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Sample records for proteolytic lumbrokinase extracted

  1. Intestinal Absorption of Fibrinolytic and Proteolytic Lumbrokinase Extracted from Earthworm, Eisenia andrei

    PubMed Central

    Yan, Xiang Mei; Kim, Chung-Hyo; Lee, Chul Kyu; Shin, Jang Sik; Cho, Il Hwan

    2010-01-01

    To investigate the intestinal absorption of a fibrinolytic and proteolytic lumbrokinase extracted from Eisenia andrei, we used rat everted gut sacs and an in situ closed-loop recirculation method. We extracted lumbrokinase from Eisenia andrei, and then raised polyclonal antibody against lumbrokinase. Fibrinolytic activity and proteolytic activity in the serosal side of rat everted gut sacs incubated with lumbrokinase showed dose- and time-dependent patterns. Immunological results obtained by western blotting serosal side solution using rat everted gut sacs method showed that lumbrokinase proteins between 33.6 and 54.7 kDa are absorbed mostly by the intestinal epithelium. Furthermore, MALDI-TOF mass spectrometric analysis of plasma fractions obtained by in situ recirculation method confirmed that lumbrokinase F1 is absorbed into blood. These results support the notion that lumbrokinase can be absorbed from mucosal lumen into blood by oral administration. PMID:20473377

  2. Intestinal Absorption of Fibrinolytic and Proteolytic Lumbrokinase Extracted from Earthworm, Eisenia andrei.

    PubMed

    Yan, Xiang Mei; Kim, Chung-Hyo; Lee, Chul Kyu; Shin, Jang Sik; Cho, Il Hwan; Sohn, Uy Dong

    2010-04-01

    To investigate the intestinal absorption of a fibrinolytic and proteolytic lumbrokinase extracted from Eisenia andrei, we used rat everted gut sacs and an in situ closed-loop recirculation method. We extracted lumbrokinase from Eisenia andrei, and then raised polyclonal antibody against lumbrokinase. Fibrinolytic activity and proteolytic activity in the serosal side of rat everted gut sacs incubated with lumbrokinase showed dose- and time-dependent patterns. Immunological results obtained by western blotting serosal side solution using rat everted gut sacs method showed that lumbrokinase proteins between 33.6 and 54.7 kDa are absorbed mostly by the intestinal epithelium. Furthermore, MALDI-TOF mass spectrometric analysis of plasma fractions obtained by in situ recirculation method confirmed that lumbrokinase F1 is absorbed into blood. These results support the notion that lumbrokinase can be absorbed from mucosal lumen into blood by oral administration. PMID:20473377

  3. Improved peripheral nerve regeneration in streptozotocin-induced diabetic rats by oral lumbrokinase.

    PubMed

    Lee, Han-Chung; Hsu, Yuan-Man; Tsai, Chin-Chuan; Ke, Cherng-Jyh; Yao, Chun-Hsu; Chen, Yueh-Sheng

    2015-01-01

    We assessed the therapeutic effects of lumbrokinase, a group of enzymes extracted from the earthworm, on peripheral-nerve regeneration using well-defined sciatic nerve lesion paradigms in diabetic rats induced by the injection of streptozotocin (STZ). We found that lumbrokinase therapy could improve the rats' circulatory blood flow and promote the regeneration of axons in a silicone rubber conduit after nerve transection. Lumbrokinase treatment could also improve the neuromuscular functions with better nerve conductive performances. Immunohistochemical staining showed that lumbrokinase could dramatically promote calcitonin gene-related peptide (CGRP) expression in the lamina I-II regions in the dorsal horn ipsilateral to the injury and cause a marked increase in the number of macrophages recruited within the distal nerve stumps. In addition, the lumbrokinase could stimulate the secretion of interleukin-1 (IL-1), nerve growth factor (NGF), platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β) in dissected diabetic sciatic nerve segments. In conclusion, the administration of lumbrokinase after nerve repair surgery in diabetic rats was found to have remarkable effects on promoting peripheral nerve regeneration and functional recovery. PMID:25787300

  4. Recombinant Protein Production of Earthworm Lumbrokinase for Potential Antithrombotic Application

    PubMed Central

    Wang, Kevin Yueju; Wang, Nan; Liu, Dehu

    2013-01-01

    Earthworms have been used as a traditional medicine in China, Japan, and other Far East countries for thousands of years. Oral administration of dry earthworm powder is considered as a potent and effective supplement for supporting healthy blood circulation. Lumbrokinases are a group of enzymes that were isolated and purified from different species of earthworms. These enzymes are recognized as fibrinolytic agents that can be used to treat various conditions associated with thrombosis. Many lumbrokinase (LK) genes have been cloned and characterized. Advances in genetic technology have provided the ability to produce recombinant LK and have made it feasible to purify a single lumbrokinase enzyme for potential antithrombotic application. In this review, we focus on expression systems that can be used for lumbrokinase production. In particular, the advantages of using a transgenic plant system to produce edible lumbrokinase are described. PMID:24416067

  5. Effect of proteolytic activities in combination with the pectolytic activities on extractability of the fat and phenolic compounds from olives.

    PubMed

    Moustakime, Youssef; Hazzoumi, Zakaria; Amrani Joutei, Khalid

    2016-01-01

    During the extraction, a portion of oil remains trapped inside the cells and its release requires the degradation of the walls and cell membranes, especially when the fruits have not reached a maximum maturity which is likely to cause an optimal embrittlement of the parietal structures and cell membrane. This can be done by specific enzymes necessary for the degradation of various cellular barriers. Three different enzyme treatments proteolytic, pectolytic or both are applied on the Moroccan Picholine olives from veraison to maturity of the fruit. The effect of these treatments is evaluated by olive oil diffusion, its phenolic content (PC) and cellular embrittlement determination of olives during ripening. The pectolytic activities lead to a significant increase in both the oil extractability (76 % at veraison and 14 % at maturity) and the PC (up to 50 % of gain compared to the control at veraison and 27 % at maturity). The proteolytic activities applied alone have no significant effect on the extractability and the polyphenols levels of oils. Furthermore, when these proteolytic activities are added in combination with the pectolytic activities, the oil extractability is doubled at veraison and its flowing up to 99 % at maturity that barely 84 % in the control in addition to a richness of polyphenols which can reach 84 % more compared to the control. This increase in polyphenols wealth is probably due to the degradation of cell walls, cellular and vacuolar membranes by enzyme activities releasing PCs that were previously associated with these structures in the drupe. PMID:27376007

  6. Effects on fibrinogen, fibrin, and blood coagulation of proteolytic extracts from fruits of Pseudananas macrodontes, Bromelia balansae, and B. hieronymi (Bromeliaceae) in comparison with bromelain.

    PubMed

    Errasti, María E; Prospitti, Anabela; Viana, Carolina A; Gonzalez, Mariana M; Ramos, Márcio V; Rotelli, Alejandra E; Caffini, Néstor O

    2016-06-01

    Extracts rich in cysteine proteases obtained from fruits of Pseudananas macrodontes (Pm), Bromelia balansae (Bb), and B. hieronymi (Bh) have previously shown an anti-inflammatory effect on animal models. Given the close relationship between hemostasis and inflammation, it is attractive to investigate therapeutic agents capable of modulating both systems. The aim of this work was to study the effect of Pm, Bb, and Bh on fibrin(ogen) and blood coagulation compared with stem bromelain (Bro). Action on fibrinogen was electrophoretically and spectrophotometrically evaluated, fibrinolytic activity was measured both electrophoretically and by the fibrin plate assay, and the effect on blood coagulation was studied by conventional coagulation tests (PT and APPT). All extracts showed the same proteolytic preference for fibrinogen subunits, that is Aα > Bβ, whereas γ was partially hydrolyzed by 100-fold concentration increase. Unlike Bro, cysteine proteases of Pm, Bb, and Bh increased absorbance at 540 nm of fibrinogen solution, suggesting thrombin-like activity, which was time-dependent and reached maximum values at lower concentration. All extracts showed the same proteolytic preference for fibrin subunits; however Pm, Bb, and Bh showed lower fibrinolytic activity than Bro at the assayed concentrations. Although Bb acted only as anticoagulant, Pm, Bh, and unexpectedly Bro showed dual action on blood coagulation: at low concentration showed procoagulant effect and at high concentration anticoagulant effect. Results reveal new plant species as potential sources of pharmacological agents for the treatment of a wide range of hemostatic disorders as well as to wound healing. PMID:26886361

  7. Effect of legume seed extracts on the inhibition of proteolytic activity and muscle degradation of fresh water prawn (Macrobrachiumrosenbergii).

    PubMed

    Sriket, Chodsana; Benjakul, Soottawat; Visessanguan, Wonnop; Hara, Kenji

    2011-12-01

    Trypsin inhibitors in the extracts from soybean (Glycine max), adzuki bean (Vigna angularis), bambara groundnut (Vigna subterranea) and red kidney bean (Phaseoulus vulgaris) varied in amount and molecular weight. The soybean extract had the highest level of trypsin inhibitor with molecular weight (MW) of 21kDa, followed by bambara groundnut extract possessing trypsin inhibitor with MW of 15kDa. Both extracts showed a more effective inhibition towards crude protease extract (CE) from the hepatopancreas of fresh water prawn (Macrobrachium rosenbergii) than the extracts from adzuki and red kidney beans. Activity staining also reconfirmed the higher inhibitory activity on CE from hepatopancreas by the extracts from both soybean and bambara groundnut. The extracts from all seeds were able to inhibit the degradation of fresh water prawn meat containing CE in a concentration dependent manner. Based on inhibitor study, the extracts from soybean and bambara groundnut can be a potential aid to suppress the muscle softening of fresh water prawn, mediated by trypsin-like proteases released from hepatopancreas, during extended iced storage. PMID:25212342

  8. Cardio Protective Effects of Lumbrokinase and Dilong on Second-Hand Smoke-Induced Apoptotic Signaling in the Heart of a Rat Model.

    PubMed

    Liao, Hung-En; Lai, Chao-Hung; Ho, Tsung-Jung; Yeh, Yu-Lan; Jong, Gwo-Ping; Kuo, Wu-Hsien; Chung, Li-Chin; Pai, Pei-ying; Wen, Su-Ying; Huang, Chih-Yang

    2015-06-30

    Exposure to second-hand tobacco smoke (SHS) has been epidemiologically linked to heart disease among non-smokers. However, the molecular mechanism behind SHS-induced cardiac disease is not well known. This study found that SD rats exposed to cigarette smoke at a dose of 10 cigarettes for 30 min twice a day for 1 month had a reduced left ventricle-to-tibia length ratio (mg/mm), increased cardiomyocyte apoptosis by TUNEL assay and a wider interstitial space by H&E staining. However, lumbrokinase and dilong both reversed the effects of SHS. Western blotting demonstrated significantly increased expression of the pro-apoptotic protein caspase-3 in the hearts of the rats exposed to SHS. Elevated protein expression levels of Fas, FADD and the apoptotic initiator activated caspase-8, a molecule in the death-receptor-dependent pathway, coupled with increased t-Bid and apoptotic initiator activated caspase-9 were found. Molecules in the mitochondria-dependent pathway, which disrupts mitochondrial membrane potential, were also found in rats exposed to SHS. These factors indicate myocardial apoptosis. However, treatment with lumbrokinase and dilong inhibited SHS-induced apoptosis. Regarding regulation of the survival pathway, we found in western blot analysis that cardiac protein expression of pAkt, Bcl2, and Bcl-xL was significantly down-regulated in rats exposed to SHS. These effects were reversed with lumbrokinase and dilong treatment. The effects of SHS on cardiomyocytes were also found to be mediated by the Fas death receptor-dependent apoptotic pathway, an unbalanced mitochondria membrane potential and decreased survival signaling. However, treatment with both lumbrokinase and dilong inhibited the effects of SHS. Our data suggest that lumbrokinase and dilong may prevent heart disease in SHS-exposed non-smokers. PMID:26014124

  9. Proteolytic properties of Funastrum clausum latex.

    PubMed

    Morcelle, Susana R; Caffini, Néstor O; Priolo, Nora

    2004-07-01

    As part of a screening of latex endopeptidases from plants growing in Argentina, the presence of proteolytic activity in the latex of Funastrum clausum stems is reported. The proteases present in the crude extract showed the main characteristics of the cysteine proteolytic class, i.e. optimum pH at alkaline range, isoelectric point (pI) higher than 9.0, and inhibition of proteolytic activity by thiol blocking reagents. A remarkable thermal stability was also evident in the crude extract. Endosterolytic preference tried on p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids was higher for the alanine, asparagine and tyrosine derivatives. Preliminary peptidase purification by two-step ionic exchange showed the presence of two proteolytic fractions with molecular masses of approximately 24.0 kDa according to SDS-PAGE. PMID:15261386

  10. Elastinolytic and proteolytic enzymes.

    PubMed

    Kessler, Efrat; Safrin, Mary

    2014-01-01

    Pseudomonas aeruginosa secretes into its environment at least seven extracellular proteases: pseudolysin (LasB protease; elastase), aeruginolysin (alkaline proteinase), staphylolysin (staphylolytic endopeptidase; LasA protease), lysyl endopeptidase (protease IV; PrpL), PASP (P. aeruginosa small protease), LepA (Large ExoProtease A), and an aminopeptidase. Their action on host proteins, both individually and synergistically, plays important roles in pathogenesis of P. aeruginosa infections. Methods to measure/detect their activities are fundamental for understanding their physiological functions, roles in pathogenesis, mechanisms of action, regulation, and secretion. Most assays for determination/detection of proteolytic activity employ modified/non-modified casein or gelatin as substrates. In the quantitative assay, fragments generated from azocasein are separated from undigested substrate by trichloroacetic acid precipitation and their absorbance is measured. In non-quantitative assays, proteolytic activity is detected as clearing zones around bacterial growth or samples of culture supernatants on casein containing solid media formed due to local casein degradation. In zymography, individual proteases are detected as clear bands in gelatin/casein containing gels after SDS-PAGE separation, renaturation and protein staining. The elastinolytic capacity of P. aeruginosa is reflected by clearing zones on nutrient agar plates containing insoluble elastin instead of casein. Mueller-Hinton agar plates on which S. aureus cells are grown as a lawn are used to assess the susceptibility of S. aureus isolates to staphylolysin. A clear zone around a staphylolysin-containing sample indicates inhibition of S. aureus growth. Methods for measuring the activity of individual proteases are based on their cleavage specificity. These include assays of elastinolytic activity of pseudolysin and/or staphylolysin using elastin-Congo red as a substrate, a method for determination of

  11. Anisi stellati fructus extract attenuates the in vitro and in vivo metastatic and angiogenic potential of malignant cancer cells by downregulating proteolytic activity and pro-angiogenic factors.

    PubMed

    Kim, Aeyung; Im, Minju; Ma, Jin Yeul

    2014-11-01

    Anisi stellati fructus (ASF), commonly known as star anise, has long been used as a traditional Chinese medicine to treat inflammation, nervousness, insomnia and pain. In recent studies, it has been demonstrated that ASF possesses anti-bacterial, anti-fungal and anti-oxidant activities, as well as exhibits inhibitory effects on capillary‑like tube formation in human umbilical vein endothelial cells (HUVECs). However, the effects of ASF extract on the metastatic potential of malignant tumor cells have not been examined. In this study, we found that daily oral administration of ASF (50 mg/kg) remarkably reduced the number of pulmonary metastatic colonies of B16F10 cells in C57BL/6J mice with no observed systemic toxicity. In an in vitro system, ASF inhibited metastatic properties, including anchorage‑independent colony formation, migration and invasion. Upon phorbol 12-myristate 13-acetate (PMA) stimulation, the mRNA levels of matrix metalloproteinases (MMPs) -9, -13, -14 and urokinase plasminogen activator (uPA) decreased in a dose-dependent manner with ASF treatment. Gelatinase, type I collagenase, and uPA activities were also suppressed efficiently by ASF treatment. In response to PMA, NF-κB and AP-1 activation as well as p38 phosphorylation, which are crucial for MMP activation, were significantly decreased by ASF. In particular, ASF considerably inhibited tumor-induced HUVEC migration and tube formation and suppressed in vivo tumor-induced angiogenesis via a reduction of pro-angiogenic factors in tumors. These results collectively indicate that ASF might be useful in the management of metastatic malignant tumors. PMID:25176510

  12. Assay for Lipolytic and Proteolytic Activity Using Marine Substrates

    PubMed Central

    Tom, Raymond A.; Crisan, Eli V.

    1975-01-01

    Nondestructive assay procedures for determining microbial lipolytic and proteolytic activity on marine substrates were developed and tested with 287 isolates of bacteria, filamentous fungi, and yeasts. A definite substrate specificity was noted when the enzymatic activities on marine and nonmarine substrates was compared. Of 170 lipolytic isolates, 14 were only active on menhaden oil, 11 could hydrolyze menhaden oil and Tween 80 and/or tributyrin, and 145 isolates could only hydrolyze one or both of the nonmarine lipids. Of the 198 proteolytic isolates, 10 were specific for codfish extract, 152 were active against the marine substrate plus casein and/or gelatin, and 36 were specific for nonmarine substrates. PMID:1167775

  13. Lactobacillus helveticus: the proteolytic system

    PubMed Central

    Griffiths, M. W.; Tellez, A. M.

    2012-01-01

    Lactobacillus helveticus is one of the species of lactic acid bacteria (LAB) most commonly used in the production of fermented milk beverages and some types of hard cheese. The versatile nature of this bacterium is based on its highly efficient proteolytic system consisting of cell-envelope proteinases (CEPs), transport system and intracellular peptidases. Besides use of L. helveticus in cheese processing, the production of fermented milk preparations with health promoting properties has become an important industrial application. Studies have shown that fermented dairy products are able to decrease blood pressure, stimulate the immune system, promote calcium absorption, and exert an anti-virulent effect against pathogens. These beneficial effects are produced by a variety of peptides released during the hydrolysis of milk proteins by the proteolytic system of L. helveticus, which provides the bacterium with its nutritional requirements for growth. In recent years, studies have focused on understanding the factors that affect the kinetics of milk protein hydrolysis by specific strains and have concentrated on the effect of pH, temperature, growth phase, and matrix composition on the bacterial enzymatic system. This review focuses on the role of the proteolytic system of L. helveticus in the production of bioactive compounds formed during fermentation of dairy products. Taking advantage of the powerful proteolytic system of this bacterium opens up future opportunities to search for novel food-derived compounds with potential health promoting properties. PMID:23467265

  14. Proteolytic Cleavage Driven by Glycosylation.

    PubMed

    Kötzler, Miriam P; Withers, Stephen G

    2016-01-01

    Proteolytic processing of human host cell factor 1 (HCF-1) to its mature form was recently shown, unexpectedly, to occur in a UDP-GlcNAc-dependent fashion within the transferase active site of O-GlcNAc-transferase (OGT) (Lazarus, M. B., Jiang, J., Kapuria, V., Bhuiyan, T., Janetzko, J., Zandberg, W. F., Vocadlo, D. J., Herr, W., and Walker, S. (2013) Science 342, 1235-1239). An interesting mechanism involving formation and then intramolecular rearrangement of a covalent glycosyl ester adduct of the HCF-1 polypeptide was proposed to account for this unprecedented proteolytic activity. However, the key intermediate remained hypothetical. Here, using a model enzyme system for which the formation of a glycosyl ester within the enzyme active site has been shown unequivocally, we show that ester formation can indeed lead to proteolysis of the adjacent peptide bond, thereby providing substantive support for the mechanism of HCF-1 processing proposed. PMID:26515062

  15. A Comparative Study of New Aspergillus Strains for Proteolytic Enzymes Production by Solid State Fermentation

    PubMed Central

    Ortiz, Gastón Ezequiel; Noseda, Diego Gabriel; Ponce Mora, María Clara; Recupero, Matías Nicolás; Blasco, Martín; Albertó, Edgardo

    2016-01-01

    A comparative study of the proteolytic enzymes production using twelve Aspergillus strains previously unused for this purpose was performed by solid state fermentation. A semiquantitative and quantitative evaluation of proteolytic activity were carried out using crude enzymatic extracts obtained from the fermentation cultures, finding seven strains with high and intermediate level of protease activity. Biochemical, thermodynamics, and kinetics features such as optimum pH and temperature values, thermal stability, activation energy (Ea), quotient energy (Q10), Km, and Vmax were studied in four enzymatic extracts from the selected strains that showed the highest productivity. Additionally, these strains were evaluated by zymogram analysis obtaining protease profiles with a wide range of molecular weight for each sample. From these four strains with the highest productivity, the proteolytic extract of A. sojae ATCC 20235 was shown to be an appropriate biocatalyst for hydrolysis of casein and gelatin substrates, increasing its antioxidant activities in 35% and 125%, respectively. PMID:26989505

  16. A Comparative Study of New Aspergillus Strains for Proteolytic Enzymes Production by Solid State Fermentation.

    PubMed

    Ortiz, Gastón Ezequiel; Noseda, Diego Gabriel; Ponce Mora, María Clara; Recupero, Matías Nicolás; Blasco, Martín; Albertó, Edgardo

    2016-01-01

    A comparative study of the proteolytic enzymes production using twelve Aspergillus strains previously unused for this purpose was performed by solid state fermentation. A semiquantitative and quantitative evaluation of proteolytic activity were carried out using crude enzymatic extracts obtained from the fermentation cultures, finding seven strains with high and intermediate level of protease activity. Biochemical, thermodynamics, and kinetics features such as optimum pH and temperature values, thermal stability, activation energy (E a), quotient energy (Q 10), K m , and V max were studied in four enzymatic extracts from the selected strains that showed the highest productivity. Additionally, these strains were evaluated by zymogram analysis obtaining protease profiles with a wide range of molecular weight for each sample. From these four strains with the highest productivity, the proteolytic extract of A. sojae ATCC 20235 was shown to be an appropriate biocatalyst for hydrolysis of casein and gelatin substrates, increasing its antioxidant activities in 35% and 125%, respectively. PMID:26989505

  17. Proteolytic Activity in the Genus Ficus 1

    PubMed Central

    Williams, Donald C.; Sgarbieri, Valdemiro C.; Whitaker, John R.

    1968-01-01

    The latices of only 13 of a total of 46 species of Ficus examined contained appreciable proteolytic activity. Therefore, high proteolytic activity in the latex is not a distinguishing feature of the genus. The latex of F. stenocarpa had the highest specific activity followed closely by the latices of F. carica and F. glabrata. Latices of 6 species of Ficus were examined by chromatography on CM-cellulose and compared with the results obtained for 9 varieties of F. carica. All of the latices were found to contain multiple proteolytic enzymes. Chromatographically, the multiple enzyme components of the several varieties of F. carica were more similar than those of the several species examined. The latices of 16 varieties of F. carica were all different as determined by free boundary electrophoresis although the specific proteolytic activity of the latices was reasonably constant. PMID:16656886

  18. Comparison of the growth kinetics and proteolytic activities of Chryseobacterium species and Pseudomonas fluorescens.

    PubMed

    Bekker, A; Steyn, L; Charimba, G; Jooste, P; Hugo, C

    2015-12-01

    The effect of temperature on the growth kinetics and proteolytic activity of Chryseobacterium joostei and Chryseobacterium bovis was determined during this study. The results were compared with the activities of Pseudomonas fluorescens, which is regarded to be a major food spoilage psychrotolerant microorganism. For the growth studies, cultures were incubated in nutrient broth in a temperature gradient incubator (from 9 to 50 °C) and separately at 4 °C, and the optical density was measured at different time intervals. Growth temperature profiles for each organism were constructed. For determination of proteolytic activity, the cultures were incubated in fat-free ultra-high temperature processed milk in the temperature gradient incubator for 72 h (temperature range as above). Cell-free extracts were used to determine the proteolytic activity using the azocasein method. Results of the growth studies showed that C. joostei had the ability to grow over a wider temperature range than C. bovis and P. fluorescens without being affected by changes in the temperature. For the proteolytic activity, C. joostei had significantly (p < 0.001) higher activity per milligram of protein at 15.5 °C, followed by C. bovis and P. fluorescens. The results showed that C. joostei potentially has an even greater spoilage capacity in milk on the basis of growth rate and proteolytic activity than did P. fluorescens. PMID:26451905

  19. Epitope extraction technique using a proteolytic magnetic reactor combined with Fourier-transform ion cyclotron resonance mass spectrometry as a tool for the screening of potential vaccine lead peptides.

    PubMed

    Bílková, Z; Stefanescu, R; Cecal, R; Korecká, L; Ouzká, S; Jezová, J; Viovy, J-L; Przybylski, M

    2005-01-01

    Epitope extraction technique is based on the specific digestion of a target protein followed by immunoaffinity isolation of a specific recognition peptide. This technique, in combination with mass spectrometry, has been efficiently used for epitope identification. The major goal of this work was to utilize newly developed enzyme and immunoaffinity magnetic reactors for the epitope extraction procedure and confirm the efficiency of this improved system for epitope screening of proteins. Alginic acid-coated magnetite microparticles with immobilized TPCK-trypsin provided high working efficiency with low non-specific adsorption, digestion time in minutes and low frequency of missed cleavages. The sensitivity and specificity of tryptic fragmentation of the beta-amyloid-peptide Abeta (1-40) as a model polypeptide was confirmed by Fourier-transform ion cyclotron resonance mass spectrometry analysis. The Sepharose reactor or immunoaffinity magnetic reactors, both with anti-amyloid-beta monoclonal antibodies, were used for specific isolation and identification of target peptides. In this way, the epitope extraction technique combined with mass spectrometric analysis is shown to be an excellent base for molecular screening of potential vaccine lead proteins. PMID:16322655

  20. LC/MS analysis of proteolytic peptides in wheat extracts for determining the content of the allergen amylase/trypsin inhibitor CM3: influence of growing area and variety.

    PubMed

    Prandi, Barbara; Faccini, Andrea; Tedeschi, Tullia; Galaverna, Gianni; Sforza, Stefano

    2013-09-01

    Food allergy from wheat is triggered by several protein classes, such as LTPs, ω5-gliadins and α-amylase/trypsin inhibitors. The latter proteins, belonging to the prolamin superfamily, are mostly involved in baker's asthma, a form of occupational allergy in which the sensitization occurs through the respiratory tract. α-Amylase/trypsin inhibitors were also found to be involved in wheat-related atopic dermatitis. In this work, the allergen Tri a 30 (the CM3 α-amylase/trypsin inhibitor) was quantified in durum wheat salt soluble extracts using a peptidomic approach. CM3 protein identification was confirmed by using LTQ-OrbiTrap analysis on peptides obtained from the enzymatically digested protein separated by gel electrophoresis. Then, marker peptides derived from the protein after enzymatic cleavage of the full wheat extracts were identified by LC-MS/MS. One of them was used as marker for quantitative determination on an UPLC/ESI-MS system by using its isotopically labelled analogue as internal standard, allowing to assess the protein content in the different samples. The CM3 allergenic proteins were found to greatly vary among different cultivation areas. PMID:23578625

  1. Proteolytic crosstalk in multi-protease networks.

    PubMed

    Ogle, Curtis T; Mather, William H

    2016-01-01

    Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete ('queue') for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics. PMID:27042892

  2. Proteolytic crosstalk in multi-protease networks

    NASA Astrophysics Data System (ADS)

    Ogle, Curtis T.; Mather, William H.

    2016-04-01

    Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete (‘queue’) for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics.

  3. Regulation of Trichophyton rubrum proteolytic activity.

    PubMed Central

    Apodaca, G; McKerrow, J H

    1989-01-01

    Trichophyton rubrum is the most common dermatophyte of humans and normally colonizes the superficial layers of the epidermis (stratum corneum). Several proteinases with a possible role in the metabolism of host proteins have been purified from this fungus. The regulation of these enzymes and their role in fungal metabolism were studied at the biochemical level. General proteolytic (azocollytic) activity was repressed when log-phase cultures of T. rubrum were grown in a minimal medium that contained readily metabolized sources of carbon, nitrogen, sulfur, and phosphorus. When either carbon, nitrogen, or sulfur was deleted from this minimal medium, azocollytic activity was derepressed. In all cases a high-molecular-weight activity (Mr, greater than 200,000) was expressed. A 71,000-Mr proteinase was observed in nitrogen-depleted cultures, and proteolytic species of Mr 124,000 and 27,000 were secreted in sulfur-depleted cultures. The addition of either inorganic (MgSO4, Na2SO3, NaS2O3) or organic (methionine, cysteine) sulfur to the sulfur-depleted medium repressed the expression of azocollytic activity. In contrast, keratinolytic activity was not repressed by carbon, nitrogen, or sulfur but instead was induced when a protein source was included in the minimal medium. Stationary-phase cultures of T. rubrum secreted all proteolytic activities constitutively. Unlike log-phase cultures, the stationary-phase cultures secreted azocollytic, elastinolytic, and keratinolytic activity in minimal medium. These activities fell in the carbon-, nitrogen-, and phosphorous-depleted media but remained high in sulfur-depleted medium. The following model is proposed for the regulation of T. rubrum proteolytic activity. In the initial stages of infection, T. rubrum grows logarithmically. In this state, proteolytic activity is derepressed whenever carbon, nitrogen, or sulfur is lacking in the fungal milieu. The general proteinases produced would act on the nonkeratinous proteins in the

  4. Expression of cholera toxin B-lumbrokinase fusion protein in Pichia pastoris--the use of transmucosal carriers in the delivery of therapeutic proteins to protect rats against thrombosis.

    PubMed

    Chunfeng, Guan; Xiaozhou, Li; Gang, Wang; Jing, Ji; Chao, Jin; Josine, Tchouopou Lontchi

    2013-01-01

    Cholera toxin B-subunit (CTB) has been widely used to facilitate antigen delivery by serving as an effective mucosal carrier molecule for the induction of oral tolerance. However, whether CTB can be used as a transmucosal carrier in the delivery of not only vaccines but also therapeutic proteins has not been widely studied. Thus, we investigate here the concept of receptor-mediated oral delivery of lumbrokinase (LK) proteins which is an important fibrinolytic enzyme derived from earthworm. CTB and LK, separated by a furin cleavage site, was expressed via Pichia pastoris. The activity and proper folding of recombinant protein in yeast were confirmed by Western blot analysis, fibrin plate assays, and G(M1)-ganglioside ELISA. Following oral administration of recombinant protein, the thrombosis model of rats and mice revealed that the oral treatment of rCTB-LK has a more significant anti-thrombotic effect on animals compared with rLK. It is possible to conclude that CTB can successfully enhance its fusion protein LK to be absorbed. The use of CTB as a transmucosal carrier in the delivery of not only vaccines but also therapeutic proteins was supported. PMID:23269637

  5. The calcium-dependent proteolytic system calpain-calpastatin in Drosophila melanogaster.

    PubMed Central

    Pintér, M; Friedrich, P

    1988-01-01

    Ca2+-dependent proteolytic activity was detected at pH 7.5 in head extracts of the fruit fly Drosophila melanogaster. This activity was abolished by iodoacetate, but was unaffected by phenylmethanesulphonyl fluoride. These properties resemble those of the Ca2+-dependent thiol-proteinase calpain. The activity appeared at Mr 280,000 on Sepharose CL-6B gel chromatography. DEAE-cellulose chromatography revealed two activity peaks, with elution positions corresponding to vertebrate calpains I and II. The fly head enzymes were inhibited by a heat-stable and trypsin-sensitive component of the fly head extract, which also inhibited calpains from rat kidney. The inhibitor emerged from Sepharose CL-6B columns at Mr 310,000 and from DEAE-cellulose at a position corresponding to the protein inhibitor calpastatin from other sources. It is concluded that Drosophila heads comprise the Ca2+-dependent calpain-calpastatin proteolytic system. PMID:2845920

  6. Proteolytic Digestion and TiO2 Phosphopeptide Enrichment Microreactor for Fast MS Identification of Proteins

    NASA Astrophysics Data System (ADS)

    Deng, Jingren; Lazar, Iulia M.

    2016-04-01

    The characterization of phosphorylation state(s) of a protein is best accomplished by using isolated or enriched phosphoprotein samples or their corresponding phosphopeptides. The process is typically time-consuming as, often, a combination of analytical approaches must be used. To facilitate throughput in the study of phosphoproteins, a microreactor that enables a novel strategy for performing fast proteolytic digestion and selective phosphopeptide enrichment was developed. The microreactor was fabricated using 100 μm i.d. fused-silica capillaries packed with 1-2 mm beds of C18 and/or TiO2 particles. Proteolytic digestion-only, phosphopeptide enrichment-only, and sequential proteolytic digestion/phosphopeptide enrichment microreactors were developed and tested with standard protein mixtures. The protein samples were adsorbed on the C18 particles, quickly digested with a proteolytic enzyme infused over the adsorbed proteins, and further eluted onto the TiO2 microreactor for enrichment in phosphopeptides. A number of parameters were optimized to speed up the digestion and enrichments processes, including microreactor dimensions, sample concentrations, digestion time, flow rates, buffer compositions, and pH. The effective time for the steps of proteolytic digestion and enrichment was less than 5 min. For simple samples, such as standard protein mixtures, this approach provided equivalent or better results than conventional bench-top methods, in terms of both enzymatic digestion and selectivity. Analysis times and reagent costs were reduced ~10- to 15-fold. Preliminary analysis of cell extracts and recombinant proteins indicated the feasibility of integration of these microreactors in more advanced workflows amenable for handling real-world biological samples.

  7. Proteolytic Digestion and TiO2 Phosphopeptide Enrichment Microreactor for Fast MS Identification of Proteins.

    PubMed

    Deng, Jingren; Lazar, Iulia M

    2016-04-01

    The characterization of phosphorylation state(s) of a protein is best accomplished by using isolated or enriched phosphoprotein samples or their corresponding phosphopeptides. The process is typically time-consuming as, often, a combination of analytical approaches must be used. To facilitate throughput in the study of phosphoproteins, a microreactor that enables a novel strategy for performing fast proteolytic digestion and selective phosphopeptide enrichment was developed. The microreactor was fabricated using 100 μm i.d. fused-silica capillaries packed with 1-2 mm beds of C18 and/or TiO2 particles. Proteolytic digestion-only, phosphopeptide enrichment-only, and sequential proteolytic digestion/phosphopeptide enrichment microreactors were developed and tested with standard protein mixtures. The protein samples were adsorbed on the C18 particles, quickly digested with a proteolytic enzyme infused over the adsorbed proteins, and further eluted onto the TiO2 microreactor for enrichment in phosphopeptides. A number of parameters were optimized to speed up the digestion and enrichments processes, including microreactor dimensions, sample concentrations, digestion time, flow rates, buffer compositions, and pH. The effective time for the steps of proteolytic digestion and enrichment was less than 5 min. For simple samples, such as standard protein mixtures, this approach provided equivalent or better results than conventional bench-top methods, in terms of both enzymatic digestion and selectivity. Analysis times and reagent costs were reduced ~10- to 15-fold. Preliminary analysis of cell extracts and recombinant proteins indicated the feasibility of integration of these microreactors in more advanced workflows amenable for handling real-world biological samples. Graphical Abstract ᅟ. PMID:26883530

  8. Activation of bean (Phaseolus vulgaris) alpha-amylase inhibitor requires proteolytic processing of the proprotein.

    PubMed Central

    Pueyo, J J; Hunt, D C; Chrispeels, M J

    1993-01-01

    Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the alpha-amylases of mammals and insects. This alpha-amylase inhibitor (alpha AI) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M(r)) 15,000 to 18,000. We report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, we found that antibodies to alpha AI recognize large (M(r) 30,000-35,000) polypeptides as well as typical alpha AI processing products (M(r) 15,000-18,000). Alpha AI activity was found in all extracts that had the typical alpha AI processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, we made a mutant alpha AI in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-alpha AI when the gene is expressed in tobacco. When pro-alpha AI was separated from mature alpha AI by gel filtration, pro-alpha AI was found not to have alpha-amylase inhibitory activity. We interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. We suggest that the polypeptide cleavage removes a conformational constraint on the precursor to produce the biochemically active molecule. PMID:8310064

  9. Activation of bean (Phaseolus vulgaris) [alpha]-amylase inhibitor requires proteolytic processing of the proprotein

    SciTech Connect

    Pueyo, J.J.; Hunt, D.C.; Chrispeels, M.J. )

    1993-04-01

    Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the [alpha]-amylases of mammals and insects. This [alpha]-amylase inhibitor ([alpha]Al) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M[sub r]) 15,000 to 18,000. The authors report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, they found that antibodies to [alpha]Al recognize large (M[sub r] 30,000-35,000) polypeptides as well as typical [alpha]Al processing products (M[sub r] 15,000-18,000). [alpha]Al activity was found in all extracts that had the typical [alpha]Al processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, they made a mutant [alpha]Al in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-[alpha]Al when the gene is expressed in tobacco. When pro-[alpha]Al was separated from mature [alpha]Al by gel filtration, pro-[alpha]Al was found not to have [alpha]-amylase inhibitory activity. The authors interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. They suggest that the polypeptide cleavage removes a conformation constraint on the precursor to produce the biochemically active molecule. 43 refs., 5 figs., 1 tab.

