RC1339/APRc from Rickettsia conorii is a novel aspartic protease with properties of retropepsin-like enzymes.

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Rui Cruz

2014-08-01

Full Text Available Members of the species Rickettsia are obligate intracellular, gram-negative, arthropod-borne pathogens of humans and other mammals. The life-threatening character of diseases caused by many Rickettsia species and the lack of reliable protective vaccine against rickettsioses strengthens the importance of identifying new protein factors for the potential development of innovative therapeutic tools. Herein, we report the identification and characterization of a novel membrane-embedded retropepsin-like homologue, highly conserved in 55 Rickettsia genomes. Using R. conorii gene homologue RC1339 as our working model, we demonstrate that, despite the low overall sequence similarity to retropepsins, the gene product of rc1339 APRc (for Aspartic Protease from Rickettsia conorii is an active enzyme with features highly reminiscent of this family of aspartic proteases, such as autolytic activity impaired by mutation of the catalytic aspartate, accumulation in the dimeric form, optimal activity at pH 6, and inhibition by specific HIV-1 protease inhibitors. Moreover, specificity preferences determined by a high-throughput profiling approach confirmed common preferences between this novel rickettsial enzyme and other aspartic proteases, both retropepsins and pepsin-like. This is the first report on a retropepsin-like protease in gram-negative intracellular bacteria such as Rickettsia, contributing to the analysis of the evolutionary relationships between the two types of aspartic proteases. Additionally, we have also shown that APRc is transcribed and translated in R. conorii and R. rickettsii and is integrated into the outer membrane of both species. Finally, we demonstrated that APRc is sufficient to catalyze the in vitro processing of two conserved high molecular weight autotransporter adhesin/invasion proteins, Sca5/OmpB and Sca0/OmpA, thereby suggesting the participation of this enzyme in a relevant proteolytic pathway in rickettsial life-cycle. As a

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

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

2016-03-01

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

Neutrophil-Derived Proteases Escalate Inflammation through Activation of IL-36 Family Cytokines

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Conor M. Henry

2016-02-01

Full Text Available Recent evidence has strongly implicated the IL-1 family cytokines IL-36α, IL-36β, and IL-36γ as key initiators of skin inflammation. Similar to the other members of the IL-1 family, IL-36 cytokines are expressed as inactive precursors and require proteolytic processing for activation; however, the responsible proteases are unknown. Here, we show that IL-36α, IL-36β, and IL-36γ are activated differentially by the neutrophil granule-derived proteases cathepsin G, elastase, and proteinase-3, increasing their biological activity ∼500-fold. Active IL-36 promoted a strong pro-inflammatory signature in primary keratinocytes and was sufficient to perturb skin differentiation in a reconstituted 3D human skin model, producing features resembling psoriasis. Furthermore, skin eluates from psoriasis patients displayed significantly elevated cathepsin G-like activity that was sufficient to activate IL-36β. These data identify neutrophil granule proteases as potent IL-36-activating enzymes, adding to our understanding of how neutrophils escalate inflammatory reactions. Inhibition of neutrophil-derived proteases may therefore have therapeutic benefits in psoriasis.

Characterization of a serine protease-mediated cell death program activated in human leukemia cells

International Nuclear Information System (INIS)

O'Connell, A.R.; Holohan, C.; Torriglia, A.; Lee, B.F.; Stenson-Cox, C.

2006-01-01

Tightly controlled proteolysis is a defining feature of apoptosis and caspases are critical in this regard. Significant roles for non-caspase proteases in cell death have been highlighted. Staurosporine causes a rapid induction of apoptosis in virtually all mammalian cell types. Numerous studies demonstrate that staurosporine can activate cell death under caspase-inhibiting circumstances. The aim of this study was to investigate the proteolytic mechanisms responsible for cell death under these conditions. To that end, we show that inhibitors of serine proteases can delay cell death in one such system. Furthermore, through profiling of proteolytic activation, we demonstrate, for the first time, that staurosporine activates a chymotrypsin-like serine protease-dependent cell death in HL-60 cells independently, but in parallel with the caspase controlled systems. Features of the serine protease-mediated system include cell shrinkage and apoptotic morphology, regulation of caspase-3, altered nuclear morphology, generation of an endonuclease and DNA degradation. We also demonstrate a staurosporine-induced activation of a putative 16 kDa chymotrypsin-like protein during apoptosis

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....

Neutrophil-Derived Proteases Escalate Inflammation through Activation of IL-36 Family Cytokines.

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Henry, Conor M; Sullivan, Graeme P; Clancy, Danielle M; Afonina, Inna S; Kulms, Dagmar; Martin, Seamus J

2016-02-02

Recent evidence has strongly implicated the IL-1 family cytokines IL-36α, IL-36β, and IL-36γ as key initiators of skin inflammation. Similar to the other members of the IL-1 family, IL-36 cytokines are expressed as inactive precursors and require proteolytic processing for activation; however, the responsible proteases are unknown. Here, we show that IL-36α, IL-36β, and IL-36γ are activated differentially by the neutrophil granule-derived proteases cathepsin G, elastase, and proteinase-3, increasing their biological activity ~500-fold. Active IL-36 promoted a strong pro-inflammatory signature in primary keratinocytes and was sufficient to perturb skin differentiation in a reconstituted 3D human skin model, producing features resembling psoriasis. Furthermore, skin eluates from psoriasis patients displayed significantly elevated cathepsin G-like activity that was sufficient to activate IL-36β. These data identify neutrophil granule proteases as potent IL-36-activating enzymes, adding to our understanding of how neutrophils escalate inflammatory reactions. Inhibition of neutrophil-derived proteases may therefore have therapeutic benefits in psoriasis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Production of rennin-like acid protease by Mucor pusillus through submerged fermentation

International Nuclear Information System (INIS)

Daudi, S.; Mukhtar, H.; Rehman, A.U.; Haq, I.U.

2015-01-01

The present study is concerned with the isolation and screening of Mucor species for the production of acid protease in shake flasks. Out of eight mould cultures evaluated, five were isolated from soil and three were provided from the Institute of Industrial Biotechnology, Government College University, Lahore. Of all the isolates tested, Mucor pusillus IHS6 was found to be the best producer of rennin-like acid protease producing 75 U/ml of the enzyme. Different agricultural byproducts were evaluated as fermentation substrates and maximum enzyme synthesis (61 U/ml) was obtained when rapeseed meal was used as a substrate. Optimum pH and fermentation period for the production of protease were 5.5 (56U/ml) and 72 hrs (55U/ml), respectively. The production of protease by Mucor pusillus IHS6 was also studied by adding different carbon and nitrogen sources to the fermentation medium. Fructose at a concentration of 1.5% (66 U/ml) and yeast extract at a concentration of 2% (68.2 U/ml) and ammonium chloride at a concentration of 0.1% (67U/ml) were found to be the best carbon and nitrogen (organic and inorganic) sources respectively. Spore inoculum at a concentration of 1% (68.4 U/ml) was found to be the best for protease production by Mucor pusillus. The fermentation broth was found to have strong milk clotting activity with 200 RU. (author)

Isolation of Lactic Acid Bacteria That Produce Protease and Bacteriocin-Like Substance From Mud Crab (Scylla sp. Digestive Tract (Isolasi Bakteri Asam Laktat yang Menghasilkan Protease dan Senyawa Bacteriocin-Like dari Saluran Pencernaan Kepiting

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Heru Pramono

2015-03-01

Kata kunci: Bakteri Asam Laktat, Bakteriosin-like substance, Protease, Scylla  sp. Digestive tract is complex environment consist of large amount of bacteria’s species. Fish intestine bacteria consist of aerobic or facultative anaerob bacteria which can produce antibacterial and enzym. The objectives of this research were to isolated lactic acid bacteria that produce bacteriocin-like and protease from mud crab digestive tract. Isolation and characterization of isolates were conducted employing media MRS.  Neutralized cell free supernatant of isolates were tested using disc diffusion agar of against pathogenic and spoilage bacteria to indicate bacteriocin-like-producing lactic acid bacteria. Protease-producing isolate was tested using disc diffusion method in casein agar. Among a hundred isolates, 96 isolates were showed clear zone in MRS+CaCO3,, catalase negative, and Gram positive bacteria. Thirty four isolates produced protease and only four isolates (i.e. IKP29, IKP30, IKP52, and IKP94 showed strong inhibition against pathogenic and spoilage bacteria. There were three patterns of inhibition among three isolates against Bacillus subtilis, Staphylococcus aureus, Eschericia coli, and Salmonella sp. All three isolates showed potential uses for produce starter culture for fishery product fermentation purpose. This is the first report of isolation lactic acid bacteria that produced protease and bacteriocin-like from digestive tract of mud crab. Keywords: Lactic acid bacteria, Bacteriocin-like substance, Protease, Scylla  sp.

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

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Evan L Pannkuk

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

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

Science.gov (United States)

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

2015-01-01

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

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

Dual function of a bee venom serine protease: prophenoloxidase-activating factor in arthropods and fibrin(ogen)olytic enzyme in mammals.

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Choo, Young Moo; Lee, Kwang Sik; Yoon, Hyung Joo; Kim, Bo Yeon; Sohn, Mi Ri; Roh, Jong Yul; Je, Yeon Ho; Kim, Nam Jung; Kim, Iksoo; Woo, Soo Dong; Sohn, Hung Dae; Jin, Byung Rae

2010-05-03

Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles for Bi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.

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

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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

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.

Detection of protease activity in cells and animals.

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Verdoes, Martijn; Verhelst, Steven H L

2016-01-01

Proteases are involved in a wide variety of biologically and medically important events. They are entangled in a complex network of processes that regulate their activity, which makes their study intriguing, but challenging. For comprehensive understanding of protease biology and effective drug discovery, it is therefore essential to study proteases in models that are close to their complex native environments such as live cells or whole organisms. Protease activity can be detected by reporter substrates and activity-based probes, but not all of these reagents are suitable for intracellular or in vivo use. This review focuses on the detection of proteases in cells and in vivo. We summarize the use of probes and substrates as molecular tools, discuss strategies to deliver these tools inside cells, and describe sophisticated read-out techniques such as mass spectrometry and various imaging applications. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of mefenamic acid on the immunity and hemostatic system of cancer patients and on the activity of cathepsin D-like protease in colonic cancer tissue

International Nuclear Information System (INIS)

Klyachkin, B.M.; Basargin, S.T.; Timofeev, I.V.; Khaliulin, Yu.G.; Dorofeev, S.A.; Alekseenko, L.D.; Gumenyuk, M.L.

1992-01-01

The study of the effect of sodium mefenaminate on radiation resistance of mice yielded positive results. Clinical investigations showed mefenamic acid to decrease the activity of cathepsin D-like protease in colonic cancer tissue. The acid field to affect the proteolytic activity of the normal mucosa. It revealed an immunomodulating activity and influenced the hemostatic system which usually manifested itself in amelioration of hypercoagulation

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

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Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

2016-07-15

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior*

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Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H.; Muyldermans, Serge; Declerck, Paul J.; Huang, Mingdong; Andreasen, Peter A.; Ngo, Jacky Chi Ki

2016-01-01

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30–40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target.

Science.gov (United States)

Withers-Martinez, Chrislaine; Suarez, Catherine; Fulle, Simone; Kher, Samir; Penzo, Maria; Ebejer, Jean-Paul; Koussis, Kostas; Hackett, Fiona; Jirgensons, Aigars; Finn, Paul; Blackman, Michael J

2012-05-15

Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Factor VII-activating protease

DEFF Research Database (Denmark)

Ramanathan, Ramshanker; Gram, Jørgen B; Sand, Niels Peter R

2017-01-01

: Factor VII-activating protease (FSAP) may regulate development of cardiovascular disease (CVD). We evaluated sex differences in FSAP measures and examined the association between FSAP and coronary artery calcification (CAC) in a middle-aged population. Participants were randomly selected citizens...

Cysteine proteases: Modes of activation and future prospects as pharmacological targets

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Sonia eVerma

2016-04-01

Full Text Available Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria and parasite to the higher organisms (mammals. Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases and metallo-proteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a pro-domain (regulatory and a mature domain (catalytic. The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein-protein interactions (PPIs and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases.

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)

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...

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 New Pepstatin-Insensitive Thermopsin-Like Protease Overproduced in Peptide-Rich Cultures of Sulfolobus solfataricus

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Marta Gogliettino

2014-02-01

Full Text Available In this study, we gain insight into the extracellular proteolytic system of Sulfolobus solfataricus grown on proteinaceous substrates, providing further evidence that acidic proteases were specifically produced in response to peptide-rich media. The main proteolytic component was the previously isolated SsMTP (Sulfolobus solfataricus multi-domain thermopsin-like protease, while the less abundant (named SsMTP-1 one was purified, characterized and identified as the sso1175 gene-product. The protein revealed a multi-domain organization shared with the cognate SsMTP with a catalytic domain followed by several tandemly-repeated motifs. Moreover, both enzymes were found spread across the Crenarchaeota phylum and belonging to the thermopsin family, although segregated into diverse phylogenetic clusters. SsMTP-1 showed a 75-kDa molecular mass and was stable in the temperature range 50–90 °C, with optimal activity at 70 °C and pH 2.0. Serine, metallo and aspartic protease inhibitors did not affect the enzyme activity, designating SsMTP-1 as a new member of the pepstatin-insensitive aspartic protease family. The peptide-bond-specificity of SsMTP-1 in the cleavage of the oxidized insulin B chain was uncommon amongst thermopsins, suggesting that it could play a distinct, but cooperative role in the protein degradation machinery. Interestingly, predictions of the transmembrane protein topology of SsMTP and SsMTP-1 strongly suggest a possible contribution in signal-transduction pathways.

Protease activated receptors (PARS) mediation in gyroxin biological activity

International Nuclear Information System (INIS)

Silva, Jose Alberto Alves da

2009-01-01

Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH 2 , respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)

Functional dissection of the alphavirus capsid protease: sequence requirements for activity.

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Thomas, Saijo; Rai, Jagdish; John, Lijo; Günther, Stephan; Drosten, Christian; Pützer, Brigitte M; Schaefer, Stephan

2010-11-18

The alphavirus capsid is multifunctional and plays a key role in the viral life cycle. The nucleocapsid domain is released by the self-cleavage activity of the serine protease domain within the capsid. All alphaviruses analyzed to date show this autocatalytic cleavage. Here we have analyzed the sequence requirements for the cleavage activity of Chikungunya virus capsid protease of genus alphavirus. Amongst alphaviruses, the C-terminal amino acid tryptophan (W261) is conserved and found to be important for the cleavage. Mutating tryptophan to alanine (W261A) completely inactivated the protease. Other amino acids near W261 were not having any effect on the activity of this protease. However, serine protease inhibitor AEBSF did not inhibit the activity. Through error-prone PCR we found that isoleucine 227 is important for the effective activity. The loss of activity was analyzed further by molecular modelling and comparison of WT and mutant structures. It was found that lysine introduced at position 227 is spatially very close to the catalytic triad and may disrupt electrostatic interactions in the catalytic site and thus inactivate the enzyme. We are also examining other sequence requirements for this protease activity. We analyzed various amino acid sequence requirements for the activity of ChikV capsid protease and found that amino acids outside the catalytic triads are important for the activity.

Moringa oleifera Lam.: Protease activity against blood coagulation cascade.

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Satish, A; Sairam, Sudha; Ahmed, Faiyaz; Urooj, Asna

2012-01-01

The present study evaluated the protease activity of aqueous extracts of Moringa oleifera (Moringaceae) leaf (MOL) and root (MOR). Protease activity was assayed using casein, human plasma clot and human fibrinogen as substrates. Caseinolytic activity of MOL was significantly higher (P ≤ 0.05) than that of MOR. Similar observations were found in case of human plasma clot hydrolyzing activity, wherein MOL caused significantly higher (P ≤ 0.05) plasma clot hydrolysis than MOR. Zymographic techniques were used to detect proteolytic enzymes following electrophoretic separation in gels. Further, both the extracts exhibited significant procoagulant activity as reflected by a significant decrease (P ≤ 0.05) in recalcification time, accompanied by fibrinogenolytic and fibrinolytic activities; clotting time was decreased from 180 ± 10 sec to 119 ± 8 sec and 143 ± 10 sec by MOL and MOR, respectively, at a concentration of 2.5 mg/mL. Fibrinogenolytic (human fibrinogen) and fibrinolytic activity (human plasma clot) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), plate method and colorimetric method. Zymographic profile indicated that both the extracts exerted their procoagulant activity by selectively hydrolyzing Aα and Bβ subunits of fibrinogen to form fibrin clot, thereby exhibiting fibrinogenolytic activity. However, prolonged incubation resulted in degradation of the formed fibrin clot, suggesting fibrinolytic like activity. These findings support the traditional usage of M. oleifera extracts for wound healing.

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.

Secretory leukocyte protease inhibitor (SLPI is, like its homologue trappin-2 (pre-elafin, a transglutaminase substrate.

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Kévin Baranger

Full Text Available Human lungs contain secretory leukocyte protease inhibitor (SLPI, elafin and its biologically active precursor trappin-2 (pre-elafin. These important low-molecular weight inhibitors are involved in controlling the potentially deleterious proteolytic activities of neutrophil serine proteases including elastase, proteinase 3 and cathepsin G. We have shown previously that trappin-2, and to a lesser extent, elafin can be linked covalently to various extracellular matrix proteins by tissue transglutaminases and remain potent protease inhibitors. SLPI is composed of two distinct domains, each of which is about 40% identical to elafin, but it lacks consensus transglutaminase sequence(s, unlike trappin-2 and elafin. We investigated the actions of type 2 tissue transglutaminase and plasma transglutaminase activated factor XIII on SLPI. It was readily covalently bound to fibronectin or elastin by both transglutaminases but did not compete with trappin-2 cross-linking. Cross-linked SLPI still inhibited its target proteases, elastase and cathepsin G. We have also identified the transglutamination sites within SLPI, elafin and trappin-2 by mass spectrometry analysis of tryptic digests of inhibitors cross-linked to mono-dansyl cadaverin or to a fibronectin-derived glutamine-rich peptide. Most of the reactive lysine and glutamine residues in SLPI are located in its first N-terminal elafin-like domain, while in trappin-2, they are located in both the N-terminal cementoin domain and the elafin moiety. We have also demonstrated that the transglutamination substrate status of the cementoin domain of trappin-2 can be transferred from one protein to another, suggesting that it may provide transglutaminase-dependent attachment properties for engineered proteins. We have thus added to the corpus of knowledge on the biology of these potential therapeutic inhibitors of airway proteases.

Characterization of active-site residues of the NIa protease from tobacco vein mottling virus.

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Hwang, D C; Kim, D H; Lee, J S; Kang, B H; Han, J; Kim, W; Song, B D; Choi, K Y

2000-10-31

Nuclear inclusion a (NIa) protease of tobacco vein mottling virus is responsible for the processing of the viral polyprotein into functional proteins. In order to identify the active-site residues of the TVMV NIa protease, the putative active-site residues, His-46, Asp-81 and Cys-151, were mutated individually to generate H46R, H46A, D81E, D81N, C151S, and C151A, and their mutational effects on the proteolytic activities were examined. Proteolytic activity was completely abolished by the mutations of H46R, H46A, D81N, and C151A, suggesting that the three residues are crucial for catalysis. The mutation of D81E decreased kcat marginally by about 4.7-fold and increased Km by about 8-fold, suggesting that the aspartic acid at position 81 is important for substrate binding but can be substituted by glutamate without any significant decrease in catalysis. The replacement of Cys-151 by Ser to mimic the catalytic triad of chymotrypsin-like serine protease resulted in the drastic decrease in kcat by about 1,260-fold. This result might be due to the difference of the active-site geometry between the NIa protease and chymotrypsin. The protease exhibited a bell-shaped pH-dependent profile with a maximum activity approximately at pH 8.3 and with the abrupt changes at the respective pKa values of approximately 6.6 and 9.2, implying the involvement of a histidine residue in catalysis. Taken together, these results demonstrate that the three residues, His-46, Asp-81, and Cys-151, play a crucial role in catalysis of the TVMV NIa protease.

Optochemical Control of Protein Localization and Activity within Cell-like Compartments.

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Caldwell, Reese M; Bermudez, Jessica G; Thai, David; Aonbangkhen, Chanat; Schuster, Benjamin S; Courtney, Taylor; Deiters, Alexander; Hammer, Daniel A; Chenoweth, David M; Good, Matthew C

2018-05-08

We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.

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'.

Chlamydial Protease-Like Activity Factor Mediated Protection Against C. trachomatis In Guinea Pigs

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Wali, Shradha; Gupta, Rishein; Yu, Jieh-Juen; Koundinya Lanka, Gopala Krishna; Chambers, James P.; Guentzel, M. Neal; Zhong, Guangming; Murthy, Ashlesh K.; Arulanandam, Bernard P.

2016-01-01

We have comprehensively demonstrated using the mouse model that intranasal immunization with recombinant chlamydial protease-like activity factor (rCPAF) leads to a significant reduction in bacterial burden, genital tract pathology and preserves fertility following intravaginal genital chlamydial challenge. In the present report, we evaluated the protective efficacy of rCPAF immunization in guinea pigs, a second animal model for genital chlamydial infection. Using a vaccination strategy similar to the mouse model, we intranasally immunized female guinea pigs with rCPAF plus CpG deoxynucleotides (CpG; as an adjuvant), and challenged intravaginally with C. trachomatis serovar D (CT-D). Immunization with rCPAF/CpG significantly reduced vaginal CT-D shedding and induced resolution of infection by day 24, compared to day 33 in CpG alone treated and challenged animals. Immunization induced robust anti-rCPAF serum IgG 2 weeks following the last immunization, and was sustained at a high level 4 weeks post challenge. Upregulation of antigen specific IFN-γ gene expression was observed in rCPAF/CpG vaccinated splenocytes. Importantly, a significant reduction in inflammation in the genital tissue in rCPAF/CpG-immunized guinea pigs compared to CpG-immunized animals was observed. Taken together, this study provides evidence of the protective efficacy of rCPAF as a vaccine candidate in a second animal model of genital chlamydial infection. PMID:27990018

The pro-coagulant fibrinogenolytic serine protease isoenzymes purified from Daboia russelii russelii venom coagulate the blood through factor V activation: role of glycosylation on enzymatic activity.

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Ashis K Mukherjee

Full Text Available Proteases from Russell's viper venom (RVV induce a variety of toxic effects in victim. Therefore, four new RVV protease isoenzymes of molecular mass 32901.044 Da, 333631.179 Da, 333571.472 Da, and 34594.776 Da, were characterized in this study. The first 10 N-terminal residues of these serine protease isoenzymes showed significant sequence homology with N-terminal sequences of snake venom thrombin-like and factor V-activating serine proteases, which was reconfirmed by peptide mass fingerprinting analysis. These proteases were found to be different from previously reported factor V activators isolated from snake venoms. These proteases showed significantly different fibrinogenolytic, BAEE-esterase and plasma clotting activities but no fibrinolytic, TAME-esterase or amidolytic activity against the chromogenic substrate for trypsin, thrombin, plasmin and factor Xa. Their Km and Vmax values towards fibrinogen were determined in the range of 6.6 to 10.5 µM and 111.0 to 125.5 units/mg protein, respectively. On the basis of fibrinogen degradation pattern, they may be classified as A/B serine proteases isolated from snake venom. These proteases contain ∼ 42% to 44% of N-linked carbohydrates by mass whereas partially deglycosylated enzymes showed significantly less catalytic activity as compared to native enzymes. In vitro these protease isoenzymes induce blood coagulation through factor V activation, whereas in vivo they provoke dose-dependent defibrinogenation and anticoagulant activity in the mouse model. At a dose of 5 mg/kg, none of these protease isoenzymes were found to be lethal in mice or house geckos, suggesting therapeutic application of these anticoagulant peptides for the prevention of thrombosis.

Effects of eye rubbing on the levels of protease, protease activity and cytokines in tears: relevance in keratoconus.

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Balasubramanian, Sivaraman A; Pye, David C; Willcox, Mark D P

2013-03-01

Proteases, protease activity and inflammatory molecules in tears have been found to be relevant in the pathogenesis of keratoconus. We sought to determine the influence of eye rubbing on protease expression, protease activity and concentration of inflammatory molecules in tears. Basal tears were collected from normal volunteers before and after 60 seconds of experimental eye rubbing. The total amount of matrix metalloproteinase (MMP)-13 and inflammatory molecules interleukin (IL)-6 and tumour necrosis factor (TNF)-α in the tear samples were measured using specific enzyme-linked immunosorbent assays (ELISA). Tear collagenase activity was investigated using a specific activity assay. The concentrations of MMP-13 (51.9 ± 34.3 versus 63 ± 36.8 pg/ml, p = 0.006), IL-6 (1.24 ± 0.98 versus 2.02 ± 1.52 pg/ml, p = 0.004) and TNF-α (1.16 ± 0.74 versus 1.44 ± 0.66 pg/ml, p = 0.003) were significantly increased in normal subjects after eye rubbing. The experimental eye rub did not alter significantly the collagenase activity (5.02 ± 3 versus 7.50 ± 3.90 fluorescent intensity units, p = 0.14) of tears. Eye rubbing for 60 seconds increased the level of tear MMP-13, IL-6 and TNF-α in normal study subjects. This increase in protease, protease activity and inflammatory mediators in tears after eye rubbing may be exacerbated even further during persistent and forceful eye rubbing seen in people with keratoconus and this in turn may contribute to the progression of the disease. © 2013 The Authors. Clinical and Experimental Optometry © 2013 Optometrists Association Australia.

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

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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.

Inhibition of SARS-CoV 3C-like Protease Activity by Theaflavin-3,3'-digallate (TF3

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Chia-Nan Chen

2005-01-01

Full Text Available SARS-CoV is the causative agent of severe acute respiratory syndrome (SARS. The virally encoded 3C-like protease (3CLPro has been presumed critical for the viral replication of SARS-CoV in infected host cells. In this study, we screened a natural product library consisting of 720 compounds for inhibitory activity against 3CLPro. Two compounds in the library were found to be inhibitive: tannic acid (IC50 = 3 µM and 3-isotheaflavin-3-gallate (TF2B (IC50 = 7 µM. These two compounds belong to a group of natural polyphenols found in tea. We further investigated the 3CLPro-inhibitory activity of extracts from several different types of teas, including green tea, oolong tea, Puer tea and black tea. Our results indicated that extracts from Puer and black tea were more potent than that from green or oolong teas in their inhibitory activities against 3CLPro. Several other known compositions in teas were also evaluated for their activities in inhibiting 3CLPro. We found that caffeine, (—-epigallocatechin gallte (EGCg, epicatechin (EC, theophylline (TP, catechin (C, epicatechin gallate (ECg and epigallocatechin (EGC did not inhibit 3CLPro activity. Only theaflavin-3,3′-digallate (TF3 was found to be a 3CLPro inhibitor. This study has resulted in the identification of new compounds that are effective 3CLPro inhibitors.

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.

Science.gov (United States)

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.

WIDE AND THICK GRAIN 1, which encodes an otubain-like protease with deubiquitination activity, influences grain size and shape in rice.

Science.gov (United States)

Huang, Ke; Wang, Dekai; Duan, Penggen; Zhang, Baolan; Xu, Ran; Li, Na; Li, Yunhai

2017-09-01

Grain size and shape are two crucial traits that influence grain yield and grain appearance in rice. Although several factors that affect grain size have been described in rice, the molecular mechanisms underlying the determination of grain size and shape are still elusive. In this study we report that WIDE AND THICK GRAIN 1 (WTG1) functions as an important factor determining grain size and shape in rice. The wtg1-1 mutant exhibits wide, thick, short and heavy grains and also shows an increased number of grains per panicle. WTG1 determines grain size and shape mainly by influencing cell expansion. WTG1 encodes an otubain-like protease, which shares similarity with human OTUB1. Biochemical analyses indicate that WTG1 is a functional deubiquitinating enzyme, and the mutant protein (wtg1-1) loses this deubiquitinating activity. WTG1 is expressed in developing grains and panicles, and the GFP-WTG1 fusion protein is present in the nucleus and cytoplasm. Overexpression of WTG1 results in narrow, thin, long grains due to narrow and long cells, further supporting the role of WTG1 in determining grain size and shape. Thus, our findings identify the otubain-like protease WTG1 to be an important factor that determines grain size and shape, suggesting that WTG1 has the potential to improve grain size and shape in rice. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Prediction of extracellular proteases of the human pathogen Helicobacter pylori reveals proteolytic activity of the Hp1018/19 protein HtrA.

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Martin Löwer

Full Text Available Exported proteases of Helicobacter pylori (H. pylori are potentially involved in pathogen-associated disorders leading to gastric inflammation and neoplasia. By comprehensive sequence screening of the H. pylori proteome for predicted secreted proteases, we retrieved several candidate genes. We detected caseinolytic activities of several such proteases, which are released independently from the H. pylori type IV secretion system encoded by the cag pathogenicity island (cagPAI. Among these, we found the predicted serine protease HtrA (Hp1019, which was previously identified in the bacterial secretome of H. pylori. Importantly, we further found that the H. pylori genes hp1018 and hp1019 represent a single gene likely coding for an exported protein. Here, we directly verified proteolytic activity of HtrA in vitro and identified the HtrA protease in zymograms by mass spectrometry. Overexpressed and purified HtrA exhibited pronounced proteolytic activity, which is inactivated after mutation of Ser205 to alanine in the predicted active center of HtrA. These data demonstrate that H. pylori secretes HtrA as an active protease, which might represent a novel candidate target for therapeutic intervention strategies.

Antimicrobial activity of an aspartic protease from Salpichroa origanifolia fruits.

Science.gov (United States)

Díaz, M E; Rocha, G F; Kise, F; Rosso, A M; Guevara, M G; Parisi, M G

2018-05-08

Plant proteases play a fundamental role in several processes like growth, development and in response to biotic and abiotic stress. In particular, aspartic proteases (AP) are expressed in different plant organs and have antimicrobial activity. Previously, we purified an AP from Salpichroa origanifolia fruits called salpichroin. The aim of this work was to determine the cytotoxic activity of this enzyme on selected plant and human pathogens. For this purpose, the growth of the selected pathogens was analysed after exposure to different concentrations of salpichroin. The results showed that the enzyme was capable of inhibiting Fusarium solani and Staphylococcus aureus in a dose-dependent manner. It was determined that 1·2 μmol l -1 of salpichroin was necessary to inhibit 50% of conidial germination, and the minimal bactericidal concentration was between 1·9 and 2·5 μmol l -1 . Using SYTOX Green dye we were able to demonstrate that salpichroin cause membrane permeabilization. Moreover, the enzyme treated with its specific inhibitor pepstatin A did not lose its antibacterial activity. This finding demonstrates that the cytotoxic activity of salpichroin is due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of the AP could represent a potential alternative for the control of pathogens that affect humans or crops of economic interest. This study provides insights into the antimicrobial activity of an aspartic protease isolated from Salpichroa origanifolia fruits on plant and human pathogens. The proteinase inhibited Fusarium solani and Staphylococcus aureus in a dose-dependent manner due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of salpichroin suggests its potential applications as an important tool for the control of pathogenic micro-organisms affecting humans and crops of economic interest. Therefore, it would

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)

The subtilisin-like protease AprV2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates.

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Ruth M Kennan

Full Text Available Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by

Defective thrombus formation in mice lacking endogenous factor VII activating protease (FSAP).

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Subramaniam, Saravanan; Thielmann, Ina; Morowski, Martina; Pragst, Ingo; Sandset, Per Morten; Nieswandt, Bernhard; Etscheid, Michael; Kanse, Sandip M

2015-04-01

Factor VII (FVII) activating protease (FSAP) is a circulating protease with a putative function in blood coagulation and fibrinolysis. Genetic epidemiological studies have implied a role for FSAP in carotid stenosis, stroke and thrombosis. To date, no in vivo evidence is available to support these claims. We have, for the first time, used FSAP-/- mice to define its role in thrombosis and haemostasis in vivo and to characterise the molecular mechanisms involved. FeCl3-induced arterial thrombosis in carotid and mesenteric artery revealed that the occlusion time was significantly increased in FSAP-/- mice (pendogenous FSAP impaired the formation of stable, occlusive thrombi in mice. The underlying in vivo effect of FSAP is more likely to be related to the modulation of TFPI rather than FVIIa.

Protease activated receptors (PARS) mediation in gyroxin biological activity; Mediacao dos receptores ativados por proteases (PARs) em atividades biologicas da giroxina

Energy Technology Data Exchange (ETDEWEB)

Silva, Jose Alberto Alves da

2009-07-01

Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH{sub 2}, respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)

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...

