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Sample records for integrating lys-n proteolysis

  1. Cell cycle-dependent adaptor complex for ClpXP-mediated proteolysis directly integrates phosphorylation and second messenger signals.

    PubMed

    Smith, Stephen C; Joshi, Kamal K; Zik, Justin J; Trinh, Katherine; Kamajaya, Aron; Chien, Peter; Ryan, Kathleen R

    2014-09-30

    The cell-division cycle of Caulobacter crescentus depends on periodic activation and deactivation of the essential response regulator CtrA. Although CtrA is critical for transcription during some parts of the cell cycle, its activity must be eliminated before chromosome replication because CtrA also blocks the initiation of DNA replication. CtrA activity is down-regulated both by dephosphorylation and by proteolysis, mediated by the ubiquitous ATP-dependent protease ClpXP. Here we demonstrate that proteins needed for rapid CtrA proteolysis in vivo form a phosphorylation-dependent and cyclic diguanylate (cdG)-dependent adaptor complex that accelerates CtrA degradation in vitro by ClpXP. The adaptor complex includes CpdR, a single-domain response regulator; PopA, a cdG-binding protein; and RcdA, a protein whose activity cannot be predicted. When CpdR is unphosphorylated and when PopA is bound to cdG, they work together with RcdA in an all-or-none manner to reduce the Km of CtrA proteolysis 10-fold. We further identified a set of amino acids in the receiver domain of CtrA that modulate its adaptor-mediated degradation in vitro and in vivo. Complex formation between PopA and CtrA depends on these amino acids, which reside on alpha-helix 1 of the CtrA receiver domain, and on cdG binding by PopA. These results reveal that each accessory factor plays an essential biochemical role in the regulated proteolysis of CtrA and demonstrate, to our knowledge, the first example of a multiprotein, cdG-dependent proteolytic adaptor.

  2. Cell cycle-dependent adaptor complex for ClpXP-mediated proteolysis directly integrates phosphorylation and second messenger signals

    PubMed Central

    Smith, Stephen C.; Joshi, Kamal K.; Zik, Justin J.; Trinh, Katherine; Kamajaya, Aron; Chien, Peter; Ryan, Kathleen R.

    2014-01-01

    The cell-division cycle of Caulobacter crescentus depends on periodic activation and deactivation of the essential response regulator CtrA. Although CtrA is critical for transcription during some parts of the cell cycle, its activity must be eliminated before chromosome replication because CtrA also blocks the initiation of DNA replication. CtrA activity is down-regulated both by dephosphorylation and by proteolysis, mediated by the ubiquitous ATP-dependent protease ClpXP. Here we demonstrate that proteins needed for rapid CtrA proteolysis in vivo form a phosphorylation-dependent and cyclic diguanylate (cdG)-dependent adaptor complex that accelerates CtrA degradation in vitro by ClpXP. The adaptor complex includes CpdR, a single-domain response regulator; PopA, a cdG-binding protein; and RcdA, a protein whose activity cannot be predicted. When CpdR is unphosphorylated and when PopA is bound to cdG, they work together with RcdA in an all-or-none manner to reduce the Km of CtrA proteolysis 10-fold. We further identified a set of amino acids in the receiver domain of CtrA that modulate its adaptor-mediated degradation in vitro and in vivo. Complex formation between PopA and CtrA depends on these amino acids, which reside on alpha-helix 1 of the CtrA receiver domain, and on cdG binding by PopA. These results reveal that each accessory factor plays an essential biochemical role in the regulated proteolysis of CtrA and demonstrate, to our knowledge, the first example of a multiprotein, cdG-dependent proteolytic adaptor. PMID:25197043

  3. Proteolysis in hyperthermophilic microorganisms

    DOE PAGES

    Ward, Donald E.; Shockley, Keith R.; Chang, Lara S.; Levy, Ryan D.; Michel, Joshua K.; Conners, Shannon B.; Kelly, Robert M.

    2002-01-01

    Proteases are found in every cell, where they recognize and break down unneeded or abnormal polypeptides or peptide-based nutrients within or outside the cell. Genome sequence data can be used to compare proteolytic enzyme inventories of different organisms as they relate to physiological needs for protein modification and hydrolysis. In this review, we exploit genome sequence data to compare hyperthermophilic microorganisms from the euryarchaeotal genus Pyrococcus , the crenarchaeote Sulfolobus solfataricus , and the bacterium Thermotoga maritima . An overview of the proteases in these organisms is given based on those proteases that have been characterized and on putativemore » proteases that have been identified from genomic sequences, but have yet to be characterized. The analysis revealed both similarities and differences in the mechanisms utilized for proteolysis by each of these hyperthermophiles and indicated how these mechanisms relate to proteolysis in less thermophilic cells and organisms.« less

  4. Plastid intramembrane proteolysis.

    PubMed

    Adam, Zach

    2015-09-01

    Progress in the field of regulated intramembrane proteolysis (RIP) in recent years has not surpassed plant biology. Nevertheless, reports on RIP in plants, and especially in chloroplasts, are still scarce. Of the four different families of intramembrane proteases, only two have been linked to chloroplasts so far, rhomboids and site-2 proteases (S2Ps). The lack of chloroplast-located rhomboid proteases was associated with reduced fertility and aberrations in flower morphology, probably due to perturbations in jasmonic acid biosynthesis, which occurs in chloroplasts. Mutations in homologues of S2P resulted in chlorophyll deficiency and impaired chloroplast development, through a yet unknown mechanism. To date, the only known substrate of RIP in chloroplasts is a PHD transcription factor, located in the envelope. Upon proteolytic cleavage by an unknown protease, the soluble N-terminal domain of this protein is released from the membrane and relocates to the nucleus, where it activates the transcription of the ABA response gene ABI4. Continuing studies on these proteases and substrates, as well as identification of the genes responsible for different chloroplast mutant phenotypes, are expected to shed more light on the roles of intramembrane proteases in chloroplast biology.

  5. Proteolysis in the secretory pathway

    SciTech Connect

    Guzowski, D.E.; Bienkowski, R.S.

    1987-05-01

    Many secretory proteins are degraded intracellularly rather than secreted, however the location of this catabolic process is not known. The authors have tested the hypothesis that the degradation occurs in the organelles of the secretory pathway. Slices of rat liver were incubated with (/sup 14/C)leucine for 3 h and then incubated under chase conditions for 30 min. The tissue was homogenized and the Golgi apparatus, smooth endoplasmic reticulum (sER) and rough endoplasmic reticulum (rER) were isolated by ultracentrifugation on a discontinuous sucrose gradient. The organelles were incubated in 0.3M sucrose-50 mM citrate (pH 4) for 8-12 h at 37 C; control samples were incubated at 4 C. Percent degradation was calculated as the amount of acid soluble radioactivity released relative to total radioactivity in the sample. Proteolysis in the organelles incubated at 37 C was as follows: Golgi: 15-25%; sER: 10-20%; rER: 10-20%. Proteolysis at 4 C was negligible in all cases. These results support the hypothesis that the compartments of the secretory pathway are capable of degrading newly synthesized secretory proteins.

  6. Endolysosomal proteolysis and its regulation.

    PubMed Central

    Pillay, Ché S; Elliott, Edith; Dennison, Clive

    2002-01-01

    The endolysosomal system comprises a unique environment for proteolysis, which is regulated in a manner that apparently does not involve protease inhibitors. The system comprises a series of membrane-bound intracellular compartments, within which endocytosed material and redundant cellular components are hydrolysed. Endocytosed material tends to flow vectorially through the system, proceeding through the early endosome, the endosome carrier vesicle, the late endosome and the lysosome. Phagocytosis and autophagy provide alternative entry points into the system. Late endosomes, lysosome/late endosome hybrid organelles, phagosomes and autophagosomes are the principal sites for proteolysis. In each case, hydrolytic competence is due to components of the endolysosomal system, i.e. proteases, lysosome-associated membrane proteins, H(+)-ATPases and possibly cysteine transporters. The view is emerging that lysosomes are organelles for the storage of hydrolases, perhaps in an inactivated form. Once a substrate has entered a proteolytically competent environment, the rate-limiting proteolytic steps are probably effected by cysteine endoproteinases. As these are affected by pH and possibly redox potential, they may be regulated by the organelle luminal environment. Regulation is probably also affected, among other factors, by organelle fusion reactions, whereby the meeting of enzyme and substrate may be controlled. Such systems would permit simultaneous regulation of a number of unrelated hydrolases. PMID:11964142

  7. Building phenomenological models that relate proteolysis in pork muscles to temperature, water and salt content.

    PubMed

    Harkouss, Rami; Safa, Hassan; Gatellier, Philippe; Lebert, André; Mirade, Pierre-Sylvain

    2014-05-15

    Throughout dry-cured ham production, salt and water content, pH and temperature are key factors affecting proteolysis, one of the main biochemical processes influencing sensory properties and final quality of the product. The aim of this study was to quantify the effect of these variables (except pH) on the time course of proteolysis in laboratory-prepared pork meat samples. Based on a Doehlert design, samples of five different types of pork muscle were prepared, salted, dried and placed at different temperatures, and sampled at different times for quantification of proteolysis. Statistical analysis of the experimental results showed that the proteolysis index (PI) was correlated positively with temperature and water content, but negatively with salt content. Applying response surface methodology and multiple linear regressions enabled us to build phenomenological models relating PI to water and salt content, and to temperature. These models could then be integrated into a 3D numerical ham model, coupling salt and water transfers to proteolysis. PMID:24423495

  8. Extracellular proteolysis in the adult murine brain.

    PubMed

    Sappino, A P; Madani, R; Huarte, J; Belin, D; Kiss, J Z; Wohlwend, A; Vassalli, J D

    1993-08-01

    Plasminogen activators are important mediators of extracellular metabolism. In the nervous system, plasminogen activators are thought to be involved in the remodeling events required for cell migration during development and regeneration. We have now explored the expression of the plasminogen activator/plasmin system in the adult murine central nervous system. Tissue-type plasminogen activator is synthesized by neurons of most brain regions, while prominent tissue-type plasminogen activator-catalyzed proteolysis is restricted to discrete areas, in particular within the hippocampus and hypothalamus. Our observations indicate that tissue-type plasminogen activator-catalyzed proteolysis in neural tissues is not limited to ontogeny, but may also contribute to adult central nervous system physiology, for instance by influencing neuronal plasticity and synaptic reorganization. The identification of an extracellular proteolytic system active in the adult central nervous system may also help gain insights into the pathogeny of neurodegenerative disorders associated with extracellular protein deposition.

  9. The discovery of ubiquitin-dependent proteolysis

    PubMed Central

    Wilkinson, Keith D.

    2005-01-01

    In early 1980, Irwin A. Rose, Avram Hershko, and Aaron Ciechanover published two papers in PNAS that reported the astounding observation that energy-dependent intracellular proteolysis was far more complicated than the previously accepted models of lysosomal proteolysis or the action of ATP-dependent proteases such as bacterial lon. In fact, it has turned out to be even more complicated than they could have suspected. The general model of covalently attaching a small protein as a targeting signal has proved to be every bit as important to eukaryotic cells as the better understood modifications such as phosphorylation or acetylation. The key player in this modification, a small protein called ubiquitin (APF-1 in these papers), is the founding member of a large family of proteins containing the β-grasp fold and is used as a posttranslational targeting signal to modify the structure, function, and/or localization of other proteins. The story of this discovery is a textbook example of the confluence of intellectual curiosity, unselfish collaboration, chance, luck, and preparation. PMID:16230621

  10. Bioelectrochemical regulation accelerates facultatively syntrophic proteolysis.

    PubMed

    Sasaki, Daisuke; Sasaki, Kengo; Morita, Masahiko; Hirano, Shin-ichi; Matsumoto, Norio; Ohmura, Naoya

    2012-07-01

    Bioelectrochemical systems can affect microbial metabolism by controlling the redox potential. We constructed bioelectrochemical cultures of the proteolytic bacterium, Coprothermobacter proteolyticus strain CT-1, both as a single-culture and as a co-culture with the hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain ∆H, to investigate the influences of bioelectrochemical regulation on facultatively syntrophic proteolysis. The co-culture and single-culture were cultivated at 55°C with an anaerobic medium containing casein as the carbon source. The working electrode potential of the bioelectrochemical system was controlled at -0.8V (vs. Ag/AgCl) for bioelectrochemical cultures and was not controlled for non-bioelectrochemical cultures. The cell densities of hydrogenotrophic methanogen and methane production in the bioelectrochemical co-culture were 3.6 and 1.5 times higher than those in the non-bioelectrochemical co-culture after 7 days of cultivation, respectively. Contrastingly, the cell density of Coprothermobacter sp. in the bioelectrochemical co-culture was only 1.3 times higher than that in the non-bioelectrochemical co-culture. The protein decomposition rates were nearly proportional to the cell density of Coprothermobacter sp. in the all types of cultures. These results indicate that bioelectrochemical regulation, particularly, affected the carbon fixation of the hydrogenotrophic methanogen and that facultatively syntrophic proteolysis was accelerated as a result of hydrogen consumption by the methanogens growing well in bioelectrochemical co-cultures. PMID:22421636

  11. Proteolysis of beta-dystroglycan in muscular diseases.

    PubMed

    Matsumura, Kiichiro; Zhong, Di; Saito, Fumiaki; Arai, Ken; Adachi, Katsuhito; Kawai, Hisaomi; Higuchi, Itsuro; Nishino, Ichizo; Shimizu, Teruo

    2005-05-01

    Alpha-dystroglycan is a cell surface peripheral membrane protein which binds to the extracellular matrix (ECM), while beta-dystroglycan is a type I integral membrane protein which anchors alpha-dystroglycan to the cell membrane via the N-terminal extracellular domain. The complex composed of alpha-and beta-dystroglycan is called the dystroglycan complex. We reported previously a matrix metalloproteinase (MMP) activity that disrupts the dystroglycan complex by cleaving the extracellular domain of beta-dystroglycan. This MMP creates a characteristic 30 kDa fragment of beta-dystroglycan that is detected by the monoclonal antibody 43DAG/8D5 directed against the C-terminus of beta-dystroglycan. We also reported that the 30 kDa fragment of beta-dystroglycan was increased in the skeletal and cardiac muscles of cardiomyopathic hamsters, the model animals of sarcoglycanopathy, and that this resulted in the disruption of the link between the ECM and cell membrane via the dystroglycan complex. In this study, we investigated the proteolysis of beta-dystroglycan in the biopsied skeletal muscles of various human muscular diseases, including sarcoglycanopathy, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, Fukuyama congenital muscular dystrophy, Miyoshi myopathy, LGMD2A, facioscapulohumeral muscular dystrophy, myotonic dystrophy and dermatomyositis/polymyositis. We show that the 30 kDa fragment of beta-dystroglycan is increased significantly in sarcoglycanopathy and DMD, but not in the other diseases. We propose that the proteolysis of beta-dystroglycan may contribute to skeletal muscle degeneration by disrupting the link between the ECM and cell membrane in sarcoglycanopathy and DMD.

  12. Pulse Proteolysis and Precipitation for Target Identification.

    PubMed

    Trindade, Rogério V; Pinto, Antônio F M; Santos, Diógenes S; Bizarro, Cristiano V

    2016-07-01

    In recent years, phenotypic screening has assumed a leading role in drug discovery efforts. However, development of new drugs from bioactive compounds obtained in screening campaigns requires identification of the cellular targets responsible for their biological activities. A new energetics-based method for target identification is presented: pulse proteolysis and precipitation for target identification (PePTID). In this method, proteins incubated with or without a ligand and submitted to a brief proteolytic pulse are directly analyzed and compared using a label-free semiquantitative mass spectrometry strategy, dispensing the SDS-PAGE readout and greatly improving the throughput. As a proof-of-concept, we applied the PePTID method to identify ATP-binding proteins in Mycobacterium smegmatis, a model system for Mycobacterium tuberculosis, the etiological agent of tuberculosis. PMID:27255303

  13. General and regulatory proteolysis in Bacillus subtilis.

    PubMed

    Molière, Noël; Turgay, Kürşad

    2013-01-01

    The soil-dwelling bacterium Bacillus subtilis is widely used as a model organism to study the Gram-positive branch of Bacteria. A variety of different developmental pathways, such as endospore formation, genetic competence, motility, swarming and biofilm formation, have been studied in this organism. These processes are intricately connected and regulated by networks containing e.g. alternative sigma factors, two-component systems and other regulators. Importantly, in some of these regulatory networks the activity of important regulatory factors is controlled by proteases. Furthermore, together with chaperones, the same proteases constitute the cellular protein quality control (PQC) network, which plays a crucial role in protein homeostasis and stress tolerance of this organism. In this review, we will present the current knowledge on regulatory and general proteolysis in B. subtilis and discuss its involvement in developmental pathways and cellular stress management.

  14. Proteolysis of Xenopus laevis egg envelope ZPA triggers envelope hardening.

    PubMed

    Lindsay, Leann L; Hedrick, Jerry L

    2004-11-12

    The egg envelope of most animal eggs is modified following fertilization, resulting in the prevention of polyspermy and hardening of the egg envelope. In frogs and mammals a prominent feature of envelope modification is N-terminal proteolysis of the envelope glycoprotein ZPA. We have purified the ZPA protease from Xenopus laevis eggs and characterized it as a zinc metalloprotease. Proteolysis of isolated egg envelopes by the isolated protease resulted in envelope hardening. The N-terminal peptide fragment of ZPA remained disulfide bond linked to the ZPA glycoprotein moiety following proteolysis. We propose a mechanism for egg envelope hardening involving ZPA proteolysis by an egg metalloprotease as a triggering event followed by induction of global conformational changes in egg envelope glycoproteins. PMID:15474476

  15. Allosteric regulation of rhomboid intramembrane proteolysis

    PubMed Central

    Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

    2014-01-01

    Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. PMID:25009246

  16. Regulatory Proteolysis in Arabidopsis-Pathogen Interactions

    PubMed Central

    Pogány, Miklós; Dankó, Tamás; Kámán-Tóth, Evelin; Schwarczinger, Ildikó; Bozsó, Zoltán

    2015-01-01

    Approximately two and a half percent of protein coding genes in Arabidopsis encode enzymes with known or putative proteolytic activity. Proteases possess not only common housekeeping functions by recycling nonfunctional proteins. By irreversibly cleaving other proteins, they regulate crucial developmental processes and control responses to environmental changes. Regulatory proteolysis is also indispensable in interactions between plants and their microbial pathogens. Proteolytic cleavage is simultaneously used both by plant cells, to recognize and inactivate invading pathogens, and by microbes, to overcome the immune system of the plant and successfully colonize host cells. In this review, we present available results on the group of proteases in the model plant Arabidopsis thaliana whose functions in microbial pathogenesis were confirmed. Pathogen-derived proteolytic factors are also discussed when they are involved in the cleavage of host metabolites. Considering the wealth of review papers available in the field of the ubiquitin-26S proteasome system results on the ubiquitin cascade are not presented. Arabidopsis and its pathogens are conferred with abundant sets of proteases. This review compiles a list of those that are apparently involved in an interaction between the plant and its pathogens, also presenting their molecular partners when available. PMID:26404238

  17. Data-driven synthesis of proteolysis-resistant peptide hormones.

    PubMed

    Prothiwa, Michaela; Syed, Ismail; Huising, Mark O; van der Meulen, Talitha; Donaldson, Cynthia J; Trauger, Sunia A; Kahn, Barbara B; Saghatelian, Alan

    2014-12-24

    Peptide hormones are key physiological regulators, and many would make terrific drugs; however, the therapeutic use of peptides is limited by poor metabolism including rapid proteolysis. To develop novel proteolysis-resistant peptide hormone analogs, we utilize a strategy that relies on data from simple mass spectrometry experiments to guide the chemical synthesis of proteolysis-resistant analogs (i.e., data-driven synthesis). Application of this strategy to oxyntomodulin (OXM), a peptide hormone that stimulates insulin secretion from islets and lowers blood glucose in vivo, defined the OXM cleavage site in serum, and this information was used to synthesize a proteolysis-resistant OXM analog (prOXM). prOXM and OXM have similar activity in binding and glucose stimulated-insulin secretion assays. Furthermore, prOXM is also active in vivo. prOXM reduces basal glucose levels and improves glucose tolerance in mice. The discovery of prOXM suggests that proteolysis-resistant variants of other important peptide hormones can also be found using this strategy to increase the number of candidate therapeutic peptides. PMID:25496053

  18. Protein oxidation affects proteolysis in a meat model system.

    PubMed

    Berardo, Alberto; Claeys, Erik; Vossen, Els; Leroy, Frédéric; De Smet, Stefaan

    2015-08-01

    The effect of hydrogen peroxide-induced protein oxidation and pH (4.8 and 5.2) on meat proteolysis was investigated in a meat model system for dry fermented sausages. In oxidised samples, increased protein carbonyl contents and decreased thiol concentrations were found. The initial concentration of protein carbonyls was significantly lower in oxidised samples at pH4.8 than in ones at pH5.2, but after ten days comparable levels were reached. The inhibition of proteolysis by the addition of a protease inhibitor cocktail did not influence protein oxidation. Yet, proteolysis was negatively affected by low pH values as well as by oxidation, resulting in a reduced release of amino acids during ripening.

  19. Biofilm recruitment of Vibrio cholerae by matrix proteolysis.

    PubMed

    Duperthuy, Marylise; Uhlin, Bernt Eric; Wai, Sun Nyunt

    2015-11-01

    The appearance of bacterial biofilms involves secretion of polysaccharides and proteins that form an extracellular matrix embedding the bacteria. Proteases have also been observed, but their role has remained unclear. Smith and co-workers have now found that proteolysis can contribute to further recruitment of bacteria to Vibrio cholerae biofilms.

  20. Effects of reduction and proteolysis on cashew allergens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Allergic reaction to cashew ingestion is frequently more severe than reaction to peanut ingestion, and food allergens are commonly resistant to digestive proteases. The purpose of this study was to characterize the sensitivity of cashew proteins to proteolysis. Cashew protein extracts and purified c...

  1. Inhibition of proteolysis in oxidized lipid-damaged proteins.

    PubMed

    Zamora, R; Hidalgo, F J

    2001-12-01

    The proteolysis of bovine serum albumin (BSA) modified by reaction with the lipid peroxidation product 4,5(E)-epoxy-2(E)-heptenal was studied to better understand the loss of digestibility observed in oxidized lipid-damaged proteins. BSA was incubated for different periods of time with eight concentrations of the epoxyalkenal and, then, treated for 24 h with chymotrypsin, pancreatin, Pronase, or trypsin. The treatment of BSA with the aldehyde always decreased its proteolysis in relation to that of native BSA, and this inhibition of the proteolysis was related to the concentration of the epoxyalkenal and the reaction time. In fact, this inhibition was correlated with the damage suffered by the protein as a consequence of its reaction with the aldehyde: mainly the development of browning, the denaturation of the protein, and the formation of the oxidized lipid/amino acid reaction product epsilon-N-pyrrolylnorleucine (p < or = 0.0011, 0.0045, and 0.0031, respectively). In addition, epsilon-N-pyrrolylnorleucine added at 0.1 or 1 mM inhibited the proteases assayed and suggested that the inhibition of the proteolysis observed in oxidized lipid-damaged proteins may be related to the formation and accumulation of pyrrolized amino acid residues. PMID:11743800

  2. Adaptor-mediated Lon proteolysis restricts Bacillus subtilis hyperflagellation.

    PubMed

    Mukherjee, Sampriti; Bree, Anna C; Liu, Jing; Patrick, Joyce E; Chien, Peter; Kearns, Daniel B

    2015-01-01

    The Lon AAA+ protease is a highly conserved intracellular protease that is considered an anticancer target in eukaryotic cells and a crucial virulence regulator in bacteria. Lon degrades both damaged, misfolded proteins and specific native regulators, but how Lon discriminates among a large pool of candidate targets remains unclear. Here we report that Bacillus subtilis LonA specifically degrades the master regulator of flagellar biosynthesis SwrA governed by the adaptor protein swarming motility inhibitor A (SmiA). SmiA-dependent LonA proteolysis is abrogated upon microbe-substrate contact causing SwrA protein levels to increase and elevate flagellar density above a critical threshold for swarming motility atop solid surfaces. Surface contact-dependent cellular differentiation in bacteria is rapid, and regulated proteolysis may be a general mechanism of transducing surface stimuli.

  3. Chronically active: activation of microglial proteolysis in ageing and neurodegeneration.

    PubMed

    Stolzing, Alexandra; Sethe, Sebastian; Grune, Tilman

    2005-01-01

    One of the microglial cell functions is the removal of modified extracellular proteins in the brain. The connection between protein oxidation, proteolysis, and microglial activation is the topic of this review. The effect of various activation agents on microglial cells with regard to changes in substrate uptake, proteolytic capacity and degradation efficiency of different types of oxidized protein materials is reviewed. It is shown that different activation stimuli initiate substrate-specific modulation for uptake and proteolysis, influencing an array of factors including receptor expression, lysosomal pH, and proteasome subunit composition. Age-related alterations in activation and proteolytic capacity in microglial cells are also discussed. In ageing, proteolytic effectiveness is diminished, while microglial cells are chronically activated and lose the oxidative burst ability, possibly supporting a 'vicious circle' of macrophage-induced neurodegeneration.

  4. Isolation and purification of versican and analysis of versican proteolysis.

    PubMed

    Foulcer, Simon J; Day, Anthony J; Apte, Suneel S

    2015-01-01

    Versican is a widely distributed chondroitin sulfate proteoglycan that forms large complexes with the glycosaminoglycan hyaluronan (HA). As a consequence of HA binding to its receptor CD44 and interactions of the versican C-terminal globular (G3) domain with a variety of extracellular matrix proteins, versican is a key component of well-defined networks in pericellular matrix and extracellular matrix. It is crucial for several developmental processes in the embryo and there is increasing interest in its roles in cancer and inflammation. Versican proteolysis by ADAMTS proteases is highly regulated, occurs at specific peptide bonds, and is relevant to several physiological and disease mechanisms. In this chapter, methods are described for the isolation and detection of intact and cleaved versican in tissues using morphologic and biochemical techniques. These, together with the methodologies for purification and analysis of recombinant versican and a versican fragment provided here, are likely to facilitate further progress on the biology of versican and its proteolysis.

  5. Protein maturation and proteolysis in plant plastids, mitochondria, and peroxisomes.

    PubMed

    van Wijk, Klaas J

    2015-01-01

    Plastids, mitochondria, and peroxisomes are key organelles with dynamic proteomes in photosynthetic eukaryotes. Their biogenesis and activity must be coordinated and require intraorganellar protein maturation, degradation, and recycling. The three organelles together are predicted to contain ∼200 presequence peptidases, proteases, aminopeptidases, and specific protease chaperones/adaptors, but the substrates and substrate selection mechanisms are poorly understood. Similarly, lifetime determinants of organellar proteins, such as N-end degrons and tagging systems, have not been identified, but the substrate recognition mechanisms likely share similarities between organelles. Novel degradomics tools for systematic analysis of protein lifetime and proteolysis could define such protease-substrate relationships, degrons, and protein lifetime. Intraorganellar proteolysis is complemented by autophagy of whole organelles or selected organellar content, as well as by cytosolic protein ubiquitination and degradation by the proteasome. This review summarizes (putative) plant organellar protease functions and substrate-protease relationships. Examples illustrate key proteolytic events.

  6. Functional significance of membrane associated proteolysis in the toxicity of Bacillus thuringiensis Cry3Aa toxin against Colorado potato beetle.

    PubMed

    García-Robles, Inmaculada; Ochoa-Campuzano, Camila; Sánchez, Jorge; Contreras, Estefanía; Real, M Dolores; Rausell, Carolina

    2012-11-01

    Bacillus thuringiensis Cry toxins are widely used as biocontrol agents in bioinsecticides and transgenic plants. In the three domain-Cry toxins, domain II has been identified as an important determinant of their highly specific activity against insects. In this work, we assessed the role in membrane associated proteolysis and toxicity in Colorado potato beetle (CPB) of a previously reported ADAM recognition motif present in Cry3Aa toxin domain II. We used site-directed mutagenesis to modify the Bacillus thuringiensis cry3A gene in amino acid residues 344, 346, 347, 351 and 353 of the ADAM recognition motif in Cry3Aa toxin. Cry3Aa toxin mutants displayed decreased toxicity when compared to the wild type toxin and impaired ability to compete CPB brush border membrane associated cleavage of an ADAM fluorogenic substrate. Although the proteolytic profile of Cry3Aa toxin mutants generated by brush border membrane associated proteases was similar to that of Cry3Aa toxin, the metalloprotease inhibitor 1,10-phenanthroline was less efficient on the proteolysis of mutants than on that of the wild type toxin. The relevance of the Cry3Aa-ADAM interaction through the predicted recognition sequence was further confirmed by analyzing the effect of membrane integrity disturbance on Cry3Aa toxin membrane associated proteolysis and CPB larvae toxicity. Data support that Cry3Aa proteolysis, as a result of the interaction with ADAM through the Cry3Aa recognition motif, is essential for Cry3Aa toxic action in CPB. Detailed knowledge of Cry3Aa interaction with CPB midgut membrane should facilitate the development of more effective Bt based products against this devastating pest and other Coleoptera. PMID:22884605

  7. Functional significance of membrane associated proteolysis in the toxicity of Bacillus thuringiensis Cry3Aa toxin against Colorado potato beetle.

    PubMed

    García-Robles, Inmaculada; Ochoa-Campuzano, Camila; Sánchez, Jorge; Contreras, Estefanía; Real, M Dolores; Rausell, Carolina

    2012-11-01

    Bacillus thuringiensis Cry toxins are widely used as biocontrol agents in bioinsecticides and transgenic plants. In the three domain-Cry toxins, domain II has been identified as an important determinant of their highly specific activity against insects. In this work, we assessed the role in membrane associated proteolysis and toxicity in Colorado potato beetle (CPB) of a previously reported ADAM recognition motif present in Cry3Aa toxin domain II. We used site-directed mutagenesis to modify the Bacillus thuringiensis cry3A gene in amino acid residues 344, 346, 347, 351 and 353 of the ADAM recognition motif in Cry3Aa toxin. Cry3Aa toxin mutants displayed decreased toxicity when compared to the wild type toxin and impaired ability to compete CPB brush border membrane associated cleavage of an ADAM fluorogenic substrate. Although the proteolytic profile of Cry3Aa toxin mutants generated by brush border membrane associated proteases was similar to that of Cry3Aa toxin, the metalloprotease inhibitor 1,10-phenanthroline was less efficient on the proteolysis of mutants than on that of the wild type toxin. The relevance of the Cry3Aa-ADAM interaction through the predicted recognition sequence was further confirmed by analyzing the effect of membrane integrity disturbance on Cry3Aa toxin membrane associated proteolysis and CPB larvae toxicity. Data support that Cry3Aa proteolysis, as a result of the interaction with ADAM through the Cry3Aa recognition motif, is essential for Cry3Aa toxic action in CPB. Detailed knowledge of Cry3Aa interaction with CPB midgut membrane should facilitate the development of more effective Bt based products against this devastating pest and other Coleoptera.

  8. Effect of high-pressure treatment on hard cheese proteolysis.

    PubMed

    Costabel, Luciana M; Bergamini, Carina; Vaudagna, Sergio R; Cuatrin, Alejandra L; Audero, Gabriela; Hynes, Erica

    2016-06-01

    The application of high hydrostatic pressure (HHP) treatment has been proposed to reduce the ripening time of cheese via modifications in the enzymatic activities or the substrate reactivity. Investigations on the effect of HHP on cheese proteolysis have been undertaken with either encouraging results or little effect according to the treatment conditions and the type of cheese, but information concerning the effect of HHP on the ripening of hard cooked cheese is still lacking. In this report, we describe the effect of HHP treatment on Reggianito cheese proteolysis. For that purpose, 1-d-old miniature cheeses (5.5-cm diameter and 6-cm height) were treated at 100 or 400MPa and 20°C for 5 or 10min, and control cheeses in the trial were not pressurized. All cheeses were ripened at 12°C during 90d. The HHP did not affect gross composition of the cheeses, but microbial load changed, especially because the starter culture count was significantly lower at the beginning of the ripening of the cheeses treated at 400MPa than in controls and cheeses treated at 100MPa. Cheeses treated at 400MPa for 10min had significantly higher plasmin activity than did the others; the residual coagulant activity was not affected by HHP. Proteolysis assessment showed that most severe treatments (400MPa) also resulted in cheeses with increased breakdown of αS1- and β-CN. In addition, nitrogen content in soluble fractions was significantly higher in cheeses treated at 400MPa, as well as soluble peptides and free AA production. Peptide profiles and individual and total content of free AA in 60-d-old treated cheese were as high as in fully ripened control cheeses (90d). Holding time had an effect only on pH-4.6-soluble nitrogen fraction and plasmin activity; cheese treated for 10min showed higher values than those treated for 5min, at both levels of pressure assayed. We concluded that HHP treatments at 400MPa applied 1d after cheesemaking increased the rate of proteolysis, leading to an

  9. Effect of high-pressure treatment on hard cheese proteolysis.

    PubMed

    Costabel, Luciana M; Bergamini, Carina; Vaudagna, Sergio R; Cuatrin, Alejandra L; Audero, Gabriela; Hynes, Erica

    2016-06-01

    The application of high hydrostatic pressure (HHP) treatment has been proposed to reduce the ripening time of cheese via modifications in the enzymatic activities or the substrate reactivity. Investigations on the effect of HHP on cheese proteolysis have been undertaken with either encouraging results or little effect according to the treatment conditions and the type of cheese, but information concerning the effect of HHP on the ripening of hard cooked cheese is still lacking. In this report, we describe the effect of HHP treatment on Reggianito cheese proteolysis. For that purpose, 1-d-old miniature cheeses (5.5-cm diameter and 6-cm height) were treated at 100 or 400MPa and 20°C for 5 or 10min, and control cheeses in the trial were not pressurized. All cheeses were ripened at 12°C during 90d. The HHP did not affect gross composition of the cheeses, but microbial load changed, especially because the starter culture count was significantly lower at the beginning of the ripening of the cheeses treated at 400MPa than in controls and cheeses treated at 100MPa. Cheeses treated at 400MPa for 10min had significantly higher plasmin activity than did the others; the residual coagulant activity was not affected by HHP. Proteolysis assessment showed that most severe treatments (400MPa) also resulted in cheeses with increased breakdown of αS1- and β-CN. In addition, nitrogen content in soluble fractions was significantly higher in cheeses treated at 400MPa, as well as soluble peptides and free AA production. Peptide profiles and individual and total content of free AA in 60-d-old treated cheese were as high as in fully ripened control cheeses (90d). Holding time had an effect only on pH-4.6-soluble nitrogen fraction and plasmin activity; cheese treated for 10min showed higher values than those treated for 5min, at both levels of pressure assayed. We concluded that HHP treatments at 400MPa applied 1d after cheesemaking increased the rate of proteolysis, leading to an

  10. [Role of defective intracellular proteolysis in human degenerative diseases].

    PubMed

    Nezelof, Christian

    2012-11-01

    Although intracellular protein synthesis has been studied extensively, protein degradation and disposal, know as proteolysis, has been relatively neglected. Modern studies which led two Nobel prizes (de Duve in 1950 and Herschko, Rose and Ciechanover in 1980) established that proteolysis is ensured by two separate but complementary mechanisms: lysosomes responsible for auto and heterophagy and the Ubiquitin-Proteasome System (UPS). The UPS involves ubiquitin, a small molecule consisting of 76 amino acids found in all eukaryotic cells that ensures the identification of the protein to be degraded and its transport to the proteasome, an intracellular complex with enzymes which degrade unneeded or damaged proteins. The proteasome, acting as a composting agent, ensures the enzymatic dissociation of the protein. In this degradation process, as infinite screw, ubiquitin, peptides and amino acids are released and made available for a new cycle. Knowledge of the UPS and its related disorders is continually expanding. Concurrent with lysosomes which work in acidic environment, it is currently known that the UPS provides 80% to 90% of the proteolysis of the short-life proteins and ensures, as chaperon-molecules, the right conformation and hence the correct function of the proteins. The proteolytic activity generates abnormal residues (tau protein, amyloid and related proteins) and various soluble and insoluble wastes. Some are precipitated as inclusion-bodies or aggregosomes, identified years ago by pathologists. These aggregosomes affect almost exclusively long-lived cells (nervous and muscular, macophages). Pigment deposits, such as lipofuscines made by the peroxydation of cell membranes, are the most abundant. Due to their diverse chemical composition, they cannot be empoyed for a scientific classification. Failures of these systems are numerous. They vary not according to the chemical nature of the abnormal protein and wastes but the life span of the targeted cells and

  11. Proteolysis of decellularized extracellular matrices results in loss of fibronectin and cell binding activity.

    PubMed

    Ramanathan, Anand; Karuri, Nancy

    2015-04-01

    Excessive inflammation in the chronic wound bed is believed to result in increased fibronectin (FN) proteolysis and poor tissue repair. However, FN fragments can prime the immune response and result in higher protease levels. The reciprocity between FN proteolysis and inflammation makes it challenging to determine the specific contribution of FN proteolysis in the extracellular matrix (ECM) on tissue responses. We studied the impact of proteolysis of decellularized extracellular matrices (dECMs) obtained from NIH 3T3 mouse fibroblasts on FN level and activity. The dECMs were treated with α chymotrypsin and proteolysis was stopped at different time points. The protease solution was obtained, the remaining dECM was scrapped and examined by immunoblotting and Bicinchoninic Acid assays. Fibronectin was 9.4 ± 1.8% of the total protein content in the dECM but was more susceptible to proteolysis. After 15 min of protease treatment there was a 67.6% and 11.1% decrease in FN and total protein, respectively, in the dECMs. Fibronectin fragments were present both in the proteolysis solution and in the dECM. Cell adhesion, spreading and actin extensions on dECMs decreased with increasing proteolysis time. Interestingly, the solutions obtained after proteolysis of the dECMs supported cell adhesion and spreading in a time dependent manner, thus demonstrating the presence of FN cell binding activity in the protease solution of dECMs. This study demonstrates the susceptibility of FN in the ECM to proteolysis and the resulting loss of cell adhesion due to the decrease of FN activity and places weight on bioengineering strategies to stabilize FN against proteolysis.

  12. Plant senescence and proteolysis: two processes with one destiny.

    PubMed

    Diaz-Mendoza, Mercedes; Velasco-Arroyo, Blanca; Santamaria, M Estrella; González-Melendi, Pablo; Martinez, Manuel; Diaz, Isabel

    2016-01-01

    Senescence-associated proteolysis in plants is a complex and controlled process, essential for mobilization of nutrients from old or stressed tissues, mainly leaves, to growing or sink organs. Protein breakdown in senescing leaves involves many plastidial and nuclear proteases, regulators, different subcellular locations and dynamic protein traffic to ensure the complete transformation of proteins of high molecular weight into transportable and useful hydrolysed products. Protease activities are strictly regulated by specific inhibitors and through the activation of zymogens to develop their proteolytic activity at the right place and at the proper time. All these events associated with senescence have deep effects on the relocation of nutrients and as a consequence, on grain quality and crop yield. Thus, it can be considered that nutrient recycling is the common destiny of two processes, plant senescence and, proteolysis. This review article covers the most recent findings about leaf senescence features mediated by abiotic and biotic stresses as well as the participants and steps required in this physiological process, paying special attention to C1A cysteine proteases, their specific inhibitors, known as cystatins, and their potential targets, particularly the chloroplastic proteins as source for nitrogen recycling. PMID:27505308

  13. Plant senescence and proteolysis: two processes with one destiny

    PubMed Central

    Diaz-Mendoza, Mercedes; Velasco-Arroyo, Blanca; Santamaria, M. Estrella; González-Melendi, Pablo; Martinez, Manuel; Diaz, Isabel

    2016-01-01

    Abstract Senescence-associated proteolysis in plants is a complex and controlled process, essential for mobilization of nutrients from old or stressed tissues, mainly leaves, to growing or sink organs. Protein breakdown in senescing leaves involves many plastidial and nuclear proteases, regulators, different subcellular locations and dynamic protein traffic to ensure the complete transformation of proteins of high molecular weight into transportable and useful hydrolysed products. Protease activities are strictly regulated by specific inhibitors and through the activation of zymogens to develop their proteolytic activity at the right place and at the proper time. All these events associated with senescence have deep effects on the relocation of nutrients and as a consequence, on grain quality and crop yield. Thus, it can be considered that nutrient recycling is the common destiny of two processes, plant senescence and, proteolysis. This review article covers the most recent findings about leaf senescence features mediated by abiotic and biotic stresses as well as the participants and steps required in this physiological process, paying special attention to C1A cysteine proteases, their specific inhibitors, known as cystatins, and their potential targets, particularly the chloroplastic proteins as source for nitrogen recycling. PMID:27505308

  14. Plant senescence and proteolysis: two processes with one destiny.

    PubMed

    Diaz-Mendoza, Mercedes; Velasco-Arroyo, Blanca; Santamaria, M Estrella; González-Melendi, Pablo; Martinez, Manuel; Diaz, Isabel

    2016-01-01

    Senescence-associated proteolysis in plants is a complex and controlled process, essential for mobilization of nutrients from old or stressed tissues, mainly leaves, to growing or sink organs. Protein breakdown in senescing leaves involves many plastidial and nuclear proteases, regulators, different subcellular locations and dynamic protein traffic to ensure the complete transformation of proteins of high molecular weight into transportable and useful hydrolysed products. Protease activities are strictly regulated by specific inhibitors and through the activation of zymogens to develop their proteolytic activity at the right place and at the proper time. All these events associated with senescence have deep effects on the relocation of nutrients and as a consequence, on grain quality and crop yield. Thus, it can be considered that nutrient recycling is the common destiny of two processes, plant senescence and, proteolysis. This review article covers the most recent findings about leaf senescence features mediated by abiotic and biotic stresses as well as the participants and steps required in this physiological process, paying special attention to C1A cysteine proteases, their specific inhibitors, known as cystatins, and their potential targets, particularly the chloroplastic proteins as source for nitrogen recycling.

  15. Calcineurin proteolysis in astrocytes: Implications for impaired synaptic function.

    PubMed

    Pleiss, Melanie M; Sompol, Pradoldej; Kraner, Susan D; Abdul, Hafiz Mohmmad; Furman, Jennifer L; Guttmann, Rodney P; Wilcock, Donna M; Nelson, Peter T; Norris, Christopher M

    2016-09-01

    Mounting evidence suggests that astrocyte activation, found in most forms of neural injury and disease, is linked to the hyperactivation of the protein phosphatase calcineurin. In many tissues and cell types, calcineurin hyperactivity is the direct result of limited proteolysis. However, little is known about the proteolytic status of calcineurin in activated astrocytes. Here, we developed a polyclonal antibody to a high activity calcineurin proteolytic fragment in the 45-48kDa range (ΔCN) for use in immunohistochemical applications. When applied to postmortem human brain sections, the ΔCN antibody intensely labeled cell clusters in close juxtaposition to amyloid deposits and microinfarcts. Many of these cells exhibited clear activated astrocyte morphology. The expression of ΔCN in astrocytes near areas of pathology was further confirmed using confocal microscopy. Multiple NeuN-positive cells, particularly those within microinfarct core regions, also labeled positively for ΔCN. This observation suggests that calcineurin proteolysis can also occur within damaged or dying neurons, as reported in other studies. When a similar ΔCN fragment was selectively expressed in hippocampal astrocytes of intact rats (using adeno-associated virus), we observed a significant reduction in the strength of CA3-CA1 excitatory synapses, indicating that the hyperactivation of astrocytic calcineurin is sufficient for disrupting synaptic function. Together, these results suggest that proteolytic activation of calcineurin in activated astrocytes may be a central mechanism for driving and/or exacerbating neural dysfunction during neurodegenerative disease and injury. PMID:27212416

  16. Localization of general and regulatory proteolysis in Bacillus subtilis cells.

    PubMed

    Kirstein, Janine; Strahl, Henrik; Molière, Noël; Hamoen, Leendert W; Turgay, Kürşad

    2008-11-01

    Protein degradation mediated by ATP-dependent proteases, such as Hsp100/Clp and related AAA+ proteins, plays an important role in cellular protein homeostasis, protein quality control and the regulation of, e.g. heat shock adaptation and other cellular differentiation processes. ClpCP with its adaptor proteins and other related proteases, such as ClpXP or ClpEP of Bacillus subtilis, are involved in general and regulatory proteolysis. To determine if proteolysis occurs at specific locations in B. subtilis cells, we analysed the subcellular distribution of the Clp system together with adaptor and general and regulatory substrate proteins, under different environmental conditions. We can demonstrate that the ATPase and the proteolytic subunit of the Clp proteases, as well as the adaptor or substrate proteins, form visible foci, representing active protease clusters localized to the polar and to the mid-cell region. These clusters could represent a compartmentalized place for protein degradation positioned at the pole close to where most of the cellular protein biosynthesis and also protein quality control are taking place, thereby spatially separating protein synthesis and degradation.

