollen fertility. Our results reveal that CEP1 is a crucial executor during tapetal PCD and that proper CEP1 expression is necessary for timely degeneration of tapetal cells and functional pollen formation. © 2014 American Society of Plant Biologists. All rights reserved.

Purification and biochemical characterization of a 22-kDa stable cysteine- like protease from the excretory-secretory product of the liver fluke Fasciola hepatica by using conventional techniques.

Science.gov (United States)

Hemici, Ahmed; Benerbaiha, Roumaila Sabrina; Bendjeddou, Dalila

2017-11-15

This study describes the purification and characterization of a stable protease activity isolated from Fasciola hepatica adult worms maintained in vitro by employing acetone precipitation (40-60%) followed by a gel filtration through Sephadex G-100 and DEAE- cellulose ion exchange column. Through this three-step purification, the enzyme was purified 11-fold with a specific activity of 1893.9U/mg and 31.5% recovery. After the final ultrafiltration step, the purification fold was increased up to 13.1 and the overall activity yield reached a rate of 18.8%. The MW of the purified protease was estimated by reducing SDS-PAGE to be 22kDa while the proteolytic activity detection was carried out by zymography on non-denaturing SDS-PAGE containing the casein as substrate. Using this substrate, the protease showed extreme proteolytic activity at pH 5.5 and temperature 35-40°C and was highly stable over a wide range of pH, from 5.0 to 10.0. In addition to its preference for the Z-Phe-Arg-AMC fluorogenic substrate resulting in maximum proteolytic activity (99.7%) at pH 7.0, the pure protease exhibited highest cleavage activity against hemoglobin and casein substrates at pH 5.5 (85.6% and 82.8%, respectively). The K m values obtained for this protease were 5.4, 13, 160 and approximately 1000μM using respectively the fluorogenic substrate Z-Phe-Arg-AMC, hemoglobin, casein and albumin. The protease activity was completely inhibited either by E-64 inhibitor (5mM) or iodoacetamide (10mM), indicating its cysteine nature. The usefulness of the purified protease as an antigen was studied by immunoblotting. Thus, sera from sheep experimentally infected with F. hepatica recognized the protease band at 2 weeks post-infection (WPI) and strongly at 7 WPI. The early detection of antibodies anti- F. hepatica suggests the application of this molecule as a specific epitope for the serodiagnosis of fascioliasis disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Cysteine protease inhibition by nitrile-based inhibitors: a computational study

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Quesne, Matthew G.; Ward, Richard A.; de Visser, Sam P.

2013-01-01

Cysteine protease enzymes are important for human physiology and catalyze key protein degradation pathways. These enzymes react via a nucleophilic reaction mechanism that involves a cysteine residue and the proton of a proximal histidine. Particularly efficient inhibitors of these enzymes are nitrile-based, however, the details of the catalytic reaction mechanism currently are poorly understood. To gain further insight into the inhibition of these molecules, we have performed a combined density functional theory and quantum mechanics/molecular mechanics study on the reaction of a nitrile-based inhibitor with the enzyme active site amino acids. We show here that small perturbations to the inhibitor structure can have dramatic effects on the catalysis and inhibition processes. Thus, we investigated a range of inhibitor templates and show that specific structural changes reduce the inhibitory efficiency by several orders of magnitude. Moreover, as the reaction takes place on a polar surface, we find strong differences between the DFT and QM/MM calculated energetics. In particular, the DFT model led to dramatic distortions from the starting structure and the convergence to a structure that would not fit the enzyme active site. In the subsequent QM/MM study we investigated the use of mechanical vs. electronic embedding on the kinetics, thermodynamics and geometries along the reaction mechanism. We find minor effects on the kinetics of the reaction but large geometric and thermodynamics differences as a result of inclusion of electronic embedding corrections. The work here highlights the importance of model choice in the investigation of this biochemical reaction mechanism. PMID:24790966

The Cysteine Protease CEP1, a Key Executor Involved in Tapetal Programmed Cell Death, Regulates Pollen Development in Arabidopsis[W][OPEN

Science.gov (United States)

Zhang, Dandan; Liu, Di; Lv, Xiaomeng; Wang, Ying; Xun, Zhili; Liu, Zhixiong; Li, Fenglan; Lu, Hai

2014-01-01

Tapetal programmed cell death (PCD) is a prerequisite for pollen grain development in angiosperms, and cysteine proteases are the most ubiquitous hydrolases involved in plant PCD. We identified a papain-like cysteine protease, CEP1, which is involved in tapetal PCD and pollen development in Arabidopsis thaliana. CEP1 is expressed specifically in the tapetum from stages 5 to 11 of anther development. The CEP1 protein first appears as a proenzyme in precursor protease vesicles and is then transported to the vacuole and transformed into the mature enzyme before rupture of the vacuole. cep1 mutants exhibited aborted tapetal PCD and decreased pollen fertility with abnormal pollen exine. A transcriptomic analysis revealed that 872 genes showed significantly altered expression in the cep1 mutants, and most of them are important for tapetal cell wall organization, tapetal secretory structure formation, and pollen development. CEP1 overexpression caused premature tapetal PCD and pollen infertility. ELISA and quantitative RT-PCR analyses confirmed that the CEP1 expression level showed a strong relationship to the degree of tapetal PCD and pollen fertility. Our results reveal that CEP1 is a crucial executor during tapetal PCD and that proper CEP1 expression is necessary for timely degeneration of tapetal cells and functional pollen formation. PMID:25035401

Characterization of Thermo- and Detergent Stable Antigenic Glycosylated Cysteine Protease of Euphorbia nivulia Buch.-Ham. and Evaluation of Its Ecofriendly Applications

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Shamkant B. Badgujar

2013-01-01

Full Text Available An antigenic glycosylated cysteine protease has been purified from the latex of Euphorbia nivulia Buch.-Ham. It exhibits remarkable protease activity in the presence of metal ions, oxidizing agents, organic solvents, and detergents. This enzyme showed potential role in leather processing industry due to its dehairing activity for animal hide without hydrolyzing fibrous proteins, producing, by this way, a better quality product. The enzyme can also be used for silver recovering from X-ray plates. In addition, the stability (temperature and surfactants and hydrolysis of blood stain data also revealed its application in detergent industries. Agriculturally, this protease finds application in biocontrol process against the infectious management of root knot nematode, Meloidogyne incognita. Biologically, it shows noticeable wound healing, haemostatic and antibacterial activity.

Compatibility in the Ustilago maydis-maize interaction requires inhibition of host cysteine proteases by the fungal effector Pit2.

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André N Mueller

2013-02-01

Full Text Available The basidiomycete Ustilago maydis causes smut disease in maize, with large plant tumors being formed as the most prominent disease symptoms. During all steps of infection, U. maydis depends on a biotrophic interaction, which requires an efficient suppression of plant immunity. In a previous study, we identified the secreted effector protein Pit2, which is essential for maintenance of biotrophy and induction of tumors. Deletion mutants for pit2 successfully penetrate host cells but elicit various defense responses, which stops further fungal proliferation. We now show that Pit2 functions as an inhibitor of a set of apoplastic maize cysteine proteases, whose activity is directly linked with salicylic-acid-associated plant defenses. Consequently, protease inhibition by Pit2 is required for U. maydis virulence. Sequence comparisons with Pit2 orthologs from related smut fungi identified a conserved sequence motif. Mutation of this sequence caused loss of Pit2 function. Consequently, expression of the mutated protein in U. maydis could not restore virulence of the pit2 deletion mutant, indicating that the protease inhibition by Pit2 is essential for fungal virulence. Moreover, synthetic peptides of the conserved sequence motif showed full activity as protease inhibitor, which identifies this domain as a new, minimal protease inhibitor domain in plant-pathogenic fungi.

Bromelain, a cysteine protease from pineapple (Ananas comosus) stem, is an inhibitor of fungal plant pathogens.

Science.gov (United States)

López-García, B; Hernández, M; Segundo, B S

2012-07-01

This study aimed to evaluate the effect of bromelain, a cysteine protease isolated from pineapple (Ananas comosus), on growth of several agronomically important fungal pathogens. Purification of bromelain from pineapple stems was carried out by chromatography techniques, and its antimicrobial activity was tested against the fungal pathogens Fusarium verticillioides, Fusarium oxysporum and Fusarium proliferatum by broth microdilution assay. A concentration of 0.3 μmol l(-1) of bromelain was sufficient for 90% growth inhibition of F. verticillioides. The capability of bromelain to inhibit fungal growth is related to its proteolytic activity. The study demonstrates that stem bromelain exhibits a potent antifungal activity against phytopathogens and suggests its potential use as an effective agent for crop protection. The results support the use of a natural protease that accumulates at high levels in pineapple stems as alternative to the use of chemical fungicides for crop protection. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

A cysteine protease (cathepsin Z) from disk abalone, Haliotis discus discus: Genomic characterization and transcriptional profiling during bacterial infections.

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Godahewa, G I; Perera, N C N; Lee, Sukkyoung; Kim, Myoung-Jin; Lee, Jehee

2017-09-05

Cathepsin Z (CTSZ) is lysosomal cysteine protease of the papain superfamily. It participates in the host immune defense via phagocytosis, signal transduction, cell-cell communication, proliferation, and migration of immune cells such as monocytes, macrophages, and dendritic cells. Hence, CTSZ is also acknowledged as an acute-phase protein in host immunity. In this study, we sought to identify the CTSZ homolog from disk abalone (AbCTSZ) and characterize it at the molecular, genomic, and transcriptional levels. AbCTSZ encodes a protein with 318 amino acids and a molecular mass of 36kDa. The structure of AbCTSZ reveals amino acid sequences that are characteristic of the signal sequence, pro-peptide, peptidase-C1 papain family cysteine protease domain, mini-loop, HIP motif, N-linked glycosylation sites, active sites, and conserved Cys residues. A pairwise comparison revealed that AbCTSZ shared the highest amino acid homology with its molluscan counterpart from Crassostrea gigas. A multiple alignment analysis revealed the conservation of functionally crucial elements of AbCTSZ, and a phylogenetic study further confirmed a proximal evolutionary relationship with its invertebrate counterparts. Further, an analysis of AbCTSZ genomic structure revealed seven exons separated by six introns, which differs from that of its vertebrate counterparts. Quantitative real time PCR (qPCR) detected the transcripts of AbCTSZ in early developmental stages and in eight different tissues. Higher levels of AbCTSZ transcripts were found in trochophore, gill, and hemocytes, highlighting its importance in the early development and immunity of disk abalone. In addition, we found that viable bacteria (Vibrio parahaemolyticus and Listeria monocytogenes) and bacterial lipopolysaccharides significantly modulated AbCTSZ transcription. Collectively, these lines of evidences suggest that AbCTSZ plays an indispensable role in the innate immunity of disk abalone. Copyright © 2017. Published by Elsevier

Small ring constrained peptidomimetics. Synthesis of epoxy peptidomimetics, inhibitors of cysteine proteases.

Science.gov (United States)

Demarcus, M; Ganadu, M L; Mura, G M; Porcheddu, A; Quaranta, L; Reginato, G; Taddei, M

2001-02-09

Different dipeptide analogues containing an oxirane ring in the place of the peptidic bond were prepared starting from naturally occurring amino acids. N-Fmoc-amino aldehydes were transformed into the corresponding methoxyvinyl derivatives through a Wittig reaction, and the addition of PhSeCl gave a series of different alpha-phenylselenyl aldehydes. Mukajiama reaction with silylketene acetals gave an intermediate product that was finally transformed into the desired oxiranyl peptidomimetics. Following this strategy we were able to control three new contiguous stereocenters starting from the enantiomerically pure amino acid. The dipeptide analogues could be used in SPPS on a SASRIN resin as the final epoxides were relatively unstable under acidic conditions. Moreover the synthesis of the single dipeptide mimetics was carried out on solid phase to generate a small library of epoxy peptidomimetics. Some of the products prepared in this work resulted as time-dependent reversible inhibitors of cysteine protease.

Pepper pathogenesis-related protein 4c is a plasma membrane-localized cysteine protease inhibitor that is required for plant cell death and defense signaling.

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Kim, Nak Hyun; Hwang, Byung Kook

2015-01-01

Xanthomonas campestris pv. vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis-related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT-triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease-inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H2 O2 and significant induction of some defense-response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT-triggered resistance responses, and enabled Xcv proliferation in infected leaves. H2 O2 accumulation, cell-death induction, and defense-response gene expression were distinctly reduced in CaPR4c-silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Transgenic soybean plants overexpressing O-acetylserine sulfhydrylase accumulate enhanced levels of cysteine and Bowman-Birk protease inhibitor in seeds.

Science.gov (United States)

Kim, Won-Seok; Chronis, Demosthenis; Juergens, Matthew; Schroeder, Amy C; Hyun, Seung Won; Jez, Joseph M; Krishnan, Hari B

2012-01-01

Soybeans provide an excellent source of protein in animal feed. Soybean protein quality can be enhanced by increasing the concentration of sulfur-containing amino acids. Previous attempts to increase the concentration of sulfur-containing amino acids through the expression of heterologous proteins have met with limited success. Here, we report a successful strategy to increase the cysteine content of soybean seed through the overexpression of a key sulfur assimilatory enzyme. We have generated several transgenic soybean plants that overexpress a cytosolic isoform of O-acetylserine sulfhydrylase (OASS). These transgenic soybean plants exhibit a four- to tenfold increase in OASS activity when compared with non-transformed wild-type. The OASS activity in the transgenic soybeans was significantly higher at all the stages of seed development. Unlike the non-transformed soybean plants, there was no marked decrease in the OASS activity even at later stages of seed development. Overexpression of cytosolic OASS resulted in a 58-74% increase in protein-bound cysteine levels compared with non-transformed wild-type soybean seeds. A 22-32% increase in the free cysteine levels was also observed in transgenic soybeans overexpressing OASS. Furthermore, these transgenic soybean plants showed a marked increase in the accumulation of Bowman-Birk protease inhibitor, a cysteine-rich protein. The overall increase in soybean total cysteine content (both free and protein-bound) satisfies the recommended levels required for the optimal growth of monogastric animals.

Purification and characterisation of a protease (tamarillin) from tamarillo fruit

KAUST Repository

Li, Zhao

2018-02-16

A protease from tamarillo fruit (Cyphomandra betacea Cav.) was purified by ammonium sulphate precipitation and diethylaminoethyl-Sepharose chromatography. Protease activity was determined on selected peak fractions using a casein substrate. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that the peak with the highest protease activity consisted of one protein of molecular mass ca. 70 kDa. The protease showed optimal activity at pH 11 and 60°C. It was sensitive to phenylmethylsulphonyl fluoride while ethylenediaminetetraacetic acid and p-chloromercuribenzoic acid had little effect on its activity, indicating that this enzyme was a serine protease. Hg2+ strongly inhibited enzyme activity, possibly due to formation of mercaptide bonds with the thiol groups of the protease, suggesting that some cysteine residues may be located close to the active site. De novo sequencing strongly indicated that the protease was a subtilisin-like alkaline serine protease. The protease from tamarillo has been named \\'tamarillin\\'.

Purification and characterisation of a protease (tamarillin) from tamarillo fruit

KAUST Repository

Li, Zhao; Scott, Ken; Hemar, Yacine; Zhang, Huoming; Otter, Don

2018-01-01

A protease from tamarillo fruit (Cyphomandra betacea Cav.) was purified by ammonium sulphate precipitation and diethylaminoethyl-Sepharose chromatography. Protease activity was determined on selected peak fractions using a casein substrate. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that the peak with the highest protease activity consisted of one protein of molecular mass ca. 70 kDa. The protease showed optimal activity at pH 11 and 60°C. It was sensitive to phenylmethylsulphonyl fluoride while ethylenediaminetetraacetic acid and p-chloromercuribenzoic acid had little effect on its activity, indicating that this enzyme was a serine protease. Hg2+ strongly inhibited enzyme activity, possibly due to formation of mercaptide bonds with the thiol groups of the protease, suggesting that some cysteine residues may be located close to the active site. De novo sequencing strongly indicated that the protease was a subtilisin-like alkaline serine protease. The protease from tamarillo has been named 'tamarillin'.

Interpain A, a cysteine proteinase from Prevotella intermedia, inhibits complement by degrading complement factor C3.

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Michal Potempa

2009-02-01

Full Text Available Periodontitis is an inflammatory disease of the supporting structures of the teeth caused by, among other pathogens, Prevotella intermedia. Many strains of P. intermedia are resistant to killing by the human complement system, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with recombinant cysteine protease of P. intermedia (interpain A resulted in a drastic decrease in bactericidal activity of the serum. Furthermore, a clinical strain 59 expressing interpain A was more serum-resistant than another clinical strain 57, which did not express interpain A, as determined by Western blotting. Moreover, in the presence of the cysteine protease inhibitor E64, the killing of strain 59 by human serum was enhanced. Importantly, we found that the majority of P. intermedia strains isolated from chronic and aggressive periodontitis carry and express the interpain A gene. The protective effect of interpain A against serum bactericidal activity was found to be attributable to its ability to inhibit all three complement pathways through the efficient degradation of the alpha-chain of C3 -- the major complement factor common to all three pathways. P. intermedia has been known to co-aggregate with P. gingivalis, which produce gingipains to efficiently degrade complement factors. Here, interpain A was found to have a synergistic effect with gingipains on complement degradation. In addition, interpain A was able to activate the C1 complex in serum, causing deposition of C1q on inert and bacterial surfaces, which may be important at initial stages of infection when local inflammatory reaction may be beneficial for a pathogen. Taken together, the newly characterized interpain A proteinase appears to be an important virulence factor of P. intermedia.

Reversible Cysteine Protease Inhibitors Show Promise for a Chagas Disease Cure

Science.gov (United States)

Beaulieu, Christian; Black, W. Cameron; Isabel, Elise; Vasquez-Camargo, Fabio; Nath-Chowdhury, Milli; Massé, Frédéric; Mellon, Christophe; Methot, Nathalie

2014-01-01

The cysteine protease cruzipain is essential for the viability, infectivity, and virulence of Trypanosoma cruzi, the causative agent of Chagas disease. Thus, inhibitors of cruzipain are considered promising anti-T. cruzi chemotherapeutic agents. Reversible cruzipain inhibitors containing a nitrile “warhead” were prepared and demonstrated 50% inhibitory concentrations (IC50s) as potent as 1 nM in baculovirus-generated cruzipain enzyme assays. In epimastigote and intracellular amastigote in vitro assays, the most potent compounds demonstrated antiparasitic behavior in the 5 to 10 μM IC50 range; however, trypomastigote production from the amastigote form was ∼90 to 95% inhibited at 2 μM. Two key compounds, Cz007 and Cz008, with IC50s of 1.1 and 1.8 nM, respectively, against the recombinant enzyme were tested in a murine model of acute T. cruzi infection, with oral dosing in chow for 28 days at doses from 3 to 50 mg/kg of body weight. At 3 mg/kg of Cz007 and 3 mg/kg of Cz008, the blood parasitemia areas under the concentration-time curves were 16% and 25% of the untreated group, respectively. At sacrifice, 24 days after immunosuppression with cyclophosphamide, parasite presence in blood, heart, and esophagus was evaluated. Based on negative quantitative PCR results in all three tissues, cure rates in surviving animals were 90% for Cz007 at 3 mg/kg, 78% for Cz008 at 3 mg/kg, and 71% for benznidazole, the control compound, at 50 mg/kg. PMID:24323474

Analysis of S-nitrosothiols via Copper Cysteine (2C) and Copper Cysteine - Carbon Monoxide (3C) Methods

Science.gov (United States)

Rogers, Stephen C.; Gibbons, Lindsey B.; Griffin, Sherraine; Doctor, Allan

2012-01-01

This chapter summarizes the principles of RSNO measurement in the gas phase, utilizing ozone-based chemiluminescence and the copper cysteine (2C) ± carbon monoxide (3C) reagent. Although an indirect method for quantifying RSNOs, this assay represents one of the most robust methodologies available. It exploits the NO• detection sensitivity of ozone based chemiluminscence, which is within the range required to detect physiological concentrations of RSNO metabolites. Additionally, the specificity of the copper cysteine (2C and 3C) reagent for RSNOs negates the need for sample pretreatment, thereby minimizing the likelihood of sample contamination (false positive results), NO species inter-conversion, or the loss of certain highly labile RSNO species. Herein, we outline the principles of this methodology, summarizing key issues, potential pitfalls and corresponding solutions. PMID:23116707

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

Science.gov (United States)

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.

A family of serine proteases of Clavibacter michiganensis subsp. michiganensis: chpC plays a role in colonization of the host plant tomato.

Science.gov (United States)

Stork, Ines; Gartemann, Karl-Heinz; Burger, Annette; Eichenlaub, Rudolf

2008-09-01

Genes for seven putative serine proteases (ChpA-ChpG) belonging to the trypsin subfamily and homologous to the virulence factor pat-1 were identified on the chromosome of Clavibacter michiganensis subsp. michiganensis (Cmm) NCPPB382. All proteases have signal peptides indicating export of these proteins. Their putative function is suggested by two motifs and an aspartate residue typical for serine proteases. Furthermore, six cysteine residues are located at conserved positions. The genes are clustered in a chromosomal region of about 50 kb with a significantly lower G + C content than common for Cmm. The genes chpA, chpB and chpD are pseudogenes as they contain frame shifts and/or in-frame stop codons. The genes chpC and chpG were inactivated by the insertion of an antibiotic resistance cassette. The chpG mutant was not impaired in virulence. However, in planta the titre of the chpC mutant was drastically reduced and only weak disease symptoms were observed. Complementation of the chpC mutant by the wild-type allele restored full virulence. ChpC is the first chromosomal gene of Cmm identified so far that affects the interaction of the pathogen with the host plant.

Identification of Cysteine Proteases and Screening of Cysteine Protease Inhibitors in Biological Samples by a Two-Dimensional Gel System of Zymography and Reverse Zymography

OpenAIRE

Saitoh, Eiichi; Yamamoto, Shinya; Okamoto, Eishiro; Hayakawa, Yoshimi; Hoshino, Takashi; Sato, Ritsuko; Isemura, Satoko; Ohtsubo, Sadami; Taniguchi, Masayuki

2007-01-01

We have developed a two-dimensional (2D-) gel system of zymography and reverse zymography for the detection and characterization of proteases and protease inhibitors. Isoelectric focusing (IEF) agarose gels with pH gradients were employed for separation in the fi rst-dimension and sodium dodecyl sulfate (SDS)-polyacrylamide gel copolymerized with gelatin used for the second dimension. Proteases and protease inhibitors separated by IEF gel were applied on the second gel without trichloroacetic...

Characterization of the secreted cathepsin B cysteine proteases family of the carcinogenic liver fluke Clonorchis sinensis.

Science.gov (United States)

Chen, Wenjun; Wang, Xiaoyun; Lv, Xiaoli; Tian, Yanli; Xu, Yanquan; Mao, Qiang; Shang, Mei; Li, Xuerong; Huang, Yan; Yu, Xinbing

2014-09-01

Clonorchis sinensis excretory/secretory products (ESP) have gained high attentions because of their potential to be vaccine candidates and drug targets in C. sinensis prevention. In this study, we extensively profiled the characteristics of four C. sinensis cathepsin B cysteine proteases (CsCB1, CsCB2, CsCB3, and CsCB4). Bioinformatics analysis showed all CsCBs contained signal peptides at the N-terminal. Functional domains and residues were found in CsCB sequences. We expressed four CsCBs and profiled immune responses followed by vaccine trials. Recombinant CsCBs could induce high IgG titers, indicating high immunogenicity of CsCB family. Additionally, ELISA results showed that both IgG1 and IgG2a levels apparently increased post-immunization with all four CsCBs, showing that combined Th1/Th2 immune responses were triggered by CsCB family. Both Real-time polymerase chain reaction (RT-PCR) and Western blotting confirmed that four CsCBs have distinct expression patterns in C. sinensis life stages. More importantly, we validated our hypothesis that CsCBs were C. sinensis excretory/secretory products. CsCBs could be recognized by C. sinensis-infected sera throughout the infection period, indicating that secreted CsCBs are immune triggers during C. sinensis infection. The protective effect was assessed by comparing the worm burden and egg per gram (EPG) between CsCB group and control group, showing that worm burden (P sinensis excretory/secretory products that may regulate host immune responses.

Light Activation of a Cysteine Protease Inhibitor: Caging of a Peptidomimetic Nitrile with RuII(bpy)2

Science.gov (United States)

Respondek, Tomasz; Garner, Robert N.; Herroon, Mackenzie K.; Podgorski, Izabela; Turro, Claudia; Kodanko, Jeremy J.

2013-01-01

A novel method for caging protease inhibitors is described. The complex [RuII(bpy)2(1)2](PF6)2 (2) was prepared from the nitrile-based peptidomimetic inhibitor Ac-Phe-NHCH2CN (1). 1H NMR, UV-vis and IR spectroscopic and mass spectrometric data confirm that two equiv of inhibitor 1 bind to RuII through the nitrile functional group. Complex 2 shows excellent stability in aqueous solution in the dark and fast release of 1 upon irradiation with visible light. Due to binding to the RuII center, the nitriles of complex 2 are caged, and 2 does not act as a potent enzyme inhibitor. However, when 2 is irradiated, it releases 1 that inhibits the cysteine proteases papain and cathepsins B, K and L, up to two times more potently than 1 alone. Ratios for IC50 values for 2 range from 6:1 to 33:1 under dark vs. light conditions, against isolated enzymes and in human cell lysates, confirming a high level of photoinduced enzyme inhibition is obtained with this method. PMID:21973207

Cysteine protease 30 (CP30) contributes to adhesion and cytopathogenicity in feline Tritrichomonas foetus

Energy Technology Data Exchange (ETDEWEB)

Gould, Emily N. [Univ. of Tennessee College of Veterinary Medicine, Knoxville, TN (United States); Giannone, Richard [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Kania, Stephen A. [The Univ. of Tennessee College of Veterinary Medicine, Knoxville, TN (United States); Tolbert, M. Katherine [Univ. of Tennessee College of Veterinary Medicine, Knoxville, TN (United States)

2017-08-01

Tritrichomonas foetus (T. foetus) is a flagellated protozoan parasite that is recognized as a significant cause of diarrhea in domestic cats with a prevalence rate as high as 30%. No drugs have been shown to consistently eliminate T. foetus infection in all cats. Cysteine proteases (CPs) have been identified as mediators of T. foetus-induced adhesion-dependent cytotoxicity to the intestinal epithelium. These CPs represent novel targets for the treatment of feline trichomonosis. However, cats also produce CPs that are part of life-critical systems. Thus, parasitic CPs need to be selectively targeted to reduce the potential for host toxicity. Previous studies have demonstrated the importance of a specific CP, CP30, in mediating bovine and human trichomonad cytopathogenicity. This CP has also recently been identified in feline T. foetus, although the function of this protease in the feline genotype remains unknown. Furthermore, the study objectives were to characterize the presence of CP30 in feline T. foetus isolates and to evaluate the effect of targeted inhibition of CP30 on feline T. foetus-induced adhesion dependent cytotoxicity.

Sequence differences in the diagnostic region of the cysteine protease 8 gene of Tritrichomonas foetus parasites of cats and cattle.

Science.gov (United States)

Sun, Zichen; Stack, Colin; Šlapeta, Jan

2012-05-25

In order to investigate the genetic variation between Tritrichomonas foetus from bovine and feline origins, cysteine protease 8 (CP8) coding sequence was selected as the polymorphic DNA marker. Direct sequencing of CP8 coding sequence of T. foetus from four feline isolates and two bovine isolates with polymerase chain reaction successfully revealed conserved nucleotide polymorphisms between feline and bovine isolates. These results provide useful information for CP8-based molecular differentiation of T. foetus genotypes. Copyright © 2011 Elsevier B.V. All rights reserved.

A Kunitz-type cysteine protease inhibitor from cauliflower and Arabidopsis

DEFF Research Database (Denmark)

Halls, C.E.; Rogers, S. W.; Ouffattole, M.

2006-01-01

proaleurain maturation protease and of papain when assayed at pH 4.5 but not at pH 6.3. In a pull-down assay, the inhibitor bound tightly to papain, but only weakly to the aspartate protease pepsin. When the cauliflower protease inhibitor was transiently expressed in tobacco suspension culture protoplasts...

Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from Asclepias fruticosa latex.

Science.gov (United States)

Trejo, Sebastián A; López, Laura M I; Caffini, Néstor O; Natalucci, Claudia L; Canals, Francesc; Avilés, Francesc X

2009-07-01

Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZalpha vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.

The structure of classical swine fever virus N(pro: a novel cysteine Autoprotease and zinc-binding protein involved in subversion of type I interferon induction.

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Keerthi Gottipati

Full Text Available Pestiviruses express their genome as a single polypeptide that is subsequently cleaved into individual proteins by host- and virus-encoded proteases. The pestivirus N-terminal protease (N(pro is a cysteine autoprotease that cleaves between its own C-terminus and the N-terminus of the core protein. Due to its unique sequence and catalytic site, it forms its own cysteine protease family C53. After self-cleavage, N(pro is no longer active as a protease. The released N(pro suppresses the induction of the host's type-I interferon-α/β (IFN-α/β response. N(pro binds interferon regulatory factor-3 (IRF3, the key transcriptional activator of IFN-α/β genes, and promotes degradation of IRF3 by the proteasome, thus preventing induction of the IFN-α/β response to pestivirus infection. Here we report the crystal structures of pestivirus N(pro. N(pro is structurally distinct from other known cysteine proteases and has a novel "clam shell" fold consisting of a protease domain and a zinc-binding domain. The unique fold of N(pro allows auto-catalysis at its C-terminus and subsequently conceals the cleavage site in the active site of the protease. Although many viruses interfere with type I IFN induction by targeting the IRF3 pathway, little information is available regarding structure or mechanism of action of viral proteins that interact with IRF3. The distribution of amino acids on the surface of N(pro involved in targeting IRF3 for proteasomal degradation provides insight into the nature of N(pro's interaction with IRF3. The structures thus establish the mechanism of auto-catalysis and subsequent auto-inhibition of trans-activity of N(pro, and its role in subversion of host immune response.

Role of Proteases in Chronic Obstructive Pulmonary Disease

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Kailash C. Pandey

2017-08-01

Full Text Available Chronic obstructive pulmonary disease (COPD is generally associated with progressive destruction of airways and lung parenchyma. Various factors play an important role in the development and progression of COPD, like imbalance of proteases, environmental and genetic factors and oxidative stress. This review is specifically focused on the role of proteases and their imbalance in COPD. There are three classes (serine, mettalo, and cysteine of proteases involved in COPD. In serine proteases, neutrophil elastase, cathepsin G, and proteinase-3 are involved in destruction of alveolar tissue. Matrix-mettaloproteinase-9, 12, 13, plays an influential role in severity of COPD. Among cysteine proteases, caspase-3, caspases-8 and caspase-9 play an important role in controlling apoptosis. These proteases activities can be regulated by inhibitors like α-1-antitrypsin, neutrophil elastase inhibitor, and leukocyte protease inhibitor. Studies suggest that neutrophil elastase may be a therapeutic target for COPD, and specific inhibitor against this enzyme has potential role to control the disease. Current study suggests that Dipeptidyl Peptidase IV is a potential marker for COPD. Since the expression of proteases and its inhibitors play an important role in COPD pathogenesis, therefore, it is worth investigating the role of proteases and their regulation. Understanding the biochemical basis of COPD pathogenesis using advanced tools in protease biochemistry and aiming toward translational research from bench-to-bedside will have great impact to deal with this health problem.

Oral delivery of Bacillus subtilis spores expressing cysteine protease of Clonorchis sinensis to grass carp (Ctenopharyngodon idellus): Induces immune responses and has no damage on liver and intestine function.

Science.gov (United States)

Tang, Zeli; Sun, Hengchang; Chen, TingJin; Lin, Zhipeng; Jiang, Hongye; Zhou, Xinyi; Shi, Cunbin; Pan, Houjun; Chang, Ouqin; Ren, Pengli; Yu, Jinyun; Li, Xuerong; Xu, Jin; Huang, Yan; Yu, Xinbing

2017-05-01

Clonorchis sinensis (C. sinensis) is a fish-borne trematode. Human can be infected by ingestion of C. sinensis metacercariae parasitized in grass carp (Ctenopharyngodon idella). For induction of effective oral immune responses, spores of Bacillus subtilis (B. subtilis) WB600 were utilized as vehicle to delivery CsCP (cysteine protease of C. sinensis) cooperated with CotC (B.s-CotC-CP), one of coat proteins, to the gastrointestinal tract. After routine culture of 8-12 h in LB medium, B. subtilis containing CotC-CsCP was transferred into the sporulation culture medium. SDS-PAGE, western blotting and the growth curve indicated that the best sporulation time of recombinant WB600 was 24-30 h at 37 °C with continuous shaking (250 rpm). Grass carp were fed with three levels of B.s-CotC-CP (1 × 10 6 , 1 × 10 7 , and 1 × 10 8  CFU g -1 ) incorporated in the basal pellets diet. The commercial pellets or supplemented with spores just expressing CotC (1 × 10 7  CFU g -1 ) were served as control diet. Our results showed that grass carp orally immunized with the feed-based B.s-CotC-CP developed a strong specific immune response with significantly (P sinensis in fish body. Therefore, this study demonstrated that the feed-based recombinant spores could trigger high levels of mucosal and humoral immunity, and would be a promising candidate vaccine against C. sinensis metacercariae formation in freshwater fish. Copyright © 2017 Elsevier Ltd. All rights reserved.

Optimization and characterization of alkaline protease and carboxymethyl-cellulase produced by Bacillus pumillus grown on Ficus nitida wastes

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Eman Zakaria Gomaa

2013-01-01

Full Text Available The potentiality of 23 bacterial isolates to produce alkaline protease and carboxymethyl-cellulase (CMCase on Ficus nitida wastes was investigated. Bacillus pumillus ATCC7061 was selected as the most potent bacterial strain for the production of both enzymes. It was found that the optimum production of protease and CMCase were recorded at 30 °C, 5% Ficus nitida leaves and incubation period of 72 h. The best nitrogen sources for protease and CMCase production were yeast extract and casein, respectively. Also maximum protease and CMCase production were reported at pH 9 and pH 10, respectively. The enzymes possessed a good stability over a pH range of 8-10, expressed their maximum activities at pH10 and temperature range of 30-50 °C, expressed their maximum activities at 50 °C. Ions of Hg2+, Fe2+ and Ag+ showed a stimulatory effect on protease activity and ions of Fe2+, Mg2+, Ca2+, Cu2+ and Ag+ caused enhancement of CMCase activity. The enzymes were stable not only towards the nonionic surfactants like Triton X-100 and Tween 80 but also the strong anionic surfactant, SDS. Moreover, the enzymes were not significantly inhibited by EDTA or cystein. Concerning biotechnological applications, the enzymes retained (51-97% of their initial activities upon incubation in the presence of commercials detergents for 1 h. The potential use of the produced enzymes in the degradation of human hair and cotton fabric samples were also assessed.

TcCYPR04, a Cacao Papain-Like Cysteine-Protease Detected in Senescent and Necrotic Tissues Interacts with a Cystatin TcCYS4.

Science.gov (United States)

Cardoso, Thyago Hermylly Santana; Freitas, Ana Camila Oliveira; Andrade, Bruno Silva; Sousa, Aurizangela Oliveira de; Santiago, André da Silva; Koop, Daniela Martins; Gramacho, Karina Peres; Alvim, Fátima Cerqueira; Micheli, Fabienne; Pirovani, Carlos Priminho

2015-01-01

The interaction amongst papain-like cysteine-proteases (PLCP) and their substrates and inhibitors, such as cystatins, can be perceived as part of the molecular battlefield in plant-pathogen interaction. In cacao, four cystatins were identified and characterized by our group. We identified 448 proteases in cacao genome, whereof 134 were cysteine-proteases. We expressed in Escherichia coli a PLCP from cacao, named TcCYSPR04. Immunoblottings with anti-TcCYSPR04 exhibited protein increases during leaf development. Additional isoforms of TcCYSPR04 appeared in senescent leaves and cacao tissues infected by Moniliophthora perniciosa during the transition from the biotrophic to the saprophytic phase. TcCYSPR04 was induced in the apoplastic fluid of Catongo and TSH1188 cacao genotypes, susceptible and resistant to M. perniciosa, respectively, but greater intensity and additional isoforms were observed in TSH1188. The fungal protein MpNEP induced PLCP isoform expression in tobacco leaves, according to the cross reaction with anti-TcCYSPR04. Several protein isoforms were detected at 72 hours after treatment with MpNEP. We captured an active PLCP from cacao tissues, using a recombinant cacao cystatin immobilized in CNBr-Sepharose. Mass spectrometry showed that this protein corresponds to TcCYSPR04. A homology modeling was obtained for both proteins. In order to become active, TcCYSPR04 needs to lose its inhibitory domain. Molecular docking showed the physical-chemical complementarities of the interaction between the cacao enzyme and its inhibitor. We propose that TcCYSPR04 and its interactions with cacao cystatins are involved in the senescence and necrosis events related to witches' broom symptoms. This molecular interaction may be the target for future interventions to control witches' broom disease.

Differential processing of Arabidopsis ubiquitin-like Atg8 autophagy proteins by Atg4 cysteine proteases

Science.gov (United States)

Woo, Jongchan; Park, Eunsook; Dinesh-Kumar, S. P.

2014-01-01

Autophagy is a highly conserved biological process during which double membrane bound autophagosomes carry intracellular cargo material to the vacuole or lysosome for degradation and/or recycling. Autophagosome biogenesis requires Autophagy 4 (Atg4) cysteine protease-mediated processing of ubiquitin-like Atg8 proteins. Unlike single Atg4 and Atg8 genes in yeast, the Arabidopsis genome contains two Atg4 (AtAtg4a and AtAtg4b) and nine Atg8 (AtAtg8a–AtAtg8i) genes. However, we know very little about specificity of different AtAtg4s for processing of different AtAtg8s. Here, we describe a unique bioluminescence resonance energy transfer-based AtAtg8 synthetic substrate to assess AtAtg4 activity in vitro and in vivo. In addition, we developed a unique native gel assay of superhRLUC catalytic activity assay to monitor cleavage of AtAtg8s in vitro. Our results indicate that AtAtg4a is the predominant protease and that it processes AtAtg8a, AtAtg8c, AtAtg8d, and AtAtg8i better than AtAtg4b in vitro. In addition, kinetic analyses indicate that although both AtAtg4s have similar substrate affinity, AtAtg4a is more active than AtAtg4b in vitro. Activity of AtAtg4s is reversibly inhibited in vitro by reactive oxygen species such as H2O2. Our in vivo bioluminescence resonance energy transfer analyses in Arabidopsis transgenic plants indicate that the AtAtg8 synthetic substrate is efficiently processed and this is AtAtg4 dependent. These results indicate that the synthetic AtAtg8 substrate is used efficiently in the biogenesis of autophagosomes in vivo. Transgenic Arabidopsis plants expressing the AtAtg8 synthetic substrate will be a valuable tool to dissect autophagy processes and the role of autophagy during different biological processes in plants. PMID:24379391

An organic solvent-, detergent-, and thermo-stable alkaline protease from the mesophilic, organic solvent-tolerant Bacillus licheniformis 3C5.

Science.gov (United States)

Rachadech, W; Navacharoen, A; Ruangsit, W; Pongtharangkul, T; Vangnai, A S

2010-01-01

Bacillus licheniformis 3C5, isolated as mesophilic bacterium, exhibited tolerance towards a wide range of non-polar and polar organic solvents at 45 degrees C. It produced an extracellular organic solvent-stable protease with an apparent molecular mass of approximately 32 kDa. The inhibitory effect of PMSF and EDTA suggested it is likely to be an alkaline serine protease. The protease was active over abroad range of temperatures (45-70 degrees C) and pH (8-10) range with an optimum activity at pH 10 and 65 degrees C. It was comparatively stable in the presence ofa relatively high concentration (35% (v/v)) of organic solvents and various types of detergents even at a relatively high temperature (45 degrees C). The protease production by B. licheniformis 3C5 was growth-dependent. The optimization of carbon and nitrogen sources for cell growth and protease production revealed that yeast extract was an important medium component to support both cell growth and the protease production. The overall properties of the protease produced by B. licheniformis 3C5 suggested that this thermo-stable, solvent-stable, detergent-stable alkaline protease is a promising potential biocatalyst for industrial and environmental applications.

Complexity of cancer protease biology: Cathepsin K expression and function in cancer progression

NARCIS (Netherlands)

Verbovšek, Urška; van Noorden, Cornelis J. F.; Lah, Tamara T.

2015-01-01

Proteases, including lysosomal cathepsins, are functionally involved in many processes in cancer progression from its initiation to invasion and metastatic spread. Only recently, cathepsin K (CatK), the cysteine protease originally reported as a collagenolytic protease produced by osteoclasts,

Cysteine Cathepsins as Regulators of the Cytotoxicity of NK and T Cells

Science.gov (United States)

Perišić Nanut, Milica; Sabotič, Jerica; Jewett, Anahid; Kos, Janko

2014-01-01

Cysteine cathepsins are lysosomal peptidases involved at different levels in the processes of the innate and adaptive immune responses. Some, such as cathepsins B, L, and H are expressed constitutively in most immune cells. In cells of innate immunity they play a role in cell adhesion and phagocytosis. Other cysteine cathepsins are expressed more specifically. Cathepsin X promotes dendritic cell maturation, adhesion of macrophages, and migration of T cells. Cathepsin S is implicated in major histocompatibility complex class II antigen presentation, whereas cathepsin C, expressed in cytotoxic T lymphocytes and natural killer (NK) cells, is involved in processing pro-granzymes into proteolytically active forms, which trigger cell death in their target cells. The activity of cysteine cathepsins is controlled by endogenous cystatins, cysteine protease inhibitors. Of these, cystatin F is the only cystatin that is localized in endosomal/lysosomal vesicles. After proteolytic removal of its N-terminal peptide, cystatin F becomes a potent inhibitor of cathepsin C with the potential to regulate pro-granzyme processing and cell cytotoxicity. This review is focused on the role of cysteine cathepsins and their inhibitors in the molecular mechanisms leading to the cytotoxic activity of T lymphocytes and NK cells in order to address new possibilities for regulation of their function in pathological processes. PMID:25520721

Kempopeptin C, a Novel Marine-Derived Serine Protease Inhibitor Targeting Invasive Breast Cancer

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Fatma H. Al-Awadhi

2017-09-01

Full Text Available Kempopeptin C, a novel chlorinated analogue of kempopeptin B, was discovered from a marine cyanobacterium collected from Kemp Channel in Florida. The structure was elucidated using NMR spectroscopy and mass spectrometry (MS. The presence of the basic Lys residue adjacent to the N-terminus of the 3-amino-6-hydroxy-2-piperidone (Ahp moiety contributed to its selectivity towards trypsin and related proteases. The antiproteolytic activity of kempopeptin C was evaluated against trypsin, plasmin and matriptase and found to inhibit these enzymes with IC50 values of 0.19, 0.36 and 0.28 μM, respectively. Due to the significance of these proteases in cancer progression and metastasis, as well as their functional redundancy with respect to targeting overlapping substrates, we examined the effect of kempopeptin C on the downstream cellular substrates of matriptase: CDCP1 and desmoglein-2 (Dsg-2. Kempopeptin C was shown to inhibit the cleavage of both substrates in vitro. Additionally, kempopeptin C reduced the cleavage of CDCP1 in MDA-MB-231 cells up to 10 µM. The functional relevance of targeting matriptase and related proteases was investigated by assessing the effect of kempopeptin C on the migration of breast cancer cells. Kempopeptin C inhibited the migration of the invasive MDA-MB-231 cells by 37 and 60% at 10 and 20 µM, respectively.