The Effect of Exogenous Protease in Broiler Diets on the Apparent Ileal Digestibility of Amino Acids and on Protease Activity in Jejunum

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Vojtěch Rada

2016-01-01

Full Text Available The objective of this study was to evaluate the effect of a mono-component commercial serine protease supplement in broiler diets on apparent ileal amino acid digestibility and protease activity. A total of 150 male (28 d old ROSS 308 were randomly placed into 30 battery pens and divided into 5 treatment groups with 6 replicates each. The experiment was performed for 7 days. Five dietary treatments were used: 2 standard protein diets without (SP and with protease (SP + P formulated 20.7 % CP, 2 lower-protein diets (19.9 % CP without (LP and with protease (LP + P and one lower‑protein diet with protease and with doubled rapeseed meal (RSM content (SP-RSM + P compared with the other treatments. Lower-protein diets were formulated with a 4 % decrease in the relative CP value compared with the standard protein diet. Enzyme protease was added to the diets at a concentration of 200 ppm (15,000 PROT units per kg. The diets contained 0.3 % Cr2O3 to facilitate the estimation of apparent AA digestibility and overall apparent ileal crude protein digestibility. Mono-component protease had no effect on apparent ileal AA digestibility or jejunum protease activity if diets contained the same level of RSM. The supplement of exogenous protease did not affect (P > 0.05 the apparent ileal AA digestibility coefficients if a higher RSM level was used. The CP level influenced (P < 0.05 only the coefficients of the apparent ileal AA digestibility of Pro and Arg. The RSM level (P < 0.01 had significant effects on protease activity in the jejunum.

Aspartic protease activities of schistosomes cleave mammalian hemoglobins in a host-specific manner

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Jeffrey W Koehler

2007-02-01

Full Text Available We examined the efficiency of digestion of hemoglobin from four mammalian species, human, cow, sheep, and horse by acidic extracts of mixed sex adults of Schistosoma japonicum and S. mansoni. Activity ascribable to aspartic protease(s from S. japonicum and S. mansoni cleaved human hemoglobin. In addition, aspartic protease activities from S. japonicum cleaved hemoglobin from bovine, sheep, and horse blood more efficiently than did the activity from extracts of S. mansoni. These findings support the hypothesis that substrate specificity of hemoglobin-degrading proteases employed by blood feeding helminth parasites influences parasite host species range; differences in amino acid sequences in key sites of the parasite proteases interact less or more efficiently with the hemoglobins of permissive or non-permissive hosts.

Understanding serine proteases implications on Leishmania spp lifecycle.

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

2018-01-01

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

Post-translational regulation and trafficking of the granulin-containing protease RD21 of Arabidopsis thaliana.

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Christian Gu

Full Text Available RD21-like proteases are ubiquitous, plant-specific papain-like proteases typified by carrying a C-terminal granulin domain. RD21-like proteases are involved in immunity and associated with senescence and various types of biotic and abiotic stresses. Here, we interrogated Arabidopsis RD21 regulation and trafficking by site-directed mutagenesis, agroinfiltration, western blotting, protease activity profiling and protein degradation. Using an introduced N-glycan sensor, deglycosylation experiments and glyco-engineered N. benthamiana plants, we show that RD21 passes through the Golgi where it becomes fucosylated. Our studies demonstrate that RD21 is regulated at three post-translational levels. Prodomain removal is not blocked in the catalytic Cys mutant, indicating that RD21 is activated by a proteolytic cascade. However, RD21 activation in Arabidopsis does not require vacuolar processing enzymes (VPEs or aleurain-like protease AALP. In contrast, granulin domain removal requires the catalytic Cys and His residues and is therefore autocatalytic. Furthermore, SDS can (re-activate latent RD21 in Arabidopsis leaf extracts, indicating the existence of a third layer of post-translational regulation, possibly mediated by endogenous inhibitors. RD21 causes a dominant protease activity in Arabidopsis leaf extracts, responsible for SDS-induced proteome degradation.

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

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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.

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

Science.gov (United States)

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

2017-05-01

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

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

Science.gov (United States)

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

2010-10-01

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

Hyper production of alkaline protease by mutagenized bacillus subtilis

International Nuclear Information System (INIS)

Qureshi, A.M.; Tanseem, F.

2010-01-01

The purpose of this work was to augment the alkaline protease production from Bacillus subtilis by using chemical mutagen (MMS) and UV mutagenesis. A number of mutants were isolated which produce high levels of extra cellular proteases. Analysis of culture supernatants of these mutants had shown that the total amounts of proteolysis activity were increased from 1 to 2 fold over the wild strain. Clones showing promote response were further characterized by analyzing different parameters; like of Temperature, pH substrate concentration and incubation period, to study the activity of protease enzyme. (author)

An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I.

Science.gov (United States)

Le, Tien; Lee, Hak Jin; Jin, Hyung Jong

2015-08-15

A method was developed to eliminate the proteases contaminating commercial DNase I, which can cause degradation of target protein during the purification process. Bio Basic DNase stock solution (in Tris-HCl buffer [pH 8.0] containing 5mM CaCl2) was first incubated at 50 °C to generate autolysis of proteases and zymogens, leading to a significant reduction in protease activity while preserving DNase activity. The residual protease activity was completely inhibited by further incubation with 2mM PMSF (phenylmethylsulfonyl fluoride) or 2× S8830 inhibitor cocktail. This approach could be readily applicable to eliminate the protease activity in any DNase products or during the preparation of commercial DNase. Copyright © 2015 Elsevier Inc. All rights reserved.

Rapid Detection of Thrombin and Other Protease Activity Directly in Whole Blood

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Yu, Johnson Chung Sing

Thrombin is a serine protease that plays a key role in the clotting cascade to promote hemostasis following injury to the endothelium. From a clinical diagnostic perspective, in-vivo thrombin activity is linked to various blood clotting disorders, as well as cardiovascular disease (DVT, arteriosclerosis, etc). Thus, the ability to rapidly measure protease activity directly in whole blood will provide important new diagnostics, and clinical researchers with a powerful tool to further elucidate the relationship between circulating protease levels and disease. The ultimate goal is to design novel point of care (POC) diagnostic devices that are capable of monitoring protease activities directly in whole blood and biological sample. A charge-changing substrate specific to the thrombin enzyme was engineered and its functionality was confirmed by a series of experiments. This led to the preliminary design, construction, and testing of two device platforms deemed fully functional for the electrophoretic separation and focusing of charged peptide fragments. The concept of using the existing charge-changing substrate platform for bacterial protease detection was also investigated. Certain strains of E coli are associated with severe symptoms such as abdominal cramps, bloody diarrhea, and vomiting. The OmpT protease is expressed on the outer membrane of E coli and plays a role in the cleavage of antimicrobial peptides, the degradation of recombinant heterologous proteins, and the activation of plasminogen in the host. Thus, a synthetic peptide substrate specific to the OmpT protease was designed and modeled for the purpose of detecting E coli in biological sample.

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.

Comparative characterization of protease activity in cultured spotted rose snapper juveniles (Lutjanus guttatus

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Emyr Peña

2015-09-01

Full Text Available Partial characterizations of digestive proteases were studied in three life stages of spotted rose snapper: early (EJ, middle (MJ and late juvenile (LJ with corresponding average weights of 21.3 ± 2.6 g (3 months after hatching, MAH, 190 ± 4.4 g (7 MAH, and 400 ± 11.5 g (12 MAH. At sampling points, the digestive tract was dissected into the stomach (St, pyloric caeca (PC, and the intestine in three sections (proximal (PI, middle (MI and distal intestine (DI. The effect of pH and temperature and specific inhibitors were evaluated for acid and alkaline proteases. Total acid and alkaline protease activity showed a tendency to increase with juvenile life stage of fish while trypsin activity decreased. Differences were found in acid and alkaline protease activities at different pH and temperatures during juvenile stages. Pepstatin A inhibited total activity in the stomach extract in all juvenile stages. Activity in total alkaline protease inhibition was significantly higher in EJ using TLCK, PMSF, SBTI, Phen and Ovo than in MJ and LJ, while no significant differences were found with TPCK inhibition. Therefore increases in protease activities with fish growth through juvenile stages in which a substitution or diversification in the type of alkaline enzymes exist. These results lead a better comprehension of changes in digestive potential of Lutjanidae fish.

The m-AAA Protease Associated with Neurodegeneration Limits MCU Activity in Mitochondria.

Science.gov (United States)

König, Tim; Tröder, Simon E; Bakka, Kavya; Korwitz, Anne; Richter-Dennerlein, Ricarda; Lampe, Philipp A; Patron, Maria; Mühlmeister, Mareike; Guerrero-Castillo, Sergio; Brandt, Ulrich; Decker, Thorsten; Lauria, Ines; Paggio, Angela; Rizzuto, Rosario; Rugarli, Elena I; De Stefani, Diego; Langer, Thomas

2016-10-06

Mutations in subunits of mitochondrial m-AAA proteases in the inner membrane cause neurodegeneration in spinocerebellar ataxia (SCA28) and hereditary spastic paraplegia (HSP7). m-AAA proteases preserve mitochondrial proteostasis, mitochondrial morphology, and efficient OXPHOS activity, but the cause for neuronal loss in disease is unknown. We have determined the neuronal interactome of m-AAA proteases in mice and identified a complex with C2ORF47 (termed MAIP1), which counteracts cell death by regulating the assembly of the mitochondrial Ca 2+ uniporter MCU. While MAIP1 assists biogenesis of the MCU subunit EMRE, the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU. Loss of the m-AAA protease results in accumulation of constitutively active MCU-EMRE channels lacking gatekeeper subunits in neuronal mitochondria and facilitates mitochondrial Ca 2+ overload, mitochondrial permeability transition pore opening, and neuronal death. Together, our results explain neuronal loss in m-AAA protease deficiency by deregulated mitochondrial Ca 2+ homeostasis. Copyright © 2016 Elsevier Inc. All rights reserved.

Crystallization of the virulent and benign subtilisin-like proteases from the ovine footrot pathogen Dichelobacter nodosus

International Nuclear Information System (INIS)

Wong, Wilson; Kennan, Ruth M.; Rosado, Carlos J.; Rood, Julian I.; Whisstock, James C.; Porter, Corrine J.

2010-01-01

The subtilisin-like proteases AprV2 and BprV from virulent strains and AprB2 and BprB from benign strains of D. nodosus have been crystallized and X-ray diffraction data have been collected to 2.0, 2.0, 1.7 and 1.8 Å resolution, respectively. Dichelobacter nodosus is the principal causative agent of ovine footrot, a disease of significant economic importance to the sheep industry. D. nodosus secretes a number of subtilisin-like serine proteases which mediate tissue damage and presumably contribute to the pathogenesis of footrot. Strains causing virulent footrot secrete the proteases AprV2, AprV5 and BprV and strains causing benign footrot secrete the closely related proteases AprB2, AprB5 and BprB. Here, the cloning, purification and crystallization of AprV2, AprB2, BprV and BprB are reported. Crystals of AprV2 and AprB2 diffracted to 2.0 and 1.7 Å resolution, respectively. The crystals of both proteases belonged to space group P1, with unit-cell parameters a = 43.1, b = 46.0, c = 47.2 Å, α = 97.8, β = 115.2, γ = 115.2° for AprV2 and a = 42.7, b = 45.8, c = 45.7 Å, α = 98.4, β = 114.0, γ = 114.6° for AprB2. Crystals of BprV and BprB diffracted to 2.0 and 1.8 Å resolution, respectively. The crystals of both proteases belonged to space group P2 1 , with unit-cell parameters a = 38.5, b = 89.6, c = 47.7 Å, β = 113.6° for BprV and a = 38.5, b = 90.5, c = 44.1 Å, β = 109.9° for BprB. The crystals of all four proteases contained one molecule in the asymmetric unit, with a solvent content ranging from 36 to 40%

Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3.

Science.gov (United States)

Zheng, Fengwei; Lu, Guoliang; Li, Ling; Gong, Peng; Pan, Zishu

2017-11-01

The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å 2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis -cleavage event. Although this conformation is incompatible with protease trans -cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis -cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different

Model building of a thermolysin-like protease by mutagenesis

NARCIS (Netherlands)

Frigerio, F; Margarit, [No Value; Nogarotto, R; Grandi, G; Vriend, G; Hardy, F; Veltman, OR; Venema, G; Eijsink, VGH

The present study concerns the use of site-directed mutagenesis experiments to optimize a three-dimensional model of the neutral protease of Bacillus subtilis (NP-sub), An initial model of NP-sub was constructed using the crystal structures of the homologous neutral proteases of Bacillus

Earthworm Protease

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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.

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...

Tunable protease-activatable virus nanonodes.

Science.gov (United States)

Judd, Justin; Ho, Michelle L; Tiwari, Abhinav; Gomez, Eric J; Dempsey, Christopher; Van Vliet, Kim; Igoshin, Oleg A; Silberg, Jonathan J; Agbandje-McKenna, Mavis; Suh, Junghae

2014-05-27

We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus-receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery.

Purification and biochemical characterization of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease

DEFF Research Database (Denmark)

Studdert, C A; Herrera Seitz, M K; Plasencia, I

2001-01-01

A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5.5-12), is rat......A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5.......5-12), is rather thermophilic (optimal activity at 60 degrees C in 1-2 M NaCl) and is dependent on high salt concentrations for activity and stability (1-2 M NaCl or KCl). Polyclonal antibodies were raised against the purified protease. In Western blots, they presented no cross-reactivity with culture medium from...... other halobacteria nor with commercial proteases except subtilisin. The amino acid sequences of three tryptic peptides obtained from Natronococcus occultus protease did not show significant similarity to other known proteolytic enzymes. This fact, in addition to its high molecular mass suggests...

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

The Inflammatory Actions of Coagulant and Fibrinolytic Proteases in Disease

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Michael Schuliga

2015-01-01

Full Text Available Aside from their role in hemostasis, coagulant and fibrinolytic proteases are important mediators of inflammation in diseases such as asthma, atherosclerosis, rheumatoid arthritis, and cancer. The blood circulating zymogens of these proteases enter damaged tissue as a consequence of vascular leak or rupture to become activated and contribute to extravascular coagulation or fibrinolysis. The coagulants, factor Xa (FXa, factor VIIa (FVIIa, tissue factor, and thrombin, also evoke cell-mediated actions on structural cells (e.g., fibroblasts and smooth muscle cells or inflammatory cells (e.g., macrophages via the proteolytic activation of protease-activated receptors (PARs. Plasmin, the principle enzymatic mediator of fibrinolysis, also forms toll-like receptor-4 (TLR-4 activating fibrin degradation products (FDPs and can release latent-matrix bound growth factors such as transforming growth factor-β (TGF-β. Furthermore, the proteases that convert plasminogen into plasmin (e.g., urokinase plasminogen activator evoke plasmin-independent proinflammatory actions involving coreceptor activation. Selectively targeting the receptor-mediated actions of hemostatic proteases is a strategy that may be used to treat inflammatory disease without the bleeding complications of conventional anticoagulant therapies. The mechanisms by which proteases of the coagulant and fibrinolytic systems contribute to extravascular inflammation in disease will be considered in this review.

Pathogen-secreted proteases activate a novel plant immune pathway.

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Cheng, Zhenyu; Li, Jian-Feng; Niu, Yajie; Zhang, Xue-Cheng; Woody, Owen Z; Xiong, Yan; Djonović, Slavica; Millet, Yves; Bush, Jenifer; McConkey, Brendan J; Sheen, Jen; Ausubel, Frederick M

2015-05-14

Mitogen-activated protein kinase (MAPK) cascades play central roles in innate immune signalling networks in plants and animals. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive. Here we report that pathogen-secreted proteases activate a previously unknown signalling pathway in Arabidopsis thaliana involving the Gα, Gβ, and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of an MAPK cascade. In this pathway, receptor for activated C kinase 1 (RACK1) functions as a novel scaffold that binds to the Gβ subunit as well as to all three tiers of the MAPK cascade, thereby linking upstream G-protein signalling to downstream activation of an MAPK cascade. The protease-G-protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signalling pathways such as that elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to an MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the new protease-mediated immune signalling pathway described here was facilitated by the use of the broad host range, opportunistic bacterial pathogen Pseudomonas aeruginosa. The ability of P. aeruginosa to infect both plants and animals makes it an excellent model to identify novel immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems.

Nepenthesin protease activity indicates digestive fluid dynamics in carnivorous nepenthes plants.

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Franziska Buch

Full Text Available Carnivorous plants use different morphological features to attract, trap and digest prey, mainly insects. Plants from the genus Nepenthes possess specialized leaves called pitchers that function as pitfall-traps. These pitchers are filled with a digestive fluid that is generated by the plants themselves. In order to digest caught prey in their pitchers, Nepenthes plants produce various hydrolytic enzymes including aspartic proteases, nepenthesins (Nep. Knowledge about the generation and induction of these proteases is limited. Here, by employing a FRET (fluorescent resonance energy transfer-based technique that uses a synthetic fluorescent substrate an easy and rapid detection of protease activities in the digestive fluids of various Nepenthes species was feasible. Biochemical studies and the heterologously expressed Nep II from Nepenthes mirabilis proved that the proteolytic activity relied on aspartic proteases, however an acid-mediated auto-activation mechanism was necessary. Employing the FRET-based approach, the induction and dynamics of nepenthesin in the digestive pitcher fluid of various Nepenthes plants could be studied directly with insect (Drosophila melanogaster prey or plant material. Moreover, we observed that proteolytic activity was induced by the phytohormone jasmonic acid but not by salicylic acid suggesting that jasmonate-dependent signaling pathways are involved in plant carnivory.

Lipase and protease extraction from activated sludge

DEFF Research Database (Denmark)

Gessesse, Amare; Dueholm, Thomas; Petersen, Steffen B.

2003-01-01

of gentle and efficient enzyme extraction methods from environmental samples is very important. In this study we present a method for the extraction of lipases and proteases from activated sludge using the non-ionic detergent Triton X-100, EDTA, and cation exchange resin (CER), alone or in combination...

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

Science.gov (United States)

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

2018-04-27

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

Bacillus amyloliquefaciens SUBSP. plantarum PROBIOTIC STRAINS AS PROTEASE PRODUCERS

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E. V. Маtseliukh

2015-04-01

Full Text Available Proteases from probiotic strains of the genus Bacillus, just like the antibiotics, bacteriocins and other hydrolytic enzymes, are one of the main factors that determine their biological activity. The aim of this work was to study the synthesis and biochemical properties of proteases from two strains Bacillus amyloliquefaciens subsp. plantarum UCM B-5139 and UCM B-5140 that included in the probiotic Endosporin. The cultivation of strains was carried out in flasks under rotating for two days. The influence of physico-chemical parameters of the reaction medium on proteolytic activity was studied on partially purified protease preparations. Lytic activity was determined by turbidimetric method. On the second day of cultivation B. amyloliquefaciens subsp. plantarum UCM В-5139 and UCM В-5140 synthesized the metal-dependent peptidase and serine protease, respectively. The optimum conditions of their action were the following: temperature 37–40 °C and pH 6.5–7.0. Isolated proteases are able to lyse the living cells of Staphylococcus aureus and Candida albicans. Thus we demonstrated that B. amyloliquefaciens subsp. plantarum UCM B-5140 and UCM B-5139, included in the probiotic veterinary preparation Endosporin, produced proteolytic enzymes that hydrolyze the native insoluble proteins (elastin, fibrin and collagen. These enzymes belong to the group of neutral metal-dependent and serine proteases. They are active under physiological conditions against gram-positive bacteria and yeasts. The application of these proteases in biotechnology is considered.

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.

Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia.

Science.gov (United States)

Pawlinski, Rafal; Pedersen, Brian; Schabbauer, Gernot; Tencati, Michael; Holscher, Todd; Boisvert, William; Andrade-Gordon, Patricia; Frank, Rolf Dario; Mackman, Nigel

2004-02-15

Sepsis is associated with a systemic activation of coagulation and an excessive inflammatory response. Anticoagulants have been shown to inhibit both coagulation and inflammation in sepsis. In this study, we used both genetic and pharmacologic approaches to analyze the role of tissue factor and protease-activated receptors in coagulation and inflammation in a mouse endotoxemia model. We used mice expressing low levels of the procoagulant molecule, tissue factor (TF), to analyze the effects of TF deficiency either in all tissues or selectively in hematopoietic cells. Low TF mice had reduced coagulation, inflammation, and mortality compared with control mice. Similarly, a deficiency of TF expression by hematopoietic cells reduced lipopolysaccharide (LPS)-induced coagulation, inflammation, and mortality. Inhibition of the down-stream coagulation protease, thrombin, reduced fibrin deposition and prolonged survival without affecting inflammation. Deficiency of either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) alone did not affect inflammation or survival. However, a combination of thrombin inhibition and PAR-2 deficiency reduced inflammation and mortality. These data demonstrate that hematopoietic cells are the major pathologic site of TF expression during endotoxemia and suggest that multiple protease-activated receptors mediate crosstalk between coagulation and inflammation.

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

Post-secretional activation of Protease IV by quorum sensing in Pseudomonas aeruginosa.

Science.gov (United States)

Oh, Jungmin; Li, Xi-Hui; Kim, Soo-Kyong; Lee, Joon-Hee

2017-06-30

Protease IV (PIV), a key virulence factor of Pseudomonas aeruginosa is a secreted lysyl-endopeptidase whose expression is induced by quorum sensing (QS). We found that PIV expressed in QS mutant has severe reduction of activity in culture supernatant (CS), even though it is overexpressed to high level. PIV purified from the QS mutant (M-PIV) had much lower activity than the PIV purified from wild type (P-PIV). We found that the propeptide cleaved from prepro-PIV was co-purified with M-PIV, but never with P-PIV. Since the activity of M-PIV was restored by adding the CS of QS-positive and PIV-deficient strain, we hypothesized that the propeptide binds to and inhibits PIV, and is degraded to activate PIV by a QS-dependent factor. In fact, the CS of the QS-positive and PIV-deficient strain was able to degrade the propeptide. Since the responsible factor should be a QS-dependently expressed extracellular protease, we tested QS-dependent proteases of P. aeruginosa and found that LasB (elastase) can degrade the propeptide and activate M-PIV. We purified the propeptide of PIV and confirmed that the propeptide can bind to and inhibit PIV. We suggest that PIV is post-secretionally activated through the extracellular degradation of the propeptide by LasB, a QS-dependent protease.

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

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.

Changes in protein metabolism after irradiation. Pt. 1. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat spleen after 600 R whole body x irradiation

Energy Technology Data Exchange (ETDEWEB)

Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

1975-12-01

The protease activity of cytoplasm and cell organelles of the rat spleen against spleen protein and hemoglobin as a substrate increases during a initial reaction phase of the organism on the first day after 600 R whole body X-irradiation. The alkaline protease in the cytoplasm and the acid protease in the cell organelles increase, whereas the protease activity against externally added hemoglobin as substrate decreases below the initial values. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the spleen is therefore not explained by an increased protease activity. Acid proteases appear in the cytoplasm which derive probably from the cell organelles. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

THE EFFECT OF FEEDING Lactobacillus ON GROWTH, SURVIVAL RATE AND PROTEASE ACTIVITY OF Litopenaeus vannamei

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Nunak Nafiqoh

2011-12-01

Full Text Available This study examined the effect of two Lactobacillus bacteria on protease activity and growth rate of Litopenaeus vannamei. An experiment was conducted to examine protease activity and growth rate. The experiment consisted of two treatment tanks, the first tank was provided with artemia immersed in 2.6 x 1016 cfu/mL of bacteria solution, the second tank served as the control tank. After 20 days, the L. vannamei in the tank that received Lactobacillus have significantly different in growth, survival rate and protease activity (P<0.05 compared to the control, but no significant difference between Lactobacillus casei and Lactobacillus plantarum treatments. Within the digestive organ, protease activity of hepatopancreas and stomach demonstrated significant higher activity (P<0.05 compared to the intestine.

Bacterial proteases and virulence

DEFF Research Database (Denmark)

Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

2013-01-01

signalling to short-circuit host cell processes. Common to both intra- and extracellular proteases is the tight control of their proteolytic activities. In general, substrate recognition by the intracellular proteases is highly selective which is, in part, attributed to the chaperone activity associated...... tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell...... with the proteases either encoded within the same polypeptide or on separate subunits. In contrast, substrate recognition by extracellular proteases is less selective and therefore these enzymes are generally expressed as zymogens to prevent premature proteolytic activity that would be detrimental to the cell...

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

Science.gov (United States)

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

Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

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Rok Gaber

2013-11-01

Full Text Available To effectively fight against the human immunodeficiency virus infection/ acquired immunodeficiency syndrome (HIV/AIDS epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity.

Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

Science.gov (United States)

Gaber, Rok; Majerle, Andreja; Jerala, Roman; Benčina, Mojca

2013-01-01

To effectively fight against the human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity. PMID:24287545

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.

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens.

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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.

Antimicrobial activity of a 48-kDa protease (AMP48) from Artocarpus heterophyllus latex.

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Siritapetawee, J; Thammasirirak, S; Samosornsuk, W

2012-01-01

Artocarpus heterophyllus (jackfruit) is a latex producing plant. Plant latex is produced from secretory cells and contains many intergradients. It also has been used in folk medicine. This study aimed to purify and characterize the biological activities of a protease from jackfruit latex. A protease was isolated and purified from crude latex of a jackfruit tree by acid precipitation and ion exchange chromatography. The proteolytic activities of protein were tested using gelatin- and casein-zymography. The molecular weight and isoelectric point (pl) of protein were analysed by SDS/12.5% PAGE and 2D-PAGE, respectively. Antimicrobial activity of protein was analysed by broth microdilution method. In addition, the antibacterial activity of protein against Pseudomonas aeruginosa ATCC 27853 was observed and measured using atomic force microscopy (AFM) technique. The purified protein contained protease activity by digesting gelatin- and casein-substrates. The protease was designated as antimicrobial protease-48 kDa or AMP48 due to its molecular mass on SDS-PAGE was approximately 48 kDa. The isoelectric point (pl) of AMP48 was approximately 4.2. In addition, AMP48 contained antimicrobial activities by it could inhibit the growths of Pseudomonas aeruginosa ATCC 27853 and clinical isolated Candida albicans at minimum inhibitory concentration (MIC) 2.2 mg/ml and Minimum microbicidal concentration (MMC) 8.8 mg/ml. AFM image also supported the antimicrobial activities of AMP48 by the treated bacterial morphology and size were altered from normal.

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

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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.

Silver-Stained Fibrin Zymography: Separation of Proteases and Activity Detection Using a Single Substrate-Containing Gel.

Science.gov (United States)

Park, Chang-Su; Kang, Dae-Ook; Choi, Nack-Shick

2017-01-01

Silver-stained fibrin zymography for separation of protease bands and activity detection using a single substrate gel was designed. The method takes advantage of the nano-scale sensitivity of both zymography and silver staining. After sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in a gel containing fibrin (protease substrate), the gel was incubated in enzyme reaction buffer and the zymogram gel was silver-stained. Bands with protease activity were stained with silver in clear areas where the protein substrate had been degraded. The molecular sizes of proteases were accurately determined.

Covalent glycoinositolphospholipid (GPI binding to hemoglobin is associated with insulin-activation of erythrocyte membrane protease

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VESNA NIKETIC

2004-05-01

Full Text Available Recently, it was demonstrated that prolonged hyperinsulinism associated with hypoglycemia, both in vivo and in vitro, caused covalent glycoinositolphospholipid (GPI binding to the C termini of both hemoglobin b-chains, which resulted in the formation of a novel, hitherto unrecognized, minor hemoglobin fraction (GPI-Hb (Niketic et al., Biochem. Biophys. Res. Commun. 239 (1997 435. In this study it was demonstrated that exposure of erythrocyte membranes to insulin causes the activation of membrane protease as well as that the formation of GPI-Hb parallels its activity. It is suggested that the insulin-activated protease is able to catalyze, albeit slowly, the transpeptidation, i.e., the replacement of the carboxy-terminal amino acid(s residues of the Hb b-chains with GPI as an exogenous nucleophile. To our knowledge the present results show for the first time that insulin stimulates protease activity in erythrocyte membranes, as well as that insulin-activated protease may be involved in post-translational GPI binding to proteins.

Papain-like protease (PLpro) inhibitory effects of cinnamic amides from Tribulus terrestris fruits.

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Song, Yeong Hun; Kim, Dae Wook; Curtis-Long, Marcus John; Yuk, Heung Joo; Wang, Yan; Zhuang, Ningning; Lee, Kon Ho; Jeon, Kwon Seok; Park, Ki Hun

2014-01-01

Tribulus terrestris fruits are well known for their usage in pharmaceutical preparations and food supplements. The methanol extract of T. terrestris fruits showed potent inhibition against the papain-like protease (PLpro), an essential proteolylic enzyme for protection to pathogenic virus and bacteria. Subsequent bioactivity-guided fractionation of this extract led to six cinnamic amides (1-6) and ferulic acid (7). Compound 6 emerged as new compound possessing the very rare carbinolamide motif. These compounds (1-7) were evaluated for severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro inhibitory activity to identify their potencies and kinetic behavior. Compounds (1-6) displayed significant inhibitory activity with IC50 values in the range 15.8-70.1 µM. The new cinnamic amide 6 was found to be most potent inhibitor with an IC50 of 15.8 µM. In kinetic studies, all inhibitors exhibited mixed type inhibition. Furthermore, the most active PLpro inhibitors (1-6) were proven to be present in the native fruits in high quantities by HPLC chromatogram and liquid chromatography with diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI/MS).

Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

Energy Technology Data Exchange (ETDEWEB)

Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

1987-08-01

The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon/sup -/ cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes (/sup 3/H)methyl-casein to acid-soluble products in the presence of ATP and Mg/sup 2 +/. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles.

Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

International Nuclear Information System (INIS)

Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

1987-01-01

The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon - cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes [ 3 H]methyl-casein to acid-soluble products in the presence of ATP and Mg 2+ . ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles

Triclabendazole Effect on Protease Enzyme Activity in the Excretory- Secretory Products of Fasciola hepatica in Vitro.

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Yosef Shrifi

2014-03-01

Full Text Available Fasciola hepatica is one of the most important helminthes parasites and triclabendazole (TCBZ is routinely used for treatment of infected people and animals. Secreted protease enzymes by the F. hepatica plays a critical role in the invasion, migration, nutrition and the survival of parasite and are key targets for novel drugs and vaccines. The aim of study was to determine the protease activity of excretory- secretory products (ESP of F. hepatica in the presence of TCBZ anthelmintic.F. hepatica helminthes were collected and cultured within RPMI 1640 [TCBZ treated (test and untreated (control] for 6 h at 37 °C. ESP of treated and control were collected, centrifuged and supernatants were stored at -20°C. Protein concentrations were measured according to Bradford method. Protease enzymes activities of ESP samples were estimated by using sigma's non-specific protease activity assay. ESP protein bands were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE.Mean protein concentrations in control and treated of ESP samples were determined 196.1 ±14.52 and 376.4 ±28.20 μg/ml, respectively. Mean protease enzymes activities in control and treated were 0.37 ±0.1 and 0.089 ±0.03 U/ml, respectively. Significant difference between proteins concentrations and protease enzymes activities of two groups was observed (P<0.05. SDS-PAGE showed different patterns of protein bands between treated and control samples.The TCBZ reduced secreted protease enzymes activities and possibly effects on invasion, migration, nutrition and particularly survival of the parasite in the host tissues.

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

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

2014-11-01

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

Mycoplasma hyopneumoniae in vitro peptidase activities: identification and cleavage of kallikrein-kinin system-like substrates.

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Moitinho-Silva, Lucas; Kondo, Marcia Y; Oliveira, Lilian C G; Okamoto, Debora N; Paes, Jéssica A; Machado, Mauricio F M; Veronez, Camila L; Motta, Guacyara; Andrade, Sheila S; Juliano, Maria A; Ferreira, Henrique B; Juliano, Luiz; Gouvea, Iuri E

2013-05-03

Bacterial proteases are important for metabolic processes and pathogenesis in host organisms. The bacterial swine pathogen Mycoplasma hyopneumoniae has 15 putative protease-encoding genes annotated, but none of them have been functionally characterized. To identify and characterize peptidases that could be relevant for infection of swine hosts, we investigated the peptidase activity present in the pathogenic 7448 strain of M. hyopneumoniae. Combinatorial libraries of fluorescence resonance energy transfer peptides, specific inhibitors and pH profiling were used to screen and characterize endopeptidase, aminopeptidase and carboxypeptidase activities in cell lysates. One metalloendopeptidase, one serine endopeptidase, and one aminopeptidase were detected. The detected metalloendopeptidase activity, prominent at neutral and basic pH ranges, was due to a thimet oligopeptidase family member (M3 family), likely an oligoendopeptidase F (PepF), which cleaved the peptide Abz-GFSPFRQ-EDDnp at the F-S bond. A chymotrypsin-like serine endopeptidase activity, possibly a subtilisin-like serine protease, was prominent at higher pH levels, and was characterized by its preference for a Phe residue at the P1 position of the substrate. The aminopeptidase P (APP) activity showed a similar profile to that of human membrane-bound APP. Genes coding for these three peptidases were identified and their transcription was confirmed in the 7448 strain. Furthermore, M. hyopneumoniae cell lysate peptidases showed effects on kallikrein-kinin system-like substrates, such as bradykinin-derived substrates and human high molecular weight kininogen. The M. hyopneumoniae peptidase activities, here characterized for the first time, may be important for bacterial survival strategies and thus represent possible targets for drug development against M. hyopneumoniae swine infections. Copyright © 2013 Elsevier B.V. All rights reserved.