  10. Silk microgels formed by proteolytic enzyme activity.

    PubMed

    Samal, Sangram K; Dash, Mamoni; Chiellini, Federica; Kaplan, David L; Chiellini, Emo

    2013-09-01

    The proteolytic enzyme α-chymotrypsin selectively cleaves the amorphous regions of silk fibroin protein (SFP) and allows the crystalline regions to self-assemble into silk microgels (SMGs) at physiological temperature. These microgels consist of lamellar crystals in the micrometer scale, in contrast to the nanometer-scaled crystals in native silkworm fibers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zeta potential results demonstrated that α-chymotrypsin utilized only the non-amorphous domains or segments of the heavy chain of SFP to form negatively charged SMGs. The SMGs were characterized in terms of size, charge, structure, morphology, crystallinity, swelling kinetics, water content and thermal properties. The results suggest that the present technique of preparing SMGs by α-chymotrypsin is simple and efficient, and that the prepared SMGs have useful features for studies related to biomaterial and pharmaceutical needs. This process is also an easy way to obtain the amorphous peptide chains for further study. PMID:23756227

  11. Detection of the apr gene in proteolytic psychrotrophic bacteria isolated from refrigerated raw milk.

    PubMed

    Martins, Maurilio L; de Araújo, Elza F; Mantovani, Hilário C; Moraes, Célia A; Vanetti, Maria C D

    2005-07-15

    Bacteria of the genus Pseudomonas have been associated with the spoilage of raw milk and dairy products due to the production of thermostable proteolytic enzymes. The apr gene encodes for alkaline metalloprotease in Pseudomonas and other related bacteria. Its presence in psychrotrophic proteolytic bacteria isolated from raw milk collected from cooling tanks was verified. A polymerase chain reaction (PCR) technique was used with degenerate primers. Total DNA from 112 isolates was pooled in different groups and then used as template for the amplification reactions. Controls consisted of DNA extracted from 26 cultures. An expected DNA fragment of 194 bp was detected in groups that contained bacteria identified as Pseudomonas. The PCR product was observed only when DNA from control cultures of Pseudomonas aeruginosa, Pseudomonas fluorescens, Serratia marcescens and Aeromonas hydrophila were used. A detection limit assay indicated that the apr gene could be directly amplified from pasteurized milk inoculated with 10(8) CFU/ml of P. fluorescens. With this method it was possible to detect proteolytic bacteria at 10(5) CFU/ml in reconstituted skim milk powder if cells were recovered for DNA extraction before amplification. PMID:15992619

  12. Characterisation of a novel proteolytic enzyme localised to goblet cells in rat and man.

    PubMed Central

    Nexø, E; Poulsen, S S; Hansen, S N; Kirkegaard, P; Olsen, P S

    1984-01-01

    A proteolytic enzyme, ingobsin , purified from rat duodenal extracts is shown to be localised to intestinal goblet cells of both man and rat. Enzyme positive cells decrease in number from duodenum to colon. The enzyme is a 33 000 Mr protein with an isoelectric point of 5.1. The pH optimum for enzymatic activity is 7.4-8.0. Based on substrate specificity for arg-x, lys-x and to a lesser degree tyr-x, on the effect of diisopropylphosphorofluoride , Trasylol and phenylmethylsulfonylfluoride and on proteolytic activity towards intact proteins, ingobsin is classified as a serine proteinase with endoproteolytic activity. Images Fig. 2 Fig. 4 Fig. 6 PMID:6735249

  13. Sequence-derived structural features driving proteolytic processing.

    PubMed

    Belushkin, Alexander A; Vinogradov, Dmitry V; Gelfand, Mikhail S; Osterman, Andrei L; Cieplak, Piotr; Kazanov, Marat D

    2014-01-01

    Proteolytic signaling, or regulated proteolysis, is an essential part of many important pathways such as Notch, Wnt, and Hedgehog. How the structure of the cleaved substrate regions influences the efficacy of proteolytic processing remains underexplored. Here, we analyzed the relative importance in proteolysis of various structural features derived from substrate sequences using a dataset of more than 5000 experimentally verified proteolytic events captured in CutDB. Accessibility to the solvent was recognized as an essential property of a proteolytically processed polypeptide chain. Proteolytic events were found nearly uniformly distributed among three types of secondary structure, although with some enrichment in loops. Cleavages in α-helices were found to be relatively abundant in regions apparently prone to unfolding, while cleavages in β-structures tended to be located at the periphery of β-sheets. Application of the same statistical procedures to proteolytic events divided into separate sets according to the catalytic classes of proteases proved consistency of the results and confirmed that the structural mechanisms of proteolysis are universal. The estimated prediction power of sequence-derived structural features, which turned out to be sufficiently high, presents a rationale for their use in bioinformatic prediction of proteolytic events. PMID:24227478

  14. SEQUENCE-DERIVED STRUCTURAL FEATURES DRIVING PROTEOLYTIC PROCESSING

    PubMed Central

    Belushkin, Alexander A.; Vinogradov, Dmitry V.; Gelfand, Mikhail S.; Osterman, Andrei L.; Cieplak, Piotr; Kazanov, Marat D.

    2014-01-01

    Proteolytic signaling, or regulated proteolysis, is an essential part of many important pathways such as Notch, Wnt, and Hedgehog. How the structure of the cleaved substrate regions influences the efficacy of proteolytic processing remains underexplored. Here, we analyzed the relative importance in proteolysis of various structural features derived from substrate sequences using a dataset of more than five thousand experimentally verified proteolytic events captured in CutDB. Accessibility to the solvent was recognized as an essential property of a proteolytically processed polypeptide chain. Proteolytic events were found nearly uniformly distributed among three types of secondary structure, although with some enrichment in loops. Cleavages in α-helices were found to be relatively abundant in regions apparently prone to unfolding, while cleavages in β-structures tended to be located at the periphery of β-sheets. Application of the same statistical procedures to proteolytic events divided into separate sets according to the catalytic classes of proteases proved consistency of the results and confirmed that the structural mechanisms of proteolysis are universal. The estimated prediction power of sequence-derived structural features, which turned out to be sufficiently high, presents a rationale for their use in bioinformatic prediction of proteolytic events. PMID:24227478

  15. Evaluation of proteolytic activity to differentiate some dematiaceous fungi.

    PubMed Central

    Espinel-Ingroff, A; Goldson, P R; McGinnis, M R; Kerkering, T M

    1988-01-01

    A total of 123 isolates of Cladosporium spp., Exophiala spp., Fonsecaea spp., Lecythophora hoffmannii, Phaeoannellomyces werneckii, Phialophora spp., Wangiella dermatitidis, and Xylohypha bantiana were tested for proteolytic activity by using 26 different formulations of gelatin, milk, casein, and Loeffler media. Other physiological properties examined included hydrolysis of tyrosine and xanthine, sodium nitrate utilization in Czapek Dox agar, and thermotolerance. Isolates of Exophiala jeanselmei, Fonsecaea compacta, Fonsecaea pedrosoi, W. dermatitidis, and X. bantiana lacked proteolytic activity. Proteolytic activity was variable among the remaining species, depending on the type of medium used. Thermotolerance had value in distinguishing some taxa. PMID:3343325

  16. Reinvestigation of the proteolytically active components of Bromelia pinguin fruit.

    PubMed

    Payrol, Juan Abreu; Obregón, Walter D; Natalucci, Claudia L; Caffini, Néstor O

    2005-09-01

    Pinguinain is the name given to a proteolytic enzyme preparation obtained from Bromelia pinguin fruits that has been scarcely studied. The present paper deals on the reexamination of the proteases present in fruits of B. pinguin grown in Cienfuegos, Cuba. The preparation (partially purified pinguinain, PPP) showed the main characteristics of the cysteine proteases, i.e., optimum pH within alkaline range (pH 7.2-8.8), inhibition of proteolytic activity by thiol blocking reagents, which is usually reverted by addition of cysteine, a remarkable thermal stability and notable stability at high ionic strength values. Isoelectric focusing and zymogram of PPP revealed the presence of several proteolytic components between pI 4.6 and 8.1. Preliminary peptidase purification by cationic exchange chromatography showed the presence of two main proteolytic fractions with molecular masses of approximately 20.0 kDa, according to SDS-PAGE. PMID:15978746

  17. Research Applications of Proteolytic Enzymes in Molecular Biology

    PubMed Central

    Mótyán, János András; Tóth, Ferenc; Tőzsér, József

    2013-01-01

    Proteolytic enzymes (also termed peptidases, proteases and proteinases) are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require their use, including production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, cell culturing and tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure-function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing and proteolytic digestion of proteins in proteomics. The aim of this paper is to review the molecular biological aspects of proteolytic enzymes and summarize their applications in the life sciences. PMID:24970197

  18. Purification and Properties of Two Proteolytic Enzymes with Carboxypeptidase Activity in Germinated Wheat 1

    PubMed Central

    Preston, Ken R.; Kruger, James E.

    1976-01-01

    Two proteolytic enzymes with carboxypeptidase activity have been isolated from a germinated wheat extract and partially characterized. Both enzymes rapidly released amino acids from hemoglobin and gluten and hydrolyzed carbobenzoxy-phenylalanylalanine. The enzymes were inhibited by diisopropylphosphofluoridate, but unaffected by salts, ethylenediaminetetraacetate, and sulfhydryl reagents at lower concentrations, and had molecular weights of approximately 55,000 and 61,000. Analysis of the hydrolysis products of hemoglobin and gluten indicated that both enzymes had broad specificities, including the ability to release proline. Images PMID:16659708

  19. Proteolytic activity during senescence of plants

    NASA Technical Reports Server (NTRS)

    Huffaker, R. C.

    1990-01-01

    Although information has rapidly developed concerning the intracellular localization of plant proteins, relatively few reports concern the intracellular location of endo- and exo-proteolytic activities. Relatively few proteases have been purified, characterized, and associated with a specific cellular location. With the exception of the processing proteases involved in transport of proteins across membranes, little progress has yet been made concerning determination of in vivo products of specific proteases. Information on the turnover of individual proteins and the assessment of rate-limiting steps in pathways as proteins are turned over is steadily appearing. Since chloroplasts are the major site of both protein synthesis and, during senescence, degradation, it was important to show unambiguously that chloroplasts can degrade their own constituents. Another important contribution was to obtain evidence that the chloroplasts contain proteases capable of degrading their constituents. This work has been more tenuous because of the low activities found and the possibility of contamination by vacuolar enzymes during the isolation of organelles. The possible targeting of cytoplasmic proteins for degradation by facilitating their transport into vacuoles is a field which hopefully will develop more rapidly in the future. Information on targeting of proteins for degradation via the ubiquitin (Ub) degradation pathway is developing rapidly. Future research must determine how much unity exists across the different eukaryotic systems. At present, it has important implications for protein turnover in plants, since apparently Ub is involved in the degradation of phytochrome. Little information has been developed regarding what triggers increased proteolysis with the onset of senescence, although it appears to involve protein synthesis. Thus far, the evidence indicates that the complement of proteases prior to senescence is sufficient to carry out the observed protein

  20. Proteolytic Processing Causes Extensive Heterogeneity of Tissue Matrilin Forms*

    PubMed Central

    Ehlen, Harald W. A.; Sengle, Gerhard; Klatt, Andreas R.; Talke, Anja; Müller, Stefan; Paulsson, Mats; Wagener, Raimund

    2009-01-01

    The matrilins are a family of multidomain extracellular matrix proteins with adapter functions. The oligomeric proteins have a bouquet-like structure and bind to a variety of different ligands whereby the avidity of their interactions is dependent on the number of subunits and domains present. Here we show the contribution of post-translational proteolytic processing to the heterogeneity of matrilins seen in tissue extracts and cell culture supernatants. A cleavage site after two glutamate residues in the hinge region close to the C-terminal coiled-coil oligomerization domain is conserved among the matrilins. Cleavage at this site yields molecules that lack almost complete subunits. The processing is least pronounced in matrilin-1 and particularly complex in matrilin-2, which contains additional cleavage sites. Replacement of the hinge region in matrilin-4 by the matrilin-1 hinge region had no marked effect on the processing. A detailed study revealed that matrilin-4 is processed already in the secretory pathway and that the activation of the responsible enzymes is dependent on proprotein convertase activity. Matrilin-3 and -4, but not matrilin-1 subunits present in matrilin-1/-3 hetero-oligomers, were identified as substrates for ADAMTS4 and ADAMTS5, whereas ADAMTS1 did not cleave any matrilin. A neo-epitope antibody raised against the N terminus of the C-terminal cleavage product of matrilin-4 detected processed matrilin-4 in cultures of primary chondrocytes as well as on cartilage sections showing that the conserved cleavage site is used in vivo. PMID:19531486

  1. Measurement of Separase Proteolytic Activity in Single Living Cells by a Fluorogenic Flow Cytometry Assay

    PubMed Central

    Haaß, Wiltrud; Kleiner, Helga; Müller, Martin C.; Hofmann, Wolf-Karsten; Fabarius, Alice; Seifarth, Wolfgang

    2015-01-01

    ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML). Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110)-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110) as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90–180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic activity in leukemic

  2. Proteolytic Pathways Induced by Herbicides That Inhibit Amino Acid Biosynthesis

    PubMed Central

    Zulet, Amaia; Gil-Monreal, Miriam; Villamor, Joji Grace; Zabalza, Ana; van der Hoorn, Renier A. L.; Royuela, Mercedes

    2013-01-01

    Background The herbicides glyphosate (Gly) and imazamox (Imx) inhibit the biosynthesis of aromatic and branched-chain amino acids, respectively. Although these herbicides inhibit different pathways, they have been reported to show several common physiological effects in their modes of action, such as increasing free amino acid contents and decreasing soluble protein contents. To investigate proteolytic activities upon treatment with Gly and Imx, pea plants grown in hydroponic culture were treated with Imx or Gly, and the proteolytic profile of the roots was evaluated through fluorogenic kinetic assays and activity-based protein profiling. Results Several common changes in proteolytic activity were detected following Gly and Imx treatment. Both herbicides induced the ubiquitin-26 S proteasome system and papain-like cysteine proteases. In contrast, the activities of vacuolar processing enzymes, cysteine proteases and metacaspase 9 were reduced following treatment with both herbicides. Moreover, the activities of several putative serine protease were similarly increased or decreased following treatment with both herbicides. In contrast, an increase in YVADase activity was observed under Imx treatment versus a decrease under Gly treatment. Conclusion These results suggest that several proteolytic pathways are responsible for protein degradation upon herbicide treatment, although the specific role of each proteolytic activity remains to be determined. PMID:24040092

  3. Amyloid-forming peptides selected proteolytically from phage display library.

    PubMed

    Koscielska-Kasprzak, Katarzyna; Otlewski, Jacek

    2003-08-01

    We demonstrated that amyloid-forming peptides could be selected from phage-displayed library via proteolysis-based selection protocol. The library of 28-residue peptides based on a sequence of the second zinc finger domain of Zif268, and computationally designed betabetaalpha peptide, FSD-1, was presented monovalently on the surface of M13 phage. The library coupled the infectivity of phage particles to proteolytic stability of a peptide introduced into the coat protein III linker. It was designed to include variants with a strong potential to fold into betabetaalpha motif of zinc finger domains, as expected from secondary structure propensities, but with no structure stabilization via zinc ion coordination. As our primary goal was to find novel monomeric betabetaalpha peptides, the library was selected for stable domains with the assumption that folded proteins are resistant to proteolysis. After less than four rounds of proteolytic selection with trypsin, chymotrypsin, or proteinase K, we obtained a number of proteolysis-resistant phage clones containing several potential sites for proteolytic attack with the proteinases. Eight peptides showing the highest proteolysis resistance were expressed and purified in a phage-free form. When characterized, the peptides possessed proteolytic resistance largely exceeding that of the second zinc finger domain of Zif268 and FSD-1. Six of the characterized peptides formed fibrils when solubilized at high concentrations. Three of them assembled into amyloids as determined through CD measurements, Congo red and thioflavin T binding, and transmission electron microscopy. PMID:12876317

  4. Sirtuins and Proteolytic Systems: Implications for Pathogenesis of Synucleinopathies

    PubMed Central

    Sampaio-Marques, Belém; Ludovico, Paula

    2015-01-01

    Insoluble and fibrillar forms of α-synuclein are the major components of Lewy bodies, a hallmark of several sporadic and inherited neurodegenerative diseases known as synucleinopathies. α-Synuclein is a natural unfolded and aggregation-prone protein that can be degraded by the ubiquitin-proteasomal system and the lysosomal degradation pathways. α-Synuclein is a target of the main cellular proteolytic systems, but it is also able to alter their function further, contributing to the progression of neurodegeneration. Aging, a major risk for synucleinopathies, is associated with a decrease activity of the proteolytic systems, further aggravating this toxic looping cycle. Here, the current literature on the basic aspects of the routes for α-synuclein clearance, as well as the consequences of the proteolytic systems collapse, will be discussed. Finally, particular focus will be given to the sirtuins’s role on proteostasis regulation, since their modulation emerged as a promising therapeutic strategy to rescue cells from α-synuclein toxicity. The controversial reports on the potential role of sirtuins in the degradation of α-synuclein will be discussed. Connection between sirtuins and proteolytic systems is definitely worth of further studies to increase the knowledge that will allow its proper exploration as new avenue to fight synucleinopathies. PMID:25946078

  5. Positive feedback of protein kinase C proteolytic activation during apoptosis.

    PubMed Central

    Leverrier, Sabrina; Vallentin, Alice; Joubert, Dominique

    2002-01-01

    In contrast with protein kinase Calpha (PKCalpha) and PKCepsilon, which are better known for promoting cell survival, PKCdelta is known for its pro-apoptotic function, which is exerted mainly through a caspase-3-dependent proteolytic activation pathway. In the present study, we used the rat GH3B6 pituitary adenoma cell line to show that PKCalpha and PKCepsilon are activated and relocalized together with PKCdelta when apoptosis is induced by a genotoxic stress. Proteolytic activation is a crucial step used by the three isoforms since: (1) the catalytic domains of the PKCalpha, PKCepsilon or PKCdelta isoforms (CDalpha, CDepsilon and CDdelta respectively) accumulated, and this accumulation was dependent on the activity of both calpain and caspase; and (2) transient expression of CDalpha, CDepsilon or CDdelta sufficed to induce apoptosis. However, following this initial step of proteolytic activation, the pathways diverge; cytochrome c release and caspase-3 activation are induced by CDepsilon and CDdelta, but not by CDalpha. Another interesting finding of the present study is the proteolysis of PKCdelta induced by CDepsilon expression that revealed the existence of a cross-talk between PKC isoforms during apoptosis. Hence the PKC family may participate in the apoptotic process of pituitary adenoma cells at two levels: downstream of caspase and calpain, and via retro-activation of caspase-3, resulting in the amplification of its own proteolytic activation. PMID:12238950

  6. Deterioration of white croaker (Pennahia argentata) meat thermally-induced gel products caused by proteolytic enzymes in the contaminated intestine and kidney.

    PubMed

    Ueki, Nobuhiko; Wan, Jianrong; Watabe, Shugo

    2016-05-15

    Thermally-induced gels were made from white croaker (Pennahia argentata) meat in the presence of its organ extracts by pre-heating at 40 and 65°C for 20 min and subsequent heating at 85°C for 20 min. The breaking strength of the gels decreased with increasing concentrations of the intestinal extracts accompanying decomposition of myosin heavy chains. However, no significant changes in the gel strength occurred when the kidney extract was added. The proteolytic activity in the intestinal extracts examined in the meat homogenate had a maximum at 60°C and pH 8.90. These results suggest that the intestinal rather than kidney proteolytic activities are responsible for gel softening known as a modori phenomenon. Thus, the removal of intestinal tracts is essential to maintain a high quality of surimi-based products. PMID:26775990

  7. Tracing the history of the ubiquitin proteolytic system: the pioneering article.

    PubMed

    Ciechanover, Aaron

    2009-09-11

    A series of findings made by several researchers during a two-decade period between the mid-1950s and mid-1970s raised the suspicion that the lysosome might not be the organelle that degrades the bulk of cellular proteins under basal conditions. These findings predicted the existence of a nonlysosomal, adenosine triphosphate (ATP)-dependent proteolytic system. Yet, following the initial discovery of such activity in a crude cell extract, it was a single article published in this journal [A. Ciechanover, Y. Hod, A. Hershko, A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes, Biochem. Biophys. Res. Commun. 81 (1978) 1100-1105], my first study as a graduate student of Avram Hershko, that made it clear that the system that catalyzes the activity is novel and complex, and does not follow the paradigm in the field of proteolysis where a single protease typically cleaves its substrate; here at least two components were required to carry out this activity, and one of them was an unusual, small, and heat-stable protein later identified as ubiquitin. PMID:19539608

  8. Antioxidative and proteolytic systems protect mitochondria from oxidative damage in S-deficient Arabidopsis thaliana.

    PubMed

    Ostaszewska-Bugajska, Monika; Rychter, Anna M; Juszczuk, Izabela M

    2015-08-15

    We examined the functioning of the antioxidative defense system in Arabidopsis thaliana under sulphur (S) deficiency with an emphasis on the role of mitochondria. In tissue extracts and in isolated mitochondria from S-deficient plants, the concentration of non-protein thiols declined but protein thiols did not change. Superoxide anion and hydrogen peroxide were accumulated in leaf blades and the generation of superoxide anion by isolated mitochondria was higher. Lower abundance of reduced (GSH) plus oxidized (GSSG) glutathione in the leaf and root tissues, and leaf mitochondria from S-deficient plants was accompanied by a decrease in the level of GSH and the changes in the GSH/GSSG ratios. In the chloroplasts, the total level of glutathione decreased. Lower levels of reduced (AsA) and oxidized (DHA) ascorbate were reflected in much higher ratios of AsA/DHA. Sulphur deficiency led to an increase in the activity of cytosolic, mitochondrial and chloroplastic antioxidative enzymes, peroxidases, catalases and superoxide dismutases. The protein carbonyl level was higher in the leaves of S-deficient plants and in the chloroplasts, while in the roots, leaf and root mitochondria it remained unchanged. Protease activity in leaf extracts of S-deficient plants was higher, but in root extracts it did not differ. The proteolytic system reflected subcellular specificity. In leaf and root mitochondria the protease activity was higher, whereas in the chloroplasts it did not change. We propose that the preferential incorporation of S to protein thiols and activation of antioxidative and proteolytic systems are likely important for the survival of S-deficient plants and that the mitochondria maintain redox homeostasis. PMID:26339750

  9. Chronic venous disease - Part II: Proteolytic biomarkers in wound healing.

    PubMed

    Ligi, Daniela; Mosti, Giovanni; Croce, Lidia; Raffetto, Joseph D; Mannello, Ferdinando

    2016-10-01

    Venous leg ulcers (VLU) are characterized by sustained proteolytic microenvironment impairing the healing process. Wound fluid (WF) reflect the biomolecular activities occurring within the wound area; however, it is unclear if WF from different healing phases have different proteolytic profiles and how VLU microenvironment affects the wound healing mechanisms. We investigated the proteolytic network of WF from distinct VLU phases, and in WF- and LPS-stimulated THP-1 monocytes treated with glycosaminoglycan sulodexide, a well known therapeutic approach for VLU healing. WF were collected from patients with VLU during inflammatory (Infl) and granulating (Gran) phases. WF and THP-1 supernatants were analyzed for nine matrix metalloproteinases (MMP) and four tissue inhibitors of metalloproteinases (TIMP) by multiplex immunoassays. Our results demonstrated that: 1) WF from Infl VLU contained significantly increased concentrations of MMP-2, MMP-9, MMP-12, TIMP-1, and TIMP-2 compared to Gran WF; 2) WF from Gran VLU showed significantly increased levels of MMP-1, MMP-7, MMP-13, and TIMP-4 compared to Infl WF; 3) LPS- and WF-stimulation of THP-1 cells significantly increased the expression of several MMP compared to untreated cells; 4) Sulodexide treatment of both LPS- and WF-stimulated THP-1 significantly down-regulated the release of several MMPs. Our study provides evidence-based medicine during treatment of patients with VLU. WF from Infl and Gran VLU have different MMP and TIMP signatures, consistent with their clinical state. The modulation of proteolytic pathways in wound microenvironment by glycosaminoglycan sulodexide, provide insights for translating research into clinical practice during VLU therapy. PMID:27460704

  10. Proteolytic components of serum IgG preparations

    PubMed Central

    Li, L; Kalaga, R; Paul, S

    2000-01-01

    Chemical catalysis, an effector mechanism utilized by fully assembled antibodies, can also be mediated by the isolated antibody subunits. Because trace amounts of free light chains (L chains) are present in IgG preparations, a detailed study was undertaken to identify the constituents responsible for the polyreactive proteolytic activity of IgG purified from human sera, determined as the extent of cleavage of the model peptide substrate Pro-Phe-Arg-methylcoumarinamide. Two proteolytic species with approximate mass of 50 kD and 150 kD were separated by repetitive gel filtration in a denaturing solvent (6 m guanidine hydrochloride). The activity of the renatured 50-kD fraction (in fluorescence units/μg protein) was more than 45-fold greater than of the 150-kD fraction. Both fractions lost the activity following immunoadsorption on immobilized anti-IgG antibody. Fab fragments prepared from the 150-kD IgG fraction retained the activity. Reducing and non-reducing SDS-electrophoresis suggested the 50-kD fraction isolated from the IgG preparations to be a mixture of heavy chain (H chain) monomers and disulphide bonded L chain dimers. Electrophoretically homogeneous monomers of 50-kD H chains and 25-kD L chains were prepared by gel filtration of reduced and alkylated IgG from seven human subjects. Each of the alkylated L chain preparations displayed the proteolytic activity. The activity in alkylated H chains was undetectable or only marginally greater than the background values. L chain dimers appear to be the major species responsible for the polyreactive proteolytic activity of serum IgG preparations, with a smaller contribution furnished by tetrameric IgG. PMID:10792374

  11. Proteolytic activity of probiotic strain Lactobacillus helveticus M92.

    PubMed

    Beganović, Jasna; Kos, Blaženka; Leboš Pavunc, Andreja; Uroić, Ksenija; Džidara, Petra; Šušković, Jagoda

    2013-04-01

    The aim of this research was to investigate the potential of previously defined probiotic strain Lactobacillus helveticus M92 as functional starter culture for fermented dairy products. Therefore, proteolytic activity of L. helveticus M92 was investigated and compared with those of different representatives of probiotic and starter culture strains. Cluster analysis of AFLP fingerprints showed a difference of L. helveticus M92 compared to five other L. helveticus strains, but the percentage of similarity confirmed the identification on species level. Casein hydrolysis by L. helveticus M92 was monitored by agar-well diffusion test, SDS-PAGE and Anson's method. L. helveticus M92 exhibited the highest proteolytic activity among tested probiotic and starter cultures strains with the fastest acidification rate and the highest pH decrease after overnight incubation in skim milk. The presence of prtH2 gene was confirmed by PCR amplification with specific primers, while PCR product was not obtained after amplification with primers specific to prtH. Furthermore, SDS-PAGE LC-MS/MS analysis of insoluble proteome of L. helveticus M92 enabled identification of several proteins involved in proteolytic system of L. helveticus such as protease PrtM as well as proteins involved in Opp peptide transport system and the intracellular peptidases PepE, PepN, and PepQ. PMID:23454496

  12. PMAP: databases for analyzing proteolytic events and pathways.

    PubMed

    Igarashi, Yoshinobu; Heureux, Emily; Doctor, Kutbuddin S; Talwar, Priti; Gramatikova, Svetlana; Gramatikoff, Kosi; Zhang, Ying; Blinov, Michael; Ibragimova, Salmaz S; Boyd, Sarah; Ratnikov, Boris; Cieplak, Piotr; Godzik, Adam; Smith, Jeffrey W; Osterman, Andrei L; Eroshkin, Alexey M

    2009-01-01

    The Proteolysis MAP (PMAP, http://www.proteolysis.org) is a user-friendly website intended to aid the scientific community in reasoning about proteolytic networks and pathways. PMAP is comprised of five databases, linked together in one environment. The foundation databases, ProteaseDB and SubstrateDB, are driven by an automated annotation pipeline that generates dynamic 'Molecule Pages', rich in molecular information. PMAP also contains two community annotated databases focused on function; CutDB has information on more than 5000 proteolytic events, and ProfileDB is dedicated to information of the substrate recognition specificity of proteases. Together, the content within these four databases will ultimately feed PathwayDB, which will be comprised of known pathways whose function can be dynamically modeled in a rule-based manner, and hypothetical pathways suggested by semi-automated culling of the literature. A Protease Toolkit is also available for the analysis of proteases and proteolysis. Here, we describe how the databases of PMAP can be used to foster understanding of proteolytic pathways, and equally as significant, to reason about proteolysis. PMID:18842634

  13. Quantum-dot-based nanosensors designed for proteolytic monitoring

    NASA Astrophysics Data System (ADS)

    Medintz, Igor L.; Clapp, Aaron R.; Brunel, Florence M.; Goldman, Ellen R.; Chang, Eddie L.; Dawson, Phillip E.; Mattoussi, Hedi

    2006-02-01

    We have previously assembled QD-based fluorescence resonance energy transfer (FRET) sensors specific for the sugar nutrient maltose and the explosive TNT. These sensors utilize several inherent benefits of QDs as FRET donors. In this report, we show that QD-FRET based sensors can also function in the monitoring of proteolytic enzyme activity. We utilize a QD with multiple dye-labeled proteins attached to the surface as a substrate for a prototypical protease. We then demonstrate how this strategy can be extended to detect protease activity by utilizing a dye-labeled peptide attached to the QD as a proteolytic substrate. Self-assembly of the peptide-dye on the QD brings the dye in close proximity to the QD and result in efficient FRET. Addition of a proteolytic enzyme that specifically recognizes and cleaves the peptide alters the FRET signature of the sensor in a concentration-dependent manner. Both qualitative and quantitative data can be derived from these sensors. The potential benefits of this type of QD sensing strategy are discussed.

  14. Proteolytic activation defines distinct lymphangiogenic mechanisms for VEGFC and VEGFD

    PubMed Central

    Bui, Hung M.; Enis, David; Robciuc, Marius R.; Nurmi, Harri J.; Cohen, Jennifer; Chen, Mei; Yang, Yiqing; Dhillon, Veerpal; Johnson, Kathy; Zhang, Hong; Kirkpatrick, Robert; Traxler, Elizabeth; Alitalo, Kari

    2016-01-01

    Lymphangiogenesis is supported by 2 homologous VEGFR3 ligands, VEGFC and VEGFD. VEGFC is required for lymphatic development, while VEGFD is not. VEGFC and VEGFD are proteolytically cleaved after cell secretion in vitro, and recent studies have implicated the protease a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS3) and the secreted factor collagen and calcium binding EGF domains 1 (CCBE1) in this process. It is not well understood how ligand proteolysis is controlled at the molecular level or how this process regulates lymphangiogenesis, because these complex molecular interactions have been difficult to follow ex vivo and test in vivo. Here, we have developed and used biochemical and cellular tools to demonstrate that an ADAMTS3-CCBE1 complex can form independently of VEGFR3 and is required to convert VEGFC, but not VEGFD, into an active ligand. Consistent with these ex vivo findings, mouse genetic studies revealed that ADAMTS3 is required for lymphatic development in a manner that is identical to the requirement of VEGFC and CCBE1 for lymphatic development. Moreover, CCBE1 was required for in vivo lymphangiogenesis stimulated by VEGFC but not VEGFD. Together, these studies reveal that lymphangiogenesis is regulated by two distinct proteolytic mechanisms of ligand activation: one in which VEGFC activation by ADAMTS3 and CCBE1 spatially and temporally patterns developing lymphatics, and one in which VEGFD activation by a distinct proteolytic mechanism may be stimulated during inflammatory lymphatic growth. PMID:27159393

  15. PMAP: databases for analyzing proteolytic events and pathways

    PubMed Central

    Igarashi, Yoshinobu; Heureux, Emily; Doctor, Kutbuddin S.; Talwar, Priti; Gramatikova, Svetlana; Gramatikoff, Kosi; Zhang, Ying; Blinov, Michael; Ibragimova, Salmaz S.; Boyd, Sarah; Ratnikov, Boris; Cieplak, Piotr; Godzik, Adam; Smith, Jeffrey W.; Osterman, Andrei L.; Eroshkin, Alexey M.

    2009-01-01

    The Proteolysis MAP (PMAP, http://www.proteolysis.org) is a user-friendly website intended to aid the scientific community in reasoning about proteolytic networks and pathways. PMAP is comprised of five databases, linked together in one environment. The foundation databases, ProteaseDB and SubstrateDB, are driven by an automated annotation pipeline that generates dynamic ‘Molecule Pages’, rich in molecular information. PMAP also contains two community annotated databases focused on function; CutDB has information on more than 5000 proteolytic events, and ProfileDB is dedicated to information of the substrate recognition specificity of proteases. Together, the content within these four databases will ultimately feed PathwayDB, which will be comprised of known pathways whose function can be dynamically modeled in a rule-based manner, and hypothetical pathways suggested by semi-automated culling of the literature. A Protease Toolkit is also available for the analysis of proteases and proteolysis. Here, we describe how the databases of PMAP can be used to foster understanding of proteolytic pathways, and equally as significant, to reason about proteolysis. PMID:18842634

  16. Recovery of Proteolytic and Collagenolytic Activities from Viscera By-products of Rayfish (Raja clavata)

    PubMed Central

    Murado, Miguel Anxo; del Pilar González, María; Vázquez, José Antonio

    2009-01-01

    The aim of this work was to study the recovery of proteolytic and collagenolytic activities from rayfish (Raja clavata) viscera wastes. Initially, different parts of the gastrointestinal tract by-products (stomach, duodenum section including pancreas, final intestine) were evaluated. The extracts from proximal intestine yielded the highest values of both enzymatic activities. Optimal conditions for protease activity quantification were established at pH = 6, T = 40 °C and incubation time ≤20 min. The mathematical equation used to model the joint effect of pH and temperature led to maximum activity at pH = 8.66 and 59.4 °C, respectively. Overcooled acetone was found to be best option for recovery of enzymatic activities in comparison with ethanol, PEG-4000, ammonium sulphate and ultrafiltration system. Finally, a simple and systematic protocol of partial purification and total recovery of proteases and collagenases was defined. PMID:20098611

  17. The role of proteolytic enzymes in degradation of plant tissues: Summary report

    SciTech Connect

    Lewosz, J.; Kelman, A.; Sequeira, L.

    1989-01-01

    The proteolytic enzymes produced by Erwinia carotovora subsp. carotovora (Ecc-strain SR 394) grown on various media were examined by isoelectrofocusing in polyacrylamide gels over a pH range of 3-10. In addition to the main protease present in culture filtrates, low concentrations of several other proteases were present in extracts from potato tubers infected by Ecc. Proteases from all these sources were similar and had the following properties: pH optimum near 8.0, calcium dependent, insensitive to serine proteinase and SH-proteinase inhibitors, inhibited by EDTA, and highly thermostable. These enzymes degraded gelatin, soluble collagen and Hide Powder Azure, and showed weak activity on casein, but did not degrade insoluble collagen or elastin.

  18. Activation and Proteolytic Activity of the Treponema pallidum Metalloprotease, Pallilysin

    PubMed Central

    Houston, Simon; Hof, Rebecca; Honeyman, Lisa; Hassler, Julia; Cameron, Caroline E.

    2012-01-01

    Treponema pallidum is a highly invasive pathogen that undergoes rapid dissemination to establish widespread infection. Previous investigations identified the T. pallidum adhesin, pallilysin, as an HEXXH-containing metalloprotease that undergoes autocatalytic cleavage and degrades laminin and fibrinogen. In the current study we characterized pallilysin's active site, activation requirements, cellular location, and fibrin clot degradation capacity through both in vitro assays and heterologous treponemal expression and degradation studies. Site-directed mutagenesis showed the pallilysin HEXXH motif comprises at least part of the active site, as introduction of three independent mutations (AEXXH [H198A], HAXXH [E199A], and HEXXA [H202A]) abolished pallilysin-mediated fibrinogenolysis but did not adversely affect host component binding. Attainment of full pallilysin proteolytic activity was dependent upon autocatalytic cleavage of an N-terminal pro-domain, a process which could not occur in the HEXXH mutants. Pallilysin was shown to possess a thrombin cleavage site within its N-terminal pro-domain, and in vitro studies confirmed cleavage of pallilysin with thrombin generates a truncated pallilysin fragment that has enhanced proteolytic activity, suggesting pallilysin can also exploit the host coagulation process to facilitate protease activation. Opsonophagocytosis assays performed with viable T. pallidum demonstrated pallilysin is a target of opsonic antibodies, consistent with a host component-interacting, surface-exposed cellular location. Wild-type pallilysin, but not the HEXXA mutant, degraded fibrin clots, and similarly heterologous expression of pallilysin in the non-invasive spirochete Treponema phagedenis facilitated fibrin clot degradation. Collectively these results identify pallilysin as a surface-exposed metalloprotease within T. pallidum that possesses an HEXXH active site motif and requires autocatalytic or host-mediated cleavage of a pro-domain to attain

  19. Effect of midgut proteolytic activity on susceptibility of lepidopteran larvae to Bacillus thuringiensis subsp. Kurstaki

    PubMed Central

    Talaei-Hassanloui, Reza; Bakhshaei, Raziyeh; Hosseininaveh, Vahid; Khorramnezhad, Ayda

    2014-01-01

    Bacillus thuringiensis (Bt) is the most effective microbial control agent for controlling numerous species from different insect orders. All subspecies and strains of B. thuringiensis can produce a spore and a crystalline parasporal body. This crystal which contains proteinaceous protoxins is dissolved in the alkaline midgut, the resulting molecule is then cleaved and activated by proteolytic enzymes and acts as a toxin. An interesting aspect of this activation process is that variations in midgut pH and protease activity have been shown to account for the spectrum of some Bt proteins activity. Thus, an important factor that could be a determinant of toxin activity is the presence of proteases in the midgut microenvironment of susceptible insects. Reciprocally, any alteration in the midgut protease composition of the host can result in resistance to Bt. Here in this paper, we reviewed this processes in general and presented our assays to reveal whether resistance mechanism to Bt in Diamondback Moth (DbM) larvae could be due to the function of the midgut proteases? We estimated LC50 for both probable susceptible and resistant populations in laboratory and greenhouse tests. Then, the midgut protease activities of the B. thuringiensis induced-resistant and susceptible populations of the DbM were assayed on Hemoglubin and on N-alpha-benzoyl-DL-arginine-p-nitroanilide (BapNA) for total and tryptic activities, respectively. Six hours after feeding on Bt treated and untreated canola leaves, the midguts of instar larvae of both populations were isolated. Following related protocols, peptides released through the activity of proteinases on Hemoglubin and BApNA were recorded using microplate reader. Control (Blank) was also considered with adding TCA to reaction mix before adding enzymatic extract. Data analysis indicated that there are significant differences for tryptic activity on BApNA and also for total proteolytic activity on Hemoglubin between susceptible and

  20. Proteolytic regulation of stress response pathways in Escherichia coli.

    PubMed

    Micevski, Dimce; Dougan, David A

    2013-01-01

    Maintaining correct cellular function is a fundamental biological process for all forms of life. A critical aspect of this process is the maintenance of protein homeostasis (proteostasis) in the cell, which is largely performed by a group of proteins, referred to as the protein quality control (PQC) network. This network of proteins, comprised of chaperones and proteases, is critical for maintaining proteostasis not only during favourable growth conditions, but also in response to stress. Indeed proteases play a crucial role in the clearance of unwanted proteins that accumulate during stress, but more importantly, in the activation of various different stress response pathways. In bacteria, the cells response to stress is usually orchestrated by a specific transcription factor (sigma factor). In Escherichia coli there are seven different sigma factors, each of which responds to a particular stress, resulting in the rapid expression of a specific set of genes. The cellular concentration of each transcription factor is tightly controlled, at the level of transcription, translation and protein stability. Here we will focus on the proteolytic regulation of two sigma factors (σ(32) and σ(S)), which control the heat and general stress response pathways, respectively. This review will also briefly discuss the role proteolytic systems play in the clearance of unwanted proteins that accumulate during stress. PMID:23479439

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

    PubMed Central

    Samac, Deborah; Storey, Richard

    1981-01-01

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

  2. Isolation and removal of proteolytic enzymes with magnetic cross-linked erythrocytes

    NASA Astrophysics Data System (ADS)

    Šafařík, Ivo; Šafaříková, Mirka

    2001-01-01

    New magnetic adsorbents for batch isolation and removal of various proteolytic enzymes were prepared by glutaraldehyde cross-linking of bovine, porcine and human erythrocytes in the presence of fine magnetic particles. Trypsin, chymotrypsin, alkaline bacterial protease and proteases present in various commercial enzyme preparations were efficiently adsorbed on these adsorbents; on the contrary, proteins without proteolytic activity were not adsorbed.