  17. Calcineurin proteolysis in astrocytes: Implications for impaired synaptic function.

    PubMed

    Pleiss, Melanie M; Sompol, Pradoldej; Kraner, Susan D; Abdul, Hafiz Mohmmad; Furman, Jennifer L; Guttmann, Rodney P; Wilcock, Donna M; Nelson, Peter T; Norris, Christopher M

    2016-09-01

    Mounting evidence suggests that astrocyte activation, found in most forms of neural injury and disease, is linked to the hyperactivation of the protein phosphatase calcineurin. In many tissues and cell types, calcineurin hyperactivity is the direct result of limited proteolysis. However, little is known about the proteolytic status of calcineurin in activated astrocytes. Here, we developed a polyclonal antibody to a high activity calcineurin proteolytic fragment in the 45-48kDa range (ΔCN) for use in immunohistochemical applications. When applied to postmortem human brain sections, the ΔCN antibody intensely labeled cell clusters in close juxtaposition to amyloid deposits and microinfarcts. Many of these cells exhibited clear activated astrocyte morphology. The expression of ΔCN in astrocytes near areas of pathology was further confirmed using confocal microscopy. Multiple NeuN-positive cells, particularly those within microinfarct core regions, also labeled positively for ΔCN. This observation suggests that calcineurin proteolysis can also occur within damaged or dying neurons, as reported in other studies. When a similar ΔCN fragment was selectively expressed in hippocampal astrocytes of intact rats (using adeno-associated virus), we observed a significant reduction in the strength of CA3-CA1 excitatory synapses, indicating that the hyperactivation of astrocytic calcineurin is sufficient for disrupting synaptic function. Together, these results suggest that proteolytic activation of calcineurin in activated astrocytes may be a central mechanism for driving and/or exacerbating neural dysfunction during neurodegenerative disease and injury.

  18. A novel ELISA-based diagnosis of acquired von Willebrand disease with increased VWF proteolysis.

    PubMed

    Rauch, Antoine; Caron, Claudine; Vincent, Flavien; Jeanpierre, Emmanuelle; Ternisien, Catherine; Boisseau, Pierre; Zawadzki, Christophe; Fressinaud, Edith; Borel-Derlon, Annie; Hermoire, Sylvie; Paris, Camille; Lavenu-Bombled, Cécile; Veyradier, Agnès; Ung, Alexandre; Vincentelli, André; van Belle, Eric; Lenting, Peter J; Goudemand, Jenny; Susen, Sophie

    2016-05-01

    Von Willebrand disease-type 2A (VWD-2A) and acquired von Willebrand syndrome (AVWS) due to aortic stenosis (AS) or left ventricular assist device (LVAD) are associated with an increased proteolysis of von Willebrand factor (VWF). Analysis of VWF multimeric profile is the most sensitive way to assess such increased VWF-proteolysis. However, several technical aspects hamper a large diffusion among routine diagnosis laboratories. This makes early diagnosis and early appropriate care of increased proteolysis challenging. In this context of unmet medical need, we developed a new ELISA aiming a quick, easy and reliable assessment of VWF-proteolysis. This ELISA was assessed successively in a LVAD-model, healthy subjects (n=39), acquired TTP-patients (n=4), VWD-patients (including VWD-2A(IIA), n=22; VWD-2B, n=26; VWD-2A(IIE), n=21; and VWD-1C, n=8) and in AVWS-patients (AS, n=9; LVAD, n=9; and MGUS, n=8). A standard of VWF-proteolysis was specifically developed. Extent of VWF-proteolysis was expressed as relative percentage and as VWF proteolysis/VWF:Ag ratio. A speed-dependent increase in VWF-proteolysis was assessed in the LVAD model whereas no proteolysis was observed in TTP-patients. In VWD-patients, VWF-proteolysis was significantly increased in VWD-2A(IIA) and VWD-2B and significantly decreased in VWD-2A(IIE) versus controls (p< 0.0001). In AVWS-patients, VWF-proteolysis was significantly increased in AS- and LVAD-patients compared to controls (p< 0.0001) and not detectable in MGUS-patients. A significant increase in VWF-proteolysis was detected as soon as three hours after LVAD implantation (p< 0.01). In conclusion, we describe a new ELISA allowing a rapid and accurate diagnosis of VWF-proteolysis validated in three different clinical situations. This assay represents a helpful alternative to electrophoresis-based assay in the diagnosis and management of AVWS with increased VWF-proteolysis. PMID:26791163

  19. A Greek Tragedy: The Growing Complexity of Alzheimer Amyloid Precursor Protein Proteolysis.

    PubMed

    Andrew, Robert J; Kellett, Katherine A B; Thinakaran, Gopal; Hooper, Nigel M

    2016-09-01

    Proteolysis of the amyloid precursor protein (APP) liberates various fragments including the proposed initiator of Alzheimer disease-associated dysfunctions, amyloid-β. However, recent evidence suggests that the accepted view of APP proteolysis by the canonical α-, β-, and γ-secretases is simplistic, with the discovery of a number of novel APP secretases (including δ- and η-secretases, alternative β-secretases) and additional metabolites, some of which may also cause synaptic dysfunction. Furthermore, various proteins have been identified that interact with APP and modulate its cleavage by the secretases. Here, we give an overview of the increasingly complex picture of APP proteolysis. PMID:27474742

  20. A Greek Tragedy: The Growing Complexity of Alzheimer Amyloid Precursor Protein Proteolysis.

    PubMed

    Andrew, Robert J; Kellett, Katherine A B; Thinakaran, Gopal; Hooper, Nigel M

    2016-09-01

    Proteolysis of the amyloid precursor protein (APP) liberates various fragments including the proposed initiator of Alzheimer disease-associated dysfunctions, amyloid-β. However, recent evidence suggests that the accepted view of APP proteolysis by the canonical α-, β-, and γ-secretases is simplistic, with the discovery of a number of novel APP secretases (including δ- and η-secretases, alternative β-secretases) and additional metabolites, some of which may also cause synaptic dysfunction. Furthermore, various proteins have been identified that interact with APP and modulate its cleavage by the secretases. Here, we give an overview of the increasingly complex picture of APP proteolysis.

  1. Circulating Peptidome to Indicate the Tumor-resident Proteolysis

    PubMed Central

    Deng, Zaian; Li, Yaojun; Fan, Jia; Wang, Guohui; Li, Yan; Zhang, Yaou; Cai, Guoping; Shen, Haifa; Ferrari, Mauro; Hu, Tony Y.

    2015-01-01

    Tumor-resident proteases (TRPs) are regarded as informative biomarkers for staging cancer progression and evaluating therapeutic efficacy. Currently in the clinic, measurement of TRP is dependent on invasive biopsies, limiting their usefulness as monitoring tools. Here we identified circulating peptides naturally produced by TRPs, and evaluated their potential to monitor the efficacy of anti-tumor treatments. We established a mouse model for ovarian cancer development and treatment by orthotopic implantation of the human drug-resistant ovarian cancer cell line HeyA8-MDR, followed by porous silicon particle- or multistage vector (MSV) - enabled EphA2 siRNA therapy. Immunohistochemistry staining of tumor tissue revealed decreased expression of matrix metallopeptidase 9 (MMP-9) in mice exhibiting positive responses to MSV-EphA2 siRNA treatment. We demonstrated, via an ex vivo proteolysis assay, that C3f peptides can act as substrates of MMP-9, which cleaves C3f at L1311-L1312 into two peptides (SSATTFRL and LWENGNLLR). Importantly, we showed that these two C3f-derived fragments detected in serum were primarily generated by tumor-resident, but not blood-circulating, MMP-9. Our results suggested that the presence of the circulating fragments specially derived from the localized cleavage in tumor microenvironment can be used to evaluate therapeutic efficacy of anti-cancer treatment, assessed through a relatively noninvasive and user-friendly proteomics approach. PMID:25788424

  2. Global Identification of Biofilm-Specific Proteolysis in Candida albicans

    PubMed Central

    Winter, Michael B.; Salcedo, Eugenia C.; Lohse, Matthew B.; Hartooni, Nairi; Gulati, Megha; Sanchez, Hiram; Takagi, Julie; Hube, Bernhard; Andes, David R.

    2016-01-01

    ABSTRACT Candida albicans is a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections. C. albicans cells proliferate in a planktonic (suspension) state, but they also form biofilms, organized and tightly packed communities of cells attached to a solid surface. Biofilms colonize many niches of the human body and persist on implanted medical devices, where they are a major source of new C. albicans infections. Here, we used an unbiased and global substrate-profiling approach to discover proteolytic activities produced specifically by C. albicans biofilms, compared to planktonic cells, with the goal of identifying potential biofilm-specific diagnostic markers and targets for therapeutic intervention. This activity-based profiling approach, coupled with proteomics, identified Sap5 (Candidapepsin-5) and Sap6 (Candidapepsin-6) as major biofilm-specific proteases secreted by C. albicans. Fluorogenic peptide substrates with selectivity for Sap5 or Sap6 confirmed that their activities are highly upregulated in C. albicans biofilms; we also show that these activities are upregulated in other Candida clade pathogens. Deletion of the SAP5 and SAP6 genes in C. albicans compromised biofilm development in vitro in standard biofilm assays and in vivo in a rat central venous catheter biofilm model. This work establishes secreted proteolysis as a promising enzymatic marker and potential therapeutic target for Candida biofilm formation. PMID:27624133

  3. EpCAM proteolysis: new fragments with distinct functions?

    PubMed Central

    Schnell, Ulrike; Kuipers, Jeroen; Giepmans, Ben N. G.

    2013-01-01

    EpCAM [epithelial cell adhesion molecule; CD326 (cluster of differentiation 326)] is highly expressed on epithelium-derived tumours and can play a role in cell proliferation. Recently, RIP (regulated intramembrane proteolysis) has been implicated as the trigger for EpCAM-mediated proliferative signalling. However, RIP does not explain all EpCAM-derived protein fragments. To shed light on how proteolytic cleavage is involved in EpCAM signalling, we characterized the protein biochemically using antibodies binding to three different EpCAM domains. Using a newly generated anti-EpCAM antibody, we find that EpCAM can be cleaved at multiple positions within its ectodomain in addition to described peptides, revealing that EpCAM is processed via distinct proteolytic pathways. Here, we report on four new peptides, but also discuss the previously described cleavage products to provide a comprehensive picture of EpCAM cleavage at multiple positions. The complex regulation of EpCAM might not only result in the absence of full-length EpCAM, but the newly formed EpCAM-derived proteins may have their own signalling properties. PMID:23409978

  4. Limited proteolysis for assaying ligand binding affinities of nuclear receptors.

    PubMed

    Benkoussa, M; Nominé, B; Mouchon, A; Lefebvre, B; Bernardon, J M; Formstecher, P; Lefebvre, P

    1997-01-01

    The binding of natural or synthetic ligands to nuclear receptors is the triggering event leading to gene transcription activation or repression. Ligand binding to the ligand binding domain of these receptors induces conformational changes that are evidenced by an increased resistance of this domain to proteases. In vitro labeled receptors were incubated with various synthetic or natural agonists or antagonists and submitted to trypsin digestion. Proteolysis products were separated by SDS-PAGE and quantified. The amount of trypsin-resistant fragments was proportional to receptor occupancy by the ligand, and allowed the determination of dissociation constants (kDa). Using the wild-type or mutated human retinoic acid receptor alpha as a model, kDa values determined by classical competition binding assays using tritiated ligands are in agreement with those measured by the proteolytic assay. This method was successfully extended to human retinoic X receptor alpha, glucocorticoid receptor, and progesterone receptor, thus providing a basis for a new, faster assay to determine simultaneously the affinity and conformation of receptors when bound to a given ligand.

  5. An affinity-directed protein missile system for targeted proteolysis

    PubMed Central

    Fulcher, Luke J.; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro

    2016-01-01

    The von Hippel–Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate. PMID:27784791

  6. Pyrrolidine dithiocarbamate and zinc inhibit proteasome-dependent proteolysis.

    PubMed

    Kim, Insook; Kim, Chul Hoon; Kim, Joo Hee; Lee, Jinu; Choi, Jun Jeong; Chen, Zheng Ai; Lee, Min Goo; Chung, Kwang Chul; Hsu, Chung Y; Ahn, Young Soo

    2004-08-01

    Proteasomes play important roles in a variety of cellular processes such as cell cycle progression, signal transduction and immune responses. Proteasome activity is important in maintaining rapid turnover of short-lived proteins, as well as preventing accumulation of misfolded or damaged proteins. Alteration in ubiquitin-proteasome function may be detrimental to its crucial role in maintaining cellular homeostasis. Here, we have found that treatment of pyrrolidine dithiocarbamate (PDTC), a zinc ionophore, resulted in the accumulation of several proteasome substrates including p53 and p21 in HeLa cells. The PDTC effect was due to an extended half-life of these proteins through the mobilization of zinc. PDTC and/or zinc also increased fluorescence intensity of Ub(G76V)-GFP fusion protein that is degraded rapidly by the ubiquitin-proteasome system. Treatment of cells with zinc induced formation of ubiquitinated inclusions in the centrosome, a histological marker of proteasome inhibition. Western blotting showed zinc-induced increase in laddering bands of polyubiquitin-conjugated proteins. In vitro study, zinc inhibited the ubiquitin-independent proteasomal degradations of p21 and alpha-synuclein. These results suggest that zinc may modulate cell functions through its action on the turnover of proteins that are susceptible to proteasome-dependent proteolysis. PMID:15242777

  7. 11S Storage globulin from pumpkin seeds: regularities of proteolysis by papain.

    PubMed

    Rudakova, A S; Rudakov, S V; Kakhovskaya, I A; Shutov, A D

    2014-08-01

    Limited proteolysis of the α- and β-chains and deep cleavage of the αβ-subunits by the cooperative (one-by-one) mechanism was observed in the course of papain hydrolysis of cucurbitin, an 11S storage globulin from seeds of the pumpkin Cucurbita maxima. An independent analysis of the kinetics of the limited and cooperative proteolyses revealed that the reaction occurs in two successive steps. In the first step, limited proteolysis consisting of detachments of short terminal peptides from the α- and β-chains was observed. The cooperative proteolysis, which occurs as a pseudo-first order reaction, started at the second step. Therefore, the limited proteolysis at the first step plays a regulatory role, impacting the rate of deep degradation of cucurbitin molecules by the cooperative mechanism. Structural alterations of cucurbitin induced by limited proteolysis are suggested to generate its susceptibility to cooperative proteolysis. These alterations are tentatively discussed on the basis of the tertiary structure of the cucurbitin subunit pdb|2EVX in comparison with previously obtained data on features of degradation of soybean 11S globulin hydrolyzed by papain.

  8. Dietary protein deficiency reduces lysosomal and nonlysosomal ATP-dependent proteolysis in muscle

    NASA Technical Reports Server (NTRS)

    Tawa, N. E. Jr; Kettelhut, I. C.; Goldberg, A. L.

    1992-01-01

    When rats are fed a protein deficient (PD) diet for 7 days, rates of proteolysis in skeletal muscle decrease by 40-50% (N. E. Tawa, Jr., and A. L. Goldberg. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E317-325, 1992). To identify the underlying biochemical adaptations, we measured different proteolytic processes in incubated muscles. The capacity for intralysosomal proteolysis, as shown by sensitivity to methylamine or lysosomal protease inhibitors, fell 55-75% in muscles from PD rats. Furthermore, extracts of muscles of PD rats showed 30-70% lower activity of many lysosomal proteases, including cathepsins B, H, and C, and carboxypeptidases A and C, as well as other lysosomal hydrolases. The fall in cathepsin B and proteolysis was evident by 3 days on the PD diet, and both returned to control levels 3 days after refeeding of the normal diet. In muscles maintained under optimal conditions, 80-90% of protein breakdown occurs by nonlysosomal pathways. In muscles of PD rats, this ATP-dependent process was also 40-60% slower. Even though overall proteolysis decreased in muscles of PD rats, their capacity for Ca(2+)-dependent proteolysis increased (by 66%), as did the activity of the calpains (+150-250%). Thus the lysosomal and the ATP-dependent processes decrease coordinately and contribute to the fall in muscle proteolysis in PD animals.

  9. Cotranslational Proteolysis Dominates Glutathione Homeostasis to Support Proper Growth and Development[C][W

    PubMed Central

    Frottin, Frédéric; Espagne, Christelle; Traverso, José A.; Mauve, Caroline; Valot, Benoît; Lelarge-Trouverie, Caroline; Zivy, Michel; Noctor, Graham; Meinnel, Thierry; Giglione, Carmela

    2009-01-01

    The earliest proteolytic event affecting most proteins is the excision of the initiating Met (NME). This is an essential and ubiquitous cotranslational process tightly regulated in all eukaryotes. Currently, the effects of NME on unknown complex cellular networks and the ways in which its inhibition leads to developmental defects and cell growth arrest remain poorly understood. Here, we provide insight into the earliest molecular mechanisms associated with the inhibition of the NME process in Arabidopsis thaliana. We demonstrate that the developmental defects induced by NME inhibition are caused by an increase in cellular proteolytic activity, primarily induced by an increase in the number of proteins targeted for rapid degradation. This deregulation drives, through the increase of the free amino acids pool, a perturbation of the glutathione homeostasis, which corresponds to the earliest limiting, reversible step promoting the phenotype. We demonstrate that these effects are universally conserved and that the reestablishment of the appropriate glutathione status restores growth and proper development in various organisms. Finally, we describe a novel integrated model in which NME, protein N-α-acylation, proteolysis, and glutathione homeostasis operate in a sequentially regulated mechanism that directs both growth and development. PMID:19855051

  10. Structural Organization of Mammalian Prions as Probed by Limited Proteolysis

    PubMed Central

    Vázquez-Fernández, Ester; Alonso, Jana; Pastrana, Miguel A.; Ramos, Adriana; Stitz, Lothar; Vidal, Enric; Dynin, Irina; Petsch, Benjamin; Silva, Christopher J.; Requena, Jesús R.

    2012-01-01

    Elucidation of the structure of PrPSc continues to be one major challenge in prion research. The mechanism of propagation of these infectious agents will not be understood until their structure is solved. Given that high resolution techniques such as NMR or X-ray crystallography cannot be used, a number of lower resolution analytical approaches have been attempted. Thus, limited proteolysis has been successfully used to pinpoint flexible regions within prion multimers (PrPSc). However, the presence of covalently attached sugar antennae and glycosylphosphatidylinositol (GPI) moieties makes mass spectrometry-based analysis impractical. In order to surmount these difficulties we analyzed PrPSc from transgenic mice expressing prion protein (PrP) lacking the GPI membrane anchor. Such animals produce prions that are devoid of the GPI anchor and sugar antennae, and, thereby, permit the detection and location of flexible, proteinase K (PK) susceptible regions by Western blot and mass spectrometry-based analysis. GPI-less PrPSc samples were digested with PK. PK-resistant peptides were identified, and found to correspond to molecules cleaved at positions 81, 85, 89, 116, 118, 133, 134, 141, 152, 153, 162, 169 and 179. The first 10 peptides (to position 153), match very well with PK cleavage sites we previously identified in wild type PrPSc. These results reinforce the hypothesis that the structure of PrPSc consists of a series of highly PK-resistant β-sheet strands connected by short flexible PK-sensitive loops and turns. A sizeable C-terminal stretch of PrPSc is highly resistant to PK and therefore perhaps also contains β-sheet secondary structure. PMID:23185550

  11. Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells

    NASA Technical Reports Server (NTRS)

    Koh, T. J.; Tidball, J. G.

    2000-01-01

    We tested the hypothesis that nitric oxide can inhibit cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many of the effects of calcium ionophore on C(2)C(12) muscle cells, including preventing talin proteolysis and release into the cytosol and reducing loss of vinculin, cell detachment, and loss of cellular protein. These results indicate that nitric oxide inhibition of calpain protected the cells from ionophore-induced proteolysis. Calpain inhibitor I and a cell-permeable calpastatin peptide also protected the cells from proteolysis, confirming that ionophore-induced proteolysis was primarily calpain mediated. The activity of m-calpain in a casein zymogram was inhibited by sodium nitroprusside, and this inhibition was reversed by dithiothreitol. Previous incubation with the active site-targeted calpain inhibitor I prevented most of the sodium nitroprusside-induced inhibition of m-calpain activity. These data suggest that nitric oxide inhibited m-calpain activity via S-nitrosylation of the active site cysteine. The results of this study indicate that nitric oxide produced endogenously by skeletal muscle and other cell types has the potential to inhibit m-calpain activity and cytoskeletal proteolysis.

  12. Proteolysis in Manchego-type cheese salted by brine vacuum impregnation.

    PubMed

    Pavia, M; Trujillo, A J; Guamis, B; Ferragut, V

    2000-07-01

    A new salting procedure based on the brine vacuum impregnation of porous products was tested on Manchego-type cheese and compared with conventional brine immersion. Its effect on cheese proteolysis throughout a 90-d ripening period was determined. Three cheese regions were evaluated (the rind, the middle, and the internal regions). The parameters analyzed were total N, water-soluble N, soluble N in trichloroacetic acid and soluble N in phosphotungstic acid by using the Kjeldahl method, casein profile by urea-PAGE, and peptide profile of the water soluble nitrogen extract by reverse-phase HPLC. Free amino acid formation was monitored with a spectrophotometric method by using a Cd-ninhydrin reagent. Globally, proteolysis was significantly affected by ripening stage (increasing throughout all the maturation period studied) and cheese region (rind showed a proteolysis pattern different from the middle and internal regions). The salting procedure only affected cheese proteolysis in the rind, whereas conventional brine-salted cheeses showed lower proteolysis than vacuum-impregnated cheeses. PMID:10908050

  13. Reelin Proteolysis Affects Signaling Related to Normal Synapse Function and Neurodegeneration.

    PubMed

    Lussier, April L; Weeber, Edwin J; Rebeck, G William

    2016-01-01

    Reelin is a neurodevelopmental protein important in adult synaptic plasticity and learning and memory. Recent evidence points to the importance for Reelin proteolysis in normal signaling and in cognitive function. Support for the dysfunction of Reelin proteolysis in neurodegeneration and cognitive dysfunction comes from postmortem analysis of Alzheimer's diseases (AD) tissues including cerebral spinal fluid (CSF), showing that levels of Reelin fragments are altered in AD compared to control. Potential key proteases involved in Reelin proteolysis have recently been defined, identifying processes that could be altered in neurodegeneration. Introduction of full-length Reelin and its proteolytic fragments into several mouse models of neurodegeneration and neuropsychiatric disorders quickly promote learning and memory. These findings support a role for Reelin in learning and memory and suggest further understanding of these processes are important to harness the potential of this pathway in treating cognitive symptoms in neuropsychiatric and neurodegenerative diseases. PMID:27065802

  14. Mass Spectrometry Guided In Situ Proteolysis to Obtain Crystals for X-ray Structure Determination

    SciTech Connect

    Gheyi, Tarun; Rodgers, Logan; Romero, Richard; Sauder, J. Michael; Burley, Stephen K.

    2012-04-30

    A strategy for increasing the efficiency of protein crystallization/structure determination with mass spectrometry has been developed. This approach combines insights from limited proteolysis/mass spectrometry and crystallization via in situ proteolysis. The procedure seeks to identify protease-resistant polypeptide chain segments from purified proteins on the time-scale of crystal formation, and subsequently crystallizing the target protein in the presence of the optimal protease at the right relative concentration. We report our experience with 10 proteins of unknown structure, two of which yielded high-resolution X-ray structures. The advantage of this approach comes from its ability to select only those structure determination candidates that are likely to benefit from application of in situ proteolysis, using conditions most likely to result in formation of a stable proteolytic digestion product suitable for crystallization.

  15. Reelin Proteolysis Affects Signaling Related to Normal Synapse Function and Neurodegeneration

    PubMed Central

    Lussier, April L.; Weeber, Edwin J.; Rebeck, G. William

    2016-01-01

    Reelin is a neurodevelopmental protein important in adult synaptic plasticity and learning and memory. Recent evidence points to the importance for Reelin proteolysis in normal signaling and in cognitive function. Support for the dysfunction of Reelin proteolysis in neurodegeneration and cognitive dysfunction comes from postmortem analysis of Alzheimer’s diseases (AD) tissues including cerebral spinal fluid (CSF), showing that levels of Reelin fragments are altered in AD compared to control. Potential key proteases involved in Reelin proteolysis have recently been defined, identifying processes that could be altered in neurodegeneration. Introduction of full-length Reelin and its proteolytic fragments into several mouse models of neurodegeneration and neuropsychiatric disorders quickly promote learning and memory. These findings support a role for Reelin in learning and memory and suggest further understanding of these processes are important to harness the potential of this pathway in treating cognitive symptoms in neuropsychiatric and neurodegenerative diseases. PMID:27065802

  16. The beneficial role of proteolysis in skeletal muscle growth and stress adaptation.

    PubMed

    Bell, Ryan A V; Al-Khalaf, Mohammad; Megeney, Lynn A

    2016-01-01

    Muscle atrophy derived from excessive proteolysis is a hallmark of numerous disease conditions. Accordingly, the negative consequences of skeletal muscle protein breakdown often overshadow the critical nature of proteolytic systems in maintaining normal cellular function. Here, we discuss the major cellular proteolysis machinery-the ubiquitin/proteosome system, the autophagy/lysosomal system, and caspase-mediated protein cleavage-and the critical role of these protein machines in establishing and preserving muscle health. We examine how ordered degradation modifies (1) the spatiotemporal expression of myogenic regulatory factors during myoblast differentiation, (2) membrane fusion during myotube formation, (3) sarcomere remodeling and muscle growth following physical stress, and (4) energy homeostasis during nutrient deprivation. Finally, we review the origin and etiology of a number of myopathies and how these devastating conditions arise from inborn errors in proteolysis.

  17. Mapping the structural domains of E. coli carbamoyl phosphate synthetase using limited proteolysis.

    PubMed

    Mareya, S M; Raushel, F M

    1995-05-01

    The structural and functional domains of Escherichia coli carbamoyl phosphate synthetase (CPS) have been identified by limited proteolysis. Incubation of CPS with several proteases, including trypsin, chymotrypsin, subtilisin and endoproteinase Asp-N, under native conditions, causes a time-dependent loss of enzymatic activity and the generation of a common fragmentation pattern. Amino-terminal sequencing studies demonstrated that the initial cleavage event by trypsin occurred at the carboxy-terminal end of the large subunit. The ultimate fragments produced in most of the proteolysis studies, 35- and 45-kDa peptides, were derived from areas corresponding to the putative ATP binding regions. Substrate protection studies showed that the addition of ligands did not affect the final fragmentation pattern of the protein. However, ornithine and UMP were found to significantly reduce the rate of inactivation by inhibition of proteolytic cleavage. MgATP and IMP provided modest protection whereas bicarbonate and glutamine showed no overall effect on proteolysis. Limited proteolysis by endoproteinase Asp-N resulted in the production of a fragment (or multiple fragments) which contained enzymatic activity but had lost all regulation by the allosteric ligands, UMP and ornithine. The small subunit has been shown to be protected from proteolysis by the large subunit. Proteolysis of the isolated small subunit resulted in the generation of a stable 31-kDa species which contained 10% of the original glutaminase activity. These studies demonstrate that a portion of the C-terminal end of the large subunit can be excised without entirely destroying the ability of CPS to catalyze the formation of carbamoyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7648201

  18. Solution structure of a soluble fragment derived from a membrane protein by shotgun proteolysis

    PubMed Central

    Allen, Mark D.; Christie, Mary; Jones, Peter; Porebski, Benjamin T.; Roome, Brendan; Freund, Stefan M.V.; Buckle, Ashley M.; Bycroft, Mark; Christ, Daniel

    2015-01-01

    We have previously reported a phage display method for the identification of protein domains on a genome-wide scale (shotgun proteolysis). Here we present the solution structure of a fragment of the Escherichia coli membrane protein yrfF, as identified by shotgun proteolysis, and determined by NMR spectroscopy. Despite the absence of computational predictions, the fragment formed a well-defined beta-barrel structure, distantly falling within the OB-fold classification. Our results highlight the potential of high-throughput experimental approaches for the identification of protein domains for structural studies. PMID:25877662

  19. Protein oxidation and proteolysis during storage and in vitro digestion of pork and beef patties.

    PubMed

    Rysman, Tine; Van Hecke, Thomas; Van Poucke, Christof; De Smet, Stefaan; Van Royen, Geert

    2016-10-15

    The effect of protein oxidation on proteolysis during meat digestion was investigated following storage and subsequent in vitro digestion of beef and pork patties. Protein oxidation was evaluated as thiol oxidation, total carbonylation, and specific carbonylation (α-amino adipic and γ-glutamic semialdehyde). Furthermore, 4-hydroxyphenylalanine, a hydroxylation product of phenylalanine, was identified and quantified as a new protein oxidation marker. After 7days of chilled illuminated storage (4°C), significant oxidative modifications were quantified and the oxidative degradation was continued during in vitro digestion. The observed effects were more abundant in beef patties. Protein oxidation before digestion resulted in impaired proteolysis during digestion.

  20. Far infrared-assisted encapsulation of filter paper strips in poly(methyl methacrylate) for proteolysis.

    PubMed

    Chen, Qiwen; Bao, Huimin; Zhang, Luyan; Chen, Gang

    2016-02-01

    Filter paper strips were enclosed between two poly(methyl methacrylate) plates to fabricate paper-packed channel microchips under pressure in the presence of far infrared irradiation. After the enclosed paper strip was oxidized by periodate, trypsin was covalently immobilized in them to fabricate microfluidic proteolysis bioreactor. The feasibility and performance of the unique bioreactor were demonstrated by digesting BSA and lysozyme. The results were comparable to those of conventional in-solution proteolysis while the digestion time was significantly reduced to ∼18 s. The suitability of the microfluidic paper-based bioreactors to complex proteins was demonstrated by digesting human serum.

  1. Proteolysis produced within biofilms of bacterial isolates from raw milk tankers.

    PubMed

    Teh, Koon Hoong; Flint, Steve; Palmer, Jon; Andrewes, Paul; Bremer, Phil; Lindsay, Denise

    2012-06-15

    In this study, six bacterial isolates that produced thermo-resistant enzymes isolated from the internal surfaces of raw milk tankers were evaluated for their ability to produce proteolysis within either single culture biofilms or co-culture biofilms. Biofilms were formed in an in vitro model system that simulated the upper internal surface of a raw milk tanker during a typical summer's day of milk collection in New Zealand. The bacterial isolates were further evaluated for their ability to form biofilms at 25, 30 and 37°C. Mutual and competitive effects were observed in some of the co-culture biofilms, with all isolates being able to form biofilms in either single culture or co-culture at the three temperatures. The proteolysis was also evaluated in both biofilms and corresponding planktonic cultures. The proteolysis per cell decreased as the temperature of incubation (20-37°C) increased. Furthermore, mutualistic interactions in terms of proteolysis were observed when cultures were grown as co-culture biofilms. This is the first study to show that proteolytic enzymes can be produced in biofilms on the internal surfaces of raw milk tankers. This has important implications for the cleaning and the temperature control of raw milk transport tankers.

  2. Effects of energy deficit, dietary protein, and feeding on intracellular regulators of skeletal muscle proteolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study examined ubiquitin-mediated proteolysis and associated gene expression in normal-23 weight adults consuming varying levels of dietary protein during short-term energy deficit. 24 Using a randomized-bock design, 32 men and 7 women were assigned to diets providing protein 25 at 0.8 (RDA), 1...

  3. Probing the structure of GPI-less PrPSc by limited proteolysis(Abstract)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Limited proteolysis is a very useful tool to pinpoint flexible regions within scrapie prion protein (PrPSc), but due to carbohydrate and glysosylphosphatidylinositol (GPI) moieties, and limitations of the analytical techniques, until now it was impossible to characterize accurately these regions. To...

  4. Halogenation and proteolysis of complement component C3 on Salmonella typhimurium during phagocytosis by human neutrophils

    SciTech Connect

    Joiner, K.A.; Schweinle, J.E.

    1989-05-01

    We examined the fate of C component C3 on the surface of Salmonella typhimurium during ingestion by human neutrophils. Initial experiments showed that C3 fragments and C3-acceptor complexes were the major serum ligands which were surface iodinated by canine myeloperoxidase on serum-incubated rough and smooth isolates of S. typhimurium. In contrast, labeled C3 was not identified when the same organisms were ingested by neutrophils in the presence of 125I-Na, a situation previously shown to iodinate particulate targets via the neutrophil myeloperoxidase-halide-H2O2 system. Pretreatment of neutrophils before phagocytosis with the lipid-soluble protease inhibitor diisopropylfluorophosphate (DFP), but not with other protease inhibitors (p-nitrophenylguanidinobenzoate, leupeptin, pepstatin), substantially blocked proteolysis of 125I-C3 on S. typhimurium strain RG108 during ingestion by neutrophils. Purification of neutrophil phagosomes containing S. typhimurium-bearing 125I-C3 showed that DFP but no other protease inhibitors blocked proteolysis of 125I-C3 within phagosomes. Iodinated C3-acceptor complexes were identified by immunoprecipitation from the detergent-insoluble fraction of phagosomes prepared from DFP-treated cells ingesting S. typhimurium in the presence of 125I-Na. These results show that C3 fragments on the surface of S. typhimurium are the major serum ligands which are halogenated and degraded by proteolysis during phagocytosis by human neutrophils, and suggest that the majority of proteolysis on the ingested target occurs within the neutrophil phagosome.

  5. Phenidone and hydroxyurea reduce sulfur mustard-increased proteolysis in hairless guinea pig skin

    SciTech Connect

    Cowan, F.M.; Bongiovanni, R.; Broomfield, C.A.; Schulz, S.M.; Smith, W.J.

    1995-12-31

    Increased proteolytic activity at the dermal-epidermal junction is postulated as being involved in sulfur-mustard-induced cutaneous injury. Homogenates of skin punch biopsy specimens from the skin of hairless guinea pig at 6, 9, 12, and 24 h after a 7 min vapor cup exposure to sulfur mustard (HD) demonstrated enhanced proteolytic activity. Homogenates from the biopsy specimens of exposed animals produced from 3 to 10 times the hydrolysis of the chromogenic peptide substrate Chromzym TH (tosyl-gly-pro-arg-p-nitrani-lide) and human elastase substrate N-methoxysuccinyl-ala- ala-pro-val-p-nitranilide than did the homogenates from control samples. In this study HD-increased proteolysis of the TH substrate by extracts of hairless guinea pig skin biopsies was nearly eliminated by systemic treatment with hydroxyurea and greatly reduced by topical application of the anti-inflammatory compound phenidone. Compounds that reduce HD-increased proteolytic activity, such as phenidone and hydroxyurea, can serve as probes to examine the role of proteolysis in HD-induced pathology. HD-increased proteolysis provides a biochemical correlate for investigating cutaneous exposure to HD. Increased proteolysis may therefore serve as a biomarker for HD-induced pathology that might be used as an index of efficacy for potential treatment compounds.

  6. Effects of insulin, biguanide antihyperglycaemic agents and beta-adrenergic agonists on pathways of myocardial proteolysis.

    PubMed

    Thorne, D P; Lockwood, T D

    1990-03-15

    Pathways of bulk protein degradation controlled by insulin and isoprenaline (isoproterenol) were distinguished in Langendorff-perfused rat hearts. Proteins were biosynthetically labelled in vitro with [3H]leucine, followed by addition of 2 mM non-radioactive leucine to competitively prevent reincorporation. Rapidly degraded proteins were eliminated during a 3 h preliminary perfusion period without insulin. One third of bulk myocardial protein degradation was inhibited by isoprenaline as described previously. An insulin concentration of 5 nM maximally inhibited proteolysis, beginning within 2 min. Inhibition reached 32% within 1.25 h and 35% after 1.5 h. The minimum effective insulin concentration was approx. 10-50 pM, which caused 10-20% inhibition. Following 3 h of perfusion without insulin, the lysosomal inhibitor, chloroquine (30 microM), inhibited 38% of bulk degradation. The 35% proteolytic inhibition caused by insulin was followed by very little further inhibition on subsequent concurrent infusion of chloroquine, i.e. the inhibitory effects of insulin and chloroquine were not additive. In contrast, prior inhibition of lysosomal proteolysis by insulin or chloroquine did not prevent the subsequent additive inhibition caused by isoprenaline. Insulin and beta-agonists additively inhibited approx. two-thirds of bulk degradation. The biguanide antihyperglycaemic agent phenformin (2 microM) inhibited 35% of bulk degradation, beginning at 2 min and reaching a near maximum at approx. 1.25-1.5 h. Following inhibition of proteolysis with phenformin (20 microM), subsequent infusion of chloroquine (30 microM) produced only a slight additional inhibition. Following inhibition of 35% of degradation by 1.5 h of perfusion with insulin (5 nM), subsequent exposure to phenformin (2 microM) produced only a slight additional inhibition which did not exceed 38% of basal proteolysis. Thus insulin and phenformin both inhibit lysosomal proteolysis; however, the adrenergic

  7. Effects of insulin, biguanide antihyperglycaemic agents and beta-adrenergic agonists on pathways of myocardial proteolysis.

    PubMed Central

    Thorne, D P; Lockwood, T D

    1990-01-01

    Pathways of bulk protein degradation controlled by insulin and isoprenaline (isoproterenol) were distinguished in Langendorff-perfused rat hearts. Proteins were biosynthetically labelled in vitro with [3H]leucine, followed by addition of 2 mM non-radioactive leucine to competitively prevent reincorporation. Rapidly degraded proteins were eliminated during a 3 h preliminary perfusion period without insulin. One third of bulk myocardial protein degradation was inhibited by isoprenaline as described previously. An insulin concentration of 5 nM maximally inhibited proteolysis, beginning within 2 min. Inhibition reached 32% within 1.25 h and 35% after 1.5 h. The minimum effective insulin concentration was approx. 10-50 pM, which caused 10-20% inhibition. Following 3 h of perfusion without insulin, the lysosomal inhibitor, chloroquine (30 microM), inhibited 38% of bulk degradation. The 35% proteolytic inhibition caused by insulin was followed by very little further inhibition on subsequent concurrent infusion of chloroquine, i.e. the inhibitory effects of insulin and chloroquine were not additive. In contrast, prior inhibition of lysosomal proteolysis by insulin or chloroquine did not prevent the subsequent additive inhibition caused by isoprenaline. Insulin and beta-agonists additively inhibited approx. two-thirds of bulk degradation. The biguanide antihyperglycaemic agent phenformin (2 microM) inhibited 35% of bulk degradation, beginning at 2 min and reaching a near maximum at approx. 1.25-1.5 h. Following inhibition of proteolysis with phenformin (20 microM), subsequent infusion of chloroquine (30 microM) produced only a slight additional inhibition. Following inhibition of 35% of degradation by 1.5 h of perfusion with insulin (5 nM), subsequent exposure to phenformin (2 microM) produced only a slight additional inhibition which did not exceed 38% of basal proteolysis. Thus insulin and phenformin both inhibit lysosomal proteolysis; however, the adrenergic

  8. Sepsis stimulates nonlysosomal, energy-dependent proteolysis and increases ubiquitin mRNA levels in rat skeletal muscle.

    PubMed Central

    Tiao, G; Fagan, J M; Samuels, N; James, J H; Hudson, K; Lieberman, M; Fischer, J E; Hasselgren, P O

    1994-01-01

    We tested the role of different intracellular proteolytic pathways in sepsis-induced muscle proteolysis. Sepsis was induced in rats by cecal ligation and puncture; controls were sham operated. Total and myofibrillar proteolysis was determined in incubated extensor digitorum longus muscles as release of tyrosine and 3-methylhistidine, respectively. Lysosomal proteolysis was assessed by using the lysosomotropic agents NH4Cl, chloroquine, leupeptin, and methylamine. Ca(2+)-dependent proteolysis was determined in the absence or presence of Ca2+ or by blocking the Ca(2+)-dependent proteases calpain I and II. Energy-dependent proteolysis was determined in muscles depleted of ATP by 2-deoxyglucose and 2.4-dinitrophenol. Muscle ubiquitin mRNA and the concentrations of free and conjugated ubiquitin were determined by Northern and Western blots, respectively, to assess the role of the ATP-ubiquitin-dependent proteolytic pathway. Total and myofibrillar protein breakdown was increased during sepsis by 50 and 440%, respectively. Lysosomal and Ca(2+)-dependent proteolysis was similar in control and septic rats. In contrast, energy-dependent total and myofibrillar protein breakdown was increased by 172% and more than fourfold, respectively, in septic muscle. Ubiquitin mRNA was increased severalfold in septic muscle. The results suggest that the increase in muscle proteolysis during sepsis is due to an increase in nonlysosomal energy-dependent protein breakdown, which may involve the ubiquitin system. Images PMID:7989581

  9. Suppression of intralysosomal proteolysis aggravates structural damage and functional impairment of liver lysosomes in rats with toxic hepatitis

    SciTech Connect

    Korolenko, T.A.; Gavrilova, N.I.; Kurysheva, N.G.; Malygin, A.E.; Pupyshev, A.B.

    1986-01-01

    This paper estimates the effect of lowering protein catabolism in the lysosomes on structural and functional properties of the latter during liver damage. For comparison, polyvinylpyrrolidone (PVP), which is inert relative to intralysosomal proteolysis, and which also accumulates largely in lysosomes of the kupffer cells of the liver, was used. The uptake of labeled bovine serum albuman (C 14-BSA) by the liver is shown and the rate of intralysosomal proteolysis is given 24 hours after administration of suramin an CCl/sub 4/ to rats. It is suggested that it is risky to use drugs which inhibit intralysosomal proteolysis in the treatment of patients with acute hepatitis.

  10. Quantitative study of the relationships among proteolysis, lipid oxidation, structure and texture throughout the dry-cured ham process.

    PubMed

    Harkouss, Rami; Astruc, Thierry; Lebert, André; Gatellier, Philippe; Loison, Olivier; Safa, Hassan; Portanguen, Stéphane; Parafita, Emilie; Mirade, Pierre-Sylvain

    2015-01-01

    Temperature, salt and water contents are key processing factors in dry-cured ham production. They affect how proteolysis, lipid oxidation, structure and texture evolve, and thus determine the sensory properties and final quality of dry-cured ham. The aim of this study was to quantify the interrelationships and the time course of (i) proteolysis, (ii) lipid oxidation, (iii) five textural parameters: hardness, fragility, cohesiveness, springiness and adhesiveness and (iv) four structural parameters: fibre numbers, extracellular spaces, cross section area, and connective tissue area, during the dry-cured ham process. Applying multiple polynomial regression enabled us to build phenomenological models relating proteolysis, salt and water contents to certain textural and structural parameters investigated. A linear relationship between lipid oxidation and proteolysis was also established. All of these models and relationships, once combined with salt penetration, water migration and heat transfer models, can be used to dynamically simulate all of these phenomena throughout dry-cured ham manufacturing.

  11. Human platelet calmodulin-binding proteins: Ca/sup 2 +/-dependent proteolysis upon platelet activation

    SciTech Connect

    Wallace, R.W.; Tallant, E.A.; McManus, M.C.

    1986-05-01

    Calmodulin (CaM)-binding proteins have been identified in human platelets using Western blotting techniques and /sup 125/I-CaM. Ten distinct proteins with molecular weights of 245, 225K, 175K, 150K, 90K, 82K(2), 60K and 41K(2) bound /sup 125/I-CaM in a Ca/sup 2 +/-dependent manner; the binding was blocked by both trifluoperazine and nonradiolabeled CaM. The 225K and 90K proteins were labeled by antisera against myosin light chain kinase (MLCK); the 60K and one of the 82K proteins were identified as the CaM-dependent phosphatase and caldesmon. The remaining proteins have not yet been identified. Most of the CaM-binding proteins were degraded upon addition of Ca/sup 2 +/ to a platelet homogenate; the degradation could be blocked by either EGTA, leupeptin or N-ethyl-maleimide which suggests that it was due to a Ca/sup 2 +/-dependent protease. Activation of intact platelets by thrombin, ADP, collagen and the Ca/sup 2 +/-ionophores A23187 and ionomycin under conditions which promote platelet aggregation (i.e. stirring with extracellular Ca/sup 2 +/) also resulted in limited proteolysis of CaM-binding proteins including those labeled with anti-MLCK and the phosphatase. Many Ca/sup 2 +//CaM-regulated enzymes have been shown to be irreversibly activated in vitro by limited proteolysis. Their data indicates that limited proteolysis also occurs in vivo; under certain conditions proteolysis may be an important physiological mechanism for irreversibly activating Ca/sup 2 +//CaM-regulated enzymes.

  12. Mapping Epitopes on a Protein Antigen by the Proteolysis of Antigen-Antibody Complexes

    NASA Astrophysics Data System (ADS)

    Jemmerson, Ronald; Paterson, Yvonne

    1986-05-01

    A monoclonal antibody bound to a protein antigen decreases the rate of proteolytic cleavage of the antigen, having the greatest effect on those regions involved in antibody contact. Thus, an epitope can be identified by the ability of the antibody to protect one region of the antigen more than others from proteolysis. By means of this approach, two distinct epitopes, both conformationally well-ordered, were characterized on horse cytochrome c.

  13. Proteolysis of the peanut allergen Ara h 1 by an endogenous aspartic protease.

    PubMed

    Wilson, Karl A; Tan-Wilson, Anna

    2015-11-01

    The 7S and 11S globulins of peanuts are subjected to proteolysis two days after seed imbibition, with Ara h 1 and the arachin acidic chains being among the first storage proteins to be mobilized. Proteolytic activity was greatest at pH 2.6-3 and is inhibited by pepstatin A, characteristic of an aspartic protease. This activity persists in seedling cotyledons up to at least 8 days after imbibition. In vitro proteolysis of Ara h 1 at pH 2.6 by extracts of cotyledons from seedlings harvested 24 h after seed imbibition generates newly appearing bands on SDS-PAGE. Partial sequences of Ara h 1 that were obtained through LC-MS/MS analysis of in-gel trypsin digests of those bands, combined with information on fragment size, suggest that proteolysis begins in the region that links the two cupin domains to produce two 33/34 kD fragments, each one encompassing an intact cupin domain. The later appearance of two 18 and 10/11 kD fragments can be explained by proteolysis within an exposed site in the cupin domains of each of the 33/34 kD fragments. The same or similar proteolytic activity was observed in developing seeds, but Ara h 1 remains intact through seed maturation. This is partly explained by the observation that acidification of the protein storage vacuoles, demonstrated by vacuolar accumulation of acridine orange that was dissipated by a membrane-permeable base, occurs only after germination. These findings suggest a method for use of the seed aspartic protease in reducing peanut allergy due to Ara h 1.