Evidence for possible involvement of an elastolytic serine protease in aspergillosis.

OpenAIRE

Kolattukudy, P E; Lee, J D; Rogers, L M; Zimmerman, P; Ceselski, S; Fox, B; Stein, B; Copelan, E A

1993-01-01

A number of isolates of Aspergillus fumigatus obtained from the hospital environment produced extracellular elastolytic activity. This activity was found to be catalyzed by a single 33-kDa protein which was purified and characterized to be a serine protease. A. fumigatus, when grown on the insoluble structural material obtained from murine and bovine lung, produced the same extracellular 33-kDa elastolytic protease, indicating that this enzyme is likely to be produced when the organism infect...

Protease-activated receptor-1 impairs host defense in murine pneumococcal pneumonia: a controlled laboratory study

NARCIS (Netherlands)

Schouten, Marcel; van't Veer, Cornelis; Roelofs, Joris J. T. H.; Levi, Marcel; van der Poll, Tom

2012-01-01

Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia. Protease-activated receptor-1 (PAR-1) is expressed by multiple cell types present in the lungs and can be activated by various proteases generated during acute inflammation. The cellular effect of PAR-1

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

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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.

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.

Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains.

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Harper, Stephen; Gratton, Hayley E; Cornaciu, Irina; Oberer, Monika; Scott, David J; Emsley, Jonas; Dreveny, Ingrid

2014-05-13

The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGFβ signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal "domain present in USPs" (DUSP) and "ubiquitin-like" (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15. A conserved VEVY motif is a signature feature at the two-domain interface that shapes a potential protein interaction site. Small angle X-ray scattering and gel filtration experiments are consistent with the USP11DU domains and full-length USP11 being monomeric. Unexpectedly, we reveal, through kinetic assays of a series of deletion mutants, that the catalytic activity of USP11 is not regulated through intramolecular autoinhibition or activation by the N-terminal DU or UBL domains. Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys(63)-, Lys(6)-, Lys(33)-, and Lys(11)-linked chains over Lys(27)-, Lys(29)-, and Lys(48)-linked and linear chains consistent with USP11's function in DNA repair pathways that is mediated by the protease domain. Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity. This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation.

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.

Soybean P34 Probable Thiol Protease Probably Has Proteolytic Activity on Oleosins.

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Zhao, Luping; Kong, Xiangzhen; Zhang, Caimeng; Hua, Yufei; Chen, Yeming

2017-07-19

P34 probable thiol protease (P34) and Gly m Bd 30K (30K) show high relationship with the protease of 24 kDa oleosin of soybean oil bodies. In this study, 9 day germinated soybean was used to separate bioprocessed P34 (P32) from bioprocessed 30K (28K). Interestingly, P32 existed as dimer, whereas 28K existed as monomer; a P32-rich sample had proteolytic activity and high cleavage site specificity (Lys-Thr of 24 kDa oleosin), whereas a 28K-rich sample showed low proteolytic activity; the P32-rich sample contained one thiol protease. After mixing with purified oil bodies, all P32 dimers were dissociated and bound to 24 kDa oleosins to form P32-24 kDa oleosin complexes. By incubation, 24 kDa oleosin was preferentially hydrolyzed, and two hydrolyzed products (HPs; 17 and 7 kDa) were confirmed. After most of 24 kDa oleosin was hydrolyzed, some P32 existed as dimer, and the other as P32-17 kDa HP. It was suggested that P32 was the protease.

Indispensable Role of Proteases in Plant Innate Immunity.

Science.gov (United States)

Balakireva, Anastasia V; Zamyatnin, Andrey A

2018-02-23

Plant defense is achieved mainly through the induction of microbe-associated molecular patterns (MAMP)-triggered immunity (MTI), effector-triggered immunity (ETI), systemic acquired resistance (SAR), induced systemic resistance (ISR), and RNA silencing. Plant immunity is a highly complex phenomenon with its own unique features that have emerged as a result of the arms race between plants and pathogens. However, the regulation of these processes is the same for all living organisms, including plants, and is controlled by proteases. Different families of plant proteases are involved in every type of immunity: some of the proteases that are covered in this review participate in MTI, affecting stomatal closure and callose deposition. A large number of proteases act in the apoplast, contributing to ETI by managing extracellular defense. A vast majority of the endogenous proteases discussed in this review are associated with the programmed cell death (PCD) of the infected cells and exhibit caspase-like activities. The synthesis of signal molecules, such as salicylic acid, jasmonic acid, and ethylene, and their signaling pathways, are regulated by endogenous proteases that affect the induction of pathogenesis-related genes and SAR or ISR establishment. A number of proteases are associated with herbivore defense. In this review, we summarize the data concerning identified plant endogenous proteases, their effect on plant-pathogen interactions, their subcellular localization, and their functional properties, if available, and we attribute a role in the different types and stages of innate immunity for each of the proteases covered.

Autoprocessing of human immunodeficiency virus type 1 protease miniprecursor fusions in mammalian cells

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Chen Chaoping

2010-07-01

Full Text Available Abstract Background HIV protease (PR is a virus-encoded aspartic protease that is essential for viral replication and infectivity. The fully active and mature dimeric protease is released from the Gag-Pol polyprotein as a result of precursor autoprocessing. Results We here describe a simple model system to directly examine HIV protease autoprocessing in transfected mammalian cells. A fusion precursor was engineered encoding GST fused to a well-characterized miniprecursor, consisting of the mature protease along with its upstream transframe region (TFR, and small peptide epitopes to facilitate detection of the precursor substrate and autoprocessing products. In HEK 293T cells, the resulting chimeric precursor undergoes effective autoprocessing, producing mature protease that is rapidly degraded likely via autoproteolysis. The known protease inhibitors Darunavir and Indinavir suppressed both precursor autoprocessing and autoproteolysis in a dose-dependent manner. Protease mutations that inhibit Gag processing as characterized using proviruses also reduced autoprocessing efficiency when they were introduced to the fusion precursor. Interestingly, autoprocessing of the fusion precursor requires neither the full proteolytic activity nor the majority of the N-terminal TFR region. Conclusions We suggest that the fusion precursors provide a useful system to study protease autoprocessing in mammalian cells, and may be further developed for screening of new drugs targeting HIV protease autoprocessing.

Functional analyses of the three simian hemorrhagic fever virus nonstructural protein 1 papain-like proteases.

Science.gov (United States)

Vatter, Heather A; Di, Han; Donaldson, Eric F; Radu, Gertrud U; Maines, Taronna R; Brinton, Margo A

2014-08-01

The N-terminal region of simian hemorrhagic fever virus (SHFV) nonstructural polyprotein 1a is predicted to encode three papain-like proteases (PLP1α, PLP1β, and PLP1γ). Catalytic residues and cleavage sites for each of the SHFV PLP1s were predicted by alignment of the SHFV PLP1 region sequences with each other as well as with those of other arteriviruses, and the predicted catalytic residues were shown to be proximal by homology modeling of the SHFV nsp1s on porcine respiratory and reproductive syndrome virus (PRRSV) nsp1 crystal structures. The functionality of the predicted catalytic Cys residues and cleavage sites was tested by analysis of the autoproteolytic products generated in in vitro transcription/translation reactions done with wild-type or mutant SHFV nsp1 constructs. Cleavage sites were also analyzed by mass spectroscopy analysis of selected immunoprecipitated cleavage products. The data showed that each of the three SHFV PLP1s is an active protease. Cys63 was identified as the catalytic Cys of SHFV PLP1α and is adjacent to an Ala instead of the canonical Tyr observed in other arterivirus PLP1s. SHFV PLP1γ is able to cleave at both downstream and upstream nsp1 junction sites. Although intermediate precursor polyproteins as well as alternative products generated by each of the SHFV PLP1s cleaving at sites within the N-terminal region of nsp1β were produced in the in vitro reactions, Western blotting of SHFV-infected, MA104 cell lysates with SHFV nsp1 protein-specific antibodies detected only the three mature nsp1 proteins. SHFV is unique among arteriviruses in having three N-terminal papain-like protease 1 (PLP1) domains. Other arteriviruses encode one or two active PLP1s. This is the first functional study of the SHFV PLP1s. Analysis of the products of in vitro autoprocessing of an N-terminal SHFV nonstructural 1a polypeptide fragment showed that each of the three SHFV PLP1s is active, and the predicted catalytic Cys residues and cleavage sites

Activation of Protease-Activated Receptor 2 Induces VEGF Independently of HIF-1

DEFF Research Database (Denmark)

Rasmussen, J.G.; Riis, Simone Elkjær; Frøbert, O.

2012-01-01

Human adipose stem cells (hASCs) can promote angiogenesis through secretion of proangiogenic factors such as vascular endothelial growth factor (VEGF). In other cell types, it has been shown that induction of VEGF is mediated by both protease activated receptor 2 (PAR2) and hypoxia inducible fact...

[Protease activity of microflora in the oral cavity of patients with periodontitis].

Science.gov (United States)

Voropaeva, E A; Baĭrakova, A L; Bichucher, A M; D'iakov, V L; Kozlov, L V

2008-01-01

Microbial spectrum and non-specific as well as specific IgA1 protease activity of isolated microorganisms were investigated in gingival liquid of patients with periodontitis. Microorganisms from the gingival liqud of these patients belonged to conditional-pathogenic obligate and facultatively anaerobic bacteria. 24 strains of microorganisms have been identified. Nonspecific proteolytic activity was found in the following microorganisms: Actinomyces israelii, Actinomyces naeslundii, Aerococcus viridans, Bifidobacterium longum, Neisseria subflave, Streptococcus parvulus, Eubacterium alactolyticum, Lactobaccilus catenoforme, Bacillus spp. Specific IgA1-protease activity and lack of proteolytic activity towards IgG was found in Streptococcus acidominimus, Streptococcus hansenii, Streptococcus salivarius, Leptotrychia buccalis, Staphylococcus haemolyticus and Neisseria sicca. No proteolytic activity was found in cultivation medium of Eubacterium alactolyticum (1 strain), Prevotella buccalis, Aerococcus viridans and Streptococcus sanguis.

Hormone therapy affects plasma measures of factor VII-activating protease in younger postmenopausal women

DEFF Research Database (Denmark)

Mathiasen, Jørn Sidelmann; Skouby, S.O.; Vitzthum, F.

2010-01-01

Objectives Current reviews indicate that hormone therapy (HT) has a protective role in coronary heart disease (CHD) in younger postmenopausal women, whereas HT contributes to CHD in older women Factor VII-activating protease (FSAP) is a serine protease that accumulates in unstable atherosclerotic...

Selection of suitable detergents for obtaining an active dengue protease in its natural form from E. coli.

Science.gov (United States)

Liew, Lynette Sin Yee; Lee, Michelle Yueqi; Wong, Ying Lei; Cheng, Jinting; Li, Qingxin; Kang, CongBao

2016-05-01

Dengue protease is a two-component enzyme and is an important drug target against dengue virus. The protease activity and protein stability of dengue nonstructural protein 3 (NS3) require a co-factor region from a four-span membrane protein NS2B. A natural form of dengue protease containing full-length NS2B and NS3 protease domain NS2BFL-NS3pro will be useful for dengue drug discovery. In current study, detergents that can be used for protease purification were tested. Using a water soluble protease construct, 39 detergents were selected for both NS2B and NS2BFL-NS3pro purification. The results showed that 18 detergents were able to sustain the activity of the natural dengue protease and 11 detergents could be used for NS2B purification. The results obtained in this study will be useful for biochemical and biophysical studies on dengue protease. Copyright © 2016 Elsevier Inc. All rights reserved.

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...

Alkaline protease production by alkaliphilic marine bacteria isolated ...

African Journals Online (AJOL)

The molecular mass determined using SDS-PAGE, was nearly 31.0 39 kDa. Some fundamental properties like effects of different temperatures, pH, metal ions (Ca2+, Mg2+, Cu2+, Pb3+, Mn2+ and Cd2+) and ethylene diamine tetraacetic acid (EDTA) on protease activity were also studied. Maximum activities were obtained ...

Aspartic Protease Zymography Case Study: Detection of Fungal Acid Proteases by Zymography.

Science.gov (United States)

Kernaghan, Gavin; Mayerhofer, Michael

2017-01-01

This chapter describes a method for the production and characterization of fungal acid proteases. Protease production is induced by growth on BSA media over a pH gradient and protein levels are monitored over time with the Bradford assay. Once protein is depleted, the media is purified and proteases are characterized by gelatin zymography using acrylamide and buffers at near-neutral pH. Maintaining pH levels below those found in traditional zymographic systems avoids the potential loss of activity that may occur in aspartic proteases under alkaline conditions.

Broad-Spectrum Inhibitors against 3C-Like Proteases of Feline Coronaviruses and Feline Caliciviruses

Science.gov (United States)

Shivanna, Vinay; Narayanan, Sanjeev; Prior, Allan M.; Weerasekara, Sahani; Hua, Duy H.; Kankanamalage, Anushka C. Galasiti; Groutas, William C.; Chang, Kyeong-Ok

2015-01-01

ABSTRACT Feline infectious peritonitis and virulent, systemic calicivirus infection are caused by certain types of feline coronaviruses (FCoVs) and feline caliciviruses (FCVs), respectively, and are important infectious diseases with high fatality rates in members of the Felidae family. While FCoV and FCV belong to two distinct virus families, the Coronaviridae and the Caliciviridae, respectively, they share a dependence on viral 3C-like protease (3CLpro) for their replication. Since 3CLpro is functionally and structurally conserved among these viruses and essential for viral replication, 3CLpro is considered a potential target for the design of antiviral drugs with broad-spectrum activities against these distinct and highly important viral infections. However, small-molecule inhibitors against the 3CLpro enzymes of FCoV and FCV have not been previously identified. In this study, derivatives of peptidyl compounds targeting 3CLpro were synthesized and evaluated for their activities against FCoV and FCV. The structures of compounds that showed potent dual antiviral activities with a wide margin of safety were identified and are discussed. Furthermore, the in vivo efficacy of 3CLpro inhibitors was evaluated using a mouse model of coronavirus infection. Intraperitoneal administration of two 3CLpro inhibitors in mice infected with murine hepatitis virus A59, a hepatotropic coronavirus, resulted in significant reductions in virus titers and pathological lesions in the liver compared to the findings for the controls. These results suggest that the series of 3CLpro inhibitors described here may have the potential to be further developed as therapeutic agents against these important viruses in domestic and wild cats. This study provides important insights into the structure and function relationships of 3CLpro for the design of antiviral drugs with broader antiviral activities. IMPORTANCE Feline infectious peritonitis virus (FIPV) is the leading cause of death in young cats

Amylase and protease inhibition activity against Fusarium verticillioides Amylase and protease inhibition activity against Fusarium verticillioides/ Atividade inibidora de amilase e protease de milho contra Fusarium verticillioides durante a germinação

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Elisa Yoko Hirooka

2006-06-01

Full Text Available The primary maize pathogen, Fusarium verticillioides (F. moniliforme Sheldon is responsible for fumonisin production, which is harmful to human and animal health. In addition, maize can be more susceptible to fungal infection after insect attack. The activity of amylase and protease inhibitors in AG 5011 and CD 307 hybrids were determined during germination with controlled and not controlled conditions of temperature and relative humidity and, they were correlated to maize resistance against Sithophilus zeamais. The inhibitory activity during corn germination was evaluated at 0, 24, 48, 72, 96 and 168 h. Amylase and protease inhibitory activity increased during germination in both hybrids, which ranged respectively from 2.8 to 39.5 UIA/g, and 550.0 to 3633.9 UIP/g. The highest levels of inhibitory activity occurred in hybrid CD 307 in germination chamber. The biologic cycle and susceptible rate were evaluated for corn resistance test. The AG 5011 hybrid was less susceptible to S. zeamais and showed higher inhibitory activity (time 0 h, demonstrating possible relationship between resistance against the insect and inhibitory enzymes. These results indicated that maize natural defense mechanism plays an important role on phytopathogen control.Fusarium verticillioides, patógeno primário do milho, destaca-se pela produção da fumonisina, prejudicial à saúde humana e animal. Considerando que os mecanismos naturais de defesa são ferramentas promissoras no controle de fitopatógenos, avaliou-se: a atividade dos inibidores de amilase e protease presente nos híbridos de milho AG 5011 e CD 307 durante a germinação em câmara de germinação (25ºC e 90-95%UR e em casa de vegetação (sem controle de temperatura e umidade contra amilase e protease de F. verticillioides. Paralelamente, avaliou-se a resistência do milho a Sitophilus zeamais. A atividade inibitória de enzimas avaliada nos tempos 0, 24, 48, 72, 96 e 168 h, aumentou durante a germina

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.

Pnserpin: A Novel Serine Protease Inhibitor from Extremophile Pyrobaculum neutrophilum

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Huan Zhang

2017-01-01

Full Text Available Serine protease inhibitors (serpins are native inhibitors of serine proteases, constituting a large protein family with members spread over eukaryotes and prokaryotes. However, only very few prokaryotic serpins, especially from extremophiles, have been characterized to date. In this study, Pnserpin, a putative serine protease inhibitor from the thermophile Pyrobaculum neutrophilum, was overexpressed in Escherichia coli for purification and characterization. It irreversibly inhibits chymotrypsin-, trypsin-, elastase-, and subtilisin-like proteases in a temperature range from 20 to 100 °C in a concentration-dependent manner. The stoichiometry of inhibition (SI of Pnserpin for proteases decreases as the temperature increases, indicating that the inhibitory activity of Pnserpin increases with the temperature. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that Pnserpin inhibits proteases by forming a SDS-resistant covalent complex. Homology modeling and molecular dynamic simulations predicted that Pnserpin can form a stable common serpin fold. Results of the present work will help in understanding the structural and functional characteristics of thermophilic serpin and will broaden the current knowledge about serpins from extremophiles.

The threonine protease activity of testes-specific protease 50 (TSP50 is essential for its function in cell proliferation.

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Yu-Yin Li

Full Text Available BACKGROUND: Testes-specific protease 50 (TSP50, a newly discovered threonine enzyme, has similar amino acid sequences and enzymatic structures to those of many serine proteases. It may be an oncogene. TSP50 is up-regulated in breast cancer epithelial cells, and ectopic expression of TSP50 in TSP50-deficient Chinese hamster ovary (CHO cells has been found to promote cell proliferation. However, the mechanisms by which TSP50 exerts its growth-promoting effects are not yet fully understood. METHODOLOGY/PRINCIPAL FINDINGS: To delineate whether the threonine protease activity of TSP50 is essential to its function in cell proliferation, we constructed and characterized a mutant TSP50, called TSP50 T310A, which was identified as a protease-dead mutant of TSP50. By a series of proliferation analyses, colony formation assays and apoptosis analyses, we showed that T310A mutation significantly depresses TSP50-induced cell proliferation in vitro. Next, the CHO stable cell line expressing either wild-type or T310A mutant TSP50 was injected subcutaneously into nude mice. We found that the T310A mutation could abolish the tumorigenicity of TSP50 in vivo. A mechanism investigation revealed that the T310A mutation prevented interaction between TSP50 and the NF-κBIκBα complex, which is necessary for TSP50 to perform its function in cell proliferation. CONCLUSION: Our data highlight the importance of threonine 310, the most critical protease catalytic site in TSP50, to TSP50-induced cell proliferation and tumor formation.

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

Science.gov (United States)

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

2012-07-01

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

Activation of the Arabidopsis membrane-bound transcription factor bZIP28 is mediated by site-2 protease, but not site-1 protease.

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Iwata, Yuji; Ashida, Makoto; Hasegawa, Chisa; Tabara, Kazuki; Mishiba, Kei-Ichiro; Koizumi, Nozomu

2017-08-01

The unfolded protein response (UPR) is a homeostatic cellular response conserved in eukaryotic cells to alleviate the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Arabidopsis bZIP28 is a membrane-bound transcription factor activated by proteolytic cleavage in response to ER stress, thereby releasing its cytosolic portion containing the bZIP domain from the membrane to translocate into the nucleus where it induces the transcription of genes encoding ER-resident molecular chaperones and folding enzymes. It has been widely recognized that the proteolytic activation of bZIP28 is mediated by the sequential cleavage of site-1 protease (S1P) and site-2 protease (S2P). In the present study we provide evidence that bZIP28 protein is cleaved by S2P, but not by S1P. We demonstrated that wild-type and s1p mutant plants produce the active, nuclear form of bZIP28 in response to the ER stress inducer tunicamycin. In contrast, tunicamycin-treated s2p mutants do not accumulate the active, nuclear form of bZIP28. Consistent with these observations, s2p mutants, but not s1p mutants, exhibited a defective transcriptional response of ER stress-responsive genes and significantly higher sensitivity to tunicamycin. Interestingly, s2p mutants accumulate two membrane-bound bZIP28 fragments with a shorter ER lumen-facing C-terminal domain. Importantly, the predicted cleavage sites are located far from the canonical S1P recognition motif previously described. We propose that ER stress-induced proteolytic activation of bZIP28 is mediated by the sequential actions of as-yet-unidentified protease(s) and S2P, and does not require S1P. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

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

Activity-Based Protein Profiling of Rhomboid Proteases in Liposomes

Czech Academy of Sciences Publication Activity Database

Wolf, E. V.; Seybold, M.; Hadravová, Romana; Stříšovský, Kvido; Verhelst, S. H. L.

2015-01-01

Roč. 16, č. 11 (2015), s. 1616-1621 ISSN 1439-4227 R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : activity-based protein profiling * chemical probes * inhibitors * intramembrane proteases * liposomes Subject RIV: CE - Biochemistry Impact factor: 2.850, year: 2015

Cross genome comparisons of serine proteases in Arabidopsis and rice

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Sowdhamini R

2006-08-01

Full Text Available Abstract Background Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis thaliana and rice (Oryza sativa genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species. Results Despite the differences in genome sizes between Arabidopsis and rice, we identified a very similar number of serine protease-like proteins in the two plant species (206 and 222, respectively. Nearly 40% of the above sequences were identified as potential orthologues. Atypical members could be identified in the plant genomes for Deg, Clp, Lon, rhomboid proteases and species-specific members were observed for the highly populated subtilisin and serine carboxypeptidase families suggesting multiple lateral gene transfers. DegP proteases, prolyl oligopeptidases, Clp proteases and rhomboids share a significantly higher percentage orthology between the two genomes indicating substantial evolutionary divergence was set prior to speciation. Single domain architectures and paralogues for several putative subtilisins, serine carboxypeptidases and rhomboids suggest they may have been recruited for additional roles in secondary metabolism with spatial and temporal regulation. The analysis reveals some domain architectures unique to either or both of the plant species and some inactive proteases, like in rhomboids and Clp proteases, which could be involved in chaperone function. Conclusion The systematic analysis of the serine protease-like proteins in the two plant species has provided some insight into the possible functional associations of previously uncharacterised serine protease-like proteins. Further investigation of these aspects may prove beneficial in our understanding of similar processes in commercially significant crop plant species.

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

Localization and activation of the Drosophila protease easter require the ER-resident saposin-like protein seele.

Science.gov (United States)

Stein, David; Charatsi, Iphigenie; Cho, Yong Suk; Zhang, Zhenyu; Nguyen, Jesse; DeLotto, Robert; Luschnig, Stefan; Moussian, Bernard

2010-11-09

Drosophila embryonic dorsal-ventral polarity is generated by a series of serine protease processing events in the egg perivitelline space. Gastrulation Defective processes Snake, which then cleaves Easter, which then processes Spätzle into the activating ligand for the Toll receptor. seele was identified in a screen for mutations that, when homozygous in ovarian germline clones, lead to the formation of progeny embryos with altered embryonic patterning; maternal loss of seele function leads to the production of moderately dorsalized embryos. By combining constitutively active versions of Gastrulation Defective, Snake, Easter, and Spätzle with loss-of-function alleles of seele, we find that Seele activity is dispensable for Spätzle-mediated activation of Toll but is required for Easter, Snake, and Gastrulation Defective to exert their effects on dorsal-ventral patterning. Moreover, Seele function is required specifically for secretion of Easter from the developing embryo into the perivitelline space and for Easter processing. Seele protein resides in the endoplasmic reticulum of blastoderm embryos, suggesting a role in the trafficking of Easter to the perivitelline space, prerequisite to its processing and function. Easter transport to the perivitelline space represents a previously unappreciated control point in the signal transduction pathway that controls Drosophila embryonic dorsal-ventral polarity. Copyright © 2010 Elsevier Ltd. All rights reserved.

Immobilization of bacterial proteases on water-solved polymer by means of electron beam

Science.gov (United States)

Gonchar, A. M.; Auslender, V. L.

1996-12-01

Possibility of electron beam usage for proteases' immobilization on 1,4-polyalkylene oxide (1,4-PAO) was studied to obtain biologically active complex for multi-purpose usage. It is shown that immobilization of Bacillus Subtilis protease takes place due to free-radical linking of enzyme and carrier with formation of mycellium-like structures. Immobilization improves heat resistance of enzyme up to 60°C without substrate and up to 80°C in presence of substrate, widens range of pH activity in comparison with non-immobilized forms. Immobilized proteases do not contain peroxides or long-live radicals. Our results permitted to create technologies for production of medical and veterinary preparations, active components for wool washing agents and leather fabrication technology.

Immobilization of bacterial proteases on water-solved polymer by means of electron beam

International Nuclear Information System (INIS)

Gonchar, A.M.; Auslender, V.L.

1996-01-01

Possibility of electron beam usage for proteases' immobilization on 1,4-polyalkylene oxide (1,4-PAO) was studied to obtain biologically active complex for multi-purpose usage. It is shown that immobilization of Bacillus Subtilis protease takes place due to free-radical linking of enzyme and carrier with formation of mycellium-like structures. Immobilization improves heat resistance of enzyme up to 60 o C without substrate and up to 80 o C in presence of substrate, widens range of pH activity in comparison with non-immobilized forms. Immobilized proteases do not contain peroxides or long-live radicals. Our results permitted to create technologies for production of medical and veterinary preparations, active components for wool washing agents and leather fabrication technology. (Author)

Chimeric exchange of coronavirus nsp5 proteases (3CLpro) identifies common and divergent regulatory determinants of protease activity.

Science.gov (United States)

Stobart, Christopher C; Sexton, Nicole R; Munjal, Havisha; Lu, Xiaotao; Molland, Katrina L; Tomar, Sakshi; Mesecar, Andrew D; Denison, Mark R

2013-12-01

Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp's 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp's at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra- and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup, genogroup 2a, with MHV, allowed for immediate viral recovery with efficient replication albeit with impaired fitness in direct competition with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types.

Molecular characterization of protease activity in Serratia sp. strain SCBI and its importance in cytotoxicity and virulence.

Science.gov (United States)

Petersen, Lauren M; Tisa, Louis S

2014-11-01

A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Antibacterial Activity of Extracellular Protease Isolated From an Algicolous Fungus Xylaria psidii KT30 Against Gram-Positive Bacteria

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Taufik Indarmawan

2016-04-01

Full Text Available Infectious diseases became more serious problem for public health in recent years. Although existing antibacterial drugs have been relatively effective, they do not rule out the emergence of resistance to the drug. Therefore, the intensive exploration of new bioactive compounds from natural, especially peptide compounds began in recent decades in order-handling infection. This study aimed to isolate, purify and test the potential application of Xylaria psidii KT30 extracellular protease as antibacterial agent against Gram-positive bacteria. X. psidii KT30, a marine fungus isolated from red seaweed Kappaphycus alvarezii showed antibacterial activity against Bacillus subtilis and Staphylococcus aureus. Antibacterial compounds of this fungus were predicted as a group of proteases. Extracellular protease exhibited an optimum activity when potato dextrose broth was used as cultivation medium. Furthermore, the highest activity of these proteases was found on fungal extract after day 15 of cultivation with value of 2.33 ± 0.19 U/mL. The partial purification of proteases using G-75 column chromatography resulted in 2 groups of fractions and showed protease activity based on zymogram assay. The extracellular proteases obtained from those fractions have 3 patterns of molecular mass based on sodium dodecyl sulfate–polyacrylamide gel electrophoresis which are 56.62, 89.12, 162.18 kDa.

Changes in protein metabolism after irradiation. Pt. 2. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat liver after 600 R whole body X-irradiation

Energy Technology Data Exchange (ETDEWEB)

Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

1976-01-01

The protease activity of cytoplasm and cell organelles of the rat liver against liver protein and hemoglobin as a substrate increases during an initial reaction phase on the first day after 600 R whole body x irradiation. This is probably a consequence of the degradation of cellular debris. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the liver is therefore not explained by an increased protease activity. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

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

Science.gov (United States)

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

2006-03-01

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

CHANGES IN LEVELS OF ACTIVITY OF SERINE PROTEASES ACCOMPANY THE EXPOSURE OF COMMON BEAN (PHASEOLUS VULGARIS L. TO WATER DEFICIT

Directory of Open Access Journals (Sweden)

M. Budič

2008-09-01

Full Text Available A wide variety of proteolytic enzymes exist in plants. On their levels depends protein turnover, a fundamental component in plant development and adaptation to environmental conditions. Cysteine proteases have frequently been reported to be influenced by drought, but only a few serine proteases (SP, among them the trypsin-like enzyme and two aminopeptidases from bean leaves (Bartels and Sunkar, 2005; Hieng et al., 2004. Our starting point was to identify proteolytic activities assigned to SPs that change with drought and then to characterize the corresponding proteases. A quantitative, analytical one-step method was used to separate endopeptidases and aminopeptidases active against a range of substrates in leaf extracts of plants grown in the field (FC. The influence of drought was determined for those of these activities which were confirmed as SPs, based on their inhibition by specific inhibitors. Under water deficit in plants grown under controlled conditions (CC their levels changed in different ways. The levels of SP activities in FC plants, observed during a period of relative drought, were similar to those measured in mildly stressed CC plants. The partial characterisations of some of these SPs will be presented. Our results point to a number of roles for different SPs in the plant response to water stress, which could range from enhanced protein turnover to limited proteolysis at specific sites.

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)

Inhibitory action of essential oils against proteases activity of Paenibacillus larvae, the etiological agent of American Foulbrood disease

Energy Technology Data Exchange (ETDEWEB)

Pellegrini, M.C.; Zalazar, L.; Fuselli, S.L.; Ponce, A.G.

2017-07-01

American foulbrood (AFB) is a disease affecting the larva of Apis mellifera. The etiological agent is Paenibacillus larvae, which releases metalloproteases involved in the degradation of larval tissues. Through quorum sensing (QS) mechanism, bacteria are able to activate specific genes such as virulence factors. The exoproteases regulation of P. larvae could be associated with QS. A promising mechanism of AFB control is to block QS mechanism with essential oils (EO). The aim of this study was to investigate the potential presence of QS signals in the regulation of P. larvae proteases and the effect of seven EOs on the exoproteases activity of P. larvae. From growth curves and evaluation of the presence of proteases by milk agar plates assay, it was observed protease activity during the late exponential phase of growth. Early production of protease activity (15 hours earlier than control) was observed when a low density culture was incubated with late exponential spent medium (SM) suggesting the presence of factor(s) inducing this activity. SM was obtained by the ultrafiltration of P. larvae cultures on late growth phase and was free of proteases. Proteolytic activity was quantified on P. larvae cultures in presence of sublethal concentration of EO by azocasein method. The EOs, except S. chilensis EO, reduced significantly protease activity (more than 50%). We report for the first time evidence on the possible role of QS on P. larvae and the antiproteolytic activity of EOs (except for S. chilensis) on exoproteases, an interesting therapeutic strategy to control AFB.

Inhibitory action of essential oils against proteases activity of Paenibacillus larvae, the etiological agent of American Foulbrood disease

International Nuclear Information System (INIS)

Pellegrini, M.C.; Zalazar, L.; Fuselli, S.L.; Ponce, A.G.

2017-01-01

American foulbrood (AFB) is a disease affecting the larva of Apis mellifera. The etiological agent is Paenibacillus larvae, which releases metalloproteases involved in the degradation of larval tissues. Through quorum sensing (QS) mechanism, bacteria are able to activate specific genes such as virulence factors. The exoproteases regulation of P. larvae could be associated with QS. A promising mechanism of AFB control is to block QS mechanism with essential oils (EO). The aim of this study was to investigate the potential presence of QS signals in the regulation of P. larvae proteases and the effect of seven EOs on the exoproteases activity of P. larvae. From growth curves and evaluation of the presence of proteases by milk agar plates assay, it was observed protease activity during the late exponential phase of growth. Early production of protease activity (15 hours earlier than control) was observed when a low density culture was incubated with late exponential spent medium (SM) suggesting the presence of factor(s) inducing this activity. SM was obtained by the ultrafiltration of P. larvae cultures on late growth phase and was free of proteases. Proteolytic activity was quantified on P. larvae cultures in presence of sublethal concentration of EO by azocasein method. The EOs, except S. chilensis EO, reduced significantly protease activity (more than 50%). We report for the first time evidence on the possible role of QS on P. larvae and the antiproteolytic activity of EOs (except for S. chilensis) on exoproteases, an interesting therapeutic strategy to control AFB.