The Tr-cp 14 cysteine protease in white clover (Trifolium repens) is localized to the endoplasmic reticulum and is associated with programmed cell death during development of tracheary elements

DEFF Research Database (Denmark)

Mulisch, Maria; Asp, Torben; Krupinska, Karin

2013-01-01

family of cysteine proteases with homology to XCP1 and XCP2 from Arabidopsis thaliana and p48h-17 from Zinnia elegans, which previously have been reported to be associated with tracheary element differentiation. The proform as well as the processed form of the protein was detected in petioles, flowers....... Immunogold studies suggest that the protease prior to the burst of the vacuole was associated to the ER cisternae. After disruption of the tonoplast, it was found in the cytoplasm, and, in later stages, associated with disintegrating material dispersed throughout the cell....

Serine Protease Variants Encoded by Echis ocellatus Venom Gland cDNA: Cloning and Sequencing Analysis

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S. S. Hasson

2010-01-01

Full Text Available Envenoming by Echis saw-scaled viper is the leading cause of death and morbidity in Africa due to snake bite. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here, we report the cloning of cDNA sequences encoding four groups or isoforms of the haemostasis-disruptive Serine protease proteins (SPs from the venom glands of Echis ocellatus. All these SP sequences encoded the cysteine residues scaffold that form the 6-disulphide bonds responsible for the characteristic tertiary structure of venom serine proteases. All the Echis ocellatus EoSP groups showed varying degrees of sequence similarity to published viper venom SPs. However, these groups also showed marked intercluster sequence conservation across them which were significantly different from that of previously published viper SPs. Because viper venom SPs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis ocellatus EoSPs on the basis of sequence alone. The extraordinary level of interspecific and intergeneric sequence conservation exhibited by the Echis ocellatus EoSPs and analogous serine proteases from other viper species leads us to speculate that antibodies to representative molecules should neutralise (that we will exploit, by epidermal DNA immunization the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East, and the Indian subcontinent.

Optimized production and characterization of a detergent-stable protease from Lysinibacillus fusiformis C250R.

Science.gov (United States)

Mechri, Sondes; Kriaa, Mouna; Ben Elhoul Berrouina, Mouna; Omrane Benmrad, Maroua; Zaraî Jaouadi, Nadia; Rekik, Hatem; Bouacem, Khelifa; Bouanane-Darenfed, Amel; Chebbi, Alif; Sayadi, Sami; Chamkha, Mohamed; Bejar, Samir; Jaouadi, Bassem

2017-08-01

In this study, we aimed to optimize the cultural and nutritional conditions for protease production by Lysinibacillus fusiformis strain C250R in submerged fermentation process using statistical methodology. The most significant factors (gruel, wheat bran, yeast extract, and FeSO 4 ) were identified by Plackett-Burman design. Response surface methodology (RSM) was used to determine the optimum levels of the screened factors and their interaction. Under the optimized conditions, protease yield 3100U/mL was 4.5 folds higher than those obtained by the use of the initial conditions (680U/mL). Additionally, a new extracellular 51kDa-protease, designated SAPLF, was purified and biochemically characterized from strain C250R. It shows optimum activity at 70°C and pH 10. Its half-life times at 70 and 80°C were 10 and 6-h, respectively. Irreversible inhibition of enzyme activity of SAPLF with serine protease inhibitors demonstrated that it belongs to the serine protease family. Interestingly, its catalytic efficiency was higher than that of SPVP from Aeribacillus pallidus strain VP3 and Alcalase Ultra 2.5L from Bacillus licheniformis. This study demonstrated that SAPLF has a high detergent compatibility and an excellent stain removal compared to Alcalase Ultra 2.5L; which offers an interesting potential for its application in the laundry detergent industry. Copyright © 2017 Elsevier B.V. All rights reserved.

Evidence of mutualism between two periodontal pathogens: co-operative haem acquisition by the HmuY haemophore of Porphyromonas gingivalis and the cysteine protease interpain A (InpA) of Prevotella intermedia.

Science.gov (United States)

Byrne, D P; Potempa, J; Olczak, T; Smalley, J W

2013-06-01

Haem (iron protoporphyrin IX) is both an essential growth factor and a virulence regulator of the periodontal pathogens Porphyromonas gingivalis and Prevotella intermedia, which acquire it through the proteolytic degradation of haemoglobin and other haem-carrying plasma proteins. The haem-binding lipoprotein HmuY haemophore and the gingipain proteases of P. gingivalis form a unique synthrophic system responsible for capture of haem from haemoglobin and methaemalbumin. In this system, methaemoglobin is formed from oxyhaemoglobin by the activities of gingipain proteases and serves as a facile substrate from which HmuY can capture haem. This study examined the possibility of cooperation between HmuY and the cysteine protease interpain A (InpA) of Pr. intermedia in the haem acquisition process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to be resistant to proteolysis and so able to cooperate with InpA to extract haem from haemoglobin, which was proteolytically converted to methaemoglobin by the protease. Spectroscopic pH titrations showed that both the iron(II) and iron(III) protoporphyrin IX-HmuY complexes were stable over the pH range 4-10, demonstrating that the haemophore could function over a range of pH that may be encountered in the dental plaque biofilm. This is the first demonstration of a bacterial haemophore working in conjunction with a protease from another bacterial species to acquire haem from haemoglobin and may represent mutualism between P. gingivalis and Pr. intermedia co-inhabiting the periodontal pocket. © 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Acid-Mediated Tumor Proteolysis: Contribution of Cysteine Cathepsins

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Jennifer M Rothberg

2013-10-01

Full Text Available One of the noncellular microenvironmental factors that contribute to malignancy of solid tumors is acidic peritumoral pH. We have previously demonstrated that extracellular acidosis leads to localization of the cysteine pro-tease cathepsin B on the tumor cell membrane and its secretion. The objective of the present study was to determine if an acidic extracellular pH such as that observed in vivo (i.e., pHe 6.8 affects the activity of proteases, e.g., cathepsin B, that contribute to degradation of collagen IV by tumor cells when grown in biologically relevant three-dimensional (3D cultures. For these studies, we used 1 3D reconstituted basement membrane overlay cultures of human carcinomas, 2 live cell imaging assays to assess proteolysis, and 3 in vivo imaging of active tumor proteases. At pHe 6.8, there were increases in pericellular active cysteine cathepsins and in degradation of dye-quenched collagen IV, which was partially blocked by a cathepsin B inhibitor. Imaging probes for active cysteine cathepsins localized to tumors in vivo. The amount of bound probe decreased in tumors in bicarbonate-treated mice, a treatment previously shown to increase peritumoral pHe and reduce local invasion of the tumors. Our results are consistent with the acid-mediated invasion hypothesis and with a role for cathepsin B in promoting degradation of a basement membrane protein substrate, i.e., type IV collagen, in an acidic peritumoral environment.

Activation of human acid sphingomyelinase through modification or deletion of C-terminal cysteine.

Science.gov (United States)

Qiu, Huawei; Edmunds, Tim; Baker-Malcolm, Jennifer; Karey, Kenneth P; Estes, Scott; Schwarz, Cordula; Hughes, Heather; Van Patten, Scott M

2003-08-29

One form of Niemann-Pick disease is caused by a deficiency in the enzymatic activity of acid sphingomyelinase. During efforts to develop an enzyme replacement therapy based on a recombinant form of human acid sphingomyelinase (rhASM), purified preparations of the recombinant enzyme were found to have substantially increased specific activity if cell harvest media were stored for several weeks at -20 degrees C prior to purification. This increase in activity was found to correlate with the loss of the single free thiol on rhASM, suggesting the involvement of a cysteine residue. It was demonstrated that a variety of chemical modifications of the free cysteine on rhASM all result in substantial activation of the enzyme, and the modified cysteine responsible for this activation was shown to be the C-terminal residue (Cys629). Activation was also achieved by copper-promoted dimerization of rhASM (via cysteine) and by C-terminal truncation using carboxypeptidase Y. The role of the C-terminal cysteine in activation was confirmed by creating mutant forms of rhASM in which this residue was either deleted or replaced by a serine, with both forms having substantially higher specific activity than wild-type rhASM. These results indicate that purified rhASM can be activated in vitro by loss of the free thiol on the C-terminal cysteine via chemical modification, dimerization, or deletion of this amino acid residue. This method of activation is similar to the cysteine switch mechanism described previously for matrix metalloproteinases and could represent a means of posttranslational regulation of ASM activity in vivo.

Development of a cysteine-deprived and C-terminally truncated GLP-1 receptor

DEFF Research Database (Denmark)

Underwood, Christina Rye; Knudsen, Lotte Bjerre; Garibay, Patrick W.

2013-01-01

The glucagon-like peptide-1 receptor (GLP-1R) belongs to family B of the G-protein coupled receptors (GPCRs), and has become a promising target for the treatment of type 2 diabetes. Here we describe the development and characterization of a fully functional cysteine-deprived and C......-terminally truncated GLP-1R. Single cysteines were initially substituted with alanine, and functionally redundant cysteines were subsequently changed simultaneously. Our results indicate that Cys174, Cys226, Cys296 and Cys403 are important for the GLP-1-mediated response, whereas Cys236, Cys329, Cys341, Cys347, Cys438...... that the membrane proximal part of the C-terminal is involved in receptor expression at the cell surface. The results show that seven cysteines and more than half of the C-terminal tail can be removed from GLP-1R without compromising GLP-1 binding or function....

Immobilised native plant cysteine proteases: packed-bed reactor for white wine protein stabilisation.

Science.gov (United States)

Benucci, Ilaria; Lombardelli, Claudio; Liburdi, Katia; Acciaro, Giuseppe; Zappino, Matteo; Esti, Marco

2016-02-01

This research presents a feasibility study of using a continuous packed-bed reactor (PBR), containing immobilised native plant cysteine proteases, as a specific and mild alternative technique relative to the usual bentonite fining for white wine protein stabilisation. The operational parameters for a PBR containing immobilised bromelain (PBR-br) or immobilised papain (PBR-pa) were optimised using model wine fortified with synthetic substrate (Bz-Phe-Val-Arg-pNA). The effectiveness of PBR-br, both in terms of hazing potential and total protein decrease, was significantly higher than PBR-pa, in all the seven unfined, white wines used. Among the wines tested, Sauvignon Blanc, given its total protein content as well as its very high intrinsic instability, was selected as a control wine to evaluate the effect of the treatment on wine as to its soluble protein profile, phenolic composition, mineral component, and sensory properties. The treatment in a PBR containing immobilised bromelain appeared effective in decreasing both wine hazing potential and total protein amount, while it did not significantly affect the phenol compounds, the mineral component nor the sensory quality of wine. The enzymatic treatment in PBR was shown to be a specific and mild technique for use as an alternative to bentonite fining for white wine protein stabilisation.

A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses.

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Rodríguez-Herva, José J; González-Melendi, Pablo; Cuartas-Lanza, Raquel; Antúnez-Lamas, María; Río-Alvarez, Isabel; Li, Ziduo; López-Torrejón, Gema; Díaz, Isabel; Del Pozo, Juan C; Chakravarthy, Suma; Collmer, Alan; Rodríguez-Palenzuela, Pablo; López-Solanilla, Emilia

2012-05-01

The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non-host tobacco, was found to also suppress the production of defence-associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His(6) -tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1(D299A) non-catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death by DC3000. Our data reveal PsbQ as a contributor to plant immunity responses and a target for pathogen suppression. © 2012 Blackwell Publishing Ltd.

Characterization of Fibrinolytic Proteases from Gloydius blomhoffii siniticus Venom

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Suk Ho Choi

2011-09-01

Full Text Available Objectives : This study was undertaken to identify fibrinolytic proteases from Gloydius blomhoffii siniticus venom and to characterize a major fibrinolytic protease purified from the venom. Methods: The venom was subjected to chromatography using columns of Q-Sepharose and Sephadex G-75. The molecular weights of fibrinolytic proteases showing fibrinolytic zone in fibrin plate assay were determined in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis The effects of inhibitors and metal ions on fibrinolytic protease and the proteolysis patterns of fibrinogen, gelatin, and bovine serum albumin were investigated. Results : 1 The fibrinolytic fractions of the three peaks isolated from Gloydius blomhoffii siniticus venom contained two polypeptides of 46 and 59 kDa and three polypeptides of 32, 18, and 15 kDa and a major polypeptide of 54 kDa, respectively. 2 The fibrinolytic activity of the purified protease of 54 kDA was inhibited by metal chelators, such as EDTA, EGTA, and 1,10-phenanthroline, and disulfhydryl-reducing compounds, such as dithiothreitol and cysteine. 3 Calcium chloride promoted the fibrinolytic activity of the protease, but mercuric chloride and cobalt(II chloride inhibited it. 4 The fibrinolytic protease cleaved preferentially A-chain and slowly B-chain of fibrinogen. It also hydrolyzed gelatin but not bovine serum albumin. Conclusions: The Gloydius blomhoffii siniticus venom contained more than three fibrinolytic proteases. The major fibrinolytic protease was a metalloprotease which hydrolyzed both fibrinogen and gelatin, but not bovine serum albumin.

Prolonged pharmacological inhibition of cathepsin C results in elimination of neutrophil serine proteases

DEFF Research Database (Denmark)

Guarino, Carla; Hamon, Yveline; Croix, Cécile

2017-01-01

cyclopropyl nitrile CatC inhibitor almost totally lack elastase. We confirmed the elimination of neutrophil elastase-like proteases by prolonged inhibition of CatC in a non-human primate. We also showed that neutrophils lacking elastase-like protease activities were still recruited to inflammatory sites....... These preclinical results demonstrate that the disappearance of neutrophil elastase-like proteases as observed in PLS patients can be achieved by pharmacological inhibition of bone marrow CatC. Such a transitory inhibition of CatC might thus help to rebalance the protease load during chronic inflammatory diseases...

Exogenous Thyropin from p41 Invariant Chain Diminishes Cysteine Protease Activity and Affects IL-12 Secretion during Maturation of Human Dendritic Cells.

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Tina Zavašnik-Bergant

Full Text Available Dendritic cells (DC play a pivotal role as antigen presenting cells (APC and their maturation is crucial for effectively eliciting an antigen-specific immune response. The p41 splice variant of MHC class II-associated chaperone, called invariant chain p41 Ii, contains an amino acid sequence, the p41 fragment, which is a thyropin-type inhibitor of proteolytic enzymes. The effects of exogenous p41 fragment and related thyropin inhibitors acting on human immune cells have not been reported yet. In this study we demonstrate that exogenous p41 fragment can enter the endocytic pathway of targeted human immature DC. Internalized p41 fragment has contributed to the total amount of the immunogold labelled p41 Ii-specific epitope, as quantified by transmission electron microscopy, in particular in late endocytic compartments with multivesicular morphology where antigen processing and binding to MHC II take place. In cell lysates of treated immature DC, diminished enzymatic activity of cysteine proteases has been confirmed. Internalized exogenous p41 fragment did not affect the perinuclear clustering of acidic cathepsin S-positive vesicles typical of mature DC. p41 fragment is shown to interfere with the nuclear translocation of NF-κB p65 subunit in LPS-stimulated DC. p41 fragment is also shown to reduce the secretion of interleukin-12 (IL-12/p70 during the subsequent maturation of treated DC. The inhibition of proteolytic activity of lysosomal cysteine proteases in immature DC and the diminished capability of DC to produce IL-12 upon their subsequent maturation support the immunomodulatory potential of the examined thyropin from p41 Ii.

Protease activation involved in resistance of human cells to x-ray cell killing

International Nuclear Information System (INIS)

Zhang, Hong-Chang; Takahashi, Shuji; Karata, Kiyonobu; Kita, Kazuko; Suzuki, Nobuo

2003-01-01

Little is known of proteases that play roles in the early steps of X-ray irradiation response. In the present study, we first searched for proteases whose activity is induced in human RSa-R cells after X-ray irradiation. The activity was identified as fibrinolytic, using 125 I-labeled fibrin as a substrate. Protease samples were prepared by lysation of cells with a buffer containing MEGA-8. RSa-R cells showed an increased level of protease activity 10 min after X-ray (up to 3 Gy) irradiation. We next examined whether this protease inducibility is causally related with the X-ray susceptibility of cells. Leupeptin, a serine-cysteine protease inhibitor, inhibited the protease activity in samples obtained from X-ray-irradiated RSa-R cells. Treatment of RSa-R cells with the inhibitor before and after X-ray irradiation resulted in an increased susceptibility of the cells to X-ray cell killing. However, the treatment of cells with other inhibitors tested did not modulate the X-ray susceptibility. These results suggest that leupeptin-sensitive proteases are involved in the resistance of human cells to X-ray cell killing. (author)

Ensemble-based ADME-Tox profiling and virtual screening for the discovery of new inhibitors of the Leishmania mexicana cysteine protease CPB2.8ΔCTE.

Science.gov (United States)

Scala, Angela; Rescifina, Antonio; Micale, Nicola; Piperno, Anna; Schirmeister, Tanja; Maes, Louis; Grassi, Giovanni

2018-02-01

In an effort to identify novel molecular warheads able to inhibit Leishmania mexicana cysteine protease CPB2.8ΔCTE, fused benzo[b]thiophenes and β,β'-triketones emerged as covalent inhibitors binding the active site cysteine residue. Enzymatic screening showed a moderate-to-excellent activity (12%-90% inhibition of the target enzyme at 20 μm). The most promising compounds were selected for further profiling including in vitro cell-based assays and docking studies. Computational data suggest that benzo[b]thiophenes act immediately as non-covalent inhibitors and then as irreversible covalent inhibitors, whereas a reversible covalent mechanism emerged for the 1,3,3'-triketones with a Y-topology. Based on the predicted physicochemical and ADME-Tox properties, compound 2b has been identified as a new drug-like, non-mutagen, non-carcinogen, and non-neurotoxic lead candidate. © 2017 John Wiley & Sons A/S.

Purification and characterization of alkaline proteases from aspergillus terreus

International Nuclear Information System (INIS)

Hussain, A.; Mannan, A.; Zubair, H.; Mirza, B.

2010-01-01

Proteases belong to an important class of enzymes known as hydrolases and catalyze hydrolysis of proteins. They act primarily to degrade proteins that are used for energy production and as biosynthetic precursors. In the following study, protease produced from Aspergillus terreus was found to be thermo stable and included in the category of alkaline serine and metallo protease. During partial purification, presence of enzyme in 60% (NH/sub 4/)/sub 2/SO/sub 4/ indicated small molecular weight polypeptide; later purification with Sephadex G-75 fractionation yielded a single proteolytic active molecule. At final purification step, the increase in specific activity of the enzyme was 7.5 fold with 23% yield. SDS-PAGE analysis revealed that alkaline protease of Aspergillus terreus is a monomer with approximate molecular weight of 35 kDa. Optimum pH for protease activity was found in the range of 7.5-11.0 (maximum at pH 8.5), thus apparently classified as an alkaline protease. The enzyme was thermo stable towards high temperature (60 deg. C), however it denatured irreversibly at 70 deg. C showing 80% loss of activity. The maximum proteolytic activity was found at 40 deg. C. The enzyme was effectively inhibited by PMSF, EDTA and urea whereas iodoacetamide and thiourea did not result in any loss in activity while cysteine was found to be activator molecule. The study with metal ions Mg/sup +2/, Mn/sup +2/ and Fe/sup +3/ (1 mM each) showed minute stimulatory effects on enzyme activity. Co/sup +2/ and Ca/sup +2/ (1 mM) had neither excitatory nor inhibitory effect while Hg/sup +2/ and Cu/sup +2/ (1 mM) slightly reduced the enzyme activity. (author)

The immunological characteristics and probiotic function of recombinant Bacillus subtilis spore expressing Clonorchis sinensis cysteine protease.

Science.gov (United States)

Tang, Zeli; Shang, Mei; Chen, Tingjin; Ren, Pengli; Sun, Hengchang; Qu, Hongling; Lin, Zhipeng; Zhou, Lina; Yu, Jinyun; Jiang, Hongye; Zhou, Xinyi; Li, Xuerong; Huang, Yan; Xu, Jin; Yu, Xinbing

2016-12-19

Clonorchiasis, a food-borne zoonosis, is caused by Clonorchis sinensis. The intestinal tract and bile ducts are crucial places for C. sinensis metacercariae to develop into adult worms. The endospore of Bacillus subtilis is an ideal oral immunization vehicle for delivery of heterologous antigens to intestine. Cysteine protease of C. sinensis (CsCP) is an endogenous key component in the excystment of metacercariae and other physiological or pathological processes. We constructed a fusion gene of CotC (a coat protein)-CsCP and obtained B. subtilis spores with recombinant plasmid of pEB03-CotC-CsCP (B.s-CotC-CsCP). CotC-CsCP expressed on spores' surface was detected by Western blotting and immunofluorescence. Immunological characteristics of recombinant spore coat protein were evaluated in a mouse model. The levels of CsCP-specific antibodies were detected by ELISA. Effects of recombinant spores on mouse intestine were evaluated by histological staining. The activities of biochemical enzymes in serum were assayed by microplate. Liver sections of infected mice were evaluated by Ishak score after Masson's trichrome. The B.s-CotC-CsCP spores displayed CsCP on their coat. Specific IgG and isotypes were significantly induced by coat proteins of B.s-CotC-CsCP spores after subcutaneous immunization. IgA levels in intestinal mucus and bile of B.s-CotC-CsCP orally treated mice significantly increased. Additionally, more IgA-secreting cells were observed in enteraden and lamina propria regions of the mouse jejunum, and an increased amount of acidic mucins in intestines were also observed. There were no significant differences in enzyme levels of serum among groups. No inflammatory injury was observed in the intestinal tissues of each group. The degree of liver fibrosis was significantly reduced after oral immunization with B.s-CotC-CsCP spores. Bacillus subtilis spores maintained the original excellent immunogenicity of CsCP expressed on their surface. Both local and systemic

Density functional theory and quantum mechanics/molecular mechanics study of cysteine protease inhibition by nitrile-based inhibitors.

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Sam P De Visser

2013-12-01

Full Text Available Cysteine protease enzymes are important for human physiology and catalyze key protein degradation pathways. These enzymes react via a nucleophilic reaction mechanism that involves a cysteine residue and the proton of a proximal histidine. Particularly efficient inhibitors of these enzymes are nitrile-based, however, the details of the catalytic reaction mechanism currently are poorly understood. To gain further insight into the inhibition of these molecules, we have performed a combined density functional theory and quantum mechanics/molecular mechanics study on the reaction of a nitrile-based inhibitor with the enzyme active site amino acids. We show here that small perturbations to the inhibitor structure can have dramatic effects on the catalysis and inhibition processes. Thus, we investigated a range of inhibitor templates and show that specific structural changes reduce the inhibitory efficiency by several orders of magnitude. Moreover, as the reaction takes place on a polar surface, we find strong differences between the DFT and QM/MM calculated energetics. In particular, the DFT model led to dramatic distortions from the starting structure and the convergence to a structure that would not fit the enzyme active site. In the subsequent QM/MM study we investigated the use of mechanical versus electronic embedding on the kinetics, thermodynamics and geometries along the reaction mechanism. We find minor effects on the kinetics of the reaction but large geometric and thermodynamics differences as a result of inclusion of electronic embedding corrections. The work here highlights the importance of model choice in the investigation of this biochemical reaction mechanism.

C-terminal domains of bacterial proteases: structure, function and the biotechnological applications.

Science.gov (United States)

Huang, J; Wu, C; Liu, D; Yang, X; Wu, R; Zhang, J; Ma, C; He, H

2017-01-01

C-terminal domains widely exist in the C-terminal region of multidomain proteases. As a β-sandwich domain in multidomain protease, the C-terminal domain plays an important role in proteolysis including regulation of the secretory process, anchoring and swelling the substrate molecule, presenting as an inhibitor for the preprotease and adapting the protein structural flexibility and stability. In this review, the diversity, structural characteristics and biological function of C-terminal protease domains are described. Furthermore, the application prospects of C-terminal domains, including polycystic kidney disease, prepeptidase C-terminal and collagen-binding domain, in the area of medicine and biological artificial materials are also discussed. © 2016 The Society for Applied Microbiology.

Functional analysis of the cathepsin-like cysteine protease genes in adult Brugia malayi using RNA interference.

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Louise Ford

Full Text Available Cathepsin-like enzymes have been identified as potential targets for drug or vaccine development in many parasites, as their functions appear to be essential in a variety of important biological processes within the host, such as molting, cuticle remodeling, embryogenesis, feeding and immune evasion. Functional analysis of Caenorhabditis elegans cathepsin L (Ce-cpl-1 and cathepsin Z (Ce-cpz-1 has established that both genes are required for early embryogenesis, with Ce-cpl-1 having a role in regulating in part the processing of yolk proteins. Ce-cpz-1 also has an important role during molting.RNA interference assays have allowed us to verify whether the functions of the orthologous filarial genes in Brugia malayi adult female worms are similar. Treatment of B. malayi adult female worms with Bm-cpl-1, Bm-cpl-5, which belong to group Ia of the filarial cpl gene family, or Bm-cpz-1 dsRNA resulted in decreased numbers of secreted microfilariae in vitro. In addition, analysis of the intrauterine progeny of the Bm-cpl-5 or Bm-cpl Pro dsRNA- and siRNA-treated worms revealed a clear disruption in the process of embryogenesis resulting in structural abnormalities in embryos and a varied differential development of embryonic stages.Our studies suggest that these filarial cathepsin-like cysteine proteases are likely to be functional orthologs of the C. elegans genes. This functional conservation may thus allow for a more thorough investigation of their distinct functions and their development as potential drug targets.

Primary structure of human pancreatic protease E determined by sequence analysis of the cloned mRNA

International Nuclear Information System (INIS)

Shen, W.; Fletcher, T.S.; Largman, C.

1987-01-01

Although protease E was isolated from human pancreas over 10 years ago, its amino acid sequence and relationship to the elastases have not been established. The authors report the isolation of a cDNA clone for human pancreatic protease E and determination of the nucleic acid sequence coding for the protein. The deduced amino acid sequence contains all of the features common to serine proteases. The substrate binding region is highly homologous to those of porcine and rat elastases 1, explaining the similar specificity for alanine reported for protease E and these elastases. However, the amino acid sequence outside the substrate binding region is less than 50% conserved, and there is a striking difference in the overall net charge for protease E (6-) and elastases 1 (8+). These findings confirm that protease E is a new member of the serine protease family. They have attempted to identify amino acid residues important for the interaction between elastases and elastin by examining the amino acid sequence differences between elastases and protease E. In addition to the large number of surface charge changes which are outside the substrate binding region, there are several changes which might be crucial for elastolysis: Leu-73/Arg-73; Arg-217A/Ala-217A; Arg-65A/Gln-65A; and the presence of two new cysteine residues (Cys-98 and Cys-99B) which computer modeling studies predict could form a new disulfide bond, not previously observed for serine proteases. They also present evidence which suggests that human pancreas does not synthesize a basic, alanine-specific elastase similar to porcine elastase 1

PROTEOLYTIC PROCESSING OF VON WILLEBRAND FACTOR BY ADAMTS13 AND LEUKOCYTE PROTEASES

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Stefano Lancellotti

2013-09-01

Full Text Available ADAMTS13 is a 190 kDa zinc protease encoded by a gene located on chromosome 9q34.   This protease specifically hydrolyzes von Willebrand factor (VWF multimers, thus causing VWF size reduction. ADAMTS13 belongs to the A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS family, involved in proteolytic processing of many matrix proteins. ADAMTS13 consists of numerous domains including a metalloprotease domain, a disintegrin domain, several thrombospondin type 1 (TSP1 repeats, a cysteine-rich domain, a spacer domain and 2 CUB (Complement c1r/c1s, sea Urchin epidermal growth factor, and Bone morphogenetic protein domains. ADAMTS13 cleaves a single peptide bond (Tyr1605-Met1606 in the central A2 domain of the VWF molecule. This proteolytic cleavage is essential to reduce the size of ultra-large VWF polymers, which, when exposed to high shear stress in the microcirculation, are prone to form with platelets clumps, which cause severe syndromes called thrombotic microangiopathies (TMAs. In this review, we a discuss the current knowledge of structure-function aspects of ADAMTS13 and its involvement in the pathogenesis of TMAs, b address the recent findings concerning proteolytic processing of VWF multimers by different proteases, such as the leukocyte-derived serine and metallo-proteases and c indicate the direction of future investigations

Comparative analysis of procoagulant and fibrinogenolytic activity of crude protease fractions of turmeric species.

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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

Delay of Iris flower senescence by protease inhibitors

NARCIS (Netherlands)

Pak, C.; Doorn, van W.G.

2005-01-01

asterisk inside a circle sign Visible senescence of the flag tepals in Iris x hollandica (cv. Blue Magic) was preceded by a large increase in endoprotease activity. Just before visible senescence about half of total endoprotease activity was apparently due to cysteine proteases, somewhat less than

Interaction of protein C inhibitor with the type II transmembrane serine protease enteropeptidase.

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Thomas A Prohaska

Full Text Available The serine protease inhibitor protein C inhibitor (PCI is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48 × 10(4 M(-1 s(-1. Low molecular weight (LMWH and unfractionated heparin (UFH slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml. By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.

Characterization and milk coagulating properties of Cynanchum otophyllum Schneid. proteases.

Science.gov (United States)

Luo, Jie; Xiao, Chen; Zhang, Hao; Ren, Fazheng; Lei, Xingen; Yang, Zibiao; Yu, Zhengquan

2018-04-01

The herbaceous plant Cynanchum otophyllum Schneid. is widely used as a milk coagulant to make a Chinese traditional milk product, milk cake. However, the milk-clotting compounds and their mechanism remain unclear. In this study, crude proteases were extracted from the dried leaves of Cynanchum otophyllum Schneid. using citric acid-phosphate buffer and then partially purified by weak anion exchange chromatography. Two proteases, QA and QC, with molecular weights of 14 and 27 kDa, respectively, were shown to exhibit milk-clotting activity. A study of the effects of pH and temperature on the milk-clotting activity and proteolytic activity of the proteases showed that they exhibited good pH stability from pH 5.5 to 7.5 and good thermal stability at temperatures from 50 to 70°C. The QA and QC were the cysteine proteases, able to hydrolyze β-casein and κ-casein completely, and α-casein partially. The cleavage site on κ-casein determined by Orbitrap (Thermo Fisher Scientific, San Jose, CA) analysis showed that QA and QC could cleave κ-casein at Ser132-Thr133. Overall, the results suggest that the Cynanchum otophyllum Schneid. proteases are a promising milk-clotting enzyme that could be used for manufacturing milk cake and cheese. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Inhibitors of cysteine cathepsin and calpain do not prevent ultraviolet-B-induced apoptosis in human keratinocytes and HeLa cells

DEFF Research Database (Denmark)

Bang, Bo; Baadsgaard, Ole; Skov, Lone

2004-01-01

been demonstrated to play a role in the execution of programmed cell death induced by other stimuli, e.g. TNF-alpha. The purpose of the present study was therefore to investigate whether inhibitors of cysteine cathepsins and calpains could prevent UVB-induced apoptosis in HeLa cells and keratinocytes....... This was done by investigating the effect of the irreversible cysteine protease inhibitor zFA-fmk, the cathepsin B inhibitor CA-074-Me and the calpain inhibitor ALLN on the viability of UVB-irradiated human keratinocytes and HeLa cells. At concentrations of 10 microM and above zVAD-fmk conferred partial dose......-dependent protection against UVB-induced apoptosis in HeLa cells and keratinocytes. Moreover, caspase-3 activity was completely blocked at zVAD-fmk concentrations of 1 microM in HeLa cells. This indicates that caspase-independent mechanisms could be involved in UVB-induced apoptosis. However, the protease inhibitors z...

A C-terminal, cysteine-rich site in poliovirus 2C(ATPase) is required for morphogenesis.

Science.gov (United States)

Wang, Chunling; Ma, Hsin-Chieh; Wimmer, Eckard; Jiang, Ping; Paul, Aniko V

2014-06-01

The morphogenesis of viruses belonging to the genus Enterovirus in the family Picornaviridae is still poorly understood despite decades-long investigations. However, we recently provided evidence that 2C(ATPase) gives specificity to poliovirus encapsidation through an interaction with capsid protein VP3. The polypeptide 2C(ATPase) is a highly conserved non-structural protein of enteroviruses with important roles in RNA replication, encapsidation and uncoating. We have identified a site (K279/R280) near the C terminus of the polypeptide that is required for morphogenesis. The aim of the current project was to search for additional functional sites near the C terminus of the 2C(ATPase) polypeptide, with particular interest in those that are required for encapsidation. We selected for analysis a cysteine-rich site of the polypeptide and constructed four mutants in which cysteines or a histidine was changed to an alanine. The RNA transcripts were transfected into HeLa cells yielding two lethal, one temperature-sensitive and one quasi-infectious mutants. All four mutants exhibited normal protein translation in vitro and three of them possessed severe RNA replication defects. The quasi-infectious mutant (C286A) yielded variants with a pseudo-reversion at the original site (A286D), but some also contained one additional mutation: A138V or M293V. The temperature-sensitive mutant (C272A/H273A) exhibited an encapsidation and possibly also an uncoating defect at 37 °C. Variants of this mutant revealed suppressor mutations at three different sites in the 2C(ATPase) polypeptide: A138V, M293V and K295R. We concluded that the cysteine-rich site near the C terminus of 2C(ATPase) is involved in encapsidation, possibly through an interaction with an upstream segment located between boxes A and B of the nucleotide-binding domain. © 2014 The Authors.

Comparative analysis of immune effects in mice model: Clonorchis sinensis cysteine protease generated from recombinant Escherichia coli and Bacillus subtilis spores.

Science.gov (United States)

Wu, Zhanshuai; Tang, Zeli; Shang, Mei; Zhao, Lu; Zhou, Lina; Kong, Xiangzhan; Lin, Zhipeng; Sun, Hengchang; Chen, Tingjin; Xu, Jin; Li, Xuerong; Huang, Yan; Yu, Xinbing

2017-07-01

Clonorchiasis remains a nonnegligible public health problem in endemic areas. Cysteine protease of Clonorchis sinensis (CsCP) plays indispensable roles in the parasitic physiology and pathology, and has been exploited as a promising drug and vaccine candidate. In recent years, development of spore-based vaccines against multiple pathogens has attracted many investigators' interest. In previous studies, the recombinant Escherichia coli (BL21) and Bacillus subtilis spores expressing CsCP have been successfully constructed, respectively. In this study, the immune effects of CsCP protein purified from recombinant BL21 (rCsCP) and B. subtilis spores presenting CsCP (B.s-CsCP) in Balb/c mice model were conducted with comparative analysis. Levels of specific IgG, IgG1 and IgG2a were significantly increased in sera from both rCsCP and B.s-CsCP intraperitoneally immunized mice. Additionally, recombinant spores expressing abundant fusion CsCP (0.03125 pg/spore) could strongly enhance the immunogenicity of CsCP with significantly higher levels of IgG and isotypes. Compared with rCsCP alone, intraperitoneal administration of mice with spores expressing CsCP achieved a better effect of fighting against C. sinensis infection by slowing down the process of fibrosis. Our results demonstrated that a combination of Th1/Th2 immune responses could be elicited by rCsCP, while spores displaying CsCP prominently induced Th1-biased specific immune responses, and the complex cytokine network maybe mediates protective immune responses against C. sinensis. This work further confirmed that the usage of B. subtilis spores displaying CsCP is an effective way to against C. sinensis.

Cysteine protease 30 (CP30) contributes to adhesion and cytopathogenicity in feline Tritrichomonas foetus.

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Gould, Emily N; Giannone, Richard; Kania, Stephen A; Tolbert, M Katherine

2017-09-15

Tritrichomonas foetus (T. foetus) is a flagellated protozoan parasite that is recognized as a significant cause of diarrhea in domestic cats with a prevalence rate as high as 30%. No drugs have been shown to consistently eliminate T. foetus infection in all cats. Cysteine proteases (CPs) have been identified as mediators of T. foetus-induced adhesion-dependent cytotoxicity to the intestinal epithelium. These CPs represent novel targets for the treatment of feline trichomonosis. However, cats also produce CPs that are part of life-critical systems. Thus, parasitic CPs need to be selectively targeted to reduce the potential for host toxicity. Previous studies have demonstrated the importance of a specific CP, CP30, in mediating bovine and human trichomonad cytopathogenicity. This CP has also recently been identified in feline T. foetus, although the function of this protease in the feline genotype remains unknown. Therefore, the study objectives were to characterize the presence of CP30 in feline T. foetus isolates and to evaluate the effect of targeted inhibition of CP30 on feline T. foetus-induced adhesion dependent cytotoxicity. The presence of CP30 in feline T. foetus isolates was identified by In gel zymography and proteomic analysis, indirect immunofluorescence (IF), and flow cytometry using a rabbit polyclonal antibody that targets bovine T. foetus CP30 (α-CP30). The effect of inhibition of CP30 activity on T. foetus adhesion and cytotoxicity was determined using CFSE-labeled feline T. foetus and crystal violet spectrophotometric assays in a previously validated co-culture model. CP30 expression was confirmed in all feline T. foetus isolates tested by all assays. Targeted inhibition of feline T. foetus CP30 resulted in decreased T. foetus adhesion to and cytotoxicity towards IPEC-J2 monolayers compared to rabbit IgG-treated T. foetus isolates. These studies establish that CP30 is expressed by feline T. foetus isolates and may be an important virulence factor

Modulation of the epithelial sodium channel (ENaC by bacterial metalloproteases and protease inhibitors.

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Michael B Butterworth

Full Text Available The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC, leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions.

Molecular cloning of the large subunit of the high-Ca2+-requiring form of human Ca2+-activated neutral protease

International Nuclear Information System (INIS)

Imajoh, Shinobu; Aoki, Kazumasa; Ohno, Shigeo; Emori, Yasufumi; Kawasaki, Hiroshi; Sugihara, Hidemitsu; Suzuki, Koichi

1988-01-01

A nearly full-length cDNA clone for the large subunit of high-Ca 2+ -requiring Ca 2+ -activated neutral protease (mCANP) from human tissues has been isolated. The deduced protein, determined for the first time as an mCANP, has essentially the same structural features as those revealed previously for the large subunits of the low-Ca 2+ -requiring form (μCANP). Namely, the protein, comprising 700 amino acid residues, is characterized by four domains, containing a cysteine protease like domain and a Ca 2+ -binding domain. The overall amino acid sequence similarities of the mCANP large subunit with those of human μCANP and chicken CANP are 62% and 66%, respectively. These values are slightly lower than that observed between μCANP and chicken CANP (70%). Local sequence similarities vary with the domain, 73-78% in the cysteine protease like domain and 48-65% in the Ca 2+ -binding domain. These results suggest that CANPs with different Ca 2+ sensitivities share a common evolutionary origin and that their regulatory mechanisms are similar except for the Ca 2+ concentrations required for activation

Complete cDNA sequence of human complement C1s and close physical linkage of the homologous genes C1s and C1r

International Nuclear Information System (INIS)

Tosi, M.; Duponchel, C.; Meo, T.; Julier, C.

1987-01-01

Overlapping molecular clones encoding the complement subcomponent C1s were isolated from a human liver cDNA library. The nucleotide sequence reconstructed from these clones spans about 85% of the length of the liver C1s messenger RNAs, which occur in three distinct size classes around 3 kilobases in length. Comparisons with the sequence of C1r, the other enzymatic subcomponent of C1, reveal 40% amino acid identity and conservation of all the cysteine residues. Beside the serine protease domain, the following sequence motifs, previously described in C1r, were also found in C1s: (a) two repeats of the type found in the Ba fragment of complement factor B and in several other complement but also noncomplement proteins, (b) a cysteine-rich segment homologous to the repeats of epidermal growth factor precursor, and (c) a duplicated segment found only in C1r and C1s. Differences in each of these structural motifs provide significant clues for the interpretation of the functional divergence of these interacting serine protease zymogens. Hybridizations of C1r and C1s probes to restriction endonuclease fragments of genomic DNA demonstrate close physical linkage of the corresponding genes. The implications of this finding are discussed with respect to the evolution of C1r and C1s after their origin by tandem gene duplication and to the previously observed combined hereditary deficiencies of Clr and Cls

SwissProt search result: AK110731 [KOME

Lifescience Database Archive (English)

Full Text Available AK110731 002-170-E10 (Q8C9S8) Cysteine protease APG4A (EC 3.4.22.-) (Autophagy 4 homolog A) (Auto...phagin-2) (Autophagy-related cysteine endopeptidase 2) (AUT-like 2 cysteine endopeptidase) APG4A_MOUSE 2e-36 ...

Producing armyworm (spodoptera sp.) Bioinsecticide based on cysteine protease of red ginger (zingiber officinale var. Rubrum)

Science.gov (United States)

Afnan, N. T.; Nur, D. F.; Utami, T. S.; Sahlan, M.; Wijanarko, A.; Hermansyah, H.

2018-03-01

Armyworm (Spodoptera sp.) is highly polyphagous defoliator on various horticulture and grain plants. Various chemical insecticides have been created to control it. There is a need to create an eco-friendly and specific insecticide which only affect armyworm’s nervous system. This research investigates cysteine-protease’s enzyme activity of red ginger (Zingiber officinale var. Rubrum) which is called zingibain. Its catalytic site matches with residue site in armyworm’s body so it can be used as bioinsecticide raw material which meets the criterias above. Fresh red ginger rhizomes were washed and extracted. The juice was then deposited in low temperature and centrifuged to get rid of its starch content. It was filtrated to remove large contaminants and poured into Potassium Phospate buffer. The liquid was then centrifuged again for 30 minutes before collecting the supernatant. Fresh leaves were then dipped into crude ginger protease extract and fed to fourth instar-armyworms. Leaves dipped into non-diluted extract were barely eaten by armyworm while the 50% and 25% dilution was half eaten and most eaten. The crude red ginger extract was not strong enough to kill them although the research showed its enzymatic activity reaches up to 169 PU. It still needs improvement to be produced as commercial bioinsecticide.

SjAPI, the first functionally characterized Ascaris-type protease inhibitor from animal venoms.

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Zongyun Chen

Full Text Available BACKGROUND: Serine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear. PRINCIPAL FINDINGS: Here, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin family, which contains four members: Scorpiops jendeki Ascaris-type protease inhibitor (SjAPI, Scorpiops jendeki Ascaris-type protease inhibitor 2 (SjAPI-2, Chaerilus tricostatus Ascaris-type protease inhibitor (CtAPI, and Buthus martensii Ascaris-type protease inhibitor (BmAPI. The detailed characterization of Ascaris-type peptide SjAPI from the venom gland of scorpion Scorpiops jendeki was carried out. The mature peptide of SjAPI contains 64 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges, different from all known protease inhibitors from venomous animals. Enzyme and inhibitor reaction kinetics experiments showed that recombinant SjAPI was a dual function peptide with α-chymotrypsin- and elastase-inhibiting properties. Recombinant SjAPI inhibited α-chymotrypsin with a Ki of 97.1 nM and elastase with a Ki of 3.7 μM, respectively. Bioinformatics analyses and chimera experiments indicated that SjAPI contained the unique short side chain functional residues "AAV" and might be a useful template to produce new serine protease inhibitors. CONCLUSIONS/SIGNIFICANCE: To our knowledge, SjAPI is the first functionally characterized animal toxin peptide with an Ascaris-type fold. The structural and functional diversity of animal toxins with protease-inhibiting properties suggested that bioactive peptides from animal venom glands might be a new source of protease inhibitors, which will accelerate the

House dust mite major allergens Der p 1 and Der p 5 activate human airway-derived epithelial cells by protease-dependent and protease-independent mechanisms

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Timmerman J André B

2006-03-01

Full Text Available Abstract House dust mite allergens (HDM cause bronchoconstriction in asthma patients and induce an inflammatory response in the lungs due to the release of cytokines, chemokines and additional mediators. The mechanism how HDM components achieve this is largely unknown. The objective of this study was to assess whether HDM components of Dermatophagoides pteronissinus with protease activity (Der p 1 and unknown enzymatic activity (Der p 2, Der p 5 induce biological responses in a human airway-derived epithelial cell line (A549, and if so, to elucidate the underlying mechanism(s of action. A549 cells were incubated with HDM extract, Der p 1, recombinant Der p 2 and recombinant Der p 5. Cell desquamation was assessed by microscopy. The proinflammatory cytokines, IL-6 and IL-8, were measured by ELISA. Intracellular Ca2+ levels were assessed in A549 cells and in mouse fibroblasts expressing the human protease activated receptor (PAR1, PAR2 or PAR4. HDM extract, Der p 1 and Der p 5 dose-dependently increased the production of IL-6 and IL-8. Added simultaneously, Der p 1 and Der p 5 further increased the production of IL-6 and IL-8. The action of Der p 1 was blocked by cysteine-protease inhibitors, while that of Der p 5 couldn't be blocked by either serine- or cysteine protease inhibitors. Der p 5 only induced cell shrinking, whereas HDM extract and Der p1 also induced cell desquamation. Der p 2 had no effect on A549 cells. Der p 1's protease activity causes desquamation and induced the release of IL6 and IL-8 by a mechanism independent of Ca2+ mobilisation and PAR activation. Der p 5 exerts a protease-independent activation of A549 that involves Ca2+ mobilisation and also leads to the production of these cytokines. Together, our data indicate that allergens present in HDM extracts can trigger protease-dependent and protease-independent signalling pathways in A549 cells.