  3. The fine-tuning of proteolytic pathways in Alzheimer's disease.

    PubMed

    Cecarini, Valentina; Bonfili, Laura; Cuccioloni, Massimiliano; Mozzicafreddo, Matteo; Angeletti, Mauro; Keller, Jeffrey N; Eleuteri, Anna Maria

    2016-09-01

    Several integrated proteolytic systems contribute to the maintenance of cellular homeostasis through the continuous removal of misfolded, aggregated or oxidized proteins and damaged organelles. Among these systems, the proteasome and autophagy play the major role in protein quality control, which is a fundamental issue in non-proliferative cells such as neurons. Disturbances in the functionality of these two pathways are frequently observed in neurodegenerative diseases, like Alzheimer's disease, and reflect the accumulation of protease-resistant, deleterious protein aggregates. In this review, we explored the sophisticated crosstalk between the ubiquitin-proteasome system and autophagy in the removal of the harmful structures that characterize Alzheimer's disease neurons. We also dissected the role of the numerous shuttle factors and chaperones that, directly or indirectly interacting with ubiquitin and LC3, are used for cargo selection and delivery to one pathway or the other. PMID:27120560

  4. Effect of dietary fiber on proteolytic pancreatic enzymes in vitro.

    PubMed

    Hansen, W E

    1986-12-01

    Chymotrypsin, trypsin, carboxypeptidase A and B, elastase and enterokinase activities were measured in buffer solutions and in human duodenal juice after incubation with wheat bran, cellulose, guar gum, pectin, psyllium and lignin. The different types of dietary fiber led to inhibition of enzymatic activity in most experiments, e.g., lignin could totally ablish the activity of isolated trypsin and chymotrypsin. Only in enterokinase was there no influence. Inhibition depended on incubation time; the effect was proportional to fiber concentration and inversely related to enzyme level. Treatment of fiber with hydrochloric acid (pH 1.5) and heat (95 degrees C) destroyed inhibitory activity in some experiments. The effect of lignin on one enzyme (trypsin) was reduced by the addition of another enzyme (chymotrypsin). It is concluded that dietary fiber could affect digestion by inhibiting proteolytic pancreatic enzymes. PMID:2824629

  5. Wound dressings for a proteolytic-rich environment.

    PubMed

    Vasconcelos, Andreia; Cavaco-Paulo, Artur

    2011-04-01

    Wound dressings have experienced continuous and significant changes over the years based on the knowledge of the biochemical events associated with chronic wounds. The development goes from natural materials used to just cover and conceal the wound to interactive materials that can facilitate the healing process, addressing specific issues in non-healing wounds. These new types of dressings often relate with the proteolytic wound environment and the bacteria load to enhance the healing. Recently, the wound dressing research is focusing on the replacement of synthetic polymers by natural protein materials to delivery bioactive agents to the wounds. This article provides an overview on the novel protein-based wound dressings such as silk fibroin keratin and elastin. The improved properties of these dressings, like the release of antibiotics and growth factors, are discussed. The different types of wounds and the effective parameters of healing process will be reviewed. PMID:21360151

  6. Electrochemical Proteolytic Beacon for Detection of Matrix Metalloproteinase Activities

    SciTech Connect

    Liu, Guodong; Wang, Jun; Wunschel, David S.; Lin, Yuehe

    2006-09-27

    This communication describes a novel method for detecting of matrix metalloproteinase-7 activity using a peptide substrate labeled with a ferrocene reporter. The substrate serves as a selective ‘electrochemical proteolytic beacon’ (EPB) for this metalloproteinase. The EPB is immobilized on a gold electrode surface to enable ‘on-off’ electrochemical signaling capability for uncleaved and cleaved events. The EPB is efficiently and selectively cleaved by MMP-7 as measured by the rate of decrease in redox current of ferrocene. Direct transduction of a signal corresponding to peptide cleavage events into an electronic signal thus provides a simple, sensitive route for detecting the MMP activity. The new method allows for identification of the activity of MMP-7 in concentrations as low as 3.4 pM. The concept can be extended to design multiple peptide substrate labeled with different electroactive reporters for assaying multiple MMPs activities.

  7. Proteolytic Cleavage of Apolipoprotein E in the Down Syndrome Brain

    PubMed Central

    Day, Ryan J.; McCarty, Katie L.; Ockerse, Kayla E.; Head, Elizabeth; Rohn, Troy T.

    2016-01-01

    Down syndrome (DS) is one of the most common genetic causes of intellectual disability and is characterized by a number of behavioral as well as cognitive symptoms. Many of the neuropathological features of early-onset Alzheimer’s disease (AD) including senile plaques and neurofibrillary tangles (NFTs) are also present in people with DS as a result of triplication of the amyloid precursor gene on chromosome 21. Evidence suggests that harboring one or both apolipoprotein E4 (APOE4) alleles may increase the risk for AD due to the proteolytic cleavage of apoE4 and a subsequent loss of function. To investigate a role for the apoE proteolysis in vivo, we compared three autopsy groups; 7 DS with AD neuropathology cases over 40 years, 5 young DS cases without AD pathology under 40 years (YDS) and 5 age-matched control cases over 40 years by immunohistochemistry utilizing an antibody that detects the amino-terminal fragment of apoE. Application of this antibody, termed the amino-terminal apoE fragment antibody (nApoECF) revealed labeling of pyramidal neurons in the frontal cortex of YDS cases, whereas in the DS-AD group, labeling with nApoECF was prominent within NFTs. NFT labeling with nApoECF was significantly greater in the hippocampus versus the frontal cortex in the same DS-AD cases, suggesting a regional distribution of truncated apoE. Colocalization immunofluorescence experiments indicated that 52.5% and 53.2% of AT8- and PHF-1-positive NFTs, respectively, also contained nApoECF. Collectively, these data support a role for the proteolytic cleavage of apoE in DS and suggest that apoE fragmentation is closely associated with NFTs. PMID:27330841

  8. Proteolytic Cleavage of Apolipoprotein E in the Down Syndrome Brain.

    PubMed

    Day, Ryan J; McCarty, Katie L; Ockerse, Kayla E; Head, Elizabeth; Rohn, Troy T

    2016-05-01

    Down syndrome (DS) is one of the most common genetic causes of intellectual disability and is characterized by a number of behavioral as well as cognitive symptoms. Many of the neuropathological features of early-onset Alzheimer's disease (AD) including senile plaques and neurofibrillary tangles (NFTs) are also present in people with DS as a result of triplication of the amyloid precursor gene on chromosome 21. Evidence suggests that harboring one or both apolipoprotein E4 (APOE4) alleles may increase the risk for AD due to the proteolytic cleavage of apoE4 and a subsequent loss of function. To investigate a role for the apoE proteolysis in vivo, we compared three autopsy groups; 7 DS with AD neuropathology cases over 40 years, 5 young DS cases without AD pathology under 40 years (YDS) and 5 age-matched control cases over 40 years by immunohistochemistry utilizing an antibody that detects the amino-terminal fragment of apoE. Application of this antibody, termed the amino-terminal apoE fragment antibody (nApoECF) revealed labeling of pyramidal neurons in the frontal cortex of YDS cases, whereas in the DS-AD group, labeling with nApoECF was prominent within NFTs. NFT labeling with nApoECF was significantly greater in the hippocampus versus the frontal cortex in the same DS-AD cases, suggesting a regional distribution of truncated apoE. Colocalization immunofluorescence experiments indicated that 52.5% and 53.2% of AT8- and PHF-1-positive NFTs, respectively, also contained nApoECF. Collectively, these data support a role for the proteolytic cleavage of apoE in DS and suggest that apoE fragmentation is closely associated with NFTs. PMID:27330841

  9. Patient specific proteolytic activity of monocyte-derived macrophages and osteoclasts predicted with temporal kinase activation states during differentiation

    PubMed Central

    Park, Keon-Young; Li, Weiwei A.; Platt, Manu O.

    2012-01-01

    Patient-to-patient variability in disease progression continues to complicate clinical decisions of treatment regimens for cardiovascular diseases, metastatic cancers and osteoporosis. Here, we investigated if monocytes, circulating white blood cells that enter tissues and contribute to disease progression by differentiating into macrophages or osteoclasts, could be useful in understanding this variability. Monocyte-derived macrophages and osteoclasts produce cysteine cathepsins, powerful extracellular matrix proteases which have been mechanistically linked to accelerated atherosclerotic, osteoporotic, and tumor progression. We hypothesized that multivariate analysis of temporal kinase activation states during monocyte differentiation could predict cathepsin proteolytic responses of monocyte-derived macrophages and osteoclasts in a patient-specific manner. Freshly isolated primary monocytes were differentiated with M-CSF or RANKL into macrophages or osteoclasts, respectively, and phosphorylation of ERK1/2, Akt, p38 MAPK, JNK, c-jun, and IκB-α were measured at days 1, 3, 6, and 9. In parallel, cell diameters and numbers of nuclei were measured, and multiplex cathepsin zymography was used to quantify cathepsins K, L, S, and V activity from cell extracts and conditioned media. There was extensive patient-to-patient variability in temporal kinase activation states, cell morphologies, and cathepsin K, L, S, and V proteolytic activity. Partial least squares regression models trained with temporal kinase activation states successfully predicted patient-specific morphological characteristics (mean cell diameter and number of nuclei) and patient-specific cathepsin proteolytic activity with predictability as high as 95%, even with the challenge of incorporating the complex, unknown cues from individual patients’ unique genetic and biochemical backgrounds. This personalized medicine approach considers patient variability in kinase signals to predict cathepsin activity

  10. Isolation and identification of thermophilic and mesophylic proteolytic bacteria from shrimp paste "Terasi"

    NASA Astrophysics Data System (ADS)

    Murwani, R.; Supriyadi, Subagio, Trianto, A.; Ambariyanto

    2015-12-01

    Terasi is a traditional product generally made of fermented shrimp. There were many studies regarding lactic acid bacteria of terasi but none regarding proteolitic bacteria. This study was conducted to isolate and identify the thermophilic and mesophylic proteolytic bacteria from terasi. In addition, the effect of different salt concentrations on the growth of the isolated proteolytic bacteria with the greatest proteolytic activity was also studied. Terasi samples were obtained from the Northern coast region of Java island i.e. Jepara, Demak and Batang. The study obtained 34 proteolytic isolates. Four isolates were identified as Sulfidobacillus, three isolates as Vibrio / Alkaligenes / Aeromonas, two isolates as Pseudomonas, 21 isolates as Bacillus, three isolates as Kurthia/ Caryophanon and one isolates as Amphibacillus. The growth of proteolytic bacteria was affected by salt concentration. The largest growth was found at 0 ppm salt concentrations and growth was declined as salt concentration increased. Maximum growth at each salt concentration tested was found at 8 hours incubation.

  11. Characterization of Protein N-Glycosylation by Analysis of ZIC-HILIC-Enriched Intact Proteolytic Glycopeptides.

    PubMed

    Pohlentz, Gottfried; Marx, Kristina; Mormann, Michael

    2016-01-01

    Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) solid-phase extraction (SPE) combined with direct-infusion nanoESI mass spectrometry (MS) and tandem MS/MS is a well-suited method for the analysis of protein N-glycosylation. A site-specific characterization of N-glycopeptides is achieved by the combination of proteolytic digestions employing unspecific proteases, glycopeptide enrichment by use of ZIC-HILIC SPE, and subsequent mass spectrometric analysis. The use of thermolysin or a mixture of trypsin and chymotrypsin leads per se to a mass-based separation, that is, small nonglycosylated peptides and almost exclusively glycopeptides at higher m/z values. As a result of their higher hydrophilicity N-glycopeptides comprising short peptide backbones are preferably accumulated by the ZIC-HILIC-based separation procedure. By employing this approach complications associated with low ionization efficiencies of N-glycopeptides resulting from signal suppression in the presence of highly abundant nonglycosylated peptides can be largely reduced. Here, we describe a simple protocol aimed at the enrichment of N-glycopeptides derived from in-solution and in-gel digestions of SDS-PAGE-separated glycoproteins preceding mass spectrometric analysis. PMID:26700048

  12. Kallikrein-related Peptidase 5 Functions in Proteolytic Processing of Profilaggrin in Cultured Human Keratinocytes*

    PubMed Central

    Sakabe, Jun-ichi; Yamamoto, Mami; Hirakawa, Satoshi; Motoyama, Akira; Ohta, Isao; Tatsuno, Kazuki; Ito, Taisuke; Kabashima, Kenji; Hibino, Toshihiko; Tokura, Yoshiki

    2013-01-01

    Filaggrin protein is synthesized in the stratum granulosum of the skin and contributes to the formation of the human skin barrier. Profilaggrin is cleaved by proteolytic enzymes and converted to functional filaggrin, but its processing mechanism remains not fully elucidated. Kallikrein-related peptidase 5 (KLK5) is a major serine protease found in the skin, which is secreted from lamellar granules following its expression in the stratum granulosum and activated in the extracellular space of the stratum corneum. Here, we searched for profilaggrin-processing protease(s) by partial purification of epidermal extracts and found KLK5 as a possible candidate. We used high performance liquid chromatography coupled with electrospray tandem mass spectrometry to show that KLK5 cleaves profilaggrin. Furthermore, based on a proximity ligation assay, immunohistochemistry, and immunoelectron microscopy analysis, we reveal that KLK5 and profilaggrin co-localize in the stratum granulosum in human epidermis. KLK5 knockdown in normal cultured human epidermal keratinocytes resulted in higher levels of profilaggrin, indicating that KLK5 potentially functions in profilaggrin cleavage. PMID:23629652

  13. Stimulation of proteolytic digestion by intestinal goblet cell mucus.

    PubMed

    Shora, W; Forstner, G G; Forstner, J F

    1975-03-01

    Intestinal goblet cell mucus (GCM) was added to incubations of casein and trypsin (or chymotrypsin) to discover whether mucus could inhibit proteolysis. Contrary to expectation, GCM stimulated casein hydrolysis, reaching a maximum effect at a GCM to casein ratio (w/w) of 0.083. GCM did not contain proteolytic enzymes or proenzymes as contaminants, nor did GCM serve as a substrate for trypsin. Stimulation was not reduced by removing 85% of the sialic acid from GCM. Harsh physical treatment (boiling and freezing) of casein decreased (50%) the GCM effect, as did partial predigestion of casein by trypsin, and elevation of trypsin concentration beyond 3 mug per ml. Thus the undegraded structure of casein appeared to be important for the stimulation of proteolysis by GCM. GCM also enhanced the hydrolysis by trypsin of intestinal brush border membrane protein, but had no effect on the hydrolysis of hemoglobin, albumin, or benzoyl arginine ethyl ester. These results suggest that GCM reacts with specific substrates, in a fashion which promotes their digestion by trypsin or chymotrypsin. PMID:1112451

  14. Addressing proteolytic efficiency in enzymatic degradation therapy for celiac disease

    PubMed Central

    Rey, Martial; Yang, Menglin; Lee, Linda; Zhang, Ye; Sheff, Joey G.; Sensen, Christoph W.; Mrazek, Hynek; Halada, Petr; Man, Petr; McCarville, Justin L; Verdu, Elena F.; Schriemer, David C.

    2016-01-01

    Celiac disease is triggered by partially digested gluten proteins. Enzyme therapies that complete protein digestion in vivo could support a gluten-free diet, but the barrier to completeness is high. Current options require enzyme amounts on the same order as the protein meal itself. In this study, we evaluated proteolytic components of the carnivorous pitcher plant (Nepenthes spp.) for use in this context. Remarkably low doses enhance gliadin solubilization rates, and degrade gliadin slurries within the pH and temporal constraints of human gastric digestion. Potencies in excess of 1200:1 (substrate-to-enzyme) are achieved. Digestion generates small peptides through nepenthesin and neprosin, the latter a novel enzyme defining a previously-unknown class of prolyl endoprotease. The digests also exhibit reduced TG2 conversion rates in the immunogenic regions of gliadin, providing a twin mechanism for evading T-cell recognition. When sensitized and dosed with enzyme-treated gliadin, NOD/DQ8 mice did not show intestinal inflammation, when compared to mice challenged with only pepsin-treated gliadin. The low enzyme load needed for effective digestion suggests that gluten detoxification can be achieved in a meal setting, using metered dosing based on meal size. We demonstrate this by showing efficient antigen processing at total substrate-to-enzyme ratios exceeding 12,000:1. PMID:27481162

  15. Cold Temperature Induces the Reprogramming of Proteolytic Pathways in Yeast.

    PubMed

    Isasa, Marta; Suñer, Clara; Díaz, Miguel; Puig-Sàrries, Pilar; Zuin, Alice; Bichman, Anne; Gygi, Steven P; Rebollo, Elena; Crosas, Bernat

    2016-01-22

    Despite much evidence of the involvement of the proteasome-ubiquitin signaling system in temperature stress response, the dynamics of the ubiquitylome during cold response has not yet been studied. Here, we have compared quantitative ubiquitylomes from a strain deficient in proteasome substrate recruitment and a reference strain during cold response. We have observed that a large group of proteins showing increased ubiquitylation in the proteasome mutant at low temperature is comprised by reverses suppressor of Ty-phenotype 5 (Rsp5)-regulated plasma membrane proteins. Analysis of internalization and degradation of plasma membrane proteins at low temperature showed that the proteasome becomes determinant for this process, whereas, at 30 °C, the proteasome is dispensable. Moreover, our observations indicate that proteasomes have increased capacity to interact with lysine 63-polyubiquitylated proteins during low temperature in vivo. These unanticipated observations indicate that, during cold response, there is a proteolytic cellular reprogramming in which the proteasome acquires a role in the endocytic-vacuolar pathway. PMID:26601941

  16. Single cell multiplexed assay for proteolytic activity using droplet microfluidics.

    PubMed

    Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Chen, Chia-Hung

    2016-07-15

    Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (~100 cells/experimental run). We characterized protease activity profiles at single cell resolution for several cancer cell lines including breast cancer cell line MDA-MB-231, lung cancer cell line PC-9, and leukemia cell line K-562 using both live-cell and in-situ cell lysis assay formats, with special focus on metalloproteinases important in metastasis. The ability to measure multiple proteases secreted from or expressed in individual cells allows us to characterize cell heterogeneity and has potential applications including systems biology, pharmacology, cancer diagnosis and stem cell biology. PMID:26995287

  17. Addressing proteolytic efficiency in enzymatic degradation therapy for celiac disease.

    PubMed

    Rey, Martial; Yang, Menglin; Lee, Linda; Zhang, Ye; Sheff, Joey G; Sensen, Christoph W; Mrazek, Hynek; Halada, Petr; Man, Petr; McCarville, Justin L; Verdu, Elena F; Schriemer, David C

    2016-01-01

    Celiac disease is triggered by partially digested gluten proteins. Enzyme therapies that complete protein digestion in vivo could support a gluten-free diet, but the barrier to completeness is high. Current options require enzyme amounts on the same order as the protein meal itself. In this study, we evaluated proteolytic components of the carnivorous pitcher plant (Nepenthes spp.) for use in this context. Remarkably low doses enhance gliadin solubilization rates, and degrade gliadin slurries within the pH and temporal constraints of human gastric digestion. Potencies in excess of 1200:1 (substrate-to-enzyme) are achieved. Digestion generates small peptides through nepenthesin and neprosin, the latter a novel enzyme defining a previously-unknown class of prolyl endoprotease. The digests also exhibit reduced TG2 conversion rates in the immunogenic regions of gliadin, providing a twin mechanism for evading T-cell recognition. When sensitized and dosed with enzyme-treated gliadin, NOD/DQ8 mice did not show intestinal inflammation, when compared to mice challenged with only pepsin-treated gliadin. The low enzyme load needed for effective digestion suggests that gluten detoxification can be achieved in a meal setting, using metered dosing based on meal size. We demonstrate this by showing efficient antigen processing at total substrate-to-enzyme ratios exceeding 12,000:1. PMID:27481162

  18. Analysis of the proteolytic degradation products of hyaline cartilage proteoglycans.

    PubMed

    Liszt, F; Schnittker-Schulze, K; Stuhlsatz, H W; Greiling, H

    1990-01-01

    The proteolytic degradation products of nasal hyaline cartilage proteoglycans produced by polymorphonuclear leukocyte lysosomal enzymes were investigated. The protein content of the degradation products is 7.0-8.6% corresponding to a peptide chain of 24-28 amino acids and the relative molecular mass of the total fragment is M(r) = 37,600-39,200. On an average, each proteoglycan fragment contains two chondroitin-sulphate chains (M(r) = 22,000-22,400), every fourth fragment contains a keratan sulphate chain (M(r) = 7000-7200) and every seventh to eighth contains an O-glycosidic oligosaccharide. The results of the disaccharide analysis show that the galactosaminoglycan chains contain 76.2-83.6% chondroitin-4-sulphate, 12.9-19.4% chondroitin-6-sulphate, 3.5-3.8% chondroitin and no dermatan sulphate. Since composition and relative molecular mass of the chondroitin sulphate and keratan sulphate chains from the degradation products resemble those from native proteoglycans, it is suggested that the degradation of the proteoglycans occurs by proteinases that attack preferably the chondroitin sulphate region of the core protein. PMID:1726643

  19. Proteolytic cleavage, trafficking, and functions of nuclear receptor tyrosine kinases.

    PubMed

    Chen, Mei-Kuang; Hung, Mien-Chie

    2015-10-01

    Intracellular localization has been reported for over three-quarters of receptor tyrosine kinase (RTK) families in response to environmental stimuli. Internalized RTK may bind to non-canonical substrates and affect various cellular processes. Many of the intracellular RTKs exist as fragmented forms that are generated by γ-secretase cleavage of the full-length receptor, shedding, alternative splicing, or alternative translation initiation. Soluble RTK fragments are stabilized and intracellularly transported into subcellular compartments, such as the nucleus, by binding to chaperone or transcription factors, while membrane-bound RTKs (full-length or truncated) are transported from the plasma membrane to the ER through the well-established Rab- or clathrin adaptor protein-coated vesicle retrograde trafficking pathways. Subsequent nuclear transport of membrane-bound RTK may occur via two pathways, INFS or INTERNET, with the former characterized by release of receptors from the ER into the cytosol and the latter characterized by release of membrane-bound receptor from the ER into the nucleoplasm through the inner nuclear membrane. Although most non-canonical intracellular RTK signaling is related to transcriptional regulation, there may be other functions that have yet to be discovered. In this review, we summarize the proteolytic processing, intracellular trafficking and nuclear functions of RTKs, and discuss how they promote cancer progression, and their clinical implications. PMID:26096795

  20. High Proteolytic Resistance of Spider-Derived Inhibitor Cystine Knots

    PubMed Central

    Kikuchi, Kyoko; Sugiura, Mika; Kimura, Tadashi

    2015-01-01

    Proteolytic stability in gastrointestinal tract and blood plasma is the major obstacle for oral peptide drug development. Inhibitor cystine knots (ICKs) are linear cystine knot peptides which have multifunctional properties and could become promising drug scaffolds. ProTx-I, ProTx-II, GTx1-15, and GsMTx-4 were spider-derived ICKs and incubated with pepsin, trypsin, chymotrypsin, and elastase in physiological conditions to find that all tested peptides were resistant to pepsin, and ProTx-II, GsMTx-4, and GTx1-15 showed resistance to all tested proteases. Also, no ProTx-II degradation was observed in rat blood plasma for 24 hours in vitro and ProTx-II concentration in circulation decreased to half in 40 min, indicating absolute stability in plasma and fast clearance from the system. So far, linear peptides are generally thought to be unsuitable in vivo, but all tested ICKs were not degraded by pepsin and stomach could be selected for the alternative site of drug absorption for fast onset of the drug action. Since spider ICKs are selective inhibitors of various ion channels which are related to the pathology of many diseases, engineered ICKs will make a novel class of peptide medicines which can treat variety of bothering symptoms. PMID:26843868

  1. Single Cell Proteolytic Assays to Investigate Cancer Clonal Heterogeneity and Cell Dynamics Using an Efficient Cell Loading Scheme

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Chih; Cheng, Yu-Heng; Ingram, Patrick; Yoon, Euisik

    2016-06-01

    Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small sample (10–100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolytic activity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolytic activity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolytic activity.

  2. Single Cell Proteolytic Assays to Investigate Cancer Clonal Heterogeneity and Cell Dynamics Using an Efficient Cell Loading Scheme

    PubMed Central

    Chen, Yu-Chih; Cheng, Yu-Heng; Ingram, Patrick; Yoon, Euisik

    2016-01-01

    Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small sample (10–100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolytic activity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolytic activity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolytic activity. PMID:27283981

  3. Proteomic identification of galectin-3 binding ligands and characterization of galectin-3 proteolytic cleavage in human prostasomes.

    PubMed

    Kovak, M R; Saraswati, S; Goddard, S D; Diekman, A B

    2013-09-01

    Galectin-3 is a multifunctional carbohydrate-binding protein that was previously characterized as a proteolytic substrate for prostate-specific antigen (PSA) and was shown to be associated with prostasomes in human semen. Prostasomes are exosome-like vesicles that are secreted by the prostatic epithelium and have multiple proposed functions in normal reproduction and prostate cancer. In the current study, galectin-3 binding ligands in human prostasomes were identified and characterized with the goal to investigate galectin-3 function in prostasomes. Galectin-3 binding proteins were isolated by affinity column chromatography. Candidate ligands identified by MS/MS were PSA, prostatic acid phosphatase (PAP), zinc alpha-2-glycoprotein (ZAG), dipeptidyl peptidase-4 (CD26), aminopeptidase N (CD13), neprilysin, clusterin, antibacterial protein (FALL-39) and alpha-1-acid glycoprotein (ORM1). Biochemical methods were used to characterize the ability of galectin-3 to bind to selected ligands, and galectin-3 cleavage assays were utilized to investigate the protease(s) in prostasomes that cleaves galectin-3. CD26, CD13, PSA, PAP and ZAG immunoreactivity were detected in extracts of purified prostasomes. One-dimensional electroblot analysis of prostasomes demonstrated that CD26, PAP and CD13 immunoreactivity co-migrated with galectin-3-reactive protein bands. PSA and ZAG were found to be associated with the surface of prostasomes. Both intact and cleaved galectin-3 were detected in prostate and prostasome extracts. Cleavage and inhibition assays indicated that PSA in prostasomes proteolytically cleaves galectin-3. The identification of these glycoproteins as galectin-3 ligands lays the groundwork for future studies of galectin-3 and prostasome function in reproduction and prostate cancer. PMID:23836758

  4. Determination of lipolytic and proteolytic activities of mycoflora isolated from dry-cured teruel ham.

    PubMed

    Alapont, C; Martínez-Culebras, P V; López-Mendoza, M C

    2015-08-01

    Fungi play a key role in dry-cured ham production because of their lipolytic and proteolytic activities. In the present study, 74 fungal strains from dry-cured Teruel hams and air chambers were tested for proteolytic and lipolytic activities, with a view to their possible use as starter cultures. Lipolytic activity of fungi was studied against lauric, palmitic, stearic and oleic acids, whereas proteolytic activity was studied against casein and myosin. Of the 74 fungal strains tested, most of them demonstrated lipolytic activity (94.59 %). Lipolytic activity against lauric and oleic acids was stronger than against palmitic and stearic acids. 39 strains (52.70 %) demonstrated proteolytic activity against casein and the 6 highest proteolytic strains were also tested for pork myosin proteolysis. Some strains belonging to Penicillium commune, Penicillium chrysogenum, Penicillium nalgiovense and Cladosporium cladosporioides were selected because of their significant proteolytic and lipolytic activities and could be suitable to use as starters in dry-cured ham. PMID:26243949

  5. Risk assessment of proteolytic Clostridium botulinum in canned foie gras.

    PubMed

    Membré, Jeanne-Marie; Diao, Moctar; Thorin, Chantal; Cordier, Grégoire; Zuber, François; André, Stéphane

    2015-10-01

    In this study, a risk assessment of proteolytic Clostridium botulinum in canned foie gras was performed, the number of illnesses per year in France due to C. botulinum in foie gras was estimated. Data on initial level in raw materials were collected at manufacturers and analysed using a Negative Binomial distribution. The effect of the usual foie gras heat treatment (equivalent time at 121 °C: F0=0.5 min) was considered at two levels: first, it led to an inactivation (estimated to 2.3 log); second it led to a spore injury and then to a spore inhibition. This latter effect was assessed by analysing data from a challenge test study carried out with Clostridium sporogenes spores in the foie gras product. The probability of spore recovering after thermal inhibition was estimated to 9.5×10(-8) (corresponding to 7.0 log). The data on the consumption pattern were collected on the French market. The Quantitative Microbiological Risk Assessment (QMRA) model and all the assumptions are reported in detail in the study. The initial contamination of raw materials, effect of thermal treatment on microbial inactivation and spore inhibition were handled mathematically using a probabilistic framework, considering only the variability dimension. The model was implemented in Excel and run through Monte Carlo simulation, using @Risk software. In parallel, epidemiological data collected from the French Institute for Public Health Surveillance during the period 2001-2012 were used to estimate an Appropriate Level Of Protection (ALOP) and then a Food Safety Objective (FSO): ALOP equalled to 2.5×10(-3) illnesses per million inhabitant per year, FSO equalled to 1.4×10(-9) foie gras portions containing C. botulinum spore (expressed in decimal logarithm, FSO=-8.9). The QMRA model output values were smaller, but on the same order of magnitude as these two figures: 8.0×10(-4) illnesses per million inhabitants per year, and, 4.5×10(-10) (-9.3 log) foie gras portions containing C

  6. A role for PACE4 in the proteolytic activation of anthrax toxin protective antigen.

    PubMed Central

    Gordon, V M; Rehemtulla, A; Leppla, S H

    1997-01-01

    Several bacterial protein toxins require activation by eukaryotic proteases. Previous studies have shown that anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are cleaved by furin C-terminal to the sequences RKKR, RQPR, and RVRR, respectively. Because furin-deficient cells retain some sensitivity to PA and DT, it is evident that other cellular proteases can activate these toxins. Whereas furin has been shown to require arginine residues at positions -1 and -4 for substrate recognition, another protease with an activity which could substitute for furin in toxin activation, the furin-related protease PACE4, requires basic residues in the -1, -2, and -4 positions of the substrate sequence. To examine the relative roles of furin and PACE4 in toxin activation, we used furin-deficient CHO cells (FD11 cells) transfected with either the furin (FD11/furin cells) or PACE4 (FD11/PACE4 cells) gene. Mutant PA proteins containing the cleavage sequence RAAR or KR were cytotoxic toward cells expressing only PACE4. In vitro cleavage data demonstrated that PACE4 can recognize RAAR and, to a much lesser extent, KR and RR. When extracts from PACE4-transfected cells were used as a source of proteases, PACE4 had minimal activity, indicating that it had been partially inactivated or did not remain associated with the cell membranes. Cleavage of iodinated PA containing the sequence RKKR or RAAR was detected on the surface of all cell types tested, but cleavage of a dibasic sequence was detected only intracellularly and only in cells that expressed furin or PACE4. The data provide evidence that PACE4 is present at the exterior of cells, that it plays a role in the proteolytic activation of anthrax toxin PA, and that PACE4 can activate substrates at the sequence RAAR or KR. PMID:9234799

  7. Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

    PubMed Central

    Kooij, Pepijn W; Rogowska-Wrzesinska, Adelina; Hoffmann, Daniel; Roepstorff, Peter; Boomsma, Jacobus J; Schiøtt, Morten

    2014-01-01

    The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fluid deposited on the newly added plant substrate. As herbivorous feeding often implies that growth is nitrogen limited, we cloned and sequenced six fungal proteases found in the fecal fluid of the leaf-cutting ant Acromyrmex echinatior and identified them as two metalloendoproteases, two serine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed significant activity in fecal fluid at pH values of 5–7, but the aspartic proteases were inactive across a pH range of 3–10. Protease activity disappeared when the ants were kept on a sugar water diet without fungus. Relative to normal mycelium, both metalloendoproteases, both serine proteases and one aspartic protease were upregulated in the gongylidia, specialized hyphal tips whose only known function is to provide food to the ants. These combined results indicate that the enzymes are derived from the ingested fungal tissues. We infer that the five proteases are likely to accelerate protein extraction from plant cells in the leaf pulp that the ants add to the fungus garden, but regulatory functions such as activation of proenzymes are also possible, particularly for the aspartic proteases that were present but without showing activity. The proteases had high sequence similarities to proteolytic enzymes of phytopathogenic fungi, consistent with previous indications of convergent evolution of decomposition enzymes in attine ant fungal symbionts and phytopathogenic fungi. PMID:24401858

  8. Proteolytic activity regarding Sarconesiopsis magellanica (Diptera: Calliphoridae) larval excretions and secretions.

    PubMed

    Pinilla, Yudi T; Moreno-Pérez, Darwin A; Patarroyo, Manuel A; Bello, Felio J

    2013-12-01

    Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Diptera maggots release proteolytic enzymes contained in larval excretion and secretion (ES) products playing a key role in digestion. Special interest in proteolytic enzymes has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue during larval therapy. This study was thus aimed at identifying and characterising S. magellanica proteolytic enzyme ES products for the first time. These products were obtained from first-, second- and third-instar larvae taken from a previously-established colony. ES proteins were separated by SDS-PAGE and their proteolytic activity was characterised by zymograms and inhibition assays involving BAPNA (Nα-benzoyl-dl-Arg-p-nitroanilide) and SAPNA substrates, using synthetic inhibitors. The protein profile ranged from ∼69kDa to ∼23kDa; several of them coincided with the Lucilia sericata ES protein profile. Serine-protease hydrolysis activity (measured by zymogram) was confirmed when a ∼25kDa band disappeared upon ES incubation with PMSF inhibitor at pH 7.8. Analysis of larval ES proteolytic activity on BAPNA and SAPNA substrates (determined by using TLCK and TPCK specific inhibitors) suggested a greater amount of trypsin-like protease. These results support the need for further experiments aimed at validating S. magellanica use in larval therapy. PMID:24076089

  9. Near Infrared Optical Proteolytic Beacons for In Vivo Imaging of Matrix Metalloproteinase Activity

    PubMed Central

    McIntyre, J. Oliver; Scherer, Randy L.; Matrisian, Lynn M.

    2010-01-01

    The exuberant expression of proteinases by tumor cells has long been associated with the breakdown of the extracellular matrix, tumor invasion, and metastasis to distant organs. There is both epidemiological and experimental data that support a causative role for proteinases of the matrix metalloproteinase (MMP) family in tumor progression. Optical imaging techniques provide an extraordinary opportunity for non-invasive “molecular imaging” of tumor-associated proteolytic activity. The application of optical proteolytic beacons for the detection of specific proteinase activities associated with tumors has several potential purposes: 1) Detection of small, early-stage tumors with increased sensitivity due to the catalytic nature of proteolytic activity, 2) Diagnosis and Prognosis to distinguished tumors that require particularly aggressive therapy or those that will not benefit from therapy, 3) Identification of tumors appropriate for specific anti-proteinase therapeutics and optimization of drug and dose based on determination of target modulation, and 4) as an indicator of efficacy of proteolytically-activated pro-drugs. This chapter describes the synthesis, characterization, and application of reagents that use visible and near infrared fluorescence resonance energy transfer (FRET) fluorophore pairs to detect and measure MMP-referable proteolytic activity in tumors in mouse models of cancer. PMID:20135290

  10. Improvement of proteolytic and oxidative stability of Chondroitinase ABC I by cosolvents.

    PubMed

    Nazari-Robati, Mahdieh; Golestani, Abolfazl; Asadikaram, GholamReza

    2016-10-01

    Recently, utilization of the enzyme Chondroitinase ABC I (cABC I) has received considerable attention in treatment of spinal cord injury. cABC I removes chondroitin sulfate proteoglycans which are inhibitory to axon growth and enhances nerve regeneration. Therefore, determination of cABC I resistance to proteolysis and oxidation provides valuable information for optimizing its clinical application. In this work, proteolytic stability of cABC I to trypsin and chymotrypsin as well as its oxidative resistance to H2O2 was measured. Moreover, the effect of cosolvents glycerol, sorbitol and trehalose on cABC I proteolytic and oxidative stability was determined. The results indicated that cABC I is highly susceptible to proteolysis and oxidation. Comparison of proteolytic patterns demonstrated a high degree of similarity which confirmed the exposure of specific regions of cABC I to proteolysis. However, proteolytic degradation was significantly reduced in the presence of cosolvents. In addition, cosolvents decreased the rate of both cABC I proteolytic and oxidative inactivation. Notably, the degree of stabilization provided by these cosolvents varied greatly. These findings indicated the high potential of cosolvents in protein stabilization to proteolysis and oxidative inactivation. PMID:27311501

  11. Proteolytic activity of molds and their metabiotic association with Salmonella in a model system.

    PubMed

    Cibelli, F; Ciccarone, C; Altieri, C; Bevilacqua, A; Sinigaglia, M

    2008-10-01

    The aim of this study was to investigate the proteolytic ability of some strains of aspergilli, fusaria, and penicillia and the metabiotic effect of Fusarium oxysporum and Penicillium expansum on Salmonella. The proteolytic activity of the target molds was determined on tomato juice agar and tomato juice, whereas the metabiotic effect of F. oxysporum and P. expansum on Salmonella was assessed in a model system consisting of tryptone soy broth with different amounts of tomato juice added. Fusaria, some aspergilli, and one strain of penicillium showed a proteolytic activity on tomato juice agar. In addition, Salmonella survival was enhanced in tryptone soy broth plus 20 or 50% tomato juice in the model system previously inoculated with F. oxysporum. PMID:18939766

  12. Proteolytic Equilibria of Vanillic Acid in the Ground and Excited States

    NASA Astrophysics Data System (ADS)

    Vusovich, O. V.; Tchaikovskaya, O. N.; Sokolova, I. V.; Vasil‧eva, N. Yu.

