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Sample records for acid protease activity

  1. Acid phosphatase and protease activities in immobilized rat skeletal muscles

    NASA Technical Reports Server (NTRS)

    Witzmann, F. A.; Troup, J. P.; Fitts, R. H.

    1982-01-01

    The effect of hind-limb immobilization on selected Iysosomal enzyme activities was studied in rat hing-limb muscles composed primarily of type 1. 2A, or 2B fibers. Following immobilization, acid protease and acid phosphatase both exhibited signifcant increases in their activity per unit weight in all three fiber types. Acid phosphatase activity increased at day 14 of immobilization in the three muscles and returned to control levels by day 21. Acid protease activity also changed biphasically, displaying a higher and earlier rise than acid phosphatase. The pattern of change in acid protease, but not acid phosphatase, closely parallels observed muscle wasting. The present data therefore demonstrate enhanced proteolytic capacity of all three fiber types early during muscular atrophy. In addition, the data suggest a dependence of basal hydrolytic and proteolytic activities and their adaptive response to immobilization on muscle fiber composition.

  2. Salicylic acid induced cysteine protease activity during programmed cell death in tomato plants.

    PubMed

    Kovács, Judit; Poór, Péter; Szepesi, Ágnes; Tari, Irma

    2016-06-01

    The hypersensitive response (HR), a type of programmed cell death (PCD) during biotic stress is mediated by salicylic acid (SA). The aim of this work was to reveal the role of proteolysis and cysteine proteases in the execution of PCD in response of SA. Tomato plants were treated with sublethal (0.1 mM) and lethal (1 mM) SA concentrations through the root system. Treatment with 1 mM SA increased the electrolyte leakage and proteolytic activity and reduced the total protein content of roots after 6 h, while the proteolytic activity did not change in the leaves and in plants exposed to 0.1 mM SA. The expression of the papain-type cysteine protease SlCYP1, the vacuolar processing enzyme SlVPE1 and the tomato metacaspase SlMCA1 was induced within the first three hours in the leaves and after 0.5 h in the roots in the presence of 1 mM SA but the transcript levels did not increase significantly at sublethal SA. The Bax inhibitor-1 (SlBI-1), an antiapoptotic gene was over-expressed in the roots after SA treatments and it proved to be transient in the presence of sublethal SA. Protease inhibitors, SlPI2 and SlLTC were upregulated in the roots by sublethal SA but their expression remained low at 1 mM SA concentration. It is concluded that in contrast to leaves the SA-induced PCD is associated with increased proteolytic activity in the root tissues resulting from a fast up-regulation of specific cysteine proteases and down-regulation of protease inhibitors.

  3. Protease activity at invadopodial focal digestive areas is dependent on NHE1-driven acidic pHe.

    PubMed

    Greco, Maria Raffaella; Antelmi, Ester; Busco, Giovanni; Guerra, Lorenzo; Rubino, Rosa; Casavola, Valeria; Reshkin, Stephan Joel; Cardone, Rosa Angela

    2014-02-01

    Degradation of the extracellular matrix (ECM) is a critical step of tumor cell invasion and requires protease-dependent proteolysis focalized at the invadopodia where the proteolysis of the ECM occurs. Most of the extracellular proteases belong to serine- or metallo-proteases and the invadopodia is where protease activity is regulated. While recent data looking at global protease activity in the growth medium reported that their activity and role in invasion is dependent on Na+/H+ exchanger 1 (NHE1)-driven extracellular acidification, there is no data on this aspect at the invadopodia, and an open question remains whether this acid extracellular pH (pHe) activation of proteases in tumor cells occurs preferentially at invadopodia. We previously reported that the NHE1 is expressed in breast cancer invadopodia and that the NHE1‑dependent acidification of the peri-invadopodial space is critical for ECM proteolysis. In the present study, using, for the first time, in situ zymography analysis, we demonstrated a concordance between NHE1 activity, extracellular acidification and protease activity at invadopodia to finely regulate ECM digestion. We demonstrated that: (i) ECM proteolysis taking place at invadopodia is driven by acidification of the peri-invadopodia microenvironment; (ii) that the proteases have a functional pHe optimum that is acidic; (iii) more than one protease is functioning to digest the ECM at these invadopodial sites of ECM proteolysis; and (iv) lowering pHe or inhibiting the NHE1 increases protease secretion while blocking protease activity changes NHE1 expression at the invadopodia.

  4. Skin Barrier Recovery by Protease-Activated Receptor-2 Antagonist Lobaric Acid

    PubMed Central

    Joo, Yeon Ah; Chung, Hyunjin; Yoon, Sohyun; Park, Jong Il; Lee, Ji Eun; Myung, Cheol Hwan; Hwang, Jae Sung

    2016-01-01

    Atopic dermatitis (AD) results from gene and environment interactions that lead to a range of immunological abnormalities and breakdown of the skin barrier. Protease-activated receptor 2 (PAR2) belongs to a family of G-protein coupled receptors and is expressed in suprabasal layers of the epidermis. PAR2 is activated by both trypsin and a specific agonist peptide, SLIGKV-NH2 and is involved in both epidermal permeability barrier homeostasis and epithelial inflammation. In this study, we investigated the effect of lobaric acid on inflammation, keratinocyte differentiation, and recovery of the skin barrier in hairless mice. Lobaric acid blocked trypsin-induced and SLIGKV-NH2-induced PAR2 activation resulting in decreased mobilization of intracellular Ca2+ in HaCaT keratinocytes. Lobaric acid reduced expression of interleukin-8 induced by SLIGKV-NH2 and thymus and activation regulated chemokine (TARC) induced by tumor necrosis factor-a (TNF-α) and IFN-γ in HaCaT keratinocytes. Lobaric acid also blocked SLIGKV-NH2-induced activation of ERK, which is a downstream signal of PAR2 in normal human keratinocytes (NHEKs). Treatment with SLIGKV-NH2 downregulated expression of involucrin, a differentiation marker protein in HaCaT keratinocytes, and upregulated expression of involucrin, transglutamase1 and filaggrin in NHEKs. However, lobaric acid antagonized the effect of SLIGKV-NH2 in HaCaT keratinocytes and NHEKs. Topical application of lobaric acid accelerated barrier recovery kinetics in a SKH-1 hairless mouse model. These results suggested that lobaric acid is a PAR2 antagonist and could be a possible therapeutic agent for atopic dermatitis. PMID:27169822

  5. Nitrated Fatty Acids Reverse Cigarette Smoke-Induced Alveolar Macrophage Activation and Inhibit Protease Activity via Electrophilic S-Alkylation.

    PubMed

    Reddy, Aravind T; Lakshmi, Sowmya P; Muchumarri, Ramamohan R; Reddy, Raju C

    2016-01-01

    Nitrated fatty acids (NFAs), endogenous products of nonenzymatic reactions of NO-derived reactive nitrogen species with unsaturated fatty acids, exhibit substantial anti-inflammatory activities. They are both reversible electrophiles and peroxisome proliferator-activated receptor γ (PPARγ) agonists, but the physiological implications of their electrophilic activity are poorly understood. We tested their effects on inflammatory and emphysema-related biomarkers in alveolar macrophages (AMs) of smoke-exposed mice. NFA (10-nitro-oleic acid or 12-nitrolinoleic acid) treatment downregulated expression and activity of the inflammatory transcription factor NF-κB while upregulating those of PPARγ. It also downregulated production of inflammatory cytokines and chemokines and of the protease cathepsin S (Cat S), a key mediator of emphysematous septal destruction. Cat S downregulation was accompanied by decreased AM elastolytic activity, a major mechanism of septal destruction. NFAs downregulated both Cat S expression and activity in AMs of wild-type mice, but only inhibited its activity in AMs of PPARγ knockout mice, pointing to a PPARγ-independent mechanism of enzyme inhibition. We hypothesized that this mechanism was electrophilic S-alkylation of target Cat S cysteines, and found that NFAs bind directly to Cat S following treatment of intact AMs and, as suggested by in silico modeling and calculation of relevant parameters, elicit S-alkylation of Cys25 when incubated with purified Cat S. These results demonstrate that NFAs' electrophilic activity, in addition to their role as PPARγ agonists, underlies their protective effects in chronic obstructive pulmonary disease (COPD) and support their therapeutic potential in this disease.

  6. Effects of L- and iso-ascorbic acid on meat protein hydrolyzing activity of four commercial plant and three microbial protease preparations.

    PubMed

    Ha, Minh; Bekhit, Alaa El-Din; Carne, Alan

    2014-04-15

    The present study investigated the effects of both l- and iso-ascorbic acid (AA) on the activity of four plant proteases (papain, bromelain, actinidin and zingibain) and three microbial proteases (Bacterial Protease G, Fungal 31,000 and Fungal 60,000) preparations using fluorescent-labelled casein, meat myofibrillar and connective tissue extracts to explore their effects on meat structure components upon treatment with individual proteases. While l-AA in the range 0.8-3.2mM inhibited the activity of papain, bromelain and zingibain, iso-AA acted as an inhibitor of papain but as an activator of zingibain and had no significant effect on bromelain. Both AA isoforms acted as an activator of the actinidin protease and the concentration of AA isoforms appeared to affect the level of activation of the protease. The effect of the two AA isoforms on collagen and myofibrillar protein hydrolyzing activity varied depending on the concentration of the two AA isoforms. The results indicate the ability to up and down regulate the activity of the investigated proteases by using an appropriate concentration of the AA isoform.

  7. Protease and protease inhibitory activity in pregnant and postpartum involuting uterus

    SciTech Connect

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

    1982-08-15

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

  8. Inhibition of Clostridium botulinum 52A toxicity and protease activity by sodium acid pyrophosphate in media systems.

    PubMed Central

    Wagner, M K; Busta, F F

    1985-01-01

    The effects of two pH levels (5.55 or 5.85) in combination with 0.4% sodium acid pyrophosphate (SAPP), NaH2PO4 X H2O, Na2HPO4 X 7H2O, or NaCl on the growth and toxicity of Clostridium botulinum 52A were studied. Absorbancy measurements at 630 nm, microscopic observations, and the mouse bioassay procedure were used to observe the effects. At pH 5.55 and 5.85 most control cultures exhibited toxicity when cell lysis began. Vegetative cell development was normal (4 micron long; 1 micron wide). SAPP-containing (0.4%) treatment cultures displayed similar growth and lysis but no or delayed (48 h) toxicity. Cells grown in the SAPP treatment culture were longer and wider (6 micron long; 1.5 micron wide) than in most other treatment cultures. Trypsinization of nontoxic supernatants from 0.4% SAPP resulted in toxicity. Addition of 0.4% SAPP to toxic C. botulinum supernatant delayed but did not prevent death of mice. The addition of various levels of SAPP to toxic supernatants resulted in a decrease in zone size with an increase in the level of SAPP (9 mm with 0.4% SAPP to 7 mm with 1.0% SAPP), using a dual substrate protease assay. A decrease in the zone size also occurred with the supernatant from cultures grown in the presence of SAPP and with Bacillus polymyxa protease dilutions containing 0.4% SAPP. Results suggest that the actual production or function of the protease responsible for toxin activation may have been inhibited by the presence of SAPP. PMID:2992374

  9. Novel 2-oxoimidazolidine-4-carboxylic acid derivatives as Hepatitis C virus NS3-4A serine protease inhibitors: synthesis, activity, and X-ray crystal structure of an enzyme inhibitor complex

    SciTech Connect

    Arasappan, Ashok; Njoroge, F. George; Parekh, Tejal N.; Yang, Xiaozheng; Pichardo, John; Butkiewicz, Nancy; Prongay, Andrew; Yao, Nanhua; Girijavallabhan, Viyyoor

    2008-06-30

    Synthesis and HCV NS3 serine protease inhibitory activity of some novel 2-oxoimidazolidine-4-carboxylic acid derivatives are reported. Inhibitors derived from this new P2 core exhibited activity in the low {micro}M range. X-ray structure of an inhibitor, 15c bound to the protease is presented.

  10. Plant cysteine proteases that evoke itch activate protease-activated receptors

    PubMed Central

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

    2013-01-01

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

  11. Biotechnology of Cold-Active Proteases

    PubMed Central

    Joshi, Swati; Satyanarayana, Tulasi

    2013-01-01

    The bulk of Earth’s biosphere is cold (<5 °C) and inhabited by psychrophiles. Biocatalysts from psychrophilic organisms (psychrozymes) have attracted attention because of their application in the ongoing efforts to decrease energy consumption. Proteinases as a class represent the largest category of industrial enzymes. There has been an emphasis on employing cold-active proteases in detergents because this allows laundry operations at ambient temperatures. Proteases have been used in environmental bioremediation, food industry and molecular biology. In view of the present limited understanding and availability of cold-active proteases with diverse characteristics, it is essential to explore Earth’s surface more in search of an ideal cold-active protease. The understanding of molecular and mechanistic details of these proteases will open up new avenues to tailor proteases with the desired properties. A detailed account of the developments in the production and applications of cold-active proteases is presented in this review. PMID:24832807

  12. Active protease mapping in 2DE gels.

    PubMed

    Zhao, Zhenjun; Russell, Pamela J

    2009-01-01

    Proteases act as the molecular mediators of many vital biological processes. To understand the function of each protease, it needs to be separated from other proteins and characterized in its natural, biologically active form. In the method described in this chapter, proteases in a biological sample are separated under nonreducing conditions in 2DE gels. A specific small protease substrate, tagged with a fluorescent dye, is copolymerized into the SDS gel in the second dimension. After electrophoresis, the proteins are renatured by washing the gel with Triton X-100 solution or Milli Q water to remove SDS. The gel is then incubated in a protease assay buffer. The hydrolysis of the tagged specific substrate by the renatured protease releases the free fluorescent dye, which fluoresces in situ. The fluorescent spots indicate the location of the specific proteases in the gel and the specificity of the proteases.

  13. Optimum production and characterization of an acid protease from marine yeast Metschnikowia reukaufii W6b

    NASA Astrophysics Data System (ADS)

    Li, Jing; Peng, Ying; Wang, Xianghong; Chi, Zhenming

    2010-12-01

    The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease. The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 °C. The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts. The optimal medium of the acid protease production was seawater containing 1.0% glucose, 1.5% casein, and 0.5% yeast extract, and the optimal cultivation conditions of the acid protease production were pH 4.0, a temperature of 25 °C and a shaking speed of 140 rmin-1. Under the optimal conditions, 72.5 UmL-1 of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level. The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecular-weight nitrogen sources. Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability. The acid protease produced by M. reukaufii W6b may have highly potential applications in cheese, food and fermentation industries.

  14. Acid protease production in fungal root endophytes.

    PubMed

    Mayerhofer, Michael S; Fraser, Erica; Kernaghan, Gavin

    2015-01-01

    Fungal endophytes are ubiquitous in healthy root tissue, but little is known about their ecosystem functions, including their ability to utilize organic nutrient sources such as proteins. Root-associated fungi may secrete proteases to access the carbon and mineral nutrients within proteins in the soil or in the cells of their plant host. We compared the protein utilization patterns of multiple isolates of the root endophytes Phialocephala fortinii s.l., Meliniomyces variabilis and Umbelopsis isabellina with those of two ectomycorrhizal (ECM) fungi, Hebeloma incarnatulum and Laccaria bicolor, and the wood-decay fungus Irpex lacteus at pH values of 2-9 on liquid BSA media. We also assessed protease activity using a fluorescently labeled casein assay and gelatin zymography and characterized proteases using specific protease inhibitors. I. lacteus and U. isabellina utilized protein efficiently, while the ECM fungi exhibited poor protein utilization. ECM fungi secreted metallo-proteases and had pH optima above 4, while other fungi produced aspartic proteases with lower pH optima. The ascomycetous root endophytes M. variabilis and P. fortinii exhibited intermediate levels of protein utilization and M. variabilis exhibited a very low pH optimum. Comparing proteolytic profiles between fungal root endophytes and fungi with well defined ecological roles provides insight into the ecology of these cryptic root associates.

  15. Proteases.

    PubMed

    Barrett, A J

    2001-05-01

    The processes of growth and remodeling of cells and tissues in multicellular organisms require the breakdown of old protein molecules, in concert with the synthesis of new ones. For example, many newly-synthesized molecules require proteolytic processing to convert them to biologically active forms. Proteolysis can terminate the activity of a protein--e.g., capsases mediate apoptosis, which is a vital step in the life cycle of the cell. Proteolysis contributes to defense systems too, as the recognition of peptide fragments of foreign proteins triggers the immune response. Proteases are the class of enzymes involved in these important reactions. This unit discusses the general categories of proteases, and sets the stage for addition of overview units on cysteine proteases, aspartic proteases, and metalloproteases, as well as protocol units featuring techniques for analyzing mammalian and yeast proteasomes and protease inhibitors, among other topics.

  16. Serine proteases, serine protease inhibitors, and protease-activated receptors: roles in synaptic function and behavior.

    PubMed

    Almonte, Antoine G; Sweatt, J David

    2011-08-17

    Serine proteases, serine protease inhibitors, and protease-activated receptors have been intensively investigated in the periphery and their roles in a wide range of processes-coagulation, inflammation, and digestion, for example-have been well characterized (see Coughlin, 2000; Macfarlane et al., 2001; Molinari et al., 2003; Wang et al., 2008; Di Cera, 2009 for reviews). A growing number of studies demonstrate that these protein systems are widely expressed in many cell types and regions in mammalian brains. Accumulating lines of evidence suggest that the brain has co-opted the activities of these interesting proteins to regulate various processes underlying synaptic activity and behavior. In this review, we discuss emerging roles for serine proteases in the regulation of mechanisms underlying synaptic plasticity and memory formation.

  17. The cooperative effect between active site ionized groups and water desolvation controls the alteration of acid/base catalysis in serine proteases.

    PubMed

    Shokhen, Michael; Khazanov, Netaly; Albeck, Amnon

    2007-08-13

    What is the driving force that alters the catalytic function of His57 in serine proteases between general base and general acid in each step along the enzymatic reaction? The stable tetrahedral complexes (TC) of chymotrypsin with trifluoromethyl ketone transition state analogue inhibitors are topologically similar to the catalytic transition state. Therefore, they can serve as a good model to study the enzyme catalytic reaction. We used DFT quantum mechanical calculations to analyze the effect of solvation and of polar factors in the active site of chymotrypsin on the pKa of the catalytic histidine in FE (the free enzyme), EI (the noncovalent enzyme inhibitor complex), and TC. We demonstrated that the acid/base alteration is controlled by the charged groups in the active site--the catalytic Asp102 carboxylate and the oxyanion. The effect of these groups on the catalytic His is modulated by water solvation of the active site.

  18. Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

    PubMed

    Thomas, Marshall P; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron J; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-06-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation.

  19. A mild pulsed electric field condition that improves acid tolerance, growth, and protease activity of Lactobacillus acidophilus LA-K and Lactobacillus delbrueckii subspecies bulgaricus LB-12.

    PubMed

    Najim, N; Aryana, Kayanush J

    2013-06-01

    Pulsed electric field (PEF) processing involves the application of pulses of voltage for less than 1 s to fluid products placed between 2 electrodes. The effect of mild PEF on beneficial characteristics of probiotic bacteria Lactobacillus acidophilus and Lactobacillus delbrueckii ssp. bulgaricus is not clearly understood. The objective of this study was to determine the influence of mild PEF conditions on acid tolerance, growth, and protease activity of Lb. acidophilus LA-K and Lactobacillus delbrueckii ssp. bulgaricus LB-12. A pilot plant PEF system (OSU-4M; The Ohio State University, Columbus) was used. The PEF treatments were positive square unipolar pulse width of 3 µs, pulse period of 0.5s, electric field strength of 1 kV/cm, delay time of 20 µs, flow rate of 60 mL/min, and 40.5°C PEF treatment temperature. Both Lb. acidophilus LA-K and Lb. bulgaricus LB-12 subjected to mild PEF conditions were acid tolerant until the end of the 120 min of incubation, unlike the Lb. bulgaricus control, which was not acid tolerant after 30 min. The mild PEF-treated Lb. acidophilus LA-K and Lb. bulgaricus LB-12 reached the logarithmic phase of growth an hour earlier than the control. Mild PEF conditions studied significantly improved acid tolerance, exponential growth, and protease activity of both Lb. acidophilus LA-K and Lb. bulgaricus LB-12 compared with the control. The mild PEF conditions studied can be recommended for pretreating cultures to enhance these desirable attributes.

  20. Homology among acid proteases: comparison of crystal structures at 3A resolution of acid proteases from Rhizopus chinensis and Endothia parasitica.

    PubMed Central

    Subramanian, E; Swan, I D; Liu, M; Davies, D R; Jenkins, J A; Tickle, I J; Blundell, T L

    1977-01-01

    The molecular structures of two fungal acid proteases at 3 A resolution have been compared, and found to have similar secondary and tertiary folding. These enzymes are bilobal and have a pronounced cleft between the two lobes. This cleft has been identified as the active site region from inhibitor binding studies. The results of the comparison are discussed in terms of homology among the acid proteases in general. Images PMID:322132

  1. Temporal dependence of cysteine protease activation following excitotoxic hippocampal injury.

    PubMed

    Berry, J N; Sharrett-Field, L J; Butler, T R; Prendergast, M A

    2012-10-11

    Excitotoxic insults can lead to intracellular signaling cascades that contribute to cell death, in part by activation of proteases, phospholipases, and endonucleases. Cysteine proteases, such as calpains, are calcium (Ca(2+))-activated enzymes which degrade cytoskeletal proteins, including microtubule-associated proteins, tubulin, and spectrin, among others. The current study used the organotypic hippocampal slice culture model to examine whether pharmacologic inhibition of cysteine protease activity inhibits N-methyl-D-aspartate- (NMDA-) induced excitotoxic (20 μM NMDA) cell death and changes in synaptophysin immunoreactivity. Significant NMDA-induced cytotoxicity (as measured by propidium iodide [PI] uptake) was found in the CA1 region of the hippocampus at all timepoints examined (24, 72, 120 h), an effect significantly attenuated by co-exposure to the selective NMDA receptor antagonist DL-2-Amino-5-phosphonopentanoic acid (APV), but not MDL-28170, a potent cysteine protease inhibitor. Results indicated sparing of NMDA-induced loss of the synaptic vesicular protein synaptophysin in all regions of the hippocampus by MDL-28170, though only at early timepoints after injury. These results suggest Ca(2+)-dependent recruitment of cysteine proteases within 24h of excitotoxic insult, but activation of alternative cellular degrading mechanisms after 24h. Further, these data suggest that synaptophysin may be a substrate for calpains and related proteases.

  2. Cell-free production of integral membrane aspartic acid proteases reveals zinc-dependent methyltransferase activity of the Pseudomonas aeruginosa prepilin peptidase PilD

    PubMed Central

    Aly, Khaled A; Beebe, Emily T; Chan, Chi H; Goren, Michael A; Sepúlveda, Carolina; Makino, Shin-ichi; Fox, Brian G; Forest, Katrina T

    2013-01-01

    Integral membrane aspartic acid proteases are receiving growing recognition for their fundamental roles in cellular physiology of eukaryotes and prokaryotes, and may be medically important pharmaceutical targets. The Gram-negative Pseudomonas aeruginosa PilD and the archaeal Methanococcus voltae FlaK were synthesized in the presence of unilamellar liposomes in a cell-free translation system. Cosynthesis of PilD with its full-length substrate, PilA, or of FlaK with its full-length substrate, FlaB2, led to complete cleavage of the substrate signal peptides. Scaled-up synthesis of PilD, followed by solubilization in dodecyl-β-d-maltoside and chromatography, led to a pure enzyme that retained both of its known biochemical activities: cleavage of the PilA signal peptide and S-adenosyl methionine-dependent methylation of the mature pilin. X-ray fluorescence scans show for the first time that PilD is a zinc-binding protein. Zinc is required for the N-terminal methylation of the mature pilin, but not for signal peptide cleavage. Taken together, our work identifies the P. aeruginosa prepilin peptidase PilD as a zinc-dependent N-methyltransferase and provides a new platform for large-scale synthesis of PilD and other integral membrane proteases important for basic microbial physiology and virulence. PMID:23255525

  3. Protease activities of rumen protozoa.

    PubMed Central

    Forsberg, C W; Lovelock, L K; Krumholz, L; Buchanan-Smith, J G

    1984-01-01

    Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids. PMID:6364968

  4. Proteases of germinating winged-bean (Psophocarpus tetragonolobus) seeds: purification and characterization of an acidic protease.

    PubMed

    Usha, R; Singh, M

    1996-01-15

    Two major classes of protease are shown to occur in germinating winged-bean (Psophocarpus tetragonolobus) seeds, by assaying extracts at pH 8.0 and pH 5.1 with [14C]gelatin as substrate. At pH 8.0, the activity profile of the enzyme shows a steady rise throughout the period of germination, whereas the activity at the acidic pH is very low up to day 5 and then increases sharply reaching a peak on day 11, followed by an equally sharp decline. The winged-bean acidic protease (WbAP) has been purified to apparent homogeneity, as attested by a single protein band on both PAGE and SDS/PAGE. WbAP is a monomeric enzyme with a molecular mass of 35 kDa and a pH optimum of 6.0. It is a thiol protease that does not belong to the papain family and it has tightly bound Ca2+ as shown by 45Ca(2+)-exchange studies. Besides gelatin and casein, it hydrolyses a 29 kDa winged-bean protein, indicating a prospective physiological role for it in storage-protein mobilization. Immunoblot analysis shows that it occurs only in the seeds and sprouting tubers of this plant and also that it is synthesized in developing seeds just before desiccation. It appears that the newly synthesized enzyme is inactive, and activation takes place around day 6 of germination. However, neither the mechanism of activation nor the signal that triggers it is clearly understood.

  5. Cloning, expression and activity analysis of a novel fibrinolytic serine protease from Arenicola cristata

    NASA Astrophysics Data System (ADS)

    Zhao, Chunling; Ju, Jiyu

    2015-06-01

    The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (< 40%) to other serine proteases. The gene encoding the active form of A. cristata serine protease was cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.

  6. Transient ECM protease activity promotes synaptic plasticity

    PubMed Central

    Magnowska, Marta; Gorkiewicz, Tomasz; Suska, Anna; Wawrzyniak, Marcin; Rutkowska-Wlodarczyk, Izabela; Kaczmarek, Leszek; Wlodarczyk, Jakub

    2016-01-01

    Activity-dependent proteolysis at a synapse has been recognized as a pivotal factor in controlling dynamic changes in dendritic spine shape and function; however, excessive proteolytic activity is detrimental to the cells. The exact mechanism of control of these seemingly contradictory outcomes of protease activity remains unknown. Here, we reveal that dendritic spine maturation is strictly controlled by the proteolytic activity, and its inhibition by the endogenous inhibitor (Tissue inhibitor of matrix metalloproteinases-1 – TIMP-1). Excessive proteolytic activity impairs long-term potentiation of the synaptic efficacy (LTP), and this impairment could be rescued by inhibition of protease activity. Moreover LTP is altered persistently when the ability of TIMP-1 to inhibit protease activity is abrogated, further demonstrating the role of such inhibition in the promotion of synaptic plasticity under well-defined conditions. We also show that dendritic spine maturation involves an intermediate formation of elongated spines, followed by their conversion into mushroom shape. The formation of mushroom-shaped spines is accompanied by increase in AMPA/NMDA ratio of glutamate receptors. Altogether, our results identify inhibition of protease activity as a critical regulatory mechanism for dendritic spines maturation. PMID:27282248

  7. Protease and Protease-Activated Receptor-2 Signaling in the Pathogenesis of Atopic Dermatitis

    PubMed Central

    Lee, Sang Eun; Jeong, Se Kyoo

    2010-01-01

    Proteases in the skin are essential to epidermal permeability barrier homeostasis. In addition to their direct proteolytic effects, certain proteases signal to cells by activating protease-activated receptors (PARs), the G-protein-coupled receptors. The expression of functional PAR-2 on human skin and its role in inflammation, pruritus, and skin barrier homeostasis have been demonstrated. Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by genetic barrier defects and allergic inflammation, which is sustained by gene-environmental interactions. Recent studies have revealed aberrant expression and activation of serine proteases and PAR-2 in the lesional skin of AD patients. The imbalance between proteases and protease inhibitors associated with genetic defects in the protease/protease inhibitor encoding genes, increase in skin surface pH, and exposure to proteolytically active allergens contribute to this aberrant protease/PAR-2 signaling in AD. The increased protease activity in AD leads to abnormal desquamation, degradation of lipid-processing enzymes and antimicrobial peptides, and activation of primary cytokines, thereby leading to permeability barrier dysfunction, inflammation, and defects in the antimicrobial barrier. Moreover, up-regulated proteases stimulate PAR-2 in lesional skin of AD and lead to the production of cytokines and chemokines involved in inflammation and immune responses, itching sensation, and sustained epidermal barrier perturbation with easier allergen penetration. In addition, PAR-2 is an important sensor for exogenous danger molecules, such as exogenous proteases from various allergens, and plays an important role in AD pathogenesis. Together, these findings suggest that protease activity or PAR-2 may be a future target for therapeutic intervention for the treatment of AD. PMID:20879045

  8. Protease and protease-activated receptor-2 signaling in the pathogenesis of atopic dermatitis.

    PubMed

    Lee, Sang Eun; Jeong, Se Kyoo; Lee, Seung Hun

    2010-11-01

    Proteases in the skin are essential to epidermal permeability barrier homeostasis. In addition to their direct proteolytic effects, certain proteases signal to cells by activating protease-activated receptors (PARs), the G-protein-coupled receptors. The expression of functional PAR-2 on human skin and its role in inflammation, pruritus, and skin barrier homeostasis have been demonstrated. Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by genetic barrier defects and allergic inflammation, which is sustained by gene-environmental interactions. Recent studies have revealed aberrant expression and activation of serine proteases and PAR-2 in the lesional skin of AD patients. The imbalance between proteases and protease inhibitors associated with genetic defects in the protease/protease inhibitor encoding genes, increase in skin surface pH, and exposure to proteolytically active allergens contribute to this aberrant protease/ PAR-2 signaling in AD. The increased protease activity in AD leads to abnormal desquamation, degradation of lipid-processing enzymes and antimicrobial peptides, and activation of primary cytokines, thereby leading to permeability barrier dysfunction, inflammation, and defects in the antimicrobial barrier. Moreover, up-regulated proteases stimulate PAR-2 in lesional skin of AD and lead to the production of cytokines and chemokines involved in inflammation and immune responses, itching sensation, and sustained epidermal barrier perturbation with easier allergen penetration. In addition, PAR-2 is an important sensor for exogenous danger molecules, such as exogenous proteases from various allergens, and plays an important role in AD pathogenesis. Together, these findings suggest that protease activity or PAR-2 may be a future target for therapeutic intervention for the treatment of AD.

  9. Identification of covalent active site inhibitors of dengue virus protease

    PubMed Central

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  10. Acanthamoeba protease activity promotes allergic airway inflammation via protease-activated receptor 2.

    PubMed

    Park, Mi Kyung; Cho, Min Kyoung; Kang, Shin Ae; Park, Hye-Kyung; Kim, Dong-Hee; Yu, Hak Sun

    2014-01-01

    Acanthamoeba is a free-living amoeba commonly present in the environment and often found in human airway cavities. Acanthamoeba possesses strong proteases that can elicit allergic airway inflammation. To our knowledge, the aeroallergenicity of Acanthamoeba has not been reported. We repeatedly inoculated mice with Acanthamoeba trophozoites or excretory-secretory (ES) proteins intra-nasally and evaluated symptoms and airway immune responses. Acanthamoeba trophozoites or ES proteins elicited immune responses in mice that resembled allergic airway inflammation. ES proteins had strong protease activity and activated the expression of several chemokine genes (CCL11, CCL17, CCL22, TSLP, and IL-25) in mouse lung epithelial cells. The serine protease inhibitor phenyl-methane-sulfonyl fluoride (PMSF) inhibited ES protein activity. ES proteins also stimulated dendritic cells and enhanced the differentiation of naive T cells into IL-4-secreting T cells. After repeated inoculation of the protease-activated receptor 2 knockout mouse with ES proteins, airway inflammation and Th2 immune responses were markedly reduced, but not to basal levels. Furthermore, asthma patients had higher Acanthamoeba-specific IgE titers than healthy controls and we found Acanthamoeba specific antigen from house dust in typical living room. Our findings suggest that Acanthamoeba elicits allergic airway symptoms in mice via a protease allergen. In addition, it is possible that Acanthamoeba may be one of the triggers human airway allergic disease.

  11. Structural determinants of MALT1 protease activity.

    PubMed

    Wiesmann, Christian; Leder, Lukas; Blank, Jutta; Bernardi, Anna; Melkko, Samu; Decock, Arnaud; D'Arcy, Allan; Villard, Frederic; Erbel, Paulus; Hughes, Nicola; Freuler, Felix; Nikolay, Rainer; Alves, Juliano; Bornancin, Frederic; Renatus, Martin

    2012-05-25

    The formation of the CBM (CARD11-BCL10-MALT1) complex is pivotal for antigen-receptor-mediated activation of the transcription factor NF-κB. Signaling is dependent on MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), which not only acts as a scaffolding protein but also possesses proteolytic activity mediated by its caspase-like domain. It remained unclear how the CBM activates MALT1. Here, we provide biochemical and structural evidence that MALT1 activation is dependent on its dimerization and show that mutations at the dimer interface abrogate activity in cells. The unliganded protease presents itself in a dimeric yet inactive state and undergoes substantial conformational changes upon substrate binding. These structural changes also affect the conformation of the C-terminal Ig-like domain, a domain that is required for MALT1 activity. Binding to the active site is coupled to a relative movement of caspase and Ig-like domains. MALT1 binding partners thus may have the potential of tuning MALT1 protease activity without binding directly to the caspase domain.

  12. Probing the segmental mobility and energy of the active zones of a protein chain (aspartic acid protease) by a coarse-grained bond-fluctuation Monte Carlo simulation

    NASA Astrophysics Data System (ADS)

    Pandey, Ras; Farmer, Barry

    2008-03-01

    A protein chain such as aspartic acid protease is described by a specific sequence of 99 residues each with its own specific characteristics. In a coarse-grained description, the backbone of a protein chain is described by nodes tethered together by peptide bonds where each node (the amino acid group) is characterized by molecular weight and hydrophobicity. A well-developed and somewhat mature computational modeling tool for the polymer chain such as the bond-fluctuation model is used to study such a specific protein chain with its constitutive amino groups and their sequence. The relative magnitude of hydrophobicity is used to develop appropriate interaction potentials for these amino acid groups in explicit solvent. The Metropolis algorithm is used to move each node and solvent constituent. Local energy and mobility of each amino group are analyzed along with global energy, mobility, and conformation of the protein chain. Effect of the solvent interaction and its concentration on these quantities will be presented.

  13. A novel serine protease with caspase- and legumain-like activities from edible basidiomycete Flammulina velutipes.

    PubMed

    Iketani, Aya; Nakamura, Mayumi; Suzuki, Yuya; Awai, Koichiro; Shioi, Yuzo

    2013-03-01

    A serine protease with caspase- and legumain-like activities from basidiocarps of the edible basidiomycete Flammulina velutipes was characterized. The protease was purified to near homogeneity by three steps of chromatography using acetyl-Tyr-Val-Ala-Asp-4-methylcoumaryl-7-amide (Ac-YVAD-MCA) as a substrate. The enzyme was termed FvSerP (F. velutipes serine protease). This enzyme activity was completely inhibited by the caspase-specific inhibitor, Ac-YVAD-CHO, as well as moderately inhibited by serine protease inhibitors. Based on the N-terminal sequence, the cDNA of FvSerP was identified. The deduced protease sequence was a peptide composed of 325 amino acids with a molecular mass of 34.5 kDa. The amino acid sequence of FvSerP showed similarity to neither caspases nor to the plant subtilisin-like serine protease with caspase-like activity called saspase. FvSerP shared identity to the functionally unknown genes from class of Agaricomycetes, with similarity to the peptidase S41 domain of a serine protease. It was thus concluded that this enzyme is likely a novel serine protease with caspase- and legumain-like activities belonging to the peptidase S41 family and distributed in the class Agaricomycetes. This enzyme possibly functions in autolysis, a type of programmed cell death that occurs in the later stages of development of basidiocarps with reference to their enzymatic functions.

  14. Yeast extracellular proteases.

    PubMed

    Ogrydziak, D M

    1993-01-01

    Many species of yeast secrete significant amounts of protease(s). In this article, results of numerous surveys of yeast extracellular protease production have been compiled and inconsistencies in the data and limitations of the methodology have been examined. Regulation, purification, characterization, and processing of yeast extracellular proteases are reviewed. Results obtained from the sequences of cloned genes, especially the Saccharomyces cerevisiae Bar protease, the Candida albicans acid protease, and the Yarrowia lipolytica alkaline protease, have been emphasized. Biotechnological applications and the medical relevance of yeast extracellular proteases are covered. Yeast extracellular proteases have potential in beer and wine stabilization, and they probably contribute to pathogenicity of Candida spp. Yeast extracellular protease genes also provide secretion and processing signals for yeast expression systems designed for secretion of heterologous proteins. Coverage of the secretion of foreign proteases such as prochymosin, urokinase, and tissue plasminogen activator by yeast in included.

  15. Potent inhibitors of HCV-NS3 protease derived from boronic acids

    SciTech Connect

    Venkatraman, Srikanth; Wu, Wanli; Prongay, Andrew; Girijavallabhan, Viyyoor; Njoroge, F. George

    2009-07-23

    Chronic hepatitis C infection is the leading causes for cirrhosis of the liver and hepatocellular carcinoma, leading to liver failure and liver transplantation. The etiological agent, HCV virus produces a single positive strand of RNA that is processed with the help of serine protease NS3 to produce mature virus. Inhibition of NS3 protease can be potentially used to develop effective drugs for HCV infections. Numerous efforts are now underway to develop potent inhibitors of HCV protease that contain ketoamides as serine traps. Herein we report the synthesis of a series of potent inhibitors that contain a boronic acid as a serine trap. The activity of these compounds were optimized to 200 pM. X-ray structure of compound 17 bound to NS3 protease is also discussed.

  16. Fibrin(ogen)olytic activity of bumblebee venom serine protease

    SciTech Connect

    Qiu Yuling; Choo, Young Moo; Yoon, Hyung Joo; Jia Jingming; Cui Zheng; Wang Dong; Kim, Doh Hoon; Sohn, Hung Dae; Jin, Byung Rae

    2011-09-01

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

  17. Serine protease activity in developmental stages of Eimeria tenella.

    PubMed

    Fetterer, R H; Miska, K B; Lillehoj, H; Barfield, R C

    2007-04-01

    A number of complex processes are involved in Eimeria spp. survival, including control of sporulation, intracellular invasion, evasion of host immune responses, successful reproduction, and nutrition. Proteases have been implicated in many of these processes, but the occurrence and functions of serine proteases have not been characterized. Bioinformatic analysis suggests that the Eimeria tenella genome contains several serine proteases that lack homology to trypsin. Using RT-PCR, a gene encoding a subtilisin-like and a rhomboid protease-like serine protease was shown to be developmentally regulated, both being poorly expressed in sporozoites (SZ) and merozoites (MZ). Casein substrate gel electrophoresis of oocyst extracts during sporulation demonstrated bands of proteolytic activity with relative molecular weights (Mr) of 18, 25, and 45 kDa that were eliminated by coincubation with serine protease inhibitors. A protease with Mr of 25 kDa was purified from extracts of unsporulated oocysts by a combination of affinity and anion exchange chromatography. Extracts of SZ contained only a single band of inhibitor-sensitive proteolytic activity at 25 kDa, while the pattern of proteases from extracts of MZ was similar to that of oocysts except for the occurrence of a 90 kDa protease, resistant to protease inhibitors. Excretory-secretory products (ESP) from MZ contained AEBSF (4-[2-Aminoethyl] benzenesulphonyl fluoride)-sensitive protease activity with a specific activity about 10 times greater than that observed in MZ extracts. No protease activity was observed in the ESP from SZ. Pretreatment of SZ with AEBSF significantly reduced SZ invasion and the release of the microneme protein, MIC2. The current results suggest that serine proteases are present in all the developmental stages examined.

  18. Detection of protease and protease activity using a single nanoscrescent SERS probe

    DOEpatents

    Liu, Gang L.; Ellman, Jonathan A.; Lee, Luke P.; Chen, Fanqing Frank

    2013-01-29

    This invention pertains to the in vitro detection of proteases using a single peptide-conjugate nanocrescent surface enhanced Raman scattering (SERS) probes with at least nanomolar sensitivity. The probe enables detection of proteolytic activity in extremely small volume and at low concentration. In certain embodiments the probes comprise an indicator for the detection of an active protease, where the indicator comprises a nanocrescent attached to a peptide, where said peptide comprises a recognition site for the protease and a Raman tag attached to the peptide.

  19. Site-Directed Mutagenesis and Structural Studies Suggest that the Germination Protease, GPR, in Spores of Bacillus Species Is an Atypical Aspartic Acid Protease

    PubMed Central

    Carroll, Thomas M.; Setlow, Peter

    2005-01-01

    Germination protease (GPR) initiates the degradation of small, acid-soluble spore proteins (SASP) during germination of spores of Bacillus and Clostridium species. The GPR amino acid sequence is not homologous to members of the major protease families, and previous work has not identified residues involved in GPR catalysis. The current work has focused on identifying catalytically essential amino acids by mutagenesis of Bacillus megaterium gpr. A residue was selected for alteration if it (i) was conserved among spore-forming bacteria, (ii) was a potential nucleophile, and (iii) had not been ruled out as inessential for catalysis. GPR variants were overexpressed in Escherichia coli, and the active form (P41) was assayed for activity against SASP and the zymogen form (P46) was assayed for the ability to autoprocess to P41. Variants inactive against SASP and unable to autoprocess were analyzed by circular dichroism spectroscopy and multiangle laser light scattering to determine whether the variant's inactivity was due to loss of secondary or quaternary structure, respectively. Variation of D127 and D193, but no other residues, resulted in inactive P46 and P41, while variants of each form were well structured and tetrameric, suggesting that D127 and D193 are essential for activity and autoprocessing. Mapping these two aspartate residues and a highly conserved lysine onto the B. megaterium P46 crystal structure revealed a striking similarity to the catalytic residues and propeptide lysine of aspartic acid proteases. These data indicate that GPR is an atypical aspartic acid protease. PMID:16199582

  20. Nonnatural amino acid incorporation into the methionine 214 position of the metzincin Pseudomonas aeruginosa alkaline protease

    PubMed Central

    Walasek, Paula; Honek, John F

    2005-01-01

    Background The alkaline protease from Pseudomonas aeruginosa (AprA) is a member of the metzincin superfamily of metalloendoproteases. A key feature of these proteases is a conserved methionine-containing 1,4-tight β turn at the base of the active site zinc binding region. Results To explore the invariant methionine position in this class of protease, incorporation of a nonnatural fluorinated methionine, L-difluoromethionine (DFM), into this site was accomplished. Although overproduction of the N-terminal catalytic fragment of AprA resulted in protein aggregates which could not be resolved, successful heterologous production of the entire AprA was accomplished in the presence and absence of the nonnatural amino acid. DFM incorporation was found to only slightly alter the enzyme kinetics of AprA. In addition, differential scanning calorimetry indicated no significant alteration in the thermal stability of the modified enzyme. Conclusion Although invariant in all metzincin proteases, the methionine 214 position in AprA can be successfully replaced by the nonnatural amino acid DFM resulting in little effect on protein structure and function. This study indicates that the increased size of the methyl group by the introduction of two fluorines is still sufficiently non-sterically demanding, and bodes well for the application of DFM to biophysical studies of protein structure and function in this class of protease. PMID:16221305

  1. Enhancement of the aspartame precursor synthetic activity of an organic solvent-stable protease.

    PubMed

    Ogino, Hiroyasu; Tsuchiyama, Shotaro; Yasuda, Masahiro; Doukyu, Noriyuki

    2010-03-01

    The PST-01 protease is highly stable and catalyzes the synthesis of the aspartame precursor with high reaction yields in the presence of organic solvents. However, the synthesis rate using the PST-01 protease was slower than that observed when thermolysin was used. Structural comparison of both enzymes showed particular amino acid differences near the active center. These few residue differences in the PST-01 protease were mutated to match those amino acid types found in thermolysin. The mutated PST-01 proteases at the 114th residue from tyrosine to phenylalanine showed enhancement of synthetic activity. This activity was found to be similar to thermolysin. In addition, mutating the residue in the PST-01 protease with arginine and serine showed more improvement of the activity. The mutant PST-01 protease should be more useful than thermolysin for the synthesis of the aspartame precursor, because this enzyme has higher stability and activity in the presence of organic solvents. The results show the potential of organic solvent-stable enzymes as industrial catalysts.

  2. Discovery of novel histidine-derived lipo-amino acids: applied in the synthesis of ultra-short antimicrobial peptidomimetics having potent antimicrobial activity, salt resistance and protease stability.

    PubMed

    Ahn, Mija; Murugan, Ravichandran N; Jacob, Binu; Hyun, Jae-Kyung; Cheong, Chaejoon; Hwang, Eunha; Park, Hyo-Nam; Seo, Ji-Hyung; Srinivasrao, G; Lee, Kyung S; Shin, Song Yub; Bang, Jeong Kyu

    2013-10-01

    Here we report for the first time the synthesis of Histidine (His) derived lipo-amino acids having pendant lipid tails at N(τ)- and N(π)-positions on imidazole group of His and applied it into synthesis of lipo-peptides. The attachment of His-derived lipo-amino acid into the very short inactive cationic peptides endows potent antimicrobial activity against Gram-positive and Gram-negative bacteria without hemolytic activity. Furthermore, our designed His-derived lipo-peptidomimetics (HDLPs) consisting of two or three residues displayed strong anti-MRSA activity and protease stability as well as retained potent antimicrobial activity under high salt concentration. Our results demonstrate that the novel lipo-amino acid is highly flexible to synthesize and carry out the extensive structure-activity relationship (SAR) on lipo-antimicrobial peptidomimetics and represents a unique amenable platform for modifying parameters important for antimicrobial activity. Through this study, we proved that the discovery of His-derived lipo-amino acid and the corresponding HDLPs are an excellent candidate as a lead compound for the development of novel antimicrobial agents.

  3. Serine protease activities in Leishmania (Leishmania) chagasi promastigotes.

    PubMed

    da Silva-López, Raquel Elisa; dos Santos, Tatiana Resende; Morgado-Díaz, José Andrés; Tanaka, Marcelo Neves; de Simone, Salvatore Giovanni

    2010-10-01

    The present work reports the isolation, biochemical characterization, and subcellular location of serine proteases from aqueous, detergent soluble, and culture supernatant of Leishmania chagasi promastigote extracts, respectively, LCSII, LCSI, and LCSIII. The active enzyme molecular masses of LCSII were about 105, 66, and 60 kDa; of LCSI, 60 and 58 kDa; and of LCSIII, approximately 76 and 68 kDa. Optimal pH for the enzymes was 7.0 for LCSI and LCSIII and 8.5 for LCSII, and the optimal temperature for all enzymes was 37°C, using α-N-ρ-tosyl-L: -arginine methyl ester as substrate. Assay of thermal stability indicated that LCSIII is the more stable enzyme. Hemoglobin, bovine serum albumin, and ovalbumin were hydrolyzed by LCSII and LCSI but not by LCSIII. Inhibition studies suggested that enzymes belong to the serine protease class modulated by divalent cations. Rabbit antiserum against 56-kDa serine protease of Leishmania amazonensis identified proteins in all extracts of L. chagasi. Furthermore, immunocytochemistry demonstrated that serine proteases are located in flagellar pocket region and cytoplasmic vesicles of L. chagasi promastigotes. These findings indicate that L. chagasi serine proteases differ from L. amazonensis proteases and all known flagellate proteases, but display some similarities with serine proteases from other Leishmania species, suggesting a conservation of this enzymatic activity in the genus.

  4. Photoactivated Spatiotemporally-Responsive Nanosensors of in Vivo Protease Activity.

    PubMed

    Dudani, Jaideep S; Jain, Piyush K; Kwong, Gabriel A; Stevens, Kelly R; Bhatia, Sangeeta N

    2015-12-22

    Proteases play diverse and important roles in physiology and disease, including influencing critical processes in development, immune responses, and malignancies. Both the abundance and activity of these enzymes are tightly regulated and highly contextual; thus, in order to elucidate their specific impact on disease progression, better tools are needed to precisely monitor in situ protease activity. Current strategies for detecting protease activity are focused on functionalizing synthetic peptide substrates with reporters that emit detection signals following peptide cleavage. However, these activity-based probes lack the capacity to be turned on at sites of interest and, therefore, are subject to off-target activation. Here we report a strategy that uses light to precisely control both the location and time of activity-based sensing. We develop photocaged activity-based sensors by conjugating photolabile molecules directly onto peptide substrates, thereby blocking protease cleavage by steric hindrance. At sites of disease, exposure to ultraviolet light unveils the nanosensors to allow proteases to cleave and release a reporter fragment that can be detected remotely. We apply this spatiotemporally controlled system to probe secreted protease activity in vitro and tumor protease activity in vivo. In vitro, we demonstrate the ability to dynamically and spatially measure metalloproteinase activity in a 3D model of colorectal cancer. In vivo, veiled nanosensors are selectively activated at the primary tumor site in colorectal cancer xenografts to capture the tumor microenvironment-enriched protease activity. The ability to remotely control activity-based sensors may offer a valuable complement to existing tools for measuring biological activity.

  5. Effects of detergents on the West Nile virus protease activity.

    PubMed

    Ezgimen, Manolya D; Mueller, Niklaus H; Teramoto, Tadahisa; Padmanabhan, R

    2009-05-01

    Detergents such as Triton X-100 are often used in drug discovery research to weed out small molecule promiscuous and non-specific inhibitors which act by aggregation in solution and undesirable precipitation in aqueous assay buffers. We evaluated the effects of commonly used detergents, Triton X-100, Tween-20, Nonidet-40 (NP-40), Brij-35, and CHAPS, on the enzymatic activity of West Nile virus (WNV) protease. Unexpectedly, Triton X-100, Tween-20, and NP-40 showed an enhancement of in vitro WNV protease activity from 2 to 2.5-fold depending on the detergent and its concentration. On the other hand, Brij-35, at 0.001% enhanced the protease activity by 1.5-fold and CHAPS had the least enhancing effect. The kinetic analysis showed that the increase in protease activity by Triton X-100 was dose-dependent. Furthermore, at Triton X-100 and Tween-20 concentrations higher than 0.001%, the inhibition of compound B, one of the lead compounds against WNV protease identified in a high throughput screen (IC(50) value of 5.7+/-2.5 microM), was reversed. However, in the presence of CHAPS, compound B still showed good inhibition of WNV protease. Our results, taken together, indicate that nonionic detergents, Triton X-100, Tween, and NP-40 are unsuitable for the purpose of discrimination of true versus promiscuous inhibitors of WNV protease in high throughput assays.

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

    PubMed Central

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-01-01

    Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macro­globulin, this protease-activation mechanism is likely to operate across the diverse members of this group. PMID:26143919

  7. Protease induced plasticity: matrix metalloproteinase-1 promotes neurostructural changes through activation of protease activated receptor 1

    PubMed Central

    Allen, Megan; Ghosh, Suhasini; Ahern, Gerard P.; Villapol, Sonia; Maguire-Zeiss, Kathleen A.; Conant, Katherine

    2016-01-01

    Matrix metalloproteinases (MMPs) are a family of secreted endopeptidases expressed by neurons and glia. Regulated MMP activity contributes to physiological synaptic plasticity, while dysregulated activity can stimulate injury. Disentangling the role individual MMPs play in synaptic plasticity is difficult due to overlapping structure and function as well as cell-type specific expression. Here, we develop a novel system to investigate the selective overexpression of a single MMP driven by GFAP expressing cells in vivo. We show that MMP-1 induces cellular and behavioral phenotypes consistent with enhanced signaling through the G-protein coupled protease activated receptor 1 (PAR1). Application of exogenous MMP-1, in vitro, stimulates PAR1 dependent increases in intracellular Ca2+ concentration and dendritic arborization. Overexpression of MMP-1, in vivo, increases dendritic complexity and induces biochemical and behavioral endpoints consistent with increased GPCR signaling. These data are exciting because we demonstrate that an astrocyte-derived protease can influence neuronal plasticity through an extracellular matrix independent mechanism. PMID:27762280

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

    PubMed

    Hu, Junjie; Liu, Fei; Ju, Huangxian

    2015-04-21

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

  9. German cockroach frass proteases modulate the innate immune response via activation of protease-activated receptor-2.

    PubMed

    Day, Scottie B; Zhou, Ping; Ledford, John R; Page, Kristen

    2010-01-01

    Allergen exposure can induce an early innate immune response; however, the mechanism by which this occurs has not been addressed. In this report, we demonstrate a role for the active serine proteases in German cockroach (GC) feces (frass) and protease-activated receptor (PAR)-2 in modulating the innate immune response. A single exposure of GC frass induced inflammatory cytokine production and cellular infiltration in the airways of mice. In comparison, exposure to protease-depleted GC frass resulted in diminution of inflammatory cytokine production and airway neutrophilia, but had no effect on macrophage infiltration. Selective activation of PAR-2 confirmed that PAR-2 was sufficient to induce airway inflammation. Exposure of GC frass to PAR-2-deficient mice led to decreased immune responses to GC frass compared to wild-type mice. Using the macrophage as an early marker of the innate immune response, we found that GC frass induced significant release of tumor necrosis factor-alpha from primary alveolar macrophages. This effect was dependent on the intrinsic proteases in GC frass. We confirmed GC frass-induced cytokine expression was mediated by activation of NF-kappaB and ERK in a macrophage cell line. Collectively, these data suggest a central role for GC frass protease-PAR-2 activation in regulating the innate immune response through the activation of alveolar macrophages. Understanding the potential role of protease-PAR-2 activation as a danger signal or adjuvant could yield attractive therapeutic targets.

  10. Characterization of the protease activity of detergents: laboratory practicals for studying the protease profile and activity of various commercial detergents.

    PubMed

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

    2011-07-01

    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 fluids, and food soils. This article describes two easy and cheap laboratory exercises to study the presence, profile, and basic enzymology of detergent proteases. These laboratory practicals are based on the determination of the detergent protease activity of various commercial detergents using the N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine p-nitroanilide method and the bovine serum albumin degradation capacity. Students are also required to elucidate the enzymatic subtype of detergent proteases by studying the inhibitory potential of several types of protease inhibitors revealed by the same experimental methodology. Additionally, the results of the exercises can be used to provide additional insights on elementary enzymology by studying the influence of several important parameters on protease activity such as temperature (in this article) and the influence of pH and effects of surfactants and oxidizers (proposed). Students also develop laboratory skills, problem-solving capacities, and the ability to write a laboratory report. The exercises are mainly designed for an advanced undergraduate project in the biochemistry and biotechnology sciences. Globally, these laboratory practicals show students the biotechnological applications of proteases in the detergent industry and also reinforce important enzymology concepts.

  11. Interplay between acid phosphatase and cysteine proteases in mediating vitellin degradation during early embryogenesis of Periplaneta americana.

    PubMed

    Oliveira, Danielle M P; Ramos, Isabela B; Reis, Flavia C G; Lima, Ana P C A; Machado, Ednildo A

    2008-05-01

    In this work, we characterized the activities of two classes of proteases and AcP during early embryogenesis of Periplaneta americana. AcP activity was first detected at day 6 and reached a maximum level at day 10 of development. Using phosphoamino acids, phosphatase activity was shown to be directed only against phosphotyrosine at day 6 while at day 10 it was also active against phosphoserine. In parallel, two classes of proteases were detected and located within yolk granules: a clan CA-cysteine protease, which was inhibited by E-64, insensitive to CA 074 and activated by acidic pH at day 3; and a neutral serine protease, which was inhibited by aprotinin at day 6. Assays of vitellin (Vt) degradation evidenced that incubations at neutral pH induced slight proteolysis, while the incubations at acidic pH did not result in Vt degradation. However, pre-incubations of Vt with AcP increased the levels of Vt acidic proteolysis and this could be inhibited by the addition of phosphatase inhibitors. On the other hand, the same pre-incubations showed no effects on the profile of degradation at neutral pH. We propose that AcP and cysteine protease cooperate to assure Vt breakdown during early embryogenesis of P. americana.

  12. Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease

    SciTech Connect

    Mittal, Seema; Cai, Yufeng; Nalam, Madhavi N.L.; Bolon, Daniel N.A.; Schiffer, Celia A.

    2012-09-11

    Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.

  13. Glutamate alteration of glutamic acid decarboxylase (GAD) in GABAergic neurons: the role of cysteine proteases.

    PubMed

    Monnerie, Hubert; Le Roux, Peter D

    2008-09-01

    Brain cell vulnerability to neurologic insults varies greatly, depending on their neuronal subpopulation. Among cells that survive a pathological insult such as ischemia or brain trauma, some may undergo morphological and/or biochemical changes that could compromise brain function. We previously reported that surviving cortical GABAergic neurons exposed to glutamate in vitro displayed an NMDA receptor (NMDAR)-mediated alteration in the levels of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67) [Monnerie, H., Le Roux, P., 2007. Reduced dendrite growth and altered glutamic acid decarboxylase (GAD) 65- and 67-kDa isoform protein expression from mouse cortical GABAergic neurons following excitotoxic injury in vitro. Exp. Neurol. 205, 367-382]. In this study, we examined the mechanisms by which glutamate excitotoxicity caused a change in cortical GABAergic neurons' GAD protein levels. Removing extracellular calcium prevented the NMDAR-mediated decrease in GAD protein levels, measured using Western blot techniques, whereas inhibiting calcium entry through voltage-gated calcium channels had no effect. Glutamate's effect on GAD protein isoforms was significantly attenuated by preincubation with the cysteine protease inhibitor N-Acetyl-L-Leucyl-L-Leucyl-L-norleucinal (ALLN). Using class-specific protease inhibitors, we observed that ALLN's effect resulted from the blockade of calpain and cathepsin protease activities. Cell-free proteolysis assay confirmed that both proteases were involved in glutamate-induced alteration in GAD protein levels. Together these results suggest that glutamate-induced excitotoxic stimulation of NMDAR in cultured cortical neurons leads to altered GAD protein levels from GABAergic neurons through intracellular calcium increase and protease activation including calpain and cathepsin. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered balance between excitation

  14. Gibberellic Acid-Induced Synthesis of Protease by Isolated Aleurone Layers of Barley 1

    PubMed Central

    Jacobsen, John V.; Varner, J. E.

    1967-01-01

    The production of protease by isolated aleurone layers of barley in response to gibberellic acid has been examined. The protease arises in the aleurone layer and is mostly released from the aleurone cells. The courses of release of amylase and protease from aleurone layers, the dose responses to gibberellic acid and the effects of inhibitors on the production of both enzymes are parallel. As is the case for amylase, protease is made de novo in response to the hormone. These data give some credence to the hypothesis that the effect of gibberellic acid is to promote the simultaneous synthesis and secretion of a group of hydrolases. PMID:16656695

  15. Effect of exchange of amino acid residues of the surface region of the PST-01 protease on its organic solvent-stability.

    PubMed

    Ogino, Hiroyasu; Uchiho, Takeshi; Doukyu, Noriyuki; Yasuda, Masahiro; Ishimi, Kosaku; Ishikawa, Haruo

    2007-07-13

    The PST-01 protease from an organic solvent tolerant Pseudomonas aeruginosa has high stability and activity in the presence of various organic solvents. The structure gene of the PST-01 protease was amplified by the error-prone PCR method. The mutated proteases were incubated in the presence of acetonitrile. By measuring remaining activities, two kinds of mutated PST-01 proteases of which the stabilities were changed were selected. These mutations hardly changed the profile of the activity and stability at various pHs. Their activity and stability at higher temperatures were slightly lower than those of the wild-type PST-01 protease. The stabilities of the mutated enzymes in the presence of various organic solvents were greatly reduced. In both the mutated PST-01 proteases, amino acids located at the surface of the enzyme had been substituted.

  16. A Tunable, Modular Approach to Fluorescent Protease-Activated Reporters

    PubMed Central

    Wu, Peng; Nicholls, Samantha B.; Hardy, Jeanne A.

    2013-01-01

    Proteases are one of the most important and historically utilized classes of drug targets. To effectively interrogate this class of proteins, which encodes nearly 2% of the human proteome, it is necessary to develop effective and cost-efficient methods that report on their activity both in vitro and in vivo. We have developed a robust reporter of caspase proteolytic activity, called caspase-activatable green fluorescent protein (CA-GFP). The caspases play central roles in homeostatic regulation, as they execute programmed cell death, and in drug design, as caspases are involved in diseases ranging from cancer to neurodegeneration. CA-GFP is a genetically encoded dark-to-bright fluorescent reporter of caspase activity in in vitro, cell-based, and animal systems. Based on the CA-GFP platform, we developed reporters that can discriminate the activities of caspase-6 and -7, two highly related proteases. A second series of reporters, activated by human rhinovirus 3C protease, demonstrated that we could alter the specificity of the reporter by reengineering the protease recognition sequence. Finally, we took advantage of the spectrum of known fluorescent proteins to generate green, yellow, cyan, and red reporters, paving the way for multiplex protease monitoring. PMID:23561537

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

    SciTech Connect

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-06-30

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

  18. Purification, characterization, and gene cloning of thermopsin, a thermostable acid protease from Sulfolobus acidocaldarius.

    PubMed

    Lin, X; Tang, J

    1990-01-25

    A thermostable, acid proteolytic activity has been found to be associated with the cells and in the culture medium of Sulfolobus acidocaldarius, an archaebacterium. This acid protease, which has been named thermopsin, was purified to homogeneity from the culture medium by a five-step procedure including column chromatographies on DEAE-Sepharose CL-6B, phenyl-Sepharose CL-4B, Sephadex G-100, monoQ (fast protein liquid chromatography), and gel filtration (high pressure liquid chromatography). The purified thermopsin produced a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the proteolytic activity was associated with the band. Thermopsin is a single-chain protein as indicated by gel electrophoresis and by a single NH2-terminal sequence. It has maximal proteolytic activity at pH 2 and 90 degrees C. A genomic library of S. acidocaldarius was prepared and screened by an oligonucleotide probe designed from the NH2-terminal sequence of thermopsin. Five positive clones were isolated. From these clones the thermopsin gene was mapped and sequenced. The nucleotide sequence showed that the thermopsin structure is encoded in 1020 bases. In the deduced protein sequence, there are 41 amino acid residues (including the initiation Met) preceding the NH2-terminal position of thermopsin. Most of these residues appear to be characteristic of a leader sequence. However, the presence in this region of a short pro sequence cannot be ruled out. Thermopsin contains a single cysteine at residue 237 that is not essential for activity (Fusek, M., Lin, X.-L., Tang, J. (1990) J. Biol. Chem. 265, 1496-1501. Thermopsin has no apparent sequence similarity to aspartic proteases of the pepsin family nor to pepstatin-insensitive acid protease (Maita, T., Nagata, S., Matsuda, G., Murata, S., Oda, K., Murao, S., and Tsura, D. (1984) J. Biochem. 95, 465-475) and thus may represent a new class of acid proteases. Also absent is the characteristic active site aspartyl sequence

  19. Potent and Selective Peptidyl Boronic Acid Inhibitors of the Serine Protease Prostate-Specific Antigen

    PubMed Central

    LeBeau, Aaron M.; Singh, Pratap; Isaacs, John T.; Denmeade, Samuel R.