Hepatitis C virus NS3/4A protease inhibits complement activation by cleaving complement component 4.

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Seiichi Mawatari

Full Text Available BACKGROUND: It has been hypothesized that persistent hepatitis C virus (HCV infection is mediated in part by viral proteins that abrogate the host immune response, including the complement system, but the precise mechanisms are not well understood. We investigated whether HCV proteins are involved in the fragmentation of complement component 4 (C4, composed of subunits C4α, C4β, and C4γ, and the role of HCV proteins in complement activation. METHODS: Human C4 was incubated with HCV nonstructural (NS 3/4A protease, core, or NS5. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then subjected to peptide sequencing. The activity of the classical complement pathway was examined using an erythrocyte hemolysis assay. The cleavage pattern of C4 in NS3/4A-expressing and HCV-infected cells, respectively, was also examined. RESULTS: HCV NS3/4A protease cleaved C4γ in a concentration-dependent manner, but viral core and NS5 did not. A specific inhibitor of NS3/4A protease reduced C4γ cleavage. NS3/4A protease-mediated cleavage of C4 inhibited classical pathway activation, which was abrogated by a NS3/4A protease inhibitor. In addition, co-transfection of cells with C4 and wild-type NS3/4A, but not a catalytic-site mutant of NS3/4A, produced cleaved C4γ fragments. Such C4 processing, with a concomitant reduction in levels of full-length C4γ, was also observed in HCV-infected cells expressing C4. CONCLUSIONS: C4 is a novel cellular substrate of the HCV NS3/4A protease. Understanding disturbances in the complement system mediated by NS3/4A protease may provide new insights into the mechanisms underlying persistent HCV infection.

Isolation, cDNA cloning, and structure-based functional characterization of oryctin, a hemolymph protein from the coconut rhinoceros beetle, Oryctes rhinoceros, as a novel serine protease inhibitor.

Science.gov (United States)

Horita, Shoichiro; Ishibashi, Jun; Nagata, Koji; Miyakawa, Takuya; Yamakawa, Minoru; Tanokura, Masaru

2010-09-24

We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant (13)C,(15)N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with K(i) values of 3.9 × 10(-10) m, 6.2 × 10(-10) m, 1.4 × 10(-9) m, and 1.2 × 10(-8) m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections.

Microenvironmental Regulation of Mammary Carcinogenesis

Science.gov (United States)

2009-06-01

F. et al., A selective activity-based probe for the papain family cysteine protease dipeptidyl peptidase I/cathepsin C. J Am Chem Soc 128 (17...based probe for the papain family cysteine protease dipeptidyl peptidase I/Cathepsin C. J Am Chem Society, 128: 5616- 5617. 39. Tan TT, Coussens LM...Bogyo, M. A selective activity-based probe for the papain family cysteine protease dipeptidyl peptidase I/cathepsin C. J Am Chem Soc 128, 5616-7

Cleavage of desmin by cysteine proteases: Calpains and cathepsin B

DEFF Research Database (Denmark)

Baron, Caroline; Jacobsen, S.; Purslow, P.P.

2004-01-01

The intermediate filament protein, desmin, was purified from pork longissimus dorsi and incubated with either P-calpain, m-calpain or cathepsin B. Proteolysis of desmin was followed using SDS-PAGE and Western blotting. After incubation of desmin with the proteases, cleavage sites on the desmin mo...

The protease degrading sperm histones post-fertilization in sea urchin eggs is a nuclear cathepsin L that is further required for embryo development.

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Violeta Morin

Full Text Available Proteolysis of sperm histones in the sea urchin male pronucleus is the consequence of the activation at fertilization of a maternal cysteine protease. We previously showed that this protein is required for male chromatin remodelling and for cell-cycle progression in the newly formed embryos. This enzyme is present in the nucleus of unfertilized eggs and is rapidly recruited to the male pronucleus after insemination. Interestingly, this cysteine-protease remains co-localized with chromatin during S phase of the first cell cycle, migrates to the mitotic spindle in M-phase and is re-located to the nuclei of daughter cells after cytokinesis. Here we identified the protease encoding cDNA and found a high sequence identity to cathepsin proteases of various organisms. A phylogenetical analysis clearly demonstrates that this sperm histone protease (SpHp belongs to the cathepsin L sub-type. After an initial phase of ubiquitous expression throughout cleavage stages, SpHp gene transcripts become restricted to endomesodermic territories during the blastula stage. The transcripts are localized in the invaginating endoderm during gastrulation and a gut specific pattern continues through the prism and early pluteus stages. In addition, a concomitant expression of SpHp transcripts is detected in cells of the skeletogenic lineage and in accordance a pharmacological disruption of SpHp activity prevents growth of skeletal rods. These results further document the role of this nuclear cathepsin L during development.

Expression and Characterization of Coprothermobacter proteolyticus Alkaline Serine Protease

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Tanveer Majeed

2013-01-01

Full Text Available A putative protease gene (aprE from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated that the enzyme had optimal activity under alkaline conditions (pH 8–10. In addition, the enzyme had an elevated optimum temperature (60°C. The protease was also stable in the presence of many surfactants and oxidant. Thus, the C. proteolyticus protease has potential applications in industries such as the detergent market.

Urinary serine proteases and activation of ENaC in kidney

DEFF Research Database (Denmark)

Svenningsen, Per; Andersen, Henrik; Nielsen, Lise Hald

2015-01-01

with albuminuria compatible with impaired renal Na(+) excretion: hypertension and volume retention is secondary to proteinuria in, e.g., preeclampsia and nephrotic syndrome; plasma concentrations of renin, angiotensin II, and aldosterone are frequently suppressed in proteinuric conditions, e.g., preeclampsia......Serine proteases, both soluble and cell-attached, can activate the epithelial sodium channel (ENaC) proteolytically through release of a putative 43-mer inhibitory tract from the ectodomain of the γ-subunit. ENaC controls renal Na(+) excretion and loss-of-function mutations lead to low blood...... pressure, while gain-of-function mutations lead to impaired Na(+) excretion, hypertension, and hypokalemia. We review an emerging pathophysiological concept that aberrant glomerular filtration of plasma proteases, e.g., plasmin, prostasin, and kallikrein, contributes to proteolytic activation of ENaC, both...

Evolutionary dynamics of hepatitis C virus NS3 protease domain during and following treatment with narlaprevir, a potent NS3 protease inhibitor

NARCIS (Netherlands)

de Bruijne, J.; Thomas, X. V.; Rebers, S. P.; Weegink, C. J.; Treitel, M. A.; Hughes, E.; Bergmann, J. F.; de Knegt, R. J.; Janssen, H. L. A.; Reesink, H. W.; Molenkamp, R.; Schinkel, J.

2013-01-01

Narlaprevir, a hepatitis C virus (HCV) NS3/4A serine protease inhibitor, has demonstrated robust antiviral activity in a placebo-controlled phase 1 study. To study evolutionary dynamics of resistant variants, the NS3 protease sequence was clonally analysed in thirty-two HCV genotype 1-infected

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

Science.gov (United States)

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

Cellular processing of the amyloidogenic cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type

DEFF Research Database (Denmark)

Benedikz, Eirikur; Merz, G S; Schwenk, V

1999-01-01

of an amyloidogenic mutation on the intracellular processing of its protein product. The protein, a mutant of the cysteine protease inhibitor cystatin C, is the amyloid precursor protein in Hereditary Cerebral Hemorrhage with Amyloidosis--Icelandic type (HCHWA-I). The amyloid fibers are composed of mutant cystatin C...... (L68Q) that lacks the first 10 amino acids. We have previously shown that processing of wild-type cystatin C entails formation of a transient intracellular dimer that dissociates prior to secretion, such that extracellular cystatin C is monomeric. We report here that the cystatin C mutation engenders...

Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents.

Science.gov (United States)

Yildirim, Vildan; Baltaci, Mustafa Ozkan; Ozgencli, Ilknur; Sisecioglu, Melda; Adiguzel, Ahmet; Adiguzel, Gulsah

2017-12-01

An extracellular thermostable alkaline serine protease enzyme from Aeribacillus pallidus C10 (GenBank No: KC333049), was purified 4.85 and 17. 32-fold with a yield of 26.9 and 19.56%, respectively, through DE52 anion exchange and Probond affinity chromatography. The molecular mass of the enzyme was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with approximately 38.35 kDa. The enzyme exhibited optimum activity at pH 9 and at temperature 60 °C. It was determined that the enzyme had remained stable at the range of pH 7.0-10.0, and that it had preserved more than 80% of its activity at a broad temperature range (20-80 °C). The enzyme activity was found to retain more than 70% and 55% in the presence of organic solvents and commercial detergents, respectively. In addition, it was observed that the enzyme activity had increased in the presence of 5% SDS. K M and V max values were calculated as 0.197 mg/mL and 7.29 μmol.mL - 1 .min - 1 , respectively.

Isolation, cDNA Cloning, and Structure-based Functional Characterization of Oryctin, a Hemolymph Protein from the Coconut Rhinoceros Beetle, Oryctes rhinoceros, as a Novel Serine Protease Inhibitor*

Science.gov (United States)

Horita, Shoichiro; Ishibashi, Jun; Nagata, Koji; Miyakawa, Takuya; Yamakawa, Minoru; Tanokura, Masaru

2010-01-01

We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant 13C,15N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with Ki values of 3.9 × 10−10 m, 6.2 × 10−10 m, 1.4 × 10−9 m, and 1.2 × 10−8 m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections. PMID:20630859

Cystatin C deficiency suppresses tumor growth in a breast cancer model through decreased proliferation of tumor cells.

Science.gov (United States)

Završnik, Janja; Butinar, Miha; Prebanda, Mojca Trstenjak; Krajnc, Aleksander; Vidmar, Robert; Fonović, Marko; Grubb, Anders; Turk, Vito; Turk, Boris; Vasiljeva, Olga

2017-09-26

Cysteine cathepsins are proteases that, in addition to their important physiological functions, have been associated with multiple pathologies, including cancer. Cystatin C (CstC) is a major endogenous inhibitor that regulates the extracellular activity of cysteine cathepsins. We investigated the role of cystatin C in mammary cancer using CstC knockout mice and a mouse model of breast cancer induced by expression of the polyoma middle T oncoprotein (PyMT) in the mammary epithelium. We showed that the ablation of CstC reduced the rate of mammary tumor growth. Notably, a decrease in the proliferation of CstC knockout PyMT tumor cells was demonstrated ex vivo and in vitro , indicating a role for this protease inhibitor in signaling pathways that control cell proliferation. An increase in phosphorylated p-38 was observed in CstC knockout tumors, suggesting a novel function for cystatin C in cancer development, independent of the TGF-β pathway. Moreover, proteomic analysis of the CstC wild-type and knockout PyMT primary cell secretomes revealed a decrease in the levels of 14-3-3 proteins in the secretome of knock-out cells, suggesting a novel link between cysteine cathepsins, cystatin C and 14-3-3 proteins in tumorigenesis, calling for further investigations.

Genome-wide comparative analysis of papain-like cysteine protease family genes in castor bean and physic nut.

Science.gov (United States)

Zou, Zhi; Huang, Qixing; Xie, Guishui; Yang, Lifu

2018-01-10

Papain-like cysteine proteases (PLCPs) are a class of proteolytic enzymes involved in many plant processes. Compared with the extensive research in Arabidopsis thaliana, little is known in castor bean (Ricinus communis) and physic nut (Jatropha curcas), two Euphorbiaceous plants without any recent whole-genome duplication. In this study, a total of 26 or 23 PLCP genes were identified from the genomes of castor bean and physic nut respectively, which can be divided into nine subfamilies based on the phylogenetic analysis: RD21, CEP, XCP, XBCP3, THI, SAG12, RD19, ALP and CTB. Although most of them harbor orthologs in Arabidopsis, several members in subfamilies RD21, CEP, XBCP3 and SAG12 form new groups or subgroups as observed in other species, suggesting specific gene loss occurred in Arabidopsis. Recent gene duplicates were also identified in these two species, but they are limited to the SAG12 subfamily and were all derived from local duplication. Expression profiling revealed diverse patterns of different family members over various tissues. Furthermore, the evolution characteristics of PLCP genes were also compared and discussed. Our findings provide a useful reference to characterize PLCP genes and investigate the family evolution in Euphorbiaceae and species beyond.

Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea.

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Nicolas Faller

Full Text Available Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functional interactions between HAI-2 and candidate membrane-bound serine proteases expressed in the gastro-intestinal tract. We found that the wild-type form of HAI-2 is a potent inhibitor of nine gastro-intestinal serine proteases. The Y163C mutation in the second Kunitz domain of HAI-2 resulted in a complete loss of inhibitory activity on two intestinal proteases, prostasin and tmprss13. The effect of the mutation of the homologous Y68C in the first Kunitz domain of HAI-2 is consistent with a differential contribution of the two Kunitz domains of HAI-2 in the inhibition of serine proteases. By contrast to the Tyr to Cys, the Tyr to Ser substitution did not change the inhibitory potency of HAI-2, indicating that the thiol-group of the cysteine rather than the Tyr deletion is responsible for the HAI-2 loss of function. Our functional assay allowed us to identify membrane-bound serine proteases as cellular target for inhibition by HAI-2 wild type and mutants, and to better define the role of the Tyr in the second Kunitz domain in the inhibitory activity of HAI-2.

Negative effect of combined cysteine and glutathione in soy lecithin-based extender on post-thawed ram spermatozoa.

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Zhandi, Mahdi; Sharafi, Mohsen

2015-09-01

This study was conducted to evaluate the effect of combined cysteine and glutathione in soy lecithin-based semen extender on post-thawed ram sperm quality. A total of 28 ejaculates were collected twice a week (from four rams) during breeding season. In each replicate, semen samples (n = 4, one ejaculate for each ram) were pooled and divided into three equal parts, and each part was diluted with one of following extender: (1) soy lecithin-based extender containing no cysteine and no glutathione (C0-G0), (2) soy lecithin-based extender containing cysteine (5 mM) and glutathione (5 mM) (C5-G5), and (3) soy lecithin-based extender containing cysteine (10 mM) and glutathione (10 mM) (C10-G10). After freeze-thawing process, motility and velocity parameters, plasma membrane integrity and functionality, mitochondrial activity, and apoptosis features of spermatozoa were evaluated. The obtained results showed that total and progressive motility, plasma membrane integrity and functionality, and live post-thawed spermatozoa was lower in C10-G10 extender compared to C0-G0 and C5-G5 extenders (P 0.05). In conclusion, it seems that high concentration of combined cysteine and glutathione in soy lecithin-based semen extender has a detrimental effect of post-thawed ram sperm quality.

Effect of poloxamer 407 administration on the serum lipids profile, anxiety level and protease activity in the heart and liver of mice

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Johnston, Thomas P.; Dubrovina, Nina I.; Kisarova, Yana A.; Zhanaeva, Svetlana Ya.; Cherkanova, Marina S.; Filjushina, Elena E.; Alexeenko, Tatyana V.; Machova, Eva; Zhukova, Natalya A.

2013-01-01

Chronic administration of the poloxamer 407 (P-407), a block copolymer, to elevate serum lipids in mice is a well-established mouse model of hyperlipidemia and atherosclerosis. We tested the hypothesis that the activity of several types of proteases in heart and liver tissue is changed in the early stages of atherosclerosis development. Additionally, we evaluated whether increased serum lipids would induce anxiety in mice, as determined by using a ‘plus-maze’ test. The mice were administered P-407 by intraperitoneal injection twice a week for one month. P-407 administration to mice resulted in a marked increase in total serum cholesterol, atherogenic non-HDL-cholesterol, and especially in total triglycerides, and it also increased anxiety. Morphological changes observed in P-407-treated mice included contractile type changes in cardiomyocytes and foamy macrophages in liver. A significant increase of cysteine proteases cathepsin B and cathepsin L (at 24 h) and aspartate protease cathepsin D (at both 24 h and 5 days) was determined in heart tissue following P-407 administration. However, no changes were noted in heart matrix metalloproteinase activity. The activity of cysteine and aspartate proteases was significantly increased in liver at both 24 hours and 5 days after P-407 administration. In conclusion, administration of P-407 to mice for one month resulted in increased anxiety, and more importantly, there was an increase in the activity of heart and liver proteases secondary to sustained dyslipidemia. It is suggested that heart and liver cysteine and aspartate proteases may represent potential therapeutic targets in the early stages of atherosclerosis. PMID:24170975

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

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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.

Characterization of detergent compatible protease from halophilic Virgibacillus sp. CD6.

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Lam, Ming Quan; Nik Mut, Nik Nurhidayu; Thevarajoo, Suganthi; Chen, Sye Jinn; Selvaratnam, Chitra; Hussin, Huszalina; Jamaluddin, Haryati; Chong, Chun Shiong

2018-02-01

A halophilic bacterium, Virgibacillus sp. strain CD6, was isolated from salted fish and its extracellular protease was characterized. Protease production was found to be highest when yeast extract was used as nitrogen source for growth. The protease exhibited stability at wide range of salt concentration (0-12.5%, w/v), temperatures (20-60 °C), and pH (4-10) with maximum activity at 10.0% (w/v) NaCl, 60 °C, pH 7 and 10, indicating its polyextremophilicity. The protease activity was enhanced in the presence of Mg 2+ , Mn 2+ , Cd 2+ , and Al 3+ (107-122% relative activity), and with retention of activity > 80% for all of other metal ions examined (K + , Ca 2+ , Cu 2+ , Co 2+ , Ni 2+ , Zn 2+ , and Fe 3+ ). Both PMSF and EDTA inhibited protease activity, denoting serine protease and metalloprotease properties, respectively. High stability (> 70%) was demonstrated in the presence of organic solvents and detergent constituents, and the extracellular protease from strain CD6 was also found to be compatible in commercial detergents. Proteinaceous stain removal efficacy revealed that crude protease of strain CD6 could significantly enhance the performance of commercial detergent. The protease from Virgibacillus sp. strain CD6 could serve as a promising alternative for various applications, especially in detergent industry.

Molecular cloning of a cysteine proteinase cDNA from the cotton boll weevil Anthonomus grandis (Coleoptera: Curculionidae).

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De Oliveira Neto, Osmundo Brilhante; Batista, João Aguiar Nogueira; Rigden, Daniel John; Franco, Octávio Luiz; Fragoso, Rodrigo Rocha; Monteiro, Ana Carolina Santos; Monnerat, Rose Gomes; Grossi-De-Sa, Maria Fátima

2004-06-01

The cotton boll weevil (Anthonomus grandis) causes severe cotton crop losses in North and South America. This report describes the presence of cysteine proteinase activity in the cotton boll weevil. Cysteine proteinase inhibitors from different sources were assayed against total A. grandis proteinases but, unexpectedly, no inhibitor tested was particularly effective. In order to screen for active inhibitors against the boll weevil, a cysteine proteinase cDNA (Agcys1) was isolated from A. grandis larvae using degenerate primers and rapid amplification of cDNA ends (RACE) techniques. Sequence analysis showed significant homologies with other insect cysteine proteinases. Northern blot analysis indicated that the mRNA encoding the proteinase was transcribed mainly in the gut of larvae. No mRNA was detected in neonatal larvae, pupae, or in the gut of the adult insect, suggesting that Agcys1 is an important cysteine proteinase for larvae digestion. The isolated gene will facilitate the search for highly active inhibitors towards boll weevil larvae that may provide a new opportunity to control this important insect pest.

Degradation of the encephalomyocarditis virus and hepatitis A virus 3C proteases by the ubiquitin/26S proteasome system in vivo

International Nuclear Information System (INIS)

Schlax, Peter E.; Zhang Jin; Lewis, Elizabeth; Planchart, Antonio; Lawson, T. Glen

2007-01-01

We have isolated stably transfected mouse embryonic fibroblast cell lines that inducibly express either the mature encephalomyocarditis virus (EMCV) or hepatitis A virus (HAV) 3C protease and have used these cells to demonstrate that both proteins are subject to degradation in vivo by the ubiquitin/26S proteasome system. The detection of 3C protease expression in these cells requires inducing conditions and the presence of one of several proteasome inhibitors. Both 3C proteases are incorporated into conjugates with ubiquitin in vivo. HAV 3C protease expression has deleterious effects on cell viability, as determined by observation and counting of cells cultured in the absence or presence of inducing conditions. The EMCV 3C protease was found to be preferentially localized to the nucleus of induced cells, while the HAV 3C protease remains in the cytoplasm. The absence of polyubiquitinated EMCV 3C protease conjugates in nuclear fraction preparations suggests that localization to the nucleus can protect this protein from ubiquitination

Characterization and identification of proteases secreted by Aspergillus fumigatus using free flow electrophoresis and MS.

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Neustadt, Madlen; Costina, Victor; Kupfahl, Claudio; Buchheidt, Dieter; Eckerskorn, Christoph; Neumaier, Michael; Findeisen, Peter

2009-06-01

Early diagnosis of life-threatening invasive aspergillosis in neutropenic patients remains challenging because current laboratory methods have limited diagnostic sensitivity and/or specificity. Aspergillus species are known to secrete various pathogenetically relevant proteases and the monitoring of their protease activity in serum specimens might serve as a new diagnostic approach.For the characterization and identification of secreted proteases, the culture supernatant of Aspergillus fumigatus was fractionated using free flow electrophoresis (Becton Dickinson). Protease activity of separated fractions was measured using fluorescently labeled reporter peptides. Fractions were also co-incubated in parallel with various protease inhibitors that specifically inhibit a distinct class of proteases e.g. metallo- or cysteine-proteases. Those fractions with high protease activity were further subjected to LC-MS/MS analysis for protease identification. The highest protease activity was measured in fractions with an acidic pH range. The results of the 'inhibitor-panel' gave a clear indication that it is mainly metallo- and serine-proteases that are involved in the degradation of reporter peptides. Furthermore, several proteases were identified that facilitate the optimization of reporter peptides for functional protease profiling as a diagnostic tool for invasive aspergillosis.

Aspartic cathepsin D degrades the cytosolic cysteine cathepsin inhibitor stefin B in the cells.

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Železnik, Tajana Zajc; Kadin, Andrey; Turk, Vito; Dolenc, Iztok

2015-09-18

Stefin B is the major general cytosolic protein inhibitor of cysteine cathepsins. Its main function is to protect the organism against the activity of endogenous potentially hazardous proteases accidentally released from lysosomes. In this study, we investigated the possible effect of endosomal/lysosomal aspartic cathepsins D and E on stefin B after membrane permeabilization. Loss of membrane integrity of lysosomes and endosomes was induced by a lysosomotropic agent L-Leucyl-L-leucine methyl ester (Leu-Leu-OMe). The rat thyroid cell line FRTL-5 was selected as a model cell line owing to its high levels of proteases, including cathepsin D and E. Permeabilization of acid vesicles from FRTL-5 cells induced degradation of stefin B. The process was inhibited by pepstatin A, a potent inhibitor of aspartic proteases. However, degradation of stefin B was prevented by siRNA-mediated silencing of cathepsin D expression. In contrast, cathepsin E silencing had no effect on stefin B degradation. These results showed that cathepsin D and not cathepsin E degrades stefin B. It can be concluded that the presence of cathepsin D in the cytosol affects the inhibitory potency of stefin B, thus preventing the regulation of cysteine cathepsin activities in various biological processes. Copyright © 2015 Elsevier Inc. All rights reserved.

Fluctuating partially native-like topologies in the acid denatured ensemble of autolysis resistant HIV-1 protease.

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Rout, Manoj Kumar; Hosur, Ramakrishna V

2009-02-01

Folding, in-vivo, starts from a denatured state and thus the nature of the denatured state would play an important role in directing the folding of a protein. We report here NMR characterization of the acid-denatured state of a mutant of HIV-1 protease, designed to prevent autolysis (Q7K, L33I, L63I) and to prevent cysteine oxidation (C67A and C95A). Secondary chemical shifts, TALOS analysis of chemical shifts and (15)N relaxation data (R(1), R(2), NOE) coupled with AABUF and hydrophobicity calculations, suggest formation of hydrophobic clusters and possibility of some partially native-like topologies in the acid denatured state of the protease. The structural and dynamics characteristics of the acid denatured PR seem to be considerably different from those of the guanidine or urea denatured states of some variants of PR. These would have implications for the folding and auto-processing of the enzyme in-vivo.

Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

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Raheem Ullah

Full Text Available Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

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Ullah, Raheem; Shah, Majid Ali; Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

2016-01-01

Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

Substrate Specificity of Cysteine Proteases Beyond the S2 Pocket: Mutagenesis and Molecular Dynamics Investigation of Fasciola hepatica Cathepsins L

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Ileana Corvo

2018-04-01

Full Text Available Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S2 subsite (pocket of the enzyme that interacts with the substrate P2 residue (Schetcher and Berger nomenclature. Although secreted FhCL1 accommodates aliphatic residues in the S2 pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S2 subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P3 specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S2 and S3 subsites that contributes to the accommodation of Gly in S3. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S2 and S3 pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity.

The role of cysteine residues in the sulphate transporter, SHST1: construction of a functional cysteine-less transporter.

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Howitt, Susan M

2005-05-20

We investigated the role of cysteine residues in the sulphate transporter, SHST1, with the aim of generating a functional cysteine-less variant. SHST1 contains five cysteine residues and none was essential for function. However, replacement of C421 resulted in a reduction in transport activity. Sulphate transport by C205 mutants was dependent on the size of the residue at this position. Alanine at position 205 resulted in a complete loss of function whereas leucine resulted in a 3-fold increase in sulphate transport relative to wild type SHST1. C205 is located in a putative intracellular loop and our results suggest that this loop may be important for sulphate transport. By replacing C205 with leucine and the other four cysteine residues with alanine, we constructed a cysteine-less variant of SHST1 that has transport characteristics indistinguishable from wild type. This construct will be useful for further structure and function studies of SHST1.

A novel nonsense mutation in cathepsin C gene in an Egyptian ...

African Journals Online (AJOL)

Background: Cathepsin C gene (CTSC) (MIM#602365) is a lysosomal cysteine proteinase coding gene which encodes for CTSC protein that plays a major role in the activation of granule serine proteases, particularly leukocyte elastase and granzymes A and B. This activity was proposed to play a role in epithelial ...

Synthetic siRNAs effectively target cystein protease 12 and α-actinin transcripts in Trichomonas vaginalis.

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Ravaee, Roya; Ebadi, Parimah; Hatam, Gholamreza; Vafafar, Arghavan; Ghahramani Seno, Mohammad Mahdi

2015-10-01

The flagellated protozoan Trichomonas vaginalis (T. vaginalis) causes trichomoniasis, a reproductive tract infection, in humans. Trichomoniasis is the most common non-viral sexually transmitted disease worldwide. In addition to direct consequences such as infertility and abortion, there are indications that trichomoniasis favours development of prostate cancer and it has also been associated with increased risk of spreading human immunodeficiency virus and papillomavirus infections. Reports from around the world show that the rate of drug resistance in T. vaginalis is increasing, and therefore new therapeutic approaches have to be developed. Studying molecular biology of T. vaginalis will be quite helpful in identifying new drugable targets. RNAi is a powerful technique which allows biologist to specifically target gene products (i.e. mRNA) helping them in unravelling gene functions and biology of systems. However, due to lack of some parts of the required intrinsic RNAi machinery, the RNAi system is not functional in all orders of life. Here, by using synthetic siRNAs targeting two genes, i.e. α-actinin and cystein protease 12 (cp12), we demonstrate T. vaginalis cells are amenable to RNAi experiments conducted by extrinsic siRNAs. Electroporation of siRNAs targeting α-actinin or cp12 into T. vaginalis cells resulted in, respectively, 48-67% and 33-72% downregulation of the cognate transcripts compared to the T. vaginalis cells received siRNAs targeting GL2 luciferase as a control. This finding is helpful in that it demonstrates the potential of using extrinsically induced RNAi in studies on molecular biology of T. vaginalis such as those aiming at identifying new drug targets. Copyright © 2015 Elsevier Inc. All rights reserved.

Optimization of Soluble Expression and Purification of Recombinant Human Rhinovirus Type-14 3C Protease Using Statistically Designed Experiments: Isolation and Characterization of the Enzyme.

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Antoniou, Georgia; Papakyriacou, Irineos; Papaneophytou, Christos

2017-10-01

Human rhinovirus (HRV) 3C protease is widely used in recombinant protein production for various applications such as biochemical characterization and structural biology projects to separate recombinant fusion proteins from their affinity tags in order to prevent interference between these tags and the target proteins. Herein, we report the optimization of expression and purification conditions of glutathione S-transferase (GST)-tagged HRV 3C protease by statistically designed experiments. Soluble expression of GST-HRV 3C protease was initially optimized by response surface methodology (RSM), and a 5.5-fold increase in enzyme yield was achieved. Subsequently, we developed a new incomplete factorial (IF) design that examines four variables (bacterial strain, expression temperature, induction time, and inducer concentration) in a single experiment. The new design called Incomplete Factorial-Strain/Temperature/Time/Inducer (IF-STTI) was validated using three GST-tagged proteins. In all cases, IF-STTI resulted in only 10% lower expression yields than those obtained by RSM. Purification of GST-HRV 3C was optimized by an IF design that examines simultaneously the effect of the amount of resin, incubation time of cell lysate with resin, and glycerol and DTT concentration in buffers, and a further 15% increase in protease recovery was achieved. Purified GST-HRV 3C protease was active at both 4 and 25 °C in a variety of buffers.

Mutant form C115H of Clostridium sporogenes methionine γ-lyase efficiently cleaves S-Alk(en)yl-l-cysteine sulfoxides to antibacterial thiosulfinates.

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Kulikova, Vitalia V; Anufrieva, Natalya V; Revtovich, Svetlana V; Chernov, Alexander S; Telegin, Georgii B; Morozova, Elena A; Demidkina, Tatyana V

2016-10-01

Pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) catalyzes the β-elimination reaction of S-alk(en)yl-l-cysteine sulfoxides to thiosulfinates, which possess antimicrobial activity. Partial inactivation of the enzyme in the course of the reaction occurs due to oxidation of active site cysteine 115 conserved in bacterial MGLs. In this work, the C115H mutant form of Clostridium sporogenes MGL was prepared and the steady-state kinetic parameters of the enzyme were determined. The substitution results in an increase in the catalytic efficiency of the mutant form towards S-substituted l-cysteine sulfoxides compared to the wild type enzyme. We used a sulfoxide/enzyme system to generate antibacterial activity in situ. Two-component systems composed of the mutant enzyme and three S-substituted l-cysteine sulfoxides were demonstrated to be effective against Gram-positive and Gram-negative bacteria and three clinical isolates from mice. © 2016 IUBMB Life, 68(10):830-835, 2016. © 2016 International Union of Biochemistry and Molecular Biology.

Processing Proteases

DEFF Research Database (Denmark)

Ødum, Anders Sebastian Rosenkrans

-terminal of the scissile bond, leaving C-terminal fusions to have non-native C-termini after processing. A solution yielding native C-termini would allow novel expression and purification systems for therapeutic proteins and peptides.The peptidyl-Lys metallopeptidase (LysN) of the fungus Armillaria mellea (Am) is one...... of few known proteases to have substrate specificity for the C-terminal side of the scissile bond. LysN exhibits specificity for lysine, and has primarily been used to complement trypsin in to proteomic studies. A working hypothesis during this study was the potential of LysN as a processing protease...

Mass spectrometric analysis of L-cysteine metabolism: physiological role and fate of L-cysteine in the enteric protozoan parasite Entamoeba histolytica.

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Jeelani, Ghulam; Sato, Dan; Soga, Tomoyoshi; Watanabe, Haruo; Nozaki, Tomoyoshi

2014-11-04

L-cysteine is essential for virtually all living organisms, from bacteria to higher eukaryotes. Besides having a role in the synthesis of virtually all proteins and of taurine, cysteamine, glutathione, and other redox-regulating proteins, L-cysteine has important functions under anaerobic/microaerophilic conditions. In anaerobic or microaerophilic protozoan parasites, such as Entamoeba histolytica, L-cysteine has been implicated in growth, attachment, survival, and protection from oxidative stress. However, a specific role of this amino acid or related metabolic intermediates is not well understood. In this study, using stable-isotope-labeled L-cysteine and capillary electrophoresis-time of flight mass spectrometry, we investigated the metabolism of L-cysteine in E. histolytica. [U-(13)C3, (15)N]L-cysteine was rapidly metabolized into three unknown metabolites, besides L-cystine and L-alanine. These metabolites were identified as thiazolidine-4-carboxylic acid (T4C), 2-methyl thiazolidine-4-carboxylic acid (MT4C), and 2-ethyl-thiazolidine-4-carboxylic acid (ET4C), the condensation products of L-cysteine with aldehydes. We demonstrated that these 2-(R)-thiazolidine-4-carboxylic acids serve for storage of L-cysteine. Liberation of L-cysteine occurred when T4C was incubated with amebic lysates, suggesting enzymatic degradation of these L-cysteine derivatives. Furthermore, T4C and MT4C significantly enhanced trophozoite growth and reduced intracellular reactive oxygen species (ROS) levels when it was added to cultures, suggesting that 2-(R)-thiazolidine-4-carboxylic acids are involved in the defense against oxidative stress. Amebiasis is a human parasitic disease caused by the protozoan parasite Entamoeba histolytica. In this parasite, L-cysteine is the principal low-molecular-weight thiol and is assumed to play a significant role in supplying the amino acid during trophozoite invasion, particularly when the parasites move from the anaerobic intestinal lumen to highly

Disruption of TLR3 signaling due to cleavage of TRIF by the hepatitis A virus protease-polymerase processing intermediate, 3CD.

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Lin Qu

2011-09-01

Full Text Available Toll-like receptor 3 (TLR3 and cytosolic RIG-I-like helicases (RIG-I and MDA5 sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF, and mitochondrial antiviral signaling protein (MAVS, respectively. Previously, we demonstrated that hepatitis A virus (HAV, a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3C(pro, that is derived by auto-processing of the P3 (3ABCD segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C-stimulated dimerization of IFN regulatory factor 3 (IRF-3, IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3C(pro protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3C(pro and downstream 3D(pol sequence, but not 3D(pol polymerase activity. Cleavage occurs at two non-canonical 3C(pro recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3D(pol sequence modulates the substrate specificity of the upstream 3C(pro protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3C(pro. HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate.

Molecular models of NS3 protease variants of the Hepatitis C virus

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Mello Isabel MVGC

2005-01-01

Full Text Available Abstract Background Hepatitis C virus (HCV currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed. Results The atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related beta-alpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures. Conclusions This project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure

Production and partial characterization of alkaline protease from bacillus subtilis mutant induced by gamma radiation

International Nuclear Information System (INIS)

Ibrahim, H.M.M.; Bashandy, A.S.

2010-01-01

Fourteen bacterial isolates belonging to B.subtilis were locally isolated from soil and screened for alkaline protease production. Only one strain, the highly potent one, was selected as alkaline protease producer and subjected to further studies to optimize its production. Alkaline protease production was maximum at 35 degree C after 72 h of incubation and at ph 10.0. molasses as a carbon source and combination of peptone and yeast extract as a nitrogen source enhanced greatly alkaline protease production. The mutant strain induced by gamma radiation showed higher alkaline protease production by 1.97 fold as compared with the parent strain. The alkaline protease enzyme was active at 40 degree C and ph 10. It was compatible with many commercial detergents and showed high stability (84 %) of its original activity with Ariel detergent. Moreover, alkaline protease enhanced the washing performance, and retained 95 % of its activity in the formulated dry powder.

Foot-and-Mouth Disease (FMD) Virus 3C Protease Mutant L127P: Implications for FMD Vaccine Development.

Science.gov (United States)

Puckette, Michael; Clark, Benjamin A; Smith, Justin D; Turecek, Traci; Martel, Erica; Gabbert, Lindsay; Pisano, Melia; Hurtle, William; Pacheco, Juan M; Barrera, José; Neilan, John G; Rasmussen, Max

2017-11-15

The foot-and-mouth disease virus (FMDV) afflicts livestock in more than 80 countries, limiting food production and global trade. Production of foot-and-mouth disease (FMD) vaccines requires cytosolic expression of the FMDV 3C protease to cleave the P1 polyprotein into mature capsid proteins, but the FMDV 3C protease is toxic to host cells. To identify less-toxic isoforms of the FMDV 3C protease, we screened 3C mutants for increased transgene output in comparison to wild-type 3C using a Gaussia luciferase reporter system. The novel point mutation 3C(L127P) increased yields of recombinant FMDV subunit proteins in mammalian and bacterial cells expressing P1-3C transgenes and retained the ability to process P1 polyproteins from multiple FMDV serotypes. The 3C(L127P) mutant produced crystalline arrays of FMDV-like particles in mammalian and bacterial cells, potentially providing a practical method of rapid, inexpensive FMD vaccine production in bacteria. IMPORTANCE The mutant FMDV 3C protease L127P significantly increased yields of recombinant FMDV subunit antigens and produced virus-like particles in mammalian and bacterial cells. The L127P mutation represents a novel advancement for economical FMD vaccine production. Copyright © 2017 Puckette et al.

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

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Luciana Bonome Zeminian

2013-02-01

Full Text Available The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3 have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

Production, Partial Purification and Characterization of Protease From Irradiated Streptomyces Spp

International Nuclear Information System (INIS)

Botros, H.W.; Ahmed, A.S.

2011-01-01

Production and partial purification of protease by the irradiated Streptomyces spp. was the aim of this study. Streptomyces spp. was allowed to grow in culture broth of 4% shrimp shells for purpose of inducing protease enzymes. Optimal conditions for protease production were 30 degree C, 0.3 kGy, ph 7, 5x10 4 /ml inoculum size and 7 days incubation period. Protease was purified by 80% ammonium sulphate saturation which exhibited 8.7 U/ml enzyme activity. Column chromatography using sephadex G-200 exerted 23.3 U/ml enzyme activity from pooled fraction (13-16). The molecular mass of protease was determined to be 39 kDa by SDS-PAGE. The enzyme was more stable over a wide range of ph 6-8 and temperature up to 40 degree C. The produced protease was activated by Ca, Mn and FeCl 2 and completely inhibited by ethylene-diamin tetraacetic acid (EDTA) at concentration of 1000 μg/ml

Cysteine peptidases of Eudiplozoon nipponicum: a broad repertoire of structurally assorted cathepsins L in contrast to the scarcity of cathepsins B in an invasive species of haematophagous monogenean of common carp

Czech Academy of Sciences Publication Activity Database

Jedličková, L.; Dvořáková, H.; Dvořák, J.; Kašný, M.; Ulrychová, Lenka; Vorel, J.; Žárský, V.; Mikeš, L.

2018-01-01

Roč. 11, Mar 6 (2018), č. článku 142. ISSN 1756-3305 R&D Projects: GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : cysteine peptidase * protease * cathepsin * S2 subsite * haematophagy * blood digestion Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.080, year: 2016 https:// parasites andvectors.biomedcentral.com/ articles /10.1186/s13071-018-2666-2

Enhanced Productivity of Serine Alkaline Protease by Bacillus sp. Using Soybean as Substrate

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Saurabh, S.

2007-01-01

Full Text Available The growth and protease production by Bacillus sp. (SBP-29 was examined for poultry processing industries. The maximum protease activity was 3028 U/mL using 1.5% (w/v of soybean meal as substrate. Soybean meal is an inexpensive and readily available, thus it can be used as the cost effective crude material for the production of an extracellular protease. Inorganic nitrogen sources proved to be less favorable, for protease production as strong catabolic repression was observed with ammonium ions. A maximum of 3208 U/mL of protease was produced in 18 h in a 10L bioreactor. The enzyme has temperature and pH optima of 60°C and 9.5 respectively. However, the temperature stability range is from 20-90 °C and pH stability range is from 6.0–12.0. The protease was completely inhibited by phenylmethylsulfonyl fluoride (PMSF and diodopropyl fluorophosphate (DFP, with little increase (10-15% in the production of upon addition of Ca++ and Mg++.

Kinetics Study of Extracellular Detergent Stable Alkaline Protease from Rhizopus oryzae

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Zareena Mushtaq

2015-04-01

Full Text Available In this study, extracellular alkaline protease was produced from Rhizopus oryzae in submerged fermentation using dairy waste (whey as a substrate. Fermentation kinetics was studied and various parameters were optimized. The strain produced maximum protease at initial medium pH of 6.0 medium depth of 26 mm, inoculum size of 2% at incubation temperature of 35ºC for 168 h of fermentation. Alkaline protease was purified to homogeneity by ammonium sulphate fractionation followed by sephadex G-100 chromatography. The molecular mass of alkaline protease was 69 kDa determined by 10% SDS-PAGE. The optimum pH and temperature of alkaline protease was 9.0 and 40ºC, respectively. Metal profile of the enzyme showed that the enzyme was non-metallic in nature. The Km , Kcat , Vmax and Kcat/Km values of purified protease were 7.0 mg/mL, 3.8 x102S-1, 54.30 µmol/min and 54.28 s-1mg -1.mL respectively, using casein as substrate. The purified alkaline protease had stability with commercial detergents.

PARTIAL PURIFICATION AND CHARACTERIZATION OF ALKALOPHILIC PROTEASE FROM PSEUDOMONAS AERUGINOSA

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R. Satheeskumar

2013-10-01

Full Text Available Partial purification and characterization of alkalophilic protease production from Pseudomonas aeruginosa was isolated from the gut of marine and coastal waters shrimp Penaeus monodon. The protease production was assayed in submerged fermentation to produce maximum protease activity (423 ± 0.09 U/ml. The enzyme was precipitated with ammonium sulphate and partially purified by ion exchange chromatography through DEAE Sephadex A-50 column. In 10th fraction showed maximum protease activity (734 ± 0.18 U/ml with increase in purification fold. The molecular weight of protease from Pseudomonas aeruginosa was recorded as 60 kDa. The stability of protease was tested at various pH and temperature; it showed maximum protease activity at pH-9 and temperature 50ºC. Among the various surfactants tested for enzyme stability, maximum activity was retained in poly ethylene glycol. The compatibility of protease enzyme with various commercial detergents; the enzyme retained maximum protease activity in tide. The results are indicated that all these properties make the bacterial proteases are most suitable for wide industrial applications.

SwissProt search result: AK242832 [KOME

Lifescience Database Archive (English)

Full Text Available AK242832 J090067L13 (Q811C2) Cysteine protease APG4C (EC 3.4.22.-) (Autophagy 4 homolog C) (Auto...phagin-3) (Autophagy-related cysteine endopeptidase 3) (AUT-like 3 cysteine endopeptidase) APG4C_MOUSE 4e-37 ...

Mature and progenitor endothelial cells perform angiogenesis also under protease inhibition: the amoeboid angiogenesis.