  14. Mass spectrometric characterization of limited proteolysis activity in human plasma samples under mild acidic conditions.

    PubMed

    Yang, Jingzhi; Röwer, Claudia; Koy, Cornelia; Ruß, Manuela; Rüger, Christopher P; Zimmermann, Ralf; von Fritschen, Uwe; Bredell, Marius; Finke, Juliane C; Glocker, Michael O

    2015-11-01

    We developed a limited proteolysis assay for estimating dynamics in plasma-borne protease activities using MALDI ToF MS analysis as readout. A highly specific limited proteolysis activity was elicited in human plasma by shifting the pH to 6. Mass spectrometry showed that two singly charged ion signals at m/z 2753.44 and m/z 2937.56 significantly increased in abundance under mild acidic conditions as a function of incubation time. For proving that a provoked proteolytic activity in mild acidic solution caused the appearance of the observed peptides, control measurements were performed (i) with pepstatin as protease inhibitor, (ii) with heat-denatured samples, (iii) at pH 1.7, and (iv) at pH 7.5. Mass spectrometric fragmentation analysis showed that the observed peptides encompass the amino acid sequences 1-24 and 1-26 from the N-terminus of human serum albumin. Investigations on peptidase specificities suggest that the two best candidates for the observed serum albumin cleavages are cathepsin D and E. Reproducibility, robustness, and sensitivity prove the potential of the developed limited proteolysis assay to become of clinical importance for estimating dynamics of plasma-borne proteases with respect to associated pathophysiological tissue conditions.

  15. The role of the ADAMTS13 cysteine-rich domain in VWF binding and proteolysis

    PubMed Central

    Lane, David A.; Crawley, James T. B.

    2015-01-01

    ADAMTS13 proteolytically regulates the platelet-tethering function of von Willebrand factor (VWF). ADAMTS13 function is dependent upon multiple exosites that specifically bind the unraveled VWF A2 domain and enable proteolysis. We carried out a comprehensive functional analysis of the ADAMTS13 cysteine-rich (Cys-rich) domain using engineered glycans, sequence swaps, and single point mutations in this domain. Mutagenesis of Cys-rich domain–charged residues had no major effect on ADAMTS13 function, and 5 out of 6 engineered glycans on the Cys-rich domain also had no effect on ADAMTS13 function. However, a glycan attached at position 476 appreciably reduced both VWF binding and proteolysis. Substitution of Cys-rich sequences for the corresponding regions in ADAMTS1 identified a hydrophobic pocket involving residues Gly471-Val474 as being of critical importance for both VWF binding and proteolysis. Substitution of hydrophobic VWF A2 domain residues to serine in a region (residues 1642-1659) previously postulated to interact with the Cys-rich domain revealed the functional importance of VWF residues Ile1642, Trp1644, Ile1649, Leu1650, and Ile1651. Furthermore, the functional deficit of the ADAMTS13 Cys-rich Gly471-Val474 variant was dependent on these same hydrophobic VWF residues, suggesting that these regions form complementary binding sites that directly interact to enhance the efficiency of the proteolytic reaction. PMID:25564400

  16. Collagen fibril architecture, domain organization, and triple-helical conformation govern its proteolysis

    SciTech Connect

    Perumal, Shiamalee; Antipova, Olga; Orgel, Joseph P.R.O.

    2008-06-24

    We describe the molecular structure of the collagen fibril and how it affects collagen proteolysis or 'collagenolysis.' The fibril-forming collagens are major components of all mammalian connective tissues, providing the structural and organizational framework for skin, blood vessels, bone, tendon, and other tissues. The triple helix of the collagen molecule is resistant to most proteinases, and the matrix metalloproteinases that do proteolyze collagen are affected by the architecture of collagen fibrils, which are notably more resistant to collagenolysis than lone collagen monomers. Until now, there has been no molecular explanation for this. Full or limited proteolysis of the collagen fibril is known to be a key process in normal growth, development, repair, and cell differentiation, and in cancerous tumor progression and heart disease. Peptide fragments generated by collagenolysis, and the conformation of exposed sites on the fibril as a result of limited proteolysis, regulate these processes and that of cellular attachment, but it is not known how or why. Using computational and molecular visualization methods, we found that the arrangement of collagen monomers in the fibril (its architecture) protects areas vulnerable to collagenolysis and strictly governs the process. This in turn affects the accessibility of a cell interaction site located near the cleavage region. Our observations suggest that the C-terminal telopeptide must be proteolyzed before collagenase can gain access to the cleavage site. Collagenase then binds to the substrate's 'interaction domain,' which facilitates the triple-helix unwinding/dissociation function of the enzyme before collagenolysis.

  17. A capillary electrophoresis method to study postmortem proteolysis in relation to pork tenderness.

    PubMed

    Kristensen, Lars; Therkildsen, Margrethe; Ertbjerg, Per

    2003-09-24

    Identification of factors that determine meat tenderness is of high priority. The aim of this work was to develop a method that can detect indicators of proteolysis in meat early postmortem. The method was validated on pork samples. A procedure to detect differences of extractable lower molecular weight compounds after a prerigor freeze/thaw cycle of meat was developed using capillary electrophoresis. The procedure was able to separate 39 peaks in the electropherograms. Eight of the peaks were correlated (P < 0.1) to Warner-Bratzler shear forces 1 day postmortem (WB1). A multiple linear regression model explained 69% of the variation in WB1 using the areas of four peaks. Several of the peaks used in modeling WB1 were related to the at-slaughter activity of the calpain system. The results presented show that the developed method is able to detect indicators of proteolysis and tenderness at an early time point after slaughter. The method is a new tool intended for studies regarding the mechanisms of postmortem proteolysis and tenderization.

  18. Alternative Surfactants for Improved Efficiency of In Situ Tryptic Proteolysis of Fingermarks

    NASA Astrophysics Data System (ADS)

    Patel, Ekta; Clench, Malcolm R.; West, Andy; Marshall, Peter S.; Marshall, Nathan; Francese, Simona

    2015-06-01

    Despite recent improvements to in situ proteolysis strategies, a higher efficiency is still needed to increase both the number of peptides detected and the associated ion intensity, leading to a complete and reliable set of biomarkers for diagnostic or prognostic purposes. In the study presented here, an extract of a systematic study is illustrated investigating a range of surfactants assisting trypsin proteolytic activity. Method development was trialled on fingermarks; this specimen results from a transfer of sweat from an individual's fingertip to a surface upon contact. As sweat carries a plethora of biomolecules, including peptides and proteins, fingermarks are, potentially, a very valuable specimen for non-invasive prognostic or diagnostic screening. A recent study has demonstrated the opportunity to quickly detect peptides and small proteins in fingermarks using Matrix Assisted Laser Desorption Ionization Mass Spectrometry Profiling (MALDI MSP). However, intact detection bears low sensitivity and does not allow species identification; therefore, a shotgun proteomic approach was employed involving in situ proteolysis. Data demonstrate that in fingermarks, further improvements to the existing method can be achieved using MEGA-8 as surfactant in higher percentages as well as combinations of different detergents. Also, for the first time, Rapigest SF, normally used in solution digestions, has been shown to successfully work also for in situ proteolysis.

  19. Extracellular proteolysis in structural and functional plasticity of mossy fiber synapses in hippocampus

    PubMed Central

    Wiera, Grzegorz; Mozrzymas, Jerzy W.

    2015-01-01

    Brain is continuously altered in response to experience and environmental changes. One of the underlying mechanisms is synaptic plasticity, which is manifested by modification of synapse structure and function. It is becoming clear that regulated extracellular proteolysis plays a pivotal role in the structural and functional remodeling of synapses during brain development, learning and memory formation. Clearly, plasticity mechanisms may substantially differ between projections. Mossy fiber synapses onto CA3 pyramidal cells display several unique functional features, including pronounced short-term facilitation, a presynaptically expressed long-term potentiation (LTP) that is independent of NMDAR activation, and NMDA-dependent metaplasticity. Moreover, structural plasticity at mossy fiber synapses ranges from the reorganization of projection topology after hippocampus-dependent learning, through intrinsically different dynamic properties of synaptic boutons to pre- and postsynaptic structural changes accompanying LTP induction. Although concomitant functional and structural plasticity in this pathway strongly suggests a role of extracellular proteolysis, its impact only starts to be investigated in this projection. In the present report, we review the role of extracellular proteolysis in various aspects of synaptic plasticity in hippocampal mossy fiber synapses. A growing body of evidence demonstrates that among perisynaptic proteases, tissue plasminogen activator (tPA)/plasmin system, β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) and metalloproteinases play a crucial role in shaping plastic changes in this projection. We discuss recent advances and emerging hypotheses on the roles of proteases in mechanisms underlying mossy fiber target specific synaptic plasticity and memory formation. PMID:26582976

  20. Drug-induced proteolysis: a correlation with oedema-reducing ability.

    PubMed Central

    Piller, N. B.

    1976-01-01

    A very strong correlation has been shown to exist between acid and neutral protease activity levels in the skin, the acid protease activity level of the oedema fluid, and the oedema-reducing ability of the benzo-pyrones and related drugs. Macrophages, which are believed to be the main cells affected by the drugs, are very common in thermally injured tissues. Their lysosomal enzymes work at an acid pH. Since the main acid protease is cathepsin D, the overall acid protease levels are representative of changes in cathepsin D levels. Elevated levels are concomitant with more complete and rapid digestion of accumulated protein. The resulting fragments then can rapidly leave the injured tissues, freeing the oedema fluid. This form of proteolysis is very much different from that which is used by pharmacologists as a measure of inflammation. Normal proteolysis in inflammation represents an estimate of tissue derangement, but the proteolysis induced by drugs such as the benzo-pyrones represents a means of lessening some of the more injurious effects of this derangement. The results presented here strongly confirm this. PMID:952727

  1. CLIC1 regulates dendritic cell antigen processing and presentation by modulating phagosome acidification and proteolysis

    PubMed Central

    Salao, Kanin; Jiang, Lele; Li, Hui; Tsai, Vicky W.-W.; Husaini, Yasmin; Curmi, Paul M. G.; Brown, Louise J.; Brown, David A.

    2016-01-01

    ABSTRACT Intracellular chloride channel protein 1 (CLIC1) participates in inflammatory processes by regulating macrophage phagosomal functions such as pH and proteolysis. Here, we sought to determine if CLIC1 can regulate adaptive immunity by actions on dendritic cells (DCs), the key professional antigen presenting cells. To do this, we first generated bone marrow-derived DCs (BMDCs) from germline CLIC1 gene-deleted (CLIC1−/−) and wild-type (CLIC1+/+) mice, then studied them in vitro and in vivo. We found phagocytosis triggered cytoplasmic CLIC1 translocation to the phagosomal membrane where it regulated phagosomal pH and proteolysis. Phagosomes from CLIC1−/− BMDCs displayed impaired acidification and proteolysis, which could be reproduced if CLIC1+/+, but not CLIC1−/− cells, were treated with IAA94, a CLIC family ion channel blocker. CLIC1−/− BMDC displayed reduced in vitro antigen processing and presentation of full-length myelin oligodendrocyte glycoprotein (MOG) and reduced MOG-induced experimental autoimmune encephalomyelitis. These data suggest that CLIC1 regulates DC phagosomal pH to ensure optimal processing of antigen for presentation to antigen-specific T-cells. Further, they indicate that CLIC1 is a novel therapeutic target to help reduce the adaptive immune response in autoimmune diseases. PMID:27113959

  2. Collagen fibril architecture, domain organization, and triple-helical conformation govern its proteolysis.

    PubMed

    Perumal, Shiamalee; Antipova, Olga; Orgel, Joseph P R O

    2008-02-26

    We describe the molecular structure of the collagen fibril and how it affects collagen proteolysis or "collagenolysis." The fibril-forming collagens are major components of all mammalian connective tissues, providing the structural and organizational framework for skin, blood vessels, bone, tendon, and other tissues. The triple helix of the collagen molecule is resistant to most proteinases, and the matrix metalloproteinases that do proteolyze collagen are affected by the architecture of collagen fibrils, which are notably more resistant to collagenolysis than lone collagen monomers. Until now, there has been no molecular explanation for this. Full or limited proteolysis of the collagen fibril is known to be a key process in normal growth, development, repair, and cell differentiation, and in cancerous tumor progression and heart disease. Peptide fragments generated by collagenolysis, and the conformation of exposed sites on the fibril as a result of limited proteolysis, regulate these processes and that of cellular attachment, but it is not known how or why. Using computational and molecular visualization methods, we found that the arrangement of collagen monomers in the fibril (its architecture) protects areas vulnerable to collagenolysis and strictly governs the process. This in turn affects the accessibility of a cell interaction site located near the cleavage region. Our observations suggest that the C-terminal telopeptide must be proteolyzed before collagenase can gain access to the cleavage site. Collagenase then binds to the substrate's "interaction domain," which facilitates the triple-helix unwinding/dissociation function of the enzyme before collagenolysis.

  3. Gemin5 proteolysis reveals a novel motif to identify L protease targets.

    PubMed

    Piñeiro, David; Ramajo, Jorge; Bradrick, Shelton S; Martínez-Salas, Encarnación

    2012-06-01

    Translation of picornavirus RNA is governed by the internal ribosome entry site (IRES) element, directing the synthesis of a single polyprotein. Processing of the polyprotein is performed by viral proteases that also recognize as substrates host factors. Among these substrates are translation initiation factors and RNA-binding proteins whose cleavage is responsible for inactivation of cellular gene expression. Foot-and-mouth disease virus (FMDV) encodes two proteases, L(pro) and 3C(pro). Widespread definition of L(pro) targets suffers from the lack of a sufficient number of characterized substrates. Here, we report the proteolysis of the IRES-binding protein Gemin5 in FMDV-infected cells, but not in cells infected by other picornaviruses. Proteolysis was specifically associated with expression of L(pro), yielding two stable products, p85 and p57. In silico search of putative L targets within Gemin5 identified two sequences whose potential recognition was in agreement with proteolysis products observed in infected cells. Mutational analysis revealed a novel L(pro) target sequence that included the RKAR motif. Confirming this result, the Fas-ligand Daxx, was proteolysed in FMDV-infected and L(pro)-expressing cells. This protein carries a RRLR motif whose substitution to EELR abrogated L(pro) recognition. Thus, the sequence (R)(R/K)(L/A)(R) defines a novel motif to identify putative targets of L(pro) in host factors.

  4. Extracellular proteolysis in structural and functional plasticity of mossy fiber synapses in hippocampus.

    PubMed

    Wiera, Grzegorz; Mozrzymas, Jerzy W

    2015-01-01

    Brain is continuously altered in response to experience and environmental changes. One of the underlying mechanisms is synaptic plasticity, which is manifested by modification of synapse structure and function. It is becoming clear that regulated extracellular proteolysis plays a pivotal role in the structural and functional remodeling of synapses during brain development, learning and memory formation. Clearly, plasticity mechanisms may substantially differ between projections. Mossy fiber synapses onto CA3 pyramidal cells display several unique functional features, including pronounced short-term facilitation, a presynaptically expressed long-term potentiation (LTP) that is independent of NMDAR activation, and NMDA-dependent metaplasticity. Moreover, structural plasticity at mossy fiber synapses ranges from the reorganization of projection topology after hippocampus-dependent learning, through intrinsically different dynamic properties of synaptic boutons to pre- and postsynaptic structural changes accompanying LTP induction. Although concomitant functional and structural plasticity in this pathway strongly suggests a role of extracellular proteolysis, its impact only starts to be investigated in this projection. In the present report, we review the role of extracellular proteolysis in various aspects of synaptic plasticity in hippocampal mossy fiber synapses. A growing body of evidence demonstrates that among perisynaptic proteases, tissue plasminogen activator (tPA)/plasmin system, β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) and metalloproteinases play a crucial role in shaping plastic changes in this projection. We discuss recent advances and emerging hypotheses on the roles of proteases in mechanisms underlying mossy fiber target specific synaptic plasticity and memory formation.

  5. Ectdomain shedding and regulated intracellular proteolysis in the central nervous system.

    PubMed

    Montes de Oca-B, Pavel

    2010-12-01

    The term Ectodomain Shedding (ES) refers to extracellular domain proteolytic release from cell membrane molecules. This proteolysis is mediated mainly by matrix metalloproteases (MMP) or disintegrin and metalloproteases (ADAM), although some other proteases may mediate it. Virtually, all functional categories of cell membrane molecules are subject of this kind of proteolysis, for this reason ES is involved in different cellular processes such as proliferation, apoptosis, migration, differentiation or pathologies such as inflammation, cancer and degeneration among others. ES releases membrane molecule's extracellular domain (or ectodomain) to the extracellular milieu where it can play different biological functions. ES of transmembrane molecules also generates membrane attached terminal fragments comprising transmembrane and intracellular domains that enable their additional processing by intracellular proteases known as Regulated Intracellular Proteolysis (RIP). This second proteolytic cleavage delivers molecule's intracellular domain (ICD) that carry out intracellular functions. RIP is mediated by the group of intracellular cleaving proteases (i-CLiPs) that include presenilin from the γ-secretase complex. In the CNS the best well known ES is that of the Amyloid Precursor Protein, although many other membrane molecules expressed by cells of the CNS are also subject to ES and RIP. In this review, these molecules are summarized, and some meaningful examples are highlighted and described. In addition, ES and RIP implications in the context of cell biology are discussed. Finally, some considerations that rise from the study of ES and RIP are formulated in view of the unexpected roles of intracellular fragments.

  6. Whole-saliva proteolysis and its impact on salivary diagnostics.

    PubMed

    Thomadaki, K; Helmerhorst, E J; Tian, N; Sun, X; Siqueira, W L; Walt, D R; Oppenheim, F G

    2011-11-01

    There is growing interest in the use of human whole saliva for diagnostics and disease monitoring as an alternative to blood samples. In contrast to blood, whole saliva is a non-sterile body fluid. Proper hand-ling and storage are required to preserve the integrity of potential biomarkers. We investigated salivary autoproteolytic degradation using a variety of approaches. We determined inhibition of protease activities by monitoring the endogenous proteome. In addition, the stability of highly protease-susceptible proteins-histatin 5, statherin, and PRP1-was assessed. Experimental variables included (a) protease inhibitors, (b) salivary pH, (c) incubation temperatures, and (d) sample heating. A cocktail containing AEBSF, aprotinin, pancreatic trypsin inhibitor, leupeptin, antipain, and EDTA could not prevent histatin 5, statherin, or PRP1 degradation in whole saliva. Among the other treatments evaluated, short-term storage of freshly collected samples on ice was effective without interfering with the chemistry of the proteome. In conclusion, whole saliva contains a unique mixture of enzymes as evidenced from their resilience to protease inhibition. Analytical evidence on protein stability is needed to ensure the validity of salivary biomarker study outcomes. Analysis of the data presented will provide help and guidance for the use of saliva samples for diagnostic purposes.

  7. Carnosine ameliorates lens protein turbidity formations by inhibiting calpain proteolysis and ultraviolet C-induced degradation.

    PubMed

    Liao, Jiahn-Haur; Lin, I-Lin; Huang, Kai-Fa; Kuo, Pei-Ting; Wu, Shih-Hsiung; Wu, Tzu-Hua

    2014-06-25

    Carnosine (CAR) is an endogenous peptide and present in lens, but there is little evidence for its effectiveness in calpain-induced proteolysis inhibition and its differential effects toward different wavelengths of ultraviolet (UV) irradiation. This study aimed to develop three in vitro cataract models to compare the mechanisms underlying the protective activities of CAR. Crude crystallins extracted from porcine lenses were used for antiproteolysis assays, and purified γ-crystallins were used for anti-UV assays. The turbidity in those in vitro models mimics cataract formation and was assayed by measuring optical density (OD) at 405 nm. The effectiveness of CAR on calpain-induced proteolysis was studied at 37 and 58 °C. Patterns of proteins were then analyzed by SDS-PAGE. The turbidity was reduced significantly (p<0.05) at 60 min measurements with the increased concentration of CAR (10-300 mM). SDS-PAGE showed that the decreased intensities at both ∼28 and ∼30 kDa protein bands in heat-enhanced assays were ameliorated by CAR at ≥10 mM concentrations. In UV-B studies, CAR (200, 300 mM) reduced the turbidity of γ-crystallin significantly (p<0.05) at 6 h observations. The turbidity of samples containing γ-crystallins was ameliorated while incubated with CAR (100, 300 mM) significantly (p<0.05) following 4 h of exposure to UV-C. SDS-PAGE showed that the presence of CAR reduced UV-B-induced aggregation of γ-crystallins at ∼44 kDa and resulted in less loss of γ-crystallin following UV-C exposure. The result of modeling also suggests that CAR acts as an inhibitor of calpain. In conclusion, CAR protects lens proteins more readily by inhibiting proteolysis and UV-C-induced degradation than aggregation induced by UV-B irradiation.

  8. Expression of SPARC during development of the chicken chorioallantoic membrane: evidence for regulated proteolysis in vivo.

    PubMed Central

    Iruela-Arispe, M L; Lane, T F; Redmond, D; Reilly, M; Bolender, R P; Kavanagh, T J; Sage, E H

    1995-01-01

    SPARC is a secreted glycoprotein that has been shown to disrupt focal adhesions and to regulate the proliferation of endothelial cells in vitro. Moreover, peptides resulting from the proteolysis of SPARC exhibit angiogenic activity. Here we describe the temporal synthesis, turnover, and angiogenic potential of SPARC in the chicken chorioallantoic membrane. Confocal immunofluorescence microscopy revealed specific expression of SPARC protein in endothelial cells, and significantly higher levels of SPARC were observed in smaller newly formed blood vessels in comparison to larger, developmentally older vessels. SPARC mRNA was detected at the earliest stages of chorioallantoic membrane morphogenesis and reached maximal levels at day 13 of embryonic development. Interestingly, steady-state levels of SPARC mRNA did not correlate directly with protein accumulation; moreover, the protein appeared to undergo limited degradation during days 10-15. Incubation of [125I]-SPARC with chorioallantoic membranes of different developmental ages confirmed that extracellular proteolysis occurred during days 9-15, but not at later stages (e.g., days 17-21). Comparison of peptides produced by incubation with chorioallantoic membranes with those generated by plasmin showed an identical pattern of proteolysis. Plasmin activity was present throughout development, and in situ zymography identified sites of plasminogen activator activity that corresponded to areas exhibiting high levels of SPARC expression. Synthetic peptides from a plasmin-sensitive region of SPARC, between amino acids 113-130, stimulated angiogenesis in the chorioallantoic membrane in a dose-dependent manner; in contrast, intact SPARC was inactive in similar assays. We have shown that SPARC is expressed in endothelial cells of newly formed blood vessels in a manner that is both temporally and spatially restricted. Between days 9 and 15 of chorioallantoic membrane development, the protein undergoes proteolytic cleavage that

  9. cAMP-dependent proteolysis of GATA-6 is linked to JNK-signaling pathway

    SciTech Connect

    Ushijima, Hironori; Maeda, Masatomo

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer A JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6. Black-Right-Pointing-Pointer Effect of a JNK activator anisomycin on the proteolysis was examined. Black-Right-Pointing-Pointer Anisomycin stimulated the export of nuclear GATA-6 into the cytoplasm. Black-Right-Pointing-Pointer JNK activated the CRM1 mediated nuclear export of GATA-6. Black-Right-Pointing-Pointer JNK further stimulated slowly the degradation of GATA-6 by cytoplasmic proteasomes. -- Abstract: A JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6 by proteasomes around its IC50. We further examined the effects of SP600125 on the degradation of GATA-6 in detail, since an activator of JNK (anisomycin) is available. Interestingly, anisomycin immediately stimulated the export of nuclear GATA-6 into the cytoplasm, and then the cytoplasmic content of GATA-6 decreased slowly through degradation by proteasomes. Such an effect of anisomycin was inhibited by SP600125, indicating that the observed phenomenon might be linked to the JNK signaling pathway. The inhibitory effect of SP600125 could not be ascribed to the inhibition of PKA, since phosphorylation of CREB occurred in the presence of dbcAMP and SP600125. The nuclear export of GATA-6 was inhibited by leptomycin B, suggesting that CRM1-mediated export could be activated by anisomycin. Furthermore, it seems likely that the JNK activated by anisomycin may stimulate not only the nuclear export of GATA-6 through CRM1 but also the degradation of GATA-6 by cytoplasmic proteasomes. In contrast, A-kinase might activate only the latter process through JNK.

  10. Effects of energy deficit, dietary protein, and feeding on intracellular regulators of skeletal muscle proteolysis.

    PubMed

    Carbone, John W; Margolis, Lee M; McClung, James P; Cao, Jay J; Murphy, Nancy E; Sauter, Edward R; Combs, Gerald F; Young, Andrew J; Pasiakos, Stefan M

    2013-12-01

    This study was undertaken to characterize the ubiquitin proteasome system (UPS) response to varied dietary protein intake, energy deficit (ED), and consumption of a mixed meal. A randomized, controlled trial of 39 adults consuming protein at 0.8 (recommended dietary allowance [RDA]), 1.6 (2×-RDA), or 2.4 (3×-RDA) g · kg(-1) · d(-1) for 31 d. A 10-d weight maintenance (WM) period was followed by 21 d of 40% ED. Ubiquitin (Ub)-mediated proteolysis and associated gene expression were assessed in the postabsorptive (fasted) and postprandial (fed; 480 kcal, 20 g protein) states after WM and ED by using muscle biopsies, fluorescence-based assays, immunoblot analysis, and real-time qRT-PCR. In the assessment of UPS responses to varied protein intakes, ED, and feeding, the RDA, WM, and fasted measures served as appropriate controls. ED resulted in the up-regulation of UPS-associated gene expression, as mRNA expression of the atrogenes muscle RING finger-1 (MuRF1) and atrogin-1 were 1.2- and 1.3-fold higher (P<0.05) for ED than for WM. However, mixed-meal consumption attenuated UPS-mediated proteolysis, independent of energy status or dietary protein, as the activities of the 26S proteasome subunits β1, β2, and β5 were lower (P<0.05) for fed than for fasted. Muscle protein ubiquitylation was also 45% lower (P<0.05) for fed than for fasted, regardless of dietary protein and energy manipulations. Independent of habitual protein intake and despite increased MuRF1 and atrogin-1 mRNA expression during ED, consuming a protein-containing mixed meal attenuates Ub-mediated proteolysis.

  11. Effects of energy deficit, dietary protein, and feeding on intracellular regulators of skeletal muscle proteolysis.

    PubMed

    Carbone, John W; Margolis, Lee M; McClung, James P; Cao, Jay J; Murphy, Nancy E; Sauter, Edward R; Combs, Gerald F; Young, Andrew J; Pasiakos, Stefan M

    2013-12-01

    This study was undertaken to characterize the ubiquitin proteasome system (UPS) response to varied dietary protein intake, energy deficit (ED), and consumption of a mixed meal. A randomized, controlled trial of 39 adults consuming protein at 0.8 (recommended dietary allowance [RDA]), 1.6 (2×-RDA), or 2.4 (3×-RDA) g · kg(-1) · d(-1) for 31 d. A 10-d weight maintenance (WM) period was followed by 21 d of 40% ED. Ubiquitin (Ub)-mediated proteolysis and associated gene expression were assessed in the postabsorptive (fasted) and postprandial (fed; 480 kcal, 20 g protein) states after WM and ED by using muscle biopsies, fluorescence-based assays, immunoblot analysis, and real-time qRT-PCR. In the assessment of UPS responses to varied protein intakes, ED, and feeding, the RDA, WM, and fasted measures served as appropriate controls. ED resulted in the up-regulation of UPS-associated gene expression, as mRNA expression of the atrogenes muscle RING finger-1 (MuRF1) and atrogin-1 were 1.2- and 1.3-fold higher (P<0.05) for ED than for WM. However, mixed-meal consumption attenuated UPS-mediated proteolysis, independent of energy status or dietary protein, as the activities of the 26S proteasome subunits β1, β2, and β5 were lower (P<0.05) for fed than for fasted. Muscle protein ubiquitylation was also 45% lower (P<0.05) for fed than for fasted, regardless of dietary protein and energy manipulations. Independent of habitual protein intake and despite increased MuRF1 and atrogin-1 mRNA expression during ED, consuming a protein-containing mixed meal attenuates Ub-mediated proteolysis. PMID:23965841

  12. Suppression of IgM Proteolysis by Conformational Stabilization Through Excipients

    PubMed Central

    Mueller, Monika; Loh, Maybelle Q. T.; Gagnon, Pete

    2015-01-01

    Protease activity from host cell lines may cause product loss or affect the quality of recombinant proteins. In this study, we showed that excipients like glycine and sorbitol reduce the proteolysis of an immunoglobulin M (IgM) in the presence of added proteases like α-chymotrypsin, papain, and pepsin. The activity of the proteases in the IgM-protective environments was conserved or even enhanced as tested using low molecular weight substrates. Thus, a higher resistance against proteolytic degradation appears to be caused by the conformational stabilization of the IgM due to preferential exclusion of sorbitol and glycine. PMID:26839826

  13. [Proteolysis of simple glyprolines by leucine aminopeptidase and enzymes from nasal slime, brain membranes, and rat blood].

    PubMed

    Shevchenko, K V; V'iunova, T V; Nagaev, I Iu; Andreeva, L A; Miasoedov, N F

    2013-01-01

    Proteolysis of Pro-Gly-Pro-Leu, Pro-Gly-Pro-Gly and Pro-Gly-Pro were studied comparatively to Met-Glu-His-Phe-Pro-Gly-Pro (semax). It is shown that all three peptides are considerably more stable to proteolysis by N-leucine-aminopeptidase (EC 3.4.11.1, Sigma, type VI, 9.2 units/mg), and by enzymes of nasal slime, brain microsomal fractions, and rat blood. Metabolites of the proteolysis showed that semax derives His-Phe-Pro-Gly-Pro only, Pro-Gly-Pro-Leu forms Gly-Pro-Leu, Pro-Gly-Pro and Gly-Pro, Pro-Gly-Pro-Gly gives Pro-Gly-Pro and Gly-Pro, and Pro-Gly-Pro forms Gly-Pro.

  14. A leucine-supplemented diet restores the defective postprandial inhibition of proteasome-dependent proteolysis in aged rat skeletal muscle

    PubMed Central

    Combaret, Lydie; Dardevet, Dominique; Rieu, Isabelle; Pouch, Marie-Noëlle; Béchet, Daniel; Taillandier, Daniel; Grizard, Jean; Attaix, Didier

    2005-01-01

    We tested the hypothesis that skeletal muscle ubiquitin–proteasome-dependent proteolysis is dysregulated in ageing in response to feeding. In Experiment 1 we measured rates of proteasome-dependent proteolysis in incubated muscles from 8- and 22-month-old rats, proteasome activities, and rates of ubiquitination, in the postprandial and postabsorptive states. Peptidase activities of the proteasome decreased in the postabsorptive state in 22-month-old rats compared with 8-month-old animals, while the rate of ubiquitination was not altered. Furthermore, the down-regulation of in vitro proteasome-dependent proteolysis that prevailed in the postprandial state in 8-month-old rats was defective in 22-month-old rats. Next, we tested the hypothesis that the ingestion of a 5% leucine-supplemented diet may correct this defect. Leucine supplementation restored the postprandial inhibition of in vitro proteasome-dependent proteolysis in 22-month-old animals, by down-regulating both rates of ubiquitination and proteasome activities. In Experiment 2, we verified that dietary leucine supplementation had long-lasting effects by comparing 8- and 22-month-old rats that were fed either a leucine-supplemented diet or an alanine-supplemented diet for 10 days. The inhibited in vitro proteolysis was maintained in the postprandial state in the 22-month-old rats fed the leucine-supplemented diet. Moreover, elevated mRNA levels for ubiquitin, 14-kDa ubiquitin-conjugating enzyme E2, and C2 and X subunits of the 20S proteasome that were characteristic of aged muscle were totally suppressed in 22-month-old animals chronically fed the leucine-supplemented diet, demonstrating an in vivo effect. Thus the defective postprandial down-regulation of in vitro proteasome-dependent proteolysis in 22-month-old rats was restored in animals chronically fed a leucine-supplemented diet. PMID:16195315

  15. Huntingtin proteolysis releases non-polyQ fragments that cause toxicity through dynamin 1 dysregulation

    PubMed Central

    El-Daher, Marie-Thérèse; Hangen, Emilie; Bruyère, Julie; Poizat, Ghislaine; Al-Ramahi, Ismael; Pardo, Raul; Bourg, Nicolas; Souquere, Sylvie; Mayet, Céline; Pierron, Gérard; Lévêque-Fort, Sandrine; Botas, Juan; Humbert, Sandrine; Saudou, Frédéric

    2015-01-01

    Cleavage of mutant huntingtin (HTT) is an essential process in Huntington’s disease (HD), an inherited neurodegenerative disorder. Cleavage generates N-ter fragments that contain the polyQ stretch and whose nuclear toxicity is well established. However, the functional defects induced by cleavage of full-length HTT remain elusive. Moreover, the contribution of non-polyQ C-terminal fragments is unknown. Using time- and site-specific control of full-length HTT proteolysis, we show that specific cleavages are required to disrupt intramolecular interactions within HTT and to cause toxicity in cells and flies. Surprisingly, in addition to the canonical pathogenic N-ter fragments, the C-ter fragments generated, that do not contain the polyQ stretch, induced toxicity via dilation of the endoplasmic reticulum (ER) and increased ER stress. C-ter HTT bound to dynamin 1 and subsequently impaired its activity at ER membranes. Our findings support a role for HTT on dynamin 1 function and ER homoeostasis. Proteolysis-induced alteration of this function may be relevant to disease. PMID:26165689

  16. How different DNA sequences are recognized by a DNA-binding protein: effects of partial proteolysis.

    PubMed

    Supakar, P C; Zhang, X Y; Githens, S; Khan, R; Ehrlich, K C; Ehrlich, M

    1989-11-11

    MDBP is a sequence-specific DNA-binding protein from mammals that recognizes a variety of DNA sequences, all of which show much homology to a partially palindromic 14 base-pair consensus sequence. MDBP subjected to limited proteolysis and then incubated with various specific oligonucleotide duplexes yielded two types of complexes. The relative concentrations of these complexes varied greatly depending on how closely the MDBP site matched the consensus sequence. No such DNA sequence-specific differences in the types of complexes formed were seen with intact MDBP. Partial proteolysis also changed the relative affinity of MDBP for several of its binding sites. The nature of the two types of complexes formed from fragmented MDBP and DNA was studied by DNA competition assays, protein titration, site-directed mutagenesis, and dimethyl sulfate and missing base interference assays. The results suggest that, for some specific DNA sequences, half-site interactions with one MDBP subunit predominate and for others, strong interaction of two subunits with both half-sites readily occur.

  17. Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression

    SciTech Connect

    Inami, Yoshihiro; Yamashina, Shunhei; Izumi, Kousuke; Ueno, Takashi; Tanida, Isei; Ikejima, Kenichi; Watanabe, Sumio

    2011-09-09

    Highlights: {yields} Acidification of autophagosome was blunted in steatotic hepatocytes. {yields} Hepatic steatosis did not disturb fusion of isolated autophagosome and lysosome. {yields} Proteinase activity of cathepsin B and L in autolysosomes was inhibited by steatosis. {yields} Hepatic expression of cathepsin B and L was suppressed by steatosis. -- Abstract: Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmented in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.

  18. Yield, changes in proteolysis, and sensory quality of Prato cheese produced with different coagulants.

    PubMed

    Alves, L S; Merheb-Dini, C; Gomes, E; da Silva, R; Gigante, M L

    2013-01-01

    The objective of this research was to compare the effect of 2 fungal proteases, one that is already commercially established as a milk-clotting agent and another produced at the laboratory scale, on Prato cheese composition, protein and fat recovery, yield, and sensory characteristics. Cheeses were produced according to the traditional protocol, using protease from the fungus Thermomucor indicae-seudaticae N31 and commercial coagulant from Rhizomucor spp. as clotting agents. A 2×6 factorial design with 3 replications was performed: 2 levels of coagulants and 6 levels of storage time. After 5, 12, 19, 33, 43, and 53d of refrigerated storage (12°C), cheeses were monitored for proteolysis, firmness, and casein degradation by capillary electrophoresis. Sensory acceptance was evaluated after 29d of manufacturing. The different coagulants did not statistically affect Prato cheese composition, protein and fat recovery, and yield. Both cheeses presented good sensory acceptance. Proteolysis increased and firmness decreased for both cheeses during the storage time, as expected for Prato cheese. Caseins were well separated by capillary electrophoresis and the results showed, with good resolution, that the cheeses exhibited similar protein hydrolysis profile. Both cheeses presented good sensory acceptance. The gathered data showed that the protease from T. indicae-seudaticae N31 presented similar action compared with the commercial enzyme, indicating its efficiency as clotting agent for Prato cheese manufacture.

  19. Much to know about proteolysis: intricate proteolytic machineries compromise essential cellular functions.

    PubMed

    Marfany, Gemma; Farràs, Rosa; Salido, Eduardo; Xirodimas, Dimitris P; Rodríguez, Manuel S

    2008-10-01

    Proteolysis has traditionally been considered as a radical way to terminate the function of a protein. However, protein destruction also is the starting point for many processes as they can only occur when the way has been cleared for the action of other proteins. Protein destruction can occur virtually in all compartments and organelles of the cell, associated with cell membranes or large protein complexes, it determines subcellular partitioning, association with positive or negative regulators which conditions the action of many critical cellular factors. The third intracellular proteolysis meeting held by the University La Laguna, Canary Islands, Spain, included speakers working with some of the most important proteolytic systems present in higher eukaryotes, such as the UPS (ubiquitin-proteasome system) and autophagy. Owing to the fact that these pathways directly or indirectly regulate many cell functions, this meeting brought together an audience with a wide range of research interests, including genetic, cell biological, biochemical and structural aspects of protein degradation. Some of these topics inspired interesting discussions and a significant number of these are developed in the issues reviewed herein.

  20. Mycobacterium tuberculosis Hip1 modulates macrophage responses through proteolysis of GroEL2.

    PubMed

    Naffin-Olivos, Jacqueline L; Georgieva, Maria; Goldfarb, Nathan; Madan-Lala, Ranjna; Dong, Lauren; Bizzell, Erica; Valinetz, Ethan; Brandt, Gabriel S; Yu, Sarah; Shabashvili, Daniil E; Ringe, Dagmar; Dunn, Ben M; Petsko, Gregory A; Rengarajan, Jyothi

    2014-05-01

    Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.

  1. Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis.

    PubMed

    Boucher, Dave; Blais, Véronique; Denault, Jean-Bernard

    2012-04-10

    During apoptosis, hundreds of proteins are cleaved by caspases, most of them by the executioner caspase-3. However, caspase-7, which shares the same substrate primary sequence preference as caspase-3, is better at cleaving poly(ADP ribose) polymerase 1 (PARP) and Hsp90 cochaperone p23, despite a lower intrinsic activity. Here, we identified key lysine residues (K(38)KKK) within the N-terminal domain of caspase-7 as critical elements for the efficient proteolysis of these two substrates. Caspase-7's N-terminal domain binds PARP and improves its cleavage by a chimeric caspase-3 by ∼30-fold. Cellular expression of caspase-7 lacking the critical lysine residues resulted in less-efficient PARP and p23 cleavage compared with cells expressing the wild-type peptidase. We further showed, using a series of caspase chimeras, the positioning of p23 on the enzyme providing us with a mechanistic insight into the binding of the exosite. In summary, we have uncovered a role for the N-terminal domain (NTD) and the N-terminal peptide of caspase-7 in promoting key substrate proteolysis.

  2. Effect of manufacturing factors, composition, and proteolysis on the functional characteristics of mozzarella cheese.

    PubMed

    Kindstedt, P S

    1993-01-01

    Shredding and melting characteristics are vital to the function of low-moisture Mozzarella cheeses that are used as ingredients for pizza and related foods. Newly manufactured Mozzarella melts to a tough, extremely elastic, and somewhat granular consistency with limited stretch that is unacceptable for pizza. However, during the first few weeks of refrigerated storage, a dramatic transformation occurs as the unmelted cheese becomes softer and the melted cheese becomes more viscous, less elastic, and highly stretchable. Thus, the cheese attains optimal functionality for pizza. Over longer periods, Mozzarella becomes excessively soft and fluid when melted and is no longer acceptable for pizza. Low-moisture Mozzarella is correctly viewed as a cheese that requires aging. The functional characteristics of low-moisture Mozzarella are due initially to the chemical composition, including fat, moisture, NaCl, and mineral contents, and the structure of the paracasein curd matrix that is established during manufacture. Changes in functional characteristics during aging are directly related to proteolysis rate and possibly proteolytic specificity. Proteolysis during aging is influenced by manufacturing factors such as starter culture, coagulant, and stretching temperature, and possibly to indigenous proteases in the cheesemilk such as plasmin.

  3. Specific effect of high-pressure treatment of milk on cheese proteolysis.

    PubMed

    Buffa, Martín; Guamis, Buenaventura; Trujillo, Antonio J

    2005-11-01

    The extent of primary and secondary proteolysis of cheeses made from raw (RA), pasteurized (PA, 72 degrees C, 15 s) or pressure-treated (PR, 500 MPa, 15 min, 20 degrees C) goats' milk was assessed. Modifications in cheese-making technology were introduced to obtain cheeses with the same moisture content, and thus studied per se the effect of milk treatment on cheese proteolysis. The PR milk cheese samples were differentiated from RA and PA milk cheeses by their elevated beta-lg content, and by the faster degradation of alphas1-, alphas2- and beta-CN throughout ripening. Non-significant differences were found in either pH 4.6 soluble-nitrogen or trichloracetic acid soluble-nitrogen contents of cheeses. However, the pasteurization of milk decreased the free amino acid production in cheese. The RA milk cheeses had the highest amount of proline and the lowest concentrations of serine, tyrosine, arginine and alpha-aminobutyric acid, whereas PR milk cheese showed higher levels of arginine.

  4. Distinct biological events generated by ECM proteolysis by two homologous collagenases.

    PubMed

    Solomonov, Inna; Zehorai, Eldar; Talmi-Frank, Dalit; Wolf, Sharon G; Shainskaya, Alla; Zhuravlev, Alina; Kartvelishvily, Elena; Visse, Robert; Levin, Yishai; Kampf, Nir; Jaitin, Diego Adhemar; David, Eyal; Amit, Ido; Nagase, Hideaki; Sagi, Irit

    2016-09-27

    It is well established that the expression profiles of multiple and possibly redundant matrix-remodeling proteases (e.g., collagenases) differ strongly in health, disease, and development. Although enzymatic redundancy might be inferred from their close similarity in structure, their in vivo activity can lead to extremely diverse tissue-remodeling outcomes. We observed that proteolysis of collagen-rich natural extracellular matrix (ECM), performed uniquely by individual homologous proteases, leads to distinct events that eventually affect overall ECM morphology, viscoelastic properties, and molecular composition. We revealed striking differences in the motility and signaling patterns, morphology, and gene-expression profiles of cells interacting with natural collagen-rich ECM degraded by different collagenases. Thus, in contrast to previous notions, matrix-remodeling systems are not redundant and give rise to precise ECM-cell crosstalk. Because ECM proteolysis is an abundant biochemical process that is critical for tissue homoeostasis, these results improve our fundamental understanding its complexity and its impact on cell behavior. PMID:27630193

  5. Cleavage Site Localization Differentially Controls Interleukin-6 Receptor Proteolysis by ADAM10 and ADAM17

    PubMed Central

    Riethmueller, Steffen; Ehlers, Johanna C.; Lokau, Juliane; Düsterhöft, Stefan; Knittler, Katharina; Dombrowsky, Gregor; Grötzinger, Joachim; Rabe, Björn; Rose-John, Stefan; Garbers, Christoph

    2016-01-01

    Limited proteolysis of the Interleukin-6 Receptor (IL-6R) leads to the release of the IL-6R ectodomain. Binding of the cytokine IL-6 to the soluble IL-6R (sIL-6R) results in an agonistic IL-6/sIL-6R complex, which activates cells via gp130 irrespective of whether the cells express the IL-6R itself. This signaling pathway has been termed trans-signaling and is thought to mainly account for the pro-inflammatory properties of IL-6. A Disintegrin And Metalloprotease 10 (ADAM10) and ADAM17 are the major proteases that cleave the IL-6R. We have previously shown that deletion of a ten amino acid long stretch within the stalk region including the cleavage site prevents ADAM17-mediated cleavage, whereas the receptor retained its full biological activity. In the present study, we show that deletion of a triple serine (3S) motif (Ser-359 to Ser-361) adjacent to the cleavage site is sufficient to prevent IL-6R cleavage by ADAM17, but not ADAM10. We find that the impaired shedding is caused by the reduced distance between the cleavage site and the plasma membrane. Positioning of the cleavage site in greater distance towards the plasma membrane abrogates ADAM17-mediated shedding and reveals a novel cleavage site of ADAM10. Our findings underline functional differences in IL-6R proteolysis by ADAM10 and ADAM17. PMID:27151651

  6. Proteolysis of AKAP121 regulates mitochondrial activity during cellular hypoxia and brain ischaemia

    PubMed Central

    Carlucci, Annalisa; Adornetto, Annagrazia; Scorziello, Antonella; Viggiano, Davide; Foca, Mariapaola; Cuomo, Ornella; Annunziato, Lucio; Gottesman, Max; Feliciello, Antonio

    2008-01-01

    A-kinase anchor protein 121 (AKAP121) assembles a multivalent signalling complex on the outer mitochondrial membrane that controls persistence and amplitude of cAMP and src signalling to mitochondria, and plays an essential role in oxidative metabolism and cell survival. Here, we show that AKAP121 levels are regulated post-translationally by the ubiquitin/proteasome pathway. Seven In-Absentia Homolog 2 (Siah2), an E3–ubiquitin ligase whose expression is induced in hypoxic conditions, formed a complex and degraded AKAP121. In addition, we show that overexpression of Siah2 or oxygen and glucose deprivation (OGD) promotes Siah2-mediated ubiquitination and proteolysis of AKAP121. Upregulation of Siah2, by modulation of the cellular levels of AKAP121, significantly affects mitochondrial activity assessed as mitochondrial membrane potential and oxidative capacity. Also during cerebral ischaemia, AKAP121 is degraded in a Siah2-dependent manner. These findings reveal a novel mechanism of attenuation of cAMP/PKA signaling, which occurs at the distal sites of signal generation mediated by proteolysis of an AKAP scaffold protein. By regulating the stability of AKAP121-signalling complex at mitochondria, cells efficiently and rapidly adapt oxidative metabolism to fluctuations in oxygen availability. PMID:18323779

  7. Regulation of Ci-SCFSlimb binding, Ci proteolysis and Hedgehog pathway activity by Ci phosphorylation

    PubMed Central

    Smelkinson, Margery G.; Zhou, Qianhe; Kalderon, Daniel

    2007-01-01

    SUMMARY Hedgehog (Hh) proteins signal by inhibiting the proteolytic processing of Ci/Gli family transcription factors and by increasing Ci/Gli specific activity. In the absence of Hh, phosphorylation of Ci/Gli triggers binding to SCF ubiquitin ligase complexes and consequent proteolysis. Here we define the principal SCFSlimb binding site in Ci as an extended variant of a canonical Slimb/β-TRCP binding motif that can be created by PKA-priming of five successive CK1 sites. GSK3 enhances binding primarily through a nearby region of Ci, which may contact an SCF component other than Slimb. Studies of Ci variants with altered CK1 and GSK3 sites suggest that the large number of phosphorylation sites that direct SCFSlimb binding confers a Hh response that is both sensitive and graded, and that in the Drosophila wing disc, morphogenetic responses involve changes in both the level and specific activity of Ci. We also show that when Ci proteolysis is compromised, its specific activity is limited principally by Su(fu) and not by Cos2 cytoplasmic tethering or PKA phosphorylation. PMID:17925225

  8. Phenylalaninylargininylarginine: a novel tripeptide exerting Zn(2+)-dependent, insulin-mimetic inhibitory action on myocardial proteolysis.