    2012-01-01

    SUMMARY Prostate cancer cells produce high (microgram to milligram/milliliter) levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the extracellular fluid surrounding prostate cancers but is found at 1,000- to 10,000-fold lower concentrations in the circulation, where it is inactivated due to binding to abundant serum protease inhibitors. The exclusive presence of high levels of active PSA within prostate cancer sites makes PSA an attractive candidate for targeted imaging and therapeutics. A synthetic approach based on a peptide substrate identified first peptide aldehyde and then boronic acid inhibitors of PSA. The best of these had the sequence Cbz-Ser-Ser-Lys-Leu-(boro)Leu, with a Ki for PSA of 65 nM. The inhibitor had a 60-fold higher Ki for chymotrypsin. A validated model of PSA’s catalytic site confirmed the critical interactions between the inhibitor and residues within the PSA enzyme. PMID:18635003

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

    PubMed

    Buch, Franziska; Kaman, Wendy E; Bikker, Floris J; Yilamujiang, Ayufu; Mithöfer, Axel

    2015-01-01

    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.

  1. Nepenthesin Protease Activity Indicates Digestive Fluid Dynamics in Carnivorous Nepenthes Plants

    PubMed Central

    Buch, Franziska; Kaman, Wendy E.; Bikker, Floris J.; Yilamujiang, Ayufu; Mithöfer, Axel

    2015-01-01

    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. PMID:25750992

  2. Increased activity of unlinked Zika virus NS2B/NS3 protease compared to linked Zika virus protease.

    PubMed

    Kuiper, Benjamin D; Slater, Kristin; Spellmon, Nicholas; Holcomb, Joshua; Medapureddy, Prasanna; Muzzarelli, Kendall M; Yang, Zhe; Ovadia, Reuben; Amblard, Franck; Kovari, Iulia A; Schinazi, Raymond F; Kovari, Ladislau C

    2017-03-22

    Zika virus (ZIKV) is a flavivirus spread by daytime-active Aedes spp. mosquitoes such as A. aegypti and A. albopictus. Previously thought to be a mild infection, the latest ZIKV outbreak in the Americas is causally associated with more severe symptoms as well as severe birth defects, such as microcephaly. Currently no vaccine or antiviral exists. However, recent progress has demonstrated the viral NS2B/NS3 protease may be a suitable target for the development of small-molecule antiviral agents. To better understand the ZIKV protease, we expressed, purified, and characterized unlinked and linked NS2B/NS3 protease corresponding to an isolate from the recent outbreak in Puerto Rico. Unlinked ZIKV protease is more active and binds substrate with greater affinity than linked ZIKV protease. Therefore, we propose that unlinked ZIKV protease be used when evaluating or designing ZIKV protease inhibitors. Additionally, potent inhibitors of related viral proteases, like West Nile Virus and Dengue virus, may serve as advanced starting points to identify and develop ZIKV protease inhibitors.

  3. The total protein content, protein fractions and proteases activities of drone prepupae of Apis mellifera due to varrosis.

    PubMed

    Zółtowska, Krystyna; Lipiński, Zbigniew; Dmitryjuk, Małgorzata

    2005-01-01

    The proteins level and activities of acid and alkaline proteases in whole body extracts of drone prepupae of Apis mellifera naturally infested with Varroa destructor were studied. The infested and a non-infested group did not differ significantly in their total protein content. However, some differences in protein profiles were found. A lack of three protein fractions of moderate and lower molecular weight in infested prepupae was noted. Moreover, some differences in the quantity of protein in most of the fractions were observed. The activity of acid proteases from infested prepupae was lower (p < 0.05) compared with the activity of these proteases from the non-infested one group. The infested drone had higher activity of alkaline proteases than non-infested but this difference was not statisticaly significant.

  4. Purification and characterization of an extracellular protease from Penicillium chrysogenum Pg222 active against meat proteins.

    PubMed

    Benito, María J; Rodríguez, Mar; Núñez, Félix; Asensio, Miguel A; Bermúdez, María E; Córdoba, Juan J

    2002-07-01

    An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60 degrees C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45 degrees C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.

  5. Purification and Characterization of an Extracellular Protease from Penicillium chrysogenum Pg222 Active against Meat Proteins

    PubMed Central

    Benito, María J.; Rodríguez, Mar; Núñez, Félix; Asensio, Miguel A.; Bermúdez, María E.; Córdoba, Juan J.

    2002-01-01

    An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products. PMID:12089038

  6. Association of circulating factor seven activating protease (FSAP) and of oral Omega-3 fatty acids supplements with clinical outcome in patients with atrial fibrillation: the OMEGA-AF study.

    PubMed

    Parahuleva, Mariana S; Kanse, Sandip; Hölschermann, Hans; Zheleva, Kirila; Zandt, Daniel; Worsch, Michael; Parviz, Behnoush; Güttler, Norbert; Tillmanns, Harald; Böning, Andreas; Erdogan, Ali

    2014-04-01

    Factor VII Activating Protease (FSAP) activates factor VII (FVII) as well as pro-urokinase (uPA). Our goal was to evaluate the relation between plasma levels of FSAP and clinical instability in atrial fibrillation (AF) and possible effects of oral omega-3 fatty acids (FA) supplements. 101 patients with persistent AF were analyzed in the OMEGA-AF Study. Plasma FSAP levels were measured at baseline and after 12 weeks of treatment with omega-3 FA. The median FSAP antigen concentration, in contrast to FSAP activity, was higher in patients with persistent AF. The maintenance of SR after successful cardioversion (CV) did not lead to a normalization of FSAP concentration. Supplementation with omega-3 FA but not placebo significantly reduced elevated FSAP concentration. Furthermore, elevated FSAP levels did not indicate a significantly increased risk of recurrence of AF after electrical CV or cardiovascular clinical events during 1 year of follow-up. Plasma FSAP concentration was increased in patients with AF and may be involved in the pathogenesis of this condition. The possible effects of omega-3 FA on clinical AF potential could be linked with modulation of circulating FSAP levels.

  7. Evaluation of a diverse set of potential P1 carboxylic acid bioisosteres in hepatitis C virus NS3 protease inhibitors.

    PubMed

    Rönn, Robert; Gossas, Thomas; Sabnis, Yogesh A; Daoud, Hanna; Kerblom, Eva; Danielson, U Helena; Sandström, Anja

    2007-06-15

    There is an urgent need for more efficient therapies for people infected with hepatitis C virus (HCV). HCV NS3 protease inhibitors have shown proof-of-concept in clinical trials, which make the virally encoded NS3 protease an attractive drug target. Product-based NS3 protease inhibitors comprising a P1 C-terminal carboxylic acid have shown to be effective and we were interested in finding alternatives to this crucial carboxylic acid group. Thus, a series of diverse P1 functional groups with different acidity and with possibilities to form a similar, or an even more powerful, hydrogen bond network as compared to the carboxylic acid were synthesized and incorporated into potential inhibitors of the NS3 protease. Biochemical evaluation of the inhibitors was performed in both enzyme and cell-based assays. Several non-acidic C-terminal groups, such as amides and hydrazides, were evaluated but failed to produce inhibitors more potent than the corresponding carboxylic acid inhibitor. The tetrazole moiety, although of similar acidity to a carboxylic acid, provided an inhibitor with mediocre potencies in both assays. However, the acyl cyanamide and the acyl sulfinamide groups rendered compounds with low nanomolar inhibitory potencies and were more potent than the corresponding carboxylic acid inhibitor in the enzymatic assay. Additionally, results from a pH-study suggest that the P(1) C-terminal of the inhibitors comprising a carboxylic acid, an acyl sulfonamide or an acyl cyanamide group binds in a similar mode in the active site of the NS3 protease.

  8. Scouring Potential of Mesophile Acidic Proteases of Pseudomonas aeruginosa for Grey Cotton Fabrics

    NASA Astrophysics Data System (ADS)

    Saravanan, D.

    2013-04-01

    Mesophile, acidic proteases were produced using the microbial source, Pseudomonas aeruginosa, with wider thermal tolerances. Process conditions of scouring treatment were optimized using Taguchi method for optimum temperature, time, pH and concentration of protease. Treatment with the protease lower weight loss values compared to the alkali scouring, however, significant improvement in the absorbency compared to the grey samples was observed. Large amounts of pectin left out in the samples resulted in higher extractable impurities, substantiated by the FTIR results. Relatively, lower reduction in the tear strengths was observed in both warp and weft directions after protease treatment of the cotton fabrics.

  9. Evaluation of a D-amino-acid-containing fluorescence resonance energy transfer peptide library for profiling prokaryotic proteases.

    PubMed

    Kaman, Wendy E; Voskamp-Visser, Ingrid; de Jongh, Denise M C; Endtz, Hubert P; van Belkum, Alex; Hays, John P; Bikker, Floris J

    2013-10-01

    Bacterial proteases play an important role in a broad spectrum of processes, including colonization, proliferation, and virulence. In this respect, bacterial proteases are potential biomarkers for bacterial diagnosis and targets for novel therapeutic protease inhibitors. To investigate these potential functions, the authors designed and used a protease substrate fluorescence resonance energy transfer (FRET) library comprising 115 short d- and l-amino-acid-containing fluorogenic substrates as a tool to generate proteolytic profiles for a wide range of bacteria. Bacterial specificity of the d-amino acid substrates was confirmed using enzymes isolated from both eukaryotic and prokaryotic organisms. Interestingly, bacterial proteases that are known to be involved in housekeeping and nutrition, but not in virulence, were able to degrade substrates in which a d-amino acid was present. Using our FRET peptide library and culture supernatants from a total of 60 different bacterial species revealed novel, bacteria-specific, proteolytic profiles, although in-species variation was observed for Pseudomonas aeruginosa, Porphyromonas gingivalis, and Staphylococcus aureus. Overall, the specific characteristic of our substrate peptide library makes it a rapid tool to high-throughput screen for novel substrates to detect bacterial proteolytic activity.

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

    PubMed

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

    2012-07-01

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

  11. Serine protease SP105 activates prophenoloxidase in Asian corn borer melanization, and is regulated by serpin-3

    PubMed Central

    Chu, Yuan; Hong, Fang; Liu, Qizhi; An, Chunju

    2017-01-01

    Melanization reaction, resulting from the activation of prophenoloxidase, is a vital immune response in insects for encapsulating and killing the invasive organisms. Prophenoloxidase needs to be proteolytically activated by its upstream prophenoloxidase-activating protease (PAP) in melanization. Identification and characterization of PAPs facilitates the understanding of the molecular mechanisms involved in insect immunity. We here cloned a full-length cDNA for a serine protease, named as SP105, from Asian corn borer, Ostrinia furnacalis (Guenée). The open reading frame of SP105 encodes 424-amino acid residue protein with a 19-residue signal peptide. Sequence comparison indicates that SP105 is most similar to Manduca sexta PAP3, a defined prophenoloxidase-activating protease. qRT-PCR analysis showed that SP105 mRNA levels increased significantly after a bacterial injection. Recombinant SP105 directly cleaved and activated Asian corn borer prophenoloxidase and therefore acted as the prophenoloxidase-activating protease. Additionally, SP105 formed SDS-stable complexes with a serine protease inhibitor, serpin-3, and its activity in activating prophenoloxidase was efficiently inhibited by serpin-3. Our work thus illustrated a prophenoloxidase-activating protease and revealed its regulation by serpin-3. The results would allow further advances in the understanding of the melanization in Asian corn borer and other insects. PMID:28358031

  12. Trypanosomatid cysteine protease activity may be enhanced by a kininogen-like moiety from host serum.

    PubMed Central

    Lonsdale-Eccles, J D; Mpimbaza, G W; Nkhungulu, Z R; Olobo, J; Smith, L; Tosomba, O M; Grab, D J

    1995-01-01

    African trypanosomes contain cysteine proteases (trypanopains) the activity of which can be measured by in vitro digestion of fibrinogen, after electrophoresis in fibrinogen-containing SDS/polyacrylamide gels. When assessed by this procedure, trypanopain from Trypanosoma brucei (trypanopain-Tb) is estimated to have a molecular mass of 28 kDa. However, two additional bands of trypanopain activity (87 kDa and 105 kDa) are observed if serum is added to the trypanopain before electrophoresis. Formation of the 87 and 105 kDa bands is frequently accompanied by a reduction in the intensity of the 28 kDa activity which suggests that the extra bands are complexes of the 28 kDa trypanopain-Tb and a molecule from rat serum called rat trypanopain moledulator (rTM). The rTM-induced activation of cysteine proteases is not restricted to T. brucei as it is also observed with proteases from other protozoan parasites such as bloodstream forms of Trypanosoma congolense and the mammalian-infective in vitro-derived promastigote forms of Leishmania donovani and Leishmania major. The physical properties of rTM resemble those of the kininogen family of cysteine protease inhibitors. rTM is an acidic (pI 4.7) heat-stable 68 kDa glycoprotein with 15 kDa protease-susceptible domains. This resemblance between rTM and kininogens was confirmed by the positive, albeit weak, immunoreactivity between anti-(human low-molecular-mass kininogen) antibody and rTM as well as anti-rTM antibody and human low-molecular-mass kininogen. Furthermore, commercial preparations of human-low-molecular-mass kininogen and chicken egg white cystatin mimicked rTM by forming extra bands of proteolytic activity in the presence of trypanopain-Tb. In some instances, low-molecular-mass kininogen was also observed to increase the rate of hydrolysis of 7-(benzyloxycarbonyl-phenylalanyl-arginyl-amido)-4- methylcoumarin by live T. brucei. Although this effect was rather erratic, in no instance was significant inhibition

  13. The fibrinolytic activity of a novel protease derived from a tempeh producing fungus, Fusarium sp. BLB.

    PubMed

    Sugimoto, Satoshi; Fujii, Tadashi; Morimiya, Tatsuo; Johdo, Osamu; Nakamura, Takumi

    2007-09-01

    Tempeh is a traditional Indonesian soybean-fermented food produced by filamentous fungi, Rhizopus sp. and Fusarium sp. We isolated and sequenced the genomic gene and a cDNA clone encoding a novel protease (FP) from Fusarium sp. BLB. The genomic gene was 856 bp in length and contained two introns. An isolated cDNA clone encoded a protein of 250 amino acids. The predicted amino acid sequence of FP showed highest homology, of 76%, with that of trypsin from Fusarium oxysporum. The hydrolysis activity of FP toward synthetic peptide was higher than that of any other protease tested, including Nattokinases. Furthermore, the thrombolytic activity of FP was about 2.1-fold higher than that of Nattokinase when the concentration of plasminogen was 24 units/ml. These results suggest that FP is superior to Nattokinases in dissolving fibrin when absorbed into the blood.

  14. Pathogen-secreted proteases activate a novel plant immune pathway.

    PubMed

    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.

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

    PubMed

    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.

  16. Luminal Cathepsin G and Protease-Activated Receptor 4

    PubMed Central

    Dabek, Marta; Ferrier, Laurent; Roka, Richard; Gecse, Krisztina; Annahazi, Anita; Moreau, Jacques; Escourrou, Jean; Cartier, Christel; Chaumaz, Gilles; Leveque, Mathilde; Ait-Belgnaoui, Afifa; Wittmann, Tibor; Theodorou, Vassilia; Bueno, Lionel

    2009-01-01

    Impairment of the colonic epithelial barrier and neutrophil infiltration are common features of inflammatory bowel disease. Luminal proteases affect colonic permeability through protease-activated receptors (PARs). We evaluated: (i) whether fecal supernatants from patients with ulcerative colitis (UC) trigger alterations of colonic paracellular permeability and inflammation, and (ii) the roles of cathepsin G (Cat-G), a neutrophil serine protease, and its selective receptor, PAR4, in these processes. Expression levels of both PAR4 and Cat-G were determined in colonic biopsies from UC and healthy subjects. The effects of UC fecal supernatants on colonic paracellular permeability were measured in murine colonic strips. Involvement of Cat-G and PAR4 was evaluated using pepducin P4pal-10 and specific Cat-G inhibitor (SCGI), respectively. In addition, the effect of PAR4-activating peptide was assessed. UC fecal supernatants, either untreated or pretreated with SCGI, were infused into mice, and myeloperoxidase activity was determined. PAR4 was found to be overexpressed in UC colonic biopsies. Increased colonic paracellular permeability that was triggered by UC fecal supernatants was blocked by both SCGI (77%) and P4pal-10 (85%). Intracolonic infusion of UC fecal supernatants into mice increased myeloperoxidase activity. This effect was abolished by SCGI. These observations support that both Cat-G and PAR4 play key roles in generating and/or amplifying relapses in UC and provide a rationale for the development of new therapeutic agents in the treatment of this disease. PMID:19528350

  17. Protease-Activated Pore-Forming Peptides for the Treatment and Imaging of Prostate Cancer

    PubMed Central

    LeBeau, Aaron M.; Denmeade, Samuel R.

    2015-01-01

    A common hallmark of cancers with highly aggressive phenotypes is increased proteolysis in the tumor and the surrounding microenvironment. Prostate cancer has a number of proteases uniquely associated with it that may play various important roles in disease progression. In this report, we utilize the peritumoral proteolytic activity of prostate cancer to activate engineered peptide constructs for the treatment and noninvasive imaging of prostate cancer. Using a modular "propeptide" approach, a cationic diastereomeric pore-forming peptide domain was linked to an inactivating acidic peptide domain. The inactivating acidic peptide domain was engineered to be a cleavable substrate for the secreted serine protease prostate-specific antigen (PSA) or the transmembrane metalloprotease prostate-specific membrane antigen (PSMA). The propeptides were then evaluated in a direct comparison study. Both the PSA and PSMA activated propeptides were found to be cytotoxic to prostate cancer cells in vitro. In vivo, however, treatment of LNCaP and CWR22Rv1 xenografts with the PSMA propeptide resulted in a pronounced cytostatic effect when compared with xenografts treated with the PSA propeptide or the cationic diastereomeric peptide alone. The PSMA activated propeptide also proved to be an effective optical imaging probe in vivo when labeled with a near-infrared fluorophore. These data suggest that protease-activated pore-forming peptides could potentially be used for both imaging and treating prostate cancer. PMID:25537662

  18. Effects of Protease, Phytase and a Bacillus sp. Direct-Fed Microbial on Nutrient and Energy Digestibility, Ileal Brush Border Digestive Enzyme Activity and Cecal Short-Chain Fatty Acid Concentration in Broiler Chickens

    PubMed Central

    Murugesan, Ganapathi R.; Romero, Luis F.; Persia, Michael E.

    2014-01-01

    Two experiments were conducted to determine the effects of protease and phytase (PP) and a Bacillus sp. direct-fed microbial (DFM) on dietary energy and nutrient utilization in broiler chickens. In the first experiment, Ross 308 broiler chicks were fed diets supplemented with PP and DFM in a 2×2 factorial arrangement. The 4 diets (control (CON), CON + PP, CON + DFM, and CON + PP + DFM) were fed from 15–21 days of age. In Experiment 1, significant interaction (P≤0.01) between PP and DFM on the apparent ileal digestibility coefficient for starch, crude protein, and amino acid indicated that both additives increased the digestibility. Both additives increased the nitrogen retention coefficient with a significant interaction (P≤0.01). Although no interaction was observed, significant main effects (P≤0.01) for nitrogen-corrected apparent ME (AMEn) for PP or DFM indicated an additive response. In a follow-up experiment, Ross 308 broiler chicks were fed the same experimental diets from 1–21 days of age. Activities of ileal brush border maltase, sucrase, and L-alanine aminopeptidase were increased (P≤0.01) by PP addition, while a trend (P = 0.07) for increased sucrase activity was observed in chickens fed DFM, in Experiment 2. The proportion of cecal butyrate was increased (P≤0.01) by DFM addition. Increased nutrient utilization and nitrogen retention appear to involve separate but complementary mechanisms for PP and DFM, however AMEn responses appear to have separate and additive mechanisms. PMID:25013936

  19. Evolution of the protease-activated receptor family in vertebrates

    PubMed Central

    JIN, MIN; YANG, HAI-WEI; TAO, AI-LIN; WEI, JI-FU

    2016-01-01

    Belonging to the G protein-coupled receptor (GPcr) family, the protease-activated receptors (Pars) consist of 4 members, PAR1-4. PARs mediate the activation of cells via thrombin, serine and other proteases. Such protease-triggered signaling events are thought to be critical for hemostasis, thrombosis and other normal pathological processes. In the present study, we examined the evolution of PARs by analyzing phylogenetic trees, chromosome location, selective pressure and functional divergence based on the 169 functional gene alignment sequences from 57 vertebrate gene sequences. We found that the 4 PARs originated from 4 invertebrate ancestors by phylogenetic trees analysis. The selective pressure results revealed that only PAR1 appeared by positive selection during its evolution, while the other PAR members did not. In addition, we noticed that although these PARs evolved separately, the results of functional divergence indicated that their evolutional rates were similar and their functions did not significantly diverge. The findings of our study provide valuable insight into the evolutionary history of the vertebrate PAR family. PMID:26820116

  20. The entomopathogenic fungus Metarhizium anisopliae alters ambient pH, allowing extracellular protease production and activity.

    PubMed

    St Leger, R J; Nelson, J O; Screen, S E

    1999-10-01

    Ambient pH regulates the expression of virulence genes of Metarhizium anisopliae, but it was unknown if M. anisopliae can regulate ambient pH. Mutants of M. anisopliae altered in production of oxalic acid were evaluated for the interrelationship of ambient pH, buffering capacity added to media, growth, and generation of extracellular proteases and ammonia. Wild-type and acid-overproducing mutants [Acid(+)] grew almost as well at pH 8 as at pH 6, but acid-non-producing [Acid(-)] mutants showed limited growth at pH 8, indicating that acid production is linked to the ability to grow at higher pH. Production of ammonia by M. anisopliae was strongly stimulated by low levels of amino acids in the medium when cells were derepressed for nitrogen and carbon. Likewise, although Aspergillus fumigatus and Neurospora crassa produced some ammonia in minimal media, addition of low levels of amino acids enhanced production. Ammonia production by A. fumigatus, N. crassa and M. anisopliae increased the pH of the medium and allowed production of subtilisin proteases, whose activities are observed only at basic pH. In contrast, protease production by the Acid(+) mutants of M. anisopliae was greatly reduced because of the acidification of the medium. This suggests that alkalinization by ammonia production is adaptive by facilitating the utilization of proteinaceous nutrients. Collectively, the data imply that ammonia may have functions related to regulation of the microenvironment and that it represents a previously unconsidered virulence factor in diverse fungi with the potential to harm tissues and disturb the host's immune system.

  1. Expression and activation of proteases in co-cultures.

    PubMed

    Paduch, Roman; Kandefer-Szerszeń, Martyna

    2011-01-01

    The present study concerned the expression and activation of metalloproteinase-2 (MMP-2), metalloproteinase-9 (MMP-9) and the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system in co-cultures of human colon carcinoma cell spheroids (HT29, LS180, SW948) with human normal colon epithelium (CCD 841 CoTr), myofibroblasts (CCD-18Co) and endothelial cells (HUVEC). Additionally, the influence of monensin on the production and function of the proteases was tested. Tumor cells expressed small amounts of MMP-2, MMP-9 and uPA. Normal cells generally produced proportionally higher concentrations of these proteases (especially MMP-2, compared with significantly smaller yields of MMP-9 and significantly lower amounts of uPAR than tumors. In co-cultures of tumor spheroids with normal cell monolayers, the concentration of the proteases was equal to the sum of the enzymes produced in monocultures of both types of cells. The highest activity of uPA, measured as the reduction of the chromogenic substrate (S-2444), was detected in supernatants and lysates of endothelial cells. Interestingly, in normal cells, the higher expression of proteases, mainly uPA, measured as the level of protein concentration, was closely linked with their lower activity and inversely, in tumor cells, the low level of the expression of the enzymes correlated with their high enzymatic activity. In zymography analysis, mainly pro-MMPs were detected both in culture supernatants and cell lysates. The highest amounts of active forms of the MMPs were detected in tumor spheroids co-cultured with endothelial cells. Monensin inhibited MMPs and uPA secretion but significantly increased uPAR release, mainly from normal cells. In conclusion, during direct interactions of tumor cells with normal cells, MMPs and the uPA/uPAR system play an important role in the degradation of ECM and tumor development, but as we found, there is a reverse relationship between the concentration and the

  2. [Chromatographic separation of activated proteases from human plasma].

    PubMed

    Lehmann, B; Taucher, M; Kühne, H; Scheuch, D W

    1988-01-01

    After separation of aceton and dextran sulfate activated human plasma by column chromatography on DEAE-cellulose three esterolytically and amidolytically active fractions, respectively, were obtained, which were assigned to the following species: plasma kallikrein (PK), PK.alpha-macroglobulin.HMW-Kininogen. Their percentage in the whole activity is variable. The proportion of free PK is low (0.11). For characterization of the products we studied inhibition by different polyvalent inhibitors. The Michaelis constant (Km) with p-toluene-sulfonyl-L-arginine methyl ester (TAME) were determined. For simulation of in vivo conditions dextran sulfate activated plasma was inactivated at 37 degrees C. The residual activity and the spontaneous activity in plasma from patients with shock are produced by different active protease inhibitor complexes.

  3. Biased signaling by peptide agonists of protease activated receptor 2.

    PubMed

    Jiang, Yuhong; Yau, Mei-Kwan; Kok, W Mei; Lim, Junxian; Wu, Kai-Chen; Liu, Ligong; Hill, Timothy A; Suen, Jacky Y; Fairlie, David P

    2017-02-07

    Protease activated receptor 2 (PAR2) is associated with metabolism, obesity, inflammatory, respiratory and gastrointestinal disorders, pain, cancer and other diseases. The extracellular N-terminus of PAR2 is a common target for multiple proteases, which cleave it at different sites to generate different N-termini that activate different PAR2-mediated intracellular signaling pathways. There are no synthetic PAR2 ligands that reproduce the same signaling profiles and potencies as proteases. Structure-activity relationships here for 26 compounds spanned a signaling bias over 3 log units, culminating in three small ligands as biased agonist tools for interrogating PAR2 functions. DF253 (2f-LAAAAI-NH2) triggered PAR2-mediated calcium release (EC50 2 μM) but not ERK1/2 phosphorylation (EC50 > 100 μM) in CHO cells transfected with hPAR2. AY77 (Isox-Cha-Chg-NH2) was a more potent calcium-biased agonist (EC50 40 nM, Ca2+; EC50 2 μM, ERK1/2), while its analogue AY254 (Isox-Cha-Chg-A-R-NH2) was an ERK-biased agonist (EC50 2 nM, ERK1/2; EC50 80 nM, Ca2+). Signaling bias led to different functional responses in human colorectal carcinoma cells (HT29). AY254, but not AY77 or DF253, attenuated cytokine-induced caspase 3/8 activation, promoted scratch-wound healing and induced IL-8 secretion, all via PAR2-ERK1/2 signaling. Different ligand components were responsible for different PAR2 signaling and functions, clues that can potentially lead to drugs that modulate different pathway-selective cellular and physiological responses.

  4. Botulinum neurotoxin devoid of receptor binding domain translocates active protease.

    PubMed

    Fischer, Audrey; Mushrush, Darren J; Lacy, D Borden; Montal, Mauricio

    2008-12-01

    Clostridium botulinum neurotoxin (BoNT) causes flaccid paralysis by disabling synaptic exocytosis. Intoxication requires the tri-modular protein to undergo conformational changes in response to pH and redox gradients across endosomes, leading to the formation of a protein-conducting channel. The approximately 50 kDa light chain (LC) protease is translocated into the cytosol by the approximately 100 kDa heavy chain (HC), which consists of two modules: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). Here we exploited the BoNT modular design to identify the minimal requirements for channel activity and LC translocation in neurons. Using the combined detection of substrate proteolysis and single-channel currents, we showed that a di-modular protein consisting only of LC and TD was sufficient to translocate active protease into the cytosol of target cells. The RBD is dispensable for cell entry, channel activity, or LC translocation; however, it determined a pH threshold for channel formation. These findings indicate that, in addition to its individual functions, each module acts as a chaperone for the others, working in concert to achieve productive intoxication.

  5. Triterpenes from Cynomorium songaricium--analysis of HCV protease inhibitory activity, quantification, and content change under the influence of heating.

    PubMed

    Ma, Chao-Mei; Wei, Ying; Wang, Zhi-Gang; Hattori, Masao

    2009-01-01

    Inhibitory activity of the three major triterpenes from the stems of Cynomorium songaricum--ursolic acid, acetyl ursolic acid, and malonyl ursolic acid hemiester--and their related compounds were tested for their inhibitory activity on HCV protease; malonyl ursolic acid hemiester was the most potent. A HPLC-PAD (photo diode array detector)-MS method was established to quantify the contents of each triterpene in C. songaricum. Using this method, the effect heating had on the contents was also investigated. It was found that among the three triterpenes, the content of malonyl ursolic acid hemiester decreased most quickly during the heating process.

  6. Cockroach protease allergen induces allergic airway inflammation via epithelial cell activation

    PubMed Central

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

    2017-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

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

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

    PubMed

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

    2014-10-24

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

  9. Conformational transition of the lid helix covering the protease active site is essential for the ATP-dependent protease activity of FtsH.

    PubMed

    Suno, Ryoji; Shimoyama, Masakazu; Abe, Akiko; Shimamura, Tatsuro; Shimodate, Natsuka; Watanabe, Yo-hei; Akiyama, Yoshinori; Yoshida, Masasuke

    2012-09-21

    When bound to ADP, ATP-dependent protease FtsH subunits adopt either an "open" or "closed" conformation. In the open state, the protease catalytic site is located in a narrow space covered by a lidlike helix. This space disappears in the closed form because the lid helix bends at Gly448. Here, we replaced Gly448 with various residues that stabilize helices. Most mutants retained low ATPase activity and bound to the substrate protein, but lost protease activity. However, a mutant proline substitution lost both activities. Our study shows that the conformational transition of the lid helix is essential for the function of FtsH.

  10. The protease activity of the paracaspase MALT1 is controlled by monoubiquitination.

    PubMed

    Pelzer, Christiane; Cabalzar, Katrin; Wolf, Annette; Gonzalez, Montserrat; Lenz, Georg; Thome, Margot

    2013-04-01

    The protease activity of the paracaspase MALT1 is central to lymphocyte activation and lymphomagenesis, but how this activity is controlled remains unknown. Here we identify a monoubiquitination of MALT1 on Lys644 that activated the protease function of MALT1. Monoubiquitinated MALT1 had enhanced protease activity, whereas a ubiquitination-deficient MALT1 mutant with replacement of that lysine with arginine (MALT1(K644R)) had less protease activity, which correlated with impaired induction of interleukin 2 (IL-2) via the T cell antigen receptor in activated T cells. Expression of MALT1(K644R) diminished the survival of cells derived from diffuse large B cell lymphoma of the activated B cell-like subtype (ABC DLBCL), which require constitutive protease activity of MALT1 for survival. Thus, monoubiquitination of MALT1 is essential for its catalytic activation and is therefore a potential target for the treatment of ABC-DLBCL and for immunomodulation.

  11. Recent developments in production and biotechnological applications of cold-active microbial proteases.

    PubMed

    Kuddus, Mohammed; Ramteke, Pramod W

    2012-11-01

    Microbial proteases that occupy a pivotal position with respect to their commercial applications are most important hydrolytic enzymes and have been studied extensively since the advent of enzymology. Cold-adapted microorganisms are potential source of cold-active proteases and they have been isolated from the cold regions. Although there are many microbial sources available for producing proteases, only few are recognized as commercial producer. Cold-active proteases along with their producing microbes are of commercial value and find multiple applications in various industrial and biotechnological sectors such as additives in detergents, additives in food industries, environmental bioremediations, biotransformation and molecular biology applications. Therefore, cold-active proteases are the enzymes of choice for many biotechnologists, microbiologists, biochemists, environmentalists and biochemical engineers. In the present review, we discuss some novel sources along with recent developments in production and biotechnological applications of cold-active microbial proteases.

  12. Extracellular protease derived from lactic acid bacteria stimulates the fermentative lactic acid production from the by-products of rice as a biomass refinery function.

    PubMed

    Watanabe, Masanori; Techapun, Charin; Kuntiya, Ampin; Leksawasdi, Noppol; Seesuriyachan, Phisit; Chaiyaso, Thanongsak; Takenaka, Shinji; Maeda, Isamu; Koyama, Masahiro; Nakamura, Kozo

    2017-02-01

    A lactic acid producing bacterium, Lactobacillus rhamnosus M-23, newly isolated from a rice washing drainage storage tank was found to produce l-(+)-lactic acid from a non-sterilized mixture of rice washing drainage and rice bran without any additions of nutrients under the simultaneous saccharification and fermentation (SSF) process. This strain has the ability to utilize the non-sterilized rice washing drainage and rice bran as a source of carbohydrate, saccharifying enzymes and nutrients for lactic acid production. Observation of extracellular protease activity in SSF culture broth showed that a higher protease activity was present in strain M-23 than in other isolated lactic acid producing bacteria (LABs). To investigate the structural changes of solid particles of rice washing drainage throughout LAB cultivation, scanning electron microscopic (SEM) observation and Fourier transform infrared-spectroscopy (FT-IR) analysis were performed. The results of the SEM observation showed that the surface material could be removed from solid particles of rice washing drainage treated by culture broth (supernatant) of strain M-23, thus exposing the crystal structure of the starch particle surface. The results of the FT-IR analysis revealed that the specific transmittance decrease of the CC and CO stretching and OH group of the solid particles of the rice washing drainage were highly correlated with the produced lactic acid concentration and extracellular protease activity, respectively. These results demonstrate the high lactic acid producing ability of strain M-23 from a non-sterilized mixture of rice washing drainage and rice bran under the SSF condition due to the removal of proteinaceous material and exposure of the starch particle surface by extracellular protease.

  13. Protease-activated receptors and prostaglandins in inflammatory lung disease

    PubMed Central

    Peters, Terence; Henry, Peter J

    2009-01-01

    Protease-activated receptors (PARs) are a novel family of G protein-coupled receptors. Signalling through PARs typically involves the cleavage of an extracellular region of the receptor by endogenous or exogenous proteases, which reveals a tethered ligand sequence capable of auto-activating the receptor. A considerable body of evidence has emerged over the past 20 years supporting a prominent role for PARs in a variety of human physiological and pathophysiological processes, and thus substantial attention has been directed towards developing drug-like molecules that activate or block PARs via non-proteolytic pathways. PARs are widely expressed within the respiratory tract, and their activation appears to exert significant modulatory influences on the level of bronchomotor tone, as well as on the inflammatory processes associated with a range of respiratory tract disorders. Nevertheless, there is debate as to whether the principal response to PAR activation is an augmentation or attenuation of airways inflammation. In this context, an important action of PAR activators may be to promote the generation and release of prostanoids, such as prostglandin E2, which have well-established anti-inflammatory effects in the lung. In this review, we primarily focus on the relationship between PARs, prostaglandins and inflammatory processes in the lung, and highlight their potential role in selected respiratory tract disorders, including pulmonary fibrosis, asthma and chronic obstructive pulmonary disease. This article is part of a themed issue on Mediators and Receptors in the Resolution of Inflammation. To view this issue visit http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009 PMID:19845685

  14. Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets

    PubMed Central

    Verma, Sonia; Dixit, Rajnikant; Pandey, Kailash C.

    2016-01-01

    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 metalloproteases. 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 prodomain (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. PMID:27199750

  15. MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses.

    PubMed

    Yu, Jong W; Hoffman, Sandy; Beal, Allison M; Dykon, Angela; Ringenberg, Michael A; Hughes, Anna C; Dare, Lauren; Anderson, Amber D; Finger, Joshua; Kasparcova, Viera; Rickard, David; Berger, Scott B; Ramanjulu, Joshi; Emery, John G; Gough, Peter J; Bertin, John; Foley, Kevin P

    2015-01-01

    CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo.

  16. A Novel Apoptotic Protease Activated in Human Breast Cancer Cells After Poisoning Topoisomerase I

    DTIC Science & Technology

    1999-10-01

    tocopherol, N- acetyl -L- cysteine ( NAC ) or pyrrolidinedithiocarbamate (PDTC)) did not significantly affect lethality caused by B-lap exposure (data not...will be required for the cloning of this novel noncaspase cysteine protease. The new hypothesis being tested is that B- lap activates calpain, which...caspase cysteine protease was activated within 4-8 hours, concomitant with the appearance of DNA fragmentation, measured by TUNEL assays; (e) protease

  17. Fluorigenic substrates for the protease activities of botulinum neurotoxins, serotypes A, B, and F.

    PubMed

    Schmidt, James J; Stafford, Robert G

    2003-01-01

    The seven botulinum neurotoxins (BoNTs) are zinc metalloproteases that cleave neuronal proteins involved in neurotransmitter release and are among the most toxic natural products known. High-throughput BoNT assays are needed for use in antibotulinum drug discovery and to characterize BoNT protease activities. Compared to other proteases, BoNTs exhibit unusually stringent substrate requirements with respect to amino acid sequences and polypeptide lengths. Nonetheless, we have devised a strategy for development of fluorigenic BoNT protease assays, based on earlier structure-function studies, that has proven successful for three of the seven serotypes: A, B, and F. In synthetic peptide substrates, the P(1) and P(3)' residues were substituted with 2,4-dinitrophenyl-lysine and S-(N-[4-methyl-7-dimethylamino-coumarin-3-yl]-carboxamidomethyl)-cysteine, respectively. By monitoring the BoNT-catalyzed increase in fluorescence over time, initial hydrolysis rates could be obtained in 1 to 2 min when BoNT concentrations were 60 ng/ml (about 1 nM) or higher. Each BoNT cleaved its fluorigenic substrate at the same location as in the neuronal target protein, and kinetic constants indicated that the substrates were selective and efficient. The fluorigenic assay for BoNT B was used to characterize a new competitive inhibitor of BoNT B protease activity with a K(i) value of 4 micro M. In addition to real-time activity measurements, toxin concentration determinations, and kinetic studies, the BoNT substrates described herein may be directly incorporated into automated high-throughput assay systems to screen large numbers of compounds for potential antibotulinum drugs.

  18. Cleavage and activation of human factor IX by serine proteases

    SciTech Connect

    Enfield, D.L.; Thompson, A.R.

    1984-10-01

    Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated /sup 125/I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa-tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of /sup 125/I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.

  19. Gastrointestinal absorption and biological activities of serine and cysteine proteases of animal and plant origin: review on absorption of serine and cysteine proteases.

    PubMed

    Lorkowski, Gerhard

    2012-01-01

    Research has confirmed that peptides and larger protein molecules pass through the mucosal barrier of the gastrointestinal tract. Orally administered serine and cysteine proteases of plant and animal origin also reach blood and lymph as intact, high molecular weight and physiologically active protein molecules. Their absorption may be supported by a self-enhanced paracellular transport mechanism resulting in sub-nanomolar concentration of transiently free protease molecules or, in a complex with anti-proteases, at higher concentrations. Data from pharmacokinetic investigations reveals dose linearity for maximum plasma levels of free proteases not unusual for body proteases and a high inter-individual variability. There is no interference with each other after oral administration of protease combinations, and absorption follows an unusual invasion and elimination kinetic due to slow velocity of absorption and a fast 100% protein binding to anti-proteases. Oral application of proteases leads to increased proteolytic serum activity and increased plasma concentrations of the corresponding anti-proteases. Their biological activity is determined by their proteolytic activity as free proteases on soluble peptides/proteins or cell surface receptors (e.g. protease activated receptors) and their activity in the complex formed with their specific and/or unspecific anti-proteases. The anti-protease-complexes, during immune reaction and injuries often loaded with different cytokines, are cleared from body fluids and tissue by receptor mediated endocytosis on hepatocytes and/or blood cells. Oral administration of enteric coated tablets containing proteolytic enzymes of plant and animal origin may be a safe method to stabilize, positively influence or enhance physiological and immunological processes during disease processes and in healthy consumers.

  20. Protease Inhibition by Oleic Acid Transfer From Chronic Wound Dressings to Albumin

    SciTech Connect

    Edwards, J. V.; Howley, Phyllis; Davis, Rachel M.; Mashchak, Andrew D.; Goheen, Steven C.

    2007-08-01

    High elastase and cathepsin G activities have been observed in chronic wounds. These levels can inhibit healing through degradation of growth factors, cytokines, and extracellular matrix proteins. Oleic acid (18:1) is a non-toxic elastase inhibitor with some potential for redressing the imbalance of elastase activity found in chronic wounds. Cotton wound dressing material was characterized as a transfer carrier for affinity uptake of 18:1 by albumin under conditions mimicking chronic wounds. 18:1-treated cotton was examined for its ability to bind and release the fatty acid in the presence of albumin. The mechanism of 18:1 uptake from cotton and binding by albumin was examined with both intact dressings and cotton fiber-designed chromatography. Raman spectra of the albumin-18:1 complexes under liquid-liquid equilibrium conditions revealed fully saturated albumin-18:1 complexes with a 1:1 weight ratio of albumin:18:1. Cotton chromatography under liquid-solid equilibrium conditions revealed oleic acid transfer from cotton to albumin at 27 mole equivalents of 18:1 per mole albumin. Cotton was contrasted with hydrogel, and hydrocolloid wound dressing for its comparative ability to lower elastase activity. Each dressing material evaluated was found to release 18:1 in the presence of albumin with significant inhibition of elastase activity. The 18:1-formulated wound dressings lowered elastase activity in a dose dependent manner in the order cotton gauze > hydrogel > hydrocolloid. In contrast the cationic serine protease Cathepsin G was inihibited by 18:1 within a narrow range of 18:1-cotton formulations. Four per cent Albumin solutions were most effective in binding cotton bound-18:1. However, 2% albumin was sufficient to transfer quantities of 18:1 necessary to achieve a significant elastase-lowering effect. Formulations with 128 mg 18:1/g cotton gauze had equivalent elastase lowering with 1 - 4% albumin. 18:1 bound to cotton wound dressings may have promise in the

  1. Design of protease-resistant myelin basic protein-derived peptides by cleavage site directed amino acid substitutions.

    PubMed

    Burster, Timo; Marin-Esteban, Viviana; Boehm, Bernhard O; Dunn, Shannon; Rotzschke, Olaf; Falk, Kirsten; Weber, Ekkehard; Verhelst, Steven H L; Kalbacher, Hubert; Driessen, Christoph

    2007-11-15

    Multiple Sclerosis (MS) is considered to be a T cell-mediated autoimmune disease. An attractive strategy to prevent activation of autoaggressive T cells in MS, is the use of altered peptide ligands (APL), which bind to major histocompatibility complex class II (MHC II) molecules. To be of clinical use, APL must be capable of resisting hostile environments including the proteolytic machinery of antigen presenting cells (APC). The current design of APL relies on cost- and labour-intensive strategies. To overcome these major drawbacks, we used a deductive approach which involved modifying proteolytic cleavage sites in APL. Cleavage site-directed amino acid substitution of the autoantigen myelin basic protein (MBP) resulted in lysosomal protease-resistant, high-affinity binding peptides. In addition, these peptides mitigated T cell activation in a similar fashion as conventional APL. The strategy outlined allows the development of protease-resistant APL and provides a universal design strategy to improve peptide-based immunotherapeutics.

  2. Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution.

    PubMed

    Martinez, Ronny; Jakob, Felix; Tu, Ran; Siegert, Petra; Maurer, Karl-Heinz; Schwaneberg, Ulrich

    2013-03-01

    Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting activity and stability properties suitable for applications in laundry and dish washing detergents. However, BgAP suffers from a significant decrease of activity at low temperatures. In order to increase BgAP activity at 15°C, a directed evolution campaign based on the SeSaM random mutagenesis method was performed. An optimized microtiter plate expression system in B. subtilis was established and classical proteolytic detection methods were adapted for high throughput screening. In parallel, the libraries were screened for increased residual proteolytic activity after incubation at 58°C. Three iterative rounds of directed BgAP evolution yielded a set of BgAP variants with increased specific activity (K(cat)) at 15°C and increased thermal resistance. Recombination of both sets of amino acid substitutions resulted finally in variant MF1 with a 1.5-fold increased specific activity (15°C) and over 100 times prolonged half-life at 60°C (224 min compared to 2 min of the WT BgAP). None of the introduced amino acid substitutions were close to the active site of BgAP. Activity-altering amino acid substitutions were from non-charged to non-charged or from sterically demanding to less demanding. Thermal stability improvements were achieved by substitutions to negatively charged amino acids in loop areas of the BgAP surface which probably fostered ionic and hydrogen bonds interactions.

  3. Activation of specific apoptotic caspases with an engineered small-molecule-activated protease.

    PubMed

    Gray, Daniel C; Mahrus, Sami; Wells, James A

    2010-08-20

    Apoptosis is a conserved cellular pathway that results in the activation of cysteine-aspartyl proteases, or caspases. To dissect the nonredundant roles of the executioner caspase-3, -6, and -7 in orchestrating apoptosis, we have developed an orthogonal protease to selectively activate each isoform in human cells. Our approach uses a split-tobacco etch virus (TEV) protease under small-molecule control, which we call the SNIPer, with caspase alleles containing genetically encoded TEV cleavage sites. These studies reveal that all three caspases are transiently activated but only activation of caspase-3 or -7 is sufficient to induce apoptosis. Proteomic analysis shown here and from others reveals that 20 of the 33 subunits of the 26S proteasome can be cut by caspases, and we demonstrate synergy between proteasome inhibition and dose-dependent caspase activation. We propose a model of proteolytic reciprocal negative regulation with mechanistic implications for the combined clinical use of proteasome inhibitors and proapoptotic drugs.

  4. Distinct contribution of Toxoplasma gondii rhomboid proteases 4 and 5 to micronemal protein protease 1 activity during invasion.

    PubMed

    Rugarabamu, George; Marq, Jean-Baptiste; Guérin, Amandine; Lebrun, Maryse; Soldati-Favre, Dominique

    2015-07-01

    Host cell entry by the Apicomplexa is associated with the sequential secretion of invasion factors from specialized apical organelles. Secretion of micronemal proteins (MICs) complexes by Toxoplasma gondii facilitates parasite gliding motility, host cell attachment and entry, as well as egress from infected cells. The shedding of MICs during these steps is mediated by micronemal protein proteases MPP1, MPP2 and MPP3. The constitutive activity of MPP1 leads to the cleavage of transmembrane MICs and is linked to the surface rhomboid protease 4 (ROM4) and possibly to rhomboid protease 5 (ROM5). To determine their importance and respective contribution to MPP1 activity, in this study ROM4 and ROM5 genes were abrogated using Cre-recombinase and CRISPR-Cas9 nuclease, respectively, and shown to be dispensable for parasite survival. Parasites lacking ROM4 predominantly engage in twirling motility and exhibit enhanced attachment and impaired invasion, whereas intracellular growth and egress is not affected. The substrates MIC2 and MIC6 are not cleaved in rom4-ko parasites, in contrast, intramembrane cleavage of AMA1 is reduced but not completely abolished. Shedding of MICs and invasion are not altered in the absence of ROM5; however, this protease responsible for the residual cleavage of AMA1 is able to cleave other AMA family members and exhibits a detectable contribution to invasion in the absence of ROM4.

  5. Development of activity-based probes for trypsin-family serine proteases.

    PubMed

    Pan, Zhengying; Jeffery, Douglas A; Chehade, Kareem; Beltman, Jerlyn; Clark, James M; Grothaus, Paul; Bogyo, Matthew; Baruch, Amos

    2006-06-01

    A series of diphenylphosphonate-based probes were developed for the trypsin-like serine proteases. These probes selectively target serine proteases rather than general serine hydrolases that are targets for fluorophosphonate-based probes. This increased selectivity allows detection of low abundance serine proteases in complex proteomes using simple SDS-PAGE methods. We present here the application of multiple probes in enzyme activity profiling of intact mast cells, a type of inflammatory cell implicated in allergy and autoimmune diseases.

  6. A novel aspartic acid protease gene from pineapple fruit (Ananas comosus): cloning, characterization and relation to postharvest chilling stress resistance.

    PubMed

    Raimbault, Astrid-Kim; Zuily-Fodil, Yasmine; Soler, Alain; Cruz de Carvalho, Maria H

    2013-11-15

    A full-length cDNA encoding a putative aspartic acid protease (AcAP1) was isolated for the first time from the flesh of pineapple (Ananas comosus) fruit. The deduced sequence of AcAP1 showed all the common features of a typical plant aspartic protease phytepsin precursor. Analysis of AcAP1 gene expression under postharvest chilling treatment in two pineapple varieties differing in their resistance to blackheart development revealed opposite trends. The resistant variety showed an up-regulation of AcAP1 precursor gene expression whereas the susceptible showed a down-regulation in response to postharvest chilling treatment. The same trend was observed regarding specific AP enzyme activity in both varieties. Taken together our results support the involvement of AcAP1 in postharvest chilling stress resistance in pineapple fruits.

  7. Soluble expression and enzymatic activity evaluation of protease from reticuloendotheliosis virus.

    PubMed

    Hu, Feng; Zhao, Yan; Qi, Xiaole; Cui, Hongyu; Gao, Yulong; Gao, Honglei; Liu, Changjun; Wang, Yongqiang; Zhang, Yanping; Li, Kai; Wang, Xiaomei; Wang, Yunfeng

    2015-10-01

    The protease (PR) encoded by most retroviruses is deeply involved in the lifecycle and infection process of retroviruses by possessing the specificity necessary to correctly cleave the viral polyproteins and host cell proteins. However, as an important representative of avian retroviruses, the enzymatic properties of PR from reticuloendotheliosis virus (REV) have not been clearly documented. The recombinant PR, its mutant fused with a His-tag, and its substrate p18-p30 fused with a GST-tag were expressed in the Escherichia coli system as soluble enzymes. The soluble PR and p18-p30 were purified using Ni-NTA His Bind Resin and Glutathione Sepharose 4B, respectively. The enzymatic activity of PR was analyzed using the substrate of p18-p30. The expressed prokaryotic protease has enzyme activity that is dependent on such conditions as temperature, pH, and ions, and its activity can be inhibited by caspase inhibitor and the divalent metal ions Ca(2+) and Ni(2+). In addition, the key role of the residue Thr (amino acids 28) for the enzymatic activity of PR was identified. Furthermore, the caspase inhibitor Z-VAD-FMK was confirmed to inhibit the PR enzymatic activity of REV. For the first time, the PR of REV was expressed in the soluble form, and the optimal enzymatic reaction system in vitro was developed and preliminarily used. This study provides essential tools and information for further understanding the infection mechanism of REV and for the development of antiviral drugs treating retroviruses.

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

    PubMed

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

    2016-03-01

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

  9. Activation of influenza viruses by proteases from host cells and bacteria in the human airway epithelium.

    PubMed

    Böttcher-Friebertshäuser, Eva; Klenk, Hans-Dieter; Garten, Wolfgang

    2013-11-01

    Influenza is an acute infection of the respiratory tract, which affects each year millions of people. Influenza virus infection is initiated by the surface glycoprotein hemagglutinin (HA) through receptor binding and fusion of viral and endosomal membranes. HA is synthesized as a precursor protein and requires cleavage by host cell proteases to gain its fusion capacity. Although cleavage of HA is crucial for virus infectivity, little was known about relevant proteases in the human airways for a long time. Recent progress in the identification and characterization of HA-activating host cell proteases has been considerable however and supports the idea of targeting HA cleavage as a novel approach for influenza treatment. Interestingly, certain bacteria have been demonstrated to support HA activation either by secreting proteases that cleave HA or due to activation of cellular proteases and thereby may contribute to virus spread and enhanced pathogenicity. In this review, we give an overview on activation of influenza viruses by proteases from host cells and bacteria with the main focus on recent progress on HA cleavage by proteases HAT and TMPRSS2 in the human airway epithelium. In addition, we outline investigations of HA-activating proteases as potential drug targets for influenza treatment.

  10. Genetic characterization and expression of the novel fungal protease, EPg222 active in dry-cured meat products.

    PubMed

    Benito, María J; Connerton, Ian F; Córdoba, Juan J

    2006-11-01

    EPg222 protease is a novel extracellular enzyme produced by Penicillium chrysogenum (Pg222) isolated from dry-cured hams that has the potential for use over a broad range of applications in industries that produce dry-cured meat products. The gene encoding EPg222 protease has been identified. Peptide sequences of EPg222 were obtained by de novo sequencing of tryptic peptides using mass spectrometry. The corresponding gene was amplified by PCR using degenerated primers based on a combination of conserved serine protease-encoding sequences and reverse translation of the peptide sequences. EPg222 is encoded as a gene of 1,361 bp interrupted by two introns. The deduced amino acid sequence indicated that the enzyme is synthesized as a preproenzyme with a putative signal sequence of 19 amino acids (aa), a prosequence of 96 aa and a mature protein of 283 aa. A cDNA encoding EPg222 has been cloned and expressed as a functionally active enzyme in Pichia pastoris. The recombinant enzyme exhibits similar activities to the native enzyme against a wide range of protein substrates including muscle myofibrillar protein. The mature sequence contains conserved aa residues characteristic of those forming the catalytic triad of serine proteases (Asp42, His76 and Ser228) but notably the food enzyme exhibits specific aa substitutions in the immunoglobulin-E recognition regions that have been identified in protein homologues that are allergenic.

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

    PubMed

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

    2016-08-01

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

  12. Inactivation of chemotactic activity of C5a by the serratial 56-kilodalton protease.

    PubMed Central

    Oda, T; Kojima, Y; Akaike, T; Ijiri, S; Molla, A; Maeda, H

    1990-01-01

    The effects of the 56-kilodalton protease (56K protease) from Serratia marcescens on complement-derived chemotactic activity were examined. Fresh human serum was incubated with zymosan to produce C5a. This activated serum was then incubated with various concentrations of 56K protease, and the chemotactic activity of mouse peritoneal exudate polymorphonuclear leukocytes (PMN) and macrophages was evaluated. A significant dose-dependent decrease of chemotactic activity was observed after protease treatment. Furthermore, treatment of human recombinant C5a with 56K protease at a dose of 1.0 microgram/ml resulted in a complete loss of chemotactic activity. When the living bacteria of the virulent strain, which produced about 10 times more protease than did the less virulent strain, were injected intraperitoneally into mice, the magnitude of infiltration of polymorphonuclear leukocytes into the peritoneal cavity was much lower than that caused by the less virulent strain. Because complement-dependent chemotactic activity is an initial response to bacterial infection, these results suggest indirect pathogenic functions of serratial proteases that suppress chemotactic activity. PMID:1691142

  13. Acidic mammalian chitinase is a proteases-resistant glycosidase in mouse digestive system

    PubMed Central

    Ohno, Misa; Kimura, Masahiro; Miyazaki, Haruko; Okawa, Kazuaki; Onuki, Riho; Nemoto, Chiyuki; Tabata, Eri; Wakita, Satoshi; Kashimura, Akinori; Sakaguchi, Masayoshi; Sugahara, Yasusato; Nukina, Nobuyuki; Bauer, Peter O.; Oyama, Fumitaka

    2016-01-01

    Chitinases are enzymes that hydrolyze chitin, a polymer of β-1, 4-linked N-acetyl-D-glucosamine (GlcNAc). Chitin has long been considered as a source of dietary fiber that is not digested in the mammalian digestive system. Here, we provide evidence that acidic mammalian chitinase (AMCase) can function as a major digestive enzyme that constitutively degrades chitin substrates and produces (GlcNAc)2 fragments in the mouse gastrointestinal environment. AMCase was resistant to endogenous pepsin C digestion and remained active in the mouse stomach extract at pH 2.0. The AMCase mRNA levels were much higher than those of four major gastric proteins and two housekeeping genes and comparable to the level of pepsinogen C in the mouse stomach tissues. Furthermore, AMCase was expressed in the gastric pepsinogen-synthesizing chief cells. The enzyme was also stable and active in the presence of trypsin and chymotrypsin at pH 7.6, where pepsin C was completely degraded. Mouse AMCase degraded polymeric colloidal and crystalline chitin substrates in the gastrointestinal environments in presence of the proteolytic enzymes. Thus, AMCase can function as a protease-resistant major glycosidase under the conditions of stomach and intestine and degrade chitin substrates to produce (GlcNAc)2, a source of carbon, nitrogen and energy. PMID:27883045

  14. Studies of a calcium-activated neutral protease from chicken skeletal muscle. I. Purification and characterization.

    PubMed

    Ishiura, S; Murofushi, H; Suzuki, K; Imahori, K

    1978-07-01

    A calcium-activated neutral protease was purified 2,700-fold over the crude extract from chicken skeletal muscle. The purified protease migrated as a single band on polyacrylamide gel electrophoresis with or without SDS. Its molecular weight was 80,000 and pH optimum for activity was 7.7. The activity required strictly the presence of calcium (optimum concentration: 1.8 mM) or strontium (optimum concentration: 10 mM) ions. The protease was inhibited by leupeptin, which is known to be a strong inhibitor of papain, cathepsin B, trypsin, and plasmin.

  15. Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki.

    PubMed

    Andrews, R E; Bibilos, M M; Bulla, L A

    1985-10-01

    Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.

  16. Overexpression of Aspergillus tubingensis faeA in protease-deficient Aspergillus niger enables ferulic acid production from plant material.

    PubMed

    Zwane, Eunice N; Rose, Shaunita H; van Zyl, Willem H; Rumbold, Karl; Viljoen-Bloom, Marinda

    2014-06-01

    The production of ferulic acid esterase involved in the release of ferulic acid side groups from xylan was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The highest activity on triticale bran as sole carbon source was observed with the A. tubingensis T8.4 strain, which produced a type A ferulic acid esterase active against methyl p-coumarate, methyl ferulate and methyl sinapate. The activity of the A. tubingensis ferulic acid esterase (AtFAEA) was inhibited twofold by glucose and induced twofold in the presence of maize bran. An initial accumulation of endoglucanase was followed by the production of endoxylanase, suggesting a combined action with ferulic acid esterase on maize bran. A genomic copy of the A. tubingensis faeA gene was cloned and expressed in A. niger D15#26 under the control of the A. niger gpd promoter. The recombinant strain has reduced protease activity and does not acidify the media, therefore promoting high-level expression of recombinant enzymes. It produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran as sole carbon source, which was threefold higher than for the A. tubingensis donor strain. The recombinant AtFAEA was able to extract 50 % of the available ferulic acid from non-pretreated maize bran, making this enzyme suitable for the biological production of ferulic acid from lignocellulosic plant material.

  17. A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival

    PubMed Central

    Kugadas, Abirami; Lamont, Elise A.; Bannantine, John P.; Shoyama, Fernanda M.; Brenner, Evan; Janagama, Harish K.; Sreevatsan, Srinand

    2016-01-01

    The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophages and MAC-T cells that coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc2 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted. PMID:27597934

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

    SciTech Connect

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

    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.

  19. Single Cell Analysis of Leukocyte Protease Activity Using Integrated Continuous-Flow Microfluidics.

    PubMed

    Jing, Tengyang; Lai, Zhangxing; Wu, Lidan; Han, Jongyoon; Lim, Chwee Teck; Chen, Chia-Hung

    2016-12-06

    Leukocytes are the essential cells of the immune system that protect the human body against bacteria, viruses, and other foreign invaders. Secretory products of individual leukocytes, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAMs), are critical for regulating the inflammatory response and mediating host defense. Conventional single cell analytical methods, such as flow cytometry for cellular surface biomarker studies, are insufficient for performing functional assays of the protease activity of individual leukocytes. Here, an integrated continuous-flow microfluidic assay is developed to effectively detect secretory protease activity of individual viable leukocytes. Leukocytes in blood are first washed on-chip with defined buffer to remove background activity, followed by encapsulating individual leukocytes with protease sensors in water-in-oil droplets and incubating for 1 h to measure protease secretion. With this design, single leukocyte protease profiles under naive and phorbol 12-myristate 13-acetate (PMA)-stimulated conditions are reliably measured. It is found that PMA treatment not only elevates the average protease activity level but also reduces the cellular heterogeneity in protease secretion, which is important in understanding immune capability and the disease condition of individual patients.

  20. Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

    SciTech Connect

    Spannaus, Ralf; Bodem, Jochen

    2014-04-15

    In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies. The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity.

  1. A fibrinolytic protease AfeE from Streptomyces sp. CC5, with potent thrombolytic activity in a mouse model.

    PubMed

    Sun, Zhibin; Liu, Pingping; Cheng, Guangyan; Zhang, Biying; Dong, Weiliang; Su, Xingli; Huang, Yan; Cui, Zhongli; Kong, Yi

    2016-04-01

    Fibrinolytic proteases have potential applications in cardiovascular disease therapy. A novel fibrinolytic protease, AfeE, with strong thrombolytic activity was purified from Streptomyces sp. CC5. AfeE displayed maximum activity at 40°C in the pH range of 7.0-12.0. It was strongly inhibited by serine protease inhibitor phenylmethanesulfonylfluoride, soybean trypsin inhibitor, tosyl-l-lysine chloromethyl ketone and tosyl-l-phenylalanine chloromethyl ketone. The activity of the enzyme was partially inhibited by Cu(2+), Co(2+) and Zn(2+). AfeE exhibited higher substrate specificity for fibrin than fibrinogen, which has rarely been reported in fibrinolytic enzymes. AfeE also showed high thrombolytic activity in a carrageenan-induced mouse tail thrombosis model. AfeE prolonged prothrombin time, activated partial thromboplastin time, and thrombin time in rat blood. A bleeding time assay revealed that AfeE did not prolong bleeding time in mice at a dose of 1mg/kg. No acute cytotoxicity was observed for AfeE at 320μg/well in human umbilical vein endothelial cells. The afeE gene was cloned from the genome of Streptomyces sp. CC5. Full-length AFE-CC5E contained 434 amino acids and was processed into a mature form consisting 284 amino acids by posttranslational modification, as revealed by high-resolution mass spectrometry analysis. These results indicate that AfeE is a prospective candidate for antithrombotic drug development.