Science.gov (United States)

Chillà, Anastasia; Margheri, Francesca; Biagioni, Alessio; Del Rosso, Mario; Fibbi, Gabriella; Laurenzana, Anna

2018-04-03

Controlling vascular growth is a challenging aim for the inhibition of tumor growth and metastasis. The amoeboid and mesenchymal types of invasiveness are two modes of migration interchangeable in cancer cells: the Rac-dependent mesenchymal migration requires the activity of proteases; the Rho-ROCK-dependent amoeboid motility is protease-independent and has never been described in endothelial cells. A cocktail of physiologic inhibitors (Ph-C) of serine-proteases, metallo-proteases and cysteine-proteases, mimicking the physiological environment that cells encounter during their migration within the angiogenesis sites was used to induce amoeboid style migration of Endothelial colony forming cells (ECFCs) and mature endothelial cells (ECs). To evaluate the mesenchymal-ameboid transition RhoA and Rac1 activation assays were performed along with immunofluorescence analysis of proteins involved in cytoskeleton organization. Cell invasion was studied in Boyden chambers and Matrigel plug assay for the in vivo angiogenesis. In the present study we showed in both ECFCs and ECs, a decrease of activated Rac1 and an increase of activated RhoA upon shifting of cells to the amoeboid conditions. In presence of Ph-C inhibitors both cell lines acquired a round morphology and Matrigel invasion was greatly enhanced with respect to that observed in the absence of protease inhibition. We also observed that the urokinase-plasminogen-activator (uPAR) receptor silencing and uPAR-integrin uncoupling with the M25 peptide abolished both mesenchymal and amoeboid angiogenesis of ECFCs and ECs in vitro and in vivo, indicating a role of the uPAR-integrin-actin axis in the regulation of amoeboid angiogenesis. Furthermore, under amoeboid conditions endothelial cells seem to be indifferent to VEGF stimulation, which induces an amoeboid signaling pattern also in mesenchymal conditions. Here we first provide a data set disclosing that endothelial cells can move and differentiate into vascular

Highly efficient synthetic method onpyroacm resin using the boc SPPS protocol for C-terminal cysteine peptide synthesis

Energy Technology Data Exchange (ETDEWEB)

Juvekar, Vinayak; Kim, Kang Tae; Gong, Young Dae [Innovative Drug Library Research Center, Dept. of Chemistry, College of Science, Dongguk University, Seoul (Korea, Republic of)

2017-01-15

A very effective process on Pyroacm resin was developed for solid-phase peptide synthesis (SPPS) of C-terminal cysteine and cysteine ester peptides. The process uses cysteine side chain anchoring to the Pyroacm resin and the Boc protocol for SPPS. The Pyroacm resin showed remarkable stability under standard trifluoromethanesulfonic acid (TFMSA) cleavage condition. TFMSA cleavage of protecting groups generates a peptide-linked resin, which can be subjected to peptide modification reactions. Finally, the peptide can be cleaved from the resin using methoxycarbonylsulfenyl chloride. The utility of this protocol was demonstrated by its applications to the synthesis of model peptides, key intermediates in the preparation of natural products riparin 1.2 and a-factor.

Production and Characterization of Alkaline Protease from a High Yielding and Moderately Halophilic Strain of SD11 Marine Bacteria

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Hongxia Cui

2015-01-01

Full Text Available A marine bacterium SD11, which was isolated from sea muds (Geziwo Qinhuangdao Sea area, China, was used to produce thermostable alkaline serine nonmetal protease in the skim milk agar plate medium with 10% NaCl. The optimal temperature about the manufacture of the extracellular protease was ~60°C. The crude enzyme was stable at 20–50°C. The activity was retained to 60% and 45% after heating for 1 h at 60 and 70°C, respectively. The protease was highly active in a wide pH scope (8.0–10.0 and maximum protease activity exhibited at pH 10.0. The activity was restrained by phenylmethylsulfonyl fluoride (PMSF but mildly increased (~107% in the presence of ethylenediaminetetraacetic acid (EDTA, indicating that the production contains serine-protease(s and nonmetal protease(s. Moreover, the crude alkaline protease was active with the 5 mM Ca2+, Mn2+, Zn2+, Cu2+, Na+, and K+ that existed separately. In addition, the protease showed superduper stability when exposed to an anionic surfactant (5 mM SDS, an oxidizing agent (1% H2O2, and several organic solvents (methanol, isopropanol, and acetone. These results suggest that the marine bacterium SD11 is significant in the industry from the prospects of its ability to produce thermally stable alkaline protease.

SHORT COMMUNICATION CHEMOSELECTIVE C-BENZOYLATION ...

African Journals Online (AJOL)

BCSE

Department of Chemistry, Savitribaiphule Pune University, Pune 411007, India ... Crafts acylation or benzoylation reactions employ acid chlorides and Lewis acids ... cyclin-dependent kinase inhibitor [14] and cysteine protease modulator.

Novel IgG-Degrading Enzymes of the IgdE Protease Family Link Substrate Specificity to Host Tropism of Streptococcus Species.

Science.gov (United States)

Spoerry, Christian; Hessle, Pontus; Lewis, Melanie J; Paton, Lois; Woof, Jenny M; von Pawel-Rammingen, Ulrich

2016-01-01

Recently we have discovered an IgG degrading enzyme of the endemic pig pathogen S. suis designated IgdE that is highly specific for porcine IgG. This protease is the founding member of a novel cysteine protease family assigned C113 in the MEROPS peptidase database. Bioinformatical analyses revealed putative members of the IgdE protease family in eight other Streptococcus species. The genes of the putative IgdE family proteases of S. agalactiae, S. porcinus, S. pseudoporcinus and S. equi subsp. zooepidemicus were cloned for production of recombinant protein into expression vectors. Recombinant proteins of all four IgdE family proteases were proteolytically active against IgG of the respective Streptococcus species hosts, but not against IgG from other tested species or other classes of immunoglobulins, thereby linking the substrate specificity to the known host tropism. The novel IgdE family proteases of S. agalactiae, S. pseudoporcinus and S. equi showed IgG subtype specificity, i.e. IgdE from S. agalactiae and S. pseudoporcinus cleaved human IgG1, while IgdE from S. equi was subtype specific for equine IgG7. Porcine IgG subtype specificities of the IgdE family proteases of S. porcinus and S. pseudoporcinus remain to be determined. Cleavage of porcine IgG by IgdE of S. pseudoporcinus is suggested to be an evolutionary remaining activity reflecting ancestry of the human pathogen to the porcine pathogen S. porcinus. The IgG subtype specificity of bacterial proteases indicates the special importance of these IgG subtypes in counteracting infection or colonization and opportunistic streptococci neutralize such antibodies through expression of IgdE family proteases as putative immune evasion factors. We suggest that IgdE family proteases might be valid vaccine targets against streptococci of both human and veterinary medical concerns and could also be of therapeutic as well as biotechnological use.

Extraction, purification and characterization of a protease from Micrococcus sp. VKMM 037.

Science.gov (United States)

Manikandan, Muthu; Kannan, Vijayaraghavan; Pasić, Lejla

2011-10-01

The haloalkaliphilic bacterium Micrococcus sp. VKMM 037, isolated from an effluent of the caustic soda industry, was found to produce a protease. Maximal proteolytic activity was observed in cell culture grown at 40 degrees C using 2% (w/v) glycerol, 2% (w/v) beef extract and 2% (w/v) peptone as nutrients in medium also containing 0.85 M NaCl with a pH of 10.0. An efficient purification procedure combining ammonium sulphate precipitation and Q-Sepharose ion-exchange chromatography was developed. The purified 41 kDa protease was stable in a temperature range between 20 degrees C and 60 degrees C. The protease remained active over a wide range of pH values (4.0-12.0) and NaCl concentrations (0-3.42 M) with an optimum at pH 10.0 and 0.85 M NaCl, respectively. Furthermore, the enzyme remained stable or was only marginally inhibited in the presence of various organic solvents, surfactants and reducing agents. The purified protease of Micrococcus sp. VKMM 037 efficiently removed blood stains within 40 minutes of treatment. Given the biochemical characteristics determined, this novel protease could be exploited as an additive in the detergent industry and also for the synthesis of biomolecules and the degradation of protein.

Protease and protease inhibitory activity in pregnant and postpartum involuting uterus

International Nuclear Information System (INIS)

Milwidsky, A.; Beller, U.; Palti, Z.; Mayer, M.

1982-01-01

The presence of two distinct proteolytic activities in the rat uterus was confirmed with 14 C-labeled globin used as a sensitive protein substrate and following release of label into the trichloroacetic acid-soluble supernatant fraction. Protease I is a cytoplasmic acid protease while protease II is associated with the pellet fraction, can be extracted by 0.6 M sodium chloride, and is active at pH 7.0. Protease I activity is low during pregnancy and markedly increases at term achieving maximal activity at day 3 post partum with a subsequent decline to preterm activity values. Lactation did not affect the uterine protease I activity. Protease II activity is not significantly different during pregnancy, at term, and post partum. The presence of an inhibitor of protease I was suggested by a decrease in enzyme activity with an increased cytosolic protein concentration. The inhibitor also lessened bovine trypsin activity but had no effect on protease II. Although its inhibitory potency on trypsin fluctuated during the various uterine physiologic stages, these changes appeared to be statistically insignificant. Human uterine samples were also found to contain the two protease activities with similar changes in protease I post partum. It is suggested that, both in the rat and in man, uterine involution post partum is associated with a marked increase in activity of acid cytosolic protease, while a particulate neutral protease and a soluble inhibitor of trypsin, which are also present in uterine cells, do not appear to play a significant role in the dissolution of uterine tissues after parturition

Biochemical characterization of a halophilic, alkalithermophilic protease from Alkalibacillus sp. NM-Da2.

Science.gov (United States)

Abdel-Hamed, Asmaa R; Abo-Elmatty, Dina M; Wiegel, Juergen; Mesbah, Noha M

2016-11-01

An extracellular, halophilic, alkalithermophilic serine protease from the halo-alkaliphilic Alkalibacillus sp. NM-Da2 was purified to homogeneity by ethanol precipitation and anion-exchange chromatography. The purified protease was a monomeric enzyme with an approximate molecular mass of 35 kDa and exhibited maximal activity at 2.7 M NaCl, pH 55 °C 9 and 56 °C. The protease showed great temperature stability, retaining greater than 80 % of initial activity after 2 h incubation at 55 °C. The protease was also extremely pH tolerant, retaining 80 % of initial activity at pH 55 °C 10.5 after 30 min incubation. Protease hydrolyzed complex substrates, displaying activity on yeast extract, tryptone, casein, gelatin and peptone. Protease activity was inhibited at casein concentrations greater than 1.2 mg/mL. The enzyme was stable and active in 40 % (v/v) solutions of isopropanol, ethanol and benzene and was stable in the presence of the polysorbate surfactant Tween 80. Activity was stimulated with the oxidizing agent hydrogen peroxide. Inhibition with phenyl methylsulfonylfluoride indicates it is a serine protease. Synthetic saline wastewater treated with the protease showed 50 % protein removal after 5 h. Being halophilic, alkaliphilic and thermophilic, in addition to being resistant to organic solvents, this protease has potential for various applications in biotechnological and pharmaceutical industries.

Alkaline protease production from industrial wastes by bacillus subtilis ML-4

International Nuclear Information System (INIS)

Sher, M.G.; Nadeem, M.; Syed, Q.; Irfan, M.; Baig, S.

2010-01-01

The influence of various culture conditions on protease production by Bacillus subtilis ML-4 was studied in the presence of growth medium containing poultry feed waste (5%), K/sub 2/HPO/sub 4/ (0.3%), CaCl/sub 2/ (0.03%) and MgSO/sub 4/ (0.015%). Maximum protease production (264.25 +- 1.86 U/ml) was observed at initial pH 9 with 3% (v/v) of inoculum size after 48 h of incubation at 37 degree C. The alkaline protease was stable over a broad range of temperature (30 to 60 degree C) and pH (8 to 11). However, maximum activity (155.45 U/ml) was observed at temperature 50 degree C and pH 10. (author)

SmCL3, a gastrodermal cysteine protease of the human blood fluke Schistosoma mansoni.

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Jan Dvorák

2009-06-01

Full Text Available Blood flukes of the genus Schistosoma are platyhelminth parasites that infect 200 million people worldwide. Digestion of nutrients from the host bloodstream is essential for parasite development and reproduction. A network of proteolytic enzymes (proteases facilitates hydrolysis of host hemoglobin and serum proteins.We identified a new cathepsin L termed SmCL3 using PCR strategies based on S. mansoni EST sequence data. An ortholog is present in Schistosoma japonicum. SmCL3 was heterologously expressed as an active enzyme in the yeast, Pichia pastoris. Recombinant SmCL3 has a broad pH activity range against peptidyl substrates and is inhibited by Clan CA protease inhibitors. Consistent with a function in degrading host proteins, SmCL3 hydrolyzes serum albumin and hemoglobin, is localized to the adult gastrodermis, and is expressed mainly in those life stages infecting the mammalian host. The predominant form of SmCL3 in the parasite exists as a zymogen, which is unusual for proteases. This zymogen includes an unusually long prodomain with alpha helical secondary structure motifs. The striking specificity of SmCL3 for amino acids with large aromatic side chains (Trp and Tyr at the P2 substrate position, as determined with positional scanning-synthetic combinatorial library, is consistent with a molecular model that shows a large and deep S2 pocket. A sequence similarity network (SSN view clusters SmCL3 and other cathepsins L in accordance with previous large-scale phylogenetic analyses that identify six super kingdoms.SmCL3 is a gut-associated cathepsin L that may contribute to the network of proteases involved in degrading host blood proteins as nutrients. Furthermore, this enzyme exhibits some unusual sequence and biophysical features that may result in additional functions. The visualization of network inter-relationships among cathepsins L suggests that these enzymes are suitable 'marker sequences' for inclusion in future phylogenetic analyses.

Survey of the rubber tree genome reveals a high number of cysteine protease-encoding genes homologous to Arabidopsis SAG12.

Science.gov (United States)

Zou, Zhi; Liu, Jianting; Yang, Lifu; Xie, Guishui

2017-01-01

Arabidopsis thaliana SAG12, a senescence-specific gene encoding a cysteine protease, is widely used as a molecular marker for the study of leaf senescence. To date, its potential orthologues have been isolated from several plant species such as Brassica napus and Nicotiana tabacum. However, little information is available in rubber tree (Hevea brasiliensis), a rubber-producing plant of the Euphorbiaceae family. This study presents the identification of SAG12-like genes from the rubber tree genome. Results showed that an unexpected high number of 17 rubber orthologues with a single intron were found, contrasting the single copy with two introns in Arabidopsis. The gene expansion was also observed in another two Euphorbiaceae plants, castor bean (Ricinus communis) and physic nut (Jatropha curcas), both of which contain 8 orthologues. In accordance with no occurrence of recent whole-genome duplication (WGD) events, most duplicates in castor and physic nut were resulted from tandem duplications. In contrast, the duplicated HbSAG12H genes were derived from tandem duplications as well as the recent WGD. Expression analysis showed that most HbSAG12H genes were lowly expressed in examined tissues except for root and male flower. Furthermore, HbSAG12H1 exhibits a strictly senescence-associated expression pattern in rubber tree leaves, and thus can be used as a marker gene for the study of senescence mechanism in Hevea.

Purification and characterization of naturally occurring HIV-1 (South African subtype C) protease mutants from inclusion bodies.

Science.gov (United States)

Maseko, Sibusiso B; Natarajan, Satheesh; Sharma, Vikas; Bhattacharyya, Neelakshi; Govender, Thavendran; Sayed, Yasien; Maguire, Glenn E M; Lin, Johnson; Kruger, Hendrik G

2016-06-01

Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Many studies have targeted HIV-1 protease for the development of drugs against AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. Along with the wild type (C-SA) we also over-expressed and characterized two mutant forms from patients that had shown resistance to protease inhibitors. Using recombinant DNA technology, we constructed three recombinant plasmids in pGEX-6P-1 and expressed them containing a sequence encoding wild type HIV protease and two mutants (I36T↑T contains 100 amino acids and L38L↑N↑L contains 101 amino acids). These recombinant proteins were isolated from inclusion bodies by using QFF anion exchange and GST trap columns. In SDS-PAGE, we obtained these HIV proteases as single bands of approximately 11.5, 11.6 and 11.7 kDa for the wild type, I36T↑Tand L38L↑N↑L mutants, respectively. The enzyme was recovered efficiently (0.25 mg protein/L of Escherichia coli culture) and had high specific activity of 2.02, 2.20 and 1.33 μmol min(-1) mg(-1) at an optimal pH of 5 and temperature of 37 °C for the wild type, I36T↑T and L38L↑N↑L, respectively. The method employed here provides an easy and rapid purification of the HIV-1(C-SA) protease from the inclusion bodies, with high yield and high specific activities. Copyright © 2016 Elsevier Inc. All rights reserved.

PfClpC Is an Essential Clp Chaperone Required for Plastid Integrity and Clp Protease Stability in Plasmodium falciparum

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Anat Florentin

2017-11-01

Full Text Available Summary: The deadly malaria parasite Plasmodium falciparum contains a nonphotosynthetic plastid, known as the apicoplast, that functions to produce essential metabolites, and drugs that target the apicoplast are clinically effective. Several prokaryotic caseinolytic protease (Clp genes have been identified in the Plasmodium genome. Using phylogenetic analysis, we focused on the Clp members that may form a regulated proteolytic complex in the apicoplast. We genetically targeted members of this complex and generated conditional mutants of the apicoplast-localized PfClpC chaperone and PfClpP protease. Conditional inhibition of the PfClpC chaperone resulted in growth arrest and apicoplast loss and was rescued by addition of the essential apicoplast-derived metabolite IPP. Using a double-conditional mutant parasite line, we discovered that the chaperone activity is required to stabilize the mature protease, revealing functional interactions. These data demonstrate the essential function of PfClpC in maintaining apicoplast integrity and its role in regulating the proteolytic activity of the Clp complex. : Plasmodium falciparum contains a unique organelle, the apicoplast. Using genetic and phenotypic assays, Florentin et al. characterize the apicoplast Clp chaperone and protease. They find that the chaperone is essential for protease stability and that together they function to maintain organelle integrity and segregation into daughter cells. Keywords: malaria, Plasmodium, apicoplast, IPP, Clp, chaperone, caseinolytic protease

Purification and biochemical characterization of asclepain c I from the latex of Asclepias curassavica L.

Science.gov (United States)

Liggieri, Constanza; Arribére, M Cecilia; Trejo, Sebastián A; Canals, Francesc; Avilés, Francesc X; Priolo, Nora S

2004-08-01

In this work we report the isolation, purification and characterization of a new protease from latex of Asclepias curassavica L. Crude extract (CE) was obtained by gathering latex on 0.1 M citric-phosphate buffer with EDTA and cysteine with subsequent ultracentrifugation. Proteolytic assays were made on casein or azocasein as substrates. Caseinolytic activity was completely inhibited by E-64. Stability at different temperatures, optimum pH and ionic strength were evaluated by measuring the residual caseinolytic activity at different times after the incubation. CE showed the highest caseinolytic activity at pH 8.5 in the presence of 12 mM cysteine. CE was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by SDS-PAGE, were isolated. The major purified protease (asclepain cI) showed a molecular mass of 23.2 kDa by mass spectrometry and a pI higher than 9.3. The N-terminal sequence showed a high similarity with those of other plant cysteine proteinases. When assayed on N-alpha-CBZ-aminoacid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative. Determinations of kinetic parameter (km and Kcat) were performed with PFLNA.

Rapid emergence of hepatitis C virus protease inhibitor resistance is expected

Energy Technology Data Exchange (ETDEWEB)

Rong, Libin [Los Alamos National Laboratory; Perelson, Alan S [Los Alamos National Laboratory; Ribeiro, Ruy M [Los Alamos National Laboratory

2009-01-01

Approximately 170 million people worldwide are infected with hepatitis C virus (HCV). Current therapy, consisting of pegylated interferon (PEG-IFN) and ribavirin (RBV), leads to sustained viral elimination in only about 45% of patients treated. Telaprevir (VX-950), a novel HCV NS3-4A serine protease inhibitor, has demonstrated substantial antiviral activity in patients with chronic hepatitis C genotype 1 infection. However, some patients experience viral breakthrough during dosing, with drug resistant variants being 5%-20% of the virus population as early as day 2 after treatment initiation. Why viral variants appear such a short time after the start of dosing is unclear, especially since this has not been seen with monotherapy for either human immunodeficiency virus or hepatitis B virus. Here, using a viral dynamic model, we explain why such rapid emergence of drug resistant variants is expected when potent HCV protease inhibitors are used as monotherapy. Surprisingly, our model also shows that such rapid emergence need not be the case with some potent HCV NS5B polymerase inhibitors. Examining the case of telaprevir therapy in detail, we show the model fits observed dynamics of both wild-type and drug-resistant variants during treatment, and supports combination therapy of direct antiviral drugs with PEG-IFN and/or RBV for hepatitis C.

The C-terminal sequence of several human serine proteases encodes host defense functions.

Science.gov (United States)

Kasetty, Gopinath; Papareddy, Praveen; Kalle, Martina; Rydengård, Victoria; Walse, Björn; Svensson, Bo; Mörgelin, Matthias; Malmsten, Martin; Schmidtchen, Artur

2011-01-01

Serine proteases of the S1 family have maintained a common structure over an evolutionary span of more than one billion years, and evolved a variety of substrate specificities and diverse biological roles, involving digestion and degradation, blood clotting, fibrinolysis and epithelial homeostasis. We here show that a wide range of C-terminal peptide sequences of serine proteases, particularly from the coagulation and kallikrein systems, share characteristics common with classical antimicrobial peptides of innate immunity. Under physiological conditions, these peptides exert antimicrobial effects as well as immunomodulatory functions by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, selected peptides are protective against lipopolysaccharide-induced shock. Moreover, these S1-derived host defense peptides exhibit helical structures upon binding to lipopolysaccharide and also permeabilize liposomes. The results uncover new and fundamental aspects on host defense functions of serine proteases present particularly in blood and epithelia, and provide tools for the identification of host defense molecules of therapeutic interest. Copyright © 2011 S. Karger AG, Basel.

SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion

International Nuclear Information System (INIS)

Madu, Ikenna G.; Belouzard, Sandrine; Whittaker, Gary R.

2009-01-01

The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1-S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.

Isolation and partial characterization of protease from Pseudomonas aeruginosa ATCC 27853

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LIDIJA IZRAEL-ŽIVKOVIĆ

2010-08-01

Full Text Available Enzymatic characteristics of a protease from a medically important, referent strain of Pseudomonas aeruginosa ATCC 27853 were determined. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE, and gel filtration, it was estimated that the molecular mass of the purified enzyme was about 15 kDa. Other enzymatic properties were found to be: pH optimum 7.1, pH stability between 6.5 and 10; temperature optimum around 60 °C while the enzyme was stable at 60 °C for 30 min. Inhibition of the enzyme was observed with metal chelators, such as EDTA and 1,10-phenanthroline, suggesting that the protease is a metalloenzyme. Furthermore, the enzyme contains one mole of zinc ion per mole of enzyme. The protease was stable in the presence of different organic solvents, which enables its potential use for the synthesis of peptides.

Electrostatic influence of local cysteine environments on disulfide exchange kinetics.

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Snyder, G H; Cennerazzo, M J; Karalis, A J; Field, D

1981-11-10

The ionic strength dependence of the bimolecular rate constant for reaction of the negative disulfide 5,5'-dithiobis (2-nitrobenzoic acid) with cysteines in fragments of naturally occurring proteins was determined by stopped-flow spectroscopy. The Debye-Hückel relationship was applied to determine the effective charge at the cysteine and thereby determine the extent to which nearby neighbors in the primary sequence influence the kinetics. Corrections for the secondary salt effect on cysteine pKs were determined by direct spectrometric pH titration of sulfhydryl groups or by observation of the ionic strength dependence of kinetics of cysteine reaction with the neutral disulfide 2,2'-dithiodipyridine. Quantitative expressions was verified by model studies with N-acetyl-cystein. At ionic strengths equal to or greater than 20 mM, the net charge at the polypeptide cysteine site is the sum of the single negative charge of the thiolate anion and the charges of the amino acids immediately preceding and following the cysteine in the primary sequence. At lower ionic strengths, more distant residues influence kinetics. At pH 7.0, 23 degree C, and an ionic strength of 20 mM, rate constants for reaction of the negative disulfide with a cysteine having two positive neighbors, one positive and one neutral neighbor, or two neutral neighbors are 132000, 3350, and 367 s-1 M-1, respectively. This corresponds to a contribution to the activation energy of 0.65- 1.1 kcal/mol per ion pair involved in collision between the cysteine and disulfide regions. The results permit the estimation that cysteine local environments may provide a means of achieving a 10(6)-fold range in rate constants in disulfide exchange reactions in random-coil proteins. This range may prove useful in developing strategies for directing disulfide pairing in synthetic proteins.

Human mast cell tryptase: Multiple cDNAs and genes reveal a multigene serine protease family

International Nuclear Information System (INIS)

Vanderslice, P.; Ballinger, S.M.; Tam, E.K.; Goldstein, S.M.; Craik, C.S.; Caughey, G.H.

1990-01-01

Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the ∼1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5' regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family

Structure of the Enterovirus 71 3C Protease in Complex with NK-1.8k and Indications for the Development of Antienterovirus Protease Inhibitor.

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Wang, Yaxin; Cao, Lin; Zhai, Yangyang; Yin, Zheng; Sun, Yuna; Shang, Luqing

2017-07-01

Hand-foot-and-mouth disease (HFMD), caused by enterovirus, is a threat to public health worldwide. To date, enterovirus 71 (EV71) has been one of the major causative agents of HFMD in the Pacific-Asia region, and outbreaks with EV71 cause millions of infections. However, no drug is currently available for clinical therapeutics. In our previous works, we developed a set of protease inhibitors (PIs) targeting the EV71 3C protease (3C pro ). Among these are NK-1.8k and NK-1.9k, which have various active groups and high potencies and selectivities. In the study described here, we determined the structures of the PI NK-1.8k in complex with wild-type (WT) and drug-resistant EV71 3C pro Comparison of these structures with the structure of unliganded EV71 3C pro and its complex with AG7088 indicated that the mutation of N69 to a serine residue destabilized the S2 pocket. Thus, the mutation influenced the cleavage activity of EV71 3C pro and the inhibitory activity of NK-1.8k in an in vitro protease assay and highlighted that site 69 is an additional key site for PI design. More information for the optimization of the P1' to P4 groups of PIs was also obtained from these structures. Together with the results of our previous works, these in-depth results elucidate the inhibitory mechanism of PIs and shed light to develop PIs for the clinical treatment of infections caused by EV71 and other enteroviruses. Copyright © 2017 American Society for Microbiology.

Inhibition of protease-inhibitor resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies

OpenAIRE

Gal-Tanamy, Meital; Zemel, Romy; Bachmatov, Larissa; Jangra, Rohit K.; Shapira, Assaf; Villanueva, Rodrigo; Yi, MinKyung; Lemon, Stanley M.; Benhar, Itai; Tur-Kaspa, Ran

2010-01-01

Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-tra...

Rapid method for measuring protease activity in milk using radiolabeled casein

International Nuclear Information System (INIS)

Christen, G.L.

1987-01-01

A rapid means to detect the presence of protease activity in raw milk could be useful in predicting keeping ability of products made from that milk. A 30-min assay has been developed and compared with three other methods of detecting protease. Casein, [methyl- 14 C]-methylated-alpha was purchased from a radioisotope supplier. Concentrations of substrate from 2 to 20 nCi gave counts per minute, which increased linearly when counted with the Charm analyzer. There was not a significant difference in counting times of 10, 20, or 30 min. A mixture of sodium acetate and acetic acid precipitated nonhydrolyzed substrate with an efficiency of 97%. Comparison of the [ 14 C] casein assay, a casein fluorescein isothiocyanate assay, trinitrobenzenesulfonic acid procedure, and the Hull procedure using protease from psychrotrophic bacteria revealed that the [ 14 C] casein and casein fluorescein isothiocyanate methods were roughly equivalent and that the radiometric procedure was 10 times more sensitive than the trinitrobenzenesulfonic acid assay. The radiometric procedure was approximately 10(4) times more sensitive than the Hull procedure. The [ 14 C] casein and casein fluorescein isothiocyanate methods were similar in time required, about 30 min, while the trinitrobenzenesulfonic acid assay and Hull method required about 1 h plus reagent preparation time. The [ 14 C] casein procedure was most expensive per test; the other three were cheaper and similar to each other in cost

Lectin, hemolysin and protease inhibitors in seed fractions with ovicidal activity against Haemonchus contortus.

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Salles, Hévila Oliveira; Braga, Ana Carolina Linhares; Nascimento, Maria Thayana dos Santos Canuto do; Sousa, Ana Márjory Paiva; Lima, Adriano Rodrigues; Vieira, Luiz da Silva; Cavalcante, Antônio Cézar Rocha; Egito, Antonio Silvio do; Andrade, Lúcia Betânia da Silva

2014-01-01

Bioactive molecules of plant species are promising alternatives for the chemical control of gastrointestinal nematodes in ruminants. Extracts of native and exotic seed species from Brazil's semi-arid region were tested in vitro in an egg hatch assay and the bioactivity of their proteins was investigated. Each seed species was subjected to three extractions with three types of solvents. All the seeds showed ovicidal activity, which varied according to the solvents. Higher ovicidal activity was found in the molecule fractions of low molecular weight (Albizia lebbeck, Ipomoea asarifolia, Jatropha curcas, Libidibia ferrea, Moringa oleifera and Ricinus communis (P0.05, Bonferroni test). Hemagglutinating activity was detected in the fractions of C. spectabilis and M. oleifera fractions, hemolysin activity in the A. lebbeck and M. oleifera fractions, serine protease inhibitory activity in the A. lebbeck, I. asarifolia, J. curcas, M. oleifera and R. communis fractions, cysteine protease inhibitor activity in the M. oleifera fraction, and no protein activity in the L. ferrea fraction. The results of this work reveal new plant species with a potential for use in controlling nematode parasites in goats, thus opening a new field of research involving plant protein molecules with ovicidal properties.

Lectin, hemolysin and protease inhibitors in seed fractions with ovicidal activity against Haemonchus contortus

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Hévila Oliveira Salles

Full Text Available Bioactive molecules of plant species are promising alternatives for the chemical control of gastrointestinal nematodes in ruminants. Extracts of native and exotic seed species from Brazil's semi-arid region were tested in vitro in an egg hatch assay and the bioactivity of their proteins was investigated. Each seed species was subjected to three extractions with three types of solvents. All the seeds showed ovicidal activity, which varied according to the solvents. Higher ovicidal activity was found in the molecule fractions of low molecular weight (0.05, Bonferroni test. Hemagglutinating activity was detected in the fractions of C. spectabilis and M. oleifera fractions, hemolysin activity in the A. lebbeck and M. oleifera fractions, serine protease inhibitory activity in the A. lebbeck, I. asarifolia, J. curcas, M. oleifera and R. communis fractions, cysteine protease inhibitor activity in the M. oleifera fraction, and no protein activity in the L. ferrea fraction. The results of this work reveal new plant species with a potential for use in controlling nematode parasites in goats, thus opening a new field of research involving plant protein molecules with ovicidal properties.

Hepatitis C virus protease inhibitor-resistance mutations: our experience and review.

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Wu, Shuang; Kanda, Tatsuo; Nakamoto, Shingo; Imazeki, Fumio; Yokosuka, Osamu

2013-12-21

Direct-acting antiviral agents (DAAs) for hepatitis C virus (HCV) infection are one of the major advances in its medical treatment. The HCV protease inhibitors boceprevir and telaprevir were the first approved DAAs in the United States, Europe, and Japan. When combined with peginterferon plus ribavirin, these agents increase sustained virologic response rates to 70%-80% in treatment-naïve patients and previous-treatment relapsers with chronic HCV genotype 1 infection. Without peginterferon plus ribavirin, DAA mono-therapies increased DAA-resistance mutations. Several new DAAs for HCV are now in clinical development and are likely to be approved in the near future. However, it has been reported that the use of these drugs also led to the emergence of DAA-resistance mutations in certain cases. Furthermore, these mutations exhibit cross-resistance to multiple drugs. The prevalence of DAA-resistance mutations in HCV-infected patients who were not treated with DAAs is unknown, and it is as yet uncertain whether such variants are sensitive to DAAs. We performed a population sequence analysis to assess the frequency of such variants in the sera of HCV genotype 1-infected patients not treated with HCV protease inhibitors. Here, we reviewed the literature on resistance variants of HCV protease inhibitors in treatment naïve patients with chronic HCV genotype 1, as well as our experience.

Characterization of an alkaline protease associated with a granulosis virus of Plodia interpunctella.

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Tweeten, K A; Bulla, L A; Consigli, R A

1978-06-01

An alkaline protease was found to be associated with the granulosis virus of the Indian meal moth. Plodia interpunctella. The protease was located within the protein matrix of the occluded virus and hydrolyzed the major constituent of this matrix, a 28,000-dalton protein (granulin), to a mixture of polypeptides ranging in molecular weight from 10,000 to 27,000. A rapid, sensitive assay for the protease was developed using radioactively labeled granulosis virus as substrate. With this assay, the proteolytic activity could be detected by measuring the release of acid-soluble peptides from the labeled virus. The protease had a pH optimum of 10.5 and a temperature optimum of 40 degrees C and was inhibited by diisopropyl phosphorofluoridate, phenylmethylsulfonyl fluoride, and L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone. Purification of the protease from matrix protein was achieved by anion-exchange and gel permeation chromatography. The molecular weight of the isolated protease, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, was approximately 14,000.

Hepatitis C Virus NS3/4A Protease Inhibitors: A Light at the End of the Tunnel

Science.gov (United States)

Chatel-Chaix, Laurent; Baril, Martin; Lamarre, Daniel

2010-01-01

Hepatitis C virus (HCV) infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease) that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection. PMID:21994705

Hepatitis C Virus NS3/4A Protease Inhibitors: A Light at the End of the Tunnel

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Laurent Chatel-Chaix

2010-08-01

Full Text Available Hepatitis C virus (HCV infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection.

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

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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

Structure of the Integral Membrane Protein CAAX Protease Ste24p

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Pryor Jr., Edward E. [Membrane Protein Structural Biology Consortium (United States); Univ. of Virginia, Charlottesville, VA (United States); Horanyi, Peter S. [Membrane Protein Structural Biology Consortium (United States); Univ. of Virginia, Charlottesville, VA (United States); Clark, Kathleen M. [Membrane Protein Structural Biology Consortium (United States); Univ. of Rochester School of Medicine and Dentistry, Rochester, NY (United States); Fedoriw, Nadia [Membrane Protein Structural Biology Consortium (United States); Univ. of Rochester School of Medicine and Dentistry, Rochester, NY (United States); Connelly, Sara M. [Membrane Protein Structural Biology Consortium (United States); Univ. of Rochester School of Medicine and Dentistry, Rochester, NY (United States); Koszelak-Rosenblum, Mary [Membrane Protein Structural Biology Consortium (United States); Hauptman-Woodward Inst., Buffalo, NY (United States); Zhu, Guangyu [Membrane Protein Structural Biology Consortium (United States); Hauptman-Woodward Inst., Buffalo, NY (United States); Malkowski, Michael G. [Membrane Protein Structural Biology Consortium (United States); Hauptman-Woodward Inst., Buffalo, NY (United States); State Univ. of New York, Buffalo, NY (United States); Wiener, Michael C. [Membrane Protein Structural Biology Consortium (United States); Univ. of Virginia, Charlottesville, VA (United States); Dumont, Mark E. [Membrane Protein Structural Biology Consortium (United States); Univ. of Rochester School of Medicine and Dentistry, Rochester, NY (United States)

2012-10-26

Posttranslational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CAAX sequence motifs (where A is an aliphatic residue and X is any residue). Isoprenylation is followed by cleavage of the AAX amino acid residues and, in some cases, by additional proteolytic cuts. We determined the crystal structure of the CAAX protease Ste24p, a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a -factor. The Ste24p core structure is a ring of seven transmembrane helices enclosing a voluminous cavity containing the active site and substrate-binding groove. The cavity is accessible to the external milieu by means of gaps between splayed transmembrane helices. We hypothesize that cleavage proceeds by means of a processive mechanism of substrate insertion, translocation, and ejection.

Per a 10 protease activity modulates CD40 expression on dendritic cell surface by nuclear factor-kappaB pathway.

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Goel, C; Kalra, N; Dwarakanath, B S; Gaur, S N; Arora, N

2015-05-01

Serine protease activity of Per a 10 from Periplaneta americana modulates dendritic cell (DC) functions by a mechanism(s) that remains unclear. In the present study, Per a 10 protease activity on CD40 expression and downstream signalling was evaluated in DCs. Monocyte-derived DCs from cockroach-allergic patients were treated with proteolytically active/heat-inactivated Per a 10. Stimulation with active Per a 10 demonstrated low CD40 expression on DCs surface (P Per a 10, suggesting cleavage of CD40. Per a 10 activity reduced the interleukin (IL)-12 and interferon (IFN)-γ secretion by DCs (P Per a 10, indicating that low CD40 expression is associated with low levels of IL-12 secretion. Active Per a 10 stimulation caused low nuclear factor-kappa B (NF-κB) activation in DCs compared to heat-inactivated Per a 10. Inhibition of the NF-κB pathway suppressed the CD40 expression and IL-12 secretion by DCs, further indicating that NF-κB is required for CD40 up-regulation. CD40 expression activated the tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), thereby suggesting its involvement in NF-κB activation. Protease activity of Per a 10 induced p38 mitogen-activated protein kinase (MAPK) activation that showed no significant effect on CD40 expression by DCs. However, inhibiting p38 MAPK or NF-κB suppressed the secretion of IL-12, IFN-γ, IL-6 and TNF-α by DCs. Such DCs further reduced the secretion of IL-4, IL-6, IL-12 and TNF-α by CD4(+) T cells. In conclusion, protease activity of Per a 10 reduces CD40 expression on DCs. CD40 down-regulation leads to low NF-κB levels, thereby modulating DC-mediated immune responses. © 2014 British Society for Immunology.

Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli

Science.gov (United States)

2012-01-01

Background Escherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli. Results Because the redox enzymes can reduce the disulfide that forms on proteins, we first tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coliL-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (ΔcysI and ΔcysJ) and the L-cysteine synthase gene (ΔcysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (ΔcysC or ΔcysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coliL-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell. Conclusions In this work, we showed that Grx1 and

Statistical medium optimization of an alkaline protease from Pseudomonas aeruginosa MTCC 10501, its characterization and application in leather processing.

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Boopathy, Naidu Ramachandra; Indhuja, Devadas; Srinivasan, Krishnan; Uthirappan, Mani; Gupta, Rishikesh; Ramudu, Kamini Numbi; Chellan, Rose

2013-04-01

Proteases are shown to have greener mode of application in leather processing for dehairing of goat skins and cow hides. Production of protease by submerged fermentation with potent activity is reported using a new isolate P. aeruginosa MTCC 10501. The production parameters were optimized by statistical methods such as Plackett-Burman and response surface methodology. The optimized production medium contained (g/L); tryptone, 2.5; yeast extract, 3.0; skim milk 30.0; dextrose 1.0; inoculum concentration 4%: initial pH 6.0; incubation temperature 30 degrees C and optimum production at 48 h with protease activity of 7.6 U/mL. The protease had the following characteristics: pH optima, 9.0; temperature optima 50 degrees C; pH stability between 5.0-10.0 and temperature stability between 10-40 degrees C. The protease was observed to have high potential for dehairing of goat skins in the pre- tanning process comparable to that of the chemical process as evidenced by histology. The method offers cleaner processing using enzyme only instead of toxic chemicals in the pre-tanning process of leather manufacture.

Studies on the Catalytic Properties of Partially Purified Alkaline Proteases from Some Selected Microorganisms

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Titilayo Olufunke Femi-Ola

2012-09-01

Full Text Available Aims: The research was done to study the conditions enhancing catalytic activities of alkaline proteases from Vibro sp., Lactobacillus brevis, Zymomonas sp., Athrobacter sp., Corynebacterium sp. and Bacillus subtilis.Methodology and Results: The proteolytic enzymes were purified in 2-step procedures involving ammonium sulphate precipitation and sephadex G-150 gel permeation chromatography. The upper and lower limits for the specific activities of proteases from the selected microorganisms were estimated at 20.63 and 47.51 units/mg protein with Zymomonas protease having the highest specific activity towards casein as its substrate and purification fold of 3.46, while that ofLactobacillus brevis protease was 8.06. The native molecular weights of these active proteins ranged from 30.4 to 45.7 kDa with Athrobacter sp. protease having the highest weight for its subunits. The proteolytic enzymes had optimum pH range of 8 to 10 and temperature range of 50 to 62 ºC accounting for the percentage relative activity range of 75 to 94% and 71 to 84 % respectively. The activities of Lactobacillus brevis and Bacillus subtilis proteases were maximum at pH 9 and 10 respectively. Lactobacillus brevis protease activity was maximum at temperature of 62 ºC, while beyond this value, a general thermal instability of these active proteins was observed. At above 70 ºC, the catalytic activities of Corynebacterium sp., Vibrio sp., Zymomonas sp. and Arthrobacter sp. proteases were progressively reduced over a period of 120 min of incubation, while Bacillus subtlis and Lactobacillus brevis proteases were relatively stable. Effect of metal ions was investigated on the catalytic activity of protease from the microorganisms. Lactobacillus brevis,Zymomonas sp., Arthrobacter sp., Corynebacterium sp. and Bacillus subtilis protease activities were strongly activated by metal ions such as Ca+2 and Mg+2. Enzyme activities were inhibited strongly by Cu2+ and Hg2+ but were not

Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

DEFF Research Database (Denmark)

Ullah, Raheem; Shah, Majid Ali; Tufail, Soban

2016-01-01

stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious...

Determination of free and total cyst(e)ine in plasma of dogs and cats.

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Tôrres, Cristina L; Miller, Joshua W; Rogers, Quinton R

2004-01-01

In human blood, the amino acid cysteine forms disulfide bonds with itself and with other sulfhydryl compounds in their free form and with sulfhydryls in protein. Protein-bound cysteine is lost when plasma proteins are removed before amino acid analysis. The purpose of this study was to assess the time course and extent of cyst(e)ine (cysteine + half-cystine) loss in dog and cat plasma. An equal volume of 6% sulfosalicylic acid was added to plasma aliquots at 0, 2, 4, 10, 16, 24, 36, 48, 60, and 72 hours after separation of blood cells. Tris-2-carboxyethyl-phosphine hydrochloride (TCEP - HCl), a reducing agent, was used to regenerate total plasma cyst(e)ine after 3 months of sample storage (-20 degrees C). Initial free cyst(e)ine concentrations (mean +/- SEM) were higher in canine plasma (77 +/- 4 micromol/L) than in feline plasma (37 +/- 3 micromol/L). Free plasma cyst(e)ine concentrations in dogs and cats decreased after first-order kinetics, with a half-life of 23 and 69 hours, respectively. Total plasma cysteine after TCEP - HCl treatment was similar for dogs (290 micromol/L) and cats (296 micromol/L), but the percentage of free cysteine was higher (P = .02) in dogs (27%) than in cats (13%). Over half of the cyst(e)ine, homocysteine, cysteinylglycine, and glutathione were bound in vivo to plasma proteins. These results emphasize the importance of removing plasma proteins within 1 hour after blood collection for reliable assay of free plasma cyst(e)ine.

Renal cysteine conjugate C-S lyase mediated toxicity of halogenated alkenes in primary cultures of human and rat proximal tubular cells.