Inhibitory action of essential oils against proteases activity of Paenibacillus larvae, the etiological agent of American Foulbrood disease

Directory of Open Access Journals (Sweden)

María C. Pellegrini

2018-02-01

Full Text Available American foulbrood (AFB is a disease affecting the larva of Apis mellifera. The etiological agent is Paenibacillus larvae, which releases metalloproteases involved in the degradation of larval tissues. Through quorum sensing (QS mechanism, bacteria are able to activate specific genes such as virulence factors. The exoproteases regulation of P. larvae could be associated with QS. A promising mechanism of AFB control is to block QS mechanism with essential oils (EO. The aim of this study was to investigate the potential presence of QS signals in the regulation of P. larvae proteases and the effect of seven EOs on the exoproteases activity of P. larvae. From growth curves and evaluation of the presence of proteases by milk agar plates assay, it was observed protease activity during the late exponential phase of growth. Early production of protease activity (15 hours earlier than control was observed when a low density culture was incubated with late exponential spent medium (SM suggesting the presence of factor(s inducing this activity. SM was obtained by the ultrafiltration of P. larvae cultures on late growth phase and was free of proteases. Proteolytic activity was quantified on P. larvae cultures in presence of sublethal concentration of EO by azocasein method. The EOs, except S. chilensis EO, reduced significantly protease activity (more than 50%. We report for the first time evidence on the possible role of QS on P. larvae and the antiproteolytic activity of EOs (except for S. chilensis on exoproteases, an interesting therapeutic strategy to control AFB.

Allicin and derivates are cysteine protease inhibitors with antiparasitic activity.

Science.gov (United States)

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.

tolerant alkaline protease from Bacillus coagulans PSB

African Journals Online (AJOL)

oyaide

2013-05-22

May 22, 2013 ... suggest the suitability of the enzyme for applications in peptide synthesis, detergent formulation and ... The cell free supernatant was recovered as crude enzyme preparation and used for further studies. Assay of protease activity. Protease activity was ... Effect of pH on growth and protease production.

Esterase and protease activities of Bacillus spp. from afitin, iru and ...

African Journals Online (AJOL)

The electrophoretic profiles of fermented African locust bean protein (ALBP), using strains presenting the highest protease activities in casein agar, were analyzed by SDS-PAGE to select strains with good ability to be used as starter cultures. All the Bacillus spp. tested showed esterase activity against tributyrin with high ...

Antiretroviral activity of protease inhibitors against Toxoplasma gondii

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Lianet Monzote

2013-02-01

Full Text Available The introduction of highly active antiretroviral therapy (HAART has caused a marked reduction in the occurrence and severity of parasitic infections, including the toxoplasmic encephalitis (TE. These changes have been attributed to the restoration of cell-mediated immunity. This study was developed to examine the activity of six antiretroviral protease inhibitors (API on Toxoplasma gondii tachyzoites. The six API showed anti-Toxoplasma activity, with IC50 value between 1.4 and 6.6 µg/mL. Further studies at the molecular level should be performed to clarify if the use of API could be beneficial or not for AIDS patients with TE.

Mechanism for Controlling the Dimer-Monomer Switch and Coupling Dimerization to Catalysis of the Severe Acute Respiratory Syndrome Coronavirus 3C-Like Protease

Energy Technology Data Exchange (ETDEWEB)

Shi,J.; Sivaraman, J.; Song, J.

2008-01-01

Unlike 3C protease, the severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CLpro) is only enzymatically active as a homodimer and its catalysis is under extensive regulation by the unique extra domain. Despite intense studies, two puzzles still remain: (i) how the dimer-monomer switch is controlled and (ii) why dimerization is absolutely required for catalysis. Here we report the monomeric crystal structure of the SARS-CoV 3CLpro mutant R298A at a resolution of 1.75 Angstroms . Detailed analysis reveals that Arg298 serves as a key component for maintaining dimerization, and consequently, its mutation will trigger a cooperative switch from a dimer to a monomer. The monomeric enzyme is irreversibly inactivated because its catalytic machinery is frozen in the collapsed state, characteristic of the formation of a short 310-helix from an active-site loop. Remarkably, dimerization appears to be coupled to catalysis in 3CLpro through the use of overlapped residues for two networks, one for dimerization and another for the catalysis.

Serum protease activity in chronic kidney disease patients: The GANI_MED renal cohort.

Science.gov (United States)

Wolke, Carmen; Teumer, Alexander; Endlich, Karlhans; Endlich, Nicole; Rettig, Rainer; Stracke, Sylvia; Fiene, Beate; Aymanns, Simone; Felix, Stephan B; Hannemann, Anke; Lendeckel, Uwe

2017-03-01

Serum or plasma proteases have been associated with various diseases including cancer, inflammation, or reno-cardiovascular diseases. We aimed to investigate whether the enzymatic activities of serum proteases are associated with the estimated glomerular filtration rate (eGFR) in patients with different stages of chronic kidney disease (CKD). Our study population comprised 268 participants of the "Greifswald Approach to Individualized Medicine" (GANI_MED) cohort. Enzymatic activity of aminopeptidase A, aminopeptidase B, alanyl (membrane) aminopeptidase, insulin-regulated aminopeptidase, puromycin-sensitive aminopeptidase, leucine aminopeptidase 3, prolyl-endopeptidase (PEP), dipeptidyl peptidase 4 (DPP4), angiotensin I-converting enzyme, and angiotensin I-converting enzyme 2 (ACE2) proteases was measured in serum. Linear regression of the respective protease was performed on kidney function adjusted for age and sex. Kidney function was modeled either by the continuous Modification of Diet in Renal Disease (MDRD)-based eGFR or dichotomized by eGFR < 15 mL/min/1.73 m 2 or <45 mL/min/1.73 m 2 , respectively. Results with a false discovery rate below 0.05 were deemed statistically significant. Among the 10 proteases investigated, only the activities of ACE2 and DPP4 were correlated with eGFR. Patients with lowest eGFR exhibited highest DPP4 and ACE2 activities. DPP4 and PEP were correlated with age, but all other serum protease activities showed no associations with age or sex. Our data indicate that ACE2 and DPP4 enzymatic activity are associated with the eGFR in patients with CKD. This finding distinguishes ACE2 and DPP4 from other serum peptidases analyzed and clearly indicates that further analyses are warranted to identify the precise role of these serum ectopeptidases in the pathogenesis of CKD and to fully elucidate underlying molecular mechanisms. Impact statement • Renal and cardiac diseases are very common and often occur concomitantly

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

Science.gov (United States)

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

2017-10-01

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

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.

Structural and functional characterization of cleavage and inactivation of human serine protease inhibitors by the bacterial SPATE protease EspPα from enterohemorrhagic E. coli.

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André Weiss

Full Text Available EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI, α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition.

Rheumatic Disease: Protease-Activated Receptor-2 in Synovial Joint Pathobiology

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Kendal McCulloch

2018-05-01

Full Text Available Protease-activated receptor-2 (PAR2 is one member of a small family of transmembrane, G-protein-coupled receptors. These receptors are activated via cleavage of their N terminus by serine proteases (e.g., tryptase, unveiling an N terminus tethered ligand which binds to the second extracellular loop of the receptor. Increasing evidence has emerged identifying key pathophysiological roles for PAR2 in both rheumatoid arthritis (RA and osteoarthritis (OA. Importantly, this includes both pro-inflammatory and destructive roles. For example, in murine models of RA, the associated synovitis, cartilage degradation, and subsequent bone erosion are all significantly reduced in the absence of PAR2. Similarly, in experimental models of OA, PAR2 disruption confers protection against cartilage degradation, subchondral bone osteosclerosis, and osteophyte formation. This review focuses on the role of PAR2 in rheumatic disease and its potential as an important therapeutic target for treating pain and joint degradation.

Salmon trypsin stimulates the expression of interleukin-8 via protease-activated receptor-2

International Nuclear Information System (INIS)

Larsen, Anett K.; Seternes, Ole-Morten; Larsen, Merethe; Aasmoe, Lisbeth; Bang, Berit

2008-01-01

In this study, we focus on salmon trypsin as an activator of inflammatory responses in airway cells in vitro. The rationale behind the investigation is that salmon industry workers are exposed to aerosols containing enzymes, which are generated during industrial processing of the fish. Knowing that serine proteases such as trypsin are highly active mediators with diverse biological activities, the stimulation of nuclear factor-kappa B (NF-κB) and interleukin (IL)-8 and the role of protease-activated receptors (PAR) in inflammatory signal mediation were investigated. Protease-activated receptors are considered important under pathological situations in the human airways, and a thorough understanding of PAR-induced cellular events and their consequences in airway inflammation is necessary. Human airway epithelial cells (A549) were exposed to trypsin isolated from fish (Salmo salar), and we observed that purified salmon trypsin could generate secretion of IL-8 in a concentration-dependent manner. Furthermore, we demonstrate that PAR-2 activation by salmon trypsin is coupled to an induction of NF-κB-mediated transcription using a PAR-2 transfected HeLa cell model. Finally, we show that the release of IL-8 from A549 following stimulation with purified salmon trypsin is mediated through activation of PAR-2 using specific small interfering RNAs (siRNAs). The results presented suggest that salmon trypsin, via activation of PAR-2, might influence inflammation processes in the airways if inhaled in sufficient amounts

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.

Characterization of a membrane-associated serine protease in Escherichia coli

International Nuclear Information System (INIS)

Palmer, S.M.; St John, A.C.

1987-01-01

Three membrane-associated proteolytic activities in Escherichia coli were resolved by DEAE-cellulose chromatography from detergent extracts of the total envelope fraction. On the basis of substrate specificity for the hydrolysis of chromogenic amino acid ester substrates, the first two eluting activities were determined previously to be protease V and protease IV, respectively. The third proteolytic activity eluting from the DEAE-cellulose column was further purified by affinity chromatography on benzamidine-Sepharose 6B. They termed this enzyme protease VI. Protease VI did not hydrolyze any of the chromogenic substrates used in the detection of protease IV and protease V. However, all three enzymes generated acid-soluble fragments from a mixture of E. coli membrane proteins which were biosynthetically labeled with radioactive amino acids. The activity of protease VI was sensitive to serine protease inhibitors. Using [ 3 H]diisopropylfluorophosphate as an active-site labeling reagent, they determined that protease VI has an apparent molecular weight of 43,000 in polyacrylamide gels. All three membrane-associated serine proteases were insensitive to inhibition by Ecotin, an endogenous, periplasmic inhibitor of trypsin

Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation

DEFF Research Database (Denmark)

Kousted, Tina Mostrup; Skjoedt, K; Petersen, S V

2013-01-01

abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between...

Mechanism of Folding and Activation of Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P)*

Science.gov (United States)

da Palma, Joel Ramos; Cendron, Laura; Seidah, Nabil Georges; Pasquato, Antonella; Kunz, Stefan

2016-01-01

The proprotein convertase subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in lipid homeostasis, the unfolded protein response, and lysosome biogenesis. The protease is further hijacked by highly pathogenic emerging viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P requires removal of an N-terminal prodomain, by a multistep process, generating the mature enzyme. Here, we uncover a modular structure of the human SKI-1/S1P prodomain and define its function in folding and activation. We provide evidence that the N-terminal AB fragment of the prodomain represents an autonomous structural and functional unit that is necessary and sufficient for folding and partial activation. In contrast, the C-terminal BC fragment lacks a defined structure but is crucial for autoprocessing and full catalytic activity. Phylogenetic analysis revealed that the sequence of the AB domain is highly conserved, whereas the BC fragment shows considerable variation and seems even absent in some species. Notably, SKI-1/S1P of arthropods, like the fruit fly Drosophila melanogaster, contains a shorter prodomain comprised of full-length AB and truncated BC regions. Swapping the prodomain fragments between fly and human resulted in a fully mature and active SKI-1/S1P chimera. Our study suggests that primordial SKI-1/S1P likely contained a simpler prodomain consisting of the highly conserved AB fragment that represents an independent folding unit. The BC region appears as a later evolutionary acquisition, possibly allowing more subtle fine-tuning of the maturation process. PMID:26645686

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.

Curcumin derivatives as HIV-1 protease inhibitors

Energy Technology Data Exchange (ETDEWEB)

Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R. [Univ. of California, San Francisco, CA (United States)

1993-12-31

Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

A Novel Aspartic Protease with HIV-1 Reverse Transcriptase Inhibitory Activity from Fresh Fruiting Bodies of the Wild Mushroom Xylaria hypoxylon

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Qing-Xiu Hu

2012-01-01

Full Text Available A novel aspartic protease with HIV-1 RT inhibitory activity was isolated and characterized from fruiting bodies of the wild mushroom Xylaria hypoxylon. The purification protocol comprised distilled water homogenization and extraction step, three ion exchange chromatographic steps (on DEAE-cellulose, Q-Sepharose, and CM-cellulose in succession, and final purification was by FPLC on Superdex 75. The protease was adsorbed on all the three ion exchangers. It was a monomeric protein with a molecular mass of 43 kDa as estimated by SDS-PAGE and FPLC. Its N-terminal amino acid sequence was HYTELLSQVV, which exhibited no sequence homology to other proteases reported. The activity of the protease was adversely affected by Pepstatin A, indicating that it is an aspartic protease. The protease activity was maximal or nearly so in the pH range 6–8 and in the temperature range 35–60°C. The purified enzyme exhibited HIV-1 RT inhibitory activity with an IC50 value of 8.3 μM, but was devoid of antifungal, ribonuclease, and hemagglutinating activities.

Engineered toxins "zymoxins" are activated by the HCV NS3 protease by removal of an inhibitory protein domain.

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Assaf Shapira

Full Text Available The synthesis of inactive enzyme precursors, also known as "zymogens," serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV as a model, we designed two HCV NS3 protease-activated "zymogenized" chimeric toxins (which we denote "zymoxins". In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA and Ricin A chain (RTA, respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the "zymoxin" approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.

Cytomegalovirus protease targeted prodrug development.

Science.gov (United States)

Sabit, Hairat; Dahan, Arik; Sun, Jing; Provoda, Chester J; Lee, Kyung-Dall; Hilfinger, John H; Amidon, Gordon L

2013-04-01

Human cytomegalovirus (HCMV) is a prevalent virus that infects up to 90% of the population. The goal of this research is to determine if small molecular prodrug substrates can be developed for a specific HCMV encoded protease and thus achieve site-specific activation. HCMV encodes a 256 amino acid serine protease that is responsible for capsid assembly, an essential process for herpes virus production. The esterase activity of the more stable HCMV A143T/A144T protease mutant was evaluated with model p-nitrophenol (ONp) esters, Boc-Xaa-ONp (Ala, Leu, Ile, Val, Gln, Phe at the Xaa position). We demonstrate that the A143T/A144T mutant has esterase activity toward specific small ester compounds, e.g., Boc-L-Ala-ONp. Mono amino acid and dipeptide prodrugs of ganciclovir (GCV) were also synthesized and evaluated for hydrolysis by the A143T/A144T protease mutant in solution. Hydrolysis of these prodrugs was also evaluated in Caco-2 cell homogenates, human liver microsomes (HLMs), and rat and human plasma. For the selectivity potential of the prodrugs, the hydrolysis ratio was evaluated as a percentage of prodrug hydrolyzed by the HCMV protease over the percentages of prodrug hydrolyses by Caco-2 cell homogenates, HLMs, and human/rat plasma. A dipeptide prodrug of ganciclovir, Ac-l-Gln-l-Ala-GCV, emerged as a potential selective prodrug candidate. The results of this research demonstrate that targeting prodrugs for activation by a specific protease encoded by the infectious HCMV pathogen may be achievable.

Cloning, characterization, expression and antifungal activity of an alkaline serine protease of Aureobasidium pullulans PL5 involved in the biological control of postharvest pathogens.

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Zhang, Dianpeng; Spadaro, Davide; Valente, Silvia; Garibaldi, Angelo; Gullino, Maria Lodovica

2012-02-15

An alkaline protease gene was amplified from genomic DNA and cDNA of the antagonistic yeast-like fungus Aureobasidium pullulans PL5, a biocontrol agent effective against Monilinia laxa on stone fruit and Botrytis cinerea and Penicillium expansum on pome fruits. An open reading frame of 1248 bp encoding a 415-amino acid (aa) protein with a calculated molecular weight (M(r)) of 42.9 kDa and an isoelectric point (pI) of 4.5 was characterized. The cDNAALP5 gene had an 18-amino acid signal peptide, one N-gylcosylation, one histidine active site, and one serine active site. The ALP5 gene with a M(r) of 1351 bp contained two introns. One intron was of 54 bp, while the other was of 50 bp. Protein BLAST and phylogenetic tree analysis of the deduced amino sequences from the cDNAALP5 gene showed that the encoded protein had 100% homology to a protease enzyme (ALP2) of a sea strain of A. pullulans, suggesting that the protein ALP5 was an alkaline serine protease. Expression of ALP5 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolysed the substrate casein and inhibited the mycelial growth of the pathogens. At its optimal pH of 10.0 and reaction temperature of 50°C, the recombinant protease exhibited the highest activity towards the substrate casein, though the highest stability was at lower temperatures and pH between 7.0 and 9.0. This study provided the direct evidence that extracellular proteases secreted by the antagonist A. pullulans PL5 played a role in the biocontrol activities against some postharvest pathogens of apple and peach. Copyright © 2011 Elsevier B.V. All rights reserved.

Structural changes in halophilic and non-halophilic proteases in response to chaotropic reagents.

Science.gov (United States)

Sinha, Rajeshwari; Khare, S K

2014-08-01

Halophilic enzymes have been established for their stability and catalytic abilities under harsh operational conditions. These have been documented to withstand denaturation at high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. The present study targets an important aspect in understanding protein-urea/GdmCl interactions using proteases from halophilic Bacillus sp. EMB9 and non-halophilic subtilisin (Carlsberg) from Bacillus licheniformis as model systems. While, halophilic protease containing 1 % (w/v) NaCl (0.17 M) retained full activity towards urea (8 M), non-halophilic protease lost about 90 % activity under similar conditions. The secondary and tertiary structure were lost in non-halophilic but preserved for halophilic protein. This effect could be due to the possible charge screening and shielding of the protein surface by Ca(2+) and Na(+) ions rendering it stable against denaturation. The dialyzed halophilic protease almost behaved like the non-halophilic counterpart. Incorporation of NaCl (up to 5 %, w/v or 0.85 M) in dialyzed EMB9 protease containing urea/GdmCl, not only helped regain of proteolytic activity but also evaded denaturing action. Deciphering the basis of this salt modulated stability amidst a denaturing milieu will provide guidelines and templates for engineering stable proteins/enzymes for biotechnological applications.

Advances in protease engineering for laundry detergents.

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Vojcic, Ljubica; Pitzler, Christian; Körfer, Georgette; Jakob, Felix; Ronny Martinez; Maurer, Karl-Heinz; Schwaneberg, Ulrich

2015-12-25

Proteases are essential ingredients in modern laundry detergents. Over the past 30 years, subtilisin proteases employed in the laundry detergent industry have been engineered by directed evolution and rational design to tailor their properties towards industrial demands. This comprehensive review discusses recent success stories in subtilisin protease engineering. Advances in protease engineering for laundry detergents comprise simultaneous improvement of thermal resistance and activity at low temperatures, a rational strategy to modulate pH profiles, and a general hypothesis for how to increase promiscuous activity towards the production of peroxycarboxylic acids as mild bleaching agents. The three protease engineering campaigns presented provide in-depth analysis of protease properties and have identified principles that can be applied to improve or generate enzyme variants for industrial applications beyond laundry detergents. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Directory of Open Access Journals (Sweden)

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.

Mechanism of Folding and Activation of Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P).

Science.gov (United States)

da Palma, Joel Ramos; Cendron, Laura; Seidah, Nabil Georges; Pasquato, Antonella; Kunz, Stefan

2016-01-29

The proprotein convertase subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in lipid homeostasis, the unfolded protein response, and lysosome biogenesis. The protease is further hijacked by highly pathogenic emerging viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P requires removal of an N-terminal prodomain, by a multistep process, generating the mature enzyme. Here, we uncover a modular structure of the human SKI-1/S1P prodomain and define its function in folding and activation. We provide evidence that the N-terminal AB fragment of the prodomain represents an autonomous structural and functional unit that is necessary and sufficient for folding and partial activation. In contrast, the C-terminal BC fragment lacks a defined structure but is crucial for autoprocessing and full catalytic activity. Phylogenetic analysis revealed that the sequence of the AB domain is highly conserved, whereas the BC fragment shows considerable variation and seems even absent in some species. Notably, SKI-1/S1P of arthropods, like the fruit fly Drosophila melanogaster, contains a shorter prodomain comprised of full-length AB and truncated BC regions. Swapping the prodomain fragments between fly and human resulted in a fully mature and active SKI-1/S1P chimera. Our study suggests that primordial SKI-1/S1P likely contained a simpler prodomain consisting of the highly conserved AB fragment that represents an independent folding unit. The BC region appears as a later evolutionary acquisition, possibly allowing more subtle fine-tuning of the maturation process. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Developpement of a photoaffinity probe for the sensitive detection of matrix metallo-protease active forms from complex biological systems

International Nuclear Information System (INIS)

Nury, Catherine

2012-01-01

A new activity-based probe able to covalently modify the active site of proteases belonging to the matrix metallo-protease family (MMPs) has been developed in this thesis project. The probe was shown to behave as potent inhibitor of several MMPs, with nanomolar Ki values. This probe was also able to modify specifically only the free active site of MMPs, with particular high yields of cross-linking varying from 50 % to 11 %, depending of the MMPs tested. Using radioactivity as means of detection, this probe was able to detect active form of MMPs with a threshold of 1 femto-mole. Applied to the study of bronchoalveolar fluids (BAL) from mice exposed to nanoparticles by a lung aspiration protocol, this probe revealed the presence of the catalytic domain of MMP-12 under its active form, but not in control animals. When used to detect active form of MMPs from extracts obtained from human arteries of patient suffering from atherosclerosis, the probe was not able to detect such MMP active forms. Despite this negative result, the detection of active form of MMP in pathological fluid like BAL has never been reported before this work. Having validated this novel MMP activity-based probe, it will be possible to use it now for detecting MMPs from other pathological fluids or tissues extracts in which MMPs can be good markers of the pathology. (author) [fr

Engineered Toxins “Zymoxins” Are Activated by the HCV NS3 Protease by Removal of an Inhibitory Protein Domain

Science.gov (United States)

Shapira, Assaf; Gal-Tanamy, Meital; Nahary, Limor; Litvak-Greenfeld, Dana; Zemel, Romy; Tur-Kaspa, Ran; Benhar, Itai

2011-01-01

The synthesis of inactive enzyme precursors, also known as “zymogens,” serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated “zymogenized” chimeric toxins (which we denote “zymoxins”). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the “zymoxin” approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected. PMID:21264238

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

Science.gov (United States)

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.

PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration

Directory of Open Access Journals (Sweden)

Alexander Jonathan S

2010-12-01

Full Text Available Abstract The normal function of poly (ADP-ribose polymerase-1 (PARP-1 is the routine repair of DNA damage by adding poly (ADP ribose polymers in response to a variety of cellular stresses. Recently, it has become widely appreciated that PARP-1 also participates in diverse physiological and pathological functions from cell survival to several forms of cell death and has been implicated in gene transcription, immune responses, inflammation, learning, memory, synaptic functions, angiogenesis and aging. In the CNS, PARP inhibition attenuates injury in pathologies like cerebral ischemia, trauma and excitotoxicity demonstrating a central role of PARP-1 in these pathologies. PARP-1 is also a preferred substrate for several 'suicidal' proteases and the proteolytic action of suicidal proteases (caspases, calpains, cathepsins, granzymes and matrix metalloproteinases (MMPs on PARP-1 produces several specific proteolytic cleavage fragments with different molecular weights. These PARP-1 signature fragments are recognized biomarkers for specific patterns of protease activity in unique cell death programs. This review focuses on specific suicidal proteases active towards PARP-1 to generate signature PARP-1 fragments that can identify key proteases and particular forms of cell death involved in pathophysiology. The roles played by some of the PARP-1 fragments and their associated binding partners in the control of different forms of cell death are also discussed.

Studies on activity, distribution, and zymogram of protease, α-amylase, and lipase in the paddlefish Polyodon spathula.

Science.gov (United States)

Ji, H; Sun, H T; Xiong, D M

2012-06-01

A series of biochemical determination and electrophoretic observations have been conducted to analyze the activities and characteristics of protease, α-amylase, and lipase of paddlefish Polyodon spathula. The results obtained have been compared with those of bighead carp (Aristichthys nobilis) and hybrid sturgeon (Huso dauricus ♀ × Acipenser schrenki Brandt ♂), in order to increase available knowledge of the physiological characteristics of this sturgeon species and to gain information with regard to its nutrition. Further, a comparative study of enzymatic activity, distribution, and characterization between commercial feed-reared paddlefish (CG) and natural live food-reared (NG) paddlefish was conducted. Results showed that higher proteolytic activity was observed in the pH range 2.5-3.0 and at a pH of 7.0 for paddlefish. Levels of acid protease activity of paddlefish were similar to that of hybrid sturgeon, and significantly higher than that of bighead carp. The inhibition assay of paddlefish showed that the rate of inhibition of tosyl-phenylalanine chloromethyl ketone was approximately 2.6-fold that of tosyl-lysine chloromethyl ketone. There was no significant difference observed for acid protease activity between PG and CG groups, whereas the activity of alkaline protease, α-amylase, and lipase in the PG group were significantly lower than those in the CG group. The substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis further showed that there were certain types of enzymes, especially α-amylase, with similar molecular mass in the paddlefish and hybrid sturgeon. It can be inferred that acid digestion was main mechanism for protein hydrolysis in paddlefish, as reported for other fishes with a stomach. This indicates that the paddlefish requires higher alkaline protease, α-amylase, and lipase activity to digest natural live food.

Molecular dynamics and docking simulation of a natural variant of Activated Protein C with impaired protease activity: implications for integrin-mediated antiseptic function.

Science.gov (United States)

D'Ursi, Pasqualina; Orro, Alessandro; Morra, Giulia; Moscatelli, Marco; Trombetti, Gabriele; Milanesi, Luciano; Rovida, Ermanna

2015-01-01

Activated Protein C (APC) is a multifunctional serine protease, primarily known for its anticoagulant function in the coagulation system. Several studies have already elucidated its role in counteracting apoptosis and inflammation in cells, while significant effort is still ongoing for defining its involvement in sepsis. Earlier literature has shown that the antiseptic function of APC is mediated by its binding to leukocyte integrins, which is due to the presence of the integrin binding motif Arg-Gly-Asp at the N-terminus of the APC catalytic chain. Many natural mutants have been identified in patients with Protein C deficiency diagnosis including a variant of specificity pocket (Gly216Asp). In this work, we present a molecular model of the complex of APC with αVβ3 integrin obtained by protein-protein docking approach. A computational analysis of this variant is hereby presented, based on molecular dynamics and docking simulations, aiming at investigating the effects of the Gly216Asp mutation on the protein conformation and inferring its functional implications. Our study shows that such mutation is likely to impair the protease activity while preserving the overall protein fold. Moreover, superposition of the integrin binding motifs in wild-type and mutant forms suggests that the interaction with integrin can still occur and thus the mutant is likely to retain its antiseptic function related to the neutrophyl integrin binding. Therapeutic applications could result in this APC mutant which retains antiseptic function without anticoagulant side effects.

Genome-wide identification and structure-function studies of proteases and protease inhibitors in Cicer arietinum (chickpea).

Science.gov (United States)

Sharma, Ranu; Suresh, C G

2015-01-01

Proteases are a family of enzymes present in almost all living organisms. In plants they are involved in many biological processes requiring stress response in situations such as water deficiency, pathogen attack, maintaining protein content of the cell, programmed cell death, senescence, reproduction and many more. Similarly, protease inhibitors (PIs) are involved in various important functions like suppression of invasion by pathogenic nematodes, inhibition of spores-germination and mycelium growth of Alternaria alternata and response to wounding and fungal attack. As much as we know, no genome-wide study of proteases together with proteinaceous PIs is reported in any of the sequenced genomes till now. Phylogenetic studies and domain analysis of proteases were carried out to understand the molecular evolution as well as gene and protein features. Structural analysis was carried out to explore the binding mode and affinity of PIs for cognate proteases and prolyl oligopeptidase protease with inhibitor ligand. In the study reported here, a significant number of proteases and PIs were identified in chickpea genome. The gene expression profiles of proteases and PIs in five different plant tissues revealed a differential expression pattern in more than one plant tissue. Molecular dynamics studies revealed the formation of stable complex owing to increased number of protein-ligand and inter and intramolecular protein-protein hydrogen bonds. The genome-wide identification, characterization, evolutionary understanding, gene expression, and structural analysis of proteases and PIs provide a framework for future analysis when defining their roles in stress response and developing a more stress tolerant variety of chickpea. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effects of Mucuna pruriens protease inhibitors on Echis carinatus venom.

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

2012-12-01

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

Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers

DEFF Research Database (Denmark)

Jakobsen, Rasmus Kragh; Ono, S.; Powers, J. C.

2005-01-01

processes that they mediate. Until only recently, the tools to conveniently address the question of where and when serine proteases are active within complex tissues have been lacking. In order to detect spatially restricted serine protease activities in Drosophila embryos and ovaries we introduce...... activity localized to the oocyte-somatic follicle cell interface of the developing egg chamber. Our results suggest that this technique holds promise to identify new spatially restricted activities in adult Drosophila tissues and developing embryos....

Deletion of protease-activated receptor 2 prolongs survival of scrapie-inoculated mice

Czech Academy of Sciences Publication Activity Database

Matěj, R.; Olejár, Tomáš; Janoušková, O.; Holada, K.

2012-01-01

Roč. 93, č. 9 (2012), s. 2057-2061 ISSN 0022-1317 Institutional support: RVO:67985823 Keywords : protease-activated receptor (PAR2) * scrapie * neurodegenerative disorders Subject RIV: FN - Epidemiology, Contagious Diseases ; Clinical Immunology Impact factor: 3.127, year: 2012

Role of the durum wheat dehydrin in the function of proteases conferring salinity tolerance in Arabidopsis thaliana transgenic lines.

Science.gov (United States)

Saibi, Walid; Zouari, Nabil; Masmoudi, Khaled; Brini, Faiçal

2016-04-01

Dehydrins are claimed to stabilize macromolecules against freezing damage, dehydration, ionic or osmotic stresses, thermal stress and re-folding yield. However, their precise function remains unknown. In this context, we report the behavior of protease activities in dehydrin transgenic Arabidopsis lines against the wild type plant under salt stress (100mM NaCl). Indeed, proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. We proved that durum wheat DHN-5 modulates the activity of some proteases, summarized on the promotion of the Cysteinyl protease and the decrease of the Aspartyl protease activity. This fact is also upgraded in salt stress conditions. We conclude that the dehydrin transgenic context encodes salinity tolerance in transgenic lines through the modulation of the interaction not only at transcriptional level but also at protein level and also with the impact of salt stress as an endogenous and exogenous effector on some biocatalysts like proteases. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of detergent compatible protease of a halophilic Bacillus sp. EMB9: differential role of metal ions in stability and activity.

Science.gov (United States)

Sinha, Rajeshwari; Khare, S K

2013-10-01

A moderately halophilic protease producer, Bacillus sp. strain isolated from sea water is described. The protease is purified to homogeneity by ammonium sulphate precipitation and CM cellulose chromatography. The serine protease has a molecular mass of 29 kDa. Enzymatic characterization of protease revealed K(m) 2.22 mg mL(-1), Vmax 1111.11 U mL(-1), pH optimum 9.0, t1/2 190 min at 60°C and salt optima 1% (w/v) NaCl. The protease is remarkably stable in hydrophilic and hydrophobic solvents at high concentrations. The purified preparation is unstable at room temperature. Ca(2+) ions are required for preventing this loss of activity. Interestingly, the activity and stability are modulated differentially. Whereas, divalent cation Ca(2+) are involved in maintaining stability in solution at room temperature by preventing unfolding, monovalent Na(+) and K(+) ions participate in regulating the activity and assist in refolding of the enzyme. Application of the protease is shown in efficient removal of blood stain. Copyright © 2012 Elsevier Ltd. All rights reserved.

Molecular characterization of 45 kDa aspartic protease of Trichinella spiralis.