  2. Inactivation of cystein-aspartic acid protease (caspase)-1 by saikosaponin A.

    PubMed

    Han, Na-Ra; Kim, Hyung-Min; Jeong, Hyun-Ja

    2011-01-01

    This work investigates the anti-inflammatory mechanism of saikosaponin A (SA), a major component of Bupleurum falcatum LINNE. SA significantly inhibited phorbol myristate acetate (PMA) plus A23187-induced the production and expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in human mast cell (HMC)-1 cells. SA suppressed PMA plus A23187-induced phosphorylation of extracellular signal-regulated kinase and p38. When HMC-1 cells were treated with SA, translocation of nuclear factor (NF)-κB/Rel A into nucleus and degradation of inhibitor of NF-κB (IκB) in cytoplasm were inhibited. SA decreased PMA plus A23187-induced cysteine-aspartic acid protease (caspase)-1 activity. IL-1β production was also inhibited by SA. Finally, SA significantly decreased the number of nasal rubs and serum TNF-α level in the ovalbumin-sensitized allergic rhinitis mouse model. The underlying mechanism involves, at least in part, inactivation of caspase-1, which provides new evidence for therapeutic application of SA to target inflammatory processes.

  3. Improvement of Functional Properties of Wheat Gluten Using Acid Protease from Aspergillus usamii

    PubMed Central

    Deng, Lingli; Wang, Zhaoxia; Yang, Sheng; Song, Junmei; Que, Fei; Zhang, Hui; Feng, Fengqin

    2016-01-01

    Hydrolysis parameters (temperature, E/S ratio, pH, and time) for acid protease (from Aspergillus usamii) hydrolysis of wheat gluten were optimized by response surface methodology (RSM) using emulsifying activity index (EAI) as the response factor. A temperature of 48.9°C, E/S ratio of 1.60%, pH 3.0, hydrolysis time of 2.5 h was found to be the optimum condition to obtain wheat gluten hydrolysate with higher EAI. The solubility of wheat gluten was greatly improved by hydrolysis and became independent of pH over the studied range. Enzymatic hydrolysis resulted in dramatically increase in EAI, water and oil holding capacity. Molecular weight distribution results showed that most of the peptides above 10 kDa have been hydrolyzed into smaller peptides. The results of FTIR spectra and disulfide bond (SS) and sulfhydryl (SH) content suggested that a more extensional conformation was formed after hydrolysis, which could account for the improved functional properties. PMID:27467884

  4. Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay

    PubMed Central

    Vosyka, Oliver; Vinothkumar, Kutti R.; Wolf, Eliane V.; Brouwer, Arwin J.; Liskamp, Rob M. J.; Verhelst, Steven H. L.

    2013-01-01

    Rhomboid proteases are evolutionary conserved intramembrane serine proteases. Because of their emerging role in many important biological pathways, rhomboids are potential drug targets. Unfortunately, few chemical tools are available for their study. Here, we describe a mass spectrometry-based assay to measure rhomboid substrate cleavage and inhibition. We have identified isocoumarin inhibitors and developed activity-based probes for rhomboid proteases. The probes can distinguish between active and inactive rhomboids due to covalent, reversible binding of the active-site serine and stable modification of a histidine residue. Finally, the structure of an isocoumarin-based inhibitor with Escherichia coli rhomboid GlpG uncovers an unusual mode of binding at the active site and suggests that the interactions between the 3-substituent on the isocoumarin inhibitor and hydrophobic residues on the protease reflect S′ subsite binding. Overall, these probes represent valuable tools for rhomboid study, and the structural insights may facilitate future inhibitor design. PMID:23359682

  5. Processing and presentation of an antigen of Mycobacterium avium require access to an acidified compartment with active proteases.

    PubMed Central

    Holsti, M A; Allen, P M

    1996-01-01

    We have generated a murine T-cell hybridoma, 1C9, which recognizes an antigen expressed by a virulent clinical isolate of Mycobacterium avium. Both peritoneal exudate macrophages and bone marrow-derived macrophages infected in vitro with M. avium process and present the antigen to the T-cell hybridoma. Gel filtration chromatography of a sonicate of M. avium followed by T-cell Western blotting (immunoblotting) demonstrated that the antigen recognized by hybridoma 1C9 is approximately 50 kDa. In addition, treatment of macrophages with the lysosomotropic agent chloroquine or with inhibitors of acid proteases inhibits processing and presentation of the antigen. These results indicate that the antigen must encounter an acidic compartment with active proteases for processing and presentation to occur. Our results are discussed in the context of our current understanding of how mycobacterial antigens are processed and presented by infected macrophages to T cells. PMID:8926074

  6. Proteolytic activation of protein kinase C delta by an ICE-like protease in apoptotic cells.

    PubMed Central

    Emoto, Y; Manome, Y; Meinhardt, G; Kisaki, H; Kharbanda, S; Robertson, M; Ghayur, T; Wong, W W; Kamen, R; Weichselbaum, R

    1995-01-01

    These studies demonstrate that treatment of human U-937 cells with ionizing radiation (IR) is associated with activation of a cytoplasmic myelin basic protein (MBP) kinase. Characterization of the kinase by gel filtration and in-gel kinase assays support activation of a 40 kDa protein. Substrate and inhibitor studies further support the induction of protein kinase C (PKC)-like activity. The results of N-terminal amino acid sequencing of the purified protein demonstrate identity of the kinase with an internal region of PKC delta. Immunoblot analysis was used to confirm proteolytic cleavage of intact 78 kDa PKC delta in control cells to the 40 kDa C-terminal fragment after IR exposure. The finding that both IR-induced proteolytic activation of PKC delta and endonucleolytic DNA fragmentation are blocked by Bcl-2 and Bcl-xL supports an association with physiological cell death (PCD). Moreover, cleavage of PKC delta occurs adjacent to aspartic acid at a site (QDN) similar to that involved in proteolytic activation of interleukin-1 beta converting enzyme (ICE). The specific tetrapeptide ICE inhibitor (YVAD) blocked both proteolytic activation of PKC delta and internucleosomal DNA fragmentation in IR-treated cells. These findings demonstrate that PCD is associated with proteolytic activation of PKC delta by an ICE-like protease. Images PMID:8557034

  7. Surface-associated MUC5B mucins promote protease activity in Lactobacillus fermentum biofilms

    PubMed Central

    2013-01-01

    Background Mucosal surfaces are coated with layers of mucus gel that protect the underlying tissues and promote colonization by members of the commensal microflora. Lactobacillus fermentum is a common inhabitant of the oral cavity, gastrointestinal and reproductive tracts and is one of the most important lactic acid bacteria contributing to the formation of a healthy intestinal microflora. We have investigated the proteolytic activity in L. fermentum in response to interactions with the MUC5B mucin, which is a major component of mucus gels at sites colonized by this micro-organism. Methods Biofilms of Lactobacillus fermentum were established in mini-flow cells in the presence or absence of human salivary MUC5B. The proteolytic activity of biofilm cells was examined in a confocal scanning laser microscope with a fluorescent protease substrate. Degradation of MUC5B by L. fermentum was analysed using SDS-PAGE followed by Western blotting with antisera raised against the MUC5B peptide. Cell surface proteins differentialy expressed in a MUC5B-rich environment were identified with the aid of comparative two-dimensional electrophoresis followed by LC-MS/MS. Results Lactobacillus fermentum adhered well to surfaces coated with MUC5B mucin and in biofilms of L. fermentum formed in a MUC5B environment, the proportion of proteolytically-active cells (47 ± 0.6% of the population), as shown by cleavage of a fluorescent casein substrate, was significantly greater (p < 0.01) than that in biofilms formed in nutrient broth (0.4 ± 0.04% of the population). Thus, the presence of MUC5B mucins enhanced bacterial protease activity. This effect was mainly attributable to contact with surface-associated mucins rather than those present in the fluid phase. Biofilms of L. fermentum were capable of degrading MUC5B mucins suggesting that this complex glycoprotein can be exploited as a nutrient source by the bacteria. Comparison of the surface proteomes of biofilm cells of L

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

    PubMed Central

    Hu, Qing-Xiu; Zhang, Guo-Qing; Zhang, Rui-Ying; Hu, Dan-Dan; Wang, He-Xiang; Ng, Tzi Bun

    2012-01-01

    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. PMID:22675256

  9. Influence of nitrogen source and pH value on undesired poly(γ-glutamic acid) formation of a protease producing Bacillus licheniformis strain.

    PubMed

    Meissner, Lena; Kauffmann, Kira; Wengeler, Timo; Mitsunaga, Hitoshi; Fukusaki, Eiichiro; Büchs, Jochen

    2015-09-01

    Bacillus spp. are used for the production of industrial enzymes but are also known to be capable of producing biopolymers such as poly(γ-glutamic acid). Biopolymers increase the viscosity of the fermentation broth, thereby impairing mixing, gas/liquid mass and heat transfer in any bioreactor system. Undesired biopolymer formation has a significant impact on the fermentation and downstream processing performance. This study shows how undesirable poly(γ-glutamic acid) formation of an industrial protease producing Bacillus licheniformis strain was prevented by switching the nitrogen source from ammonium to nitrate. The viscosity was reduced from 32 to 2.5 mPa s. A constant or changing pH value did not influence the poly(γ-glutamic acid) production. Protease production was not affected: protease activities of 38 and 46 U mL(-1) were obtained for ammonium and nitrate, respectively. With the presented results, protease production with industrial Bacillus strains is now possible without the negative impact on fermentation and downstream processing by undesired poly(γ-glutamic acid) formation.

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

    PubMed

    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.

  11. Basis Tetrapeptides as Potent Intracellular Inhibitors of type A Botulinum Neurotoxin Protease Activity

    SciTech Connect

    Hale, M.; Swaminathan, S.; Oyler, G.; Ahmed, S. A.

    2011-01-21

    Botulinum neurotoxins (BoNT) are the most potent of all toxins that cause flaccid muscle paralysis leading to death. They are also potential biothreat agents. A systematic investigation of various short peptide inhibitors of the BoNT protease domain with a 17-residue peptide substrate led to arginine-arginine-glycine-cysteine having a basic tetrapeptide structure as the most potent inhibitor. When assayed in the presence of dithiothreitol (DTT), the inhibitory effect was drastically reduced. Replacing the terminal cysteine with one hydrophobic residue eliminated the DTT effect but with two hydrophobic residues made the pentapeptide a poor inhibitor. Replacing the first arginine with cysteine or adding an additional cysteine at the N terminus did not improve inhibition. When assessed using mouse brain lysates, the tetrapeptides also inhibited BoNT/A cleavage of the endogenous SNAP-25. The peptides penetrated the neuronal cell lines, N2A and BE(2)-M17, without adversely affecting metabolic functions as measured by ATP production and P-38 phosphorylation. Biological activity of the peptides persisted within cultured chick motor neurons and rat and mouse cerebellar neurons for more than 40 h and inhibited BoNT/A protease action inside the neurons in a dose- and time-dependent fashion. Our results define a tetrapeptide as the smallest peptide inhibitor in the backdrop of a large substrate protein of 200+ amino acids having multiple interaction regions with its cognate enzyme. The inhibitors should also be valuable candidates for drug development.

  12. Characterization of senescence-associated protease activities involved in the efficient protein remobilization during leaf senescence of winter oilseed rape.

    PubMed

    Poret, Marine; Chandrasekar, Balakumaran; van der Hoorn, Renier A L; Avice, Jean-Christophe

    2016-05-01

    Oilseed rape (Brassica napus L.) is a crop plant characterized by a poor nitrogen (N) use efficiency that is mainly due to low N remobilization efficiency during the sequential leaf senescence of the vegetative stage. As a high leaf N remobilization efficiency was strongly linked to a high remobilization of proteins during leaf senescence of rapeseed, our objective was to identify senescence-associated protease activities implicated in the protein degradation. To reach this goal, leaf senescence processes and protease activities were investigated in a mature leaf becoming senescent in plants subjected to ample or low nitrate supply. The characterization of protease activities was performed by using in vitro analysis of RuBisCO degradation with or without inhibitors of specific protease classes followed by a protease activity profiling using activity-dependent probes. As expected, the mature leaf became senescent regardless of the nitrate treatment, and nitrate limitation enhanced the senescence processes associated with an enhanced degradation of soluble proteins. The characterization of protease activities revealed that: (i) aspartic proteases and the proteasome were active during senescence regardless of nitrate supply, and (ii) the activities of serine proteases and particularly cysteine proteases (Papain-like Cys proteases and vacuolar processing enzymes) increased when protein remobilization associated with senescence was accelerated by nitrate limitation. Short statement: Serine and particularly cysteine proteases (both PLCPs and VPEs) seem to play a crucial role in the efficient protein remobilization when leaf senescence of oilseed rape was accelerated by nitrate limitation.

  13. The prototype foamy virus protease is active independently of the integrase domain

    PubMed Central

    2012-01-01

    Background Recently, contradictory results on foamy virus protease activity were published. While our own results indicated that protease activity is regulated by the viral RNA, others suggested that the integrase is involved in the regulation of the protease. Results To solve this discrepancy we performed additional experiments showing that the protease-reverse transcriptase (PR-RT) exhibits protease activity in vitro and in vivo, which is independent of the integrase domain. In contrast, Pol incorporation, and therefore PR activity in the viral context, is dependent on the integrase domain. To further analyse the regulation of the protease, we incorporated Pol in viruses by expressing a GagPol fusion protein, which supported near wild-type like infectivity. A GagPR-RT fusion, lacking the integrase domain, also resulted in wild-type like Gag processing, indicating that the integrase is dispensable for viral Gag maturation. Furthermore, we demonstrate with a trans-complementation assays that the PR in the context of the PR-RT protein supports in trans both, viral maturation and infectivity. Conclusion We provide evidence that the FV integrase is required for Pol encapsidation and that the FV PR activity is integrase independent. We show that an active PR can be encapsidated in trans as a GagPR-RT fusion protein. PMID:22574974

  14. Purification and characterization of serine proteases that exhibit caspase-like activity and are associated with programmed cell death in Avena sativa.

    PubMed

    Coffeen, Warren C; Wolpert, Thomas J

    2004-04-01

    Victoria blight of Avena sativa (oat) is caused by the fungus Cochliobolus victoriae, which is pathogenic because of the production of the toxin victorin. The victorin-induced response in sensitive A. sativa has been characterized as a form of programmed cell death (PCD) and displays morphological and biochemical features similar to apoptosis, including chromatin condensation, DNA laddering, cell shrinkage, altered mitochondrial function, and ordered, substrate-specific proteolytic events. Victorin-induced proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is shown to be prevented by caspase-specific and general protease inhibitors. Evidence is presented for a signaling cascade leading to Rubisco proteolysis that involves multiple proteases. Furthermore, two proteases that are apparently involved in the Rubisco proteolytic cascade were purified and characterized. These proteases exhibit caspase specificity and display amino acid sequences homologous to plant subtilisin-like Ser proteases. The proteases are constitutively present in an active form and are relocalized to the extracellular fluid after induction of PCD by either victorin or heat shock. The role of the enzymes as processive proteases involved in a signal cascade during the PCD response is discussed.

  15. Identification of proteolytic bacteria from the Arctic Chukchi Sea expedition cruise and characterization of cold-active proteases.

    PubMed

    Park, Ha Ju; Lee, Yung Mi; Kim, Sunghui; Wi, Ah Ram; Han, Se Jong; Kim, Han-Woo; Kim, Il-Chan; Yim, Joung Han; Kim, Dockyu

    2014-10-01

    Following collection of seawater samples during an Arctic Chukchi Sea expedition cruise of the Korean icebreaker Araon in 2012, a total of 15,696 bacteria were randomly isolated from Marine Broth 2216 agar plates. Of these, 2,526 (16%) showed proteolytic activity and were identified as mainly Alteromonas (31%), Staphylococcus (27%), and Pseudoalteromonas (14%). Among the proteolytic strains, seven were selected based on their significant ability to grow and produce a halo on skim milk plates at low temperatures (<5°C) owing to cold-active proteases. These strains were affiliated with the genus Pseudoalteromonas and were divided into three groups based on phylogenetic analysis of the 16S rRNA genes. Profiling cell membrane fatty acids confirmed the 16S rRNA-based differentiation and revealed the accordance between the two analyses. Seven genes for serine protease precursors were amplified from the corresponding strains, and based on sequence similarities, these genes were divided into three groups that were identical to those identified by the 16S rRNA phylogenetic analysis. Three protease genes from the representative strains of each group were composed of 2,127-2,130 bp, encoding 708-709 amino acids, and these genes yielded products with calculated molecular weights of approximately 72.3-72.8 kDa. Amino acid sequence analysis suggested that the precursors are members of the subtilase serine endo- and exo-peptidase clan and contain four domains (signal peptide, N-terminal prosequence, catalytic domain, and two pre-peptidase C-terminal domains). Upon expression in E. coli, each recombinant protease exhibited proteolytic activity on zymogram gels.

  16. Design, synthesis, and activity of nanocellulosic protease sensors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we contrast the molecular assembly, and biochemical utility of nanocellulosic materials prepared from cotton and wood as protease sensors. The cotton-based nanocellulosic substrates were prepared in a variety of ways to produce nanocrystals, films and aerogels, which were derivatized with eithe...

  17. Analysis of 16S rRNA gene lactic acid bacteria (LAB) isolate from Markisa fruit (Passiflora sp.) as a producer of protease enzyme and probiotics

    NASA Astrophysics Data System (ADS)

    Hidayat, Habibi

    2017-03-01

    16S rRNA gene analysis of bacteria lactic acid (LAB) isolate from Markisa Kuning Fruit (Passiflora edulis var. flavicarpa) as a producer of protease enzyme and probiotics has been done. The aim of the study is to determine the protease enzyme activity and 16S rRNA gene amplification using PCR. The calculation procedure was done to M4 isolate bacteria lactic acid (LAB) Isolate which has been resistant to acids with pH 2.0 in the manner of screening protease enzyme activity test result 6.5 to clear zone is 13 mm againts colony diametre is 2 mm. The results of study enzyme activity used spectrophotometer UV-Vis obtainable the regression equation Y=0.02983+0.001312X, with levels of protein M4 isolate is 0.6594 mg/mL and enzyme activity of obtainable is 0.8626 unit/ml while the spesific enzyme activity produced is 1.308 unit/mg. Then, 16S rRNA gene amplificatiom and DNA sequencing has been done. The results of study showed that the bacteria species contained from M4 bacteria lactic acid (LAB) isolate is Weisella cibiria strain II-I-59. Weisella cibiria strain II-I-59 is one of bacteria could be utilized in the digestive tract.

  18. Purification and characterization of a prothrombin-activating protease from Nephila clavata.

    PubMed

    Joo, Han-Seung; Park, Gun-Chun; Cho, Woo Ri; Tak, Eunsik; Paik, Seung R; Chang, Chung-Soon

    2002-03-01

    We report upon the purification and characterization of a novel prothrombin-activating enzyme from the body fluid (total homogenates of isolated digestive tract without eggs, spinnerets and silk glands) of the spider, Nephila clavata by a combination of acetone fractionation, ion exchange, and Soybean trypsin inhibitor-Sepharose chromatography. Analysis of the purified enzyme with SDS-PAGE and gel filtration revealed a single polypeptide chain with an apparent molecular weight of 24kDa. The proteolytic activity of the enzyme was stable up to 50 degrees C, however, it became unstable over 55 degrees C. The enzyme had an optimum pH of 8, and Ca(2+) was not required for the enzyme activity. According to inhibition profiles obtained with several serine protease inhibitors such as PMSF and benzamidine, the purified protease is a member of the serine proteases. Bz-Ile-Glu(gamma-OR)- Gly-Arg-pNA and Z-Arg-Gly-Arg-pNA which are known as substrates for factor Xa, were hydrolyzed favorably by the enzyme. And the Nephila protease could produce thrombin from prothrombin at nM range, and form the turbid ring using fibrinogen-agarose plate. The results obtained confirmed that the purified protease is a potent prothrombin-activating activity belonging to the family of serine protease.

  19. Preferential packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles.

    PubMed

    Elhenawy, Wael; Debelyy, Mykhaylo O; Feldman, Mario F

    2014-03-11

    Outer membrane vesicles (OMV) are spherical membranous structures released from the outer membrane (OM) of Gram-negative bacteria. OMV have been proposed to play several different roles during both pathogenesis and symbiosis. Despite the fact that OMV were described several decades ago, their biogenesis is a poorly characterized process. Whether OMV are produced by an active mechanism or by passive disintegration of the OM is a still matter of controversy. Bacteroides fragilis and Bacteroides thetaiotaomicron are important members of the human microbiota. In this work, we determined and compared the protein compositions of OM and OMV from B. fragilis and B. thetaiotaomicron. SDS-PAGE analysis of both fractions revealed dramatically different protein profiles. Proteomic analysis of OM and OMV in B. fragilis identified more than 40 proteins found exclusively in OMV and more than 30 proteins detectable only in the OM. The OMV-specific proteome showed a high prevalence of glycosidases and proteases, some of which were shown to be active in vitro. Similar results were obtained for B. thetaiotaomicron. Most of the OMV-exclusive proteins were acidic. Based on these results, we propose that these species possess machinery devoted to selectively pack acidic proteins into the OMV. These OMV equipped with hydrolytic enzymes could help in securing nutrients for the benefit of the whole bacterial community present in the microbiota, uncovering a novel function for bacterial OMV. IMPORTANCE The members of genus Bacteroides are key players in the symbiosis between the human host and the gut microbiota. It is known for its ability to degrade a wide variety of glycans that are not substrates for human glycosidases. The cleaved glycans can be utilized by Bacteroides and other microbiota members, resulting in the production of short-chain fatty acids that are beneficial for the host. Although members of the genus Bacteroides are known to secrete different hydrolases, their secretion

  20. [Production of a concentrate of Mucor bacilliformis acid protease].

    PubMed

    Bottaro Castilla, H R; Waehner, R S; Meinardi, C A; Zalazar, C A; Fraile, E R

    1982-01-01

    A concentrate of milk-clotting enzyme was produced by culture of Mucor bacilliformis on wheat bran medium moistened to 120% water on dry bases with HC1 2 N solution. The wheat bran was autoclaved, spread on trays and inoculated with 5.10(6) spore/gr of dry bran. After 10 days of culture at 21 degrees C, the enzyme produced was extracted with water and adjusted to pH 4.4. The precipitation was performed with ethanol. The precipitate was dissolved in HCl solution (pH 4.5) and it was concentrated by dialysis against polyethylene glycol 20.000. The enzyme solution had a specific activity of 1123 units/mg. and it was tested in the elaboration of cream cheese.

  1. Evaluation on Potential Contributions of Protease Activated Receptors Related Mediators in Allergic Inflammation

    PubMed Central

    Zhang, Huiyun; Zeng, Xiaoning; He, Shaoheng

    2014-01-01

    Protease activated receptors (PARs) have been recognized as a distinctive four-member family of seven transmembrane G protein-coupled receptors (GPCRs) that can be cleaved by certain serine proteases. In recent years, there has been considerable interest in the role of PARs in allergic inflammation, the fundamental pathologic changes of allergy, but the potential roles of PARs in allergy remain obscure. Since many of these proteases are produced and actively involved in the pathologic process of inflammation including exudation of plasma components, inflammatory cell infiltration, and tissue damage and repair, PARs appear to make important contribution to allergy. The aim of the present review is to summarize the expression of PARs in inflammatory and structural cells, the influence of agonists or antagonists of PARs on cell behavior, and the involvement of PARs in allergic disorders, which will help us to better understand the roles of serine proteases and PARs in allergy. PMID:24876677

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

    ERIC Educational Resources Information Center

    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…

  3. Approaches for the generation of active papain-like cysteine proteases from inclusion bodies of Escherichia coli.

    PubMed

    Ling, Chunfang; Zhang, Junyan; Lin, Deqiu; Tao, Ailin

    2015-05-01

    Papain-like cysteine proteases are widely expressed, fulfill specific functions in extracellular matrix turnover, antigen presentation and processing events, and may represent viable drug targets for major diseases. In depth and rigorous studies of the potential for these proteins to be targets for drug development require sufficient amounts of protease protein that can be used for both experimental and therapeutic purposes. Escherichia coli was widely used to express papain-like cysteine proteases, but most of those proteases are produced in insoluble inclusion bodies that need solubilizing, refolding, purifying and activating. Refolding is the most critical step in the process of generating active cysteine proteases and the current approaches to refolding include dialysis, dilution and chromatography. Purification is mainly achieved by various column chromatography. Finally, the attained refolded proteases are examined regarding their protease structures and activities.

  4. Multiplex Detection of Protease Activity with Quantum Dot Nanosensors Prepared by Intein-Mediated Specific Bioconjugation

    PubMed Central

    Xia, Zuyong; Xing, Yun; So, Min-Kyung; Koh, Ai Leen; Sinclair, Robert; Rao, Jianghong

    2009-01-01

    We report here a protease sensing nanoplatform based on semiconductor nanocrystals or quantum dots (QDs) and bioluminescence resonance energy transfer (QD-BRET) to detect the protease activity in complex biological samples. These nanosensors consist of bioluminescent proteins as the BRET donor, quantum dots as the BRET acceptor, and protease substrates sandwiched between the two as a sensing group. An intein-mediated conjugation strategy was developed for site-specific conjugation of proteins to QDs in preparing these QD nanosensors. In this traceless ligation, the intein itself is spliced out and excluded from the final conjugation product. With this method, we have synthesized a series of QD nanosensors for highly sensitive detection of an important class of protease matrix metalloproteinase (MMP) activity. We demonstrated that these nanosensors can detect the MMP activity in buffers and in mouse serum with the sensitivity to a few ng/ml, and secreted proteases by tumor cells. The suitability of these nanosensors for a multiplex protease assay has also been shown. PMID:18922019

  5. Effects of Glycosylation on the Enzymatic Activity and Mechanisms of Proteases

    PubMed Central

    Goettig, Peter

    2016-01-01

    Posttranslational modifications are an important feature of most proteases in higher organisms, such as the conversion of inactive zymogens into active proteases. To date, little information is available on the role of glycosylation and functional implications for secreted proteases. Besides a stabilizing effect and protection against proteolysis, several proteases show a significant influence of glycosylation on the catalytic activity. Glycans can alter the substrate recognition, the specificity and binding affinity, as well as the turnover rates. However, there is currently no known general pattern, since glycosylation can have both stimulating and inhibiting effects on activity. Thus, a comparative analysis of individual cases with sufficient enzyme kinetic and structural data is a first approach to describe mechanistic principles that govern the effects of glycosylation on the function of proteases. The understanding of glycan functions becomes highly significant in proteomic and glycomic studies, which demonstrated that cancer-associated proteases, such as kallikrein-related peptidase 3, exhibit strongly altered glycosylation patterns in pathological cases. Such findings can contribute to a variety of future biomedical applications. PMID:27898009

  6. Proteases as Insecticidal Agents

    PubMed Central

    Harrison, Robert L.; Bonning, Bryony C.

    2010-01-01

    Proteases from a variety of sources (viruses, bacteria, fungi, plants, and insects) have toxicity towards insects. Some of these insecticidal proteases evolved as venom components, herbivore resistance factors, or microbial pathogenicity factors, while other proteases play roles in insect development or digestion, but exert an insecticidal effect when over-expressed from genetically engineered plants or microbial pathogens. Many of these proteases are cysteine proteases, although insect-toxic metalloproteases and serine proteases have also been examined. The sites of protease toxic activity range from the insect midgut to the hemocoel (body cavity) to the cuticle. This review discusses these insecticidal proteases along with their evaluation and use as potential pesticides. PMID:22069618

  7. Proteaselike sequence in hepatitis B virus core antigen is not required for e antigen generation and may not be part of an aspartic acid-type protease.

    PubMed Central

    Nassal, M; Galle, P R; Schaller, H

    1989-01-01

    The hepatitis B virus (HBV) C gene directs the synthesis of two major gene products: HBV core antigen (HBcAg[p21c]), which forms the nucleocapsid, and HBV e antigen (HBeAg [p17e]), a secreted antigen that is produced by several processing events during its maturation. These proteins contain an amino acid sequence similar to the active-site residues of aspartic acid and retroviral proteases. On the basis of this sequence similarity, which is highly conserved among mammalian hepadnaviruses, a model has been put forward according to which processing to HBeAg is due to self-cleavage of p21c involving the proteaselike sequence. Using site-directed mutagenesis in conjunction with transient expression of HBV proteins in the human hepatoma cell line HepG2, we tested this hypothesis. Our results with HBV mutants in which one or two of the conserved amino acids have been replaced by others suggest strongly that processing to HBeAg does not depend on the presence of an intact proteaselike sequence in the core protein. Attempts to detect an influence of this sequence on the processing of HBV P gene products into enzymatically active viral polymerase also gave no conclusive evidence for the existence of an HBV protease. Mutations replacing the putatively essential aspartic acid showed little effect on polymerase activity. Additional substitution of the likewise conserved threonine residue by alanine, in contrast, almost abolished the activity of the polymerase. We conclude that an HBV protease, if it exists, is functionally different from aspartic acid and retroviral proteases. Images PMID:2657101

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

    NASA Astrophysics Data System (ADS)

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

    1986-04-01

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

  9. [Peptide hydrolases with catalytic dyad Ser-Lys. Similarity and distinctions of the active centers of ATP-dependent Lon proteases, LexA repressors, signal peptidases and C-terminal processing proteases].

    PubMed

    Rotanova, T V

    2002-01-01

    It is established that ATP-dependent protease Lon family belongs to the serine-lysine peptide hydrolases clan. Significant similarity of amino acid sequences of proteases Lon and repressors LexA in the regions including the catalytic serine and lysine residues is revealed by comparing primary structures of different families of the enzymes with Ser-Lys catalytic dyad. The both Lon and LexA families are shown to be divided into two subfamilies in accordance with the nature of amino acids in the catalytically active serine environment. Putative DNA binding sites are revealed in proteolytic domains of Lon A subfamily. Similarities and distinctions of the all families peptide hydrolases of the clan in the regions of their active centers are discussed.

  10. MALT1 Protease Activity Controls the Expression of Inflammatory Genes in Keratinocytes upon Zymosan Stimulation.

    PubMed

    Schmitt, Anja; Grondona, Paula; Maier, Tabea; Brändle, Marc; Schönfeld, Caroline; Jäger, Günter; Kosnopfel, Corinna; Eberle, Franziska C; Schittek, Birgit; Schulze-Osthoff, Klaus; Yazdi, Amir S; Hailfinger, Stephan

    2016-04-01

    The protease activity of the paracaspase mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) plays an important role in antigen receptor-mediated lymphocyte activation by controlling the activity of the transcription factor nuclear factor-κB and is thus essential for the expression of inflammatory target genes. MALT1 is not only present in cells of the hematopoietic lineage, but is ubiquitously expressed. Here we report that stimulation with zymosan or Staphylococcus aureus induced MALT1 protease activity in human primary keratinocytes. Inhibition of the Src family of kinases or novel protein kinase C isoforms as well as silencing of CARMA2 or BCL10 interfered with activation of MALT1 protease. Silencing or inhibition of MALT1 protease strongly decreased the expression of important inflammatory genes such as TNFα, IL-17C, CXCL8 and HBD-2. MALT1-inhibited cells were unable to mount an antimicrobial response upon zymosan stimulation or phorbolester/ionomycin treatment, demonstrating a central role of MALT1 protease activity in keratinocyte immunity and suggesting MALT1 as a potential target in inflammatory skin diseases.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-05-01

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

  13. Searching for discrimination rules in protease proteolytic cleavage activity using genetic programming with a min-max scoring function.

    PubMed

    Yang, Zheng Rong; Thomson, Rebecca; Hodgman, T Charles; Dry, Jonathan; Doyle, Austin K; Narayanan, Ajit; Wu, XiKun

    2003-11-01

    This paper presents an algorithm which is able to extract discriminant rules from oligopeptides for protease proteolytic cleavage activity prediction. The algorithm is developed using genetic programming. Three important components in the algorithm are a min-max scoring function, the reverse Polish notation (RPN) and the use of minimum description length. The min-max scoring function is developed using amino acid similarity matrices for measuring the similarity between an oligopeptide and a rule, which is a complex algebraic equation of amino acids rather than a simple pattern sequence. The Fisher ratio is then calculated on the scoring values using the class label associated with the oligopeptides. The discriminant ability of each rule can therefore be evaluated. The use of RPN makes the evolutionary operations simpler and therefore reduces the computational cost. To prevent overfitting, the concept of minimum description length is used to penalize over-complicated rules. A fitness function is therefore composed of the Fisher ratio and the use of minimum description length for an efficient evolutionary process. In the application to four protease datasets (Trypsin, Factor Xa, Hepatitis C Virus and HIV protease cleavage site prediction), our algorithm is superior to C5, a conventional method for deriving decision trees.

  14. Supermarket Proteases.

    ERIC Educational Resources Information Center

    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)

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Pathomimetic cancer avatars for live-cell imaging of protease activity.

    PubMed

    Ji, Kyungmin; Heyza, Joshua; Cavallo-Medved, Dora; Sloane, Bonnie F

    2016-03-01

    Proteases are essential for normal physiology as well as multiple diseases, e.g., playing a causative role in cancer progression, including in tumor angiogenesis, invasion, and metastasis. Identification of dynamic alterations in protease activity may allow us to detect early stage cancers and to assess the efficacy of anti-cancer therapies. Despite the clinical importance of proteases in cancer progression, their functional roles individually and within the context of complex protease networks have not yet been well defined. These gaps in our understanding might be addressed with: 1) accurate and sensitive tools and methods to directly identify changes in protease activities in live cells, and 2) pathomimetic avatars for cancer that recapitulate in vitro the tumor in the context of its cellular and non-cellular microenvironment. Such avatars should be designed to facilitate mechanistic studies that can be translated to animal models and ultimately the clinic. Here, we will describe basic principles and recent applications of live-cell imaging for identification of active proteases. The avatars optimized by our laboratory are three-dimensional (3D) human breast cancer models in a matrix of reconstituted basement membrane (rBM). They are designated mammary architecture and microenvironment engineering (MAME) models as they have been designed to mimic the structural and functional interactions among cell types in the normal and cancerous human breast. We have demonstrated the usefulness of these pathomimetic avatars for following dynamic and temporal changes in cell:cell interactions and quantifying changes in protease activity associated with these interactions in real-time (4D). We also briefly describe adaptation of the avatars to custom-designed and fabricated tissue architecture and microenvironment engineering (TAME) chambers that enhance our ability to analyze concomitant changes in the malignant phenotype and the associated tumor microenvironment.

  17. The circadian Clock gene regulates acrosin activity of sperm through serine protease inhibitor A3K

    PubMed Central

    Cheng, Shuting; Liang, Xin; Wang, Yuhui; Jiang, Zhou; Liu, Yanyou; Hou, Wang; Li, Shiping; Zhang, Jing

    2015-01-01

    Our previous study found that CLOCK knockdown in the testes of male mice led to a reduced fertility, which might be associated with the lower acrosin activity. In this present study, we examined the differential expression in proteins of CLOCK knockdown sperm. Clock gene expression was knocked down in cells to confirm those differentially expressions and serine protease inhibitor SERPINA3K was identified as a potential target. The up-regulated SERPINA3K revealed an inverse relationship with Clock knockdown. Direct treatment of normal sperm with recombinant SERPINA3K protein inhibited the acrosin activity and reduced in vitro fertilization rate. The luciferase reporter gene assay showed that the down-regulated of Clock gene could activate the Serpina3k promoter, but this activation was not affected by the mutation of E-box core sequence. Co-IP demonstrated a natural interaction between SERPIAN3K and RORs (α and β). Taken together, these results demonstrated that SERPINA3K is involved in the Clock gene-mediated male fertility by regulating acrosin activity and provide the first evidence that SERPINA3K could be regulated by Clock gene via retinoic acid-related orphan receptor response elements. PMID:26264441

  18. A potential Kazal-type serine protease inhibitor involves in kinetics of protease inhibition and bacteriostatic activity.

    PubMed

    Kumaresan, Venkatesh; Harikrishnan, Ramaswamy; Arockiaraj, Jesu

    2015-02-01

    Kazal-type serine protease inhibitor (KSPI) is a pancreatic secretary trypsin inhibitor which involves in various cellular component regulations including development and defense process. In this study, we have characterized a KSPI cDNA sequence of freshwater striped murrel fish Channa striatus (Cs) at molecular level. Cellular location analysis predicted that the CsKSPI was an extracellular protein. The domain analysis showed that the CsKSPI contains a Kazal domain at 47-103 along with its family signature between 61 and 83. Phylogenetically, CsKSPI is closely related to KSPI from Maylandia zebra and formed a sister group with mammals. The 2D structure of CsKSPI showed three α-helical regions which are connected with random coils, one helix at signal sequence and two at the Kazal domain region. The relative gene expression showed that the CsKSPI was highly expressed in gills and its expression was induced upon fungus (Aphanomyces invadans), bacteria (Aeromonas hydrophila) and poly I:C (a viral analogue) challenge. The CsKSPI recombinant protein was produced to characterize and study the CsKSPI gene specific functions. The recombinant CsKSPI strongly inhibited trypsin compared to other tested proteases. The results of the kinetic activity of CsKSPI against trypsin was V(max)s = 1.62 nmol/min, K(M)s = 0.21 mM and K(i)s = 15.37 nM. Moreover, the recombinant CsKSPI inhibited the growth of Gram-negative bacteria A. hydrophila at 20 μM and Gram-positive bacteria Bacillus subtilis at the MIC50 of 15 μM. Overall, the study indicated that the CsKSPI was a potential trypsin inhibitor which involves in antimicrobial activity.

  19. Antioxidant and Anticancer Activities of Walnut (Juglans regia L.) Protein Hydrolysates Using Different Proteases.

    PubMed

    Jahanbani, Raheleh; Ghaffari, S Mahmood; Salami, Maryam; Vahdati, Kourosh; Sepehri, Houri; Sarvestani, Nazanin Namazi; Sheibani, Nader; Moosavi-Movahedi, Ali Akbar

    2016-12-01

    Walnut (Juglans regia L.) contains approximately 20-25 % protein with abundant essential amino acids. The enzymatic hydrolysate of Persian walnut (Chandler) seed proteins was prepared by incubation with three different proteases, including pancreatic chymotrypsin and trypsin, and a microbial enzyme proteinase K. The hydrolysates were found to possess excellent antioxidant capacities. The peptide fractions scavenged the 2, 2'-anizo-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) free radicals and inhibited the activity of reactive oxygen species. Walnut protein hydrolysates were also tested, for the first time, against the viability of human breast (MDA-MB231) and colon (HT-29) cancer cell lines. MTT, [3-(4, 5dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide], assay was used to assess in vitro cancer cell viability upon treatment with the peptide fractions. The peptide fractions showed cell growth inhibition of 63 ± 1.73 % for breast cancer and 51 ± 1.45 % for colon cancer cells. Thus, a direct correlation between antioxidant and anticancer activities of walnut peptide fractions exists and supports their potential therapeutic benefit.

  20. Active site amino acid sequence of human factor D.

    PubMed

    Davis, A E

    1980-08-01

    Factor D was isolated from human plasma by chromatography on CM-Sephadex C50, Sephadex G-75, and hydroxylapatite. Digestion of reduced, S-carboxymethylated factor D with cyanogen bromide resulted in three peptides which were isolated by chromatography on Sephadex G-75 (superfine) equilibrated in 20% formic acid. NH2-Terminal sequences were determined by automated Edman degradation with a Beckman 890C sequencer using a 0.1 M Quadrol program. The smallest peptide (CNBr III) consisted of the NH2-terminal 14 amino acids. The other two peptides had molecular weights of 17,000 (CNBr I) and 7000 (CNBr II). Overlap of the NH2-terminal sequence of factor D with the NH2-terminal sequence of CNBr I established the order of the peptides. The NH2-terminal 53 residues of factor D are somewhat more homologous with the group-specific protease of rat intestine than with other serine proteases. The NH2-terminal sequence of CNBr II revealed the active site serine of factor D. The typical serine protease active site sequence (Gly-Asp-Ser-Gly-Gly-Pro was found at residues 12-17. The region surrounding the active site serine does not appear to be more highly homologous with any one of the other serine proteases. The structural data obtained point out the similarities between factor D and the other proteases. However, complete definition of the degree of relationship between factor D and other proteases will require determination of the remainder of the primary structure.

  1. Proteases from Canavalia ensiformis: Active and Thermostable Enzymes with Potential of Application in Biotechnology

    PubMed Central

    Gonçalves, Rayane Natshe; Gozzini Barbosa, Suellen Duarte

    2016-01-01

    Extracts of leaves, seeds, roots, and stem from a tropical legume, C. ensiformis, were prepared employing buffers and detergent in aqueous solution. Leaf extracts had the highest protein content and the most pronounced peptidase activity with optimal pH in the neutral to alkaline range. All extracts exhibited peaks of activity at various pH values, suggesting the presence of distinctive classes of proteases. N-α-Tosyl-L-arginine methyl ester hydrolysis was maximal at 30°C to 60°C and peptidase activity from all extracts presented very good thermal stability after 24 h incubation at 70°C. C. ensiformis proteases exhibited molecular masses of about 200–57, 40–37, and 20–15 kDa by SDS-PAGE analysis. These enzymes cleaved hemoglobin, bovine serum albumin, casein, and gelatin at different levels. Serine and metalloproteases are the major proteases in C. ensiformis extracts, modulated by divalent cations, stable at 1% of surfactant Triton X-100 and at different concentrations of the reducing agent β-mercaptoethanol. Thus, C. ensiformis expresses a particular set of proteases in distinctive organs with high activity and stability, making this legume an important source of proteases with biotechnological potential. PMID:27630776

  2. Activation of Bacteroides fragilis toxin by a novel bacterial protease contributes to anaerobic sepsis

    PubMed Central

    Choi, Vivian M.; Herrou, Julien; Hecht, Aaron L.; Teoh, Wei Ping; Turner, Jerrold R.; Crosson, Sean; Wardenburg, Juliane Bubeck

    2016-01-01

    Bacteroides fragilis is the leading cause of anaerobic bacteremia and sepsis 1. Enterotoxigenic strains producing B. fragilis toxin (BFT, fragilysin) contribute to colitis 2 and intestinal malignancy 3, yet are also isolated in bloodstream infection 4,5. It is not known whether these strains harbor unique genetic determinants that confer virulence in extra-intestinal disease. We demonstrate that BFT contributes to sepsis and identify a B. fragilis protease, fragipain (Fpn), which is required for endogenous activation of BFT through removal of its auto-inhibitory prodomain. Structural analysis of Fpn reveals a His-Cys catalytic dyad characteristic of C11 family cysteine proteases that are conserved in multiple pathogenic Bacteroides spp and Clostridium spp. Fpn-deficient enterotoxigenic B. fragilis is attenuated in its ability to induce sepsis, however Fpn is dispensable in B. fragilis colitis wherein host proteases mediate BFT activation. Our findings define a role for B. fragilis enterotoxin and its activating protease in the pathogenesis of bloodstream infection, indicating a greater complexity of cellular targeting and action of BFT than previously appreciated. The expression of fpn by both toxigenic and non-toxigenic strains suggests this protease may contribute to anaerobic sepsis beyond its role in toxin activation, potentially serving as a target for disease modification. PMID:27089515

  3. Regulation of Foamy Virus Protease Activity by Viral RNA: a Novel and Unique Mechanism among Retroviruses▿†

    PubMed Central

    Hartl, Maximilian J.; Bodem, Jochen; Jochheim, Fabian; Rethwilm, Axel; Rösch, Paul; Wöhrl, Birgitta M.

    2011-01-01

    Foamy viruses (FVs) synthesize the Pol precursor protein from a specific transcript. Thus, in contrast to what was found for orthoretroviruses, e.g., human immunodeficiency virus, no Gag-Pol precursor protein is synthesized. Foamy viral Pol consists of a protease (PR) domain, a reverse transcriptase domain, and an integrase domain and is processed into a mature protease-reverse transcriptase (PR-RT) fusion protein and the integrase. Protease activity has to be strictly regulated in order to avoid premature Gag and Pol processing before virus assembly. We have demonstrated recently that FV protease is an inactive monomer with a very weak dimerization tendency and postulated protease activation through dimerization. Here, we identify a specific protease-activating RNA motif (PARM) located in the pol region of viral RNA which stimulates PR activity in vitro and in vivo, revealing a novel and unique mechanism of retroviral protease activation. This mechanism is strikingly different to that of orthoretroviruses, where the protease can be activated even in the absence of viral RNA during the assembly of virus-like particles. Although it has been shown that the integrase domain is important for Pol uptake, activation of the foamy virus protease is integrase independent. We show that at least two foamy virus PR-RT molecules bind to the PARM and only RNAs containing the PARM result in significant activation of the protease. DNA harboring the PARM is not capable of protease activation. Structure determination of the PARM by selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE) revealed a distinct RNA folding, important for protease activation and thus virus maturation. PMID:21325405

  4. Reactive Carbonyl Species Activate Caspase-3-Like Protease to Initiate Programmed Cell Death in Plants.

    PubMed

    Biswas, Md Sanaullah; Mano, Jun'ichi

    2016-07-01

    Reactive oxygen species (ROS)-triggered programmed cell death (PCD) is a typical plant response to biotic and abiotic stressors. We have recently shown that lipid peroxide-derived reactive carbonyl species (RCS), downstream products of ROS, mediate oxidative signal to initiate PCD. Here we investigated the mechanism by which RCS initiate PCD. Tobacco Bright Yellow-2 cultured cells were treated with acrolein, one of the most potent RCS. Acrolein at 0.2 mM caused PCD in 5 h (i.e. lethal), but at 0.1 mM it did not (sublethal). Specifically, these two doses caused critically different effects on the cells. Both lethal and sublethal doses of acrolein exhausted the cellular glutathione pool in 30 min, while the lethal dose only caused a significant ascorbate decrease and ROS increase in 1-2 h. Prior to such redox changes, we found that acrolein caused significant increases in the activities of caspase-1-like protease (C1LP) and caspase-3-like protease (C3LP), the proteases which trigger PCD. The lethal dose of acrolein increased the C3LP activity 2-fold more than did the sublethal dose. In contrast, C1LP activity increments caused by the two doses were not different. Acrolein and 4-hydroxy-(E)-2-nonenal, another RCS, activated both proteases in a cell-free extract from untreated cells. H2O2 at 1 mM added to the cells increased C1LP and C3LP activities and caused PCD, and the RCS scavenger carnosine suppressed their activation and PCD. However, H2O2 did not activate the proteases in a cell-free extract. Thus the activation of caspase-like proteases, particularly C3LP, by RCS is an initial biochemical event in oxidative signal-stimulated PCD in plants.

  5. Essential role of platelet activation via protease activated receptor 4 in tissue factor-initiated inflammation

    PubMed Central

    Busso, Nathalie; Chobaz-Péclat, Veronique; Hamilton, Justin; Spee, Pieter; Wagtmann, Nicolai; So, Alexander

    2008-01-01

    Introduction Tissue factor (TF) activation of the coagulation proteases enhances inflammation in animal models of arthritis and endotoxemia, but the mechanism of this effect is not yet fully understood – in particular, whether this is primarily due to fibrin formation or through activation of protease activated receptors (PARs). Methods We induced extravascular inflammation by injection of recombinant soluble murine TF (sTF1–219) in the hind paw. The effects of thrombin inhibition, fibrinogen and platelet depletion were evaluated, as well as the effects of PAR deficiency using knockout mice deficient for each of the PARs. Results Injection of soluble TF provoked a rapid onset of paw swelling. Inflammation was confirmed histologically and by increased serum IL-6 levels. Inflammation was significantly reduced by depletion of fibrinogen (P < 0.05) or platelets (P = 0.015), and by treatment with hirudin (P = 0.04) or an inhibitor of activated factor VII (P < 0.001) compared with controls. PAR-4-deficient mice exhibited significantly reduced paw swelling (P = 0.003). In contrast, a deficiency in either PAR-1, PAR-2 or PAR-3 did not affect the inflammatory response to soluble TF injection. Conclusion Our results show that soluble TF induces acute inflammation through a thrombin-dependent pathway and both fibrin deposition and platelet activation are essential steps in this process. The activation of PAR-4 on platelets is crucial and the other PARs do not play a major role in soluble TF-induced inflammation. PMID:18412955

  6. Assessing Activity and Inhibition of Middle East Respiratory Syndrome Coronavirus Papain-Like and 3C-Like Proteases Using Luciferase-Based Biosensors

    PubMed Central

    Kilianski, Andy; Mielech, Anna M.; Deng, Xufang

    2013-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with an outbreak of more than 90 cases of severe pneumonia with high mortality (greater than 50%). To date, there are no antiviral drugs or specific therapies to treat MERS-CoV. To rapidly identify potential inhibitors of MERS-CoV replication, we expressed the papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro) from MERS-CoV and developed luciferase-based biosensors to monitor protease activity in cells. We show that the expressed MERS-CoV PLpro recognizes and processes the canonical CoV-PLpro cleavage site RLKGG in the biosensor. However, existing CoV PLpro inhibitors were unable to block MERS-CoV PLpro activity, likely due to the divergence of the amino acid sequence in the drug binding site. To investigate MERS-CoV 3CLpro activity, we expressed the protease in context with flanking nonstructural protein 4 (nsp4) and the amino-terminal portion of nsp6 and detected processing of the luciferase-based biosensors containing the canonical 3CLpro cleavage site VRLQS. Importantly, we found that a small-molecule inhibitor that blocks replication of severe acute respiratory syndrome (SARS) CoV and murine CoV also inhibits the activity of MERS-CoV 3CLpro. Overall, the protease expression and biosensor assays developed here allow for rapid evaluation of viral protease activity and the identification of protease inhibitors. These biosensor assays can now be used to screen for MERS-CoV-specific or broad-spectrum coronavirus PLpro and 3CLpro inhibitors. PMID:23986593

  7. Screening of protease producing fungi for microbial digestion of seed proteins and synthesis of amino acids-metalnutrient chelates.

    PubMed

    Deore, G B; Limaye, A S; Dushing, Y A; Dhobale, S B; Kale, S; Laware, S L

    2013-01-15

    The problem of metalnutrient deficiency is becoming more serious with the introduction of modern agricultural practices. As a result, metalnutrient deficiency is recognized as one of the critical yield limiting factors. Metalnutrients are generally offered in their sulphate or oxide forms. However, it is reported that organically bound minerals generally have a higher bioavailability than inorganic minerals. Chelation makes otherwise unavailable metalnutrients plant available. Amino acids are well known among various chelating agents. In present investigation the fungus Paecilomyces variotii PR-4 was isolated from soil and was used for production of protease and determination of its activity. Proteins from germinating seeds of chick pea, mung bean, soybean and cowpea were hydrolyzed for the production of amino acids. Amino acids were recovered, estimated and utilized for chelation of metalnutrients viz., Zn, Cu, Fe, Mn, Mg, B and Mo. The resultant chelates were employed to detect with Fourier Transform Infra-Red Spectrophotometer (FTIR) analysis. The peaks of most intensive bands in the IR spectra of ligands recorded were present in the intervals of the wave numbers 3500-3300 and 1720-1700 cm(-1). Chelation of metalnutrients led to the broadening of peak and changes of the peak position of hydroxyl groups, which indicated the binding of the carboxylic groups and primary amine groups of amino acids to the metalnutrients. The resultant amino acids-metalnutrient chelates can be utilized as organic fertilizer.

  8. Expression of protease-activated receptor 1 and 2 and anti-tubulogenic activity of protease-activated receptor 1 in human endothelial colony-forming cells.

    PubMed

    Fortunato, Tiago M; Vara, Dina S; Wheeler-Jones, Caroline P; Pula, Giordano

    2014-01-01

    Endothelial colony-forming cells (ECFCs) are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC) fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR) 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK) 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin). The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2) in response to PAR1 stimulation. Moreover, the addition of VEGF (50-100 ng/ml) but not basic Fibroblast Growth Factor (FGF) (25-100 ng/ml) rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade.

  9. The role of AAA+ proteases in mitochondrial protein biogenesis, homeostasis and activity control.

    PubMed

    Voos, Wolfgang; Ward, Linda A; Truscott, Kaye N

    2013-01-01

    Mitochondria are specialised organelles that are structurally and functionally integrated into cells in the vast majority of eukaryotes. They are the site of numerous enzymatic reactions, some of which are essential for life. The double lipid membrane of the mitochondrion, that spatially defines the organelle and is necessary for some functions, also creates a physical but semi-permeable barrier to the rest of the cell. Thus to ensure the biogenesis, regulation and maintenance of a functional population of proteins, an autonomous protein handling network within mitochondria is required. This includes resident mitochondrial protein translocation machinery, processing peptidases, molecular chaperones and proteases. This review highlights the contribution of proteases of the AAA+ superfamily to protein quality and activity control within the mitochondrion. Here they are responsible for the degradation of unfolded, unassembled and oxidatively damaged proteins as well as the activity control of some enzymes. Since most knowledge about these proteases has been gained from studies in the eukaryotic microorganism Saccharomyces cerevisiae, much of the discussion here centres on their role in this organism. However, reference is made to mitochondrial AAA+ proteases in other organisms, particularly in cases where they play a unique role such as the mitochondrial unfolded protein response. As these proteases influence mitochondrial function in both health and disease in humans, an understanding of their regulation and diverse activities is necessary.

  10. Impact of an acid fungal protease in high gravity fermentation for ethanol production using Indian sorghum as a feedstock.

    PubMed

    Gohel, V; Duan, G; Maisuria, V B

    2013-01-01

    This study evaluated the conventional jet cooking liquefaction process followed by simultaneous saccharification and fermentation (SSF) at 30% and 35% dry solids (DS) concentration of Indian sorghum feedstock for ethanol production, with addition of acid fungal protease or urea. To evaluate the efficacy of thermostable α-amylase in liquefaction at 30% and 35% DS concentration of Indian sorghum, liquefact solubility, higher dextrins, and fermentable sugars were analyzed at the end of the process. The liquefact was further subjected to SSF using yeast. In comparison with urea, addition of an acid fungal protease during SSF process was observed to accelerate yeast growth (μ), substrate consumption (Q(s)), ultimately ethanol yield based on substrate (Y(p/s)) and ethanol productivity based on fermentation time (Q(p)). The fermentation efficiency and ethanol recovery were determined for both concentrations of Indian sorghum and found to be increased with use of acid fungal protease in SSF process.

  11. Full length and protease domain activity of chikungunya virus nsP2 differ from other alphavirus nsP2 proteases in recognition of small peptide substrates.

    PubMed

    Saisawang, Chonticha; Sillapee, Pornpan; Sinsirimongkol, Kwanhathai; Ubol, Sukathida; Smith, Duncan R; Ketterman, Albert J

    2015-04-22

    Alphavirus nsP2 proteins are multifunctional and essential for viral replication. The protease role of nsP2 is critical for virus replication as only the virus protease activity is used for processing of the viral non-structural polypeptide. Chikungunya virus is an emerging disease problem that is becoming a world-wide health issue. We have generated purified recombinant chikungunya virus nsP2 proteins, both full length and a truncated protease domain from the C-terminus of the nsP2 protein. Enzyme characterization shows that the protease domain alone has different properties compared with the full length nsP2 protease. We also show chikungunya nsP2 protease possesses different substrate specificity to the canonical alphavirus nsP2 polyprotein cleavage specificity. Moreover, the chikungunya nsP2 also appears to differ from other alphavirus nsP2 in its distinctive ability to recognize small peptide substrates.

  12. Full length and protease domain activity of chikungunya virus nsP2 differ from other alphavirus nsP2 proteases in recognition of small peptide substrates

    PubMed Central

    Saisawang, Chonticha; Sillapee, Pornpan; Sinsirimongkol, Kwanhathai; Ubol, Sukathida; Smith, Duncan R.; Ketterman, Albert J.

    2015-01-01

    Alphavirus nsP2 proteins are multifunctional and essential for viral replication. The protease role of nsP2 is critical for virus replication as only the virus protease activity is used for processing of the viral non-structural polypeptide. Chikungunya virus is an emerging disease problem that is becoming a world-wide health issue. We have generated purified recombinant chikungunya virus nsP2 proteins, both full length and a truncated protease domain from the C-terminus of the nsP2 protein. Enzyme characterization shows that the protease domain alone has different properties compared with the full length nsP2 protease. We also show chikungunya nsP2 protease possesses different substrate specificity to the canonical alphavirus nsP2 polyprotein cleavage specificity. Moreover, the chikungunya nsP2 also appears to differ from other alphavirus nsP2 in its distinctive ability to recognize small peptide substrates. PMID:26182358

  13. Advances in protease engineering for laundry detergents.

    PubMed

    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.

  14. Activity of Protease-Activated Receptors in Primary Cultured Human Myenteric Neurons

    PubMed Central

    Kugler, Eva M.; Mazzuoli, Gemma; Demir, Ihsan E.; Ceyhan, Güralp O.; Zeller, Florian; Schemann, Michael

    2012-01-01

    Activity of the four known protease-activated receptors (PARs) has been well studied in rodent enteric nervous system and results in animal models established an important role for neuronal PAR2. We recently demonstrated that, unlike in rodents, PAR1 is the dominant neuronal protease receptor in the human submucous plexus. With this study we investigated whether this also applies to the human myenteric plexus. We used voltage sensitive dye recordings to detect action potential discharge in primary cultures of human myenteric neurons in response to PAR activating peptides (APs). Application of the PAR1-AP (TFLLR) or PAR4-AP (GYPGQV) evoked spike discharge in 79 or 23% of myenteric neurons, respectively. The PAR1-AP response was mimicked by the endogenous PAR1 activator thrombin and blocked by the PAR1 antagonists SCH79797. Human myenteric neurons did not respond to PAR2-AP. This was not due to culture conditions because all three PAR-APs evoked action potentials in cultured guinea pig myenteric neurons. Consecutive application of PAR-APs revealed coexpression (relative to the population responding to PAR-APs) of PAR1/PAR2 in 51%, PAR1/PAR4 in 43%, and of PAR2/PAR4 in 29% of guinea pig myenteric neurons. Our study provided further evidence for the prominent role of neuronal PAR1 in the human enteric nervous system. PMID:22988431

  15. Cysteine protease gene expression and proteolytic activity during senescence of Alstroemeria petals.

    PubMed

    Wagstaff, Carol; Leverentz, Michael K; Griffiths, Gareth; Thomas, Brian; Chanasut, Usawadee; Stead, Anthony D; Rogers, Hilary J

    2002-02-01

    The functional life of the flower is terminated by senescence and/or abscission. Multiple processes contribute to produce the visible signs of petal wilting and inrolling that typify senescence, but one of the most important is that of protein degradation and remobilization. This is mediated in many species through protein ubiquitination and the action of specific protease enzymes. This paper reports the changes in protein and protease activity during development and senescence of Alstroemeria flowers, a Liliaceous species that shows very little sensitivity to ethylene during senescence and which shows perianth abscission 8-10 d after flower opening. Partial cDNAs of ubiquitin (ALSUQ1) and a putative cysteine protease (ALSCYP1) were cloned from Alstroemeria using degenerate PCR primers and the expression pattern of these genes was determined semi-quantitatively by RT-PCR. While the levels of ALSUQ1 only fluctuated slightly during floral development and senescence, there was a dramatic increase in the expression of ALSCYP1 indicating that this gene may encode an important enzyme for the proteolytic process in this species. Three papain class cysteine protease enzymes showing different patterns of activity during flower development were identified on zymograms, one of which showed a similar expression pattern to the cysteine protease cDNA.

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

    PubMed Central

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

    2017-01-01

    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. PMID:28222138

  17. Relationship between free radicals produced by entamoeba histolytica and its proteases complex activity.

    PubMed

    Crisóstomo-Vázquez, M P; Cervantes-Cervantes, M P; Jiménez-Cardoso, E; Muñoz-Sánchez, J L

    2002-01-01

    Entamoeba histolytica is a parasite which causes health problems and there has been many approaches to know what is the factor causing its pathogenicity. In the present work, we assayed if the production of free radicals by the amoeba, has a relationship with the proteases activity. When we test the DMSO action (free radicals quenching activity) the specific activity of the proteases complex of the parasite were affected also. At 33.3% (V/V) concentration of DMSO it was present a maximal decrease of the initial activity (about 46% decrease), for to a higher concentrations existing a trend to recuperate the original activity, suggesting that the free radicals are an important factor for the hydrolysis grade of the protein substrate. All the differences except those between 46.7 and 66.6%, were significantly different compared with the control without any addition. The effects of Probucol and Probucol plus DMSO, compared to those caused by Metronidazol (MZ). We can observe that the quenchers caused a decrease on proteases activity similar to that of MZ (which is an antiparasite drug) and it was of c.a. 58% of activity decrease. These data suggest that the action of both, free radicals and proteases complex of Entamoeba histolytica, can account for the pathogenicity of the parasite.

  18. Characterization of the S1 binding site of the glutamic acid-specific protease from Streptomyces griseus.

    PubMed Central

    Stennicke, H. R.; Birktoft, J. J.; Breddam, K.

    1996-01-01

    The glutamic acid-specific protease from Streptomyces griseus (SGPE) is an 18.4-kDa serine protease with a distinct preference for Glu in the P1 position. Other enzymes characterized by a strong preference for negatively charged residues in the P1 position, e.g., interleukin-1 beta converting enzyme (ICE), use Arg or Lys residues as counterions within the S1 binding site. However, in SGPE, this function is contributed by a His residue (His 213) and two Ser residues (Ser 192 and S216). It is demonstrated that proSGPE is activated autocatalytically and dependent on the presence of a Glu residue in the -1 position. Based on this observation, the importance of the individual S1 residues is evaluated considering that enzymes unable to recognize a Glu in the P1 position will not be activated. Among the residues constituting the S1 binding site, it is demonstrated that His 213 and Ser 192 are essential for recognition of Glu in the P1 position, whereas Ser 216 is less important for catalysis out has an influence on stabilization of the ground state. From the three-dimensional structure, it appears that His 213 is linked to two other His residues (His 199 and His 228), forming a His triad extending from the S1 binding site to the back of the enzyme. This hypothesis has been tested by substitution of His 199 and His 228 with other amino acid residues. The catalytic parameters obtained with the mutant enzymes, as well as the pH dependence, do not support this theory; rather, it appears that His 199 is responsible for orienting His 213 and that His 228 has no function associated with the recognition of Glu in P1. PMID:8931145

  19. Single amino acid mutation alters thermostability of the alkaline protease from Bacillus pumilus: thermodynamics and temperature dependence.