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McGoldrick, Trevor A; Lock, Edward A; Rodilla, Vicente; Hawksworth, Gabrielle M

2003-07-01

Proximal tubular cells from human (HPT) and rat (RPT) kidneys were isolated, grown to confluence and incubated with S-(1,2-dichlorovinyl)- l-cysteine (DCVC), S-(1,2,2-trichlorovinyl)- l-cysteine (TCVC), S-(1,1,2,2-tetrafluoroethyl)- l-cysteine (TFEC) and S-(2-chloro-1,1-difluorethyl)- l-cysteine (CDFEC), the cysteine conjugates of nephrotoxicants. The cultures were exposed to the conjugates for 12, 24 and 48 h and the toxicity determined using the MTT assay. All four conjugates caused dose-dependent toxicity to RPT cells over the range 50-1,000 microM, the order of toxicity being DCVC>TCVC>TFEC=CDFEC. The inclusion of aminooxyacetic acid (AOAA; 250 microM), an inhibitor of pyridoxal phosphate-dependent enzymes such as C-S lyase, afforded protection, indicating that C-S lyase has a role in the bioactivation of these conjugates. In HPT cultures only DCVC caused significant time- and dose-dependent toxicity. Exposure to DCVC (500 microM) for 48 h decreased cell viability to 7% of control cell values, whereas co-incubation of DCVC (500 microM) with AOAA (250 microM) resulted in cell viability of 71%. Human cultures were also exposed to S-(1,2-dichlorovinyl)-glutathione (DCVG). DCVG was toxic to HPT cells, but the onset of toxicity was delayed compared with the corresponding cysteine conjugate. AOAA afforded almost complete protection from DCVG toxicity. Acivicin (250 microM), an inhibitor of gamma-glutamyl transferase (gamma-GT), partially protected against DCVG (500 microM)-induced toxicity at 48 h (5% viability and 53% viability in the absence and presence of acivicin, respectively). These results suggest that DCVG requires processing by gamma-GT prior to bioactivation by C-S lyase in HPT cells. The activity of C-S lyase, using TFEC as a substrate, and glutamine transaminase K (GTK) was measured in rat and human cells with time in culture. C-S lyase activity in RPT and HPT cells decreased to approximately 30% of fresh cell values by the time the cells reached

Enterovirus 71 3C protease cleaves a novel target CstF-64 and inhibits cellular polyadenylation.

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Kuo-Feng Weng

2009-09-01

Full Text Available Identification of novel cellular proteins as substrates to viral proteases would provide a new insight into the mechanism of cell-virus interplay. Eight nuclear proteins as potential targets for enterovirus 71 (EV71 3C protease (3C(pro cleavages were identified by 2D electrophoresis and MALDI-TOF analysis. Of these proteins, CstF-64, which is a critical factor for 3' pre-mRNA processing in a cell nucleus, was selected for further study. A time-course study to monitor the expression levels of CstF-64 in EV71-infected cells also revealed that the reduction of CstF-64 during virus infection was correlated with the production of viral 3C(pro. CstF-64 was cleaved in vitro by 3C(pro but neither by mutant 3C(pro (in which the catalytic site was inactivated nor by another EV71 protease 2A(pro. Serial mutagenesis was performed in CstF-64, revealing that the 3C(pro cleavage sites are located at position 251 in the N-terminal P/G-rich domain and at multiple positions close to the C-terminus of CstF-64 (around position 500. An accumulation of unprocessed pre-mRNA and the depression of mature mRNA were observed in EV71-infected cells. An in vitro assay revealed the inhibition of the 3'-end pre-mRNA processing and polyadenylation in 3C(pro-treated nuclear extract, and this impairment was rescued by adding purified recombinant CstF-64 protein. In summing up the above results, we suggest that 3C(pro cleavage inactivates CstF-64 and impairs the host cell polyadenylation in vitro, as well as in virus-infected cells. This finding is, to our knowledge, the first to demonstrate that a picornavirus protein affects the polyadenylation of host mRNA.

Inhibition of protease-inhibitor resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies

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Gal-Tanamy, Meital; Zemel, Romy; Bachmatov, Larissa; Jangra, Rohit K.; Shapira, Assaf; Villanueva, Rodrigo; Yi, MinKyung; Lemon, Stanley M.; Benhar, Itai; Tur-Kaspa, Ran

2015-01-01

Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons resistant to small molecule inhibitors of the NS3/4A protease, and replicons derived from different HCV genotypes. The combination of intrabodies and interferon-α had an additive inhibitory effect on RNA replication in the replicon model. Intrabody expression also inhibited production of infectious HCV in a cell culture system. The NS3 protease activity was inhibited by the intrabodies in NS3-expressing cells. In contrast, cell-free synthesis of HCV RNA by preformed replicase complexes was not inhibited by intrabodies, suggesting that the major mode of inhibition of viral replication is inhibition of NS3/4A protease activity and subsequent suppression of viral polyprotein processing. PMID:20705106

Structure Determination of Mycobacterium tuberculosis Serine Protease Hip1 (Rv2224c)

Energy Technology Data Exchange (ETDEWEB)

Naffin-Olivos, Jacqueline L.; Daab, Andrew; White, Andre; Goldfarb, Nathan E.; Milne, Amy C.; Liu, Dali; Baikovitz, Jacqueline; Dunn, Ben M.; Rengarajan, Jyothi; Petsko, Gregory A.; Ringe, Dagmar

2017-04-07

The Mycobacterium tuberculosis (Mtb) serine protease Hip1 (hydrolase important for pathogenesis; Rv2224c) promotes tuberculosis (TB) pathogenesis by impairing host immune responses through proteolysis of a protein substrate, Mtb GroEL2. The cell surface localization of Hip1 and its immunomodulatory functions make Hip1 a good drug target for new adjunctive immune therapies for TB. Here, we report the crystal structure of Hip1 to a resolution of 2.6 Å and the kinetic studies of the enzyme against model substrates and the protein GroEL2. The structure shows a two-domain protein, one of which contains the catalytic residues that are the signature of a serine protease. Surprisingly, a threonine is located within the active site close enough to hydrogen bond with the catalytic residues Asp463 and His490. Mutation of this residue, Thr466, to alanine established its importance for function. Our studies provide insights into the structure of a member of a novel family of proteases. Knowledge of the Hip1 structure will aid in designing inhibitors that could block Hip1 activity

The macromolecular complex of ICP and falcipain-2 from Plasmodium: preparation, crystallization and preliminary X-ray diffraction analysis

International Nuclear Information System (INIS)

Hansen, Guido; Schwarzloh, Britta; Rennenberg, Annika; Heussler, Volker T.; Hilgenfeld, Rolf

2011-01-01

The macromolecular complex of ICP (inhibitor of cysteine proteases) from P. berghei and falcipain-2 from P. falciparum has been prepared and crystallized, and a diffraction data set has been collected to a resolution of 2.6 Å. The malaria parasite Plasmodium depends on the tight control of cysteine-protease activity throughout its life cycle. Recently, the characterization of a new class of potent inhibitors of cysteine proteases (ICPs) secreted by Plasmodium has been reported. Here, the recombinant production, purification and crystallization of the inhibitory C-terminal domain of ICP from P. berghei in complex with the P. falciparum haemoglobinase falcipain-2 is described. The 1:1 complex was crystallized in space group P4 3 , with unit-cell parameters a = b = 71.15, c = 120.09 Å. A complete diffraction data set was collected to a resolution of 2.6 Å

The molecular basis of drug resistance against hepatitis C virus NS3/4A protease inhibitors.

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Keith P Romano

Full Text Available Hepatitis C virus (HCV infects over 170 million people worldwide and is the leading cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer. Available antiviral therapies cause severe side effects and are effective only for a subset of patients, though treatment outcomes have recently been improved by the combination therapy now including boceprevir and telaprevir, which inhibit the viral NS3/4A protease. Despite extensive efforts to develop more potent next-generation protease inhibitors, however, the long-term efficacy of this drug class is challenged by the rapid emergence of resistance. Single-site mutations at protease residues R155, A156 and D168 confer resistance to nearly all inhibitors in clinical development. Thus, developing the next-generation of drugs that retain activity against a broader spectrum of resistant viral variants requires a comprehensive understanding of the molecular basis of drug resistance. In this study, 16 high-resolution crystal structures of four representative protease inhibitors--telaprevir, danoprevir, vaniprevir and MK-5172--in complex with the wild-type protease and three major drug-resistant variants R155K, A156T and D168A, reveal unique molecular underpinnings of resistance to each drug. The drugs exhibit differential susceptibilities to these protease variants in both enzymatic and antiviral assays. Telaprevir, danoprevir and vaniprevir interact directly with sites that confer resistance upon mutation, while MK-5172 interacts in a unique conformation with the catalytic triad. This novel mode of MK-5172 binding explains its retained potency against two multi-drug-resistant variants, R155K and D168A. These findings define the molecular basis of HCV N3/4A protease inhibitor resistance and provide potential strategies for designing robust therapies against this rapidly evolving virus.

Viral kinetics in patients with chronic hepatitis C treated with the serine protease inhibitor BILN 2061

NARCIS (Netherlands)

Herrmann, Eva; Zeuzem, Stefan; Sarrazin, Christoph; Hinrichsen, Holger; Benhamou, Yves; Manns, Michael P.; Reiser, Markus; Reesink, Henk; Calleja, José L.; Forns, Xavier; Steinmann, Gerhard G.; Nehmiz, Gerhard

2006-01-01

We analysed viral kinetics from a 2-day treatment with BILN 2061, a serine protease inhibitor of hepatitis C virus, in patients chronically infected with genotype 1 hepatitis C virus. The efficiency (E), describing inhibition of viral production, was above 99.45% in all patients with minor or

Ultrahigh and High Resolution Structures and Mutational Analysis of Monomeric Streptococcus pyogenes SpeB Reveal a Functional Role for the Glycine-rich C-terminal Loop

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González-Páez, Gonzalo E.; Wolan, Dennis W. (Scripps)

2012-09-05

Cysteine protease SpeB is secreted from Streptococcus pyogenes and has been studied as a potential virulence factor since its identification almost 70 years ago. Here, we report the crystal structures of apo mature SpeB to 1.06 {angstrom} resolution as well as complexes with the general cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and a novel substrate mimetic peptide inhibitor. These structures uncover conformational changes associated with maturation of SpeB from the inactive zymogen to its active form and identify the residues required for substrate binding. With the use of a newly developed fluorogenic tripeptide substrate to measure SpeB activity, we determined IC{sub 50} values for trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and our new peptide inhibitor and the effects of mutations within the C-terminal active site loop. The structures and mutational analysis suggest that the conformational movements of the glycine-rich C-terminal loop are important for the recognition and recruitment of biological substrates and release of hydrolyzed products.

Reconstruction of Cysteine Biosynthesis Using Engineered Cysteine-Free and Methionine-Free Enzymes

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Wang, Kendrick; Fujishima, Kosuke; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J.

2016-01-01

Ten of the proteinogenic amino acids can be generated abiotically while the remaining thirteen require biology for their synthesis. Paradoxically, the biosynthesis pathways observed in nature require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine. Here, we substituted alternate amino acids for cysteine and also methionine, which is biosynthesized from cysteine, in serine acetyl transferase (CysE) and O-acetylserine sulfhydrylase (CysM). CysE function was rescued by cysteine-and-methionine-free enzymes and CysM function was rescued by cysteine-free enzymes. Structural modeling suggests that methionine stabilizes CysM and is present in the active site of CysM. Cysteine is not conserved among CysE and CysM protein orthologs, suggesting that cysteine is not functionally important for its own synthesis. Engineering biosynthetic enzymes that lack the amino acids being synthesized provides insights into the evolution of amino acid biosynthesis and pathways for bioengineering.

THERMOPHILIC BACILLUS LICHENIFORMIS RBS 5 ISOLATED FROM HOT TUNISIAN SPRING CO-PRODUCING ALKALINE AND THERMOSTABLE α-AMYLASE AND PROTEASE ENZYMES

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Rakia Ben Salem

2016-06-01

Full Text Available Bacillus licheniformis RBS 5 was isolated from thermal spring in Tunisia. The isolate coproduce α-amylase and protease enzymes. The α-amylase activity showed an optimal activity at approximately 65°C and in wide pH interval ranging from 4 to 9. This enzyme was stable over the range of 45 to 70°C after 30 min of incubation and in the pH range of 8 to 10. Protease activity was optimal; at 80°C, pH 12. This enzyme was stable until 60°C over the pH range of 10 to 12. EDTA at concentration of 5 mM reduces slightly both activities evoking the serine alkaline protease. Cationic ions (Ca2+, Cu2+, Zn2+, and Mg 2+ have an inhibition effect on α-amylase. However, protease activity was enhanced by Ca2+, Cu2+ and Mg 2+; the other cations reduce slightly the proteolytic activity. SDS and H2O2 were found as inhibitors for both activities whereas Triton X-100 and perfume have no effect. Taken together, these traits make protease activity of B. licheniformis RBS 5 as efficient for use in detergent industry.

Modulating effects of the protease inhibitor Antipain on x-ray induced transformations

International Nuclear Information System (INIS)

Borek, C.; Miller, R.C.

1979-01-01

Protease inhibitors have been shown to inhibit the expression of mutations in bacteria and to inhibit the tumor-promoting effect of phorbol esters in mice. We have investigated the effect of the protease inhibitor Antipain on cell transformation by x-irradiation in two in vitro systems; namely short-term cultures of freshly explanted hamster embryo cells and in the 10T1/2 cell line derived and cloned from C3H mouse embryo

Cysteine Cathepsins in the secretory vesicle produce active peptides: Cathepsin L generates peptide neurotransmitters and cathepsin B produces beta-amyloid of Alzheimer's disease.

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Hook, Vivian; Funkelstein, Lydiane; Wegrzyn, Jill; Bark, Steven; Kindy, Mark; Hook, Gregory

2012-01-01

Recent new findings indicate significant biological roles of cysteine cathepsin proteases in secretory vesicles for production of biologically active peptides. Notably, cathepsin L in secretory vesicles functions as a key protease for proteolytic processing of proneuropeptides (and prohormones) into active neuropeptides that are released to mediate cell-cell communication in the nervous system for neurotransmission. Moreover, cathepsin B in secretory vesicles has been recently identified as a β-secretase for production of neurotoxic β- amyloid (Aβ) peptides that accumulate in Alzheimer's disease (AD), participating as a notable factor in the severe memory loss in AD. These secretory vesicle functions of cathepsins L and B for production of biologically active peptides contrast with the well-known role of cathepsin proteases in lysosomes for the degradation of proteins to result in their inactivation. The unique secretory vesicle proteome indicates proteins of distinct functional categories that provide the intravesicular environment for support of cysteine cathepsin functions. Features of the secretory vesicle protein systems insure optimized intravesicular conditions that support the proteolytic activity of cathepsins. These new findings of recently discovered biological roles of cathepsins L and B indicate their significance in human health and disease. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. Copyright © 2011 Elsevier B.V. All rights reserved.

Expression and characterization of plant aspartic protease nepenthesin-1 from Nepenthes gracilis.

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Kadek, Alan; Tretyachenko, Vyacheslav; Mrazek, Hynek; Ivanova, Ljubina; Halada, Petr; Rey, Martial; Schriemer, David C; Man, Petr

2014-03-01

Carnivorous plants of the genus Nepenthes produce their own aspartic proteases, nepenthesins, to digest prey trapped in their pitchers. Nepenthesins differ significantly in sequence from other aspartic proteases in the animal or even plant kingdoms. This difference, which also brings more cysteine residues into the structure of these proteases, can be a cause of uniquely high temperature and pH stabilities of nepenthesins. Their detailed structure characterization, however, has not previously been possible due to low amounts of protease present in the pitcher fluid and also due to limited accessibility of Nepenthes plants. In the present study we describe a convenient way for obtaining high amounts of nepenthesin-1 from Nepenthes gracilis using heterologous production in Escherichia coli. The protein can be easily refolded in vitro and its characteristics are very close to those described for a natural enzyme isolated from the pitcher fluid. Similarly to the natural enzyme, recombinant nepenthesin-1 is sensitive to denaturing and reducing agents. It also has maximal activity around pH 2.5, shows unusual stability at high pH and its activity is not irreversibly inhibited even after prolonged incubation in the basic pH range. On the other hand, temperature stability of the recombinant enzyme is lower in comparison with the natural enzyme, which can be attributed to missing N-glycosylation in the recombinant protein. Copyright © 2013 Elsevier Inc. All rights reserved.

Evaluation of the Effects of S-Allyl-L-cysteine, S-Methyl-L-cysteine, trans-S-1-Propenyl-L-cysteine, and Their N-Acetylated and S-Oxidized Metabolites on Human CYP Activities.

Science.gov (United States)

Amano, Hirotaka; Kazamori, Daichi; Itoh, Kenji

2016-01-01

Three major organosulfur compounds of aged garlic extract, S-allyl-L-cysteine (SAC), S-methyl-L-cysteine (SMC), and trans-S-1-propenyl-L-cysteine (S1PC), were examined for their effects on the activities of five major isoforms of human CYP enzymes: CYP1A2, 2C9, 2C19, 2D6, and 3A4. The metabolite formation from probe substrates for the CYP isoforms was examined in human liver microsomes in the presence of organosulfur compounds at 0.01-1 mM by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Allicin, a major component of garlic, inhibited CYP1A2 and CYP3A4 activity by 21-45% at 0.03 mM. In contrast, a CYP2C9-catalyzed reaction was enhanced by up to 1.9 times in the presence of allicin at 0.003-0.3 mM. SAC, SMC, and S1PC had no effect on the activities of the five isoforms, except that S1PC inhibited CYP3A4-catalyzed midazolam 1'-hydroxylation by 31% at 1 mM. The N-acetylated metabolites of the three compounds inhibited the activities of several isoforms to a varying degree at 1 mM. N-Acetyl-S-allyl-L-cysteine and N-acetyl-S-methyl-L-cysteine inhibited the reactions catalyzed by CYP2D6 and CYP1A2, by 19 and 26%, respectively, whereas trans-N-acetyl-S-1-propenyl-L-cysteine showed weak to moderate inhibition (19-49%) of CYP1A2, 2C19, 2D6, and 3A4 activities. On the other hand, both the N-acetylated and S-oxidized metabolites of SAC, SMC, and S1PC had little effect on the reactions catalyzed by the five isoforms. These results indicated that SAC, SMC, and S1PC have little potential to cause drug-drug interaction due to CYP inhibition or activation in vivo, as judged by their minimal effects (IC 50 >1 mM) on the activities of five major isoforms of human CYP in vitro.

SwissProt search result: AK242832 [KOME

Lifescience Database Archive (English)

Full Text Available AK242832 J090067L13 (Q8C9S8) Cysteine protease APG4A (EC 3.4.22.-) (Autophagy 4 homolog A) (Auto...phagin-2) (Autophagy-related cysteine endopeptidase 2) (AUT-like 2 cysteine endopeptidase) APG4A_MOUSE 2e-47 ...

SwissProt search result: AK069012 [KOME

Lifescience Database Archive (English)

Full Text Available AK069012 J023002M16 (Q8C9S8) Cysteine protease APG4A (EC 3.4.22.-) (Autophagy 4 homolog A) (Auto...phagin-2) (Autophagy-related cysteine endopeptidase 2) (AUT-like 2 cysteine endopeptidase) APG4A_MOUSE 4e-48 ...

Boceprevir: a protease inhibitor for the treatment of hepatitis C.

Science.gov (United States)

Chang, Mei H; Gordon, Lori A; Fung, Horatio B

2012-10-01

Boceprevir is a protease inhibitor indicated for the treatment of chronic hepatitis C virus (HCV) genotype 1 infection in combination with peginterferon and ribavirin for treatment-naive patients and those who previously failed to improve with interferon and ribavirin treatment. This article provides an overview of the mechanism of action, pharmacologic and pharmacokinetic properties, clinical efficacy, and tolerability of boceprevir. Relevant information was identified through a search of PubMed (1990-July 2012), EMBASE (1990-July 2012), International Pharmaceutical Abstracts (1970-July 2012), and Google Scholar using the key words boceprevir, SCH 503034, non-structural protein 3 (NS3) serine protease inhibitor, and direct-acting antiviral agent (DAA). Additional information was obtained from the US Food and Drug Administration's Web site, review of the reference lists of identified articles, and posters and abstracts from scientific meetings. Clinical efficacy of boceprevir was assessed in 2 Phase III trials, Serine Protease Inhibitor Therapy-2 (SPRINT-2) for treatment-naive patients and Retreatment with HCV Serine Protease Inhibitor Boceprevir and PegIntron/Rebetol 2 (RESPOND-2) for treatment-experienced patients. In SPRINT-2, patients were randomized to receive peginterferon + ribavirin (PR) or peginterferon + ribavirin + boceprevir (PRB); duration of boceprevir therapy varied from 24, 32, to 44 weeks on the basis of HCV RNA results. The primary endpoint was achievement of sustained virologic response (SVR; lower limit of detection, 9.3 IU/mL). The addition of boceprevir was shown to be superior, with overall SVR rates ranging from 63% to 66% compared with 38% with PR (P < 0.001). Results of SVR in SPRINT-2 were also reorganized to monitor SVRs in black and non-black patients. Treatment-experienced patients were assessed in RESPOND-2; however, null responders were excluded. Patients were again randomized to PR or PRB; duration of boceprevir therapy varied from

Protease-Mediated Suppression of DRG Neuron Excitability by Commensal Bacteria.

Science.gov (United States)

Sessenwein, Jessica L; Baker, Corey C; Pradhananga, Sabindra; Maitland, Megan E; Petrof, Elaine O; Allen-Vercoe, Emma; Noordhof, Curtis; Reed, David E; Vanner, Stephen J; Lomax, Alan E

2017-11-29

Peripheral pain signaling reflects a balance of pronociceptive and antinociceptive influences; the contribution by the gastrointestinal microbiota to this balance has received little attention. Disorders, such as inflammatory bowel disease and irritable bowel syndrome, are associated with exaggerated visceral nociceptive actions that may involve altered microbial signaling, particularly given the evidence for bacterial dysbiosis. Thus, we tested whether a community of commensal gastrointestinal bacteria derived from a healthy human donor (microbial ecosystem therapeutics; MET-1) can affect the excitability of male mouse DRG neurons. MET-1 reduced the excitability of DRG neurons by significantly increasing rheobase, decreasing responses to capsaicin (2 μm) and reducing action potential discharge from colonic afferent nerves. The increase in rheobase was accompanied by an increase in the amplitude of voltage-gated K + currents. A mixture of bacterial protease inhibitors abrogated the effect of MET-1 effects on DRG neuron rheobase. A serine protease inhibitor but not inhibitors of cysteine proteases, acid proteases, metalloproteases, or aminopeptidases abolished the effects of MET-1. The serine protease cathepsin G recapitulated the effects of MET-1 on DRG neurons. Inhibition of protease-activated receptor-4 (PAR-4), but not PAR-2, blocked the effects of MET-1. Furthermore, Faecalibacterium prausnitzii recapitulated the effects of MET-1 on excitability of DRG neurons. We conclude that serine proteases derived from commensal bacteria can directly impact the excitability of DRG neurons, through PAR-4 activation. The ability of microbiota-neuronal interactions to modulate afferent signaling suggests that therapies that induce or correct microbial dysbiosis may impact visceral pain. SIGNIFICANCE STATEMENT Commercially available probiotics have the potential to modify visceral pain. Here we show that secretory products from gastrointestinal microbiota derived from a human

Synthesis and Application of Aurophilic Poly(Cysteine and Poly(Cysteine-Containing Copolymers

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David Ulkoski

2017-10-01

Full Text Available The redox capacity, as well as the aurophilicity of the terminal thiol side groups, in poly(Cysteine lend a unique characteristic to this poly(amino acid or polypeptide. There are two major application fields for this polymer: (i biomedical applications in drug delivery and surface modification of biomedical devices and (ii as coating for electrodes to enhance their electrochemical sensitivity. The intended application determines the synthetic route for p(Cysteine. Polymers to be used in biomedical applications are typically polymerized from the cysteine N-carboxyanhydride by a ring-opening polymerization, where the thiol group needs to be protected during the polymerization. Advances in this methodology have led to conditions under which the polymerization progresses as living polymerization, which allows for a strict control of the molecular architecture, molecular weight and polydispersity and the formation of block copolymers, which eventually could display polyphilic properties. Poly(Cysteine used as electrode coating is typically polymerized onto the electrode by cyclic voltammetry, which actually produces a continuous, pinhole-free film on the electrode via the formation of covalent bonds between the amino group of Cysteine and the carbon of the electrode. This resulting coating is chemically very different from the well-defined poly(Cysteine obtained by ring-opening polymerizations. Based on the structure of cysteine a significant degree of cross-linking within the coating deposited by cyclic voltammetry can be assumed. This manuscript provides a detailed discussion of the ring-opening polymerization of cysteine, a brief consideration of the role of glutathione, a key cysteine-containing tripeptide, and examples for the utilization of poly(Cysteine and poly(Cysteine-containing copolymers, in both, the biomedical as well as electrochemical realm.

Detection of proteases from Sporosarcina aquimarina and Algoriphagus antarcticus isolated from Antarctic soil

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Anderson F. Santos

2015-03-01

Full Text Available Two psychrophilic bacterial samples were isolated from King George Island soil, in Antarctica. The phylogenetic analysis based on the 16S rRNA (rrs gene led to the correlation with the closest related isolates as Sporosarcina aquimarina (99% and Algoriphagus antarcticus(99%, with query coverage of 99% and 98%, respectively.The spent culture media from both isolates displayed proteolytic activities detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis containing gelatin as protein substrate. Under the employed conditions, S. aquimarina showed a 55 kDa protease with the best activity detected at pH 7.0 and at 27°C. A. antarcticusalso showed a single extracellular protease, however its molecular mass was around 90kDa and its best activity was detected at pH 9.0 and at 37°C. The proteases from both isolates were inhibited by 1,10-phenanthroline and EDTA, two metalloprotease inhibitors. This is the first record of protease detection in both species, and our results may contribute to broaden the basic knowledge of proteases from the Antarctica environment and may help prospecting future biotechnological applications of these enzymes.

Some Investigations on Protease Enzyme Production Kinetics Using Bacillus licheniformis BBRC 100053 and Effects of Inhibitors on Protease Activity

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Zahra Ghobadi Nejad

2014-01-01

Full Text Available Due to great commercial application of protease, it is necessary to study kinetic characterization of this enzyme in order to improve design of enzymatic reactors. In this study, mathematical modeling of protease enzyme production kinetics which is derived from Bacillus licheniformis BBRC 100053 was studied (at 37°C, pH 10 after 73 h in stationary phase, and 150 rpm. The aim of the present paper was to determine the best kinetic model and kinetic parameters for production of protease and calculating Ki (inhibition constant of different inhibitors to find the most effective one. The kinetic parameters Km (Michaelis-Menten constant and Vm (maximum rate were calculated 0.626 mM and 0.0523 mM/min. According to the experimental results, using DFP (diisopropyl fluorophosphate and PMSF (phenylmethanesulfonyl fluoride as inhibitors almost 50% of the enzyme activity could be inhibited when their concentrations were 0.525 and 0.541 mM, respectively. Ki for DFP and PMSF were 0.46 and 0.56 mM, respectively. Kinetic analysis showed that the Lineweaver-Burk model was the best fitting model for protease production kinetics DFP was more effective than PMSF and both of them should be covered in the group of noncompetitive inhibitors.

Lifespan extension and increased resistance to environmental stressors by N-Acetyl-L-Cysteine in Caenorhabditis elegans

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Seung-Il Oh

2015-05-01

Full Text Available OBJECTIVE: This study was performed to determine the effect of N-acetyl-L-cysteine, a modified sulfur-containing amino acid that acts as a strong cellular antioxidant, on the response to environmental stressors and on aging in C. elegans. METHOD: The survival of worms under oxidative stress conditions induced by paraquat was evaluated with and without in vivo N-acetyl-L-cysteine treatment. The effect of N-acetyl-L-cysteine on the response to other environmental stressors, including heat stress and ultraviolet irradiation (UV, was also monitored. To investigate the effect on aging, we examined changes in lifespan, fertility, and expression of age-related biomarkers in C. elegans after N-acetyl-L-cysteine treatment. RESULTS: Dietary N-acetyl-L-cysteine supplementation significantly increased resistance to oxidative stress, heat stress, and UV irradiation in C. elegans. In addition, N-acetyl-L-cysteine supplementation significantly extended both the mean and maximum lifespan of C. elegans. The mean lifespan was extended by up to 30.5% with 5 mM N-acetyl-L-cysteine treatment, and the maximum lifespan was increased by 8 days. N-acetyl-L-cysteine supplementation also increased the total number of progeny produced and extended the gravid period of C. elegans. The green fluorescent protein reporter assay revealed that expression of the stress-responsive genes, sod-3 and hsp-16.2, increased significantly following N-acetyl-L-cysteine treatment. CONCLUSION: N-acetyl-L-cysteine supplementation confers a longevity phenotype in C. elegans, possibly through increased resistance to environmental stressors.

The effect of L-cysteine on the portion-selective uptake of cadmium in the renal proximal tubule

International Nuclear Information System (INIS)

Murakami, Masataka; Sano, Kenichi; Webb, M.

1987-01-01

Cadmium (Cd), co-administered with an excess of L-cysteine, accumulates rapidly in the kidneys of the rat. After subcutaneous (s.c.) injection of 3 μmol CdCl 2 /kg body wt the concentrations of Cd in the blood and kidneys increase with the dose of cysteine over the range 0.06-5.0 mmol/kg body wt. At cysteine doses of less than 1.5 mmol/kg body wt the ratio of the concentrations of Cd in the outer medulla and cortex of the kidney remains the same as that after the injection of Cd alone. This ratio, however, is more than doubled at dose levels of 5-10 mmol cysteine/kg body wt. Hepatic uptake of Cd is unaffected by doses of cysteine below 1.5 mmol/kg body wt but decreases markedly at higher doses. In animals that are dosed simultaneously with 5 mmol cysteine/kg body wt, renal uptake of 109 Cd is known to occur in the straight segments of the proximal tubules. At a dose level of less than 1.5 mmol cysteine/kg body wt the present autoradiographical studies show that 109 Cd is taken up predominantly by the proximal convoluted tubules of the kidney cortex. At the critical dose level (1.5 mmol/kg body wt), cysteine decreases the retention of Cd at the s.c. injection site, but probably has little effect on the distribution of Cd between protein and other carrier molecules in the blood. This distribution, however, is altered at higher cysteine dose levels. It is suggested that, under the latter conditions, stable Cd-cysteine complexes are formed in the blood and are filtered readily through the glomeruli. These complexes are taken up in the kidney at the sites of cysteine reabsorption which, by studies with L-[ 35 S]-cysteine, are identified as the straight segments of the proximal tubules. (orig.)

Purification and characterization of protease enzyme from ...

African Journals Online (AJOL)

The enzyme was active in pH range 5 to11 and temperature of 30 to 80°C. The optimum pH and the temperature for protease activity were recorded to be pH 8 and 50°C, respectively. The enzyme was stable up to 40°C and pH 9. The protease activity was inhibited by Zn2+, Ni2+ and Sn2+ and increased by Ca2+, Mg2+ ...

SwissProt search result: AK110731 [KOME

Lifescience Database Archive (English)

Full Text Available AK110731 002-170-E10 (Q8BGV9) Cysteine protease APG4D (EC 3.4.22.-) (Autophagy 4 homolog D) (Auto...phagin-4) (Autophagy-related cysteine endopeptidase 4) (AUT-like 4 cysteine endopeptidase) APG4D_MOUSE 7e-33 ...

Exploration of peptides that fit into the thermally vibrating active site of cathepsin K protease by alternating artificial intelligence and molecular simulation

Science.gov (United States)

Nishiyama, Katsuhiko

2017-08-01

Eighteen tripeptides that fit into the thermally vibrating active site of cathepsin K were discovered by alternating artificial intelligence and molecular simulation. The 18 tripeptides fit the active site better than the cysteine protease inhibitor E64, and a better inhibitor of cathepsin K could be designed considering these tripeptides. Among the 18 tripeptides, Phe-Arg-Asp and Tyr-Arg-Asp fit the active site the best and their structural similarity should be considered in the design process. Interesting factors emerged from the structure of the decision tree, and its structural information will guide exploration of potential inhibitor molecules for proteases.

Proteases from Latex of Euphorbia spp. and Its Application on Milk Clot Formation

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Fidia Fibriana

2015-09-01

Full Text Available Crude proteases were extracted from Euphorbiaceae family, i.e. E. milii var imperata, E. trigona, and E. maculata. Among those three crude proteases, the activity of protease from E. trigona was the highest (812.50 U/ml, whereas E. milii and E. maculata crude proteases activity were 298.60 U/ml and 95.80 U/ml, respectively. E. maculata protein concentration was the highest among those three crude enzymes (1.206 mg/ml. The optimum pH and temperature of the enzymes were pH 7.0, pH 6.0, pH 6.5 and 60 °C, 50 °C, and 50 °C, respectively. Crude protease from E. milii var imperata, E. trigona, and E. maculata retained proteolytic activity over a wide range of pH (5.0–9.0 and temperature (up to 65 °C with casein as substrate. All crude proteases showed milk clotting activity ranged from 0.58 U/ml to 1.01 U/ml. Thus, these crude proteases are potential to be applied in dairy industries. However, further study on enzyme purification and characterization are necessary to obtain high purity of proteases before its application.Protease kasar berhasil diekstrak dari tanaman family Euphorbiaceae, yaitu E. milii var imperata, E. trigona, dan E. maculata. Diantara ketiga protease tersebut, aktivitas protease tertinggi diperoleh dari E. trigona (812,50 U/ml, sedangkan aktivitas protease dari E. milii dan E. maculata adalah 298,60 U/ml dan 95,80 U/ml, berturut-turut. Konsentrasi total protein tertinggi terdapat pada protease kasar E. maculata (1,206 mg/ml. pH dan suhu optimum ketiga enzim tersebut adalah pH 7.0, pH 6.0, pH 6.5 dan suhu 60 °C, 50 °C, and 50 °C, berturut-turut. Protease kasar dari E. milii var imperata, E. trigona, dan E. maculata menunjukkan aktivitas proteolitik pada rentang pH 5.0–9.0 dan rentang suhu sampai 65 °C menggunakan kasein sebagai substrat. Semua protease kasar menunjukkan aktivitas penggumpalan susu dengan rentang dari 0,58 U/ml sampai 1,01 U/ml. Berdasarkan hasil yang diperoleh, protease kasar dari ketiga jenis tanaman ini

Diversity of both the cultivable protease-producing bacteria and bacterial extracellular proteases in the coastal sediments of King George Island, Antarctica.

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Ming-Yang Zhou

Full Text Available Protease-producing bacteria play a vital role in degrading sedimentary organic nitrogen. However, the diversity of these bacteria and their extracellular proteases in most regions remain unknown. In this paper, the diversity of the cultivable protease-producing bacteria and of bacterial extracellular proteases in the sediments of Maxwell Bay, King George Island, Antarctica was investigated. The cultivable protease-producing bacteria reached 10(5 cells/g in all 8 sediment samples. The cultivated protease-producing bacteria were mainly affiliated with the phyla Actinobacteria, Firmicutes, Bacteroidetes, and Proteobacteria, and the predominant genera were Bacillus (22.9%, Flavobacterium (21.0% and Lacinutrix (16.2%. Among these strains, Pseudoalteromonas and Flavobacteria showed relatively high protease production. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria were serine proteases or metalloproteases. These results begin to address the diversity of protease-producing bacteria and bacterial extracellular proteases in the sediments of the Antarctic Sea.

Emission of hydrogen sulfide by leaf tissue in response to L-cysteine

International Nuclear Information System (INIS)

Sekiya, J.; Schmidt, A.; Wilson, L.G.; Filner, P.

1982-01-01

Leaf discs and detached leaves exposed to L-cysteine emitted a volatile sulfur compound which was proven by gas chromatography to be H 2 S. This phenomenon was demonstrated in all nine species tested (Cucumis sativus, Cucurbita pepo, Nicotiana tabacum, Coleus blumei, Beta vulgaris, Phaseolus vulgaris, Medicago sativa, Hordeum vulgare, and Gossypium hirsutum). The emission of volatile sulfur by cucumber leaves occurred in the dark at a similar rate to that in the light. The emission of leaf discs reached the maximal rate, more than 40 picomoles per minute per square centimeter, 2 to 4 hours after starting exposure to L-cysteine; then it decreased. In the case of detached leaves, the maximum occurred 5 to 10 h after starting exposure. The average emission rate of H 2 S during the first 4 hours from leaf discs of cucurbits in response to 10 millimolar L-cysteine, was usually more than 40 picomoles per minute per square centimeter, i.e. 0.24 micromoles per hour per square decimeter. Leaf discs exposed to 1 millimolar L-cysteine emitted only 2% as much as did the discs exposed to 10 millimolar L-cysteine. The emission from leaf discs and from detached leaves lasted for at least 5 and 15 hours, respectively. However, several hours after the maximal emission, injury of the leaves, manifested as chlorosis, was evident. H 2 S emission was a specific consequence of exposure to L-cysteine; neither D-cysteine nor L-cysteine elicited H 2 S emission. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate dependent enzymes, inhibited the emission. In a cell free system from cucumber leaves, H 2 S formation and its release occurred in response to L-cysteine. Feeding experiments with [ 35 S]t-cysteine showed that most of the sulfur in H 2 S was derived from sulfur in the L-cysteine supplied

Effects of protease inhibitors on radiation transformation in vitro

International Nuclear Information System (INIS)

Kennedy, A.R.; Little, J.B.

1981-01-01

We have investigated the effects of three protease inhibitors, antipain, leupeptin, and soybean trypsin inhibitor, on the induction of oncogenic transformation in mouse C3H10T 1/2 cells by X-rays. The patterns of inhibition by the three protease inhibitors were different. Antipain was the most effective, having the ability to suppress completely radiation transformation as well as radiation transformation enhanced by the phorbol ester promoting agent 12-O-tetradecanoylphorbol-13-acetate. The fact that antipain could suppress transformation when present for only 1 day following irradiation suggests that an effect on a DNA repair process might be important in its action. Leupeptin was less effective than antipain in its inhibition of radiation transformation. Soybean trypsin inhibitor suppressed only the promotional effects of 12-O-tetradecanoylphorbol-13-acetate on transformation. Our results suggest that there may be more than one protease involved in carcinogenesis

Crystal Structure of a Hidden Protein, YcaC, a Putative Cysteine Hydrolase from Pseudomonas aeruginosa, with and without an Acrylamide Adduct

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Morten K. Grøftehauge

2015-07-01

Full Text Available As part of the ongoing effort to functionally and structurally characterize virulence factors in the opportunistic pathogen Pseudomonas aeruginosa, we determined the crystal structure of YcaC co-purified with the target protein at resolutions of 2.34 and 2.56 Å without a priori knowledge of the protein identity or experimental phases. The three-dimensional structure of YcaC adopts a well-known cysteine hydrolase fold with the putative active site residues conserved. The active site cysteine is covalently bound to propionamide in one crystal form, whereas the second form contains an S-mercaptocysteine. The precise biological function of YcaC is unknown; however, related prokaryotic proteins have functions in antibacterial resistance, siderophore production and NADH biosynthesis. Here, we show that YcaC is exceptionally well conserved across both bacterial and fungal species despite being non-ubiquitous. This suggests that whilst YcaC may not be part of an integral pathway, the function could confer a significant evolutionary advantage to microbial life.

Assessment of FIV-C infection of cats as a function of treatment with the protease inhibitor, TL-3

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de Rozières Sohela

2004-11-01

Full Text Available Abstract Background The protease inhibitor, TL-3, demonstrated broad efficacy in vitro against FIV, HIV and SIV (simian immunodeficiency virus, and exhibited very strong protective effects on early neurologic alterations in the CNS of FIV-PPR infected cats. In this study, we analyzed TL-3 efficacy using a highly pathogenic FIV-C isolate, which causes a severe acute phase immunodeficiency syndrome, with high early mortality rates. Results Twenty cats were infected with uncloned FIV-C and half were treated with TL-3 while the other half were left untreated. Two uninfected cats were used as controls. The general health and the immunological and virological status of the animals was monitored for eight weeks following infection. All infected animals became viremic independent of TL-3 treatment and seven of 20 FIV-C infected animals developed severe immunodepletive disease in conjunction with significantly (p ≤ 0.05 higher viral RNA loads as compared to asymptomatic animals. A marked and progressive increase in CD8+ T lymphocytes in animals surviving acute phase infection was noted, which was not evident in symptomatic animals (p ≤ 0.05. Average viral loads were lower in TL-3 treated animals and of the 6 animals requiring euthanasia, four were from the untreated cohort. At eight weeks post infection, half of the TL-3 treated animals and only one of six untreated animals had viral loads below detection limits. Analysis of protease genes in TL-3 treated animals with higher than average viral loads revealed sequence variations relative to wild type protease. In particular, one mutant, D105G, imparted 5-fold resistance against TL-3 relative to wild type protease. Conclusions The findings indicate that the protease inhibitor, TL-3, when administered orally as a monotherapy, did not prevent viremia in cats infected with high dose FIV-C. However, the modest lowering of viral loads with TL-3 treatment, the greater survival rate in symptomatic animals of

Intracellular alkaline proteases produced by thermoacidophiles: detection of protease heterogeneity by gelatin zymography and polymerase chain reaction (PCR)

Energy Technology Data Exchange (ETDEWEB)

Kocab, S.; Erdem, B. [Middle East Technical University, Ankara (Turkey). Dept. of Biological Sciences

2002-08-01

In this study 24 thermoacidophilic archeal and bacterial strains isolated from hot-springs and hot-soils were screened for their ability to produce intracellular alkaline proteases. The protease activities of the strains, based on azocasein hydrolysis, showed a variation from 0.6 to 5.1 U. The cell extracts of three most potent producers were further examined and it was found that their proteases exhibited maximum activity at 60-70{sup o}C and showed a pH optimum over a range of pH 7.0-8.5. Gelatin zymography revealed that two of the selected archeal strains produced multiple active SDS-resistant proteases. On the other hand, PCR amplification of alkaline serine protease gene sequences of total DNA from all isolates yielded four distinct amplification fragments of 650, 450, 400 and 300 bp, which might have been derived from different serine protease genes. (author)

Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

Science.gov (United States)

Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Sun, Kaiji; Zhang, Qing; Wu, Guoyao

2016-01-01

High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P L-cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P L-cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

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

Science.gov (United States)

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

Formation of Hg(II) Tetrathiolate Complexes with Cysteine at Neutral pH.

Science.gov (United States)

Warner, Thomas; Jalilehvand, Farideh

2016-04-01

Mercury(II) ions precipitate from aqueous cysteine (H 2 Cys) solutions containing H 2 Cys/Hg(II) mole ratio ≥ 2.0 as Hg( S -HCys) 2 . In absence of additional cysteine, the precipitate dissolves at pH ~12 with the [Hg( S,N -Cys) 2 ] 2- complex dominating. With excess cysteine (H 2 Cys/Hg(II) mole ratio ≥ 4.0), higher complexes form and the precipitate dissolves at lower pH values. Previously, we found that tetrathiolate [Hg( S -Cys) 4 ] 6- complexes form at pH = 11.0; in this work we extend the investigation to pH values of physiological interest. We examined two series of Hg(II)-cysteine solutions in which C Hg(II) varied between 8 - 9 mM and 80 - 100 mM, respectively, with H 2 Cys/Hg(II) mole ratios from 4 to ~20. The solutions were prepared in the pH range 7.1 - 8.8, at the pH at which the initial Hg( S -HCys) 2 precipitate dissolved. The variations in the Hg(II) speciation were followed by 199 Hg NMR, X-ray absorption and Raman spectroscopic techniques. Our results show that in the dilute solutions ( C Hg(II) = 8 - 9 mM), mixtures of di-, tri- (major) and tetrathiolate complexes exist at moderate cysteine excess ( C H2Cys ~ 0.16 M) at pH 7.1. In the more concentrated solutions ( C Hg(II) = 80 - 100 mM) with high cysteine excess ( C H2Cys > 0.9 M), tetrathiolate [Hg( S -cysteinate) 4 ] m -6 ( m = 0 - 4) complexes dominate in the pH range 7.3 - 7.8, with lower charge than for the [Hg( S -Cys) 4 ] 6- complex due to protonation of some ( m ) of the amino groups of the coordinated cysteine ligands. The results of this investigation could provide a key to the mechanism of biosorption and accumulation of Hg(II) ions in biological / environmental systems.