    PubMed Central

    Zhang, L; Lockwood, T D

    1993-01-01

    A novel tripeptide, Phe-Arg-Arg, was found to exert a potent, insulin-mimetic inhibitory action on lysosomal proteolysis in the Langendorff-perfused rat heart. This tripeptide was synthesized based upon its partial structural analogy to the biguanide anti-hyperglycaemic agent, phenformin (phenylethylbiguanide), which has previously been found to exert a Zn(2+)-dependent inhibitory action on lysosomal proteolysis. Hearts were biosynthetically labelled with [3H]leucine in vitro. The percentage change in subsequent release of [3H]leucine (2 mM non-radioactive leucine) was determined in non-recirculating perfusate. The background Zn2+ content of the perfusate was determined to be 20 nM. Major endogenous Zn2+ buffers were present in molar excess of Zn2+: 0.1 mM citrate, 0.2% BSA, and complete physiological amino acids. Infusion of maximally effective levels of chloroquine (30 microM) or insulin (5 nM) caused a 38% inhibition of total proteolysis, which corresponds to the lysosomal subcomponent. In the presence of background levels of perfusate Zn2+ the infusion of Phe-Arg-Arg (10 microM), insulin (5 nM), or phenformin (2 microM) maximally caused a 39% inhibition of [3H]leucine release. Combined infusion of maximally effective levels of insulin and Phe-Arg-Arg, or maximal levels of chloroquine and Phe-Arg-Arg did not cause additive inhibition of [3H]leucine release greater than the 39% inhibition caused by either agent alone, regardless of the order of infusion. Addition of physiological concentrations of Zn2+ (1 microM) to the background perfusate Zn2+ accelerated the insulin-mimetic action of submaximally effective levels of Phe-Arg-Arg, and increased its potency. Prior chelation of background Zn2+ by a 3 h perfusion with CaNa2 EDTA (2 microM) reversibly delayed the time course of Phe-Arg-Arg action and decreased its potency at submaximal concentrations. PMID:8352749

  9. Enzymatically structured emulsions in simulated gastrointestinal environment: impact on interfacial proteolysis and diffusion in intestinal mucus.

    PubMed

    Macierzanka, Adam; Böttger, Franziska; Rigby, Neil M; Lille, Martina; Poutanen, Kaisa; Mills, E N Clare; Mackie, Alan R

    2012-12-18

    Fundamental knowledge of physicochemical interactions in the gastrointestinal environment is required in order to support rational designing of protein-stabilized colloidal food and pharmaceutical delivery systems with controlled behavior. In this paper, we report on the colloidal behavior of emulsions stabilized with the milk protein sodium caseinate (Na-Cas), and exposed to conditions simulating the human upper gastrointestinal tract. In particular, we looked at how the kinetics of proteolysis was affected by adsorption to an oil-water interface in emulsion and whether the proteolysis and the emulsion stability could be manipulated by enzymatic structuring of the interface. After cross-linking with the enzyme transglutaminase, the protein was digested with use of an in vitro model of gastro-duodenal proteolysis in the presence or absence of physiologically relevant surfactants (phosphatidylcholine, PC; bile salts, BS). Significant differences were found between the rates of digestion of Na-Cas cross-linked in emulsion (adsorbed protein) and in solution. In emulsion, the digestion of a population of polypeptides of M(r) ca. 50-100 kDa was significantly retarded through the gastric digestion. The persistent interfacial polypeptides maintained the original emulsion droplet size and prevented the system from phase separating. Rapid pepsinolysis of adsorbed, non-cross-linked Na-Cas and its displacement by PC led to emulsion destabilization. These results suggest that structuring of emulsions by enzymatic cross-linking of the interfacial protein may affect the phase behavior of emulsion in the stomach and the gastric digestion rate in vivo. Measurements of ζ-potential revealed that BS displaced the remaining protein from the oil droplets during the simulated duodenal phase of digestion. Diffusion of the postdigestion emulsion droplets through ex vivo porcine intestinal mucus was only significant in the presence of BS due to the high negative charge these

  10. Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes.

    PubMed

    Jahic, Mehmedalija; Gustavsson, Malin; Jansen, Ann-Katrin; Martinelle, Mats; Enfors, Sven-Olof

    2003-04-10

    A fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced by Pichia pastoris Mut(+) in high-cell density bioreactor cultures. The production was induced by switching from growth on glycerol to growth on methanol. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 1.3 g x l(-1) of CBM-CALB. However, only about 40% of the product was of full-length according to Western blot analysis. This loss was due to a cleavage of the protein in the linker between the CBM and the CALB moieties. The cleavage was catalyzed by serine proteases in the culture supernatant. The CALB-moiety was subjected to further slow degradation by cell-associated proteolysis. Different strategies were used to reduce the proteolysis. Previous efforts to shorten the linker region resulted in a stable protein but with ten times reduced product concentration in bioreactor cultures (Gustavsson et al. 2001, Protein Eng. 14, 711-715). Addition of rich medium for protease substrate competition had no effect on the proteolysis of CBM-CALB. The kinetics for the proteolytic reactions, with and without presence of cells were shown to be influenced by pH. The fastest reaction, cleavage in the linker, was substantially reduced at pH values below 5.0. Decreasing the pH from 5.0 to 4.0 in bioreactor cultures resulted in an increase of the fraction of full-length product from 40 to 90%. Further improvement was achieved by decreasing the temperature from 30 to 22 degrees C during the methanol feed phase. By combining the optimal pH and the low temperature almost all product (1.5 g x l(-1)) was obtained as full-length protein with a considerably higher purity in the culture supernatant compared with the original cultivation. PMID:12668313

  11. Age-related homeostatic midchannel proteolysis of neuronal L-type voltage-gated Ca²⁺ channels.

    PubMed

    Michailidis, Ioannis E; Abele-Henckels, Kathryn; Zhang, Wei K; Lin, Bochao; Yu, Yong; Geyman, Lawrence S; Ehlers, Michael D; Pnevmatikakis, Eftychios A; Yang, Jian

    2014-06-01

    Neural circuitry and brain activity depend critically on proper function of voltage-gated calcium channels (VGCCs), whose activity must be tightly controlled. We show that the main body of the pore-forming α1 subunit of neuronal L-type VGCCs, Cav1.2, is proteolytically cleaved, resulting in Cav1.2 fragment channels that separate but remain on the plasma membrane. This "midchannel" proteolysis is regulated by channel activity, involves the Ca(2+)-dependent protease calpain and the ubiquitin-proteasome system, and causes attenuation and biophysical alterations of VGCC currents. Recombinant Cav1.2 fragment channels mimicking the products of midchannel proteolysis do not form active channels on their own but, when properly paired, produce currents with distinct biophysical properties. Midchannel proteolysis increases dramatically with age and can be attenuated with an L-type VGCC blocker in vivo. Midchannel proteolysis represents a novel form of homeostatic negative-feedback processing of VGCCs that could profoundly affect neuronal excitability, neurotransmission, neuroprotection, and calcium signaling in physiological and disease states. PMID:24908485

  12. Age-related homeostatic mid-channel proteolysis of neuronal L-type voltage-gated Ca2+ channels

    PubMed Central

    Michailidis, Ioannis E.; Abele-Henckels, Kathryn; Zhang, Wei K.; Lin, Bochao; Yu, Yong; Geyman, Larry; Ehlers, Michael D.; Pnevmatikakis, Eftychios A.; Yang, Jian

    2014-01-01

    SUMMARY Neural circuitry and brain activity depend critically on proper function of voltage-gated calcium channels (VGCCs), whose activity must be tightly controlled. We show that the main body of the pore-forming α1 subunit of neuronal L-type VGCCs, Cav1.2, is proteolytically cleaved, resulting in Cav1.2 fragment-channels that separate but remain on the plasma membrane. This “gmid-channel” proteolysis is regulated by channel activity, involves the Ca2+-dependent protease calpain and the ubiquitin-proteasome system, and causes attenuation and biophysical alterations of VGCC currents. Recombinant Cav1.2 fragment-channels mimicking the products of mid-channel proteolysis do not form active channels on their own, but when properly paired, produce currents with distinct biophysical properties. Mid-channel proteolysis increases dramatically with age and can be attenuated with an L-type VGCC blocker in vivo. Mid-channel proteolysis represents a novel form of homeostatic negative-feedback processing of VGCCs that could profoundly affect neuronal excitability, neurotransmission, neuroprotection, and calcium signaling in physiological and disease states. PMID:24908485

  13. Ligand binding to an Allergenic Lipid Transfer Protein Enhances Conformational Flexibility resulting in an Increase in Susceptibility to Gastroduodenal Proteolysis.

    PubMed

    Abdullah, Syed Umer; Alexeev, Yuri; Johnson, Philip E; Rigby, Neil M; Mackie, Alan R; Dhaliwal, Balvinder; Mills, E N Clare

    2016-01-01

    Non-specific lipid transfer proteins (LTPs) are a family of lipid-binding molecules that are widely distributed across flowering plant species, many of which have been identified as allergens. They are highly resistant to simulated gastroduodenal proteolysis, a property that may play a role in determining their allergenicity and it has been suggested that lipid binding may further increase stability to proteolysis. It is demonstrated that LTPs from wheat and peach bind a range of lipids in a variety of conditions, including those found in the gastroduodenal tract. Both LTPs are initially cleaved during gastroduodenal proteolysis at three major sites between residues 39-40, 56-57 and 79-80, with wheat LTP being more resistant to cleavage than its peach ortholog. The susceptibility of wheat LTP to proteolyic cleavage increases significantly upon lipid binding. This enhanced digestibility is likely to be due to the displacement of Tyr79 and surrounding residues from the internal hydrophobic cavity upon ligand binding to the solvent exposed exterior of the LTP, facilitating proteolysis. Such knowledge contributes to our understanding as to how resistance to digestion can be used in allergenicity risk assessment of novel food proteins, including GMOs. PMID:27458082

  14. Chemical and proteolysis-derived changes during long-term storage of lactose-hydrolyzed ultrahigh-temperature (UHT) milk.

    PubMed

    Jansson, Therese; Jensen, Hanne B; Sundekilde, Ulrik K; Clausen, Morten R; Eggers, Nina; Larsen, Lotte B; Ray, Colin; Andersen, Henrik J; Bertram, Hanne C

    2014-11-19

    Proteolytic activity in milk may release bitter-tasting peptides and generate free amino terminals that react with carbohydrates, which initiate Maillard reaction. Ultrahigh temperature (UHT) heat treatment inactivates the majority of proteolytic enzymes in milk. In lactose-hydrolyzed milk a β-galactosidase preparation is applied to the milk after heat treatment, which has proteolytic side activities that may induce quality deterioration of long-term-stored milk. In the present study proteolysis, glycation, and volatile compound formation were investigated in conventional (100% lactose), filtered (60% lactose), and lactose-hydrolyzed (<1% lactose) UHT milk using reverse phase high-pressure liquid chromatography-mass spectrometry, proton nuclear magnetic resonance, and gas chromatography-mass spectrometry. Proteolysis was observed in all milk types. However, the degree of proteolysis was significantly higher in the lactose-hydrolyzed milk compared to the conventional and filtered milk. The proteins most prone to proteolysis were β-CN and αs1-CN, which were clearly hydrolyzed after approximately 90 days of storage in the lactose-hydrolyzed milk.

  15. The Putative O-Linked N-Acetylglucosamine Transferase SPINDLY Inhibits Class I TCP Proteolysis to Promote Sensitivity to Cytokinin.

    PubMed

    Steiner, Evyatar; Livne, Sivan; Kobinson-Katz, Tammy; Tal, Lior; Pri-Tal, Oded; Mosquna, Assaf; Tarkowská, Danuše; Mueller, Bruno; Tarkowski, Petr; Weiss, David

    2016-06-01

    Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) is a putative serine and threonine O-linked N-acetylglucosamine transferase (OGT). While SPY has been shown to suppress gibberellin signaling and to promote cytokinin (CK) responses, its catalytic OGT activity was never demonstrated and its effect on protein fate is not known. We previously showed that SPY interacts physically and functionally with TCP14 and TCP15 to promote CK responses. Here, we aimed to identify how SPY regulates TCP14/15 activities and how these TCPs promote CK responses. We show that SPY activity is required for TCP14 stability. Mutation in the putative OGT domain of SPY (spy-3) stimulated TCP14 proteolysis by the 26S proteasome, which was reversed by mutation in CULLIN1 (CUL1), suggesting a role for SKP, CUL1, F-box E3 ubiquitin ligase in TCP14 proteolysis. TCP14 proteolysis in spy-3 suppressed all TCP14 misexpression phenotypes, including the enhanced CK responses. The increased CK activity in TCP14/15-overexpressing flowers resulted from increased sensitivity to the hormone and not from higher CK levels. TCP15 overexpression enhanced the response of the CK-induced synthetic promoter pTCS to CK, suggesting that TCP14/15 affect early steps in CK signaling. We propose that posttranslational modification of TCP14/15 by SPY inhibits their proteolysis and that the accumulated proteins promote the activity of the CK phosphorelay cascade in developing Arabidopsis leaves and flowers. PMID:27208284

  16. Proteolysis approach without chemical modification for a simple and rapid analysis of disulfide bonds using thermostable protease-immobilized microreactors.

    PubMed

    Yamaguchi, Hiroshi; Miyazaki, Masaya; Maeda, Hideaki

    2010-08-01

    Disulfide bonds in proteins are important not only for the conformational stability of the protein but also for the regulation of oxidation-reduction in signal transduction. The conventional method for the assignment of disulfide bond by chemical cleavage and/or proteolysis is a time-consuming multi-step procedure. In this study, we report a simple and rapid analysis of disulfide bond from protein digests that were prepared by the thermostable protease-immobilized microreactors. The feasibility and performance of this approach were evaluated by digesting lysozyme and BSA at several temperatures. The proteins which stabilize their conformations by disulfide bonds were thermally denatured during proteolysis and were efficiently digested by the immobilized protease but not by free protease. The digests were directly analyzed by ESI-TOF MS without any purification or concentration step. All four disulfide bonds on lysozyme and 10 of 17 on BSA were assigned from the digests by the trypsin-immobilized microreactor at 50 degrees C. The procedure for proteolysis and the assignment were achieved within 2 h without any reduction and alkylation procedure. From the present results, the proteolysis approach by the thermostable protease-immobilized microreactor provides a strategy for the high-throughput analysis of disulfide bond in proteomics.

  17. Ligand binding to an Allergenic Lipid Transfer Protein Enhances Conformational Flexibility resulting in an Increase in Susceptibility to Gastroduodenal Proteolysis

    PubMed Central

    Abdullah, Syed Umer; Alexeev, Yuri; Johnson, Philip E.; Rigby, Neil M.; Mackie, Alan R.; Dhaliwal, Balvinder; Mills, E. N. Clare

    2016-01-01

    Non-specific lipid transfer proteins (LTPs) are a family of lipid-binding molecules that are widely distributed across flowering plant species, many of which have been identified as allergens. They are highly resistant to simulated gastroduodenal proteolysis, a property that may play a role in determining their allergenicity and it has been suggested that lipid binding may further increase stability to proteolysis. It is demonstrated that LTPs from wheat and peach bind a range of lipids in a variety of conditions, including those found in the gastroduodenal tract. Both LTPs are initially cleaved during gastroduodenal proteolysis at three major sites between residues 39–40, 56–57 and 79–80, with wheat LTP being more resistant to cleavage than its peach ortholog. The susceptibility of wheat LTP to proteolyic cleavage increases significantly upon lipid binding. This enhanced digestibility is likely to be due to the displacement of Tyr79 and surrounding residues from the internal hydrophobic cavity upon ligand binding to the solvent exposed exterior of the LTP, facilitating proteolysis. Such knowledge contributes to our understanding as to how resistance to digestion can be used in allergenicity risk assessment of novel food proteins, including GMOs. PMID:27458082

  18. A model of proteolysis and amino acid biosynthesis for Lactobacillus delbrueckii subsp. bulgaricus in whey.

    PubMed

    Liu, Enuo; Zheng, Huajun; Hao, Pei; Konno, Tomonobu; Yu, Yao; Kume, Hisae; Oda, Munehiro; Ji, Zai-Si

    2012-12-01

    Lactobacillus delbrueckii subsp. bulgaricus 2038 (L. bulgaricus 2038) is a bacterium that is used as a starter for dairy products by Meiji Co., Ltd of Japan. Culturing L. bulgaricus 2038 with whey as the sole nitrogen source results in a shorter lag phase than other milk proteins under the same conditions (carbon source, minerals, and vitamins). Microarray results of gene expression revealed characteristics of amino acid anabolism with whey as the nitrogen source and established a model of proteolysis and amino acid biosynthesis for L. bulgaricus. Whey peptides and free amino acids are readily metabolized, enabling rapid entry into the logarithmic growth phase. The oligopeptide transport system is the primary pathway for obtaining amino acids. Amino acid biosynthesis maintains the balance between amino acids required for cell growth and the amount obtained from environment. The interconversion of amino acids is also important for L. bulgaricus 2038 growth.

  19. Effect of Flavourzyme on proteolysis, antioxidant capacity and sensory attributes of Chinese sausage.

    PubMed

    Feng, Li; Qiao, Yan; Zou, Yufeng; Huang, Ming; Kang, Zhuangli; Zhou, Guanghong

    2014-09-01

    The objective of this study was to investigate the effect of Flavourzyme, at levels of 0 (control) 4, 8, 12, 16 and 20 LAPU/kg raw meat, on the proteolysis, antioxidant capacity and sensory attributes of Chinese sausage made at 50 °C for 48 h. Results showed that Flavourzyme addition in Chinese sausage accelerated protein degradation, which was reflected by the increase of non-protein nitrogen and appearance of new protein bands in both water-soluble and salt-soluble proteins. By adding Flavourzyme, texture profile analysis (TPA) parameters decreased significantly, and aroma, taste and texture scores were enhanced, respectively. The best sensory attributes were obtained at 8 and 12 LAPU/kg Flavourzyme dose. Besides, Flavourzyme addition enhanced antioxidant capacity, lowered water activity and TBARS values of Chinese sausage. Therefore, moderate Flavourzyme addition is a novel method with great potential to improve eating properties and storage stability of Chinese sausage. PMID:24831062

  20. Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets.

    PubMed

    McLauchlan, John; Lemberg, Marius K; Hope, Graham; Martoglio, Bruno

    2002-08-01

    Hepatitis C virus (HCV) is the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma. The virus has a positive-sense RNA genome encoding a single polyprotein with the virion components located in the N-terminal portion. During biosynthesis of the polyprotein, an internal signal sequence between the core protein and the envelope protein E1 targets the nascent polypeptide to the endoplasmic reticulum (ER) membrane for translocation of E1 into the ER. Following membrane insertion, the signal sequence is cleaved from E1 by signal peptidase. Here we provide evidence that after cleavage by signal peptidase, the signal peptide is further processed by the intramembrane-cleaving protease SPP that promotes the release of core protein from the ER membrane. Core protein is then free for subsequent trafficking to lipid droplets. This study represents an example of a potential role for intramembrane proteolysis in the maturation of a viral protein. PMID:12145199

  1. MBTPS2 mutations cause defective regulated intramembrane proteolysis in X-linked osteogenesis imperfecta

    PubMed Central

    Lindert, Uschi; Cabral, Wayne A.; Ausavarat, Surasawadee; Tongkobpetch, Siraprapa; Ludin, Katja; Barnes, Aileen M.; Yeetong, Patra; Weis, Maryann; Krabichler, Birgit; Srichomthong, Chalurmpon; Makareeva, Elena N.; Janecke, Andreas R.; Leikin, Sergey; Röthlisberger, Benno; Rohrbach, Marianne; Kennerknecht, Ingo; Eyre, David R.; Suphapeetiporn, Kanya; Giunta, Cecilia; Marini, Joan C.; Shotelersuk, Vorasuk

    2016-01-01

    Osteogenesis imperfecta (OI) is a collagen-related bone dysplasia. We identified an X-linked recessive form of OI caused by defects in MBTPS2, which encodes site-2 metalloprotease (S2P). MBTPS2 missense mutations in two independent kindreds with moderate/severe OI cause substitutions at highly conserved S2P residues. Mutant S2P has normal stability, but impaired functioning in regulated intramembrane proteolysis (RIP) of OASIS, ATF6 and SREBP transcription factors, consistent with decreased proband secretion of type I collagen. Further, hydroxylation of the collagen lysine residue (K87) critical for crosslinking is reduced in proband bone tissue, consistent with decreased lysyl hydroxylase 1 in proband osteoblasts. Reduced collagen crosslinks presumptively undermine bone strength. Also, proband osteoblasts have broadly defective differentiation. These mutations provide evidence that RIP plays a fundamental role in normal bone development. PMID:27380894

  2. A redox switch shapes the Lon protease exit pore to facultatively regulate proteolysis.

    PubMed

    Nishii, Wataru; Kukimoto-Niino, Mutsuko; Terada, Takaho; Shirouzu, Mikako; Muramatsu, Tomonari; Kojima, Masaki; Kihara, Hiroshi; Yokoyama, Shigeyuki

    2015-01-01

    The Lon AAA+ protease degrades damaged or misfolded proteins in its intramolecular chamber. Its activity must be precisely controlled, but the mechanism by which Lon is regulated in response to different environments is not known. Facultative anaerobes in the Enterobacteriaceae family, mostly symbionts and pathogens, encounter both anaerobic and aerobic environments inside and outside the host's body, respectively. The bacteria characteristically have two cysteine residues on the Lon protease (P) domain surface that unusually form a disulfide bond. Here we show that the cysteine residues act as a redox switch of Lon. Upon disulfide bond reduction, the exit pore of the P-domain ring narrows by ∼30%, thus interrupting product passage and decreasing activity by 80%; disulfide bonding by oxidation restores the pore size and activity. The redox switch (E°' = -227 mV) is appropriately tuned to respond to variation between anaerobic and aerobic conditions, thus optimizing the cellular proteolysis level for each environment.

  3. Interstitial collagenase is a Brownian ratchet driven by proteolysis of collagen.

    PubMed

    Saffarian, Saveez; Collier, Ivan E; Marmer, Barry L; Elson, Elliot L; Goldberg, Gregory

    2004-10-01

    We show that activated collagenase (MMP-1) moves processively on the collagen fibril. The mechanism of movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. Inactivation of the enzyme by a single amino acid residue substitution in the active center eliminates the bias without noticeable effect on rate of diffusion. Monte Carlo simulations using a model similar to a "burnt bridge" Brownian ratchet accurately describe our experimental results and previous observations on kinetics of collagen digestion. The biological implications of MMP-1 acting as a molecular ratchet tethered to the cell surface suggest new mechanisms for its role in tissue remodeling and cell-matrix interaction.

  4. The effect of proteolysis on the induction of cell death by monomeric alpha-lactalbumin.

    PubMed

    Brück, Wolfram M; Gibson, Glenn R; Brück, Thomas B

    2014-02-01

    α-Lactalbumin (α-la) is a major whey protein found in milk. Previous data suggested that α-la has antiproliferative effects in human adenocarcinoma cell lines such as Caco-2 and HT-29. However, the cell death inducing α-la was not a naturally occurring monomer but either a multimeric variant or an α-la:oleic acid complex (HAMLET/BAMLET). Proteolysis showed that both human and bovine α-la are susceptible to digestion. ELISA assays assessing cell death with the native undigested α-la fractions showed that undigested protein fractions did have a significant cell death effect on CaCo-2 cells. Bovine α-la was also more effective than human α-la. A reduction in activity corresponded with lower concentrations of the protein and partial digestion and fragmentation of the protein using trypsin and pepsin. This suggests that the tertiary structure is vital for the apoptotic effect.

  5. Effect of Flavourzyme on proteolysis, antioxidant capacity and sensory attributes of Chinese sausage.

    PubMed

    Feng, Li; Qiao, Yan; Zou, Yufeng; Huang, Ming; Kang, Zhuangli; Zhou, Guanghong

    2014-09-01

    The objective of this study was to investigate the effect of Flavourzyme, at levels of 0 (control) 4, 8, 12, 16 and 20 LAPU/kg raw meat, on the proteolysis, antioxidant capacity and sensory attributes of Chinese sausage made at 50 °C for 48 h. Results showed that Flavourzyme addition in Chinese sausage accelerated protein degradation, which was reflected by the increase of non-protein nitrogen and appearance of new protein bands in both water-soluble and salt-soluble proteins. By adding Flavourzyme, texture profile analysis (TPA) parameters decreased significantly, and aroma, taste and texture scores were enhanced, respectively. The best sensory attributes were obtained at 8 and 12 LAPU/kg Flavourzyme dose. Besides, Flavourzyme addition enhanced antioxidant capacity, lowered water activity and TBARS values of Chinese sausage. Therefore, moderate Flavourzyme addition is a novel method with great potential to improve eating properties and storage stability of Chinese sausage.

  6. Improvement of the antimicrobial and antioxidant activities of camel and bovine whey proteins by limited proteolysis.

    PubMed

    Salami, Maryam; Moosavi-Movahedi, Ali Akbar; Ehsani, Mohammad Reza; Yousefi, Reza; Haertlé, Thomas; Chobert, Jean-Marc; Razavi, Seyed Hadi; Henrich, Robert; Balalaie, Saeed; Ebadi, Seyed Ahmad; Pourtakdoost, Samineh; Niasari-Naslaji, Amir

    2010-03-24

    The compositions and structures of bovine and camel milk proteins are different, which define their functional and biological properties. The aim of this study was to investigate the effects of enzymatic hydrolysis of camel and bovine whey proteins (WPs) on their antioxidant and antimicrobial properties. After enzymatic treatment, both the antioxidant and the antimicrobial activities of bovine and camel WPs were improved. The significantly higher antioxidant activity of camel WPs and their hydrolysates as compared with that of bovine WPs and their hydrolysates may result from the differences in amounts and/or in accessibilities of antioxidant amino acid residues present in their primary structures and from the prevalence of alpha-lactalbumin and beta-lactoglobulin as proteolytic substrates in camel and bovine whey, respectively. The results of this study reveal differences in antimicrobial and antioxidant activities between WP hydrolysates of bovine and camel milk and the effects of limited proteolysis on these activities.

  7. Mechanisms of accelerated proteolysis in rat soleus muscle atrophy induced by unweighting or denervation

    NASA Technical Reports Server (NTRS)

    Tischler, Marc E.; Kirby, Christopher; Rosenberg, Sara; Tome, Margaret; Chase, Peter

    1991-01-01

    A hypothesis proposed by Tischler and coworkers (Henriksen et al., 1986; Tischler et al., 1990) concerning the mechanisms of atrophy induced by unweighting or denervation was tested using rat soleus muscle from animals subjected to hindlimb suspension and denervation of muscles. The procedure included (1) measuring protein degradation in isolated muscles and testing the effects of lysosome inhibitors, (2) analyzing the lysosome permeability and autophagocytosis, (3) testing the effects of altering calcium-dependent proteolysis, and (4) evaluating in vivo the effects of various agents to determine the physiological significance of the hypothesis. The results obtained suggest that there are major differences between the mechanisms of atrophies caused by unweighting and denervation, though slower protein synthesis is an important feature common for both.

  8. Ubiquitin-Like Proteasome System Represents a Eukaryotic-Like Pathway for Targeted Proteolysis in Archaea

    PubMed Central

    Fu, Xian; Liu, Rui; Sanchez, Iona; Silva-Sanchez, Cecilia; Hepowit, Nathaniel L.; Cao, Shiyun; Chen, Sixue

    2016-01-01

    ABSTRACT The molecular mechanisms of targeted proteolysis in archaea are poorly understood, yet they may have deep evolutionary roots shared with the ubiquitin-proteasome system of eukaryotic cells. Here, we demonstrate in archaea that TBP2, a TATA-binding protein (TBP) modified by ubiquitin-like isopeptide bonds, is phosphorylated and targeted for degradation by proteasomes. Rapid turnover of TBP2 required the functions of UbaA (the E1/MoeB/ThiF homolog of archaea), AAA ATPases (Cdc48/p97 and Rpt types), a type 2 JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) homolog (JAMM2), and 20S proteasomes. The ubiquitin-like protein modifier small archaeal modifier protein 2 (SAMP2) stimulated the degradation of TBP2, but SAMP2 itself was not degraded. Analysis of the TBP2 fractions that were not modified by ubiquitin-like linkages revealed that TBP2 had multiple N termini, including Met1-Ser2, Ser2, and Met1-Ser2(p) [where (p) represents phosphorylation]. The evidence suggested that the Met1-Ser2(p) form accumulated in cells that were unable to degrade TBP2. We propose a model in archaea in which the attachment of ubiquitin-like tags can target proteins for degradation by proteasomes and be controlled by N-terminal degrons. In support of a proteolytic mechanism that is energy dependent and recycles the ubiquitin-like protein tags, we find that a network of AAA ATPases and a JAMM/MPN+ metalloprotease are required, in addition to 20S proteasomes, for controlled intracellular proteolysis. PMID:27190215

  9. The Improvement of The Endogenous Antioxidant Property of Stone Fish (Actinopyga lecanora) Tissue Using Enzymatic Proteolysis

    PubMed Central

    Bordbar, Sara; Ebrahimpour, Afshin; Abdul Hamid, Azizah; Abdul Manap, Mohd Yazid; Anwar, Farooq; Saari, Nazamid

    2013-01-01

    The stone fish (Actinopyga lecanora) ethanolic and methanolic tissue extracts were investigated for total phenolic contents (TPCs) as well as antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging activity and ferric reducing antioxidant power (FRAP) assays. Both extracts showed low amount of phenolics (20.33 to 17.03 mg of gallic acid equivalents/100 g dried sample) and moderate antioxidant activity (39% to 34%  DPPH• radical scavenging activity and 23.95 to 22.30 mmol/100 mL FeSO4 FRAP value). Enzymatic proteolysis was carried out in order to improve the antioxidant activity using six commercially available proteases under their optimum conditions. The results revealed that the highest increase in antioxidant activity up to 85% was obtained for papain-generated proteolysate, followed by alcalase (77%), trypsin (75%), pepsin (68%), bromelain (68%), and flavourzyme (50%) as measured by DPPH• radical scavenging activity, whilst for the FRAP value, the highest increase in the antioxidant activity up to 39.2 mmol/100 mL FeSO4 was obtained for alcalase-generated proteolysate, followed by papain (29.5 mmol/100 mL FeSO4), trypsin (23.2 mmol/100 mL FeSO4), flavourzyme (24.7 mmol/100 mL FeSO4), bromelain (22.9 mmol/100 mL FeSO4), and pepsin (20.8 mmol/100 mL FeSO4). It is obvious that proteolysis of stone fish tissue by proteolytic enzymes can considerably enhance its antioxidant activity. PMID:23586061

  10. In vitro regulation of pericellular proteolysis in prostatic tumor cells treated with bombesin.

    PubMed

    Festuccia, C; Guerra, F; D'Ascenzo, S; Giunciuglio, D; Albini, A; Bologna, M

    1998-01-30

    Bombesin is a potent inducer of signal trasduction pathways involved in the proliferation and invasion of androgen-insensitive prostatic tumor cells. This study examines the bombesin-mediated modulation of pericellular proteolysis, monitoring cell capability to migrate and invade basement membranes, using a chemo-invasion assay and analyzing protease production. The results suggest that bombesin could modulate the invasive potential of prostatic cell lines regulating secretion and cell-surface uptake of uPA and MMP-9 activation. In fact, in PC3 and DU145 cells but not in LNCaP cells, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) are induced by bombesin treatment. Bombesin also stimulates cell proliferation and this effect can be inhibited blocking uPA by antibodies and/or uPA inhibitor p-aminobenzamidine. Moreover, HMW-uPA induces cell proliferation in LNCaP cells, which do not produce uPA in the basal conditions, while PC3 and DU145 cell growth is supported by autocrine production of uPA. The increment of uPA activity on the external plasma membrane causes an increased pericellular plasmin activation. This effect is inhibited by antibodies against uPA and by p-aminobenzamidine. Similarly to EGF, bombesin stimulates secretion and activation of MMP-9 and TIMP-1 production. MMP-9 activation can be also obtained by HMW-uPA treatment, suggesting that plasma-membrane-bound uPA can start a proteolytic cascade involving MMP-9. Therefore, in in vitro assays, bombesin is able to modulate pericellular proteolysis and cell proliferation, differently distributing and activating proteolytic activities. This effect can be related to the "non-random" degradation of the extracellular matrix in which membrane uPA-uPAreceptor complexes could start bombesin-induced directional protein degradation during metastatic spread. PMID:9455804

  11. Structural analysis of covalently labeled estrogen receptors by limited proteolysis and monoclonal antibody reactivity

    SciTech Connect

    Katzenellenbogen, B.S.; Elliston, J.F.; Monsma, F.J. Jr.; Springer, P.A.; Ziegler, Y.S.

    1987-04-21

    The authors have used limited proteolysis of affinity-labeled estrogen receptors (ER), coupled with antireceptor antibody immunoreactivity, to assess structural features of ER and the relatedness of ER from MCF-7 human breast cancer and rat uterine cells. MCF-7 ER preparations covalently labeled with (/sup 3/H)tamoxifen aziridine ((/sup 3/H)TAZ) were treated with trypsin (T), ..cap alpha..-chymotrypsin (C), or Staphylococcus aureus V8 protease prior to electrophoresis on sodium dodecyl sulfate gels. Fluorography revealed a distinctive ladder of ER fragments containing TAZ for each protease generated from the M/sub r/ 66,000 ER. Immunoblot detection with the primate-specific antibody D75P3..gamma.. revealed that all immunoreactive fragments corresponded to TAZ-labeled fragments but that some small TAZ-labeled fragments were no longer immunoreactive. In contrast, use of the antibody H222SP..gamma.. revealed a correspondence between TAZ-labeled and immunoreactive fragments down to the smallest fragments generated, ca. 6K for T and C and 28K for V8. MCF-7 nuclear and cytosol ER showed very similar digest patterns, and there was a remarkable similarity in the TAZ-labeled and H222-immunoreactive fragments generated by proteolysis of both MCF-7 and rat uterine ER. These findings reveal great structural similarities between the human (breast cancer) and rat (uterine) ER and between nuclear and cytosol ER, indicate charge heterogeneity of ER, and allow a comparison of the immunoreactive and hormone attachment site domains of the ER. The observation that T and C generate a ca. 6K TAZ-labeled fragment that is also detectable with the H222 antibody should be of interest inn studies determining the hormone binding domain of the ER and in amino acid sequencing of this region.

  12. Hemin inhibits ubiquitin-dependent proteolysis in both a higher plant and yeast

    SciTech Connect

    Vierstra, R.D.; Sullivan, M.L.

    1988-05-03

    In eukaryotes, a major route for ATP-dependent protein breakdown proceeds through covalent intermediates of target proteins destined for degradation and the highly conserved, 76 amino acid protein ubiquitin. In rabbit reticulocytes, it has been shown that hemin effectively inhibits this pathway by blocking the catabolism of ubiquitin-protein conjugates. Here the authors demonstrate that hemin is also an effective inhibitor of the ubiquitin-dependent proteolytic pathway in both a higher plant, oats (Avena sativa), and yeast (Saccharomyces cerevisiae). Hemin inhibits all stages of the pathway in vitro, including ATP-dependent formation of ubiquitin-protein conjugates, disassembly of conjugates by ubiquitin-protein lyase(s) (or isopeptidases), and degradation of ubiquitin-protein conjugates by ATP-dependent protease(s). Using ubiquitin-/sup 125/I-lysozyme conjugates synthesized in vitro as substrates, they determined the specific effects of hemin on the rates of disassembly and degradation separately. The concentration of hemin required for half-maximal inhibition of both processes was identical in each species, approx. 60 ..mu..M in oats and approx. 50 ..mu..M in yeast. Similar inhibitory effects were observed when two hemin analogues, mesoheme or protoporphyrin IX, were employed. These results demonstrate that the effect of hemin on ubiquitin-dependent proteolysis is not restricted to erythroid cells and as a result hemin may be a useful tool in studies of this pathway in all eukaryotic cells. These results also question models where hemin serves as a specific negative modulator of proteolysis in erythroid cells.

  13. Matrix Metalloproteinase (MMP) Proteolysis of the Extracellular Loop of Voltage-gated Sodium Channels and Potential Alterations in Pain Signaling*

    PubMed Central

    Remacle, Albert G.; Kumar, Sonu; Motamedchaboki, Khatereh; Cieplak, Piotr; Hullugundi, Swathi; Dolkas, Jennifer; Shubayev, Veronica I.; Strongin, Alex Y.

    2015-01-01

    Congenital insensitivity to pain (CIP) or congenital analgesia is a rare monogenic hereditary condition. This disorder is characterized by the inability to perceive any form of pain. Nonsense mutations in Nav.1.7, the main pain signaling voltage-gated sodium channel, lead to its truncations and, consequently, to the inactivation of the channel functionality. However, a non-truncating homozygously inherited missense mutation in a Bedouin family with CIP (Nav1.7-R907Q) has also been reported. Based on our currently acquired in-depth knowledge of matrix metalloproteinase (MMP) cleavage preferences, we developed the specialized software that predicts the presence of the MMP cleavage sites in the peptide sequences. According to our in silico predictions, the peptide sequence of the exposed extracellular unstructured region linking the S5–S6 transmembrane segments in the DII domain of the human Nav1.7 sodium channel is highly sensitive to MMP-9 proteolysis. Intriguingly, the CIP R907Q mutation overlaps with the predicted MMP-9 cleavage site sequence. Using MMP-9 proteolysis of the wild-type, CIP, and control peptides followed by mass spectrometry of the digests, we demonstrated that the mutant sequence is severalfold more sensitive to MMP-9 proteolysis relative to the wild type. Because of the substantial level of sequence homology among sodium channels, our data also implicate MMP proteolysis in regulating the cell surface levels of the Nav1.7, Nav1.6, and Nav1.8 channels, but not Nav1.9. It is likely that the aberrantly accelerated MMP-9 proteolysis during neurogenesis is a biochemical rational for the functional inactivation in Nav1.7 and that the enhanced cleavage of the Nav1.7-R907Q mutant is a cause of CIP in the Bedouin family. PMID:26283785

  14. Matrix Metalloproteinase (MMP) Proteolysis of the Extracellular Loop of Voltage-gated Sodium Channels and Potential Alterations in Pain Signaling.

    PubMed

    Remacle, Albert G; Kumar, Sonu; Motamedchaboki, Khatereh; Cieplak, Piotr; Hullugundi, Swathi; Dolkas, Jennifer; Shubayev, Veronica I; Strongin, Alex Y

    2015-09-18

    Congenital insensitivity to pain (CIP) or congenital analgesia is a rare monogenic hereditary condition. This disorder is characterized by the inability to perceive any form of pain. Nonsense mutations in Nav.1.7, the main pain signaling voltage-gated sodium channel, lead to its truncations and, consequently, to the inactivation of the channel functionality. However, a non-truncating homozygously inherited missense mutation in a Bedouin family with CIP (Nav1.7-R907Q) has also been reported. Based on our currently acquired in-depth knowledge of matrix metalloproteinase (MMP) cleavage preferences, we developed the specialized software that predicts the presence of the MMP cleavage sites in the peptide sequences. According to our in silico predictions, the peptide sequence of the exposed extracellular unstructured region linking the S5-S6 transmembrane segments in the DII domain of the human Nav1.7 sodium channel is highly sensitive to MMP-9 proteolysis. Intriguingly, the CIP R907Q mutation overlaps with the predicted MMP-9 cleavage site sequence. Using MMP-9 proteolysis of the wild-type, CIP, and control peptides followed by mass spectrometry of the digests, we demonstrated that the mutant sequence is severalfold more sensitive to MMP-9 proteolysis relative to the wild type. Because of the substantial level of sequence homology among sodium channels, our data also implicate MMP proteolysis in regulating the cell surface levels of the Nav1.7, Nav1.6, and Nav1.8 channels, but not Nav1.9. It is likely that the aberrantly accelerated MMP-9 proteolysis during neurogenesis is a biochemical rational for the functional inactivation in Nav1.7 and that the enhanced cleavage of the Nav1.7-R907Q mutant is a cause of CIP in the Bedouin family. PMID:26283785

  15. The proteolysis adaptor, NblA, initiates protein pigment degradation by interacting with the cyanobacterial light-harvesting complexes.

    PubMed

    Sendersky, Eleonora; Kozer, Noga; Levi, Mali; Garini, Yuval; Shav-Tal, Yaron; Schwarz, Rakefet

    2014-07-01

    Degradation of the cyanobacterial protein pigment complexes, the phycobilisomes, is a central acclimation response that controls light energy capture. The small protein, NblA, is essential for proteolysis of these large complexes, which may reach a molecular mass of up to 4 MDa. Interactions of NblA in vitro supported the suggestion that NblA is a proteolysis adaptor that labels the pigment proteins for degradation. The mode of operation of NblA in situ, however, remained unresolved. Particularly, it was unclear whether NblA interacts with phycobilisome proteins while part of the large complex, or alternatively interaction with NblA, necessitates dissociation of pigment subunits from the assembly. Fluorescence intensity profiles demonstrated the preferential presence of NblA::GFP (green fluorescent protein) at the photosynthetic membranes, indicating co-localization with phycobilisomes. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for interaction of NblA with phycobilisome protein pigments. Additionally, we demonstrated the role of NblA in vivo as a proteolysis tag based on the rapid degradation of the fusion protein NblA::GFP compared with free GFP. Taken together, these observations demonstrated in vivo the role of NblA as a proteolysis adaptor. Additionally, the interaction of NblA with phycobilisomes indicates that the dissociation of protein pigment subunits from the large complex is not a prerequisite for interaction with this adaptor and, furthermore, implicates NblA in the disassembly of the protein pigment complex. Thus, we suggest that, in the case of proteolysis of the phycobilisome, the adaptor serves a dual function: undermining the complex stability and designating the dissociated pigments for degradation.

  16. Homologous desensitization of HEL cell thrombin receptors. Distinguishable roles for proteolysis and phosphorylation.