    PubMed

    Huang, Rong; Yang, Qingjun; Feng, Hong

    2015-02-01

    Dehairing alkaline protease (DHAP) from Bacillus pumilus BA06 has been demonstrated to have high catalytic efficiency and good thermostability, with potential application in leather processing. In order to get insights into its catalytic mechanism, two mutants with single amino acid substitution according to the homology modeling and multiple sequence alignment were characterized in thermodynamics of thermal denaturation and temperature dependence of substrate hydrolysis. The results showed that both mutants of V149I and R249E have a systematic increase in catalytic efficiency (kcat/Km) in a wide range of temperatures, mainly due to an increase of k1 (substrate diffusion) and k2 (acylation) for V149I and of k2 and k3 (deacylation) for R249E. In comparison with the wild-type DHAP, the thermostability is increased for V149I and decreased for R249E. Thermodynamic analysis indicated that the free energy (ΔGa°) of activation for thermal denaturation may govern the thermostability. The value of ΔGa° is increased for V149I and decreased for R249E. Based on these data and the structural modeling, it is suggested that substitution of Val149 with Ile may disturb the local flexibility in the substrate-binding pocket, leading to enhancement of binding affinity for the substrate. In contrast, substitution of Arg249 with Glu leads to interruption of interaction with the C-terminal of enzyme, thus resulting in less thermostability. This study indicates that amino acid residues in the active center or in the substrate-binding pocket may disturb the catalytic process and can be selected as the target for protein engineering in the bacterial alkaline proteases.

  20. Activity of calcium activated protease in skeletal muscles and its changes in atrophy and stretch

    NASA Technical Reports Server (NTRS)

    Ellis, S.; Nagainis, P. A.

    1984-01-01

    The reduction of protein content in skeletal muscle undergoing disuse-induced atrophy is correlated with accelerated rates of protein degradation and reduced rates of protein synthesis (Goldspink, 1977). It is not known in what manner myofibers are partially disassembled during disuse atrophy to fibers of smaller diameter; nor is it known which proteases are responsible for this morphological change in contractile protein mass. Dayton and colleagues (1975) have suggested that the Ca(2+)-activated protease (CaP) may initiate myofibril degradation. The discovery of a form of CaP that is activatable by nano-molar concentrations of Ca(2+) indicates that CaP activity may be regulated by physiological concentrations of Ca(2+) (Mellgren, 1980). The enhancement of proteolysis by the Ca(2+) ionophore A23187, reported by Etlinger (1979), is consistent with a significant role for CaP in protein degradation. It was of interest, therefore, to measure the levels of CaP activity and the CaP inhibitor in extracts obtained from skeletal muscles of rat and chicken limbs undergoing disuse atrophy or stretch hypertrophy, respectively.

  1. Silver-stained fibrin zymography: separation of proteases and activity detection using a single substrate-containing gel.

    PubMed

    Chung, Dong-Min; Kim, Ki Eun; Ahn, Keug-Hyun; Park, Chan-Sun; Kim, Dong-Ho; Koh, Hong Bum; Chun, Hyo Kon; Yoon, Byung-Dae; Kim, Hong Jib; Kim, Min-Soo; Choi, Nack-Shick

    2011-08-01

    A new zymogram method, silver-stained fibrin zymography, for separation of protease bands and activity detection using a single substrate gel, was developed. The method takes advantage of the nanoscale sensitivity of both zymography and silver staining. After SDS-PAGE in a gel containing fibrin, the gel was incubated in enzyme reaction buffer and the zymogram 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. Furthermore, proteases of high molecular weight were clearly and sharply resolved.

  2. Identification and functional characterizations of serpin8, a potential prophenoloxidase-activating protease inhibitor in Pacific white shrimp, Litopenaeus vannamei.

    PubMed

    Liu, Yongjie; Sun, Yuhang; Wang, Qiai; Hou, Fujun; Liu, Xiaolin

    2017-01-01

    Serpins have been characterized from varieties of organisms by their inhibitory roles on serine or cysteine proteases. However, research for the functional study of serpins in crustacean is relatively small. To fully clarify the immune characterizations of serpin, a novel serpin (named Lvserpin8) encoding 414 amino acids with a 19-amino acid signal peptide and a serpin domain was identified from the Pacific white shrimp Litopenaeus vannamei. Sequence analysis indicated that the genomic Lvserpin8 gene contains 5 exons and 4 introns. The P1 residues of the predicted scissile bond in the reactive center loop (RCL) region represented for Lysine (Lys), which is in accordance with Pmserpin8, Dmserpin27A, Ofserpin3, Bmserpin3 and Msserpin3. Quantitative results showed that high mRNA expression of Lvserpin8 was detected in hepatopancreas and testis. Notably, a significant increase of Lvserpin8 was appeared post injection of Vibrio anguillarum, and Micrococcus lysodeikticus. Moreover, Lvserpin8 was knocked down in vivo by double-stranded RNA (dsRNA) mediated RNA interference (RNAi). Suppression of Lvserpin8 led to a significant increase in the transcripts of LvPPAE2 (Prophenoloxidase-activating Enzyme 2) and cumulative mortality. What's more, recombinant Lvserpin8 protein (rLvserpin8) displayed inhibition roles on trypsin activity, and prophenoloxidase activation. Taken together, the results implied that Lvserpin8 may be involved in shrimp innate immunity via the inhibition of prophenoloxidase-activating proteases.

  3. Distinguishing Functional Amino Acid Covariation from Background Linkage Disequilibrium in HIV Protease and Reverse Transcriptase

    PubMed Central

    Wang, Qi; Lee, Christopher

    2007-01-01

    Correlated amino acid mutation analysis has been widely used to infer functional interactions between different sites in a protein. However, this analysis can be confounded by important phylogenetic effects broadly classifiable as background linkage disequilibrium (BLD). We have systematically separated the covariation induced by selective interactions between amino acids from background LD, using synonymous (S) vs. amino acid (A) mutations. Covariation between two amino acid mutations, (A,A), can be affected by selective interactions between amino acids, whereas covariation within (A,S) pairs or (S,S) pairs cannot. Our analysis of the pol gene — including the protease and the reverse transcriptase genes — in HIV reveals that (A,A) covariation levels are enormously higher than for either (A,S) or (S,S), and thus cannot be attributed to phylogenetic effects. The magnitude of these effects suggests that a large portion of (A,A) covariation in the HIV pol gene results from selective interactions. Inspection of the most prominent (A,A) interactions in the HIV pol gene showed that they are known sites of independently identified drug resistance mutations, and physically cluster around the drug binding site. Moreover, the specific set of (A,A) interaction pairs was reproducible in different drug treatment studies, and vanished in untreated HIV samples. The (S,S) covariation curves measured a low but detectable level of background LD in HIV. PMID:17726544

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

    PubMed Central

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

    2014-01-01

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

  5. Pressure-enhanced activity and stability of a hyperthermophilic protease from a deep-sea methanogen.

    PubMed

    Michels, P C; Clark, D S

    1997-10-01

    We describe the properties of a hyperthermophilic, barophilic protease from Methanococcus jannaschii, an extremely thermophilic deep-sea methanogen. This enzyme is the first protease to be isolated from an organism adapted to a high-pressure-high-temperature environment. The partially purified enzyme has a molecular mass of 29 kDa and a narrow substrate specificity with strong preference for leucine at the P1 site of polypeptide substrates. Enzyme activity increased up to 116(deg)C and was measured up to 130(deg)C, one of the highest temperatures reported for the function of any enzyme. In addition, enzyme activity and thermostability increased with pressure: raising the pressure to 500 atm increased the reaction rate at 125(deg)C 3.4-fold and the thermostability 2.7-fold. Spin labeling of the active-site serine revealed that the active-site geometry of the M. jannaschii protease is not grossly different from that of several mesophilic proteases; however, the active-site structure may be relatively rigid at moderate temperatures. The barophilic and thermophilic behavior of the enzyme is consistent with the barophilic growth of M. jannaschii observed previously (J. F. Miller et al., Appl. Environ. Microbiol. 54:3039-3042, 1988).

  6. Regulation of factor XIa activity by platelets and alpha 1-protease inhibitor.

    PubMed Central

    Walsh, P N; Sinha, D; Kueppers, F; Seaman, F S; Blankstein, K B

    1987-01-01

    We have studied the complex interrelationships between platelets, Factor XIa, alpha 1-protease inhibitor and Factor IX activation. Platelets were shown to secrete an inhibitor of Factor XIa, and to protect Factor XIa from inactivation in the presence of alpha 1-protease inhibitor and the secreted platelet inhibitor. This protection of Factor XIa did not arise from the binding of Factor XIa to platelets, the presence of high molecular weight kininogen, or the inactivation of alpha 1-protease inhibitor by platelets. The formation of a complex between alpha 1-protease inhibitor and the active-site-containing light chain of Factor XIa was inhibited by activated platelets and by platelet releasates, but not by high molecular weight kininogen. These results support the hypothesis that platelets can regulate Factor XIa-catalyzed Factor IX activation by secreting an inhibitor of Factor XIa that may act primarily outside the platelet microenvironment and by protecting Factor XIa from inhibition, thereby localizing Factor IX activation to the platelet plug. Images PMID:3500185

  7. Learning and memory deficits in mice lacking protease activated receptor-1.

    PubMed

    Almonte, Antoine G; Hamill, Cecily E; Chhatwal, Jasmeer P; Wingo, Thomas S; Barber, Jeremy A; Lyuboslavsky, Polina N; David Sweatt, J; Ressler, Kerry J; White, David A; Traynelis, Stephen F

    2007-10-01

    The roles of serine proteases and protease activated receptors have been extensively studied in coagulation, wound healing, inflammation, and neurodegeneration. More recently, serine proteases have been suggested to influence synaptic plasticity. In this context, we examined the role of protease activated receptor 1 (PAR1), which is activated following proteolytic cleavage by thrombin and plasmin, in emotionally motivated learning. We were particularly interested in PAR1 because its activation enhances the function of NMDA receptors, which are required for some forms of synaptic plasticity. We examined several baseline behavioral measures, including locomotor activity, expression of anxiety-like behavior, motor task acquisition, nociceptive responses, and startle responses in C57Bl/6 mice in which the PAR1 receptor has been genetically deleted. In addition, we evaluated learning and memory in these mice using two memory tasks, passive avoidance and cued fear-conditioning. Whereas locomotion, pain response, startle, and measures of baseline anxiety were largely unaffected by PAR1 removal, PAR1-/- animals showed significant deficits in a passive avoidance task and in cued fear conditioning. These data suggest that PAR1 may play an important role in emotionally motivated learning.

  8. Purification and characterization of a serine protease (CPM-2) with fibrinolytic activity from the dung beetles.

    PubMed

    Ahn, Mi Young; Hahn, Bum-Soo; Ryu, Kang Sun; Hwang, Jae Sam; Kim, Yeong Shik

    2005-07-01

    Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gln Asp Phe Val Glu Glu Ile Leu. CPM-2 was inactivated by Cu2+ and Zn2+ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and alpha1-antitrypsin. However, EDTA, EGTA, cysteine, beta-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved Aalpha- and Bbeta-chains of fibrinogen and fibrin, and gamma-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

  9. Peptide-based inhibitors of the hepatitis C virus NS3 protease: structure-activity relationship at the C-terminal position.

    PubMed

    Rancourt, Jean; Cameron, Dale R; Gorys, Vida; Lamarre, Daniel; Poirier, Martin; Thibeault, Diane; Llinàs-Brunet, Montse

    2004-05-06

    The structure-activity relationship at the C-terminal position of peptide-based inhibitors of the hepatitis C virus NS3 protease is presented. The observation that the N-terminal cleavage product (DDIVPC-OH) of a substrate derived from the NS5A/5B cleavage site was a competitive inhibitor of the NS3 protease was previously described. The chemically unstable cysteine residue found at the P1 position of these peptide-based inhibitors could be replaced with a norvaline residue, at the expense of a substantial drop in the enzymatic activity. The fact that an aminocyclopropane carboxylic acid (ACCA) residue at the P1 position of a tetrapeptide such as 1 led to a significant gain in the inhibitory enzymatic activity, as compared to the corresponding norvaline derivative 2, prompted a systematic study of substituent effects on the three-membered ring. We report herein that the incorporation of a vinyl group with the proper configuration onto this small cycle produced inhibitors of the protease with much improved in vitro potency. The vinyl-ACCA is the first reported carboxylic acid containing a P1 residue that produced NS3 protease inhibitors that are significantly more active than inhibitors containing a cysteine at the same position.

  10. Biofluid proteases profiling in diabetes mellitus.

    PubMed

    Trindade, Fábio; Ferreira, Rita; Amado, Francisco; Vitorino, Rui

    2015-01-01

    The investigation of protease relevance in biologic systems beyond catabolism of proteins and peptides to amino acids has stimulated interest as to their role in the pathogenesis of several disorders including diabetes mellitus (DM). Evaluation of proteases and the assessment of their activity in biofluids are fundamental to elucidate these proteolytic systems in DM and its related complications. In contrast to traditional immunoassay or substrate based approaches that targeted specific proteases and their inhibitors, the field of degradomics has provided a comprehensive approach to study these enzymes. Although the degradome contains over 500 proteases, very few have been associated with DM and its micro- and macrovascular complications. In this paper, we review these proteases and their respective inhibitors with emphasis on DM. It is likely that future research will expand these initial studies and look to develop high throughput automated technologies to identify and characterize biofluid proteases of diagnostic and prognostic value in other pathologies.

  11. Improved protease stability of the antimicrobial peptide Pin2 substituted with D-amino acids.

    PubMed

    Carmona, G; Rodriguez, A; Juarez, D; Corzo, G; Villegas, E

    2013-08-01

    Cationic antimicrobial peptides (AMPs) have attracted a great interest as novel class of antibiotics that might help in the treatment of infectious diseases caused by pathogenic bacteria. However, some AMPs with high antimicrobial activities are also highly hemolytic and subject to proteolytic degradation from human and bacterial proteases that limit their pharmaceutical uses. In this work a D-diastereomer of Pandinin 2, D-Pin2, was constructed to observe if it maintained antimicrobial activity in the same range as the parental one, but with the purpose of reducing its hemolytic activity to human erythrocytes and improving its ability to resist proteolytic cleavage. Although, the hydrophobic and secondary structure characteristics of L- and D-Pin2 were to some extent similar, an important reduction in D-Pin2 hemolytic activity (30-40 %) was achieved compared to that of L-Pin2 over human erythrocytes. Furthermore, D-Pin2 had an antimicrobial activity with a MIC value of 12.5 μM towards Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and two strains of Pseudomonas aeruginosa in agar diffusion assays, but it was half less potent than that of L-Pin2. Nevertheless, the antimicrobial activity of D-Pin2 was equally effective as that of L-Pin2 in microdilution assays. Yet, when D- and L-Pin2 were incubated with trypsin, elastase and whole human serum, only D-Pin2 kept its antimicrobial activity towards all bacteria, but in diluted human serum, L- and D-Pin2 maintained similar peptide stability. Finally, when L- and D-Pin2 were incubated with proteases from P. aeruginosa DFU3 culture, a clinical isolated strain, D-Pin2 kept its antibiotic activity while L-Pin2 was not effective.

  12. A novel carboxyl-terminal protease derived from Paenibacillus lautus CHN26 exhibiting high activities at multiple sites of substrates

    PubMed Central

    2013-01-01

    Background Carboxyl-terminal protease (CtpA) plays essential functions in posttranslational protein processing in prokaryotic and eukaryotic cells. To date, only a few bacterial ctpA genes have been characterized. Here we cloned and characterized a novel CtpA. The encoding gene, ctpAp (ctpA of Paenibacillus lautus), was derived from P. lautus CHN26, a Gram-positive bacterium isolated by functional screening. Recombinant protein was obtained from protein over-expression in Escherichia coli and the biochemical properties of the enzyme were investigated. Results Screening of environmental sediment samples with a skim milk-containing medium led to the isolation of a P. lautus CHN26 strain that exhibited a high proteolytic activity. A gene encoding a carboxyl-terminal protease (ctpAp) was cloned from the isolate and characterized. The deduced mature protein contains 466 aa with a calculated molecular mass of 51.94 kDa, displaying 29-38% amino acid sequence identity to characterized bacterial CtpA enzymes. CtpAp contains an unusual catalytic dyad (Ser309-Lys334) and a PDZ substrate-binding motif, characteristic for carboxyl-terminal proteases. CtpAp was expressed as a recombinant protein and characterized. The purified enzyme showed an endopeptidase activity, which effectively cleaved α S1- and β- casein substrates at carboxyl-terminus as well as at multiple internal sites. Furthermore, CtpAp exhibited a high activity at room temperature and strong tolerance to conventional protease inhibitors, demonstrating that CtpAp is a novel endopeptidase. Conclusions Our work on CtpA represents the first investigation of a member of Family II CtpA enzymes. The gene was derived from a newly isolated P. lautus CHN26 strain exhibiting a high protease activity in the skim milk assay. We have demonstrated that CtpAp is a novel endopeptidase with distinct cleavage specificities, showing a strong potential in biotechnology and industry applications. PMID:24161150

  13. Structural Insight into Serine Protease Rv3671c that Protects M. tuberculosis from Oxidative and Acidic Stress

    SciTech Connect

    Biswas, Tapan; Small, Jennifer; Vandal, Omar; Odaira, Toshiko; Deng, Haiteng; Ehrt, Sabine; Tsodikov, Oleg V.

    2010-11-15

    Rv3671c, a putative serine protease, is crucial for persistence of Mycobacterium tuberculosis in the hostile environment of the phagosome. We show that Rv3671c is required for M. tuberculosis resistance to oxidative stress in addition to its role in protection from acidification. Structural and biochemical analyses demonstrate that the periplasmic domain of Rv3671c is a functional serine protease of the chymotrypsin family and, remarkably, that its activity increases on oxidation. High-resolution crystal structures of this protease in an active strained state and in an inactive relaxed state reveal that a solvent-exposed disulfide bond controls the protease activity by constraining two distant regions of Rv3671c and stabilizing it in the catalytically active conformation. In vitro biochemical studies confirm that activation of the protease in an oxidative environment is dependent on this reversible disulfide bond. These results suggest that the disulfide bond modulates activity of Rv3671c depending on the oxidative environment in vivo.

  14. Proteolytic activity of Plasmodium falciparum subtilisin-like protease 3 on parasite profilin, a multifunctional protein.

    PubMed

    Alam, Asrar; Bhatnagar, Raj K; Relan, Udbhav; Mukherjee, Paushali; Chauhan, Virander S

    2013-10-01

    Subtilisin-like proteases of malaria parasite Plasmodium falciparum (PfSUB1, 2 and 3) are expressed at late asexual blood stages. PfSUB1 and 2 are considered important drug targets due to their essentiality for parasite blood stages and role in merozoite egress and invasion of erythrocytes. We have earlier shown the in vitro serine protease activity of PfSUB3 and its localization at asexual blood stages. In this study, we attempted to identify the biological substrate(s) of PfSUB3 and found parasite profilin (PfPRF) as a substrate of the protease. Eukaryotic profilins are multifunctional proteins with primary role in regulation of actin filament assembly. PfPRF possesses biochemical features of eukaryotic profilins and its rodent ortholog is essential in blood stages. Profilin from related apicomplexan parasite Toxoplasma gondii (TgPRF) is known to be involved in parasite motility, host cell invasion, active egress from host cell, immune evasion and virulence in mice. In this study, mature PfSUB3 proteolysed recombinant PfPRF in a dose-dependent manner in in vitro assays. Recombinant PfPRF was assessed for its proinflammatory activity and found to induce high level of TNF-α and low but significant level of IL-12 from mouse bone marrow-derived dendritic cells. Proteolysis of PfPRF by PfSUB3 is suggestive of the probable role of the protease in the processes of motility, virulence and immune evasion.

  15. Protease Gene Duplication and Proteolytic Activity in Drosophila Female Reproductive Tracts

    PubMed Central

    Kelleher, Erin S.; Pennington, James E.

    2009-01-01

    Secreted proteases play integral roles in sexual reproduction in a broad range of taxa. In the genetic model Drosophila melanogaster, these molecules are thought to process peptides and activate enzymes inside female reproductive tracts, mediating critical postmating responses. A recent study of female reproductive tract proteins in the cactophilic fruit fly Drosophila arizonae, identified pervasive, lineage-specific gene duplication amongst secreted proteases. Here, we compare the evolutionary dynamics, biochemical nature, and physiological significance of secreted female reproductive serine endoproteases between D. arizonae and its congener D. melanogaster. We show that D. arizonae lower female reproductive tract (LFRT) proteins are significantly enriched for recently duplicated secreted proteases, particularly serine endoproteases, relative to D. melanogaster. Isolated lumen from D. arizonae LFRTs, furthermore, exhibits significant trypsin-like and elastase-like serine endoprotease acitivity, whereas no such activity is seen in D. melanogaster. Finally, trypsin- and elastase-like activity in D. arizonae female reproductive tracts is negatively regulated by mating. We propose that the intense proteolytic environment of the D. arizonae female reproductive tract relates to the extraordinary reproductive physiology of this species and that ongoing gene duplication amongst these proteases is an evolutionary consequence of sexual conflict. PMID:19546158

  16. Active site conformational changes of prostasin provide a new mechanism of protease regulation by divalent cations

    SciTech Connect

    Spraggon, Glen; Hornsby, Michael; Shipway, Aaron; Tully, David C.; Bursulaya, Badry; Danahay, Henry; Harris, Jennifer L.; Lesley, Scott A.

    2010-01-12

    Prostasin or human channel-activating protease 1 has been reported to play a critical role in the regulation of extracellular sodium ion transport via its activation of the epithelial cell sodium channel. Here, the structure of the extracellular portion of the membrane associated serine protease has been solved to high resolution in complex with a nonselective d-FFR chloromethyl ketone inhibitor, in an apo form, in a form where the apo crystal has been soaked with the covalent inhibitor camostat and in complex with the protein inhibitor aprotinin. It was also crystallized in the presence of the divalent cation Ca{sup +2}. Comparison of the structures with each other and with other members of the trypsin-like serine protease family reveals unique structural features of prostasin and a large degree of conformational variation within specificity determining loops. Of particular interest is the S1 subsite loop which opens and closes in response to basic residues or divalent ions, directly binding Ca{sup +2} cations. This induced fit active site provides a new possible mode of regulation of trypsin-like proteases adapted in particular to extracellular regions with variable ionic concentrations such as the outer membrane layer of the epithelial cell.

  17. The Race against Protease Activation Defines the Role of ESCRTs in HIV Budding

    PubMed Central

    Bendjennat, Mourad; Saffarian, Saveez

    2016-01-01

    HIV virions assemble on the plasma membrane and bud out of infected cells using interactions with endosomal sorting complexes required for transport (ESCRTs). HIV protease activation is essential for maturation and infectivity of progeny virions, however, the precise timing of protease activation and its relationship to budding has not been well defined. We show that compromised interactions with ESCRTs result in delayed budding of virions from host cells. Specifically, we show that Gag mutants with compromised interactions with ALIX and Tsg101, two early ESCRT factors, have an average budding delay of ~75 minutes and ~10 hours, respectively. Virions with inactive proteases incorporated the full Gag-Pol and had ~60 minutes delay in budding. We demonstrate that during budding delay, activated proteases release critical HIV enzymes back to host cytosol leading to production of non-infectious progeny virions. To explain the molecular mechanism of the observed budding delay, we modulated the Pol size artificially and show that virion release delays are size-dependent and also show size-dependency in requirements for Tsg101 and ALIX. We highlight the sensitivity of HIV to budding “on-time” and suggest that budding delay is a potent mechanism for inhibition of infectious retroviral release. PMID:27280284

  18. Inhibition of ribonuclease and protease activities in arsenic exposed rice seedlings: role of proline as enzyme protectant.

    PubMed

    Mishra, Shruti; Dubey, Rama Shanker

    2006-09-01

    When seedlings of two rice (Oryza sativa L.) cvs. Malviya-36 and Pant-12 were raised under 25 and 50 microM As2O3 in the medium an increase in the level of RNA, proteins and proline accompanied with a decline in the level of free amino acid pool was observed under arsenic supplementation compared to controls. In situ As3+ treatment caused a marked inhibition in activities of ribonuclease (RNase, EC 3.1.27.1), protease and leucine aminopeptidase (LAP, EC 3.4.11.1) whereas the activity level of carboxypeptidase (EC 3.4.16.5) was enhanced. In vitro supply of As2O3 in the enzyme assay medium beyond 400 microM resulted in gradual inhibition of RNase and beyond 5 microM inhibition of LAP activities. Addition of 1M proline in the assay medium significantly restored the loss in RNase activity due to in vitro arsenic treatment or due to osmotic stress created by incorporation of polyethylene glycol (PEG). Isoform pattern of RNase extracted from As3+ -exposed seedlings showed a significant alteration compared to its pattern in unexposed seedlings. Results suggest that arsenic exposure impairs hydrolysis of RNA and proteins in rice seedlings due to inhibition of RNase and proteases activities and that proline accumulating under As3+ toxicity appears to serve as enzyme protectant.

  19. [Methods of increasing the activity of extracellular esterase, beta-fructofuranosidase and proteases of wine yeast].

    PubMed

    Abdurazakova, S Kh; Salomov, Kh T

    1975-01-01

    Upon regular fermentation changes in the activity of the enzymes esterase, beta-fructofuranosidase and protease of the yeast Saccharomyces mini of the Parkent I race were examined. The maximum activity of the enzymes occurred in the stationary phase of the yeast growth. An increase in the activity of the above enzymes was shown possible during a prolonged stabilization of the stationary conditions in the process of a continuous chemostat cultivation of wine yeast.

  20. Protease and lipase activities of fungal and bacterial strains derived from an artisanal raw ewe's milk cheese.

    PubMed

    Ozturkoglu-Budak, Sebnem; Wiebenga, Ad; Bron, Peter A; de Vries, Ronald P

    2016-11-21

    We previously identified the microbiota present during cheese ripening and observed high protease and lipase activity in Divle Cave cheese. To determine the contribution of individual isolates to enzyme activities, we investigated a range of species representing this microbiota for their proteolytic and lipolytic ability. In total, 17 fungal, 5 yeast and 18 bacterial strains, previously isolated from Divle Cave cheese, were assessed. Qualitative protease and lipase activities were performed on skim-milk agar and spirit-blue lipase agar, respectively, and resulted in a selection of strains for quantitative assays. For the quantitative assays, the strains were grown on minimal medium containing irradiated Divle Cave cheese, obtained from the first day of ripening. Out of 16 selected filamentous fungi, Penicillium brevicompactum, Penicillium cavernicola and Penicillium olsonii showed the highest protease activity, while Mucor racemosus was the best lipase producer. Yarrowia lipolytica was the best performing yeast with respect to protease and lipase activity. From the 18 bacterial strains, 14 and 11 strains, respectively showed protease and lipase activity in agar plates. Micrococcus luteus, Bacillus stratosphericus, Brevibacterium antiquum, Psychrobacter glacincola and Pseudomonas proteolytica displayed the highest protease and lipase activity. The proteases of yeast and filamentous fungi were identified as mainly aspartic protease by specific inhibition with Pepstatin A, whereas inhibition by PMSF (phenylmethylsulfonyl fluoride) indicated that most bacterial enzymes belong to serine type protease. Our results demonstrate that aspartic proteases, which usually have high milk clotting activity, are predominantly derived from fungal strains, and therefore fungal enzymes appear to be more suitable for use in the cheese industry. Microbial enzymes studied in this research might be alternatives for rennin (chymosin) from animal source because of their low cost and stable

  1. Active subtilisin-like protease from a hyperthermophilic archaeon in a form with a putative prosequence.

    PubMed

    Kannan, Y; Koga, Y; Inoue, Y; Haruki, M; Takagi, M; Imanaka, T; Morikawa, M; Kanaya, S

    2001-06-01

    The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly(-82) to Gly(316)), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca(2+) ion with an optimal pH and temperature of pH 9.5 and 80 degrees C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80 degrees C, 20 min at 90 degrees C, and 7 min at 100 degrees C.

  2. Proteases from psychrotrophs: an overview.

    PubMed

    Kasana, Ramesh Chand

    2010-05-01

    Proteases are hydrolytic enzymes which catalyze the total hydrolysis of proteins in to amino acids. Although proteolytic enzymes can be obtained from animals and plants but microorganisms are the preferred source for industrial applications in view of scientific and economical advantage. Among various groups of microbes, psychrotrophs are ideal candidates for enzymes production keeping in mind that enzymes active at low temperature and stable under alkaline condition, in presence of oxidants and detergents are in large demand as laundry additive. The proteases from psychrotrophs also find application in environmental bioremediation, food and molecular biology. During the previous two decades, proteases from psychrotrophs have received increased attention because of their wide range of applications, but the full potential of psychrotrophic proteases has not been exploited. This review focuses attention on the present status of knowledge on the production, optimization, molecular characteristics, applications, substrate specificity, and crystal structure of psychrotrophic proteases. The review will help in making strategies for exploitation of psychrotrophic protease resources and improvement of enzymes to obtain more robust proteases of industrial and biotechnological significance.

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

    PubMed Central

    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. PMID:27093053

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

    PubMed

    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.

  5. Role of enteric nerves in immune-mediated changes in protease activated receptor 2 effects on gut function

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protease activated receptors (PARs) are expressed on structural cells and immune cells. Control of the initiation, duration, and magnitude of the PAR effects are linked to the level of receptor expression, the availability of proteases, and the intracellular signal transduction machinery. We inve...

  6. Collagenolytic activity related to metalloproteases (and serine proteases) in the fish parasite Hysterothylacium aduncum (Nematoda: Anisakidae).

    PubMed

    Malagón, David; Adroher, Francisco Javier; Díaz-López, Manuel; Benítez, Rocío

    2010-06-11

    Proteases play a vital role in both the life cycle of parasites and the parasite-host relationship and are considered important virulence factors. In the present study, the presence of proteases with collagenolytic activity was investigated in the fish nematode Hysterothylacium aduncum during in vitro development. Collagenolytic activity was found in all studied developmental stages of the nematode (third [L3] and fourth [L4] larval stages and adults). In L3, the activity was maximum at pH 6.5 and, in the other stages, at 7.0. Pepsin is known to favour in vitro development of the worm, but, in this study, collagenolytic activity was shown to be significantly greater when no pepsin was added to the culture medium (at pH 6.5, p = 0.011). At pH 7.0, most activity was observed in the immature adult, after the final moult, suggesting that the collagenolytic activity may be involved in remodelling of the cuticle and in sexual maturity. On the other hand, at pH 6.5, activity may be related to tissue migration by L3 within the host. Using specific inhibitors, it was demonstrated that most of the collagenolytic activity detected in all the developmental stages was due to metalloproteases (40 to 100%), although serine proteases were also detected in L4 and adults (10 to 30%).

  7. Investigations with Protease.

    ERIC Educational Resources Information Center

    Yip, Din Yan

    1997-01-01

    Presents two simple and reliable ways for measuring protease activity that can be used for a variety of investigations in a range of biology class levels. The investigations use protease from a variety of sources. (DDR)

  8. Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity—A Structural and Biochemical Approach

    PubMed Central

    Dutta, Sruti; Choudhury, Debi; Roy, Sumana; Dattagupta, Jiban Kanti; Biswas, Sampa

    2016-01-01

    Papain-like proteases contain an N-terminal pro-peptide in their zymogen form that is important for correct folding and spatio-temporal regulation of the proteolytic activity of these proteases. Catalytic removal of the pro-peptide is required for the protease to become active. In this study, we have generated three different mutants of papain (I86F, I86L and I86A) by replacing the residue I86 in its pro-peptide region, which blocks the specificity determining S2-subsite of the catalytic cleft of the protease in its zymogen form with a view to investigate the effect of mutation on the catalytic activity of the protease. Steady-state enzyme kinetic analyses of the corresponding mutant proteases with specific peptide substrates show significant alteration of substrate specificity—I86F and I86L have 2.7 and 29.1 times higher kcat/Km values compared to the wild-type against substrates having Phe and Leu at P2 position, respectively, while I86A shows lower catalytic activity against majority of the substrates tested. Far-UV CD scan and molecular mass analyses of the mature form of the mutant proteases reveal similar CD spectra and intact masses to that of the wild-type. Crystal structures of zymogens of I86F and I86L mutants suggest that subtle reorganization of active site residues, including water, upon binding of the pro-peptide may allow the enzyme to achieve discriminatory substrate selectivity and catalytic efficiency. However, accurate and reliable predictions on alteration of substrate specificity require atomic resolution structure of the catalytic domain after zymogen activation, which remains a challenging task. In this study we demonstrate that through single amino acid substitution in pro-peptide, it is possible to modify the substrate specificity of papain and hence the pro-peptide of a protease can also be a useful target for altering its catalytic activity/specificity. PMID:27352302

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

    PubMed

    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.

  10. [Effects of Tagetes erecta extracts on glutathione S-transferase and protease activities and protein content in Tetranychus viennensis].

    PubMed

    Shi, Guang-lu; Wang, You-nian; Wang, Hong-lei; Zhao, Li-lin; Liu, Su-qi; Cao, Hui; Yu, Tong-quan; Lu, Ping

    2007-02-01

    With in vivo and in vitro Tagetes erecta roots under light and dark as test materials, this paper studied the effects of their extracts on the glutathione S-transferase and protease activities and protein content in Tetranychus viennensis. The results showed that the chloroform extract of T. erecta roots had the highest light-activated activity, followed by water extract, and methanol extract. After treated with chloroform extract, the glutathione S-transferase and protease activities in T. viennensis increased markedly, while its protein content decreased obviously. The variation degree of T. viennensis protease activity and protein content was significantly higher when the chloroform extract came from the T. erecta roots under light, suggesting that there existed active matters in the extract, which could promote the activation of protease, and thus, the decomposition of protein in T. viennensis. The bioactivity of T. erecta metabolites was mainly of light-activated one.

  11. TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus.

    PubMed

    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.

  12. In vitro anti-human immunodeficiency virus (HIV) activities of transition state mimetic HIV protease inhibitors containing allophenylnorstatine.

    PubMed Central

    Kageyama, S; Mimoto, T; Murakawa, Y; Nomizu, M; Ford, H; Shirasaka, T; Gulnik, S; Erickson, J; Takada, K; Hayashi, H

    1993-01-01

    Transition state mimetic tripeptide human immunodeficiency virus (HIV) protease inhibitors containing allophenylnorstatine [(2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] were synthesized and tested for activity against HIV in vitro. Two compounds, KNI-227 and KNI-272, which were highly potent against HIV protease with little inhibition of other aspartic proteases, showed the most potent activity against the infectivity and cytopathic effect of a wide spectrum of HIV strains. As tested in target CD4+ ATH8 cells, the 50% inhibitory concentrations of KNI-227 against HIV type 1 LAI (HIV-1LAI), HIV-1RF, HIV-1MN, and HIV-2ROD were 0.1, 0.02, 0.03, and 0.1 microM, respectively, while those of KNI-272 were 0.1, 0.02, 0.04, and 0.1 microM, respectively. Both agents completely blocked the replication of 3'-azido-2',3'-dideoxythymidine-sensitive and -insensitive clinical HIV-1 isolates at 0.08 microM as tested in target phytohemagglutinin-activated peripheral blood mononuclear cells. The ratios of 50% cytotoxic concentrations to 50% inhibitory concentrations for KNI-227 and KNI-272 were approximately 2,500 and > 4,000, respectively, as assessed in peripheral blood mononuclear cells. Both compounds blocked the posttranslational cleavage of the p55 precursor protein to generate the mature p24 Gag protein in stably HIV-1-infected cells. The n-octanol-water partition coefficients of KNI-227 and KNI-272 were high, with log Po/w values of 3.79 and 3.56, respectively. Degradation of KNI-227 and KNI-272 in the presence of pepsin (1 mg/ml, pH 2.2) at 37 degrees C for 24 h was negligible. Current data warrant further careful investigations toward possible clinical application of these two novel compounds. Images PMID:8494379

  13. Potential antimicrobial and antiproliferative activities of autochthonous starter cultures and protease EPg222 in dry-fermented sausages.

    PubMed

    Fernández, Margarita; Ruiz-Moyano, Santiago; Benito, María José; Martín, Alberto; Hernández, Alejandro; Córdoba, María de Guía

    2016-05-18

    This work studied the presence of nitrogen compounds with bioactive properties in Iberian pork sausages that were manufactured using different autochthonous starter cultures (Pediococcus acidilactici MS200 and Staphylococcus vitulus RS34) and protease EPg222. Nitrogen compounds were extracted and evaluated for their antimicrobial effect against spoilage and pathogenic bacteria, such as Bacillus cereus, Escherichia coli, Salmonella choleraesuis, Staphylococcus aureus and Listeria monocytogenes, and antiproliferative activity on the HT-29 colon adenocarcinoma cell line. Dry-fermented sausages elaborated with starter cultures P200S34 and protease EPg222 generate extracts that cause inhibition of the growth of pathogens reaching 25% inhibition of Bacillus cereus, making this a promising tool for biocontrol in the meat industry. On the other hand, the inoculation of well-adapted starter cultures with high proteolytic activity also increased the antiproliferative activity of these extracts, around 45% inhibition at 72 h, mainly due to an increase in free amino acids, such as Lys and Pro, but also small peptides.

  14. A novel TMPRSS6 mutation that prevents protease auto-activation causes IRIDA

    PubMed Central

    Altamura, Sandro; D'Alessio, Flavia; Selle, Barbara; Muckenthaler, Martina U.

    2010-01-01

    IRIDA (iron-refractory iron-deficiency anaemia) is a rare autosomal-recessive disorder hallmarked by hypochromic microcytic anaemia, low transferrin saturation and high levels of the iron-regulated hormone hepcidin. The disease is caused by mutations in the transmembrane serine protease TMPRSS6 (transmembrane protease serine 6) that prevent inactivation of HJV (haemojuvelin), an activator of hepcidin transcription. In the present paper, we describe a patient with IRIDA who carries a novel mutation (Y141C) in the SEA domain of the TMPRSS6 gene. Functional characterization of the TMPRSS6(Y141C) mutant protein in cultured cells showed that it localizes to similar subcellular compartments as wild-type TMPRSS6 and binds HJV, but fails to auto-catalytically activate itself. As a consequence, hepcidin mRNA expression is increased, causing the clinical symptoms observed in this IRIDA patient. The present study provides important mechanistic insight into how TMPRSS6 is activated. PMID:20704562

  15. Differential Expression of Extracellular Lipase and Protease Activities of Mycelial and Yeast Forms in Malassezia furfur.

    PubMed

    Juntachai, Weerapong; Kajiwara, Susumu

    2015-10-01

    Malassezia furfur is a dimorphic yeast that is part of the human skin microflora. This fungus is a pathogen of a certain skin diseases, such as pityriasis versicolor, and in rare cases causes systemic infection in neonates. However, the role of dimorphism in the pathogenicity remains unclear. A modified induction medium (IM) was successfully able to induce mycelial growth of M. furfur under both solid and liquid condition. Filamentous elements with branching hyphae were observed when cultured in the IM. Furthermore, addition of bovine fetus serum into the liquid IM did not promote hyphal formation; on the contrary, it retrograded hyphae to the yeast form. Plate-washing assay showed that M. furfur hyphae did not possess the ability of invasive growth. Secretory proteins from both yeast and hyphal forms were isolated, and lipase and protease activities were analyzed. Intriguingly, the hyphal form showed higher activities than those of the yeast form, particularly the protease activity.

  16. Conditions influencing the synthesis of acid protease by Mucor pusillus Lindt.

    PubMed

    Somkuti, G A; Babel, F J

    1967-11-01

    Protease synthesis by Mucor pusillus Lindt, in a wheat bran medium under submerged conditions, was influenced by substrate concentration, initial pH of the medium, and temperature of incubation. A 4% wheat bran (dry weight) concentration was satisfactory for enzyme production. The initial pH of the medium had a substantial effect on enzyme synthesis; adjustment of the enzyme production medium to pH 5.0 prior to sterilization was desirable. Incubation at 35 C resulted in the best enzyme yields. Under optimal conditions of enzyme production, maximal activity was detected after 5 days of incubation. The enrichment of the medium with glucose increased the yield of mycelia but lowered the amount of enzyme produced.

  17. Complete amino acid sequence of ananain and a comparison with stem bromelain and other plant cysteine proteases.

    PubMed Central

    Lee, K L; Albee, K L; Bernasconi, R J; Edmunds, T

    1997-01-01

    The amino acid sequences of ananain (EC3.4.22.31) and stem bromelain (3.4.22.32), two cysteine proteases from pineapple stem, are similar yet ananain and stem bromelain possess distinct specificities towards synthetic peptide substrates and different reactivities towards the cysteine protease inhibitors E-64 and chicken egg white cystatin. We present here the complete amino acid sequence of ananain and compare it with the reported sequences of pineapple stem bromelain, papain and chymopapain from papaya and actinidin from kiwifruit. Ananain is comprised of 216 residues with a theoretical mass of 23464 Da. This primary structure includes a sequence insert between residues 170 and 174 not present in stem bromelain or papain and a hydrophobic series of amino acids adjacent to His-157. It is possible that these sequence differences contribute to the different substrate and inhibitor specificities exhibited by ananain and stem bromelain. PMID:9355753

  18. Delivery of a Protease-Activated Cytolytic Peptide Prodrug by Perfluorocarbon Nanoparticles.

    PubMed

    Jallouk, Andrew P; Palekar, Rohun U; Marsh, Jon N; Pan, Hua; Pham, Christine T N; Schlesinger, Paul H; Wickline, Samuel A

    2015-08-19

    Melittin is a cytolytic peptide derived from bee venom that inserts into lipid membranes and oligomerizes to form membrane pores. Although this peptide is an attractive candidate for treatment of cancers and infectious processes, its nonspecific cytotoxicity and hemolytic activity have limited its therapeutic applications. Several groups have reported the development of cytolytic peptide prodrugs that only exhibit cytotoxicity following activation by site-specific proteases. However, systemic administration of these constructs has proven difficult because of their poor pharmacokinetic properties. Here, we present a platform for the design of protease-activated melittin derivatives that may be used in conjunction with a perfluorocarbon nanoparticle delivery system. Although native melittin was substantially hemolytic (HD50: 1.9 μM) and cytotoxic (IC50: 2.4 μM), the prodrug exhibited 2 orders of magnitude less hemolytic activity (HD50: > 100 μM) and cytotoxicity (IC50: > 100 μM). Incubation with matrix metalloproteinase-9 (MMP-9) led to cleavage of the prodrug at the expected site and restoration of hemolytic activity (HD50: 3.4 μM) and cytotoxicity (IC50: 8.1 μM). Incubation of the prodrug with perfluorocarbon nanoparticles led to stable loading of 10,250 peptides per nanoparticle. Nanoparticle-bound prodrug was also cleaved and activated by MMP-9, albeit at a fourfold slower rate. Intravenous administration of prodrug-loaded nanoparticles in a mouse model of melanoma significantly decreased tumor growth rate (p = 0.01). Because MMPs and other proteases play a key role in cancer invasion and metastasis, this platform holds promise for the development of personalized cancer therapies directed toward a patient's individual protease expression profile.

  19. Antimicrobial activity of protease inhibitor from leaves of Coccinia grandis (L.) Voigt.

    PubMed

    Satheesh, L Shilpa; Murugan, K

    2011-05-01

    Antimicrobial activity of protease inhibitor isolated from Coccinia grandis (L.) Voigt. has been reported. A 14.3 kDa protease inhibitor (PI) was isolated and purified to homogeneity by ammonium sulfate precipitation (20-85% saturation), sephadex G-75, DEAE sepharose column and trypsin-sepharose affinity chromatography from the leaves of C. grandis. The purity was checked by reverse phase high performance liquid chromatography. PI exhibited marked growth inhibitory effects on colon cell lines in a dose-dependent manner. PI was thermostable and showed antimicrobial activity without hemolytic activity. PI strongly inhibited pathogenic microbial strains, including Staphylococcus aureus, Klebsiella pneumoniae, Proteus vulgaris, Eschershia coli, Bacillus subtilis and pathogenic fungus Candida albicans, Mucor indicus, Penicillium notatum, Aspergillus flavus and Cryptococcus neoformans. Examination by bright field microscopy showed inhibition of mycelial growth and sporulation. Morphologically, PI treated fungus showed a significant shrinkage of hyphal tips. Reduced PI completely lost its activity indicating that disulfide bridge is essential for its protease inhibitory and antifungal activity. Results reported in this study suggested that PI may be an excellent candidate for development of novel oral or other anti-infective agents.

  20. Crystal structure of Clostridium botulinum neurotoxin protease in a product-bound state: Evidence for noncanonical zinc protease activity.

    PubMed

    Segelke, Brent; Knapp, Mark; Kadkhodayan, Saloumeh; Balhorn, Rod; Rupp, Bernhard

    2004-05-04

    Clostridium botulinum neurotoxins (BoNTs), the most potent toxins known, disrupt neurotransmission through proteolysis of proteins involved in neuroexocytosis. The light chains of BoNTs are unique zinc proteases that have stringent substrate specificity and require exceptionally long substrates. We have determined the crystal structure of the protease domain from BoNT serotype A (BoNT/A). The structure reveals a homodimer in a product-bound state, with loop F242-V257 from each monomer deeply buried in its partner's catalytic site. The loop, which acts as a substrate, is oriented in reverse of the canonical direction for other zinc proteases. The Y249-Y250 peptide bond of the substrate loop is hydrolyzed, leaving the Y249 product carboxylate coordinated to the catalytic zinc. From the crystal structure of the BoNT/A protease, detailed models of noncanonical binding and proteolysis can be derived which we propose are also consistent with BoNT/A binding and proteolysis of natural substrate synaptosome-associated protein of 25 kDa (SNAP-25). The proposed BoNT/A substrate-binding mode and catalytic mechanism are markedly different from those previously proposed for the BoNT serotype B.

  1. Thalidomide combined with irradiation alters the activity of two proteases.

    PubMed

    Şimşek, Ece; Aydemir, Esra; Korcum, Aylin Fidan; Fişkın, Kayahan

    2015-02-01

    The aim of the present study was to investigate the effects of thalidomide, a drug known for its anti‑angiogenic and antitumor properties, at its cytotoxic dose previously determined as 40 µg/ml (according to four cytotoxic test results). The effect of the drug alone and in combination with radiotherapy using Cobalt 60 (60Co) at 45 Gy on the enzymatic activity of substance‑P degrading A disintegrin and metalloproteinase (ADAM)10 and neprilysin (NEP) was investigated in the mouse breast cancer cell lines 4T1 and 4T1 heart metastases post‑capsaicin (4THMpc). Thalidomide (40 µg/ml) exerted differing effects on the activities of ADAM10 and NEP enzymes. In 4T1 cells, 40 µg/ml thalidomide alone did not alter ADAM10 enzyme activity. 60Co irradiation at 45 Gy alone caused a 42% inhibition in ADAM10 activity, however, the inhibition increased to 89% when combined therapy was used. By contrast, in the 4THMpc cell line, 40 µg/ml thalidomide alone induced a 66.6% increase in ADAM10 enzyme activity. Radiotherapy alone and thalidomide with 60Co combined therapy caused a 33.3 and 40% inhibition of ADAM10 activity, respectively. In 4T1 cells, thalidomide alone caused a 40.9% increase in NEP activity. Radiation therapy alone or in combination with the drug caused a 40.7% increase in NEP activity. In more aggressive 4THMpc cells, thalidomide alone caused a 26.6% increase in NEP activity. Radiotherapy alone and combined therapy caused a 33.3 and 37% increase in enzyme activity, respectively. To the best of our knowledge, the present study is the first to demonstrate that thalidomide alone or in combination with radiotherapy exhibits significant cytotoxic effects on 4T1 and 4THMpc mouse breast cancer cell lines indicating that this drug affects the enzymatic activity of ADAM10 and NEP in vitro.

  2. Discovery of novel protease activated receptors 1 antagonists with potent antithrombotic activity in vivo.

    PubMed

    Perez, Michel; Lamothe, Marie; Maraval, Catherine; Mirabel, Etienne; Loubat, Chantal; Planty, Bruno; Horn, Clemens; Michaux, Julien; Marrot, Sebastien; Letienne, Robert; Pignier, Christophe; Bocquet, Arnaud; Nadal-Wollbold, Florence; Cussac, Didier; de Vries, Luc; Le Grand, Bruno

    2009-10-08

    Protease activated receptors (PARs) or thrombin receptors constitute a class of G-protein-coupled receptors (GPCRs) implicated in the activation of many physiological mechanisms. Thus, thrombin activates many cell types such as vascular smooth muscle cells, leukocytes, endothelial cells, and platelets via activation of these receptors. In humans, thrombin-induced platelet aggregation is mediated by one subtype of these receptors, termed PAR1. This article describes the discovery of new antagonists of these receptors and more specifically two compounds: 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)penta-1,3-dienyl]benzonitrile 36 (F 16618) and 3-(2-chlorophenyl)-1-[4-(4-fluorobenzyl)piperazin-1-yl]propenone 39 (F 16357), obtained after optimization. Both compounds are able to inhibit SFLLR-induced human platelet aggregation and display antithrombotic activity in an arteriovenous shunt model in the rat after iv or oral administration. Furthermore, these compounds are devoid of bleeding side effects often observed with other types of antiplatelet drugs, which constitutes a promising advantage for this new class of antithrombotic agents.

  3. Nucleotide and deduced amino acid sequences of a subtilisin-like serine protease from a deep-sea bacterium, Alkalimonas collagenimarina AC40(T).

    PubMed

    Kurata, Atsushi; Uchimura, Kohsuke; Shimamura, Shigeru; Kobayashi, Tohru; Horikoshi, Koki

    2007-11-01

    The acpI gene encoding an alkaline protease (AcpI) from a deep-sea bacterium, Alkalimonas collagenimarina AC40(T), was shotgun-cloned and sequenced. It had a 1,617-bp open reading frame encoding a protein of 538 amino acids. Based on analysis of the deduced amino acid sequence, AcpI is a subtilisin-like serine protease belonging to subtilase family A. It consists of a prepropeptide, a catalytic domain, and a prepeptidase C-terminal domain like other serine proteases from the genera Pseudomonas, Shewanella, Alteromonas, and Xanthomonas. Heterologous expression of the acpI gene in Escherichia coli cells yielded a 28-kDa recombinant AcpI (rAcpI), suggesting that both the prepropeptide and prepeptidase C-terminal domains were cleaved off to give the mature form. Analysis of N-terminal and C-terminal amino acid sequences of purified rAcpI showed that the mature enzyme would be composed of 273 amino acids. The optimal pH and temperature for the caseinolytic activity of the purified rAcpI were 9.0-9.5 and 45 degrees C in 100 mM glycine-NaOH buffer. Calcium ions slightly enhanced the enzyme activity and stability. The enzyme favorably hydrolyzed gelatin, collagen, and casein. AcpI from A. collagenimarina AC40(T) was also purified from culture broth, and its molecular mass was around 28 kDa, indicating that the cleavage manner of the enzyme is similar to that in E. coli cells.

  4. Tobacco etch virus protease retains its activity in various buffers and in the presence of diverse additives.

    PubMed

    Sun, Changsheng; Liang, Jiongqiu; Shi, Rui; Gao, Xuna; Zhang, Ruijuan; Hong, Fulin; Yuan, Qihang; Wang, Shengbin

    2012-03-01

    Tobacco etch virus (TEV) protease is widely used to remove tags from recombinant fusion proteins because of its stringent sequence specificity. It is generally accepted that the high concentrations of salts or other special agents in most protein affinity chromatography buffers can affect enzyme activity, including that of TEV protease. Consequently, tedious desalination or the substitution of standard TEV reaction buffer for elution buffer are often needed to ensure TEV protease activity when removing fusion tags after purifying target proteins using affinity chromatography. To address this issue, we used SOE PCR technology to synthesize a TEV protease gene with a codon pattern adapted to the codon usage bias of Escherichia coli, recovered the purified recombinant TEV protease, and examined its activity in various elution buffers commonly used in affinity chromatography as well as the effects of selected additives on its activity. Our results showed that the rTEV protease maintained high activity in all affinity chromatography elution buffers tested and tolerated high concentrations of additives commonly used in protein purification procedures, such as ethylene glycol, EGTA, Triton X-100, Tween-20, NP-40, CHAPS, urea, SDS, guanidine hydrochloride and β-mercaptoethanol. These results will facilitate the use of rTEV protease in removing tags from fusion proteins.

  5. Sclerotiamide: The First Non-Peptide-Based Natural Product Activator of Bacterial Caseinolytic Protease P.

    PubMed

    Lavey, Nathan P; Coker, Jesse A; Ruben, Eliza A; Duerfeldt, Adam S

    2016-04-22

    Caseinolytic protease P (ClpP) maintains essential roles in bacterial homeostasis. As such, both the inhibition and activation of this enzyme result in bactericidal activity, making ClpP a promising target for antibacterial drug development. Herein, we report the results of a fluorescence-based screen of ∼450 structurally diverse fungal and bacterial secondary metabolites. Sclerotiamide (1), a paraherquamide-related indolinone, was identified as the first non-peptide-based natural product activator of ClpP. Structure-activity relationships arising from the initial screen, preliminary biochemical evaluation of 1, and rationale for the exploitation of this chemotype to develop novel ClpP activators are presented.

  6. HIV-Protease Inhibitors Suppress Skeletal Muscle Fatty Acid Oxidation by Reducing CD36 and CPT-I Fatty Acid Transporters

    PubMed Central

    Richmond, Scott R.; Carper, Michael J.; Lei, Xiaoyong; Zhang, Sheng; Yarasheski, Kevin E.; Ramanadham, Sasanka

    2010-01-01

    Infection with human immunodeficiency virus (HIV) and treatment with HIV-protease inhibitor (PI)-based highly active antiretroviral therapies (HAART) is associated with dysregulated fatty acid and lipid metabolism. Enhanced lipolysis, increased circulating fatty acid levels, and hepatic and intramuscular lipid accumulation appear to contribute to insulin resistance in HIV-infected people treated with PI-based HAART. However, it is unclear whether currently prescribed HIV-PIs directly alter skeletal muscle fatty acid transport, oxidation, and storage. We find that ritonavir (r, 5 μmol/l) plus 20 μmol/l of atazanavir (ATV), lopinavir (LPV), or darunavir (DRV) reduce palmitate oxidation(16-21%) in differentiated C2C12 myotubes. Palmitate oxidation was increased following exposure to high fatty acid media but this effect was blunted when myotubes were pre-exposed to the HIV-PIs. However, LPV/r and DRV/r, but not ATV/r suppressed palmitate uptake into myotubes. We found no effect of the HIV-PIs on FATP1, FATP4, or FABPpm but both CD36/FAT and carnitine palmitoyltransferase I (CPTI) were reduced by all three regimens though ATV/r caused only a small decrease in CPT1, relative to LPV/r or DRV/r. In contrast, sterol regulatory element binding protein-1 was increased by all 3 HIV-PIs. These findings suggest that HIV-PIs suppress fatty acid oxidation in murine skeletal muscle cells and that this may be related to decreases in cytosolic- and mitochondrial-associated fatty acid transporters. HIV-PIs may also directly impair fatty acid handling and partitioning in skeletal muscle, and this may contribute to the cluster of metabolic complications that occur in people living with HIV. PMID:20117238

  7. Protease activity of MALT1: a mystery unravelled.

    PubMed

    Kirchhofer, Daniel; Vucic, Domagoj

    2012-06-01

    Constitutive NF-κB (nuclear factor κB) activation in B-cell lymphomas relies greatly on the CARMA1 [CARD (caspase recruitment domain)-containing MAGUK (membrane-associated guanylate kinase) 1]-Bcl10-MALT1 (mucosa-associated lymphoid tissue translocation gene 1) signalling complex. Within this protein complex, MALT1 possesses a rather unique enzymatic activity, which allows it to cleave Bcl10, RelB and CYLD, among other substrates. The catalytic activity of MALT1 promotes activation of canonical and non-canonical NF-κB as well as other signalling pathways. However, even after a decade of intense research on MALT1, many mechanistic aspects of its enzymatic activity remain elusive. A recent article by Hachmann, Snipas, van Raam, Cancino, Houlihan, Poreba, Kasperkiewicz, Drag and Salvesen [(2012) Biochem. J. 443, 287-295] provides novel insight into the activation mechanism and the substrate specificity of MALT1. These intriguing findings convincingly demonstrate the importance of MALT1 dimerization for its catalytic activity and pave the way for novel therapeutic approaches that target this crucial regulator of lymphoma survival and proliferation.

  8. Hemoglobinase activity of the lysine gingipain protease (Kgp) of Porphyromonas gingivalis W83.

    PubMed

    Lewis, J P; Dawson, J A; Hannis, J C; Muddiman, D; Macrina, F L

    1999-08-01

    Porphyromonas gingivalis, an important periodontal disease pathogen, forms black-pigmented colonies on blood agar. Pigmentation is believed to result from accumulation of iron protoporphyrin IX (FePPIX) derived from erythrocytic hemoglobin. The Lys-X (Lys-gingipain) and Arg-X (Arg-gingipain) cysteine proteases of P. gingivalis bind and degrade erythrocytes. We have observed that mutations abolishing activity of the Lys-X-specific cysteine protease, Kgp, resulted in loss of black pigmentation of P. gingivalis W83. Because the hemagglutinating and hemolytic potentials of mutant strains were reduced but not eliminated, we hypothesized that this protease played a role in acquisition of FePPIX from hemoglobin. In contrast to Arg-gingipain, Lys-gingipain was not inhibited by hemin, suggesting that this protease played a role near the cell surface where high concentrations of hemin confer the black pigmentation. Human hemoglobin contains 11 Lys residues in the alpha chain and 10 Lys residues in the beta chain. In contrast, there are only three Arg residues in each of the alpha and beta chains. These observations are consistent with human hemoglobin being a preferred substrate for Lys-gingipain but not Arg-gingipain. The ability of the Lys-gingipain to cleave human hemoglobin at Lys residues was confirmed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry of hemoglobin fragments resulting from digestion with the purified protease. We were able to detect several of the predicted hemoglobin fragments rendered by digestion with purified Lys-gingipain. Thus, we postulate that the Lys-gingipain of P. gingivalis is a hemoglobinase which plays a role in heme and iron uptake by effecting the accumulation of FePPIX on the bacterial cell surface.

  9. Activation of protease activated receptor 1 increases the excitability of the dentate granule neurons of hippocampus

    PubMed Central

    2011-01-01

    Protease activated receptor-1 (PAR1) is expressed in multiple cell types in the CNS, with the most prominent expression in glial cells. PAR1 activation enhances excitatory synaptic transmission secondary to the release of glutamate from astrocytes following activation of astrocytically-expressed PAR1. In addition, PAR1 activation exacerbates neuronal damage in multiple in vivo models of brain injury in a manner that is dependent on NMDA receptors. In the hippocampal formation, PAR1 mRNA appears to be expressed by a subset of neurons, including granule cells in the dentate gyrus. In this study we investigate the role of PAR activation in controlling neuronal excitability of dentate granule cells. We confirm that PAR1 protein is expressed in neurons of the dentate cell body layer as well as in astrocytes throughout the dentate. Activation of PAR1 receptors by the selective peptide agonist TFLLR increased the intracellular Ca2+ concentration in a subset of acutely dissociated dentate neurons as well as non-neuronal cells. Bath application of TFLLR in acute hippocampal slices depolarized the dentate gyrus, including the hilar region in wild type but not in the PAR1-/- mice. PAR1 activation increased the frequency of action potential generation in a subset of dentate granule neurons; cells in which PAR1 activation triggered action potentials showed a significant depolarization. The activation of PAR1 by thrombin increased the amplitude of NMDA receptor-mediated component of EPSPs. These data suggest that activation of PAR1 during normal function or pathological conditions, such as during ischemia or hemorrhage, can increase the excitability of dentate granule cells. PMID:21827709

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

    Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

    2017-01-01

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

  12. L-chicoric acid, an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase, improves on the in vitro anti-HIV-1 effect of Zidovudine plus a protease inhibitor (AG1350).

    PubMed

    Robinson, W E

    1998-08-01

    Combinations of anti-human immunodeficiency virus (HIV) drugs, including reverse transcriptase inhibitors and protease inhibitors, have proven immensely potent in the therapy of acquired immune deficiency syndrome (AIDS). To determine whether HIV integrase is a suitable target for combination therapy, the ability of an HIV integrase inhibitor, L-chicoric acid, to work in combination with a protease inhibitor and Zidovudine was tested in vitro. The addition of L-chicoric acid to either Zidovudine or protease inhibitor improved upon the observed anti-HIV activity of either compound alone. When all three drugs were combined, the anti-HIV activity was substantially better than either of the three compounds alone or any combination of two inhibitors. Doses of both Zidovudine and protease inhibitor could be reduced by more than 33% for an equivalent anti-HIV effect if L-chicoric acid was added. The improved anti-HIV activity was observed with a tissue culture adapted strain of HIV (HIV(LAI)) and with limited passage clinical isolates of HIV (HIV(R19) and HIV(R45)). These data demonstrate that a first generation HIV integrase inhibitor, L-chicoric acid, is at least additive in combination with existing multi-drug regimens and suggest that HIV integrase will be an excellent target for combination therapy of HIV infection.

  13. Influence of skin penetration enhancers on skin barrier function and skin protease activity.

    PubMed

    Mohammed, Diar; Hirata, Kazumasa; Hadgraft, Jonathan; Lane, Majella E

    2014-01-23

    In order to overcome the skin's excellent barrier function formulation scientists often employ skin penetration enhancers (SPEs) in topical and transdermal formulations. The effects of these compounds on skin health is still not well understood at the molecular level. The aim of the present work was to probe the effects of some common SPEs on desquamatory protease activity in healthy skin. The SPEs studied were isopropyl myristate (IPM), propylene glycol, (PG), propylene glycol laurate (PGL) and Transcutol™ (TC). Occluded infinite doses of each SPE were applied to human volunteers for 24 h. Transepidermal water loss (TEWL) measurements were taken before and after application of SPEs. Tape strips were collected from the treated sites to determine protein content and the activity of two desquamatory proteases kallikrein 5 (KLK5) and kallikrein 7 (KLK7). TEWL values were also measured after tape stripping. PG was found to elevate both TEWL values and KLK7 activity to a significant extent (p<0.05). No significant effects were observed for the other SPEs. The ability of PG to alter the skin barrier at the macroscopic level and the influence of the molecule on protease activity reported here may have implications for its use in topical formulations used for the management of impaired skin barrier function such as atopic eczema or psoriasis.

  14. Single-site substitutions improve cold activity and increase thermostability of the dehairing alkaline protease (DHAP).