    2016-03-01

    Proteolytic equilibria of vanillic acid in aqueous solutions were studied using electronic spectroscopy. The pH ranges for anionic, dianionic, cationic, and neutral forms of vanillic acid in the ground and excited states were determined. The electron density distribution on atoms in the proteolytic forms was determined using quantum-chemistry methods. The anion formed as a result of dissociation of the carboxylic acid. The dianion formed in the presence of two and more equivalents of alkali as a result of proton loss from the phenol and carboxylic acid. The vanillic acid cation formed via protonation of the carbonyl oxygen. Differences in spectral features of the proteolytic forms in the ground and excited states were observed.

  13. Resin-assisted Enrichment of N-terminal Peptides for Characterizing Proteolytic Processing

    SciTech Connect

    Kim, Jong Seo; Dai, Ziyu; Aryal, Uma K.; Moore, Ronald J.; Camp, David G.; Baker, Scott E.; Smith, Richard D.; Qian, Weijun

    2013-06-17

    Proteolytic processing is a ubiquitous, irreversible posttranslational modification that plays an important role in cellular regulation in all living organisms. Herein we report a resin-assisted positive selection method for specifically enriching protein N-terminal peptides to facilitate the characterization of proteolytic processing events by liquid chromatography-tandem mass spectrometry. In this approach, proteins are initially reduced and alkylated and their lysine residues are converted to homoarginines. Then, protein N-termini are selectively converted to reactive thiol groups. We demonstrate that these sequential reactions were achieved with nearly quantitative efficiencies. Thiol-containing N-terminal peptides are then captured (>98% efficiency) by a thiol-affinity resin, a significant improvement over the traditional avidin/biotin enrichment. Application to cell lysates of Aspergillus niger, a filamentous fungus of interest for biomass degradation, enabled the identification of 1672 unique protein N-termini and proteolytic cleavage sites from 690 unique proteins.

  14. Epitope Structure of the Carbohydrate Recognition Domain of Asialoglycoprotein Receptor to a Monoclonal Antibody Revealed by High-Resolution Proteolytic Excision Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Stefanescu, Raluca; Born, Rita; Moise, Adrian; Ernst, Beat; Przybylski, Michael

    2011-01-01

    Recent studies suggest that the H1 subunit of the carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor is used as an entry site into hepatocytes by hepatitis A and B viruses and Marburg virus. Thus, molecules binding specifically to the CRD might exert inhibition towards these diseases by blocking the virus entry site. We report here the identification of the epitope structure of H1CRD to a monoclonal antibody by proteolytic epitope excision of the immune complex and high-resolution MALDI-FTICR mass spectrometry. As a prerequisite of the epitope determination, the primary structure of the H1CRD antigen was characterised by ESI-FTICR-MS of the intact protein and by LC-MS/MS of tryptic digest mixtures. Molecular mass determination and proteolytic fragments provided the identification of two intramolecular disulfide bridges (seven Cys residues), and a Cys-mercaptoethanol adduct formed by treatment with β-mercaptoethanol during protein extraction. The H1CRD antigen binds to the monoclonal antibody in both native and Cys-alkylated form. For identification of the epitope, the antibody was immobilized on N-hydroxysuccinimide (NHS)-activated Sepharose. Epitope excision and epitope extraction with trypsin and FTICR-MS of affinity-bound peptides provided the identification of two specific epitope peptides (5-16) and (17-23) that showed high affinity to the antibody. Affinity studies of the synthetic epitope peptides revealed independent binding of each peptide to the antibody.

  15. Neuropathogenic Escherichia coli K1 does not exhibit proteolytic activities to exert its pathogenicity

    PubMed Central

    2013-01-01

    Background Proteases are well-known virulence factors that promote survival, pathogenesis and immune evasion of many pathogens. Several lines of evidence suggest that the blood–brain barrier permeability is a prerequisite in microbial invasion of the central nervous system. Because proteases are frequently associated with vascular permeability by targeting junctional proteins, here it is hypothesized that neuropathogenic Escherichia coli K1 exhibit proteolytic activities to exert its pathogenicity. Methods Zymographic assays were performed using collagen and gelatin as substrates. The lysates of whole E. coli K1 strain E44, or E. coli K-12 strain HB101 were tested for proteolytic activities. The conditioned media were prepared by incubating bacteria in RPMI-1640 in the presence or absence of serum. The cell-free supernatants were collected and tested for proteases in zymography as mentioned above. Additionally, proteolytic degradation of host immune factors was determined by co-incubating conditioned media with albumin/immunoglobulins using protease assays. Results When collagen or gelatin were used as substrates in zymographic assays, neither whole bacteria nor conditioned media exhibited proteolytic activities. The conditioned media of neuropathogenic E. coli K1 strain E44, or E. coli K-12 strain HB101 did not affect degradation of albumin and immunoglobulins using protease assays. Conclusions Neither zymographic assays nor protease assays detected proteolytic activities in either the whole bacteria or conditioned media of E. coli K1 strain E44 and E. coli K-12 strain HB101. These findings suggest that host cell monolayer disruptions and immune evasion strategies are likely independent of proteolytic activities of neuropathogenic E. coli K1. PMID:23634997

  16. Seasonal variation in the temperature sensitivity of proteolytic enzyme activity in temperate forest soils

    NASA Astrophysics Data System (ADS)

    Brzostek, Edward R.; Finzi, Adrien C.

    2012-03-01

    Increasing soil temperature has the potential to alter the activity of the extracellular enzymes that mobilize nitrogen (N) from soil organic matter (SOM) and ultimately the availability of N for primary production. Proteolytic enzymes depolymerize N from proteinaceous components of SOM into amino acids, and their activity is a principal driver of the within-system cycle of soil N. The objectives of this study were to investigate whether the soils of temperate forest tree species differ in the temperature sensitivity of proteolytic enzyme activity over the growing season and the role of substrate limitation in regulating temperature sensitivity. Across species and sampling dates, proteolytic enzyme activity had relatively low sensitivity to temperature with a mean activation energy (Ea) of 33.5 kJ mol-1. Ea declined in white ash, American beech, and eastern hemlock soils across the growing season as soils warmed. By contrast, Eain sugar maple soil increased across the growing season. We used these data to develop a species-specific empirical model of proteolytic enzyme activity for the 2009 calendar year and studied the interactive effects of soil temperature (ambient or +5°C) and substrate limitation (ambient or elevated protein) on enzyme activity. Declines in substrate limitation had a larger single-factor effect on proteolytic enzyme activity than temperature, particularly in the spring. There was, however, a large synergistic effect of increasing temperature and substrate supply on proteolytic enzyme activity. Our results suggest limited increases in N availability with climate warming unless there is a parallel increase in the availability of protein substrates.

  17. Inhibition of proteolytic processing of adenoviral proteins by epsilon-aminocaproic acid and ambenum in adenovirus-infected cells.

    PubMed

    Nosach, Lidiya; Dyachenko, Nataliya; Zhovnovataya, Valentina; Lozinskiy, Miron; Lozitsky, Victor

    2002-01-01

    Maturation of adenovirus particles is markedly affected by proteolytic processing. The possibility for blocking the conversion of precursor structural core protein (preVII) into mature structure protein VII by officinal drugs epsilon-aminocaproic acid and ambenum has been demonstrated in Hep-2 cells infected with adenovirus. Proteolytic processing may be regarded as one of the targets for inhibiting adenovirus reproduction. PMID:12545207

  18. Changes in expression of proteolytic genes in response to anabolic and catabolic signals in rainbow trout

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rates of protein accrual are largely affected by rates of protein degradation. Determining how proteolytic pathways are affected by catabolic and anabolic signals will contribute to the understanding of the impact and regulation these pathways have on protein turnover. Real time RT-PCR was used to...

  19. Site-specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants.

    PubMed

    Hehle, Verena K; Lombardi, Raffaele; van Dolleweerd, Craig J; Paul, Mathew J; Di Micco, Patrizio; Morea, Veronica; Benvenuto, Eugenio; Donini, Marcello; Ma, Julian K-C

    2015-02-01

    Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis. PMID:25283551

  20. Alleviation of Proteolytic Sensitivity To Enhance Recombinant Lipase Production in Escherichia coli▿

    PubMed Central

    Narayanan, Niju; Chou, C. Perry

    2009-01-01

    Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, which was resolved by DsbA coexpression. PMID:19542329

  1. TAILS N-terminomics of human platelets reveals pervasive metalloproteinase-dependent proteolytic processing in storage.

    PubMed

    Prudova, Anna; Serrano, Katherine; Eckhard, Ulrich; Fortelny, Nikolaus; Devine, Dana V; Overall, Christopher M

    2014-12-18

    Proteases, and specifically metalloproteinases, have been linked to the loss of platelet function during storage before transfusion, but the underlying mechanisms remain unknown. We used a dedicated N-terminomics technique, iTRAQ terminal amine isotopic labeling of substrates (TAILS), to characterize the human platelet N-terminome, proteome, and posttranslational modifications throughout platelet storage over 9 days under blood-banking conditions. From the identified 2938 proteins and 7503 unique peptides, we characterized N-terminal methionine excision, co- and posttranslational Nα acetylation, protein maturation, and proteolytic processing of proteins in human platelets. We also identified for the first time 10 proteins previously classified by the Human Proteome Organization as "missing" in the human proteome. Most N termini (77%) were internal neo-N termini (105 were novel potential alternative translation start sites, and 2180 represented stable proteolytic products), thus highlighting a prominent yet previously uncharacterized role of proteolytic processing during platelet storage. Protease inhibitor studies revealed metalloproteinases as being primarily responsible for proteolytic processing (as opposed to degradation) during storage. System-wide identification of metalloproteinase and other proteinase substrates and their respective cleavage sites suggests novel mechanisms of the effect of proteases on protein activity and platelet function during storage. All data sets and metadata are available through ProteomeXchange with the data set identifier PXD000906. PMID:25331112

  2. Proteolytic Enzymes in Detergents: Evidence of Their Presence through Activity Measurements Based on Electrophoresis

    ERIC Educational Resources Information Center

    Saperas, Nuria; Fonfria-Subiros, Elsa

    2011-01-01

    This laboratory exercise uses a problem-based approach to expose students to some basic concepts relating to proteins and enzymes. One of the main applications of enzymes at the industrial level is their use in the detergent market. The students examine a detergent sample to ascertain whether proteolytic enzymes are a component and, if so, which…

  3. Splicing and proteolytic processing in VEGF signaling: now it is the coreceptor's turn.

    PubMed

    Yao, Xiaolan; Bouyain, Samuel

    2015-04-01

    Alternative splicing and proteolytic processing of VEGFs generate proteins with distinct physiological roles. In this issue of Structure, Parker et al. show that proteolysis of an isoform of the VEGF-C coreceptor Nrp2 produces a soluble receptor that inhibits VEGF-C/Nrp2 interactions. PMID:25862932

  4. Extracellular thermostable proteolytic activity of the milk spoilage bacterium Pseudomonas fluorescens PS19 on bovine caseins.

    PubMed

    Stuknytė, M; Decimo, M; Colzani, M; Silvetti, T; Brasca, M; Cattaneo, S; Aldini, G; De Noni, I

    2016-06-01

    We studied the thermostable proteolytic activity of Pseudomonas fluorescens PS19 isolated from raw bovine milk. The heat-treated cell-free supernatant (HT-CFS) contained a thermostable protease of approximately 45 kDa, as revealed by casein zymography. We assigned this enzyme to P. fluorescens AprX metalloprotease (UniProtKB Acc. No. C9WKP6). After concentration by ultrafiltration at 10 kDa, the HT-CFS showed 2 other thermostable proteolytic bands on zymogram, with molecular masses of approximately 15 and 25 kDa. The former resulted a fragment of the AprX protease, whereas the 25-kDa protease was not homologous to any known protein of Pseudomonas spp. Subsequently, we assessed the proteolytic activity of the HT-CFS on bovine αS-, β-, and κ-casein during in vitro incubation at 7 or 22°C. By means of ultra-performance liquid chromatography-tandem mass spectrometry we identified the released peptides (n=591). Some of them resisted proteolysis during the whole incubation period at both incubation temperatures and, therefore, they could be assumed as indicators of the proteolytic action of P. fluorescens PS19 on bovine caseins. PMID:26995139

  5. Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting

    NASA Technical Reports Server (NTRS)

    Wing, S. S.; Goldberg, A. L.; Goldberger, A. L. (Principal Investigator)

    1993-01-01

    Glucocorticoids are essential for the increase in protein breakdown in skeletal muscle normally seen during fasting. To determine which proteolytic pathway(s) are activated upon fasting, leg muscles from fed and fasted normal rats were incubated under conditions that block or activate different proteolytic systems. After food deprivation (1 day), the nonlysosomal ATP-dependent process increased by 250%, as shown in experiments involving depletion of muscle ATP. Also, the maximal capacity of the lysosomal process increased 60-100%, but no changes occurred in the Ca(2+)-dependent or the residual energy-independent proteolytic processes. In muscles from fasted normal and adrenalectomized (ADX) rats, the protein breakdown sensitive to inhibitors of the lysosomal or Ca(2+)-dependent pathways did not differ. However, the ATP-dependent process was 30% slower in muscles from fasted ADX rats. Administering dexamethasone to these animals or incubating their muscles with dexamethasone reversed this defect. During fasting, when the ATP-dependent process rises, muscles show a two- to threefold increase in levels of ubiquitin (Ub) mRNA. However, muscles of ADX animals failed to show this response. Injecting dexamethasone into the fasted ADX animals increased muscle Ub mRNA within 6 h. Thus glucocorticoids activate the ATP-Ub-dependent proteolytic pathway in fasting apparently by enhancing the expression of components of this system such as Ub.

  6. Proteolytic Products of the Porcine Reproductive and Respiratory Syndrome Virus Nsp2 Replicase Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The nsp2 replicase protein of porcine reproductive and respiratory syndrome virus (PRRSV) was recently demonstrated to be processed from its precursor by the PL2 protease at or near the G1196|G1197 dipeptide in transfected CHO cells. Here, the proteolytic cleavage of PRRSV nsp2 was further investiga...

  7. Cellulolytic and proteolytic ability of bacteria isolated from gastrointestinal tract and composting of a hippopotamus.

    PubMed

    da Cruz Ramos, Geomárcia Feitosa; Ramos, Patricia Locosque; Passarini, Michel Rodrigo Zambrano; Vieira Silveira, Marghuel A; Okamoto, Débora Noma; de Oliveira, Lilian Caroline Gonçalves; Zezzo, Larissa Vieira; Marem, Alyne; Santos Rocha, Rafael Costa; da Cruz, João Batista; Juliano, Luiz; de Vasconcellos, Suzan Pantaroto

    2016-03-01

    The bioprospection for cellulase and protease producers is a promise strategy for the discovery of potential biocatalysts for use in hydrolysis of lignocellulosic materials as well as proteic residues. These enzymes can increment and turn viable the production of second generation ethanol from different and alternative sources. In this context, the goal of this study was the investigation of cellulolytic and proteolytic abilities of bacteria isolated from the gastrointestinal tract of a hippopotamus as well as from its composting process. It is important to highlight that hippopotamus gastrointestinal samples were a non-typical sources of efficient hydrolytic bacteria with potential for application in biotechnological industries, like biofuel production. Looking for this, a total of 159 bacteria were isolated, which were submitted to qualitative and quantitative enzymatic assays. Proteolytic analyzes were conducted through the evaluation of fluorescent probes. Qualitative assays for cellulolytic abilities revealed 70 positive hits. After quantitative analyzes, 44 % of these positive hits were selected, but five (5) strains showed cellulolytic activity up to 11,8 FPU/mL. Regarding to proteolytic activities, six (6) strains showed activity above 10 %, which overpassed results described in the literature. Molecular analyzes based on the identification of 16S rDNA, revealed that all the selected bacterial isolates were affiliated to Bacillus genus. In summary, these results strongly indicate that the isolated bacteria from a hippopotamus can be a potential source of interesting biocatalysts with cellulolytic and proteolytic activities, with relevance for industrial applications. PMID:26931430

  8. Proteolytic processing of Porcine Reproductive and Respiratory Syndrome Virus nsp2 replicase protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One critical step in porcine reproductive and respiratory syndrome virus (PRRSV) replication is the proteolytic processing of the ORF1 polyprotein (replicase). The replicase polyprotein is generally believed to be processed to generate at least 12 smaller nonstructural proteins (nsps) involved in r...

  9. Adenovirus coded deoxyribonucleic acid binding protein. Isolation, physical properties, and effects of proteolytic digestion

    SciTech Connect

    Schechter, N.M.; Davies, W.; Anderson, C.W.

    1980-01-01

    A procedure has been developed for the purification of adenovirus type 2 DNA-binding protein (DBP) from nuclei of infected HeLa cells. This procedure routinely yields 0.2 to 0.6 mg of protein per 10/sup 9/ cells that is greater than 98% DBP. Binding protein so prepared does not precipitate at low ionic strength, interacts with both single- and double-stranded DNA, and complements Ad5 ts125 function in an in vitro DNA synthesizing system dependent upon exogenous DBP. An examination of the hydrodynamic properties of Ad2 DBP indicated that DBP undergoes a concentration-dependent self-association process. In high ionic strength solutions (1.0 M NaCl), self-association is a limited process observed at DBP concentrations above about 0.1 mg/mL; the product is a unit having a molecular weight of a trimer. At low ionic strengths (0.1 M NaCl), self-association is more extensive and is observed at lower protein concentrations. Our findings suggest that units other than the 72,000 molecular weight monomer may interact with DNA in the cell. Purified Ad2 DBP was digested with several proteolytic enzymes to determine if smaller DNA-binding products could be generated that resemble the 48,000 molecular weight species observed in extracts of infected cells. Digestion of purified DBP with Pronase or chymotrypsin produced relatively stable fragments with molecular weights of 45,000 and 53,000, respectively. Trypsin cleavage produced a 51,000 molecular weight fragment that upon continued incubation was further digested to produce a 35,000-M/sub r/ peptide. The production of the 35,000-M/sub r/ peptide by trypsin cleavage of the 51,000-M/sub r/ fragment was not observed if a sufficient amount of DNA was added to the DBP solution prior to trypsin digestion. This result indicates that bound DNA protects a trypsin-sensitive site(s) in the 51,000-M/sub r/ fragment, and it suggests that the 51,000-M/sub r/ fragment contains at least a part of the binding site for single-stranded DNA.

  10. Role of proteolytic enzymes in degradation of plant tissues

    SciTech Connect

    Lewosz, J.; Kelman, A.; Sequeira, L.

    1991-01-01

    Strain SR 394 of Erwinia carotovora (Ecc) produced proteases constitutively in all media tested. Growth of Ecc and production of protease were enhanced significantly by the presence of poetic materials and/or plant call walls in the test media. After electrofocusing, one major and one minor protease bands, at PI 4.8 and PI 5.1, respectively, were detected. Only one band of 43 kDa was detected on SDS gels. Only one protease band was detected in SDS gels of infected plant extracts. This protease was purified to homogeneity. It in a highly thermostable metal protease; it degrades gelatin, soluble collagen and hide powderazure, shows weak activity on casein and azocasein, but does not degrade insoluble collagen or elastin.

  11. Enhanced Proteolytic Processing of Recombinant Human Coagulation Factor VIII B-Domain Variants by Recombinant Furins.

    PubMed

    Demasi, Marcos A; de S Molina, Erika; Bowman-Colin, Christian; Lojudice, Fernando H; Muras, Angelita; Sogayar, Mari C

    2016-06-01

    Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg(1648) and Glu(1649). By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg(1648) FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg(1648) FVIII processing site by PCSK6. PMID:27126696

  12. Proteolytic and antimicrobial activity of lactic acid bacteria grown in goat milk

    PubMed Central

    Atanasova, Jivka; Moncheva, Penka; Ivanova, Iskra

    2014-01-01

    We examined 62 strains and 21 trade starter cultures from the collection of LB Bulgaricum PLC for proteolytic and antimicrobial activity of lactic acid bacteria (LAB) grown in goat milk. The aim of this study was to investigate the fermentation of caseins, α-lactalbumin and β-lactoglobulin by LAB, using the o-phthaldialdehyde (OPA) spectrophotometric assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteolysis targeted mainly caseins, especially β-casein. Whey proteins were proteolyzed, essentially β-lactoglobulin. The proteolytic activity of Lactococcus lactis l598, Streptococcus thermophilus t3D1, Dt1, Lactobacillus lactis 1043 and L. delbrueckii subsp. bulgaricus b38, b122 and b24 was notably high. The proteolysis process gave rise to medium-sized peptide populations. Most of the examined strains showed antimicrobial activity against some food pathogens, such as Escherichia coli, Staphylococcus aureus, Salmonella cholere enteridis, Listeria monocytogenes, Listeria innocua and Enterobacter aerogenes. The most active producers of antimicrobial-active peptides were strains of L. delbrueckii subsp. bulgaricus and S. thermophilus, which are of practical importance. The starter cultures containing the examined species showed high proteolytic and antimicrobial activity in skimmed goat milk. The greatest antimicrobial activity of the cultures was detected against E. aerogenes. The obtained results demonstrated the significant proteolytic potential of the examined strains in goat milk and their potential for application in the production of dairy products from goat's milk. The present results could be considered as the first data on the proteolytic capacity of strains and starter cultures in goat milk for the purposes of trade interest of LB Bulgaricum PLC. PMID:26019593

  13. Combinatorial protein engineering of proteolytically resistant mesotrypsin inhibitors as candidates for cancer therapy.

    PubMed

    Cohen, Itay; Kayode, Olumide; Hockla, Alexandra; Sankaran, Banumathi; Radisky, Derek C; Radisky, Evette S; Papo, Niv

    2016-05-15

    Engineered protein therapeutics offer advantages, including strong target affinity, selectivity and low toxicity, but like natural proteins can be susceptible to proteolytic degradation, thereby limiting their effectiveness. A compelling therapeutic target is mesotrypsin, a protease up-regulated with tumour progression, associated with poor prognosis, and implicated in tumour growth and progression of many cancers. However, with its unique capability for cleavage and inactivation of proteinaceous inhibitors, mesotrypsin presents a formidable challenge to the development of biological inhibitors. We used a powerful yeast display platform for directed evolution, employing a novel multi-modal library screening strategy, to engineer the human amyloid precursor protein Kunitz protease inhibitor domain (APPI) simultaneously for increased proteolytic stability, stronger binding affinity and improved selectivity for mesotrypsin inhibition. We identified a triple mutant APPIM17G/I18F/F34V, with a mesotrypsin inhibition constant (Ki) of 89 pM, as the strongest mesotrypsin inhibitor yet reported; this variant displays 1459-fold improved affinity, up to 350 000-fold greater specificity and 83-fold improved proteolytic stability compared with wild-type APPI. We demonstrated that APPIM17G/I18F/F34V acts as a functional inhibitor in cell-based models of mesotrypsin-dependent prostate cancer cellular invasiveness. Additionally, by solving the crystal structure of the APPIM17G/I18F/F34V-mesotrypsin complex, we obtained new insights into the structural and mechanistic basis for improved binding and proteolytic resistance. Our study identifies a promising mesotrypsin inhibitor as a starting point for development of anticancer protein therapeutics and establishes proof-of-principle for a novel library screening approach that will be widely applicable for simultaneously evolving proteolytic stability in tandem with desired functionality for diverse protein scaffolds. PMID:26957636

  14. Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells’ Migration

    PubMed Central

    Salamone, Monica; Carfì Pavia, Francesco

    2016-01-01

    In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a “resting” phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process. PMID:27152413

  15. C1q-TNF-related protein-9, a novel cardioprotetcive cardiokine, requires proteolytic cleavage to generate a biologically active globular domain isoform.

    PubMed

    Yuan, Yuexing; Lau, Wayne Bond; Su, Hui; Sun, Yang; Yi, Wei; Du, Yunhui; Christopher, Theodore; Lopez, Bernard; Wang, Yajing; Ma, Xin-Liang

    2015-05-15

    Prevalence and severity of postmyocardial infarction heart failure continually escalate in type 2 diabetes via incompletely understood mechanisms. The discovery of the cardiac secretomes, collectively known as "cardiokines", has significantly enhanced appreciation of the local microenvironment's influence on disease development. Recent studies demonstrated that C1q-TNF-related protein-9 (CTRP9), a newly discovered adiponectin (APN) paralog, is highly expressed in the heart. However, its relationship with APN (concerning diabetic cardiovascular injury in particular) remains unknown. Plasma CTRP9 levels are elevated in APN knockout and reduced in diabetic mice. In contrast to APN, which circulates as full-length multimers, CTRP9 circulates in the plasma primarily in the globular domain isoform (gCTRP9). Recombinant full-length CTRP9 (fCTRP9) was cleaved when incubated with cardiac tissue extracts, generating gCTRP9, a process inhibited by protease inhibitor cocktail. gCTRP9 rapidly activates cardiac survival kinases, including AMPK, Akt, and endothelial NOS. However, fCTRP9-mediated kinase activation is much less potent and significantly delayed. Kinase activation by fCTRP9, but not gCTRP9, is inhibited by protease inhibitor cocktail. These results demonstrate for the first time that the novel cardiokine CTRP9 undergoes proteolytic cleavage to generate gCTRP9, the dominant circulatory and actively cardioprotective isoform. Enhancing cardiac CTRP9 production and/or its proteolytic posttranslational modification are of therapeutic potential, attenuating diabetic cardiac injury. PMID:25783894

  16. Isolation and evaluation of proteolytic actinomycete isolates as novel inducers of pearl millet downy mildew disease protection.

    PubMed

    Jogaiah, Sudisha; Kurjogi, Mahantesh; Govind, Sharathchandra Ramasandra; Huntrike, Shekar Shetty; Basappa, Vedamurthy Ankala; Tran, Lam-Son Phan

    2016-01-01

    Native endophytic actinomycetes isolated from pearl millet roots were examined for their efficacy to protect pearl millet against downy mildew. Nineteen of 39 isolates were found to be proteolytic, of which 7 strains could directly suppress the sporangium formation of Sclerospora graminicola, the pearl millet downy mildew pathogen. Thus, mycelial suspensions containing either spores or cell-free extract of these 7 isolates were used for seed-coating and -soaking treatments to test for their induction of downy mildew resistance. Results indicated that seed-coating overall provided better protection to downy mildew than seed-soaking. In both treatments, the tested isolates demonstrated differential abilities in downy mildew disease protection, with Streptomyces griseus SJ_UOM-07-09 and Streptosporangium roseum SJ_UOM-18-09 showing the highest protection rates. Additionally, the levels of disease protection conferred by the actinomycetes were just slightly lower than that of the systemic fungicide Apron, suggesting their effectiveness. Further studies revealed that the more rapid root colonization by SJ_UOM-18-09 resulted in faster and higher induced resistance in comparison with SJ_UOM-07-09 under greenhouse conditions, indicating that SJ_UOM-18-09 was superior than SJ_UOM-07-09 in inducing resistance. Results from this study provide comprehensive information on biocontrol functions of SJ_UOM- 18-09 with great potential to control downy mildew disease in pearl millet. PMID:27499196

  17. Isolation and evaluation of proteolytic actinomycete isolates as novel inducers of pearl millet downy mildew disease protection

    PubMed Central

    Jogaiah, Sudisha; Kurjogi, Mahantesh; Govind, Sharathchandra Ramasandra; Huntrike, Shekar Shetty; Basappa, Vedamurthy Ankala; Tran, Lam-Son Phan

    2016-01-01

    Native endophytic actinomycetes isolated from pearl millet roots were examined for their efficacy to protect pearl millet against downy mildew. Nineteen of 39 isolates were found to be proteolytic, of which 7 strains could directly suppress the sporangium formation of Sclerospora graminicola, the pearl millet downy mildew pathogen. Thus, mycelial suspensions containing either spores or cell-free extract of these 7 isolates were used for seed-coating and -soaking treatments to test for their induction of downy mildew resistance. Results indicated that seed-coating overall provided better protection to downy mildew than seed-soaking. In both treatments, the tested isolates demonstrated differential abilities in downy mildew disease protection, with Streptomyces griseus SJ_UOM-07-09 and Streptosporangium roseum SJ_UOM-18-09 showing the highest protection rates. Additionally, the levels of disease protection conferred by the actinomycetes were just slightly lower than that of the systemic fungicide Apron, suggesting their effectiveness. Further studies revealed that the more rapid root colonization by SJ_UOM-18-09 resulted in faster and higher induced resistance in comparison with SJ_UOM-07-09 under greenhouse conditions, indicating that SJ_UOM-18-09 was superior than SJ_UOM-07-09 in inducing resistance. Results from this study provide comprehensive information on biocontrol functions of SJ_UOM- 18-09 with great potential to control downy mildew disease in pearl millet. PMID:27499196

  18. A new method for monitoring the extracellular proteolytic activity of wine yeasts during alcoholic fermentation of grape must.

    PubMed

    Chasseriaud, Laura; Miot-Sertier, Cécile; Coulon, Joana; Iturmendi, Nerea; Moine, Virginie; Albertin, Warren; Bely, Marina

    2015-12-01

    The existing methods for testing proteolytic activity are time consuming, quite difficult to perform, and do not allow real-time monitoring. Proteases have attracted considerable interest in winemaking and some yeast species naturally present in grape must, such as Metschnikowia pulcherrima, are capable of expressing this activity. In this study, a new test is proposed for measuring proteolytic activity directly in fermenting grape must, using azocasein, a chromogenic substrate. Several yeast strains were tested and differences in proteolytic activity were observed. Moreover, analysis of grape must proteins in wines revealed that protease secreted by Metschnikowia strains may be active against wine proteins. PMID:26529648

  19. [A micromethod for the rapid detection of proteolytic activity of microorganisms].

    PubMed

    Kuznetsova, G G

    1989-01-01

    The author has developed a micromodification of the rapid photoemulsion method for the detection of the microorganism proteolytic activity. The test is carried out on polystyrene plates, meat-peptone broth is used as the suspension fluid. Rectangular film strips are vertically placed into the wells, thus preventing false-negative reactions possible in case of an erroneous horizontal position of the film with the emulsion layer turned upwards if square or round film fragments are used. The proteolytic activities of 120 microorganism strains (95 of these hydrolyze gelatin) have been examined by the micromethod and the routine test with gelatin. The suggested test is 2-4-fold more rapid than the routine one used in investigations of the cultures slowly hydrolyzing gelatin. The fact that the test is carried out on the plates considerably reduces the nutrient medium and the number of laboratory glassware and helps obtain more accurate results. PMID:2468029

  20. When activity requires breaking up: LEKTI proteolytic activation cascade for specific proteinase inhibition.

    PubMed

    Furio, Laetitia; Hovnanian, Alain

    2011-11-01

    Lymphoepithelial Kazal-type related inhibitor (LEKTI) is a multidomain proteinase inhibitor whose defective expression causes Netherton syndrome (NS). LEKTI is encoded by SPINK5, which is also a susceptibility gene for atopic disease. In this issue, Fortugno et al. report an elegant and thorough study of the LEKTI proteolytic activation process in which they identify the precise nature of the cleavage sites used and the bioactive fragments generated. They propose a proteolytic activation model in human skin and confirm differential inhibition of kallikrein (KLK) 5, 7, and 14 by the major physiological LEKTI fragments. They show that these bioactive fragments inhibit KLK-mediated proteolysis of desmoglein 1 (DSG1) and suggest a fine-tuned inhibition process controlling target serine proteinase (SP) activity. PMID:21997416

  1. Loss of proteolytically processed filaggrin caused by epidermal deletion of Matriptase/MT-SP1

    PubMed Central

    List, Karin; Szabo, Roman; Wertz, Philip W.; Segre, Julie; Haudenschild, Christian C.; Kim, Soo-Youl; Bugge, Thomas H.

    2003-01-01

    Profilaggrin is a large epidermal polyprotein that is proteolytically processed during keratinocyte differentiation to release multiple filaggrin monomer units as well as a calcium-binding regulatory NH2-terminal filaggrin S-100 protein. We show that epidermal deficiency of the transmembrane serine protease Matriptase/MT-SP1 perturbs lipid matrix formation, cornified envelope morphogenesis, and stratum corneum desquamation. Surprisingly, proteomic analysis of Matriptase/MT-SP1–deficient epidermis revealed the selective loss of both proteolytically processed filaggrin monomer units and the NH2-terminal filaggrin S-100 regulatory protein. This was associated with a profound accumulation of profilaggrin and aberrant profilaggrin-processing products in the stratum corneum. The data identify keratinocyte Matriptase/MT-SP1 as an essential component of the profilaggrin-processing pathway and a key regulator of terminal epidermal differentiation. PMID:14638864

  2. In vivo sensing of proteolytic activity with an NSET-based NIR fluorogenic nanosensor.

    PubMed

    Ku, Minhee; Hong, Yoochan; Heo, Dan; Lee, Eugene; Hwang, Seungyeon; Suh, Jin-Suck; Yang, Jaemoon

    2016-03-15

    Biomedical in vivo sensing methods in the near-infrared (NIR) range, which that provide relatively high photon transparency, separation from auto-fluorescence background, and extended sensitivity, are being used increasingly for non-invasive mapping and monitoring of molecular events in cancer cells. In this study, we fabricated an NIR fluorogenic nanosensor based on the nanoparticle surface energy transfer effect, by conjugation of fluorescent proteolytic enzyme-specific cleavable peptides with gold nanorods (GNRs). Membrane-anchored membrane type 1-matrix metalloproteinases (MT1-MMPs), a family of zinc-dependent proteolytic enzymes, can induce the metastatic potential of cancer cells by promoting degradation of the extracellular matrix. Therefore, sensitive detection of MT1-MMP activity can provide essential information in the clinical setting. We have applied in vivo NIR sensing to evaluate MT1-MMP activity, as an NIR imaging target, in an MT1-MMP-expressing metastatic tumor mouse model. PMID:26454829

  3. N-Terminal Enrichment: Developing a Protocol to Detect Specific Proteolytic Fragments

    SciTech Connect

    Schepmoes, Athena A.; Zhang, Qibin; Petritis, Brianne O.; Qian, Weijun; Smith, Richard D.

    2009-12-01

    Proteolytic processing events are essential to physiological processes such as reproduction, development, and host responses, as well as regulating proteins in cancer; therefore, there is a significant need to develop robust approaches for characterizing such events. The current mass spectrometry (MS)-based proteomics techniques employs a “bottom-up” strategy, which does not allow for identification of different proteolytic proteins since the strategy measures all the small peptides from any given protein. The aim of this development is to enable the effective identification of specific proteolytic fragments. The protocol utilizes an acetylation reaction to block the N-termini of a protein, as well as any lysine residues. Following digestion, N-terminal peptides are enriched by removing peptides that contain free amines, using amine-reactive silica-bond succinic anhydride beads. The resulting enriched sample has one N-terminal peptide per protein, which reduces sample complexity and allows for increased analytical sensitivity compared to global proteomics.1 We initially compared the peptide identification and efficiency of blocking lysine using acetic anhydride (a 42 Da modification) or propionic anhydride (a 56 Da modification) in our protocol. Both chemical reactions resulted in comparable peptide identifications and *95 percent efficiency for blocking lysine residues. However, the use of propionic anhydride allowed us to distinguish in vivo acetylated peptides from chemically-tagged peptides.2 In an initial experiment using mouse plasma, we were able to identify *300 unique N-termini peptides, as well as many known cleavage sites. This protocol holds potential for uncovering new information related to proteolytic pathways, which will assist our understanding about cancer biology and efforts to identify potential biomarkers for various diseases.

  4. N-Terminal Enrichment: Developing a Protocol to Detect Specific Proteolytic Fragments

    PubMed Central

    Schepmoes, Athena A.; Zhang, Qibin; Petritis, Brianne O.; Qian, Wei-Jun; Smith, Richard D.

    2009-01-01

    Proteolytic processing events are essential to physiological processes such as reproduction, development, and host responses, as well as regulating proteins in cancer; therefore, there is a significant need to develop robust approaches for characterizing such events. The current mass spectrometry (MS)-based proteomics techniques employs a “bottom-up” strategy, which does not allow for identification of different proteolytic proteins since the strategy measures all the small peptides from any given protein. The aim of this development is to enable the effective identification of specific proteolytic fragments. The protocol utilizes an acetylation reaction to block the N-termini of a protein, as well as any lysine residues. Following digestion, N-terminal peptides are enriched by removing peptides that contain free amines, using amine-reactive silica-bond succinic anhydride beads. The resulting enriched sample has one N-terminal peptide per protein, which reduces sample complexity and allows for increased analytical sensitivity compared to global proteomics.1 We initially compared the peptide identification and efficiency of blocking lysine using acetic anhydride (a 42 Da modification) or propionic anhydride (a 56 Da modification) in our protocol. Both chemical reactions resulted in comparable peptide identifications and ∼95 percent efficiency for blocking lysine residues. However, the use of propionic anhydride allowed us to distinguish in vivo acetylated peptides from chemically-tagged peptides.2 In an initial experiment using mouse plasma, we were able to identify >300 unique N-termini peptides, as well as many known cleavage sites. This protocol holds potential for uncovering new information related to proteolytic pathways, which will assist our understanding about cancer biology and efforts to identify potential biomarkers for various diseases. PMID:19949699

  5. Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum

    PubMed Central

    Carter, Andrew T; Paul, Catherine J; Mason, David R; Twine, Susan M; Alston, Mark J; Logan, Susan M; Austin, John W; Peck, Michael W

    2009-01-01

    Background Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray. Results Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs) present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes), and the flagellar glycosylation island (FGI). These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5) has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI. Conclusion Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism outbreaks. PMID:19298644

  6. Proteolytic cleavage and shedding of the bovine prion protein in two cell culture systems.

    PubMed

    Zhao, Hongxing; Klingeborn, Mikael; Simonsson, Magnus; Linné, Tommy

    2006-01-01

    We have compared the processing, turnover and release of bovine PrP (boPrP) in transfected baby hamster kidney (BHK) and mouse neuroblastoma (N2a) cells. In BHK cells, boPrP was subjected to two distinct proteolytic cleavage events, the first was mapped between K(121) and H(122) generating an N-terminal and a C-terminal PrP fragment. Transport block experiments, cell surface biotinylation and PIPLC analyses showed that the bulk of boPrP on the cell surface was the C-terminal fragment and indicated that the first cleavage of boPrP took place prior to or very soon after it appears at the cell surface. The second cleavage was situated at the extreme C-terminus of the boPrP GPI-anchored C-terminal fragment and as a result of this was shed into the medium rapidly. The kinetics, the migration in SDS-PAGE of the released fragment and protease inhibition studies indicate that a proteolytic activity was responsible for the release of the boPrP fragment from its GPI-anchor. Both N- and C-terminal fragments of boPrP could be detected in the medium. Moreover, in normal bovine brain, a C-terminal fragment was identified, suggesting that similar proteolytic processing events occur in vivo. In N2a cells, the majority of boPrP was subjected to a more complete degradation process, and only trace amounts of full length boPrP was shed into cell culture medium in a process which also indicated a release by proteolytic cleavage. PMID:16140411

  7. Composition and proteolytic processing of corneal deposits associated with mutations in the TGFBI gene

    PubMed Central

    Karring, Henrik; Runager, Kasper; Thøgersen, Ida B.; Klintworth, Gordon K.; Højrup, Peter; Enghild, Jan J.