    PubMed

    Brass, L F

    1992-03-25

    Loss of sensitivity to thrombin following an initial response is characteristic of a number of cell types, including platelets. It has recently been proposed that thrombin receptors resemble other G protein-coupled receptors, but that activation involves a novel mechanism in which thrombin cleaves the receptor, exposing a new N terminus that serves as the ligand for the receptor. Based upon this model, we have examined the mechanism of thrombin receptor desensitization by comparing the effects of thrombin with those of a peptide corresponding to the N-terminal sequence of the receptor following proteolysis by thrombin: SFLLRNPNDKYEPF or TRP42/55. Like thrombin, TRP42/55 stimulated pertussis toxin-sensitive inositol 1,4,5-trisphosphate formation, raised cytosolic Ca2+, and inhibited cAMP formation in the megakaryoblastic HEL cell line. Exposure to either thrombin or TRP42/55 desensitized the cells to both, but not to a third agonist, neuropeptide Y. The rate of recovery after desensitization depended upon the order of agonist addition. Resensitization of the cell to thrombin following a brief exposure to thrombin required up to 24 h and could be inhibited with cycloheximide. Resensitization to TRP42/55 after exposure to thrombin, or to thrombin after exposure to TRP42/55, on the other hand, was detectable within 30 min and could be inhibited by serine/threonine phosphatase inhibitors, but not by cycloheximide. Loss of responsiveness to thrombin and TRP42/55 was also observed following addition of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). However, while the protein kinase inhibitor staurosporine completely prevented the desensitization caused by TPA, it had only a limited effect on the desensitization caused by TRP42/55. These results demonstrate that the G protein-mediated effects of thrombin can be reproduced by a receptor-derived peptide and suggest that desensitization occurs by at least two mechanisms. The first, which is seen with thrombin

  17. Identification and Characterization of a Cleavage Site in the Proteolysis of Orf Virus 086 Protein

    PubMed Central

    Wang, Xiaoping; Xiao, Bin; Zhang, Jiafeng; Chen, Daxiang; Li, Wei; Li, Ming; Hao, Wenbo; Luo, Shuhong

    2016-01-01

    The orf virus (ORFV) is among the parapoxvirus genus of the poxviridae family, but little is known about the proteolytic pathways of ORFV encoding proteins. By contrast, the proteolysis mechanism of the vaccinia virus (VV) has been extensively explored. Vaccinia virus core protein P4a undergoes a proteolytic process that takes place at a conserved cleavage site Ala-Gly-X (where X is any amino acid) and participates in virus assembly. Bioinformatics analysis revealed that an ORFV encoding protein, ORFV086, has a similar structure to the vaccinia virus P4a core protein. In this study, we focus on the kinetic analysis and proteolysis mechanism of ORFV086. We found, via kinetic analysis, that ORFV086 is a late gene that starts to express at 8 h post infection at mRNA level and 12–24 h post infection at the protein level. The ORFV086 precursor and a 21 kDa fragment can be observed in mature ORFV virions. The same bands were detected at only 3 h post infection, suggesting that both the ORFV086 precursor and the 21 kDa fragment are viral structural proteins. ORFV086 was cleaved from 12 to 24 h post infection. The cleavage took place at different sites, resulting in seven bands with differing molecular weights. Sequence alignment revealed that five putative cleavage sites were predicted at C-terminal and internal regions of ORFV086. To investigate whether those cleavage sites are involved in proteolytic processing, full length and several deletion mutant ORFV086 recombinant proteins were expressed and probed. The GGS site that produced a 21 kDa cleavage fragment was confirmed by identification of N/C-terminal FLAG epitope recombinant proteins, site-directed mutagenesis and pulse-chase analysis. Interestingly, chase results demonstrated that, at late times, ORFV086 is partially cleaved. Taken together, we concluded that GGS is a cleavage site in ORFV086 and produces a 21 kDa fragment post infection. Both ORFV086 precursor and the 21 kDa fragment are structural proteins of

  18. Effects of in vitro ozone treatment on proteolysis of purified rubisco from two hybrid poplar clones. [Populus maximowizii x trichocarpa

    SciTech Connect

    Landry, L.G.; Pell, E.J. )

    1989-04-01

    Plants exposed to ozone (O{sub 3}) exhibited symptoms of premature senescence, including early decline in quantity of rubisco. O{sub 3}-induced oxidation may cause changes in protein conformation of rubisco, resulting in enhanced proteolysis. To test this hypothesis, rubisco was purified from two hybrid clones of Populus maximowizii x trichocarpa, clones 388 and 245, and treated in vitro with O{sub 3} or air. Rubisco was then challenged with bromelain, papain, chymotrypsin, carboxypeptidase A, or endoproteinase Glu-C and percent degradation measured by SDS-PAGE and densitometric scanning of the gels. Degree of rubisco sensitivity to oxidation may be related to available sulfhydryl (SH) groups on the protein. The number of SH groups in native and denatured rubisco was measured for purified rubisco of both clones by DTNB titration method. The relationship between sensitivity to proteolysis and number and availability of SH groups is discussed.

  19. Prevention of unloading-induced atrophy by vitamin E supplementation: links between oxidative stress and soleus muscle proteolysis?

    PubMed Central

    Servais, Stéphane; Letexier, Dominique; Favier, Roland; Duchamp, Claude; Desplanches, Dominique

    2007-01-01

    Exposure to reduced activity induces skeletal muscle atrophy. Oxidative stress might contribute to muscle wasting via proteolysis activation. This study aimed to test two hypotheses in rats. Firstly, supplementation of the antioxidant vitamin E, prior and during the phase of unloading, would partly counteract unloading-induced soleus muscle atrophy. Secondly, vitamin E supplementation would decrease the rate of muscle proteolysis by reducing expression of calpains, caspase-3, -9, -12 and E3 ubiquitin ligases (MuRF1 and MAFbx). Soleus muscle atrophy (− 49%) induced by fourteen days of hindlimb unloading was reduced to only 32 % under vitamin E. Vitamin E partly prevented the decrease in type I and IIa fiber size. Supplementation increased HSP72 content, suppressed the rise in muscle level of thiobarbituric acid-reactive substance caused by unloading but failed to modify the lower ratio of reduced vs. oxidized glutathione, the higher uncoupling proteins mRNA and the antioxidant enzyme activities (superoxide dismutase, catalase, glutathione peroxidase) observed after unloading. Vitamin E treatment abolished the large upregulation of caspase 9, 12 and MuRF1 transcripts in unloaded muscle and greatly decreased the upregulation of μ-calpain, caspase 3 and MAFbx mRNA. In conclusion, the protective effect of vitamin E might be due to modulation of muscle proteolysis-related genes rather than to its antioxidant function. PMID:17291986

  20. A Novel Yeast Screen for Mitotic Arrest Mutants Identifies DOC1, a New Gene Involved in Cyclin Proteolysis

    PubMed Central

    Hwang, Lena H.; Murray, Andrew W.

    1997-01-01

    B-type cyclins are rapidly degraded at the transition between metaphase and anaphase and their ubiquitin-mediated proteolysis is required for cells to exit mitosis. We used a novel enrichment to isolate new budding mutants that arrest the cell cycle in mitosis. Most of these mutants lie in the CDC16, CDC23, and CDC27 genes, which have already been shown to play a role in cyclin proteolysis and encode components of a 20S complex (called the cyclosome or anaphase promoting complex) that ubiquitinates mitotic cyclins. We show that mutations in CDC26 and a novel gene, DOC1, also prevent mitotic cyclin proteolysis. Mutants in either gene arrest as large budded cells with high levels of the major mitotic cyclin (Clb2) protein at 37°C and cannot degrade Clb2 in G1-arrested cells. Cdc26 associates in vivo with Doc1, Cdc16, Cdc23, and Cdc27. In addition, the majority of Doc1 cosediments at 20S with Cdc27 in a sucrose gradient, indicating that Cdc26 and Doc1 are components of the anaphase promoting complex. PMID:9348530

  1. Importance of thioredoxin in the proteolysis of an immunoglobulin G as antigen by lysosomal Cys-proteases

    PubMed Central

    KERBLAT, I; DROUET, C; CHESNE, S; MARCHE, P N

    1999-01-01

    For disulphide-bonded antigens, reduction has been postulated to be a prerequisite for proteolytic antigen processing, with subsequent production of major histocompatibility complex (MHC) class II binding fragments. The murine monoclonal immunoglobulin G (IgG) CE25/B7 was used as a multimeric antigen in a human model. Native IgG is highly resistant to proteolysis and has been previously found to be partially reduced at early steps of cell processing to become a suitable substrate for endopeptidases. The role of the oxidoreductase thioredoxin (Trx) was assessed in the reduction of the IgG by cleavage of H–L and H–H disulphide bonds. Recombinant human Trx (rTrx) has been assayed in a proteolytic in vitro system on IgG using endosomal and lysosomal subcellular fractions from B lymphoblastoid cells. rTrx is required in a dose-dependent manner for development of efficient proteolysis, catalysed by thiol-dependent Cys-proteases, such as cathepsin B. We demonstrated that cathepsin B activity was stimulated by the addition of rTrx. Thus, we propose that Trx-dependent IgG proteolysis occurred, on the one hand by means of the unfolding of the IgG after disulphide reduction, becoming a substrate of lysosomal proteases, and on the other hand by Cys-proteases such as cathepsin B that are fully active upon the regeneration of their activity by hydrogen donors. PMID:10447715

  2. DNA Stimulates ATP-Dependent Proteolysis and Protein-Dependent ATPase Activity of Protease La from Escherichia coli

    NASA Astrophysics Data System (ADS)

    Chung, Chin Ha; Goldberg, Alfred L.

    1982-02-01

    The product of the lon gene in Escherichia coli is an ATP-dependent protease, protease La, that also binds strongly to DNA. Addition of double-stranded or single-stranded DNA to the protease in the presence of ATP was found to stimulate the hydrolysis of casein or globin 2- to 7-fold, depending on the DNA concentration. Native DNA from several sources (plasmid pBR322, phage T7, or calf thymus) had similar effects, but after denaturation the DNA was 20-100% more effective than the native form. Although poly(rA), globin mRNA, and various tRNAs did not stimulate proteolysis, poly(rC) and poly(rU) were effective. Poly(dT) was stimulatory but (dT)10 was not. In the presence of DNA as in its absence, proteolysis required concomitant ATP hydrolysis, and the addition of DNA also enhanced ATP hydrolysis by protease La 2-fold, but only in the presence of casein. At much higher concentrations, DNA inhibited proteolysis as well as ATP cleavage. Thus, association of this enzyme with DNA may regulate the degradation of cell proteins in vivo.

  3. Locked and proteolysis-based transcription activator-like effector (TALE) regulation.

    PubMed

    Lonzarić, Jan; Lebar, Tina; Majerle, Andreja; Manček-Keber, Mateja; Jerala, Roman

    2016-02-18

    Development of orthogonal, designable and adjustable transcriptional regulators is an important goal of synthetic biology. Their activity has been typically modulated through stimulus-induced oligomerization or interaction between the DNA-binding and activation/repression domain. We exploited a feature of the designable Transcription activator-like effector (TALE) DNA-binding domain that it winds around the DNA which allows to topologically prevent it from binding by intramolecular cyclization. This new approach was investigated through noncovalent ligand-induced cyclization or through a covalent split intein cyclization strategy, where the topological inhibition of DNA binding by cyclization and its restoration by a proteolytic release of the topologic constraint was expected. We show that locked TALEs indeed have diminished DNA binding and regain full transcriptional activity by stimulation with the rapamycin ligand or site-specific proteolysis of the peptide linker, with much higher level of activation than rapamycin-induced heterodimerization. Additionally, we demonstrated reversibility, activation of genomic targets and implemented logic gates based on combinations of protein cyclization, proteolytic cleavage and ligand-induced dimerization, where the strongest fold induction was achieved by the proteolytic cleavage of a repression domain from a linear TALE.

  4. Organization of a multifunctional protein in pyrimidine biosynthesis. A domain hypersensitive to proteolysis.

    PubMed

    Rumsby, P C; Campbell, P C; Niswander, L A; Davidson, J N

    1984-01-15

    When the multifunctional protein that catalyses the first three steps of pyrimidine biosynthesis in hamster cells is treated with staphylococcal V8 proteinase, a single cleavage takes place. The activities of carbamoyl-phosphate synthetase (EC 6.3.5.5), aspartate carbamoyltransferase (EC 2.1.3.2) and dihydro-orotase (EC 3.5.2.3) and the allosteric inhibition by UTP are unaffected. One fragment, of Mr 182000, has the first and third enzyme activities, whereas the other fragment, of Mr 42000, has aspartate carbamoyltransferase activity and an aggregation site. A similar small fragment is observed in protein digested with low concentrations of trypsin. A similar large fragment is seen after digestion with trypsin and as the predominating form of this protein in certain mutants defective in pyrimidine biosynthesis. These results indicate that a region located adjacent to the aspartate carbamoyltransferase domain is hypersensitive to proteinase action in vitro and may also be sensitive to proteolysis in vivo. PMID:6365086

  5. Quantitative proteomics and terminomics to elucidate the role of ubiquitination and proteolysis in adaptive immunity

    PubMed Central

    Klein, Theo; Viner, Rosa I.

    2016-01-01

    Adaptive immunity is the specialized defence mechanism in vertebrates that evolved to eliminate pathogens. Specialized lymphocytes recognize specific protein epitopes through antigen receptors to mount potent immune responses, many of which are initiated by nuclear factor-kappa B activation and gene transcription. Most, if not all, pathways in adaptive immunity are further regulated by post-translational modification (PTM) of signalling proteins, e.g. phosphorylation, citrullination, ubiquitination and proteolytic processing. The importance of PTMs is reflected by genetic or acquired defects in these pathways that lead to a dysfunctional immune response. Here we discuss the state of the art in targeted proteomics and systems biology approaches to dissect the PTM landscape specifically regarding ubiquitination and proteolysis in B- and T-cell activation. Recent advances have occurred in methods for specific enrichment and targeted quantitation. Together with improved instrument sensitivity, these advances enable the accurate analysis of often rare PTM events that are opaque to conventional proteomics approaches, now rendering in-depth analysis and pathway dissection possible. We discuss published approaches, including as a case study the profiling of the N-terminome of lymphocytes of a rare patient with a genetic defect in the paracaspase protease MALT1, a key regulator protease in antigen-driven signalling, which was manifested by elevated linear ubiquitination. This article is part of the themed issue ‘Quantitative mass spectrometry’. PMID:27644975

  6. Regulated proteolysis of a transcription factor complex is critical to cell cycle progression in Caulobacter crescentus.

    PubMed

    Gora, Kasia G; Cantin, Amber; Wohlever, Matthew; Joshi, Kamal K; Perchuk, Barrett S; Chien, Peter; Laub, Michael T

    2013-03-01

    Cell cycle transitions are often triggered by the proteolysis of key regulatory proteins. In Caulobacter crescentus, the G1-S transition involves the degradation of an essential DNA-binding response regulator, CtrA, by the ClpXP protease. Here, we show that another critical cell cycle regulator, SciP, is also degraded during the G1-S transition, but by the Lon protease. SciP is a small protein that binds directly to CtrA and prevents it from activating target genes during G1. We demonstrate that SciP must be degraded during the G1-S transition so that cells can properly activate CtrA-dependent genes following DNA replication initiation and the reaccumulation of CtrA. These results indicate that like CtrA, SciP levels are tightly regulated during the Caulobacter cell cycle. In addition, we show that formation of a complex between CtrA and SciP at target promoters protects both proteins from their respective proteases. Degradation of either protein thus helps trigger the destruction of the other, facilitating a cooperative disassembly of the complex. Collectively, our results indicate that ClpXP and Lon each degrade an important cell cycle regulator, helping to trigger the onset of S phase and prepare cells for the subsequent programmes of gene expression critical to polar morphogenesis and cell division.

  7. The role of limited proteolysis of thyrotropin-releasing hormone in thermoregulation. Final report

    SciTech Connect

    Prasad, C.

    1982-01-01

    Cyclo (His-Pro) is a biologiclly active cyclic dipeptide derived from thyrotropin-releasing hormone by its limited proteolysis. We have developed a specific radioimmunoassay for this cyclic peptide and shown its presence throughout rat and monkey brains. The normal rat brain concentration of cyclo (His-Pro) ranged from 35-61 pmols/brain. The elution profiles of rat brain cyclo (His-Pro)-like immunoreactivity and synthetic radioactive cyclo (His-Pro) following gel filtration, ion-exchange chromatography and high pressure liquid chromatography were similar. An analysis of the regional distribution of cyclo (His-Pro) and TRH in rat and monkey brains exhibited no apparent precursor-product relationship. Studies on the neuroanatomic sites for the thermoregulatory effects of cyclo (His-Pro) suggested that the neural loci responsible for cyclo (His-Pro)-induced hypothermia resides within POA/AHA. The endogenous levels of brain cyclo (His-Pro) were elevated when rats were made either hypothyroid by surgical thyroidectomy or forced to drink alcohol for six weeks. These studies demonstrate that cyclo (His-Pro) is present throughout the central nervous system in physiologically relevant concentrations which can be modified by appropriate physiological and pharamacological manipulations. These data in conjunction with earlier reports of multiple biological activities of exogenous cyclo (His-Pro), suggest that endogenous cyclo (His-Pro) is a biological active peptide and it may play a neurotransmitter or neuromodulator role in the central nervous system.

  8. Study of proteolysis in river buffalo mozzarella cheese using a proteomics approach.

    PubMed

    Petrella, G; Pati, S; Gagliardi, R; Rizzuti, A; Mastrorilli, P; la Gatta, B; Di Luccia, A

    2015-11-01

    The guarantee of the origin and quality of raw material is essential for the protection and valorization of Campana buffalo mozzarella cheese. The risk of utilization of semifinished products and stored milk in substitution for fresh milk is increasing, due to the continuous desire to reduce production costs. A proteomics approach and electrophoresis survey of retail mozzarella cheeses indicated different rates of proteolysis in the production of dairy industries. The use of fresh milk and correct cheesemaking protocol yielded only γ-caseins, which are derived from β-casein by plasmin, and para-κ-casein, which is derived from κ-casein by chymosin. The detection of abnormal hydrolysis resulting in β- and αS1-casein fragments, identified by mass spectrometry, indicates the use of stored milk or stored and pressed curd, or the reuse of unsold mozzarella cheese, to produce mozzarella. The formation of γ-caseins and other fragments during a long storage of raw materials at room or refrigeration temperature was ascribed to plasmin (endogenous milk enzyme), whereas formation of αS1-casein fragments, mainly αS1-I(6P)- and αS1-I(7P)-casein during the storage of curd was ascribed to the action of chymosin (exogenous enzyme) from rennet. Sodium dodecyl sulfate-PAGE and alkaline urea-PAGE permitted us to evaluate the freshness of the raw materials used in the manufacturing of buffalo mozzarella cheese and to reveal possible inappropriate preservation.

  9. Quantitative proteomics and terminomics to elucidate the role of ubiquitination and proteolysis in adaptive immunity.

    PubMed

    Klein, Theo; Viner, Rosa I; Overall, Christopher M

    2016-10-28

    Adaptive immunity is the specialized defence mechanism in vertebrates that evolved to eliminate pathogens. Specialized lymphocytes recognize specific protein epitopes through antigen receptors to mount potent immune responses, many of which are initiated by nuclear factor-kappa B activation and gene transcription. Most, if not all, pathways in adaptive immunity are further regulated by post-translational modification (PTM) of signalling proteins, e.g. phosphorylation, citrullination, ubiquitination and proteolytic processing. The importance of PTMs is reflected by genetic or acquired defects in these pathways that lead to a dysfunctional immune response. Here we discuss the state of the art in targeted proteomics and systems biology approaches to dissect the PTM landscape specifically regarding ubiquitination and proteolysis in B- and T-cell activation. Recent advances have occurred in methods for specific enrichment and targeted quantitation. Together with improved instrument sensitivity, these advances enable the accurate analysis of often rare PTM events that are opaque to conventional proteomics approaches, now rendering in-depth analysis and pathway dissection possible. We discuss published approaches, including as a case study the profiling of the N-terminome of lymphocytes of a rare patient with a genetic defect in the paracaspase protease MALT1, a key regulator protease in antigen-driven signalling, which was manifested by elevated linear ubiquitination.This article is part of the themed issue 'Quantitative mass spectrometry'. PMID:27644975

  10. Influence of curd heating on proteolysis and volatiles of Kashkaval cheese.

    PubMed

    Sulejmani, Erhan; Hayaloglu, Ali A

    2016-11-15

    Kashkaval is the most popular hard cheese in Macedonia and other countries of Balkan peninsula. The aim of this research is to assess the differences of heat treatments effect (60, 70 and 90°C for 5min) in several biochemical and technological characteristics of Kashkaval cheese. Proteolysis was observed to take place at a faster rate in the Kashkaval cheeses made using the lower heat treatment. The residual enzyme activity of cheeses averaged 67.7, 43.7 and 8.4% for the cheeses heated at 60, 70 and 90°C, respectively. Acids and esters constituted the main chemical class of the cheeses during ripening (mean abundances of these were 57.1% and 26.8% w/w of total volatiles, respectively). The colour (L(∗)) and meltability values decreased significantly during ripening. In conclusion, powerful correlations were observed between extents of the heat treatment and levels of residual coagulant activity, breakdown of proteins and formation of volatiles. PMID:27283619

  11. HEMERA Couples the Proteolysis and Transcriptional Activity of PHYTOCHROME INTERACTING FACTORs in Arabidopsis Photomorphogenesis

    PubMed Central

    Qiu, Yongjian; Li, Meina; Pasoreck, Elise K.; Long, Lingyun; Shi, Yiting; Galvão, Rafaelo M.; Chou, Conrad L.; Wang, He; Sun, Amanda Y.; Zhang, Yiyin C.; Jiang, Anna; Chen, Meng

    2015-01-01

    Phytochromes (phys) are red and far-red photoreceptors that control plant development and growth by promoting the proteolysis of a family of antagonistically acting basic helix-loop-helix transcription factors, the PHYTOCHROME-INTERACTING FACTORs (PIFs). We have previously shown that the degradation of PIF1 and PIF3 requires HEMERA (HMR). However, the biochemical function of HMR and the mechanism by which it mediates PIF degradation remain unclear. Here, we provide genetic evidence that HMR acts upstream of PIFs in regulating hypocotyl growth. Surprisingly, genome-wide analysis of HMR- and PIF-dependent genes reveals that HMR is also required for the transactivation of a subset of PIF direct-target genes. We show that HMR interacts with all PIFs. The HMR-PIF interaction is mediated mainly by HMR’s N-terminal half and PIFs’ conserved active-phytochrome B binding motif. In addition, HMR possesses an acidic nine-amino-acid transcriptional activation domain (9aaTAD) and a loss-of-function mutation in this 9aaTAD impairs the expression of PIF target genes and the destruction of PIF1 and PIF3. Together, these in vivo results support a regulatory mechanism for PIFs in which HMR is a transcriptional coactivator binding directly to PIFs and the 9aaTAD of HMR couples the degradation of PIF1 and PIF3 with the transactivation of PIF target genes. PMID:25944101

  12. The structure of infant formulas impacts their lipolysis, proteolysis and disintegration during in vitro gastric digestion.

    PubMed

    Bourlieu, Claire; Ménard, Olivia; De La Chevasnerie, Alix; Sams, Laura; Rousseau, Florence; Madec, Marie-Noëlle; Robert, Benoît; Deglaire, Amélie; Pezennec, Stéphane; Bouhallab, Saïd; Carrière, Frédéric; Dupont, Didier

    2015-09-01

    Milk lipids supply most of the calories necessary for newborn growth in maternal milk or infant formulas. The chemical composition of infant formulas has been optimized but not the structure of the emulsion. There is still a major difference between the native emulsions of milk fat globules and processed submicronic emulsions in infant formulas. This difference may modify the kinetics of digestion of emulsions in newborns and influence lipid metabolism. To check this, semi-dynamic gastric in vitro digestions were conducted on three matrices: a standardized milk emulsion containing native milk fat globules referred to as minimally-processed emulsion and two processed model infant formulas (homogenized or homogenized/pasteurized). Gastric conditions mimicked those reported in newborns. The minimally-processed emulsion was lipolyzed and proteolyzed slower than processed formulas. The difference in initial structure persisted during digestion. The surface of the droplets was the key parameter to control gastric lipolysis kinetics, the pattern of released fatty acids and proteolysis by faster hydrolysis of adsorbed proteins. PMID:25842331

  13. Proteolysis in dry fermented sausages: The effect of selected exogenous proteases.

    PubMed

    Díaz, O; Fernandez, M; De Fernando, G D; de la Hoz, L; Ordoñez, J A

    1997-05-01

    The effect of three commercial proteases (pronase E from Streptomyces griseus, aspartyl proteinase from Aspergillus oryzae and papain) on protein breakdown and the sensory characteristics of dry fermented sausages was investigated. Water soluble, non-protein, 5% phosphotungstic acid soluble, 5% sulphosalicylic acid soluble and total volatile basic nitrogen contents increased during fermentation, stabilizing later until the end of ripening (26th day). Nitrogen values were always greater in the aspartyl proteinase added batch in comparison with the other protease added batches. Total free amino acid changes showed a similar pattern to those observed for the 5% sulphosalicylic acid soluble nitrogen. The electrophoretic studies demonstrated that proteolysis of high molecular weight myofibrillar and sarcoplasmic proteins was more prominent in protease added batches. It was especially intensive in papain one. The dominant amino acids at the end of ripening were similar in all batches. Tyramine and histamine increased throughout ripening. No significant differences in sensory properties were found between control and pronase E and papain added batches, but they were significantly different (p < 0.01) from the sausages containing aspartyl proteinase, due to an excessive softening. The effect of exogenous enzyme addition on the flavour potentiation of dry fermented sausage is discussed. PMID:22061850

  14. Limited proteolysis of myoglobin opens channel in ferrochelatase-globin complex for iron to zinc transmetallation.

    PubMed

    Paganelli, Marcella O; Grossi, Alberto B; Dores-Silva, Paulo R; Borges, Julio C; Cardoso, Daniel R; Skibsted, Leif H

    2016-11-01

    Recombinant ferrochelatase (BsFECH) from Bacillus subtilis expressed in Escherichia coli BL21(DE3) was found by UV-visible spectroscopy to bind the model substrate tetraphenylporphyrin-sulfonate, TPPS, with Ka=3.8 10(5)mol/L in aqueous phosphate buffer pH 5.7 at 30°C, and to interact with metmyoglobin with Ka=1.07±0.13 10(5)mol/L at 30°C. The iron/zinc exchange in myoglobin occurring during maturation of Parma hams seems to depend on such substrate binding to BsFECH and was facilitated by limited pepsin proteolysis of myoglobin to open a reaction channel for metal exchange still with BsFECH associated to globin. BsFECH increased rate of zinc insertion in TPPS significantly and showed saturation kinetics with an apparent binding constant of Zn(II) to the [enzyme-TPPS] complex of 1.3 10(4)mol/L and a first-order rate constant of 6.6 10(-1)s(-1) for dissociation of the tertiary complex, a similar pattern was found for zinc/iron transmetallation in myoglobin. PMID:27211675

  15. A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome

    PubMed Central

    Huber, Eva M.; Heinemeyer, Wolfgang; Li, Xia; Arendt, Cassandra S.; Hochstrasser, Mark; Groll, Michael

    2016-01-01

    Biogenesis of the 20S proteasome is tightly regulated. The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease. PMID:26964885

  16. Controlled intracellular proteolysis during postpartal involution of the uterus: characterization and regulation of an alkaline proteinase.

    PubMed

    Roth, M; Hoechst, M; Afting, E G

    1981-01-01

    The postpartal involution of the uterus is predominantly due to cellular hypotrophy. This implies an intracellular proteolytic system which must be carefully controlled pre and post partum. We have characterized and partially purified a proteinase with an alkaline pH-optimum of activity and a proteinase inhibitor protein which inhibits this proteinase very strongly. The alkaline proteinase copurifies with the actomyosin complex of the uterine myometrium and degrades the actomyosin complex with a concomitant loss of its myosin-ATPase activity. The alkaline proteinase is a very labile enzyme, markedly sensitive to SH-group modifying agents and has very high molecular weight at the present state of purification. This proteolytic enzyme could specifically be separated from the main components of the actomyosin complex by extraction with low ionic strength phosphate buffers. The proteinase inhibitor protein may control the activity of this alkaline proteinase during pregnancy and involution. The inhibitor protein raises 15-fold during pregnancy, possibly blocks important steps of intracellular proteolysis and permits organ growth. The dramatic fall of the inhibitor protein activity after parturition, which precedes the loss of weight, could release the proteolytic system, including the alkaline proteinase, and permits controlled intracellular degradation.

  17. Locked and proteolysis-based transcription activator-like effector (TALE) regulation.

    PubMed

    Lonzarić, Jan; Lebar, Tina; Majerle, Andreja; Manček-Keber, Mateja; Jerala, Roman

    2016-02-18

    Development of orthogonal, designable and adjustable transcriptional regulators is an important goal of synthetic biology. Their activity has been typically modulated through stimulus-induced oligomerization or interaction between the DNA-binding and activation/repression domain. We exploited a feature of the designable Transcription activator-like effector (TALE) DNA-binding domain that it winds around the DNA which allows to topologically prevent it from binding by intramolecular cyclization. This new approach was investigated through noncovalent ligand-induced cyclization or through a covalent split intein cyclization strategy, where the topological inhibition of DNA binding by cyclization and its restoration by a proteolytic release of the topologic constraint was expected. We show that locked TALEs indeed have diminished DNA binding and regain full transcriptional activity by stimulation with the rapamycin ligand or site-specific proteolysis of the peptide linker, with much higher level of activation than rapamycin-induced heterodimerization. Additionally, we demonstrated reversibility, activation of genomic targets and implemented logic gates based on combinations of protein cyclization, proteolytic cleavage and ligand-induced dimerization, where the strongest fold induction was achieved by the proteolytic cleavage of a repression domain from a linear TALE. PMID:26748097

  18. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis

    PubMed Central

    Fearnley, Gareth W.; Smith, Gina A.; Abdul-Zani, Izma; Yuldasheva, Nadira; Mughal, Nadeem A.; Homer-Vanniasinkam, Shervanthi; Kearney, Mark T.; Zachary, Ian C.; Tomlinson, Darren C.; Harrison, Michael A.; Wheatcroft, Stephen B.; Ponnambalam, Sreenivasan

    2016-01-01

    ABSTRACT Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A–VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor–ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes. PMID:27044325

  19. Exposure of fibrinogen receptors in human platelets by surface proteolysis with elastase.

    PubMed Central

    Kornecki, E; Ehrlich, Y H; De Mars, D D; Lenox, R H

    1986-01-01

    Human platelets that were preincubated with porcine elastase aggregated spontaneously upon the addition of fibrinogen. Maximal aggregation to fibrinogen was observed with platelets pretreated with an elastase concentration of 111 micrograms/ml, and half-maximal aggregation occurred after treatment with 11 micrograms/ml elastase. Binding of radiolabeled fibrinogen to elastase-treated platelets was specific, saturable, and showed a single class of 48,400 +/- 9,697 fibrinogen-binding sites per platelet with a dissociation constant of 6.30 +/- 1.48 X 10(-7) M. ATP, apyrase, and the stimulators of platelet adenylate cyclase forskolin, prostaglandin E1, prostacyclin, and N6, 2'-O-dibutyryl cyclic AMP did not inhibit the fibrinogen-induced aggregation of elastase-treated platelets. EDTA completely blocked the initiation of aggregation and reversed the fibrinogen-induced aggregation of elastase-treated platelets. Monoclonal and polyclonal antibodies directed against glycoproteins (GP) IIb and IIIa completely blocked the fibrinogen-induced aggregation of elastase-treated platelets. Immunoprecipitates with these antibodies obtained from detergent extracts of surface-radiolabeled, intact, and elastase-treated platelets contained the glycoproteins IIb and IIIa. We conclude that surface proteolysis by low concentrations of elastase can expose fibrinogen-binding sites associated with GPIIb and GPIIIa on the platelet surface, resulting in spontaneous aggregation upon the addition of fibrinogen. These findings may be relevant to hemostatic changes observed in patients with increased levels of circulating elastase. Images PMID:3005363

  20. Proteolysis in goat "coalho" cheese supplemented with probiotic lactic acid bacteria.

    PubMed

    Bezerra, Taliana Kênia Alves; de Araujo, Ana Rita Ribeiro; do Nascimento, Edilza Santos; de Matos Paz, José Eduardo; Gadelha, Carlos Alberto; Gadelha, Tatiane Santi; Pacheco, Maria Teresa Bertoldo; do Egypto Queiroga, Rita de Cássia Ramos; de Oliveira, Maria Elieidy Gomes; Madruga, Marta Suely

    2016-04-01

    This study aimed to analyse the proteolytic effects of adding isolated and combined probiotic strains to goat "coalho" cheese. The cheeses were: QS - with culture Start, composed by Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris (R704); QLA - with Lactobacillus acidophilus (LA-5); QLP - with Lactobacillus paracasei subsp. paracasei (L. casei 01); QB - with Bifidobacterium animalis subsp. lactis (BB 12); and QC, co-culture with the three probiotic microorganisms. The cheeses were analysed during 28 days of storage at 10°C. The probiotic cell count was higher than 6.5 and 7 log colony-forming units (CFU) g(-1) of cheese at the 1st and 28th days of storage, respectively. The addition of co-culture influenced (p<0.01) proteolysis in the cheese and resulted in a higher content of soluble protein and release of amino acids at the 1st day after processing. However, over all 28 days, the cheese supplemented with Bifidobacterium lactis in its isolated form showed the highest proteolytic activity, particularly in the hydrolysis of the alpha-s2 and kappa-casein fractions.

  1. Immobilization of trypsin via graphene oxide-silica composite for efficient microchip proteolysis.

    PubMed

    Bao, Huimin; Zhang, Luyan; Chen, Gang

    2013-10-01

    In this report, trypsin was covalently immobilized in the graphene oxide (GO)-silica composite coating on the channel wall of poly(methyl methacrylate) (PMMA) microchips to fabricate microfluidic bioreactors for highly efficient proteolysis. A mixture solution containing GO nanosheets and silica gel was injected into the channels to form coating. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were used as carboxyl activating agents to crosslink the primary amino groups of trypsin to the carboxyl groups of the entrapped GO sheets in the composite to realize covalent immobilization. The feasibility and performance of the novel GO-based microfluidic bioreactors were demonstrated by the digestion of hemoglobin (HEM), cytochrome c (Cyt-c), myoglobin (MYO), and ovalbumin (OVA) and the digestion time was significantly reduced to 5s. The obtained digests were identified by MALDI-TOF MS with the sequence coverages of 95%, 76%, 69%, and 55% for HEM, Cyt-c, MYO, and OVA, respectively. The suitability of the prepared bioreactors to complex proteins was demonstrated by digesting human serum.

  2. The structure of infant formulas impacts their lipolysis, proteolysis and disintegration during in vitro gastric digestion.

    PubMed

    Bourlieu, Claire; Ménard, Olivia; De La Chevasnerie, Alix; Sams, Laura; Rousseau, Florence; Madec, Marie-Noëlle; Robert, Benoît; Deglaire, Amélie; Pezennec, Stéphane; Bouhallab, Saïd; Carrière, Frédéric; Dupont, Didier

    2015-09-01

    Milk lipids supply most of the calories necessary for newborn growth in maternal milk or infant formulas. The chemical composition of infant formulas has been optimized but not the structure of the emulsion. There is still a major difference between the native emulsions of milk fat globules and processed submicronic emulsions in infant formulas. This difference may modify the kinetics of digestion of emulsions in newborns and influence lipid metabolism. To check this, semi-dynamic gastric in vitro digestions were conducted on three matrices: a standardized milk emulsion containing native milk fat globules referred to as minimally-processed emulsion and two processed model infant formulas (homogenized or homogenized/pasteurized). Gastric conditions mimicked those reported in newborns. The minimally-processed emulsion was lipolyzed and proteolyzed slower than processed formulas. The difference in initial structure persisted during digestion. The surface of the droplets was the key parameter to control gastric lipolysis kinetics, the pattern of released fatty acids and proteolysis by faster hydrolysis of adsorbed proteins.

  3. Extensive in vivo human milk peptidomics reveals specific proteolysis yielding protective antimicrobial peptides

    PubMed Central

    Dallas, David C.; Guerrero, Andres; Khaldi, Nora; Castillo, Patricia A.; Martin, William F.; Smilowitz, Jennifer T.; Bevins, Charles L.; Barile, Daniela; German, J. Bruce; Lebrilla, Carlito B.

    2013-01-01

    Milk is traditionally considered an ideal source of the basic elemental nutrients required by infants. More detailed examination is revealing that milk represents a more functional ensemble of components with benefits to both infants and mothers. A comprehensive peptidomics method was developed and used to analyze human milk yielding an extensive array of protein products present in the fluid. Over 300 milk peptides were identified originating from major and many minor protein components of milk. As expected, the majority of peptides derived from β-casein, however no peptide fragments from the major milk proteins lactoferrin, α-lactalbumin and secretory immunoglobulin A were identified. Proteolysis in the mammary gland is selective—released peptides were drawn only from specific proteins and typically from only select parts of the parent sequence. A large number of the peptides showed significant sequence overlap with peptides with known antimicrobial or immunomodulatory functions. Antibacterial assays showed the milk peptide mixtures inhibited the growth of Escherichia coli and Staphylococcus aureus. The pre-digestion of milk proteins and the consequent release antibacterial peptides may provide a selective advantage through evolution by protecting both the mother's mammary gland and her nursing offspring from infection. PMID:23586814

  4. Calpain A modulates Toll responses by limited Cactus/IκB proteolysis

    PubMed Central

    Fontenele, Marcio; Lim, Bomyi; Oliveira, Danielle; Buffolo, Márcio; Perlman, David H.; Schupbach, Trudi; Araujo, Helena

    2013-01-01

    Calcium-dependent cysteine proteases of the calpain family are modulatory proteases that cleave their substrates in a limited manner. Among their substrates, calpains target vertebrate and invertebrate IκB proteins. Because proteolysis by calpains potentially generates novel protein functions, it is important to understand how this affects NFκB activity. We investigate the action of Calpain A (CalpA) on the Drosophila melanogaster IκB homologue Cactus in vivo. CalpA alters the absolute amounts of Cactus protein. Our data indicate, however, that CalpA uses additional mechanisms to regulate NFκB function. We provide evidence that CalpA interacts physically with Cactus, recognizing a Cactus pool that is not bound to Dorsal, a fly NFκB/Rel homologue. We show that proteolytic cleavage by CalpA generates Cactus fragments lacking an N-terminal region required for Toll responsiveness. These fragments are generated in vivo and display properties distinct from those of full-length Cactus. We propose that CalpA targets free Cactus, which is incorporated into and modulates Toll-responsive complexes in the embryo and immune system. PMID:23864715

  5. Structural changes in emulsion-bound bovine beta-lactoglobulin affect its proteolysis and immunoreactivity.

    PubMed

    Marengo, Mauro; Miriani, Matteo; Ferranti, Pasquale; Bonomi, Francesco; Iametti, Stefania; Barbiroli, Alberto

    2016-07-01

    Adsorption on the surface of sub-micrometric oil droplets resulted in significant changes in the tertiary structure of bovine beta-lactoglobulin (BLG), a whey protein broadly used as a food ingredient and a major food allergen. The adsorbed protein had increased sensitivity to trypsin, and increased immunoreactivity towards specific monoclonal antibodies. In spite of the extensive tryptic breakdown of emulsion-bound BLG, some sequence stretches in BLG became trypsin-insensitive upon absorption of the protein on the fat droplets. As a consequence - at contrast with free BLG - proteolysis of emulsion-bound BLG did not decrease the immunoreactivity of the protein, and some of the large peptides generated by trypsinolysis of emulsion-bound BLG were still recognizable by specific monoclonal antibodies. Structural changes occurring in emulsion-bound BLG and their consequences are discussed in comparison with those occurring when the tertiary structure of BLG is modified by lipophilic salts, by urea, or upon interaction with solid hydrophobic surfaces. Such a comparison highlights the relevance of situation-specific structural modifications, that in turn may affect physiologically relevant features of the protein. PMID:27085639

  6. Altered glycosylation of platelet-derived von Willebrand factor confers resistance to ADAMTS13 proteolysis.

    PubMed

    McGrath, Rachel T; van den Biggelaar, Maartje; Byrne, Barry; O'Sullivan, Jamie M; Rawley, Orla; O'Kennedy, Richard; Voorberg, Jan; Preston, Roger J S; O'Donnell, James S

    2013-12-12

    Platelet-von Willebrand factor (VWF) is stored within α-granules and accounts for ∼20% of total VWF in platelet-rich plasma. This platelet-VWF pool is distinct from plasma-VWF and is enriched in high molecular weight multimers (HMWM). Previous studies have described significant functional discrepancies between platelet-VWF and plasma-VWF; however, the molecular basis of these differences is not well understood. We have characterized terminal glycan expression on platelet-VWF. Our findings demonstrate that platelet-VWF exists as a distinct natural glycoform. In particular, N-linked sialylation is markedly reduced (>50%) compared with plasma-VWF. Moreover, unlike plasma-VWF, platelet-VWF does not express AB blood group determinants, although precursor H antigen expression is similar to that on plasma-VWF. Because of this differential glycosylation, platelet-VWF exhibits specific resistance to ADAMTS13 proteolysis. Thus platelet activation at sites of vascular injury results in the release of high local concentrations of HMWM platelet-VWF that is more resistant to ADAMTS13, thereby facilitating platelet-plug formation. PMID:24106205

  7. A protease substrate profiling method that links site-specific proteolysis with antibiotic resistance.

    PubMed

    Sandersjöö, Lisa; Kostallas, George; Löfblom, John; Samuelson, Patrik

    2014-01-01

    Proteases are involved in many biological processes and have become important tools in biomedical research and industry. Technologies for engineering and characterization of, for example, proteolytic activity and specificity are essential in protease research. Here, we present a novel method for assessment of site-specific proteolysis. The assay utilizes plasmid-encoded reporters that, upon processing by a co-expressed protease, confer antibiotic resistance to bacteria in proportion to the cleavage efficiency. We have demonstrated that cells co-expressing cleavable reporters together with tobacco etch virus protease (TEVp) could be discriminated from cells with non-cleavable reporters by growth in selective media. Importantly, the resistance to antibiotics proved to correlate with the substrate processing efficiency. Thus, by applying competitive growth of a mock library in antibiotic-containing medium, we could show that the substrate preferred by TEVp was enriched relative to less-efficient substrates. We believe that this simple methodology will facilitate protease substrate identification, and hold great promise for directed evolution of proteases and protease recognition sequences towards improved or even new functionality.

  8. Three-dimensional migration of macrophages requires Hck for podosome organization and extracellular matrix proteolysis

    PubMed Central

    Cougoule, Céline; Le Cabec, Véronique; Poincloux, Renaud; Al Saati, Talal; Mège, Jean-Louis; Tabouret, Guillaume; Lowell, Clifford A.; Laviolette-Malirat, Nathalie

    2010-01-01

    Tissue infiltration of phagocytes exacerbates several human pathologies including chronic inflammations or cancers. However, the mechanisms involved in macrophage migration through interstitial tissues are poorly understood. We investigated the role of Hck, a Src-family kinase involved in the organization of matrix adhesion and degradation structures called podosomes. In Hck−/− mice submitted to peritonitis, we found that macrophages accumulated in interstitial tissues and barely reached the peritoneal cavity. In vitro, 3-dimensional (3D) migration and matrix degradation abilities, 2 protease-dependent properties of bone marrow–derived macrophages (BMDMs), were affected in Hck−/− BMDMs. These macrophages formed few and undersized podosome rosettes and, consequently, had reduced matrix proteolysis operating underneath despite normal expression and activity of matrix metalloproteases. Finally, in fibroblasts unable to infiltrate matrix, ectopic expression of Hck provided the gain–of–3D migration function, which correlated positively with formation of podosome rosettes. In conclusion, spatial organization of podosomes as large rosettes, proteolytic degradation of extracellular matrix, and 3D migration appeared to be functionally linked and regulated by Hck in macrophages. Hck, as the first protein combining a phagocyte-limited expression with a role in 3D migration, could be a target for new anti-inflammatory and antitumor molecules. PMID:19897576

  9. EXTRACELLULAR PROTEOLYSIS OF REELIN BY TISSUE PLASMINOGEN ACTIVATOR FOLLOWING SYNAPTIC POTENTIATION

    PubMed Central

    TROTTER, J. H.; LUSSIER, A. L.; PSILOS, K. E.; MAHONEY, H. L.; SPONAUGLE, A. E.; HOE, H.-S.; REBECK, G. W.; WEEBER, E. J.

    2015-01-01

    The secreted glycoprotein reelin plays an indispensable role in neuronal migration during development and in regulating adult synaptic functions. The upstream mechanisms responsible for initiating and regulating the duration and magnitude of reelin signaling are largely unknown. Here we report that reelin is cleaved between EGF-like repeats 6–7 (R6–7) by tissue plasminogen activator (tPA) under cell-free conditions. No changes were detected in the level of reelin and its fragments in the brains of tPA knockouts, implying that other unknown proteases are responsible for generating reelin fragments found constitutively in the adult brain. Induction of NMDAR-independent long-term potentiation with the potassium channel blocker tetraethylammonium chloride (TEA-Cl) led to a specific up-regulation of reelin processing at R6–7 in wild-type mice. In contrast, no changes in reelin expression and processing were observed in tPA knockouts following TEA-Cl treatment. These results demonstrate that synaptic potentiation results in tPA-dependent reelin processing and suggest that extracellular proteolysis of reelin may regulate reelin signaling in the adult brain. PMID:24892761

  10. Sulfur mustard-increased proteolysis following in vitro and in vivo exposures

    SciTech Connect

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

    1993-05-13

    The pathologic mechanisms underlying sulfur mustard (HD)-induced skin vesication are as yet undefined. Papirmeister et al. (1) postulate enhanced proteolytic activity as a proximate cause of HD-induced cutaneous injury. Using a chromogenic peptide substrate assay, we previously reported that in vitro exposure of cell cultures to HD enhances proteolytic activity. We have continued our investigation of HD-increased proteolytic activity in vitro and have expanded our studies to include an in vivo animal model for HD exposure. In vitro exposure of human peripheral blood lymphocytes (PBL) to HD demonstrated that the increase in proteolytic activity is both time- and temperature-dependent. Using a panel of 10 protease substrates, we established that the HD-increased proteolysis was markedly different from that generated by plasminogen activator. The hairless guinea pig is an animal model used for the study of HD-induced dermal pathology. When control and HD-exposed human PBL and hairless guinea pig skin where examined, similarities in their protease substrate reactivities were observed. HD-exposed hairless guinea pig skin biopsies demonstrated increased proteolytic activity that was time-dependent. The HD-increased proteolytic response was similar in both in vitro and in vivo studies and may be useful for elucidating both the mechanism of HD-induced vesication and potential treatment compounds.