    PubMed

    Zhao, Hong-Yan; Wu, Li-Ying; Liu, Gang; Feng, Hong

    2016-12-01

    To engineer dehairing alkaline protease (DHAP) variants to improve cold activity and increase thermostability so these variants are suitable for the leather processing industry. Based on previous studies with bacterial alkaline proteases, double-site mutations (W106K/V149I and W106K/M124L) were introduced into the DHAP from Bacillus pumilus. Compared with the wild-type DHAP hydrolytic activity, the double-site variant W106K/V149I showed an increase in specific hydrolytic activity at 15 °C by 2.3-fold toward casein in terms of hydrolytic rate and 2.7-fold toward the synthetic peptide AAPF-pN by means of kcat/Km value. The thermostability of the variant (W106K/V149I) was improved with the half-life at 60 and 70 °C increased by 2.7- and 5.0-fold, respectively, when compared with the thermostability of the wild-type DHAP. Conclusively, an increase in the cold activity and thermostability of a bacterial alkaline protease was achieved by protein engineering.

  15. Small Molecule-Induced Allosteric Activation of the Vibrio Cholerae RTX Cysteine Protease Domain

    SciTech Connect

    Lupardus, P.J.; Shen, A.; Bogyo, M.; Garcia, K.C.

    2009-05-19

    Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP{sub 6}), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP{sub 6}. InsP{sub 6} binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP{sub 6} binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.

  16. Synthesis of Arylpiperazine Derivatives As Protease Activated Receptor 1 Antagonists And Their Evaluation As Antiproliferative Agents.

    PubMed

    Zotti, Andrea Ilaria; Di Gennaro, Elena; Corvino, Angela; Frecentese, Francesco; Magli, Elisa; Perissutti, Elisa; Cirino, Giuseppe; Roviezzo, Fiorentina; Terranova-Barberio, Manuela; Iannelli, Federica; Caliendo, Giuseppe; Santagada, Vincenzo; Fiorino, Ferdinando; Budillon, Alfredo; Severino, Beatrice

    2016-09-26

    Protease activated receptor-1 (PAR1) is a G-coupled receptor activated by α-thrombin and other proteases. Several reports demonstrate PAR1 involvement in tumorigenesis and tumor progression. In order to investigate on potential use of PAR1 antagonists as antiproliferative agents, we have identified a series of arylpiperazine derivatives acting as PAR1 antagonists; the selected molecules have been evaluated for their antiproliferative properties. All the compounds inhibited the growth of a panel of cell lines expressing PAR1; two of them, compounds 13 and 15, were able to inhibit, in a dose dependent manner, the growth of the selected cell lines with the lowest IC50 values, and were further characterized to define the mechanism responsible for the observed antiproliferative effect. This study directed us to the identification of two interesting leads that may help to further validate PAR1 as an important therapeutic target for cancer treatment.

  17. Inhibition of human natural killer cell activity by Pseudomonas aeruginosa alkaline protease and elastase.

    PubMed Central

    Pedersen, B K; Kharazmi, A

    1987-01-01

    The present study was designed to examine the effect of Pseudomonas aeruginosa alkaline protease (AP) and elastase (Ela) on human natural killer (NK) cell activity in vitro. AP and Ela were found to inhibit NK cell function. Addition of alpha interferon and interleukin-2 did not abolish this inhibition of NK cell activity. Adhesion of effector to target cells was studied in a single-cell agarose assay of monocyte-depleted NK-cell-enriched cell populations. AP and Ela were shown to inhibit effector/target cell conjugate formation. Furthermore, AP and Ela inhibited the binding of the monoclonal antibody Leu-11, which reacts with the Fc receptor of NK cells. The inhibition of NK cell binding to the target cell by P. aeruginosa proteases is most likely due to proteolytic cleavage of the surface receptors involved in the binding of the effector cell to the target cell. PMID:3030937

  18. Design, Synthesis and Biological Evaluation of a Library of Thiocarbazates and their Activity as Cysteine Protease Inhibitors

    PubMed Central

    Liu, Zhuqing; Myers, Michael C.; Shah, Parag P.; Beavers, Mary Pat; Benedetti, Phillip A.; Diamond, Scott L.

    2010-01-01

    Recently, we identified a novel class of potent cathepsin L inhibitors, characterized by a thiocarbazate warhead. Given the potential of these compounds to inhibit other cysteine proteases, we designed and synthesized a library of thiocarbazates containing diversity elements at three positions. Biological characterization of this library for activity against a panel proteases indicated a significant preference for members of the papain family of cysteine proteases over serine, metallo-, and certain classes of cysteine proteases, such as caspases. Several very potent inhibitors of Cathepsin L and S were identified. The SAR data was employed in docking studies in an effort to understand the structural elements required for Cathepsin S inhibition. This study provides the basis for the design of highly potent and selective inhibitors of the papain family of cysteine proteases. PMID:20438448

  19. The Role of Protease-Activated Receptors for the Development of Myocarditis: Possible Therapeutic Implications.

    PubMed

    Weithauser, Alice; Witkowski, Marco; Rauch, Ursula

    2016-01-01

    Protease-activated receptors (PARs) are a unique group of four G-protein coupled receptors. They are widely expressed within the cardiovascular system and the heart. PARs are activated via cleavage by serine proteases. In vitro and in vivo studies showed that the activation of PAR1 and PAR2 plays a crucial role in virus induced inflammatory diseases. The receptors enable cells to recognize pathogen-derived changes in the extracellular environment. An infection with Coxsackie-virus B3 (CVB3) can cause myocarditis. Recent studies have been shown that PAR1 signaling enhanced the antiviral innate immune response via interferon β (IFNβ) and thus limited the virus replication and cardiac damage. In contrast, PAR2 signaling decreased the antiviral innate immune response via IFNβ und thus increased the virus replication, which caused severe myocarditis. Along with CVB3 other viruses such as influenza A virus (IAV) and herpes simplex virus (HSV) can induce myocarditis. The role of PAR signaling in IAV infections is contrarily discussed. During HSV infections PARs facilitate the virus infection of the host cell. These studies show that PARs might be interesting drug targets for the treatment of virus infections and inflammatory heart diseases. First studies with PAR agonists, antagonists, and serine protease inhibitors have been conducted in mice. The inhibition of thrombin the main PAR1 activating protease decreased the IFNβ response and increased the virus replication in CVB3-induced myocarditis. This indicates that further studies with direct PAR agonists and antagonists are needed to determine whether PARs are useful drug targets for the therapy of virus-induced heart diseases.

  20. The Zinc-Dependent Protease Activity of the Botulinum Neurotoxins

    PubMed Central

    Lebeda, Frank J.; Cer, Regina Z.; Mudunuri, Uma; Stephens, Robert; Singh, Bal Ram; Adler, Michael

    2010-01-01

    The botulinum neurotoxins (BoNT, serotypes A-G) are some of the most toxic proteins known and are the causative agents of botulism. Following exposure, the neurotoxin binds and enters peripheral cholinergic nerve endings and specifically and selectively cleaves one or more SNARE proteins to produce flaccid paralysis. This review centers on the kinetics of the Zn-dependent proteolytic activities of these neurotoxins, and briefly describes inhibitors, activators and factors underlying persistence of toxin action. Some of the structural, enzymatic and inhibitor data that are discussed here are available at the botulinum neurotoxin resource, BotDB (http://botdb.abcc.ncifcrf.gov). PMID:22069621

  1. Synergism of Selective Tumor Vascular Thrombosis and Protease Activated Prodrug

    DTIC Science & Technology

    2008-05-01

    cells of the hair follicles , and cells of the reproductive and digestive tracts. Chemotherapy can also cause nausea, vomiting, 14 W81XWH-07-1-0389......selected sites promises reduced toxicity and enhanced efficacy. We propose to investigate and validate a bipartite drug delivery-activation system that

  2. A new autocatalytic activation mechanism for cysteine proteases revealed by Prevotella intermedia interpain A

    PubMed Central

    Mallorquí-Fernández, Noemí; Manandhar, Surya P.; Mallorquí-Fernández, Goretti; Usón, Isabel; Wawrzonek, Katarzyna; Kantyka, Tomasz; Solà, Maria; Thøgersen, Ida B.; Enghild, Jan J.; Potempa, Jan; Gomis-Rüth, F.Xavier

    2009-01-01

    Prevotella intermedia is a major periodontopathogen contributing to human gingivitis and periodontitis. Such pathogens release proteases as virulence factors that cause deterrence of host defences and tissue destruction. A new cysteine protease from the cysteine-histidine-dyad class, interpain A, was studied in its zymogenic and its self-processed mature form. The latter consists of a bivalved moiety made up by two subdomains. In the structure of a catalytic cysteine-to-alanine zymogen variant, the right subdomain interacts with an unusual prodomain, thus contributing to latency. Unlike the catalytic cysteine residue, already in its competent conformation in the zymogen, the catalytic histidine is swung out from its active conformation and trapped in a cage shaped by a backing helix, a zymogenic hairpin and a latency flap in the zymogen. Dramatic rearrangement of up to 20Å of these elements triggered by a tryptophan switch occurs during activation and accounts for a new activation mechanism for proteolytic enzymes. These findings can be extrapolated to related potentially pathogenic cysteine proteases such as Streprococcus pyogenes SpeB and Porphyromonas gingivalis periodontain. PMID:17993455

  3. Protein Degradation and Protease Activity During the Life Cycle of Blastocladiella emersonii

    PubMed Central

    Lodi, W. R.; Sonneborn, D. R.

    1974-01-01

    Analysis of protein degradation during the life cycle of Blastocladiella emersonii showed that (i) protein degradation is especially high during two phases of differentiation (sporulation, 12%/h and germination, 5%/h) in contrast with a much smaller degradation rate in the other phases (growth and zoospores, less than 1%/hr); (ii) protein degradation during germination in growth medium, as well as most of the germination process, is quantitatively unaffected by cycloheximide; (iii) a caseinolytic protease (pH optimum 5.5, apparent molecular weight 55,000 to 60,000) is present in extracts of zoospores and germinating cells; (iv) this protease activity is very low (perhaps absent) in extracts of late growth phase cells, but reappears during induced sporulation; (v) a different class of caseinolytic protease activity (pH optima 7 and 10; apparent molecular weight 25,000 to 30,000) is found in cellular extracts of late growth phase and early phases of sporulation; (vi) the latter class of enzyme activity is released into the medium during later phases of sporulation and is replaced in the cells by the former class. Speculations as to the roles of protein degradation in cell differentiation are discussed. PMID:4813892

  4. Fullerene Derivatives Strongly Inhibit HIV-1 Replication by Affecting Virus Maturation without Impairing Protease Activity

    PubMed Central

    Martinez, Zachary S.; Castro, Edison; Seong, Chang-Soo; Cerón, Maira R.

    2016-01-01

    Three compounds (1, 2, and 3) previously reported to inhibit HIV-1 replication and/or in vitro activity of reverse transcriptase were studied, but only fullerene derivatives 1 and 2 showed strong antiviral activity on the replication of HIV-1 in human CD4+ T cells. However, these compounds did not inhibit infection by single-round infection vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped viruses, indicating no effect on the early steps of the viral life cycle. In contrast, analysis of single-round infection VSV-G-pseudotyped HIV-1 produced in the presence of compound 1 or 2 showed a complete lack of infectivity in human CD4+ T cells, suggesting that the late stages of the HIV-1 life cycle were affected. Quantification of virion-associated viral RNA and p24 indicates that RNA packaging and viral production were unremarkable in these viruses. However, Gag and Gag-Pol processing was affected, as evidenced by immunoblot analysis with an anti-p24 antibody and the measurement of virion-associated reverse transcriptase activity, ratifying the effect of the fullerene derivatives on virion maturation of the HIV-1 life cycle. Surprisingly, fullerenes 1 and 2 did not inhibit HIV-1 protease in an in vitro assay at the doses that potently blocked viral infectivity, suggesting a protease-independent mechanism of action. Highlighting the potential therapeutic relevance of fullerene derivatives, these compounds block infection by HIV-1 resistant to protease and maturation inhibitors. PMID:27431232

  5. A new autocatalytic activation mechanism for cysteine proteases revealed by Prevotella intermedia interpain A.

    PubMed

    Mallorquí-Fernández, Noemí; Manandhar, Surya P; Mallorquí-Fernández, Goretti; Usón, Isabel; Wawrzonek, Katarzyna; Kantyka, Tomasz; Solà, Maria; Thøgersen, Ida B; Enghild, Jan J; Potempa, Jan; Gomis-Rüth, F Xavier

    2008-02-01

    Prevotella intermedia is a major periodontopathogen contributing to human gingivitis and periodontitis. Such pathogens release proteases as virulence factors that cause deterrence of host defenses and tissue destruction. A new cysteine protease from the cysteine-histidine-dyad class, interpain A, was studied in its zymogenic and self-processed mature forms. The latter consists of a bivalved moiety made up by two subdomains. In the structure of a catalytic cysteine-to-alanine zymogen variant, the right subdomain interacts with an unusual prodomain, thus contributing to latency. Unlike the catalytic cysteine residue, already in its competent conformation in the zymogen, the catalytic histidine is swung out from its active conformation and trapped in a cage shaped by a backing helix, a zymogenic hairpin, and a latency flap in the zymogen. Dramatic rearrangement of up to 20A of these elements triggered by a tryptophan switch occurs during activation and accounts for a new activation mechanism for proteolytic enzymes. These findings can be extrapolated to related potentially pathogenic cysteine proteases such as Streprococcus pyogenes SpeB and Porphyromonas gingivalis periodontain.

  6. Protease-activated receptor-2 (PAR2) in cardiovascular system.

    PubMed

    Bucci, Mariarosaria; Roviezzo, Fiorentina; Cirino, Giuseppe

    2005-10-01

    Vascular system is constituted by a complex and articulate network, e.g. arteries, arterioles, venules and veins, that requires a high degree of coordination between different elemental cell types. Proteinase-activated receptors (PARs) constitute a recent described family of 7-transmembrane G protein-coupled receptors that are activated by proteolysis. In recent years several evidence have been accumulated for an involvement of this receptor in the response to endothelial injury in vitro and in vivo experimental settings suggesting a role for PAR2 in the pathophysiology of cardiovascular system. This review will deal with the role of PAR2 receptor in the cardiovascular system analyzing both in vivo and in vitro published data. In particular this review will deal with the role of this receptor in vascular reactivity, ischemia/reperfusion injury, coronary atherosclerotic lesions and angiogenesis.

  7. Evaluation of trypanocidal activity of combinations of anti-sleeping sickness drugs with cysteine protease inhibitors.

    PubMed

    Steverding, Dietmar

    2015-01-01

    Chemotherapy of human African trypanosomiasis (HAT) is unsatisfactory because only a few drugs, with serious side effects and poor efficacy, are available. As drug combination regimes often achieve greater therapeutic efficacy than monotherapies, here the trypanocidal activity of the cysteine protease inhibitor K11777 in combination with current anti-HAT drugs using bloodstream forms of Trypanosoma brucei was investigated. Isobolographic analysis was used to determine the interaction between cysteine protease inhibitors (K11777, CA-074Me and CAA0225) and anti-HAT drugs (suramin, pentamidine, melarsoprol and eflornithine). Bloodstream forms of T. brucei were incubated in culture medium containing cysteine protease inhibitors or anti-HAT drugs alone or in combination at a 1:1 fixed-dose ratio. After 48 h incubation, live cells were counted, the 50% growth inhibition values determined and combination indices calculated. The general cytotoxicity of drug combinations was evaluated with human leukaemia HL-60 cells. Combinations of K11777 with suramin, pentamidine and melarsoprol showed antagonistic effects while with eflornithine a synergistic effect was observed. Whereas eflornithine antagonises with CA-074Me, an inhibitor inactivating the targeted TbCATL only under reducing conditions, it synergises with CAA0255, an inhibitor structurally related to CA-074Me which inactivates TbCATL independently of thiols. These findings indicate an essential role of thiols for the synergistic interaction between K11777 and eflornithine. Encouragingly, the K11777/eflornithine combination displayed higher trypanocidal than cytotoxic activity. The results of this study suggest that the combination of the cysteine protease inhibitor K11777 and eflornithine display promising synergistic trypanocidal activity that warrants further investigation of the drug combination as possible alternative treatment of HAT.

  8. Activity of purified hepatitis C virus protease NS3 on peptide substrates.

    PubMed Central

    Steinkühler, C; Urbani, A; Tomei, L; Biasiol, G; Sardana, M; Bianchi, E; Pessi, A; De Francesco, R

    1996-01-01

    The protease domain of the hepatitis C virus (HCV) protein NS3 was expressed in Escherichia coli, purified to homogeneity, and shown to be active on peptides derived from the sequence of the NS4A-NS4B junction. Experiments were carried out to optimize protease activity. Buffer requirements included the presence of detergent, glycerol, and dithiothreitol, pH between 7.5 and 8.5, and low ionic strength. C- and N-terminal deletion experiments defined a peptide spanning from the P6 to the P4' residue as a suitable substrate. Cleavage kinetics were subsequently measured by using decamer P6-P4' peptides corresponding to all intermolecular cleavage sites of the HCV polyprotein. The following order of cleavage efficiency, in terms of kcat/Km, was determined: NS5A-NS5B > NS4A-NS4B >> NS4B-NS5A. A 14-mer peptide containing residues 21 to 34 of the protease cofactor NS4A (Pep4A 21-34), when added in stoichiometric amounts, was shown to increase cleavage rates of all peptides, the largest effect (100-fold) being observed on the hydrolysis of the NS4B-NS5A decamer. From the kinetic analysis of cleavage data, we conclude that (i) primary structure is an important determinant of the efficiency with which each site is cleaved during polyprotein processing, (ii) slow cleavage of the NS4B-NS5A site in the absence of NS4A is due to low binding affinity of the enzyme for this site, and (iii) formation of a 1:1 complex between the protease and Pep4A 21-34 is sufficient and required for maximum activation. PMID:8794305

  9. An 11-kDa form of human immunodeficiency virus protease expressed in Escherichia coli is sufficient for enzymatic activity.

    PubMed Central

    Graves, M C; Lim, J J; Heimer, E P; Kramer, R A

    1988-01-01

    In order to define the protease domain of human immunodeficiency virus 1, various regions of the pol open reading frame were cloned and expressed in Escherichia coli. Antiserum directed against the conserved retroviral protease active site was used to identify pol precursor and processed species containing the presumed protease domain. The smallest product that accumulates is about 11 kDa as measured by NaDodSO4/PAGE. This size agrees with that predicted from the presence in this region of two Phe-Pro sequences, which is one of the cleavage sites recognized by HIV protease. DNA encoding only the predicted 11-kDa protein was cloned, bypassing the need for autoprocessing, and the protein was expressed to a high level in E. coli. This form is active as demonstrated by its ability to specifically cleave protease-deficient pol protein in vivo in E. coli. Extracts of E. coli containing the 11-kDa protease also process human immunodeficiency virus gag substrates in vitro. These results demonstrate that the 11-kDa protease is sufficient for enzymatic activity and are consistent with a major role for this form in virus maturation. Images PMID:3282230

  10. [Clinical and diagnostic importance of the evaluation of the Ig proteases activity in children with intestinal dysbacteriosis].

    PubMed

    Zinkevich, O D; Bondarenko, V M; Tiurin, Iu A; Safina, N A; Anokhin, V A

    2004-01-01

    The specific activity of serine, metal dependent and thiolic Ig proteases in the coprofiltrates of children with manifestations of intestinal dysbacteriosis was determined by the enzyme immunoassay. 56 children with pronounced symptoms of intestinal disorders (37 children aged up to 1 year and 19 children over 1 year) were examined. A group of 25 clinically healthy children was used as control. Simultaneously with protease activity of coprofiltrates, there was detected the level of Ig-degrading activity of the opportunistic bacteria islolates of different taxonomic groups from feces of children with dysbacteriosis of different severity (as determined by the classical bacteriological method). The evaluation of the Ig-proteolytic activity of fecal supernatants, associated with the presence of serine, metal-dependent and thiolic proteases in the intestine, as well as detection of such proteases in microbial isolates, seems to be highly important for the diagnosis of intestinal disorders in children and is recommended for screening of intestinal dysbacteriosis.

  11. Extended substrate specificity and first potent irreversible inhibitor/activity-based probe design for Zika virus NS2B-NS3 protease.

    PubMed

    Rut, Wioletta; Zhang, Linlin; Kasperkiewicz, Paulina; Poreba, Marcin; Hilgenfeld, Rolf; Drąg, Marcin

    2017-03-01

    Zika virus is spread by Aedes mosquitoes and is linked to acute neurological disorders, especially to microcephaly in newborn children and Guillan-Barré Syndrome. The NS2B-NS3 protease of this virus is responsible for polyprotein processing and therefore considered an attractive drug target. In this study, we have used the Hybrid Combinatorial Substrate Library (HyCoSuL) approach to determine the substrate specificity of ZIKV NS2B-NS3 protease in the P4-P1 positions using natural and a large spectrum of unnatural amino acids. Obtained data demonstrate a high level of specificity of the S3-S1 subsites, especially for basic amino acids. However, the S4 site exhibits a very broad preference toward natural and unnatural amino acids with selected D-amino acids being favored over L enantiomers. This information was used for the design of a very potent phosphonate inhibitor/activity-based probe of ZIKV NS2B-NS3 protease.

  12. Activation of the epithelial sodium channel (ENaC) by the alkaline protease from Pseudomonas aeruginosa.

    PubMed

    Butterworth, Michael B; Zhang, Liang; Heidrich, Elisa M; Myerburg, Michael M; Thibodeau, Patrick H

    2012-09-21

    Pseudomonas aeruginosa is an opportunistic pathogen that significantly contributes to the mortality of patients with cystic fibrosis. Chronic infection by Pseudomonas induces sustained immune and inflammatory responses and damage to the airway. The ability of Pseudomonas to resist host defenses is aided, in part, by secreted proteases, which act as virulence factors in multiple modes of infection. Recent studies suggest that misregulation of protease activity in the cystic fibrosis lung may alter fluid secretion and pathogen clearance by proteolytic activation of the epithelial sodium channel (ENaC). To evaluate the possibility that proteolytic activation of ENaC may contribute to the virulence of Pseudomonas, primary human bronchial epithelial cells were exposed to P. aeruginosa and ENaC function was assessed by short circuit current measurements. Apical treatment with a strain known to express high levels of alkaline protease (AP) resulted in an increase in basal ENaC current and a loss of trypsin-inducible ENaC current, consistent with sustained activation of ENaC. To further characterize this AP-induced ENaC activation, AP was purified, and its folding, activity, and ability to activate ENaC were assessed. AP folding was efficient under pH and calcium conditions thought to exist in the airway surface liquid of normal and cystic fibrosis (CF) lungs. Short circuit measurements of ENaC in polarized monolayers indicated that AP activated ENaC in immortalized cell lines as well as post-transplant, primary human bronchial epithelial cells from both CF and non-CF patients. This activation was mapped to the γ-subunit of ENaC. Based on these data, patho-mechanisms associated with AP in the CF lung are proposed wherein secretion of AP leads to decreased airway surface liquid volume and a corresponding decrease in mucocilliary clearance of pulmonary pathogens.

  13. ATP-dependent Lon protease controls tumor bioenergetics by reprogramming mitochondrial activity.

    PubMed

    Quirós, Pedro M; Español, Yaiza; Acín-Pérez, Rebeca; Rodríguez, Francisco; Bárcena, Clea; Watanabe, Kenta; Calvo, Enrique; Loureiro, Marta; Fernández-García, M Soledad; Fueyo, Antonio; Vázquez, Jesús; Enríquez, José Antonio; López-Otín, Carlos

    2014-07-24

    We generated mice deficient in Lon protease (LONP1), a major enzyme of the mitochondrial quality control machinery. Homozygous deletion of Lonp1 causes early embryonic lethality, whereas its haploinsufficiency protects against colorectal and skin tumors. Furthermore, LONP1 knockdown inhibits cellular proliferation and tumor and metastasis formation, whereas its overexpression increases tumorigenesis. Clinical studies indicate that high levels of LONP1 are a poor prognosis marker in human colorectal cancer and melanoma. Additionally, functional analyses show that LONP1 plays a key role in metabolic reprogramming by remodeling OXPHOS complexes and protecting against senescence. Our findings demonstrate the relevance of LONP1 for cellular and organismal viability and identify this protease as a central regulator of mitochondrial activity in oncogenesis.

  14. 7-hydroxycalamenene Effects on Secreted Aspartic Proteases Activity and Biofilm Formation of Candida spp.

    PubMed Central

    Azevedo, Mariana M. B.; Almeida, Catia A.; Chaves, Francisco C. M.; Rodrigues, Igor A.; Bizzo, Humberto R.; Alviano, Celuta S.; Alviano, Daniela S.

    2016-01-01

    Background: The 7-hydroxycalamenenene-rich essential oil (EO) obtained from the leaves of Croton cajucara (red morphotype) have been described as active against bacteria, protozoa, and fungi species. In this work, we aimed to evaluate the effectiveness of 7-hydroxycalamenenene against Candida albicans and nonalbicans species. Materials and Methods: C. cajucara EO was obtained by hydrodistillation and its major compound, 7-hydroxycalamenene, was purified using preparative column chromatography. The anti-candidal activity was investigated by minimum inhibitory concentration (MIC) and secreted aspartic proteases (SAP) and biofilm inhibition assays. Results: 7-hydroxycalamenene (98% purity) displayed anti-candidal activity against all Candida species tested. Higher activity was observed against Candida dubliniensis, Candida parapsilosis and Candida albicans, showing MIC values ranging from 39.06 μg/ml to 78.12 μg/ml. The purified 7-hydroxycalamenene was able to inhibit 58% of C. albicans ATCC 36801 SAP activity at MIC concentration (pH 7.0). However, 7-hydroxycalamenene demonstrated poor inhibitory activity on C. albicans ATCC 10231 biofilm formation even at the highest concentration tested (2500 μg/ml). Conclusion: The bioactive potential of 7-hydroxycalamenene against planktonic Candida spp. further supports its use for the development of antimicrobials with anti-candidal activity. SUMMARY Croton cajucara Benth. essential oil provides high amounts of 7-hydroxycalamenene7-Hydroxycalameneneisolated from C. cajucarais active against Candida spp7-Hydroxycalameneneinhibits C. albicans aspartic protease activity7-Hydroxycalamenene was not active against C. albicans biofilm formation. Figure PMID:27019560

  15. A biosensor for the protease TACE reveals actin damage induced TACE activation

    PubMed Central

    Chapnick, Douglas A.; Bunker, Eric; Liu, Xuedong

    2016-01-01

    Ligand shedding has gained increased attention as a major posttranslational modification mechanism used by cells to respond to diverse environmental conditions. The TACEadam17 protease is a critical mediator of such ligand shedding, regulating the maturation and release of an impressive range of extracellular substrates that drive diverse cellular responses. Exactly how this protease is itself activated remains unclear, in part due to the lack of available tools to measure TACE activity with temporal and spatial resolution in live cells. We have developed a FRET based biosensor for TACE activity (TSen), which is capable of reporting TACE activation kinetics in live cells with a high degree of specificity. TSen was used in combination with chemical biology to probe the dependence of various means of TACE activation on p38 and Erk kinase activities, as well as to identify a novel connection between actin cytoskeletal disruption and TACE activation. Such cytoskeletal disruption leads to rapid and robust TACE activation in some cell types and accumulation of TACE at the plasma membrane, allowing for increased cleavage of endogenous substrates. Our study highlights both the versatility of TSen as a tool to understand the mechanisms of TACE activation in live cells and the importance of actin cytoskeletal integrity as a modulator of TACE activity. PMID:25714465

  16. Cysteine protease activity of feline Tritrichomonas foetus promotes adhesion-dependent cytotoxicity to intestinal epithelial cells.

    PubMed

    Tolbert, M K; Stauffer, S H; Brand, M D; Gookin, J L

    2014-07-01

    Trichomonads are obligate protozoan parasites most renowned as venereal pathogens of the reproductive tract of humans and cattle. Recently, a trichomonad highly similar to bovine venereal Tritrichomonas foetus but having a unique tropism for the intestinal tract was recognized as a significant cause of colitis in domestic cats. Despite a high prevalence, worldwide distribution, and lack of consistently effective drugs for treatment of the infection, the cellular mechanisms of T. foetus pathogenicity in the intestinal tract have not been examined. The aims of this study were to determine the pathogenic effect of feline T. foetus on porcine intestinal epithelial cells, the dependence of T. foetus pathogenicity on adhesion of T. foetus to the intestinal epithelium, and the identity of mediators responsible for these effects. Using an in vitro coculture approach to model feline T. foetus infection of the intestinal epithelium, these studies demonstrate that T. foetus promotes a direct contact-dependent activation of intestinal epithelial cell apoptosis signaling and progressive monolayer destruction. Moreover, these pathological effects were demonstrated to be largely dependent on T. foetus cell-associated cysteine protease activity. Finally, T. foetus cysteine proteases were identified as enabling cytopathic effects by promoting adhesion of T. foetus to the intestinal epithelium. The present studies are the first to examine the cellular mechanisms of pathogenicity of T. foetus toward the intestinal epithelium and support further investigation of the cysteine proteases as virulence factors in vivo and as potential therapeutic targets for ameliorating the pathological effects of intestinal trichomonosis.

  17. Expression, purification and molecular modeling of the NIa protease of Cardamom mosaic virus.

    PubMed

    Jebasingh, T; Pandaranayaka, Eswari P J; Mahalakshmi, A; Kasin Yadunandam, A; Krishnaswamy, S; Usha, R

    2013-01-01

    The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8 M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease.

  18. The crystal structure of the cysteine protease Xylellain from Xylella fastidiosa reveals an intriguing activation mechanism.

    PubMed

    Leite, Ney Ribeiro; Faro, Aline Regis; Dotta, Maria Amélia Oliva; Faim, Livia Maria; Gianotti, Andreia; Silva, Flavio Henrique; Oliva, Glaucius; Thiemann, Otavio Henrique

    2013-02-14

    Xylella fastidiosa is responsible for a wide range of economically important plant diseases. We report here the crystal structure and kinetic data of Xylellain, the first cysteine protease characterized from the genome of the pathogenic X. fastidiosa strain 9a5c. Xylellain has a papain-family fold, and part of the N-terminal sequence blocks the enzyme active site, thereby mediating protein activity. One novel feature identified in the structure is the presence of a ribonucleotide bound outside the active site. We show that this ribonucleotide plays an important regulatory role in Xylellain enzyme kinetics, possibly functioning as a physiological mediator.

  19. β-Amyloid induces nuclear protease-mediated lamin fragmentation independent of caspase activation.

    PubMed

    Ramasamy, Vijay Sankar; Islam, Md Imamul; Haque, Md Aminul; Shin, Song Yub; Park, Il-Seon

    2016-06-01

    β-Amyloid (Aβ), a hallmark peptide of Alzheimer's disease, induces both caspase-dependent apoptosis and non-apoptotic cell death. In this study, we examined caspase-independent non-apoptotic cell death preceding caspase activation in Aβ42-treated cells. We first determined the optimal treatment conditions for inducing cell death without caspase activation and selected a double-treatment method involving the incubation of cells with Aβ42 for 4 and 6 h (4+6 h sample). We observed that levels of lamin A (LA) and lamin B (LB) were reduced in the 4+6 h samples. This reduction was decreased by treatment with suc-AAPF-CMK, an inhibitor of nuclear scaffold (NS) protease, but not by treatment with z-VAD-FMK, a pan-caspase inhibitor. In addition, suc-AAPF-CMK decreased the changes in nuclear morphology observed in cells in the 4+6 h samples, which were different from nuclear fragmentation observed in STS-treated cells. Furthermore, suc-AAPF-CMK inhibited cell death in the 4+6 h samples. LA and LB fragmentation occurred in the isolated nuclei and was also inhibited by suc-AAPF-CMK. Together, these data indicated that the fragmentation of LA and LB in the Aβ42-treated cells was induced by an NS protease, whose identity is not clearly determined yet. A correlation between Aβ42 toxicity and the lamin fragmentation by NS protease suggests that inhibition of the protease could be an effective method for controlling the pathological process of AD.

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

    PubMed

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

    2015-09-01

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

  1. Inhibitory Activity of Human Immunodeficiency Virus Aspartyl Protease Inhibitors against Encephalitozoon intestinalis Evaluated by Cell Culture-Quantitative PCR Assay

    PubMed Central

    Menotti, Jean; Santillana-Hayat, Maud; Cassinat, Bruno; Sarfati, Claudine; Derouin, Francis; Molina, Jean-Michel

    2005-01-01

    Immune reconstitution might not be the only factor contributing to the low prevalence of microsporidiosis in human immunodeficiency virus (HIV)-infected patients treated with protease inhibitors, as these drugs may exert a direct inhibitory effect against fungi and protozoa. In this study, we developed a cell culture-quantitative PCR assay to quantify Encephalitozoon intestinalis growth in U-373-MG human glioblastoma cells and used this assay to evaluate the activities of six HIV aspartyl protease inhibitors against E. intestinalis. A real-time quantitative PCR assay targeted the E. intestinalis small-subunit rRNA gene. HIV aspartyl protease inhibitors were tested over serial concentrations ranging from 0.2 to 10 mg/liter, with albendazole used as a control. Ritonavir, lopinavir, and saquinavir were able to inhibit E. intestinalis growth, with 50% inhibitory concentrations of 1.5, 2.2, and 4.6 mg/liter, respectively, whereas amprenavir, indinavir, and nelfinavir had no inhibitory effect. Pepstatin A, a reference aspartyl protease inhibitor, could also inhibit E. intestinalis growth, suggesting that HIV protease inhibitors may act through the inhibition of an E. intestinalis-encoded aspartyl protease. These results showed that some HIV protease inhibitors can inhibit E. intestinalis growth at concentrations that are achievable in vivo and that the real-time quantitative PCR assay that we used is a valuable tool for the in vitro assessment of the activities of drugs against E. intestinalis. PMID:15917534

  2. Determination of activable proacrosin/acrosin in bovine sperm using an irreversible isocoumarin serine protease inhibitor.

    PubMed

    Palencia, D D; Garner, D L; Hudig, D; Holcombe, D W; Burner, C A; Redelman, D; Fernandez, G C; Abuelyaman, A S; Kam, C M; Powers, J C

    1996-09-01

    The activable proacrosin/acrosin levels in bovine sperm were examined using fluorescent staining and flow cytometry. The proportion of sperm with active acrosin were determined using the biotinylated isocoumarin serine protease inhibitor, Bi-Aca-Aca-OMe-IC (BIC). The presence of bound inhibitor on sperm was then determined by secondary labeling with avidin fluorescein conjugate. The proportion of sperm with activable proacrosin/acrosin was assessed by using detergent treatment to expose the active acrosin in intact sperm. The difference between untreated and detergent-treated aliquots was used to estimate the proportion of sperm with activable proacrosin/acrosin. In the 24-h stored samples from six bulls, the mean proportion of sperm with activable proacrosin/acrosin was 78.8 +/- 2.8%, whereas the mean proportion with exposed acrosin after cryopreservation of these samples was 55.8 +/- 4.1%. Significant differences (p < 0.05) were found among bulls in the proportion of sperm with activable proacrosin/acrosin both before and after cryopreservation. Activable proacrosin/acrosin levels in samples of cryopreserved sperm from five bulls were not correlated with fertility. These results do indicate, however, that the irreversible isocoumarin serine protease inhibitor BIC can be used to determine the proportion of sperm cells that retain activable proacrosin/acrosin after cryopreservation and thawing.

  3. Effects of tricyclic compounds on membrane binding of bivalent cations, activities of acetylcholinesterase and some tissue proteases.

    PubMed

    Molnar, J; Sohar, I; Kovacs, J; Rakonczay, Z; Rausch, H

    1993-01-01

    A tricyclic compound tetrahydroaminoacridine is known to improve the cognitive function in Alzheimer's disease. The possible mechanism of action of acridine and structurally related tricyclic compounds was studied on the bivalent cation content of bacterial membrane, rat brain acetylcholinesterase and some tissue proteases in model experiments. Acridine orange and disubstituted chlorpromazine (CPZ) derivatives lowered Ca2+ and Mg2+ binding and membrane polarization in the simplest biological membrane (E. coli), as revealed by reactor neutron activation analysis. Acetylcholinesterase (AChE) was inhibited by CPZ, 3,7,8-trihydroxy-CPZ, acridine orange partially saturated desipramine, imipramine, trans-clopenthixol and tetrahydrocannabidiolic at 10(-4) to 10(-5). A metalloproteinase, MMP-7-ase, was inhibited by tetrahydrocannabidiolic acid, 3,7,8-trihydroxy-CPZ, acridine orange but other tissue proteinases, ATN-ase and cathepsin B, were less sensitive to these compounds. (ATN-ase is an acetyltyrosine-p-nitroanilide splitting enzyme, a serine protease). The chelate complex forming ability and electron donor capacity of the compounds may play a role in the biological effects tested. It is assumed that compounds which do not displace bivalent cations in membranes may exert an inhibitory effect on AChE, and that metalloproteinase enzymes may be promising for the treatment of degenerative brain diseases.

  4. Effect of sulfur mustard exposure on protease activity in human peripheral blood lymphocytes. (Reannouncement with new availability information)

    SciTech Connect

    Cowan, F.M.; Broomfield, C.A.; Smith, W.J.

    1991-12-31

    Sulfur mustard is a chemical warfare blistering agent for which neither the mechanism of action nor an antidote is known. Papirmeister et al. (1985) have postulated a biochemical hypothesis for mustard-induced cutaneous injury involving a sequelae of DNA alkylation, metabolic disruption and activation of protease. Human peripheral blood lymphocytes in cell cultures were employed as an in vitro model for alkylating agent toxicity. A chromogenic peptide substrate assay was used for detection of protease in lymphocytes treated with sulfur mustard or chloroethyl sulfide. Exposure of human peripheral blood lymphocytes from normal donors to these alkylating agents resulted in an increase in cell associated protease activity. This increase in protease activity may contribute to the pathology or act as an indicator to predict methods of therapeutic intervention for sulfur mustard toxicity.

  5. Activation of Protease Activated Receptor 2 by Exogenous Agonist Exacerbates Early Radiation Injury in Rat Intestine

    SciTech Connect

    Wang Junru; Boerma, Marjan; Kulkarni, Ashwini; Hollenberg, Morley D.; Hauer-Jensen, Martin

    2010-07-15

    Purpose: Protease-activated receptor-2 (PAR{sub 2}) is highly expressed throughout the gut and regulates the inflammatory, mitogenic, fibroproliferative, and nociceptive responses to injury. PAR{sub 2} is strikingly upregulated and exhibits increased activation in response to intestinal irradiation. We examined the mechanistic significance of radiation enteropathy development by assessing the effect of exogenous PAR{sub 2} activation. Methods and Materials: Rat small bowel was exposed to localized single-dose radiation (16.5 Gy). The PAR{sub 2} agonist (2-furoyl-LIGRLO-NH{sub 2}) or vehicle was injected intraperitoneally daily for 3 days before irradiation (before), for 7 days after irradiation (after), or both 3 days before and 7 days after irradiation (before-after). Early and delayed radiation enteropathy was assessed at 2 and 26 weeks after irradiation using quantitative histologic examination, morphometry, and immunohistochemical analysis. Results: The PAR{sub 2} agonist did not elicit changes in the unirradiated (shielded) intestine. In contrast, in the irradiated intestine procured 2 weeks after irradiation, administration of the PAR{sub 2} agonist was associated with more severe mucosal injury and increased intestinal wall thickness in all three treatment groups (p <.05) compared with the vehicle-treated controls. The PAR{sub 2} agonist also exacerbated the radiation injury score, serosal thickening, and mucosal inflammation (p <.05) in the before and before-after groups. The short-term exogenous activation of PAR{sub 2} did not affect radiation-induced intestinal injury at 26 weeks. Conclusion: The results of the present study support a role for PAR{sub 2} activation in the pathogenesis of early radiation-induced intestinal injury. Pharmacologic PAR{sub 2} antagonists might have the potential to reduce the intestinal side effects of radiotherapy and/or as countermeasures in radiologic accidents or terrorism scenarios.

  6. Intraspecies differences in hemostatic venom activities of the South American rattlesnakes, Crotalus durissus cumanensis, as revealed by a range of protease inhibitors.

    PubMed

    Salazar, Ana M; Aguilar, Irma; Guerrero, Belsy; Girón, María E; Lucena, Sara; Sánchez, Elda E; Rodríguez-Acosta, Alexis

    2008-09-01

    Crotalus durissus cumanensis is an endemic rattlesnake found in Venezuela and Colombia. In this study, a comparative analysis of hemorrhagic, coagulation and fibrino(geno)lytic activities in the presence or absence of protease inhibitors was performed with venoms of the same species Crotalus durissus cumanensis, from seven geographical regions of Venezuela (Lagunetica, Santa Teresa, Carrizales, Guarenas, Anzoátegui, Margarita and Maracay). Lagunetica, Carrizales and Anzoátegui venoms induced hemorrhagic activity. All venoms, except that of snakes from the Carrizales region presented thrombin-like activity, which was inhibited completely by phenylmethanesulfonyl fluoride and ethylene glycol-bis-N, N,N',N'-tetraacetic acid. This effect of the latter could be explained by the high chelant calcium effect, which is a cofactor for the fibrin polymerization process. Soybean trypsin inhibitor was effective on Santa Teresa venom. Antithrombin III/Hep complex and phenantroline partially inhibited this activity in all venoms except Margarita and Anzoátegui, respectively, which were not inhibited. Serine protease inhibitors were more effective against thrombin, kallikrein and plasmin-like amidolytic activities. Additionally, metalloprotease inhibitors significantly inhibited the t-PA-like amidolytic activity and completely the hemorrhagic and fibrino(geno)lytic activities. In conclusion, the thrombin-like activity observed in these venoms was partially reduced by serine protease inhibitors, indicating the possible presence of catalytic domains in those enzymes that do not interact with these inhibitors. Using protease inhibitors on venom hemostatic activities could contribute to our understanding of the active components of snake venom on the hemostatic system, and further reveal the intraspecies variation of venoms, which is important in the treatment of envenomation, and in addition represents an interesting model for the study of venom in hemostasis.

  7. Secreted fungal aspartic proteases: A review.

    PubMed

    Mandujano-González, Virginia; Villa-Tanaca, Lourdes; Anducho-Reyes, Miguel Angel; Mercado-Flores, Yuridia

    2016-01-01

    The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application.

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

    PubMed

    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 (<12 kDa) for Albizia lebbeck, Ipomoea asarifolia, Jatropha curcas, Libidibia ferrea, Moringa oleifera and Ricinus communis (P<0.05, Bonferroni test). The two fractions of Crotalaria spectabilis showed the same ovicidal activity (P>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.

  9. Role of protease-activated receptors 2 (PAR2) in ocular infections and inflammation

    PubMed Central

    Tripathi, Trivendra; Alizadeh, Hassan

    2015-01-01

    Protease-activated receptors (PARs) belong to a unique family of G protein-coupled receptors (GPCRs) that are cleaved at an activation site within the N-terminal exodomain by a variety of proteinases, essentially of the serine (Ser) proteinase family. After cleavage, the new N-terminal sequence functions as a tethered ligand, which binds intramolecularly to activate the receptor and initiate signaling. Cell signals induced through the activation of PARs appear to play a significant role in innate and adoptive immune responses of the cornea, which is constantly exposed to proteinases under physiological or pathophysiological conditions. Activation of PARs interferes with all aspects of the corneal physiology such as barrier function, transports, innate and adoptive immune responses, and functions of corneal nerves. It is not known whether the proteinase released from the microorganism can activate PARs and triggers the inflammatory responses. The role of PAR2 expressed by the corneal epithelial cells and activation by serine protease released from microorganism is discussed here. Recent evidences suggest that activation of PAR2, by the serine proteinases, play an important role in innate and inflammatory responses of the corneal infection. PMID:26078987

  10. Assessing the impact of long term frozen storage of faecal samples on protein concentration and protease activity

    PubMed Central

    Morris, Laura S.; Marchesi, Julian R.

    2016-01-01

    Background The proteome is the second axis of the microbiome:host interactome and proteases are a significant aspect in this interaction. They interact with a large variety of host proteins and structures and in many situations are implicated in pathogenesis. Furthermore faecal samples are commonly collected and stored frozen so they can be analysed at a later date. So we were interested to know whether long term storage affected the integrity of proteases and total protein and whether historical native faecal samples were still a viable option for answering research questions around the functional proteome. Methods Faecal samples were collected from 3 healthy volunteers (3 biological replicates) and processed in order to be stored at both − 20 °C and − 80 °C and in a variety of storage buffers. Protein extraction, protein content and protease activity were assessed at the time of collection, after 24 h, 1 week, 1 month, 3 months 6 months and finally 1 year. Results Beadbeating impacted the quantity of protein extracted, while sodium azide did not impact protease assays. Long term storage of extracted proteins showed that both total protein and protease activity were affected when they were stored as extracted protein. Intact faecal samples were shown to maintain both protein levels and protease activity regardless of time and temperature. Conclusions Beadbeating increases the protein and protease activity when extracting from a faecal sample, however, the extracted protein is not stable and activity is lost, even with a suitable storage buffer. The most robust solution is to store the proteins in an intact frozen native faecal matrix and extract at the time of assay or analysis, this approach was shown to be suitable for samples in which, there are low levels of protease activity and which had been frozen for a year. PMID:26853125

  11. Transcriptional activation by heat and cold of a thiol protease gene in tomato. [Lycopersicon esculentum

    SciTech Connect

    Schaffer, M.A.; Fischer, R.L. )

    1990-08-01

    We previously determined that low temperature induces the accumulation in tomato (Lycopersicon esculentum) fruit of a cloned mRNA, designated C14, encoding a polypeptide related to thiol proteases. We now demonstrate that C14 mRNA accumulation is a response common to both high (40{degree}C) and low (4{degree}C) temperature stresses. Exposure of tomato fruit to 40{degree}C results in the accumulation of C14 mRNA, by 8 hours. This response is more rapid than that to 4{degree}C, but slower than the induction of many heat shock messages by 40{degree}C, and therefore unique. We have also studied the mechanism by which heat and cold exposure activate C14 gene expression. Both high and low temperature regulate protease gene expression through transcriptional induction of a single C14 gene. A hypothesis for the function of C14 thiol protease gene expression in response to heat and cold is discussed.

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

    PubMed Central

    Cotârleţ, Mihaela; Negoiţă, Teodor Gh.; Bahrim, Gabriela E.; Stougaard, Peter

    2011-01-01

    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 20°C, 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 20°C. 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. PMID:24031702

  13. Antiviral activities of peptide-based covalent inhibitors of the Enterovirus 71 3C protease

    PubMed Central

    Tan, Yong Wah; Ang, Melgious Jin Yan; Lau, Qiu Ying; Poulsen, Anders; Ng, Fui Mee; Then, Siew Wen; Peng, Jianhe; Hill, Jeffrey; Hong, Wan Jin; Chia, Cheng San Brian; Chu, Justin Jang Hann

    2016-01-01

    Hand, Foot and Mouth Disease is a highly contagious disease caused by a range of human enteroviruses. Outbreaks occur regularly, especially in the Asia-Pacific region, putting a burden on public healthcare systems. Currently, there is no antiviral for treating this infectious disease and the only vaccines are limited to circulation in China, presenting an unmet medical need that needs to be filled urgently. The human enterovirus 3 C protease has been deemed a plausible drug target due to its essential roles in viral replication. In this study, we designed and synthesized 10 analogues of the Rhinovirus 3 C protease inhibitor, Rupintrivir, and tested their 3 C protease inhibitory activities followed by a cellular assay using human enterovirus 71 (EV71)-infected human RD cells. Our results revealed that a peptide-based compound containing a trifluoromethyl moiety to be the most potent analogue, with an EC50 of 65 nM, suggesting its potential as a lead for antiviral drug discovery. PMID:27645381

  14. Structural Insights into the Activation and Inhibition of Histo-Aspartic Protease from Plasmodium falciparum

    SciTech Connect

    Bhaumik, Prasenjit; Xiao, Huogen; Hidaka, Koushi; Gustchina, Alla; Kiso, Yoshiaki; Yada, Rickey Y.; Wlodawer, Alexander

    2012-09-17

    Histo-aspartic protease (HAP) from Plasmodium falciparum is a promising target for the development of novel antimalarial drugs. The sequence of HAP is highly similar to those of pepsin-like aspartic proteases, but one of the two catalytic aspartates, Asp32, is replaced with histidine. Crystal structures of the truncated zymogen of HAP and of the complex of the mature enzyme with inhibitor KNI-10395 have been determined at 2.1 and 2.5 {angstrom} resolution, respectively. As in other proplasmepsins, the propeptide of the zymogen interacts with the C-terminal domain of the enzyme, forcing the N- and C-terminal domains apart, thereby separating His32 and Asp215 and preventing formation of the mature active site. In the inhibitor complex, the enzyme forms a tight domain-swapped dimer, not previously seen in any aspartic proteases. The inhibitor is found in an unprecedented conformation resembling the letter U, stabilized by two intramolecular hydrogen bonds. Surprisingly, the location and conformation of the inhibitor are similar to those of the fragment of helix 2 comprising residues 34p-38p in the prosegments of the zymogens of gastric aspartic proteases; a corresponding helix assumes a vastly different orientation in proplasmepsins. Each inhibitor molecule is in contact with two molecules of HAP, interacting with the carboxylate group of the catalytic Asp215 of one HAP protomer through a water molecule, while also making a direct hydrogen bond to Glu278A' of the other protomer. A comparison of the shifts in the positions of the catalytic residues in the inhibitor complex presented here with those published previously gives further hints regarding the enzymatic mechanism of HAP.

  15. Transfecting deoxyribonucleic acid of Bacillus bacteriophage phi 29 that is protease sensitive.

    PubMed

    Hirokawa, H

    1972-06-01

    The transfecting activity of Bacillus phage varphi29 DNA, extracted either by sodium lauroyl sarcosine-phenol or by 2 M perchlorate, was destroyed by treatment with proteolytic enzymes, although these enzymes did not effect transfecting DNAs of SPP1, SPO1, and SP50. These facts suggest that a protein is associated with transfective varphi29 DNA. Stabilization of protease-resistance during transfection appeared earlier than that of DNaseresistance, indicating that the protein associated with varphi29 DNA is necessary for initiation of the incorporation of DNA molecules into competent cells. The physical nature of varphi29 DNA before and after the trypsin treatment was investigated by sucrose and CsCl density gradient centrifugations. The trypsin treatment did not alter the sedimentation rate of the unit varphi29 DNA; however, it did convert the sedimentation rate of the aggregated material in the untreated DNA to that of the unit varphi29 DNA. The density of the trypsinized DNA was 0.009 g/cm(3) greater than that of the untreated DNA. The possible location of the protein on the DNA is discussed.

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

    PubMed

    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

  17. Cytomegalovirus protease targeted prodrug development.

    PubMed

    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.

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

    PubMed

    Yonamine, Camila M; Kondo, Marcia Y; Nering, Marcela B; Gouvêa, Iuri E; Okamoto, Débora; Andrade, Douglas; da Silva, José Alberto A; Prieto da Silva, Alvaro R B; Yamane, Tetsuo; Juliano, Maria A; Juliano, Luiz; Lapa, Antônio J; Hayashi, Mirian A F; Lima-Landman, Maria Teresa R

    2014-03-01

    Gyroxin is a serine protease displaying a thrombin-like activity found in the venom of the South American rattlesnake Crotalus durissus terrificus. Typically, intravenous injection of purified gyroxin induces a barrel rotation syndrome in mice. The serine protease thrombin activates platelets aggregation by cleaving and releasing a tethered N-terminus peptide from the G-protein-coupled receptors, known as protease-activated receptors (PARs). Gyroxin also presents pro-coagulant activity suggested to be dependent of PARs activation. In the present work, the effects of these serine proteases, namely gyroxin and thrombin, on PARs were comparatively studied by characterizing the hydrolytic specificity and kinetics using PARs-mimetic FRET peptides. We show for the first time that the short (sh) and long (lg) peptides mimetizing the PAR-1, -2, -3, and -4 activation sites are all hydrolyzed by gyroxin exclusively after the Arg residues. Thrombin also hydrolyzes PAR-1 and -4 after the Arg residue, but hydrolyzes sh and lg PAR-3 after the Lys residue. The kcat/KM values determined for gyroxin using sh and lg PAR-4 mimetic peptides were at least 2150 and 400 times smaller than those determined for thrombin, respectively. For the sh and lg PAR-2 mimetic peptides the kcat/KM values determined for gyroxin were at least 6500 and 2919 times smaller than those determined for trypsin, respectively. The kcat/KM values for gyroxin using the PAR-1 and -3 mimetic peptides could not be determined due to the extreme low hydrolysis velocity. Moreover, the functional studies of the effects of gyroxin on PARs were conducted in living cells using cultured astrocytes, which express all PARs. Despite the ability to cleavage the PAR-1, -2, -3, and -4 peptides, gyroxin was unable to activate the PARs expressed in astrocytes as determined by evaluating the cytosolic calcium mobilization. On the other hand, we also showed that gyroxin is able to interfere with the activation of PAR-1 by thrombin or

  19. 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.

    PubMed

    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.

  20. The effects of bioprocess parameters on extracellular proteases in a recombinant Aspergillus niger B1-D.

    PubMed

    Li, Qiang; Harvey, Linda M; McNeil, Brian

    2008-02-01

    Although host proteases are often considered to have a negative impact upon heterologous protein production by filamentous fungi, relatively little is known about the pattern of their appearance in recombinant fungal bioprocesses. In the present study, we investigated extracellular proteases from a filamentous fungus, Aspergillus niger B1-D, genetically modified to secrete hen egg white lysozyme (HEWL). Our findings indicate that extracellular protease activity is only detected after the carbon source is completely utilised in batch cultures. The proteases are predominantly acid proteases and have optimal temperature for activity at around 45 degrees C. Their activity could be partially inhibited by protease inhibitors, indicating the existence of at least four kinds of proteases in these culture fluids, aspartic-, serine-, cysteine-, and metallo-proteases. Oxygen enrichment does not have any noticeable effects on extracellular protease activity except that the onset of protease activity appears earlier in oxygen enrichment runs. Oxygen enrichment stimulates HEWL production substantially, and we propose that it is related to fungal morphology. Thermal stress imposed by raising process temperature (from 25 to 30 and 35 degrees C) in early exponential phase, led to appearance of protease activity in the medium following the heat shock. Continued cultivation at high temperatures significantly reduced HEWL production, which was associated with increased activity of the extracellular proteases in these cultures.

  1. Synthesis and characterization of a new fluorogenic active-site titrant of serine proteases.

    PubMed

    Livingston, D C; Brocklehurst, J R; Cannon, J F; Leytus, S P; Wehrly, J A; Peltz, S W; Peltz, G A; Mangel, W F

    1981-07-21

    The molecule 3',6'-bis(4-guanidinobenzoyloxy)-5-[N'-(4-carboxyphenyl)thioureido[spirop]isobenzofuran-1-(3H),9'-[9H]xanthen]-3-one, abbreviated FDE, was designed and synthesized as a fluorogenic active-site titrant for serine proteases. It is an analogue of p-nitrophenyl p-guanidino-benzoate (NPGB) in which a fluorescein derivative is substituted for p-nitrophenol. FDE and NPGB exhibit similar kinetic characteristics in an active-site titration of trypsin in phosphate-buffered saline, pH 7.2. The rate of acylation with FDE is extremely fast (k2 = 1.05 s-1) and the rate of deacylation extremely slow (k3 = 1.66 X 10(-5) s-1). The Ks is 3.06 X 10(-6) M, and the Km(app) is 4.85 X 10(-11) M. With two of the serine proteases involved in fibrinolysis, the rate of acylation with FDE is also fast, K2 = 0.112 s-1 for urokinase and 0.799 s-1 for plasmin, and the rate of deacylation is slow, k3 = 3.64 X 10(-4) s-1 for urokinase and 6.27 X 10(-6) s-1 for plasmin. The solubility limit of FDE in phosphate-buffered saline is 1.3 X 10(-5) M, and the first-order rate constant for spontaneous hydrolysis is 5.1 X 10(-6) s-1. The major difference between FDE and NPGB is the detectability of the product in an active-site titration. p-Nitrophenol can be detected at concentrations no lower than 10(-6) M whereas fluorescein can be detected at concentrations as low as 10(-12) M. Thus, FDE should be useful in quantitatively assaying serine proteases as very low concentrations.

  2. Protoporphyrins Enhance Oligomerization and Enzymatic Activity of HtrA1 Serine Protease

    PubMed Central

    Jo, Hakryul; Patterson, Victoria; Stoessel, Sean; Kuan, Chia-Yi; Hoh, Josephine

    2014-01-01

    High temperature requirement protein A1 (HtrA1), a secreted serine protease of the HtrA family, is associated with a multitude of human diseases. However, the exact functions of HtrA1 in these diseases remain poorly understood. We seek to unravel the mechanisms of HtrA1 by elucidating its interactions with chemical or biological modulators. To this end, we screened a small molecule library of 500 bioactive compounds to identify those that alter the formation of extracellular HtrA1 complexes in the cell culture medium. An initial characterization of two novel hits from this screen showed that protoporphyrin IX (PPP-IX), a precursor in the heme biosynthetic pathway, and its metalloporphyrin (MPP) derivatives fostered the oligomerization of HtrA1 by binding to the protease domain. As a result of the interaction with MPPs, the proteolytic activity of HtrA1 against Fibulin-5, a specific HtrA1 substrate in age-related macular degeneration (AMD), was increased. This physical interaction could be abolished by the missense mutations of HtrA1 found in patients with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Furthermore, knockdown of HtrA1 attenuated apoptosis induced by PPP-IX. These results suggest that PPP-IX, or its derivatives, and HtrA1 may function as co-factors whereby porphyrins enhance oligomerization and the protease activity of HtrA1, while active HtrA1 elevates the pro-apoptotic actions of porphyrin derivatives. Further analysis of this interplay may shed insights into the pathogenesis of diseases such as AMD, CARASIL and protoporphyria, as well as effective therapeutic development. PMID:25506911

  3. Endosomal deubiquitinating enzymes control ubiquitination and down-regulation of protease-activated receptor 2.

    PubMed

    Hasdemir, Burcu; Murphy, Jane E; Cottrell, Graeme S; Bunnett, Nigel W

    2009-10-09

    The E3 ubiquitin ligase c-Cbl ubiquitinates the G protein-coupled receptor protease-activated receptor 2 (PAR(2)), which is required for postendocytic sorting of activated receptors to lysosomes, where degradation terminates signaling. The mechanisms of PAR(2) deubiquitination and its importance in trafficking and signaling of endocytosed PAR(2) are unknown. We report that receptor deubiquitination occurs between early endosomes and lysosomes and involves the endosomal deubiquitinating proteases AMSH and UBPY. Expression of the catalytically inactive mutants, AMSH(D348A) and UBPY(C786S), caused an increase in PAR(2) ubiquitination and trapped the receptor in early endosomes, thereby preventing lysosomal trafficking and degradation. Small interfering RNA knockdown of AMSH or UBPY also impaired deubiquitination, lysosomal trafficking, and degradation of PAR(2). Trapping PAR(2) in endosomes through expression of AMSH(D348A) or UBPY(C786S) did not prolong the association of PAR(2) with beta-arrestin2 or the duration of PAR(2)-induced ERK2 activation. Thus, AMSH and UBPY are essential for trafficking and down-regulation of PAR(2) but not for regulating PAR(2) dissociation from beta-arrestin2 or PAR(2)-mediated ERK2 activation.

  4. Engineered tobacco etch virus (TEV) protease active in the secretory pathway of mammalian cells.

    PubMed

    Cesaratto, Francesca; López-Requena, Alejandro; Burrone, Oscar R; Petris, Gianluca

    2015-10-20

    Tobacco etch virus protease (TEVp) is a unique endopeptidase with stringent substrate specificity. TEVp has been widely used as a purified protein for in vitro applications, but also as a biological tool directly expressing it in living cells. To adapt the protease to diverse applications, several TEVp mutants with different stability and enzymatic properties have been reported. Herein we describe the development of a novel engineered TEVp mutant designed to be active in the secretory pathway. While wild type TEVp targeted to the secretory pathway of mammalian cells is synthetized as an N-glycosylated and catalytically inactive enzyme, a TEVp mutant with selected mutations at two verified N-glycosylation sites and at an exposed cysteine was highly efficient. This mutant was very active in the endoplasmic reticulum (ER) of living cells and can be used as a biotechnological tool to cleave proteins within the secretory pathway. As an immediate practical application we report the expression of a complete functional monoclonal antibody expressed from a single polypeptide, which was cleaved by our TEVp mutant into the two antibody chains and secreted as an assembled and functional molecule. In addition, we show active TEVp mutants lacking auto-cleavage activity.

  5. Protease-activated receptor-1 modulates hippocampal memory formation and synaptic plasticity.

    PubMed

    Almonte, Antoine G; Qadri, Laura H; Sultan, Faraz A; Watson, Jennifer A; Mount, Daniel J; Rumbaugh, Gavin; Sweatt, J David

    2013-01-01

    Protease-activated receptor-1 (PAR1) is an unusual G-protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. Although previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1's contribution to normal brain function. Here, we used PAR1-/- mice to investigate the contribution of PAR1 function to memory formation and synaptic function. We demonstrate that PAR1-/- mice have deficits in hippocampus-dependent memory. We also show that while PAR1-/- mice have normal baseline synaptic transmission at Schaffer collateral-CA1 synapses, they exhibit severe deficits in N-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). Mounting evidence indicates that activation of PAR1 leads to potentiation of NMDAR-mediated responses in CA1 pyramidal cells. Taken together, this evidence and our data suggest an important role for PAR1 function in NMDAR-dependent processes subserving memory formation and synaptic plasticity.

  6. Characterization of the protease activity that cleaves the extracellular domain of {beta}-dystroglycan

    SciTech Connect

    Zhong Di; Saito, Fumiaki; Saito, Yuko; Nakamura, Ayami; Shimizu, Teruo; Matsumura, Kiichiro . E-mail: k-matsu@med.teikyo-u.ac.jp

    2006-06-30

    Dystroglycan (DG) complex, composed of {alpha}DG and {beta}DG, provides a link between the extracellular matrix (ECM) and cortical cytoskeleton. Although the proteolytic processing of {beta}DG 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 {beta}DG 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 {beta}DG. These results indicate (1) that RT4 cells secrete the protease activity that degrades the extracellular domain of {beta}DG specifically and (2) that MMP-2 and MMP-9 may be involved in this process.

  7. Protease-Activated Receptors and other G-Protein-Coupled Receptors: the Melanoma Connection.

    PubMed

    Rosero, Rebecca A; Villares, Gabriel J; Bar-Eli, Menashe

    2016-01-01

    The vast array of G-protein-coupled receptors (GPCRs) play crucial roles in both physiological and pathological processes, including vision, coagulation, inflammation, autophagy, and cell proliferation. GPCRs also affect processes that augment cell proliferation and metastases in many cancers including melanoma. Melanoma is the deadliest form of skin cancer, yet limited therapeutic modalities are available to patients with metastatic melanoma. Studies have found that both chemokine receptors and protease-activated receptors, both of which are GPCRs, are central to the metastatic melanoma phenotype and may serve as potential targets in novel therapies against melanoma and other cancers.