    2012-01-01

    Different types of granular corneal dystrophy (GCD)1 and lattice corneal dystrophy (LCD) are associated with mutations in the transforming growth factor beta induced gene (TGFBI). These dystrophies are characterized by the formation of non-amyloid granular deposits (GCDs) and amyloid (LCD type 1 and its variants) in the cornea. Typical corneal non-amyloid deposits from GCD type 2 (R124H), amyloid from a variant of LCD type 1 (V624M) and disease-free tissue controls were procured by laser capture microdissection and analyzed by tandem mass spectrometry. Label-free quantitative comparisons of deposits and controls suggested that the non-amyloid sample (R124H) specifically accumulated transforming growth factor beta induced protein (TGFBIp/keratoepithelin/βig-h3), serum amyloid P-component, clusterin, type III collagen, keratin 3, and histone H3-like protein. The amyloid (V624M) similarly accumulated serum amyloid P-component and clusterin but also a C-terminal fragment of TGFBIp containing residues Y571-R588 derived from the fourth fasciclin-1 domain (FAS1-4), apolipoprotein E and apolipoprotein A-IV. Significantly, analyses of the amyloid sample also revealed the presence of the serine protease Htr (High-temperature requirement) A1 and a number of proteolytic cleavage sites in the FAS1-4 domain of TGFBIp. These cleavage sites were consistent with the ligand binding and proteolytic activity of HtrA1 suggesting that it plays a role in the proteolytic processing of the amyloidogenic FAS1-4 domain. Taken together, the data suggest that the amyloidogenic-prone region of the fourth FAS1 domain of TGFBIp encompasses the Y571-R588 peptide and that HtrA1 is involved in the proteolytic processing of TGFBIp-derived amyloid in vivo. PMID:22155582

  8. [Activity of proteolytic and nucleolytic enzymes from the gonades of hydrobionts].

    PubMed

    Pozdniakova, Iu M; Pivnenko, T N; Epshteĭn, L M

    2004-01-01

    The activity of nucleolytic and proteolytic enzymes in milt of nine kinds of fishes belonging to various families and of three kinds invertebrates is determined. There is carried out electrophoreses division of preparations DNA, received from milts by various methods; there are determined structure and molecular weights of oligonucleotides. The influence of activity tissue enzymes on a destruction degree of DNA is established at addition enzymes of exogenic origin. PMID:19621738

  9. The Proteolytic Activity of Philibertia gilliesii Latex. Purification of Philibertain g II.

    PubMed

    Sequeiros, Cynthia; Torres, María J; Nievas, Marina L; Caffini, Néstor O; Natalucci, Claudia L; López, Laura M I; Trejo, Sebastián A

    2016-05-01

    The latex from the patagonic plant Philibertia gilliesii Hook. et Arn. (Apocynaceae) is a milky-white suspension containing a proteolytic system constituted by several cysteine endopeptidases. A proteolytic preparation (philibertain g) from the latex of P. gilliesii fruits was obtained and characterized to evaluate its potential use in bioprocesses. Philibertain g contained 1.2 g/L protein and a specific (caseinolytic) activity of 7.0 Ucas/mg protein. It reached 80 % of its maximum caseinolytic activity in the pH 7-10 range, retained 80 % of the original activity after 2 h of incubation at temperatures ranging from 25 to 45 °C and could be fully inactivated after 5 min at 75 °C. Philibertain g retained 60 % of the initial activity even at 1 M NaCl and was able to hydrolyze proteins from stickwater one, of the main waste effluents generated during fishmeal production. Furthermore, as a contribution to the knowledge of the proteolytic system of P. gilliesii, we are reporting the purification of a new peptidase, named philibertain g II (pI 9.4, molecular mass 23,977 Da, N-terminus LPESVDWREKGVVFPXRNQ) isolated from philibertain g through a purification scheme including acetone fractionation, cation exchange, molecular exclusion chromatography, and ultrafiltration. PMID:26852027

  10. On the agglutinogens of red cells developed with proteolytic enzymes and neuraminidase.

    PubMed

    Sagisaka, K; Takahashi, K

    1976-10-01

    It has been known that the agglutinability of human red cells is changed or enhanced by treatments with proteolytic enzymes or neuraminidase. In this paper, the serological properties of agglutinogens developed by proteolytic enzymes (bromelin, ficin, papain, trypsin and pronase) and neuraminidase are investigated by using antisera to trypsin- and neuraminidase-treated red cells. The adsorptions of the antiserum to trypsinized red cells with the cells treated with each of the proteolytic enzymes showed that the agglutinogens uncovered by bromelin, ficin and papain were different from those by pronase and trypsin. It was demonstrated that pronase was the most effective enzyme to uncover the agglutinogen located on deeper site of red cell membrane. This was confirmed by the agglutination with the test cells treated twice with two kinds of the enzymes. The reactions of the antiserum to neuraminidase-treated red cells treated with six kinds of the enzymes indicated that the agglutinogens developed by neuraminidase resembled those by bromelin, ficin and papain more than those by trypsin and pronase. PMID:982434

  11. Analyzing Protease Specificity and Detecting in Vivo Proteolytic Events Using Tandem Mass Spectrometry

    SciTech Connect

    Gupta, Nitin; Hixson, Kim K.; Culley, David E.; Smith, Richard D.; Pevzner, Pavel A.

    2010-07-01

    While trypsin remains the most commonly used protease in mass spectrometry, other proteases may be employed for increasing peptide-coverage or generating overlapping peptides. Knowledge of the accurate specifcity rules of these proteases is helpful for database search tools to detect peptides, and becomes crucial when mass spectrometry is used to discover in vivo proteolytic cleavages. In this study, we use tandem mass spectrometry to analyze the specifcity rules of selected proteases and describe MS- Proteolysis, a software tool for identifying putative sites of in vivo proteolytic cleavage. Our analysis suggests that the specifcity rules for some commonly used proteases can be improved, e.g., we find that V8 protease cuts not only after Asp and Glu, as currently expected, but also shows a smaller propensity to cleave after Gly for the conditions tested in this study. Finally, we show that comparative analysis of multiple proteases can be used to detect putative in vivo proteolytic sites on a proteome-wide scale.

  12. Leucoagglutinating phytohemagglutinin: purification, characterization, proteolytic digestion and assessment for allergenicity potential in BALB/c mice.

    PubMed

    Kumar, Sandeep; Sharma, Akanksha; Das, Mukul; Jain, S K; Dwivedi, Premendra D

    2014-04-01

    Red kidney bean (Phaseolus vulgaris) is consumed worldwide as a vegetarian protein source. But, at the same time the allergenicity potential of red kidney bean is a matter of concern. This study is aimed towards purification, characterization, thermal stability, proteolytic digestion and allergenicity assessment of one of the clinically relevant allergens of red kidney bean. The purification of red kidney bean allergic protein was carried out with the help of column chromatography, IgE immunoblotting and reverse phase high-pressure liquid chromatography (RP-HPLC). The purified protein was characterized by peptide mass finger printing (PMF) and studied for its thermal stability, and proteolytic resistance using simulated gastric fluid (SGF) assay. The allergenicity potential of the purified protein was studied in BALB/c mice. The purified protein was identified as leucoagglutinating phytohemagglutinin (PHA-L) with molecular weight 29.5 kDa. The PHA-L showed resistance to heat as well as proteolytic enzyme. Higher levels of total IgE, specific IgE, and histamine were observed in PHA-L treated BALB/c mice when compared to control. Overall, PHA-L possesses characteristics of allergens and may play a potential role in the red kidney bean induced allergy. PMID:24548135

  13. Use of proteolytic enzymes as an additional tool for trypanosomatid identification.

    PubMed

    Santos, A L S; Abreu, C M; Alviano, C S; Soares, R M A

    2005-01-01

    The expression of proteolytic activities in the Trypanosomatidae family was explored as a potential marker to discriminate between the morphologically indistinguishable flagellates isolated from insects and plants. We have comparatively analysed the proteolytic profiles of 19 monoxenous trypanosomatids (Herpetomonas anglusteri, H. samuelpessoai, H. mariadeanei, H. roitmani, H. muscarum ingenoplastis, H. muscarum muscarum, H. megaseliae, H. dendoderi, Herpetomoas sp., Crithidia oncopelti, C. deanei, C. acanthocephali, C. harmosa, C. fasciculata, C. guilhermei, C. luciliae, Blastocrithidia culicis, Leptomonas samueli and Lept. seymouri) and 4 heteroxenous flagellates (Phytomonas serpens, P. mcgheei, Trypanosoma cruzi and Leishmania amazonensis) by in situ detection of enzyme activities on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE ) containing co-polymerized gelatine as substrate, in association with specific proteinase inhibitors. All 23 trypanosomatids expressed at least 1 acidic proteolytic enzyme. In addition, a characteristic and specific pattern of cell-associated metallo and/or cysteine proteinases was observed, except for the similar profiles detected in 2 Herpetomonas (H. anglusteri and H. samuelpessoai) and 3 Crithidia (C. fasciculata, C. guilhermei and C. luciliae) species. However, these flagellates released distinct secretory proteinase profiles into the extracellular medium. These findings strongly suggest that the association of cellular and secretory proteinase pattern could represent a useful marker to help trypanosomatid identification. PMID:15700759

  14. The genetic and molecular bases of monogenic disorders affecting proteolytic systems

    PubMed Central

    Richard, I

    2005-01-01

    Complete and limited proteolysis represents key events that regulate many biological processes. At least 5% of the human genome codes for components of proteolytic processes if proteases, inhibitors, and cofactors are taken into account. Accordingly, disruption of proteolysis is involved in numerous pathological conditions. In particular, molecular genetic studies have identified a growing number of monogenic disorders caused by mutations in protease coding genes, highlighting the importance of this class of enzymes in development, organogenesis, immunity, and brain function. This review provides insights into the current knowledge about the molecular genetic causes of these disorders. It should be noted that most are due to loss of function mutations, indicating absolute requirement of proteolytic activities for normal cellular functions. Recent progress in understanding the function of the implicated proteins and the disease pathogenesis is detailed. In addition to providing important clues to the diagnosis, treatment, and pathophysiology of disease, functional characterisation of mutations in proteolytic systems emphasises the pleiotropic functions of proteases in the body homeostasis. PMID:15994873

  15. Curcumin cross-linked collagen aerogels with controlled anti-proteolytic and pro-angiogenic efficacy.

    PubMed

    Dharunya, G; Duraipandy, N; Lakra, Rachita; Korapatti, Purna Sai; Jayavel, R; Kiran, Manikantan Syamala

    2016-01-01

    This paper elucidates the development of a curcumin cross-linked collagen aerogel system with controlled anti-proteolytic activity and pro-angiogenic efficacy. The results of this study showed that in situ cross-linking of curcumin with collagen leads to the development of aerogels with enhanced physical and mechanical properties. The integrity of collagen after cross-linking with curcumin was studied via FTIR spectroscopy. The results confirmed that the cross-linking with curcumin did not induce any structural changes in the collagen. The curcumin cross-linked collagen aerogels exhibited potent anti-proteolytic and anti-microbial activity. Scanning electron and atomic force microscopic analysis of curcumin cross-linked collagen aerogels showed a 3D microstructure that enhanced the adhesion and proliferation of cells. The highly organized geometry of collagen-curcumin aerogels enhanced the permeability and water-retaining ability required for the diffusion of nutrients that aid cellular growth. The pro-angiogenic properties of collagen-curcumin aerogels were ascribed to the cumulative effect of the nutraceutical and the collagen molecule, which augmented the restoration of damaged tissue. Further, these aerogels exhibited controlled anti-proteolytic activity, which makes them suitable 3D scaffolds for biomedical applications. This study provides scope for the development of biocompatible and bioresorbable collagen aerogel systems that use a nutraceutical as a cross-linker for biomedical applications. PMID:27509047

  16. A potent reporter applicable to the monitoring of caspase-3-dependent proteolytic cleavage.

    PubMed

    Park, Kyoungsook; Kang, Hyo-Jin; Ahn, Junhyoung; Yi, So Yeon; Han, Sang Hee; Park, Hye-Jung; Chung, Sang J; Chung, Bong Hyun; Kim, Moonil

    2008-11-01

    In this study, we developed a chimeric caspase-3 substrate (GST:DEVD:EGFP) comprised of glutathione-S transferase (GST) and enhanced green fluorescent protein (EGFP) with a specialized linker peptide harboring the caspase-3 cleavage sequence, DEVD. Using this reporter, we assessed the proteolytic cleavage of the artificial caspase-3 substrate for caspase-3. The common feature of this approach is that the presence of the DEVD sequence between GST and EGFP allows for caspase-3-dependent cleavage after the Asp (D) residue, resulting in the elimination of EGFP from the GST:DEVD:EGFP reporter. To the best of our knowledge, this study reports the first application employing a chimeric protein substrate, with the similar accuracy level compared to the conventional methods such as fluorometric assays. As a result, using this GST:DEVD:EGFP reporter, caspase-3 activation based on proteolytic properties could be monitored via a variety of bioanalytical techniques such as immunoblot analysis, glutathione-agarose bead assay, and on-chip visualization, providing both technical and economical advantages over the extensively utilized fluorogenic peptide assay. Our results convincingly showed that this versatile reporter (GST:DEVD:EGFP) constitutes a useful system for the monitoring of caspase-3 activation, potentially enabling the monitoring of the proteolytic activities of different intra-cellular proteases via the substitution of the cleavage sequence within the same schematic construct. PMID:18775457

  17. Longterm persistence of proteolytic activities in frass of Blattella germanica increases its allergenic potential.

    PubMed

    Erban, T; Hubert, J

    2011-06-01

    Chromogenic microplate assays in 96 wells were used to determine the stability of enzyme activity in frass of Blattella germanica (Blattodea: Blattellidae). Frass samples were exposed to controlled conditions [temperature 15-35 °C and/or 53-100% relative humidity (RH)] and to household conditions (apartment). Exposure times were 0 (control), 90, 183 and 276 days. Starch digestion and cellulolytic activities decreased during exposure. Non-specific proteolytic activities were affected by changes in selective proteolytic activities. Activities towards AAPpNA and SA(3) pNA strongly increased at 100% RH, indicating the possible influence of microorganisms growing on frass. Activities towards BApNA and ArgpNA decreased with increasing decomposition time, whereas activity towards ZRRpNA was not influenced by exposure time. The largest decrease in activities towards ArgpNA and BApNA occurred at temperatures of 15 °C, 30 °C and 35 °C and at 100% RH. Activities towards BApNA and ZRRpNA were very stable under different temperature and RH conditions; this was confirmed by findings showing that these activities were stable in the experimental apartment. In comparison with the control, activities towards ZRRpNA and BApNA after 276 days decreased by 1% and 19%, respectively. The longterm persistence of proteolytic activities in cockroach frass increases their allergenic hazard potential. PMID:21198710

  18. Effects of Pleurotus eryngii polysaccharides on bacterial growth, texture properties, proteolytic capacity, and angiotensin-I-converting enzyme-inhibitory activities of fermented milk.

    PubMed

    Li, Siqian; Shah, Nagendra P

    2015-05-01

    Pleurotus eryngii is one of the most favored oyster mushrooms and contains various beneficial bioactive compounds. Polysaccharide extracted from P. eryngii (PEPS) was added as a natural-source ingredient to milk before fermentation, and the effects of additional PEPS on fermented milk were investigated in this study. The PEPS were extracted and added to reconstituted skim milk (12%, wt/vol) at 0.5, 0.25, and 0.125% (wt/vol) and fermented by a non-exopolysaccharide-producing strain, Streptococcus thermophilus Australian Starter Culture Collection (ASCC) 1303 (ST 1303), or an exopolysaccharide-producing Strep. thermophilus ASCC 1275 (ST 1275). Bacterial growth, texture properties, microstructure, proteolytic capacity, and angiotensin-I-converting enzyme-inhibitory activities of fermented milk (FM) were determined during refrigerated storage at 4°C for 21d. Viable counts of starter bacteria in FM with 0.5% PEPS added were the highest. Changes in pH were consistent with changes in titratable acidities for all samples. The FM samples with added PEPS showed denser protein aggregates containing larger serum pores in confocal micrographs compared with those without PEPS at d 0 and 21during refrigerated storage. The values for spontaneous whey separation of FM with added PEPS were significantly higher than those of FM fermented by ST 1303 or ST 1275 without PEPS. The proteolytic activities of ST 1303 of FM with added PEPS were higher than those of FM fermented by ST 1303 without PEPS. The FM with added 0.125% PEPS had similar angiotensin-I-converting enzyme-inhibitory activity to that fermented by ST 1303 without PEPS; both were higher than those of other samples during refrigerated storage. Firmness and gumminess values of FM with added PEPS were higher than those of FM fermented by ST 1303 or ST 1275 without PEPS. PMID:25747830

  19. Unmasking Proteolytic Activity for Adult Visual Cortex Plasticity by the Removal of Lynx1

    PubMed Central

    Bukhari, Noreen; Burman, Poromendro N.; Hussein, Ayan; Demars, Michael P.; Sadahiro, Masato; Brady, Daniel M.; Tsirka, Stella E.; Russo, Scott J.

    2015-01-01

    Experience-dependent cortical plasticity declines with age. At the molecular level, experience-dependent proteolytic activity of tissue plasminogen activator (tPA) becomes restricted in the adult brain if mice are raised in standard cages. Understanding the mechanism for the loss of permissive proteolytic activity is therefore a key link for improving function in adult brains. Using the mouse primary visual cortex (V1) as a model, we demonstrate that tPA activity in V1 can be unmasked following 4 d of monocular deprivation when the mice older than 2 months are raised in standard cages by the genetic removal of Lynx1, a negative regulator of adult plasticity. This was also associated with the reduction of stubby and thin spine density and enhancement of ocular dominance shift in adult V1 of Lynx1 knock-out (KO) mice. These structural and functional changes were tPA-dependent because genetic removal of tPA in Lynx1 KO mice can block the monocular deprivation-dependent reduction of dendritic spine density, whereas both genetic and adult specific inhibition of tPA activity can ablate the ocular dominance shift in Lynx1 KO mice. Our work demonstrates that the adult brain has an intrinsic potential for experience-dependent elevation of proteolytic activity to express juvenile-like structural and functional changes but is effectively limited by Lynx1 if mice are raised in standard cages. Insights into the Lynx1-tPA plasticity mechanism may provide novel therapeutic targets for adult brain disorders. SIGNIFICANCE STATEMENT Experience-dependent proteolytic activity of tissue plasminogen activator (tPA) becomes restricted in the adult brain in correlation with the decline in cortical plasticity when mice are raised in standard cages. We demonstrated that removal of Lynx1, one of negative regulators of plasticity, unmasks experience-dependent tPA elevation in visual cortex of adult mice reared in standard cages. This proteolytic elevation facilitated dendritic spine reduction

  20. Evidence that the glucoamylases and alpha-amylase secreted by Aspergillus niger are proteolytically processed products of a precursor enzyme.

    PubMed

    Dubey, A K; Suresh, C; Kavitha, R; Karanth, N G; Umesh-Kumar, S

    2000-04-14

    A 125-kDa starch hydrolysing enzyme of Aspergillus niger characterised by its ability to dextrinise and saccharify starch [Suresh et al. (1999) Appl. Microbiol. Biotechnol. 51, 673-675] was also found to possess activity towards raw starch. Segregation of these activities in the 71-kDa glucoamylase and a 53-kDa alpha-amylase-like enzyme supported by antibody cross-reactivity studies and the isolation of mutants based on assay screens for the secretion of particular enzyme forms revealed the 125-kDa starch hydrolysing enzyme as their precursor. N-terminal sequence analysis further revealed that the 71-kDa glucoamylase was the N-terminal product of the precursor enzyme. Immunological cross reactivity of the 53-kDa amylase with antibodies raised against the precursor enzyme but not with the 71- and 61-kDa glucoamylase antibodies suggested that this enzyme activity is represented by the C-terminal fragment of the precursor. The N-terminal sequence of the 53-kDa protein showed similarity to the reported Taka amylase of Aspergillus oryzae. Antibody cross-reactivity to a 10-kDa non-enzymic peptide and a 61-kDa glucoamylase described these proteins as products of the 71-kDa glucoamylase. Identification of only the precursor starch hydrolysing enzyme in the protein extracts of fungal protoplasts suggested proteolytic processing in the cellular periplasmic space as the cause for the secretion of multiple forms of amylases by A. niger. PMID:10767433

  1. Comparison of proteolytic activity of Candida sp. strains depending on their origin.

    PubMed

    Modrzewska, B; Kurnatowski, P; Khalid, K

    2016-06-01

    The aim of the research was to evaluate the proteolytic activity of various Candida strains isolated from the oral cavity of persons without clinical symptoms of fungal infection, outpatients with oral cavity disorders and patients hospitalized due to head and neck tumors. A secondary aim was to confirm the presence of secreted aspartyl protease (SAP) genes in the isolated strains and then to compare it depending on the fungal species. Material consisted of 134 fungal strains that were analysed by a modified Staib method and polymerase chain reaction (PCR) with the use of specific primer pairs. The greatest proteolytic activity of fungi was observed at pH 3.5. The proteolysis were the strongest for strains isolated from dental patients and the weakest from persons without changes in the oral cavity. In total, 61.9% of the strains exhibited the presence of at least one of the SAP1-3 genes in all examined groups, SAP1 being the most common; SAP4-6 genes were not observed. All genes were more frequent in the strains isolated from the dental patients than from other groups. SAP1-3 genes were present in Candida albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. humicola and C. lipolytica, but were not noted in other isolated species. The lowest activity of proteolytic enzymes and the least number of aspartyl protease genes are observed among strains isolated from patients without clinical symptoms of mycosis. SAP1-3 genes are most frequently detected in the strains isolated from the oral cavity; their presence varies depending on the species of the fungi. PMID:26922385

  2. Complex Negative Regulation of TLR9 by Multiple Proteolytic Cleavage Events.

    PubMed

    Sinha, Siddhartha S; Cameron, Jody; Brooks, James C; Leifer, Cynthia A

    2016-08-15

    TLR9 is an innate immune receptor important for recognizing DNA of host and foreign origin. A mechanism proposed to prevent excessive response to host DNA is the requirement for proteolytic cleavage of TLR9 in endosomes to generate a mature form of the receptor (TLR9(471-1032)). We previously described another cleavage event in the juxtamembrane region of the ectodomain that generated a dominant-negative form of TLR9. Thus, there are at least two independent cleavage events that regulate TLR9. In this study, we investigated whether an N-terminal fragment of TLR9 could be responsible for regulation of the mature or negative-regulatory form. We show that TLR9(471-1032), corresponding to the proteolytically cleaved form, does not function on its own. Furthermore, activity is not rescued by coexpression of the N-terminal fragment (TLR9(1-440)), inclusion of the hinge region (TLR9(441-1032)), or overexpression of UNC93B1, the last of which is critical for trafficking and cleavage of TLR9. TLR9(1-440) coimmunoprecipitates with full-length TLR9 and TLR9(471-1032) but does not rescue the native glycosylation pattern; thus, inappropriate trafficking likely explains why TLR9(471-1032) is nonfunctional. Lastly, we show that TLR9(471-1032) is also a dominant-negative regulator of TLR9 signaling. Together, these data provide a new perspective on the complexity of TLR9 regulation by proteolytic cleavage and offer potential ways to inhibit activity through this receptor, which may dampen autoimmune inflammation. PMID:27421483

  3. Neutrophil proteolytic activation cascades: a possible mechanistic link between chronic periodontitis and coronary heart disease.

    PubMed

    Alfakry, Hatem; Malle, Ernst; Koyani, Chintan N; Pussinen, Pirkko J; Sorsa, Timo

    2016-01-01

    Cardiovascular diseases are chronic inflammatory diseases that affect a large segment of society. Coronary heart disease (CHD), the most common cardiovascular disease, progresses over several years and affects millions of people worldwide. Chronic infections may contribute to the systemic inflammation and enhance the risk for CHD. Periodontitis is one of the most common chronic infections that affects up to 50% of the adult population. Under inflammatory conditions the activation of endogenous degradation pathways mediated by immune responses leads to the release of destructive cellular molecules from both resident and immigrant cells. Matrix metalloproteinases (MMPs) and their regulators can activate each other and play an important role in immune response via degrading extracellular matrix components and modulating cytokines and chemokines. The action of MMPs is required for immigrant cell recruitment at the site of inflammation. Stimulated neutrophils represent the major pathogen-fighting immune cells that upregulate expression of several proteinases and oxidative enzymes, which can degrade extracellular matrix components (e.g. MMP-8, MMP-9 and neutrophil elastase). The activity of MMPs is regulated by endogenous inhibitors and/or candidate MMPs (e.g. MMP-7). The balance between MMPs and their inhibitors is thought to mirror the proteolytic burden. Thus, neutrophil-derived biomarkers, including myeloperoxidase, may activate proteolytic destructive cascades that are involved in subsequent immune-pathological events associated with both periodontitis and CHD. Here, we review the existing studies on the contribution of MMPs and their regulators to the infection-related pathology. Also, we discuss the possible proteolytic involvement and role of neutrophil-derived enzymes as an etiological link between chronic periodontitis and CHD. PMID:26608308

  4. Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B.

    PubMed

    Fan, Yongfeng; Dong, Jianbo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Geren, Isin N; Garcia-Rodriguez, Consuelo; Smith, Theresa J; Smith, Leonard A; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A; Marks, James D

    2015-09-01

    Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors. PMID:26343720

  5. Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B

    PubMed Central

    Fan, Yongfeng; Dong, Jianbo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Geren, Isin N.; Garcia-Rodriguez, Consuelo; Smith, Theresa J.; Smith, Leonard A.; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A.; Marks, James D.

    2015-01-01

    Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors. PMID:26343720

  6. Interaction of Leptospira interrogans with Human Proteolytic Systems Enhances Dissemination through Endothelial Cells and Protease Levels

    PubMed Central

    Vieira, Monica L.; Alvarez-Flores, Miryam P.; Kirchgatter, Karin; Romero, Eliete C.; Alves, Ivy J.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Chudzinski-Tavassi, Ana M.

    2013-01-01

    We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date. PMID:23478319

  7. Modulation of bovine microvascular endothelial cell proteolytic properties by inhibitors of angiogenesis.

    PubMed

    Pepper, M S; Vassalli, J D; Wilks, J W; Schweigerer, L; Orci, L; Montesano, R

    1994-08-01

    A tightly controlled increase in extracellular proteolysis, restricted both in time and space, is an important component of the angiogenic process, while anti-proteolysis is effective in inhibiting angiogenesis. By focussing on the plasminogen activator (PA)-plasmin system, the objective of the present studies was to assess whether previously described inhibitors of angiogenesis modify bovine microvascular endothelial cell proteolytic properties. We demonstrate that although synthetic angiostatic steroids (U-24067 and U-42129), heparin, suramin, interferon alpha-2a, and retinoic acid are all inhibitors of in vitro angiogenesis, each of these agents has distinct effects on the plasminogen-dependent proteolytic system. Specifically, angiostatic steroids and interferon alpha-2a reduce urokinase-type PA (u-PA) and PA inhibitor-1 activity, while heparin and retinoic acid increase u-PA activity. Suramin reduces cell-associated u-PA activity and greatly increases PAI-1 production at doses which induce monolayer disruption. These findings demonstrate that a spectrum of alterations in extracellular proteolysis is associated with anti-angiogenesis, and that anti-angiogenesis and anti-proteolysis are not necessarily correlated. A reduction in extracellular proteolysis would be expected to reduce invasion, whereas an increase in proteolysis might modulate the activity of inhibitory cytokines, which in turn could reduce endothelial cell proliferation and migration and inhibit angiogenesis. The spectrum of effects on different elements of the PA system observed in response to the agents assessed suggests that the role of modulations in extracellular proteolytic activity in anti-angiogenesis is likely to be varied and complex. PMID:7525617

  8. Sensitive microplate assay for the detection of proteolytic enzymes using radiolabeled gelatin

    SciTech Connect

    Robertson, B.D.; Kwan-Lim, G.E.; Maizels, R.M.

    1988-07-01

    A sensitive, microplate assay is described for the detection of a wide range of proteolytic enzymes, using radio-iodine-labeled gelatin as substrate. The technique uses the Bolton-Hunter reagent to label the substrate, which is then coated onto the wells of polyvinyl chloride microtiter plates. By measuring the radioactivity released the assay is able to detect elastase, trypsin, and collagenase in concentrations of 1 ng/ml or less, while the microtiter format permits multiple sample handling and minimizes sample volumes required for analysis.

  9. Colon tumour secretopeptidome: insights into endogenous proteolytic cleavage events in the colon tumour microenvironment.

    PubMed

    Greening, David W; Kapp, Eugene A; Ji, Hong; Speed, Terry P; Simpson, Richard J

    2013-11-01

    The secretopeptidome comprises endogenous peptides derived from proteins secreted into the tumour microenvironment through classical and non-classical secretion. This study characterised the low-Mr (<3kDa) component of the human colon tumour (LIM1215, LIM1863) secretopeptidome, as a first step towards gaining insights into extracellular proteolytic cleavage events in the tumour microenvironment. Based on two biological replicates, this secretopeptidome isolation strategy utilised differential centrifugal ultrafiltration in combination with analytical RP-HPLC and nanoLC-MS/MS. Secreted peptides were identified using a combination of Mascot and post-processing analyses including MSPro re-scoring, extended feature sets and Percolator, resulting in 474 protein identifications from 1228 peptides (≤1% q-value, ≤5% PEP) - a 36% increase in peptide identifications when compared with conventional Mascot (homology ionscore thresholding). In both colon tumour models, 122 identified peptides were derived from 41 cell surface protein ectodomains, 23 peptides (12 proteins) from regulated intramembrane proteolysis (RIP), and 12 peptides (9 proteins) generated from intracellular domain proteolysis. Further analyses using the protease/substrate database MEROPS, (http://merops.sanger.ac.uk/), revealed 335 (71%) proteins classified as originating from classical/non-classical secretion, or the cell membrane. Of these, peptides were identified from 42 substrates in MEROPS with defined protease cleavage sites, while peptides generated from a further 205 substrates were fragmented by hitherto unknown proteases. A salient finding was the identification of peptides from 88 classical/non-classical secreted substrates in MEROPS, implicated in tumour progression and angiogenesis (FGFBP1, PLXDC2), cell-cell recognition and signalling (DDR1, GPA33), and tumour invasiveness and metastasis (MACC1, SMAGP); the nature of the proteases responsible for these proteolytic events is unknown. To

  10. A radiolabel-release microwell assay for proteolytic enzymes present in cell culture media

    SciTech Connect

    Rucklidge, G.J.; Milne, G. )

    1990-03-01

    A modified method for the measurement of proteolytic enzyme activity in cell culture-conditioned media has been developed. Using the release of 3H-labeled peptides from 3H-labeled gelatin the method is performed in microwell plates. The substrate is insolubilized and attached to the wells by glutaraldehyde treatment, thus eliminating the need for a precipitation step at the end of the assay. The assay is sensitive, reproducible, and convenient for small sample volumes. The effect of different protease inhibitors on activity can be assessed rapidly allowing an early characterization of the enzyme. It can also be adapted to microplate spectrophotometric analysis by staining residual substrate with Coomassie blue.

  11. Proteolytic yeasts isolated from raw, ripe tomatoes and metabiotic association of Geotrichum candidum with Salmonella.

    PubMed

    Wade, Wendy N; Vasdinnyei, Rita; Deak, Tibor; Beuchat, Larry R

    2003-09-01

    Metabiotic associations between food-borne fungi and bacteria capable of causing human diseases are a public health concern. A survey of decayed and damaged, uncooked, ripe tomatoes was done to determine the presence and prevalence of yeasts capable of increasing the pH pulp tissue, thus creating a more favorable environment for survival and growth of enteric pathogens. Sixty-two of the 371 (16.7%) fungi isolated from 215 decayed or damaged tomatoes; 12 of the 62 (19.4%) yeasts showed proteolytic activity on gelatin agar (GA) and/or standard methods caseinate (SMC) agar. The pH of tomato pericarp (pulp) tissue from which 9 of the 12 yeasts were isolated ranged from 4.3 to 7.5 (mean=5.3) compared to 4.2-5.1 (mean=4.8) for sound pulp tissue in the same tomatoes. The 12 proteolytic yeasts consisted of four strains of Cryptococcus albidus, two strains each of Debaryomyces hansenii and Trichosporon pullulans, and one strain each of Cryptococcus humicolus, Cryptococcus laurentii, Geotrichum candidum, and Sporidiobolus pararoseus. Survival and growth characteristics of a five-serotype mixture of Salmonella co-inoculated with G. candidum into sound (not chill injured) and chill-injured tomatoes were studied. Storage of sound tomatoes at 15 degrees C for 10 days resulted in an increase in population of 7.6 log(10) cfu of Salmonella/g of a 2-g sample of co-infected pulp tissue. Increases were less in tissue inoculated with Salmonella only, Salmonella on day 0 followed by G. candidum on day 3, or G. candidum on day 0 followed by Salmonella on day 3. Trends were similar in sound inoculated tomatoes stored at 25 degrees C. Growth of Salmonella was enhanced in chill-injured tomatoes compared to sound tomatoes; a population of 10 log(10) cfu/g of chill-injured pulp tissue was reached within 10 days at 25 degrees C. Results clearly show that growth of a proteolytic, alkalinizing yeast such as G. candidum in raw tomatoes enhances conditions for growth of Salmonella. The removal of

  12. Functionalized Biopolymer Particles Enhance Performance of a Tissue-Protective Peptide under Proteolytic and Thermal Stress.

    PubMed

    Dooley, Kevin; Devalliere, Julie; Uygun, Basak E; Yarmush, Martin L

    2016-06-13

    Cutaneous burns are often exacerbated by poor perfusion and subsequent necrosis of the microvasculature surrounding the primary injury. Preservation of these vessels can reduce necrotic tissue expansion and increase success rates of skin graft procedures. Recent work has identified a peptide derived from erythropoietin, ARA290, with the ability to mediate tissue protection in a variety of cell types. Here we demonstrate the advantages of fusing ARA290 to an elastin-like polypeptide (ELP) to salvage microvascular endothelial cells in harsh proteolytic conditions following thermal shock. These fusion proteins were expressed recombinantly in bacterial hosts and rapidly purified by inverse transition cycling. They were shown to spontaneously aggregate into particles at subphysiological temperatures. The bifunctional submicron particles were resistant to digestion in enzymes upregulated after burn injury. Furthermore, the data strongly suggest these ARA290-functionalized particles were superior to treatment with the peptide alone in preventing microvascular cell death in these conditions. The results bring to light an efficient and cost-effective strategy for the delivery therapeutic peptides to proteolytically active wound sites. PMID:27219509

  13. Biochemical and Functional Characterization of Parawixia bistriata Spider Venom with Potential Proteolytic and Larvicidal Activities

    PubMed Central

    Gimenez, Gizeli S.; Coutinho-Neto, Antonio; Kayano, Anderson M.; Simões-Silva, Rodrigo; Trindade, Frances; de Almeida e Silva, Alexandre; Marcussi, Silvana; da Silva, Saulo L.; Fernandes, Carla F. C.; Zuliani, Juliana P.; Calderon, Leonardo A.; Soares, Andreimar M.; Stábeli, Rodrigo G.