  11. Study of proteolysis in river buffalo mozzarella cheese using a proteomics approach.

    PubMed

    Petrella, G; Pati, S; Gagliardi, R; Rizzuti, A; Mastrorilli, P; la Gatta, B; Di Luccia, A

    2015-11-01

    The guarantee of the origin and quality of raw material is essential for the protection and valorization of Campana buffalo mozzarella cheese. The risk of utilization of semifinished products and stored milk in substitution for fresh milk is increasing, due to the continuous desire to reduce production costs. A proteomics approach and electrophoresis survey of retail mozzarella cheeses indicated different rates of proteolysis in the production of dairy industries. The use of fresh milk and correct cheesemaking protocol yielded only γ-caseins, which are derived from β-casein by plasmin, and para-κ-casein, which is derived from κ-casein by chymosin. The detection of abnormal hydrolysis resulting in β- and αS1-casein fragments, identified by mass spectrometry, indicates the use of stored milk or stored and pressed curd, or the reuse of unsold mozzarella cheese, to produce mozzarella. The formation of γ-caseins and other fragments during a long storage of raw materials at room or refrigeration temperature was ascribed to plasmin (endogenous milk enzyme), whereas formation of αS1-casein fragments, mainly αS1-I(6P)- and αS1-I(7P)-casein during the storage of curd was ascribed to the action of chymosin (exogenous enzyme) from rennet. Sodium dodecyl sulfate-PAGE and alkaline urea-PAGE permitted us to evaluate the freshness of the raw materials used in the manufacturing of buffalo mozzarella cheese and to reveal possible inappropriate preservation. PMID:26364106

  12. Dietary L-Lysine Suppresses Autophagic Proteolysis and Stimulates Akt/mTOR Signaling in the Skeletal Muscle of Rats Fed a Low-Protein Diet.

    PubMed

    Sato, Tomonori; Ito, Yoshiaki; Nagasawa, Takashi

    2015-09-23

    Amino acids, especially L-leucine, regulate protein turnover in skeletal muscle and have attracted attention as a means of increasing muscle mass in people suffering from malnutrition, aging (sarcopenia), or a bedridden state. We previously showed that oral administration of L-lysine (Lys) by gavage suppressed proteolysis in skeletal muscles of fasted rats. However, the intake of Lys in the absence of other dietary components is unlikely in a non-experimental setting, and other dietary components may interfere with the suppressive effect of Lys on proteolysis. We supplemented Lys to a 10% casein diet and investigated the effect of Lys on proteolysis and autophagy, a major proteolytic system, in the skeletal muscle of rats. The rate of proteolysis was evaluated from 3-methylhisitidine (MeHis) released from isolated muscles, in plasma, and excreted in urine. Supplementing lysine with the 10% casein diet decreased the rate of proteolysis induced by intake of a low-protein diet. The upregulated autophagy activity [light chain 3 (LC3)-II/total LC3] caused by a low-protein diet was reduced, and the Akt/mTOR signaling pathway was activated by Lys. Importantly, continuous feeding of a Lys-rich 10% casein diet for 15 days increased the masses of the soleus and gastrocnemius muscles. Taken together, supplementation of Lys to a low-protein diet suppresses autophagic proteolysis through the Akt/mTOR signaling pathway, and continuous feeding of a Lys-rich diet may increase skeletal muscle mass.

  13. Matriptase activation connects tissue factor-dependent coagulation initiation to epithelial proteolysis and signaling.

    PubMed

    Le Gall, Sylvain M; Szabo, Roman; Lee, Melody; Kirchhofer, Daniel; Craik, Charles S; Bugge, Thomas H; Camerer, Eric

    2016-06-23

    The coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and other extravascular cells is exposed to plasma, sequential activation of coagulation proteases coordinates hemostasis and contributes to host defense and tissue repair. Membrane-anchored serine proteases (MASPs) play critical roles in the development and homeostasis of epithelial barrier tissues; how MASPs are activated in mature epithelia is unknown. We here report that proteases of the extrinsic pathway of blood coagulation transactivate the MASP matriptase, thus connecting coagulation initiation to epithelial proteolysis and signaling. Exposure of TF-expressing cells to factors (F) VIIa and Xa triggered the conversion of latent pro-matriptase to an active protease, which in turn cleaved the pericellular substrates protease-activated receptor-2 (PAR2) and pro-urokinase. An activation pathway-selective PAR2 mutant resistant to direct cleavage by TF:FVIIa and FXa was activated by these proteases when cells co-expressed pro-matriptase, and matriptase transactivation was necessary for efficient cleavage and activation of wild-type PAR2 by physiological concentrations of TF:FVIIa and FXa. The coagulation initiation complex induced rapid and prolonged enhancement of the barrier function of epithelial monolayers that was dependent on matriptase transactivation and PAR2 signaling. These observations suggest that the coagulation cascade engages matriptase to help coordinate epithelial defense and repair programs after injury or infection, and that matriptase may contribute to TF-driven pathogenesis in cancer and inflammation. PMID:27114461

  14. Impact of microbial cultures on proteolysis and release of bioactive peptides in fermented milk.

    PubMed

    Chaves-López, Clemencia; Serio, Annalisa; Paparella, Antonello; Martuscelli, Maria; Corsetti, Aldo; Tofalo, Rosanna; Suzzi, Giovanna

    2014-09-01

    This study aimed at evaluating co-cultures of selected microorganisms for their proteolytic activity and capability to produce fermented milk enriched with ACE-inhibitory (ACEI) peptides. Selected yeasts (Torulaspora delbruekii KL66A, Galactomyces geotrichum KL20B, Pichia kudriavzevii KL84A and Kluyveromyces marxianus KL26A) and lactic acid bacteria strains (Lactobacillus plantarum LAT03, Lb. plantarum KLAT01 and the not virulent Enterococcus faecalis KE06) were screened as single cultures for their capacity of releasing ACEI peptides without producing bitter taste. Three strains cultures (yeast, Lb. plantarum and E. faecalis) were performed to evaluate the combined impact on microbial growth, lactic acid production, citric acid consumption, proteolysis, ACEI activity, and bitter taste after 36 h of fermentation at 28 °C. While G. geotrichum KL20B showed a strong stimulating effect on Lb. plantarum strains and the production of peptides with ACEI activity, the presence of T. delbruekii KL26A in the cultures was deleterious both to ACEI activity and product taste. The most effective combination was P. kudriavzevii KL84A, Lb. plantarum LAT3, E. faecalis KL06, which showed the highest ACEI activity (IC50 = 30.63 ± 1.11 μg ml(-1)) and gave no bitter taste for 7 days at 6 °C. Our results highlight the importance of choosing the strains combination carefully, to obtain a high yield of ACEI activity without bitter taste.

  15. Impact of microbial cultures on proteolysis and release of bioactive peptides in fermented milk.

    PubMed

    Chaves-López, Clemencia; Serio, Annalisa; Paparella, Antonello; Martuscelli, Maria; Corsetti, Aldo; Tofalo, Rosanna; Suzzi, Giovanna

    2014-09-01

    This study aimed at evaluating co-cultures of selected microorganisms for their proteolytic activity and capability to produce fermented milk enriched with ACE-inhibitory (ACEI) peptides. Selected yeasts (Torulaspora delbruekii KL66A, Galactomyces geotrichum KL20B, Pichia kudriavzevii KL84A and Kluyveromyces marxianus KL26A) and lactic acid bacteria strains (Lactobacillus plantarum LAT03, Lb. plantarum KLAT01 and the not virulent Enterococcus faecalis KE06) were screened as single cultures for their capacity of releasing ACEI peptides without producing bitter taste. Three strains cultures (yeast, Lb. plantarum and E. faecalis) were performed to evaluate the combined impact on microbial growth, lactic acid production, citric acid consumption, proteolysis, ACEI activity, and bitter taste after 36 h of fermentation at 28 °C. While G. geotrichum KL20B showed a strong stimulating effect on Lb. plantarum strains and the production of peptides with ACEI activity, the presence of T. delbruekii KL26A in the cultures was deleterious both to ACEI activity and product taste. The most effective combination was P. kudriavzevii KL84A, Lb. plantarum LAT3, E. faecalis KL06, which showed the highest ACEI activity (IC50 = 30.63 ± 1.11 μg ml(-1)) and gave no bitter taste for 7 days at 6 °C. Our results highlight the importance of choosing the strains combination carefully, to obtain a high yield of ACEI activity without bitter taste. PMID:24929726

  16. Fragmentation of myofibrils, limited proteolysis and water holding capacity of meat.

    PubMed

    Kołodziejska, I; Sikorski, Z E; Lewandowska, T; Niecikowska, C

    1986-01-01

    Protein changes in ageing meat result in increased vulnerability of the myofibrils to fragmentation, caused possibly by limited proteolysis. It was investigated which groups of muscle proteases, if any, were involved and what was the relation between fragmentation and hydration of beef meat. In samples ranging in natural pH from 5.4 to 7.0 the least fragmentation after 3 days at 2 degrees C was at pH 6. This could suggest the role of both the cathepsins and neutral proteases. In samples aged in the presence of EDTA fragmentation was significantly lower than in the controls. This could indicate the role of Ca2+ activated neutral proteases, or support the hypothesis on the nonenzymatic mechanism involving Ca2+. The results of PAG electrophoresis could not have been due to the neutral proteases, as the 30,000 g X mol-1 component, resulting from the hydrolysis of troponin T, did not accumulate at pH 7 until the 9th day of ageing, but at pH 5.4 the intensity of this band increased markedly already after 3 days. There was no correlation between the fragmentation and the hydration of the aged meat after cooking. The addition of 0.001% of trypsin or 0.0005% of papain to minced meat did not cause after 9 days any increase in the contents of free amino acids and peptides or significant changes in the PAGE pattern as compared to those in the controls. However, the fragmentation and hydration of the raw meat was larger in the samples containing added enzymes. After cooking the hydration of the samples did not differ.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3092054

  17. Topology of sarcoplasmic reticulum Ca2+-ATPase: an infrared study of thermal denaturation and limited proteolysis.

    PubMed

    Echabe, I; Dornberger, U; Prado, A; Goñi, F M; Arrondo, J L

    1998-05-01

    Sarcoplasmic reticulum Ca2+-ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as alpha-helix, beta-sheet, etc...., two unusual bands, one at 1,645 cm(-1) in H2O buffer and the other at 1,625 cm(-1) in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm(-1) is tentatively assigned to alpha-helical segments located in the cytoplasmic domain and coupled to beta-sheet structure, whereas the band at 1,625 cm(-1) arises probably from monomer-monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% alpha-helical segments in the transmembrane plus stalk domain; 20% alpha-helix and 22% beta-sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca2+-ATPase is a complex process that cannot be described as a two-state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm(-1) band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm(-1) band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37 degrees C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.

  18. Topology of sarcoplasmic reticulum Ca2+-ATPase: an infrared study of thermal denaturation and limited proteolysis.

    PubMed Central

    Echabe, I.; Dornberger, U.; Prado, A.; Goñi, F. M.; Arrondo, J. L.

    1998-01-01

    Sarcoplasmic reticulum Ca2+-ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as alpha-helix, beta-sheet, etc...., two unusual bands, one at 1,645 cm(-1) in H2O buffer and the other at 1,625 cm(-1) in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm(-1) is tentatively assigned to alpha-helical segments located in the cytoplasmic domain and coupled to beta-sheet structure, whereas the band at 1,625 cm(-1) arises probably from monomer-monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% alpha-helical segments in the transmembrane plus stalk domain; 20% alpha-helix and 22% beta-sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca2+-ATPase is a complex process that cannot be described as a two-state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm(-1) band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm(-1) band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37 degrees C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited. PMID:9605321

  19. Prostate-specific membrane antigen (PSMA)-mediated laminin proteolysis generates a pro-angiogenic peptide.

    PubMed

    Conway, Rebecca E; Rojas, Camilo; Alt, Jesse; Nováková, Zora; Richardson, Spencer M; Rodrick, Tori C; Fuentes, Julio L; Richardson, Noah H; Attalla, Jonathan; Stewart, Samantha; Fahmy, Beshoy; Barinka, Cyril; Ghosh, Mallika; Shapiro, Linda H; Slusher, Barbara S

    2016-10-01

    Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase expressed in a number of tissues. PSMA participates in various biological functions depending on the substrate available in the particular tissue; in the brain, PSMA cleaves the abundant neuropeptide N-acetyl-aspartyl-glutamate to regulate release of key neurotransmitters, while intestinal PSMA cleaves polyglutamated peptides to supply dietary folate. PSMA expression is also progressively upregulated in prostate cancer where it correlates with tumor progression as well as in tumor vasculature, where it regulates angiogenesis. The previous research determined that PSMA cleavage of small peptides generated via matrix metalloprotease-mediated proteolysis of the extracellular matrix protein laminin potently activated endothelial cells, integrin signaling and angiogenesis, although the specific peptide substrates were not identified. Herein, using enzymatic analyses and LC/MS, we unequivocally demonstrate that several laminin-derived peptides containing carboxy-terminal glutamate moieties (LQE, IEE, LNE) are bona fide substrates for PSMA. Subsequently, the peptide products were tested for their effects on angiogenesis in various models. We report that LQ, the dipeptide product of PSMA cleavage of LQE, efficiently activates endothelial cells in vitro and enhances angiogenesis in vivo. Importantly, LQE is not cleaved by an inactive PSMA enzyme containing an active site mutation (E424S). Endothelial cell activation by LQ was dependent on integrin beta-1-induced activation of focal adhesion kinase. These results characterize a novel PSMA substrate, provide a functional rationale for the upregulation of PSMA in cancer cells and tumor vasculature and suggest that inhibition of PSMA could lead to the development of new angiogenic therapies. PMID:27387982

  20. Proteolysis of EphA2 converts it from a tumor suppressor to an oncoprotein

    PubMed Central

    KOSHIKAWA, Naohiko; HOSHINO, Daisuke; TANIGUCHI, Hiroaki; MINEGISHI, Tomoko; TOMARI, Taizo; NAM, Sung-Ouk; AOKI, Mikiko; SUETA, Takayuki; NAKAGAWA, Takashi; MIYAMOTO, Shingo; NABESHIMA, Kazuki; WEAVER, Alissa M.; SEIKI, Motoharu

    2015-01-01

    Eph receptor tyrosine kinases are considered candidate therapeutic targets in cancer, but they can exert opposing effects on cell growth. In presence of its ligands, Eph receptor EphA2 suppresses signaling by other growth factor receptors, including ErbB, whereas ligand-independent activation of EphA2 augments ErbB signaling. To deploy EphA2-targeting drugs effectively in tumors, the anti-oncogenic ligand-dependent activation state of EphA2 must be discriminated from its oncogenic ligand-independent state. Since the molecular basis for the latter is little understood, we investigated how the activation state of EphA2 can be switched in tumor tissue. We found that ligand-binding domain of EphA2 is cleaved frequently by the membrane metalloproteinase MT1-MMP, a powerful modulator of the pericellular environment in tumor cells. EphA2 immunostaining revealed a significant loss of the N-terminal portion of EphA2 in areas of tumor tissue that expressed MT1-MMP. Moreover, EphA2 phosphorylation patterns that signify ligand-independent activation were observed specifically in these areas of tumor tissue. Mechanistic experiments revealed that processing of EphA2 by MT1-MMP promoted ErbB signaling, anchorage-independent growth, and cell migration. Conversely, expression of a proteolysis-resistant mutant of EphA2 prevented tumorigenesis and metastasis of human tumor xenografts in mice. Overall, our results showed how the proteolytic state of EphA2 in tumors determines its effector function and influences its status as a candidate biomarker for targeted therapy. PMID:26130649

  1. Differential Proteolysis of Glycinin and beta-Conglycinin Polypeptides during Soybean Germination and Seedling Growth.

    PubMed

    Wilson, K A; Rightmire, B R; Chen, J C; Tan-Wilson, A L

    1986-09-01

    The degradation of the major seed storage globulins of the soybean (Glycine max [L.] Merrill) was examined during the first 12 days of germination and seedling growth. The appearance of glycinin and beta-conglycinin degradation products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cotyledon extracts followed by electroblotting to nitrocellulose and immunostaining using glycinin and beta-conglycinin specific antibodies. The three subunits of beta-conglycinin were preferentially metabolized. Of the three subunits of beta-conglycinin, the larger alpha and alpha' subunits are rapidly degraded, generating new beta-conglycinin cross-reactive polypeptides of 51,200 molecular weight soon after imbibition of the seed. After 6 days of growth the beta-subunit is also hydrolyzed. At least six polypeptides, ranging from 33,100 to 24,000 molecular weight, appear as apparent degradation products of beta-conglycinin. The metabolism of the glycinin acidic chains begins early in growth. The glycinin acidic chains present at day 3 have already been altered from the native form in the ungerminated seed, as evidenced by their higher mobility in an alkaline-urea polyacrylamide gel electrophoresis system. However, no change in the molecular weight of these chains is detectable by sodium dodecyl sulfate-polyarylamide gel electrophoresis. Examination of the glycinin polypeptide amino-termini by dansylation suggests that this initial modification of the acidic chains involves limited proteolysis at the carboxyl-termini, deamidation, or both. After 3 days of growth the acidic chains are rapidly hydrolyzed to a smaller (21,900 molecular weight) form. The basic polypeptides of glycinin appear to be unaltered during the first 8 days of growth, but are rapidly degraded thereafter to unidentified products. All of the original glycinin basic chains have been destroyed by day 10 of growth.

  2. Oral branched-chain amino acids decrease whole-body proteolysis

    NASA Technical Reports Server (NTRS)

    Ferrando, A. A.; Williams, B. D.; Stuart, C. A.; Lane, H. W.; Wolfe, R. R.

    1995-01-01

    BACKGROUND: This study reports the effects of ingesting branched-chain amino acids (leucine, valine, and isoleucine) on protein metabolism in four men. METHODS: To calculate leg protein synthesis and breakdown, we used a new model that utilized the infusion of L-[ring-13C6]phenylalanine and the sampling of the leg arterial-venous difference and muscle biopsies. In addition, protein-bound enrichments provided for the direct calculation of muscle fractional synthetic rate. Four control subjects ingested an equivalent amount of essential amino acids (threonine, methionine, and histidine) to discern the effects of branched-chain amino acid nitrogen vs the effects of essential amino acid nitrogen. Each drink also included 50 g of carbohydrate. RESULTS: Consumption of the branched-chain and the essential amino acid solutions produced significant threefold and fourfold elevations in their respective arterial concentrations. Protein synthesis and breakdown were unaffected by branched-chain amino acids, but they increased by 43% (p < .05) and 36% (p < .03), respectively, in the group consuming the essential amino acids. However, net leg balance of phenylalanine was unchanged by either drink. Direct measurement of protein synthesis by tracer incorporation into muscle protein (fractional synthetic rate) revealed no changes within or between drinks. Whole-body phenylalanine flux was significantly suppressed by each solution but to a greater extent by the branched-chain amino acids (15% and 20%, respectively) (p < .001). CONCLUSIONS: These results suggest that branched-chain amino acid ingestion suppresses whole-body proteolysis in tissues other than skeletal muscle in normal men.

  3. The targeting, docking and anti-proteolysis functions of the secretin chaperone PulS.

    PubMed

    Collin, Séverine; Krehenbrink, Martin; Guilvout, Ingrid; Pugsley, Anthony P

    2013-06-01

    The Klebsiella oxytoca lipoprotein PulS might function as either or both a pilot and a docking factor in the outer membrane targeting and assembly of the Type II secretion system secretin PulD. In the piloting model, PulS binds to PulD monomers and targets them to the outer membrane via the lipoprotein sorting pathway components LolA and LolB. In this model, PulS also protects the PulD monomers from proteolysis during transit through the periplasm. In the docking model, PulS is targeted alone to the outer membrane, where it acts as a receptor for PulD monomers, allowing them to accumulate and assemble specifically in this membrane. PulS was shown to dissociate from and/or re-associate freely with PulD multimers in zwitterionic detergent, making it difficult to determine whether PulS remains associated with PulD dodecamers in the outer membrane by co-purification. However, PulD protomers in the dodecamer were shown to be stable in the absence of PulS, indicating that PulS is only required to protect the protease-susceptible monomer. DegP was identified as one of the proteases that could contribute to PulD degradation in the absence of PulS. Studies on the in vitro assembly and targeting of PulD into Escherichia coli membrane vesicles demonstrated its strong preference to insert into the inner membrane, as is the case in vivo in the absence of PulS. However, PulD could be targeted to outer membrane fragments in vitro if they were preloaded with PulS, indicating the technical feasibility of the docking model. We conclude that both modes of action might contribute to efficient outer membrane targeting of PulD in vivo, although the piloting function is likely to predominate.

  4. Prostate-specific membrane antigen (PSMA)-mediated laminin proteolysis generates a pro-angiogenic peptide.

    PubMed

    Conway, Rebecca E; Rojas, Camilo; Alt, Jesse; Nováková, Zora; Richardson, Spencer M; Rodrick, Tori C; Fuentes, Julio L; Richardson, Noah H; Attalla, Jonathan; Stewart, Samantha; Fahmy, Beshoy; Barinka, Cyril; Ghosh, Mallika; Shapiro, Linda H; Slusher, Barbara S

    2016-10-01

    Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase expressed in a number of tissues. PSMA participates in various biological functions depending on the substrate available in the particular tissue; in the brain, PSMA cleaves the abundant neuropeptide N-acetyl-aspartyl-glutamate to regulate release of key neurotransmitters, while intestinal PSMA cleaves polyglutamated peptides to supply dietary folate. PSMA expression is also progressively upregulated in prostate cancer where it correlates with tumor progression as well as in tumor vasculature, where it regulates angiogenesis. The previous research determined that PSMA cleavage of small peptides generated via matrix metalloprotease-mediated proteolysis of the extracellular matrix protein laminin potently activated endothelial cells, integrin signaling and angiogenesis, although the specific peptide substrates were not identified. Herein, using enzymatic analyses and LC/MS, we unequivocally demonstrate that several laminin-derived peptides containing carboxy-terminal glutamate moieties (LQE, IEE, LNE) are bona fide substrates for PSMA. Subsequently, the peptide products were tested for their effects on angiogenesis in various models. We report that LQ, the dipeptide product of PSMA cleavage of LQE, efficiently activates endothelial cells in vitro and enhances angiogenesis in vivo. Importantly, LQE is not cleaved by an inactive PSMA enzyme containing an active site mutation (E424S). Endothelial cell activation by LQ was dependent on integrin beta-1-induced activation of focal adhesion kinase. These results characterize a novel PSMA substrate, provide a functional rationale for the upregulation of PSMA in cancer cells and tumor vasculature and suggest that inhibition of PSMA could lead to the development of new angiogenic therapies.

  5. Oxidization without substrate unfolding triggers proteolysis of the peroxide-sensor, PerR.

    PubMed

    Ahn, Bo-Eun; Baker, Tania A

    2016-01-01

    Peroxide operon regulator (PerR) is a broadly conserved hydrogen peroxide sensor in bacteria, and oxidation of PerR at its regulatory metal-binding site is considered irreversible. Here, we tested whether this oxidation specifically targets PerR for proteolysis. We find that oxidizing conditions stimulate PerR degradation in vivo, and LonA is the principal AAA+ (ATPases associated with diverse cellular activities) protease that degrades PerR. Degradation of PerR by LonA is recapitulated in vitro, and biochemical dissection of this degradation reveals that the presence of regulatory metal and PerR-binding DNA dramatically extends the half-life of the protein. We identified a LonA-recognition site critical for oxidation-controlled PerR turnover. Key residues for LonA-interaction are exposed to solvent in PerR lacking metal, but are buried in the metal-bound form. Furthermore, one residue critical for Lon recognition is also essential for specific DNA-binding by PerR, thus explaining how both the metal and DNA ligands prevent PerR degradation. This ligand-controlled allosteric mechanism for protease recognition provides a compelling explanation for how the oxidation-induced conformational change in PerR triggers degradation. Interestingly, the critical residues recognized by LonA and exposed by oxidation do not function as a degron, because they are not sufficient to convert a nonsubstrate protein into a LonA substrate. Rather, these residues are a conformation-discriminator sequence, which must work together with other residues in PerR to evoke efficient degradation. This mechanism provides a useful example of how other proteins with only mild or localized oxidative damage can be targeted for degradation without the need for extensive oxidation-dependent protein denaturation. PMID:26677871

  6. HtrA1 Proteolysis of ApoE In Vitro Is Allele Selective.

    PubMed

    Chu, Qian; Diedrich, Jolene K; Vaughan, Joan M; Donaldson, Cynthia J; Nunn, Michael F; Lee, Kuo-Fen; Saghatelian, Alan

    2016-08-01

    Apolipoprotein E (ApoE) belongs to a large class of proteins that solubilize lipids for physiological transport. Humans have three different APOE alleles, APOE ε2, APOE ε3, and APOE ε4, and genetic studies identified ApoE4 as the strongest genetic risk factor for Alzheimer's disease (AD). People who are homozygous for ApoE4 (i.e., ApoE4/E4) are an order of magnitude more likely to develop late-onset AD (LOAD) than ApoE3/E3 carriers. Several differences between ApoE3 and ApoE4 may contribute to AD including the observation that ApoE4 is degraded to a greater extent than ApoE3 in the human brain. Experiments with high-temperature requirement serine peptidase A1 (HtrA1), which is found in the nervous system, demonstrate that HtrA1 is an allele-selective ApoE-degrading enzyme that degrades ApoE4 more quickly than ApoE3. This activity is specific to HtrA1, as similar assays with HtrA2 showed minimal ApoE4 proteolysis and trypsin had no preference between ApoE4 and ApoE3. HtrA1 has also been reported to cleave the tau protein (Tau) and the amyloid protein precursor (APP) to hinder the formation of toxic amyloid deposits associated with AD. Competition assays with ApoE4, ApoE3, and Tau revealed that ApoE4 inhibits Tau degradation. Thus, the identification of ApoE4 as an in vitro HtrA1 substrate suggests a potential biochemical mechanism that links ApoE4 regulation of AD proteins such as Tau.

  7. Human platelet calmodulin-binding proteins: identification and Ca/sup 2 +/-dependent proteolysis upon platelet activation

    SciTech Connect

    Wallace, R.W.; Tallant, E.A.; McManus, M.C.

    1987-05-19

    Calmodulin-binding proteins have been identified in human platelets by using Western blotting techniques and /sup 125/I-calmodulin. Ten distinct proteins of 245, 225, 175, 150, 90, 82 (2), 60, and 41 (2) kilodaltons (kDa) bound /sup 125/I-calmodulin in a Ca/sup 2 +/-dependent manner; the binding was blocked by ethylene glycol bis(..beta..-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), trifluoperazine, and nonradiolabeled calmodulin. Proteins of 225 and 90 kDa were labeled by antisera against myosin light chain kinase; 60- and 82-kDa proteins were labeled by antisera against the calmodulin-dependent phosphatase and caldesmon, respectively. The remaining calmodulin-binding proteins have not been identified. Calmodulin-binding proteins were degraded upon addition of Ca/sup 2 +/ to a platelet homogenate; the degradation could be blocked by either EGTA, leupeptin, or N-ethylmaleimide which suggests that the degradation was due to a Ca/sup 2 +/-dependent protease. Activation of intact platelets by thrombin, adenosine 5'-diphosphate, and collagen under conditions which promote platelet aggregation also resulted in limited proteolysis of calmodulin-binding proteins including those labeled with antisera against myosin light chain kinase and the calmodulin-dependent phosphatase. Activation by the Ca/sup 2 +/ ionophores A23187 and ionomycin also promoted degradation of the calmodulin-binding proteins in the presence of extracellular Ca/sup 2 +/. The data indicate that limited proteolysis of Ca/sup 2 +//calmodulin-regulated enzymes also occurs in the intact platelet and suggest that the proteolysis is triggered by an influx of extracellular Ca/sup 2 +/ associated with platelet aggregation.

  8. Proteolysis of Virulence Regulator ToxR Is Associated with Entry of Vibrio cholerae into a Dormant State

    PubMed Central

    Almagro-Moreno, Salvador; Kim, Tae K.; Skorupski, Karen; Taylor, Ronald K.

    2015-01-01

    Vibrio cholerae O1 is a natural inhabitant of aquatic environments and causes the diarrheal disease, cholera. Two of its primary virulence regulators, TcpP and ToxR, are localized in the inner membrane. TcpP is encoded on the Vibrio Pathogenicity Island (VPI), a horizontally acquired mobile genetic element, and functions primarily in virulence gene regulation. TcpP has been shown to undergo regulated intramembrane proteolysis (RIP) in response to environmental conditions that are unfavorable for virulence gene expression. ToxR is encoded in the ancestral genome and is present in non-pathogenic strains of V. cholerae, indicating it has roles outside of the human host. In this study, we show that ToxR undergoes RIP in V. cholerae in response to nutrient limitation at alkaline pH, a condition that occurs during the stationary phase of growth. This process involves the site-2 protease RseP (YaeL), and is dependent upon the RpoE-mediated periplasmic stress response, as deletion mutants for the genes encoding these two proteins cannot proteolyze ToxR under nutrient limitation at alkaline pH. We determined that the loss of ToxR, genetically or by proteolysis, is associated with entry of V. cholerae into a dormant state in which the bacterium is normally found in the aquatic environment called viable but nonculturable (VBNC). Strains that can proteolyze ToxR, or do not encode it, lose culturability, experience a change in morphology associated with cells in VBNC, yet remain viable under nutrient limitation at alkaline pH. On the other hand, mutant strains that cannot proteolyze ToxR remain culturable and maintain the morphology of cells in an active state of growth. Overall, our findings provide a link between the proteolysis of a virulence regulator and the entry of a pathogen into an environmentally persistent state. PMID:25849031

  9. The core of tau-paired helical filaments studied by scanning transmission electron microscopy and limited proteolysis.

    PubMed

    von Bergen, Martin; Barghorn, Stefan; Müller, Shirley A; Pickhardt, Marcus; Biernat, Jacek; Mandelkow, Eva-Maria; Davies, Peter; Aebi, Ueli; Mandelkow, Eckhard

    2006-05-23

    In Alzheimer's disease and frontotemporal dementias the microtubule-associated protein tau forms intracellular paired helical filaments (PHFs). The filaments formed in vivo consist mainly of full-length molecules of the six different isoforms present in adult brain. The substructure of the PHF core is still elusive. Here we applied scanning transmission electron microscopy (STEM) and limited proteolysis to probe the mass distribution of PHFs and their surface exposure. Tau filaments assembled from the three repeat domain have a mass per length (MPL) of approximately 60 kDa/nm and filaments from full-length tau (htau40DeltaK280 mutant) have approximately 160 kDa/nm, compared with approximately 130 kDa/nm for PHFs from Alzheimer's brain. Polyanionic cofactors such as heparin accelerate assembly but are not incorporated into PHFs. Limited proteolysis combined with N-terminal sequencing and mass spectrometry of fragments reveals a protease-sensitive N-terminal half and semiresistant PHF core starting in the first repeat and reaching to the C-terminus of tau. Continued proteolysis leads to a fragment starting at the end of the first repeat and ending in the fourth repeat. PHFs from tau isoforms with four repeats revealed an additional cleavage site within the middle of the second repeat. Probing the PHFs with antibodies detecting epitopes either over longer stretches in the C-terminal half of tau or in the fourth repeat revealed that they grow in a polar manner. These data describe the physical parameters of the PHFs and enabled us to build a model of the molecular arrangement within the filamentous structures.

  10. Calcitonin gene-related peptide inhibits autophagic-lysosomal proteolysis through cAMP/PKA signaling in rat skeletal muscles.

    PubMed

    Machado, Juliano; Manfredi, Leandro H; Silveira, Wilian A; Gonçalves, Dawit A P; Lustrino, Danilo; Zanon, Neusa M; Kettelhut, Isis C; Navegantes, Luiz C

    2016-03-01

    Calcitonin gene-related peptide (CGRP) is a neuropeptide released by motor neuron in skeletal muscle and modulates the neuromuscular transmission by induction of synthesis and insertion of acetylcholine receptor on postsynaptic muscle membrane; however, its role in skeletal muscle protein metabolism remains unclear. We examined the in vitro and in vivo effects of CGRP on protein breakdown and signaling pathways in control skeletal muscles and muscles following denervation (DEN) in rats. In isolated muscles, CGRP (10(-10) to 10(-6)M) reduced basal and DEN-induced activation of overall proteolysis in a concentration-dependent manner. The in vitro anti-proteolytic effect of CGRP was completely abolished by CGRP8-37, a CGRP receptor antagonist. CGRP down-regulated the lysosomal proteolysis, the mRNA levels of LC3b, Gabarapl1 and cathepsin L and the protein content of LC3-II in control and denervated muscles. In parallel, CGRP elevated cAMP levels, stimulated PKA/CREB signaling and increased Foxo1 phosphorylation in both conditions. In denervated muscles and starved C2C12 cells, Rp-8-Br-cAMPs or PKI, two PKA inhibitors, completely abolished the inhibitory effect of CGRP on Foxo1, 3 and 4 and LC3 lipidation. A single injection of CGRP (100 μg kg(-1)) in denervated rats increased the phosphorylation levels of CREB and Akt, inhibited Foxo transcriptional activity, the LC3 lipidation as well as the mRNA levels of LC3b and cathepsin L, two bona fide targets of Foxo. This study shows for the first time that CGRP exerts a direct inhibitory action on autophagic-lysosomal proteolysis in control and denervated skeletal muscle by recruiting cAMP/PKA signaling, effects that are related to inhibition of Foxo activity and LC3 lipidation. PMID:26718975

  11. The Brucella abortus General Stress Response System Regulates Chronic Mammalian Infection and Is Controlled by Phosphorylation and Proteolysis*

    PubMed Central

    Kim, Hye-Sook; Caswell, Clayton C.; Foreman, Robert; Roop, R. Martin; Crosson, Sean

    2013-01-01

    Brucella spp. are adept at establishing a chronic infection in mammals. We demonstrate that core components of the α-proteobacterial general stress response (GSR) system, PhyR and σE1, are required for Brucella abortus stress survival in vitro and maintenance of chronic murine infection in vivo. ΔphyR and ΔrpoE1 null mutants exhibit decreased survival under acute oxidative and acid stress but are not defective in infection of primary murine macrophages or in initial colonization of BALB/c mouse spleens. However, ΔphyR and ΔrpoE1 mutants are attenuated in spleens beginning 1 month postinfection. Thus, the B. abortus GSR system is dispensable for colonization but is required to maintain chronic infection. A genome-scale analysis of the B. abortus GSR regulon identified stress response genes previously linked to virulence and genes that affect immunomodulatory components of the cell envelope. These data support a model in which the GSR system affects both stress survival and the interface between B. abortus and the host immune system. We further demonstrate that PhyR proteolysis is a unique feature of GSR control in B. abortus. Proteolysis of PhyR provides a mechanism to avoid spurious PhyR protein interactions that inappropriately activate GSR-dependent transcription. We conclude that the B. abortus GSR system regulates acute stress adaptation and long term survival within a mammalian host and that PhyR proteolysis is a novel regulatory feature in B. abortus that ensures proper control of GSR transcription. PMID:23546883

  12. The splicing factor U2AF65 stabilizes TRF1 protein by inhibiting its ubiquitin-dependent proteolysis

    SciTech Connect

    Kim, Jeonghee; Chung, In Kwon

    2014-01-17

    Highlights: •Identification of U2AF65 as a novel TRF1-interacting protein. •U2AF65 stabilizes TRF1 protein by inhibiting its ubiquitin-dependent proteolysis. •U2AF65 interferes with the interaction between TRF1 and Fbx4. •U2AF65 represents a new route for modulating TRF1 function at telomeres. -- Abstract: The human telomeric protein TRF1 is a component of the six-subunit protein complex shelterin, which provides telomere protection by organizing the telomere into a high-order structure. TRF1 functions as a negative regulator of telomere length by controlling the access of telomerase to telomeres. Thus, the cellular abundance of TRF1 at telomeres should be maintained and tightly regulated to ensure proper telomere function. Here, we identify U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 (U2AF65), an essential pre-mRNA splicing factor, as a novel TRF1-interacting protein. U2AF65 interacts with TRF1 in vitro and in vivo and is capable of stabilizing TRF1 protein by inhibiting its ubiquitin-dependent proteolysis. We also found that U2AF65 interferes with the interaction between TRF1 and Fbx4, an E3 ubiquitin ligase for TRF1. Depletion of endogenous U2AF65 expression by short interfering RNA (siRNA) reduced the stability of endogenous TRF1 whereas overexpression of U2AF65 significantly extended the half-life of TRF1. These findings demonstrate that U2AF65 plays a critical role in regulating the level of TRF1 through physical interaction and ubiquitin-mediated proteolysis. Hence, U2AF65 represents a new route for modulating TRF1 function at telomeres.

  13. The role of N-linked glycosylation in the protection of human and bovine lactoferrin against tryptic proteolysis.

    PubMed

    van Veen, Harrie A; Geerts, Marlieke E J; van Berkel, Patrick H C; Nuijens, Jan H

    2004-02-01

    Lactoferrin (LF) is an iron-binding glycoprotein of the innate host defence system. To elucidate the role of N-linked glycosylation in protection of LF against proteolysis, we compared the tryptic susceptibility of human LF (hLF) variants from human milk, expressed in human 293(S) cells or in the milk of transgenic mice and cows. The analysis revealed that recombinant hLF (rhLF) with mutations Ile130-->Thr and Gly404-->Cys was about twofold more susceptible than glycosylated and unglycosylated variants with the naturally occurring Ile130 and Gly404. Hence, N-linked glycosylation is not involved in protection of hLF against tryptic proteolysis. Apparently, the previously reported protection by N-linked glycosylation of hLF [van Berkel, P.H.C., Geerts, M.E.J., van Veen, H.A., Kooiman, P.M., Pieper, F., de Boer, H.A. & Nuijens, J.H. (1995) Biochem. J. 312, 107-114] is restricted to rhLF containing the Thr130 and Cys404. Comparison of the tryptic proteolysis of hLF and bovine LF (bLF) revealed that hLF is about 100-fold more resistant than bLF. Glycosylation variants A and B of bLF differed by about 10-fold in susceptibility to trypsin. This difference is due to glycosylation at Asn281 in bLF-A. Hence, glycosylation at Asn281 protects bLF against cleavage by trypsin at Lys282. PMID:14764083

  14. Matrix metalloproteinase proteolysis of the mycobacterial HSP65 protein as a potential source of immunogenic peptides in human tuberculosis.

    PubMed

    Shiryaev, Sergey A; Cieplak, Piotr; Aleshin, Alexander E; Sun, Qing; Zhu, Wenhong; Motamedchaboki, Khatereh; Sloutsky, Alexander; Strongin, Alex Y

    2011-09-01

    Mycobacterium tuberculosis is the causative agent of human tuberculosis (TB). Mycobacterial secretory protein ESAT-6 induces matrix metalloproteinase (MMP)-9 in epithelial cells neighboring infected macrophages. MMP-9 then enhances recruitment of uninfected macrophages, which contribute to nascent granuloma maturation and bacterial growth. Disruption of MMP-9 function attenuates granuloma formation and bacterial growth. The abundant mycobacterial 65 kDa heat shock protein (HSP65) chaperone is the major target for the immune response and a critical component in M. tuberculosis adhesion to macrophages. We hypothesized that HSP65 is susceptible to MMP-9 proteolysis and that the resulting HSP65 immunogenic peptides affect host adaptive immunity. To identify MMPs that cleave HSP65, we used MMP-2 and MMP-9 gelatinases, the simple hemopexin domain MMP-8, membrane-associated MMP-14, MMP-15, MMP-16 and MMP-24, and glycosylphosphatidylinositol-linked MMP-17 and MMP-25. We determined both the relative cleavage efficiency of MMPs against the HSP65 substrate and the peptide sequence of the cleavage sites. Cleavage of the unstructured PAGHG474L C-terminal region initiates the degradation of HSP65 by MMPs. This initial cleavage destroys the substrate-binding capacity of the HSP65 chaperone. Multiple additional cleavages of the unfolded HSP65 then follow. MMP-2, MMP-8, MMP-14, MMP-15 and MMP-16, in addition to MMP-9, generate the known highly immunogenic N-terminal peptide of HSP65. Based on our biochemical data, we now suspect that MMP proteolysis of HSP65 in vivo, including MMP-9 proteolysis, also results in the abundant generation of the N-terminal immunogenic peptide and that this peptide, in addition to intact HSP65, contributes to the complex immunomodulatory interplay in the course of TB infection.

  15. Time-Course of Muscle Mass Loss, Damage, and Proteolysis in Gastrocnemius following Unloading and Reloading: Implications in Chronic Diseases

    PubMed Central

    Chacon-Cabrera, Alba; Lund-Palau, Helena; Gea, Joaquim; Barreiro, Esther

    2016-01-01

    Background Disuse muscle atrophy is a major comorbidity in patients with chronic diseases including cancer. We sought to explore the kinetics of molecular mechanisms shown to be involved in muscle mass loss throughout time in a mouse model of disuse muscle atrophy and recovery following immobilization. Methods Body and muscle weights, grip strength, muscle phenotype (fiber type composition and morphometry and muscle structural alterations), proteolysis, contractile proteins, systemic troponin I, and mitochondrial content were assessed in gastrocnemius of mice exposed to periods (1, 2, 3, 7, 15 and 30 days) of non-invasive hindlimb immobilization (plastic splint, I cohorts) and in those exposed to reloading for different time-points (1, 3, 7, 15, and 30 days, R cohorts) following a seven-day period of immobilization. Groups of control animals were also used. Results Compared to non-exposed controls, muscle weight, limb strength, slow- and fast-twitch cross-sectional areas, mtDNA/nDNA, and myosin content were decreased in mice of I cohorts, whereas tyrosine release, ubiquitin-proteasome activity, muscle injury and systemic troponin I levels were increased. Gastrocnemius reloading following splint removal improved muscle mass loss, strength, fiber atrophy, injury, myosin content, and mtDNA/nDNA, while reducing ubiquitin-proteasome activity and proteolysis. Conclusions A consistent program of molecular and cellular events leading to reduced gastrocnemius muscle mass and mitochondrial content and reduced strength, enhanced proteolysis, and injury, was seen in this non-invasive mouse model of disuse muscle atrophy. Unloading of the muscle following removal of the splint significantly improved the alterations seen during unloading, characterized by a specific kinetic profile of molecular events involved in muscle regeneration. These findings have implications in patients with chronic diseases including cancer in whom physical activity may be severely compromised. PMID

  16. Cell cycle -dependent proteolysis in plants. Identification Of the destruction box pathway and metaphase arrest produced by the proteasome inhibitor mg132

    PubMed Central

    Genschik, P; Criqui, MC; Parmentier, Y; Derevier, A; Fleck, J

    1998-01-01

    It is widely assumed that mitotic cyclins are rapidly degraded during anaphase, leading to the inactivation of the cell cycle-dependent protein kinase Cdc2 and allowing exit from mitosis. The proteolysis of mitotic cyclins is ubiquitin/26S proteasome mediated and requires the presence of the destruction box motif at the N terminus of the proteins. As a first attempt to study cyclin proteolysis during the plant cell cycle, we investigated the stability of fusion proteins in which the N-terminal domains of an A-type and a B-type tobacco mitotic cyclin were fused in frame with the chloramphenicol acetyltransferase (CAT ) reporter gene and constitutively expressed in transformed tobacco BY2 cells. For both cyclin types, the N-terminal domains led the chimeric cyclin-CAT fusion proteins to oscillate in a cell cycle-specific manner. Mutations within the destruction box abolished cell cycle-specific proteolysis. Although both fusion proteins were degraded after metaphase, cyclin A-CAT proteolysis was turned off during S phase, whereas that of cyclin B-CAT was turned off only during the late G2 phase. Thus, we demonstrated that mitotic cyclins in plants are subjected to post-translational control (e.g., proteolysis). Moreover, we showed that the proteasome inhibitor MG132 blocks BY2 cells during metaphase in a reversible way. During this mitotic arrest, both cyclin-CAT fusion proteins remained stable. PMID:9836745

  17. ADAMTS4 and ADAMTS5 Knockout Mice Are Protected from Versican but Not Aggrecan or Brevican Proteolysis during Spinal Cord Injury

    PubMed Central

    Demircan, Kadir; Topcu, Vehap; Takigawa, Tomoyuki; Akyol, Sumeyya; Yonezawa, Tomoko; Ozturk, Gulfer; Ugurcu, Veli; Hasgul, Rukiye; Yigitoglu, M. Ramazan

    2014-01-01

    The chondroitin sulfate proteoglycans (CSPGs) aggrecan, versican, and brevican are large aggregating extracellular matrix molecules that inhibit axonal growth of the mature central nervous system (CNS). ADAMTS proteoglycanases, including ADAMTS4 and ADAMTS5, degrade CSPGs, representing potential targets for ameliorating axonal growth-inhibition by CSPG accumulation after CNS injury. We investigated the proteolysis of CSPGs in mice homozygous for Adamts4 or Adamts5 null alleles after spinal cord injury (SCI). ADAMTS-derived 50–60 kDa aggrecan and 50 kDa brevican fragments were observed in Adamts4−/−, Adamts5−/−, and wt mice but not in the sham-operated group. By contrast Adamts4−/− and Adamts5−/− mice were both protected from versican proteolysis with an ADAMTS-generated 70 kDa versican fragment predominately observed in WT mice. ADAMTS1, ADAMTS9, and ADAMTS15 were detected by Western blot in Adamts4−/− mice' spinal cords after SCI. Immunohistochemistry showed astrocyte accumulation at the injury site. These data indicate that aggrecan and brevican proteolysis is compensated in Adamts4−/− or Adamts5−/− mice by ADAMTS proteoglycanase family members but a threshold of versican proteolysis is sensitive to the loss of a single ADAMTS proteoglycanase during SCI. We show robust ADAMTS activity after SCI and exemplify the requirement for collective proteolysis for effective CSPG clearance during SCI. PMID:25101296

  18. Powering a burnt bridges Brownian ratchet: a model for an extracellular motor driven by proteolysis of collagen.

    PubMed

    Saffarian, Saveez; Qian, Hong; Collier, Ivan; Elson, Elliot; Goldberg, Gregory

    2006-04-01

    Biased diffusion of collagenase on collagen fibrils may represent the first observed adenosine triphosphate-independent extracellular molecular motor. The magnitude of force generated by the enzyme remains unclear. We propose a propulsion mechanism based on a burnt bridges Brownian ratchet model with a varying degree of coupling of the free energy from collagen proteolysis to the enzyme motion. When constrained by experimental observations, our model predicts 0.1 pN stall force for individual collagenase molecules. A dimer, surprisingly, can generate a force in the range of 5 pN, suggesting that the motor can be of biological significance.