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

    PubMed

    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.

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

    PubMed Central

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

    2014-01-01

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

  10. Aspartic acid protease from Botrytis cinerea removes haze-forming proteins during white winemaking.

    PubMed

    Van Sluyter, Steven C; Warnock, Nicholas I; Schmidt, Simon; Anderson, Peter; van Kan, Jan A L; Bacic, Antony; Waters, Elizabeth J

    2013-10-09

    White wines suffer from heat-induced protein hazes during transport and storage unless the proteins are removed prior to bottling. Bentonite fining is by far the most commonly used method, but it is inefficient and creates several other process challenges. An alternative to bentonite is the enzymatic removal of haze-forming grape pathogenesis-related proteins using added proteases. The major problem with this approach is that grape pathogenesis-related proteins are highly protease resistant unless they are heat denatured in combination with enzymatic treatment. This paper demonstrates that the protease BcAP8, from the grape fungal pathogen Botrytis cinerea , is capable of degrading chitinase, a major class of haze-forming proteins, without heat denaturation. Because BcAP8 effectively removes haze-forming proteins under normal winemaking conditions, it could potentially benefit winemakers by reducing bentonite requirements.

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

    PubMed

    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.

  12. Fungal fermentation of whey incorporated with certain supplements for the production of proteases.

    PubMed

    Ashour, S A; el-Shora, H M; Metwally, M; Habib, S A

    1996-01-01

    The pattern and the extent of formation of proteases and secretion varied with the fungus, age and/or the nature of the co-supplement. Addition of yeast extract induced the best yield of proteases from both Aspergillus niger and A. terreus. Proteases from A. niger were highly induced by glutamic acid, alanine or albumin, with minor differences, whereas gelatin, peptone, aspartic acid, casein or acetamide stimulated the accumulation of proteases in the culture medium of A. terreus. Galactose stimulated the yield of the enzyme particularly with A. terreus while most C-supplements inhibited such processes, more prominently in the presence of cellulose or starch. Incorporation of nicotinic acid and wheat bran initiated the maximum productivity of proteases from A. niger and A. terreus, respectively. Co+2 and Cu+2 highly stimulated the output of proteases from A. niger and A. terreus, respectively. Co+2 and Ca+2 stimulated the enzyme activity of alkaline and acid proteases from A. terreus and acid proteases from A. niger. The other six cations and DTT inhibited variably the three proteases particularly alkaline proteases from A. terreus indicating that proteases from various sources even from closely related species showed different properties.

  13. Inhibition of sulfur mustard-increased protease activity by niacinamide, N-acetyl-L-cysteine or dexamethasone

    SciTech Connect

    Cowan, F.M.; Broomfield, C.A.; Smith, W.J.

    1991-03-11

    The pathologic mechanism of sulfur mustard-induced skin vesication is as yet undefined. Papirmeister et al. have postulated a biochemical mechanism for sulfur mustard-induced cutaneous injury involving sequelae of DNA alkylation, metabolic disruption resulting in NAD+ depletion and activation of protease. The authors have utilized a chromogenic peptide substrate assay to establish that human peripheral blood lymphocytes exposed 24 hr previously to sulfur mustard exhibited an increase in proteolytic activity. Doses of compounds known to alter the biochemical events associated with sulfur mustard exposure or reduce protease activity were tested in this system for their ability to block the sulfur mustard-induced protease activity. Treatment with niacinamide 1 hr after or with N-acetyl-L-cysteine or dexamethasone 24 hr prior to sulfur mustard exposure resulted in a decrease of 39%, 33% and 42% respectively of sulfur mustard-increased protease activity. These data suggest that therapeutic intervention into the biochemical pathways that culminate in protease activation might serve as an approach to treatment of sulfur mustard-induced pathology.

  14. Amino acid sequence and some properties of phytolacain G, a cysteine protease from growing fruit of pokeweed, Phytolacca americana.

    PubMed

    Uchikoba, T; Arima, K; Yonezawa, H; Shimada, M; Kaneda, M

    2000-10-18

    A protease, phytolacain G, has been found to appear on CM-Sepharose ion-exchange chromatography of greenish small-size fruits of pokeweed, Phytolacca americana L, from ca. 2 weeks after flowering, and increases during fruit enlargement. Reddish ripe fruit of the pokeweed contained both phytolacain G and R. The molecular mass of phytolacain G was estimated to be 25.5 kDa by SDS-PAGE. Its amino acid sequence was reconstructed by automated sequence analysis of the peptides obtained after cleavage with Achromobacter protease I, chymotrypsin, and cyanogen bromide. The enzyme is composed of 216 amino acid residues, of which it shares 152 identical amino acid residues (70%) with phytolacain R, 126 (58%) with melain G, 108 (50%) with papain, 106 (49%) with actinidain, and 96 (44%) with stem bromelain. The amino acid residues forming the substrate binding S(2) pocket of papain, Tyr67, Pro68, Trp69, Val133, and Phe207, were predicted to be replaced by Trp, Met, His, Ala, and Ser in phytolacain G, respectively. As a consequence of these substitutions, the S(2) pocket is expected to be less hydrophobic in phytolacain G than in papain.

  15. Effects of Hypothermia and Self-Warming on Activity of Ca(2+)-Dependent Neutral Proteases in Tissues of Ground Squirrels and Rats.

    PubMed

    Nurmagomedova, P M; Emirbekov, E Z; Abasova, M M

    2016-04-01

    Effects of hypothermia and subsequent self-warming on activity of Ca(2+)-dependent neutral proteases were studied in tissues of ground squirrels and rats. Moderate hypothermia did not significantly change activity of Ca(2+)-dependent neutral proteases in the analyzed tissues of ground squirrels, but reduced protease activity in rat heart. Severe hypothermia reduced enzyme activity in the analyzed tissues of rats and ground squirrels. Differences in activity of Ca(2+)-dependent neutral proteases after long-term hypothermia and subsequent self-warming were found only in the heart.

  16. Protease with collagenolytic activity produced by Bacillus sp. DPUA 1728 from Amazonian soil

    PubMed Central

    Lima, Lorena A.; Cruz, Raimundo F.; dos Santos, Januário G.; Silva, Wilson C.

    2015-01-01

    Qualitative analyses were carried out on solid medium with insoluble collagen 0.25% (w/v) to detect proteases with collagenolytic activity produced by Bacillus sp. In cultures incubated for 24 h, a 23 full factorial design with four repetitions at the center point was developed to analyze the effects and interactions between initial pH, temperature and the concentration of gelatin. Based on the results of the first 23 full factorial design, a successive 23 full factorial design was performed. The most favorable production conditions were found to be 1.5% (w/v) gelatin, pH 9.0 and 37 °C with enzymatic activity of 86.27 U/mL. The enzyme showed optimal activity at 50 °C and pH 9.0, and it was stable over wide pH (7.2-10.0) and temperature (45 °C-60 °C) ranges. These results indicate that Bacillus sp DPUA 1728 is a potential source for producing collagenolytic protease with possible biotechnological applications, such as in the food, cosmetics and leather industries. PMID:26691484

  17. The aspartyl protease DDI2 activates Nrf1 to compensate for proteasome dysfunction

    PubMed Central

    Koizumi, Shun; Irie, Taro; Hirayama, Shoshiro; Sakurai, Yasuyuki; Yashiroda, Hideki; Naguro, Isao; Ichijo, Hidenori; Hamazaki, Jun; Murata, Shigeo

    2016-01-01

    In response to proteasome dysfunction, mammalian cells upregulate proteasome gene expression by activating Nrf1. Nrf1 is an endoplasmic reticulum-resident transcription factor that is continually retrotranslocated and degraded by the proteasome. Upon proteasome inhibition, Nrf1 escapes degradation and is cleaved to become active. However, the processing enzyme for Nrf1 remains obscure. Here we show that the aspartyl protease DNA-damage inducible 1 homolog 2 (DDI2) is required to cleave and activate Nrf1. Deletion of DDI2 reduced the cleaved form of Nrf1 and increased the full-length cytosolic form of Nrf1, resulting in poor upregulation of proteasomes in response to proteasome inhibition. These defects were restored by adding back wild-type DDI2 but not protease-defective DDI2. Our results provide a clue for blocking compensatory proteasome synthesis to improve cancer therapies targeting proteasomes. DOI: http://dx.doi.org/10.7554/eLife.18357.001 PMID:27528193

  18. The Prc and RseP proteases control bacterial cell-surface signalling activity.

    PubMed

    Bastiaansen, Karlijn C; Ibañez, Aurelia; Ramos, Juan L; Bitter, Wilbert; Llamas, María A

    2014-08-01

    Extracytoplasmic function (ECF) sigma factors play a key role in the regulation of vital functions in the bacterial response to the environment. In Gram-negative bacteria, activity of these sigma factors is often controlled by cell-surface signalling (CSS), a regulatory system that also involves an outer membrane receptor and a transmembrane anti-sigma factor. To get more insight into the molecular mechanism behind CSS regulation, we have focused on the unique Iut system of Pseudomonas putida. This system contains a hybrid protein containing both a cytoplasmic ECF sigma domain and a periplasmic anti-sigma domain, apparently leading to a permanent interaction between the sigma and anti-sigma factor. We show that the Iut ECF sigma factor regulates the response to aerobactin under iron deficiency conditions and is activated by a proteolytic pathway that involves the sequential action of two proteases: Prc, which removes the periplasmic anti-sigma domain, and RseP, which subsequently removes the transmembrane domain and thereby generates the ECF active transcriptional form. We furthermore demonstrate the role of these proteases in the regulation of classical CSS systems in which the sigma and anti-sigma factors are two different proteins.

  19. Oral administration of protease inhibits enterotoxigenic Escherichia coli receptor activity in piglet small intestine.

    PubMed Central

    Mynott, T L; Luke, R K; Chandler, D S

    1996-01-01

    The virulence of enterotoxigenic Escherichia coli (ETEC) is attributed to their ability to adhere via fimbrial adhesins to specific receptors located on the intestinal mucosa. A novel approach to preventing ETEC induced diarrhoea would be to prevent attachment of ETEC to intestine by proteolytically modifying the receptor attachment sites. This study aimed to examine the effect of bromelain, a proteolytic extract obtained from pineapple stems, on ETEC receptor activity in porcine small intestine. Bromelain was administered orally to piglets and K88+ ETEC attachment to small intestine was measured at 50 cm intervals using an enzyme immunoassay. K88+ ETEC attachment to intestinal sections that were not treated with bromelain varied appreciably between sampling sites. Variability in receptor activity along the intestinal surface is though to be caused by the localised effects of endogenous proteases. Oral administration of exogenous protease inhibited K88+ ETEC attachment to pig small intestine in a dose dependent manner (p < 0.05). Attachment of K88+ ETEC was negligible after treatment, resembling the levels of attachment of K88 to piglets of the genetically determined non-adhesive phenotype, which are resistant to K88+ ETEC infection. Serum biochemical analysis and histopathological examination of treated piglets showed no adverse effects of the bromelain treatment. It is concluded that administration of bromelain can inhibit ETEC receptor activity in vivo and may therefore be useful for prevention of K88+ ETEC induced diarrhoea. PMID:8566855

  20. Entomotoxicity, protease and chitinase activity of Bacillus thuringiensis fermented wastewater sludge with a high solids content.

    PubMed

    Brar, Satinder K; Verma, M; Tyagi, R D; Valéro, J R; Surampalli, R Y

    2009-10-01

    This study investigated the production of biopesticides, protease and chitinase activity by Bacillus thuringiensis grown in raw wastewater sludge at high solids concentration (30 g/L). The rheology of wastewater sludge was modified with addition of Tween-80 (0.2% v/v). This addition resulted in 1.6 and 1.3-fold increase in cell and spore count, respectively. The maximum specific growth rate (micro(max)) augmented from 0.17 to 0.22 h(-1) and entomotoxicity (Tx) increased by 29.7%. Meanwhile, volumetric mass transfer coefficient (k(L)a) showed marked variations during fermentation, and oxygen uptake rate (OUR) increased 2-fold. The proteolytic activity increased while chitinase decreased for Tween amended wastewater sludge, but the entomotoxicity increased. The specific entomotoxicity followed power law when plotted against spore concentration and the relation between Tx and protease activity was linear. The viscosity varied and volume percent of particles increased in Tween-80 amended wastewater sludge and particle size (D(50)) decreased at the end of fermentation. Thus, there was an increase in entomotoxicity at higher suspended solids (30 g/L) as Tween addition improved rheology (viscosity, particle size, surface tension); enhanced maximum growth rate and OUR.

  1. Effects of worts treated with proteases on the assimilation of free amino acids and fermentation performance of lager yeast.

    PubMed

    Lei, Hongjie; Zheng, Liye; Wang, Chenxia; Zhao, Haifeng; Zhao, Mouming

    2013-02-01

    The objective of this study was to investigate the changes in free amino acids (FAA) composition by supplementing three commercial proteases (Neutrase, Flavorzyme and Protamex) at the beginning of wort mashing, and monitoring the effects on the assimilation pattern of FAA and fermentation performance of lager yeast (Saccharomyces pastorianus) during normal and high gravity fermentations. Proteases supplementation significantly improved the extract yield and FAA level of mashed worts. Normal gravity worts treated with Flavorzyme and Neutrase exhibited higher fermentability, ethanol production and flavor volatiles concentration compared to the control worts, while these beneficial effects were observed in high gravity worts treated with Protamex and Neutrase. The reason for the above results is proposed to be the change in the assimilation pattern of FAA in lager yeast with increased wort gravity, especially for the improved assimilation ratios of Leu, Arg, Phe, His, Asp and Val. In normal gravity fermentations, there were strong correlations between the assimilation amounts of Lys, Leu, Arg and His and fermentability, while in high gravity fermentations, these good correlations were found with only Lys and His. The present study suggested that optimizing the composition of FAA by supplementing proteases during wort mashing was beneficial to beer brewing for improving fermentation performance of lager yeast and flavor volatiles formation.

  2. Cloning and analysis of WF146 protease, a novel thermophilic subtilisin-like protease with four inserted surface loops.

    PubMed

    Wu, Jiang; Bian, Yan; Tang, Bing; Chen, Xiangdong; Shen, Ping; Peng, Zhenrong

    2004-01-30

    Cloning and sequencing of the gene encoding WF146 protease, an extracellular subtilisin-like protease from the thermophile Bacillus sp. WF146, revealed that the WF146 protease was translated as a 416-amino acid precursor consisting of a putative 18-amino acid signal peptide, a 10-kDa N-terminal propeptide and a 32-kDa mature protease region. The mature WF146 protease shares a high degree of amino acid sequence identity with two psychrophilic subtilisins, S41 (68.2%) and S39 (65.4%), and a mesophilic subtilisin, SSII (67.1%). Significantly, these closely related proteases adapted to different temperatures all had four inserted surface loops not found in other subtilisins. However, unlike those of S41, S39 and SSII, the inserted loops of the WF146 protease possessed stabilizing features, such as the introduction of Pro residues into the loop regions. Interestingly, the WF146 protease contained five of the seven mutations previously found in a hyperstable variant of subtilisin S41 obtained by directed evolution. The proform of WF146 protease (pro-WF146 protease) was overexpressed in Escherichia coli in an inactive soluble form. After heat treatment, the 42-kDa pro-WF146 protease converted to a 32-kDa active mature form by processing the N-terminal propeptide. The purified mature WF146 protease hydrolyzed casein with an optimum temperature of 85 degrees C, and lost activity with a half-life of 30 min at 80 degrees C in the presence of 10 mM CaCl2.

  3. Serpin1 and WSCP differentially regulate the activity of the cysteine protease RD21 during plant development in Arabidopsis thaliana.

    PubMed

    Rustgi, Sachin; Boex-Fontvieille, Edouard; Reinbothe, Christiane; von Wettstein, Diter; Reinbothe, Steffen

    2017-02-28

    Proteolytic enzymes (proteases) participate in a vast range of physiological processes, ranging from nutrient digestion to blood coagulation, thrombosis, and beyond. In plants, proteases are implicated in host recognition and pathogen infection, induced defense (immunity), and the deterrence of insect pests. Because proteases irreversibly cleave peptide bonds of protein substrates, their activity must be tightly controlled in time and space. Here, we report an example of how nature evolved alternative mechanisms to fine-tune the activity of a cysteine protease dubbed RD21 (RESPONSIVE TO DESICCATION-21). One mechanism in the model plant Arabidopsis thaliana studied here comprises irreversible inhibition of RD21's activity by Serpin1, whereas the other mechanism is a result of the reversible inhibition of RD21 activity by a Kunitz protease inhibitor named water-soluble chlorophyll-binding protein (WSCP). Activity profiling, complex isolation, and homology modeling data revealed unique interactions of RD21 with Serpin1 and WSCP, respectively. Expression studies identified only partial overlaps in Serpin1 and WSCP accumulation that explain how RD21 contributes to the innate immunity of mature plants and arthropod deterrence of seedlings undergoing skotomorphogenesis and greening.

  4. Toxin-Antitoxin Modules Are Pliable Switches Activated by Multiple Protease Pathways

    PubMed Central

    Muthuramalingam, Meenakumari; White, John C.; Bourne, Christina R.

    2016-01-01

    Toxin-antitoxin (TA) modules are bacterial regulatory switches that facilitate conflicting outcomes for cells by promoting a pro-survival phenotypic adaptation and/or by directly mediating cell death, all through the toxin activity upon degradation of antitoxin. Intensive study has revealed specific details of TA module functions, but significant gaps remain about the molecular details of activation via antitoxin degradation used by different bacteria and in different environments. This review summarizes the current state of knowledge about the interaction of antitoxins with cellular proteases Lon and ClpP to mediate TA module activation. An understanding of these processes can answer long-standing questions regarding stochastic versus specific activation of TA modules and provide insight into the potential for manipulation of TA modules to alter bacterial growth. PMID:27409636

  5. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion.

    PubMed

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W; Liu, Yan; Walter, Nils G; Yan, Hao

    2016-02-10

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

  6. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

    PubMed Central

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

    2016-01-01

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology. PMID:26861509

  7. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

    NASA Astrophysics Data System (ADS)

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

    2016-02-01

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

  8. Synthesis, crystal structure, structure-activity relationships, and antiviral activity of a potent SARS coronavirus 3CL protease inhibitor.

    PubMed

    Yang, Syaulan; Chen, Shu-Jen; Hsu, Min-Feng; Wu, Jen-Dar; Tseng, Chien-Te K; Liu, Yu-Fan; Chen, Hua-Chien; Kuo, Chun-Wei; Wu, Chi-Shen; Chang, Li-Wen; Chen, Wen-Chang; Liao, Shao-Ying; Chang, Teng-Yuan; Hung, Hsin-Hui; Shr, Hui-Lin; Liu, Cheng-Yuan; Huang, Yu-An; Chang, Ling-Yin; Hsu, Jen-Chi; Peters, Clarence J; Wang, Andrew H-J; Hsu, Ming-Chu

    2006-08-10

    A potent SARS coronavirus (CoV) 3CL protease inhibitor (TG-0205221, Ki = 53 nM) has been developed. TG-0205221 showed remarkable activity against SARS CoV and human coronavirus (HCoV) 229E replications by reducing the viral titer by 4.7 log (at 5 microM) for SARS CoV and 5.2 log (at 1.25 microM) for HCoV 229E. The crystal structure of TG-0205221 (resolution = 1.93 A) has revealed a unique binding mode comprising a covalent bond, hydrogen bonds, and numerous hydrophobic interactions. Structural comparisons between TG-0205221 and a natural peptide substrate were also discussed. This information may be applied toward the design of other 3CL protease inhibitors.

  9. Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63.

    PubMed

    Chen, Zhongbin; Wang, Yanhua; Ratia, Kiira; Mesecar, Andrew D; Wilkinson, Keith D; Baker, Susan C

    2007-06-01

    Human coronavirus NL63 (HCoV-NL63), a common human respiratory pathogen, is associated with both upper and lower respiratory tract disease in children and adults. Currently, no antiviral drugs are available to treat CoV infections; thus, potential drug targets need to be identified and characterized. Here, we identify HCoV-NL63 replicase gene products and characterize two viral papain-like proteases (PLPs), PLP1 and PLP2, which process the viral replicase polyprotein. We generated polyclonal antisera directed against two of the predicted replicase nonstructural proteins (nsp3 and nsp4) and detected replicase proteins from HCoV-NL63-infected LLC-MK2 cells by immunofluorescence, immunoprecipitation, and Western blot assays. We found that HCoV-NL63 replicase products can be detected at 24 h postinfection and that these proteins accumulate in perinuclear sites, consistent with membrane-associated replication complexes. To determine which viral proteases are responsible for processing these products, we generated constructs representing the amino-terminal end of the HCoV-NL63 replicase gene and established protease cis-cleavage assays. We found that PLP1 processes cleavage site 1 to release nsp1, whereas PLP2 is responsible for processing both cleavage sites 2 and 3 to release nsp2 and nsp3. We expressed and purified PLP2 and used a peptide-based assay to identify the cleavage sites recognized by this enzyme. Furthermore, by using K48-linked hexa-ubiquitin substrate and ubiquitin-vinylsulfone inhibitor specific for deubiquitinating enzymes (DUBs), we confirmed that, like severe acute respiratory syndrome (SARS) CoV PLpro, HCoV-NL63 PLP2 has DUB activity. The identification of the replicase products and characterization of HCoV-NL63 PLP DUB activity will facilitate comparative studies of CoV proteases and aid in the development of novel antiviral reagents directed against human pathogens such as HCoV-NL63 and SARS-CoV.

  10. TRY-5 Is a Sperm-Activating Protease in Caenorhabditis elegans Seminal Fluid

    PubMed Central

    Smith, Joseph R.; Stanfield, Gillian M.

    2011-01-01

    Seminal fluid proteins have been shown to play important roles in male reproductive success, but the mechanisms for this regulation remain largely unknown. In Caenorhabditis elegans, sperm differentiate from immature spermatids into mature, motile spermatozoa during a process termed sperm activation. For C. elegans males, sperm activation occurs during insemination of the hermaphrodite and is thought to be mediated by seminal fluid, but the molecular nature of this activity has not been previously identified. Here we show that TRY-5 is a seminal fluid protease that is required in C. elegans for male-mediated sperm activation. We observed that TRY-5::GFP is expressed in the male somatic gonad and is transferred along with sperm to hermaphrodites during mating. In the absence of TRY-5, male seminal fluid loses its potency to transactivate hermaphrodite sperm. However, TRY-5 is not required for either hermaphrodite or male fertility, suggesting that hermaphrodite sperm are normally activated by a distinct hermaphrodite-specific activator to which male sperm are also competent to respond. Within males, TRY-5::GFP localization within the seminal vesicle is antagonized by the protease inhibitor SWM-1. Together, these data suggest that TRY-5 functions as an extracellular activator of C. elegans sperm. The presence of TRY-5 within the seminal fluid couples the timing of sperm activation to that of transfer of sperm into the hermaphrodite uterus, where motility must be rapidly acquired. Our results provide insight into how C. elegans has adopted sex-specific regulation of sperm motility to accommodate its male-hermaphrodite mode of reproduction. PMID:22125495

  11. TRY-5 is a sperm-activating protease in Caenorhabditis elegans seminal fluid.

    PubMed

    Smith, Joseph R; Stanfield, Gillian M

    2011-11-01

    Seminal fluid proteins have been shown to play important roles in male reproductive success, but the mechanisms for this regulation remain largely unknown. In Caenorhabditis elegans, sperm differentiate from immature spermatids into mature, motile spermatozoa during a process termed sperm activation. For C. elegans males, sperm activation occurs during insemination of the hermaphrodite and is thought to be mediated by seminal fluid, but the molecular nature of this activity has not been previously identified. Here we show that TRY-5 is a seminal fluid protease that is required in C. elegans for male-mediated sperm activation. We observed that TRY-5::GFP is expressed in the male somatic gonad and is transferred along with sperm to hermaphrodites during mating. In the absence of TRY-5, male seminal fluid loses its potency to transactivate hermaphrodite sperm. However, TRY-5 is not required for either hermaphrodite or male fertility, suggesting that hermaphrodite sperm are normally activated by a distinct hermaphrodite-specific activator to which male sperm are also competent to respond. Within males, TRY-5::GFP localization within the seminal vesicle is antagonized by the protease inhibitor SWM-1. Together, these data suggest that TRY-5 functions as an extracellular activator of C. elegans sperm. The presence of TRY-5 within the seminal fluid couples the timing of sperm activation to that of transfer of sperm into the hermaphrodite uterus, where motility must be rapidly acquired. Our results provide insight into how C. elegans has adopted sex-specific regulation of sperm motility to accommodate its male-hermaphrodite mode of reproduction.

  12. Antidermatophytic and Protease-inhibiting Activities of Zerumbone: A Natural Sesquiterpene from the Rhizome of Zingiber zerumbet (L.) Roscoe ex J.E; Smith

    PubMed Central

    Jyothilakshmi, Madhavankutty; Jyothis, Mathew; Narayanan, Gokulanathan Nair Hari; Latha, Mukalel Sankunni

    2017-01-01

    the development of new antidermatophytic drug.Cytotoxicity of zerumbone against Chang liver cell line was found to be very low compared with the reference drug doxorubicin. SUMMARY Zerumbone isolated from the rhizome of Zingiber zerumbet exhibited antidermatophytic activity against E. floccosum and M. canis (MIC 8 mg/L) and M. gypseum and T. Rubrum (MIC 16 mg/L).Zerumbone exhibited remarkable protease-inhibiting activity.Zerumbone is a novel candidate for the development of new antidermatophytic drug.Cytotoxicity of zerumbone against Chang liver cell line was found to be very low compared with the reference drug doxorubicin. Abbreviations used: CFU: colony forming unit, CLSI: Clinical and Laboratory Standards Institute, COX: cyclooxygenase, DMSO: dimethyl sulphoxide, EDTA: ethylene diamine tetra acetic acid, FT-IR: Fourier transform–infra red spectroscopy, HPLC: high-performance liquid chromatography, LOX: lipoxygenase, IMTECH: Institute of Microbial Technology, LCMS: liquid chromatography mass spectrometry, MTT: 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTCC: microbial type culture collection, MFC: minimum fungicidal concentration, MIC: minimum inhibitory concentration, MPO: myeloperoxidase, NMR: nuclear magnetic resonance spectroscopy, PAR: proteinase-activated receptor, PBS: phosphate-buffered saline, TCA: trichloro acetic acid PMID:28216875

  13. Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement

    PubMed Central

    Ortmann, Weronika; Kolaczkowska, Elzbieta

    2016-01-01

    Formation of extracellular traps (ETs) capturing and immobilizing pathogens is now a well-established defense mechanism added to the repertoire of vertebrate phagocytes. These ETs are composed of extracellular DNA (extDNA), histones and antimicrobial proteins. Formation of mouse and human ETs depends on enzymes (i) facilitating decondensation of chromatin by citrullination of histones, and (ii) serine proteases degrading histones. In invertebrates, initial reports revealed existence of ETs composed of extDNA and histones, and here we document for the first time that also coelomocytes, immunocompetent cells of an earthworm Eisenia andrei, cast ETs which successfully trap bacteria in a reactive oxygen species (ROS)-dependent and -independent manner. Importantly, the formation of ETs was observed not only when coelomocytes were studied ex vivo, but also in vivo, directly in the earthworm coelom. These ETs were composed of extDNA, heat shock proteins (HSP27) and H3 histones. Furthermore, the formation of E. andrei ETs depended on activity of serine proteases, including elastase-like activity. Moreover, ETs interconnected and hold together aggregating coelomocytes, a processes proceeding encapsulation. In conclusion, the study confirms ET formation by earthworms, and unravels mechanisms leading to ET formation and encapsulation in invertebrates. PMID:27416067

  14. Mapping inhibitor binding modes on an active cysteine protease via nuclear magnetic resonance spectroscopy.

    PubMed

    Lee, Gregory M; Balouch, Eaman; Goetz, David H; Lazic, Ana; McKerrow, James H; Craik, Charles S

    2012-12-18

    Cruzain is a member of the papain/cathepsin L family of cysteine proteases, and the major cysteine protease of the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We report an autoinduction methodology that provides soluble cruzain in high yields (>30 mg/L in minimal medium). These increased yields provide sufficient quantities of active enzyme for use in nuclear magnetic resonance (NMR)-based ligand mapping. Using circular dichroism and NMR spectroscopy, we also examined the solution-state structural dynamics of the enzyme in complex with a covalently bound vinyl sulfone inhibitor (K777). We report the backbone amide and side chain carbon chemical shift assignments of cruzain in complex with K777. These resonance assignments were used to identify and map residues located in the substrate binding pocket, including the catalytic Cys25 and His162. Selective [(15)N]Cys, [(15)N]His, and [(13)C]Met labeling was performed to quickly assess cruzain-ligand interactions for a set of eight low-molecular weight compounds exhibiting micromolar binding or inhibition. Chemical shift perturbation mapping verified that six of the eight compounds bind to cruzain at the active site. Three different binding modes were delineated for the compounds, namely, covalent, noncovalent, and noninteracting. These results provide examples of how NMR spectroscopy can be used to screen compounds for fast evaluation of enzyme-inhibitor interactions to facilitate lead compound identification and subsequent structural studies.

  15. Cellular aspartyl proteases promote the unconventional secretion of biologically active HIV-1 matrix protein p17

    PubMed Central

    Caccuri, Francesca; Iaria, Maria Luisa; Campilongo, Federica; Varney, Kristen; Rossi, Alessandro; Mitola, Stefania; Schiarea, Silvia; Bugatti, Antonella; Mazzuca, Pietro; Giagulli, Cinzia; Fiorentini, Simona; Lu, Wuyuan; Salmona, Mario; Caruso, Arnaldo

    2016-01-01

    The human immune deficiency virus type 1 (HIV-1) matrix protein p17 (p17), although devoid of a signal sequence, is released by infected cells and detected in blood and in different organs and tissues even in HIV-1-infected patients undergoing successful combined antiretroviral therapy (cART). Extracellularly, p17 deregulates the function of different cells involved in AIDS pathogenesis. The mechanism of p17 secretion, particularly during HIV-1 latency, still remains to be elucidated. A recent study showed that HIV-1-infected cells can produce Gag without spreading infection in a model of viral latency. Here we show that in Gag-expressing cells, secretion of biologically active p17 takes place at the plasma membrane and occurs following its interaction with phosphatidylinositol-(4,5)-bisphosphate and its subsequent cleavage from the precursor Gag (Pr55Gag) operated by cellular aspartyl proteases. These enzymes operate a more complex Gag polypeptide proteolysis than the HIV-1 protease, thus hypothetically generating slightly truncated or elongated p17s in their C-terminus. A 17 C-terminal residues excised p17 was found to be structurally and functionally identical to the full-length p17 demonstrating that the final C-terminal region of p17 is irrelevant for the protein’s biological activity. These findings offer new opportunities to identify treatment strategies for inhibiting p17 release in the extracellular microenvironment. PMID:27905556

  16. Calcium and adenosine triphosphate control of cellular pathology: asparaginase-induced pancreatitis elicited via protease-activated receptor 2

    PubMed Central

    Peng, Shuang; Gerasimenko, Julia V.; Tsugorka, Tatiana; Gryshchenko, Oleksiy; Samarasinghe, Sujith; Gerasimenko, Oleg V.

    2016-01-01

    Exocytotic secretion of digestive enzymes from pancreatic acinar cells is elicited by physiological cytosolic Ca2+ signals, occurring as repetitive short-lasting spikes largely confined to the secretory granule region, that stimulate mitochondrial adenosine triphosphate (ATP) production. By contrast, sustained global cytosolic Ca2+ elevations decrease ATP levels and cause necrosis, leading to the disease acute pancreatitis (AP). Toxic Ca2+ signals can be evoked by products of alcohol and fatty acids as well as bile acids. Here, we have investigated the mechanism by which l-asparaginase evokes AP. Asparaginase is an essential element in the successful treatment of acute lymphoblastic leukaemia, the most common type of cancer affecting children, but AP is a side-effect occurring in about 5–10% of cases. Like other pancreatitis-inducing agents, asparaginase evoked intracellular Ca2+ release followed by Ca2+ entry and also substantially reduced Ca2+ extrusion because of decreased intracellular ATP levels. The toxic Ca2+ signals caused extensive necrosis. The asparaginase-induced pathology depended on protease-activated receptor 2 and its inhibition prevented the toxic Ca2+ signals and necrosis. We tested the effects of inhibiting the Ca2+ release-activated Ca2+ entry by the Ca2+ channel inhibitor GSK-7975A. This markedly reduced asparaginase-induced Ca2+ entry and also protected effectively against the development of necrosis. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377732

  17. The Effectiveness of Negative Pressure Therapy in Diabetic Foot Ulcers with Elevated Protease Activity: A Case Series

    PubMed Central

    Izzo, Valentina; Meloni, Marco; Giurato, Laura; Ruotolo, Valeria; Uccioli, Luigi

    2017-01-01

    Objective: Despite several works have described the usefulness of negative pressure therapy (NPT) in the treatment of diabetic foot ulcers (DFUs), no studies have reported its ability in the proteases modulation in DFUs. The aim of this work was to evaluate the role of NPT as a protease-modulating treatment in DFUs. Approach: We conducted a prospective study of a series of diabetic patients affected by chronic DFUs. Each ulcer was assessed for matrix metalloproteinases (MMPs) activity with a protease status diagnostic test at the baseline and after 2 weeks of NPT. Results: Four patients were included. All patients had type 2 diabetes with a disease duration of ≈20 years. A1c was 79.5 ± 15.3 mmol/mol. Ulcer area was >5 cm2 in all cases. All wounds showed elevated protease activity (EPA) at the baseline. After 2 weeks, all patients showed a normalization of MMPs activity. Innovation: NPT showed its effectiveness in the reduction of EPA in chronic DFUs. Conclusion: This study confirms the role of NPT in the positive modulation of protease activity also in chronic DFUs. PMID:28116227

  18. Peptide Mass Fingerprinting and N-Terminal Amino Acid Sequencing of Glycosylated Cysteine Protease of Euphorbia nivulia Buch.-Ham.

    PubMed Central

    Badgujar, Shamkant B.; Mahajan, Raghunath T.

    2013-01-01

    A new cysteine protease named Nivulian-II has been purified from the latex of Euphorbia nivulia Buch.-Ham. The apparent molecular mass of Nivulian-II is 43670.846 Da (MALDI TOF/MS). Peptide mass fingerprint analysis revealed peptide matches to Maturase K (Q52ZV1_9MAGN) of Banksia quercifolia. The N-terminal sequence (DFPPNTCCCICC) showed partial homology with those of other cysteine proteinases of biological origin. This is the first paper to characterize a Nivulian-II of E. nivulia latex with respect to amino acid sequencing. PMID:23476742

  19. Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres

    PubMed Central

    Ibrahim, Abdelnasser Salah Shebl; Al-Salamah, Ali A.; El-Toni, Ahmed M.; Almaary, Khalid S.; El-Tayeb, Mohamed A.; Elbadawi, Yahya B.; Antranikian, Garabed

    2016-01-01

    The stability and reusability of soluble enzymes are of major concerns, which limit their industrial applications. Herein, alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto hollow core-mesoporous shell silica (HCMSS) nanospheres. Subsequently, the properties of immobilized proteases were evaluated. Non-, ethane- and amino-functionalized HCMSS nanospheres were synthesized and characterized. NPST-AK15 was immobilized onto the synthesized nano-supports by physical and covalent immobilization approaches. However, protease immobilization by covalent attachment onto the activated HCMSS–NH2 nanospheres showed highest immobilization yield (75.6%) and loading capacity (88.1 μg protein/mg carrier) and was applied in the further studies. In comparison to free enzyme, the covalently immobilized protease exhibited a slight shift in the optimal pH from 10.5 to 11.0, respectively. The optimum temperature for catalytic activity of both free and immobilized enzyme was seen at 60 °C. However, while the free enzyme was completely inactivated when treated at 60 °C for 1 h the immobilized enzyme still retained 63.6% of its initial activity. The immobilized protease showed higher Vmax, kcat and kcat/Km, than soluble enzyme by 1.6-, 1.6- and 2.4-fold, respectively. In addition, the immobilized protease affinity to the substrate increased by about 1.5-fold. Furthermore, the enzyme stability in various organic solvents was significantly enhanced upon immobilization. Interestingly, the immobilized enzyme exhibited much higher stability in several commercial detergents including OMO, Tide, Ariel, Bonux and Xra by up to 5.2-fold. Finally, the immobilized protease maintained significant catalytic efficiency for twelve consecutive reaction cycles. These results suggest the effectiveness of the developed nanobiocatalyst as a candidate for detergent formulation and peptide synthesis in non-aqueous media. PMID:26840303

  20. Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres.

    PubMed

    Ibrahim, Abdelnasser Salah Shebl; Al-Salamah, Ali A; El-Toni, Ahmed M; Almaary, Khalid S; El-Tayeb, Mohamed A; Elbadawi, Yahya B; Antranikian, Garabed

    2016-01-29

    The stability and reusability of soluble enzymes are of major concerns, which limit their industrial applications. Herein, alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto hollow core-mesoporous shell silica (HCMSS) nanospheres. Subsequently, the properties of immobilized proteases were evaluated. Non-, ethane- and amino-functionalized HCMSS nanospheres were synthesized and characterized. NPST-AK15 was immobilized onto the synthesized nano-supports by physical and covalent immobilization approaches. However, protease immobilization by covalent attachment onto the activated HCMSS-NH₂ nanospheres showed highest immobilization yield (75.6%) and loading capacity (88.1 μg protein/mg carrier) and was applied in the further studies. In comparison to free enzyme, the covalently immobilized protease exhibited a slight shift in the optimal pH from 10.5 to 11.0, respectively. The optimum temperature for catalytic activity of both free and immobilized enzyme was seen at 60 °C. However, while the free enzyme was completely inactivated when treated at 60 °C for 1 h the immobilized enzyme still retained 63.6% of its initial activity. The immobilized protease showed higher V(max), k(cat) and k(cat)/K(m), than soluble enzyme by 1.6-, 1.6- and 2.4-fold, respectively. In addition, the immobilized protease affinity to the substrate increased by about 1.5-fold. Furthermore, the enzyme stability in various organic solvents was significantly enhanced upon immobilization. Interestingly, the immobilized enzyme exhibited much higher stability in several commercial detergents including OMO, Tide, Ariel, Bonux and Xra by up to 5.2-fold. Finally, the immobilized protease maintained significant catalytic efficiency for twelve consecutive reaction cycles. These results suggest the effectiveness of the developed nanobiocatalyst as a candidate for detergent formulation and peptide synthesis in non-aqueous media.

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

    PubMed

    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.

  2. Activation of prothrombin by two subtilisin-like serine proteases from Acremonium sp.

    PubMed

    Liu, Chunli; Matsushita, Yasuhiko; Shimizu, Kosuke; Makimura, Koichi; Hasumi, Keiji

    2007-06-22

    Two novel subtilisin-like serine proteases (AS-E1 and -E2) that activate prothrombin have been identified in a culture of the fungus Acremonium sp. The enzymes were purified through repeated hydrophobic interaction chromatography. The N-terminal sequences of AS-E1 (34.4 kDa) and AS-E2 (32 kDa) showed high similarity to the internal sequences of two distinct subtilisin-like hypothetical proteins from Chaetomium globosum. Both enzymes proteolytically activated prothrombin to meizothrombin(desF1)-like molecules, while the activation cleavage seemed to occur at a site (Tyr(316)-Ile(317)) that is four residues proximal to the canonical Xa cleavage site (Arg(320)-Ile(321)). Both enzymes inhibited plasma clotting, possibly due to extensive degradation of fibrinogen and production of meizothrombin(desF1)-like molecule.

  3. A Cell-based Fluorescence Resonance Energy Transfer (FRET) Sensor Reveals Inter- and Intragenogroup Variations in Norovirus Protease Activity and Polyprotein Cleavage.

    PubMed

    Emmott, Edward; Sweeney, Trevor R; Goodfellow, Ian

    2015-11-13

    The viral protease represents a key drug target for the development of antiviral therapeutics. Because many protease inhibitors mimic protease substrates, differences in substrate recognition between proteases may affect their sensitivity to a given inhibitor. Here we use a cell-based FRET sensor to investigate the activity of different norovirus proteases upon cleavage of various norovirus cleavage sites inserted into a linker region separating cyan fluorescent protein and yellow fluorescent protein. Using this system, we demonstrate that differences in substrate processing exist between proteases from human noroviruses (genogroups I (GI) and II) and the commonly used murine norovirus (MNV, genogroup V) model. These altered the cleavage efficiency of specific cleavage sites both within and between genogroups. The differences observed between these proteases may affect sensitivity to protease inhibitors and the suitability of MNV as a model system for testing such molecules against the human norovirus protease. Finally, we demonstrate that replacement of MNV polyprotein cleavage sites with the GI or GII equivalents, with the exception of the NS6-7 junction, leads to the production of infectious virus when the MNV NS6 protease, but not the GI or GII proteases, are present.

  4. β-Amino acid catalyzed asymmetric Michael additions: design of organocatalysts with catalytic acid/base dyad inspired by serine proteases.

    PubMed

    Yang, Hui; Wong, Ming Wah

    2011-09-16

    A new type of chiral β-amino acid catalyst has been computationally designed, mimicking the enzyme catalysis of serine proteases. Our catalyst approach is based on the bioinspired catalytic acid/base dyad, namely, a carboxyl and imidazole pair. DFT calculations predict that this designed organocatalyst catalyzes Michael additions of aldehydes to nitroalkenes with excellent enantioselectivities and remarkably high anti diastereoselectivities. The unusual stacked geometry of the enamine intermediate, hydrogen bonding network, and the adoption of an exo transition state are the keys to understand the stereoselectivity.

  5. Maintenance of luminal pH and protease activity in lysosomes/late endosomes by vacuolar ATPase in chlorpromazine-treated RAW264 cells accumulating phospholipids.

    PubMed

    Hamaguchi, Ryohei; Haginaka, Jun; Tanimoto, Toshiko; Kuroda, Yukihiro

    2014-02-01

    Cationic amphiphilic drugs (CADs) inhibit phospholipases competitively/uncompetitively. It has also been reported that CADs spontaneously accumulate in acidic organelles and increase their luminal pH, which may lead to deactivation of phospholipid-metabolising enzymes, causing cellular phospholipid accumulation. Recently, however, contradictory results have also been reported in that the luminal pH is not increased by CAD treatment. In this study, we examined whether the lysosomal/late endosomal acidic pH was maintained by vacuolar ATPase (v-ATPase) after treatment with chlorpromazine (CPZ) as a model CAD. The activity of lysosomal protease after CPZ treatment was also measured. Oregon Green-dextran-tetramethylrhodamine conjugate was employed to determine the luminal pH of the lysosomes/late endosomes in RAW264 cells. The luminal pH remained acidic after treatment with CPZ for 23 h, and the lysosomal protease activity was not decreased by 5-min CPZ treatment. Co-treatment with CPZ and bafilomycin A1 (v-ATPase inhibitor) raised the luminal pH. These results suggest that the lysosomal/late endosomal pH is not affected by a 23-h CPZ treatment. In addition, lysosomal enzymes presumably maintain their activity when CPZ accumulates. Our results imply that the pH homeostasis in lysosomes/late endosomes is strictly maintained even after a longer treatment with CADs.

  6. The ClpP N-Terminus Coordinates Substrate Access with Protease Active Site Reactivity

    SciTech Connect

    Jennings, L.; Bohon, J; Chance, M; Licht, S

    2008-01-01

    Energy-dependent protein degradation machines, such as the Escherichia coli protease ClpAP, require regulated interactions between the ATPase component (ClpA) and the protease component (ClpP) for function. Recent studies indicate that the ClpP N-terminus is essential in these interactions, yet the dynamics of this region remain unclear. Here, we use synchrotron hydroxyl radical footprinting and kinetic studies to characterize functionally important conformational changes of the ClpP N-terminus. Footprinting experiments show that the ClpP N-terminus becomes more solvent-exposed upon interaction with ClpA. In the absence of ClpA, deletion of the ClpP N-terminus increases the initial degradation rate of large peptide substrates 5-15-fold. Unlike ClpAP, ClpP?N exhibits a distinct slow phase of product formation that is eliminated by the addition of hydroxylamine, suggesting that truncation of the N-terminus leads to stabilization of the acyl-enzyme intermediate. These results indicate that (1) the ClpP N-terminus acts as a 'gate' controlling substrate access to the active sites, (2) binding of ClpA opens this 'gate', allowing substrate entry and formation of the acyl-enzyme intermediate, and (3) closing of the N-terminal 'gate' stimulates acyl-enzyme hydrolysis.

  7. Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses

    PubMed Central

    2010-01-01

    Background Baculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricutural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL), and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat), were constructed. and their insecticidal properties analysed against Spodoptera frugiperda larvae. Results Bioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill. Conclusions Baculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs. PMID:20587066

  8. Regulation of protease production in Clostridium sporogenes.

    PubMed Central

    Allison, C; Macfarlane, G T

    1990-01-01

    The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions. PMID:2268158

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

    PubMed

    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.

  10. Effects of Coated Compound Proteases on Apparent Total Tract Digestibility of Nutrients and Apparent Ileal Digestibility of Amino Acids for Pigs

    PubMed Central

    Pan, L.; Zhao, P. F.; Yang, Z. Y.; Long, S. F.; Wang, H. L.; Tian, Q. Y.; Xu, Y. T.; Xu, X.; Zhang, Z. H.; Piao, X. S.

    2016-01-01

    Two experiments were conducted to evaluate effects of coated compound proteases (CC protease) on apparent total tract digestibility (ATTD) of nitrogen (N) and energy, and apparent ileal digestibility (AID) of amino acids (AA) and nutrients in diets for pigs. In Exp. 1, 12 crossbred barrows (initial body weight: 20.14±1.71 kg) were housed in individual metabolism crates and allotted into 2 treatments with 6 piglets per treatment according to weight in a randomized complete block design. The 2 diets were corn-soybean meal basal diets with (0.2 g/kg) or without CC protease supplementation. The CC protease supplementation increased (p<0.05) the digestible and metabolizable N and energy values and the digestibility and retention rate of N in the diet. The ATTD of energy and nutrients had been improved (p<0.05) in the diet supplemented with CC protease. In Exp. 2, 12 crossbred barrows (initial body weight: 20.79±1.94 kg), fitted with T-cannulas at the distal ileum, were blocked by body weight into 2 groups with 6 pigs each. The diets were the same as those in Exp. 1. The CC protease increased (p<0.05) the AID of crude protein and some essential AA including arginine, isoleucine and leucine. The AID and ATTD of energy and nutrients had been improved (p<0.05) by supplemental CC protease, but the hindgut digestibility of nutrients was unaffected. Overall, the CC protease improved the ATTD of N and energy and AID of some indispensible AA and nutrients in the corn-soybean meal diet for pigs. Therefore, the CC protease supplement could improve the utilization of protein in the corn-soybean meal diet and thus contribute to lower N excretion to the environment. PMID:27004811

  11. Effects of Coated Compound Proteases on Apparent Total Tract Digestibility of Nutrients and Apparent Ileal Digestibility of Amino Acids for Pigs.

    PubMed

    Pan, L; Zhao, P F; Yang, Z Y; Long, S F; Wang, H L; Tian, Q Y; Xu, Y T; Xu, X; Zhang, Z H; Piao, X S

    2016-12-01

    Two experiments were conducted to evaluate effects of coated compound proteases (CC protease) on apparent total tract digestibility (ATTD) of nitrogen (N) and energy, and apparent ileal digestibility (AID) of amino acids (AA) and nutrients in diets for pigs. In Exp. 1, 12 crossbred barrows (initial body weight: 20.14±1.71 kg) were housed in individual metabolism crates and allotted into 2 treatments with 6 piglets per treatment according to weight in a randomized complete block design. The 2 diets were corn-soybean meal basal diets with (0.2 g/kg) or without CC protease supplementation. The CC protease supplementation increased (p<0.05) the digestible and metabolizable N and energy values and the digestibility and retention rate of N in the diet. The ATTD of energy and nutrients had been improved (p<0.05) in the diet supplemented with CC protease. In Exp. 2, 12 crossbred barrows (initial body weight: 20.79±1.94 kg), fitted with T-cannulas at the distal ileum, were blocked by body weight into 2 groups with 6 pigs each. The diets were the same as those in Exp. 1. The CC protease increased (p<0.05) the AID of crude protein and some essential AA including arginine, isoleucine and leucine. The AID and ATTD of energy and nutrients had been improved (p<0.05) by supplemental CC protease, but the hindgut digestibility of nutrients was unaffected. Overall, the CC protease improved the ATTD of N and energy and AID of some indispensible AA and nutrients in the corn-soybean meal diet for pigs. Therefore, the CC protease supplement could improve the utilization of protein in the corn-soybean meal diet and thus contribute to lower N excretion to the environment.

  12. OMP peptides activate the DegS stress-sensor protease by a relief of inhibition mechanism.

    PubMed

    Sohn, Jungsan; Grant, Robert A; Sauer, Robert T

    2009-10-14

    In the E. coli periplasm, C-terminal peptides of misfolded outer-membrane porins (OMPs) bind to the PDZ domains of the trimeric DegS protease, triggering cleavage of a transmembrane regulator and transcriptional activation of stress genes. We show that an active-site DegS mutation partially bypasses the requirement for peptide activation and acts synergistically with mutations that disrupt contacts between the protease and PDZ domains. Biochemical results support an allosteric model, in which these mutations, active-site modification, and peptide/substrate binding act in concert to stabilize proteolytically active DegS. Cocrystal structures of DegS in complex with different OMP peptides reveal activation of the protease domain with varied conformations of the PDZ domain and without specific contacts from the bound OMP peptide. Taken together, these results indicate that the binding of OMP peptides activates proteolysis principally by relieving inhibitory contacts between the PDZ domain and the protease domain of DegS.

  13. OMP Peptides Activate the DegS Stress-Sensor Protease by a Relief of Inhibition Mechanism

    SciTech Connect

    Sohn, Jungsan; Grant, Robert A.; Sauer, Robert T.; MIT

    2010-03-19

    In the E. coli periplasm, C-terminal peptides of misfolded outer-membrane porins (OMPs) bind to the PDZ domains of the trimeric DegS protease, triggering cleavage of a transmembrane regulator and transcriptional activation of stress genes. We show that an active-site DegS mutation partially bypasses the requirement for peptide activation and acts synergistically with mutations that disrupt contacts between the protease and PDZ domains. Biochemical results support an allosteric model, in which these mutations, active-site modification, and peptide/substrate binding act in concert to stabilize proteolytically active DegS. Cocrystal structures of DegS in complex with different OMP peptides reveal activation of the protease domain with varied conformations of the PDZ domain and without specific contacts from the bound OMP peptide. Taken together, these results indicate that the binding of OMP peptides activates proteolysis principally by relieving inhibitory contacts between the PDZ domain and the protease domain of DegS.

  14. Labile carbon regulates protease activity and nitrogen acquisition in boreal forest topsoil.

    NASA Astrophysics Data System (ADS)

    Lindén, A.; Heinonsalo, J.; Oinonen, M.; Sonninen, E.; Hilasvuori, E.; Pumpanen, J.

    2012-04-01

    In boreal zone, soil organic matter (SOM) contains a substantial amount of recalcitrant material and forms a large nitrogen pool. However, this pool is to a great extent inaccessible to plants, due to its low decomposability. Although, the nitrogen supply is the most limiting factor of net ecosystem production (NEP) in boreal forests, it has been speculated that as a result of the accelerated decomposition of SOM induced by climate warming, part of this nitrogen pool could be released. It has also been shown that a substantial proportion of gross primary production (GPP) is allocated below ground and acts as an energy source for decomposing rhizomicrobial organisms, and that changes in the GPP rate could therefore increase the belowground turn over rate of otherwise recalcitrant nitrogen-rich SOM. We were studying the effects of increasing labile carbon input on the symbiotic microbial N acquisition and protease activities in a controlled microcosm experiment. We compared the natural abundance of isotope ratios of 13C and 14C in soil CO2efflux, protease enzyme activity, natural abundance of 15N in the needles, and microbial biomass in microcosms containing bare soil and tree seedlings. In addition, we had treatments were additional energy was given to the bare soil and seedling microcosms in the form of glucose. The age of the CO2 originating from the decomposition process of SOM was older in all treatments where easily decomposable carbon (energy) was available for soil microorganisms. The increased natural abundance of 15N in the needles of the seedlings treated with glucose, suggests a shift in nitrogen acquisition to different SOM pool, which was reflected strongly to the total N content of the SOM and evolving 13C signature in soil CO2 efflux. The protease activity was highest in treatments with artificial glucose addition. Our results suggest that the increased input of easily available carbon from aboveground enables the decomposition of recalcitrant

  15. Novel antiviral activity and mechanism of bromocriptine as a Zika virus NS2B-NS3 protease inhibitor.

    PubMed

    Chan, Jasper Fuk-Woo; Chik, Kenn Ka-Heng; Yuan, Shuofeng; Yip, Cyril Chik-Yan; Zhu, Zheng; Tee, Kah-Meng; Tsang, Jessica Oi-Ling; Chan, Chris Chung-Sing; Poon, Vincent Kwok-Man; Lu, Gang; Zhang, Anna Jinxia; Lai, Kin-Kui; Chan, Kwok-Hung; Kao, Richard Yi-Tsun; Yuen, Kwok-Yung

    2017-02-07

    Zika virus (ZIKV) infection is associated with congenital malformations in infected fetuses and severe neurological and other systemic complications in adults. There are currently limited anti-ZIKV treatment options that are readily available and safe for use in pregnancy. In this drug repurposing study, bromocriptine was found to have inhibitory effects on ZIKV replication in cytopathic effect inhibition, virus yield reduction, and plaque reduction assays. Time-of-drug-addition assay showed that bromocriptine exerted anti-ZIKV activity between 0 and 12 h post-ZIKV inoculation, corroborating with post-entry events in the virus replication cycle prior to budding. Our docking model showed that bromocriptine interacted with several active site residues of the proteolytic cavity involving H51 and S135 in the ZIKV-NS2B-NS3 protease protein, and might occupy the active site and inhibit the protease activity of the ZIKV-NS2B-NS3 protein. A fluorescence-based protease inhibition assay confirmed that bromocriptine inhibited ZIKV protease activity. Moreover, bromocriptine exhibited synergistic effect with interferon-α2b against ZIKV replication in cytopathic effect inhibition assay. The availability of per vagina administration of bromocriptine as suppositories or vaginoadhesive discs and the synergistic anti-ZIKV activity between bromocriptine and type I interferon may make bromocriptine a potentially useful and readily available treatment option for ZIKV infection. The anti-ZIKV effects of bromocriptine should be evaluated in a suitable animal model.

  16. Neutrophil maturation rate determines the effects of dipeptidyl peptidase 1 inhibition on neutrophil serine protease activity

    PubMed Central

    Wikell, C; Clifton, S; Shearer, J; Benjamin, A; Peters, S A

    2016-01-01

    Background and Purpose Neutrophil serine proteases (NSPs) are activated by dipeptidyl peptidase 1 (DPP1) during neutrophil maturation. The effects of neutrophil turnover rate on NSP activity following DPP1 inhibition was studied in a rat pharmacokinetic/pharmacodynamic model. Experimental Approach Rats were treated with a DPP1 inhibitor twice daily for up to 14 days; NSP activity was measured in onset or recovery studies, and an indirect response model was fitted to the data to estimate the turnover rate of the response. Key Results Maximum NSP inhibition was achieved after 8 days of treatment and a reduction of around 75% NSP activity was achieved at 75% in vitro DPP1 inhibition. Both the rate of inhibition and recovery of NSP activity were consistent with a neutrophil turnover rate of between 4–6 days. Using human neutrophil turnover rate, it is predicted that maximum NSP inhibition following DPP1 inhibition takes around 20 days in human. Conclusions and Implications Following inhibition of DPP1 in the rat, the NSP activity was determined by the amount of DPP1 inhibition and the turnover of neutrophils and is thus supportive of the role of neutrophil maturation in the activation of NSPs. Clinical trials to monitor the effect of a DPP1 inhibitor on NSPs should take into account the delay in maximal response on the one hand as well as the potential delay in a return to baseline NSP levels following cessation of treatment. PMID:27186823

  17. The effect of the P1 side chain on the binding of optimized carboxylate and activated carbonyl inhibitors of the hepatitis C virus NS3 protease.

    PubMed

    Kawai, Stephen H; LaPlante, Steven R; Llinàs-Brunet, Montse; Hucke, Oliver

    2010-07-01

    Peptidyl inhibitors of the hepatitis C virus NS3 protease hold much promise as direct-acting antiviral agents against hepatitis C infection. The optimization of N-terminal cleavage products, found to exhibit activity (product inhibition) against the enzyme, has led to potent tripeptide inhibitors that bear free C-terminal carboxylate groups. An analogous activated carbonyl compound (pentafluoroethyl ketone) bearing a P1 norvaline (Nva) was found to possess comparable activity against hepatitis C virus protease. However, an analogue bearing an aminocyclopropylcarboxylic acid (Acca) P1 residue exhibited very poor activity. (19)F-NMR studies indicate that the propensity of the Acca-derived activated carbonyl to form hemiketals is only slightly reduced compared with that of a P1 Nva equivalent. These results, as well as molecular modeling studies, argue against steric hindrance of the nucleophilic attack of Ser-139 accounting for the poor mechanism-based inhibition by the former. We hypothesize that the conformational properties of the respective C-termini in the context of an adaptable active site account for the divergent P1 structure-activity relationships.

  18. Fibrin(ogen)olytic and antiplatelet activities of a subtilisin-like protease from Solanum tuberosum (StSBTc-3).

    PubMed

    Pepe, Alfonso; Frey, María Eugenia; Muñoz, Fernando; Fernández, María Belén; Pedraza, Anabela; Galbán, Gustavo; García, Diana Noemí; Daleo, Gustavo Raúl; Guevara, María Gabriela

    2016-06-01

    Plant serine proteases have been widely used in food science and technology as well as in medicine. In this sense, several plant serine proteases have been proposed as potential anti-coagulants and anti-platelet agents. Previously, we have reported the purification and identification of a plant serine protease from Solanum tuberosum leaves. This potato enzyme, named as StSBTc-3, has a molecular weight of 72 kDa and it was characterized as a subtilisin like protease. In this work we determine and characterize the biochemical and medicinal properties of StSBTc-3. Results obtained show that, like the reported to other plant serine proteases, StSBTc-3 is able to degrade all chains of human fibrinogen and to produces fibrin clot lysis in a dose dependent manner. The enzyme efficiently hydrolyzes β subunit followed by partially hydrolyzed α and γ subunits of human fibrinogen. Assays performed to determine StSBTc-3 substrate specificity using oxidized insulin β-chain as substrate, show seven cleavage sites: Asn3-Gln4; Cys7-Gly8; Glu13-Ala14; Leu15-Tyr16; Tyr16-Leu17; Arg22-Gly23 and Phe25-Tyr26, all of them were previously reported for other serine proteases with fibrinogenolytic activity. The maximum StSBTc-3 fibrinogenolytic activity was determined at pH 8.0 and at 37 C. Additionally, we demonstrate that StSBTc-3 is able to inhibit platelet aggregation and is unable to exert cytotoxic activity on human erythrocytes in vitro at all concentrations assayed. These results suggest that StSBTc-3 could be evaluated as a new agent to be used in the treatment of thromboembolic disorders such as strokes, pulmonary embolism and deep vein thrombosis.

  19. T Cell Determinants Incorporating [beta]-Amino Acid Residues Are Protease Resistant and Remain Immunogenic In Vivo

    SciTech Connect

    Webb, Andrew I.; Dunstone, Michelle A.; Williamson, Nicholas A.; Price, Jason D.; Kauwe, Andreade; Chen, Weisan; Oakley, Aaron; Perlmutter, Patrick; McCluskey, James; Aguilar, Marie-Isabel; Rossjohn, Jamie; Purcell, Anthony W.

    2010-07-20

    A major hurdle in designing successful epitope-based vaccines resides in the delivery, stability, and immunogenicity of the peptide immunogen. The short-lived nature of unmodified peptide-based vaccines in vivo limits their therapeutic application in the immunotherapy of cancers and chronic viral infections as well as their use in generating prophylactic immunity. The incorporation of {beta}-amino acids into peptides decreases proteolysis, yet its potential application in the rational design of T cell mimotopes is poorly understood. To address this, we have replaced each residue of the SIINFEKL epitope individually with the corresponding {beta}-amino acid and examined the resultant efficacy of these mimotopes. Some analogs displayed similar MHC binding and superior protease stability compared with the native epitope. Importantly, these analogs were able to generate cross-reactive CTLs in vivo that were capable of lysing tumor cells that expressed the unmodified epitope as a surrogate tumor Ag. Structural analysis of peptides in which anchor residues were substituted with {beta}-amino acids revealed the basis for enhanced MHC binding and retention of immunogenicity observed for these analogs and paves the way for future vaccine design using {beta}-amino acids. We conclude that the rational incorporation of {beta}-amino acids into T cell determinants is a powerful alternative to the traditional homologous substitution of randomly chosen naturally occurring {alpha}-amino acids, and these mimotopes may prove particularly useful for inclusion in epitope-based vaccines.

  20. Dipeptidyl peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis

    PubMed Central

    Adkison, April M.; Raptis, Sofia Z.; Kelley, Diane G.; Pham, Christine T.N.

    2002-01-01

    Leukocyte recruitment in inflammation is critical for host defense, but excessive accumulation of inflammatory cells can lead to tissue damage. Neutrophil-derived serine proteases (cathepsin G [CG], neutrophil elastase [NE], and proteinase 3 [PR3]) are expressed specifically in mature neutrophils and are thought to play an important role in inflammation. To investigate the role of these proteases in inflammation, we generated a mouse deficient in dipeptidyl peptidase I (DPPI) and established that DPPI is required for the full activation of CG, NE, and PR3. Although DPPI–/– mice have normal in vitro neutrophil chemotaxis and in vivo neutrophil accumulation during sterile peritonitis, they are protected against acute arthritis induced by passive transfer of monoclonal antibodies against type II collagen. Specifically, there is no accumulation of neutrophils in the joints of DPPI–/– mice. This protective effect correlates with the inactivation of neutrophil-derived serine proteases, since NE–/– × CG–/– mice are equally resistant to arthritis induction by anti-collagen antibodies. In addition, protease-deficient mice have decreased response to zymosan- and immune complex–mediated inflammation in the subcutaneous air pouch. This defect is accompanied by a decrease in local production of TNF-α and IL-1β. These results implicate DPPI and polymorphonuclear neutrophil–derived serine proteases in the regulation of cytokine production at sites of inflammation. PMID:11827996

  1. A novel alkaline protease with antiproliferative activity from fresh fruiting bodies of the toxic wild mushroom Amanita farinosa.