    2014-01-01

    Toxins purified from the venom of spiders have high potential to be studied pharmacologically and biochemically. These biomolecules may have biotechnological and therapeutic applications. This study aimed to evaluate the protein content of Parawixia bistriata venom and functionally characterize its proteins that have potential for biotechnological applications. The crude venom showed no phospholipase, hemorrhagic, or anti-Leishmania activities attesting to low genotoxicity and discrete antifungal activity for C. albicans. However the following activities were observed: anticoagulation, edema, myotoxicity and proteolysis on casein, azo-collagen, and fibrinogen. The chromatographic and electrophoretic profiles of the proteins revealed a predominance of acidic, neutral, and polar proteins, highlighting the presence of proteins with high molecular masses. Five fractions were collected using cation exchange chromatography, with the P4 fraction standing out as that of the highest purity. All fractions showed proteolytic activity. The crude venom and fractions P1, P2, and P3 showed larvicidal effects on A. aegypti. Fraction P4 showed the presence of a possible metalloprotease (60 kDa) that has high proteolytic activity on azo-collagen and was inhibited by EDTA. The results presented in this study demonstrate the presence of proteins in the venom of P. bistriata with potential for biotechnological applications. PMID:24895632

  14. CleavPredict: A Platform for Reasoning about Matrix Metalloproteinases Proteolytic Events

    PubMed Central

    Kumar, Sonu; Ratnikov, Boris I.; Kazanov, Marat D.; Smith, Jeffrey W.; Cieplak, Piotr

    2015-01-01

    CleavPredict (http://cleavpredict.sanfordburnham.org) is a Web server for substrate cleavage prediction for matrix metalloproteinases (MMPs). It is intended as a computational platform aiding the scientific community in reasoning about proteolytic events. CleavPredict offers in silico prediction of cleavage sites specific for 11 human MMPs. The prediction method employs the MMP specific position weight matrices (PWMs) derived from statistical analysis of high-throughput phage display experimental results. To augment the substrate cleavage prediction process, CleavPredict provides information about the structural features of potential cleavage sites that influence proteolysis. These include: secondary structure, disordered regions, transmembrane domains, and solvent accessibility. The server also provides information about subcellular location, co-localization, and co-expression of proteinase and potential substrates, along with experimentally determined positions of single nucleotide polymorphism (SNP), and posttranslational modification (PTM) sites in substrates. All this information will provide the user with perspectives in reasoning about proteolytic events. CleavPredict is freely accessible, and there is no login required. PMID:25996941

  15. Interaction specificity between the chaperone and proteolytic components of the cyanobacterial Clp protease.

    PubMed

    Tryggvesson, Anders; Ståhlberg, Frida M; Mogk, Axel; Zeth, Kornelius; Clarke, Adrian K

    2012-09-01

    The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC-ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity. PMID:22657732

  16. Effects of Ghrelin on the Proteolytic Pathways of Alzheimer's Disease Neuronal Cells.

    PubMed

    Cecarini, Valentina; Bonfili, Laura; Cuccioloni, Massimiliano; Keller, Jeffrey N; Bruce-Keller, Annadora J; Eleuteri, Anna Maria

    2016-07-01

    Ghrelin is an orexigenic hormone with a role in the onset and progression of neurodegenerative disorders. It has been recently associated to Alzheimer's disease (AD) for its neuroprotective and anti-apoptotic activity. In the present study, we dissected the effect of ghrelin treatment on the two major intracellular proteolytic pathways, the ubiquitin-proteasome system (UPS) and autophagy, in cellular models of AD (namely SH-SY5Y neuroblastoma cells stably transfected with either the wild-type AβPP gene or the 717 valine-to-glycine AβPP-mutated gene). Ghrelin showed a growth-promoting effect on neuronal cells inducing also time-dependent modifications of the growth hormone secretagogue receptor type 1 (GHS-R1) expression. Interestingly, we demonstrated for the first time that ghrelin was able to activate the proteasome in neural cells playing also a role in the interplay between the UPS and autophagy. Our data provide a novel mechanism by which circulating hormones control neural homeostasis through the regulation of proteolytic pathways implicated in AD. PMID:26033219

  17. Investigation of plant latices of Asteraceae and Campanulaceae regarding proteolytic activity.

    PubMed

    Sytwala, Sonja; Domsalla, André; Melzig, Matthias F

    2015-12-01

    Occurrence of plant latices is widespread, there are more than 40 families of plants characterized to establish lactiferous structures. The appearance of hydrolytic active proteins, incorporated in latices is already characterized, and hydrolytic active proteins are considerable, and for several plant families, the occurrence of hydrolytic active proteins is already specified e.g. Apocynaceae Juss., Caricaceae Dumort, Euphorbiaceae Juss., Moraceae Gaudich and Papaveraceae Juss. In our investigation, focused on latex bearing plants of order Asterales, Asteraceae and Campanulaceae in particular. The present outcomes represent a comprehensive study, relating to the occurrence of proteolytic active enzymes of order Asterales for the first time. 131 different species of Asteraceae and Campanulaceae were tested, and the appearance of plant latex proteases were determined in different quantities. Proteolytic activity was investigated by inhibitory studies and determination of residual activity in the following, enable us to characterize the proteases. Most of the considered species exhibit a serine protease activity and a multiplicity of species exhibited two or more subclasses of proteases. PMID:26458257

  18. Structural identification and proteolytic effects of the hatching enzyme from starfish Asterias amurensis.

    PubMed

    Li, Zhi Jiang; Kim, Sang Moo

    2014-07-01

    Hatching enzyme (HE) is secreted from the blastula stage during fertilization and can cleave the egg membrane. The structural identification and proteolytic effects on the collagen and fibrinogen were investigated in this study. Approximate 20 kDa of Asn-linked oligosaccharides were attached to the HE. Five peptide fragments of the starfish HE were homogenous to those of the coat matrix protein of starfish Patiria pectinifera. Amino acids of the starfish HE consisted of mainly Leu (10.0%), Asp (12.5%), and Glu (12.8%). Collagenolytic and fibrinolytic activities of the starfish HE were weaker than those of collagenase and α-chymotrypsin. The degree values of hydrolysis for collagenase and α- chymotrypsin were significantly higher than those of HE in a dose- and time-dependent manner. The peptide mappings of the starfish HE on the collagenolysis (110.7, 84.7, and 20.8 kDa) and fibrinogenolysis (34, 30, and 29 kDa) were different from those of collagenase and α-chymotrypsin. Based on the proteolytic effects on the collagen and fibrinogen, the starfish hatching enzyme might have the potential application to remove the matrix composition in scar or keloid tissue. PMID:24559163

  19. Histone H3.3 and its proteolytically processed form drive a cellular senescence program

    PubMed Central

    Duarte, Luis F.; Young, Andrew R. J.; Wang, Zichen; Wu, Hsan-Au; Panda, Taniya; Kou, Yan; Kapoor, Avnish; Hasson, Dan; Mills, Nicholas R.; Ma’ayan, Avi; Narita, Masashi; Bernstein, Emily

    2014-01-01

    The process of cellular senescence generates a repressive chromatin environment, however, the role of histone variants and histone proteolytic cleavage in senescence remains unclear. Using models of oncogene-induced and replicative senescence, here we report novel histone H3 tail cleavage events mediated by the protease Cathepsin L. We find that cleaved forms of H3 are nucleosomal and the histone variant H3.3 is the preferred cleaved form of H3. Ectopic expression of H3.3 and its cleavage product (H3.3cs1), which lacks the first twenty-one amino acids of the H3 tail, is sufficient to induce senescence. Further, H3.3cs1 chromatin incorporation is mediated by the HUCA histone chaperone complex. Genome-wide transcriptional profiling revealed that H3.3cs1 facilitates transcriptional silencing of cell cycle regulators including RB/E2F target genes, likely via the permanent removal of H3K4me3. Collectively, our study identifies histone H3.3 and its proteolytically processed forms as key regulators of cellular senescence. PMID:25394905

  20. Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw.

    PubMed

    Orlandelli, Ravely Casarotti; de Almeida, Tiago Tognolli; Alberto, Raiani Nascimento; Polonio, Julio Cesar; Azevedo, João Lúcio; Pamphile, João Alencar

    2015-06-01

    Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes. PMID:26273250

  1. Proteolytic processing of Alzheimer’s β-amyloid precursor protein

    PubMed Central

    Zhang, Han; Ma, Qilin; Zhang, Yun-wu; Xu, Huaxi

    2011-01-01

    β–amyloid precursor protein (APP) is a critical factor in the pathogenesis of Alzheimer’s disease (AD). APP undergoes posttranslational proteolysis/processing to generate the hydrophobic β-amyloid (Aβ) peptides. Deposition of Aβ in the brain, forming oligomeric Aβ and plaques, is identified as one of the key pathological hallmarks of AD. The processing of APP to generate Aβ is executed by β- and γ-secretase and is highly regulated. Aβ toxicity can lead to synaptic dysfunction, neuronal cell death, impaired learning/memory and abnormal behaviors in AD models in vitro and in vivo. Aside from Aβ, proteolytic cleavages of APP can also give rise to the APP intracellular domain (AICD), reportedly involved in multiple types of cellular events such as gene transcription and apoptotic cell death. In addition to amyloidogenic processing, APP can also be cleaved by α-secretase to form a soluble or secreted APP ectodomain (sAPP-α) that has been shown to be mostly neuro-protective. In this review, we describe the mechanisms involved in APP metabolism and the likely functions of its various proteolytic products to give a better understanding of the patho/physiological functions of APP. PMID:22122372

  2. Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes.

    PubMed Central

    Khan, A. R.; James, M. N.

    1998-01-01

    Proteolytic enzymes are synthesized as inactive precursors, or "zymogens," to prevent unwanted protein degradation, and to enable spatial and temporal regulation of proteolytic activity. Upon sorting or appropriate compartmentalization, zymogen conversion to the active enzyme typically involves limited proteolysis and removal of an "activation segment." The sizes of activation segments range from dipeptide units to independently folding domains comprising more than 100 residues. A common form of the activation segment is an N-terminal extension of the mature enzyme, or "prosegment," that sterically blocks the active site, and thereby prevents binding of substrates. In addition to their inhibitory role, prosegments are frequently important for the folding, stability, and/or intracellular sorting of the zymogen. The mechanisms of conversion to active enzymes are diverse in nature, ranging from enzymatic or nonenzymatic cofactors that trigger activation, to a simple change in pH that results in conversion by an autocatalytic mechanism. Recent X-ray crystallographic studies of zymogens and comparisons with their active counterparts have identified the structural changes that accompany conversion. This review will focus upon the structural basis for inhibition by activation segments, as well as the molecular events that lead to the conversion of zymogens to active enzymes. PMID:9568890

  3. Intrinsic Tau Acetylation Is Coupled to Auto-Proteolytic Tau Fragmentation

    PubMed Central

    Cohen, Todd J.; Constance, Brian H.; Hwang, Andrew W.; James, Michael; Yuan, Chao-Xing

    2016-01-01

    Tau proteins are abnormally aggregated in a range of neurodegenerative tauopathies including Alzheimer’s disease (AD). Recently, tau has emerged as an extensively post-translationally modified protein, among which lysine acetylation is critical for normal tau function and its pathological aggregation. Here, we demonstrate that tau isoforms have different propensities to undergo lysine acetylation, with auto-acetylation occurring more prominently within the lysine-rich microtubule-binding repeats. Unexpectedly, we identified a unique intrinsic property of tau in which auto-acetylation induces proteolytic tau cleavage, thereby generating distinct N- and C-terminal tau fragments. Supporting a catalytic reaction-based mechanism, mapping and mutagenesis studies showed that tau cysteines, which are required for acetyl group transfer, are also essential for auto-proteolytic tau processing. Further mass spectrometry analysis identified the C-terminal 2nd and 4th microtubule binding repeats as potential sites of auto-cleavage. The identification of acetylation-mediated auto-proteolysis provides a new biochemical mechanism for tau self-regulation and warrants further investigation into whether auto-catalytic functions of tau are implicated in AD and other tauopathies. PMID:27383765

  4. Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw

    PubMed Central

    Orlandelli, Ravely Casarotti; de Almeida, Tiago Tognolli; Alberto, Raiani Nascimento; Polonio, Julio Cesar; Azevedo, João Lúcio; Pamphile, João Alencar

    2015-01-01

    Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes. PMID:26273250

  5. Endosomal localization of TLR8 confers distinctive proteolytic processing on human myeloid cells.

    PubMed

    Ishii, Noriko; Funami, Kenji; Tatematsu, Megumi; Seya, Tsukasa; Matsumoto, Misako

    2014-11-15

    Nucleic acid-sensing TLRs are involved in both antimicrobial immune responses and autoimmune inflammation. TLR8 is phylogenetically and structurally related to TLR7 and TLR9, which undergo proteolytic processing in the endolysosomes to generate functional receptors. Recent structural analyses of human TLR8 ectodomain and its liganded form demonstrated that TLR8 is also cleaved, and both the N- and C-terminal halves contribute to ligand binding. However, the structures and ssRNA recognition mode of endogenous TLR8 in human primary cells are largely unknown. In this study, we show that proteolytic processing of TLR8 occurs in human monocytes and macrophages in a different manner compared with TLR7/9 cleavage. The insertion loop between leucine-rich repeats 14 and 15 in TLR8 is indispensable for the cleavage and stepwise processing that occurs in the N-terminal fragment. Both furin-like proprotein convertase and cathepsins contribute to TLR8 cleavage in the early/late endosomes. TLR8 recognizes viral ssRNA and endogenous RNA, such as microRNAs, resulting in the production of proinflammatory cytokines. Hence, localization sites of the receptors are crucial for the nucleic acid-sensing mode and downstream signaling. PMID:25297876

  6. Electrochemical sensing system employing fructosamine 6-kinase enables glycated albumin measurement requiring no proteolytic digestion.

    PubMed

    Kameya, Miho; Tsugawa, Wakako; Yamada-Tajima, Mayumi; Hatada, Mika; Suzuki, Keita; Sakaguchi-Mikami, Akane; Ferri, Stefano; Klonoff, David C; Sode, Koji

    2016-06-01

    Currently available enzymatic methods for the measurement of glycated proteins utilize fructosyl amino acid/peptide oxidases (FAOXs/FPOXs) as sensing elements. FAOXs/FPOXs oxidize glycated amino acids or glycated dipeptides but they are not able to accept longer glycated peptides or intact glycated proteins as substrates. Therefore, pretreatment via proteolytic digestion is unavoidable with the current enzymatic methods, and there remains a need for simpler measurement methods for glycated proteins. In this study, in order to develop a novel sensing system for glycated albumin (GA), a marker for diabetes, with no requirement for proteolytic digestion, we created an electrochemical sensor based on fructosamine 6-kinase (FN6K) from Escherichia coli. Uniquely, FN6K can react directly with intact GA unlike FAOXs/FPOXs. The concentration of GA in samples was measured using a carbon-printed disposable electrode upon which FN6K as well as two additional enzymes, pyruvate kinase and pyruvate dehydrogenase were overlaid. A clear correlation between the response current and the concentration of GA was observed in the range of 20-100 µM GA, which is suitable for measurement of GA in diluted blood samples from both healthy individuals and patients with diabetes. The sensing system reported here could be applied to point-of-care-testing devices for measurement of glycated proteins. PMID:27067959

  7. Proteolytic activity of Enterococcus faecalis VB63F for reduction of allergenicity of bovine milk proteins.

    PubMed

    Biscola, V; Tulini, F L; Choiset, Y; Rabesona, H; Ivanova, I; Chobert, J-M; Todorov, S D; Haertlé, T; Franco, B D G M

    2016-07-01

    With the aim of screening proteolytic strains of lactic acid bacteria to evaluate their potential for the reduction of allergenicity of the major bovine milk proteins, we isolated a new proteolytic strain of Enterococcus faecalis (Ent. faecalis VB63F) from raw bovine milk. The proteases produced by this strain had strong activity against caseins (αS1-, αS2-, and β-casein), in both skim milk and sodium caseinate. However, only partial hydrolysis of whey proteins was observed. Proteolysis of Na-caseinate and whey proteins, observed after sodium dodecyl sulfate-PAGE, was confirmed by analysis of peptide profiles by reversed-phase HPLC. Inhibition of proteolysis with EDTA indicated that the proteases produced by Ent. faecalis VB63F belonged to the group of metalloproteases. The optimal conditions for their activity were 42°C and pH 6.5. The majority of assessed virulence genes were absent in Ent. faecalis VB63F. The obtained results suggest that Ent. faecalis VB63F could be efficient in reducing the immunoreactivity of bovine milk proteins. PMID:27179865

  8. Thrombin stimulates fibroblast procollagen production via proteolytic activation of protease-activated receptor 1.

    PubMed Central

    Chambers, R C; Dabbagh, K; McAnulty, R J; Gray, A J; Blanc-Brude, O P; Laurent, G J

    1998-01-01

    Thrombin is a multifunctional serine protease that has a crucial role in blood coagulation. It is also a potent mesenchymal cell mitogen and chemoattractant and might therefore have an important role in the recruitment and local proliferation of mesenchymal cells at sites of tissue injury. We hypothesized that thrombin might also affect the deposition of connective tissue proteins at these sites by directly stimulating fibroblast procollagen production. To address this hypothesis, the effect of thrombin on procollagen production and gene expression by human foetal lung fibroblasts was assessed over 48 h. Thrombin stimulated procollagen production at concentrations of 1 nM and above, with maximal increases of between 60% and 117% at 10 nM thrombin. These effects of thrombin were, at least in part, due to increased steady-state levels of alpha1(I) procollagen mRNA. They could furthermore be reproduced with thrombin receptor-activating peptides for the protease-activated receptor 1 (PAR-1) and were completely abolished when thrombin was rendered proteolytically inactive with the specific inhibitors d-Phe-Pro-ArgCH2Cl and hirudin, indicating that thrombin is mediating these effects via the proteolytic activation of PAR-1. These results suggest that thrombin might influence the deposition of connective tissue proteins during normal wound healing and the development of tissue fibrosis by stimulating fibroblast procollagen production. PMID:9639571

  9. CleavPredict: A Platform for Reasoning about Matrix Metalloproteinases Proteolytic Events.

    PubMed

    Kumar, Sonu; Ratnikov, Boris I; Kazanov, Marat D; Smith, Jeffrey W; Cieplak, Piotr

    2015-01-01

    CleavPredict (http://cleavpredict.sanfordburnham.org) is a Web server for substrate cleavage prediction for matrix metalloproteinases (MMPs). It is intended as a computational platform aiding the scientific community in reasoning about proteolytic events. CleavPredict offers in silico prediction of cleavage sites specific for 11 human MMPs. The prediction method employs the MMP specific position weight matrices (PWMs) derived from statistical analysis of high-throughput phage display experimental results. To augment the substrate cleavage prediction process, CleavPredict provides information about the structural features of potential cleavage sites that influence proteolysis. These include: secondary structure, disordered regions, transmembrane domains, and solvent accessibility. The server also provides information about subcellular location, co-localization, and co-expression of proteinase and potential substrates, along with experimentally determined positions of single nucleotide polymorphism (SNP), and posttranslational modification (PTM) sites in substrates. All this information will provide the user with perspectives in reasoning about proteolytic events. CleavPredict is freely accessible, and there is no login required. PMID:25996941

  10. Preliminary investigation of intrinsic UV fluorescence spectroscopic changes associated with proteolytic digestion of bovine articular cartilage

    NASA Astrophysics Data System (ADS)

    Lewis, William; Padilla-Martinez, Juan-Pablo; Ortega-Martinez, Antonio; Franco, Walfre

    2016-03-01

    Degradation and destruction of articular cartilage is the etiology of osteoarthritis (OA), an entity second only to cardiovascular disease as a cause of disability in the United States. Joint mechanics and cartilage biochemistry are believed to play a role in OA; an optical tool to detect structural and chemical changes in articular cartilage might offer benefit for its early detection and treatment. The objective of the present study was to identify the spectral changes in intrinsic ultraviolet (UV) fluorescence of cartilage that occur after proteolytic digestion of cartilage. Bovine articular cartilage samples were incubated in varying concentrations of collagenase ranging from 10ug/mL up to 5mg/mL for 18 hours at 37°C, a model of OA. Pre- and post-incubation measurements were taken of the UV excitation-emission spectrum of each cartilage sample. Mechanical tests were performed to determine the pre- and post-digestion force/displacement ratio associated with indentation of each sample. Spectral changes in intrinsic cartilage fluorescence and stiffness of the cartilage were associated with proteolytic digestion. In particular, changes in the relative intensity of fluorescence peaks associated with pentosidine crosslinks (330 nm excitation, 390 nm emission) and tryptophan (290 nm excitation, 340 nm emission) were found to correlate with different degrees of cartilage digestion and cartilage stiffness. In principle, it may be possible to use UV fluorescence spectral data for early detection of damage to articular cartilage, and as a surrogate measure for cartilage stiffness.

  11. [Proteolytic enzymes in the prevention and treatment of early ventilatory complications following lung resection in patients with tuberculosis].

    PubMed

    Ershov, V N; Krylov, V V; Zolotarev, V P

    1989-01-01

    The authors examined 259 patients with tuberculosis of the lungs before and after partial resection of the lungs. Proteolytic enzymes were administered endotracheally in the pre- and postoperative periods to 132 patients (main group), 127 patients (control group) received a placebo. Endotracheal administration of proteolytic enzymes in the main group reduced the number of early pleuropulmonary complications of the type of atelectasis and hypoventilation by half to one third and accelerated their resolution by 3-4 times as compared to these values in the control group. Fibrous changes and pleural adhesions on the side of the operation formed from the early pleuropulmonary complications more often in the control than in the main group. The function of external respiration was restored under the effect of the proteolytic enzymes in the control group 1-2 months after segmental and combined resection, in the control group it was restored 3-4 months and later after the operation. PMID:2687135

  12. The proteolytic fragments of the Alzheimer's disease-associated presenilin-1 form heterodimers and occur as a 100-150-kDa molecular mass complex.

    PubMed

    Capell, A; Grünberg, J; Pesold, B; Diehlmann, A; Citron, M; Nixon, R; Beyreuther, K; Selkoe, D J; Haass, C

    1998-02-01

    Mutations in the presenilin (PS) genes are linked to early onset familial Alzheimer's disease (FAD). PS-1 proteins are proteolytically processed by an unknown protease to two stable fragments of approximately 30 kDa (N-terminal fragment (NTF)) and approximately 20 kDa (C-terminal fragment (CTF)) (Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. H., Spitzer, L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M., Hardy, J., Levey, A. I., Gandy, S. E., Jenkins, N. A., Copeland, N. G., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 181-190). Here we show that the CTF and NTF of PS-1 bind to each other. Fractionating proteins from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-extracted membrane preparations by velocity sedimentation reveal a high molecular mass SDS and Triton X-100-sensitive complex of approximately 100-150 kDa. To prove if both proteolytic fragments of PS-1 are bound to the same complex, we performed co-immunoprecipitations using multiple antibodies specific to the CTF and NTF of PS-1. These experiments revealed that both fragments of PS-1 occur as a tightly bound non-covalent complex. Upon overexpression, unclipped wild type PS-1 sediments at a lower molecular weight in glycerol velocity gradients than the endogenous fragments. In contrast, the non-cleavable, FAD-associated PS-1 Deltaexon 9 sediments at a molecular weight similar to that observed for the endogenous proteolytic fragments. This result may indicate that the Deltaexon 9 mutation generates a mutant protein that exhibits biophysical properties similar to the naturally occurring PS-1 fragments. This could explain the surprising finding that the Deltaexon 9 mutation is functionally active, although it cannot be proteolytically processed (Baumeister, R., Leimer, U., Zweckbronner, I., Jakubek, C., Grünberg, J., and Haass, C. (1997) Genes & Function 1, 149-159; Levitan, D., Doyle, T., Brousseau, D., Lee, M., Thinakaran, G., Slunt, H., Sisodia, S., and

  13. Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2

    PubMed Central

    2011-01-01

    Background Caseinolytic proteases (ClpPs) are barrel-shaped self-compartmentalized peptidases involved in eliminating damaged or short-lived regulatory proteins. The Mycobacterium tuberculosis (MTB) genome contains two genes coding for putative ClpPs, ClpP1 and ClpP2 respectively, that are likely to play a role in the virulence of the bacterium. Results We report the first biochemical characterization of ClpP1 and ClpP2 peptidases from MTB. Both proteins were produced and purified in Escherichia coli. Use of fluorogenic model peptides of diverse specificities failed to show peptidase activity with recombinant mycobacterial ClpP1 or ClpP2. However, we found that ClpP1 had a proteolytic activity responsible for its own cleavage after the Arg8 residue and cleavage of ClpP2 after the Ala12 residue. In addition, we showed that the absence of any peptidase activity toward model peptides was not due to an obstruction of the entry pore by the N-terminal flexible extremity of the proteins, nor to an absolute requirement for the ClpX or ClpC ATPase complex. Finally, we also found that removing the putative propeptides of ClpP1 and ClpP2 did not result in cleavage of model peptides. We have also shown that recombinant ClpP1 and ClpP2 do not assemble in the conventional functional tetradecameric form but in lower order oligomeric species ranging from monomers to heptamers. The concomitant presence of both ClpP1 and ClpP2 did not result in tetradecameric assembly. Deleting the amino-terminal extremity of ClpP1 and ClpP2 (the putative propeptide or entry gate) promoted the assembly in higher order oligomeric species, suggesting that the flexible N-terminal extremity of mycobacterial ClpPs participated in the destabilization of interaction between heptamers. Conclusion Despite the conservation of a Ser protease catalytic triad in their primary sequences, mycobacterial ClpP1 and ClpP2 do not have conventional peptidase activity toward peptide models and display an unusual

  14. Four proteolytic processes of myocardium, one insensitive to thiol reactive agents and thiol protease inhibitor.

    PubMed

    Thorne, D P; Lockwood, T D

    1993-07-01

    Four distinct processes mediating protein degradation were identified in the Langendorff perfused rat heart. Hearts were biosynthetically labeled in vitro with [3H]leucine for 10 min. The subsequent release of [3H]leucine at 1.5-min intervals (2 mM nonradioactive leucine) was determined from 20 min to 8 h after labeling in rhythmically contracting hearts. Rapid turnover proteins were eliminated during the first 3 h; this degradation was not inhibited by insulin (5 nM) or isoproterenol (0.5 microM). However, the nontoxic thiol reactive agent diamide (100 microM) caused a complete inhibition of the [3H]leucine release from rapidly degraded proteins. After the elimination of rapidly degraded proteins at 3 h, the release of [3H]leucine was inhibited 35-40% by insulin (5 nM) or the lysosomal inhibitor chloroquine (30 microM), thereby defining a second vesicular process. The beta-agonist isoproterenol (0.5 microM) or the nonselective alpha-agonist naphazoline (100 microM) caused 30-35% proteolytic inhibitions, defining a third adrenergic-responsive process. The inhibitory effects of simultaneously combined insulin and chloroquine did not exceed the effect of either agent alone. However, the combined effects of insulin and isoproterenol were additive, inhibiting two-thirds of basal degradation. Beginning at 3 h after labeling a 75% proteolytic inhibition resulted from the thiol reactive agents diamide (100 microM) or N-ethylmaleimide (10 microM); the thiol protease active site inhibitor trans-epoxysuccinly-L-leucylamino-(4-quinidino)butane (50 microM) caused 65% inhibition. The 75% inhibition caused by diamide includes both the insulin-responsive and beta-adrenergic-responsive pathways. A novel fourth proteolytic process (25% of proteolysis) was thereby distinguished from the above three by its resistance to inhibition by insulin, adrenergic agonists, thiol reactive agents, or thiol protease inhibitor. Only the adrenergic-responsive process was correlated with changes in

  15. Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells

    PubMed Central

    Cerqueira, Carla; Samperio Ventayol, Pilar; Vogeley, Christian

    2015-01-01

    ABSTRACT The entry of human papillomaviruses into host cells is a complex process. It involves conformational changes at the cell surface, receptor switching, internalization by a novel endocytic mechanism, uncoating in endosomes, trafficking of a subviral complex to the Golgi complex, and nuclear entry during mitosis. Here, we addressed how the stabilizing contacts in the capsid of human papillomavirus 16 (HPV16) may be reversed to allow uncoating of the viral genome. Using biochemical and cell-biological analyses, we determined that the major capsid protein L1 underwent proteolytic cleavage during entry. In addition to a dispensable cathepsin-mediated proteolysis that occurred likely after removal of capsomers from the subviral complex in endosomes, at least two further proteolytic cleavages of L1 were observed, one of which was independent of the low-pH environment of endosomes. This cleavage occurred extracellularly. Further analysis showed that the responsible protease was the secreted trypsin-like serine protease kallikrein-8 (KLK8) involved in epidermal homeostasis and wound healing. Required for infection, the cleavage was facilitated by prior interaction of viral particles with heparan sulfate proteoglycans. KLK8-mediated cleavage was crucial for further conformational changes exposing an important epitope of the minor capsid protein L2. Occurring independently of cyclophilins and of furin that mediate L2 exposure, KLK8-mediated cleavage of L1 likely facilitated access to L2, located in the capsid lumen, and potentially uncoating. Since HPV6 and HPV18 also required KLK8 for entry, we propose that the KLK8-dependent entry step is conserved. IMPORTANCE Our analysis of the proteolytic processing of incoming HPV16, an etiological agent of cervical cancer, demonstrated that the capsid is cleaved extracellularly by a serine protease active during wound healing and that this cleavage was crucial for infection. The cleavage of L1 is one of at least four structural

  16. Activities of indigenous proteolytic enzymes in caprine milk of different somatic cell counts.

    PubMed

    Albenzio, M; Santillo, A; Kelly, A L; Caroprese, M; Marino, R; Sevi, A

    2015-11-01

    Individual caprine milk with different somatic cell counts (SCC) were studied with the aim of investigating the percentage distribution of leukocyte cell types and the activities of indigenous proteolytic enzymes; proteolysis of casein was also studied in relation to cell type following recovery from milk. The experiment was conducted on 5 intensively managed dairy flocks of Garganica goats; on the basis of SCC, the experimental groups were denoted low (L-SCC; <700,000 cells/mL), medium (M-SCC; from 701,000 to 1,500,000 cells/mL), and high (H-SCC; >1,501,000 cells/mL) SCC. Leukocyte distribution differed between groups; polymorphonuclear neutrophilic leukocytes were higher in M-SCC and H-SCC milk samples, the percentage macrophages was the highest in H-SCC, and levels of nonviable cells significantly decreased with increasing SCC. Activities of all the main proteolytic enzymes were affected by SCC; plasmin activity was the highest in H-SCC milk and the lowest in L-SCC, and elastase and cathepsin D activities were the highest in M-SCC. Somatic cell count influenced casein hydrolysis patterns, with less intact α- and β-casein in H-SCC milk. Higher levels of low electrophoretic mobility peptides were detected in sodium caseinate incubated with leukocytes isolated from L-SCC milk, independent of cell type, whereas among cells recovered from M-SCC milk, macrophages yielded the highest levels of low electrophoretic mobility peptides from sodium caseinate. The level of high electrophoretic mobility peptides was higher in sodium caseinate incubated with polymorphonuclear neutrophilic leukocytes and macrophages isolated from M-SCC, whereas the same fraction of peptides was always the highest, independent of leukocyte type, for cells recovered from H-SCC milk. In caprine milk, a level of 700,000 cells/mL represented the threshold for changes in leukocyte distribution, which is presumably related to the immune status of the mammary gland. Differences in the profile of

  17. Effect of the proteolytic enzyme serrapeptase on swelling, pain and trismus after surgical extraction of mandibular third molars.

    PubMed

    Al-Khateeb, T H; Nusair, Y

    2008-03-01

    The aim of this study was to investigate the ability of serrapeptase to reduce postoperative swelling, pain and trismus after third molar surgery. Twenty-four healthy individuals with symmetrically impacted mandibular third molars underwent surgical removal in a prospective, intra-individual, randomized, double-blind, cross-over study. Teeth were removed in 2 sessions by the same surgeon. At each session, one third molar was removed under local anaesthesia via a buccal osteotomy. All patients received a combination of either serrapeptase 5mg or placebo tablets and 1000 mg paracetamol tablets at either the 1st or 2nd operation in accordance with the randomization plan. Cheek thickness, pain and interincisal distance were measured preoperatively, and on the 1st, 2nd, 3rd and 7th postoperative days. Cheek thickness and maximum interincisal distance were measured using calipers. Pain intensity was assessed clinically using a numeric scale. There was a significant reduction in the extent of cheek swelling and pain intensity in the serrapeptase group at the 2nd, 3rd and 7th postoperative days (P<0.05), but no significant difference in mean maximal interincisal distance was found between the 2 groups (P>0.05). PMID:18272344

  18. The effects of Capn1 gene inactivation on skeletal muscle growth, development, and atrophy, and the compensatory role of other proteolytic systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myofibrillar protein turnover is a key component of muscle growth and degeneration, requiring proteolytic enzymes to degrade the skeletal muscle proteins. The objective of this study was to investigate the role of the calpain proteolytic system in muscle growth development using µ-calpain knockout (...

  19. Trichomonas vaginalis Cysteine Proteinases: Iron Response in Gene Expression and Proteolytic Activity.

    PubMed

    Arroyo, Rossana; Cárdenas-Guerra, Rosa Elena; Figueroa-Angulo, Elisa Elvira; Puente-Rivera, Jonathan; Zamudio-Prieto, Olga; Ortega-López, Jaime

    2015-01-01

    We focus on the iron response of Trichomonas vaginalis to gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes. PMID:26090464

  20. Fibrin Clots Are Equilibrium Polymers That Can Be Remodeled Without Proteolytic Digestion

    NASA Astrophysics Data System (ADS)

    Chernysh, Irina N.; Nagaswami, Chandrasekaran; Purohit, Prashant K.; Weisel, John W.

    2012-11-01

    Fibrin polymerization is a necessary part of hemostasis but clots can obstruct blood vessels and cause heart attacks and strokes. The polymerization reactions are specific and controlled, involving strong knob-into-hole interactions to convert soluble fibrinogen into insoluble fibrin. It has long been assumed that clots and thrombi are stable structures until proteolytic digestion. On the contrary, using the technique of fluorescence recovery after photobleaching, we demonstrate here that there is turnover of fibrin in an uncrosslinked clot. A peptide representing the knobs involved in fibrin polymerization can compete for the holes and dissolve a preformed fibrin clot, or increase the fraction of soluble oligomers, with striking rearrangements in clot structure. These results imply that in vivo clots or thrombi are more dynamic structures than previously believed that may be remodeled as a result of local environmental conditions, may account for some embolization, and suggest a target for therapeutic intervention.

  1. Influence of Amitraz and Oxalic Acid on the Cuticle Proteolytic System of Apis mellifera L. Workers

    PubMed Central

    Strachecka, Aneta; Paleolog, Jerzy; Olszewski, Krzysztof; Borsuk, Grzegorz

    2012-01-01

    This work verifies that amitraz and oxalic acid treatment affect honeybee cuticle proteolytic enzymes (CPE). Three bee groups were monitored: oxalic acid treatment, amitraz treatment, control. Electrophoresis of hydrophilic and hydrophobic CPE was performed. Protease and protease inhibitor activities (in vitro) and antifungal/antibacterial efficiencies (in vivo), were analyzed. Amitraz and oxalic acid treatment reduced hydrophobic, but did not affect hydrophilic, protein concentrations and reduced both hydrophilic and hydrophobic body surface asparagine and serine protease activities in relation to most substrates and independently of pH. The activities of natural cuticle inhibitors of acidic, neutral, and alkaline proteases were suppressed as a result of the treatments, corresponding with reduced antifungal and antibacterial activity. Electrophoretic patterns of low-, medium-, and high-molecular-weight proteases and protease inhibitors were also affected by the treatments. PMID:26466630

  2. Changes in nitrogen fractions and proteolytic activities in the cotyledons of germinating lentils.

    PubMed

    Guerra, H; Nicolás, G

    1983-09-01

    Changes in ninhydrin positive material, free amino acids and protein content during germination of seeds of Lens culinaris Med. have been studied. Ninhydrin positive material and free amino acids reached their highest concentration at the fifth day of germination. Total protein which represents 21% of the total dry weight of the lentil cotyledons, suffers a degradation of only 24% in seven days of germination; in the same period of time, reserve proteins underwent a degradation of 69%, legumin being the more abundant at the start, and the more rapidly depleted. Five different classes of proteolytic activities have been reported in lentil cotyledons: caseinolytic, active against the reserve proteins of the lentil cotyledons themselves, aminopeptidase, peptidehydrolase, carboxypeptidase and dipeptidase. The removal of the axis did not seem to exert any significant influence on the enzymatic activity. PMID:6361930

  3. Sequencing Lys-N proteolytic peptides by ESI and MALDI tandem mass spectrometry.

    PubMed

    Dupré, Mathieu; Cantel, Sonia; Verdié, Pascal; Martinez, Jean; Enjalbal, Christine

    2011-02-01

    In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments were investigated. PMID:21472586

  4. Sequencing Lys-N Proteolytic Peptides by ESI and MALDI Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Dupré, Mathieu; Cantel, Sonia; Verdié, Pascal; Martinez, Jean; Enjalbal, Christine

    2011-02-01

    In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments were investigated.

  5. Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system.

    PubMed Central

    Ciechanover, A; Heller, H; Katz-Etzion, R; Hershko, A

    1981-01-01

    It had been shown previously that the heat-stable polypeptide of the ATP-dependent proteolytic system of reticulocytes, designated APF-1, forms covalent conjugates with protein substrates in an ATP-requiring process. We now describe an enzyme that carries out the activation by ATP of the polypeptide with pyrophosphate displacement. The formation of AMP-polypeptide and transfer of the polypeptide to a secondary acceptor are suggested by an APF-1 requirement for ATP-PPi and ATP-AMP exchange reactions, respectively. With radiolabeled polypeptide, an ATP-dependent labeling of the enzyme was shown to be by a linkage that is acid stable but is labile to treatment with mild alkali, hydroxylamine, borohydride, or mercuric salts. It therefore appears that the AMP-polypeptide undergoes attack by an -SH group of the enzyme to form a thiolester. PMID:6262770

  6. Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines.

    PubMed

    Gerdes, Florian; Tatsuta, Takashi; Langer, Thomas

    2012-01-01

    Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines. PMID:22001671

  7. Lipoprotein receptors and cholesterol in APP trafficking and proteolytic processing, implications for Alzheimer's disease.

    PubMed

    Marzolo, Maria-Paz; Bu, Guojun

    2009-04-01

    Amyloid-beta (Abeta) peptide accumulation in the brain is central to the pathogenesis of Alzheimer's disease (AD). Abeta is produced through proteolytic processing of a transmembrane protein, beta-amyloid precursor protein (APP), by beta- and gamma-secretases. Mounting evidence has demonstrated that alterations in APP cellular trafficking and localization directly impact its processing to Abeta. Members of the low-density lipoprotein receptor family, including LRP, LRP1B, SorLA/LR11, and apoER2, interact with APP and regulate its endocytic trafficking. Additionally, APP trafficking and processing are greatly affected by cellular cholesterol content. In this review, we summarize the current understanding of the roles of lipoprotein receptors and cholesterol in APP trafficking and processing and their implication for AD pathogenesis and therapy. PMID:19041409

  8. Proteolytic activation of latent Paraguaya peach PPO. Characterization of monophenolase activity.

    PubMed

    Laveda, F; Núñez-Delicado, E; García-Carmona, F; Sánchez-Ferrer, A

    2001-02-01

    The kinetics of the activation process of latent peach PPO by trypsin was studied. By coupling this activation process to the oxidation of 4-tert-butylcatechol (TBC) to its corresponding quinone, it was possible to evaluate the specific rate constant of active PPO formation, k(3), which showed a value of 0.04 s(-1). This proteolytic activation of latent peach PPO permitted us to characterize the monophenolase activity of peach PPO for the first time using p-cresol as substrate, and it showed the characteristic lag period of the kinetic mechanism of monophenols hydroxylation, which depended on the enzyme and substrate concentration, the pH and the presence of catalytic amounts of o-diphenol (4-methylcatechol). The enzyme activation constant, k(act), was 2 microM. PMID:11262063

  9. Mechanical Allostery: Evidence for a Force Requirement in the Proteolytic Activation of Notch

    PubMed Central

    Gordon, Wendy R.; Zimmerman, Brandon; He, Li; Miles, Laura J.; Huang, Jiuhong; Tiyanont, Kittichoat; McArthur, Debbie G.; Aster, Jon C.; Perrimon, Norbert; Loparo, Joseph J.; Blacklow, Stephen C.