Science.gov (United States)

Park, Jong Nam; Park, Sang Kyun; Cho, Min Kyoung; Park, Mi-Kyung; Kang, Shin Ae; Kim, Dong-Hee; Yu, Hak Sun

2012-12-21

In a previous study, we identified an aspartic protease gene (Ts-Asp) from the Trichinella spiralis muscle stage larva cDNA library. The gene sequence of Ts-Asp was 1281 bp long and was found to encode a protein consisting of 405 amino acids, with a molecular mass of 45.248 kD and a pI of 5.95. The deduced Ts-Asp has a conserved catalytic motif with catalytic aspartic acid residues in the active site, a common characteristic of aspartic proteases. In addition, the deduced amino acid sequence of Ts-Asp was found to possess significant homology (above 50%) with aspartic proteases from nematode parasites. Results of phylogenetic analysis indicated a close relationship of Ts-Asp with cathepsin D aspartic proteases. For production of recombinant Ts-Asp (rTs-Asp), the pGEX4T expression system was used. Like other proteases, the purified rTs-Asp was able to digest collagen matrix in vitro. Abundant expression of Ts-Asp was observed in muscle stage larva. Ts-Asp was detected in ES proteins, and was able to elicit the production of specific antibodies. It is the first report of molecular characterization of aspartic protease isolated from T. spiralis. Copyright © 2012 Elsevier B.V. All rights reserved.

Analysis of Milk from Mothers Who Delivered Prematurely Reveals Few Changes in Proteases and Protease Inhibitors across Gestational Age at Birth and Infant Postnatal Age.

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Demers-Mathieu, Veronique; Nielsen, Søren Drud; Underwood, Mark A; Borghese, Robyn; Dallas, David C

2017-06-01

Background: Peptidomics research has demonstrated that protease activity is higher in breast milk from preterm-delivering mothers than from term-delivering mothers. However, to our knowledge, the effect of the degree of prematurity and postnatal age on proteases and protease inhibitors in human milk remains unknown. Objective: We aimed to determine the change of proteases and protease inhibitors in milk from mothers who delivered prematurely across gestational age (GA) and postnatal age. Methods: Milk samples were collected from 18 mothers aged 26-40 y who delivered preterm infants and who lacked mastitis. For analysis, samples were separated into 2 groups: 9 from early GA (EGA) (24-26 wk GA)-delivering mothers and 9 from late GA (LGA) (27-32 wk GA)-delivering mothers. Within the 9 samples in each group, the collection time ranged from postnatal days 2 to 47. The activity and predicted activity of proteases in preterm milk were determined with the use of fluorometric and spectrophotometric assays and peptidomics, respectively. Protease and protease inhibitor concentrations were determined with the use of ELISA. Linear mixed models were applied to compare enzymes across GA and postnatal age. Results: Carboxypeptidase B2, kallikrein, plasmin, elastase, thrombin, and cytosol aminopeptidase were present and active in the milk of preterm-delivering mothers. Most milk protease and antiprotease concentrations did not change with GA or postnatal age. However, the concentration and activity of kallikrein, the most abundant and active protease in preterm milk, increased by 25.4 ng · mL -1 · d -1 and 0.454 μg · mL -1 · d -1 postnatally, respectively, in EGA milk samples while remaining stable in LGA milk samples. Conclusions: This research demonstrates that proteases are active in human milk and begin to degrade milk protein within the mammary gland before consumption by infants. Proteases and protease inhibitors in milk from mothers of premature infants mostly did not

Skeletal muscle protease activities in the early growth and development of wild Atlantic salmon (Salmo salar L.).

Science.gov (United States)

Lysenko, Liudmila A; Kantserova, Nadezda P; Kaivarainen, Elena I; Krupnova, Marina Yu; Nemova, Nina N

2017-09-01

Growth-related dynamics of intracellular protease activities in four year classes of the Atlantic salmon (Salmo salar L. 1758) parr and smolts inhabiting salmon rivers of northwestern Russia (the White Sea basin) were studied. Cathepsin B, cathepsin D, proteasome, and calpain activities in the skeletal muscles of salmon were assessed to investigate their relative contribution to the total protein degradation as well as to young fish growth process. It was confirmed that calpain activity dominates in salmon muscles while proteasome plays a minor role, in contrast to terrestrial vertebrates. Calpain and proteasome activities were maximal at the early post-larval stage (in parrs 0+) and declined with age (parrs 1+ through 2+) dropping to the lowest level in salmon smolts. Annual growth increments and proteolytic activities of calpains and proteasome in the muscles of salmon juveniles changed with age in an orchestrated manner, while lysosomal cathepsin activities increased with age. Comparing protease activities and growth increments in salmon parr and smolts we suggested that the partial suppression of the protein degradation could be a mechanism stimulating efficient growth in smoltifying salmon. Growth and smoltification-related dynamics of protease activities was quite similar in salmon populations from studied spawning rivers, such as Varzuga and Indera; however, some habitat-related differences were observed. Growth increments and protease activities varied in salmon parr 0+ (but not on later ages) inhabiting either main rivers or small tributaries apparently due to habitat difference on the resources for fish growth. Copyright © 2017 Elsevier Inc. All rights reserved.

Antimalarial activity of HIV-1 protease inhibitor in chromone series.

Science.gov (United States)

Lerdsirisuk, Pradith; Maicheen, Chirattikan; Ungwitayatorn, Jiraporn

2014-12-01

Increasing parasite resistance to nearly all available antimalarial drugs becomes a serious problem to human health and necessitates the need to continue the search for new effective drugs. Recent studies have shown that clinically utilized HIV-1 protease (HIV-1 PR) inhibitors can inhibit the in vitro and in vivo growth of Plasmodium falciparum. In this study, a series of chromone derivatives possessing HIV-1 PR inhibitory activity has been tested for antimalarial activity against P. falciparum (K1 multi-drug resistant strain). Chromone 15, the potent HIV-1 PR inhibitor (IC50=0.65μM), was found to be the most potent antimalarial compound with IC50=0.95μM while primaquine and tafenoquine showed IC50=2.41 and 1.95μM, respectively. Molecular docking study of chromone compounds against plasmepsin II, an aspartic protease enzyme important in hemoglobin degradation, revealed that chromone 15 exhibited the higher binding affinity (binding energy=-13.24kcal/mol) than the known PM II inhibitors. Thus, HIV-1 PR inhibitor in chromone series has the potential to be a new class of antimalarial agent. Copyright © 2014 Elsevier Inc. 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.

Insecticide resistance and intracellular proteases.

Science.gov (United States)

Wilkins, Richard M

2017-12-01

Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases

Science.gov (United States)

Vriend, Gert; Eijsink, Vincent

1993-08-01

Bacillus neutral proteases (NPs) form a group of well-characterized homologous enzymes, that exhibit large differences in thermostability. The three-dimensional (3D) structures of several of these enzymes have been modelled on the basis of the crystal structures of the NPs of B. thermoproteolyticus (thermolysin) and B. cercus. Several new techniques have been developed to improve the model-building procedures. Also a model-building by mutagenesis' strategy was used, in which mutants were designed just to shed light on parts of the structures that were particularly hard to model. The NP models have been used for the prediction of site-directed mutations aimed at improving the thermostability of the enzymes. Predictions were made using several novel computational techniques, such as position-specific rotamer searching, packing quality analysis and property-profile database searches. Many stabilizing mutations were predicted and produced: improvement of hydrogen bonding, exclusion of buried water molecules, capping helices, improvement of hydrophobic interactions and entropic stabilization have been applied successfully. At elevated temperatures NPs are irreversibly inactivated as a result of autolysis. It has been shown that this denaturation process is independent of the protease activity and concentration and that the inactivation follows first-order kinetics. From this it has been conjectured that local unfolding of (surface) loops, which renders the protein susceptible to autolysis, is the rate-limiting step. Despite the particular nature of the thermal denaturation process, normal rules for protein stability can be applied to NPs. However, rather than stabilizing the whole protein against global unfolding, only a small region has to be protected against local unfolding. In contrast to proteins in general, mutational effects in proteases are not additive and their magnitude is strongly dependent on the location of the mutation. Mutations that alter the stability

Trypsin- and Chymotrypsin-Like Serine Proteases in Schistosoma mansoni - 'The Undiscovered Country'

Czech Academy of Sciences Publication Activity Database

Horn, Martin; Fajtová, Pavla; Arreola, L. R.; Ulrychová, Lenka; Bartošová-Sojková, Pavla; Franta, Zdeněk; Protasio, A. V.; Opavský, David; Vondrášek, Jiří; McKerrow, J. H.; Mareš, Michael; Caffrey, C. R.; Dvořák, Jan

2014-01-01

Roč. 8, č. 3 (2014), e2766/1-e2766/13 ISSN 1935-2735 R&D Projects: GA ČR(CZ) GAP302/11/1481; GA MŠk(CZ) ME10011 EU Projects: European Commission(XE) 248642 - SCHISTOSOMA PROTEASE Institutional support: RVO:61388963 ; RVO:68378050 ; RVO:60077344 Keywords : schistosomiasis * blood fluke * serine protease Subject RIV: CE - Biochemistry; EB - Genetics ; Molecular Biology (UMG-J); FN - Epidemiology, Contagious Diseases ; Clinical Immunology (BC-A) Impact factor: 4.446, year: 2014 http://www.plosntds.org/article/info%3Adoi%2F10.1371%2Fjournal.pntd.0002766

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.

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.

An enzyme from the earthworm Eisenia fetida is not only a protease but also a deoxyribonuclease.

Science.gov (United States)

Pan, Rong; Zhou, Yuan; He, Hai-Jin; He, Rong-Qiao

2011-04-01

The earthworm enzyme Eisenia fetida Protease-III-1 (EfP-III-1) is known as a trypsin-like protease which is localized in the alimentary canal of the earthworm. Here, we show that EfP-III-1 also acts as a novel deoxyribonuclease. Unlike most DNases, this earthworm enzyme recognizes 5'-phosphate dsDNA (5'P DNA) and degrades it without sequence specificity, but does not recognize 5'OH DNA. As is the case for most DNases, Mg(2+) was observed to markedly enhance the DNase activity of EfP-III-1. Whether the earthworm enzyme functioned as a DNase or as a protease depended on the pH values of the enzyme solution. The protein acted as a protease under alkaline conditions whereas it exhibited DNase activity under acid conditions. At pH 7.0, the enzyme could work as either a DNase or a protease. Given the complex living environment of the earthworm, this dual function of EfP-III-1 may play an important role in the alimentary digestion of the earthworm. Copyright © 2011 Elsevier Inc. All rights reserved.

Cathepsin K in Lymphangioleiomyomatosis: LAM Cell-Fibroblast Interactions Enhance Protease Activity by Extracellular Acidification.

Science.gov (United States)

Dongre, Arundhati; Clements, Debbie; Fisher, Andrew J; Johnson, Simon R

2017-08-01

Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells and fibroblasts form lung nodules and it is hypothesized that LAM nodule-derived proteases cause cyst formation and tissue damage. On protease gene expression profiling in whole lung tissue, cathepsin K gene expression was 40-fold overexpressed in LAM compared with control lung tissue (P ≤ 0.0001). Immunohistochemistry confirmed cathepsin K protein was expressed in LAM but not control lungs. Cathepsin K gene expression and protein and protease activity were detected in LAM-associated fibroblasts but not the LAM cell line 621-101. In lung nodules, cathepsin K immunoreactivity predominantly co-localized with LAM-associated fibroblasts. In vitro, fibroblast extracellular cathepsin K activity was minimal at pH 7.5 but significantly enhanced at pH 7 and 6. 621-101 cells reduced extracellular pH with acidification dependent on 621-101 mechanistic target of rapamycin activity and net hydrogen ion exporters, particularly sodium bicarbonate co-transporters and carbonic anhydrases, which were also expressed in LAM lung tissue. In LAM cell-fibroblast co-cultures, acidification paralleled cathepsin K activity, and both were reduced by sodium bicarbonate co-transporter (P ≤ 0.0001) and carbonic anhydrase inhibitors (P = 0.0021). Our findings suggest that cathepsin K activity is dependent on LAM cell-fibroblast interactions, and inhibitors of extracellular acidification may be potential therapies for LAM. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Reverse zymography alone does not confirm presence of a protease inhibitor.

Science.gov (United States)

Dutta, Sangita; Bhattacharyya, Debasish

2013-03-01

Reverse zymography is applied for identification and semi-quantification of protease inhibitors that are of protein in nature. However, a protein that shows band in reverse zymography against a protease used for digestion of the gel need not be an inhibitor; it might be resistant to degradation by the protease. We demonstrate that in reverse zymography, avidin, streptavidin and the leaf extract of Catharanthus roseus behave like inhibitors of proteases like papain, ficin, bromelain extracts from pineapple leaf, stem and fruit and trypsin. Still, they do not act as inhibitors of those proteases when enzyme assays were done in solution. In reverse zymography, the extract of pineapple crown leaf shows two major inhibitor bands against its own proteases. Identification of these proteins from sequences derived from MALDI TOF MS analysis indicated that they are fruit and stem bromelains. Avidin, streptavidin and bromelains are 'kinetically stable proteins' that are usually resistant to proteolysis. Thus, it is recommended that identification of an inhibitor of a protease by reverse zymography should be supported by independent assay methods for confirmation.

Monocytes can be induced by lipopolysaccharide-triggered T lymphocytes to express functional factor VII/VIIa protease activity

OpenAIRE

1984-01-01

In the present study we demonstrate that human monocytes can be induced by the model stimulus, lipopolysaccharide (LPS), to produce and assemble on their surface functional Factor VII/VIIa. This protease was not induced in relatively purified monocytes alone following exposure to LPS; but was induced in the presence of Leu-3a positive helper/inducer T cells. The Factor VII/VIIa protease activity represented 35-40% of the potential initiating activity for the extrinsic coagulation pathway and ...

A transcription activator-like effector (TALE) induction system mediated by proteolysis.

Science.gov (United States)

Copeland, Matthew F; Politz, Mark C; Johnson, Charles B; Markley, Andrew L; Pfleger, Brian F

2016-04-01

Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications owing to their customizable DNA-binding specificity. In this work we expanded the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded after induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator-agnostic.

Factor VII-activating protease : Unraveling the release and regulation of dead cell nuclear damps

NARCIS (Netherlands)

Marsman, G.

2017-01-01

Upon inflammation, uncleared dying cells are an important source of pro-inflammatory damage-associated molecular patterns (DAMPs). Major DAMPs are histones and double-stranded DNA, which together form chromatin. Factor VII-activating protease (FSAP) is activated upon contact with dead cells, and its

Supermarket Proteases.

Science.gov (United States)

Hagar, William G.; Bullerwell, Lornie D.

2003-01-01

Presents a laboratory activity on enzymes. Uses common items found in the supermarket that contain protease enzymes, such as contact lens cleaner and meat tenderizer. Demonstrates the digestion of gelatin proteins as part of enzymatic reactions. (Author/SOE)

Multifunctional Mitochondrial AAA Proteases.

Science.gov (United States)

Glynn, Steven E

2017-01-01

Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle.

Effect of Seed Proteinaceous Inhibitors on Digestive Protease Activity of Tomato Leaf Miner Tuta absoluta (Meyrick (Lep.: Gelechiidae

Directory of Open Access Journals (Sweden)

M. University of Tehran

2017-01-01

Full Text Available Introduction: Tomato leaf miner Tuta absoluta Meyrick (Lep.: Gelechiidae is one of the most important pest of tomato around the world which have been introduced to Iran in recent years. Pesticides can usually reduce the damage caused by the pest, however the extensive use of pesticides substantially increase production costs and lead to development of insecticide resistance. Thus, pest resistance and also adverse effects of pesticides on the human health and the environment make search for alternative control method/s inevitable. The aim of this study was to examine the effect of proteinaceous extracts of Datura (Datura stramonium L., Bean (Phaseolus vulgaris L., Wild Oat (Avena fatua L., Broad bean (Vicia faba L., Amaranth (Amaranthus retroflexus L., Mung bean (Vigna radiate L., Pea (Pisum sativum L., Canola (Brassica napus L., Triticale (Triticosecale wittmack and Sivand, Aflak and Alborz wheat (Triticum aestivum L. cultivars seeds on protease activity of tomato leaf miner. Also in this study optimum pH for protease activity and effects of pH on inhibitory activity of extracts were determined. Materials and Methods: For this work fourth instar larvae of Tuta absoluta was used for enzyme extraction. Plant proteinaceous extracts were obtained using 0.1 M NaCl. For protease activity assay 10 μl enzyme extract and 50 μl substrate solution (Azocasein 2% were mixed with 40 μl of the 20 mM Glycine-NaOH buffer at pH 10.0 After 60 min incubation, 100 μl of 30% trichloroacetic acid (TCA was added to the reaction mixture, and kept at 4 °C for 30 min, followed by centrifugation at 15,000gfor 15 min to precipitate non-hydrolysis substrate. 100 μl of 1 M NaOH was added to 100μl supernatant and the absorbance at 405 nm was measured. To determine the effect of the seed proteinaceous extracts on the protease activities, enzymes were pre-incubated with each seed extracts for 30 min at 35 °C followed by determination of the residual enzyme activity as

Structural basis for the ATP-independent proteolytic activity of LonB proteases and reclassification of their AAA+ modules.

Science.gov (United States)

An, Young Jun; Na, Jung-Hyun; Kim, Myung-Il; Cha, Sun-Shin

2015-10-01

Lon proteases degrade defective or denature proteins as well as some folded proteins for the control of cellular protein quality. There are two types of Lon proteases, LonA and LonB. Each consists of two functional components: a protease component and an ATPase associated with various cellular activities (AAA+ module). Here, we report the 2.03 -resolution crystal structure of the isolated AAA+ module (iAAA+ module) of LonB from Thermococcus onnurineus NA1 (TonLonB). The iAAA+ module, having no bound nucleotide, adopts a conformation virtually identical to the ADP-bound conformation of AAA+ modules in the hexameric structure of TonLonB; this provides insights into the ATP-independent proteolytic activity observed in a LonB protease. Structural comparison of AAA+ modules between LonA and LonB revealed that the AAA+ modules of Lon proteases are separated into two distinct clades depending on their structural features. The AAA+ module of LonB belongs to the -H2 & Ins1 insert clade (HINS clade)- defined for the first time in this study, while the AAA+ module of LonA is a member of the HCLR clade.

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

Directory of Open Access Journals (Sweden)

Lin Yuan

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

Isolasi, Seleksi Dan Opttmasi Produksi Protease Daribeberapaisolat Bakteri*(isolation, Selection and Optimalization of Protease Production of Some Bacterial Isolates)

OpenAIRE

Naiola, Elidar; Widhyastuti, Nunuk

2002-01-01

Thirty-seven out of sixty-one bacterial isolates from various sources of samples were screened for protease production. The isolate of ISO PL3 could produce the highest enzyme activity, and it was used as a standard bacterial strain in this observation. For any reason,we implemented ISO PL2 to study the optimum condition for producing bacterial protease. Result shows that the maximum protease activity was obtained in a medium containing 100 gram of rice brand in a liter tofu liquid waste. The...

Studies on screening, isolation and purification of a fibrinolytic protease from an isolate (VK12) of Ganoderma lucidum and evaluation of its antithrombotic activity.

Science.gov (United States)

Kumaran, Sekar; Palani, Perumal; Nishanthi, Ramasami; Kaviyarasan, Venkatesan

2011-01-01

Antithrombotic activity of a protease purified from a medicinal mushroom, Ganoderma lucidum, has been evaluated platelet aggregation in vitro and pulmonary thrombosis in vivo. The purified protease exhibited concentration dependent inhibitory effects on platelet aggregation induced by ADP (adenosine diphosphate), with an IC(50) value of 2.4 mg/mL. The purified protease protected mice against thrombotic death or paralysis induced by collagen and epinephrine in a dose dependent manner when administered orally. It produced a significant inhibition of thrombotic death or paralysis at 60 µg/kg body weight, while aspirin produced a significant inhibition of thrombosis at 10-20 mg/kg body weight. The purified protease also has showed fibrinolytic activity and alters coagulation parameters such as activated partial thromboplastin time (APTT), and thrombin time (TT) in rat platelet. These results suggested that the antithrombotic activity of Ganoderma lucidum protease might be due to antiplatelet activity rather than anticoagulation activity.

Hyaluronidase and protease activities from Indian snake venoms: neutralization by Mimosa pudica root extract.

Science.gov (United States)

Girish, K S; Mohanakumari, H P; Nagaraju, S; Vishwanath, B S; Kemparaju, K

2004-06-01

The aqueous root extract of Mimosa pudica dose dependently inhibited the hyaluronidase and protease activities of Indian snakes (Naja naja, Vipera russelii and Echis carinatus) venom. Copyright 2004 Elsevier B.V.

Extracellular Neutrophil Proteases Are Efficient Regulators of IL-1, IL-33, and IL-36 Cytokine Activity but Poor Effectors of Microbial Killing.

Science.gov (United States)

Clancy, Danielle M; Sullivan, Graeme P; Moran, Hannah B T; Henry, Conor M; Reeves, Emer P; McElvaney, Noel G; Lavelle, Ed C; Martin, Seamus J

2018-03-13

Neutrophil granule proteases are thought to function as anti-microbial effectors, cooperatively hydrolyzing microorganisms within phagosomes, or upon deployment into the extracellular space. However, evidence also suggests that neutrophil proteases play an important role in the coordination and escalation of inflammatory reactions, but how this is achieved has been obscure. IL-1 family cytokines are important initiators of inflammation and are typically released via necrosis but require proteolytic processing for activation. Here, we show that proteases liberated from activated neutrophils can positively or negatively regulate the activity of six IL-1 family cytokines (IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ) with exquisite sensitivity. In contrast, extracellular neutrophil proteases displayed very poor bactericidal activity, exhibiting 100-fold greater potency toward cytokine processing than bacterial killing. Thus, in addition to their classical role as phagocytes, neutrophils play an important immunoregulatory role through deployment of their granule proteases into the extracellular space to process multiple IL-1 family cytokines. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Apical serine protease activity is necessary for assembly of a high-resistance renal collecting duct epithelium

DEFF Research Database (Denmark)

Steensgaard, Mette; Svenningsen, Per; Tinning, Anne R

2010-01-01

Abstract AIM: We hypothesized that the serine protease prostasin is necessary for differentiation of a high resistance renal collecting duct epithelium governed by glucocorticoid. METHODS: Postnatal rat kidney and adult human kidney was used to study expression and localization of prostasin......-cadherin distribution did not change. CONCLUSION: Apical, GPI-anchored, lipid raft-associated serine protease activity, compatible with prostasin, is necessary for development of a high-resistance collecting duct epithelium....

Identification and isoforms specificity of barley (Hordeum vulgare) grain proteinaceous inhibitors of commercial feed protease

DEFF Research Database (Denmark)

Dionisio, Giuseppe; Brinch-Pedersen, Henrik

2016-01-01

Protease is commonly used as feed additive. Ronozyme® ProAct, a subtilisin-like serine feed protease is different from the already characterized Bacillus subtilisin-like serine protease. When used in wheat and barley based feed, its degree of efficiency differs according to the cultivar in analys...

High throughput in vivo protease inhibitor selection platform

DEFF Research Database (Denmark)

2017-01-01

The invention relates to a recombinant microbial cell comprising a selection platform for screening for a protease inhibitor, wherein the platform comprises transgenes encoding a protease having selective peptide bond cleavage activity at a recognition site amino acid sequence; and transgenes...... platform for screening for a protease inhibitor....

Two-Dimensional Zymography of Proteases from Steatotic Duck Liver.

Science.gov (United States)

Wilkesman, Jeff; Padrón, María Fernanda; Kurz, Liliana; Rémignon, Hervé

2017-01-01

Protease activity present in liver cells with steatosis can be electrophoretically characterized. Zymographic techniques allow semi-quantitative results, successfully detecting cathepsin and metalloprotease activity using polyacrylamide gels copolymerized with gelatin and quantified by densitometry. By using specific inhibitors, the identity of the proteases can be confirmed. 2D zymography allows the determination of both M r. and pI of the metalloprotease and cathepsin activity present in the homogenates. The analysis of liver proteases activities in force fed ducks may elucidate the mechanisms behind steatosis development.

Heterologous expression of Hordeum vulgare cysteine protease in yeast

DEFF Research Database (Denmark)

Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben B

Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins during germination. Several Cysteine proteases have been identified in barley. One of the key enzymes, Hordeum vulgare endoprotease B2 (HvEPB2) was cloned with and w......Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins during germination. Several Cysteine proteases have been identified in barley. One of the key enzymes, Hordeum vulgare endoprotease B2 (HvEPB2) was cloned...

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

Science.gov (United States)

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.

Cathepsin F cysteine protease of the human liver fluke, Opisthorchis viverrini.

Directory of Open Access Journals (Sweden)

Porntip Pinlaor

Full Text Available The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities.Here, we describe the cDNA, gene organization, phylogenetic relationships, immunolocalization, and functional characterization of the cathepsin F cysteine protease gene, here termed Ov-cf-1, from O. viverrini. The full length mRNA of 1020 nucleotides (nt encoded a 326 amino acid zymogen consisting of a predicted signal peptide (18 amino acids, aa, prosegment (95 aa, and mature protease (213 aa. BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes, including Clonorchis sinensis (81%, Paragonimus westermani (58%, Schistosoma mansoni and S. japonicum (52%, and with vertebrate cathepsin F (51%. Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host. The Ov-cf-1 gene, of approximately 3 kb in length, included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length, the introns from 43 to 1,060 bp. The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene, although the gene structure of human cathepsin F is more complex. Unlike Ov-CF-1, human cathepsin F zymogen includes a cystatin domain in the prosegment region. Phylogenetic analysis revealed that the fluke, human, and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases. A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris. Although the recombinant protease did not autocatalytically process and

Comparative Detection of Alkaline Protease Production in Exiguobacterium acetylicum

International Nuclear Information System (INIS)

Gomaa, O.M.; EI Shafey, H.M.

2009-01-01

Alkaline protease is one of the most important enzymes in industry, medicine, and research. In the present work, a comparative detection for alkaline protease activity was established for instant detection of enzyme activity. Eight different alkalophilic bacterial isolates were compared based on the clear zone they produced on skim milk agar. One strain gave an absolute clear zone in 16 hours and was used for alkaline protease detection. The result of Phenotypic identification using Biology Microlog 3 identified the isolate as Exiguobacterium acetylicum. The isolate under study showed slightly different characteristics from a known Exiguobacterium acetylicum strain. The isolate tolerated alkaline conditions up to ph 11, while good growth was evident at ph 7, the maximum alkaline protease activity was observed at ph 9 which reached up to 109.01 U/ml. The alkaline activity assay using alkaline protease enzyme assay were coordinating with those obtained by conductivity; there was a relevant decrease in conductivity at the maximum increase in enzyme activity, which proved the cell membrane conductivity has a close relation to alkaline protease production. This isolate has tolerated gamma radiation, the increase in dose (up to 4 Gy) gave wider clear zones in terms of diameter and this was relevant to the conductivity measurements

Lyso-myristoyl phosphatidylcholine micelles sustain the activity of Dengue non-structural (NS) protein 3 protease domain fused with the full-length NS2B.

Science.gov (United States)

Huang, Qiwei; Li, Qingxin; Joy, Joma; Chen, Angela Shuyi; Ruiz-Carrillo, David; Hill, Jeffrey; Lescar, Julien; Kang, Congbao

2013-12-01

Dengue virus (DENV), a member of the flavivirus genus, affects 50-100 million people in tropical and sub-tropical regions. The DENV protease domain is located at the N-terminus of the NS3 protease and requires for its enzymatic activity a hydrophilic segment of the NS2B that acts as a cofactor. The protease is an important antiviral drug target because it plays a crucial role in virus replication by cleaving the genome-coded polypeptide into mature functional proteins. Currently, there are no drugs to inhibit DENV protease activity. Most structural and functional studies have been conducted using protein constructs containing the NS3 protease domain connected to a soluble segment of the NS2B membrane protein via a nine-residue linker. For in vitro structural and functional studies, it would be useful to produce a natural form of the DENV protease containing the NS3 protease domain and the full-length NS2B protein. Herein, we describe the expression and purification of a natural form of DENV protease (NS2BFL-NS3pro) containing the full-length NS2B protein and the protease domain of NS3 (NS3pro). The protease was expressed and purified in detergent micelles necessary for its folding. Our results show that this purified protein was active in detergent micelles such as lyso-myristoyl phosphatidylcholine (LMPC). These findings should facilitate further structural and functional studies of the protease and will facilitate drug discovery targeting DENV. Copyright © 2013 Elsevier Inc. All rights reserved.

A cathepsin L-like protease from Strongylus vulgaris: an orthologue of Caenorhabditis elegans CPL-1.

Science.gov (United States)

Ultaigh, Sinéad Nic An; Carolan, James C; Britton, Collette; Murray, Linda; Ryan, Michael F

2009-04-01

Cathespin L-like proteases (CPLs), characterized from a wide range of helminths, are significant in helminth biology. For example, in Caenorhabditis elegans CPL is essential for embryogenesis. Here, we report a cathepsin L-like gene from three species of strongyles that parasitize the horse, and describe the isolation of a cpl gene (Sv-cpl-1) from Strongylus vulgaris, the first such from equine strongyles. It encodes a protein of 354 amino acids with high similarity to other parasitic Strongylida (90-91%), and C.elegans CPL-1 (87%), a member of the same Clade. As S.vulgaris cpl-1 rescued the embryonic lethal phenotype of the C.elegans cpl-1 mutant, these genes may be orthologues, sharing the same function in each species. Targeting Sv-CPL-1 might enable novel control strategies by decreasing parasite development and transmission.

Dimerization-Induced Allosteric Changes of the Oxyanion-Hole Loop Activate the Pseudorabies Virus Assemblin pUL26N, a Herpesvirus Serine Protease.

Directory of Open Access Journals (Sweden)

Martin Zühlsdorf

2015-07-01

Full Text Available Herpesviruses encode a characteristic serine protease with a unique fold and an active site that comprises the unusual triad Ser-His-His. The protease is essential for viral replication and as such constitutes a promising drug target. In solution, a dynamic equilibrium exists between an inactive monomeric and an active dimeric form of the enzyme, which is believed to play a key regulatory role in the orchestration of proteolysis and capsid assembly. Currently available crystal structures of herpesvirus proteases correspond either to the dimeric state or to complexes with peptide mimetics that alter the dimerization interface. In contrast, the structure of the native monomeric state has remained elusive. Here, we present the three-dimensional structures of native monomeric, active dimeric, and diisopropyl fluorophosphate-inhibited dimeric protease derived from pseudorabies virus, an alphaherpesvirus of swine. These structures, solved by X-ray crystallography to respective resolutions of 2.05, 2.10 and 2.03 Å, allow a direct comparison of the main conformational states of the protease. In the dimeric form, a functional oxyanion hole is formed by a loop of 10 amino-acid residues encompassing two consecutive arginine residues (Arg136 and Arg137; both are strictly conserved throughout the herpesviruses. In the monomeric form, the top of the loop is shifted by approximately 11 Å, resulting in a complete disruption of the oxyanion hole and loss of activity. The dimerization-induced allosteric changes described here form the physical basis for the concentration-dependent activation of the protease, which is essential for proper virus replication. Small-angle X-ray scattering experiments confirmed a concentration-dependent equilibrium of monomeric and dimeric protease in solution.

Enzymatic activity of proteases and its isoenzymes in fermentation process in cultivars of cocoa (Theobroma cacao L. produced in southern Bahia, Brazil

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Luciane Santos SOUSA

Full Text Available Abstract The fermentation of cocoa seeds envolves microbial processes and the action of enzymes. To identify the possible differences in the cocoa fermentation process, with regards to proteolysis, this study has the objective of determining protease activity (under predetermined conditions and its isoenzymes in two cocoa cultivars (PH-16 and HRT-1188 in different cocoa fermentation times, in addition to establishing the microbial load (molds and yeasts and aerobic mesophilic. Protease and its isoenzymes were extracted and partially purified and the enzymatic activities determined by spectrophotometry. The results showed that the proteases activity was higher at 66h of fermentation for both cultivars. When the isoenzymes activity was evaluated, the results demonstrated similar activity behavior for both cultivars, with regards to the isoenzymes aminopeptidase and carboxypeptidase, although the behavior of the endoprotease isoenzyme activity proved to be a little different for TSH-1188 cultivar. Concerning microbiological analyses, the results indicate that the period after molds and yeast counting reduction is consistent with the period of protease activity increase.

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

Oxidative Stress: Promoter of Allergic Sensitization to Protease Allergens?