    PubMed

    Sun, Jian; Zhao, Yongchang; Chai, Hongmei; Wang, Hexiang; Ng, Tzi Bun

    2011-01-01

    A novel protease with a molecular mass of 15 kDa was purified from fresh fruiting bodies of the wild mushroom Amanita farinosa. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and SP-Sepharose. It demonstrated a single 15-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and a 15-kDa peak in gel filtration. The optimal pH and optimal temperature of the protease were pH 8.0 and 65 °C, respectively. Proliferation of human hepatoma HepG2 cells was inhibited by the protease with an IC(50) of 25 µM. The protease did not have antifungal or ribonuclease activity.

  2. Construction, expression, and characterization of a novel fully activated recombinant single-chain hepatitis C virus protease.

    PubMed Central

    Taremi, S. S.; Beyer, B.; Maher, M.; Yao, N.; Prosise, W.; Weber, P. C.; Malcolm, B. A.

    1998-01-01

    Efficient proteolytic processing of essential junctions of the hepatitis C virus (HCV) polyprotein requires a heterodimeric complex of the NS3 bifunctional protease/helicase and the NS4A accessory protein. A single-chain recombinant form of the protease has been constructed in which NS4A residues 21-32 (GSVVIVGRIILS) were fused in frame to the amino terminus of the NS3 protease domain (residues 3-181) through a tetrapeptide linker. The single-chain recombinant protease has been overexpressed as a soluble protein in E. coli and purified to homogeneity by a combination of metal chelate and size-exclusion chromatography. The single-chain recombinant protease domain shows full proteolytic activity cleaving the NS5A-5B synthetic peptide substrate, DTEDVVCCSMSYTWTGK with a Km and k(cat) of 20.0 +/- 2.0 microM and 9.6 +/- 2.0 min(-1), respectively; parameters identical to those of the authentic NS3(1-631)/NS4A(1-54) protein complex generated in eukaryotic cells (Sali DL et al., 1998, Biochemistry 37:3392-3401). PMID:9792101

  3. MALT1 Protease Activation Triggers Acute Disruption of Endothelial Barrier Integrity via CYLD Cleavage.

    PubMed

    Klei, Linda R; Hu, Dong; Panek, Robert; Alfano, Danielle N; Bridwell, Rachel E; Bailey, Kelly M; Oravecz-Wilson, Katherine I; Concel, Vincent J; Hess, Emily M; Van Beek, Matthew; Delekta, Phillip C; Gu, Shufang; Watkins, Simon C; Ting, Adrian T; Gough, Peter J; Foley, Kevin P; Bertin, John; McAllister-Lucas, Linda M; Lucas, Peter C

    2016-09-27

    Microvascular endothelial cells maintain a tight barrier to prevent passage of plasma and circulating immune cells into the extravascular tissue compartment, yet endothelial cells respond rapidly to vasoactive substances, including thrombin, allowing transient paracellular permeability. This response is a cornerstone of acute inflammation, but the mechanisms responsible are still incompletely understood. Here, we demonstrate that thrombin triggers MALT1 to proteolytically cleave cylindromatosis (CYLD). Fragmentation of CYLD results in microtubule disruption and a cascade of events leading to endothelial cell retraction and an acute permeability response. This finding reveals an unexpected role for the MALT1 protease, which previously has been viewed mostly as a driver of pro-inflammatory NF-κB signaling in lymphocytes. Thus, MALT1 not only promotes immune cell activation but also acutely regulates endothelial cell biology, actions that together facilitate tissue inflammation. Pharmacologic inhibition of MALT1 may therefore have synergistic impact by targeting multiple disparate steps in the overall inflammatory response.

  4. Purification and characterization of the cold-active alkaline protease from marine cold-adaptive Penicillium chrysogenum FS010.

    PubMed

    Zhu, Hui-Yuan; Tian, Yong; Hou, Yun-Hua; Wang, Tian-Hong

    2009-11-01

    An extracellular cold-active alkaline serine protease from Penicillium chrysogenum FS010 has been purified. The purification procedure involved: ammonium sulfate precipitation, DEAE ion-exchange chromatography and sephadex G-100 gel chromatography. SDS-PAGE of the purified enzyme indicated a molecular weight of 41,000 +/- 1,000 Da. The protease is stable in a pH range of 7.0-9.0 and has a maximum activity at pH 9.0. Compared with other industrial proteases, the enzyme shows a high hydrolytic activities at lower temperatures and a high sensitivity at a temperature over 50 degrees C. The isoelectric point of the enzyme is approximate to 6.0. Enzymatic activity is enhanced by the addition of divalent cations such as Mg(2+) and Ca(2+) and inhibited by addition of Cu(2+)and Co(2+). PMSF and DFP are its specific inhibitors. The application of the cold-active alkaline protease is extremely extensive, and widely used in detergents, feed, food, leather and many other industries.

  5. A mouse-human phase 1 co-clinical trial of a protease-activated fluorescent probe for imaging cancer

    PubMed Central

    Whitley, Melodi Javid; Cardona, Diana M.; Lazarides, Alexander L.; Spasojevic, Ivan; Ferrer, Jorge M.; Cahill, Joan; Lee, Chang-Lung; Snuderl, Matija; Blazer, Dan G.; Hwang, E. Shelley; Greenup, Rachel A.; Mosca, Paul J.; Mito, Jeffrey K.; Cuneo, Kyle C.; Larrier, Nicole A.; O’Reilly, Erin K.; Riedel, Richard F.; Eward, William C.; Strasfeld, David B.; Fukumura, Dai; Jain, Rakesh K.; Lee, W. David; Griffith, Linda G.; Bawendi, Moungi G.; Kirsch, David G.; Brigman, Brian E.

    2016-01-01

    Local recurrence is a common cause of treatment failure for patients with solid tumors. Intraoperative detection of microscopic residual cancer in the tumor bed could be used to decrease the risk of a positive surgical margin, reduce rates of reexcision, and tailor adjuvant therapy. We used a protease-activated fluorescent imaging probe, LUM015, to detect cancer in vivo in a mouse model of soft tissue sarcoma (STS) and ex vivo in a first-in-human phase 1 clinical trial. In mice, intravenous injection of LUM015 labeled tumor cells, and residual fluorescence within the tumor bed predicted local recurrence. In 15 patients with STS or breast cancer, intravenous injection of LUM015 before surgery was well tolerated. Imaging of resected human tissues showed that fluorescence from tumor was significantly higher than fluorescence from normal tissues. LUM015 biodistribution, pharmacokinetic profiles, and metabolism were similar in mouse and human subjects. Tissue concentrations of LUM015 and its metabolites, including fluorescently labeled lysine, demonstrated that LUM015 is selectively distributed to tumors where it is activated by proteases. Experiments in mice with a constitutively active PEGylated fluorescent imaging probe support a model where tumor-selective probe distribution is a determinant of increased fluorescence in cancer. These co-clinical studies suggest that the tumor specificity of protease-activated imaging probes, such as LUM015, is dependent on both biodistribution and enzyme activity. Our first-in-human data support future clinical trials of LUM015 and other protease-sensitive probes. PMID:26738797

  6. A mouse-human phase 1 co-clinical trial of a protease-activated fluorescent probe for imaging cancer.

    PubMed

    Whitley, Melodi Javid; Cardona, Diana M; Lazarides, Alexander L; Spasojevic, Ivan; Ferrer, Jorge M; Cahill, Joan; Lee, Chang-Lung; Snuderl, Matija; Blazer, Dan G; Hwang, E Shelley; Greenup, Rachel A; Mosca, Paul J; Mito, Jeffrey K; Cuneo, Kyle C; Larrier, Nicole A; O'Reilly, Erin K; Riedel, Richard F; Eward, William C; Strasfeld, David B; Fukumura, Dai; Jain, Rakesh K; Lee, W David; Griffith, Linda G; Bawendi, Moungi G; Kirsch, David G; Brigman, Brian E

    2016-01-06

    Local recurrence is a common cause of treatment failure for patients with solid tumors. Intraoperative detection of microscopic residual cancer in the tumor bed could be used to decrease the risk of a positive surgical margin, reduce rates of reexcision, and tailor adjuvant therapy. We used a protease-activated fluorescent imaging probe, LUM015, to detect cancer in vivo in a mouse model of soft tissue sarcoma (STS) and ex vivo in a first-in-human phase 1 clinical trial. In mice, intravenous injection of LUM015 labeled tumor cells, and residual fluorescence within the tumor bed predicted local recurrence. In 15 patients with STS or breast cancer, intravenous injection of LUM015 before surgery was well tolerated. Imaging of resected human tissues showed that fluorescence from tumor was significantly higher than fluorescence from normal tissues. LUM015 biodistribution, pharmacokinetic profiles, and metabolism were similar in mouse and human subjects. Tissue concentrations of LUM015 and its metabolites, including fluorescently labeled lysine, demonstrated that LUM015 is selectively distributed to tumors where it is activated by proteases. Experiments in mice with a constitutively active PEGylated fluorescent imaging probe support a model where tumor-selective probe distribution is a determinant of increased fluorescence in cancer. These co-clinical studies suggest that the tumor specificity of protease-activated imaging probes, such as LUM015, is dependent on both biodistribution and enzyme activity. Our first-in-human data support future clinical trials of LUM015 and other protease-sensitive probes.

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

    PubMed Central

    Edwards, J. Vincent; Fontenot, Krystal R.; Prevost, Nicolette T.; Pircher, Nicole; Liebner, Falk; Condon, Brian D.

    2016-01-01

    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 diseases. The

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

    PubMed

    Edwards, J Vincent; Fontenot, Krystal R; Prevost, Nicolette T; Pircher, Nicole; Liebner, Falk; Condon, Brian D

    2016-10-26

    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 diseases. The

  9. Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes.

    PubMed

    Withana, Nimali P; Garland, Megan; Verdoes, Martijn; Ofori, Leslie O; Segal, Ehud; Bogyo, Matthew

    2016-01-01

    Active enzymes, such as proteases, often serve as valuable biomarkers for various disease pathologies. Therefore, methods to detect specific enzyme activities in biological samples can provide information to guide disease detection and diagnosis and to increase our understanding of the biological roles of specific enzyme targets. In this protocol, we outline methods for the topical application of fluorescently quenched activity-based probes (qABPs) to fresh-frozen tissue samples. This technique enables rapid imaging of enzyme activity at cellular resolution, and it can be combined with antibody labeling for immunodiagnosis. In this method, fresh-frozen tissue sections are fixed, incubated with the probe and imaged using fluorescence microscopy. This provides an advance over classical immunohistochemistry (IHC) in that it is rapid (4-8 h) and inexpensive, and it provides information on enzyme activity. Furthermore, it can be used with any of the growing number of fluorescent ABPs to provide data for more effective disease monitoring and diagnosis.

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

    PubMed

    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.

  11. Serine proteases inhibiting cyanopeptides.

    PubMed

    Radau, G

    2000-08-01

    There are many compounds inhibiting serine proteases which play an important role in the human organism. This article reviews publications on the low-molecular weight, serine protease inhibitory cyanopeptides and reports on new developments in establishing structure-activity relationships.

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

    PubMed

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

    2016-10-15

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

  13. Inherent chaperone-like activity of aspartic proteases reveals a distant evolutionary relation to double-ψ barrel domains of AAA-ATPases

    PubMed Central

    Hulko, Michael; Lupas, Andrei N.; Martin, Jörg

    2007-01-01

    Chaperones and proteases share the ability to interact with unfolded proteins. Here we show that enzymatically inactive forms of the aspartic proteases HIV-1 protease and pepsin have inherent chaperone-like activity and can prevent the aggregation of denatured substrate proteins. In contrast to proteolysis, which requires dimeric enzymes, chaperone-like activity could be observed also with monomeric domains. The involvement of the active site cleft in the chaperone-like function was demonstrated by the inhibitory effect of peptide substrate inhibitors. The high structural similarity between aspartic proteases and the N-terminal double-ψ barrels of Cdc48-like proteins, which are involved in the unfolding and dissociation of proteins, suggests that they share a common ancestor. The latent chaperone-like activity in aspartic proteases can be seen as a relic that has further evolved to serve substrate binding in the context of proteolytic activity. PMID:17384229

  14. Effects of temperature and sodium chloride concentration on the activities of proteases and amylases in soy sauce koji.

    PubMed

    Su, Nan-Wei; Wang, Mei-Ling; Kwok, Kam-Fu; Lee, Min-Hsiung

    2005-03-09

    This study investigated the effects of temperature and sodium chloride concentration on the proteolytic and amylolytic activities of soy sauce koji. The optimal temperatures for both protease and amylase were found in the range of 50-55 degrees C. The protease was not stable at 55 degrees C and retained only approximately 20% residual activity after incubation at 55 degrees C for 4 h. The protease was labile in sodium chloride solution, whereas the amylase was quite stable. The residual protease activity in an 18% NaCl solution was only approximately 3%. The harvested koji was mixed with 1.5 volumes of water (v/w) and incubated at 45 degrees C for 48 h; the total nitrogen and amino nitrogen contents were 1.3 and 0.56%, respectively. The results indicated that the hydrolysis of koji at the critical temperature of 45 degrees C could be employed as a rapid fermentation method to reduce the time for soy sauce manufacturing. According to this study, the combination of 5% sodium chloride and fermentation at 45 degrees C was considered as the best condition for the prohydrolysis of koji for making soy sauce. In addition, the critical temperature of 45 degrees C was very important when used in the preparation of protein hydrolysates for the flavoring industry and for the preparation of biologically active peptides.

  15. The serine protease motif of Pic mediates a dose-dependent mucolytic activity after binding to sugar constituents of the mucin substrate.

    PubMed

    Gutiérrez-Jiménez, Javier; Arciniega, Ivonne; Navarro-García, Fernando

    2008-08-01

    The pic gene is harbored on the chromosomes of three important pathogens: enteroaggregative Escherichia coli (EAEC), uropathogenic E. coli (UPEC), and Shigella flexneri. Since Pic is secreted into the intestinal lumen during EAEC infection, we sought to identify intestinal-mucosal substrates for Pic. Pic did not damage epithelial cells, cleave fodrin, or degrade host defense proteins embedded in the mucus layer (sIgA, lactoferrin and lysozyme). However, by using a solid-phase assay to evaluate the mucinolytic activity of EAEC Pic, we documented a specific, dose-dependent mucinolytic activity. A serine protease inhibitor and an enzymatically inactive variant of Pic were used to show that the Pic serine protease motif is required for mucinolytic activity. Pic binds mucin, and this binding was blocked in competition assays using monosaccharide constituents of the oligosaccharide side chains of mucin. Moreover, Pic mucinolytic activity decreased when sialic acid was removed from mucin. Thus, Pic is a mucinase with lectin-like activity that can be related to its reported hemagglutinin activity. Our results suggest that EAEC may secrete Pic into the intestinal lumen as a strategy for penetrating the gel-like mucus layer during EAEC colonization.

  16. Digestive system development and study of acid and alkaline protease digestive capacities using biochemical and molecular approaches in totoaba (Totoaba macdonaldi) larvae.

    PubMed

    Galaviz, Mario A; López, Lus M; García Gasca, Alejandra; Álvarez González, Carlos Alfonso; True, Conal D; Gisbert, Enric

    2015-10-01

    The present study aimed to describe and understand the development of the digestive system in totoaba (Totoaba macdonaldi) larvae from hatching to 40 days post-hatch (dph) from morphological and functional perspectives. At hatch, the digestive system of totoaba was undifferentiated. The anus and the mouth opened at 4 and 5 dph, respectively. During exogenous feeding, development of the esophagus, pancreas, liver and intestine was observed with a complete differentiation of all digestive organs. Expression and activity of trypsin and chymotrypsin were observed as early as at 1 dph, and increments in their expression and activity coincided with changes in food items (live and compound diets) and morpho-physiological development of the accessory digestive glands. In contrast, pepsin was detected later during development, which includes the appearance of the gastric glands between 24 and 28 dph. One peak in gene expression was detected at 16 dph, few days before the initial development of the stomach at 20 dph. A second peak of pepsin expression was detected at day 35, followed by a peak of activity at day 40, coinciding with the change from live to artificial food. Totoaba larvae showed a fully morphologically developed digestive system between 24 and 28 dph, as demonstrated by histological observations. However, gene expression and activity of alkaline and acid proteases were detected earlier, indicating the functionality of the exocrine pancreas and stomach before the complete morphological development of the digestive organs. These results showed that integrative studies are needed to fully understand the development of the digestive system from a morphological and functional point of views, since the histological organization of digestive structures does not reflect their real functionality. These results indicate that the digestive system of totoaba develops rapidly during the first days post-hatch, especially for alkaline proteases, and the stomach

  17. Protease-activated receptor 2 modulates proliferation and invasion of oral squamous cell carcinoma cells.

    PubMed

    Al-Eryani, Kamal; Cheng, Jun; Abé, Tatsuya; Maruyama, Satoshi; Yamazaki, Manabu; Babkair, Hamzah; Essa, Ahmed; Saku, Takashi

    2015-07-01

    Based on our previous finding that protease-activated receptor 2 (PAR-2) regulates hemophagocytosis of oral squamous cell carcinoma (SCC) cells, which induces their heme oxygenase 1-dependent keratinization, we have formulated a hypothesis that PAR-2 functions in wider activities of SCC cells. To confirm this hypothesis, we investigated immunohistochemical profiles of PAR-2 in oral SCC tissues and its functional roles in cell proliferation and invasion in SCC cells in culture. The PAR-2 expression modes were determined in 48 surgical tissue specimens of oral SCC. Using oral SCC-derived cell systems, we determined both gene and protein expression levels of PAR-2. SCC cell proliferation and invasive properties were also examined in conditions in which PAR-2 was activated by the synthetic peptide SLIGRL. PAR-2 was immunolocalized in oral SCC and carcinoma in situ cells, especially in those on the periphery of carcinoma cell foci (100% of cases), but not in normal oral epithelia. Its expression at both gene and protein levels was confirmed in 3 oral SCC cell lines including ZK-1. Activation of PAR-2 induced ZK-1 cell proliferation in a dose-dependent manner. PAR-2-activated ZK-1 cells invaded faster than nonactivated ones. The expression of PAR-2 is specific to oral malignancies, and PAR-2 regulates the growth and invasion of oral SCC cells.

  18. RbdB, a Rhomboid Protease Critical for SREBP Activation and Virulence in Aspergillus fumigatus

    PubMed Central

    Dhingra, Sourabh; Kowlaski, Caitlin H.; Thammahong, Arsa; Beattie, Sarah R.; Bultman, Katherine M.

    2016-01-01

    ABSTRACT SREBP transcription factors play a critical role in fungal virulence; however, the mechanisms of sterol regulatory element binding protein (SREBP) activation in pathogenic fungi remains ill-defined. Screening of the Neurospora crassa whole-genome deletion collection for genes involved in hypoxia responses identified a gene for an uncharacterized rhomboid protease homolog, rbdB, required for growth under hypoxic conditions. Loss of rbdB in Aspergillus fumigatus also inhibited growth under hypoxic conditions. In addition, the A. fumigatus ΔrbdB strain also displayed phenotypes consistent with defective SREBP activity, including increased azole drug susceptibility, reduced siderophore production, and full loss of virulence. Expression of the basic helix-loop-helix (bHLH) DNA binding domain of the SREBP SrbA in ΔrbdB restored all of the phenotypes linking RdbB activity with SrbA function. Furthermore, the N-terminal domain of SrbA containing the bHLH DNA binding region was absent from ΔrbdB under inducing conditions, suggesting that RbdB regulates the protein levels of this important transcription factor. As SrbA controls clinically relevant aspects of fungal pathobiology in A. fumigatus, understanding the mechanisms of SrbA activation provides opportunities to target this pathway for therapeutic development. IMPORTANCE Aspergillus fumigatus causes life-threatening infections, and treatment options remain limited. Thus, there is an urgent need to find new therapeutic targets to treat this deadly disease. Previously, we have shown that SREBP transcription factors and their regulatory components are critical for the pathobiology of A. fumigatus. Here we identify a role for RbdB, a rhomboid protease, as an essential component of SREBP activity. Our results indicate that mutants lacking rbdB have growth defects under hypoxic conditions, are hypersusceptible to voriconazole, lack extracellular siderophore production, and fail to cause disease in a murine

  19. Metalloproteinase-9 contributes to endothelial dysfunction in atherosclerosis via protease activated receptor-1

    PubMed Central

    Florence, Jon M.; Booshehri, Laela M.; Allen, Timothy C.; Kurdowska, Anna K.

    2017-01-01

    The atherosclerotic process begins when vascular endothelial cells undergo pro-inflammatory changes such as aberrant activation to dysfunctional phenotypes and apoptosis, leading to loss of vascular integrity. Our laboratory has demonstrated that exposure of mice to second hand smoke triggers an increase in expression of metalloproteinase-9. Further, metalloproteinase-9 released by second hand smoke—activated leukocytes may propagate pro-atherogenic alterations in endothelial cells. We have shown that levels of metalloproteinase-9 were increased in the plasma from apolipoprotein E deficient (ApoE-/-) mice exposed to second hand smoke relative to non-exposed controls. Moreover, we have collected data from two different, but complementary, treatments of second hand smoke exposed atherosclerotic mice. Animals received either cell specific metalloproteinase-9 directed siRNA to minimize metalloproteinase-9 expression in neutrophils and endothelial cells, or a pharmacological inhibitor of Bruton’s tyrosine kinase which indirectly limits metalloproteinase-9 production in neutrophils. These treatments reduced atherosclerotic changes in mice and improved overall vascular health. We also demonstrated that metalloproteinase-9 could activate endothelial cells and induce their apoptosis via cleavage of protease activated receptor-1. In summary, better understanding of metalloproteinase-9’s pathogenic capabilities as well as novel signaling pathways involved may lead to development of treatments which may provide additional benefits to atherosclerosis patients with a history of second hand smoke exposure. PMID:28166283

  20. Hydroxyproline substitutions stabilize non-glycosylated drosocin against serum proteases without challenging its antibacterial activity.

    PubMed

    Knappe, Daniel; Cassone, Marco; Nollmann, Friederike Inga; Otvos, Laszlo; Hoffmann, Ralf

    2014-04-01

    The increasing incidence of multi- and pan-resistant pathogens demands novel compounds to fight Grampositive and especially Gram-negative bacteria. Among the currently investigated compound classes, antimicrobial peptides (AMPs) inhibiting specific bacterial targets appear especially promising for systemic therapy of infections, although unmodified linear peptides are typically rapidly degraded by serum proteases. Proline-rich AMPs have been heavily investigated in recent years due to their low toxicity and proven in vivo efficacy. Here, we report novel unglycosylated drosocin analogs with extended half-life in mouse serum and improved activity against Gram-negative pathogens Escherichia coli and Klebsiella pneumoniae. Substituting proline (Pro) residues in positions 3, 5, 10, and 14 with trans-4-hydroxy-Lproline ((t)Hyp) improved the antibacterial activity, whereas substitution of Pro-16 reduced the activity. Drosocin analogs with (t)Hyp in positions 3 and 5 were also four to eight times more stable in mouse serum than the unmodified analog. The new compounds were not toxic against human HeLa, HEK293, and HepG2 cell lines and showed no hemolytic activity against human erythrocytes at peptide concentrations of at least 600 µg/mL.

  1. Cyclic strain increases protease-activated receptor-1 expression in vascular smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Nguyen, K. T.; Frye, S. R.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

    2001-01-01

    Cyclic strain regulates many vascular smooth muscle cell (VSMC) functions through changing gene expression. This study investigated the effects of cyclic strain on protease-activated receptor-1 (PAR-1) expression in VSMCs and the possible signaling pathways involved, on the basis of the hypothesis that cyclic strain would enhance PAR-1 expression, reflecting increased thrombin activity. Uniaxial cyclic strain (1 Hz, 20%) of cells cultured on elastic membranes induced a 2-fold increase in both PAR-1 mRNA and protein levels. Functional activity of PAR-1, as assessed by cell proliferation in response to thrombin, was also increased by cyclic strain. In addition, treatment of cells with antioxidants or an NADPH oxidase inhibitor blocked strain-induced PAR-1 expression. Preincubation of cells with protein kinase inhibitors (staurosporine or Ro 31-8220) enhanced strain-increased PAR-1 expression, whereas inhibitors of NO synthase, tyrosine kinase, and mitogen-activated protein kinases had no effect. Cyclic strain in the presence of basic fibroblast growth factor induced PAR-1 mRNA levels beyond the effect of cyclic strain alone, whereas no additive effect was observed between cyclic strain and platelet-derived growth factor-AB. Our findings that cyclic strain upregulates PAR-1 mRNA expression but that shear stress downregulates this gene in VSMCs provide an opportunity to elucidate signaling differences by which VSMCs respond to different mechanical forces.

  2. Signalling pathways induced by protease-activated receptors and integrins in T cells.

    PubMed

    Bar-Shavit, Rachel; Maoz, Miriam; Yongjun, Yin; Groysman, Maya; Dekel, Idit; Katzav, Shulamit

    2002-01-01

    Recent characterization of the thrombin receptor indicates that it plays a role in T-cell signalling pathways. However, little is known regarding the signalling events following stimulation of additional members of the protease-activated receptor (PAR) family, i.e. PAR2 and PAR3. Most of the postligand cascades are largely unknown. Here, we illustrate that in Jurkat T-leukaemic cells, activation of PAR1, PAR2 and PAR3 induce tyrosine phosphorylation of Vav1. This response was impaired in Jurkat T cells deficient in p56lck (JCaM1.6). Activation of PARs also led to an increase in tyrosine phosphorylation of ZAP-70 and SLP-76, two key proteins in T-cell receptor (TCR) signalling. We also demonstrated that p56lck is meaningful for integrin signalling. Thus, JCaM1.6 cells exhibited a marked reduction in their adherence to fibronectin-coated plates, as compared to the level of adherence of Jurkat T cells. While the phosphorylation of Vav1 in T cells is augmented following adhesion, no additional increase was noted following treatment of the adhered cells with PARs. Altogether, we have identified key components in the postligand-signalling cascade of PARs and integrins. Furthermore, we have identified Lck as a critical and possibly upstream component of PAR-induced Vav1 phosphorylation, as well as integrin activation, in Jurkat T cells.

  3. Protease-activated receptors (PARs)--biology and role in cancer invasion and metastasis.

    PubMed

    Wojtukiewicz, Marek Z; Hempel, Dominika; Sierko, Ewa; Tucker, Stephanie C; Honn, Kenneth V

    2015-12-01

    Although many studies have demonstrated that components of the hemostatic system may be involved in signaling leading to cancer progression, the potential mechanisms by which they contribute to cancer dissemination are not yet precisely understood. Among known coagulant factors, tissue factor (TF) and thrombin play a pivotal role in cancer invasion. They may be generated in the tumor microenvironment independently of blood coagulation and can induce cell signaling through activation of protease-activated receptors (PARs). PARs are transmembrane G-protein-coupled receptors (GPCRs) that are activated by a unique proteolytic mechanism. They play important roles in vascular physiology, neural tube closure, hemostasis, and inflammation. All of these agents (TF, thrombin, PARs-mainly PAR-1 and PAR-2) are thought to promote cancer invasion and metastasis at least in part by facilitating tumor cell migration, angiogenesis, and interactions with host vascular cells, including platelets, fibroblasts, and endothelial cells lining blood vessels. Here, we discuss the role of PARs and their activators in cancer progression, focusing on TF- and thrombin-mediated actions. Therapeutic options tailored specifically to inhibit PAR-induced signaling in cancer patients are presented as well.

  4. Protease-activated receptor-1 deficiency protects against streptozotocin-induced diabetic nephropathy in mice

    PubMed Central

    Waasdorp, Maaike; Duitman, JanWillem; Florquin, Sandrine; Spek, C. Arnold

    2016-01-01

    Endogenously administered activated protein C ameliorates diabetic nephropathy (DN) in a protease-activated receptor-1 (PAR-1)-dependent manner, suggesting that PAR-1 activation limits the progression of DN. Activation of PAR-1 in fibroblast-like cells, however, induces proliferation and extracellular matrix production, thereby driving fibrotic disease. Considering the key role of mesangial proliferation and extracellular matrix production during DN, PAR-1 may in fact potentiate diabetes-induced kidney injury. To determine the net effect of PAR-1 in DN, streptozotocin-induced DN was studied in wild type and PAR-1 deficient mice. Subsequent mechanistic insight was obtained by assessing profibrotic responses of mesangial and tubular epithelial cells in vitro, following PAR-1 stimulation and inhibition. Despite having similar glucose levels, PAR-1 deficient mice developed less kidney damage after induction of diabetes, as evidenced by diminished proteinuria, plasma cystatin C levels, expansion of the mesangial area, and tubular atrophy. In vitro, PAR-1 signaling in mesangial cells led to increased proliferation and expression of matrix proteins fibronectin and collagen IV. Conversely, a reduction in both proliferation and fibronectin deposition was observed in diabetic PAR-1 deficient mice. Overall, we show that PAR-1 plays an important role in the development of DN and PAR-1 might therefore be an attractive therapeutic target to pursue in DN. PMID:27618774

  5. Protease modulation of the activity of the epithelial sodium channel expressed in Xenopus oocytes.

    PubMed

    Chraïbi, A; Vallet, V; Firsov, D; Hess, S K; Horisberger, J D

    1998-01-01

    We have investigated the effect of extracellular proteases on the amiloride-sensitive Na+ current (INa) in Xenopus oocytes expressing the three subunits alpha, beta, and gamma of the rat or Xenopus epithelial Na+ channel (ENaC). Low concentrations of trypsin (2 microg/ml) induced a large increase of INa within a few minutes, an effect that was fully prevented by soybean trypsin inhibitor, but not by amiloride. A similar effect was observed with chymotrypsin, but not with kallikrein. The trypsin-induced increase of INa was observed with Xenopus and rat ENaC, and was very large (approximately 20-fold) with the channel obtained by coexpression of the alpha subunit of Xenopus ENaC with the beta and gamma subunits of rat ENaC. The effect of trypsin was selective for ENaC, as shown by the absence of effect on the current due to expression of the K+ channel ROMK2. The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-gammaS, suggesting that this effect was not mediated by G proteins. Measurement of the channel protein expression at the oocyte surface by antibody binding to a FLAG epitope showed that the effect of trypsin was not accompanied by an increase in the channel protein density, indicating that proteolysis modified the activity of the channel present at the oocyte surface rather than the cell surface expression. At the single channel level, in the cell-attached mode, more active channels were observed in the patch when trypsin was present in the pipette, while no change in channel activity could be detected when trypsin was added to the bath solution around the patch pipette. We conclude that extracellular proteases are able to increase the open probability of the epithelial sodium channel by an effect that does not occur through activation of a G protein-coupled receptor, but rather through proteolysis of a protein that is either a constitutive part of the channel itself or closely associated with it.

  6. Transcriptional activation of the human cytotoxic serine protease gene CSP-B in T lymphocytes.

    PubMed Central

    Hanson, R D; Ley, T J

    1990-01-01

    The cytotoxic serine protease B (CSP-B) gene is activated during cytotoxic T-lymphocyte maturation. In this report, we demonstrate that the PEER T-cell line (bearing gamma/delta T-cell receptors) accumulates CSP-B mRNA following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) and N6-2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (bt2cAMP) because of transcriptional activation of the CSP-B gene. TPA and bt2cAMP act synergistically to induce CSP-B expression, since neither agent alone causes activation of CSP-B transcription or mRNA accumulation. Chromatin upstream from the CSP-B gene is resistant to DNase I digestion in untreated PEER cells, but becomes sensitive following TPA-bt2cAMP treatment. Upon activation of PEER cells, a DNase I-hypersensitive site forms upstream from the CSP-B gene within a region that is highly conserved in the mouse. Transient transfection of CSP-B promoter constructs identified two regulatory regions in the CSP-B 5'-flanking sequence, located at positions -609 to -202 and positions -202 to -80. The region from -615 to -63 is sufficient to activate a heterologous promoter in activated PEER cells, but activation is orientation specific, suggesting that this region behaves as an upstream promoter element rather than a classical enhancer. Consensus AP-1, AP-2, and cAMP response elements are found upstream from the CSP-B gene (as are several T-cell-specific consensus elements), but the roles of these elements in CSP-B gene activation have yet to be determined. Images PMID:2233710

  7. Protease inhibitor monotherapy is associated with a higher level of monocyte activation, bacterial translocation and inflammation

    PubMed Central

    Torres, Berta; Guardo, Alberto C; Leal, Lorna; Leon, Agathe; Lucero, Constanza; Alvarez-Martinez, Míriam J; Martinez, Miguel J; Vila, Jordi; Martínez-Rebollar, María; González-Cordón, Ana; Gatell, Josep M; Plana, Montserrat; García, Felipe

    2014-01-01

    Introduction Monotherapy with protease-inhibitors (MPI) may be an alternative to cART for HIV treatment. We assessed the impact of this strategy on immune activation, bacterial translocation and inflammation. Methods We performed a cross-sectional study comparing patients on successful MPI (n=40) with patients on cART (n=20). Activation, senescence, exhaustion and differentiation stage in CD4+ and CD8+ T lymphocyte subsets, markers of monocyte activation, microbial translocation, inflammation, coagulation and low-level viremia were assessed. Results CD4+ or CD8+ T lymphocyte subset parameters were not significantly different between both groups. Conversely, as compared with triple cART, MPI patients showed a higher proportion of activated monocytes (CD14+ CD16−CD163+ cells, p=0.031), soluble markers of monocyte activation (sCD14 p=0.004, sCD163 p=0.002), microbial translocation (lipopolysaccharide (LPS)-binding protein; LBP p=0.07), inflammation (IL-6 p=0.04) and low-level viremia (p=0.035). In a multivariate model, a higher level of CD14+ CD16−CD163+ cells and sCD14, and presence of very low-level viremia were independently associated with MPI. Monocyte activation was independently associated with markers of inflammation (IL-6, p=0.006), microbial translocation (LBP, p=0.01) and low-level viremia (p=0.01). Conclusions Patients on MPI showed a higher level of monocyte activation than patients on standard therapy. Microbial translocation and low-level viremia were associated with the high level of monocyte activation observed in patients on MPI. The long-term clinical consequences of these findings should be assessed. PMID:25280865

  8. Assessment of protease activity in hydrolysed extracts from SSF of hair waste by and indigenous consortium of microorganisms.

    PubMed

    Abu Yazid, Noraziah; Barrena, Raquel; Sánchez, Antoni

    2016-03-01

    Hair wastes from the tannery industry were assessed for its suitability as substrates for protease production by solid-state fermentation (SSF) using a pilot-batch mode operation and anaerobically digested sludge as co-substrate. Maximum protease activity (52,230±1601 U g(-1) DM) was observed at the 14th day of SSF. Single step purification resulted in 2 fold purification with 74% of recovery by ultrafiltration with 10 kDa cut-off. The recovered enzyme was stable at a temperature of 30°C and pH 11; optimal conditions that were determined by a central composite full factorial experimental design. The enzyme activity was inhibited by phenylmethylsulfonyl fluoride, which indicates that it belongs to serine protease group. The remaining solid material after protease extraction could be easily stabilized to obtain a final good quality compost-like material as the final dynamic respiration index was lower than 1 g O2 kg(-1) OM h(-1). The lyophilized recovered enzymes were a good alternative in the process of cowhides dehairing with respect to the current chemical treatment, avoiding the production of solid wastes and highly polluted wastewaters. In conclusion, the entire process can be considered a low-cost sustainable technology for the dehairing process, closing the organic matter cycle in the form of value added product and a compost-like material from a waste.

  9. Characterization of the in vitro activities of the P1 and helper component proteases of Soybean mosaic virus Strain G2 and Tobacco vein mottling virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potyviruses express their RNA genomes through the production of polyproteins that are processed in host cells by three virus-encoded proteases. Soybean plants produce large amounts of protease inhibitors during seed development and in response to wounding that could affect the activities of these pr...

  10. Protease-Activated Receptor 4 Deficiency Offers Cardioprotection after Acute Ischemia Reperfusion Injury

    PubMed Central

    Kolpakov, Mikhail A.; Rafiq, Khadija; Guo, Xinji; Hooshdaran, Bahman; Wang, Tao; Vlasenko, Liudmila; Bashkirova, Yulia V.; Zhang, Xiaoxiao; Chen, Xiongwen; Iftikhar, Sahar; Libonati, Joseph R.; Kunapuli, Satya P.; Sabri, Abdelkarim

    2016-01-01

    Protease-activated receptor (PAR)4 is a low affinity thrombin receptor with less understood function relative to PAR1. PAR4 is involved in platelet activation and hemostasis, but its specific actions on myocyte growth and cardiac function remain unknown. This study examined the role of PAR4 deficiency on cardioprotection after myocardial ischemia-reperfusion (IR) injury in mice. When challenged by in vivo or ex vivo IR, PAR4 knockout (KO) mice exhibited increased tolerance to injury, which was manifest as reduced infarct size and a more robust functional recovery compared to wild-type mice. PAR4 KO mice also showed reduced cardiomyocyte apoptosis and putative signaling shifts in survival pathways in response to IR. Inhibition of PAR4 expression in isolated cardiomyocytes by shRNA offered protection against thrombin and PAR4-agonist peptide-induced apoptosis, while overexpression of wild-type PAR4 significantly enhanced the susceptibility of cardiomyocytes to apoptosis, even under low thrombin concentrations. Further studies implicate Src- and epidermal growth factor receptor-dependent activation of JNK on the proapoptotic effect of PAR4 in cardiomyocytes. These findings reveal a pivotal role for PAR4 as a regulator of cardiomyocyte survival and point to PAR4 inhibition as a therapeutic target offering cardioprotection after acute IR injury. PMID:26643815

  11. Comparative Study on Biochemical Properties and Antioxidative Activity of Cuttlefish (Sepia officinalis) Protein Hydrolysates Produced by Alcalase and Bacillus licheniformis NH1 Proteases

    PubMed Central

    Balti, Rafik; Bougatef, Ali; El Hadj Ali, Nedra; Ktari, Naourez; Jellouli, Kemel; Nedjar-Arroume, Naima; Dhulster, Pascal; Nasri, Moncef

    2011-01-01

    Antioxidative activities and biochemical properties of protein hydrolysates prepared from cuttlefish (Sepia officinalis) using Alcalase 2.4 L and Bacillus licheniformis NH1 proteases with different degrees of hydrolysis (DH) were determined. For the biochemical properties, hydrolysis by both enzymes increased protein solubility to above 75% over a wide pH range. The antioxidant activities of cuttlefish protein hydrolysates (CPHs) increase with increasing DH. In addition, all CPHs exhibited antioxidative activity in a concentration-dependent manner. NH1-CPHs generally showed greater antioxidative activity than Alcalase protein hydrolysates (P < 0.05) as indicated by the higher 1,1-diphenyl-1-picryhydrazyl (DPPH) radical scavenging activity and ferrous chelating activity. Both Alcalase and NH1 protein hydrolysates were able to retard lipid peroxidation and β-carotene-linoleic acid oxidation. Alcalase-CPH (DH = 12.5%) and NH1-CPH (DH = 15%) contained 75.36% and 80.11% protein, respectively, with histidine and arginine as the major amino acids, followed by glutamic acid/glutamine, serine, lysine, and leucine. In addition, CPHs have a high percentage of essential amino acids made up 48.85% and 50.04%. Cuttlefish muscle protein hydrolysates had a high nutritional value and could be used as supplement to poorly balanced dietary proteins. PMID:22312455

  12. 'Pergularain e I'--a plant cysteine protease with thrombin-like activity from Pergularia extensa latex.

    PubMed

    Shivaprasad, Holenarasipura V; Rajaiah, Rajesh; Frey, Brigitte M; Frey, Felix J; Vishwanath, Bannikuppe S

    2010-03-01

    Pergularain e I, a cysteine protease with thrombin-like activity, was purified by ion exchange chromatography from the latex of Pergularia extensa. Its homogeneity was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of pergularain e I by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was found to be 23.356 kDa and the N-terminal sequence is L-P-H-D-V-E. Pergularain e I is a glycoprotein containing approximately 20% of carbohydrate. Pergularain e I constituted 6.7% of the total protein with a specific activity of 9.5 units/mg/min with a 2.11-fold increased purity. Proteolytic activity of the pergularain e I was completely inhibited by iodoacetic acid (IAA). Pergularain e I exhibited procoagulant activity with citrated plasma and fibrinogen similar to thrombin. Pergularain e I increases the absorbance of fibrinogen solution in concentration-dependent and time-dependent manner. At 10 microg concentration, an absorbance of 0.48 was reached within 10 min of incubation time. Similar absorbance was observed when 0.2 NIH units of thrombin were used. Thrombin-like activity of pergularain e I is because of the selective hydrolysis of A alpha and B beta chains of fibrinogen and gamma-chain was observed to be insusceptible to hydrolysis. Molecular masses of the two peptide fragments released from fibrinogen due to the hydrolysis by pergularain e I at 5-min incubation time were found to be 1537.21 and 1553.29 and were in close agreement with the molecular masses of 16 amino acid sequence of fibrinopeptide A and 14 amino acid sequence of fibrinopeptide B, respectively. Prolonged fibrinogen-pergularain e I incubation releases additional peptides and their sequence comparison of molecular masses of the released peptides suggested that pergularain e I hydrolyzes specifically after arginine residues.

  13. Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13

    PubMed Central

    Gogia, Shobhit; Lo, Chi Y.; Neelamegham, Sriram

    2015-01-01

    Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594–1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly

  14. Characterization, cloning, and heterologous expression of a subtilisin-like serine protease gene VlPr1 from Verticillium lecanii.

    PubMed

    Yu, Gang; Liu, Jin-Liang; Xie, Li-Qin; Wang, Xue-Liang; Zhang, Shi-Hong; Pan, Hong-Yu

    2012-12-01

    The entomopathogenic fungus Verticillium lecanii is a well-known biocontrol agent. V. lecanii produces subtilisin-like serine protease (Pr1), which is important in the biological control activity of some insect pests by degrading insect cuticles. In this study, a subtilisin-like serine protease gene VlPr1 was cloned from the fungus and the VlPr1 protein was expressed in Escherichia coli. The VlPr1 gene contains an open reading frame (ORF) interrupted by three short introns, and encodes a protein of 379 amino acids. Protein sequence analysis revealed high homology with subtilisin serine proteases. The molecular mass of the protease was 38 kDa, and the serine protease exhibited its maximal activity at 40°C and pH 9.0. Protease activity was also affected by Mg(2+) and Ca(2+) concentration. The protease showed inhibitory activity against several plant pathogens, especially towards Fusarium moniliforme.

  15. Large lateral movement of transmembrane helix S5 is not required for substrate access to the active site of rhomboid intramembrane protease.

    PubMed

    Xue, Yi; Ha, Ya

    2013-06-07

    Rhomboids represent an evolutionarily ancient protease family. Unlike most other proteases, they are polytopic membrane proteins and specialize in cleaving transmembrane protein substrates. The polar active site of rhomboid protease is embedded in the membrane and normally closed. For the bacterial rhomboid GlpG, it has been proposed that one of the transmembrane helices (S5) of the protease can rotate to open a lateral gate, enabling substrate to enter the protease from inside the membrane. Here, we studied the conformational change in GlpG by solving the cocrystal structure of the protease with a mechanism-based inhibitor. We also examined the lateral gating model by cross-linking S5 to a neighboring helix (S2). The crystal structure shows that inhibitor binding displaces a capping loop (L5) from the active site but causes only minor shifts in the transmembrane helices. Cross-linking S5 and S2, which not only restricts the lateral movement of S5 but also prevents substrate from passing between the two helices, does not hinder the ability of the protease to cleave a membrane protein substrate in detergent solution and in reconstituted membrane vesicles. Taken together, these data suggest that a large lateral movement of the S5 helix is not required for substrate access to the active site of rhomboid protease.

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

    PubMed Central

    2011-01-01

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

  17. Bacterial proteases and virulence.

    PubMed

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    Bacterial pathogens rely on proteolysis for variety of purposes during the infection process. In the cytosol, the main proteolytic players are the conserved Clp and Lon proteases that directly contribute to virulence through the timely degradation of virulence regulators and indirectly by providing 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 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 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. These extracellular proteases are activated in complex cascades involving auto-processing and proteolytic maturation. Thus, proteolysis has been adopted by bacterial pathogens at multiple levels to ensure the success of the pathogen in contact with the human host.

  18. Plasmin induces in vivo monocyte recruitment through protease-activated receptor-1-, MEK/ERK-, and CCR2-mediated signaling.

    PubMed

    Carmo, Aline A F; Costa, Bruno R C; Vago, Juliana P; de Oliveira, Leonardo C; Tavares, Luciana P; Nogueira, Camila R C; Ribeiro, Ana Luíza C; Garcia, Cristiana C; Barbosa, Alan S; Brasil, Bruno S A F; Dusse, Luci M; Barcelos, Lucíola S; Bonjardim, Cláudio A; Teixeira, Mauro M; Sousa, Lirlândia P

    2014-10-01

    The plasminogen (Plg)/plasmin (Pla) system is associated with a variety of biological activities beyond the classical dissolution of fibrin clots, including cell migration, tissue repair, and inflammation. Although the capacity of Plg/Pla to induce cell migration is well defined, the mechanism underlying this process in vivo is elusive. In this study, we show that Pla induces in vitro migration of murine fibroblasts and macrophages (RAW 264.7) dependent on the MEK/ERK pathway and by requiring its proteolytic activity and lysine binding sites. Plasmin injection into the pleural cavity of BALB/c mice induced a time-dependent influx of mononuclear cells that was associated with augmented ERK1/2 and IκB-α phosphorylation and increased levels of CCL2 and IL-6 in pleural exudates. The inhibition of protease activity by using a serine protease inhibitor leupeptin or two structurally different protease-activated receptor-1 antagonists (SCH79797 and RWJ56110) abolished Pla-induced mononuclear recruitment and ERK1/2 and IκB-α phosphorylation. Interestingly, inhibition of the MEK/ERK pathway abolished Pla-induced CCL2 upregulation and mononuclear cell influx. In agreement with a requirement for the CCL2/CCR2 axis to Pla-induced cell migration, the use of a CCR2 antagonist (RS504393) prevented the Plg/Pla-induced recruitment of mononuclear cells to the pleural cavity and migration of macrophages at transwell plates. Therefore, Pla-induced mononuclear cell recruitment in vivo was dependent on protease-activated receptor-1 activation of the MEK/ERK/NF-κB pathway, which led to the release of CCL2 and activation of CCR2.

  19. Bacterial diversity and bioprospecting for cold-active lipases, amylases and proteases, from culturable bacteria of kongsfjorden and Ny-alesund, Svalbard, Arctic.

    PubMed

    Srinivas, T N R; Nageswara Rao, S S S; Vishnu Vardhan Reddy, P; Pratibha, M S; Sailaja, B; Kavya, B; Hara Kishore, K; Begum, Z; Singh, S M; Shivaji, S

    2009-11-01

    Culturable bacterial diversity of seven marine sediment samples of Kongsfjorden and a sediment and a soil sample from Ny-Alesund, Svalbard, Arctic was studied. The bacterial abundance in the marine sediments of Kongsfjorden varied marginally (0.5 x 10(3)-1.3 x 10(4) cfu/g sediment) and the bacterial number in the two samples collected from the shore of Ny-Alesund also was very similar (0.6 x 10(4) and 3.4 x 10(4), respectively). From the nine samples a total of 103 bacterial isolates were obtained and these isolates could be grouped in to 47 phylotypes based on the 16S rRNA gene sequence belonging to 4 phyla namely Actinobacteria, Bacilli, Bacteroidetes and Proteobacteria. Representatives of the 47 phylotypes varied in their growth temperature range (4-37 degrees C), in their tolerance to NaCl (0.3-2 M NaCl) and growth pH range (2-11). Representatives of 26 phylotypes exhibited amylase and lipase activity either at 5 or 20 degrees C or at both the temperatures. A few of the representatives exhibited amylase and/or lipase activity only at 5 degrees C. None of the phylotypes exhibited protease activity. Most of the phylotypes (38) were pigmented. Fatty acid profile studies indicated that short chain fatty acids, unsaturated fatty acids, branched fatty acids, the cyclic and the cis fatty acids are predominant in the psychrophilic bacteria.

  20. Role of calcium and calcium-activated proteases in CYP2E1-dependent toxicity in HEPG2 cells.

    PubMed

    Caro, Andres A; Cederbaum, Arthur I

    2002-01-04

    The objective of this work was to investigate whether CYP2E1- and oxidative stress-dependent toxicity in HepG2 cells is mediated by an increase of cytosolic Ca2+ and activation of Ca2+-modulated processes. HepG2 cells expressing CYP2E1 (E47 cells) or control cells not expressing CYP2E1 (C34 cells) were preloaded with arachidonic acid (AA, up to 10 microm) and, after washing, incubated with iron-nitrilotriacetic acid (up to 100 microm) for variable periods (up to 12 h). Toxicity was greater in E47 cells than in C34 cells at all times and combinations of iron/AA tested. Cytosolic calcium increased with incubation time in both cell lines, but the increase was higher in E47 cells than in C34 cells. The rise in calcium was an early event and preceded the developing toxicity. Toxicity in E47 cells and the increase in Ca2+ were inhibited by omission of Ca2+ from the extracellular medium, and toxicity was restored by reincorporation of Ca2+. An inhibitor of Ca2+ release from intracellular stores did not prevent the toxicity or the increase in Ca2+, reflecting a role for the influx of extracellular Ca2+ in the toxicity. Reactive oxygen production was similar in media with or without calcium, indicating that calcium was not modulating CYP2E1-dependent oxidative stress. Toxicity, lipid peroxidation, and the increase of Ca2+ in E47 cells exposed to iron-AA were inhibited by alpha-tocopherol. E47 cells (but not C34 cells) exposed to iron-AA showed increased calpain activity in situ (40-fold). The toxicity in E47 cells mirrored calpain activation and was inhibited by calpeptin, suggesting that calpain activation plays a causal role in toxicity. These results suggest that CYP2E1-dependent toxicity in this model depends on the activation of lipid peroxidation, followed by an increased influx of extracellular Ca2+ and activation of Ca2+-dependent proteases.

  1. Decolorization of crude latex by activated charcoal, purification and physico-chemical characterization of religiosin, a milk-clotting serine protease from the latex of Ficus religiosa.

    PubMed

    Kumari, Moni; Sharma, Anurag; Jagannadham, M V

    2010-07-14

    The crude latex of Ficus religiosa is decolorized by activated charcoal. Decolorization follows the Freundlich and Langmuir equations. A serine protease, named religiosin, has been purified to homogeneity from the decolorized latex using anion exchange chromatography. Religiosin is a glycoprotein with a molecular mass of 43.4 kDa by MALDI-TOF. Religiosin is an acidic protein with a pI value of 3.8 and acts optimally at pH 8.0-8.5 and temperature 50 degrees C. The proteolytic activity of religiosin is strongly inhibited by PMSF and chymostatin indicating that the enzyme is a serine protease. The extinction coefficient (epsilon(1%)(280)) of religiosin is 29.47 M(-1) cm(-1)with 16 tryptophan, 26 tyrosine, and 11 cysteine residues per molecule. The enzyme shows broad substrate specificity against natural as well as synthetic substrates with an apparent K(m) of 0.066 mM and 6.25 mM using casein and Leu-pNA, respectively. MS/MS analysis confirms the novelty of the enzyme. Religiosin is highly stable against denaturants, metal ions, and detergents as well as over a wide range of pH and temperature. In addition, the enzyme exhibits milk-clotting as well as detergent activity.

  2. Real time in vivo imaging and measurement of serine protease activity in the mouse hippocampus using a dedicated complementary metal-oxide semiconductor imaging device.

    PubMed

    Ng, David C; Tamura, Hideki; Tokuda, Takashi; Yamamoto, Akio; Matsuo, Masamichi; Nunoshita, Masahiro; Ishikawa, Yasuyuki; Shiosaka, Sadao; Ohta, Jun

    2006-09-30

    The aim of the present study is to demonstrate the application of complementary metal-oxide semiconductor (CMOS) imaging technology for studying the mouse brain. By using a dedicated CMOS image sensor, we have successfully imaged and measured brain serine protease activity in vivo, in real-time, and for an extended period of time. We have developed a biofluorescence imaging device by packaging the CMOS image sensor which enabled on-chip imaging configuration. In this configuration, no optics are required whereby an excitation filter is applied onto the sensor to replace the filter cube block found in conventional fluorescence microscopes. The fully packaged device measures 350 microm thick x 2.7 mm wide, consists of an array of 176 x 144 pixels, and is small enough for measurement inside a single hemisphere of the mouse brain, while still providing sufficient imaging resolution. In the experiment, intraperitoneally injected kainic acid induced upregulation of serine protease activity in the brain. These events were captured in real time by imaging and measuring the fluorescence from a fluorogenic substrate that detected this activity. The entire device, which weighs less than 1% of the body weight of the mouse, holds promise for studying freely moving animals.

  3. A tobacco etch virus protease with increased substrate tolerance at the P1' position.

    PubMed

    Renicke, Christian; Spadaccini, Roberta; Taxis, Christof

    2013-01-01

    Site-specific proteases are important tools for in vitro and in vivo cleavage of proteins. They are widely used for diverse applications, like protein purification, assessment of protein-protein interactions or regulation of protein localization, abundance or activity. Here, we report the development of a procedure to select protease variants with altered specificity based on the well-established Saccharomyces cerevisiae adenine auxotrophy-dependent red/white colony assay. We applied this method on the tobacco etch virus (TEV) protease to obtain a protease variant with altered substrate specificity at the P1' Position. In vivo experiments with tester substrates showed that the mutated TEV protease still efficiently recognizes the sequence ENLYFQ, but has almost lost all bias for the amino acid at the P1' Position. Thus, we generated a site-specific protease for synthetic approaches requiring in vivo generation of proteins or peptides with a specific N-terminal amino acid.

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

    PubMed

    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.

  5. Mechanisms of protease-activated receptor 2-evoked hyperexcitability of nociceptive neurons innervating the mouse colon

    PubMed Central

    Kayssi, Ahmed; Amadesi, Silvia; Bautista, Francisco; Bunnett, Nigel W; Vanner, Stephen

    2007-01-01

    Agonists of protease-activated receptor 2 (PAR2) evoke hyperexcitability of dorsal root ganglia (DRG) neurons by unknown mechanisms. We examined the cellular mechanisms underlying PAR2-evoked hyperexcitability of mouse colonic DRG neurons to determine their potential role in pain syndromes such as visceral hyperalgesia. Colonic DRG neurons were identified by injecting Fast Blue and DiI retrograde tracers into the mouse colon. Using immunofluorescence, we found that DiI-labelled neurons contained PAR2 immunoreactivity, confirming the presence of receptors on colonic neurons. Whole-cell current-clamp recordings of acutely dissociated neurons demonstrated that PAR2 activation with a brief application (3 min) of PAR2 agonists, SLIGRL-NH2 and trypsin, evoked sustained depolarizations (up to 60 min) which were associated with increased input resistance and a marked reduction in rheobase (50% at 30 min). In voltage clamp, SLIGRL-NH2 markedly suppressed delayed rectifier IK currents (55% at 10 min), but had no effect on the transient IA current or TTX-resistant Na+ currents. In whole-cell current-clamp recordings, the sustained excitability evoked by PAR2 activation was blocked by the PKC inhibitor, calphostin, and the ERK1/2 inhibitor PD98059. Studies of ERK1/2 phosphorylation using confocal microscopy demonstrated that SLIGRL-NH2 increased levels of immunoreactive pERK1/2 in DRG neurons, particularly in proximity to the plasma membrane. Thus, activation of PAR2 receptors on colonic nociceptive neurons causes sustained hyperexcitability that is related, at least in part, to suppression of delayed rectifier IK currents. Both PKC and ERK1/2 mediate the PAR2-induced hyperexcitability. These studies describe a novel mechanism of sensitization of colonic nociceptive neurons that may be implicated in conditions of visceral hyperalgesia such as irritable bowel syndrome. PMID:17289784

  6. N-Linked Glycosylation of Protease-activated Receptor-1 Second Extracellular Loop

    PubMed Central

    Soto, Antonio G.; Trejo, JoAnn

    2010-01-01

    Protease-activated receptor-1 (PAR1) contains five N-linked glycosylation consensus sites as follows: three residing in the N terminus and two localized on the surface of the second extracellular loop (ECL2). To study the effect of N-linked glycosylation in the regulation of PAR1 signaling and trafficking, we generated mutants in which the critical asparagines of the consensus sites were mutated. Here, we report that both the PAR1 N terminus and ECL2 serve as sites for N-linked glycosylation but have different functions in the regulation of receptor signaling and trafficking. N-Linked glycosylation of the PAR1 N terminus is important for transport to the cell surface, whereas the PAR1 mutant lacking glycosylation at ECL2 (NA ECL2) trafficked to the cell surface like the wild-type receptor. However, activated PAR1 NA ECL2 mutant internalization was impaired compared with wild-type receptor, whereas constitutive internalization of unactivated receptor remained intact. Remarkably, thrombin-activated PAR1 NA ECL2 mutant displayed an enhanced maximal signaling response compared with wild-type receptor. The increased PAR1 NA ECL2 mutant signaling was not due to defects in the ability of thrombin to cleave the receptor or signal termination mechanisms. Rather, the PAR1 NA ECL2 mutant displayed a greater efficacy in thrombin-stimulated G protein signaling. Thus, N-linked glycosylation of the PAR1 extracellular surface likely influences ligand docking interactions and the stability of the active receptor conformation. Together, these studies strongly suggest that N-linked glycosylation of PAR1 at the N terminus versus the surface of ECL2 serves distinct functions critical for proper regulation of receptor trafficking and the fidelity of thrombin signaling. PMID:20368337

  7. Functional regulation of PVBV Nuclear Inclusion protein-a protease activity upon interaction with Viral Protein genome-linked and phosphorylation

    SciTech Connect

    Mathur, C.; Jimsheena, V.K.; Banerjee, S.; Makinen, K.; Gowda, L.R.; Savithri, H.S.

    2012-01-20

    Regulation of NIa-Pro is crucial for polyprotein processing and hence, for successful infection of potyviruses. We have examined two novel mechanisms that could regulate NIa-Pro activity. Firstly, the influence of VPg domain on the proteolytic activity of NIa-Pro was investigated. It was shown that the turnover number of the protease increases when these two domains interact (cis: two-fold; trans: seven-fold) with each other. Secondly, the protease activity of NIa-Pro could also be modulated by phosphorylation at Ser129. A mutation of this residue either to aspartate (phosphorylation-mimic) or alanine (phosphorylation-deficient) drastically reduces the protease activity. Based on these observations and molecular modeling studies, we propose that interaction with VPg as well as phosphorylation of Ser129 could relay a signal through Trp143 present at the protein surface to the active site pocket by subtle conformational changes, thus modulating protease activity of NIa-Pro.

  8. Effects of dietary soybean stachyose and phytic acid on gene expressions of serine proteases in Japanese flounder ( Paralichthys olivaceus)

    NASA Astrophysics Data System (ADS)

    Mi, Haifeng; Mai, Kangsen; Zhang, Wenbing; Wu, Chenglong; Cai, Yinghua

    2011-09-01

    Soybean stachyose (SBS) and phytic acid (PA) are anti-nutritional factors (ANF) which have deleterious effects on the growth and digestibility in fish. The present research studied the effects of dietary SBS and PA on the expression of three serine protease genes in the liver of Japanese flounder ( Paralichthys olivaceus). These genes are trypsinogen 1 (poTRY), elastase 1 (poEL) and chymotrypsinogen 1 (poCTRY). Eight artificial diets with graded levels of supplemented ANFs were formulated to 4 levels of SBS (0.00, 0.40, 0.80 and 1.50%), 4 levels of PA (0.00, 0.20, 0.40 and 0.80), respectively. Japanese flounder (initial weight 2.45 g ± 0.01 g) were fed with these diets for 10 weeks with three replications per treatment. At the end of 10 weeks, supplementation of 0.40% of dietary SBS or PA significantly increased the gene expression of poTRY and poCTRY ( P<0.05). The same level of dietary SBS significantly decreased the gene expression of poEL. In comparison with the control group (ANF-free), dietary PA (0.2% and 0.8%) significantly decreased the gene expression of poTRY, poCTRY and poEL ( P<0.05). However, excessive supplement of dietary SBS (1.5%) has no significant effects on these gene expressions ( P>0.05). These results suggested that dietary SBS and dietary PA could directly affect the serine protease genes at the transcriptional level in Japanese flounder, and these genes' expression was more sensitive to dietary PA than to SBS under the current experimental conditions.

  9. Integral membrane protease fibroblast activation protein sensitizes fibrosarcoma to chemotherapy and alters cell death mechanisms.

    PubMed

    Baird, Sarah K; Rigopoulos, Angela; Cao, Diana; Allan, Laura; Renner, Christoph; Scott, Fiona E; Scott, Andrew M

    2015-11-01

    Fibroblast activation protein (FAP), an integral membrane serine protease, is found on fibro- and osteo-sarcoma and on myofibroblasts in epithelial carcinoma, but rarely on other adult tissue. FAP has been demonstrated to be an excellent target for tumor imaging in clinical trials, and antibodies and other FAP-targeting drugs are in development. Here we have shown that FAP overexpression increased the growth of HT1080 fibrosarcoma cells in vitro and in vivo, and found that the expression of FAP affects response to chemotherapy. When treated with doxorubicin, expression of FAP increased susceptibility to the drug. In spite of this, FAP-HT1080 cells had fewer markers of classical apoptosis than HT1080 cells and neither necrosis nor necroptosis were enhanced. However, levels of early mitochondrial and lysosomal membrane permeability markers were increased, and autophagy switched from a protective function in HT1080 cells to part of the cell death mechanism with FAP expression. Therefore, FAP may affect how the tumor responds to chemotherapeutic drugs overall, which should be considered in targeted drug development. The overexpression of FAP also alters cell signaling and responses to the environment in this cell line. This includes cell death mechanisms, changing the response of HT1080 cells to doxorubicin from classical apoptosis to an organelle membrane permeability-dependent form of cell death.