An Alkaline Protease from Bacillus pumilus MP 27: Functional Analysis of its Binding Model towards its Applications as Detergent Additive

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Mehak Baweja

2016-08-01

Full Text Available A proteolytic strain of Bacillus pumilus MP 27 was isolated from water samples of Southern ocean produced alkaline protease. Since protease production need expensive ingredients, an economically viable process was developed by using low cost carbon source, wheat straw, supplemented with peptone. This protease was active within temperature ranges 10˚C -70˚C at pH 9. This process was optimized by response surface methodology using a Box Bekhman design by Design Expert 7.0 software that increased the protease activity to 776.5 U/ml. Moreover, the enzyme was extremely stable at a broad range of temperature and pH retaining 69% of its activity at 50 ºC and 70% at pH 11. The enzyme exhibited excellent compatibility with surfactants and commercial detergents, showing 87% stability with triton X-100 and ̴ 100% stability with Tide commercial detergent. The results of the wash performance analysis demonstrated considerably good de-staining at 50ºC and 4ºC with low supplementation (109 U/ml. Molecular modeling of the protease revealed the presence of serine proteases, subtilase family and serine active site and further docking supported the association of catalytic site with the various substrates. Certainly, such protease can be considered as a good detergent additive in detergent industry with a possibility to remove the stains effectively even in a cold wash.

A novel potentiometric biosensor for selective L-cysteine determination using L-cysteine-desulfhydrase producing Trichosporon jirovecii yeast cells coupled with sulfide electrode

International Nuclear Information System (INIS)

Hassan, Saad S.M.; El-Baz, Ashraf F.; Abd-Rabboh, Hisham S.M.

2007-01-01

Trichosporon jirovecii yeast cells are used for the first time as a source of L-cysteine desulfhydrase enzyme (EC 4.4.1.1) and incorporated in a biosensor for determining L-cysteine. The cells are grown under cadmium stress conditions to increase the expression level of the enzyme. The intact cells are immobilized on the membrane of a solid-state Ag 2 S electrode to provide a simple L-cysteine responsive biosensor. Upon immersion of the sensor in L-cysteine containing solutions, L-cysteine undergoes enzymatic hydrolysis into pyruvate, ammonia and sulfide ion. The rate of sulfide ion formation is potentiometrically measured as a function of L-cysteine concentration. Under optimized conditions (phosphate buffer pH 7, temperature 37 ± 1 deg. C and actual weight of immobilized yeast cells 100 mg), a linear relationship between L-cysteine concentration and the initial rate of sulfide liberation (dE/dt) is obtained. The sensor response covers the concentration range of 0.2-150 mg L -1 (1.7-1250 μmol L -1 ) L-cysteine. Validation of the assay method according to the quality control/quality assurance standards (precision, accuracy, between-day variability, within-day reproducibility, range of measurements and lower limit of detection) reveals remarkable performance characteristics of the proposed biosensor. The sensor is satisfactorily utilized for determination of L-cysteine in some pharmaceutical formulations. The lower limit of detection is ∼1 μmol L -1 and the accuracy and precision of the method are 97.5% and ±1.1%, respectively. Structurally similar sulfur containing compounds such as glutathione, cystine, methionine, and D-cysteine do no interfere

Structure and function of C-terminal catalytic region of pasteurella multocida toxin

International Nuclear Information System (INIS)

Kitadokoro, Kengo; Kamitami, Shigeki; Horiguchi, Yasuhiko

2008-01-01

Pasteurella multocida toxin (PMT) is one of virulence factors responsible for the pathogenesis in some Pasteurellosis. We determined the crystal structure of the C-terminal region of PMT (C-PMT), which carries an intracellularly active moiety. The overall structure of C-PMT displays three different domains designated C1, C2 and C3. We found in the C3 domain the Cys-His-Asp catalytic triad that is organized only when the Cys is released from a disulfide bond. The steric alignment of the triad corresponded well to that of papain or other enzymes carrying the Cys-His-Asp triad. Our results demonstrate that PMT is an enzymatic toxin carrying the cysteine-protease like catalytic triad, which is organized only under reducing conditions. (author)

Mosaic serine proteases in the mammalian central nervous system.

Science.gov (United States)

Mitsui, Shinichi; Watanabe, Yoshihisa; Yamaguchi, Tatsuyuki; Yamaguchi, Nozomi

2008-01-01

We review the structure and function of three kinds of mosaic serine proteases expressed in the mammalian central nervous system (CNS). Mosaic serine proteases have several domains in the proenzyme fragment, which modulate proteolytic function, and a protease domain at the C-terminus. Spinesin/TMPRSS5 is a transmembrane serine protease whose presynaptic distribution on motor neurons in the spinal cord suggests that it is significant for neuronal plasticity. Cell type-specific alternative splicing gives this protease diverse functions by modulating its intracellular localization. Motopsin/PRSS12 is a mosaic protease, and loss of its function causes mental retardation. Recent reports indicate the significance of this protease for cognitive function. We mention the fibrinolytic protease, tissue plasminogen activator (tPA), which has physiological and pathological functions in the CNS.

Characterization of novel extracellular protease produced by marine bacterial isolate from the Indian Ocean

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Rachana Fulzele

2011-12-01

Full Text Available Out of the vast pool of enzymes, proteolytic enzymes from microorganisms are the most widely used in different industries such as detergent, food, peptide production etc. Several marine microorganisms are known to produce proteases with commercially desirable characteristics. We have isolated nine different cultures from marine samples of the Indian Ocean. All of them were i motile ii rod shaped iii non spore forming iv catalase and amylase positive v able to grow in presence of 10 % NaCl. They produced acid from glucose, fructose and maltose and grew optimally at 30 0C temperature and pH 7.0-8.0. None of them could grow above 45 0C and below 15 0C. Only one of them (MBRI 7 exhibited extracellular protease activity on skim milk agar plates. Based on 16S rDNA sequencing, it belonged to the genus Marinobacter (98% sequence similarity, 1201 bp. The cell free extract was used to study effects of temperature and pH on protease activity. The optimum temperature and pH for activity were found to be 40 0C and 7.0 respectively. The crude enzyme was stable at temperature range of 30-80 0C and pH 5.0-9.0. It retained 60 % activity at 80 0C after 4 h and more than 70 % activity at 70 0C after 1 h. D value was found to be 342 minutes and 78 minutes for 40 0C and 80 0C respectively. Interestingly the enzyme remained 50 % active at pH 9.0 after 1 h. Comparison with other proteases from different microbial sources indicated that the neutral protease from the halotolerant marine isolate MBRI 7 is a novel enzyme with high thermostability.

Dual functionality of β-tryptase protomers as both proteases and cofactors in the active tetramer.

Science.gov (United States)

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.

Purification and characterization of an extreme halothermophilic protease from a halophilic bacterium Chromohalobacter sp. TVSP101

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Malashetty Vidyasagar

2009-03-01

Full Text Available An extreme halophilic bacterium was isolated from solar saltern samples and identified based on biochemical tests and 16S r RNA sequencing as Chromohalobacter sp. strain TVSP101. The halophilic protease was purified using ultrafiltration, ethanol precipitation, hydrophobic interaction column chromatography and gel permeation chromatography to 180 fold with 22% yield. The molecular mass of the protease determined by SDS PAGE was 66 kDa. The purified enzyme was salt dependent for its activity and stability with an optimum of 4.5 M NaCl. The optimum temperature for maximum protease activity was 75°C. The protease was optimally active at pH 8 and retained more than 80% of its activity in the range of pH 7-10. Sucrose and glycine at 10% (w/v were the most effective osmolytes, retained 100% activity in the absence of NaCl. The activity was completely inhibited by ZnCl2 (2 mM, 0.1% SDS and PMSF (1mM. The enzyme was not inhibited by 1mM of pepstatin, EDTA and PCMB. The protease was active and retained 100% it activity in 10% (v/v DMSO, DMF, ethanol and acetone.

Optimization of Nutrients and Culture Conditions for Alkaline Protease Production Using Two Endophytic Micrococci: Micrococcus aloeverae and Micrococcus yunnanensis.

Science.gov (United States)

Prakash, Om; Nimonkar, Yogesh; Chavadar, Mahesh S; Bharti, Nidhi; Pawar, Shrikant; Sharma, Ashutosh; Shouche, Yogesh S

2017-06-01

An endophytic species of Micrococcus was isolated from Aloe vera leaf (syn. Aloe barbadensis ) and screened for protease production with five other species of Micrococcus . Data indicated that endophytic Micrococcus aloeverae AE-6 MCC 2184 T and Micrococcus yunnanensis DSM 21948 T showed efficient protease production potential and secreted active protease at high salt (10%), temperature (40 °C) and in wide range of pH 8-10. Unlike M . yunnanensis DSM 21948 T , protease production by M . aloeverae AE-6 MCC 2184 T was stringently controlled by pH. Protease induction study using different group of peptides, peptide carbohydrates and peptide macronutrient combinations showed variable response with both the organisms. Result indicated that the amount of protease was not directly related to cell biomass but it depends on nature of inducible peptides. In this study we also developed a modified agar-well assay for semi-quantitative data from large number of replicates.

[Protective Effect of S-isopentenyl-L-cysteine against DNA Damage in Irradiated Mice].

Science.gov (United States)

Zheng, Qi-sheng; Yu, Guang-yun; He, Xin; Jiang, Ming; Chu, Xiao-fei; Zhao, Shu-yi; Fan, Sai-jun; Liu, Pei-xun

2015-10-01

To evaluate the protective effect of S-isopentenyl-L-cysteine,a new cysteine derivative,on DNA damage induced by radiation by using acute radiation injury animal models. Forty ICR mice were randomly divided into five groups:the control group,1.0Gy gamma irradiation group,1.0Gy gamma irradiation combined with S-isopentenyl-L-cysteine group,7.2Gy gamma irradiation group,and 7.2Gy gamma irradiation combined with S-isopentenyl-L-cysteine group,with 8 mice in each group.The comet assay and bone marrow polychromatic micronucleus experiments were performed to evaluate the double-strand DNA breaks in ICR mice exposed to 1.0 and 7.2Gy gamma-ray, respectively. The tail DNA percentage,tail length,tail moment,and olive tail moment of peripheral blood lymphocytes in 7.2Gy gamma irradiation group were significantly higher than that of the control group (PL-cysteine group was significantly less than that of 7.2Gy gamma irradiation group (PL-cysteine before irradiation,the micronucleus rate of ICR mice exposed to 1.0 and 7.2Gy gamma-ray decreased from (39.5000 ± 3.3141)‰ to (28.1667±4.1345)‰ (P=0.033) and from (76.5000 ± 4.6242)‰ to (22.8333 ± 3.6553)‰(P=0.000),respectively. The bone marrow polychromatic micronucleus experiment indicated that the value of polychromatic erythrocyte (PCE)/normochromatic erythrocyte(NCE) of ICR mice exposed to 1.0 and 7.2Gy gamma-ray was less than the control group(PL-cysteine before irradiation was significantly higher than the corresponding groups (PL-cysteine has a good protective effect against DNA damage induced by radiation.

The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite.

Science.gov (United States)

Putrianti, Elyzana D; Schmidt-Christensen, Anja; Arnold, Iris; Heussler, Volker T; Matuschewski, Kai; Silvie, Olivier

2010-06-01

Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called 'serine repeat antigens' (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs.

A biotechnology perspective of fungal proteases

Directory of Open Access Journals (Sweden)

Paula Monteiro de Souza

2015-06-01

Full Text Available Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications.

Purification and Characterization of Fibrinolytic Proteases from Mushroom Volvariela volvaceae Extract.

Directory of Open Access Journals (Sweden)

Willy Praira

2010-11-01

Full Text Available Purification and Characterization of Fibrinolytic Proteases from Mushroom Volvariela volvaceae Extract. Ediblestraw mushroom (V. volvaceae has been known used for improvement of blood circulation due to its fibrinolyticcontent. The objective of the study is to purify and characterize fibrinolytic protease from straw mushroom extract.Purification were performed through several steps, i.e. precipitation using ammonium sulphate 75%, dialyzed membran(cut-off 10 kDa, and ion-exchange chromatography using DEAE Sepharose. The active fraction of DEAE-Sepharosecontains two purified protein bands with molecular weight of 12.9 and 15.8 kDa. The active fraction has specificactivity of 0.383 U/mg with 2.7 fold higher purification compared to its crude extract. Both crude and purified enzymeshad optimum activity at temperature of 50 ºC and pH 7 in 10 minutes of incubation. Fibrin zymographic profiledemonstrated that the enzyme hydrolyzed fibrin, as well as casein, indicating their potent fibrinolytic activity. Theenzyme was strongly inhibited by phenilmethylsulphonyl fluoride and N-p-tosil-L-lysinchloromethyl keton. Thissuggested that it was a serine protease. In summary, these results showed that crude and purified protease of strawmushroom (V. volvaceae has fibrinolytic activities that can be applied for alternative thrombolytic therapy.

Genome-wide analysis of regulatory proteases sequences identified through bioinformatics data mining in Taenia solium.

Science.gov (United States)

Yan, Hong-Bin; Lou, Zhong-Zi; Li, Li; Brindley, Paul J; Zheng, Yadong; Luo, Xuenong; Hou, Junling; Guo, Aijiang; Jia, Wan-Zhong; Cai, Xuepeng

2014-06-04

. Phylogenetic analysis using Bayes approach provided support for inferring functional divergence among regulatory cysteine and serine proteases. Numerous putative proteases were identified for the first time in T. solium, and important regulatory proteases have been predicted. This comprehensive analysis not only complements the growing knowledge base of proteolytic enzymes, but also provides a platform from which to expand knowledge of cestode proteases and to explore their biochemistry and potential as intervention targets.

Process optimization by response surface methodology for extracellular alkaline protease production from bacillus subtilis

International Nuclear Information System (INIS)

Mushtaq, Z.; Adnan, A.; Mehmood, Z.

2014-01-01

Three microbial cultures Bacillus subtilis DSM 1970, Bacillus subtilis GCU-8 and Bacillus licheniformis DSM 1969 were screened for protease production by casein agar plate method. Among these Bacillus subtilis GCU-8 was found to be the most potent protease producer in wide pH range (5.0 to 8.0). Fermentation conditions were optimized for the production of alkaline protease using two statistical tools: Placket Burmen Model for linear regression study and Response Surface Model for interactive effects of significant factors on production. The alkaline protease was optimally produced after 48 hours of incubation at 37 degree C in fermentation media containing equal amounts of substrates (soybean meal and wheat bran, 7.5 g), MgSO/sub 4/ 7H/sub 2/O, 0.10 g and yeast extract 0.55 g. The protease was purified to homogeneity by salt precipitation, ion-exchange chromatography and size exclusion chromatography. The homogeneity and molecular weights were checked by SDS-PAGE. The protease was 45 KDa protein, predominantly alkaline and optimally active at pH 8.0. (author)

Optimization of alkaline protease production from Pseudomonas ...

African Journals Online (AJOL)

PRECIOUS

2009-12-15

Dec 15, 2009 ... protease production was 37°C at pH 9, with 2% inoculum in the medium for 24 h. .... Positive. Catalase test. Positive ... The enzyme activity gradually decreases from ... Effect of temperature on protease production by Pseudomonas fluorescens. 0 .... between RNA polymerase and upstream promotes DNA.

Pengaruh PH dan Suhu terhadap Aktivitas Protease Penicillium SP.

OpenAIRE

Yusriah, Yusriah; Kuswytasari, Nengah Dwianita

2013-01-01

Tujuan penelitian ini adalah untuk mengetahui pengaruh pH dan suhu terhadap aktivitas protease pada Penicillium sp.3 T3f2. Selanjutnya, isolat Penicillium sp. di kultur dalam media produksi protease untuk menghasilkan protease. Suhu yang digunakan adalah 300 – 500C sedangkan pH-nya 4 – 8. Aktivitas protease ditentukan dan diukur dengan spektrofotometer pada panjang gelombang 275 nm, dengan kasein sebagai substrat. Berdasarkan uji ANOVA yang dilanjutkan dengan uji Duncan dengan taraf kepercaya...

Activity, specificity, and probe design for the smallpox virus protease K7L.

Science.gov (United States)

Aleshin, Alexander E; Drag, Marcin; Gombosuren, Naran; Wei, Ge; Mikolajczyk, Jowita; Satterthwait, Arnold C; Strongin, Alex Y; Liddington, Robert C; Salvesen, Guy S

2012-11-16

The K7L gene product of the smallpox virus is a protease implicated in the maturation of viral proteins. K7L belongs to protease Clan CE, which includes distantly related cysteine proteases from eukaryotes, pathogenic bacteria, and viruses. Here, we describe its recombinant high level expression, biochemical mechanism, substrate preference, and regulation. Earlier studies inferred that the orthologous I7L vaccinia protease cleaves at an AG-X motif in six viral proteins. Our data for K7L suggest that the AG-X motif is necessary but not sufficient for optimal cleavage activity. Thus, K7L requires peptides extended into the P7 and P8 positions for efficient substrate cleavage. Catalytic activity of K7L is substantially enhanced by homodimerization, by the substrate protein P25K as well as by glycerol. RNA and DNA also enhance cleavage of the P25K protein but not of synthetic peptides, suggesting that nucleic acids augment the interaction of K7L with its protein substrate. Library-based peptide preference analyses enabled us to design an activity-based probe that covalently and selectively labels K7L in lysates of transfected and infected cells. Our study thus provides proof-of-concept for the design of inhibitors and probes that may contribute both to a better understanding of the role of K7L in the virus life cycle and the design of novel anti-virals.

Novel tetra-peptide insertion in Gag-p6 ALIX-binding motif in HIV-1 subtype C associated with protease inhibitor failure in Indian patients.

Science.gov (United States)

Neogi, Ujjwal; Rao, Shwetha D; Bontell, Irene; Verheyen, Jens; Rao, Vasudev R; Gore, Sagar C; Soni, Neelesh; Shet, Anita; Schülter, Eugen; Ekstrand, Maria L; Wondwossen, Amogne; Kaiser, Rolf; Madhusudhan, Mallur S; Prasad, Vinayaka R; Sonnerborg, Anders

2014-09-24

A novel tetra-peptide insertion was identified in Gag-p6 ALIX-binding region, which appeared in protease inhibitor failure Indian HIV-1C sequences (odds ratio=17.1, P < 0.001) but was naturally present in half of untreated Ethiopian HIV-1C sequences. The insertion is predicted to restore ALIX-mediated virus release pathway, which is lacking in HIV-1C. The clinical importance of the insertion needs to be evaluated in HIV-1C dominating regions wherein the use of protease inhibitor drugs are being scaled up.

Dynamic hepatitis C virus genotypic and phenotypic changes in patients treated with the protease inhibitor telaprevir

NARCIS (Netherlands)

Sarrazin, Christoph; Kieffer, Tara L.; Bartels, Doug; Hanzelka, Brian; Müh, Ute; Welker, Martin; Wincheringer, Dennis; Zhou, Yi; Chu, Hui-May; Lin, Chao; Weegink, Christine; Reesink, Henk; Zeuzem, Stefan; Kwong, Ann D.

2007-01-01

BACKGROUND & AIMS: Telaprevir (VX-950), a hepatitis C virus (HCV) NS3.4A protease inhibitor, has shown strong antiviral activity in phase 1 clinical studies. Because of high levels of HCV replication and the low fidelity of HCV polymerase, selection of resistant isolates during therapy may occur.

Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus.

Science.gov (United States)

Cuenca-Soria, C A; Álvarez-González, C A; Ortiz-Galindo, J L; Nolasco-Soria, H; Tovar-Ramírez, D; Guerrero-Zárate, R; Castillo-Domínguez, A; Perera-García, M A; Hernández-Gómez, R; Gisbert, E

2014-06-01

The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2–3 and at temperatures of 35–45 °C, whereas the alkaline proteases were most stable at pH values of 6–9 and at 25–55 °C. The inhibition assays recorded a residual activity of 4% with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80% with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8% with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.

DC type 2 polarization depends on both the allergic status of the individual and protease activity of Per a 10.

Science.gov (United States)

Goel, Chhavi; Gaur, S N; Bhati, Gaurav; Arora, Naveen

2015-10-01

Cockroach proteases are important risk factors for asthma development in predisposed individuals. In the present study, effect of allergic status of patients on DCs polarization in response to protease allergen Per a 10 was investigated. Cockroach-allergic, other-allergic patients and healthy individuals were selected following the guidelines of ATS/ARIA. Monocyte-derived dendritic cells (DCs) were generated from the selected individuals and stimulated with Per a 10. Flow cytometric analysis showed a significantly high expression of CD80 and CD86 on DCs from cockroach-allergic patients after Per a 10 stimulation as compared to healthy individuals or other-allergic patients (PPer a 10 induced comparable level of CD83 expression on DCs from all the 3 groups, showing it was irrespective of the allergic status. CD40 expression was significantly low (PPer a 10 induced lower CD40 expression on DCs than the heat-inactivated Per a 10 (PPer a 10 stimulated DC cultures was significantly higher than in heat-inactivated Per a 10 (PPer a 10-stimulated DCs than heat-inactivated Per a 10-stimulated DCs. Per a 10-stimulated DCs from cockroach-allergic patients secreted high levels of IL-5, IL-6, TNF-α than that from healthy individuals or other-allergic patients (PPer a 10-stimulated DCs from cockroach-allergic patients induced increased secretions of IL-4, IL-5, IL-6, TNF-α and low IL-12 by T cells as compared to those from other groups (PPer a 10 allergen, polarization of DCs shifts toward type 2 in cockroach-allergic patients but not in the healthy individuals or other-allergic patients. In conclusion, both allergic status of the individual and protease activity of Per a 10 are important parameters that participate in DCs polarization. Copyright © 2015 Elsevier GmbH. All rights reserved.

Simultaneous electrochemical determination of L-cysteine and L-cysteine disulfide at carbon ionic liquid electrode.

Science.gov (United States)

Safavi, Afsaneh; Ahmadi, Raheleh; Mahyari, Farzaneh Aghakhani

2014-04-01

A linear sweep voltammetric method is used for direct simultaneous determination of L-cysteine and L-cysteine disulfide (cystine) based on carbon ionic liquid electrode. With carbon ionic liquid electrode as a high performance electrode, two oxidation peaks for L-cysteine (0.62 V) and L-cysteine disulfide (1.3 V) were observed with a significant separation of about 680 mV (vs. Ag/AgCl) in phosphate buffer solution (pH 6.0). The linear ranges were obtained as 1.0-450 and 5.0-700 μM and detection limits were estimated to be 0.298 and 4.258 μM for L-cysteine and L-cysteine disulfide, respectively. This composite electrode was applied for simultaneous determination of L-cysteine and L-cysteine disulfide in two real samples, artificial urine and nutrient broth. Satisfactory results were obtained which clearly indicate the applicability of the proposed electrode for simultaneous determination of these compounds in complex matrices.

Effect of (L)-cysteine on acetaldehyde self-administration.

Science.gov (United States)

Peana, Alessandra T; Muggironi, Giulia; Fois, Giulia R; Zinellu, Manuel; Sirca, Donatella; Diana, Marco

2012-08-01

Acetaldehyde (ACD), the first metabolite of ethanol, has been implicated in several behavioural actions of alcohol, including its reinforcing effects. Recently, we reported that l-cysteine, a sequestrating agent of ACD, reduced oral ethanol self-administration and that ACD was orally self-administered. This study examined the effects of l-cysteine pre-treatment during the acquisition and maintenance phases of ACD (0.2%) self-administration as well as on the deprivation effect after ACD extinction and on a progressive ratio (PR) schedule of reinforcement. In a separate PR schedule of reinforcement, the effect of l-cysteine was assessed on the break-point produced by ethanol (10%). Furthermore, we tested the effect of l-cysteine on saccharin (0.2%) reinforcement. Wistar rats were trained to self-administer ACD by nose poking on a fixed ratio (FR1) schedule in 30-min daily sessions. Responses on an active nose-poke caused delivery of ACD solution, whereas responses on an inactive nose-poke had no consequences. l-cysteine reduced the acquisition (40 mg/kg), the maintenance and the deprivation effect (100 mg/kg) of ACD self-administration. Furthermore, at the same dose, l-cysteine (120 mg/kg) decreased both ACD and ethanol break point. In addition, l-cysteine was unable to suppress the different responses for saccharin, suggesting that its effect did not relate to an unspecific decrease in a general motivational state. Compared to saline, l-cysteine did not modify responses on inactive nose-pokes, suggesting an absence of a non-specific behavioural activation. Taken together, these results could support the hypotheses that ACD possesses reinforcing properties and l-cysteine reduces motivation to self-administer ACD. Copyright © 2012 Elsevier Inc. All rights reserved.

The potential of P1 site alterations in peptidomimetic protease inhibitors as suggested by virtual screening and explored by the use of C-C-coupling reagents.

Science.gov (United States)

Weik, Steffen; Luksch, Torsten; Evers, Andreas; Böttcher, Jark; Sotriffer, Christoph A; Hasilik, Andrej; Löffler, Hans-Gerhard; Klebe, Gerhard; Rademann, Jörg

2006-04-01

A synthetic concept is presented that allows the construction of peptide isostere libraries through polymer-supported C-acylation reactions. A phosphorane linker reagent is used as a carbanion equivalent; by employing MSNT as a coupling reagent, the C-acylation can be conducted without racemization. Diastereoselective reduction was effected with L-selectride. The reagent linker allows the preparation of a norstatine library with full variation of the isosteric positions including the P1 side chain that addresses the protease S1 pocket. Therefore, the concept was employed to investigate the P1 site specificity of peptide isostere inhibitors systematically. The S1 pocket of several aspartic proteases including plasmepsin II and cathepsin D was modeled and docked with approximately 500 amino acid side chains. Inspired by this virtual screen, a P1 site mutation library was designed, synthesized, and screened against three aspartic proteases (plasmepsin II, HIV protease, and cathepsin D). The potency of norstatine inhibitors was found to depend strongly on the P1 substituent. Large, hydrophobic residues such as biphenyl, 4-bromophenyl, and 4-nitrophenyl enhanced the inhibitory activity (IC50) by up to 70-fold against plasmepsin II. In addition, P1 variation introduced significant selectivity, as up to 9-fold greater activity was found against plasmepsin II relative to human cathepsin D. The active P1 site residues did not fit into the crystal structure; however, molecular dynamics simulation suggested a possible alternative binding mode.

Hereditary thrombophilia: identification of nonsense and missense mutations in the protein C gene

International Nuclear Information System (INIS)

Romeo, G.; Hassan, H.J.; Staempfli, S.

1987-01-01

The structure of the gene for protein C, an anticoagulant serine protease, was analyzed in 29 unrelated patients with hereditary thrombophilia and protein C deficiency. Gene deletion(s) or gross rearrangement(s) was not demonstrable by Southern blot hybridization to cDNA probes. However, two unrelated patients showed a variant restriction pattern after Pvu II or BamHi digestion, due to mutations in the last exon: analysis of their pedigrees, including three or seven heterozygotes, respectively, with ∼50% reduction of both enzymatic and antigen level, showed the abnormal restriction pattern in all heterozygous individuals, but not in normal relatives. Cloning of protein C gene and sequencing of the last exon allowed the authors to identify a nonsense and a missense mutation, respectively. In the first case, codon 306 (CGA, arginine) is mutated to an inframe stop codon, thus generating a new Pvu II recognition site. In the second case, a missense mutation in the BamHI palindrome (GGATCC → GCATCC) leads to substitution of a key amino acid (a tryptophan to cysteine substitution at position 402), invariantly conserved in eukaryotic serine proteases. These point mutations may explain the protein C-deficiency phenotype of heterozygotes in the two pedigrees

Saccharomyces boulardii protease inhibits Clostridium difficile toxin A effects in the rat ileum.

Science.gov (United States)

Castagliuolo, I; LaMont, J T; Nikulasson, S T; Pothoulakis, C

1996-01-01

Saccharomyces boulardii, a nonpathogenic yeast, is effective in treating some patients with Clostridium difficile diarrhea and colitis. We have previously reported that S. boulardii inhibits rat ileal secretion in response to C. difficile toxin A possibly by releasing a protease that digests the intestinal receptor for this toxin (C. Pothoulakis, C. P. Kelly, M. A. Joshi, N. Gao, C. J. O'Keane, I. Castagliuolo, and J. T. LaMont, Gastroenterology 104: 1108-1115, 1993). The aim of this study was to purify and characterize this protease. S. boulardii protease was partially purified by gel filtration on Sephadex G-50 and octyl-Sepharose. The effect of S. boulardii protease on rat ileal secretion, epithelial permeability, and morphology in response to toxin A was examined in rat ileal loops in vivo. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified S. boulardii protease revealed a major band at 54 kDa. Pretreatment of rat ileal brush border (BB) membranes with partially purified protease reduced specific toxin A receptor binding (by 26%). Partially purified protease digested the toxin A molecule and significantly reduced its binding to BB membranes in vitro (by 42%). Preincubation of toxin A with S. boulardii protease inhibited ileal secretion (46% inhibition, P < 0.01), mannitol permeability (74% inhibition, P < 0.01), and histologic damage caused by toxin A. Thus, S. boulardii protease inhibits the intestinal effects of C. difficile toxin A by proteolysis of the toxin and inhibition of toxin A binding to its BB receptor. Our results may be relevant to the mechanism by which S. boulardii exerts its protective effects in C. difficile infection in humans. PMID:8945570

Biodegradation of a keratin waste and the concomitant production of detergent stable serine proteases from Paecilomyces lilacinus.

Science.gov (United States)

Cavello, I A; Cavalitto, S F; Hours, R A

2012-07-01

Paecilomyces lilacinus (LPS 876) efficiently degraded keratin in chicken feather during submerged cultivation producing extracellular proteases. Characterization of crude protease activity was done including its compatibility in commercial detergents. Optimum pH and temperature were 10.0 and 60 °C, respectively. Protease activity was enhanced by Ca²⁺ but was strongly inhibited by PMSF and by Hg²⁺ suggesting the presence of thiol-dependent serine proteases. The crude protease showed extreme stability toward non-ionic (Tween 20, Tween 85, and Triton X-100) and anionic (SDS) surfactants, and relative stability toward oxidizing agent (H₂O₂ and sodium perborate). In addition, it showed excellent stability and compatibility with various solid and liquid commercial detergents from 30 to 50 °C. The enzyme preparation retained more than 95% of its initial activity with solid detergents (Ariel™ and Drive™) and 97% of its original activity with a liquid detergent (Ace™) after pre-incubation at 40 °C. The protective effect of polyols (propylene glycol, PEG 4000, and glycerol) on the heat inactivation was also examined and the best results were obtained with glycerol from 50 to 60 °C. Considering its promising properties, P. lilacinus enzymatic preparation may be considered as a candidate for use in biotechnological processes (i.e., as detergent additive) and in the processing of keratinous wastes.

Cathepsins: Proteases that are vital for survival but can also be fatal.

Science.gov (United States)

Patel, Seema; Homaei, Ahmad; El-Seedi, Hesham R; Akhtar, Nadeem

2018-06-06

The state of enzymes in the human body determines the normal physiology or pathology, so all the six classes of enzymes are crucial. Proteases, the hydrolases, can be of several types based on the nucleophilic amino acid or the metal cofactor needed for their activity. Cathepsins are proteases with serine, cysteine, or aspartic acid residues as the nucleophiles, which are vital for digestion, coagulation, immune response, adipogenesis, hormone liberation, peptide synthesis, among a litany of other functions. But inflammatory state radically affects their normal roles. Released from the lysosomes, they degrade extracellular matrix proteins such as collagen and elastin, mediating parasite infection, autoimmune diseases, tumor metastasis, cardiovascular issues, and neural degeneration, among other health hazards. Over the years, the different types and isoforms of cathepsin, their optimal pH and functions have been studied, yet much information is still elusive. By taming and harnessing cathepsins, by inhibitors and judicious lifestyle, a gamut of malignancies can be resolved. This review discusses these aspects, which can be of clinical relevance. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

SwissProt search result: AK242832 [KOME

Lifescience Database Archive (English)

Full Text Available AK242832 J090067L13 (Q6PZ02) Cysteine protease APG4B (EC 3.4.22.-) (Autophagy 4 homolog B) (Auto...phagy-related cysteine endopeptidase 2B) (Autophagin-2B) (cAut2B) APG4B_CHICK 2e-44 ...

Host regulation of lysogenic decision in bacteriophage lambda: transmembrane modulation of FtsH (HflB), the cII degrading protease, by HflKC (HflA).

Science.gov (United States)

Kihara, A; Akiyama, Y; Ito, K

1997-05-27

The cII gene product of bacteriophage lambda is unstable and required for the establishment of lysogenization. Its intracellular amount is important for the decision between lytic growth and lysogenization. Two genetic loci of Escherichia coli are crucial for these commitments of infecting lambda genome. One of them, hflA encodes the HflKC membrane protein complex, which has been believed to be a protease degrading the cII protein. However, both its absence and overproduction stabilized cII in vivo and the proposed serine protease-like sequence motif in HflC was dispensable for the lysogenization control. Moreover, the HflKC protein was found to reside on the periplasmic side of the plasma membrane. In contrast, the other host gene, ftsH (hflB) encoding an integral membrane ATPase/protease, is positively required for degradation of cII, since loss of its function stabilized cII and its overexpression accelerated the cII degradation. In vitro, purified FtsH catalyzed ATP-dependent proteolysis of cII and HflKC antagonized the FtsH action. These results, together with our previous finding that FtsH and HflKC form a complex, suggest that FtsH is the cII degrading protease and HflKC is a modulator of the FtsH function. We propose that this transmembrane modulation differentiates the FtsH actions to different substrate proteins such as the membrane-bound SecY protein and the cytosolic cII protein. This study necessitates a revision of the prevailing view about the host control over lambda lysogenic decision.

Soft Cysteine Signaling Network: The Functional Significance of Cysteine in Protein Function and the Soft Acids/Bases Thiol Chemistry That Facilitates Cysteine Modification.

Science.gov (United States)

Wible, Ryan S; Sutter, Thomas R

2017-03-20

The unique biophysical and electronic properties of cysteine make this molecule one of the most biologically critical amino acids in the proteome. The defining sulfur atom in cysteine is much larger than the oxygen and nitrogen atoms more commonly found in the other amino acids. As a result of its size, the valence electrons of sulfur are highly polarizable. Unique protein microenvironments favor the polarization of sulfur, thus increasing the overt reactivity of cysteine. Here, we provide a brief overview of the endogenous generation of reactive oxygen and electrophilic species and specific examples of enzymes and transcription factors in which the oxidation or covalent modification of cysteine in those proteins modulates their function. The perspective concludes with a discussion of cysteine chemistry and biophysics, the hard and soft acids and bases model, and the proposal of the Soft Cysteine Signaling Network: a hypothesis proposing the existence of a complex signaling network governed by layered chemical reactivity and cross-talk in which the chemical modification of reactive cysteine in biological networks triggers the reorganization of intracellular biochemistry to mitigate spikes in endogenous or exogenous oxidative or electrophilic stress.

Purification and characterisation of a salt-stable protease from the halophilic archaeon Halogranum rubrum.

Science.gov (United States)

Gao, Ruichang; Shi, Tong; Liu, Xiangdong; Zhao, Mengqin; Cui, Henglin; Yuan, Li

2017-03-01

Because proteases play an important role in the fermentation of fish sauce, the purification and characterisation of an extracellular protease from the halophilic archaeon Halogranum rubrum was investigated. The molecular mass of the protease was estimated to be approximately 47 kDa based on sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) and native-PAGE analysis. The optimum conditions for catalytic activity were pH 8.0 and 50°C. The protease showed alkaline stability (pH 7.0-10.0). The protease also exhibited novel catalytic ability over a broad range of salinity (NaCl 0-3 mol L -1 ). Calcium ion enhanced the proteolytic activity of the enzyme. The K m and V max values of the purified protease for casein were calculated to be 4.89 mg mL -1 and 1111.11 U mL -1 , respectively. The protease was strongly inhibited by ethylenediamine tetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Meanwhile, the protease was stable in the presence of Triton X-100, isopropanol, ethanol or dithio-bis-nitrobenzoic (DTNB), but was inhibited by sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO) or methanol. MALDI -TOF/TOF MS analysis revealed that the protease shared some functional traits with protease produced by Halogranum salarium. Furthermore, it exhibited high hydrolytic activity on silver carp myosin protein. The protease is an alkaline and salt-tolerant enzyme that hydrolyses silver carp myosin with high efficiency. These excellent characteristics make this protease an attractive candidate for industrial use in low-salt fish sauce fermentation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Concomitant production of two proteases and alpha-amylase by a novel strain of Bacillus subtilis in a microprocessor controlled bioreactor

Directory of Open Access Journals (Sweden)

Hamid Mukhtar

2012-09-01

Full Text Available We describe the simultaneous production of Bacillus subtilis based proteases and alpha amylase using a computer controlled laboratory scale 7.5 L batch bioreactor. The present strain is the first to be reported that concomitantly produces these two industrially important enzymes. The growth and sporulation of Bacillus subtilis was monitored and maximum production of alkaline protease and alpha amylase was found to coincide with maximum sporulation. Two types of proteases were detected in the fermentation broth; a neutral and an alkaline protease most active in a pH range of 7.0-8.0 and 8.0-10, respectively. Maximum production of proteases was observed at an incubation temperature of 37ºC while that of alpha amylase was observed at 40ºC. The optimum aeration and agitation levels for protease production were 0.6 L/L/min and 200rpm, respectively, and for alpha amylase were 0.6 L/L/min and 150 rpm. The kinetic parameters Yp/x and qp were also found to be significant at the given fermentation conditions.

Effect of semen extender supplementation with cysteine on postthaw sperm quality, DNA damage, and fertilizing ability in the common carp (Cyprinus carpio).

Science.gov (United States)

Öğretmen, Fatih; İnanan, Burak Evren; Kutluyer, Filiz; Kayim, Murathan

2015-06-01

Amino acids have an important biological role for prevention of cell damage during cryopreservation. The objective of this study is to determine the effects of cysteine on postthaw sperm motility, duration of sperm motility, DNA damage, and fertility in the common carp (Cyprinus carpio). Sperm collected from 10 individuals was cryopreserved in extenders containing different cysteine concentrations (2.5, 5, 10, and 20 mM). Semen samples diluted at the ratio of 1:9 by the extenders were subjected to cryopreservation. After dilution, the semen was aspirated into 0.25-mL straws; the straws were placed on the tray, frozen in nitrogen vapor, and plunged into liquid nitrogen. DNA damage was evaluated by comet assay after cryopreservation. Our results indicated that an increase in the concentration of cysteine caused a significant increase in the motility rate and duration of sperm in the common carp (C carpio; P < 0.05). Comparing all concentrations of cysteine, the best concentration of cysteine was 20 mM. Higher postthaw motility (76.00 ± 1.00%) and fertilization (97.00 ± 1.73%) rates were obtained with the extender at the concentration of 20 mM. Supplementation of the extender with cysteine was increased the fertilization and hatching rate and decreased DNA damage. Consequently, cysteine affected the motility, fertilization, and DNA damage positively, and extenders could be supplemented with cysteine. Copyright © 2015 Elsevier Inc. All rights reserved.

Yeast genes involved in regulating cysteine uptake affect production of hydrogen sulfide from cysteine during fermentation.

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Huang, Chien-Wei; Walker, Michelle E; Fedrizzi, Bruno; Gardner, Richard C; Jiranek, Vladimir

2017-08-01

An early burst of hydrogen sulfide (H2S) produced by Saccharomyces cerevisiae during fermentation could increase varietal thiols and therefore enhance desirable tropical aromas in varieties such as Sauvignon Blanc. Here we attempted to identify genes affecting H2S formation from cysteine by screening yeast deletion libraries via a colony colour assay on media resembling grape juice. Both Δlst4 and Δlst7 formed lighter coloured colonies and produced significantly less H2S than the wild type on high concentrations of cysteine, likely because they are unable to take up cysteine efficiently. We then examined the nine known cysteine permeases and found that deletion of AGP1, GNP1 and MUP1 led to reduced production of H2S from cysteine. We further showed that deleting genes involved in the SPS-sensing pathway such as STP1 and DAL81 also reduced H2S from cysteine. Together, this study indirectly confirms that Agp1p, Gnp1p and Mup1p are the major cysteine permeases and that they are regulated by the SPS-sensing and target of rapamycin pathways under the grape juice-like, cysteine-supplemented, fermentation conditions. The findings highlight that cysteine transportation could be a limiting factor for yeast to generate H2S from cysteine, and therefore selecting wine yeasts without defects in cysteine uptake could maximise thiol production potential. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Enterovirus type 71 2A protease functions as a transcriptional activator in yeast

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Lai Meng-Jiun

2010-08-01

Full Text Available Abstract Enterovirus type 71 (EV71 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost this activity after truncation of 60 amino acids at the N-terminus or deletion of 20 amino acids at the C-terminus. Thus, the acidic domain at the C-terminus of this protein is essential for its transcriptional activity. Indeed, deletion of amino acids from 146 to 149 (EAME in this acidic domain lost the transcriptional activity of EV71 2A protein though still retained its protease activity. EV71 2A protease was detected both in the cytoplasm and nucleus using confocal microscopy analysis. Coxsackie virus B3 2A protease also exhibited transcriptional activity in yeast cells. As expected, an acidic domain in the C-terminus of Coxsackie virus B3 2A protease was also identified. Truncation of this acidic domain resulted in the loss of transcriptional activity. Interestingly, this acidic region of poliovirus 2A protease is critical for viral RNA replication. The transcriptional activity of the EV71 or Coxsackie virus B3 2A protease should play a role in viral replication and/or pathogenesis.

Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

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Madhusudhan Budatha

Full Text Available 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.

Mechanisms of Mitochondrial Holocytochrome c Synthase and the Key Roles Played by Cysteines and Histidine of the Heme Attachment Site, Cys-XX-Cys-His*

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Babbitt, Shalon E.; San Francisco, Brian; Mendez, Deanna L.; Lukat-Rodgers, Gudrun S.; Rodgers, Kenton R.; Bretsnyder, Eric C.; Kranz, Robert G.

2014-01-01

Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19. PMID:25170082

An Alkaline Protease from Bacillus pumilus MP 27: Functional Analysis of Its Binding Model toward Its Applications As Detergent Additive.

Science.gov (United States)

Baweja, Mehak; Tiwari, Rameshwar; Singh, Puneet K; Nain, Lata; Shukla, Pratyoosh

2016-01-01

A proteolytic strain of Bacillus pumilus MP 27 was isolated from water samples of Southern ocean produced alkaline protease. Since protease production need expensive ingredients, an economically viable process was developed by using low cost carbon source, wheat straw, supplemented with peptone. This protease was active within temperature ranges 10-70°C at pH 9. This process was optimized by response surface methodology using a Box Bekhman design by Design Expert 7.0 software that increased the protease activity to 776.5 U/ml. Moreover, the enzyme was extremely stable at a broad range of temperature and pH retaining 69% of its activity at 50°C and 70% at pH 11. The enzyme exhibited excellent compatibility with surfactants and commercial detergents, showing 87% stability with triton X-100 and 100% stability with Tide commercial detergent. The results of the wash performance analysis demonstrated considerably good de-staining at 50 and 4°C with low supplementation (109 U/ml). Molecular modeling of the protease revealed the presence of serine proteases, subtilase family and serine active site and further docking supported the association of catalytic site with the various substrates. Certainly, such protease can be considered as a good detergent additive in detergent industry with a possibility to remove the stains effectively even in a cold wash.