NARCIS (Netherlands)

van Rijt, Leonie S.; Utsch, Lara; Lutter, René; van Ree, Ronald

2017-01-01

Allergies arise from aberrant T helper type 2 responses to allergens. Several respiratory allergens possess proteolytic activity, which has been recognized to act as an adjuvant for the development of a Th2 response. Allergen source-derived proteases can activate the protease-activated receptor-2,

Preparation, Characterization and Activity of a Peptide-Cellulosic Aerogel Protease Sensor from Cotton

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J. Vincent Edwards

2016-10-01

Full Text Available Nanocellulosic aerogels (NA provide a lightweight biocompatible material with structural properties, like interconnected high porosity and specific surface area, suitable for biosensor design. We report here the preparation, characterization and activity of peptide-nanocellulose aerogels (PepNA made from unprocessed cotton and designed with protease detection activity. Low-density cellulosic aerogels were prepared from greige cotton by employing calcium thiocyanate octahydrate/lithium chloride as a direct cellulose dissolving medium. Subsequent casting, coagulation, solvent exchange and supercritical carbon dioxide drying afforded homogeneous cellulose II aerogels of fibrous morphology. The cotton-based aerogel had a porosity of 99% largely dominated by mesopores (2–50 nm and an internal surface of 163 m2·g−1. A fluorescent tripeptide-substrate (succinyl-alanine-proline-alanine-4-amino-7-methyl-coumarin was tethered to NA by (1 esterification of cellulose C6 surface hydroxyl groups with glycidyl-fluorenylmethyloxycarbonyl (FMOC, (2 deprotection and (3 coupling of the immobilized glycine with the tripeptide. Characterization of the NA and PepNA included techniques, such as elemental analysis, mass spectral analysis, attenuated total reflectance infrared imaging, nitrogen adsorption, scanning electron microscopy and bioactivity studies. The degree of substitution of the peptide analog attached to the anhydroglucose units of PepNA was 0.015. The findings from mass spectral analysis and attenuated total reflectance infrared imaging indicated that the peptide substrate was immobilized on to the surface of the NA. Nitrogen adsorption revealed a high specific surface area and a highly porous system, which supports the open porous structure observed from scanning electron microscopy images. Bioactivity studies of PepNA revealed a detection sensitivity of 0.13 units/milliliter for human neutrophil elastase, a diagnostic biomarker for inflammatory

In-cell protease assay systems based on trans-localizing molecular beacon proteins using HCV protease as a model system.

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Jeong Hee Kim

Full Text Available This study describes a sensitive in-cell protease detection system that enables direct fluorescence detection of a target protease and its inhibition inside living cells. This live-cell imaging system provides a fluorescent molecular beacon protein comprised of an intracellular translocation signal sequence, a protease-specific cleavage sequence, and a fluorescent tag sequence(s. The molecular beacon protein is designed to change its intracellular localization upon cleavage by a target protease, i.e., from the cytosol to a subcellular organelle or from a subcellular organelle to the cytosol. Protease activity can be monitored at the single cell level, and accordingly the entire cell population expressing the protease can be accurately enumerated. The clear cellular change in fluorescence pattern makes this system an ideal tool for various life science and drug discovery research, including high throughput and high content screening applications.

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

Science.gov (United States)

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

Directory of Open Access Journals (Sweden)

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.

Cell-based fluorescence assay for human immunodeficiency virus type 1 protease activity

Czech Academy of Sciences Publication Activity Database

Lindsten, K.; Kondrová, Taťána; Konvalinka, Jan; Masucci, M. G.; Dantuma, N. P.

2001-01-01

Roč. 45, č. 9 (2001), s. 2616-2622 ISSN 0066-4804 R&D Projects: GA ČR GA303/98/1559 Grant - others:HHMI(US) 75195-540801; ECTM(XE) ERBFMRXCT960026 Institutional research plan: CEZ:AV0Z4055905 Keywords : HIV-1 * protease activity Subject RIV: CE - Biochemistry Impact factor: 4.562, year: 2001

Characterization of the protease activity that cleaves the extracellular domain of β-dystroglycan

International Nuclear Information System (INIS)

Zhong Di; Saito, Fumiaki; Saito, Yuko; Nakamura, Ayami; Shimizu, Teruo; Matsumura, Kiichiro

2006-01-01

Dystroglycan (DG) complex, composed of αDG and βDG, provides a link between the extracellular matrix (ECM) and cortical cytoskeleton. Although the proteolytic processing of βDG was reported in various physiological and pathological conditions, its exact mechanism remains unknown. In this study, we addressed this issue using the cell culture system of rat schwannoma cell line RT4. We found that the culture medium of RT4 cells was enriched with the protease activity that degrades the fusion protein construct of the extracellular domain of βDG specifically. This activity was suppressed by the inhibitor of matrix metalloproteinase-2 (MMP-2) and MMP-9, but not by the inhibitors of MMP-1, MMP-3, MMP-8, and MMP-13. Zymography and RT-PCR analysis showed that RT4 cells secreted MMP-2 and MMP-9 into the culture medium. Finally, active MMP-2 and MMP-9 enzymes degraded the fusion protein construct of the extracellular domain of βDG. These results indicate (1) that RT4 cells secrete the protease activity that degrades the extracellular domain of βDG specifically and (2) that MMP-2 and MMP-9 may be involved in this process

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+ ...

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.

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.

Role of protease-activated receptor-2 in inflammation, and its possible implications as a putative mediator of periodontitis

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M Holzhausen

2005-03-01

Full Text Available Proteinase-activated receptor-2 (PAR2 belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. This receptor is widely distributed throughout the body and seems to be importantly involved in inflammatory processes. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and bacterial proteases, such as gingipain produced by Porphyromonas gingivalis. This review describes the current stage of knowledge of the possible mechanisms that link PAR2 activation with periodontal disease, and proposes future therapeutic strategies to modulate the host response in the treatment of periodontitis.

Isolation and molecular characterization of cathepsin L-like cysteine protease cDNAs from Western flower thrips (Frankliniella occidentalis)

NARCIS (Netherlands)

Kuipers, A.G.J.; Jongsma, M.A.

2004-01-01

Cysteine proteases are predominant in thrips guts (TGs) and, therefore, a suitable target for selecting effective protease inhibitors against western flower thrips (Frankliniella occidentalis). We report the isolation of four full-length cysteine protease cDNA clones from thrips in a two-step PCR

Crystal structure of a cold-active protease (Pro21717) from the psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, at 1.4 Å resolution: Structural adaptations to cold and functional analysis of a laundry detergent enzyme.

Science.gov (United States)

Park, Ha Ju; Lee, Chang Woo; Kim, Dockyu; Do, Hackwon; Han, Se Jong; Kim, Jung Eun; Koo, Bon-Hun; Lee, Jun Hyuck; Yim, Joung Han

2018-01-01

Enzymes isolated from organisms found in cold habitats generally exhibit higher catalytic activity at low temperatures than their mesophilic homologs and are therefore known as cold-active enzymes. Cold-active proteases are very useful in a variety of biotechnological applications, particularly as active ingredients in laundry and dishwashing detergents, where they provide strong protein-degrading activity in cold water. We identified a cold-active protease (Pro21717) from a psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, and determined the crystal structure of its catalytic domain (CD) at a resolution of 1.4 Å. The Pro21717-CD structure shows a conserved subtilisin-like fold with a typical catalytic triad (Asp185, His244, and Ser425) and contains four calcium ions and three disulfide bonds. Interestingly, we observed an unexpected electron density at the substrate-binding site from a co-purified peptide. Although the sequence of this peptide is unknown, analysis of the peptide-complexed structure nonetheless provides some indication of the substrate recognition and binding mode of Pro21717. Moreover, various parameters, including a wide substrate pocket size, an abundant active-site loop content, and a flexible structure provide potential explanations for the cold-adapted properties of Pro21717. In conclusion, this is first structural characterization of a cold-adapted subtilisin-like protease, and these findings provide a structural and functional basis for industrial applications of Pro21717 as a cold-active laundry or dishwashing detergent enzyme.

Optimization of alkaline protease production and its fibrinolytic ...

African Journals Online (AJOL)

Optimization of alkaline protease production and its fibrinolytic activity from the ... nitrogen sources and sodium chloride concentration for protease production by the ... exploited to assist in protein degradation in various industrial processes.

Isolation of Mucorales from processed maize (Zea mays L.) and screening for protease activity

OpenAIRE

de Azevedo Santiago, Andr? Luiz Cabral Monteiro; de Souza Motta, Cristina Maria

2008-01-01

Mucorales were isolated from maize flour, corn meal and cooked cornflakes using surface and depth plate methods. Rhizopus oryzae, Circinella muscae, Mucor subtilissimus, Mucor hiemalis f. hiemalis, Syncephalastrum racemosum, Rhizopus microsporus var. chinensis and Absidia cylindrospora showed protease activity.

Protease-activated receptor (PAR)-2 is required for PAR-1 signalling in pulmonary fibrosis

NARCIS (Netherlands)

Lin, Cong; von der Thüsen, Jan; Daalhuisen, Joost; ten Brink, Marieke; Crestani, Bruno; van der Poll, Tom; Borensztajn, Keren; Spek, C. Arnold

2015-01-01

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease-activated receptor (PAR)-1 and PAR-2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin-induced

Pharmacological targeting of protease-activated receptor 2 affords protection from bleomycin-induced pulmonary fibrosis

NARCIS (Netherlands)

C. Lin (Cong); J. von der Thusen (Jan); J. Daalhuisen (Joost); M. Ten Brink (Marieke); B. Crestani (Bruno); T. van der Poll (Tom); K. Borensztajn (Keren); C. Arnold Spek (C.)

2015-01-01

textabstractIdiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease that remains refractory to therapy. Despite increasing evidence that protease-activated receptor 2 (PAR-2) contributes to fibrosis, its importance in pulmonary fibrosis is under debate. We addressed

Isolation of Mucorales from processed maize (Zea mays L.) and screening for protease activity

Science.gov (United States)

de Azevedo Santiago, André Luiz Cabral Monteiro; de Souza Motta, Cristina Maria

2008-01-01

Mucorales were isolated from maize flour, corn meal and cooked cornflakes using surface and depth plate methods. Rhizopus oryzae, Circinella muscae, Mucor subtilissimus, Mucor hiemalis f. hiemalis, Syncephalastrum racemosum, Rhizopus microsporus var. chinensis and Absidia cylindrospora showed protease activity. PMID:24031292

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

Science.gov (United States)

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.

Partial characterization of cold active amylases and proteases of Streptomyces sp. from Antarctica

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Mihaela Cotârleţ

2011-09-01

Full Text Available The aim of this study was to isolate novel enzyme-producing bacteria from vegetation samples from East Antarctica and also to characterize them genetically and biochemically in order to establish their phylogeny. The ability to grow at low temperature and to produce amylases and proteases cold-active was also tested. The results of the 16S rRNA gene sequence analysis showed that the 4 Alga rRNA was 100% identical to the sequences of Streptomyces sp. rRNA from Norway and from the Solomon Islands. The Streptomyces grew well in submerged system at 20ºC, cells multiplication up to stationary phase being drastically increased after 120 h of submerged cultivation. The beta-amylase production reached a maximum peak after seven days, while alpha-amylase and proteases were performing biosynthesis after nine days of submerged cultivation at 20ºC. Newly Streptomyces were able to produce amylase and proteases in a cold environment. The ability to adapt to low temperature of these enzymes could make them valuable ingredients for detergents, the food industry and bioremediation processes which require low temperatures.

The protease inhibitor HAI-2, but not HAI-1, regulates matriptase activation and shedding through prostasin

DEFF Research Database (Denmark)

Friis, Stine; Sales, Katiuchia Uzzun; Schafer, Jeffrey Martin

2014-01-01

cells. Whereas ablation of HAI-1 did not affect matriptase in epithelial cells of the small or large intestine, ablation of HAI-2 resulted in the loss of matriptase from both tissues. Gene silencing studies in intestinal Caco-2 cell monolayers revealed that this loss of cell-associated matriptase......The membrane-anchored serine proteases, matriptase and prostasin, and the membrane-anchored serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2, are critical effectors of epithelial development and postnatal epithelial homeostasis. Matriptase and prostasin...... form a reciprocal zymogen activation complex that results in the formation of active matriptase and prostasin that are targets for inhibition by HAI-1 and HAI-2. Conflicting data, however, have accumulated as to the existence of auxiliary functions for both HAI-1 and HAI-2 in regulating...

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

Science.gov (United States)

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.

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)

Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro

International Nuclear Information System (INIS)

Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas; Choi, Kyung H.

2014-01-01

Pestivirus N pro is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N pro blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N pro' s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N pro -GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N pro proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N pro' s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N pro does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N pro' s autoproteolysis is studied using N pro -GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N pro prefers small amino acids with non-branched beta carbons at the P1 position

Effect of Gastrointestinal Protease Digestion on Bioactivity of Marine Peptides

DEFF Research Database (Denmark)

Jensen, Ida-Johanne; Andersen, Lisa Lystbæk; Ossum, Carlo Gunnar

2014-01-01

executed without concerning subsequent digestion after intake and the aim of this work was hence to investigate how the in vitro antioxidative, antihypertensive and caspase activating activities of peptides are affected by digestion with gastrointestinal (GI) proteases. Five different fish protein...... hydrolysates were chosen to study the effect of in vitro digestion on bioactivity. The protein concentration decreased in all samples during digestion and the molecular weight distribution of the peptides shifted towards lower values. Thus, in vitro digestion with GI proteases resulted in a further degradation...... of the peptides obtained by hydrolysis. The antihypertensive effect increased in all samples after digestion with GI proteases whereas the antioxidative capacity decreased. The effect on the caspase activity depended on the proteases used in the preparation of hydrolysates. In conclusion, the caspase activity...

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.

Pharmacological Targeting of Protease-Activated Receptor 2 Affords Protection from Bleomycin-Induced Pulmonary Fibrosis

NARCIS (Netherlands)

Lin, Cong; von der Thüsen, Jan; Daalhuisen, Joost; ten Brink, Marieke; Crestani, Bruno; van der Poll, Tom; Borensztajn, Keren; Spek, C. Arnold

2015-01-01

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease that remains refractory to therapy. Despite increasing evidence that protease-activated receptor 2 (PAR-2) contributes to fibrosis, its importance in pulmonary fibrosis is under debate. We addressed whether PAR-2

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.

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

Regulation of cuticle-degrading subtilisin proteases from the entomopathogenic fungi, Lecanicillium spp: implications for host specificity.

Science.gov (United States)

Bye, Natasha J; Charnley, A Keith

2008-01-01

The ability to produce cuticle-degrading proteases to facilitate host penetration does not distinguish per se entomopathogenic fungi from saprophytes. However, adapted pathogens may produce host-protein specific enzymes in response to cues. This possibility prompted an investigation of the regulation of isoforms of the subtilisin Pr1-like proteases from five aphid-pathogenic isolates of Lecanicillium spp. Significant differences were found in substrate specificity and regulation of Pr1-like proteases between isoforms of the same isolate and between different isolates. For example, the pI 8.6 isoform from KV71 was considerably more active against aphid than locust cuticle and was induced specifically by N-acetylglucosamine (NAG). Isoform pI 9.1 from the same isolate was only produced on insect cuticle while most other isoforms were more prominent on chitin containing substrates but not induced by NAG. The ability to regulate isoforms independently may allow production at critical points in host penetration. Appearance of proteases (not subtilisins) with pI 4.2 and 4.4 only on aphid cuticle was a possible link with host specificity of KV71. The absence of C or N metabolite repression in subtilisins from KV42 is unusual for pathogen proteases and may help to account for differences in virulence strategy between aphid-pathogenic isolates of Lecanicillium longisporum (unpublished data).

Antioxidant and ACE-inhibitory activities of hemp (Cannabis sativa L.) protein hydrolysates produced by the proteases AFP, HT, Pro-G, actinidin and zingibain.

Science.gov (United States)

Teh, Sue-Siang; Bekhit, Alaa El-Din A; Carne, Alan; Birch, John

2016-07-15

Hemp protein isolates (HPIs) were hydrolysed by proteases (AFP, HT, ProG, actinidin and zingibain). The enzymatic hydrolysis of HPIs was evaluated through the degree of hydrolysis and SDS-PAGE profiles. The bioactive properties of the resultant hydrolysates (HPHs) were accessed through ORAC, DPPḢ scavenging and ACE-inhibitory activities. The physical properties of the resultant HPHs were evaluated for their particle sizes, zeta potential and surface hydrophobicity. HT had the highest rate of caseinolytic activity at the lowest concentration (0.1 mg mL(-1)) compared to other proteases that required concentration of 100 mg mL(-1) to achieve their maximum rate of caseinolytic activity. This led to the highest degree of hydrolysis of HPIs by HT in the SDS-PAGE profiles. Among all proteases and substrates, HT resulted in the highest bioactivities (ORAC, DPPḢ scavenging and ACE-inhibitory activities) generated from alkali extracted HPI in the shortest time (2 h) compared to the other protease preparations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Exposure to ozone modulates human airway protease/antiprotease balance contributing to increased influenza A infection.

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Matthew J Kesic

Full Text Available Exposure to oxidant air pollution is associated with increased respiratory morbidities and susceptibility to infections. Ozone is a commonly encountered oxidant air pollutant, yet its effects on influenza infections in humans are not known. The greater Mexico City area was the primary site for the spring 2009 influenza A H1N1 pandemic, which also coincided with high levels of environmental ozone. Proteolytic cleavage of the viral membrane protein hemagglutinin (HA is essential for influenza virus infectivity. Recent studies suggest that HA cleavage might be cell-associated and facilitated by the type II transmembrane serine proteases (TTSPs human airway trypsin-like protease (HAT and transmembrane protease, serine 2 (TMPRSS2, whose activities are regulated by antiproteases, such as secretory leukocyte protease inhibitor (SLPI. Based on these observations, we sought to determine how acute exposure to ozone may modulate cellular protease/antiprotease expression and function, and to define their roles in a viral infection. We utilized our in vitro model of differentiated human nasal epithelial cells (NECs to determine the effects of ozone on influenza cleavage, entry, and replication. We show that ozone exposure disrupts the protease/antiprotease balance within the airway liquid. We also determined that functional forms of HAT, TMPRSS2, and SLPI are secreted from human airway epithelium, and acute exposure to ozone inversely alters their expression levels. We also show that addition of antioxidants significantly reduces virus replication through the induction of SLPI. In addition, we determined that ozone-induced cleavage of the viral HA protein is not cell-associated and that secreted endogenous proteases are sufficient to activate HA leading to a significant increase in viral replication. Our data indicate that pre-exposure to ozone disrupts the protease/antiprotease balance found in the human airway, leading to increased influenza susceptibility.

Characterization of a secreted Chlamydia protease

DEFF Research Database (Denmark)

Shaw, A.C.; Vandahl, B.B.; Larsen, M.R.

2002-01-01

Chlamydiae are obligate intracellular bacteria that are important human pathogens. The Chlamydia genomes contain orthologues to secretion apparatus proteins from other intracellular bacteria, but only a few secreted proteins have been identified. Most likely, effector proteins are secreted in order...... to promote infection. Effector proteins cannot be identified by motif or similarity searches. As a new strategy for identification of secreted proteins we have compared 2D-PAGE profiles of [35S]-labelled Chlamydia proteins from whole lysates of infected cells to 2D-PAGE profiles of proteins from purified...... Chlamydia. Several secretion candidates from Chlamydia trachomatis D and Chlamydia pneumoniae were detected by this method. Two protein spots were identified among the candidates. These represent fragments of the 'chlamydial protease- or proteasome-like activity factor' (CPAF) and were clearly present in 2D...

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.

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

Science.gov (United States)

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

Effects of temperature, pH and NaCl on protease activity in digestive tract of young turbot, Scophthalmus maximus

Science.gov (United States)

Chen, Muyan; Zhang, Xiumei; Gao, Tianxiang; Chen, Chao

2006-09-01

The protease activity in digestive tract of young turbot Scophthalmus maximum was studied, and the optimal pH, temperature and NaCl concentration were determined for different portions of the fish's internal organs. The optimal activity in the fish's stomach was at pH of 2.2, while that in the intestinal extracts was within the alkaline range from 9.5 to 10.0. In hepatopancreas, the optimal pH was in low alkalinity at 8.5. The optimal reaction temperature was above 40°C in stomach, intestine and hepatopancreas. With increasing temperature, the pH value increased in stomach, while in the intestine, an opposite tendency was observed due to combined effect of pH and temperature. NaCl concentration showed inhibitory impact on protein digestion in hepatopancreas. The main protease for protein digestion in turbot seemed to be pepsin. Moreover, the maximum protease activity in different segments of intestine existed in the hindgut.

Production of alkaline proteases by alkalophilic Bacillus subtilis ...

African Journals Online (AJOL)

Tuoyo Aghomotsegin

2016-11-23

Nov 23, 2016 ... Key words: Production, alkaline protease, Bacillus subtilis, animal wastes, enzyme activity. ... Generally, alkaline proteases are produced using submerged fermentation .... biopolymer concentrations were reported to have an influence ... adding nitrogenous compounds stimulate microorganism growth and ...

Cysteine Protease Zymography: Brief Review.

Science.gov (United States)

Wilkesman, Jeff

2017-01-01

Cysteine proteases play multiple roles in basically all aspects of physiology and development. In plants, they are involved in growth and development and in accumulation and mobilization of storage proteins. Furthermore, they are engaged in signalling pathways and in the response to biotic and abiotic stresses. In animals and also in humans, they are responsible for senescence and apoptosis, prohormone processing, and ECM remodelling. When analyzed by zymography, the enzyme must be renaturated after SDS-PAGE. SDS must be washed out and substituted by Triton X-100. Gels are then further incubated under ideal conditions for activity detection. Cysteine proteases require an acidic pH (5.0-6.0) and a reducing agent, usually DTT. When screening biological samples, there is generally no previous clue on what peptidase class will be present, neither optimal proteolysis conditions are known. Hence, it is necessary to assess several parameters, such as incubation time, pH, temperature, influence of ions or reducing agents, and finally evaluate the inhibition profile. For detection of cysteine peptidase activity, the use of specific inhibitors, such as E-64, can be used to prevent the development of cysteine peptidase activity bands and positively confirm its presence. Here four different protocols to assess cysteine protease activity from different sources are presented.

Post-secretional activation of Protease IV by quorum sensing in Pseudomonas aeruginosa

OpenAIRE

Oh, Jungmin; Li, Xi-Hui; Kim, Soo-Kyong; Lee, Joon-Hee

2017-01-01

Protease IV (PIV), a key virulence factor of Pseudomonas aeruginosa is a secreted lysyl-endopeptidase whose expression is induced by quorum sensing (QS). We found that PIV expressed in QS mutant has severe reduction of activity in culture supernatant (CS), even though it is overexpressed to high level. PIV purified from the QS mutant (M-PIV) had much lower activity than the PIV purified from wild type (P-PIV). We found that the propeptide cleaved from prepro-PIV was co-purified with M-PIV, bu...

Studies on detection and analysis of proteases in leaf extract of medicinally important plants.

Science.gov (United States)

Chinnadurai, Gandhi Shree; Krishnan, Sivakumar; Perumal, Palani

2018-02-01

The whole plant or the extracts obtained from them have long been used as medicine to treat various human diseases and disorders. Notably, those plants endowed with protease activity have been traditionally used as the agents for treating tumors, digestion disorders, swelling, blood coagulation, fibrinolysis and also for immune-modulation. Proteases occupy a pivotal position in enzyme based industries. Plant proteases have been increasingly exploited for pharmaceutical, food, leather and textile processing industries. Earlier investigations have focused on the occurrence of proteases in medicinally unimportant plants. Therefore it has been aimed to study the occurrence of proteolytic enzymes from medicinally important plants establish any correlation exists between protease activity and medicinal use of individual plants. Crude extract were obtained from the leaves of 80 different medicinal plants. Tris-HCl buffer was used as the extraction buffer and the supernatants obtained were used for determination of total protein and protease activity using spectrophotometric methods. Qualitative screening for the presence of protease was carried out with agar diffusion method by incorporating the substrate. SDS-PAGE was used to analyse the isoforms of protease and for determination of relative molecular mass. Relatively higher protease activities were observed in the extracts of leaves of Pongamia pinnata (Fabaceae), Wrightia tinctoria (Apocyanaceae) Acalypha indica (Euphorbiaceae), Adhatoda vasica (Acanthaceae) and Curcuma longa (Zingiberaceae). No correlation was found between the total protein content and protease activity in individual plant species. SDS-PAGE analysis indicated the presence of multiple forms of protease of higher molecular weight range in several plant species. We found a strong correlation between the protease activity and medicinal application of the plant CONCLUSION: The present study has unequivocally revealed that the leaves of medicinal plants

Purification and characterization of protease enzyme from ...

African Journals Online (AJOL)

user

2013-03-20

Mar 20, 2013 ... Full Length Research Paper. Purification and ... ting into small peptides and free amino acids, which can ... Isolated strain was cultured in synthetic medium- casein (SMC; ... Protease activity was assayed by sigma's non-specific protease ... following buffers: 0.05 M citrate-phosphate buffer (pH 5 to 6), Tris-.

Differential Response of Extracellular Proteases of Trichoderma Harzianum Against Fungal Phytopathogens.

Science.gov (United States)

Sharma, Vivek; Salwan, Richa; Sharma, Prem N

2016-09-01

In the present study, production of extracellular proteases by Trichoderma harzianum was evaluated based on the relative gene expression and spectrophotometric assay. The fungal isolates were grown in Czapek Dox Broth medium supplemented with deactivated mycelium of plant fungal pathogens such as Fusarium oxysporum, Colletotrichum capsici, Gloeocercospora sorghi, and Colletotrichum truncatum. The maximum protease activity was detected after 48 h of incubation against Colletotrichum spp. Similarly in qRT-PCR, the relative gene expression of four proteases varied from 48 to 96 h against host pathogens in a time-independent manner. Among proteases, statistically significant upregulation of asp, asp, and srp was observed against Colletotrichum spp., followed by F. oxysporum. But in the case of pepM22, maximum upregulation was observed against F. oxysporum. The variation in enzyme assay and qRT-PCR of proteases at different time intervals against various fungal phytopathogens could be due to the limitation of using casein as a substrate for all types of proteases or protease-encoding transcripts selected for qRT-PCR, which may not be true representative of total protease activity.

Targeting protease activated receptor-1 with P1pal-12 limits bleomycin-induced pulmonary fibrosis

NARCIS (Netherlands)

Lin, Cong; Duitman, Janwillem; Daalhuisen, Joost; ten Brink, Marieke; von der Thüsen, Jan; van der Poll, Tom; Borensztajn, Keren; Spek, C. Arnold

2014-01-01

Idiopathic pulmonary fibrosis is the most devastating fibrotic diffuse parenchymal lung disease which remains refractory to pharmacological therapies. Therefore, novel treatments are urgently required. Protease-activated receptor (PAR)-1 is a G-protein-coupled receptor that mediates critical

Thermolysin damages animal life through degradation of plasma proteins enhanced by rapid cleavage of serpins and activation of proteases.

Science.gov (United States)

Kong, Lulu; Lu, Anrui; Guan, Jingmin; Yang, Bing; Li, Muwang; Hillyer, Julián F; Ramarao, Nalini; Söderhäll, Kenneth; Liu, Chaoliang; Ling, Erjun

2015-01-01

Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians. © 2014 Wiley Periodicals, Inc.

Kinetic intermediates en route to the final serpin-protease complex: studies of complexes of α1-protease inhibitor with trypsin.

Science.gov (United States)

Maddur, Ashoka A; Swanson, Richard; Izaguirre, Gonzalo; Gettins, Peter G W; Olson, Steven T

2013-11-01

Serpin protein protease inhibitors inactivate their target proteases through a unique mechanism in which a major serpin conformational change, resulting in a 70-Å translocation of the protease from its initial reactive center loop docking site to the opposite pole of the serpin, kinetically traps the acyl-intermediate complex. Although the initial Michaelis and final trapped acyl-intermediate complexes have been well characterized structurally, the intermediate stages involved in this remarkable transformation are not well understood. To better characterize such intermediate steps, we undertook rapid kinetic studies of the FRET and fluorescence perturbation changes of site-specific fluorophore-labeled derivatives of the serpin, α1-protease inhibitor (α1PI), which report the serpin and protease conformational changes involved in transforming the Michaelis complex to the trapped acyl-intermediate complex in reactions with trypsin. Two kinetically resolvable conformational changes were observed in the reactions, ascribable to (i) serpin reactive center loop insertion into sheet A with full protease translocation but incomplete protease distortion followed by, (ii) full conformational distortion and movement of the protease and coupled serpin conformational changes involving the F helix-sheet A interface. Kinetic studies of calcium effects on the labeled α1PI-trypsin reactions demonstrated both inactive and low activity states of the distorted protease in the final complex that were distinct from the intermediate distorted state. These studies provide new insights into the nature of the serpin and protease conformational changes involved in trapping the acyl-intermediate complex in serpin-protease reactions and support a previously proposed role for helix F in the trapping mechanism.

HIV protease drug resistance and its impact on inhibitor design.

Science.gov (United States)

Ala, P J; Rodgers, J D; Chang, C H

1999-07-01

The primary cause of resistance to the currently available HIV protease inhibitors is the accumulation of multiple mutations in the viral protease. So far more than 20 substitutions have been observed in the active site, dimer interface, surface loops and flaps of the homodimer. While many mutations reduce the protease's affinity for inhibitors, others appear to enhance its catalytic efficiency. This high degree of genetic flexibility has made the protease an elusive drug target. The design of the next generation of HIV protease inhibitors will be discussed in light of the current structural information.

The Cysteine Protease–Cysteine Protease Inhibitor System Explored in Soybean Nodule Development

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Marian Dorcas Quain

2013-08-01

Full Text Available Almost all protease families have been associated with plant development, particularly senescence, which is the final developmental stage of every organ before cell death. Proteolysis remobilizes and recycles nitrogen from senescent organs that is required, for example, seed development. Senescence-associated expression of proteases has recently been characterized using large-scale gene expression analysis seeking to identify and characterize senescence-related genes. Increasing activities of proteolytic enzymes, particularly cysteine proteases, are observed during the senescence of legume nodules, in which a symbiotic relationship between the host plant and bacteria (Rhizobia facilitate the fixation of atmospheric nitrogen. It is generally considered that cysteine proteases are compartmentalized to prevent uncontrolled proteolysis in nitrogen-fixing nodules. In addition, the activities of cysteine proteases are regulated by endogenous cysteine protease inhibitors called cystatins. These small proteins form reversible complexes with cysteine proteases, leading to inactivation. However, very little is currently known about how the cysteine protease-cysteine protease inhibitor (cystatin system is regulated during nodule development. Moreover, our current understanding of the expression and functions of proteases and protease inhibitors in nodules is fragmented. To address this issue, we have summarized the current knowledge and techniques used for studying proteases and their inhibitors including the application of “omics” tools, with a particular focus on changes in the cysteine protease-cystatin system during nodule development.

The dissemination of C10 cysteine protease genes in Bacteroides fragilis by mobile genetic elements

LENUS (Irish Health Repository)

Thornton, Roibeard F

2010-04-23

Abstract Background The C10 family of cysteine proteases includes enzymes that contribute to the virulence of bacterial pathogens, such as SpeB in Streptococcus pyogenes. The presence of homologues of cysteine protease genes in human commensal organisms has not been examined. Bacteroides fragilis is a member of the dominant Bacteroidetes phylum of the human intestinal microbiota, and is a significant opportunistic pathogen. Results Four homologues of the streptococcal virulence factor SpeB were identified in the B. fragilis genome. These four protease genes, two were directly contiguous to open reading frames predicted to encode staphostatin-like inhibitors, with which the protease genes were co-transcribed. Two of these protease genes are unique to B. fragilis 638R and are associated with two large genomic insertions. Gene annotation indicated that one of these insertions was a conjugative Tn-like element and the other was a prophage-like element, which was shown to be capable of excision. Homologues of the B. fragilis C10 protease genes were present in a panel of clinical isolates, and in DNA extracted from normal human faecal microbiota. Conclusions This study suggests a mechanism for the evolution and dissemination of an important class of protease in major members of the normal human microbiota.

The dissemination of C10 cysteine protease genes in Bacteroides fragilis by mobile genetic elements

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Kagawa Todd F

2010-04-01

Full Text Available Abstract Background The C10 family of cysteine proteases includes enzymes that contribute to the virulence of bacterial pathogens, such as SpeB in Streptococcus pyogenes. The presence of homologues of cysteine protease genes in human commensal organisms has not been examined. Bacteroides fragilis is a member of the dominant Bacteroidetes phylum of the human intestinal microbiota, and is a significant opportunistic pathogen. Results Four homologues of the streptococcal virulence factor SpeB were identified in the B. fragilis genome. These four protease genes, two were directly contiguous to open reading frames predicted to encode staphostatin-like inhibitors, with which the protease genes were co-transcribed. Two of these protease genes are unique to B. fragilis 638R and are associated with two large genomic insertions. Gene annotation indicated that one of these insertions was a conjugative Tn-like element and the other was a prophage-like element, which was shown to be capable of excision. Homologues of the B. fragilis C10 protease genes were present in a panel of clinical isolates, and in DNA extracted from normal human faecal microbiota. Conclusions This study suggests a mechanism for the evolution and dissemination of an important class of protease in major members of the normal human microbiota.

Functional protease profiling for diagnosis of malignant disease.