  10. Shear stress reduces protease activated receptor-1 expression in human endothelial cells

    NASA Technical Reports Server (NTRS)

    Nguyen, K. T.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

    2001-01-01

    Shear stress has been shown to regulate several genes involved in the thrombotic and proliferative functions of endothelial cells. Thrombin receptor (protease-activated receptor-1: PAR-1) increases at sites of vascular injury, which suggests an important role for PAR-1 in vascular diseases. However, the effect of shear stress on PAR-1 expression has not been previously studied. This work investigates effects of shear stress on PAR-1 gene expression in both human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs). Cells were exposed to different shear stresses using a parallel plate flow system. Northern blot and flow cytometry analysis showed that shear stress down-regulated PAR-1 messenger RNA (mRNA) and protein levels in both HUVECs and HMECs but with different thresholds. Furthermore, shear-reduced PAR-1 mRNA was due to a decrease of transcription rate, not increased mRNA degradation. Postshear stress release of endothelin-1 in response to thrombin was reduced in HUVECs and HMECs. Moreover, inhibitors of potential signaling pathways applied during shear stress indicated mediation of the shear-decreased PAR-1 expression by protein kinases. In conclusion, shear stress exposure reduces PAR-1 gene expression in HMECs and HUVECs through a mechanism dependent in part on protein kinases, leading to altered endothelial cell functional responses to thrombin.

  11. Crystal structure of the caseinolytic protease gene regulator, a transcriptional activator in actinomycetes.

    PubMed

    Russo, Santina; Schweitzer, Jens-Eric; Polen, Tino; Bott, Michael; Pohl, Ehmke

    2009-02-20

    Human pathogens of the genera Corynebacterium and Mycobacterium possess the transcriptional activator ClgR (clp gene regulator) which in Corynebacterium glutamicum has been shown to regulate the expression of the ClpCP protease genes. ClgR specifically binds to pseudo-palindromic operator regions upstream of clpC and clpP1P2. Here, we present the first crystal structure of a ClgR protein from C. glutamicum. The structure was determined from two different crystal forms to resolutions of 1.75 and 2.05 A, respectively. ClgR folds into a five-helix bundle with a helix-turn-helix motif typical for DNA-binding proteins. Upon dimerization the two DNA-recognition helices are arranged opposite to each other at the protein surface in a distance of approximately 30 A, which suggests that they bind into two adjacent major grooves of B-DNA in an anti-parallel manner. A binding pocket is situated at a strategic position in the dimer interface and could possess a regulatory role altering the positions of the DNA-binding helices.

  12. Quantitation of fibroblast activation protein (FAP)-specific protease activity in mouse, baboon and human fluids and organs.

    PubMed

    Keane, Fiona M; Yao, Tsun-Wen; Seelk, Stefanie; Gall, Margaret G; Chowdhury, Sumaiya; Poplawski, Sarah E; Lai, Jack H; Li, Youhua; Wu, Wengen; Farrell, Penny; Vieira de Ribeiro, Ana Julia; Osborne, Brenna; Yu, Denise M T; Seth, Devanshi; Rahman, Khairunnessa; Haber, Paul; Topaloglu, A Kemal; Wang, Chuanmin; Thomson, Sally; Hennessy, Annemarie; Prins, John; Twigg, Stephen M; McLennan, Susan V; McCaughan, Geoffrey W; Bachovchin, William W; Gorrell, Mark D

    2013-01-01

    The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.

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

    SciTech Connect

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

    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.

  14. Inhibition of Protease-activated Receptor 1 Ameliorates Intestinal Radiation Mucositis in a Preclinical Rat Model

    SciTech Connect

    Wang, Junru; Kulkarni, Ashwini; Chintala, Madhu; Fink, Louis M.; Hauer-Jensen, Martin

    2013-01-01

    Purpose: To determine, using a specific small-molecule inhibitor of protease-activated receptor 1 (PAR1) signaling, whether the beneficial effect of thrombin inhibition on radiation enteropathy development is due to inhibition of blood clotting or to cellular (PAR1-mediated) thrombin effects. Methods and Materials: Rats underwent fractionated X-irradiation (5 Gy Multiplication-Sign 9) of a 4-cm small-bowel segment. Early radiation toxicity was evaluated in rats receiving PAR1 inhibitor (SCH602539, 0, 10, or 15 mg/kg/d) from 1 day before to 2 weeks after the end of irradiation. The effect of PAR1 inhibition on development of chronic intestinal radiation fibrosis was evaluated in animals receiving SCH602539 (0, 15, or 30 mg/kg/d) until 2 weeks after irradiation, or continuously until termination of the experiment 26 weeks after irradiation. Results: Blockade of PAR1 ameliorated early intestinal toxicity, with reduced overall intestinal radiation injury (P=.002), number of myeloperoxidase-positive (P=.03) and proliferating cell nuclear antigen-positive (P=.04) cells, and collagen III accumulation (P=.005). In contrast, there was no difference in delayed radiation enteropathy in either the 2- or 26-week administration groups. Conclusion: Pharmacological blockade of PAR1 seems to reduce early radiation mucositis but does not affect the level of delayed intestinal radiation fibrosis. Early radiation enteropathy is related to activation of cellular thrombin receptors, whereas platelet activation or fibrin formation may play a greater role in the development of delayed toxicity. Because of the favorable side-effect profile, PAR1 blockade should be further explored as a method to ameliorate acute intestinal radiation toxicity in patients undergoing radiotherapy for cancer and to protect first responders and rescue personnel in radiologic/nuclear emergencies.

  15. Protease-Activated Receptor 2 Mediates Mucus Secretion in the Airway Submucosal Gland

    PubMed Central

    Lee, Hyun Jae; Yang, Yu-Mi; Kim, Kyubo; Shin, Dong Min; Yoon, Joo-Heon; Cho, Hyung-Ju; Choi, Jae Young

    2012-01-01

    Protease-activated receptor 2 (PAR2), a G protein-coupled receptor expressed in airway epithelia and smooth muscle, plays an important role in airway inflammation. In this study, we demonstrated that activation of PAR2 induces mucus secretion from the human airway gland and examined the underlying mechanism using the porcine and murine airway glands. The mucosa with underlying submucosal glands were dissected from the cartilage of tissues, pinned with the mucosal side up at the gas/bath solution interface of a physiological chamber, and covered with oil so that secretions from individual glands could be visualized as spherical bubbles in the oil. Secretion rates were determined by optical monitoring of the bubble diameter. The Ca2+-sensitive dye Fura2-AM was used to determine intracellular Ca2+ concentration ([Ca2+]i) by means of spectrofluorometry. Stimulation of human tracheal mucosa with PAR2-activating peptide (PAR2-AP) elevated intracellular Ca2+ and induced glandular secretion equal to approximately 30% of the carbachol response in the human airway. Porcine gland tissue was more sensitive to PAR2-AP, and this response was dependent on Ca2+ and anion secretion. When the mouse trachea were exposed to PAR2-AP, large amounts of secretion were observed in both wild type and ΔF508 cystic fibrosis transmembrane conductance regulator mutant mice but there is no secretion from PAR-2 knock out mice. In conclusion, PAR2-AP is an agonist for mucus secretion from the airway gland that is Ca2+-dependent and cystic fibrosis transmembrane conductance regulator-independent. PMID:22916223

  16. The Lon protease from the haloalkaliphilic archaeon Natrialba magadii is transcriptionally linked to a cluster of putative membrane proteases and displays DNA-binding activity.

    PubMed

    Sastre, Diego E; Paggi, Roberto A; De Castro, Rosana E

    2011-05-20

    The ATP-dependent Lon protease is universally distributed in bacteria, eukaryotic organelles and archaea. In comparison with bacterial and eukaryal Lon proteases, the biology of the archaeal Lon has been studied to a limited extent. In this study, the gene encoding the Lon protease of the alkaliphilic haloarchaeon Natrialba magadii (Nmlon) was cloned and sequenced, and the genetic organization of Nmlon was examined at the transcriptional level. Nmlon encodes a 84 kDa polypeptide with a pI of 4.42 which contains the ATPase, protease and membrane targeting domains of the archaeal-type LonB proteases. Nmlon is part of an operon that encodes membrane proteases and it is transcribed as a polycistronic mRNA in N. magadii cells at different growth stages. Accordingly, NmLon was detected in cell membranes of N. magadii throughout growth by Western blot analysis using specific anti-NmLon antibodies. Interestingly, in electrophoretic mobility shift assays, purified NmLon bound double stranded as well as single stranded DNA in the presence of elevated salt concentrations. This finding shows that DNA-binding is conserved in the LonA and LonB subfamilies and suggests that Lon-DNA interaction may be relevant for its function in haloarchaea.

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

    SciTech Connect

    Hodder, Anthony N.; Malby, Robyn L.; Clarke, Oliver B.; Fairlie, W. Douglas; Colman, Peter M.; Crabb, Brendan S.; Smith, Brian J.

    2009-08-28

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

  18. Derivation of rat embryonic stem cells and generation of protease-activated receptor-2 knockout rats.

    PubMed

    Yamamoto, Satoshi; Nakata, Mitsugu; Sasada, Reiko; Ooshima, Yuki; Yano, Takashi; Shinozawa, Tadahiro; Tsukimi, Yasuhiro; Takeyama, Michiyasu; Matsumoto, Yoshio; Hashimoto, Tadatoshi

    2012-08-01

    One of the remarkable achievements in knockout (KO) rat production reported during the period 2008-2010 is the derivation of authentic embryonic stem (ES) cells from rat blastocysts using a novel culture medium containing glycogen synthase kinase 3 and mitogen-activated protein kinase kinase inhibitors (2i medium). Here, we report gene-targeting technology via homologous recombination in rat ES cells, demonstrating its use through production of a protease-activated receptor-2 gene (Par-2) KO rat. We began by generating germline-competent ES cells from Dark Agouti rats using 2i medium. These ES cells, which differentiate into cardiomyocytes in vitro, can produce chimeras with high ES cell contribution when injected into blastocysts. We then introduced a targeting vector with a neomycin-resistant gene driven by the CAG promoter to disrupt Par-2. After a 7-day drug selection, 489 neomycin-resistant colonies were obtained. Following screening by polymerase chain reaction (PCR) genotyping and quantitative PCR analysis, we confirmed three homologous recombinant clones, resulting in chimeras that transmitted the Par-2 targeted allele to offspring. Par-2 KO rats showed a loss of Par-2 messenger RNA expression in their stomach cells and a lack of PAR-2 mediated smooth muscle relaxation in the aorta as indicated by pharmacological testing. Compared with mice, rats offer many advantages in biomedical research, including a larger body size; consequently, they are widely used in scientific investigation. Thus, the establishment of a gene-targeting technology using rat ES cells will be a valuable tool in human disease model production and drug discovery.

  19. Role of Fibrinogen and Protease-Activated Receptors in Acute Xenobiotic-Induced Cholestatic Liver Injury

    PubMed Central

    Luyendyk, James P.; Mackman, Nigel; Sullivan, Bradley P.

    2011-01-01

    Alpha-naphthylisothiocyanate (ANIT)–induced cholestatic liver injury causes tissue factor (TF)–dependent coagulation in mice, and TF deficiency reduces ANIT-induced liver injury. However, the mechanism whereby TF contributes to hepatotoxicity in this model is not known. Utilizing pharmacological and genetic strategies, we evaluated the contribution of fibrinogen and two distinct receptors for thrombin, protease-activated receptor-1 (PAR-1) and PAR-4, in a model of acute ANIT hepatotoxicity. ANIT administration (60 mg/kg, po) caused a marked induction of the genes encoding the three fibrinogen chains (α, β, and γ) in liver, an increase in plasma fibrinogen, and concurrent deposition of thrombin-cleaved fibrin in liver. Partial depletion of circulating fibrinogen with ancrod did not impact ANIT hepatotoxicity. However, complete fibrin(ogen) deficiency significantly reduced serum alanine aminotransferase activity and hepatocellular necrosis in ANIT-treated mice. ANIT-induced hepatocellular necrosis was similar in PAR-1−/− mice compared with PAR-1+/+ mice. Interestingly, the progression of ANIT-induced hepatocellular necrosis was significantly reduced in PAR-4−/− mice and by administration of an inhibitory PAR-4 pepducin (P4Pal-10, 0.5 mg/kg, sc) to wild-type mice 8 h after ANIT treatment. Interestingly, a distinct lesion, parenchymal-type peliosis, was also observed in PAR-4−/− mice treated with ANIT and in mice that were given P4Pal-10 prior to ANIT administration. The results suggest that fibrin(ogen), but not PAR-1, contributes to the progression of ANIT hepatotoxicity in mice. Moreover, the data suggest a dual role for PAR-4 in ANIT hepatotoxicity, both mediating an early protection against peliosis and contributing to the progression of hepatocellular necrosis. PMID:20974703

  20. Macelignan inhibits melanosome transfer mediated by protease-activated receptor-2 in keratinocytes.

    PubMed

    Choi, Eun-Jung; Kang, Young-Gyu; Kim, Jaekyung; Hwang, Jae-Kwan

    2011-01-01

    Skin pigmentation is the result of melanosome transfer from melanocytes to keratinocytes. Protease-activated receptor-2 (PAR-2) is a key mediator of melanosome transfer, which occurs as the melanocyte extends its dendrite toward surrounding keratinocytes that take up melanosomes by phagocytosis. We investigated the effects of macelignan isolated from Myristica fragrans HOUTT. (nutmeg) on melanosome transfer and the regulation of PAR-2 in human keratinocytes (HaCaT). HaCaT cells stimulated by the PAR-2-activating peptide Ser-Leu-Ile-Gly-Arg-Leu-NH₂ (SLIGRL) were treated with macelignan; PAR-2 expression was then determined by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry. We evaluated the effects of macelignan on calcium mobilization and keratinocyte phagocytosis. In addition, B16F10 melanoma cells and keratinocytes were co-cultured to assess the effects of macelignan on prostaglandin E₂ (PGE₂) secretion and subsequent dendrite formation. Macelignan decreased HaCaT PAR-2 mRNA and protein levels in a dose-dependent manner. Furthermore, macelignan markedly reduced intracellular calcium mobilization and significantly downregulated keratinocyte phagocytosis, as shown by decreased ingestion of Escherichia coli bioparticles and fluorescent microspheres. In co-culture experiments, macelignan reduced keratinocyte PGE₂ secretion, thereby preventing dendrite formation in B16F10 melanoma cells compared with SLIGRL-treated controls. Macelignan inhibits melanosome transfer by downregulating PAR-2, thereby reducing keratinocyte phagocytosis and PGE₂ secretion, which in turn inhibits dendrite formation in B16F10 melanoma cells. Taken together, our findings suggest that macelignan could be used as a natural depigmenting agent to ameliorate hyperpigmentation.

  1. Effects of catechin polyphenols and preparations from the plant-parasitic nematode Heterodera glycines on protease activity and behavior in three nematode species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protease activities in preparations from the plant-parasitic nematodes Heterodera glycines and Meloidogyne incognita and the free-living nematode Panagrellus redivivus were inhibited by exposure to a series of 8 catechin polyphenol analogs, (+)-catechin, (-)- epicatechin (EC), (-)-gallocatechin (GC)...

  2. Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion

    SciTech Connect

    Simmons, Graham; Bertram, Stephanie; Glowacka, Ilona; Steffen, Imke; Chaipan, Chawaree; Agudelo, Juliet; Lu Kai; Rennekamp, Andrew J.; Hofmann, Heike; Bates, Paul; Poehlmann, Stefan

    2011-05-10

    Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation.

  3. HCV NS3 protease enhances liver fibrosis via binding to and activating TGF-β type I receptor

    NASA Astrophysics Data System (ADS)

    Sakata, Kotaro; Hara, Mitsuko; Terada, Takaho; Watanabe, Noriyuki; Takaya, Daisuke; Yaguchi, So-Ichi; Matsumoto, Takehisa; Matsuura, Tomokazu; Shirouzu, Mikako; Yokoyama, Shigeyuki; Yamaguchi, Tokio; Miyazawa, Keiji; Aizaki, Hideki; Suzuki, Tetsuro; Wakita, Takaji; Imoto, Masaya; Kojima, Soichi

    2013-11-01

    Viruses sometimes mimic host proteins and hijack the host cell machinery. Hepatitis C virus (HCV) causes liver fibrosis, a process largely mediated by the overexpression of transforming growth factor (TGF)-β and collagen, although the precise underlying mechanism is unknown. Here, we report that HCV non-structural protein 3 (NS3) protease affects the antigenicity and bioactivity of TGF-β2 in (CAGA)9-Luc CCL64 cells and in human hepatic cell lines via binding to TGF-β type I receptor (TβRI). Tumor necrosis factor (TNF)-α facilitates this mechanism by increasing the colocalization of TβRI with NS3 protease on the surface of HCV-infected cells. An anti-NS3 antibody against computationally predicted binding sites for TβRI blocked the TGF-β mimetic activities of NS3 in vitro and attenuated liver fibrosis in HCV-infected chimeric mice. These data suggest that HCV NS3 protease mimics TGF-β2 and functions, at least in part, via directly binding to and activating TβRI, thereby enhancing liver fibrosis.

  4. CONTRIBUTION OF PROTEASE-ACTIVATED RECEPTOR 1 IN STATUS EPILEPTICUS-INDUCED EPILEPTOGENESIS

    PubMed Central

    Isaev, D.; Lushnikova, I.; Lunko, O.; Zapukhliak, O.; Maximyuk, O.; Romanov, A.; Skibo, G.G.; Tian, C.; Holmes, G.L.; Isaeva, E.

    2015-01-01

    Clinical observations and studies on different animal models of acquired epilepsy consistently demonstrate that blood-brain barrier (BBB) leakage can be an important risk factor for developing recurrent seizures. However, the involved signaling pathways remain largely unclear. Given the important role of thrombin and its major receptor in the brain, protease-activated receptor 1 (PAR1), in the pathophysiology of neurological injury, we hypothesized that PAR1 may contribute to status epilepticus (SE)-induced epileptogenesis and that its inhibition shortly after SE will have neuroprotective and antiepileptogenic effects. Adult rats subjected to lithium-pilocarpine SE were administrated SCH79797 (a PAR1 selective antagonist) after SE termination. Thrombin and PAR1 levels and neuronal cell survival were evaluated 48 hr following SE. The effect of PAR1 inhibition on animal survival, interictal spikes (IIS) and electrographic seizures during the first two weeks after SE and behavioral seizures during the chronic period were evaluated. SE resulted in a high mortality rate and incidence of IIS and seizures in the surviving animals. There was a marked increase in thrombin, decrease in PAR1 immunoreactivity and hippocampal cell loss in the SE-treated rats. Inhibition of PAR1 following SE resulted in a decrease in mortality and morbidity, increase in neuronal cell survival in the hippocampus and suppression of IIS, electrographic and behavioral seizures following SE. These data suggest that the PAR1 signaling pathway contributes to epileptogenesis following SE. Because breakdown of the BBB occurs frequently in brain injuries, PAR1 inhibition may have beneficial effects in a variety of acquired injuries leading to epilepsy. PMID:25843668

  5. Assessment of the Fusion Tags on Increasing Soluble Production of the Active TEV Protease Variant and Other Target Proteins in E. coli.

    PubMed

    Yu, Xuelian; Sun, Jiaqi; Wang, Weiyu; Jiang, Li; Cheng, Beijiu; Fan, Jun

    2016-12-17

    In this study, five fusion tags affecting soluble production and cleavage activity of the tobacco etch virus (TEV) protease (TEVp) variant in Escherichia coli strains BL21 (DE3) and Rosetta™ (DE3) are investigated. Combination of the augmenting rare transfer RNAs (tRNAs) and the fused expressivity tag (N-terminal seven amino acid residues of E. coli translation initiation factor II) promotes the soluble TEVp partner expressed at relatively high level. Attachment of the maltose-binding protein (MBP) tag increases soluble expression of the protease released from the fusion protein in E. coli cells, but the incorporated TEVp recognition sequence slightly decreases expressivity of the fusion construct. Except for the green fluorescent protein, the attached expressivity tag shows less efficiency than the MBP tag in enhancing expression levels of the selected five target proteins in the Rosetta™ (DE3) cells under different induction conditions. Our results identified that high-level production of the functional target protein as the fusion partner in E. coli is combined with the intrinsic property of fusion tag, fusion protein stability, inherent folding of target protein, rare tRNA abundance, and the incorporated linker. Purified TEVp fusion constructs with the N-terminal expressivity tag, as well as the MBP partner, are the ideal alternatives for removing fusion tag.

  6. BmSI-7, a novel subtilisin inhibitor from Boophilus microplus, with activity toward Pr1 proteases from the fungus Metarhizium anisopliae.

    PubMed

    Sasaki, Sergio D; de Lima, Cássia A; Lovato, Diogo V; Juliano, Maria A; Torquato, Ricardo J S; Tanaka, Aparecida S

    2008-02-01

    BmSI-7 and BmSI-6, two Boophilus microplus subtilisin inhibitors (BmSI) were purified and characterized from eggs. The inhibitors isolated by classical purification methods presented molecular masses of 7408 and 7271Da, respectively, by MALDI-TOF-MS. Both BmSI-7 and BmSI-6 inhibited neutrophil elastase (K(i) 0.4 and 0.3nM) and subtilisin A (K(i) 1.4nM for both inhibitors). They also strongly inhibited Pr1 proteases from the fungus Metarhizium anisopliae; BmSI-7 (K(i) 50nM) and BmSI-6 (K(i) 2.2nM). The BmSI-7 full length cDNA was obtained using amino acid sequence information of BmSI-7 peptides generated by proteolytic digestion. BmSI-7 belongs to trypsin inhibitor like cysteine rich domain family (TIL), and it is transcribed in ovary, fat body, gut, salivary gland and haemocytes. BmSI-7 is the first TIL inhibitor described with inhibitory activity toward subtilisin A and Pr1 proteases of entomopathogenic fungi.

  7. A Biocompatible “Split Luciferin” Reaction and its Application for Non-Invasive Bioluminescent Imaging of Protease Activity in Living Animals

    PubMed Central

    Godinat, Aurélien; Budin, Ghyslain; Molares, Alma R.; Park, Hyo Min; Sanman, Laura E.; Bogyo, Matthew; Yu, Allen; Stahl, Andreas; Dubikovskaya, Elena A.

    2014-01-01

    The great complexity of many human pathologies such as cancer, diabetes, and neurodegenerative diseases requires new tools for studies of biological processes on the whole organism level. The discovery of novel biocompatible reactions has tremendously advanced our understanding of basic biology, however, no efficient tools exist for real-time non-invasive imaging of many human proteases that play very important roles in multiple human disorders. We recently reported that “split luciferin” biocompatible reaction represents a valuable tool for evaluation of protease activity directly in living animals using bioluminescence imaging (BLI). Since BLI is the most sensitive in vivo imaging modality known to date, this method can be widely applied for the evaluation of multiple proteases activity as well as identification of their new peptide-specific substrates. In this protocol we describe several applications of this “split luciferin” reaction for quantification of protease activities in test tube assays and living animals. PMID:25205565

  8. Amino Acid Prodrugs: An Approach to Improve the Absorption of HIV-1 Protease Inhibitor, Lopinavir

    PubMed Central

    Patel, Mitesh; Mandava, Nanda; Gokulgandhi, Mitan; Pal, Dhananjay; Mitra, Ashim K.

    2014-01-01

    Poor systemic concentrations of lopinavir (LPV) following oral administration occur due to high cellular efflux by P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs) and extensive metabolism by CYP3A4 enzymes. In this study, amino acid prodrugs of LPV were designed and investigated for their potential to circumvent efflux processes and first pass effects. Three amino acid prodrugs were synthesized by conjugating isoleucine, tryptophan and methionine to LPV. Prodrug formation was confirmed by the LCMS/MS and NMR technique. Interaction of LPV prodrugs with efflux proteins were carried out in P-gp (MDCK-MDR1) and MRP2 (MDCK-MRP2) transfected cells. Aqueous solubility studies demonstrated that prodrugs generate higher solubility relative to LPV. Prodrugs displayed higher stability under acidic conditions and degraded significantly with rise in pH. Uptake and transport data suggested that prodrugs carry significantly lower affinity towards P-gp and MRP2 relative to LPV. Moreover, prodrugs exhibited higher liver microsomal stability relative to LPV. Hence, amino acid prodrug modification might be a viable approach for enhancing LPV absorption across intestinal epithelial and brain endothelial cells which expresses high levels of P-gp and MRP2. PMID:24727459

  9. Serine protease inhibitors suppress pancreatic endogenous proteases and modulate bacterial neutral proteases.

    PubMed

    Nduaguibe, Chikodili C; Bentsi-Barnes, Kwamina; Mullen, Yoko; Kandeel, Fouad; Al-Abdullah, Ismail

    2010-01-01

    Pefabloc, Trasylol and Urinary Trypsin Inhibitor (UTI) have been reported to be effective serine protease inhibitors that impair pancreatic endogenous proteases resulting in improved islet yield. Here we evaluated the effect of these inhibitors on endogenous proteases (trypsin, chymotrypsin and elastase), bacterial neutral proteases (thermolysin and neutral protease) and islet isolation digestion samples. Protease activity was measured using a fluorimetric assay and islet function was assessed by dynamic perifusion. Trypsin, chymotrypsin and elastase were significantly inhibited by Pefabloc and UTI. Trasylol showed strong inhibitory effects on trypsin and chymotrypsin but also decreased thermolysin activity. UTI was found to inhibit the activity of endogenous proteases and increase the activity of bacterial neutral proteases. Human islets exposed to Pefabloc had reduced insulin response, unlike Trasylol or UTI, which had no detrimental effect on insulin secretion. Although Trasylol was an effective inhibitor of endogenous proteases, FDA regulatory issues preclude its use in clinical application and thus in the isolation process. UTI has the greatest potential because it impairs endogenous pancreatic proteases and enhances digestion enzymes.

  10. Localization and enzymatic activity profiles of the proteases responsible for tachykinin-directed oocyte growth in the protochordate, Ciona intestinalis.

    PubMed

    Aoyama, Masato; Kawada, Tsuyoshi; Satake, Honoo

    2012-03-01

    We previously substantiated that Ci-TK, a tachykinin of the protochordate, Ciona intestinalis (Ci), triggered oocyte growth from the vitellogenic stage (stage II) to the post-vitellogenic stage (stage III) via up-regulation of the gene expression and enzymatic activity of the proteases: cathepsin D, carboxypeptidase B1, and chymotrypsin. In the present study, we have elucidated the localization, gene expression and activation profile of these proteases. In situ hybridization showed that the Ci-cathepsin D mRNA was present exclusively in test cells of the stage II oocytes, whereas the Ci-carboxypeptidase B1 and Ci-chymotrypsin mRNAs were detected in follicular cells of the stage II and stage III oocytes. Double-immunostaining demonstrated that the immunoreactivity of Ci-cathepsin D was largely colocalized with that of the receptor of Ci-TK, Ci-TK-R, in test cells of the stage II oocytes. Ci-cathepsin D gene expression was detected at 2h after treatment with Ci-TK, and elevated for up to 5h, and then slightly decreased. Gene expression of Ci-carboxypeptidase B1 and Ci-chymotrypsin was observed at 5h after treatment with Ci-TK, and then decreased. The enzymatic activities of Ci-cathepsin D, Ci-carboxypeptidase B1, and Ci-chymotrypsin showed similar alterations with 1-h lags. These gene expression and protease activity profiles verified that Ci-cathepsin D is initially activated, which is followed by the activation of Ci-carboxypeptidase B1 and Ci-chymotrypsin. Collectively, the present data suggest that Ci-TK directly induces Ci-cahtepsin D in test cells expressing Ci-TK receptor, leading to the secondary activation of Ci-chymotrypsin and Ci-carboxypeptidase B1 in the follicle in the tachykininergic oocyte growth pathway.

  11. Suppression of Helicobacter pylori protease activity towards growth factors by sulglycotide.

    PubMed

    Piotrowski, J; Slomiany, A; Slomiany, B L

    1997-09-01

    Infection with H. pylori is now recognized as a major factor in the pathogenesis of gastric disease. Here, we examined the susceptibility of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF beta) and platelet derived growth factor (PDGF) to degradation by H. pylori protease, and assessed the effect of a cytoprotective agent, sulglycotide, on this process. The 125I-labeled EGF, bFGF, TGF beta and PDGF were incubatet with H. pylori protease, obtained from the filtrates of saline washes of the bacterium culture, in the presence of 0-100 micrograms sulglycotide. The results showed that, under the assay conditions, H. pylori protease caused only 5% degradation of EGF and 7% degradation of bFGF. However, the protease evoked a 61.7% degradation of PDGF and a 62.3% degradation of TGF beta. Introduction of sulglycotide to the reaction assay system caused a dose-dependent inhibition in PDGF and TGF beta proteolysis by the H. pylori enzyme. The maximal inhibitory effect was obtained with sulglycotide at 100 micrograms/ml, at which dose an 84.4% decrease in PDGF and 88.3% decrease in TGF beta degradation was achieved. The results provide a strong evidence for the effectiveness of sulglycotide in the protection of gastric mucosal growth factors against degradation by H. pylori.

  12. Microplate fluorescence protease assays test the inhibition of select North American snake venoms' activities with an anti-proteinase library.

    PubMed

    Price, Joseph A

    2015-09-01

    Snake envenomation is a relatively neglected significant world health problem, designated an orphan disease by the WHO. While often effective, antivenins are insufficient. Could another approach greatly aid inhibition of the venom toxins? New fluorescent substrates for measuring protease activity in microplate assays suitable for high throughput screening were tested and found reproducible with snake venom. Representative North American venoms showed relatively strong proteinase and collagenase, but weaker elastase activities. Caseinolytic activity is inhibited by the nonspecific proteinase inhibitor 1,10-phenanthroline and by EDTA, as is collagenase activity, consistent with the action of metalloproteinases. Both general protease and collagenase assays CV average 3%, and Km measured were above normal working conditions. Using a library of anti -proteinase compounds with multiple venoms revealed high inhibitor activity by three agents with known multiple metalloproteinase inhibitor activity (Actinonin, GM6001, and NNGH), which incidentally supports the concept that much of the degradative activity of certain venoms is due to metalloproteinases with collagenase activity. These results together support the use of microplate proteinase assays, particularly this collagenase assay, in future drug repurposing studies leading to the development of new treatments for those envenomations that have a major proteolytic component in their pathophysiology.

  13. House dust mite potentiates capsaicin-evoked Ca2+ transients in mouse pulmonary sensory neurons via activation of protease-activated receptor-2.

    PubMed

    Gu, Qihai; Lee, Lu-Yuan

    2012-04-01

    House dust mite (HDM) is a major source of allergen in house dust and has been suggested to be involved in the pathogenesis of asthma. In this study, we aimed to investigate whether HDM can modulate the sensitivity of pulmonary sensory neurons and, if so, to elucidate the underlying mechanism. Fura-2-based ratiometric Ca(2+) imaging was carried out to determine the effect of HDM extract on the capsaicin-evoked Ca(2+) transient in mouse vagal pulmonary sensory neurons. Pretreatment with HDM (50 μg ml(-1), 5 min) significantly enhanced the Ca(2+) transient evoked by capsaicin in these neurons isolated from wild-type mice. This potentiating effect of HDM was not antagonized by E-64, a selective cysteine protease inhibitor, but was completely prevented by AEBSF, a specific serine protease inhibitor. In addition, the potentiating effect of HDM on capsaicin-evoked Ca(2+) transient was absent in the pulmonary sensory neurons isolated from protease-activated receptor-2 (PAR(2)) knockout mice. Furthermore, the sensitizing effect of HDM was completely abolished by U73122, a phosholipase C inhibitor, or chelerythrine, a protein kinase C inhibitor. In summary, our results demonstrate that HDM, mainly through its serine protease activity, potentiates capsaicin-evoked Ca(2+) transient in mouse pulmonary sensory neurons via the activation of PAR(2) and the phosholipase C-protein kinase C intracellular transduction cascade.

  14. Prediction of Extracellular Proteases of the Human Pathogen Helicobacter pylori Reveals Proteolytic Activity of the Hp1018/19 Protein HtrA

    PubMed Central

    Löwer, Martin; Weydig, Christiane; Metzler, Dirk; Reuter, Andreas; Starzinski-Powitz, Anna

    2008-01-01

    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. PMID:18946507

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

    PubMed

    Löwer, Martin; Weydig, Christiane; Metzler, Dirk; Reuter, Andreas; Starzinski-Powitz, Anna; Wessler, Silja; Schneider, Gisbert

    2008-01-01

    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.

  16. The stromal cell-surface protease fibroblast activation protein-α localizes to lipid rafts and is recruited to invadopodia.

    PubMed

    Knopf, Julia D; Tholen, Stefan; Koczorowska, Maria M; De Wever, Olivier; Biniossek, Martin L; Schilling, Oliver

    2015-10-01

    Fibroblast activation protein alpha (FAPα) is a cell surface protease expressed by cancer-associated fibroblasts in the microenvironment of most solid tumors. As there is increasing evidence for proteases having non-catalytic functions, we determined the FAPα interactome in cancer-associated fibroblasts using the quantitative immunoprecipitation combined with knockdown (QUICK) method. Complex formation with adenosin deaminase, erlin-2, stomatin, prohibitin, Thy-1 membrane glycoprotein, and caveolin-1 was further validated by immunoblotting. Co-immunoprecipitation (co-IP) of the known stoichiometric FAPα binding partner dipeptidyl-peptidase IV (DPPIV) corroborated the proteomic strategy. Reverse co-IPs validated the FAPα interaction with caveolin-1, erlin-2, and stomatin while co-IP upon RNA-interference mediated knock-down of DPPIV excluded adenosin deaminase as a direct FAPα interaction partner. Many newly identified FAPα interaction partners localize to lipid rafts, including caveolin-1, a widely-used marker for lipid raft localization. We hypothesized that this indicates a recruitment of FAPα to lipid raft structures. In density gradient centrifugation, FAPα co-fractionates with caveolin-1. Immunofluorescence optical sectioning microscopy of FAPα and lipid raft markers further corroborates recruitment of FAPα to lipid rafts and invadopodia. FAPα is therefore an integral component of stromal lipid rafts in solid tumors. In essence, we provide one of the first interactome analyses of a cell surface protease and translate these results into novel biological aspects of a marker protein for cancer-associated fibroblasts.

  17. A Serine Protease Isolated from the Bristles of the Amazonic Caterpillar, Premolis semirufa, Is a Potent Complement System Activator

    PubMed Central

    Villas Boas, Isadora Maria; Pidde-Queiroz, Giselle; Magnoli, Fabio Carlos; Gonçalves-de-Andrade, Rute M.; van den Berg, Carmen W.; Tambourgi, Denise V.

    2015-01-01

    Background The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called “Pararamose”, characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa’s bristles extract could interfere with the human complement system. Results The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly

  18. Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells.

    PubMed

    Tillery, Lakeisha C; Epperson, Tenille A; Eguchi, Satoru; Motley, Evangeline D

    2016-03-01

    Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632

  19. Molecular docking and structure-based virtual screening studies of potential drug target, CAAX prenyl proteases, of Leishmania donovani.

    PubMed

    Singh, Shalini; Vijaya Prabhu, Sitrarasu; Suryanarayanan, Venkatesan; Bhardwaj, Ruchika; Singh, Sanjeev Kumar; Dubey, Vikash Kumar

    2016-11-01

    Targeting CAAX prenyl proteases of Leishmania donovani can be a good approach towards developing a drug molecule against Leishmaniasis. We have modeled the structure of CAAX prenyl protease I and II of L. donovani, using homology modeling approach. The structures were further validated using Ramachandran plot and ProSA. Active site prediction has shown difference in the amino acid residues present at the active site of CAAX prenyl protease I and CAAX prenyl protease II. The electrostatic potential surface of the CAAX prenyl protease I and II has revealed that CAAX prenyl protease I has more electropositive and electronegative potentials as compared CAAX prenyl protease II suggesting significant difference in their activity. Molecular docking with known bisubstrate analog inhibitors of protein farnesyl transferase and peptidyl (acyloxy) methyl ketones reveals significant binding of these molecules with CAAX prenyl protease I, but comparatively less binding with CAAX prenyl protease II. New and potent inhibitors were also found using structure-based virtual screening. The best docked compounds obtained from virtual screening were subjected to induced fit docking to get best docked configurations. Prediction of drug-like characteristics has revealed that the best docked compounds are in line with Lipinski's rule. Moreover, best docked protein-ligand complexes of CAAX prenyl protease I and II are found to be stable throughout 20 ns simulation. Overall, the study has identified potent drug molecules targeting CAAX prenyl protease I and II of L. donovani whose drug candidature can be verified further using biochemical and cellular studies.

  20. Comparison of helper component-protease RNA silencing suppression activity, subcellular localization, and aggregation of three Korean isolates of Turnip mosaic virus.

    PubMed

    Han, Jae-Yeong; Chung, Jinsoo; Kim, Jungkyu; Seo, Eun-Young; Kilcrease, James P; Bauchan, Gary R; Lim, Seungmo; Hammond, John; Lim, Hyoun-Sub

    2016-08-01

    In 2014, we performed a nationwide survey in Korean radish fields to investigate the distribution and variability of Turnip mosaic virus (TuMV). Brassica rapa ssp. pekinensis sap-inoculated with three isolates of TuMV from infected radish tissue showed different symptom severities, whereas symptoms in Raphanus sativus were similar for each isolate. The helper component-protease (HC-Pro) genes of each isolate were sequenced, and phylogenetic analysis showed that the three Korean isolates were clustered into the basal-BR group. The HC-Pro proteins of these isolates were tested for their RNA silencing suppressor (VSR) activity and subcellular localization in Nicotiana benthamiana. A VSR assay by co-agroinfiltration of HC-Pro with soluble-modified GFP (smGFP) showed that HC-Pro of isolate R007 and R041 showed stronger VSR activity than R065. The HC-Pros showed 98.25 % amino acid identity, and weak VSR isolate (R065) has a single variant residue in the C-terminal domain associated with protease activity and self-interaction compared to isolates with strong VSR activity. Formation of large subcellular aggregates of GFP:HC-Pro fusion proteins in N. benthamiana was only observed for HC-Pro from isolates with strong VSR activity, suggesting that R065 'weak' HC-Pro may have diminished self-association; substitution of the variant C-terminal residue largely reversed the HC-Pro aggregation and silencing suppressor characteristics. The lack of correlation between VSR efficiency and induction of systemic necrosis (SN) suggests that differences in viral accumulation due to HC-Pro are not responsible for SN.

  1. Effect of solvent on the conformation and dynamics of Aspartic Acid Protease by a coarse-grained bond-fluctuating Monte Carlo simulation

    NASA Astrophysics Data System (ADS)

    Pandey, Ras; Farmer, Barry

    2007-03-01

    In a coarse-grained description of a protein chain, all of the 20 amino acid residues can be broadly divided into three groups, hydrophobic (H), polar (P), and electrostatic (E). A protein can be described by tethered nodes in a chain with a node representing the amino acid group. Aspartic acid protease consists of 99 residues in a well-defined sequence. The specific sequence of H, P and E nodes tethered together by fluctuating bonds is placed on a cubic lattice where empty lattice sites constitute an effective solvent medium. The amino groups (nodes) interact with the solvent (S) sites with appropriate attractive (HS) and repulsive (PS) interactions with the solvent. Each node executes its stochastic movement with the Metropolis algorithm. Variations of the root mean square displacements of the center of mass and that of its center node of the protease chain, and its gyration radius with the time steps are examined for different solvent strength. The structure of the protease swells on increasing the solvent interaction strength which tends to enhance the relaxation time to reach diffusive behavior of the chain.

  2. Conformation of a coarse-grained protein chain (an aspartic acid protease) model in effective solvent by a bond-fluctuating Monte Carlo simulation

    NASA Astrophysics Data System (ADS)

    Pandey, R. B.; Farmer, B. L.

    2008-03-01

    In a coarse-grained description of a protein chain, all of the 20 amino acid residues can be broadly divided into three groups: Hydrophobic (H) , polar (P) , and electrostatic (E) . A protein can be described by nodes tethered in a chain with a node representing an amino acid group. Aspartic acid protease consists of 99 residues in a well-defined sequence of H , P , and E nodes tethered together by fluctuating bonds. The protein chain is placed on a cubic lattice where empty lattice sites constitute an effective solvent medium. The amino groups (nodes) interact with the solvent (S) sites with appropriate attractive (PS) and repulsive (HS) interactions with the solvent and execute their stochastic movement with the Metropolis algorithm. Variations of the root mean square displacements of the center of mass and that of its center node of the protease chain and its gyration radius with the time steps are examined for different solvent strength. The structure of the protease swells on increasing the solvent interaction strength which tends to enhance the relaxation time to reach the diffusive behavior of the chain. Equilibrium radius of gyration increases linearly on increasing the solvent strength: A slow rate of increase in weak solvent regime is followed by a faster swelling in stronger solvent. Variation of the gyration radius with the time steps suggests that the protein chain moves via contraction and expansion in a somewhat quasiperiodic pattern particularly in strong solvent.

  3. NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies

    PubMed Central

    Boukadida, Célia; Marnata, Caroline; Montserret, Roland; Cohen, Lisette; Blumen, Brigitte; Gouttenoire, Jérôme; Moradpour, Darius; Penin, François

    2014-01-01

    ABSTRACT GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion

  4. The influence of repeated administration of poloxamer 407 on serum lipoproteins and protease activity in mouse liver and heart.

    PubMed

    Korolenko, Tatyana A; Tuzikov, Fedor V; Johnston, Thomas P; Tuzikova, Natalia A; Kisarova, Yana A; Zhanaeva, Svetlana Ya; Alexeenko, Tatyana V; Zhukova, Natalia A; Brak, Ivan V; Spiridonov, Victor K; Filjushina, Elena E; Cherkanova, Marina S; Monoszon, Anna A

    2012-11-01

    The effects of repeated administration of poloxamer 407 (P-407) on lipoprotein-cholesterol (LP-C) and lipoprotein-triglyceride (LP-TG) fractions and subfractions, as well as the effect on liver and heart proteases, were studied. Repeated administration of P-407 to male CBA mice resulted in a model of atherosclerosis with increased diastolic blood pressure; there was a drastic increase in total serum cholesterol and especially TG. A novel small-angle X-ray scattering method for the determination of the fractional and subfractional composition of LP-C and LP-TG was used. In chronically P-407-treated mice, P-407 significantly increased atherogenic low-density lipoprotein C (LDL-C) fractions, as well as intermediate-density lipoprotein C (IDL-C), and LDL₁₋₃-C subfractions, and very-low-density lipoprotein-C (VLDL-C) fractions, as well as VLDL₁₋₂-C and VLDL₃₋₅-C subfractions), to a lesser extent, the total anti-atherogenic high-density lipoprotein C (HDL-C) fraction, as well as HDL₂-C and HDL₃-C subfractions. Additionally, we demonstrated an increase in the serum chitotriosidase activity, without significant changes in serum matrix metalloprotease (MMP) activity. Morphological changes observed in P-407-treated mice included atherosclerosis in the heart and storage syndrome in the liver macrophages. P-407 significantly increased the activity of cysteine, aspartate proteases, and MMPs in the heart, and only the activity of cathepsin B and MMPs in the liver of mice. Thus, repeated administration of P-407 to mice induced atherosclerosis secondary to sustained dyslipidemia and formation of foamy macrophages in liver, and also modulated the activity of heart and liver proteases.

  5. Evaluation of cytotoxic and anti-tumor activity of partially purified serine protease isolate from the Indian earthworm Pheretima posthuma

    PubMed Central

    Verma, Mahendra Kumar; Xavier, Francies; Verma, Yogendra Kumar; Sobha, Kota

    2013-01-01

    Objective To isolate, partially purify and evaluate the cytotoxic and antitumor activity of a serine protease from the chosen Indian earthworm Pheretima posthuma. Methods Whole animal extract was prepared and purified its protein constituents by size and charge based chromatographic separation techniques using Sephadex G-50 and DEAE-Cellulose resin respectively. Average molecular weight of the protein isolate was determined and analyzed for its cytotoxic property against Vero cells in different dilutions (1: 20 and 1: 40) and anti-tumor activity by MTT assay (a colorimetric assay) using breast cancer cell line MCF-7, with tamoxifen as standard. Results One of the protein constituents after purification was characterized as serine protease by Caseinolytic plate diffusion assay. Average molecular weight of this purified isolate was determined, by SDS-PAGE analysis with standard protein ladder, as of 15 kDa. The performed tests suggested that the 15kDa fraction has potent cytotoxic activity and satisfactory antitumor activity as well in vitro. Conclusions Exact molecular mechanism of the cytotoxic and antitumor activities is yet to be explored and currently we are working on ultra-purification and biophysical characterization of this fraction. Further investigation into the mechanism(s) of cytotoxic and antitumor activities at molecular level would be useful in treatment of various classes of cancer and viral infections in future.

  6. Protease activated receptor 4 limits bacterial growth and lung pathology during late stage Streptococcus pneumoniae induced pneumonia in mice.

    PubMed

    de Stoppelaar, S F; Van't Veer, C; van den Boogaard, F E; Nieuwland, R; Hoogendijk, A J; de Boer, O J; Roelofs, J J T H; van der Poll, T

    2013-09-01

    Streptococcus pneumoniae is a common causative pathogen of pneumonia and sepsis. Pneumonia and sepsis are associated with enhanced activation of coagulation, resulting in the production of several host-derived proteases at the primary site of infection and in the circulation. Serine proteases cleave protease activated receptors (PARs), which form a molecular link between coagulation and inflammation. PAR4 is one of four subtypes of PARs and is widely expressed by multiple cell types in the respiratory tract implicated in pulmonary inflammation, by immune cells and by platelets. In mice, mouse (m)PAR4 is the only thrombin receptor expressed by platelets. We here sought to determine the contribution of mPAR4 to the host response during pneumococcal pneumonia. Pneumonia was induced by intranasal inoculation with S. pneumoniae in mPAR4-deficient (par4-/-) and wild-type mice. Mice were sacrificed after 6, 24 or 48 hours (h). Blood, lungs, liver and spleen were collected for analyses. Ex vivo stimulation assays were performed with S. pneumoniae and mPAR4 activating peptides. At 48 h after infection, higher bacterial loads were found in the lungs and blood of par4-/- mice (p < 0.05), accompanied by higher histopathology scores and increased cytokine levels (p < 0.05) in the lungs. Ex vivo, co-stimulation with mPAR4 activating peptide enhanced the whole blood cytokine response to S. pneumoniae. Thrombin inhibition resulted in decreased cytokine release after S. pneumoniae stimulation in human whole blood. Our findings suggest that mPAR4 contributes to antibacterial defence during murine pneumococcal pneumonia.

  7. Characterisation of faecal protease activity in irritable bowel syndrome with diarrhoea: origin and effect of gut transit

    PubMed Central

    Tooth, David; Garsed, Klara; Singh, Gulzar; Marciani, Luca; Lam, Ching; Fordham, Imogen; Fields, Annie; Banwait, Rawinder; Lingaya, Melanie; Layfield, Robert; Hastings, Maggie; Whorwell, Peter; Spiller, Robin

    2014-01-01

    Objectives Faecal serine proteases (FSPs) may play a role in irritable bowel syndrome with diarrhoea (IBS-D), but their origin is unclear. We aimed to structurally characterise them and define the impact of colonic cleansing and transit time. Design Faecal samples were obtained from 30 healthy volunteers (HV) and 79 patients with IBS-D participating in a trial of ondansetron versus placebo. Colonic transit was measured using radio-opaque markers. Samples were also obtained from 24 HV before and after colonic cleansing with the osmotic laxative MoviPrep. FSPs were purified from faecal extracts using benzamidine-Sepharose affinity chromatography. SDS-PAGE profiled components were identified using trypsinolysis and tandem mass spectrometry. Functional protease activity in faecal extracts was measured using a colorimetric assay based on the proteolysis of azo-casein. Results Protein analysis identified the most abundant FSPs as being of human origin and probably derived from pancreatic juice. Functional assays showed increased faecal protease (FP) and amylase in patients with IBS-D compared with HV. Those with higher amylase had significantly higher FP and greater anxiety. FP activity correlated negatively with whole gut transit in patients with IBS-D (Spearman r=−0.32, p=0.005) and HV (r=−0.55, p=0.014). Colon cleansing caused a significant rise in FP activity in HV from a baseline of median (IQR) 253 (140–426) to 1031 (435–2296), levels similar to those seen in patients with IBS-D. FSP activity correlated positively with days/week with urgency. Conclusions The most abundant FSPs are of human origin. Rapid transit through the colon and/or decreased (possibly bacterial) proteolytic degradation increases their faecal concentration and could contribute to visceral hypersensitivity in patients with IBS-D. ClinicalTrials.gov NCT00745004. PMID:23911555

  8. Protease-activated receptors modulate excitability of murine colonic smooth muscles by differential effects on interstitial cells

    PubMed Central

    Sung, Tae Sik; Kim, Heung Up; Kim, Jeong Hwan; Lu, Hongli; Sanders, Kenton M; Koh, Sang Don

    2015-01-01

    Abstract Protease-activated receptors (PARs) are G protein-coupled receptors activated by proteolytic cleavage at their amino termini by serine proteases. PAR activation contributes to the inflammatory response in the gastrointestinal (GI) tract and alters GI motility, but little is known about the specific cells within the tunica muscularis that express PARs and the mechanisms leading to contractile responses. Using real time PCR, we found PARs to be expressed in smooth muscle cells (SMCs), interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor α positive (PDGFRα+) cells. The latter cell-type showed dominant expression of F2r (encodes PAR1) and F2rl1 (encodes PAR2). Contractile and intracellular electrical activities were measured to characterize the integrated responses to PAR activation in whole muscles. Cells were isolated and ICC and PDGFRα+ cells were identified by constitutive expression of fluorescent reporters. Thrombin (PAR1 agonist) and trypsin (PAR2 agonist) caused biphasic responses in colonic muscles: transient hyperpolarization and relaxation followed by repolarization and excitation. The inhibitory phase was blocked by apamin, revealing a distinct excitatory component. Patch clamp studies showed that the inhibitory response was mediated by activation of small conductance calcium-activated K+ channels in PDGFRα+ cells, and the excitatory response was mediated by activation of a Cl− conductance in ICC. SMCs contributed little to PAR responses in colonic muscles. In summary, PARs regulate the excitability of colonic muscles; different conductances are activated in each cell type of the SMC–ICC–PDGFRα+ cell (SIP) syncytium. Motor responses to PAR agonists are integrated responses of the SIP syncytium. Key points Activation of protease-activated receptors (PAR) regulates gastrointestinal (GI) motility but little is known about the cells and mechanisms in GI muscles responsible for PAR responses. Using mouse cells, we

  9. PLGA-PEG Nanoparticles Coated with Anti-CD45RO and Loaded with HDAC Plus Protease Inhibitors Activate Latent HIV and Inhibit Viral Spread

    NASA Astrophysics Data System (ADS)

    Tang, Xiaolong; Liang, Yong; Liu, Xinkuang; Zhou, Shuping; Liu, Liang; Zhang, Fujina; Xie, Chunmei; Cai, Shuyu; Wei, Jia; Zhu, Yongqiang; Hou, Wei

    2015-10-01

    Activating HIV-1 proviruses in latent reservoirs combined with inhibiting viral spread might be an effective anti-HIV therapeutic strategy. Active specific delivery of therapeutic drugs into cells harboring latent HIV, without the use of viral vectors, is a critical challenge to this objective. In this study, nanoparticles of poly(lactic-co-glycolic acid)-polyethylene glycol diblock copolymers conjugated with anti-CD45RO antibody and loaded with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and/or protease inhibitor nelfinavir (Nel) were tested for activity against latent virus in vitro. Nanoparticles loaded with SAHA, Nel, and SAHA + Nel were characterized in terms of size, surface morphology, zeta potential, entrapment efficiency, drug release, and toxicity to ACH-2 cells. We show that SAHA- and SAHA + Nel-loaded nanoparticles can target latently infected CD4+ T-cells and stimulate virus production. Moreover, nanoparticles loaded with SAHA + NEL were capable of both activating latent virus and inhibiting viral spread. Taken together, these data demonstrate the potential of this novel reagent for targeting and eliminating latent HIV reservoirs.

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

    PubMed

    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.

  11. Conformational Lability in Serine Protease Active Sites: Structures of Hepatocyte Growth Factor Activator (HGFA) Alone and with the Inhibitory Domain from HGFA Inhibitor-1B

    SciTech Connect

    Shia, Steven; Stamos, Jennifer; Kirchhofer, Daniel; Fan, Bin; Wu, Judy; Corpuz, Raquel T.; Santell, Lydia; Lazarus, Robert A.; Eigenbrot, Charles

    2010-07-20

    Hepatocyte growth factor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. When activated HGF binds its cognate receptor Met, cellular signals lead to cell growth, differentiation, and migration, activities which promote tissue regeneration in liver, kidney and skin. Intervention in the conversion of HGF to its active form has the potential to provide therapeutic benefit where HGF/Met activity is associated with tumorigenesis. To help identify ways to moderate HGF/Met effects, we have determined the molecular structure of the protease domain of HGFA. The structure we determined, at 2.7 {angstrom} resolution, with no pseudo-substrate or inhibitor bound is characterized by an unconventional conformation of key residues in the enzyme active site. In order to find whether this apparently non-enzymatically competent arrangement would persist in the presence of a strongly-interacting inhibitor, we also have determined, at 2.6 {angstrom} resolution, the X-ray structure of HGFA complexed with the first Kunitz domain (KD1) from the physiological inhibitor hepatocyte growth factor activator inhibitor 1B (HAI-1B). In this complex we observe a rearranged substrate binding cleft that closely mirrors the cleft of other serine proteases, suggesting an extreme conformational dynamism. We also characterize the inhibition of 16 serine proteases by KD1, finding that the previously reported enzyme specificity of the intact extracellular region of HAI-1B resides in KD1 alone. We find that HGFA, matriptase, hepsin, plasma kallikrein and trypsin are potently inhibited, and use the complex structure to rationalize the structural basis of these results.

  12. Determining Protease Activity In Vivo by Fluorescence Cross-Correlation Analysis

    PubMed Central

    Kohl, Tobias; Haustein, Elke; Schwille, Petra

    2005-01-01

    To date, most biochemical approaches to unravel protein function have focused on purified proteins in vitro. Whereas they analyze enzyme performance under assay conditions, they do not necessarily tell us what is relevant within a living cell. Ideally, cellular functions should be examined in situ. In particular, association/dissociation reactions are ubiquitous, but so far there is no standard technique permitting online analysis of these processes in vivo. Featuring single-molecule sensitivity combined with intrinsic averaging, fluorescence correlation spectroscopy is a minimally invasive technique ideally suited to monitor proteins. Moreover, endogenous fluorescence-based assays can be established by genetically encoding fusions of autofluorescent proteins and cellular proteins, thus avoiding the disadvantages of in vitro protein labeling and subsequent delivery to cells. Here, we present an in vivo protease assay as a model system: Green and red autofluorescent proteins were connected by Caspase-3- sensitive and insensitive protein linkers to create double-labeled protease substrates. Then, dual-color fluorescence cross-correlation spectroscopy was employed to study the protease reaction in situ. Allowing assessment of multiple dynamic parameters simultaneously, this method provided internal calibration and improved experimental resolution for quantifying protein stability. This approach, which is easily extended to reversible protein-protein interactions, seems very promising for elucidating intracellular protein functions. PMID:16055538

  13. In vitro ANTIGIARDIAL ACTIVITY OF THE CYSTEINE PROTEASE INHIBITOR E-64

    PubMed Central

    de Carvalho, Thaís Batista; Oliveira-Sequeira, Teresa Cristina Goulart; Guimarães, Semíramis

    2014-01-01

    The quest for new antiparasitic alternatives has led researchers to base their studies on insights into biology, host-parasite interactions and pathogenesis. In this context, proteases and their inhibitors are focused, respectively, as druggable targets and new therapy alternatives. Herein, we proposed to evaluate the in vitro effect of the cysteine protease inhibitor E-64 on Giardia trophozoites growth, adherence and viability. Trophozoites (105) were exposed to E-64 at different final concentrations, for 24, 48 and 72 h at 37 °C. In the growth and adherence assays, the number of trophozoites was estimated microscopically in a haemocytometer, whereas cell viability was evaluated by a dye-reduction assay using MTT. The E-64 inhibitor showed effect on growth, adherence and viability of trophozoites, however, its better performance was detected in the 100 µM-treated cultures. Although metronidazole was more effective, the E-64 was shown to be able to inhibit growth, adherence and viability rates by ≥ 50%. These results reveal that E-64 can interfere in some crucial processes to the parasite survival and they open perspectives for future investigations in order to confirm the real antigiardial potential of the protease inhibitors. PMID:24553607

  14. Proteochemometrics mapping of the interaction space for retroviral proteases and their substrates.

    PubMed

    Kontijevskis, Aleksejs; Petrovska, Ramona; Yahorava, Sviatlana; Komorowski, Jan; Wikberg, Jarl E S

    2009-07-15

    Understanding the complex interactions of retroviral proteases with their ligands is an important scientific challenge in efforts to achieve control of retroviral infections. Development of drug resistance because of high mutation rates and extensive polymorphisms causes major problems in treating the deadly diseases these viruses cause, and prompts efforts to identify new strategies. Here we report a comprehensive analysis of the interaction of 63 retroviral proteases from nine different viral species with their substrates and inhibitors based on publicly available data from the past 17years of retroviral research. By correlating physico-chemical descriptions of retroviral proteases and substrates to their biological activities we constructed a highly statistically valid 'proteochemometric' model for the interactome of retroviral proteases. Analysis of the model indicated amino acid positions in retroviral proteases with the highest influence on ligand activity and revealed general physicochemical properties essential for tight binding of substrates across multiple retroviral proteases. Hexapeptide inhibitors developed based on the discovered general properties effectively inhibited HIV-1 proteases in vitro, and some exhibited uniformly high inhibitory activity against all HIV-1 proteases mutants evaluated. A generalized proteochemometric model for retroviral proteases interactome has been created and analysed in this study. Our results demonstrate the feasibility of using the developed general strategy in the design of inhibitory peptides that can potentially serve as templates for drug resistance-improved HIV retardants.

  15. Genetic and biochemical analysis of gonococcal IgA1 protease: cloning in Escherichia coli and construction of mutants of gonococci that fail to produce the activity.

    PubMed Central

    Koomey, J M; Gill, R E; Falkow, S

    1982-01-01

    The biological significance of bacterial extracellular proteases that specifically cleave human IgA1 is unknown. We have prepared a gene bank of gonococcal chromosomal DNA in Escherichia coli K-12 using a cosmid cloning system. Among these clones, we have identified and characterized an E. coli strain that elaborates an extracellular endopeptidase that is indistinguishable from gonococcal IgA1 protease in its substrate specificity and action on human IgA1. Analysis of recombinant plasmids and examination of plasmid-specific peptides in minicells have shown that the IgA1 protease activity in E. coli is associated with expression of a Mr 140,000 peptide. We have isolated IgA1 protease-deficient mutants of Neisseria gonorrhoeae by reintroduction of physically defined deletions of the cloned gene into the gonococcal chromosome by transformation. Images PMID:6818556

  16. Protease-activated receptor 2 activation is sufficient to induce the transition to a chronic pain state.

    PubMed

    Tillu, Dipti V; Hassler, Shayne N; Burgos-Vega, Carolina C; Quinn, Tammie L; Sorge, Robert E; Dussor, Gregory; Boitano, Scott; Vagner, Josef; Price, Theodore J

    2015-05-01

    Protease-activated receptor type 2 (PAR2) is known to play an important role in inflammatory, visceral, and cancer-evoked pain based on studies using PAR2 knockout (PAR2(-/-)) mice. We have tested the hypothesis that specific activation of PAR2 is sufficient to induce a chronic pain state through extracellular signal-regulated kinase (ERK) signaling to protein synthesis machinery. We have further tested whether the maintenance of this chronic pain state involves a brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (trkB)/atypical protein kinase C (aPKC) signaling axis. We observed that intraplantar injection of the novel highly specific PAR2 agonist, 2-aminothiazol-4-yl-LIGRL-NH2 (2-at), evokes a long-lasting acute mechanical hypersensitivity (median effective dose ∼12 pmoles), facial grimacing, and causes robust hyperalgesic priming as revealed by a subsequent mechanical hypersensitivity and facial grimacing to prostaglandin E2 (PGE2) injection. The promechanical hypersensitivity effect of 2-at is completely absent in PAR2(-/-) mice as is hyperalgesic priming. Intraplantar injection of the upstream ERK inhibitor, U0126, and the eukaryotic initiation factor (eIF) 4F complex inhibitor, 4EGI-1, prevented the development of acute mechanical hypersensitivity and hyperalgesic priming after 2-at injection. Systemic injection of the trkB antagonist ANA-12 similarly inhibited PAR2-mediated mechanical hypersensitivity, grimacing, and hyperalgesic priming. Inhibition of aPKC (intrathecal delivery of ZIP) or trkB (systemic administration of ANA-12) after the resolution of 2-at-induced mechanical hypersensitivity reversed the maintenance of hyperalgesic priming. Hence, PAR2 activation is sufficient to induce neuronal plasticity leading to a chronic pain state, the maintenance of which is dependent on a BDNF/trkB/aPKC signaling axis.

  17. Protease-Activated Receptor-1 Supports Locomotor Recovery by Biased Agonist Activated Protein C after Contusive Spinal Cord Injury

    PubMed Central

    Whetstone, William D.; Walker, Breset; Trivedi, Alpa; Lee, Sangmi; Noble-Haeusslein, Linda J.; Hsu, Jung-Yu C.

    2017-01-01

    Thrombin-induced secondary injury is mediated through its receptor, protease activated receptor-1 (PAR-1), by "biased agonism." Activated protein C (APC) acts through the same PAR-1 receptor but functions as an anti-coagulant and anti-inflammatory protein, which counteracts many of the effects of thrombin. Although the working mechanism of PAR-1 is becoming clear, the functional role of PAR-1 and its correlation with APC in the injured spinal cord remains to be elucidated. Here we investigated if PAR-1 and APC are determinants of long-term functional recovery after a spinal cord contusive injury using PAR-1 null and wild-type mice. We found that neutrophil infiltration and disruption of the blood-spinal cord barrier were significantly reduced in spinal cord injured PAR-1 null mice relative to the wild-type group. Both locomotor recovery and ability to descend an inclined grid were significantly improved in the PAR-1 null group 42 days after injury and this improvement was associated with greater long-term sparing of white matter and a reduction in glial scarring. Wild-type mice treated with APC acutely after injury showed a similar level of improved locomotor recovery to that of PAR-1 null mice. However, improvement of APC-treated PAR-1 null mice was indistinguishable from that of vehicle-treated PAR-1 null mice, suggesting that APC acts through PAR-1. Collectively, ou