Protease signaling through protease activated receptor 1 mediate nerve activation by mucosal supernatants from irritable bowel syndrome but not from ulcerative colitis patients.

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Buhner, Sabine; Hahne, Hannes; Hartwig, Kerstin; Li, Qin; Vignali, Sheila; Ostertag, Daniela; Meng, Chen; Hörmannsperger, Gabriele; Braak, Breg; Pehl, Christian; Frieling, Thomas; Barbara, Giovanni; De Giorgio, Roberto; Demir, Ihsan Ekin; Ceyhan, Güralp Onur; Zeller, Florian; Boeckxstaens, Guy; Haller, Dirk; Kuster, Bernhard; Schemann, Michael

2018-01-01

The causes of gastrointestinal complaints in irritable bowel syndrome (IBS) remain poorly understood. Altered nerve function has emerged as an important pathogenic factor as IBS mucosal biopsy supernatants consistently activate enteric and sensory neurons. We investigated the neurally active molecular components of such supernatants from patients with IBS and quiescent ulcerative colitis (UC). Effects of supernatants from 7 healthy controls (HC), 20 IBS and 12 UC patients on human and guinea pig submucous neurons were studied with neuroimaging techniques. We identify differentially expressed proteins with proteome analysis. Nerve activation by IBS supernatants was prevented by the protease activated receptor 1 (PAR1) antagonist SCHE79797. UC supernatants also activated enteric neurons through protease dependent mechanisms but without PAR1 involvement. Proteome analysis of the supernatants identified 204 proteins, among them 17 proteases as differentially expressed between IBS, UC and HC. Of those the four proteases elastase 3a, chymotrypsin C, proteasome subunit type beta-2 and an unspecified isoform of complement C3 were significantly more abundant in IBS compared to HC and UC supernatants. Of eight proteases, which were upregulated in IBS, the combination of elastase 3a, cathepsin L and proteasome alpha subunit-4 showed the highest prediction accuracy of 98% to discriminate between IBS and HC groups. Elastase synergistically potentiated the effects of histamine and serotonin-the two other main neuroactive substances in the IBS supernatants. A serine protease inhibitor isolated from the probiotic Bifidobacterium longum NCC2705 (SERPINBL), known to inhibit elastase-like proteases, prevented nerve activation by IBS supernatants. Proteases in IBS and UC supernatants were responsible for nerve activation. Our data demonstrate that proteases, particularly those signalling through neuronal PAR1, are biomarker candidates for IBS, and protease profiling may be used to

SCREENING OF PROTEASE INHIBITORS RESISTANCE MUTATIONS IN HEPATITIS C VIRUS ISOLATES INFECTING ROMANIAN PATIENTS UNEXPOSED TO TRIPLE THERAPY.

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Dinu, Sorin; Calistru, Petre-Iacob; Ceauşu, Emanoil; Târdeil, Graţiela; Oprişan, Gabriela

2015-01-01

Although the European recommendations include the use of new antiviral drugs for the treatment of hepatitis C, in Romania the current treatment remains interferon plus ribavirin. First generation viral protease inhibitors (i.e. boceprevir, telaprevir), which have raised the chances of obtaining viral clearance in up to 70% of infection cases produced by genotype 1 isolates, have not been introduced yet as standard treatment in our country. The success of these new antivirals is limited by the occurrence and selection of resistance mutations during therapy. We set-up a molecular study aiming to detect any resistance mutations to boceprevir and telaprevir harbored by hepatitis C isolates infecting Romanian patients naïve to viral protease inhibitors. Since these new antivirals are efficient and approved for genotype 1 infection, viral samples were genotyped following a protocol previously developed by our research group. We analyzed by both population sequencing and molecular cloning and sequencing the NS3 protease region of hepatitis C virus isolates infecting patients which were not previously exposed to boceprevir and telaprevir. All the analyzed samples were subtype 1b and resembled the samples collected in recent years from Romanian patients. Molecular cloning followed by sequencing showed great intra-host diversity, which is known to represent the source of isolates with different resistance phenotypes. Both population sequencing and molecular cloning followed by clone sequencing revealed two boceprevir resistance mutations (T54S and V55A), respectively, a telaprevir resistance mutation (T54S) in the sequences obtained from a patient with chronic hepatitis C. To our knowledge, this is the first study indicating the existence of pre-treatment resistance mutations to boceprevir and telaprevir in hepatitis C virus isolates infecting Romanian patients.

Ebselen inhibits hepatitis C virus NS3 helicase binding to nucleic acid and prevents viral replication.

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Mukherjee, Sourav; Weiner, Warren S; Schroeder, Chad E; Simpson, Denise S; Hanson, Alicia M; Sweeney, Noreena L; Marvin, Rachel K; Ndjomou, Jean; Kolli, Rajesh; Isailovic, Dragan; Schoenen, Frank J; Frick, David N

2014-10-17

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 μM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 μM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure-activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines.

Isolation of alkaline protease from Bacillus subtilis AKRS3

African Journals Online (AJOL)

ashok

2012-08-28

Aug 28, 2012 ... production proved high protease production than the other tested ... Crude alkaline protease was most active at 55°C, pH 9 with casein as ... 13416 Afr. J. Biotechnol. ... The Gram-positive, aerobic, rod-shaped endospore-.

Cow dung: a potential biomass substrate for the production of detergent-stable dehairing protease by alkaliphilic Bacillus subtilis strain VV.

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Vijayaraghavan, Ponnuswamy; Vijayan, Aija; Arun, Arumugaperumal; Jenisha, John Kennady; Vincent, Samuel Gnana Prakash

2012-01-01

Cow dung, a cheap and easily available source of energy, was used as the substrate for the production of alkaline protease by solid-state fermentation using the Bacillus subtilis strain VV. In order to achieve the maximum yield of this enzyme, the following optimum process parameters are needed: fermentation period (72 h), pH (10.0), moisture content (140%), inoculum (25%), temperature (30-40°C), carbon source (2% (w/w) maltose) and nitrogen source (1% (w/w) urea). The protease was stable over a broad temperature range (30-50°C) and pH (8.0-10.0), with maximum activity at 50°C and pH 10.0. Among the divalent ions tested, Ca(2+) (0.01 M) increased enzyme activity. The purified protease, after being subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was found to have a molecular mass of 38.5 kDa. The enzyme was solvent-and surfactant-stable and showed activity even after 24 h incubation along with various commercially available detergents. This enzyme possessed dehairing properties for animal hide after 16 h of incubation at room temperature. From these results it is evident that cow dung is a potential substrate for the production of a detergent-stable, dehairing protease by B. subtilis. This enzyme has a lot of potential applications in the detergent and leather-processing industries.

Characterization of the gene encoding serine acetyltransferase, a regulated enzyme of cysteine biosynthesis from the protist parasites Entamoeba histolytica and Entamoeba dispar. Regulation and possible function of the cysteine biosynthetic pathway in Entamoeba.

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Nozaki, T; Asai, T; Sanchez, L B; Kobayashi, S; Nakazawa, M; Takeuchi, T

1999-11-05

The enteric protist parasites Entamoeba histolytica and Entamoeba dispar possess a cysteine biosynthetic pathway, unlike their mammalian host, and are capable of de novo production of L-cysteine. We cloned and characterized cDNAs that encode the regulated enzyme serine acetyltransferase (SAT) in this pathway from these amoebae by genetic complementation of a cysteine-auxotrophic Escherichia coli strain with the amoebic cDNA libraries. The deduced amino acid sequences of the amoebic SATs exhibited, within the most conserved region, 36-52% identities with the bacterial and plant SATs. The amoebic SATs contain a unique insertion of eight amino acids, also found in the corresponding region of a plasmid-encoded SAT from Synechococcus sp., which showed the highest overall identities to the amoebic SATs. Phylogenetic reconstruction also revealed a close kinship of the amoebic SATs with cyanobacterial SATs. Biochemical characterization of the recombinant E. histolytica SAT revealed several enzymatic features that distinguished the amoebic enzyme from the bacterial and plant enzymes: 1) inhibition by L-cysteine in a competitive manner with L-serine; 2) inhibition by L-cystine; and 3) no association with cysteine synthase. Genetically engineered amoeba strains that overproduced cysteine synthase and SAT were created. The cysteine synthase-overproducing amoebae had a higher level of cysteine synthase activity and total thiol content and revealed increased resistance to hydrogen peroxide. These results indicate that the cysteine biosynthetic pathway plays an important role in antioxidative defense of these enteric parasites.

Protease activity of Per a 10 potentiates Th2 polarization by increasing IL-23 and OX40L.

Science.gov (United States)

Agrawal, Komal; Kale, Sagar L; Arora, Naveen

2015-12-01

Proteases are implicated in exacerbation of allergic diseases. In this study, the role of proteolytic activity of Per a 10 was evaluated on Th2 polarization. Intranasal administration of Per a 10 in mice led to allergic airway inflammation as seen by higher IgE levels, cellular infiltration, IL-17A, and Th2 cytokines, whereas, inactive (Δ)Per a 10 showed attenuated response. There was an increased OX40L expression on lung and lymph node dendritic cells in Per a 10 immunized group and on Per a 10 stimulated BMDCs. Reduction in CD40 expression without any change at transcript level in lungs of Per a 10 immunized mice suggested CD40 cleavage. BMDCs pulsed with Per a 10 showed reduced CD40 expression with lower IL-12p70 secretion as compared to heat inactivated Per a 10. IL-23, TNF-α, and IL-6 levels were significantly higher in Per a 10 stimulated BMDCs supernatant. In DC-T cell coculture studies, Per a 10 pulsed BMDCs showed higher levels of IL-4 and IL-13 that were reduced on blocking of either IL-23 or OX40L. In conclusion, the data suggests a critical role of protease activity of Per a 10 in promoting Th2 polarization by increasing IL-23 secretion and OX40L expression on dendritic cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cold denaturation of the HIV-1 protease monomer

DEFF Research Database (Denmark)

Rösner, Heike Ilona; Caldarini, Martina; Prestel, Andreas

2017-01-01

The HIV-1-protease is a complex protein which in its active form adopts a homodimer dominated by -sheet structures. We have discovered a cold-denatured state of the monomeric subunit of HIV-1-protease which is populated above 0ºC and therefore directly accessible to various spectroscopic approac...

Effect of the culture conditions on the production of an extracellular protease by thermophilic Bacillus sp and some properties of the enzymatic activity Efeito das condições de cultivo sobre a produção de proteases extracelulares pelo termofílico Bacillus sp e algumas propriedades da atividade enzimática

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Camila Rocha da Silva

2007-06-01

Full Text Available Protease production by thermophilic Bacillus sp strain SMIA-2 cultivated in liquid cultures containing 1% maltose as a carbon source and supplemented with whey protein (0.1% and corn steep liquor (0.3% reached a maximum at 14 h, with levels of 42 U/mg protein. The microorganism was capable of utilizing a wide range of carbon sources, but protease activity varied according the carbon source. Starch and maltose were the best carbon sources in the present study for protease secretion, while lactose and sucrose were less effective. Increasing maltose concentration in the medium until 1%, improved the growth of the organism and the enzyme activity. Regarding the amounts of corn steep liquor and whey protein in the medium, the concentrations of 0.2% and 0.1% respectively, were considered the most effective for protease secretion by the organism. Studies on the protease characterization revealed that the optimum temperature of this enzyme was 70ºC. Thermostability profile indicated that the enzyme retained 80% of the original activity after 2 h heat treatment at 60ºC. At 70ºC, 70% of the original activity was retained after 15 min heat treatment. The optimum pH of the enzyme was found to be 8.5. After incubation of crude enzyme solution at room temperature for 2 h at pH 6.0-10.0, a decreased of about 15% of its original activity at pH 8.5 was observed. At pH 10.0, the decrease was 24%. In the presence of 1.0 M and 5.0 M NaCl, 76% and 37% of protease activity was retained after 2 h incubating at 45ºC respectively.A produção de proteases pelo termofílico Bacillus sp cepa SMIA-2 cultivado em culturas líquidas contendo maltose (1% e suplementada com proteínas de soro (0,1% e água de maceração de milho (0,3% alcançou o máximo em 14 h, com níveis de 42 U/mg proteína. O microrganismo foi capaz de utilizar várias fontes de carbono, mas a atividade da protease variou com cada fonte. Amido e maltose foram as melhores fontes para a secreção da

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

Science.gov (United States)

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.

S-Substituted cysteine derivatives and thiosulfinate formation in Petiveria alliacea-part II.

Science.gov (United States)

Kubec, Roman; Kim, Seokwon; Musah, Rabi A

2002-11-01

Three cysteine derivatives, (R)-S-(2-hydroxyethyl)cysteine, together with (R(S)R(C))- and (S(S)R(C))-S-(2-hydroxyethyl)cysteine sulfoxides, have been isolated from the roots of Petiveria alliacea. Furthermore, three additional amino acids, S-methyl-, S-ethyl-, and S-propylcysteine derivatives, were detected. They were present only in trace amounts (<3 microg g(-1) fr. wt), precluding determination of their absolute configurations and oxidation states. In addition, four thiosulfinates, S-(2-hydroxyethyl) (2-hydroxyethane)-, S-(2-hydroxyethyl) phenylmethane-, S-benzyl (2-hydroxyethane)- and S-benzyl phenylmethanethiosulfinates, have been found in a homogenate of the roots. The formation pathways of various benzyl/phenyl-containing compounds previously found in the plant were also discussed.

A new method for the characterization of strain-specific conformational stability of protease-sensitive and protease-resistant PrPSc.

Directory of Open Access Journals (Sweden)

Laura Pirisinu

Full Text Available Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrP(Sc, a disease-associated isoform of the host-encoded cellular protein (PrP(C. Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrP(Sc. However, PrP(Sc is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrP(Sc aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrP(C and PrP(Sc by means of differential centrifugation. The conformational solubility and stability assay (CSSA was then developed by measuring PrP(Sc solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl](1/2 values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl](1/2 values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M, followed by sheep scrapie (2.2 M and by MM2 sCJD (1.6 M. In order to test the ability of CSSA to characterise protease-sensitive PrP(Sc, we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrP(Sc aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrP(Sc conformational stabilities of protease-resistant and protease-sensitive PrP(Sc and that it is a valuable tool

Antimalarial Activity of Azadipeptide Nitriles

OpenAIRE

Löser, Reik; Gut, Jiri; Rosenthal, Philip J.; Frizler, Maxim; Gütschow, Michael; Andrews, Katherine T.

2009-01-01

Azadipeptide nitriles – novel cysteine protease inhibitors – display structure-dependent antimalarial activity against both chloroquine-sensitive and chloroquine-resistant lines of cultured Plasmodium falciparum malaria parasites. Inhibition of parasite’s haemoglobin-degrading cysteine proteases was also investigated, revealing the azadipeptide nitriles as potent inhibitors of falcipain-2 and -3. A correlation between the cysteine protease-inhibiting activity and the antimalarial potential of...

Construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors.

Science.gov (United States)

Lai, Huiguo; Teramoto, Tadahisa; Padmanabhan, Radhakrishnan

2014-01-01

Dengue virus serotypes 1-4 (DENV1-4) are mosquito-borne human pathogens of global significance causing ~390 million cases annually worldwide. The virus infections cause in general a self-limiting disease, known as dengue fever, but occasionally also more severe forms, especially during secondary infections, dengue hemorrhagic fever and dengue shock syndrome causing ~25,000 deaths annually. The DENV genome contains a single-strand positive sense RNA, approximately 11 kb in length. The 5'-end has a type I cap structure. The 3'-end has no poly(A) tail. The viral RNA has a single long open reading frame that is translated by the host translational machinery to yield a polyprotein precursor. Processing of the polyprotein precursor occurs co-translationally by cellular proteases and posttranslationally by the viral serine protease in the endoplasmic reticulum (ER) to yield three structural proteins (capsid (C), precursor membrane (prM), and envelope (E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The active viral protease consists of both NS2B, an integral membrane protein in the ER, and the N-terminal part of NS3 (180 amino acid residues) that contains the trypsin-like serine protease domain having a catalytic triad of H51, D75, and S135. The C-terminal part of NS3, ~170-618 amino acid residues, encodes an NTPase/RNA helicase and 5'-RNA triphosphatase activities; the latter enzyme is required for the first step in 5'-capping. The cleavage sites of the polyprotein by the viral protease consist of two basic amino acid residues such as KR, RR, or QR, followed by short chain amino acid residues, G, S, or T. Since the cleavage of the polyprotein by the viral protease is absolutely required for assembly of the viral replicase, blockage of NS2B/NS3pro activity provides an effective means for designing dengue virus (DENV) small-molecule therapeutics. Here we describe the screening of small-molecule inhibitors against DENV2 protease.

C1q protein binds to the apoptotic nucleolus and causes C1 protease degradation of nucleolar proteins.

Science.gov (United States)

Cai, Yitian; Teo, Boon Heng Dennis; Yeo, Joo Guan; Lu, Jinhua

2015-09-11

In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r2C1s2), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Efficient Absorption of X-Hydroxyproline (Hyp)-Gly after Oral Administration of a Novel Gelatin Hydrolysate Prepared Using Ginger Protease.

Science.gov (United States)

Taga, Yuki; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

2016-04-13

Recent studies have reported that oral intake of gelatin hydrolysate has various beneficial effects, such as reduction of joint pain and lowering of blood sugar levels. In this study, we produced a novel gelatin hydrolysate using a cysteine-type ginger protease having unique substrate specificity with preferential peptide cleavage with Pro at the P2 position. Substantial amounts of X-hydroxyproline (Hyp)-Gly-type tripeptides were generated up to 2.5% (w/w) concomitantly with Gly-Pro-Y-type tripeptides (5%; w/w) using ginger powder. The in vivo absorption of the ginger-degraded gelatin hydrolysate was estimated using mice. The plasma levels of collagen-derived oligopeptides, especially X-Hyp-Gly, were significantly high (e.g., 2.3-fold for Glu-Hyp-Gly, p < 0.05) compared with those of the control gelatin hydrolysate, which was prepared using gastrointestinal proteases and did not contain detectable X-Hyp-Gly. This study demonstrated that orally administered X-Hyp-Gly was effectively absorbed into the blood, probably due to the high protease resistance of this type of tripeptide.

Potent Inhibitors of the Hepatitis C Virus NS3 Protease: Design and Synthesis of Macrocyclic Substrate-Based [beta]-Strand Mimics

Energy Technology Data Exchange (ETDEWEB)

Goudreau, Nathalie; Brochu, Christian; Cameron, Dale R.; Duceppe, Jean-Simon; Faucher, Anne-Marie; Ferland, Jean-Marie; Grand-Maître, Chantal; Poirier, Martin; Simoneau, Bruno; Tsantrizos, Youla S. (Boehringer)

2008-06-30

The virally encoded NS3 protease is essential to the life cycle of the hepatitis C virus (HCV), an important human pathogen causing chronic hepatitis, cirrhosis of the liver, and hepatocellular carcinoma. The design and synthesis of 15-membered ring {beta}-strand mimics which are capable of inhibiting the interactions between the HCV NS3 protease enzyme and its polyprotein substrate will be described. The binding interactions between a macrocyclic ligand and the enzyme were explored by NMR and molecular dynamics, and a model of the ligand/enzyme complex was developed.

Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

DEFF Research Database (Denmark)

Chen, Z. W.; Jiang, C. Y.; She, Qunxin

2005-01-01

). The thio-modifying reagent N-ethylmaleimide and Zn2+ strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant......Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C31 and C101-X-X-C104; numbering according to the Acidianus tengchongensis numbering system...... proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have...

Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

Energy Technology Data Exchange (ETDEWEB)

Amand, Helene L., E-mail: helene.amand@chalmers.se [Chalmers University of Technology, Department of Chemical and Biological Engineering/Physical Chemistry, SE-412 96 Gothenburg (Sweden); Norden, Bengt, E-mail: norden@chalmers.se [Chalmers University of Technology, Department of Chemical and Biological Engineering/Physical Chemistry, SE-412 96 Gothenburg (Sweden); Fant, Kristina, E-mail: kristina.fant@sp.se [Chalmers University of Technology, Department of Chemical and Biological Engineering/Physical Chemistry, SE-412 96 Gothenburg (Sweden)

2012-02-17

Highlights: Black-Right-Pointing-Pointer Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. Black-Right-Pointing-Pointer Dimer formation enhances peptiplex stability, resulting in increased transfection. Black-Right-Pointing-Pointer By dimerisation, the CPP EB1 even gain endosomal escape properties while lowering cytotoxicity. -- Abstract: Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes ('peptiplexes') enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the 'chelate effect' and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide

Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

International Nuclear Information System (INIS)

Åmand, Helene L.; Nordén, Bengt; Fant, Kristina

2012-01-01

Highlights: ► Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. ► Dimer formation enhances peptiplex stability, resulting in increased transfection. ► By dimerisation, the CPP EB1 even gain endosomal escape properties while lowering cytotoxicity. -- Abstract: Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes (“peptiplexes”) enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the “chelate effect” and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide dimer resulting from its stronger binding to DNA.

A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication

Energy Technology Data Exchange (ETDEWEB)

Ratia, Kiira; Pegan, Scott; Takayama, Jun; Sleeman, Katrina; Coughlin, Melissa; Baliji, Surendranath; Chaudhuri, Rima; Fu, Wentao; Prabhakar, Bellur S.; Johnson, Michael E.; Baker, Susan C.; Ghosh, Arun K.; Mesecar, Andrew D. (Loyola); (Purdue); (UIC)

2008-10-27

We report the discovery and optimization of a potent inhibitor against the papain-like protease (PLpro) from the coronavirus that causes severe acute respiratory syndrome (SARS-CoV). This unique protease is not only responsible for processing the viral polyprotein into its functional units but is also capable of cleaving ubiquitin and ISG15 conjugates and plays a significant role in helping SARS-CoV evade the human immune system. We screened a structurally diverse library of 50,080 compounds for inhibitors of PLpro and discovered a noncovalent lead inhibitor with an IC{sub 50} value of 20 {mu}M, which was improved to 600 nM via synthetic optimization. The resulting compound, GRL0617, inhibited SARS-CoV viral replication in Vero E6 cells with an EC{sub 50} of 15 {mu}M and had no associated cytotoxicity. The X-ray structure of PLpro in complex with GRL0617 indicates that the compound has a unique mode of inhibition whereby it binds within the S4-S3 subsites of the enzyme and induces a loop closure that shuts down catalysis at the active site. These findings provide proof-of-principle that PLpro is a viable target for development of antivirals directed against SARS-CoV, and that potent noncovalent cysteine protease inhibitors can be developed with specificity directed toward pathogenic deubiquitinating enzymes without inhibiting host DUBs.

Effect of Medium Composition on Commercially Important Alkaline Protease Production by Bacillus licheniformis N-2

Directory of Open Access Journals (Sweden)

Javed Iqbal Qazi

2008-01-01

Full Text Available Protease production by alkalophilic B. licheniformis N-2 was investigated in 50 mL of the growth medium consisting of (in g/L: glucose 10.0, soybean meal 10.0, K2HPO4 3.0, MgSO4·7H2O 0.5, NaCl 0.5 and CaCl2·2H2O 0.5 at pH=10. Different carbon and nitrogen sources in the form of fine powder of organic, inorganic and defatted meals were studied to select the suitable substrate for alkaline protease production. The highest level of alkaline protease (677.64 U/mL was obtained in the medium containing glucose followed by soluble starch and wheat bran. Among various nitrogen sources, defatted soybean meal was found to be the best inducer of alkaline protease, while inorganic nitrogen sources in the form of ammonium salts repressed the enzyme activity up to 96 %. Thermostability studies showed that the enzyme in the presence of 10 mM Ca2+ ions retained its residual activity up to 80 % even after incubation at 40 °C for 12 h. The enzyme was found stable over a broad range of pH (8–11 and lost 52 % of its residual activity at pH=12. After the treatment with Tween 20, Tween 45, Tween 65, Triton X-405, H2O2 and sodium perborate, each at 1.0 % concentration, the enzyme showed residual activity of 105, 82, 116, 109, 135 and 126 %, respectively. The application of alkaline protease for removal of blood stains from cotton fabric also indicates its potential use in detergent formulations.

SwissProt search result: AK110731 [KOME

Lifescience Database Archive (English)

Full Text Available AK110731 002-170-E10 (Q6PZ05) Cysteine protease APG4A (EC 3.4.22.-) (Autophagy 4 homolog A) (Auto...phagy-related cysteine endopeptidase 2A) (Autophagin-2A) (bAut2A) APG4A_BOVIN 5e-35 ...

Invasion of melanoma cells into dermal connective tissue in vitro: evidence for an important role of cysteine proteases.

NARCIS (Netherlands)

Dennhofer, R.; Kurschat, P.; Zigrino, P.; Klose, A.; Bosserhoff, A.; Muijen, G.N.P. van; Krieg, T.; Mauch, C.; Hunzelmann, N.

2003-01-01

Invasion of melanoma cells into the dermal connective tissue is a major characteristic in the complex process of metastasis. Proteases play an important role in tumor cell invasion as these enzymes are able to degrade most components of the extracellular matrix (ECM), and thus enable cells to

Novel inexpensive fungi proteases: Production by solid state fermentation and characterization.

Science.gov (United States)

Novelli, Paula Kern; Barros, Margarida Maria; Fleuri, Luciana Francisco

2016-05-01

A comparative study was carried out for proteases production using agroindustrial residues as substrate for solid state fermentation (SSF) of several fungal strains. High protease production was observed for most of the microorganisms studied, as well as very different biochemical characteristics, including activities at specific temperatures and a wide range of pH values. The enzymes produced were very different regarding optimum pH and they showed stability at 50 °C. Aspergillus oryzae showed stability at all pH values studied. Penicillium roquefortii and Aspergillus flavipes presented optimum activity at temperatures of 50 °C and 90 °C, respectively. Lyophilized protease from A. oryzae reached 1251.60 U/g and yield of 155010.66 U/kg of substrate. Therefore, the substrate as well as the microorganism strain can modify the biochemical character of the enzyme produced. The high protease activity and stability established plus the low cost of substrates, make these fungal proteases potential alternatives for the biotechnological industry. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mechanisms of mitochondrial holocytochrome c synthase and the key roles played by cysteines and histidine of the heme attachment site, Cys-XX-Cys-His.

Science.gov (United States)

Babbitt, Shalon E; San Francisco, Brian; Mendez, Deanna L; Lukat-Rodgers, Gudrun S; Rodgers, Kenton R; Bretsnyder, Eric C; Kranz, Robert G

2014-10-17

Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Crystal Structure of Mammalian Cysteine dioxygenase: A Novel Mononuclear Iron Center for Cysteine Thiol Oxidation

Energy Technology Data Exchange (ETDEWEB)

Simmons,C.; Liu, Q.; Huang, Q.; Hao, Q.; Begley, T.; Karplus, P.; Stipanuk, M.

2006-01-01

Cysteine dioxygenase is a mononuclear iron-dependent enzyme responsible for the oxidation of cysteine with molecular oxygen to form cysteinesulfinate. This reaction commits cysteine to either catabolism to sulfate and pyruvate or to the taurine biosynthetic pathway. Cysteine dioxygenase is a member of the cupin superfamily of proteins. The crystal structure of recombinant rat cysteine dioxygenase has been determined to 1.5 Angstroms resolution, and these results confirm the canonical cupin {beta}-sandwich fold and the rare cysteinyl-tyrosine intramolecular crosslink (between Cys93 and Tyr157) seen in the recently reported murine cysteine dioxygenase structure. In contrast to the catalytically inactive mononuclear Ni(II) metallocenter present in the murine structure, crystallization of a catalytically competent preparation of rat cysteine dioxygenase revealed a novel tetrahedrally coordinated mononuclear iron center involving three histidines (His86, His88, and His140) and a water molecule. Attempts to acquire a structure with bound ligand using either co-crystallization or soaks with cysteine revealed the formation of a mixed disulfide involving Cys164 near the active site, which may explain previously observed substrate inhibition. This work provides a framework for understanding the molecular mechanisms involved in thiol dioxygenation and sets the stage for exploring the chemistry of both the novel mononuclear iron center and the catalytic role of the cysteinyl-tyrosine linkage.

The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

Science.gov (United States)

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.

The effect of cysteine on the corrosion of 304L stainless steel in sulphuric acid

International Nuclear Information System (INIS)

Silva, A.B.; Agostinho, S.M.L.; Barcia, O.E.; Cordeiro, G.G.O.; D'Elia, E.

2006-01-01

The effect of cysteine on the corrosion of 304L stainless steel in 1 mol l -1 H 2 SO 4 was studied using open-circuit potential measurements, anodic polarization curves, electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). All the electrochemical measurements obtained in the presence of low cysteine concentration (10 -6 -10 -5 mol l -1 ) presented the same behaviour as those obtained in the absence of cysteine, a passivated steel surface. However, for higher cysteine concentrations (10 -4 -10 -2 mol l -1 ), a different behaviour was observed: the corrosion potential stabilized at a more negative value; an active region was observed in the anodic polarization curves and the electrochemical impedance diagrams showed an inductive loop at lower frequencies and a much lower polarization resistance. These results show that the presence of cysteine at high concentration turns the surface of 304L stainless steel electrochemically active, probably dissolving the passivation layer and promoting the stainless steel anodic dissolution. SEM experiments performed after immersion experiments at corrosion potential were in good agreement with the electrochemical results

Partial purification and characterization of alkaline proteases from ...

African Journals Online (AJOL)

Alkaline proteases from the digestive tract of anchovy were partially purified by ammonium sulfate fractionation, dialysis and Sephadex G-75 gel filtration. The purification fold and yield were 6.23 and 4.49%, respectively. The optimum activities of partially purified alkaline proteases were observed at 60°C and at pH 11.0.

GlyGly-CTERM and rhombosortase: a C-terminal protein processing signal in a many-to-one pairing with a rhomboid family intramembrane serine protease.

Directory of Open Access Journals (Sweden)

Daniel H Haft

Full Text Available The rhomboid family of serine proteases occurs in all domains of life. Its members contain at least six hydrophobic membrane-spanning helices, with an active site serine located deep within the hydrophobic interior of the plasma membrane. The model member GlpG from Escherichia coli is heavily studied through engineered mutant forms, varied model substrates, and multiple X-ray crystal studies, yet its relationship to endogenous substrates is not well understood. Here we describe an apparent membrane anchoring C-terminal homology domain that appears in numerous genera including Shewanella, Vibrio, Acinetobacter, and Ralstonia, but excluding Escherichia and Haemophilus. Individual genomes encode up to thirteen members, usually homologous to each other only in this C-terminal region. The domain's tripartite architecture consists of motif, transmembrane helix, and cluster of basic residues at the protein C-terminus, as also seen with the LPXTG recognition sequence for sortase A and the PEP-CTERM recognition sequence for exosortase. Partial Phylogenetic Profiling identifies a distinctive rhomboid-like protease subfamily almost perfectly co-distributed with this recognition sequence. This protease subfamily and its putative target domain are hereby renamed rhombosortase and GlyGly-CTERM, respectively. The protease and target are encoded by consecutive genes in most genomes with just a single target, but far apart otherwise. The signature motif of the Rhombo-CTERM domain, often SGGS, only partially resembles known cleavage sites of rhomboid protease family model substrates. Some protein families that have several members with C-terminal GlyGly-CTERM domains also have additional members with LPXTG or PEP-CTERM domains instead, suggesting there may be common themes to the post-translational processing of these proteins by three different membrane protein superfamilies.

Earthworm Protease

Directory of Open Access Journals (Sweden)

Rong Pan

2010-01-01

Full Text Available The alimentary tract of earthworm secretes a group of proteases with a relative wide substrate specificity. In 1983, six isozymes were isolated from earthworm with fibrinolytic activities and called fibriniolytic enzymes. So far, more isozymes have been found from different earthworm species such as Lumbricus rubellus and Eisenia fetida. For convenience, the proteases are named on the basis of the earthworm species and the protein function, for instance, Eisenia fetida protease (EfP. The proteases have the abilities not only to hydrolyze fibrin and other protein, but also activate proenzymes such as plasminogen and prothrombin. In the light of recent studies, eight of the EfPs contain oligosaccharides chains which are thought to support the enzyme structure. Interestingly, EfP-II has a broader substrate specificity presenting alkaline trypsin, chymotrypsin and elastase activities, but EfP-III-1 has a stricter specificity. The protein crystal structures show the characteristics in their specificities. Earthworm proteases have been applied in several areas such as clinical treatment of clotting diseases, anti-tumor study, environmental protection and nutritional production. The current clinical utilizations and some potential new applications of the earthworm protease will be discussed in this paper.

Earthworm Protease

International Nuclear Information System (INIS)

Pan, R.; Zhang, Z.; He, R.

2010-01-01

The alimentary tract of earthworm secretes a group of proteases with a relative wide substrate specificity. In 1983, six isozymes were isolated from earthworm with fibrinolytic activities and called fibrinolytic enzymes. So far, more isozymes have been found from different earthworm species such as Lumbricus rubellus and Eisenia fetida. For convenience, the proteases are named on the basis of the earthworm species and the protein function, for instance, Eisenia fetida protease (EfP). The proteases have the abilities not only to hydrolyze fibrin and other protein, but also activate pro enzymes such as plasminogen and prothrombin. In the light of recent studies, eight of the EfPs contain oligosaccharides chains which are thought to support the enzyme structure. Interestingly, EfP-II has a broader substrate specificity presenting alkaline trypsin, chymotrypsin and elastase activities, but EfP-III-1 has a stricter specificity. The protein crystal structures show the characteristics in their specificities. Earthworm proteases have been applied in several areas such as clinical treatment of clotting diseases, anti-tumor study, environmental protection and nutritional production. The current clinical utilizations and some potential new applications of the earthworm protease will be discussed in this paper.

Impact of cysteine variants on the structure, activity, and stability of recombinant human α-galactosidase A

Science.gov (United States)

Qiu, Huawei; Honey, Denise M; Kingsbury, Jonathan S; Park, Anna; Boudanova, Ekaterina; Wei, Ronnie R; Pan, Clark Q; Edmunds, Tim

2015-01-01

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity. PMID:26044846

Proteolysis of complement factors iC3b and C5 by the serine protease prostate-specific antigen in prostatic fluid and seminal plasma.

Science.gov (United States)

Manning, Michael L; Williams, Simon A; Jelinek, Christine A; Kostova, Maya B; Denmeade, Samuel R

2013-03-15

Prostate-specific Ag (PSA) is a serine protease that is expressed exclusively by normal and malignant prostate epithelial cells. The continued high-level expression of PSA by the majority of men with both high- and low-grade prostate cancer throughout the course of disease progression, even in the androgen-ablated state, suggests that PSA has a role in the pathogenesis of disease. Current experimental and clinical evidence suggests that chronic inflammation, regardless of the cause, may predispose men to prostate cancer. The responsibility of the immune system in immune surveillance and eventually tumor progression is well appreciated but not completely understood. In this study, we used a mass spectrometry-based evaluation of prostatic fluid obtained from diseased prostates after removal by radical prostatectomy to identify potential immunoregulatory proteins. This analysis revealed the presence of Igs and the complement system proteins C3, factor B, and clusterin. Verification of these findings by Western blot confirmed the high-level expression of C3 in the prostatic fluid and the presence of a previously uncharacterized C-terminal C3 cleavage product. Biochemical analysis of this C3 cleavage fragment revealed a putative PSA cleavage site after tyrosine-1348. Purified PSA was able to cleave iC3b and the related complement protein C5. These results suggest a previously uncharacterized function of PSA as an immunoregulatory protease that could help to create an environment hospitable to malignancy through proteolysis of the complement system.

Indirect flow injection determination of N-acetyl-L-cysteine using cerium(IV) and ferroin

International Nuclear Information System (INIS)

Vieira, Heberth Juliano; Fatibello-Filho, Orlando

2005-01-01

An indirect flow injection spectrophotometric procedure is proposed for the determination of N-acetyl-L-cysteine in pharmaceutical formulations. In this system, ferroin ([Fe(II)-(fen) 2 ] 2+ ) in excess, with a strong absorption at 500 nm, is oxidized by cerium(IV) yielding cerium(III) and [Fe(III)-(fen) 2 ] 3+ (colorless), thus producing a baseline. When N-acetyl-L-cysteine solution is introduced into the flow injection system, it reacts with cerium(IV) increasing the analytical signal in proportion to the drug concentration. Under optimal experimental conditions, the linearity of the analytical curve for N-acetyl-L-cysteine ranged from 6.5x10 -6 to 1.3x10 -4 mol L -1 . The detection limit was 5.0x10 -6 mol L -1 and recoveries between 98.0 and 106% were obtained. The sampling frequency was 60 determinations per hour and the RSD was smaller than 1.4% for 2.2x10 -5 mol L -1 N-acetyl-L-cysteine. (author)

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens.

Science.gov (United States)

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.

Correlation of secreted protein acidic and rich in cysteine with diabetic nephropathy

OpenAIRE

Li, Lei; Song, Hai-Yan; Liu, Kai; An, Meng-Meng

2015-01-01

To detect the serum concentrations of secreted protein acidic and rich in cysteine (SPARC) in patients with diabetic nephropathy and SPARC mRNA and protein expressions in renal tissue of db/db mice (C57BL/KsJ, diabetic nephropathy mice), thus preliminary exploration on the role of secreted protein acidic riches in cysteine in the development of diabetic nephropathy were carried out. Serum SPARC levels in normal subjects, patients with type 2 diabetes mellitus (without diabetic nephropathy), c...

[Analysis of salivary protease spectrum in chronic periodontitis].

Science.gov (United States)

Qian, Li; Xuedong, Zhou; Yaping, Fan; Tengyu, Yang; Songtao, Wu; Yu, Yu; Jiao, Chen; Ping, Zhang; Yun, Feng

2017-02-01

This study aimed to investigate the difference in salivary protease expression in patients with chronic periodontitis and normal individuals. The stimulating saliva in patients with chronic periodontitis and normal individuals were collected. Protein chip technology was adapted to analyze salivary protease spectrum. Among the 34 proteases in the chip, disintegrin and metalloproteinase (ADAM)8, matrix metalloproteinase (MMP)-8, MMP-12, neprilysin/CD10, and uridylyl phosphate adenosine/urokinase showed a significantly increased concentration in the saliva of chronic periodontitis patients compared with those in the saliva of normal individuals (Pchronic periodontitis patients and normal individuals significantly differed. Analysis of salivary protease spectrum is a potential clinical method to examine, diagnose, and monitor chronic periodontitis.

21 CFR 184.1271 - L-Cysteine.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true L-Cysteine. 184.1271 Section 184.1271 Food and... Substances Affirmed as GRAS § 184.1271 L-Cysteine. (a) L-Cysteine is the chemical L-2-amino-3... of total L-cysteine per 100 parts of flour in dough as a dough strengthener as defined in § 170.3(o...

Characterization of a novel protease from Aeribacillus pallidus strain VP3 with potential biotechnological interest.

Science.gov (United States)

Mechri, Sondes; Ben Elhoul Berrouina, Mouna; Omrane Benmrad, Maroua; Zaraî Jaouadi, Nadia; Rekik, Hatem; Moujehed, Emna; Chebbi, Alif; Sayadi, Sami; Chamkha, Mohamed; Bejar, Samir; Jaouadi, Bassem

2017-01-01

The present study investigates the purification and physico-chemical characterization of an extracellular protease from the Aeribacillus pallidus strain VP3 previously isolated from a geothermal oil-field (Sfax, Tunisia). The maximum protease activity recorded after 22h of incubation at 45°C was 3000U/ml. Pure enzyme, designated as SPVP, was obtained after ammonium sulfate fractionation (40-60%)-dialysis followed by heat-treatment (70°C for 30min) and UNO Q-6 FPLC anion-exchange chromatography. The purified enzyme is a monomer of molecular mass about 29kDa. The sequence of the 25 NH 2 -terminal residues of SPVP showed a high homology with those of Bacillus proteases. The almost complete inhibition by PMSF and DIFP confirmed that SPVP is a member of serine protease family. Its optima of pH and temperature were pH 10 and 60°C, respectively. Its half-life times at 70 and 80°C were 8 and 4h, respectively. Its catalytic efficiency was higher than those of SAPCG, Alcalase Ultra 2.5L, and Thermolysin type X. SPVP exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively and high resistance against organic solvents. These properties make SPVP a potential candidate for applications in detergent formulations and non-aqueous peptide biocatalysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Nationwide experience of treatment with protease inhibitors in chronic hepatitis C patients in Denmark: identification of viral resistance mutations.

Science.gov (United States)

Sølund, Christina; Krarup, Henrik; Ramirez, Santseharay; Thielsen, Peter; Røge, Birgit T; Lunding, Suzanne; Barfod, Toke S; Madsen, Lone G; Tarp, Britta; Christensen, Peer B; Gerstoft, Jan; Laursen, Alex L; Bukh, Jens; Weis, Nina

2014-01-01

The first standard of care in treatment of chronic HCV genotype 1 infection involving directly acting antivirals was protease inhibitors telaprevir or boceprevir combined with pegylated-interferon and ribavirin (triple therapy). Phase III studies include highly selected patients. Thus, treatment response and development of viral resistance during triple therapy in a routine clinical setting needs to be determined. The aims of this study were to investigate treatment outcome and identify sequence variations after triple therapy in patients with chronic HCV genotype 1 infection in a routine clinical setting. 80 patients, who initiated and completed triple therapy in Denmark between May 2011 and November 2012, were included. Demographic data and treatment response were obtained from the Danish Database for Hepatitis B and C. Direct sequencing and clonal analysis of the RT-PCR amplified NS3 protease were performed in patients without cure following triple therapy. 38 (47%) of the patients achieved cure, 15 (19%) discontinued treatment due to adverse events and remained infected, and 27 (34%) experienced relapse or treatment failure of whom 15 of 21 analyzed patients had well-described protease inhibitor resistance variants detected. Most frequently detected protease variants were V36M and/or R155K, and V36M, in patients with genotype 1a and 1b infection, respectively. The cure rate after triple therapy in a routine clinical setting was 47%, which is substantially lower than in clinical trials. Resistance variants towards protease inhibitors were seen in 71% of patients failing therapy indicating that resistance could have an important role in treatment response.

Resolution of oxidative stress by thioredoxin reductase: Cysteine versus selenocysteine

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Brian Cunniff

2014-01-01

Full Text Available Thioredoxin reductase (TR catalyzes the reduction of thioredoxin (TRX, which in turn reduces mammalian typical 2-Cys peroxiredoxins (PRXs 1–4, thiol peroxidases implicated in redox homeostasis and cell signaling. Typical 2-Cys PRXs are inactivated by hyperoxidation of the peroxidatic cysteine to cysteine-sulfinic acid, and regenerated in a two-step process involving retro-reduction by sulfiredoxin (SRX and reduction by TRX. Here transient exposure to menadione and glucose oxidase was used to examine the dynamics of oxidative inactivation and reactivation of PRXs in mouse C10 cells expressing various isoforms of TR, including wild type cytoplasmic TR1 (Sec-TR1 and mitochondrial TR2 (Sec-TR2 that encode selenocysteine, as well as mutants of TR1 and TR2 in which the selenocysteine codon was changed to encode cysteine (Cys-TR1 or Cys-TR2. In C10 cells endogenous TR activity was insensitive to levels of hydrogen peroxide that hyperoxidize PRXs. Expression of Sec-TR1 increased TR activity, reduced the basal cytoplasmic redox state, and increased the rate of reduction of a redox-responsive cytoplasmic GFP probe (roGFP, but did not influence either the rate of inactivation or the rate of retro-reduction of PRXs. In comparison to roGFP, which was reduced within minutes once oxidants were removed reduction of 2-Cys PRXs occurred over many hours. Expression of wild type Sec-TR1 or Sec-TR2, but not Cys-TR1 or TR2, increased the rate of reduction of PRXs and improved cell survival after menadione exposure. These results indicate that expression levels of TR do not reduce the severity of initial oxidative insults, but rather govern the rate of reduction of cellular factors required for cell viability. Because Sec-TR is completely insensitive to cytotoxic levels of hydrogen peroxide, we suggest TR functions at the top of a redox pyramid that governs the oxidation state of peroxiredoxins and other protein factors, thereby dictating a hierarchy of phenotypic

Activated protein C (APC) can increase bone anabolism via a protease-activated receptor (PAR)1/2 dependent mechanism.