Science.gov (United States)

Findeisen, Peter; Neumaier, Michael

2012-01-01

Clinical proteomic profiling by mass spectrometry (MS) aims at uncovering specific alterations within mass profiles of clinical specimens that are of diagnostic value for the detection and classification of various diseases including cancer. However, despite substantial progress in the field, the clinical proteomic profiling approaches have not matured into routine diagnostic applications so far. Their limitations are mainly related to high-abundance proteins and their complex processing by a multitude of endogenous proteases thus making rigorous standardization difficult. MS is biased towards the detection of low-molecular-weight peptides. Specifically, in serum specimens, the particular fragments of proteolytically degraded proteins are amenable to MS analysis. Proteases are known to be involved in tumour progression and tumour-specific proteases are released into the blood stream presumably as a result of invasive progression and metastasis. Thus, the determination of protease activity in clinical specimens from patients with malignant disease can offer diagnostic and also therapeutic options. The identification of specific substrates for tumour proteases in complex biological samples is challenging, but proteomic screens for proteases/substrate interactions are currently experiencing impressive progress. Such proteomic screens include peptide-based libraries, differential isotope labelling in combination with MS, quantitative degradomic analysis of proteolytically generated neo-N-termini, monitoring the degradation of exogenous reporter peptides with MS, and activity-based protein profiling. In the present article, we summarize and discuss the current status of proteomic techniques to identify tumour-specific protease-substrate interactions for functional protease profiling. Thereby, we focus on the potential diagnostic use of the respective approaches. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Extracellular proteases of Trichoderma species. A review.

Science.gov (United States)

Kredics, L; Antal, Zsuzsanna; Szekeres, A; Hatvani, L; Manczinger, L; Vágvölgyi, Cs; Nagy, Erzsébet

2005-01-01

Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed.

A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease

International Nuclear Information System (INIS)

Campos-Gomez, Javier; Ahmad, Fahim; Rodriguez, Efrain; Saeed, Mohammad F.

2016-01-01

The encephalitic alphaviruses encode nsP2 protease (nsP2pro), which because of its vital role in virus replication, represents an attractive target for therapeutic intervention. To facilitate the discovery of nsP2 inhibitors we have developed a novel assay for quantitative measurement of nsP2pro activity in a cell-based format. The assay is based on a substrate fusion protein consisting of eGFP and Gaussia luciferase (Gluc) linked together by a small peptide containing a VEEV nsp2pro cleavage sequence. The expression of the substrate protein in cells along with recombinant nsP2pro results in cleavage of the substrate protein resulting in extracellular release of free Gluc. The Gluc activity in supernatants corresponds to intracellular nsP2pro-mediated substrate cleavage; thus, providing a simple and convenient way to quantify nsP2pro activity. Here, we demonstrate potential utility of the assay in identification of nsP2pro inhibitors, as well as in investigations related to molecular characterization of nsP2pro. - Highlights: • A novel cell-based assay to measure VEEV nsP2 protease activity was developed. • Assay utility was demonstrated for antiviral screening. • .The assay also proved to be useful in basic mechanistic studies of nsP2 protease.

A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease

Energy Technology Data Exchange (ETDEWEB)

Campos-Gomez, Javier; Ahmad, Fahim; Rodriguez, Efrain; Saeed, Mohammad F., E-mail: saeed@southernresearch.org

2016-09-15

The encephalitic alphaviruses encode nsP2 protease (nsP2pro), which because of its vital role in virus replication, represents an attractive target for therapeutic intervention. To facilitate the discovery of nsP2 inhibitors we have developed a novel assay for quantitative measurement of nsP2pro activity in a cell-based format. The assay is based on a substrate fusion protein consisting of eGFP and Gaussia luciferase (Gluc) linked together by a small peptide containing a VEEV nsp2pro cleavage sequence. The expression of the substrate protein in cells along with recombinant nsP2pro results in cleavage of the substrate protein resulting in extracellular release of free Gluc. The Gluc activity in supernatants corresponds to intracellular nsP2pro-mediated substrate cleavage; thus, providing a simple and convenient way to quantify nsP2pro activity. Here, we demonstrate potential utility of the assay in identification of nsP2pro inhibitors, as well as in investigations related to molecular characterization of nsP2pro. - Highlights: • A novel cell-based assay to measure VEEV nsP2 protease activity was developed. • Assay utility was demonstrated for antiviral screening. • .The assay also proved to be useful in basic mechanistic studies of nsP2 protease.

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.

HIV-1 protease-substrate coevolution in nelfinavir resistance.

Science.gov (United States)

Kolli, Madhavi; Ozen, Ayşegül; Kurt-Yilmaz, Nese; Schiffer, Celia A

2014-07-01

Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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

Highly potent fibrinolytic serine protease from Streptomyces.

Science.gov (United States)

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

2011-01-05

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

Proteases and antiproteases in chronic neutrophilic lung disease - relevance to drug discovery.

LENUS (Irish Health Repository)

Greene, Catherine M

2009-10-01

Chronic inflammatory lung diseases such as cystic fibrosis and emphysema are characterized by higher-than-normal levels of pulmonary proteases. While these enzymes play important roles such as bacterial killing, their dysregulated expression or activity can adversely impact on the inflammatory process. The existence of efficient endogenous control mechanisms that can dampen or halt this overexuberant protease activity in vivo is essential for the effective resolution of inflammatory lung disease. The function of pulmonary antiproteases is to fulfil this role. Interestingly, in addition to their antiprotease activity, protease inhibitors in the lung also often possess other intrinsic properties that contribute to microbial killing or termination of the inflammatory process. This review will outline important features of chronic inflammation that are regulated by pulmonary proteases and will describe the various mechanisms by which antiproteases attempt to counterbalance exaggerated protease-mediated inflammatory events. These proteases, antiproteases and their modifiers represent interesting targets for therapeutic intervention.

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.

Serine protease inhibitors of parasitic helminths.

Science.gov (United States)

Molehin, Adebayo J; Gobert, Geoffrey N; McManus, Donald P

2012-05-01

Serine protease inhibitors (serpins) are a superfamily of structurally conserved proteins that inhibit serine proteases and play key physiological roles in numerous biological systems such as blood coagulation, complement activation and inflammation. A number of serpins have now been identified in parasitic helminths with putative involvement in immune regulation and in parasite survival through interference with the host immune response. This review describes the serpins and smapins (small serine protease inhibitors) that have been identified in Ascaris spp., Brugia malayi, Ancylostoma caninum Onchocerca volvulus, Haemonchus contortus, Trichinella spiralis, Trichostrongylus vitrinus, Anisakis simplex, Trichuris suis, Schistosoma spp., Clonorchis sinensis, Paragonimus westermani and Echinococcus spp. and discusses their possible biological functions, including roles in host-parasite interplay and their evolutionary relationships.

The coagulation factor Xa/protease activated receptor-2 axis in the progression of liver fibrosis : a multifaceted paradigm

NARCIS (Netherlands)

Borensztajn, Keren; von der Thusen, Jan H.; Peppelenbosch, Maikel P.; Spek, C. Arnold

2010-01-01

Introduction Activation of the coagulation cascade during liver fibrosis: a puzzling paradox Protease-activated receptors: the link between coagulation cascade activation and liver fibrosis Expression and distribution of human PAR-2 in normal and pathological liver tissue FXa signalling on PAR-2

Production, purification and characterization of an aspartic protease from Aspergillus foetidus.

Science.gov (United States)

Souza, Paula Monteiro; Werneck, Gabriela; Aliakbarian, Bahar; Siqueira, Felix; Ferreira Filho, Edivaldo Ximenes; Perego, Patrizia; Converti, Attilio; Magalhães, Pérola Oliveira; Junior, Adalberto Pessoa

2017-11-01

An acidic thermostable protease was extracellularly produced either in shake flask or in stirred tank bioreactor by an Aspergillus foetidus strain isolated from the Brazilian savanna soil using different nitrogen sources. Its maximum activity (63.7 U mL -1 ) was obtained in a medium containing 2% (w/v) peptone. A cultivation carried out in a 5.0 L stirred-tank bioreactor provided a maximum protease activity 9% lower than that observed in Erlenmeyer flasks, which was obtained after a significantly shorter (by 16-29%) time. Protease purification by a combination of gel-filtration chromatography resulted in a 16.9-fold increase in specific activity (248.1 U g -1 ). The estimated molecular weight of the purified enzyme was 50.6 kDa, and the optimal pH and temperature were 5.0 and 55 °C, respectively. The enzyme was completely inhibited by pepstatin A, and its activity enhanced by some metals. According to the inhibition profiles, it was confirmed that the purified acid protease belongs to the aspartic protease type. These results are quite promising for future development of large-scale production of such protease, which can be useful in biotechnological applications requiring high enzyme activity and stability under acidic conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Molecular cloning and immunochemical characterization of a novel major Japanese cedar pollen allergen belonging to the aspartic protease family.

Science.gov (United States)

Ibrahim, Ahmed Ragaa Nour; Kawamoto, Seiji; Aki, Tsunehiro; Shimada, Yayoi; Rikimaru, Satoshi; Onishi, Nobukazu; Babiker, Elfadil Elfadl; Oiso, Isao; Hashimoto, Kunihiko; Hayashi, Takaharu; Ono, Kazuhisa

2010-01-01

Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis in Japan. Protease activity in the pollen grains may trigger pro-allergic responses but no such proteases have yet been identified as pollen allergens. We report the molecular cloning and immunochemical characterization of a novel C. japonica pollen allergen belonging to the aspartic protease family. We focused on the C. japonica pollen allergen spot No. 63 (CPA63, 47.5% IgE binding frequency) on our 2-dimensional IgE immunoblot map. The internal amino acid sequences were determined using time-of-flight mass spectrometry. Full-length cpa63 cDNA was cloned by rapid amplification of cDNA ends (RACE)-PCR. Recombinant CPA63 (r-CPA63) was expressed using the baculovirus-insect cell culture system and its IgE binding capacity was analyzed by enzyme-linked immunosorbent assay (ELISA). The proteolytic activity of r-CPA63 was also assessed using a putative mature enzyme produced upon autolysis. cpa63 cDNA encoded a 472 amino acid polypeptide showing about 40% sequence identity to members of the plant atypical aspartic protease family. ELISA showed that r-CPA63 was recognized by IgE antibodies in the serum of 58% (18/31) of Japanese cedar pollinosis patients. We also demonstrated an aspartic protease-like enzyme activity of the putative mature r-CPA63. We have identified the first plant aspartic protease allergen from Japanese cedar pollen. The availability of the CPA63 sequence and its recombinant allergen production system are useful not only for pharmaceutical applications but also for further examination of the role of protease activity in the pathogenesis of cedar pollinosis. 2010 S. Karger AG, Basel.

Molecular adaptation of a plant-bacterium outer membrane protease towards plague virulence factor Pla

Science.gov (United States)

2011-01-01

Background Omptins are a family of outer membrane proteases that have spread by horizontal gene transfer in Gram-negative bacteria that infect vertebrates or plants. Despite structural similarity, the molecular functions of omptins differ in a manner that reflects the life style of their host bacteria. To simulate the molecular adaptation of omptins, we applied site-specific mutagenesis to make Epo of the plant pathogenic Erwinia pyrifoliae exhibit virulence-associated functions of its close homolog, the plasminogen activator Pla of Yersinia pestis. We addressed three virulence-associated functions exhibited by Pla, i.e., proteolytic activation of plasminogen, proteolytic degradation of serine protease inhibitors, and invasion into human cells. Results Pla and Epo expressed in Escherichia coli are both functional endopeptidases and cleave human serine protease inhibitors, but Epo failed to activate plasminogen and to mediate invasion into a human endothelial-like cell line. Swapping of ten amino acid residues at two surface loops of Pla and Epo introduced plasminogen activation capacity in Epo and inactivated the function in Pla. We also compared the structure of Pla and the modeled structure of Epo to analyze the structural variations that could rationalize the different proteolytic activities. Epo-expressing bacteria managed to invade human cells only after all extramembranous residues that differ between Pla and Epo and the first transmembrane β-strand had been changed. Conclusions We describe molecular adaptation of a protease from an environmental setting towards a virulence factor detrimental for humans. Our results stress the evolvability of bacterial β-barrel surface structures and the environment as a source of progenitor virulence molecules of human pathogens. PMID:21310089

In silico insights into protein-protein interactions and folding dynamics of the saposin-like domain of Solanum tuberosum aspartic protease.

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Dref C De Moura

Full Text Available The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L. plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family.

Protease-activated receptor-2 stimulates intestinal epithelial chloride transport through activation of PLC and selective PKC isoforms.

Science.gov (United States)

van der Merwe, Jacques Q; Moreau, France; MacNaughton, Wallace K

2009-06-01

Serine proteases play important physiological roles through their activity at G protein-coupled protease-activated receptors (PARs). We examined the roles that specific phospholipase (PL) C and protein kinase (PK) C (PKC) isoforms play in the regulation of PAR(2)-stimulated chloride secretion in intestinal epithelial cells. Confluent SCBN epithelial monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short-circuit current (I(sc)) responses to basolateral application of the selective PAR(2) activating peptide, SLIGRL-NH(2), were monitored as a measure of net electrogenic ion transport caused by PAR(2) activation. SLIGRL-NH(2) induced a transient I(sc) response that was significantly reduced by inhibitors of PLC (U73122), phosphoinositol-PLC (ET-18), phosphatidylcholine-PLC (D609), and phosphatidylinositol 3-kinase (PI3K; LY294002). Immunoblot analysis revealed the phosphorylation of both PLCbeta and PLCgamma following PAR(2) activation. Pretreatment of the cells with inhibitors of PKC (GF 109203X), PKCalpha/betaI (Gö6976), and PKCdelta (rottlerin), but not PKCzeta (selective pseudosubstrate inhibitor), also attenuated this response. Cellular fractionation and immunoblot analysis, as well as confocal immunocytochemistry, revealed increases of PKCbetaI, PKCdelta, and PKCepsilon, but not PKCalpha or PKCzeta, in membrane fractions following PAR(2) activation. Pretreatment of the cells with U73122, ET-18, or D609 inhibited PKC activation. Inhibition of PI3K activity only prevented PKCdelta translocation. Immunoblots revealed that PAR(2) activation induced phosphorylation of both cRaf and ERK1/2 via PKCdelta. Inhibition of PKCbetaI and PI3K had only a partial effect on this response. We conclude that basolateral PAR(2)-induced chloride secretion involves activation of PKCbetaI and PKCdelta via a PLC-dependent mechanism resulting in the stimulation of cRaf and ERK1/2 signaling.

Barley (Hordeum vulgare L.) cysteine proteases: heterologous expression, purification and characterization

DEFF Research Database (Denmark)

Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach

2011-01-01

During germination of barley seeds, mobilization of protein is essential and cysteine proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins. Cysteine proteases exist as pro-enzyme and is activated through reduction of the active...... site cysteines and by removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and efficient eukaryotic expression platform is highly desirable. A cDNA clone of the barley key cysteine endoprotease...

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-.

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

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...

The mimivirus R355 gene product: preliminary crystallographic analysis of a putative ubiquitin-like protein-specific protease

International Nuclear Information System (INIS)

Jeudy, Sandra; Lartigue, Audrey; Mansuelle, Pascal; Ogata, Yuki; Abergel, Chantal

2010-01-01

The genome sequence of mimivirus, the largest known double-stranded DNA virus, encodes a putative protease: the R355 gene product. Its expression in E. coli, its crystallization and the preliminary phasing of a MAD data set using the selenium signal present in a crystal of recombinant selenomethionine-substituted protein are reported. The complete genome sequence of the largest known double-stranded DNA virus, mimivirus, reveals the presence of a gene (denoted R355) that potentially encodes a cysteine protease that is expressed late (after 6 h) in the infectious cycle of the virus. In order to verify a sequence-based functional prediction and understand its role during the infectious process, the R355 protein was produced to assay its proteolytic activity and solve its three-dimensional structure. Here, the preliminary crystallographic analysis of the recombinant viral protein is reported. The crystals belonged to the orthorhombic space group P2 1 2 1 2 1 , with a monomer in the asymmetric unit. A MAD data set was used for preliminary phasing using the selenium signal from a selenomethionine-substituted protein crystal

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.

Isolation of microorganisms with chinitase, protease and keratinase activities from petroleum contaminated soils

International Nuclear Information System (INIS)

Cervantes-Gonzalez, E.; Rojas-Avelizapa, L.; Cruz-Camarillo, R.; Rojas-Avelizapa, N.G.

2005-01-01

The most important part in one process of bio-remediation are the microorganisms with the capacities to degrade target compounds, this research is based to find microorganisms hydrocarbon-clastic with enzyme activities to degrade chicken feather (keratinolytic activity) which is also a contaminant and has been used such as sorbent of petroleum and can be composted after the oil spill cleanup is complete, the isolation was also to degrade shrimp waste (chitinolitic and proteolitic activity) which is waste material that can be used in compost or such as sorbent of petroleum too. We isolated mesofilic aerobic microorganisms from mexican soils located in Tabasco, Mexico. We achieved to isolate 105 bacteria from 10 soils, 90% was Bacillus Gram (-) which are common in soils and all were hydrocarbon-clastic, only 7 different bacteria had protease and chitinase activity and 12 bacteria had keratinase activity. So we found three fungi and one actinomycete with capacity to degrade hydrocarbons and presence of chitinase activity. The results of growth and enzyme activities in liquid culture showed that the protease activity was produced between 18 and 48 h in almost all bacteria, the chitinase activity started at 12 h but was slight , only 0.5 U/ml, and the keratinase activity was produced after 6 h of incubation and there were correlation between logarithmic phase of growth and enzymes production. With this study we showed the existence of some enzyme activities from microorganisms that live in hostile habitats. This, can be useful in bio-treatment soils by the possible use of this type of residues that can be bio-degraded at the same time that the hydrocarbons increasing the speed or the quality of cleanup in soils. (authors)

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.

Cloned Bacillus subtilis alkaline protease (aprA) gene showing high level of keratinolytic activity.

Science.gov (United States)

Zaghloul, T I

1998-01-01

The Bacillus subtilis alkaline protease(aprA) gene was previously cloned on a pUBHO-derivative plasmid. High levels of expression and gene stability were demonstrated when B. subtilis cells were grown on the laboratory medium 2XSG. B. subtilis cells harboring the multicopy aprA gene were grown on basal medium, supplemented with 1 % chicken feather as a source of energy, carbon, and nitrogen. Proteolytic and keratinolytic activities were monitored throughout the cultivation time. A high level of keratinolytic activity was obtained, and this indicates that alkaline protease is acting as a keratinase. Furthermore, considerable amounts of soluble proteins and free amino acids were obtained as a result of the enzymatic hydrolysis of feather. Biodegradation of feather waste using these cells represents an alternative way to improve the nutritional value of feather, since feather waste is currently utilized on a limited basis as a dietary protein supplement for animal feedstuffs. Moreover, the release of free amino acids from feather and the secreted keratinase enzyme would promote industries based on feather waste.

Factor VII-activating protease in patients with acute deep venous thrombosis

DEFF Research Database (Denmark)

Sidelmann, Johannes J; Vitzthum, Frank; Funding, Eva

2008-01-01

Factor VII-activating protease (FSAP) is involved in haemostasis and inflammation. FSAP cleaves single chain urokinase-type plasminogen activator (scu-PA). The 1601GA genotype of the 1601G/A polymorphism in the FSAP gene leads to the expression of a FSAP variant with reduced ability to activate scu......-PA, without affecting the ability to activate coagulation Factor VII (FVII). Previous studies have investigated the association of the 1601GA genotype with incidence and progression of carotid stenosis and deep venous thrombosis (DVT). The present study is the first to evaluate the potential association...... between the FSAP phenotype and DVT. We studied the association between the 1601G/A polymorphism, FSAP activity, FSAP antigen, Factor VIIa (FVIIa), prothrombin fragment 1+2 (F1+2), and C-reactive protein (CRP) in plasmas of 170 patients suspected for DVT. FSAP genotypes were equally distributed in patients...

Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: comparative analysis of structure and activities.

Science.gov (United States)

Qian, Ye-Qing; Li, Ye; Yang, Fan; Yu, Yan-Qin; Yang, Jin-Shu; Yang, Wei-Jun

2012-03-01

Kazal-type inhibitors (KPIs) play important roles in many biological and physiological processes, such as blood clotting, the immune response and reproduction. In the present study, two male reproductive tract KPIs, termed Man-KPI and Ers-KPI, were identified in Macrobrachium nipponense and Eriocheir sinensis, respectively. The inhibitory activities of recombinant Man-KPI and Ers-KPI against chymotrypsin, elastase, trypsin and thrombin were determined. The results showed that both of them strongly inhibit chymotrypsin and elastase. Kinetic studies were performed to elucidate their inhibition mechanism. Furthermore, individual domains were also expressed to learn further which domain contributes to the inhibitory activities of intact KPIs. Only Man-KPI_domain3 is active in the inhibition of chymotrypsin and elastase. Meanwhile, Ers-KPI_domain2 and 3 are responsible for inhibition of chymotrypsin, and Ers-KPI_domains2, 3 and 4 are responsible for the inhibition of elastase. Meanwhile, the inhibitory activities of these two KPIs toward Macrobrachium rosenbergii, M. nipponense and E. sinensis sperm were compared with that of the Kazal-type peptidase inhibitor (MRPINK) characterized from the M. rosenbergii reproductive tract in a previous study. The results demonstrated that KPIs can completely inhibit the gelatinolytic activities of sperm proteases from their own species, while different levels of cross-inhibition were observed between KPI and proteases from different species. These results may provide new perspective to further clarify the mechanism of KPI-proteases interaction in the male reproductive system. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

ISOLASI DAN KARAKTERISASI PROTEASE ALKALIN DARI ISOLAT BAKTERI LIMBAH TERNAK DI EXFARM FAKULTAS PETERNAKAN UNSOED

Directory of Open Access Journals (Sweden)

Zusfahair

2011-05-01

Full Text Available Protease is one of the widely used enzymes for the industry. The potential resource of microorganism that produced protease is milk cow waste. In this research, isolation and characterization has been done toward isolated protease from milk cow waste of the Exfarm’s Animal Husbandry Faculty at University of Jenderal Soedirman, Purwokerto. The research used experiment method and the parameters observed were the genus of bacteria which produce protease and the activity of protease. The characterizations of protease were determination of optimum pH and temperature, the influence of metal ions, EDTA, surfactant, and commercial detergent toward enzyme activity, and also the study of enzyme stability. The results from the research showed that the isolated bacteria from the Exfarm’s of Animal Husbandry Faculty of UNSOED, which produced protease was Salmonella sp. Characterization of isolated Salmonella sp. from 45% ammonium sulphate fraction indicated that the optimum temperature was 50 ºC, optimum pH was 8, the enzyme was activated by Ca2+ dan Mg2+ ion, whereas it was inhibited by Zn2+, Cu2+ ions and EDTA. The addition of Tween-80 with the concentration of 0.2% and 0.4% increased protease activity, however the addition of Tween-80 with concentration higher than 0.6% decreased the protease activity. Enzyme protease from isolated Salmonella sp. was relatively stable with the addition of commercial detergent such as Attack, Surf, and Bukrim.

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

Hepatitis C Virus NS3/4A Protease Inhibitors Incorporating Flexible P2 Quinoxalines Target Drug Resistant Viral Variants.

Science.gov (United States)

Matthew, Ashley N; Zephyr, Jacqueto; Hill, Caitlin J; Jahangir, Muhammad; Newton, Alicia; Petropoulos, Christos J; Huang, Wei; Kurt-Yilmaz, Nese; Schiffer, Celia A; Ali, Akbar

2017-07-13

A substrate envelope-guided design strategy is reported for improving the resistance profile of HCV NS3/4A protease inhibitors. Analogues of 5172-mcP1P3 were designed by incorporating diverse quinoxalines at the P2 position that predominantly interact with the invariant catalytic triad of the protease. Exploration of structure-activity relationships showed that inhibitors with small hydrophobic substituents at the 3-position of P2 quinoxaline maintain better potency against drug resistant variants, likely due to reduced interactions with residues in the S2 subsite. In contrast, inhibitors with larger groups at this position were highly susceptible to mutations at Arg155, Ala156, and Asp168. Excitingly, several inhibitors exhibited exceptional potency profiles with EC 50 values ≤5 nM against major drug resistant HCV variants. These findings support that inhibitors designed to interact with evolutionarily constrained regions of the protease, while avoiding interactions with residues not essential for substrate recognition, are less likely to be susceptible to drug resistance.

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.

Science.gov (United States)

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.

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.

Characterization of the Protease Activity of Detergents: Laboratory Practicals for Studying the Protease Profile and Activity of Various Commercial Detergents

Science.gov (United States)

Valls, Cristina; Pujadas, Gerard; Garcia-Vallve, Santi; Mulero, Miquel

2011-01-01

Detergent enzymes account for about 30% of the total worldwide production of enzymes and are one of the largest and most successful applications of modern industrial biotechnology. Proteases can improve the wash performance of household, industrial, and institutional laundry detergents used to remove protein-based stains such as blood, grass, body…

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.

Extracellular Protease of Pseudomonas fluorescens CHA0, a Biocontrol Factor with Activity against the Root-Knot Nematode Meloidogyne incognita

OpenAIRE

Siddiqui, Imran Ali; Haas, Dieter; Heeb, Stephan

2005-01-01

In Pseudomonas fluorescens CHA0, mutation of the GacA-controlled aprA gene (encoding the major extracellular protease) or the gacA regulatory gene resulted in reduced biocontrol activity against the root-knot nematode Meloidogyne incognita during tomato and soybean infection. Culture supernatants of strain CHA0 inhibited egg hatching and induced mortality of M. incognita juveniles more strongly than did supernatants of aprA and gacA mutants, suggesting that AprA protease contributes to biocon...

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.

Profiling gene expression induced by protease-activated receptor 2 (PAR2 activation in human kidney cells.

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Jacky Y Suen

Full Text Available Protease-Activated Receptor-2 (PAR2 has been implicated through genetic knockout mice with cytokine regulation and arthritis development. Many studies have associated PAR2 with inflammatory conditions (arthritis, airways inflammation, IBD and key events in tumor progression (angiogenesis, metastasis, but they have relied heavily on the use of single agonists to identify physiological roles for PAR2. However such probes are now known not to be highly selective for PAR2, and thus precisely what PAR2 does and what mechanisms of downstream regulation are truly affected remain obscure. Effects of PAR2 activation on gene expression in Human Embryonic Kidney cells (HEK293, a commonly studied cell line in PAR2 research, were investigated here by comparing 19,000 human genes for intersecting up- or down-regulation by both trypsin (an endogenous protease that activates PAR2 and a PAR2 activating hexapeptide (2f-LIGRLO-NH(2. Among 2,500 human genes regulated similarly by both agonists, there were clear associations between PAR2 activation and cellular metabolism (1,000 genes, the cell cycle, the MAPK pathway, HDAC and sirtuin enzymes, inflammatory cytokines, and anti-complement function. PAR-2 activation up-regulated four genes more than 5 fold (DUSP6, WWOX, AREG, SERPINB2 and down-regulated another six genes more than 3 fold (TXNIP, RARG, ITGB4, CTSD, MSC and TM4SF15. Both PAR2 and PAR1 activation resulted in up-regulated expression of several genes (CD44, FOSL1, TNFRSF12A, RAB3A, COPEB, CORO1C, THBS1, SDC4 known to be important in cancer. This is the first widespread profiling of specific activation of PAR2 and provides a valuable platform for better understanding key mechanistic roles of PAR2 in human physiology. Results clearly support the development of both antagonists and agonists of human PAR2 as potential disease modifying therapeutic agents.

Microbial alkaline proteases: Optimization of production parameters and their properties

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Kanupriya Miglani Sharma

2017-06-01

Full Text Available Proteases are hydrolytic enzymes capable of degrading proteins into small peptides and amino acids. They account for nearly 60% of the total industrial enzyme market. Proteases are extensively exploited commercially, in food, pharmaceutical, leather and detergent industry. Given their potential use, there has been renewed interest in the discovery of proteases with novel properties and a constant thrust to optimize the enzyme production. This review summarizes a fraction of the enormous reports available on various aspects of alkaline proteases. Diverse sources for isolation of alkaline protease producing microorganisms are reported. The various nutritional and environmental parameters affecting the production of alkaline proteases in submerged and solid state fermentation are described. The enzymatic and physicochemical properties of alkaline proteases from several microorganisms are discussed which can help to identify enzymes with high activity and stability over extreme pH and temperature, so that they can be developed for industrial applications.

Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target

Science.gov (United States)

Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; McKerrow, James H.

1999-09-01

Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

Analysis of serine proteases from marine sponges by 2-D zymography.

Science.gov (United States)

Wilkesman, Jeff G; Schröder, Heinz C

2007-02-01

Proteolytic activities isolated from the marine demosponges Geodia cydonium and Suberites domuncula were analyzed by 2-D zymography, a technique that combines IEF and zymography. After purification, a 200 kDa proteolytically active protein band was obtained from G. cydonium when analyzed in gelatin copolymerized 1-D zymograms. The enzymatic activity was quantified using alpha-N-benzoyl-D-arginine p-nitroanilide (BAPNA) as a substrate and corresponded to a serine protease. The protease activity was resistant to urea and SDS. DTT and 2-mercaptoethanol (2-ME) did not significantly change the protease activity, but induced a shift in molecular mass of the proteolytic band to lower M(r) values as detected by zymography. Under mild denaturing conditions, lower M(r) bands (zymography, the protease from G. cydonium revealed a pI of 8.0 and an M(r) shift from 200 to 66 kDa. To contrast these results, a cytosolic sample from S. domuncula was analyzed. The proteolytic activity of this sponge after 2-D zymography corresponded to an M(r) of 40 kDa and a pI of 4.0. The biological function of both sponge proteases is not yet known. This study demonstrates that mild denaturing conditions required for IEF may alter the interpretation of the 2-D zymography, and care must be taken during sample preparation.

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...

High-resolution structure of a retroviral protease folded as a monomer

International Nuclear Information System (INIS)

Gilski, Miroslaw; Kazmierczyk, Maciej; Krzywda, Szymon; Zábranská, Helena; Cooper, Seth; Popović, Zoran; Khatib, Firas; DiMaio, Frank; Thompson, James; Baker, David; Pichová, Iva; Jaskolski, Mariusz

2011-01-01

The crystal structure of Mason–Pfizer monkey virus protease folded as a monomer has been solved by molecular replacement using a model generated by players of the online game Foldit. The structure shows at high resolution the details of a retroviral protease folded as a monomer which can guide rational design of protease dimerization inhibitors as retroviral drugs. Mason–Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerization of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the C α deviations are large and the active-site ‘DTG’ loop (here NTG) deviates up to 2.7 Å from the standard conformation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections

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.

High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13.

Science.gov (United States)

Kretz, Colin A; Tomberg, Kärt; Van Esbroeck, Alexander; Yee, Andrew; Ginsburg, David

2018-02-12

We have combined random 6 amino acid substrate phage display with high throughput sequencing to comprehensively define the active site specificity of the serine protease thrombin and the metalloprotease ADAMTS13. The substrate motif for thrombin was determined by >6,700 cleaved peptides, and was highly concordant with previous studies. In contrast, ADAMTS13 cleaved only 96 peptides (out of >10 7 sequences), with no apparent consensus motif. However, when the hexapeptide library was substituted into the P3-P3' interval of VWF73, an exosite-engaging substrate of ADAMTS13, 1670 unique peptides were cleaved. ADAMTS13 exhibited a general preference for aliphatic amino acids throughout the P3-P3' interval, except at P2 where Arg was tolerated. The cleaved peptides assembled into a motif dominated by P3 Leu, and bulky aliphatic residues at P1 and P1'. Overall, the P3-P2' amino acid sequence of von Willebrand Factor appears optimally evolved for ADAMTS13 recognition. These data confirm the critical role of exosite engagement for substrates to gain access to the active site of ADAMTS13, and define the substrate recognition motif for ADAMTS13. Combining substrate phage display with high throughput sequencing is a powerful approach for comprehensively defining the active site specificity of proteases.

Modularly Constructed Synthetic Granzyme B Molecule Enables Interrogation of Intracellular Proteases for Targeted Cytotoxicity.

Science.gov (United States)

Ho, Patrick; Ede, Christopher; Chen, Yvonne Y

2017-08-18

Targeted therapies promise to increase the safety and efficacy of treatments against diseases ranging from cancer to viral infections. However, the vast majority of targeted therapeutics relies on the recognition of extracellular biomarkers, which are rarely restricted to diseased cells and are thus prone to severe and sometimes-fatal off-target toxicities. In contrast, intracellular antigens present a diverse yet underutilized repertoire of disease markers. Here, we report a protein-based therapeutic platform-termed Cytoplasmic Oncoprotein VErifier and Response Trigger (COVERT)-which enables the interrogation of intracellular proteases to trigger targeted cytotoxicity. COVERT molecules consist of the cytotoxic protein granzyme B (GrB) fused to an inhibitory N-terminal peptide, which can be removed by researcher-specified proteases to activate GrB function. We demonstrate that fusion of a small ubiquitin-like modifier 1 (SUMO1) protein to GrB yields a SUMO-GrB molecule that is specifically activated by the cancer-associated sentrin-specific protease 1 (SENP1). SUMO-GrB selectively triggers apoptotic phenotypes in HEK293T cells that overexpress SENP1, and it is highly sensitive to different SENP1 levels across cell lines. We further demonstrate the rational design of additional COVERT molecules responsive to enterokinase (EK) and tobacco etch virus protease (TEVp), highlighting the COVERT platform's modularity and adaptability to diverse protease targets. As an initial step toward engineering COVERT-T cells for adoptive T-cell therapy, we verified that primary human T cells can express, package, traffic, and deliver engineered GrB molecules in response to antigen stimulation. Our findings set the foundation for future intracellular-antigen-responsive therapeutics that can complement surface-targeted therapies.