  19. Sequence of Fibrinogen Proteolysis and Platelet Release after Intrauterine Infusion of Hypertonic Saline

    PubMed Central

    Nossel, H. L.; Wasser, J.; Kaplan, K. L.; Lagamma, K. S.; Yudelman, I.; Canfield, R. E.

    1979-01-01

    platelets. Later plasmin cleaves Bβ1-42 from fibrin I to produce fragment X, which is further degraded to form serum fibrinogen degradation products. This sequence of proteolysis indicates that plasmin action on fibrin I serves as a mechanism that regulates fibrin II formation by removing the Bβ chain cleavage site, which is required for thrombin action in converting fibrin I to fibrin II. PMID:500818

  20. On-chip enzymatic microreactor using trypsin-immobilized superparamagnetic nanoparticles for highly efficient proteolysis.

    PubMed

    Liu, Junyan; Lin, Shuang; Qi, Dawei; Deng, Chunhui; Yang, Pengyuan; Zhang, Xiangmin

    2007-12-28

    An easily replaceable microchip enzymatic microreactor has been fabricated based on the glass microchip with trypsin-immobilized superparamagnetic nanoparticles. Magnetic nanoparticles with small size (50 nm in diameter) and strong magnetism were synthesized. At first, amine-functionalized magnetic nanoparticles with high magnetic responsivity and excellent dispersibility were prepared through a facile one-pot strategy. Then, magnetic nanoparticles were functionalized with numerous aldehyde (-CHO) groups by treating the as-synthesized, amine-functionalized magnetic nanoparticles with glutaraldehyde. Finally, immobilization of trypsin onto the aldehyde-functionalized magnetic nanoparticles was achieved through reaction of the aldehyde groups with amine groups of trypsin. The prepared magnetic nanoparticles were then locally packed onto the glass microchip by the application of a strong magnetic field using a magnet to form an on-chip magnetic nanoparticles packing bed. Capability of the proteolytic microreactor was demonstrated by cytochrome c, bovine serum albumin and myoglobin as model proteins. The digestion products were characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with sequence coverage of 83%, 43% and 79% observed, respectively. Complete protein digestion was achieved in a short time (10 s) under the flow rate of 5 microL/min. These results are expected to open up a new possibility for the proteolysis analysis as well as a new application of magnetic nanoparticles. It is easy to replace the nanoparticles and make the new microreactor. It takes less than 1 min under the condition of extra magnetic to form a new packing bed. The packing bed can be used for at least five times without any treatments. Additionally, since the preparation and surface functionality of magnetic nanoparticles is low-cost and reproducible, the preparation method and application approach of the magnetic nanoparticles may find much

  1. The role of citric acid in oral peptide and protein formulations: relationship between calcium chelation and proteolysis inhibition.

    PubMed

    Welling, Søren H; Hubálek, František; Jacobsen, Jette; Brayden, David J; Rahbek, Ulrik L; Buckley, Stephen T

    2014-04-01

    The excipient citric acid (CA) has been reported to improve oral absorption of peptides by different mechanisms. The balance between its related properties of calcium chelation and permeation enhancement compared to a proteolysis inhibition was examined. A predictive model of CA's calcium chelation activity was developed and verified experimentally using an ion-selective electrode. The effects of CA, its salt (citrate, Cit) and the established permeation enhancer, lauroyl carnitine chloride (LCC) were compared by measuring transepithelial electrical resistance (TEER) and permeability of insulin and FD4 across Caco-2 monolayers and rat small intestinal mucosae mounted in Ussing chambers. Proteolytic degradation of insulin was determined in rat luminal extracts across a range of pH values in the presence of CA. CA's capacity to chelate calcium decreased ~10-fold for each pH unit moving from pH 6 to pH 3. CA was an inferior weak permeation enhancer compared to LCC in both in vitro models using physiological buffers. At pH 4.5 however, degradation of insulin in rat luminal extracts was significantly inhibited in the presence of 10mM CA. The capacity of CA to chelate luminal calcium does not occur significantly at the acidic pH values where it effectively inhibits proteolysis, which is its dominant action in oral peptide formulations. On account of insulin's low basal permeability, inclusion of alternative permeation enhancers is likely to be necessary to achieve sufficient oral bioavailability since this is a weak property of CA.

  2. Phosphorylation-Coupled Proteolysis of the Transcription Factor MYC2 Is Important for Jasmonate-Signaled Plant Immunity

    PubMed Central

    Zhai, Qingzhe; Yan, Liuhua; Tan, Dan; Chen, Rong; Sun, Jiaqiang; Gao, Liyan; Dong, Meng-Qiu; Wang, Yingchun; Li, Chuanyou

    2013-01-01

    As a master regulator of jasmonic acid (JA)–signaled plant immune responses, the basic helix-loop-helix (bHLH) Leu zipper transcription factor MYC2 differentially regulates different subsets of JA–responsive genes through distinct mechanisms. However, how MYC2 itself is regulated at the protein level remains unknown. Here, we show that proteolysis of MYC2 plays a positive role in regulating the transcription of its target genes. We discovered a 12-amino-acid element in the transcription activation domain (TAD) of MYC2 that is required for both the proteolysis and the transcriptional activity of MYC2. Interestingly, MYC2 phosphorylation at residue Thr328, which facilitates its turnover, is also required for the MYC2 function to regulate gene transcription. Together, these results reveal that phosphorylation-coupled turnover of MYC2 stimulates its transcription activity. Our results exemplify that, as with animals, plants employ an “activation by destruction” mechanism to fine-tune their transcriptome to adapt to their ever-changing environment. PMID:23593022

  3. The multifaceted role of Lon proteolysis in seedling establishment and maintenance of plant organelle function: living from protein destruction.

    PubMed

    Rigas, Stamatis; Daras, Gerasimos; Tsitsekian, Dikran; Hatzopoulos, Polydefkis

    2012-05-01

    Intracellular selective proteolysis is an important post-translational regulatory mechanism maintaining protein quality control by removing defective, damaged or even deleterious protein aggregates. The ATP-dependent Lon protease is a key component of protein quality control that is highly conserved across the kingdoms of living organisms. Major advancements have been made in bacteria and in non-plant organisms to understand the role of Lon in protection against protein oxidation, ageing and neurodegenerative diseases. This review presents the progress currently made in plants. The Lon gene family in Arabidopsis consists of four members that produce distinct protein isoforms localized in several organelles. Lon1 and Lon4 that potentially originate from a recent gene duplication event are dual-targeted to mitochondria and chloroplasts through distinct mechanisms revealing divergent evolution. Arabidopsis mutant analysis showed that mitochondria and peroxisomes biogenesis or maintenance of function is modulated by Lon1 and Lon2, respectively. Consequently, the lack of Lon selective proteolysis leading to growth retardation and impaired seedling establishment can be attributed to defects in the oil reserve mobilization pathway. The current progress in Arabidopsis research uncovers the role of Lon in the proteome homeostasis of plant organelles and stimulates biotechnology scenarios of plant tolerance against harsh abiotic conditions because of climate instability.

  4. Effect of monoclonal antibodies on limited proteolysis of native glycoprotein gD of herpes simplex virus type 1

    SciTech Connect

    Eisenberg, R.J.; Long, D.; Pereira, L.; Hampar, B.; Zweig, M.; Cohen, G.H.

    1982-02-01

    We examined the properties of 17 monoclonal antibodies to glycoprotein gD of herpes simplex type 1 (HSV-1) (gD-1) and HSV-2 (gD-2). The antibodies recognized eight separate determinants of gD, based on differences in radioimmuno-precipitation and neutralization assays. The determinants were distributed as follows: three were gD-1 specific, one was gD-2 specific, and four were type common. Several type-specific and type-common determinants appeared to be involved in neutralization. We developed a procedure for examining the effect that binding of monoclonal antibody has on proteolysis of native gD-1 by Staphylococcus aureus protease V8. We showed that several different patterns of protease V8 cleavage were obtained, depending on the monoclonal antibody used. The proteolysis patterns were generally consistent with the immunological groupings. With four groups of antibodies, we found that fragments of gD-1 remained bound to antibody after V8 treatment. A 38,000-dalton fragment remained bound to antibodies in three different groups of monoclonal antibodies. This fragment appeared to contain one type-common and two type-specific determinants. A 12,000-dalton fragment remained bound to antibodies belonging to one type-common group of monoclonal antibodies. Tryptic peptide analysis revealed that the 12,000-dalton fragment represented a portion of the 38,000-dalton fragment and was enriched in a type-common arginine tryptic peptide.

  5. Cathepsin B-like proteolysis and MARCKS degradation in sub-lethal NMDA-induced collapse of dendritic spines.

    PubMed

    Graber, S; Maiti, S; Halpain, Shelley

    2004-10-01

    Sub-lethal excitotoxic injury to dendrites can elicit loss or shrinkage of dendritic spines. Here, we used a cell culture model of sub-lethal NMDA-induced injury to investigate a role for proteolysis in spine collapse. Transient incubation with NMDA-induced spine collapse and spine F-actin loss within 10 min, an effect not mimicked by the actin assembly inhibitor latrunculin A. NMDA-induced spine collapse was significantly attenuated by preincubation with broad-spectrum cysteine protease inhibitors. Results obtained using several class-specific protease inhibitors suggested that this protective effect was due to specific blockade of cathepsin B/L type protease activity, since selective inhibitors of only these proteases significantly attenuated spine loss. Cathepsin B-like immunoreactivity was observed at synaptic sites, but lysosomes were not. Immunoblot analysis showed that MARCKS (myristoylated-alanine-rich C-kinase substrate), a known substrate of cathepsin B, was specifically degraded in response to intense NMDA receptor stimulation. This effect was blocked by preincubation with a cathepsin B-selective inhibitor. Together these data suggest a model in which NMDA-induced spine collapse involves cathepsin B-like proteolysis of MARCKS, and possibly other proteins that regulate the actin-based cytoskeleton.

  6. Effect of pulsed electric field on the proteolysis of cold boned beef M. Longissimus lumborum and M. Semimembranosus.

    PubMed

    Suwandy, Via; Carne, Alan; van de Ven, Remy; Bekhit, Alaa El-Din A; Hopkins, David L

    2015-02-01

    The effects of pulsed electric field (PEF) and ageing (3, 7, 14 and 21 days) on the shear force, protein profile, and post-mortem proteolysis of beef loins (M. Longissimus lumborum, LL) and topsides (M. Semimembranosus, SM) were investigated using a range of pulsed electric field treatments [voltages (5 and 10 kV) and frequencies (20, 50, and 90 Hz)]. PEF treatment decreased the shear force of beef LL and SM muscles by up to 19%. The reduction in the shear force in the LL was not affected by the treatment intensity whereas the reduction in the SM was dependent on PEF frequency. PEF treated beef loins showed increased proteolysis, both early post-mortem and during subsequent post-mortem storage reflected by increased degradation of troponin-T and desmin. The most prominent troponin-T degradation was found in samples treated with 5 kV-90 Hz, 10 kV-20 Hz at day 3 and day 7 post-treatment in addition to 10 kV-50 Hz in subsequent post-treatment times. The degradation of desmin in PEF treated beef loins increased with ageing time.

  7. The Growth and Tumor Suppressors NORE1A and RASSF1A Are Targets for Calpain-Mediated Proteolysis

    PubMed Central

    Kuznetsov, Sergey; Khokhlatchev, Andrei V.

    2008-01-01

    Background NORE1A and RASSF1A are growth and tumour suppressors inactivated in a variety of cancers. Methylation of NORE1A and RASSF1A promoters is the predominant mechanism for downregulation of these proteins; however, other mechanisms are likely to exist. Methodology/Principal Findings Here we describe a proteolysis of NORE1A and RASSF1A by calpains as alternative mechanism of their downregulation. Extracts of H358 cell line, a human bronchoalveolar carcinoma, and H460, a large cell carcinoma, were capable of proteolysis of NORE1A protein in the calpain-dependent manner. Likewise, RASSF1A tumor suppressor was proteolyzed by the H358 cell extract. Addition of calpain inhibitor to H358 and H460 cells growing in tissue culture resulted in re-expression of endogenous NORE1A. A survey of 10 human lung tumours revealed that three of them contain an activity capable of inducing NORE1A degradation. Conclusions/Significance Thus, degradation by calpains is a novel mechanism for downregulation of NORE1A and RASSF1A proteins and might be the mechanism allowing cancer cells to escape growth suppression. PMID:19098985

  8. Site-specific proteolysis of the transcriptional coactivator HCF-1 can regulate its interaction with protein cofactors.

    PubMed

    Vogel, Jodi L; Kristie, Thomas M

    2006-05-01

    Limited proteolytic processing is an important transcriptional regulatory mechanism. In various contexts, proteolysis controls the cytoplasmic-to-nuclear transport of important transcription factors or removes domains to produce factors with altered activities. The transcriptional coactivator host cell factor-1 (HCF-1) is proteolytically processed within a unique domain consisting of 20-aa reiterations. Site-specific cleavage within one or more repeats generates a family of amino- and carboxyl-terminal subunits that remain tightly associated. However, the consequences of HCF-1 processing have been undefined. In this study, it was determined that the HCF-1-processing domain interacts with several proteins including the transcriptional coactivator/corepressor four-and-a-half LIM domain-2 (FHL2). Analysis of this interaction has uncovered specificity with both sequence and context determinants within the reiterations of this processing domain. In cells, FHL2 interacts exclusively with the nonprocessed coactivator and costimulates transcription of an HCF-1-dependent target gene. The functional interaction of HCF-1 with FHL2 supports a model in which site-specific proteolysis regulates the interaction of HCF-1 with protein partners and thus can modulate the activity of this coactivator. This paradigm expands the biological significance of limited proteolytic processing as a regulatory mechanism in gene transcription.

  9. Substrate Recognition by the Cdh1 Destruction Box Receptor Is a General Requirement for APC/CCdh1-mediated Proteolysis.

    PubMed

    Qin, Liang; Guimarães, Dimitrius Santiago P S F; Melesse, Michael; Hall, Mark C

    2016-07-22

    The anaphase-promoting complex, or cyclosome (APC/C), is a ubiquitin ligase that selectively targets proteins for degradation in mitosis and the G1 phase and is an important component of the eukaryotic cell cycle control system. How the APC/C specifically recognizes its substrates is not fully understood. Although well characterized degron motifs such as the destruction box (D-box) and KEN-box are commonly found in APC/C substrates, many substrates apparently lack these motifs. A variety of alternative APC/C degrons have been reported, suggesting either that multiple modes of substrate recognition are possible or that our definitions of degron structure are incomplete. We used an in vivo yeast assay to compare the G1 degradation rate of 15 known substrates of the APC/C co-activator Cdh1 under normal conditions and conditions that impair binding of D-box, KEN-box, and the recently identified ABBA motif degrons to Cdh1. The D-box receptor was required for efficient proteolysis of all Cdh1 substrates, despite the absence of canonical D-boxes in many. In contrast, the KEN-box receptor was only required for normal proteolysis of a subset of substrates and the ABBA motif receptor for a single substrate in our system. Our results suggest that binding to the D-box receptor may be a shared requirement for recognition and processing of all Cdh1 substrates.

  10. Effect of pulsed electric field on the proteolysis of cold boned beef M. Longissimus lumborum and M. Semimembranosus.

    PubMed

    Suwandy, Via; Carne, Alan; van de Ven, Remy; Bekhit, Alaa El-Din A; Hopkins, David L

    2015-02-01

    The effects of pulsed electric field (PEF) and ageing (3, 7, 14 and 21 days) on the shear force, protein profile, and post-mortem proteolysis of beef loins (M. Longissimus lumborum, LL) and topsides (M. Semimembranosus, SM) were investigated using a range of pulsed electric field treatments [voltages (5 and 10 kV) and frequencies (20, 50, and 90 Hz)]. PEF treatment decreased the shear force of beef LL and SM muscles by up to 19%. The reduction in the shear force in the LL was not affected by the treatment intensity whereas the reduction in the SM was dependent on PEF frequency. PEF treated beef loins showed increased proteolysis, both early post-mortem and during subsequent post-mortem storage reflected by increased degradation of troponin-T and desmin. The most prominent troponin-T degradation was found in samples treated with 5 kV-90 Hz, 10 kV-20 Hz at day 3 and day 7 post-treatment in addition to 10 kV-50 Hz in subsequent post-treatment times. The degradation of desmin in PEF treated beef loins increased with ageing time. PMID:25460129

  11. Acute alcohol intoxication increases atrogin-1 and MuRF1 mRNA without increasing proteolysis in skeletal muscle

    PubMed Central

    Vary, Thomas C.; Frost, Robert A.; Lang, Charles H.

    2008-01-01

    Acute alcohol intoxication decreases muscle protein synthesis, but there is a paucity of data on the ability of alcohol to regulate muscle protein degradation. Furthermore, various types of atrophic stimuli appear to regulate ubiquitin-proteasome-dependent proteolysis by increasing the muscle-specific E3 ligases atrogin-1 and MuRF1 (i.e., “atrogenes”). Therefore, the present study was designed to test the hypothesis that acute alcohol intoxication increases atrogene expression leading to an elevated rate of muscle protein breakdown. In male rats, the intraperitoneal injection of alcohol dose- and time-dependently increased atrogin-1 and MuRF1 mRNA in gastrocnemius, the latter of which was most pronounced. A comparable change was absent in the soleus and heart. The ability of in vivo-administered ethanol to increase atrogene expression was independent of the route of alcohol administration (intraperitoneal vs. oral), as well as of nutritional status (fed vs. fasted) and gender (male vs. female). The increase in atrogin-1 and MuRF1 was independent of alcohol metabolism, and the overproduction of endogenous glucocorticoids and could not be prevented by maintaining the circulating concentration of insulin-like growth factor-I. Despite marked changes in atrogene expression, acute alcohol in vivo did not alter the release of either 3-methylhistidine (MH) or tyrosine from the isolated perfused hindlimb, suggesting that the rate of muscle proteolysis remains unchanged. Moreover, alcohol did not increase the directly determined rate of protein degradation in isolated epitrochlearis muscles or cultured myocytes. Finally, no increase in atrogene expression or 3-MH release was detected in muscle from rats fed an alcohol-containing diet. Our results indicate that although acute alcohol intoxication increases atrogin-1 and MuRF1 mRNA preferentially in fast-twitch skeletal muscle, this change was not associated with increased rates of muscle proteolysis. Therefore, the loss

  12. Limited proteolysis and peptide mapping for comparability of biopharmaceuticals: An evaluation of repeatability, intra-assay precision and capability to detect structural change.

    PubMed

    Perrin, Camille; Burkitt, Will; Perraud, Xavier; O'Hara, John; Jone, Carl

    2016-05-10

    The use of limited proteolysis followed by peptide mapping for the comparability of the higher-order structure of biopharmaceuticals was investigated. In this approach the proteolysis is performed under non-reducing and non-denaturing conditions, and the resulting peptide map is determined by the samples primary and higher order structures. This allows comparability of biopharmaceuticals to be made in terms of their higher order structure, using a method that is relatively simple to implement. The digestion of a monoclonal antibody under non-denaturing conditions was analyzed using peptide mapping, circular dichroism (CD) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This allowed an optimal digestion time to be chosen. This method was then assessed for its ability to detect structural change using a monoclonal antibody, which had been subjected to a range of stresses; deglycosylation, mild denaturation and a batch that had failed specifications due to in-process reduction. The repeatability and inter-assay precision were assessed. It was demonstrated that the limited proteolysis peptide maps of the three stressed samples were significantly different to control samples and that the differences observed were consistent between the occasions when the assays were run. A combination of limited proteolysis and CD or SDS-PAGE analysis was shown to enhance the capacity of these techniques to detect structural change, which otherwise would not have been observed.

  13. Reducing fat levels in cheddar-like goat cheese: impact on proteolysis and rheological properties over 6 months of refrigerated storage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Development of low-fat goat cheeses that appeal to health conscious consumers requires information on how the reduction of fat affects the quality traits of the cheese, such as its proteolysis and rheology. Goat milk samples containing 3.6, 2.0, 1.0, and <0.5% fat were processed into full-fat (F...

  14. Proteasome-mediated proteolysis of the polyglutamine-expanded androgen receptor is a late event in spinal and bulbar muscular atrophy (SBMA) pathogenesis.

    PubMed

    Heine, Erin M; Berger, Tamar R; Pluciennik, Anna; Orr, Christopher R; Zboray, Lori; Merry, Diane E

    2015-05-15

    Proteolysis of polyglutamine-expanded proteins is thought to be a required step in the pathogenesis of several neurodegenerative diseases. The accepted view for many polyglutamine proteins is that proteolysis of the mutant protein produces a "toxic fragment" that induces neuronal dysfunction and death in a soluble form; toxicity of the fragment is buffered by its incorporation into amyloid-like inclusions. In contrast to this view, we show that, in the polyglutamine disease spinal and bulbar muscular atrophy, proteolysis of the mutant androgen receptor (AR) is a late event. Immunocytochemical and biochemical analyses revealed that the mutant AR aggregates as a full-length protein, becoming proteolyzed to a smaller fragment through a process requiring the proteasome after it is incorporated into intranuclear inclusions. Moreover, the toxicity-predicting conformational antibody 3B5H10 bound to soluble full-length AR species but not to fragment-containing nuclear inclusions. These data suggest that the AR is toxic as a full-length protein, challenging the notion of polyglutamine protein fragment-associated toxicity by redefining the role of AR proteolysis in spinal and bulbar muscular atrophy pathogenesis.

  15. Impact of pasteurization of human milk on preterm newborn in vitro digestion: Gastrointestinal disintegration, lipolysis and proteolysis.

    PubMed

    de Oliveira, Samira C; Bourlieu, Claire; Ménard, Olivia; Bellanger, Amandine; Henry, Gwénaële; Rousseau, Florence; Dirson, Emelyne; Carrière, Frédéric; Dupont, Didier; Deglaire, Amélie

    2016-11-15

    Human milk feeding is an important recommendation for preterm newborns considering their vulnerability and digestive immaturity. Holder pasteurization (62.5°C, 30min) applied in milk banks modifies its biological quality and its microstructure. We investigated the impact of pasteurization of preterm human milk on its gastrointestinal kinetics of lipolysis, proteolysis and structural disintegration. An in vitro dynamic system was set up to simulate the gastrointestinal digestion of preterm newborns. A pool of preterm human milk was digested as raw or after Holder pasteurization. Pasteurization impacted the microstructure of undigested human milk, its gastrointestinal disintegration and tended to limit the intestinal lipolysis. Furthermore, the gastrointestinal bioaccessibility of some fatty acids was decreased by pasteurization, while the intestinal bioaccessibility of some amino acids was selectively modulated. The impact of pasteurization on the digestion of human milk may have nutritional relevance in vivo and potentially modulates preterm development and growth.

  16. Hair follicle aging is driven by transepidermal elimination of stem cells via COL17A1 proteolysis.

    PubMed

    Matsumura, Hiroyuki; Mohri, Yasuaki; Binh, Nguyen Thanh; Morinaga, Hironobu; Fukuda, Makoto; Ito, Mayumi; Kurata, Sotaro; Hoeijmakers, Jan; Nishimura, Emi K

    2016-02-01

    Hair thinning and loss are prominent aging phenotypes but have an unknown mechanism. We show that hair follicle stem cell (HFSC) aging causes the stepwise miniaturization of hair follicles and eventual hair loss in wild-type mice and in humans. In vivo fate analysis of HFSCs revealed that the DNA damage response in HFSCs causes proteolysis of type XVII collagen (COL17A1/BP180), a critical molecule for HFSC maintenance, to trigger HFSC aging, characterized by the loss of stemness signatures and by epidermal commitment. Aged HFSCs are cyclically eliminated from the skin through terminal epidermal differentiation, thereby causing hair follicle miniaturization. The aging process can be recapitulated by Col17a1 deficiency and prevented by the forced maintenance of COL17A1 in HFSCs, demonstrating that COL17A1 in HFSCs orchestrates the stem cell-centric aging program of the epithelial mini-organ.

  17. Proteolysis of His-Phe-Arg-Trp-Pro-Gly-Pro in the blood and brain of rats in vivo.

    PubMed

    Shevchenko, K V; Nagaev, I Yu; Babakov, V N; Andreeva, L A; Shevchenko, V P; Radilov, A S; Myasoedov, N F

    2015-01-01

    The kinetics of the content of His-Phe-Arg-Trp-Pro-Gly-Pro (ACTH (6-9)PGP) and its hydrolysis products in the blood and brain of rats in the case of intranasal administration and intravenous injection of tritiated ACTH(6-9)PGP was studied. The parameters of bioavailability of ACTH(6-9)PGP administered intranasally were higher, indicating certain prospects in the intranasal application in clinical practice. We also found that the factor that determines ACTH(6-9)PGP proteolysis in experiments both in vivo and in vitro is aminopeptidases. The main products of ACTH(6-9)PGP during its metabolism in rats are short peptides and amino acids.

  18. Deregulated proteolysis by the F-box proteins SKP2 and β-TrCP: tipping the scales of cancer

    PubMed Central

    Frescas, David; Pagano, Michele

    2009-01-01

    The maintenance and preservation of distinct phases during the cell cycle is a highly complex and coordinated process. It is regulated by phosphorylation — through the activity of cyclin-dependent kinases (CDKs) — and protein degradation, which occurs through ubiquitin ligases such as SCF (SKP1–CUL1–F-box protein) complexes and APC/C (anaphase-promoting complex/cyclosome). Here, we explore the functionality and biology of the F-box proteins, SKP2 (S-phase kinase-associated protein 2) and β-TrCP (β-transducin repeat-containing protein), which are emerging as important players in cancer biogenesis owing to the deregulated proteolysis of their substrates. PMID:18500245

  19. Hair follicle aging is driven by transepidermal elimination of stem cells via COL17A1 proteolysis.

    PubMed

    Matsumura, Hiroyuki; Mohri, Yasuaki; Binh, Nguyen Thanh; Morinaga, Hironobu; Fukuda, Makoto; Ito, Mayumi; Kurata, Sotaro; Hoeijmakers, Jan; Nishimura, Emi K

    2016-02-01

    Hair thinning and loss are prominent aging phenotypes but have an unknown mechanism. We show that hair follicle stem cell (HFSC) aging causes the stepwise miniaturization of hair follicles and eventual hair loss in wild-type mice and in humans. In vivo fate analysis of HFSCs revealed that the DNA damage response in HFSCs causes proteolysis of type XVII collagen (COL17A1/BP180), a critical molecule for HFSC maintenance, to trigger HFSC aging, characterized by the loss of stemness signatures and by epidermal commitment. Aged HFSCs are cyclically eliminated from the skin through terminal epidermal differentiation, thereby causing hair follicle miniaturization. The aging process can be recapitulated by Col17a1 deficiency and prevented by the forced maintenance of COL17A1 in HFSCs, demonstrating that COL17A1 in HFSCs orchestrates the stem cell-centric aging program of the epithelial mini-organ. PMID:26912707

  20. Matriptase Complexes and Prostasin Complexes with HAI-1 and HAI-2 in Human Milk: Significant Proteolysis in Lactation.

    PubMed

    Lai, Chih-Hsin; Lai, Ying-Jung J; Chou, Feng-Pai; Chang, Hsiang-Hua D; Tseng, Chun-Che; Johnson, Michael D; Wang, Jehng-Kang; Lin, Chen-Yong

    2016-01-01

    Significant proteolysis may occur during milk synthesis and secretion, as evidenced by the presence of protease-protease inhibitor complex containing the activated form of the type 2 transmembrane serine protease matriptase and the transmembrane Kunitz-type serine protease inhibitor HAI-1. In order to identify other proteolysis events that may occur during lactation, human milk was analyzed for species containing HAI-1 and HAI-2 which is closely related to HAI-1. In addition to the previously demonstrated matriptase-HAI-1 complex, HAI-1 was also detected in complex with prostasin, a glycosylphosphatidylinositol (GPI)-anchored serine protease. HAI-2 was also detected in complexes, the majority of which appear to be part of higher-order complexes, which do not bind to ionic exchange columns or immunoaffinity columns, suggesting that HAI-2 and its target proteases may be incorporated into special protein structures during lactation. The small proportion HAI-2 species that could be purified contain matriptase or prostasin. Human mammary epithelial cells are the likely cellular sources for these HAI-1 and HAI-2 complexes with matriptase and prostasin given that these protease-inhibitor complexes with the exception of prostasin-HAI-2 complex were detected in milk-derived mammary epithelial cells. The presence of these protease-inhibitor complexes in human milk provides in vivo evidence that the proteolytic activity of matriptase and prostasin are significantly elevated at least during lactation, and possibly contribute to the process of lactation, and that they are under tight control by HAI-1 and HAI-2.

  1. Matriptase Complexes and Prostasin Complexes with HAI-1 and HAI-2 in Human Milk: Significant Proteolysis in Lactation

    PubMed Central

    Lai, Chih-Hsin; Lai, Ying-Jung J.; Chou, Feng-Pai; Chang, Hsiang-Hua D.; Tseng, Chun-Che; Johnson, Michael D.; Wang, Jehng-Kang; Lin, Chen-Yong

    2016-01-01

    Significant proteolysis may occur during milk synthesis and secretion, as evidenced by the presence of protease-protease inhibitor complex containing the activated form of the type 2 transmembrane serine protease matriptase and the transmembrane Kunitz-type serine protease inhibitor HAI-1. In order to identify other proteolysis events that may occur during lactation, human milk was analyzed for species containing HAI-1 and HAI-2 which is closely related to HAI-1. In addition to the previously demonstrated matriptase-HAI-1 complex, HAI-1 was also detected in complex with prostasin, a glycosylphosphatidylinositol (GPI)-anchored serine protease. HAI-2 was also detected in complexes, the majority of which appear to be part of higher-order complexes, which do not bind to ionic exchange columns or immunoaffinity columns, suggesting that HAI-2 and its target proteases may be incorporated into special protein structures during lactation. The small proportion HAI-2 species that could be purified contain matriptase or prostasin. Human mammary epithelial cells are the likely cellular sources for these HAI-1 and HAI-2 complexes with matriptase and prostasin given that these protease-inhibitor complexes with the exception of prostasin-HAI-2 complex were detected in milk-derived mammary epithelial cells. The presence of these protease-inhibitor complexes in human milk provides in vivo evidence that the proteolytic activity of matriptase and prostasin are significantly elevated at least during lactation, and possibly contribute to the process of lactation, and that they are under tight control by HAI-1 and HAI-2. PMID:27043831

  2. Mannose-binding lectin (MBL) mutants are susceptible to matrix metalloproteinase proteolysis: potential role in human MBL deficiency.

    PubMed

    Butler, Georgina S; Sim, Derek; Tam, Eric; Devine, Dana; Overall, Christopher M

    2002-05-17

    Mannose-binding lectin (MBL) plays a critical role in innate immunity. Point mutations in the collagen-like domain (R32C, G34D, or G37E) of MBL cause a serum deficiency, predisposing patients to infections and diseases such as rheumatoid arthritis. We examined whether MBL mutants show enhanced susceptibility to proteolysis by matrix metalloproteinases (MMPs), which are important mediators in inflammatory tissue destruction. Human and rat MBL were resistant to proteolysis in the native state but were cleaved selectively within the collagen-like domain by multiple MMPs after heat denaturation. In contrast, rat MBL with mutations homologous to those of the human variants (R23C, G25D, or G28E) was cleaved efficiently without denaturation in the collagen-like domain by MMP-2 and MMP-9 (gelatinases A and B) and MMP-14 (membrane type-1 MMP), as well as by MMP-1 (collagenase-1), MMP-8 (neutrophil collagenase), MMP-3 (stromelysin-1), neutrophil elastase, and bacterial collagenase. Sites and order of cleavage of the rat MBL mutants for MMP-2 and MMP-9 were: Gly(45)-Lys(46) --> Gly(51)-Ser(52) --> Gly(63)-Gln(64) --> Asn(80)-Met(81) which differed from that of MMP-14, Gly(39)-Leu(40) --> Asn(80)-Met(81), revealing that the MMPs were not functionally interchangeable. These sites were homologous to those cleaved in denatured human MBL. Hence, perturbation of the collagen-like structure of MBL by natural mutations or by denaturation renders MBL susceptible to MMP cleavage. MMPs are likely to contribute to MBL deficiency in individuals with variant alleles and may also be involved in clearance of MBL and modulation of the host response in normal individuals.

  3. Ascorbate attenuates pulmonary emphysema by inhibiting tobacco smoke and Rtp801-triggered lung protein modification and proteolysis.

    PubMed

    Gupta, Indranil; Ganguly, Souradipta; Rozanas, Christine R; Stuehr, Dennis J; Panda, Koustubh

    2016-07-19

    Cigarette smoking causes emphysema, a fatal disease involving extensive structural and functional damage of the lung. Using a guinea pig model and human lung cells, we show that oxidant(s) present in tobacco smoke not only cause direct oxidative damage of lung proteins, contributing to the major share of lung injury, but also activate Rtp801, a key proinflammatory cellular factor involved in tobacco smoke-induced lung damage. Rtp801 triggers nuclear factor κB and consequent inducible NOS (iNOS)-mediated overproduction of NO, which in combination with excess superoxide produced during Rtp801 activation, contribute to increased oxido-nitrosative stress and lung protein nitration. However, lung-specific inhibition of iNOS with a iNOS-specific inhibitor, N6-(1-iminoethyl)-L-lysine, dihydrochloride (L-NIL) solely restricts lung protein nitration but fails to prevent or reverse the major tobacco smoke-induced oxidative lung injury. In comparison, the dietary antioxidant, ascorbate or vitamin C, can substantially prevent such damage by inhibiting both tobacco smoke-induced lung protein oxidation as well as activation of pulmonary Rtp801 and consequent iNOS/NO-induced nitration of lung proteins, that otherwise lead to increased proteolysis of such oxidized or nitrated proteins by endogenous lung proteases, resulting in emphysematous lung damage. Vitamin C also restricts the up-regulation of matrix-metalloproteinase-9, the major lung protease involved in the proteolysis of such modified lung proteins during tobacco smoke-induced emphysema. Overall, our findings implicate tobacco-smoke oxidant(s) as the primary etiopathogenic factor behind both the noncellular and cellular damage mechanisms governing emphysematous lung injury and demonstrate the potential of vitamin C to accomplish holistic prevention of such damage. PMID:27382160

  4. Amino acid sequences of neuropeptides in the sinus gland of the land crab Cardisoma carnifex: a novel neuropeptide proteolysis site.

    PubMed

    Newcomb, R W

    1987-08-01

    The sinus gland is a major neurosecretory structure in Crustacea. Five peptides, labeled C, D, E, F, and I, isolated from the sinus gland of the land crab have been hypothesized to arise from the incomplete proteolysis at two internal sites on a single biosynthetic intermediate peptide "H", based on amino acid composition additivities and pulse-chase radiolabeling studies. The presence of only a single major precursor for the sinus gland peptides implies that peptide H may be synthesized on a common precursor with crustacean hyperglycemic hormone forms, "J" and "L," and a peptide, "K," similar to peptides with molt inhibiting activity. Here I report amino acid sequences of these peptides. The amino terminal sequence of the parent peptide, H, (and the homologous fragments) proved refractory to Edman degradation. Data from amino acid analysis and carboxypeptidase digestion of the naturally occurring fragments and of fragments produced by endopeptidase digestion were used together with Edman degradation to obtain the sequences. Amino acid analysis of fragments of the naturally occurring "overlap" peptides (those produced by internal cleavage at one site on H) was used to obtain the sequences across the cleavage sites. The amino acid sequence of the land crab peptide H is Arg-Ser-Ala-Asp-Gly-Phe-Gly-Arg-Met-Glu-Ser-Leu-Leu-Thr-Ser-Leu-Arg-Gly- Ser-Ala-Glu- Ser-Pro-Ala-Ala-Leu-Gly-Glu-Ala-Ser-Ala-Ala-His-Pro-Leu-Glu. In vivo cleavage at one site involves excision of arginine from the sequence Leu-Arg-Gly, whereas cleavage at the other site involves excision of serine from the sequence Glu-Ser-Leu. Proteolysis at the latter sequence has not been previously reported in intact secretory granules. The aspartate at position 4 is possibly covalently modified.

  5. The roles of the actin-myosin interaction and proteolysis in tenderization during the aging of chicken muscle.

    PubMed

    Li, S; Xu, X; Zhou, G

    2012-01-01

    The objective of this study was to investigate the contribution of the changes in the actin-myosin interaction and proteolysis on meat tenderization during postmortem storage. Following slaughter, chicken breast muscles were removed and stored at 4°C. Changes in the actin-myosin interaction over 48 h of aging were determined by monitoring the Mg(2+)- and Ca(2+)-ATPase activities. Shear force values, pH, protein degradation, calpain activities, and myofibrillar ultrastructures were also investigated. Results showed that the initial weak actin-myosin interaction strengthened at 12 h postmortem followed by a gradual weakening, which was supported by a decrease in Mg(2+)-ATPase activities and a lengthening of the sarcomeres. According to SDS-PAGE and Western blotting analyses, the 30-kDa troponin-T fragment could not be readily detected until 12 h, whereas, at the same time, desmin had been rapidly degraded. However, there was a gradual decline in μ-calpain activity, commencing after about 6 h. Meanwhile, the largest decline in shear force was observed between 12 and 24 h postmortem. These findings suggest that weakening of the strong actin-myosin interaction formed at rigor may play a large role in meat tenderization during the early period of storage. It is proposed that weakening of the actin-myosin interaction results in lengthening of the sarcomeres, and then activated calpains are more able to reach their targeted sites, enabling proteolysis. These 2 factors may be involved in the conversion of muscle to tender meat during postmortem storage.

  6. Regulation of limited N-terminal proteolysis of APE1 in tumor via acetylation and its role in cell proliferation

    PubMed Central

    Bhakat, Kishor K.; Sengupta, Shiladitya; Adeniyi, Victor F.; Roychoudhury, Shrabasti; Nath, Somsubhra; Bellot, Larry J.; Feng, Dan; Mantha, Anil K.; Sinha, Mala; Qiu, Suimin; Luxon, Bruce A.

    2016-01-01

    Mammalian apurinic/apyrimidinic (AP) endonuclease 1 (APE1), a ubiquitous and multifunctional protein, plays an essential role in the repair of both endogenous and drug-induced DNA damages in the genome. Unlike its E.coli counterpart Xth, mammalian APE1 has a unique N-terminal domain and possesses both DNA damage repair and transcriptional regulatory functions. Although the overexpression of APE1 in diverse cancer types and the association of APE1 expression with chemotherapy resistance and poor prognosis are well documented, the cellular and molecular mechanisms that alter APE1 functions during tumorigenesis are largely unknown. Here, we show the presence of full-length APE1 and N-terminal truncated isoforms of APE1 in tumor tissue samples of various cancer types. However, primary tumor tissue has higher levels of acetylated APE1 (AcAPE1) as well as full-length APE1 compared to adjacent non-tumor tissue. We found that APE1 is proteolytically cleaved by an unknown serine protease at its N-terminus following residue lysine (Lys) Lys6 and/or Lys7 and after Lys27 and Lys31 or Lys32. Acetylation of these Lys residues in APE1 prevents this proteolysis. The N-terminal domain of APE1 and its acetylation are required for modulation of the expression of hundreds of genes. Importantly, we found that AcAPE1 is essential for sustained cell proliferation. Together, our study demonstrates that increased acetylation levels of APE1 in tumor cells inhibit the limited N-terminal proteolysis of APE1 and thereby maintain the functions of APE1 to promote tumor cells' sustained proliferation and survival. PMID:26981776

  7. DnaB proteolysis in vivo regulates oligomerization and its localization at oriC in Bacillus subtilis

    PubMed Central

    Grainger, William H.; Machón, Cristina; Scott, David J.; Soultanas, Panos

    2010-01-01

    Initiation of bacterial DNA replication at oriC is mediated by primosomal proteins that act cooperatively to melt an AT-rich region where the replicative helicase is loaded prior to the assembly of the replication fork. In Bacillus subtilis, the dnaD, dnaB and dnaI genes are essential for initiation of DNA replication. We established that their mRNAs are maintained in fast growing asynchronous cultures. DnaB is truncated at its C-terminus in a growth phase-dependent manner. Proteolysis is confined to cytosolic, not to membrane-associated DnaB, and affects oligomerization. Truncated DnaB is depleted at the oriC relative to the native protein. We propose that DNA-induced oligomerization is essential for its action at oriC and proteolysis regulates its localization at oriC. We show that DnaB has two separate ssDNA-binding sites one located within residues 1–300 and another between residues 365–428, and a dsDNA-binding site within residues 365–428. Tetramerization of DnaB is mediated within residues 1–300, and DNA-dependent oligomerization within residues 365–428. Finally, we show that association of DnaB with the oriC is asymmetric and extensive. It encompasses an area from the middle of dnaA to the end of yaaA that includes the AT-rich region melted during the initiation stage of DNA replication. PMID:20071750

  8. Ascorbate attenuates pulmonary emphysema by inhibiting tobacco smoke and Rtp801-triggered lung protein modification and proteolysis.

    PubMed

    Gupta, Indranil; Ganguly, Souradipta; Rozanas, Christine R; Stuehr, Dennis J; Panda, Koustubh

    2016-07-19

    Cigarette smoking causes emphysema, a fatal disease involving extensive structural and functional damage of the lung. Using a guinea pig model and human lung cells, we show that oxidant(s) present in tobacco smoke not only cause direct oxidative damage of lung proteins, contributing to the major share of lung injury, but also activate Rtp801, a key proinflammatory cellular factor involved in tobacco smoke-induced lung damage. Rtp801 triggers nuclear factor κB and consequent inducible NOS (iNOS)-mediated overproduction of NO, which in combination with excess superoxide produced during Rtp801 activation, contribute to increased oxido-nitrosative stress and lung protein nitration. However, lung-specific inhibition of iNOS with a iNOS-specific inhibitor, N6-(1-iminoethyl)-L-lysine, dihydrochloride (L-NIL) solely restricts lung protein nitration but fails to prevent or reverse the major tobacco smoke-induced oxidative lung injury. In comparison, the dietary antioxidant, ascorbate or vitamin C, can substantially prevent such damage by inhibiting both tobacco smoke-induced lung protein oxidation as well as activation of pulmonary Rtp801 and consequent iNOS/NO-induced nitration of lung proteins, that otherwise lead to increased proteolysis of such oxidized or nitrated proteins by endogenous lung proteases, resulting in emphysematous lung damage. Vitamin C also restricts the up-regulation of matrix-metalloproteinase-9, the major lung protease involved in the proteolysis of such modified lung proteins during tobacco smoke-induced emphysema. Overall, our findings implicate tobacco-smoke oxidant(s) as the primary etiopathogenic factor behind both the noncellular and cellular damage mechanisms governing emphysematous lung injury and demonstrate the potential of vitamin C to accomplish holistic prevention of such damage.

  9. SUMO-targeted ubiquitin ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin

    PubMed Central

    Ohkuni, Kentaro; Takahashi, Yoshimitsu; Fulp, Alyona; Lawrimore, Josh; Au, Wei-Chun; Pasupala, Nagesh; Levy-Myers, Reuben; Warren, Jack; Strunnikov, Alexander; Baker, Richard E.; Kerscher, Oliver; Bloom, Kerry; Basrai, Munira A.

    2016-01-01

    Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast and flies and tumorigenesis in human cells; thus defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast shows that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by the small ubiquitin-related modifier (SUMO)-targeted ubiquitin ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1, since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization than either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability. PMID:26960795

  10. In vivo effect of an antilipolytic drug (3,5'-dimethylpyrazole) on autophagic proteolysis and autophagy-related gene expression in rat liver

    SciTech Connect

    Donati, Alessio; Ventruti, Annamaria; Cavallini, Gabriella; Masini, Matilde; Vittorini, Simona; Chantret, Isabelle; Codogno, Patrice; Bergamini, Ettore

    2008-02-15

    Autophagy is an intracellular pathway induced by starvation, inhibited by nutrients, that is responsible for degradation of long-lived proteins and altered cell organelles. This process is involved in cell maintenance could be induced by antilipolytic drugs and may have anti-aging effects [A. Donati, The involvement of macroautophagy in aging and anti-aging interventions, Mol. Aspects Med. 27 (2006) 455-470]. We analyzed the effect of an intraperitoneal injection of an antilipolytic agent (3,5'-dimethylpyrazole, DMP, 12 mg/kg b.w.), that mimics nutrient shortage on autophagy and expression of autophagic genes in the liver of male 3-month-old Sprague-Dawley albino rats. Autophagy was evaluated by observing electron micrographs of the liver autophagosomal compartment and by monitoring protein degradation assessed by the release of valine into the bloodstream. LC3 gene expression, whose product is one of the best known markers of autophagy, was also monitored. As expected, DMP decreased the plasma levels of free fatty acids, glucose, and insulin and increased autophagic vacuoles and proteolysis. DMP treatment caused an increase in the expression of the LC3 gene although this occurred later than the induction of authophagic proteolysis caused by DMP. Glucose treatment rescued the effects caused by DMP on glucose and insulin plasma levels and negatively affected the rate of autophagic proteolysis, but did not suppress the positive regulatory effect on LC3 mRNA levels. In conclusion, antilipolytic drugs may induce both autophagic proteolysis and higher expression of an autophagy-related gene and the effect on autophagy gene expression might not be secondary to the stimulation of autophagic proteolysis.