    2015-01-01

    Summary Ligands stimulate Notch receptors by inducing regulated intramembrane proteolysis (RIP) to produce a transcriptional effector. Notch activation requires unmasking of a metalloprotease cleavage site remote from the site of ligand binding, raising the question of how proteolytic sensitivity is achieved. Here, we show that application of physiologically relevant forces to the regulatory switch results in sensitivity to metalloprotease cleavage, and that bound ligands induce Notch signal transduction in cells only in the presence of applied mechanical force. Synthetic receptor-ligand systems that remove the native ligand-receptor interaction also activate Notch by inducing proteolysis of the regulatory switch. Together, these studies show that mechanical force exerted by signal-sending cells is required for ligand-induced Notch activation, and establish that force-induced proteolysis can act as a mechanism of cellular mechanotransduction. PMID:26051539

  10. The Xanthomonas campestris Type III Effector XopJ Proteolytically Degrades Proteasome Subunit RPT61[OPEN

    PubMed Central

    2015-01-01

    Many animal and plant pathogenic bacteria inject type III effector (T3E) proteins into their eukaryotic host cells to suppress immunity. The Yersinia outer protein J (YopJ) family of T3Es is a widely distributed family of effector proteins found in both animal and plant pathogens, and its members are highly diversified in virulence functions. Some members have been shown to possess acetyltransferase activity; however, whether this is a general feature of YopJ family T3Es is currently unknown. The T3E Xanthomonas outer protein J (XopJ), a YopJ family effector from the plant pathogen Xanthomonas campestris pv vesicatoria, interacts with the proteasomal subunit Regulatory Particle AAA-ATPase6 (RPT6) in planta to suppress proteasome activity, resulting in the inhibition of salicylic acid-related immune responses. Here, we show that XopJ has protease activity to specifically degrade RPT6, leading to reduced proteasome activity in the cytoplasm as well as in the nucleus. Proteolytic degradation of RPT6 was dependent on the localization of XopJ to the plasma membrane as well as on its catalytic triad. Mutation of the Walker B motif of RPT6 prevented XopJ-mediated degradation of the protein but not XopJ interaction. This indicates that the interaction of RPT6 with XopJ is dependent on the ATP-binding activity of RPT6, but proteolytic cleavage additionally requires its ATPase activity. Inhibition of the proteasome impairs the proteasomal turnover of Nonexpressor of Pathogenesis-Related1 (NPR1), the master regulator of salicylic acid responses, leading to the accumulation of ubiquitinated NPR1, which likely interferes with the full induction of NPR1 target genes. Our results show that YopJ family T3Es are not only highly diversified in virulence function but also appear to possess different biochemical activities. PMID:25739698

  11. Multiplexed homogeneous assays of proteolytic activity using a smartphone and quantum dots.

    PubMed

    Petryayeva, Eleonora; Algar, W Russ

    2014-03-18

    Semiconductor quantum dot (QD) bioconjugates, with their unique and highly advantageous physicochemical and optical properties, have been extensively utilized as probes for bioanalysis and continue to generate widespread interest for these applications. An important consideration for expanding the utility of QDs and making their use routine is to make assays with QDs more accessible for laboratories that do not specialize in nanomaterials. Here, we show that digital color imaging of QD photoluminescence (PL) with a smartphone camera is a viable, easily accessible readout platform for quantitative, multiplexed, and real-time bioanalyses. Red-, green-, and blue-emitting CdSeS/ZnS QDs were conjugated with peptides that were labeled with a deep-red fluorescent dye, Alexa Fluor 647, and the dark quenchers, QSY9 and QSY35, respectively, to generate Förster resonance energy transfer (FRET) pairs sensitive to proteolytic activity. Changes in QD PL caused by the activity of picomolar to nanomolar concentrations of protease were detected as changes in the red-green-blue (RGB) channel intensities in digital color images. Importantly, measurements of replicate samples made with smartphone imaging and a sophisticated fluorescence plate reader yielded the same quantitative results, including initial proteolytic rates and specificity constants. Homogeneous two-plex and three-plex assays for the activity of trypsin, chymotrypsin, and enterokinase were demonstrated with RGB imaging. Given the ubiquity of smartphones, this work largely removes any instrumental impediments to the adoption of QDs as routine tools for bioanalysis in research laboratories and is a critical step toward the use of QDs for point-of-care diagnostics. This work also adds to the growing utility of smartphones in analytical methods by enabling multiplexed fluorimetric assays within a single sample volume and across multiple samples in parallel. PMID:24571675

  12. Role of proteolytic activation of protein kinase Cδ in the pathogenesis of prion disease

    PubMed Central

    Harischandra, Dilshan S; Kondru, Naveen; Martin, Dustin P; Kanthasamy, Arthi; Jin, Huajun; Anantharam, Vellareddy; Kanthasamy, Anumantha G

    2014-01-01

    Prion diseases are infectious and inevitably fatal neurodegenerative diseases characterized by prion replication, widespread protein aggregation and spongiform degeneration of major brain regions controlling motor function. Oxidative stress has been implicated in prion-related neuronal degeneration, but the molecular mechanisms underlying prion-induced oxidative damage are not well understood. In this study, we evaluated the role of oxidative stress-sensitive, pro-apoptotic protein kinase Cδ (PKCδ) in prion-induced neuronal cell death using cerebellar organotypic slice cultures (COSC) and mouse models of prion diseases. We found a significant upregulation of PKCδ in RML scrapie-infected COSC, as evidenced by increased levels of both PKCδ protein and its mRNA. We also found an enhanced regulatory phosphorylation of PKCδ at its two regulatory sites, Thr505 in the activation loop and Tyr311 at the caspase-3 cleavage site. The prion infection also induced proteolytic activation of PKCδ in our COSC model. Immunohistochemical analysis of scrapie-infected COSC revealed loss of PKCδ positive Purkinje cells and enhanced astrocyte proliferation. Further examination of PKCδ signaling in the RML scrapie adopted in vivo mouse model showed increased proteolytic cleavage and Tyr 311 phosphorylation of the kinase. Notably, we observed a delayed onset of scrapie-induced motor symptoms in PKCδ knockout (PKCδ−/−) mice as compared with wild-type (PKCδ+/+) mice, further substantiating the role of PKCδ in prion disease. Collectively, these data suggest that PKCδ signaling likely plays a role in the neurodegenerative processes associated with prion diseases. PMID:24576946

  13. Tumor-Specific Proteolytic Processing of Cyclin E Generates Hyperactive Lower-Molecular-Weight Forms

    PubMed Central

    Porter, Donald C.; Zhang, Ning; Danes, Christopher; McGahren, Mollianne J.; Harwell, Richard M.; Faruki, Shamsa; Keyomarsi, Khandan

    2001-01-01

    Cyclin E is a G1 cyclin essential for S-phase entry and has a profound role in oncogenesis. Previously this laboratory found that cyclin E is overexpressed and present in lower-molecular-weight (LMW) isoforms in breast cancer cells and tumor tissues compared to normal cells and tissues. Such alteration of cyclin E is linked to poor patient outcome. Here we report that the LMW forms of cyclin E are hyperactive biochemically and they can more readily induce G1-to-S progression in transfected normal cells than the full-length form of the protein can. Through biochemical and mutational analyses we have identified two proteolytically sensitive sites in the amino terminus of human cyclin E that are cleaved to generate the LMW isoforms found in tumor cells. Not only are the LMW forms of cyclin E functional, as they phosphorylate substrates such as histone H1 and GST-Rb, but also their activities are higher than the full-length cyclin E. These nuclear localized LMW forms of cyclin E are also biologically functional, as their overexpression in normal cells increases the ability of these cells to enter S and G2/M. Lastly, we show that cyclin E is selectively cleaved in vitro by the elastase class of serine proteases to generate LMW forms similar to those observed in tumor cells. These studies suggest that the defective entry into and exit from S phase by tumor cells is in part due to the proteolytic processing of cyclin E, which generates hyperactive LMW isoforms whose activities have been modified from that of the full-length protein. PMID:11509668

  14. A novel synthetic quinolinone inhibitor presents proteolytic and hemorrhagic inhibitory activities against snake venom metalloproteases.

    PubMed

    Baraldi, Patrícia T; Magro, Angelo J; Matioli, Fábio F; Marcussi, Silvana; Lemke, Ney; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M; Correa, Arlene G; Fontes, Marcos R M

    2016-02-01

    Metalloproteases play a fundamental role in snake venom envenomation inducing hemorrhagic, fibrigen(ogen)olytic and myotoxic effects in their victims. Several snake venoms, such as those from the Bothrops genus, present important local effects which are not efficiently neutralized by conventional serum therapy. Consequently, these accidents may result in permanent sequelae and disability, creating economic and social problems, especially in developing countries, leading the attention of the World Health Organization that considered ophidic envenomations a neglected tropical disease. Aiming to produce an efficient inhibitor against bothropic venoms, we synthesized different molecules classified as quinolinones - a group of low-toxic chemical compounds widely used as antibacterial and antimycobacterial drugs - and tested their inhibitory properties against hemorrhage caused by bothropic venoms. The results from this initial screening indicated the molecule 2-hydroxymethyl-6-methoxy-1,4-dihydro-4-quinolinone (Q8) was the most effective antihemorrhagic compound among all of the assayed synthetic quinolinones. Other in vitro and in vivo experiments showed this novel compound was able to inhibit significantly the hemorrhagic and/or proteolytic activities of bothropic crude venoms and isolated snake venom metalloproteases (SVMPs) even at lower concentrations. Docking and molecular dynamic simulations were also performed to get insights into the structural basis of Q8 inhibitory mechanism against proteolytic and hemorrhagic SVMPs. These structural studies demonstrated that Q8 may form a stable complex with SVMPs, impairing the access of substrates to the active sites of these toxins. Therefore, both experimental and structural data indicate that Q8 compound is an interesting candidate for antiophidic therapy, particularly for the treatment of the hemorrhagic and necrotic effects induced by bothropic venoms. PMID:26700145

  15. Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

    SciTech Connect

    M Salameh; A Soares; D Navaneetham; D Sinha; P Walsh; E Radisky

    2011-12-31

    An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P{sub 1} (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P'{sub 2} favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P{sub 1} and P'{sub 2} substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin-APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.

  16. A Familial Mutation Renders Atrial Natriuretic Peptide Resistant to Proteolytic Degradation*

    PubMed Central

    Dickey, Deborah M.; Yoder, Andrea R.; Potter, Lincoln R.

    2009-01-01

    A heterozygous frameshift mutation causing a 12-amino acid extension to the C terminus of atrial natriuretic peptide (ANP) was recently genetically linked to patients with familial atrial fibrillation (Hodgson-Zingman, D. M., Karst, M. L., Zingman, L. V., Heublein, D. M., Darbar, D., Herron, K. J., Ballew, J. D., de Andrade, M., Burnett, J. C., Jr., and Olson, T. M. (2008) N. Engl. J. Med. 359, 158–165). The frameshift product (fsANP), but not wild-type ANP (wtANP), was elevated in the serum of affected patients, but the molecular basis for the elevated peptide concentrations was not determined. Here, we measured the ability of fsANP to interact with natriuretic peptide receptors and to be proteolytically degraded. fsANP and wtANP bound and activated human NPR-A and NPR-C similarly, whereas fsANP had a slightly increased efficacy for human NPR-B. Proteolytic susceptibility was addressed with novel bioassays that measure the time required for kidney membranes or purified neutral endopeptidase to abolish ANP-dependent activation of NPR-A. The half-life of fsANP was markedly greater than that of wtANP in both assays. Additional membrane proteolysis studies indicated that wtANP and fsANP are preferentially degraded by neutral endopeptidase and serine peptidases, respectively. These data indicate that the familial ANP mutation associated with atrial fibrillation has only minor effects on natriuretic peptide receptor interactions but markedly modifies peptide proteolysis. PMID:19458086

  17. High stability of self-assembled peptide nanowires against thermal, chemical, and proteolytic attacks.

    PubMed

    Ryu, Jungki; Park, Chan Beum

    2010-02-01

    Understanding the self-assembly of peptides into ordered nanostructures is recently getting much attention since it can provide an alternative route for fabricating novel bio-inspired materials. In order to realize the potential of the peptide-based nanofabrication technology, however, more information is needed regarding the integrity or stability of peptide nanostructures under the process conditions encountered in their applications. In this study, we investigated the stability of self-assembled peptide nanowires (PNWs) and nanotubes (PNTs) against thermal, chemical, proteolytic attacks, and their conformational changes upon heat treatment. PNWs and PNTs were grown by the self-assembly of diphenylalanine (Phe-Phe), a peptide building block, on solid substrates at different chemical atmospheres and temperatures. The incubation of diphenylalanine under aniline vapor at 150 degrees C led to the formation of PNWs, while its incubation with water vapor at 25 degrees C produced PNTs. We analyzed the stability of peptide nanostructures using multiple tools, such as electron microscopy, thermal analysis tools, circular dichroism, and Fourier-transform infrared spectroscopy. Our results show that PNWs are highly stable up to 200 degrees C and remain unchanged when incubated in aqueous solutions (from pH 1 to 14) or in various chemical solvents (from polar to non-polar). In contrast, PNTs started to disintegrate even at 100 degrees C and underwent a conformational change at an elevated temperature. When we further studied their resistance to a proteolytic environment, we discovered that PNWs kept their initial structure while PNTs fully disintegrated. We found that the high stability of PNWs originates from their predominant beta-sheet conformation and the conformational change of diphenylalanine nanostructures. Our study suggests that self-assembled PNWs are suitable for future nano-scale applications requiring harsh processing conditions. PMID:19777585

  18. Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

    SciTech Connect

    Salameh, M.A.; Soares, A.; Navaneetham, D.; Sinha, D.; Walsh, P. N.; Radisky, E. S.

    2010-11-19

    An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P{sub 1} (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P'{sub 2} favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P1 and P'{sub 2} substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin {center_dot} APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.

  19. Development of a solid-phase assay for measurement of proteolytic enzyme activity

    SciTech Connect

    Varani, J.; Johnson, K.; Kaplan, J.

    1980-09-15

    A solid-phase, plate assay was developed for the measurement of proteolytic enzyme activity. In this assay procedure, radiolabeled substrates were dried onto the surface of microtiter wells. Following drying, the wells were washed two times with saline to remove the nonadherent substrate. When proteolytic enzymes were added to the wells, protein hydrolysis occurred, releasing radioactivity into the supernatant fluid. The amount of protein hydrolysis that occurred was reflected by the amount of radioactivity in the supernatant fluid. When /sup 125/I-hemoglobin was used as the substrate, it was as susceptible to hydrolysis by trypsin in the solid-phase assay as it was in solution in a standard assay procedure. Protease activity from a variety of sources (including from viable cells as well as from extracellular sources) were also able to hydrolyze the hemoglobin on the plate. /sup 125/I-Labeled serum albumen, fibrinogen, and rat pulmonary basement membrane were also susceptible to hydrolysis by trypsin in the solid phase. When (/sup 14/C)elastin was dried onto the plate, it behaved in a similar manner to elastin in solution. It was resistant to hydrolysis by nonspecific proteases such as trypsin and chymotrypsin but was highly susceptible to hydrolysis by elastase. The solid-phase plate assay has several features which recommended it for routine use. It is as sensitive as standard tube assays (and much more sensitive than routinely used colormetric assays). It is quick and convenient; there are no precipitation, centrifugation, or filtration steps. In addition, very small volumes of radioactive wastes are generated. Another advantage of the solid-phase plate assay is the resistance of the dried substrates to spontaneous breakdown and to microbial contamination. Finally, this assay is suitable for use with viable cells as well as for extracellular proteases.

  20. Proteolytic activation of both components of the cation stress-responsive Slt pathway in Aspergillus nidulans.

    PubMed

    Mellado, Laura; Arst, Herbert N; Espeso, Eduardo A

    2016-08-15

    Tolerance of Aspergillus nidulans to alkalinity and elevated cation concentrations requires both SltA and SltB. Transcription factor SltA and the putative pseudokinase/protease signaling protein SltB comprise a regulatory pathway specific to filamentous fungi. In vivo, SltB is proteolytically cleaved into its two principal domains. Mutational analysis defines a chymotrypsin-like serine protease domain that mediates SltB autoproteolysis and proteolytic cleavage of SltA. The pseudokinase domain might modulate the protease activity of SltB. Three forms of the SltA transcription factor coexist in cells: a full-length, 78-kDa version and a processed, 32-kDa form, which is found in phosphorylated and unphosphorylated states. The SltA32kDa version mediates transcriptional regulation of sltB and, putatively, genes required for tolerance to cation stress and alkalinity. The full-length form, SltA78kDa, apparently has no transcriptional function. In the absence of SltB, only the primary product of SltA is detectable, and its level equals that of SltA78kDa. Mutations in sltB selected as suppressors of null vps alleles and resulting in cation/alkalinity sensitivity either reduced or eliminated SltA proteolysis. There is no evidence for cation or alkalinity regulation of SltB cleavage, but activation of sltB expression requires SltA. This work identifies the molecular mechanisms governing the Slt pathway. PMID:27307585

  1. Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis

    PubMed Central

    Mangione, P. Patrizia; Porcari, Riccardo; Gillmore, Julian D.; Pucci, Piero; Monti, Maria; Porcari, Mattia; Giorgetti, Sofia; Marchese, Loredana; Raimondi, Sara; Serpell, Louise C.; Chen, Wenjie; Relini, Annalisa; Marcoux, Julien; Clatworthy, Innes R.; Taylor, Graham W.; Tennent, Glenys A.; Robinson, Carol V.; Hawkins, Philip N.; Stoppini, Monica; Wood, Stephen P.; Pepys, Mark B.; Bellotti, Vittorio

    2014-01-01

    The Ser52Pro variant of transthyretin (TTR) produces aggressive, highly penetrant, autosomal-dominant systemic amyloidosis in persons heterozygous for the causative mutation. Together with a minor quantity of full-length wild-type and variant TTR, the main component of the ex vivo fibrils was the residue 49-127 fragment of the TTR variant, the portion of the TTR sequence that previously has been reported to be the principal constituent of type A, cardiac amyloid fibrils formed from wild-type TTR and other TTR variants [Bergstrom J, et al. (2005) J Pathol 206(2):224–232]. This specific truncation of Ser52Pro TTR was generated readily in vitro by limited proteolysis. In physiological conditions and under agitation the residue 49-127 proteolytic fragment rapidly and completely self-aggregates into typical amyloid fibrils. The remarkable susceptibility to such cleavage is likely caused by localized destabilization of the β-turn linking strands C and D caused by loss of the wild-type hydrogen-bonding network between the side chains of residues Ser52, Glu54, Ser50, and a water molecule, as revealed by the high-resolution crystallographic structure of Ser52Pro TTR. We thus provide a structural basis for the recently hypothesized, crucial pathogenic role of proteolytic cleavage in TTR amyloid fibrillogenesis. Binding of the natural ligands thyroxine or retinol-binding protein (RBP) by Ser52Pro variant TTR stabilizes the native tetrameric assembly, but neither protected the variant from proteolysis. However, binding of RBP, but not thyroxine, inhibited subsequent fibrillogenesis. PMID:24474780

  2. Autosomal ichthyosis with hypotrichosis syndrome displays low matriptase proteolytic activity and is phenocopied in ST14 hypomorphic mice.

    PubMed

    List, Karin; Currie, Brooke; Scharschmidt, Tiffany C; Szabo, Roman; Shireman, Jessica; Molinolo, Alfredo; Cravatt, Benjamin F; Segre, Julia; Bugge, Thomas H

    2007-12-14

    Human autosomal recessive ichthyosis with hypotrichosis (ARIH) is an inherited disorder recently linked to homozygosity for a point mutation in the ST14 gene that causes a G827R mutation in the matriptase serine protease domain (G216 in chymotrypsin numbering). Here we show that human G827R matriptase has strongly reduced proteolytic activity toward small molecule substrates, as well as toward its candidate epidermal target, prostasin. To further investigate the possible contribution of low matriptase activity to ARIH, we generated an ST14 hypomorphic mouse strain that displays a 100-fold reduction in epidermal matriptase mRNA levels. Interestingly, unlike ST14 null mice, ST14 hypomorphic mice were viable and fertile but displayed a spectrum of abnormalities that strikingly resembled ARIH. Thus, ST14 hypomorphic mice developed hyperproliferative and retention ichthyosis with impaired desquamation, hypotrichosis with brittle, thin, uneven, and sparse hair, and tooth defects. Biochemical analysis of ST14 hypomorphic epidermis revealed reduced prostasin proteolytic activation and profilaggrin proteolytic processing, compatible with a primary role of matriptase in this process. This work strongly indicates that reduced activity of a matriptase-prostasin proteolytic cascade is the etiological origin of human ARIH and provides an important mouse model for the exploration of matriptase function in ARIH, as well as multiple other physiological and pathological processes. PMID:17940283

  3. Opportunistic proteolytic processing of carbonic anhydrase 1 from Chlamydomonas in Arabidopsis reveals a novel route for protein maturation.

    PubMed

    Juvale, Parijat S; Wagner, Ryan L; Spalding, Martin H

    2016-04-01

    Proteolytic processing of secretory proteins to yield an active form generally involves specific proteolytic cleavage of a pre-protein. Multiple specific proteases have been identified that target specific pre-protein processing sites in animals. However, characterization of site-specific proteolysis of plant pre-proteins is still evolving. In this study, we characterized proteolytic processing of Chlamydomonas periplasmic carbonic anhydrase 1 (CAH1) in Arabidopsis. CAH1 pre-protein undergoes extensive post-translational modification in the endomembrane system, including glycosylation, disulfide bond formation and proteolytic removal of a peptide 'spacer' region, resulting in a mature, heterotetrameric enzyme with two large and two small subunits. We generated a series of small-scale and large-scale modifications to the spacer and flanking regions to identify potential protease target motifs. Surprisingly, we found that the endoproteolytic removal of the spacer from the CAH1 pre-protein proceeded via an opportunistic process apparently followed by further maturation via amino and carboxy peptidases. We also discovered that the spacer itself is not required for processing, which appears to be dependent only on the number of amino acids separating two key disulfide-bond-forming cysteines. Our data suggest a novel, opportunistic route for pre-protein processing of CAH1. PMID:26917556

  4. Opportunistic proteolytic processing of carbonic anhydrase 1 from Chlamydomonas in Arabidopsis reveals a novel route for protein maturation

    PubMed Central

    Juvale, Parijat S.; Wagner, Ryan L.; Spalding, Martin H.

    2016-01-01

    Proteolytic processing of secretory proteins to yield an active form generally involves specific proteolytic cleavage of a pre-protein. Multiple specific proteases have been identified that target specific pre-protein processing sites in animals. However, characterization of site-specific proteolysis of plant pre-proteins is still evolving. In this study, we characterized proteolytic processing of Chlamydomonas periplasmic carbonic anhydrase 1 (CAH1) in Arabidopsis. CAH1 pre-protein undergoes extensive post-translational modification in the endomembrane system, including glycosylation, disulfide bond formation and proteolytic removal of a peptide ‘spacer’ region, resulting in a mature, heterotetrameric enzyme with two large and two small subunits. We generated a series of small-scale and large-scale modifications to the spacer and flanking regions to identify potential protease target motifs. Surprisingly, we found that the endoproteolytic removal of the spacer from the CAH1 pre-protein proceeded via an opportunistic process apparently followed by further maturation via amino and carboxy peptidases. We also discovered that the spacer itself is not required for processing, which appears to be dependent only on the number of amino acids separating two key disulfide-bond-forming cysteines. Our data suggest a novel, opportunistic route for pre-protein processing of CAH1. PMID:26917556

  5. The Caspase Proteolytic System in Callipyge and Normal Lambs in Longissimus, Semimembranosus, and Infraspinatus Muscles During Postmortem Storage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this experiment was to determine whether the caspase proteolytic system has a role in postmortem tenderization. Six ewes and six wethers that were non-carriers and six ewes and six wethers that were expressing the callipyge gene were used for this study. Caspase activities were de...

  6. Investigation of the types and characteristics of the proteolytic enzymes formed by diverse strains of Proteus species.

    PubMed

    Senior, B W

    1999-07-01

    Many diverse clinical isolates of Proteus mirabilis (48 strains), P. penneri (25), P. vulgaris biogroup 2 (48) and P. vulgaris biogroup 3 (21) from man were examined for their ability to produce proteolytic enzymes and the nature and characteristics of the proteases were studied. All the P. penneri isolates, most (94-90%) of the P. mirabilis and P. vulgaris biogroup 2 isolates, but only 71% of the P. vulgaris biogroup 3 isolates, secreted proteolytic enzymes. These were detected most readily at pH 8 with gelatin as substrate. A strong correlation was found between the ability of a strain to form swarming growth and its ability to secrete proteases. Non-swarming isolates invariably appeared to be non-proteolytic. However, some isolates, particularly of P. vulgaris biogroup 3, were non-proteolytic even when they formed swarming growth. Analysis of the secreted enzymes of the different Proteus spp. on polyacrylamide-gelatin gels under various constraints of pH and other factors showed that they were all EDTA-sensitive metalloproteinases. Analysis of the kinetics of production of the proteases revealed the formation of an additional protease of undefined type and function that was cell-associated and formed before the others were secreted. The secreted protease was subsequently modified to two isoforms whose mass (53-46 kDa) varied with the Proteus spp. and the strain. There was no evidence that the secreted proteases of strains of Proteus spp. were of types other than metalloproteinases. PMID:10403412

  7. Screening of Rubiaceae and Apocynaceae extracts for mosquito larvicidal potential.

    PubMed

    Suryawanshi, Rahul; Patil, Chandrashekhar; Borase, Hemant; Narkhede, Chandrakant; Patil, Satish

    2015-01-01

    Rubiaceae and Apocynaceae families are well known for the expression of cyclotides having insecticidal properties. Leaves and flowers extracts of plants from the families Rubiaceae (Ixora coccinea) and Apocynaceae (Allamanda violacea) were evaluated for mosquito larvicidal effect against early IVth instars of Aedes aegypti and Anopheles stephensi. Two forms of plant extracts, one untreated and the other treated with heat and proteolytic enzyme were used for assay. After primary assay, the extract showing more than 50% inhibition was further used for quantification purpose. LC50 and LC90 values of all the extracts were found to be reduced with the treated form. Phytochemical analysis of plant extracts was performed. Primary confirmation for the presence of cyclotides was done by Lowry test, thin layer chromatography and haemolytic assay. This novel approach merits use of plant extracts in mosquito control programmes. PMID:25317964

  8. Nrf2 silencing to inhibit proteolytic defense induced by hyperthermia in HT22 cells.

    PubMed

    Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

    2016-08-01

    Nrf2 pathway has been known to be protective against cancer progression however recent studies have revealed that the antioxidant activity of Nrf2 contributes to chemotherapy resistance. For many years, hyperthermia has been used as an additional therapy to increase the efficiency of chemotherapy and radiotherapy. Besides the positive effects of hyperthermia during treatment procedure, thermotolerance has been found to develop against heat treatment. Although the involved molecular mechanisms have not been fully clarified, heat shock proteins (HSP) and proteasome activity are known to be involved in the acquisition of thermotolerance. The aim of this study was to investigate the potential beneficial effects of combining hyperthermia with Nrf2 silencing to inhibit molecular mechanisms leading to induction of defense mechanisms in transcription level. Following heat treatment of HT22 cells, HSP70 and the proteasome levels and as well as proteasome activity were found to be elevated in the nucleus. Our results demonstrated that Nrf2 silencing reduced defense mechanisms against heat treatment both in antioxidant and proteolytic manner and Nrf2 may be a potential target for therapeutic approach in order to improve the beneficial effects of hyperthermia in cancer therapy. PMID:26966891

  9. Hypoxia Associated Proteolytic Processing of OS-9 by the Metalloproteinase Meprin β

    PubMed Central

    Martin, Barry Lee; Conley, Sabena Michelle; Harris, Regine Simone; Stanley, Corshe Devon

    2016-01-01

    Meprin metalloproteases play a role in the pathology of ischemia/reperfusion- (IR-) induced renal injury. The endoplasmic reticulum-associated protein, osteosarcoma-9 (OS-9), has been shown to interact with the carboxyl-terminal tail of meprin β. More importantly, OS-9 interacts with the hypoxia inducible factor-1α (HIF-1α) and the prolyl-hydroxylase, proteins which mediate the cell's response to hypoxia. To determine if OS-9 is a meprin substrate, kidney proteins from meprin αβ knockout mice (αβKO) (which lack endogenous meprins) and purified human OS-9 were incubated with activated forms of meprin A and meprin B, and Western blot analysis was used to evaluate proteolytic processing of OS-9. Fragmentation of OS-9 was observed in reactions with meprin B, but not meprin A. To determine whether meprin B cleaves OS-9 in vivo, wild-type (WT) and meprin αβKO mice were subjected to IR-induced renal injury. Fragmentation of OS-9 was observed in kidney proteins from WT mice subjected to IR, but not in meprin αβKO counterparts. Transfection of kidney cells (MDCK and HEK293) with meprin β cDNA prevented accumulation of OS-9 following exposure to the hypoxia mimic, CoCl2. These data suggest that meprin β interaction with OS-9 plays a role in the hypoxia response associated with IR-induced renal injury. PMID:27478637

  10. The Impact of Proteolytic Pork Hydrolysate on Microbial, Flavor and Free Amino Acids Compounds of Yogurt

    PubMed Central

    Lin, Jinzhong; Hua, Baozhen; Xu, Zhiping; Li, Sha; Ma, Chengjie

    2016-01-01

    The aim of this study was to investigate the influence of proteolytic pork hydrolysate (PPH) on yoghurt production by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. Fresh lean pork was cut into pieces and mixed with deionized water and dealt with protease, then the resulting PPH was added to milk to investigate the effects of PPH on yoghurt production. The fermentation time, the viable cell counts, the flavor, free amino acids compounds, and sensory evaluation of yoghurt were evaluated. These results showed that PPH significantly stimulated the growth and acidification of the both bacterial strains. When the content of PPH reached 5% (w/w), the increased acidifying rate occurred, which the fermentation time was one hour less than that of the control, a time saving of up to 20% compared with the control. The viable cell counts, the total free amino acids, and the scores of taste, flavor and overall acceptability in PPH-supplemented yoghurt were higher than the control. Furthermore, the contents of some characteristic flavor compounds including acids, alcohols, aldehydes, ketones and esters were richer than the control. We concluded that the constituents of PPH such as small peptide, vitamins, and minerals together to play the stimulatory roles and result in beneficial effect for the yoghurt starter cultures growth. PMID:27621698

  11. Structural analysis and proteolytic activation of Enterococcus faecalis cytolysin, a novel lantibiotic.

    PubMed

    Booth, M C; Bogie, C P; Sahl, H G; Siezen, R J; Hatter, K L; Gilmore, M S

    1996-09-01

    Clinical isolates of Enterococcus faecalis more commonly produce a cytolysin than do commensal isolates. Epidemiologic evidence and animal-model studies have established a role for the cytolysin in the pathogenesis of enterococcal disease. The cytolysin consists of two structural subunits, CylLL and CylLS, that are activated by a third component, CylA. Genetic and biochemical characterization of CylA indicate that it is a serine protease, and that activation putatively results from cleavage of one or both cytolysin subunits. Genetic evidence also suggests that the cytolysin subunits are related to the rapidly growing class of bacteriocins termed lantibiotics. However, unlike lantibiotics, the cytolysin is lytic for eukaryotic as well as prokaryotic cells, and it consists of two structural subunits. This report describes the purification and characterization of the cytolysin subunits and detection of lanthionine-type post-translational modifications within their structures. Furthermore, the cleavage specificity of the CylA activator is reported and it is shown that proteolytic activation of both subunits is essential for activity. PMID:8898386

  12. Proteolytic Isoforms of SPARC Induce Adipose Stromal Cell Mobilization in Obesity.

    PubMed

    Tseng, Chieh; Kolonin, Mikhail G

    2016-01-01

    Adipose stromal cells (ASC) are mesenchymal adipocyte progenitors that reside in the peri-endothelium of fat tissue. ASC mobilization and migration accompany white adipose tissue (WAT) remodeling and pathological conditions. Mechanisms regulating ASC trafficking are largely unknown. We previously reported that binding of the matricellular protein secreted protein acidic and rich in cysteine (SPARC) to β1 integrin on ASC surface induces their motility. Here, we show that SPARC is required for ASC mobilization. We report two SPARC proteolytic isoforms, C-SPARC (lacking the N terminus) and N-SPARC (lacking the C terminus), generated in mesenteric WAT of obese mice. C-SPARC, but not N-SPARC, binds to β1 integrin on ASC, while N-SPARC preferentially binds to the extracellular matrix (ECM) and blocks ECM/integrin interaction. Interestingly, both C-SPARC and N-SPARC induce ASC deadhesion from the ECM, which is associated with modulation of integrin-dependent FAK-ERK signaling and integrin-independent ILK-Akt signaling. We show that these SPARC isoforms, acting on ASC through distinct mechanisms, have an additive effect in inducing ASC migration. PMID:26381424

  13. Complete amino acid sequence of a histidine-rich proteolytic fragment of human ceruloplasmin.

    PubMed

    Kingston, I B; Kingston, B L; Putnam, F W

    1979-04-01

    The complete amino acid sequence has been determined for a fragment of human ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1]. The fragment (designated Cp F5) contains 159 amino acid residues and has a molecular weight of 18,650; it lacks carbohydrate, is rich in histidine, and contains one free cysteine that may be part of a copper-binding site. This fragment is present in most commercial preparations of ceruloplasmin, probably owing to proteolytic degradation, but can also be obtained by limited cleavage of single-chain ceruloplasmin with plasmin. Cp F5 probably is an intact domain attached to the COOH-terminal end of single-chain ceruloplasmin via a labile interdomain peptide bond. A model of the secondary structure predicted by empirical methods suggests that almost one-third of the amino acid residues are distributed in alpha helices, about a third in beta-sheet structure, and the remainder in beta turns and unidentified structures. Computer analysis of the amino acid sequence has not demonstrated a statistically significant relationship between this ceruloplasmin fragment and any other protein, but there is some evidence for an internal duplication. PMID:287005

  14. Proteolytic processing of Escherichia coli twin-arginine signal peptides by LepB.

    PubMed

    Lüke, Iris; Handford, Jennifer I; Palmer, Tracy; Sargent, Frank

    2009-12-01

    The twin-arginine translocation (Tat) apparatus is a protein targeting system found in the cytoplasmic membranes of many prokaryotes. Substrate proteins of the Tat pathway are synthesised with signal peptides bearing SRRxFLK 'twin-arginine' amino acid motifs. All Tat signal peptides have a common tripartite structure comprising a polar N-terminal region, followed by a hydrophobic region of variable length and a polar C-terminal region. In Escherichia coli, Tat signal peptides are proteolytically cleaved after translocation. The signal peptide C-terminal regions contain conserved AxA motifs, which are possible recognition sequences for leader peptidase I (LepB). In this work, the role of LepB in Tat signal peptide processing was addressed directly. Deliberate repression of lepB expression prevented processing of all Tat substrates tested, including SufI, AmiC, and a TorA-23K reporter protein. In addition, electron microscopy revealed gross defects in cell architecture and membrane integrity following depletion of cellular LepB protein levels. PMID:19809807

  15. Characterization of the sites of proteolytic activation of Newcastle disease virus membrane glycoprotein precursors.

    PubMed

    Gorman, J J; Nestorowicz, A; Mitchell, S J; Corino, G L; Selleck, P W

    1988-09-01

    The F1- and F2-polypeptide components of the fusion proteins and the hemagglutinin/neuraminidase proteins of the avirulent Queensland (V4) and virulent Australia-Victoria (AuV) strains of Newcastle disease virus have been isolated and subjected to extensive primary structural analysis including amino-terminal sequence analysis and fast atom bombardment-mass spectrometry mapping. Nucleotide sequence analysis was performed on the gene which encodes the V4 hemagglutinin/neuraminidase protein. Signal peptidase cleavage was found to have occurred at the Ser31-Leu32 peptide bond of the primary translation products of the fusion protein genes. Activation cleavage of the V4 fusion protein precursor generated a sequence of -Gly-Lys-Gln-Gly84 at the carboxyl terminus of the F2-polypeptide and an amino-terminal sequence of the F1-polypeptide commencing with 86Leu-Ile-Gly-. The V4 hemagglutinin/neuraminidase protein gene was found to encode a primary translation product 45 amino acids longer at the carboxyl terminus than obtainable from the corresponding gene of the AuV strain (McGinnes, L. W., and Morrison, T. G. (1986) Virus Res. 5, 343-356). However, post-translational proteolytic processing, exclusive to the primary translation product of the V4 hemagglutinin/neuraminidase protein gene, was found to have removed the last 42 residues of this carboxyl-terminal appendage. PMID:3045120

  16. Membrane-associated proteolytic activity in Escherichia coli that is stimulated by ATP

    SciTech Connect

    Klemes, Y.; Voellmy, R.W.; Goldberg, A.L.

    1986-05-01

    The degradation of proteins in bacteria requires metabolism energy. One important enzyme in this process is protease La, a soluble ATP-dependent protease encoded by the lon gene. However, lon mutants that lack a functional protease La still show some ATP-dependent protein breakdown. The authors have reported an ATP-stimulated endoproteolytic activity associated with the inner membrane of E. coli. This ATP-stimulated activity is found in normal levels in membranes derived from lon mutants, including strains carrying insertions in the lon gene. The membrane-bound activity hydrolyzes /sup 14/C-methylglobin at a linear rate for up to 3 hours. These fractions also contain appreciable proteolytic activity that is not affected by ATP. The stimulation by ATP requires the presence of Mg/sup 2 +/. Nonhydrolyzable ATP analogs (e.g. AMPPNP or ATP-..gamma..-S) and ADP do not enhance proteolysis. Unlike protease La, the membrane-associated enzyme does not degrade the fluorometric substrate, Glt-Ala-Ala-Phe-MNA, in an ATP-stimulated fashion, and its level is not influenced by high temperature of by the gene which regulates the heat-shock response. The enzyme is inhibited by dichloroisocoumarin and certain peptide chloromethyl ketones. They conclude that E. coli contain at least two ATP-dependent proteases with distinct specificities: one is soluble and the other is membrane-associated.