Plasma factor VII-activating protease is increased by oral contraceptives and induces factor VII activation in-vivo

DEFF Research Database (Denmark)

Sidelmann, Johannes J; Skouby, Sven O; Kluft, Cornelis

2011-01-01

Oral contraceptive (OC) use influences the hemostatic system significantly and is a risk factor for development of cardiovascular disease. Factor VII-activating protease (FSAP) has potential effects on hemostasis. The 1601GA genotype of the 1601G/A polymorphism in the FSAP gene expresses a FSAP...... progestins. FSAP genotypes, FSAP and factor VII (FVII) plasma measures were assessed at baseline and after 6 cycles of OC. The 1601GA genotype was present in 49 (8.3%) of the women and was associated with significantly reduced levels of FSAP (P=0.001). OC use increased FSAP antigen by 25% and FSAP activity...

Isolation, identification and characterization of organic solvent tolerant protease from Bacillus sp. DAF-01

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Arastoo Badoei-Dalfard

2012-01-01

Full Text Available Introduction: Organic solvent-tolerant bacteria are relatively novel extermophilic microorganisms, which can produce organic tolerant protease with capacity of being used in industrial biotechnology for producing high-value compounds. Therefore, finding of these bacteria has drawn much researchers attention nowadays. Materials and Methods: In this project, samples were collected from a hot spring, located in Jiroft. Samples were incubated in medium supplemented with cyclohexane and toluene for 3 days. Screening of protease producing bacteria was performed on the specific media, SKM (Skim milk agar, based on clear area diameter. The best bacterium was identified based on 16s rDNA gene. Protease activity was considered in different temperatures, pH and organic solvents.Results: Sequence alignment and phylogenetic tree results showed that this bacteria was closely related to Bacillus niacini, with 97% homology. Enzymatic studies showed that, this enzyme was active at a wide range of temperatures, 20-90 °C and it,s optimal activity was in 60 °C. In addition, maximum protease activity was obtained in the 8-9 range of pH, and optimal stability was also at pH 9.0. Protease activity in the presence of methanol, toluene, isopropanol, cyclohexane and DMF ‏showed that, remaining activity was at least 80% compared to the control (without organic solvent Discussion and Conclusion: Thermopilic capacity, being active in alkaline protease and high protease stability in the presence of organic solvents all herald a remarkable application for using in different industries.

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

Science.gov (United States)

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

2002-09-25

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

Extracellular Protease of Pseudomonas fluorescens CHA0, a Biocontrol Factor with Activity against the Root-Knot Nematode Meloidogyne incognita

Science.gov (United States)

Siddiqui, Imran Ali; Haas, Dieter; Heeb, Stephan

2005-01-01

In Pseudomonas fluorescens CHA0, mutation of the GacA-controlled aprA gene (encoding the major extracellular protease) or the gacA regulatory gene resulted in reduced biocontrol activity against the root-knot nematode Meloidogyne incognita during tomato and soybean infection. Culture supernatants of strain CHA0 inhibited egg hatching and induced mortality of M. incognita juveniles more strongly than did supernatants of aprA and gacA mutants, suggesting that AprA protease contributes to biocontrol. PMID:16151170

Human eosinophils constitutively express a unique serine protease, PRSS33.

Science.gov (United States)

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

2017-07-01

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

Structural and Functional Analysis of Human HtrA3 Protease and Its Subdomains.

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Przemyslaw Glaza

Full Text Available Human HtrA3 protease, which induces mitochondria-mediated apoptosis, can be a tumor suppressor and a potential therapeutic target in the treatment of cancer. However, there is little information about its structure and biochemical properties. HtrA3 is composed of an N-terminal domain not required for proteolytic activity, a central serine protease domain and a C-terminal PDZ domain. HtrA3S, its short natural isoform, lacks the PDZ domain which is substituted by a stretch of 7 C-terminal amino acid residues, unique for this isoform. This paper presents the crystal structure of the HtrA3 protease domain together with the PDZ domain (ΔN-HtrA3, showing that the protein forms a trimer whose protease domains are similar to those of human HtrA1 and HtrA2. The ΔN-HtrA3 PDZ domains are placed in a position intermediate between that in the flat saucer-like HtrA1 SAXS structure and the compact pyramidal HtrA2 X-ray structure. The PDZ domain interacts closely with the LB loop of the protease domain in a way not found in other human HtrAs. ΔN-HtrA3 with the PDZ removed (ΔN-HtrA3-ΔPDZ and an N-terminally truncated HtrA3S (ΔN-HtrA3S were fully active at a wide range of temperatures and their substrate affinity was not impaired. This indicates that the PDZ domain is dispensable for HtrA3 activity. As determined by size exclusion chromatography, ΔN-HtrA3 formed stable trimers while both ΔN-HtrA3-ΔPDZ and ΔN-HtrA3S were monomeric. This suggests that the presence of the PDZ domain, unlike in HtrA1 and HtrA2, influences HtrA3 trimer formation. The unique C-terminal sequence of ΔN-HtrA3S appeared to have little effect on activity and oligomerization. Additionally, we examined the cleavage specificity of ΔN-HtrA3. Results reported in this paper provide new insights into the structure and function of ΔN-HtrA3, which seems to have a unique combination of features among human HtrA proteases.

Structural and Functional Analysis of Human HtrA3 Protease and Its Subdomains.

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Glaza, Przemyslaw; Osipiuk, Jerzy; Wenta, Tomasz; Zurawa-Janicka, Dorota; Jarzab, Miroslaw; Lesner, Adam; Banecki, Bogdan; Skorko-Glonek, Joanna; Joachimiak, Andrzej; Lipinska, Barbara

2015-01-01

Human HtrA3 protease, which induces mitochondria-mediated apoptosis, can be a tumor suppressor and a potential therapeutic target in the treatment of cancer. However, there is little information about its structure and biochemical properties. HtrA3 is composed of an N-terminal domain not required for proteolytic activity, a central serine protease domain and a C-terminal PDZ domain. HtrA3S, its short natural isoform, lacks the PDZ domain which is substituted by a stretch of 7 C-terminal amino acid residues, unique for this isoform. This paper presents the crystal structure of the HtrA3 protease domain together with the PDZ domain (ΔN-HtrA3), showing that the protein forms a trimer whose protease domains are similar to those of human HtrA1 and HtrA2. The ΔN-HtrA3 PDZ domains are placed in a position intermediate between that in the flat saucer-like HtrA1 SAXS structure and the compact pyramidal HtrA2 X-ray structure. The PDZ domain interacts closely with the LB loop of the protease domain in a way not found in other human HtrAs. ΔN-HtrA3 with the PDZ removed (ΔN-HtrA3-ΔPDZ) and an N-terminally truncated HtrA3S (ΔN-HtrA3S) were fully active at a wide range of temperatures and their substrate affinity was not impaired. This indicates that the PDZ domain is dispensable for HtrA3 activity. As determined by size exclusion chromatography, ΔN-HtrA3 formed stable trimers while both ΔN-HtrA3-ΔPDZ and ΔN-HtrA3S were monomeric. This suggests that the presence of the PDZ domain, unlike in HtrA1 and HtrA2, influences HtrA3 trimer formation. The unique C-terminal sequence of ΔN-HtrA3S appeared to have little effect on activity and oligomerization. Additionally, we examined the cleavage specificity of ΔN-HtrA3. Results reported in this paper provide new insights into the structure and function of ΔN-HtrA3, which seems to have a unique combination of features among human HtrA proteases.

Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities.

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Pelliccia, Sveva; Wu, Yu-Hsuan; Coluccia, Antonio; La Regina, Giuseppe; Tseng, Chin-Kai; Famiglini, Valeria; Masci, Domiziana; Hiscott, John; Lee, Jin-Ching; Silvestri, Romano

2017-12-01

Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes - NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases.

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.

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

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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.

The HtrA-like serine protease PepD interacts with and modulates the Mycobacterium tuberculosis 35-kDa antigen outer envelope protein.

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Mark J White

2011-03-01

Full Text Available Mycobacterium tuberculosis remains a significant global health concern largely due to its ability to persist for extended periods within the granuloma of the host. While residing within the granuloma, the tubercle bacilli are likely to be exposed to stress that can result in formation of aberrant proteins with altered structures. Bacteria encode stress responsive determinants such as proteases and chaperones to deal with misfolded or unfolded proteins. pepD encodes an HtrA-like serine protease and is thought to process proteins altered following exposure of M. tuberculosis to extra-cytoplasmic stress. PepD functions both as a protease and chaperone in vitro, and is required for aspects of M. tuberculosis virulence in vivo. pepD is directly regulated by the stress-responsive two-component signal transduction system MprAB and indirectly by extracytoplasmic function (ECF sigma factor SigE. Loss of PepD also impacts expression of other stress-responsive determinants in M. tuberculosis. To further understand the role of PepD in stress adaptation by M. tuberculosis, a proteomics approach was taken to identify binding proteins and possible substrates of this protein. Using subcellular fractionation, the cellular localization of wild-type and PepD variants was determined. Purified fractions as well as whole cell lysates from Mycobacterium smegmatis or M. tuberculosis strains expressing a catalytically compromised PepD variant were immunoprecipitated for PepD and subjected to LC-MS/MS analyses. Using this strategy, the 35-kDa antigen encoding a homolog of the PspA phage shock protein was identified as a predominant binding partner and substrate of PepD. We postulate that proteolytic cleavage of the 35-kDa antigen by PepD helps maintain cell wall homeostasis in Mycobacterium and regulates specific stress response pathways during periods of extracytoplasmic stress.

Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells.

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Mandal, Manoj K; Fischer, Rainer; Schillberg, Stefan; Schiermeyer, Andreas

2014-08-01

Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease-deficient tobacco BY-2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY-2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full-length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV-1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four-fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N-terminal sequencing data revealed that the antibody has two cleavage sites within the CDR-H3 and one site at the end of the H4-framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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).

Mycoplasma hyorhinis-Contaminated Cell Lines Activate Primary Innate Immune Cells via a Protease-Sensitive Factor.

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Simon Heidegger

Full Text Available Mycoplasma are a frequent and occult contaminant of cell cultures, whereby these prokaryotic organisms can modify many aspects of cell physiology, rendering experiments that are conducted with such contaminated cells problematic. Chronic Mycoplasma contamination in human monocytic cells lines has been associated with suppressed Toll-like receptor (TLR function. In contrast, we show here that components derived from a Mycoplasma hyorhinis-infected cell line can activate innate immunity in non-infected primary immune cells. Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2. Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment. We further show that in plasmacytoid dendritic cells, soluble factors from Mycoplasma hyorhinis-infected cells induce the production of large amounts of IFN-α. We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments.

Inactivation of brain mitochondrial Lon protease by peroxynitrite precedes electron transport chain dysfunction.

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Stanyer, Lee; Jorgensen, Wenche; Hori, Osamu; Clark, John B; Heales, Simon J R

2008-09-01

The accumulation of oxidatively modified proteins has been shown to be a characteristic feature of many neurodegenerative disorders and its regulation requires efficient proteolytic processing. One component of the mitochondrial proteolytic system is Lon, an ATP-dependent protease that has been shown to degrade oxidatively modified aconitase in vitro and may thus play a role in defending against the accumulation of oxidized matrix proteins in mitochondria. Using an assay system that allowed us to distinguish between basal and ATP-stimulated Lon protease activity, we have shown in isolated non-synaptic rat brain mitochondria that Lon protease is highly susceptible to oxidative inactivation by peroxynitrite (ONOO(-)). This susceptibility was more pronounced with regard to ATP-stimulated activity, which was inhibited by 75% in the presence of a bolus addition of 1mM ONOO(-), whereas basal unstimulated activity was inhibited by 45%. Treatment of mitochondria with a range of peroxynitrite concentrations (10-1000 microM) revealed that a decline in Lon protease activity preceded electron transport chain (ETC) dysfunction (complex I, II-III and IV) and that ATP-stimulated activity was approximately fivefold more sensitive than basal Lon protease activity. Furthermore, supplementation of mitochondrial matrix extracts with reduced glutathione, following ONOO(-) exposure, resulted in partial restoration of basal and ATP-stimulated activity, thus suggesting possible redox regulation of this enzyme complex. Taken together these findings suggest that Lon protease may be particularly vulnerable to inactivation in conditions associated with GSH depletion and elevated oxidative stress.

Detection of protease activity by fluorescent protein FRET sensors: from computer simulation to live cells

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Goryashchenko, Alexander S.; Khrenova, Maria G.; Savitsky, Alexander P.

2018-04-01

Förster resonance energy transfer (FRET) sensors are widely used for the detection of protease activity in vitro and in vivo. Usually they consist of a FRET pair connected with a polypeptide linker containing a specific cleavage site for the relevant protease. Use of the fluorescent proteins as components of the FRET pair allows genetic encoding of such sensors and solves the problem of their delivery into live cells and animals. There are several ways to improve the properties of such sensors, mainly to increase FRET efficiency and therefore the dynamic range. One of the ways to achieve this is to use a non-fluorescent chromoprotein as an acceptor. Molecular dynamic simulations may assist in the construction of linker structures connecting donor and acceptor molecules. Estimation of the orientation factor κ 2 can be obtained by methods based on quantum theory and combined quantum mechanics/molecular mechanics approaches. The linker can be structured by hydrophobic interactions, bringing it into a closed conformation that shortens the distance between donor and acceptor and, consequently, increases FRET efficiency. We analyzed the effects of different linker structures on the detection of caspase-3 activity using a non-fluorescent acceptor. Also we have constructed the Tb3+- TagRFP sensor in which a complex of the terbium ion and terbium-binding peptide is used as a donor. This allowed us to use the unique property of lanthanide ions—fluorescence lifetime up to milliseconds—to perform measurements with time delay and exclude the nanosecond-order fluorescence. Using our systems as a starting point, by changing the recognition site in the linker it is possible to perform imaging of different protease activity in vitro or in vivo.

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

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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.

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

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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

Nucleic Acid Aptamers Against Proteases

DEFF Research Database (Denmark)

Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø

2011-01-01

, directed against blood coagulation factors, are in clinical trials as anticoagulant drugs. Several of the studies on protease-binding aptamers have been pioneering and trend-setting in the field. The work with protease-binding aptamers also demonstrates many interesting examples of non-standard selection......Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing...... small molecule protease inhibitors of sufficient specificity has proved a daunting task. Aptamers seem to represent a promising alternative. In our review, we concentrate on biochemical mechanisms of aptamer selection, proteinaptamer recognition, protease inhibition, and advantages of aptamers...

Vorapaxar: The Current Role and Future Directions of a Novel Protease-Activated Receptor Antagonist for Risk Reduction in Atherosclerotic Disease.

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Gryka, Rebecca J; Buckley, Leo F; Anderson, Sarah M

2017-03-01

Despite the current standard of care, patients with cardiovascular disease remain at a high risk for recurrent events. Inhibition of thrombin-mediated platelet activation through protease-activated receptor-1 antagonism may provide reductions in atherosclerotic disease beyond those achievable with the current standard of care. Our primary objective is to evaluate the clinical literature regarding the role of vorapaxar (Zontivity™) in the reduction of cardiovascular events in patients with a history of myocardial infarction and peripheral artery disease. In particular, we focus on the potential future directions for protease-activating receptor antagonists in the treatment of a broad range of atherosclerotic diseases. A literature search of PubMed and EBSCO was conducted to identify randomized clinical trials from August 2005 to June 2016 using the search terms: 'vorapaxar', 'SCH 530348', 'protease-activated receptor-1 antagonist', and 'Zontivity™'. Bibliographies were searched and additional resources were obtained. Vorapaxar is a first-in-class, protease-activated receptor-1 antagonist. The Thrombin Receptor Antagonist for Clinical Event Reduction (TRACER) trial did not demonstrate a significant reduction in a broad primary composite endpoint. However, the Thrombin-Receptor Antagonist in Secondary Prevention of Atherothrombotic Ischemic Events (TRA 2°P-TIMI 50) trial examined a more traditional composite endpoint and found a significant benefit with vorapaxar. Vorapaxar significantly increased bleeding compared with standard care. Ongoing trials will help define the role of vorapaxar in patients with peripheral arterial disease, patients with diabetes mellitus, and other important subgroups. The use of multivariate modeling may enable the identification of subgroups with maximal benefit and minimal harm from vorapaxar. Vorapaxar provides clinicians with a novel mechanism of action to further reduce the burden of ischemic heart disease. Identification of

Identification of cysteine proteases and screening of cysteine protease inhibitors in biological samples by a two-dimensional gel system of zymography and reverse zymography.

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

2007-11-18

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 first-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 acid (TCA) fixation. Protease activity in the 2D-gel was visualized as transparent spots where gelatin substrate was digested after commassie brilliant blue (CBB) staining. Some of the transparent spots from the skin mucus extract of rainbow trout were determined to be a cysteine protease through use of E-64 or CA-074. In the reverse zymography technique, the gel was incubated with papain solution at 37 degrees C for 18 h. Cysteine protease inhibitors from broad bean seeds were detected as clear blue spots after CBB staining. The amino (N-) terminal sequences of four papain inhibitor spots thus detected were demonstrated to be identical to that of favin beta chain, a broad bean lectin. Taken together, our system can be considered to be an efficient technique for discovering and characterizing new proteases and protease inhibitors in biological samples. This is the first report describing a 2D-gel system of zymography and reverse zymography.

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.

Interdependence of Inhibitor Recognition in HIV-1 Protease.

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Paulsen, Janet L; Leidner, Florian; Ragland, Debra A; Kurt Yilmaz, Nese; Schiffer, Celia A

2017-05-09

Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1' and S2' subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1' subsite highly influences other subsites: the extension of the hydrophobic P1' moiety results in 1) reduced van der Waals contacts in the P2' subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor.

Proteases and protease inhibitors of urinary extracellular vesicles in diabetic nephropathy.

Science.gov (United States)

Musante, Luca; Tataruch, Dorota; Gu, Dongfeng; Liu, Xinyu; Forsblom, Carol; Groop, Per-Henrik; Holthofer, Harry

2015-01-01

Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes.

Proteases and Protease Inhibitors of Urinary Extracellular Vesicles in Diabetic Nephropathy

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Luca Musante

2015-01-01

Full Text Available Diabetic nephropathy (DN is one of the major complications of diabetes mellitus (DM, leads to chronic kidney disease (CKD, and, ultimately, is the main cause for end-stage kidney disease (ESKD. Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes.

TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus.

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Yun, Bingling; Zhang, Yao; Liu, Yongzhen; Guan, Xiaolu; Wang, Yongqiang; Qi, Xiaole; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Gao, Honglei; Gao, Li; Li, Kai; Gao, Yulong; Wang, Xiaomei

2016-12-15

The entry of avian metapneumovirus (aMPV) into host cells initially requires the fusion of viral and cell membranes, which is exclusively mediated by fusion (F) protein. Proteolysis of aMPV F protein by endogenous proteases of host cells allows F protein to induce membrane fusion; however, these proteases have not been identified. Here, we provide the first evidence that the transmembrane serine protease TMPRSS12 facilitates the cleavage of subtype B aMPV (aMPV/B) F protein. We found that overexpression of TMPRSS12 enhanced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. Subsequently, knockdown of TMPRSS12 with specific small interfering RNAs (siRNAs) reduced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. We also found a cleavage motif in the aMPV/B F protein (amino acids 100 and 101) that was recognized by TMPRSS12. The histidine, aspartic acid, and serine residue (HDS) triad of TMPRSS12 was shown to be essential for the proteolysis of aMPV/B F protein via mutation analysis. Notably, we observed TMPRSS12 mRNA expression in target organs of aMPV/B in chickens. Overall, our results indicate that TMPRSS12 is crucial for aMPV/B F protein proteolysis and aMPV/B infectivity and that TMPRSS12 may serve as a target for novel therapeutics and prophylactics for aMPV. Proteolysis of the aMPV F protein is a prerequisite for F protein-mediated membrane fusion of virus and cell and for aMPV infection; however, the proteases used in vitro and vivo are not clear. A combination of analyses, including overexpression, knockdown, and mutation methods, demonstrated that the transmembrane serine protease TMPRSS12 facilitated cleavage of subtype B aMPV (aMPV/B) F protein. Importantly, we located the motif in the aMPV/B F protein recognized by TMPRSS12 and the catalytic triad in TMPRSS12 that facilitated proteolysis of the aMPV/B F protein. This is the first report on TMPRSS12 as a protease for proteolysis of viral envelope

Optimizing PHB and Protease Production by Box Behnken Design

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Amro Abd al fattah Amara

2013-04-01

Full Text Available Mixed culture is more suitable to adapt more flexible fermentation process and produce different product simultaneously. In this study a mixed Bacillus culture was investigated for their ability to produce the bioplastic "Polyhydroxybutyrate" and both of the mesophilic and the thermophilic proteases in one flask. Box-Behnken experimental design was used. The produced amount of PHB has been increased significantly. Meanwhile there is a competition between PHB and proteases. The maximum produced amount of PHB using Box-Behnken design was 2.82 g/l/48 h with protease activity equal to 41.9 Units/ml/48 h for thermophilic proteases and 99.65 Units/ml/48 h for mesophilic proteases. Excel solver was used for extra-optimization for the optimum conditions obtained from Box-Behnken experiments and its model. The maximum PHB obtained after using Excel solver was 2.88 g/l/48 h. The maximum mesophilic and thermophilic activities obtained at the same PHB production conditions were 175.68 and 243.38 Units/ml respectively. The model accuracy as obtained from Excel solver was 118.8%, which prove the power of the experimental design in optimizing such complicated process. The strategies used in this study are recommended for the production of PHB and different proteases simultaneously using Bacillus mixed culture. ABSTRAK: Kultur campuran adalah lebih sesuai bagi proses penapaian yang fleksibel dan ia boleh menghasilkan produk yang berbeza secara serentak. Dalam kajian ini keupayaan  menghasilkan "Polyhydroxybutyrate" bioplastik serta mesofilik dan termofilik protease dalam satu flask oleh  kultur Bacillus campuran telah disiasat. Eksperimen rekabentuk Box-Behnken telah digunakan. Jumlah PHB yang dikeluarkan meningkat dengan ketara dan terdapat persaingan antara PHB dan protease. Jumlah keluaran PHB maksima menggunakan rekabentuk Box-Behnken adalah 2.82 g/l/48 jam dengan aktiviti protease sama dengan 41.9 Unit/ml/48 jam untuk protease termofilik dan 99.65 Unit

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.

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

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

2011-01-01

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

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

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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.

Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

Energy Technology Data Exchange (ETDEWEB)

Gottipati, Keerthi; Acholi, Sudheer [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States); Ruggli, Nicolas [Institute of Virology and Immunology, CH-3147 Mittelhäusern (Switzerland); Choi, Kyung H., E-mail: kychoi@utmb.edu [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States)

2014-03-15

Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.

Production and partial characterization of proteases from Mucor hiemalis URM3773

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Roana Cecília dos Santos Ribeiro

2015-03-01

Full Text Available The current study evaluated the proteases production from 11 fungal species belonging to the genera Mucor, Rhizomucor and Absidia. The species were obtained from the Collection of Cultures URM at the Mycology Department-UFPE, Brazil. The best producing species was Mucor hiemalis URM 3773 (1.689 U mL-1. Plackett-Burman design methodology was employed to select the most effective parameter for protease production out of 11 medium components, including: concentration of filtrate soybean, glucose, incubation period, yeast extract, tryptone, pH, aeration, rotation, NH4Cl, MgSO4 and K2HPO4. Filtrated soybean concentration was the significant variable over the response variable, which was the specific protease activity. The crude enzyme extract showed optimal activity in pH 7.5 and at 50ºC. The enzyme was stable within a wide pH range from 5.8 to 8.0, in the phosphate buffer 0.1M and in stable temperature variation of 40-70ºC, for 180 minutes. The ions FeSO4, NaCl, MnCl2, MgCl2 and KCl stimulated the protease activity, whereas ZnCl2 ion inhibited the activity in 2.27%. Iodoacetic acid at 1mM was the proteases inhibitor that presented greater action.The results indicate that the studied enzyme have great potential for industrial application.

Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

International Nuclear Information System (INIS)

Jin Xin; Li Jufang; Huang Pingying; Dong Xuyan; Guo Lulu; Yang Liang; Cao Yuancheng; Wei Fang; Zhao Yuandi

2010-01-01

(3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63±2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

Energy Technology Data Exchange (ETDEWEB)

Jin Xin [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Li Jufang [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Huang Pingying [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Dong Xuyan [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Guo Lulu [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Yang Liang; Cao Yuancheng [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Wei Fang [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Zhao Yuandi, E-mail: zydi@mail.hust.edu.c [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China)

2010-07-15

(3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63+-2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

Enhanced Effector Function of CD8+ T Cells From Healthy Controls and HIV-Infected Patients Occurs Through Thrombin Activation of Protease-Activated Receptor 1

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Hurley, Amanda; Smith, Mindy; Karpova, Tatiana; Hasley, Rebecca B.; Belkina, Natalya; Shaw, Stephen; Balenga, Nariman; Druey, Kirk M.; Nickel, Erin; Packard, Beverly; Imamichi, Hiromi; Hu, Zonghui; Follmann, Dean; McNally, James; Higgins, Jeanette; Sneller, Michael; Lane, H. Clifford; Catalfamo, Marta

2013-01-01

Disruption of vascular integrity by trauma and other tissue insults leads to inflammation and activation of the coagulation cascade. The serine protease thrombin links these 2 processes. The proinflammatory function of thrombin is mediated by activation of protease-activated receptor 1 (PAR-1). We found that peripheral blood effector memory CD4+ and CD8+ T lymphocytes expressed PAR-1 and that expression was increased in CD8+ T cells from human immunodeficiency virus (HIV)–infected patients. Thrombin enhanced cytokine secretion in CD8+ T cells from healthy controls and HIV-infected patients. In addition, thrombin induced chemokinesis, but not chemotaxis, of CD8+ T cells, which led to structural changes, including cell polarization and formation of a structure rich in F-actin and phosphorylated ezrin-radexin-moesin proteins. These findings suggest that thrombin mediates cross-talk between the coagulation system and the adaptive immune system at sites of vascular injury through increased T-cell motility and production of proinflammatory cytokines. PMID:23204166

Enhanced effector function of CD8(+) T cells from healthy controls and HIV-infected patients occurs through thrombin activation of protease-activated receptor 1.

Science.gov (United States)

Hurley, Amanda; Smith, Mindy; Karpova, Tatiana; Hasley, Rebecca B; Belkina, Natalya; Shaw, Stephen; Balenga, Nariman; Druey, Kirk M; Nickel, Erin; Packard, Beverly; Imamichi, Hiromi; Hu, Zonghui; Follmann, Dean; McNally, James; Higgins, Jeanette; Sneller, Michael; Lane, H Clifford; Catalfamo, Marta

2013-02-15

Disruption of vascular integrity by trauma and other tissue insults leads to inflammation and activation of the coagulation cascade. The serine protease thrombin links these 2 processes. The proinflammatory function of thrombin is mediated by activation of protease-activated receptor 1 (PAR-1). We found that peripheral blood effector memory CD4(+) and CD8(+) T lymphocytes expressed PAR-1 and that expression was increased in CD8(+) T cells from human immunodeficiency virus (HIV)-infected patients. Thrombin enhanced cytokine secretion in CD8(+) T cells from healthy controls and HIV-infected patients. In addition, thrombin induced chemokinesis, but not chemotaxis, of CD8(+) T cells, which led to structural changes, including cell polarization and formation of a structure rich in F-actin and phosphorylated ezrin-radexin-moesin proteins. These findings suggest that thrombin mediates cross-talk between the coagulation system and the adaptive immune system at sites of vascular injury through increased T-cell motility and production of proinflammatory cytokines.

Mass spectrometric analysis of electrophoretically separated allergens and proteases in grass pollen diffusates

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Geczy Carolyn L

2003-09-01

Full Text Available Abstract Background Pollens are important triggers for allergic asthma and seasonal rhinitis, and proteases released by major allergenic pollens can injure airway epithelial cells in vitro. Disruption of mucosal epithelial integrity by proteases released by inhaled pollens could promote allergic sensitisation. Methods Pollen diffusates from Kentucky blue grass (Poa pratensis, rye grass (Lolium perenne and Bermuda grass (Cynodon dactylon were assessed for peptidase activity using a fluorogenic substrate, as well as by gelatin zymography. Following one- or two-dimensional gel electrophoresis, Coomassie-stained individual bands/spots were excised, subjected to tryptic digestion and analysed by mass spectrometry, either MALDI reflectron TOF or microcapillary liquid chromatography MS-MS. Database searches were used to identify allergens and other plant proteins in pollen diffusates. Results All pollen diffusates tested exhibited peptidase activity. Gelatin zymography revealed high Mr proteolytic activity at ~ 95,000 in all diffusates and additional proteolytic bands in rye and Bermuda grass diffusates, which appeared to be serine proteases on the basis of inhibition studies. A proteolytic band at Mr ~ 35,000 in Bermuda grass diffusate, which corresponded to an intense band detected by Western blotting using a monoclonal antibody to the timothy grass (Phleum pratense group 1 allergen Phl p 1, was identified by mass spectrometric analysis as the group 1 allergen Cyn d 1. Two-dimensional analysis similarly demonstrated proteolytic activity corresponding to protein spots identified as Cyn d 1. Conclusion One- and two-dimensional electrophoretic separation, combined with analysis by mass spectrometry, is useful for rapid determination of the identities of pollen proteins. A component of the proteolytic activity in Bermuda grass diffusate is likely to be related to the allergen Cyn d 1.

Extracellular Protease Activity of Enteropathogenic Escherechia coli on Mucin Substrate

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SRI BUDIARTI

2007-03-01

Full Text Available Enteropathogenic Escherichia coli (EPEC causes gastrointestinal infections in human. EPEC invasion was initiated by attachment and aggressive colonization on intestinal surface. Attachment of EPEC alter the intestine mucosal cells. Despite this, the pathogenic mechanism of EPEC infectior has not been fully understood. This research hypothesizes that extracellular proteolytic enzymes is necessary for EPEC colonization. The enzyme is secreted into gastrointestinal milieu and presumably destroy mucus layer cover the gastrointestinal tract. The objective of this study was to assay EPEC extracellular protease enzyme by using mucin substrate. The activity of EPEC extracellular proteolytic enzyme on 1% mucin substrate was investigated. Non-pathogenic E. coli was used as a negative control. Positive and tentative controls were Yersinia enterocolitica and Salmonella. Ten EPEC strains were assayed, seven of them were able to degrade mucin, and the highest activity was produced by K1.1 strain. Both positive and tentative controls also showed the ability to digest 0.20% mucin.

The role of protease-activated receptor type 2 in nociceptive signaling and pain

Czech Academy of Sciences Publication Activity Database

Mrózková, Petra; Paleček, Jiří; Špicarová, Diana

2016-01-01

Roč. 65, č. 3 (2016), s. 357-367 ISSN 0862-8408 R&D Projects: GA MŠk(CZ) LH12058; GA ČR(CZ) GBP304/12/G069; GA ČR(CZ) GA15-11138S; GA MŠk(CZ) LH15279; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : protease-activated receptor (PAR2) * signaling pathways * nociception * pain * spinal cord Subject RIV: FH - Neurology Impact factor: 1.461, year: 2016

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

Science.gov (United States)

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

1999-08-05

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

Characterization and isolation of an extracellular serine protease from the tomato pathogen Colletotrichum coccodes, and it's role in pathogenicity

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Redman, Regina S.; Rodriguez, Rusty J.

2002-01-01

Extracellular enzymes play an important role in the pathogenicity and virulence of phytopathogenic fungi. Several isolates of Colletotrichum coccodes causal agent of anthracnose on tomato, were screened to determine the relationship between protease activity and virulence. A direct relationship was observed between extracellular protease activity and the induction of disease symptoms of fruit and mortality in plants. Isolate Cc155 exhibited the highest protease activity after five days of growth in protease induction medium and produced an extracellular serine protease (sp78) that was 78 kDa, auto-degradative, glucose repressible, and non-glycosylated. To determine the role of sp78 in pathogenicity, a UV-induced extracellular protease deficient mutant (np155) was generated from the wildtype isolate Cc155. Np155 maintained growth rates comparable to Cc155 and produced wildtype levels of extracellular cellulase but did not produce extracellular protease. Unlike Cc155, np155 caused no disease symptoms on tomato fruit and 0% mortality on tomato seedlings. These results suggest that extracellular protease activity is required for pathogenicity and virulence of C. coccodes and that the elimination of protease activity transforms a virulent pathogen to a non-pathogenic endophyte.