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Shen, Kaitlin; Murphy, Ciara M; Chan, Ben; Kolind, Mille; Cheng, Tegan L; Mikulec, Kathy; Peacock, Lauren; Xue, Meilang; Park, Sang-Youel; Little, David G; Jackson, Chris J; Schindeler, Aaron

2014-12-01

Activated Protein C (APC) is an anticoagulant with strong cytoprotective properties that has been shown to promote wound healing. In this study APC was investigated for its potential orthopedic application using a Bone Morphogenetic Protein 2 (rhBMP-2) induced ectopic bone formation model. Local co-administration of 10 µg rhBMP-2 with 10 µg or 25 µg APC increased bone volume at 3 weeks by 32% (N.S.) and 74% (pAPC are largely mediated by its receptors endothelial protein C receptor (EPCR) and protease-activated receptors (PARs). Cultured pre-osteoblasts and bone nodule tissue sections were shown to express PAR1/2 and EPCR. When pre-osteoblasts were treated with APC, cell viability and phosphorylation of ERK1/2, Akt, and p38 were increased. Inhibition with PAR1 and sometimes PAR2 antagonists, but not with EPCR blocking antibodies, ameliorated the effects of APC on cell viability and kinase phosphorylation. These data indicate that APC can affect osteoblast viability and signaling, and may have in vivo applications with rhBMP-2 for bone repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Effect of hydrolysis enzymatic process of corn using protease crude (Rhizopus oligosporus-C1) to produce corn hydrolisate rich folic acid

Science.gov (United States)

Maryati, Yati; Susilowati, Agustine; Melanie, Hakiki; Lotulung, Puspa D.

2017-11-01

Corn hydrolyzate (Zea mays L) as a functional food fortificant derived from natural folic acid has been evaluated through a hydrolysis process using protease enzyme Rhizopus oligosporus strain C1. Enzymatic hydrolysis was carried out on two types of corn; yellow and white pearl variety corn, at concentration of protease enzyme (rough) 0.025; 0.125; and 0.225% (v/w of soluble nixtamal corn protein) with a hydrolysis time of 24 h at 30 °C, and pH 5.0. The results showed that the concentration of protease enzymes can increase the folic acid to the optimum condition, from the beginning to the end of the process time. Folic acid optimization of hydrolysis results in each corn was at the concentration of protease enzyme 0.225% (v/w of soluble nixtamal corn protein) in white corn and yellow corn at 24 hours hydrolysis, with folic acid composition, 283.56 µg/mL and 412.52 µg/mL, 1.07 and 1.04 mg/mL of soluble proteins, proteolytic activity 2.09 and 2.06 U/mL, total solids of 21.74 and 17.85%, total sugars of 0.56 and 2.22 mg/mL, and reducing sugar 91.72 and 48.47 mg/mL. In this condition, the increase of optimum folic acid for white corn was 33.57% and for yellow corn was 71.60% after hydrolysis.

The mitochondrial toxicity of cysteine-S-conjugates: Studies with pentachlorobutadienyl-L-cysteine

International Nuclear Information System (INIS)

Wallin, A.

1990-01-01

Nephrotoxic cysteine conjugates, arising from mercapturate biosynthesis, can perturb the mitochondrial membrane potential and calcium homeostasis in renal epithelial cells. Activation of these cysteine conjugates to reactive species by mitochondrial β-lyases results in covalent binding and mitochondrial damage. PCBC and related cysteine conjugates inhibit ADP-stimulated respiration in mitochondria respiring on alpha-ketoglutrate/malate and succinate indicating that both dehydrogenases may be targets. The respiratory inhibition is blocked by aminooxyacetic acid, an inhibitor of the β-lyase. Hence, metabolic activation is required implying that covalent binding of reactive intermediates may be important to the mitochondrial injury. Binding of 35 S-fragments has been found for 5 conjugates with varying degrees of mitochondrial toxicity. PCBC is more lipophilic and has a higher affinity for cellular membranes than other cysteine conjugates. PCBC rapidly depolarizes the inner membrane potential resulting in an inhibition of mitochondrial oxidative phosphorylation and calcium upon sequestration. Consequently, mitochondria and renal epithelial cells exposed to PCBC show a sudden release of calcium upon exposure to PCBC which is followed by a later increase in state 4 respiration leading to an inhibition of oxidative phosphorylation. The primary effect of other cysteine conjugates is an inhibition of the dehydrogenases, thus inhibiting state 3 respiration

Anti-oxidant supplementation improves boar sperm characteristics and fertility after cryopreservation: comparison between cysteine and rosemary (Rosmarinus officinalis).

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Malo, C; Gil, L; Gonzalez, N; Martínez, F; Cano, R; de Blas, I; Espinosa, E

2010-08-01

Anti-oxidants partially ameliorated the detrimental effects of reactive oxidative substances produced during cryopreservation. The objective of the study was to determine the effect of anti-oxidant addition to the freezing extender on boar semen qualities and fertility capacity. Ejaculates were collected from a previously selected boar and semen samples were processed using the straw freezing procedure. In experiment 1, semen samples were cryopreserved in lactose-egg yolk solution supplemented with various concentrations of cysteine (0, 5 and 10mM) to determinate a cysteine concentration capable of producing a protective effect during cryopreservation. Semen quality (total motility, progressive motility, viability, acrosome integrity and hypoosmotic swelling test) was evaluated after freezing and thawing and then every hour for 3h. In experiment 2, ejaculates were cryopreserved with lactose-egg yolk extender with or without the following anti-oxidants: cysteine, rosemary (Rosmarinus officinalis) and cysteine plus rosemary. Semen quality was evaluated. In the experiment 3, fertility capacity of semen frozen in anti-oxidant supplementation extenders was examined in vitro. A total of 2232 oocytes were in vitro matured and inseminated with frozen-thawed sperm. In summary: (i) the effective concentration of cysteine in freezing extender was 10mM; (ii) the addition of exogenous rosemary or cysteine to the freezing extender positively affected post-thawed viability and acrosome integrity. Only rosemary supplementation improved total motility at 3h and progressive motility at any time; (iii) the inclusion of rosemary into the extender was effective in penetration and cleavage rate and also in the efficiency of the fertilization system. (c) 2010 Elsevier Inc. All rights reserved.

Co-evolution of insect proteases and plant protease inhibitors.

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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.

Isolation of protease producing novel Bacillus cereus and detection ...

African Journals Online (AJOL)

user

2011-02-14

Feb 14, 2011 ... The highest protease activity was determined at 30°C temperature and 6.4 pH conditions and after the 18th hour, it decreased evidently. Key words: Protease, production, optimization, Bacillus sp. INTRODUCTION. Enzymes have been produced in large industrial scale for several decades (Falch, 1991).

Purification and characterization of an alkaline protease from Micrococcus sp. isolated from the South China Sea

Science.gov (United States)

Hou, Enling; Xia, Tao; Zhang, Zhaohui; Mao, Xiangzhao

2017-04-01

Protease is wildly used in various fields, such as food, medicine, washing, leather, cosmetics and other industrial fields. In this study, an alkaline protease secreted by Micrococcus NH54PC02 isolated from the South China Sea was purified and characterized. The growth curve and enzyme activity curve indicated that the cell reached a maximum concentration at the 30th hour and the enzyme activity reached the maximum value at the 36th hour. The protease was purified with 3 steps involving ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic chromatography with 8.22-fold increase in specific activity and 23.68% increase in the recovery. The molecular mass of the protease was estimated to be 25 kDa by SDS-PAGE analysis. The optimum temperature and pH for the protease activity were 50°C and pH 10.0, respectively. The protease showed a strong stability in a wide range of pH values ranging from 6.0-11.0, and maintained 90% enzyme activity in strong alkaline environment with pH 11.0. Inhibitor trials indicated that the protease might be serine protease. But it also possessed the characteristic of metalloprotease as it could be strongly inhibited by EDTA and strongly stimulated by Mn2+. Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) showed that the protease might belong to the peptidase S8 family.

Studies on the stability of protease from Bacillus sp. and its compatibility with commercial detergent Estudos sobre a estabilidade de uma protease de Bacillus sp. e sua compatibilidade com detergentes comerciais

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Wellingta Cristina Almeida do Nascimento

2006-09-01

Full Text Available Enzymes, and particularly proteases, have become an important and indispensable part of industrial processes such as laundry detergents, pharmaceuticals and food products. Detergents such as Tide®, Ariel® and Biz® contain proteolytic enzymes, most of them produced by members of the genus Bacillus. This paper describes the compatibility of protease produced by the thermophilic Bacillus sp, with commercial laundry detergent. Stability studies indicated that this enzyme retained about 95% and 74% of its maximum activity after 1h at 60ºC in the presence of glycine in combination with MnSO4 and CaCl2, respectively. No inhibitory effect was observed at 1.0-5.0 mM of EDTA. Triton X-100 inhibited the enzyme in all the concentrations tested. The enzyme was unstable in a 5% (v/v concentration of peroxide solution. The protease retained more than 80% and 65% of its activity after 30 min incubation at 60ºC in the presence of Tide® and Cheer® detergents, respectively. After supplementation of CaCl2 (10 mM and glycine (1 mM, the enzyme in Tide® detergent retained more than 85% of its activity after 1h. Based on these findings, Bacillus sp. protease shows a good potential for application in laundry detergents.As enzimas, principalmente as proteases, têm uma participação importante e indispensável em muitos processos industriais tais como na indústria farmacêutica, de alimentos e de detergentes. Alguns detergentes como Tide®, Ariel® e Biz® contem enzimas proteolíticas em sua formulação, sendo a maioria produzida por bactérias do gênero Bacillus sp. Neste artigo, foi avaliada a compatibilidade de uma protease produzida por um microrganismo termofílico, Bacillus sp., com alguns detergentes comerciais. Estudos sobre a estabilidade mostraram que a enzima reteve cerca de 95% e 74% de sua máxima atividade após 1h a 60ºC na presença de glicina em combinação com MnSO4 e CaCl2 respectivamente. A enzima não foi inibida em presença de 1.0

Effect of L-cysteine on remote organ injury in rats with severe acute pancreatitis induced by bile-pancreatic duct obstruction.

Science.gov (United States)

Yang, Li-Juan; Wan, Rong; Shen, Jia-Qing; Shen, Jie; Wang, Xing-Peng

2013-08-01

Remote organ failure occurs in cases of acute pancreatitis (AP); however, the reports on AP induced by pancreatic duct obstruction are rare. In this study we determined the effect of L-cysteine on pancreaticobiliary inflammation and remote organ damage in rats after pancreaticobiliary duct ligation (PBDL). AP was induced by PBDL in rats with 5/0 silk. Sixty rats were randomly divided into 4 groups. Groups A and B were sham-operated groups that received injections of saline or L-cysteine (10 mg/kg) intraperitoneally (15 rats in each group). Groups C and D were PBDL groups that received injections of saline or L-cysteine (10 mg/kg) intraperitoneally (15 rats in each group). The tissue samples of the pancreas and remote organs such as the lung, liver, intestine and kidney were subsequently examined for pathological changes under a light microscope. The samples were also stored for the determination of malondialdehyde and glutathione levels. Blood urea nitrogen (BUN), plasma amylase, ALT and AST levels were determined spectrophotometrically using an automated analyzer. Also, we evaluated the effect of L-cysteine on remote organ injury in rats with AP induced by retrograde infusion of 3.5% sodium taurocholate (NaTc) into the bile-pancreatic duct. Varying degrees of injury in the pancreas, lung, liver, intestine and kidney were observed in the rats 24 hours after PBDL. The severity of injury to the lung, liver and intestine was attenuated, while injury status was not changed significantly in the pancreas and kidney after L-cysteine treatment. Oxidative stress was also affected by L-cysteine in PBDL-treated rats. The concentration of tissue malondialdehyde decreased in the pancreas and remote organs of PBDL and L-cysteine administrated rats, and the concentration of glutathione increased more significantly than that of the model control group. However, L-cysteine administration reduced the severity of injury in remote organs but not in the pancreas in rats with Na

Dataset of cocoa aspartic protease cleavage sites

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Katharina Janek

2016-09-01

Full Text Available The data provide information in support of the research article, “The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors” (Janek et al., 2016 [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans. Keywords: Aspartic protease, Cleavage sites, Cocoa, In-vitro proteolysis, Mass spectrometry, Peptides

Structural Basis for Specificity of Propeptide-Enzyme Interaction in Barley C1A Cysteine Peptidases

Science.gov (United States)

Cambra, Inés; Hernández, David; Diaz, Isabel; Martinez, Manuel

2012-01-01

C1A cysteine peptidases are synthesized as inactive proenzymes. Activation takes place by proteolysis cleaving off the inhibitory propeptide. The inhibitory capacity of propeptides from barley cathepsin L and B-like peptidases towards commercial and barley cathepsins has been characterized. Differences in selectivity have been found for propeptides from L-cathepsins against their cognate and non cognate enzymes. Besides, the propeptide from barley cathepsin B was not able to inhibit bovine cathepsin B. Modelling of their three-dimensional structures suggests that most propeptide inhibitory properties can be explained from the interaction between the propeptide and the mature cathepsin structures. Their potential use as biotechnological tools is discussed. PMID:22615948

Electrochemical behavior of cysteine at a CuGeO3 nanowires modified glassy carbon electrode

International Nuclear Information System (INIS)

Dong Yongping; Pei Lizhai; Chu Xiangfeng; Zhang Wangbing; Zhang Qianfeng

2010-01-01

A CuGeO 3 nanowire modified glassy carbon electrode was fabricated and characterized by scanning electron microscopy. The results of electrochemical impedance spectroscopy reveal that electron transfer through nanowire film is facile compared with that of bare glassy carbon electrode. The modified electrode exhibited a novel electrocatalytic behavior to the electrochemical reactions of L-cysteine in neutral solution, which was not reported previously. Two pairs of semi-reversible electrochemical peaks were observed and assigned to the processes of oxidation/reduction and adsorption/desorption of cysteine at the modified electrode, respectively. The electrochemical response of cysteine is poor in alkaline condition and is enhanced greatly in acidic solution, suggesting that hydrogen ions participate in the electrochemical oxidation process of cysteine. The intensities of two anodic peaks varied linearly with the concentration of cysteine in the range of 1 x 10 -6 to 1 x 10 -3 mol L -1 , which make it possible to sensitive detection of cysteine with the CuGeO 3 nanowire modified electrode. Furthermore, the modified electrode exhibited good reproducibility and stability.

Protease-sensitive synthetic prions.

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David W Colby

2010-01-01

Full Text Available Prions arise when the cellular prion protein (PrP(C undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP(Sc. Frequently, PrP(Sc is protease-resistant but protease-sensitive (s prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164, denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174 did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrP(Sc and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600-750 days in Tg4053 mice, which exhibited sPrP(Sc. These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrP(Sc.

Knockout of the murine cysteine dioxygenase gene results in severe impairment in ability to synthesize taurine and an increased catabolism of cysteine to hydrogen sulfide

Science.gov (United States)

Ueki, Iori; Roman, Heather B.; Valli, Alessandro; Fieselmann, Krista; Lam, Jimmy; Peters, Rachel; Hirschberger, Lawrence L.

2011-01-01

Cysteine homeostasis is dependent on the regulation of cysteine dioxygenase (CDO) in response to changes in sulfur amino acid intake. CDO oxidizes cysteine to cysteinesulfinate, which is further metabolized to either taurine or to pyruvate plus sulfate. To gain insight into the physiological function of CDO and the consequence of a loss of CDO activity, mice carrying a null CDO allele (CDO+/− mice) were crossed to generate CDO−/−, CDO+/−, and CDO+/+ mice. CDO−/− mice exhibited postnatal mortality, growth deficit, and connective tissue pathology. CDO−/− mice had extremely low taurine levels and somewhat elevated cysteine levels, consistent with the lack of flux through CDO-dependent catabolic pathways. However, plasma sulfate levels were slightly higher in CDO−/− mice than in CDO+/− or CDO+/+ mice, and tissue levels of acid-labile sulfide were elevated, indicating an increase in cysteine catabolism by cysteine desulfhydration pathways. Null mice had lower hepatic cytochrome c oxidase levels, suggesting impaired electron transport capacity. Supplementation of mice with taurine improved survival of male pups but otherwise had little effect on the phenotype of the CDO−/− mice. H2S has been identified as an important gaseous signaling molecule as well as a toxicant, and pathology may be due to dysregulation of H2S production. Control of cysteine levels by regulation of CDO may be necessary to maintain low H2S/sulfane sulfur levels and facilitate the use of H2S as a signaling molecule. PMID:21693692

A novel protease activity assay using a protease-responsive chaperone protein

International Nuclear Information System (INIS)

Sao, Kentaro; Murata, Masaharu; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto

2009-01-01

Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

A novel protease activity assay using a protease-responsive chaperone protein

Energy Technology Data Exchange (ETDEWEB)

Sao, Kentaro [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Murata, Masaharu, E-mail: m-murata@dem.med.kyushu-u.ac.jp [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Fujisaki, Yuri; Umezaki, Kaori [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Nishi-ku Fukuoka 819-0395 (Japan); Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Hashizume, Makoto [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan)

2009-06-05

Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

L-Cysteine Metabolism and Fermentation in Microorganisms.

Science.gov (United States)

Takagi, Hiroshi; Ohtsu, Iwao

L-Cysteine is an important amino acid both biologically and commercially. Although most amino acids are industrially produced by microbial fermentation, L-cysteine has been mainly produced by protein hydrolysis. Due to environmental and safety problems, synthetic or biotechnological products have been preferred in the market. Here, we reviewed L-cysteine metabolism, including biosynthesis, degradation, and transport, and biotechnological production (including both enzymatic and fermentation processes) of L-cysteine. The metabolic regulation of L-cysteine including novel sulfur metabolic pathways found in microorganisms is also discussed. Recent advancement in biochemical studies, genome sequencing, structural biology, and metabolome analysis has enabled us to use various approaches to achieve direct fermentation of L-cysteine from glucose. For example, worldwide companies began to supply L-cysteine and its derivatives produced by bacterial fermentation. These companies successfully optimized the original metabolism of their private strains. Basically, a combination of three factors should be required for improving L-cysteine fermentation: that is, (1) enhancing biosynthesis: overexpression of the altered cysE gene encoding feedback inhibition-insensitive L-serine O-acetyltransferase (SAT), (2) weakening degradation: knockout of the genes encoding L-cysteine desulfhydrases, and (3) exploiting export system: overexpression of the gene involved in L-cysteine transport. Moreover, we found that "thiosulfate" is much more effective sulfur source than commonly used "sulfate" for L-cysteine production in Escherichia coli, because thiosulfate is advantageous for saving consumption of NADPH and relating energy molecules.

Collagenolytic serine protease PC and trypsin PC from king crab Paralithodes camtschaticus: cDNA cloning and primary structure of the enzymes

Directory of Open Access Journals (Sweden)

Rebrikov Denis V

2004-01-01

Full Text Available Abstract Background In this paper, we describe cDNA cloning of a new anionic trypsin and a collagenolytic serine protease from king crab Paralithodes camtschaticus and the elucidation of their primary structures. Constructing the phylogenetic tree of these enzymes was undertaken in order to prove the evolutionary relationship between them. Results The mature trypsin PC and collagenolytic protease PC contain 237 (Mcalc 24.8 kDa and 226 amino acid residues (Mcalc 23.5 kDa, respectively. Alignments of their amino acid sequences revealed a high degree of the trypsin PC identity to the trypsin from Penaeus vannamei (approximately 70% and of the collagenolytic protease PC identity to the collagenase from fiddler crab Uca pugilator (76%. The phylogenetic tree of these enzymes was constructed. Conclusions Primary structures of the two mature enzymes from P. camtschaticus were obtained and compared with those of other proteolytic proteins, including some enzymes from brachyurans. A phylogenetic analysis was also carried out. These comparisons revealed that brachyurins are closely related to their vertebrate and bacterial congeners, occupy an intermediate position between them, and their study significantly contributes to the understanding of the evolution and function of serine proteases.

Human cystatin C forms an inactive dimer during intracellular trafficking in transfected CHO cells

DEFF Research Database (Denmark)

Merz, G S; Benedikz, Eirikur; Schwenk, V

1997-01-01

To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C...... that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin......, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through...

Dicty_cDB: CHR769 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available r cysteine) proteinase inhibitor clade B (ovalbumin) family member. 32 0.51 2 CX072513 |CX072513.1 UCRCS08_28E10_g Parent...is cDNA clone UCRCS08-28E10-J20-1-4.g, mRNA sequence. 46 0.97 1 CX072512 |CX072512.1 UCRCS08_28E10_b Parent

Structural Dynamics Investigation of Human Family 1 & 2 Cystatin-Cathepsin L1 Interaction: A Comparison of Binding Modes.

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Suman Kumar Nandy

Full Text Available Cystatin superfamily is a large group of evolutionarily related proteins involved in numerous physiological activities through their inhibitory activity towards cysteine proteases. Despite sharing the same cystatin fold, and inhibiting cysteine proteases through the same tripartite edge involving highly conserved N-terminal region, L1 and L2 loop; cystatins differ widely in their inhibitory affinity towards C1 family of cysteine proteases and molecular details of these interactions are still elusive. In this study, inhibitory interactions of human family 1 & 2 cystatins with cathepsin L1 are predicted and their stability and viability are verified through protein docking & comparative molecular dynamics. An overall stabilization effect is observed in all cystatins on complex formation. Complexes are mostly dominated by van der Waals interaction but the relative participation of the conserved regions varied extensively. While van der Waals contacts prevail in L1 and L2 loop, N-terminal segment chiefly acts as electrostatic interaction site. In fact the comparative dynamics study points towards the instrumental role of L1 loop in directing the total interaction profile of the complex either towards electrostatic or van der Waals contacts. The key amino acid residues surfaced via interaction energy, hydrogen bonding and solvent accessible surface area analysis for each cystatin-cathepsin L1 complex influence the mode of binding and thus control the diverse inhibitory affinity of cystatins towards cysteine proteases.

Pressor response to L-cysteine injected into the cisterna magna of conscious rats involves recruitment of hypothalamic vasopressinergic neurons.

Science.gov (United States)

Takemoto, Yumi

2013-03-01

The sulfur-containing non-essential amino acid L-cysteine injected into the cisterna magna of adult conscious rats produces an increase in blood pressure. The present study examined if the pressor response to L-cysteine is stereospecific and involves recruitment of hypothalamic vasopressinergic neurons and medullary noradrenergic A1 neurons. Intracisternally injected D-cysteine produced no cardiovascular changes, while L-cysteine produced hypertension and tachycardia in freely moving rats, indicating the stereospecific hemodynamic actions of L-cysteine via the brain. The double labeling immunohistochemistry combined with c-Fos detection as a marker of neuronal activation revealed significantly higher numbers of c-Fos-positive vasopressinergic neurons both in the supraoptic and paraventricular nuclei and tyrosine hydroxylase containing medullary A1 neurons, of L-cysteine-injected rats than those injected with D-cysteine as iso-osmotic control. The results indicate that the cardiovascular responses to intracisternal injection of L-cysteine in the conscious rat are stereospecific and include recruitment of hypothalamic vasopressinergic neurons both in the supraoptic and paraventricular nuclei, as well as of medullary A1 neurons. The findings may suggest a potential function of L-cysteine as an extracellular signal such as neuromodulators in central regulation of blood pressure.

Transcription factor DecR (YbaO) controls detoxification of L-cysteine in Escherichia coli.

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Shimada, Tomohiro; Tanaka, Kan; Ishihama, Akira

2016-09-01

YbaO is an uncharacterized AsnC-family transcription factor of Escherichia coli. In both Salmonella enterica and Pantoea ananatis, YbaO homologues were identified to regulate the adjacent gene encoding cysteine desulfhydrase for detoxification of cysteine. Using the genomic SELEX (systematic evolution of ligands by exponential enrichment) screening system, we identified the yhaOM operon, located far from the ybaO gene on the E. coli genome, as a single regulatory target of YbaO. In both gel shift assay in vitro and reporter and Northern blot assays in vivo, YbaO was found to regulate the yhaOM promoter. The growth of mutants lacking either ybaO or its targets yhaOM was delayed in the presence of cysteine, indicating involvement of these genes in cysteine detoxification. In the major pathway of cysteine degradation, hydrogen sulfide is produced in wild-type E. coli, but its production was not observed in each of the ybaO, yhaO and yhaM mutants. The yhaOM promoter was activated in the presence of cysteine, implying the role of cysteine in activation of YbaO. Taken together, we propose that YbaO is the cysteine-sensing transcriptional activator of the yhaOM operon, which is involved in the detoxification of cysteine. We then propose the naming of ybaO as decR (regulator of detoxification of cysteine).

Identification of a Degradation Signal Sequence within Substrates of the Mitochondrial i-AAA Protease.

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Rampello, Anthony J; Glynn, Steven E

2017-03-24

The i-AAA protease is a component of the mitochondrial quality control machinery that regulates respiration, mitochondrial dynamics, and protein import. The protease is required to select specific substrates for degradation from among the diverse complement of proteins present in mitochondria, yet the rules that govern this selection are unclear. Here, we reconstruct the yeast i-AAA protease, Yme1p, to examine the in vitro degradation of two intermembrane space chaperone subunits, Tim9 and Tim10. Yme1p degrades Tim10 more rapidly than Tim9 despite high sequence and structural similarity, and loss of Tim10 is accelerated by the disruption of conserved disulfide bonds within the substrate. An unstructured N-terminal region of Tim10 is necessary and sufficient to target the substrate to the protease through recognition of a short phenylalanine-rich motif, and the presence of similar motifs in other small Tim proteins predicts robust degradation by the protease. Together, these results identify the first specific degron sequence within a native i-AAA protease substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development of potent inhibitors of the coxsackievirus 3C protease

International Nuclear Information System (INIS)

Lee, Eui Seung; Lee, Won Gil; Yun, Soo-Hyeon; Rho, Seong Hwan; Im, Isak; Yang, Sung Tae; Sellamuthu, Saravanan; Lee, Yong Jae; Kwon, Sun Jae; Park, Ohkmae K.; Jeon, Eun-Seok; Park, Woo Jin; Kim, Yong-Chul

2007-01-01

Coxsackievirus B3 (CVB3) 3C protease (3CP) plays essential roles in the viral replication cycle, and therefore, provides an attractive therapeutic target for treatment of human diseases caused by CVB3 infection. CVB3 3CP and human rhinovirus (HRV) 3CP have a high degree of amino acid sequence similarity. Comparative modeling of these two 3CPs revealed one prominent distinction; an Asn residue delineating the S2' pocket in HRV 3CP is replaced by a Tyr residue in CVB3 3CP. AG7088, a potent inhibitor of HRV 3CP, was modified by substitution of the ethyl group at the P2' position with various hydrophobic aromatic rings that are predicted to interact preferentially with the Tyr residue in the S2' pocket of CVB3 3CP. The resulting derivatives showed dramatically increased inhibitory activities against CVB3 3CP. In addition, one of the derivatives effectively inhibited the CVB3 proliferation in vitro

Synthesis of glycinamides using protease immobilized magnetic nanoparticles

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Abha Sahu

2016-12-01

Full Text Available In the present investigation, Bacillus subtilis was isolated from slaughterhouse waste and screened for the production of protease enzyme. The purified protease was successfully immobilized on magnetic nanoparticles (MNPs and used for the synthesis of series of glycinamides. The binding and thermal stability of protease on MNPs was confirmed by FTIR spectroscopy and TGA analysis. The surface morphology of MNPs before and after protease immobilization was carried out using SEM analysis. XRD pattern revealed no phase change in MNPs after enzyme immobilization. The processing parameters for glycinamides synthesis viz. temperature, pH, and time were optimized using Response Surface Methodology (RSM by using Design Expert (9.0.6.2. The maximum yield of various amides 2 butyramidoacetic acid (AMD-1,83.4%, 2-benzamidoacetic acid (AMD-2,80.5% and 2,2′((carboxymethyl amino-2-oxoethyl-2-hydroxysuccinylbis(azanediyldiacetic acid (AMD-3,80.8% formed was observed at pH-8, 50 °C and 30 min. The synthesized immobilized protease retained 70% of the initial activity even after 8 cycles of reuse.

A novel cysteine-rich antimicrobial peptide from the mucus of the snail of Achatina fulica.

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Zhong, Jian; Wang, Wenhong; Yang, Xiaomei; Yan, Xiuwen; Liu, Rui

2013-01-01

Antimicrobial peptides (AMPs) are important components of the innate immunity. Many antimicrobial peptides have been found from marine mollusks. Little information about AMPs of mollusks living on land is available. A novel cysteine-rich antimicrobial peptide (mytimacin-AF) belonging to the peptide family of mytimacins was purified and characterized from the mucus of the snail of Achatina fulica. Its cDNA was also cloned from the cDNA library. Mytimacin-AF is composed of 80 amino acid residues including 10 cysteines. Mytimacin-AF showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria and the fungus Candida albicans. Among tested microorganisms, it exerted strongest antimicrobial activity against Staphylococcus aureus with a minimal peptide concentration (MIC) of 1.9 μg/ml. Mytimacin-AF had little hemolytic activity against human blood red cells. The current work confirmed the presence of mytimacin-like antimicrobial peptide in land-living mollusks. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

Attachment Site Cysteine Thiol pKa Is a Key Driver for Site-Dependent Stability of THIOMAB Antibody-Drug Conjugates.

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Vollmar, Breanna S; Wei, Binqing; Ohri, Rachana; Zhou, Jianhui; He, Jintang; Yu, Shang-Fan; Leipold, Douglas; Cosino, Ely; Yee, Sharon; Fourie-O'Donohue, Aimee; Li, Guangmin; Phillips, Gail L; Kozak, Katherine R; Kamath, Amrita; Xu, Keyang; Lee, Genee; Lazar, Greg A; Erickson, Hans K

2017-10-18

The incorporation of cysteines into antibodies by mutagenesis allows for the direct conjugation of small molecules to specific sites on the antibody via disulfide bonds. The stability of the disulfide bond linkage between the small molecule and the antibody is highly dependent on the location of the engineered cysteine in either the heavy chain (HC) or the light chain (LC) of the antibody. Here, we explore the basis for this site-dependent stability. We evaluated the in vivo efficacy and pharmacokinetics of five different cysteine mutants of trastuzumab conjugated to a pyrrolobenzodiazepine (PBD) via disulfide bonds. A significant correlation was observed between disulfide stability and efficacy for the conjugates. We hypothesized that the observed site-dependent stability of the disulfide-linked conjugates could be due to differences in the attachment site cysteine thiol pK a . We measured the cysteine thiol pK a using isothermal titration calorimetry (ITC) and found that the variants with the highest thiol pK a (LC K149C and HC A140C) were found to yield the conjugates with the greatest in vivo stability. Guided by homology modeling, we identified several mutations adjacent to LC K149C that reduced the cysteine thiol pK a and, thus, decreased the in vivo stability of the disulfide-linked PBD conjugated to LC K149C. We also present results suggesting that the high thiol pK a of LC K149C is responsible for the sustained circulation stability of LC K149C TDCs utilizing a maleimide-based linker. Taken together, our results provide evidence that the site-dependent stability of cys-engineered antibody-drug conjugates may be explained by interactions between the engineered cysteine and the local protein environment that serves to modulate the side-chain thiol pK a . The influence of cysteine thiol pK a on stability and efficacy offers a new parameter for the optimization of ADCs that utilize cysteine engineering.

H1-A, a compound isolated from Fusarium oxysporum inhibits hepatitis C virus (HCV) NS3 serine protease.

Science.gov (United States)

Yang, Li-Yuan; Lin, Jun; Zhou, Bin; Liu, Yan-Gang; Zhu, Bao-Quan

2016-04-01

The present study was aimed to isolate the active compounds from the fermentation products of Fusarium oxysporum, which had hepatitis C virus (HCV) NS3 protease inhibitory activity. A bioactive compound was isolated by reverse-phase silica-gel column chromatography, silica-gel column chromatography, semi-preparative reverse-phase High Performance Liquid Chromatography (HPLC), and then its molecular structure was elucidated based on the spectrosopic analysis. As a result, the compound (H1-A, 1) Ergosta-5, 8 (14), 22-trien-7-one, 3-hydroxy-,(3β, 22E) was isolated and identified. To the best of our knowledge, this was the first report on the isolation of H1-A from microorganisms with the inhibitory activity of NS3 protease. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Role of conserved cysteine residues in Herbaspirillum seropedicae NifA activity.

Science.gov (United States)

Oliveira, Marco A S; Baura, Valter A; Aquino, Bruno; Huergo, Luciano F; Kadowaki, Marco A S; Chubatsu, Leda S; Souza, Emanuel M; Dixon, Ray; Pedrosa, Fábio O; Wassem, Roseli; Monteiro, Rose A

2009-01-01

Herbaspirillum seropedicae is an endophytic diazotrophic bacterium that associates with economically important crops. NifA protein, the transcriptional activator of nif genes in H. seropedicae, binds to nif promoters and, together with RNA polymerase-sigma(54) holoenzyme, catalyzes the formation of open complexes to allow transcription initiation. The activity of H. seropedicae NifA is controlled by ammonium and oxygen levels, but the mechanisms of such control are unknown. Oxygen sensitivity is attributed to a conserved motif of cysteine residues in NifA that spans the central AAA+ domain and the interdomain linker that connects the AAA+ domain to the C-terminal DNA binding domain. Here we mutagenized this conserved motif of cysteines and assayed the activity of mutant proteins in vivo. We also purified the mutant variants of NifA and tested their capacity to bind to the nifB promoter region. Chimeric proteins between H. seropedicae NifA, an oxygen-sensitive protein, and Azotobacter vinelandii NifA, an oxygen-tolerant protein, were constructed and showed that the oxygen response is conferred by the central AAA+ and C-terminal DNA binding domains of H. seropedicae NifA. We conclude that the conserved cysteine motif is essential for NifA activity, although single cysteine-to-serine mutants are still competent at binding DNA.

Surface-modified CdS nanoparticles as a fluorescent probe for the selective detection of cysteine

International Nuclear Information System (INIS)

Negi, Devendra P S; Chanu, T Inakhunbi

2008-01-01

We present a novel method for the selective detection of cysteine, a sulfur-containing amino acid, which plays a crucial role in many important biological functions such as protein folding. Surface-modified colloidal CdS nanoparticles have been used as a fluorescent probe to selectively detect cysteine in the presence of other amino acids in the micromolar concentration range. Cysteine quenches the emission of CdS in the 0.5-10 μM concentration range, whereas the other amino acids do not affect its emission. Among the other amino acids, histidine is most efficient in quenching the emission of the CdS nanoparticles. The sulfur atom of cysteine plays a crucial role in the quenching process in the 0.5-10 μM concentration range. Cysteine is believed to quench the emission of the CdS nanoparticles by binding to their surface via its negatively charged sulfur atom. This method can potentially be applied for its detection in biological samples.

Production and some properties of crude alkaline proteases of indigenous Central Amazonian rhizobia strains

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Arlem Nascimento de Oliveira

2010-10-01

Full Text Available Two rhizobia strains isolated from soils of the Central Amazonian floodplain produced appreciable quantities of crude alkaline protease extracts with inexpensive carbon and nitrogen sources. These protease crude extracts were optimally active at pH 9.0-11.0. The optimum temperatures were 35 ºC for Rhizobium sp. strain R-986 and 55 ºC for Bradyrhizobium sp. strain R-993. Protease activities in the crude extracts were enhanced in the presence of 5 mM metal ions, such as Na+, Ca2+, Mg2+ and Mn2+. Rhizobia proteases were strongly inhibited by PMSF, a serine-protease inhibitor. The enzymes were active in the presence of surfactants (SDS and Triton X-100 and stable in oxidizing (H2O2 and reducing agents (β-mercaptoethanol, and organic solvents (acetone, hexane, methanol, 1-propanol and toluene.Duas estirpes de rizóbia isoladas de solos de várzea da Amazônia Central produziram grandes quantidades de proteases alcalinas extracelulares, usando fontes baratas de carbono e nitrogênio. Os extratos brutos de proteases foram ativos em pH 9,0-11,0. As temperaturas ótimas foram de 35 ºC para a enzima do Rhizobium R-986 e de 55 ºC para a do Bradyrhizobium R-993. As atividades proteolíticas aumentaram na presença de 5 mM dos íons Na+, Ca2+ , Mg2+ e Mn2+ . As proteases secretadas pelos rizóbios foram fortemente inibidas por PMSF, um inibidor de serina protease. As enzimas foram ativas na presença de surfactantes (SDS e Triton X-100, e estáveis na presença de agentes oxidantes (H2O2 e redutores (β-mercaptoetanol e solventes orgânicos (acetona, hexano, metanol, 1-propanol e tolueno.

Different contributions of HtrA protease and chaperone activities to Campylobacter jejuni stress tolerance and physiology

DEFF Research Database (Denmark)

Bæk, Kristoffer Torbjørn; Vegge, Christina Skovgaard; Skórko-Glonek, Joanna

2011-01-01

activity is sufficient for growth at high temperature or oxidative stress, whereas the HtrA protease activity is only essential at conditions close to the growth limit for C. jejuni. However, the protease activity was required to prevent induction of the cytoplasmic heat-shock response even at optimal......The microaerophilic bacterium Campylobacter jejuni is the most common cause of bacterial food-borne infections in the developed world. Tolerance to environmental stress relies on proteases and chaperones in the cell envelope such as HtrA and SurA. HtrA displays both chaperone and protease activity......, but little is known about how each of these activities contributes to stress tolerance in bacteria. In vitro experiments showed temperature dependent protease and chaperone activities of C. jejuni HtrA. A C. jejuni mutant lacking only the protease activity of HtrA was used to show that the HtrA chaperone...

21 CFR 184.1272 - L-Cysteine monohydrochloride.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true L-Cysteine monohydrochloride. 184.1272 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1272 L-Cysteine monohydrochloride. (a) L-Cysteine... ingredient is used to supply up to 0.009 part of total L-cysteine per 100 parts of flour in dough as a dough...

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia.

Science.gov (United States)

Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M

2012-01-01

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

Portulaca oleracea L. as a Prospective Candidate Inhibitor of Hepatitis C Virus NS3 Serine Protease.

Science.gov (United States)

Noreen, Sobia; Hussain, Ishtiaq; Tariq, Muhammad Ilyas; Ijaz, Bushra; Iqbal, Shahid; Qamar-ul-Zaman; Ashfaq, Usman Ali; Husnain, Tayyab

2015-06-01

Hepatitis C virus (HCV) infection is a worldwide health problem affecting about 300 million individuals. HCV causes chronic liver disease, liver cirrhosis, hepatocellular carcinoma, and death. Many side effects are associated with the current treatment options. Natural products that can be used as anti-HCV drugs are thus of considerable potential significance. NS3 serine protease (NS3-SP) is a target for the screening of antiviral activity against HCV. The present work explores plants with anti-HCV potential, isolating possible lead compounds. Ten plants, used for medicinal purposes against different infections in rural areas of Pakistan, were collected. The cellular toxicity effects of methanolic extracts of the plants on the viability of Huh-7 cells were studied through the Trypan blue dye exclusion method. Following this, the anti-HCV potential of phytoextracts was assessed by infecting liver cells with HCV-3a-infected serum inoculum. Only the methanolic extract of Portulaca oleracea L. (PO) exhibited more than 70% inhibition. Four fractions were obtained through bioassay-guided extraction of PO. Subsequent inhibition of all organic extract fractions against NS3 serine protease was checked to track the specific target in the virus. The results showed that the PO methanolic crude and ethyl acetate extract specifically abridged the HCV NS3 protease expression in a dose-dependent fashion. Hence, PO extract and its constituents either alone or with interferon could offer a future option to treat chronic HCV.

Assay of cysteine dioxygenase activity

International Nuclear Information System (INIS)

Bagley, P.J.; Stipanuk, M.H.

1990-01-01

It has been proposed that rat liver contains two cysteine dioxygenase enzymes which convert cysteine to cysteinesulfinic acid, one which is stimulated by NAD + and has a pH optimum of 6.8 and one which is not stimulated by NAD + and has a pH optimum of 9.0. This led the authors to reinvestigate assay conditions for measuring cysteine dioxygenase activity in rat liver homogenate. An HPLC method, using an anion exchange column (Dionex Amino-Pac trademark PA1 (4x250 mm)) was used to separate the [ 35 S]cysteinesulfinic acid produced from [ 35 S]cysteine in the incubation mixture. They demonstrated that inclusion of hydroxylamine prevented further metabolism of cysteinesulfinic acid. which occurred rapidly in the absence of hydroxylamine

On the Dynamical Behavior of the Cysteine Dioxygenase-l-Cysteine Complex in the Presence of Free Dioxygen and l-Cysteine.

Science.gov (United States)

Pietra, Francesco

2017-11-01

In this work, viable models of cysteine dioxygenase (CDO) and its complex with l-cysteine dianion were built for the first time, under strict adherence to the crystal structure from X-ray diffraction studies, for all atom molecular dynamics (MD). Based on the CHARMM36 FF, the active site, featuring an octahedral dummy Fe(II) model, allowed us observing water exchange, which would have escaped attention with the more popular bonded models. Free dioxygen (O 2 ) and l-cysteine, added at the active site, could be observed being expelled toward the solvating medium under Random Accelerated Molecular Dynamics (RAMD) along major and minor pathways. Correspondingly, free dioxygen (O 2 ), added to the solvating medium, could be observed to follow the same above pathways in getting to the active site under unbiased MD. For the bulky l-cysteine, 600 ns of trajectory were insufficient for protein penetration, and the molecule was stuck at the protein borders. These models pave the way to free energy studies of ligand associations, devised to better clarify how this cardinal enzyme behaves in human metabolism. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

Mixing compatibilities of Aspergillus and American cockroach allergens with other high-protease fungal and insect extracts.

Science.gov (United States)

Grier, Thomas J; Hall, Dawn M; Duncan, Elizabeth A; Coyne, Terrance C

2015-03-01

Recent studies have shown that Alternaria and German cockroach allergens can be degraded by endogenous proteases from other insect and fungal extracts when combined for immunotherapy, but data supporting the compatibilities of other high-protease products in comparable mixtures have not been reported. To assess the stabilities and compatibilities of Aspergillus fumigatus and American cockroach allergens after mixing with protease-rich extracts from other insects or fungi at concentrations similar to those recommended for subcutaneous immunotherapy. Mixtures containing A fumigatus, American cockroach, and other fungal or insect extracts were evaluated by quantitative (enzyme-linked immunosorbent assays) and qualitative (immunoblotting) methods. Test mixtures and control samples at 10% to 50% glycerin concentrations were analyzed after storage for up to 12 months at 2°C to 8°C. Moderate to high recoveries of Aspergillus extract activities were retained in control samples and extract mixtures under all conditions examined. American cockroach extract controls were partly degraded at 10% to 25% glycerin, and cockroach allergen compatibilities were decreased significantly in mixtures with several fungal extracts at 25% glycerin. Mixing with other insects did not compromise the stability of American cockroach allergens at 25% to 50% glycerin. Aspergillus extracts exhibited favorable stabilities after mixing with other high-protease products. American cockroach extract potencies were unstable in less than 50% glycerin, even in the absence of other protease-containing allergens, and were destabilized in mixtures with several fungal extracts. Addition of fungal and insect extracts to separate treatment vials or preparation of fungal-insect mixtures at elevated glycerin concentrations might be necessary to produce compatible patient formulations for allergen immunotherapy injections. Copyright © 2015 American College of Allergy, Asthma & Immunology. Published by Elsevier

π-Clamp-mediated cysteine conjugation