  17. Anti-proteolytic capacity and bonding durability of proanthocyanidin-biomodified demineralized dentin matrix

    PubMed Central

    Liu, Rui-Rui; Fang, Ming; Zhang, Ling; Tang, Cheng-Fang; Dou, Qi; Chen, Ji-Hua

    2014-01-01

    Our previous studies showed that biomodification of demineralized dentin collagen with proanthocyanidin (PA) for a clinically practical duration improves the mechanical properties of the dentin matrix and the immediate resin–dentin bond strength. The present study sought to evaluate the ability of PA biomodification to reduce collagenase-induced biodegradation of demineralized dentin matrix and dentin/adhesive interfaces in a clinically relevant manner. The effects of collagenolytic and gelatinolytic activity on PA-biomodified demineralized dentin matrix were analysed by hydroxyproline assay and gelatin zymography. Then, resin-/dentin-bonded specimens were prepared and challenged with bacterial collagenases. Dentin treated with 2% chlorhexidine and untreated dentin were used as a positive and negative control, respectively. Collagen biodegradation, the microtensile bond strengths of bonded specimens and the micromorphologies of the fractured interfaces were assessed. The results revealed that both collagenolytic and gelatinolytic activity on demineralized dentin were notably inhibited in the PA-biomodified groups, irrespective of PA concentration and biomodification duration. When challenged with exogenous collagenases, PA-biomodified bonded specimens exhibited significantly less biodegradation and maintained higher bond strengths than the untreated control. These results suggest that PA biomodification was effective at inhibiting proteolytic activity on demineralized dentin matrix and at stabilizing the adhesive/dentin interface against enzymatic degradation, is a new concept that has the potential to improve bonding durability. PMID:24810807

  18. Proteolytic Activation of the Porcine Epidemic Diarrhea Coronavirus Spike Fusion Protein by Trypsin in Cell Culture

    PubMed Central

    Wicht, Oliver; Li, Wentao; Willems, Lione; Meuleman, Tom J.; Wubbolts, Richard W.; van Kuppeveld, Frank J. M.; Rottier, Peter J. M.

    2014-01-01

    ABSTRACT Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. IMPORTANCE Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV. PMID:24807723

  19. Reciprocal Degradation of YME1L and OMA1 Adapts Mitochondrial Proteolytic Activity During Stress

    PubMed Central

    Rainbolt, T. Kelly; Lebeau, Justine; Puchades, Cristina; Wiseman, R. Luke

    2016-01-01

    SUMMARY The mitochondrial inner membrane proteases YME1L and OMA1 are critical regulators of essential mitochondrial functions including inner membrane proteostasis maintenance and mitochondrial dynamics. Here, we show that YME1L and OMA1 are reciprocally degraded in response to distinct types of cellular stress. OMA1 is degraded through a YME1L-dependent mechanism in response to toxic insults that depolarize the mitochondrial membrane. Alternatively, insults that depolarize mitochondria and deplete cellular ATP stabilize active OMA1 and promote YME1L degradation. We show that the differential degradation of YME1L and OMA1 alters their proteolytic processing of the dynamin-like GTPase OPA1, a critical regulator of mitochondrial inner membrane morphology, which influences the recovery of tubular mitochondria following membrane depolarization-induced fragmentation. Our results reveal the differential stress-induced degradation of YME1L and OMA1 as a mechanism to sensitively adapt mitochondrial inner membrane protease activity and function in response to distinct types of cellular insults. PMID:26923599

  20. Effect of wine inhibitors on the proteolytic activity of papain from Carica papaya L. latex.

    PubMed

    Benucci, Ilaria; Esti, Marco; Liburdi, Katia

    2015-01-01

    The influence of potential inhibitors naturally present in wine on the proteolytic activity of papain from Carica papaya latex was investigated to evaluate its applicability in white wine protein haze stabilization. Enzymatic activity was tested against a synthetic tripeptide chromogenic substrate in wine-like acidic medium that consisted of tartaric buffer (pH 3.2) supplemented with ethanol, free sulfur dioxide (SO2 ), grape skin and seed tannins within the average ranges of concentrations that are typical in wine. The diagnosis of inhibition type, performed with the graphical method, demonstrated that all of tested wine constituents were reversible inhibitors of papain. The strongest inhibition was exerted by free SO2 , which acted as a mixed-type inhibitor, similar to grape skin and seed tannins. Finally, when tested in table white wines, the catalytic activity of papain, even when if it was ascribable to the hyperbolic behavior of Michaelis-Menten equation, was determined to be strongly affected by free SO2 and total phenol level. PMID:25376439

  1. Proteolytic processing of Atg32 by the mitochondrial i-AAA protease Yme1 regulates mitophagy.

    PubMed

    Wang, Ke; Jin, Meiyan; Liu, Xu; Klionsky, Daniel J

    2013-11-01

    Mitophagy, the autophagic removal of mitochondria, occurs through a highly selective mechanism. In the yeast Saccharomyces cerevisiae, the mitochondrial outer membrane protein Atg32 confers selectivity for mitochondria sequestration as a cargo by the autophagic machinery through its interaction with Atg11, a scaffold protein for selective types of autophagy. The activity of mitophagy in vivo must be tightly regulated considering that mitochondria are essential organelles that produce most of the cellular energy, but also generate reactive oxygen species that can be harmful to cell physiology. We found that Atg32 was proteolytically processed at its C terminus upon mitophagy induction. Adding an epitope tag to the C terminus of Atg32 interfered with its processing and caused a mitophagy defect, suggesting the processing is required for efficient mitophagy. Furthermore, we determined that the mitochondrial i-AAA protease Yme1 mediated Atg32 processing and was required for mitophagy. Finally, we found that the interaction between Atg32 and Atg11 was significantly weakened in yme1∆ cells. We propose that the processing of Atg32 by Yme1 acts as an important regulatory mechanism of cellular mitophagy activity. PMID:24025448

  2. Self-assembled quantum dot-bioconjugates: characterization and use for sensing proteolytic activity

    NASA Astrophysics Data System (ADS)

    Medintz, Igor L.; Pons, Thomas; Sapsford, Kim E.; Dawson, Philip E.; Mattoussi, Hedi

    2008-04-01

    We present a characterization of the metal-affinity driven self-assembly between luminescent CdSe-ZnS core-shell semiconductor quantum dots (QDs) and either peptides or proteins appended with various length terminal polyhistidine tags. We first monitor the kinetics of self-assembly between surface-immobilized QDs and proteins/peptides under flow conditions (immobilized). To accomplish this, the QDs were immobilized onto functionalized substrates and then exposed to dye-labeled peptides/proteins. By using evanescent wave excitation of the substrate, self-assembly was assessed by monitoring the time-dependent changes in the dye fluorescence. This configuration was complemented with experiments using freely diffusing QDs and proteins/peptides (solution-phase) via energy transfer between QDs and dye-labeled proteins/peptides. Cumulatively, these measurements allowed determination of kinetic parameters, including association and dissociation rates (k on and k off) and the binding constant (K d). We find that self-assembly is rapid with an equilibrium constant K d -1 in the low nM. We next demonstrate the importance of understanding this self-assembly by creating QD-peptide bioconjugates which we employ as substrates to monitor the cleavage activity of proteolytic enzymes. This confirms that metal-affinity interactions can provide QD-bioconjugates that are functional and stable.

  3. A Peptide-Based Mechano-sensitive, Proteolytically Stable Hydrogel with Remarkable Antibacterial Properties.

    PubMed

    Baral, Abhishek; Roy, Subhasish; Ghosh, Srabanti; Hermida-Merino, Daniel; Hamley, Ian W; Banerjee, Arindam

    2016-02-23

    A long-chain amino acid containing dipeptide has been found to form a hydrogel in phosphate buffer whose pH ranges from 6.0 to 8.8. The hydrogel formed at pH 7.46 has been characterized by small-angle X-ray scattering (SAXS), wide-angle powder X-ray diffraction (PXRD), Fourier transform infrared (FT-IR) spectroscopy, field-emission scanning electron microscopy (FE-SEM), high-resolution transmission electron microscopy (HR-TEM) imaging and rheological analyses. The microscopic imaging studies suggest the formation of a nanofibrillar three-dimensional (3D) network for the hydrogel. As observed visually and confirmed rheologically, the hydrogel at pH 7.46 exhibits thixotropy. This thixotropic property can be exploited to inject the peptide. Furthermore, the hydrogel exhibits remarkable antibacterial activity against Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, which are responsible for many common diseases. The hydrogel has practical applicability due to its biocompatibility with human red blood cells and human fibroblast cells. Interestingly, this hydrogel shows high resistance toward proteolytic enzymes, making it a new potential antimicrobial agent for future applications. It has also been observed that a small change in molecular structure of the gelator peptide not only turns the gelator into a nongelator molecule under similar conditions, but it also has a significant negative impact on its bactericidal character. PMID:26818698

  4. Nrf2 silencing to inhibit proteolytic defense induced by hyperthermia in HT22 cells

    PubMed Central

    Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

    2016-01-01

    Nrf2 pathway has been known to be protective against cancer progression however recent studies have revealed that the antioxidant activity of Nrf2 contributes to chemotherapy resistance. For many years, hyperthermia has been used as an additional therapy to increase the efficiency of chemotherapy and radiotherapy. Besides the positive effects of hyperthermia during treatment procedure, thermotolerance has been found to develop against heat treatment. Although the involved molecular mechanisms have not been fully clarified, heat shock proteins (HSP) and proteasome activity are known to be involved in the acquisition of thermotolerance. The aim of this study was to investigate the potential beneficial effects of combining hyperthermia with Nrf2 silencing to inhibit molecular mechanisms leading to induction of defense mechanisms in transcription level. Following heat treatment of HT22 cells, HSP70 and the proteasome levels and as well as proteasome activity were found to be elevated in the nucleus. Our results demonstrated that Nrf2 silencing reduced defense mechanisms against heat treatment both in antioxidant and proteolytic manner and Nrf2 may be a potential target for therapeutic approach in order to improve the beneficial effects of hyperthermia in cancer therapy. PMID:26966891

  5. 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. PMID:11807127

  6. Reduced secretion and altered proteolytic processing caused by missense mutations in progranulin.

    PubMed

    Kleinberger, Gernot; Capell, Anja; Brouwers, Nathalie; Fellerer, Katrin; Sleegers, Kristel; Cruts, Marc; Van Broeckhoven, Christine; Haass, Christian

    2016-03-01

    Progranulin (GRN) is a secreted growth factor involved in various cellular functions, and loss-of-function mutations are a major cause of frontotemporal lobar degeneration (FTLD) with TDP-43 positive pathology. Most FTLD-related GRN mutations are nonsense mutations resulting in reduced GRN expression. Nonsynonymous GRN missense mutations have been described as risk factor for neurodegenerative brain diseases, but their pathogenic nature remains largely elusive. We identified a double missense mutation in GRN leading to amino acid changes p.D33E and p.G35R in an FTLD patient from Turkish origin. Biochemical and cell biological analysis of the double-mutation together with 2 so-far uncharacterized GRN missense mutations (p.C105R and p.V514M) revealed a reduced secretion efficiency of the GRN p.D33E/p.G35R and p.C105R proteins. Furthermore, loss of the conserved cysteine residue affects protein folding and altered proteolytic processing by neutrophil elastase and proteinase 3. Our data indicate that the described variants may cause a loss-of-function, albeit to a lesser extent than GRN null mutations, and hence could be considered as low-penetrant risk factors for neurodegenerative diseases. PMID:26811050

  7. The Impact of Proteolytic Pork Hydrolysate on Microbial, Flavor and Free Amino Acids Compounds of Yogurt.

    PubMed

    Lin, Jinzhong; Hua, Baozhen; Xu, Zhiping; Li, Sha; Ma, Chengjie

    2016-01-01

    The aim of this study was to investigate the influence of proteolytic pork hydrolysate (PPH) on yoghurt production by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. Fresh lean pork was cut into pieces and mixed with deionized water and dealt with protease, then the resulting PPH was added to milk to investigate the effects of PPH on yoghurt production. The fermentation time, the viable cell counts, the flavor, free amino acids compounds, and sensory evaluation of yoghurt were evaluated. These results showed that PPH significantly stimulated the growth and acidification of the both bacterial strains. When the content of PPH reached 5% (w/w), the increased acidifying rate occurred, which the fermentation time was one hour less than that of the control, a time saving of up to 20% compared with the control. The viable cell counts, the total free amino acids, and the scores of taste, flavor and overall acceptability in PPH-supplemented yoghurt were higher than the control. Furthermore, the contents of some characteristic flavor compounds including acids, alcohols, aldehydes, ketones and esters were richer than the control. We concluded that the constituents of PPH such as small peptide, vitamins, and minerals together to play the stimulatory roles and result in beneficial effect for the yoghurt starter cultures growth. PMID:27621698

  8. Small-molecule-mediated rescue of protein function by an inducible proteolytic shunt

    PubMed Central

    Pratt, Matthew R.; Schwartz, Edmund C.; Muir, Tom W.

    2007-01-01

    Controlling protein function through posttranslational manipulations has emerged as an attractive complementary technology to existing genetic systems. Often these methods involve developing pharmacological agents to probe protein function without the need to generate a unique compound for each protein family. One common strategy uses small molecules that act as chemical inducers of dimerization by mediating the interaction of two proteins. Herein we report the use of a chemical inducer of dimerization for the development of a posttranslational technology for the manipulation of protein function. This system, split ubiquitin for the rescue of function (SURF), places the complementation of genetically split ubiquitin under the control of rapamycin-induced dimerization of FK506-binding protein and FKBP12-rapamycin-binding protein. Before complementation a “degron” dooms a protein of interest for destruction by the proteasome. Addition of rapamycin results in a proteolytic shunt away from degradation by inducing ubiquitin complementation and cleavage of the protein of interest from the degron. Importantly, the native protein is rescued. We characterized this system with firefly luciferase and went on to apply it to members of three important classes of proteins: proteases (caspase-3), kinases (v-Src), and transcription factors (Smad3). This general strategy should allow for inducible rescue of a variety of proteins in such a way that their native structure and function are maintained. PMID:17563385

  9. Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development.

    PubMed

    Uchiyama, Kazuhiko; Sakiyama, Toshio; Hasebe, Takumu; Musch, Mark W; Miyoshi, Hiroyuki; Nakagawa, Yasushi; He, Tong-Chuan; Lichtenstein, Lev; Naito, Yuji; Itoh, Yoshito; Yoshikawa, Toshikazu; Jabri, Bana; Stappenbeck, Thaddeus; Chang, Eugene B

    2016-01-01

    Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation. PMID:27561676

  10. Proteolytic cleavage of ataxin-7 promotes SCA7 retinal degeneration and neurological dysfunction.

    PubMed

    Guyenet, Stephan J; Mookerjee, Shona S; Lin, Amy; Custer, Sara K; Chen, Sylvia F; Sopher, Bryce L; La Spada, Albert R; Ellerby, Lisa M

    2015-07-15

    The neurodegenerative disorder spinocerebellar ataxia type 7 (SCA7) is caused by a polyglutamine (polyQ) expansion in the ataxin-7 protein, categorizing SCA7 as one member of a large class of heritable neurodegenerative proteinopathies. Cleavage of ataxin-7 by the protease caspase-7 has been demonstrated in vitro, and the accumulation of proteolytic cleavage products in SCA7 patients and mouse models has been identified as an early pathological change. However, it remains unknown whether a causal relationship exists between ataxin-7 proteolysis and in vivo SCA7 disease progression. To determine whether caspase cleavage is a critical event in SCA7 disease pathogenesis, we generated transgenic mice expressing polyQ-expanded ataxin-7 with a second-site mutation (D266N) to prevent caspase-7 proteolysis. When we compared SCA7-D266N mice with SCA7 mice lacking the D266N mutation, we found that SCA7-D266N mice exhibited improved motor performance, reduced neurodegeneration and substantial lifespan extension. Our findings indicate that proteolysis at the D266 caspase-7 cleavage site is an important mediator of ataxin-7 neurotoxicity, suggesting that inhibition of caspase-7 cleavage of polyQ-ataxin-7 may be a promising therapeutic strategy for this untreatable disorder. PMID:25859008

  11. Low-volume multiplexed proteolytic activity assay and inhibitor analysis through a pico-injector array.

    PubMed

    Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Lauffenburger, Doug A; Chen, Chia-Hung

    2015-02-21

    Secreted active proteases, from families of enzymes such as matrix metalloproteinases (MMPs) and ADAMs (a disintegrin and metalloproteinases), participate in diverse pathological processes. To simultaneously measure multiple specific protease activities, a series of parallel enzyme reactions combined with a series of inhibitor analyses for proteolytic activity matrix analysis (PrAMA) are essential but limited due to the sample quantity requirements and the complexity of performing multiple reactions. To address these issues, we developed a pico-injector array to generate 72 different reactions in picoliter-volume droplets by controlling the sequence of combinational injections, which allowed simultaneous recording of a wide range of multiple enzyme reactions and measurement of inhibitor effects using small sample volumes (~10 μL). Multiple MMP activities were simultaneously determined by 9 different substrates and 2 inhibitors using injections from a pico-injector array. Due to the advantages of inhibitor analysis, the MMP/ADAM activities of MDA-MB-231, a breast cancer cell line, were characterized with high MMP-2, MMP-3 and ADAM-10 activity. This platform could be customized for a wide range of applications that also require multiple reactions with inhibitor analysis to enhance the sensitivity by encapsulating different chemical sensors. PMID:25553996

  12. Mitochondrial proteolytic stress induced by loss of mortalin function is rescued by Parkin and PINK1

    PubMed Central

    Burbulla, L F; Fitzgerald, J C; Stegen, K; Westermeier, J; Thost, A-K; Kato, H; Mokranjac, D; Sauerwald, J; Martins, L M; Woitalla, D; Rapaport, D; Riess, O; Proikas-Cezanne, T; Rasse, T M; Krüger, R

    2014-01-01

    The mitochondrial chaperone mortalin was implicated in Parkinson's disease (PD) because of its reduced levels in the brains of PD patients and disease-associated rare genetic variants that failed to rescue impaired mitochondrial integrity in cellular knockdown models. To uncover the molecular mechanisms underlying mortalin-related neurodegeneration, we dissected the cellular surveillance mechanisms related to mitochondrial quality control, defined the effects of reduced mortalin function at the molecular and cellular levels and investigated the functional interaction of mortalin with Parkin and PINK1, two PD-related proteins involved in mitochondrial homeostasis. We found that reduced mortalin function leads to: (1) activation of the mitochondrial unfolded protein response (UPR(mt)), (2) increased susceptibility towards intramitochondrial proteolytic stress, (3) increased autophagic degradation of fragmented mitochondria and (4) reduced mitochondrial mass in human cells in vitro and ex vivo. These alterations caused increased vulnerability toward apoptotic cell death. Proteotoxic perturbations induced by either partial loss of mortalin or chemical induction were rescued by complementation with native mortalin, but not disease-associated mortalin variants, and were independent of the integrity of autophagic pathways. However, Parkin and PINK1 rescued loss of mortalin phenotypes via increased lysosomal-mediated mitochondrial clearance and required intact autophagic machinery. Our results on loss of mortalin function reveal a direct link between impaired mitochondrial proteostasis, UPR(mt) and PD and show that effective removal of dysfunctional mitochondria via either genetic (PINK1 and Parkin overexpression) or pharmacological intervention (rapamycin) may compensate mitochondrial phenotypes. PMID:24743735

  13. Proteolytic Cleavage of the Plasmodium falciparum Circumsporozoite Protein Is a Target of Protective Antibodies.

    PubMed

    Espinosa, Diego A; Gutierrez, Gabriel M; Rojas-López, Maricarmen; Noe, Amy R; Shi, Lirong; Tse, Sze-Wah; Sinnis, Photini; Zavala, Fidel

    2015-10-01

    Studies in animals and human volunteers demonstrate that antibodies against the repeat-region of the Plasmodium circumsporozoite protein (CSP) abrogate sporozoite infection. However, the realization that the N- and C- terminal regions flanking the repeats play essential roles in parasite infectivity raised the possibility that they could be targeted by protective antibodies. We characterized a monoclonal antibody (mAb5D5) specific for the N-terminus of the P. falciparum CSP, which inhibits the proteolytic cleavage of the CSP, a key requirement for parasite infection of hepatocytes. Adoptive transfer of mAb5D5 strongly inhibits the in vivo infection of sporozoites expressing the N-terminus of P. falciparum CSP, and this protection is greatly enhanced when combined with antirepeat antibodies. Our results show that antibodies interfering with molecular processes required for parasite infectivity can exert a strong in vivo protective activity and indicate that pre-erythrocytic vaccines against Plasmodium should include the CSP N-terminal region. PMID:25762791

  14. Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development

    PubMed Central

    Uchiyama, Kazuhiko; Sakiyama, Toshio; Hasebe, Takumu; Musch, Mark W.; Miyoshi, Hiroyuki; Nakagawa, Yasushi; He, Tong-Chuan; Lichtenstein, Lev; Naito, Yuji; Itoh, Yoshito; Yoshikawa, Toshikazu; Jabri, Bana; Stappenbeck, Thaddeus; Chang, Eugene B.

    2016-01-01

    Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation. PMID:27561676

  15. Doxorubicin blocks proliferation of cancer cells through proteolytic activation of CREB3L1

    PubMed Central

    Denard, Bray; Lee, Ching; Ye, Jin

    2012-01-01

    Doxorubicin is used extensively for chemotherapy of diverse types of cancer, yet the mechanism through which it inhibits proliferation of cancer cells remains unclear. Here we report that doxorubicin stimulates de novo synthesis of ceramide, which in turn activates CREB3L1, a transcription factor synthesized as a membrane-bound precursor. Doxorubicin stimulates proteolytic cleavage of CREB3L1 by Site-1 Protease and Site-2 Protease, allowing the NH2-terminal domain of CREB3L1 to enter the nucleus where it activates transcription of genes encoding inhibitors of the cell cycle, including p21. Knockdown of CREB3L1 mRNA in human hepatoma Huh7 cells and immortalized human fibroblast SV589 cells conferred increased resistance to doxorubicin, whereas overexpression of CREB3L1 in human breast cancer MCF-7 cells markedly enhanced the sensitivity of these cells to doxorubicin. These results suggest that measurement of CREB3L1 expression may be a useful biomarker in identifying cancer cells sensitive to doxorubicin. DOI: http://dx.doi.org/10.7554/eLife.00090.001 PMID:23256041

  16. Hypoxia Associated Proteolytic Processing of OS-9 by the Metalloproteinase Meprin β.

    PubMed

    Martin, Barry Lee; Conley, Sabena Michelle; Harris, Regine Simone; Stanley, Corshe Devon; Niyitegeka, Jean-Marie Vianney; Ongeri, Elimelda Moige

    2016-01-01

    Meprin metalloproteases play a role in the pathology of ischemia/reperfusion- (IR-) induced renal injury. The endoplasmic reticulum-associated protein, osteosarcoma-9 (OS-9), has been shown to interact with the carboxyl-terminal tail of meprin β. More importantly, OS-9 interacts with the hypoxia inducible factor-1α (HIF-1α) and the prolyl-hydroxylase, proteins which mediate the cell's response to hypoxia. To determine if OS-9 is a meprin substrate, kidney proteins from meprin αβ knockout mice (αβKO) (which lack endogenous meprins) and purified human OS-9 were incubated with activated forms of meprin A and meprin B, and Western blot analysis was used to evaluate proteolytic processing of OS-9. Fragmentation of OS-9 was observed in reactions with meprin B, but not meprin A. To determine whether meprin B cleaves OS-9 in vivo, wild-type (WT) and meprin αβKO mice were subjected to IR-induced renal injury. Fragmentation of OS-9 was observed in kidney proteins from WT mice subjected to IR, but not in meprin αβKO counterparts. Transfection of kidney cells (MDCK and HEK293) with meprin β cDNA prevented accumulation of OS-9 following exposure to the hypoxia mimic, CoCl2. These data suggest that meprin β interaction with OS-9 plays a role in the hypoxia response associated with IR-induced renal injury. PMID:27478637

  17. Proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture.

    PubMed

    Wicht, Oliver; Li, Wentao; Willems, Lione; Meuleman, Tom J; Wubbolts, Richard W; van Kuppeveld, Frank J M; Rottier, Peter J M; Bosch, Berend Jan

    2014-07-01

    Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. Importance: Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV. PMID:24807723

  18. Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine

    PubMed Central

    Aubin-Tam, Marie-Eve; Olivares, Adrian O.; Sauer, Robert T.; Baker, Tania A.; Lang, Matthew J.

    2011-01-01

    All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5–8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP. PMID:21496645

  19. Internalization and proteolytic action of botulinum toxins in CNS neurons and astrocytes.

    PubMed

    Verderio, C; Coco, S; Rossetto, O; Montecucco, C; Matteoli, M

    1999-07-01

    Tetanus and botulinum toxins bind and are internalized at the neuromuscular junction. Botulinum neurotoxins (BoNTs) enter the cytosol at the motor nerve terminal; tetanus neurotoxin (TeNT) proceeds retroaxonally inside the motor axon to reach the spinal cord inhibitory interneurons. Although the major target of BoNTs is the peripheral cholinergic terminals, CNS neurons are susceptible to intoxication as well. We investigated the route of entry and the proteolytic activity of BoNT/B and BoNT/F in cultured hippocampal neurons and astrocytes. We show that, differently from TeNT, which enters hippocampal neurons via the process of synaptic vesicle (SV) recycling, BoNTs are internalized and cleave the substrate synaptobrevin/VAMP2 via a process independent of synaptic activity. Labeling of living neurons with Texas Red-conjugated BoNTs and fluoresceinated dextran revealed that these toxins enter hippocampal neurons via endocytic processes not mediated by SV recycling. Botulinum toxins also exploit endocytosis to enter cultured astrocytes, where they partially cleave cellubrevin, a ubiquitous synaptobrevin/VAMP isoform. These results indicate that, in spite of their closely related protein structure, TeNT and BoNTs use different routes to penetrate hippocampal neurons. These findings bear important implications for the identification of the protein receptors of clostridial toxins. PMID:10386990

  20. Haemolytic and proteolytic activity of coagulase-negative staphylococci isolated from mastitis cows.

    PubMed

    Bochniarz, M; Wawron, W

    2012-01-01

    The aim of the present study was to assess the haemolytic and proteolytic activity of coagulase-negative staphylococci (CNS) isolated from cows with mastitis. The study was conducted on 100 CNS strains: S. xylosus (n=28), S. chromogenes (n=26), S. haemolyticus (n=25), S. sciuri (n=14), S. warneri (n=4), S. hominis (n=2), S. saprophyticus (n=1); 22 CNS were isolated from cows with clinical mastitis and 78 from those with subclinical mastitis. The CNS studied showed the ability to produce only alpha-haemolysin and belonged to one strain - S. haemolyticus (21.0% of isolated CNS strains). Haemolysin-positive CNS were responsible for both clinical and subclinical mastitis (22.7% and 20.5%, respectively). The ability to produce protease was found in 31.0% of CNS belonging to two strains: S. chromogenes and S. sciuri. Protease-positive CNS were the etiological factor of both clinical and subclinical mastitis (31.8% and 30.8%, respectively). All S. xylosus, S. warneri, S. hominis, and S. saprophyticus strains were found protease-negative and haemolysin-negative, irrespective of whether they caused clinical or subclinical mastitis in cows. PMID:22708359

  1. Substrate specificity of proteolytic activity in the testes fluid and seminal plasma of the common carp Cyprinus carpio.

    PubMed

    Cejko, B I; Słowińska, M; Judycka, S; Kowalski, R K

    2016-05-01

    Substrate specificity in the seminal plasma and testes fluids of the common carp Cyprinus carpio was determined using gelatin, casein, albumin and haemoglobin. Proteolytic profiles of the testes and seminal plasma were compared. Different ranges of pH (5·5-9·5) and temperature (4-37° C) were used during incubations of seminal plasma proteinases. Differences in proteolytic activity between testes and seminal plasma may reflect specific functions of the testes and sperm ducts in semen production. Seminal plasma metalloproteinases were characterized by higher substrate specificity than were serine proteinases. Zymography optimization for seminal plasma indicated that pH 7·5 and 22° C were the optimal conditions for gel incubations. PMID:27001550

  2. A new insight into phagocytosis of apoptotic cells: proteolytic enzymes divert the recognition and clearance of polymorphonuclear leukocytes by macrophages.

    PubMed

    Guzik, K; Bzowska, M; Smagur, J; Krupa, O; Sieprawska, M; Travis, J; Potempa, J

    2007-01-01

    The recognition of phosphatidylserine (PS) on the surface of any apoptotic cell is considered to be a key event for its clearance. We challenge this concept by showing that pretreatment of neutrophils with either host or bacterial protease affects their uptake by human monocyte-derived macrophages without having an effect on cell-surface PS presentation. Specifically, whereas preincubation of apoptotic neutrophils with cathepsin G or thrombin significantly inhibited their uptake, gingipains R or clostripain enhanced phagocytosis by macrophages. Moreover, bacterial proteinases sensitized healthy neutrophils for uptake by macrophages, whereas endogenous proteinases were unable to elicit this effect. This stimulation was apparently owing to the combined effect of proteolytic cleavage of an antiphagocytic signal (CD31) and the generation of a novel 'eat-me' signal on the neutrophil surface. These results argue that neutrophil recognition and phagocytosis by macrophages is mediated by a protein ligand whose proteolytic modification could affect the local inflammatory process. PMID:16628232

  3. A psychrotrophic Burkholderia cepacia strain isolated from refrigerated raw milk showing proteolytic activity and adhesion to stainless steel.

    PubMed

    Nörnberg, Maria de Fátima Barros Leal; Mentges, Marilene Lenz; Silveira, Silvana Terra; Tondo, Eduardo César; Brandelli, Adriano

    2011-08-01

    The proteolytic activity of a psychrotrophic strain of Burkholderia cepacia isolated from refrigerated raw milk was characterized. Bur. cepacia produced proteolytic activity during growth at refrigeration temperature, with maximum activity at pH 6-7. The enzyme showed relative thermal stability in the range 40-50°C during 25 min, and maintained 80% its initial activity at 76°C/30 s. Milk coagulation assay showed that the crude protease from Bur. cepacia caused coagulation from day 2 for skimmed milk, whereas coagulation was observed from day 5 for whole milk. The adherence of this strain to stainless steel was evaluated, and the substrata had around 107 CFU/cm2 after 15 to 60 min incubation. Results on biofilm development suggest that this bacterium could adhere and to form biofilms even at refrigeration temperatures. These results indicate that Bur. cepacia may represent a potential hazardous to milk and dairy products. PMID:21457588

  4. [The permeability of the hemato-encephalic barrier and the proteolytic potential of the cerebrospinal fluid in severe craniocerebral trauma].

    PubMed

    Churliaev, Iu A; Nikiforova, N V; Lutsik, A A; Kuksinskiĭ, V A; Lykova, O F; Martynenkov, V Ia; Karpenko, V S

    1999-01-01

    To study blood-brain barrier permeability and proteolytic changes in in patients with severe brain injury and to evaluate their impact on its course and outcome, the concentrations of albumin, plasminogen (plasmin), alpha 2-macroglobulin, alpha 2-antiplasmin, and alpha 1-antitrypsin were examined in 58 victims by enzyme immunoassay. The control group comprised 20 patients examined for lumbar discal hernia. The studies indicate that early severe brain injury showed blood-brain barrier dysfunction whose severity can be detected by the spinal fluid levels of albumin, plasminogen, and alpha 2-macroglobulin. Proteolytic changes in spinal fluid are determined by its albumin, plasminogen (plasmin), alpha 2-macroglobulin, alpha 2-antiplasmin, and alpha 1-antitrypsin concentrations and affect the development of secondary brain lesion and they are of practical value. PMID:10696680

  5. Calmodulin inhibitors trigger the proteolytic processing of membrane type-1 matrix metalloproteinase, but not its shedding in glioblastoma cells.

    PubMed Central

    Annabi, B; Pilorget, A; Bousquet-Gagnon, N; Gingras, D; Béliveau, R

    2001-01-01

    Most transmembrane proteins are subjected to limited proteolysis by cellular proteases, and stimulation of cleavage of membrane proteins by calmodulin (CaM) inhibitors was recently shown. The present study investigated the ability of several CaM inhibitors to induce the proteolytic cleavage of the membrane type-1 matrix metalloproteinase (MT1-MMP) from the cell surface of highly invasive U-87 glioblastoma cells. Although no shedding of a soluble MT1-MMP form was induced by CaM inhibitors in the conditioned media, we showed that these inhibitors induced MT1-MMP proteolytic processing to the 43 kDa membrane-bound inactive form that was not correlated with an increase in proMMP-2 activation but rather with an increase in tissue inhibitor of MMPs (TIMP)-2 expression levels. Moreover, this proteolytic processing was sensitive to marimastat suggesting the involvement of MMPs. Interestingly, CaM inhibitors antagonized concanavalin A- and cytochalasin D-induced proMMP-2 activation, and affected the cytoskeletal actin organization resulting in the loss of migratory potential of U-87 glioblastoma cells. Cytoplasmic tail-truncated MT1-MMP constructs expressed in COS-7 cells were also affected by CaM inhibitors suggesting that these inhibitors stimulated MT1-MMP proteolytic processing by mechanisms independent of the CaM-substrate interaction. We also propose that TIMP-2 acts as a negative regulator of MT1-MMP-dependent activities promoted by the action of CaM inhibitors in U-87 glioblastoma cells. PMID:11583578

  6. Effects of reducing fat content on the proteolytic and rheological properties of Cheddar-like caprine milk cheese

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High-moisture Cheddar-like cheeses made from caprine milk containing 3.6, 2.0, 1.0, and 0.1-0.5% fat were manufactured and their proteolytic and rheological properties compared after 1, 3, and 6 mo of aging at 4 deg C. The full-fat (FF), reduced fat (RF), low-fat (LF), and non-fat (NF) cheeses conta...

  7. Influence of Selective Fluorination on the Biological Activity and Proteolytic Stability of Glucagon-like Peptide-1

    PubMed Central

    Meng, He; Krishnaji, Subrahmanian Tarakkad; Beinborn, Martin; Kumar, Krishna

    2009-01-01

    The relative simplicity and high specificity of peptide therapeutics has fueled recent interest. However, peptide and protein drugs generally require injection and suffer from low metabolic stability. We report here the design, synthesis and characterization of fluorinated analogues of the gut hormone peptide, GLP-1. Overall, fluorinated GLP-1 analogues displayed higher proteolytic stability with simultaneous retention of biological activity (efficacy). Fluorinated amino acids are useful for engineering peptide drug candidates and probing ligand-receptor interactions. PMID:18950150

  8. Effect of culturing conditions on the expression of key enzymes in the proteolytic system of Lactobacillus bulgaricus *

    PubMed Central

    Hou, Jun-cai; Liu, Fei; Ren, Da-xi; Han, Wei-wei; Du, Yue-ou

    2015-01-01

    The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions (different phases, initial pH values, temperatures, and nitrogen sources) using real-time polymerase chain reaction (RT-PCR). The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase. The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains (P<0.05). All these results indicated that the culturing conditions affected the expression of the proteolytic system genes in Lactobacillus bulgaricus at the transcription level. PMID:25845365

  9. An evaluation of the proteolytic and lipolytic potential of Penicillium spp. isolated from traditional Greek sausages in submerged fermentation.

    PubMed

    Papagianni, Maria

    2014-01-01

    A number of novel Penicillium strains belonging to Penicillium nalgiovense, Penicillium solitum, Penicillium commune, Penicillium olsonii, and Penicillium oxalicum species, isolated from the surface of traditional Greek sausages, were evaluated for their proteolytic and lipolytic potential in a solid substrate first and next in submerged fermentations, using complex media. Extracellular proteolytic activity was assessed at acid, neutral, and alkaline pH, while the lipolytic activity was assessed using olive oil, the short-chain triacylglycerol tributyrin, and the long-chain triolein, as substrates. The study revealed that although closely related, the tested strains produce enzymes of distinct specificities. P. nalgiovense PNA9 produced the highest alkaline proteolytic activity (13.2 unit (U)/ml) and the highest lipolytic activity with tributyrin (92 U/ml). Comparisons with known sources show that proteases and/or lipases can be secreted effectively by some Penicillia (P. nalgiovense PNA4, PNA7, and PNA9 and P. solitum PSO1), and further investigations on their properties and characteristics would be promising. PMID:24122629

  10. Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF-R1 receptosomes

    PubMed Central

    Edelmann, Bärbel; Bertsch, Uwe; Tchikov, Vladimir; Winoto-Morbach, Supandi; Perrotta, Cristiana; Jakob, Marten; Adam-Klages, Sabine; Kabelitz, Dieter; Schütze, Stefan

    2011-01-01

    We previously demonstrated that tumour necrosis factor (TNF)-induced ceramide production by endosomal acid sphingomyelinase (A-SMase) couples to apoptosis signalling via activation of cathepsin D and cleavage of Bid, resulting in caspase-9 and caspase-3 activation. The mechanism of TNF-mediated A-SMase activation within the endolysosomal compartment is poorly defined. Here, we show that TNF-induced A-SMase activation depends on functional caspase-8 and caspase-7 expression. The active forms of all three enzymes, caspase-8, caspase-7 and A-SMase, but not caspase-3, colocalize in internalized TNF receptosomes. While caspase-8 and caspase-3 are unable to induce activation of purified pro-A-SMase, we found that caspase-7 mediates A-SMase activation by direct interaction resulting in proteolytic cleavage of the 72-kDa pro-A-SMase zymogen at the non-canonical cleavage site after aspartate 253, generating an active 57 kDa A-SMase molecule. Caspase-7 down modulation revealed the functional link between caspase-7 and A-SMase, confirming proteolytic cleavage as one further mode of A-SMase activation. Our data suggest a signalling cascade within TNF receptosomes involving sequential activation of caspase-8 and caspase-7 for induction of A-SMase activation by proteolytic cleavage of pro-A-SMase. PMID:21157428