  11. Acute interleukin-6 infusion increases IGFBP-1 but has no short-term effect on IGFBP-3 proteolysis in healthy men.

    PubMed

    Pihl, S; Carlsson-Skwirut, C; Berg, U; Ekström, K; Bang, P

    2006-01-01

    Human conditions of elevated interleukin-6 (IL-6) and transgenic mice overexpressing IL-6 have increased proteolytic degradation of insulin-like growth factor binding protein (IGFBP)-3. In addition, IL-6 alters the hepatic expression of insulin-like growth factor-I (IGF-I) and the IGFBPs in vitro. The aim of the present study was to investigate whether moderately elevated IL-6 levels have short-term effects on circulating IGF-I, IGFBP-1 and IGFBP-3 proteolysis in vivo. Healthy men received a 3-h IL-6 (n = 6) or saline (n = 6) infusion and blood samples were collected prior to and up to 8 h after the start of infusion. Free IGF-I, total IGF-I, IGFBP-1, insulin and cortisol were measured using immunoassays. Serum IGFBP-3 proteolysis was analyzed by Western immunoblot and by in vitro degradation of (125)I-IGFBP-3. We found that IL-6 concentrations reaching approximately 100 pg/ml significantly increased IGFBP-1 after the end of infusion in the absence of changes in insulin. In addition, plasma levels of cortisol were increased in response to IL-6 during and after infusion compared to saline. There was no effect of IL-6 on IGFBP-3 proteolysis, total IGF-I or free dissociable IGF-I. These data suggest that moderately elevated levels of IL-6 such as in the post-operative state or after exercise may contribute to increased levels of IGFBP-1. Although this study does not exclude that high levels and/or prolonged exposure to IL-6 may induce IGFBP-3 proteolysis in sepsis or chronic inflammatory disease, it suggests that IL-6 released from exercising skeletal muscle is not directly involved in proteolysis of circulating IGFBP-3.

  12. The E104D mutation increases the susceptibility of human triosephosphate isomerase to proteolysis. Asymmetric cleavage of the two monomers of the homodimeric enzyme.

    PubMed

    De La Mora-De La Mora, Ignacio; Torres-Larios, Alfredo; Mendoza-Hernández, Guillermo; Enriquez-Flores, Sergio; Castillo-Villanueva, Adriana; Mendez, Sara T; Garcia-Torres, Itzhel; Torres-Arroyo, Angélica; Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Oria-Hernández, Jesús; López-Velázquez, Gabriel; Reyes-Vivas, Horacio

    2013-12-01

    The deficiency of human triosephosphate isomerase (HsTIM) generates neurological alterations, cardiomyopathy and premature death. The mutation E104D is the most frequent cause of the disease. Although the wild type and mutant exhibit similar kinetic parameters, it has been shown that the E104D substitution induces perturbation of an interfacial water network that, in turn, reduces the association constant between subunits promoting enzyme inactivation. To gain further insight into the effects of the mutation on the structure, stability and function of the enzyme, we measured the sensitivity of recombinant E104D mutant and wild type HsTIM to limited proteolysis. The mutation increases the susceptibility to proteolysis as consequence of the loss of rigidity of its overall 3-D structure. Unexpectedly, it was observed that proteolysis of wild type HsTIM generated two different stable nicked dimers. One was formed in relatively short times of incubation with proteinase K; as shown by spectrometric and crystallographic data, it corresponded to a dimer containing a nicked monomer and an intact monomer. The formation of the other nicked species requires relatively long incubation times with proteinase K and corresponds to a dimer with two clipped subunits. The first species retains 50% of the original activity, whereas the second species is inactive. Collectively, we found that the E104D mutant is highly susceptible to proteolysis, which in all likelihood contributes to the pathogenesis of enzymopathy. In addition, the proteolysis data on wild type HsTIM illustrate an asymmetric conduct of the two monomers.

  13. Validated LC/MS Bioanalysis of Rituximab CDR Peptides Using Nano-surface and Molecular-Orientation Limited (nSMOL) Proteolysis.

    PubMed

    Iwamoto, Noriko; Takanashi, Megumi; Hamada, Akinobu; Shimada, Takashi

    2016-07-01

    Presently, monoclonal antibodies (mAbs) therapeutics have big global sales and are starting to receive competition from biosimilars. We previously reported that the nano-surface and molecular-orientation limited (nSMOL) proteolysis which is optimal method for bioanalysis of antibody drugs in plasma. The nSMOL is a Fab-selective limited proteolysis, which utilize the difference of protease nanoparticle diameter (200 nm) and antibody resin pore diameter (100 nm). In this report, we have demonstrated that the full validation for chimeric antibody Rituximab bioanalysis in human plasma using nSMOL proteolysis. The immunoglobulin fraction was collected using Protein A resin from plasma, which was then followed by the nSMOL proteolysis using the FG nanoparticle-immobilized trypsin under a nondenaturing condition at 50°C for 6 h. After removal of resin and nanoparticles, Rituximab signature peptides (GLEWIGAIYPGNGDTSYNQK, ASGYTFTSYNMHWVK, and FSGSGSGTSYSLTISR) including complementarity-determining region (CDR) and internal standard P14R were simultaneously quantified by multiple reaction monitoring (MRM). This quantification of Rituximab using nSMOL proteolysis showed lower limit of quantification (LLOQ) of 0.586 µg/mL and linearity of 0.586 to 300 µg/mL. The intra- and inter-assay precision of LLOQ, low quality control (LQC), middle quality control (MQC), and high quality control (HQC) was 5.45-12.9% and 11.8, 5.77-8.84% and 9.22, 2.58-6.39 and 6.48%, and 2.69-7.29 and 4.77%, respectively. These results indicate that nSMOL can be applied to clinical pharmacokinetics study of Rituximab, based on the precise analysis. PMID:27150271

  14. Advances in nanomaterial-based microwaves and infrared wave-assisted tryptic digestion for ultrafast proteolysis and rapid detection by MALDI-MS.

    PubMed

    Kailasa, Suresh Kumar; Wu, Hui-Fen

    2014-01-01

    The unique physical/chemical properties of nanomaterials have significant impacts in electromagnetic waves (microwave and infrared waves)-assisted tryptic digestion approaches by using them as heat absorbers to expedite digestion and as affinity probes to enrich digested proteins prior to MALDI-MS analysis. We review recent developments in electromagnetic waves (microwaves and infrared waves)-assisted proteolysis using nanomaterials as heat absorbers and as affinity probes for analysis of digested proteins in MALDI-MS. New trends in ultrafast proteolysis (nonphosphoproteins- lysozyme, cytochrome c, myoglobin and bovine serum albumin (BSA); phosphoproteins- α- and β- caseins) using nanomaterials based microwaves and infrared (IR) waves assisted digestion approaches for rapid identification of digested proteins in the MALDI-MS.

  15. Effect of nitric oxide on μ-calpain activation, protein proteolysis, and protein oxidation of pork during post-mortem aging.

    PubMed

    Li, Yu-pin; Liu, Rui; Zhang, Wan-gang; Fu, Qing-quan; Liu, Nian; Zhou, Guang-hong

    2014-06-25

    The aim of the current research was to examine the influence of nitric oxide (NO) on calpain activation, protein proteolysis, and oxidation in post-mortem pork. Five longissimus muscles were removed from carcass after slaughter, and samples were incubated with water, nitric oxide synthase (NOS) inhibitor, or NO donor for 24 h at 4 °C. The samples were taken out and then stored under 4 °C for 1, 4, and 7 d. Results showed that autolysis of μ-calpain increased by incubation with NOS inhibitor after storage for 1 d (P<0.05). Degradation of titin and nebulin increased by treatment of NOS inhibitor among three treatments (P<0.05). Higher levels of protein oxidation were observed after samples incubated with NO donor than treatment of NOS inhibitor (P<0.05). These data indicated that NO could participate in regulating calpain activation and its proteolysis activity during post-mortem aging.

  16. The UPEC pore-forming toxin α-hemolysin triggers proteolysis of host proteins to disrupt cell adhesion, inflammatory, and survival pathways.

    PubMed

    Dhakal, Bijaya K; Mulvey, Matthew A

    2012-01-19

    Uropathogenic Escherichia coli (UPEC), which are the leading cause of both acute and chronic urinary tract infections, often secrete a labile pore-forming toxin known as α-hemolysin (HlyA). We show that stable insertion of HlyA into epithelial cell and macrophage membranes triggers degradation of the cytoskeletal scaffolding protein paxillin and other host regulatory proteins, as well as components of the proinflammatory NFκB signaling cascade. Proteolysis of these factors requires host serine proteases, and paxillin degradation specifically involves the serine protease mesotrypsin. The induced activation of mesotrypsin by HlyA is preceded by redistribution of mesotrypsin precursors from the cytosol into foci along microtubules and within nuclei. HlyA intoxication also stimulated caspase activation, which occurred independently of effects on host serine proteases. HlyA-induced proteolysis of host proteins likely allows UPEC to not only modulate epithelial cell functions, but also disable macrophages and suppress inflammatory responses.

  17. Asn347 Glycosylation of Corticosteroid-binding Globulin Fine-tunes the Host Immune Response by Modulating Proteolysis by Pseudomonas aeruginosa and Neutrophil Elastase.

    PubMed

    Sumer-Bayraktar, Zeynep; Grant, Oliver C; Venkatakrishnan, Vignesh; Woods, Robert J; Packer, Nicolle H; Thaysen-Andersen, Morten

    2016-08-19

    Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues upon elastase-based proteolysis of the exposed reactive center loop (RCL). However, the molecular mechanisms that regulate the RCL proteolysis by co-existing host and bacterial elastases in inflamed/infected tissues remain unknown. We document that RCL-localized Asn(347) glycosylation fine-tunes the RCL cleavage rate by human neutrophil elastase (NE) and Pseudomonas aeruginosa elastase (PAE) by different mechanisms. NE- and PAE-generated fragments of native and exoglycosidase-treated blood-derived CBG of healthy individuals were monitored by gel electrophoresis and LC-MS/MS to determine the cleavage site(s) and Asn(347) glycosylation as a function of digestion time. The site-specific (Val(344)-Thr(345)) and rapid (seconds to minutes) NE-based RCL proteolysis was significantly antagonized by several volume-enhancing Asn(347) glycan features (i.e. occupancy, triantennary GlcNAc branching, and α1,6-fucosylation) and augmented by Asn(347) NeuAc-type sialylation (all p < 0.05). In contrast, the inefficient (minutes to hours) PAE-based RCL cleavage, which occurred equally well at Thr(345)-Leu(346) and Asn(347)-Leu(348), was abolished by the presence of Asn(347) glycosylation but was enhanced by sialoglycans on neighboring CBG N-sites. Molecular dynamics simulations of various Asn(347) glycoforms of uncleaved CBG indicated that multiple Asn(347) glycan features are modulating the RCL digestion efficiencies by NE/PAE. Finally, high concentrations of cortisol showed weak bacteriostatic effects toward virulent P. aeruginosa, which may explain the low RCL potency of the abundantly secreted PAE during host infection. In conclusion, site-specific CBG N-glycosylation regulates the bioavailability of cortisol in inflamed environments by fine-tuning the RCL proteolysis by endogenous and exogenous elastases. This study offers new molecular insight into host- and pathogen

  18. Cytoplasmic domain of the beta-amyloid protein precursor of Alzheimer's disease: function, regulation of proteolysis, and implications for drug development.

    PubMed

    Kerr, Megan L; Small, David H

    2005-04-15

    The beta-amyloid protein precursor (APP) has been extensively studied for its role in amyloid production and the pathogenesis of Alzheimer's disease (AD). However, little is known about the normal function of APP and its biological interactions. In this Mini-Review, the role of the cytoplasmic domain of APP in APP trafficking and proteolysis is described. These studies suggest that proteins that bind to the cytoplasmic domain may be important targets for drug development in AD. PMID:15672415

  19. Identification of Ypk1 as a Novel Selective Substrate for Nitrogen Starvation-triggered Proteolysis Requiring Autophagy System and Endosomal Sorting Complex Required for Transport (ESCRT) Machinery Components*

    PubMed Central

    Shimobayashi, Mitsugu; Takematsu, Hiromu; Eiho, Kazuo; Yamane, Yukari; Kozutsumi, Yasunori

    2010-01-01

    Nitrogen starvation-mediated reduction of Ypk1 is suggested to suppress translational initiation, possibly in parallel with the target of rapamycin complex 1 (TORC1) signaling. However, the molecular mechanism that regulates Ypk1 in nitrogen-starved cells is poorly understood. Here we report that Ypk1 is a novel selective substrate for nitrogen starvation-triggered proteolysis requiring autophagy system. Among various nutrient starvation methods used to elicit autophagy, rapid Ypk1 degradation was specific to nitrogen starvation. In screening genes required for such nitrogen starvation-specific vacuolar proteolysis, we found that autophagy-related degradation of Ypk1 depended on the endosomal sorting complex required for transport (ESCRT) machinery, which is conventionally thought to function in endosomal trafficking. In microscopic analyses, the disruption of ESCRT subunits resulted in the accumulation of both Ypk1 and autophagosomal Atg8 at a perivacuolar site that was distinct from conventional endosomes. ESCRT machinery was not involved in autophagic flux induced by the TORC1 inhibitor rapamycin, thus suggesting that ESCRT represents an exclusive mechanism of nitrogen starvation-specific proteolysis of Ypk1. Overall, we propose a novel regulation of Ypk1 that is specific to nitrogen limitation. PMID:20855891

  20. A small unstructured region in Vibrio cholerae ToxT mediates the response to positive and negative effectors and ToxT proteolysis.

    PubMed

    Thomson, Joshua J; Plecha, Sarah C; Withey, Jeffrey H

    2015-02-01

    Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. The production of the virulence factors that are required for human disease is controlled by a complex network of transcriptional and posttranscriptional regulators. ToxT is the transcription regulator that directly controls the production of the two major virulence factors, toxin-coregulated pilus (TCP) and cholera toxin (CT). The solved crystal structure of ToxT revealed an unstructured region in the N-terminal domain between residues 100 and 110. This region and the surrounding amino acids have been previously implicated in ToxT proteolysis, resistance to inhibition by negative effectors, and ToxT dimerization. To better characterize this region, site-directed mutagenesis was performed to assess the effects on ToxT proteolysis and bile sensitivity. This analysis identified specific mutations within this unstructured region that prevent ToxT proteolysis and other mutations that reduce inhibition by bile and unsaturated fatty acids. In addition, we found that mutations that affect the sensitivity of ToxT to bile also affect the sensitivity of ToxT to its positive effector, bicarbonate. These results suggest that a small unstructured region in the ToxT N-terminal domain is involved in multiple aspects of virulence gene regulation and response to human host signals.

  1. Proteolysis and sensory properties of dry-cured bacon as affected by the partial substitution of sodium chloride with potassium chloride.

    PubMed

    Wu, Haizhou; Zhang, Yingyang; Long, Men; Tang, Jing; Yu, Xiang; Wang, Jiamei; Zhang, Jianhao

    2014-03-01

    Quadriceps femoris muscle samples (48) from 24 pigs were processed into dry-cured bacon. This study investigated the influence of partial substitution of sodium chloride (NaCl) with potassium chloride (KCl) on proteolysis and sensory properties of dry-cured bacon. Three salt treatments were considered, namely, I (100% NaCl), II (60% NaCl, 40% KCl), and III (30% NaCl, 70% KCl). No significant differences were observed among treatments in the proteolysis, which was reflected by SDS-PAGE, proteolysis index, amino acid nitrogen, and peptide nitrogen contents. Furthermore, there were no significant differences in the moisture content between control and treatment II, whereas the moisture content in treatment III was significantly higher (p<0.05) in comparison with control (treatment I). The sensory analysis indicated that it was possible to reduce NaCl by 40% without adverse effects on sensory properties, but 70% replacement of NaCl with KCl resulted in bacon with less hardness and saltiness and higher (p<0.05) juiciness and bitterness.

  2. The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase.

    PubMed

    Brandeis, M; Hunt, T

    1996-10-01

    We have studied how the cell cycle-specific oscillations of mitotic B-type cyclins are generated in mouse fibroblasts. A reporter enzyme comprising the N-terminus of a B-type cyclin fused to bacterial chloramphenicol acetyl transferase (CAT) was degraded at the end of mitosis like endogenous cyclins. Point mutations in the destruction box of this construct completely abolished its mitotic instability. When the destructible reporter was driven by the cyclin B2 promoter, CAT activity mimicked the oscillations in the level of the endogenous cyclin B2. These oscillations were largely conserved when the reporter was transcribed constitutively from the SV40 promoter. Pulse-chase experiments or addition of the proteasome inhibitors lactacystin and ALLN showed that cyclin synthesis continued after the end of mitosis. The destruction box-specific degradation of cyclins normally ceases at the onset of S phase, and is active in fibroblasts arrested in G0 and in differentiated C2 myoblasts. We were able to reproduce this proteolysis in vitro in extracts of synchronized cells. Extracts of G1 cells degraded cyclin B1 whereas p27Kip1 was stable, in contrast, cyclin B1 remained stable and p27Kip1 was degraded in extracts of S phase cells. PMID:8895573

  3. Limited proteolysis of human leukocyte interferon-. cap alpha. 2 and localization of the monoclonal antibody-binding antigenic determinant

    SciTech Connect

    Kostrov, S.V.; Chernovskaya, T.V.; Khodova, O.M.; Borukhov, S.I.; Ryzhavskaya, A.S.; Izotova, L.S.; Strongin, A.Ya.

    1986-05-20

    Large peptide fragments of human leukocyte interferon-..cap alpha..2 (INF-..cap alpha..2) were produced by limited proteolysis with trypsin, pepsin, thermolysin, and Bacillus amyloliquefaciens serine proteinase, and the ability of the fragments to react with murine monoclonal antibodies NK2, directed toward INF-..cap alpha..2, was studied by the immunoblotting technique. The region of the sequence 110-149 is the most sensitive to proteinase attack and evidently is exposed on the surface of the INF-..cap alpha..2 molecule. The INF-..cap alpha..2 fragments 1-139, 1-147, and 1-149 react with antibodies, whereas the fragments 1-109 and 1-112 do not bind NK2 antibodies. A comparison of the primary structure of the families of human leukocyte and murine leukocyte INF in the region of the sequence 110-139 and an analysis of the ability of human INF differing in amino acid sequence to interact with NK2 antibodies suggested that the antigenic determinant that binds monoclonal antibodies NK2 is the sequence Glu/sub 114/-Asp/sub 115/-Ser/sub 116/-He/sub 117/ of the INF-..cap alpha..2 molecule.

  4. Secretion and proteolysis of heterologous proteins fused to the Escherichia coli maltose binding protein in Pichia pastoris.

    PubMed

    Li, Zhiguo; Leung, Wilson; Yon, Amy; Nguyen, John; Perez, Vincent C; Vu, Jane; Giang, William; Luong, Linda T; Phan, Tracy; Salazar, Kate A; Gomez, Seth R; Au, Colin; Xiang, Fan; Thomas, David W; Franz, Andreas H; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P

    2010-07-01

    The Escherichia coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. When located N-terminal to its cargo protein, MBP increases the solubility of intracellular proteins and improves the export of secreted proteins in bacterial systems. We initially explored whether MBP would have the same effect in the methylotrophic yeast Pichia pastoris, a popular eukaryotic host for heterologous protein expression. When MBP was fused as an N-terminal partner to several C-terminal cargo proteins expressed in this yeast, proteolysis occurred between the two peptides, and MBP reached the extracellular region unattached to its cargo. However, in two of three instances, the cargo protein reached the extracellular region as well, and its initial attachment to MBP enhanced its secretion from the cell. Extensive mutagenesis of the spacer region between MBP and its C-terminal cargo protein could not inhibit the cleavage although it did cause changes in the protease target sites in the fusion proteins, as determined by mass spectrometry. Taken together, these results suggested that an uncharacterized P. pastoris protease attacked at different locations in the region C-terminal of the MBP domain, including the spacer and cargo regions, but the MBP domain could still act to enhance the secretion of certain cargo proteins. PMID:20230898

  5. Secretion and Proteolysis of Heterologous Proteins Fused to the Escherichia coli Maltose Binding Protein in Pichia pastoris

    PubMed Central

    Li, Zhiguo; Leung, Wilson; Yon, Amy; Nguyen, John; Perez, Vincent C.; Vu, Jane; Giang, William; Luong, Linda T.; Phan, Tracy; Salazar, Katherine A.; Gomez, Seth R.; Au, Colin; Xiang, Fan; Thomas, David W.; Franz, Andreas H.; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P.

    2010-01-01

    The E. coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. When located N-terminal to its cargo protein, MBP increases the solubility of intracellular proteins and improves the export of secreted proteins in bacterial systems. We initially explored whether MBP would have the same effect in the methylotrophic yeast Pichia pastoris, a popular eukaryotic host for heterologous protein expression. When MBP was fused as an N-terminal partner to several C-terminal cargo proteins expressed in this yeast, proteolysis occurred between the two peptides, and MBP reached the extracellular region unattached to its cargo. However, in two of three instances, the cargo protein reached the extracellular region as well, and its initial attachment to MBP enhanced its secretion from the cell. Extensive mutagenesis of the spacer region between MBP and its C-terminal cargo protein could not inhibit the cleavage although it did cause changes in the protease target sites in the fusion proteins, as determined by mass spectrometry. Taken together, these results suggested that an uncharacterized P. pastoris protease attacked at different locations in the region C-terminal of the MBP domain, including the spacer and cargo regions, but the MBP domain could still act to enhance the secretion of certain cargo proteins. PMID:20230898

  6. Proteolysis in heat-stressed HeLa cells. Stabilization of ubiquitin correlates with the loss of proline endopeptidase.

    PubMed

    Pratt, G; Hough, R; Rechsteiner, M

    1989-07-25

    When intact HeLa cells were incubated at 45 degrees C, there was progressive inactivation of proline endopeptidase. Rapid loss of the enzyme did not occur in extracts maintained at 45 degrees C. Since Western blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no decrease in the immunoreactive 70-kDa proline endopeptidase band, its in vivo disappearance apparently results from irreversible denaturation or modification. Loss of proline endopeptidase activity was paralleled by reduced degradation of injected ubiquitin and bovine serum albumin. In contrast, proteolysis of injected lysozyme or pancreatic trypsin inhibitor was barely affected. Electrophoretic analysis of ubiquitin or bovine serum albumin retrieved from heated HeLa cells showed that the injected proteins were intact. Thus, the presence of proline endopeptidase appears to be required for initial cleavage of these two substrates, but it has not been shown that the enzyme is directly responsible. Selective stabilization of a subset of the injected proteins does, however, demonstrate the existence of distinct proteolytic pathways in HeLa cytosol. PMID:2545710

  7. Identification of ClpP substrates in Caulobacter crescentus reveals a role for regulated proteolysis in bacterial development.

    PubMed

    Bhat, Nowsheen H; Vass, Robert H; Stoddard, Patrick R; Shin, Dong K; Chien, Peter

    2013-06-01

    Energy-dependent proteases ensure the timely removal of unwanted proteins in a highly selective fashion. In Caulobacter crescentus, protein degradation by the ClpXP protease is critical for cell cycle progression; however, only a handful of substrates are currently known. Here, we use a trapping approach to identify putative substrates of the ClpP associated proteases in C. crescentus. Biochemical validation of several of these targets reveals specific protease recognition motifs and suggests a need for ClpXP-specific degradation beyond degradation of known cell cycle regulators. We focus on a particular instance of regulated proteolysis in Caulobacter by exploring the role of ClpXP in degrading the stalk synthesis transcription factor TacA. We show that TacA degradation is controlled during the cell cycle dependent on the ClpXP regulator CpdR and that stabilization of TacA increases degradation of another ClpXP substrate, CtrA, while restoring deficiencies associated with prolific CpdR activity. Together, our work reveals a number of new validated ClpXP substrates, clarifies rules of protease substrate selection, and demonstrates how regulated protein degradation is critical for Caulobacter development and cell cycle progression.

  8. Bombay phenotype is associated with reduced plasma-VWF levels and an increased susceptibility to ADAMTS13 proteolysis.

    PubMed

    O'Donnell, James S; McKinnon, Thomas A J; Crawley, James T B; Lane, David A; Laffan, Michael A

    2005-09-15

    ABO blood group is an important determinant of plasma von Willebrand factor antigen (VWF:Ag) levels, with lower levels in group O. Previous reports have suggested that ABO(H) sugars affect the susceptibility of VWF to ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type-1 repeats-13) cleavage. To further test this hypothesis, we collected plasma from individuals with the rare Bombay blood group. VWF:Ag levels were significantly lower in Bombay patients (median, 0.69 IU/mL) than in groups AB, A, or B (P < .05) and lower than in group O individuals (median, 0.82 IU/mL). Susceptibility of purified VWF fractions to recombinant ADAMTS13 cleavage, assessed using VWF collagen-binding assay (VWF:CB), was increased in Bombays compared with either group O or AB. Increasing urea concentration (0.5 to 2 M) increased the cleavage rate for each blood group but eliminated the differences between groups. We conclude that reduction in the number of terminal sugars on N-linked glycan increases susceptibility of globular VWF to ADAMTS13 proteolysis and is associated with reduced plasma VWF:Ag and VWF:CB levels.

  9. In situ proteolysis, crystallization and preliminary X-ray diffraction analysis of a VHH that binds listeria internalin B.

    PubMed

    Huh, Ian; Gene, Robert; Kumaran, Jyothi; MacKenzie, C Roger; Brooks, Cory L

    2014-11-01

    The variable region of camelid heavy-chain antibodies produces the smallest known antibody fragment with antigen-binding capability (a VHH). The VHH R303 binds internalin B (InlB), a virulence factor expressed by the pathogen Listeria monocytogenes. InlB is critical for initiation of Listeria infection, as it binds a receptor (c-Met) on epithelial cells, triggering the entry of bacteria into host cells. InlB is surface-exposed and is required for virulence, hence a VHH targeting InlB has potential applications for pathogen detection or therapeutic intervention. Here, the expression, purification, crystallization and X-ray diffraction of R303 are reported. Crystals of R303 were obtained following in situ proteolysis with trypsin. Gel filtration and SDS-PAGE revealed that trypsin removed the C-terminal tag region of R303, facilitating crystal formation. Crystals of R303 diffracted to 1.3 Å resolution and belonged to the monoclinic space group P2₁, with unit-cell parameters a=46.4, b=31.2, c=74.8 Å, β=93.8°. The crystals exhibited a Matthews coefficient of 1.95 Å3 Da(-1) with two molecules in the asymmetric unit.

  10. The cellular response to vascular endothelial growth factors requires co-ordinated signal transduction, trafficking and proteolysis

    PubMed Central

    Smith, Gina A.; Fearnley, Gareth W.; Tomlinson, Darren C.; Harrison, Michael A.; Ponnambalam, Sreenivasan

    2015-01-01

    VEGFs (vascular endothelial growth factors) are a family of conserved disulfide-linked soluble secretory glycoproteins found in higher eukaryotes. VEGFs mediate a wide range of responses in different tissues including metabolic homoeostasis, cell proliferation, migration and tubulogenesis. Such responses are initiated by VEGF binding to soluble and membrane-bound VEGFRs (VEGF receptor tyrosine kinases) and co-receptors. VEGF and receptor splice isoform diversity further enhances complexity of membrane protein assembly and function in signal transduction pathways that control multiple cellular responses. Different signal transduction pathways are simultaneously activated by VEGFR–VEGF complexes with membrane trafficking along the endosome–lysosome network further modulating signal output from multiple enzymatic events associated with such pathways. Balancing VEGFR–VEGF signal transduction with trafficking and proteolysis is essential in controlling the intensity and duration of different intracellular signalling events. Dysfunction in VEGF-regulated signal transduction is important in chronic disease states including cancer, atherosclerosis and blindness. This family of growth factors and receptors is an important model system for understanding human disease pathology and developing new therapeutics for treating such ailments. PMID:26285805

  11. In situ proteolysis, crystallization and preliminary X-ray diffraction analysis of a VHH that binds listeria internalin B

    PubMed Central

    Huh, Ian; Gene, Robert; Kumaran, Jyothi; MacKenzie, C. Roger; Brooks, Cory L.

    2014-01-01

    The variable region of camelid heavy-chain antibodies produces the smallest known antibody fragment with antigen-binding capability (a VHH). The VHH R303 binds internalin B (InlB), a virulence factor expressed by the pathogen Listeria monocytogenes. InlB is critical for initiation of Listeria infection, as it binds a receptor (c-Met) on epithelial cells, triggering the entry of bacteria into host cells. InlB is surface-exposed and is required for virulence, hence a VHH targeting InlB has potential applications for pathogen detection or therapeutic intervention. Here, the expression, purification, crystallization and X-ray diffraction of R303 are reported. Crystals of R303 were obtained following in situ proteolysis with trypsin. Gel filtration and SDS–PAGE revealed that trypsin removed the C-terminal tag region of R303, facilitating crystal formation. Crystals of R303 diffracted to 1.3 Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 46.4, b = 31.2, c = 74.8 Å, β = 93.8°. The crystals exhibited a Matthews coefficient of 1.95 Å3 Da−1 with two molecules in the asymmetric unit. PMID:25372824

  12. Identification of a novel 4 kDa immunoglobulin-A-binding peptide obtained by the limited proteolysis of jacalin.

    PubMed

    Kabir, S; Aebersold, R; Daar, A S

    1993-02-13

    Jacalin, an IgA-binding lectin from jackfruit (Artocarpus heterophyllus) seeds, was isolated by the passage of PBS extracts of seeds over an affinity matrix containing IgA-Sepharose-4B. It was further purified by HPLC. When analyzed by SDS-PAGE under both reducing and nonreducing conditions, the native jacalin was dissociated into two subunits of 12 and 15.4 kDa. Both the subunits could bind IgA. Peptide mapping performed with radioiodinated jacalin indicated that both the subunits were susceptible to proteolysis by Staphylococcus aureus V8 proteinase. One degradation product was a small peptide of 4 kDa. This small proteolytic fragment also bound IgA. The amino-termini of the two major IgA binding subunits, 12 and 15.4 kDa, were identical. The 4 kDa IgA-binding proteolytic fragment of jacalin had a different amino-terminal sequence, suggesting that the region of jacalin which binds IgA does not remain close to the amino-terminus of the peptide.

  13. SCFCdc4 acts antagonistically to the PGC-1alpha transcriptional coactivator by targeting it for ubiquitin-mediated proteolysis.

    PubMed

    Olson, Brian L; Hock, M Benjamin; Ekholm-Reed, Susanna; Wohlschlegel, James A; Dev, Kumlesh K; Kralli, Anastasia; Reed, Steven I

    2008-01-15

    Peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator-1alpha (PGC-1alpha) is a highly regulated transcriptional coactivator that coordinates energy metabolism in mammals. Misregulation of PGC-1alpha has been implicated in the pathogenesis of several human diseases, including diabetes, obesity, and neurological disorders. We identified SCF(Cdc4) as an E3 ubiquitin ligase that regulates PGC-1alpha through ubiquitin-mediated proteolysis. PGC-1alpha contains two Cdc4 phosphodegrons that bind Cdc4 when phosphorylated by Glycogen Synthase Kinase 3beta (GSK3beta) and p38 MAPK, leading to SCF(Cdc4)-dependent ubiquitylation and proteasomal degradation of PGC-1alpha. Furthermore, SCF(Cdc4) negatively regulates PGC-1alpha-dependent transcription. We demonstrate that RNAi-mediated reduction of Cdc4 in primary neurons results in an increase of endogenous PGC-1alpha protein, while ectopic expression of Cdc4 leads to a reduction of endogenous PGC-1alpha protein. Finally, under conditions of oxidative stress in neurons, Cdc4 levels are decreased, leading to an increase in PGC-1alpha protein and PGC-1alpha-dependent transcription. These results suggest that attenuation of SCF(Cdc4)-dependent proteasomal degradation of PGC-1alpha has a role in mediating the PGC-1alpha-dependent transcriptional response to oxidative stress.

  14. Insulin-degrading enzyme in brain microvessels: proteolysis of amyloid {beta} vasculotropic variants and reduced activity in cerebral amyloid angiopathy.

    PubMed

    Morelli, Laura; Llovera, Ramiro E; Mathov, Irina; Lue, Lih-Fen; Frangione, Blas; Ghiso, Jorge; Castaño, Eduardo M

    2004-12-31

    The accumulation of amyloid beta (Abeta) in the walls of small vessels in the cerebral cortex is associated with diseases characterized by dementia or stroke. These include Alzheimer's disease, Down syndrome, and sporadic and hereditary cerebral amyloid angiopathies (CAAs) related to mutations within the Abeta sequence. A higher tendency of Abeta to aggregate, a defective clearance to the systemic circulation, and insufficient proteolytic removal have been proposed as mechanisms that lead to Abeta accumulation in the brain. By using immunoprecipitation and mass spectrometry, we show that insulin-degrading enzyme (IDE) from isolated human brain microvessels was capable of degrading (125)I-insulin and cleaved Abeta-(1-40) wild type and the genetic variants Abeta A21G (Flemish), Abeta E22Q (Dutch), and Abeta E22K (Italian) at the predicted sites. In microvessels from Alzheimer's disease cases with CAA, IDE protein levels showed a 44% increase as determined by sandwich enzyme-linked immunosorbent assay and Western blot. However, the activity of IDE upon radiolabeled insulin was significantly reduced in CAA as compared with age-matched controls. These results support the notion that a defect in Abeta proteolysis by IDE contributes to the accumulation of this peptide in the cortical microvasculature. Moreover they raise the possibility that IDE inhibition or inactivation is a pathogenic mechanism that may open novel strategies for the treatment of cerebrovascular Abeta amyloidoses. PMID:15489232

  15. SCFCdc4 acts antagonistically to the PGC-1α transcriptional coactivator by targeting it for ubiquitin-mediated proteolysis

    PubMed Central

    Olson, Brian L.; Hock, M. Benjamin; Ekholm-Reed, Susanna; Wohlschlegel, James A.; Dev, Kumlesh K.; Kralli, Anastasia; Reed, Steven I.

    2008-01-01

    Peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) is a highly regulated transcriptional coactivator that coordinates energy metabolism in mammals. Misregulation of PGC-1α has been implicated in the pathogenesis of several human diseases, including diabetes, obesity, and neurological disorders. We identified SCFCdc4 as an E3 ubiquitin ligase that regulates PGC-1α through ubiquitin-mediated proteolysis. PGC-1α contains two Cdc4 phosphodegrons that bind Cdc4 when phosphorylated by Glycogen Synthase Kinase 3β (GSK3β) and p38 MAPK, leading to SCFCdc4-dependent ubiquitylation and proteasomal degradation of PGC-1α. Furthermore, SCFCdc4 negatively regulates PGC-1α-dependent transcription. We demonstrate that RNAi-mediated reduction of Cdc4 in primary neurons results in an increase of endogenous PGC-1α protein, while ectopic expression of Cdc4 leads to a reduction of endogenous PGC-1α protein. Finally, under conditions of oxidative stress in neurons, Cdc4 levels are decreased, leading to an increase in PGC-1α protein and PGC-1α-dependent transcription. These results suggest that attenuation of SCFCdc4-dependent proteasomal degradation of PGC-1α has a role in mediating the PGC-1α-dependent transcriptional response to oxidative stress. PMID:18198341

  16. Proteolysis process in fermented sausage model systems as studied by NMR relaxometry.

    PubMed

    García García, Ana Belén; Larsen, Lotte Bach; Cambero Rodríguez, María Isabel; Cruz Díaz, Karen Paola; Bertram, Hanne Christine

    2015-03-25

    Proton NMR relaxation analyses were performed in sausage model systems (SMS) at different manufacturing times (0, 1, 3, 5, 7, and 9 days) to evaluate changes in water distribution and mobility. Three different water populations were identified, T2b (5-10 ms), T21 (30-70 ms), and T22 (100-300 ms), and the progress of ripening could be followed as a shift toward shorter relaxation times. In addition, the combined effect of adding commercial proteases (Pronase E and aspartyl proteinase) on protein breakdown and structural integrity of sausage models (SMS+P) was investigated, resulting in the formation of a more fluid and less organized meat matrix that led to changes in water populations T2b2 and T22 compared with SMS. A very different protein degradation pattern between SMS and SMS+P was observed by means of SDS-PAGE and fluorescamine assay, supporting that some degree of protein aggregation is needed for the presence of the T22 population in fermented sausages.

  17. Extracellular ATP triggers proteolysis and cytosolic Ca2+ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites

    PubMed Central

    2012-01-01

    Background Plasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host. Methods Using the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca2+ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated. Results The protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 μM) and PPADS (50 μM) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 μM), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 μM) to isolated parasites previously loaded with Fluo4/AM in a Ca2+-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 μM), TNP-ATP (50 μM) or the purinergic blockers KN-62 (10 μM) and Ip5I (10 μM). Incubating P. berghei infected cells with KN-62 (200 μM) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 μM) led to an increase in rings forms (82% ± 4, n = 11) and a decrease in trophozoite forms (18% ± 4, n = 11). Conclusions The data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway. PMID:22420332

  18. Stimulation of glioma cell motility by expression, proteolysis, and release of the L1 neural cell recognition molecule

    PubMed Central

    Yang, Muhua; Adla, Shalini; Temburni, Murali K; Patel, Vivek P; Lagow, Errin L; Brady, Owen A; Tian, Jing; Boulos, Magdy I; Galileo, Deni S

    2009-01-01

    Background Malignant glioma cells are particularly motile and can travel diffusely through the brain parenchyma, apparently without following anatomical structures to guide their migration. The neural adhesion/recognition protein L1 (L1CAM; CD171) has been implicated in contributing to stimulation of motility and metastasis of several non-neural cancer types. We explored the expression and function of L1 protein as a stimulator of glioma cell motility using human high-grade glioma surgical specimens and established rat and human glioma cell lines. Results L1 protein expression was found in 17 out of 18 human high-grade glioma surgical specimens by western blotting. L1 mRNA was found to be present in human U-87/LacZ and rat C6 and 9L glioma cell lines. The glioma cell lines were negative for surface full length L1 by flow cytometry and high resolution immunocytochemistry of live cells. However, fixed and permeablized cells exhibited positive staining as numerous intracellular puncta. Western blots of cell line extracts revealed L1 proteolysis into a large soluble ectodomain (~180 kDa) and a smaller transmembrane proteolytic fragment (~32 kDa). Exosomal vesicles released by the glioma cell lines were purified and contained both full-length L1 and the proteolyzed transmembrane fragment. Glioma cell lines expressed L1-binding αvβ5 integrin cell surface receptors. Quantitative time-lapse analyses showed that motility was reduced significantly in glioma cell lines by 1) infection with an antisense-L1 retroviral vector and 2) L1 ectodomain-binding antibodies. Conclusion Our novel results support a model of autocrine/paracrine stimulation of cell motility in glioma cells by a cleaved L1 ectodomain and/or released exosomal vesicles containing L1. This mechanism could explain the diffuse migratory behavior of high-grade glioma cancer cells within the brain. PMID:19874583

  19. HtrC is involved in proteolysis of YpeB during germination of Bacillus anthracis and Bacillus subtilis spores.

    PubMed

    Bernhards, Casey B; Chen, Yan; Toutkoushian, Hannah; Popham, David L

    2015-01-01

    Bacterial endospores can remain dormant for decades yet can respond to nutrients, germinate, and resume growth within minutes. An essential step in the germination process is degradation of the spore cortex peptidoglycan wall, and the SleB protein in Bacillus species plays a key role in this process. Stable incorporation of SleB into the spore requires the YpeB protein, and some evidence suggests that the two proteins interact within the dormant spore. Early during germination, YpeB is proteolytically processed to a stable fragment. In this work, the primary sites of YpeB cleavage were identified in Bacillus anthracis, and it was shown that the stable products are comprised of the C-terminal domain of YpeB. Modification of the predominant YpeB cleavage sites reduced proteolysis, but cleavage at other sites still resulted in loss of full-length YpeB. A B. anthracis strain lacking the HtrC protease did not generate the same stable YpeB products. In B. anthracis and Bacillus subtilis htrC mutants, YpeB was partially stabilized during germination but was still degraded at a reduced rate by other, unidentified proteases. Purified HtrC cleaved YpeB to a fragment similar to that observed in vivo, and this cleavage was stimulated by Mn(2+) or Ca(2+) ions. A lack of HtrC did not stabilize YpeB or SleB during spore formation in the absence of the partner protein, indicating other proteases are involved in their degradation during sporulation.

  20. Probing the 3-D Structure, Dynamics, and Stability of Bacterial Collagenase Collagen Binding Domain (apo- versus holo-) by Limited Proteolysis MALDI-TOF MS

    NASA Astrophysics Data System (ADS)

    Sides, Cynthia R.; Liyanage, Rohana; Lay, Jackson O.; Philominathan, Sagaya Theresa Leena; Matsushita, Osamu; Sakon, Joshua

    2012-03-01

    Pairing limited proteolysis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to probe clostridial collagenase collagen binding domain (CBD) reveals the solution dynamics and stability of the protein, as these factors are crucial to CBD effectiveness as a drug-delivery vehicle. MS analysis of proteolytic digests indicates initial cleavage sites, thereby specifying the less stable and highly accessible regions of CBD. Modulation of protein structure and stability upon metal binding is shown through MS analysis of calcium-bound and cobalt-bound CBD proteolytic digests. Previously determined X-ray crystal structures illustrate that calcium binding induces secondary structure transformation in the highly mobile N-terminal arm and increases protein stability. MS-based detection of exposed residues confirms protein flexibility, accentuates N-terminal dynamics, and demonstrates increased global protein stability exported by calcium binding. Additionally, apo- and calcium-bound CBD proteolysis sites correlate well with crystallographic B-factors, accessibility, and enzyme specificity. MS-observed cleavage sites with no clear correlations are explained either by crystal contacts of the X-ray crystal structures or by observed differences between Molecules A and B in the X-ray crystal structures. The study newly reveals the absence of the βA strand and thus the very dynamic N-terminal linker, as corroborated by the solution X-ray scattering results. Cobalt binding has a regional effect on the solution phase stability of CBD, as limited proteolysis data implies the capture of an intermediate-CBD solution structure when cobalt is bound.

  1. Effect of the beta-adrenergic agonist L644,969 on muscle growth, endogenous proteinase activities, and postmortem proteolysis in wether lambs.

    PubMed

    Koohmaraie, M; Shackelford, S D; Muggli-Cockett, N E; Stone, R T

    1991-12-01

    To examine the effect of a beta-adrenergic agonist (BAA) on muscle growth, proteinase activities, and postmortem proteolysis, 16 wether lambs were randomly assigned to receive 0 or 4 ppm of L644,969 in a completely mixed high-concentrate diet for 6 wk. Weight of the biceps femoris was 18.6% heavier in treated lambs. At 0 h after slaughter, treated lambs had higher cathepsin B (35.6%), cathepsins B + L (19.1%), calpastatin (62.8%), and m-calpain (24.6%) than control lambs, but both groups had similar mu-calpain activities. In both longissimus and biceps femoris muscles, treated lambs had higher protein and RNA and lower DNA concentrations. However, total DNA was not affected, indicating that the increase in muscle mass was probably due to muscle hypertrophy rather than to hyperplasia. The pattern of postmortem proteolysis was significantly altered by BAA feeding. In treated lambs, postmortem storage had no effect on the myofibril fragmentation index and degradation of desmin and troponin-T. These results indicate that the ability of the muscle to undergo postmortem proteolysis has been dramatically reduced with BAA feeding. Similar proteolytic systems are thought to be involved in antemortem and postmortem degradation of myofibrillar proteins, so BAA-mediated protein accretion is probably due, at least in part, to reduced protein degradation. To examine whether protein synthesis was altered with BAA feeding, the level of skeletal muscle alpha-actin mRNA was quantified. Longissimus muscle alpha-actin mRNA abundance was 30% greater in BAA-fed lambs. Collectively, these results indicate that dietary administration of BAA increases muscle mass through hypertrophy and that the increase in muscle protein accretion is due to reduced degradation and possibly to increased synthesis of muscle proteins.

  2. Proteolysis, NaOH and ultrasound-enhanced extraction of anticoagulant and antioxidant sulfated polysaccharides from the edible seaweed, Gracilaria birdiae.

    PubMed

    Fidelis, Gabriel Pereira; Camara, Rafael Barros Gomes; Queiroz, Moacir Fernandes; Santos Pereira Costa, Mariana Santana; Santos, Pablo Castro; Rocha, Hugo Alexandre Oliveira; Costa, Leandro Silva

    2014-11-13

    The sulfated polysaccharides (SP) from the edible red seaweed, Gracilaria birdiae, were obtained using five different extraction conditions: Gracilaria birdiae 1 (GB1)-water; GB1s-water/sonication; GB1sp-water/sonication/proteolysis; GB2s-NaOH/sonication; and GB2sp-NaOH/sonication/proteolysis. The yield (g) increased in the following order: GB2sp>GB1sp>GB2s>GB1s>GB1. However, the amount of SP extracted increased in a different way: GB2sp>GB1>GB1sp>GB1s>GB2s. Infrared and electrophoresis analysis showed that all conditions extracted the same SP. In addition, monosaccharide composition showed that ultrasound promotes the extraction of polysaccharides other than SP. In the prothrombin time (PT) test, which evaluates the extrinsic coagulation pathway, none of the samples showed anticoagulant activity. While in the activated partial thromboplastin time (aPTT) test, which evaluates the intrinsic coagulation pathway, all samples showed anticoagulant activity, except GB2s. The aPTT activity decreased in the order of GB1sp>GB2sp>GB1>GB1s>GB2s. The total capacity antioxidant (TCA) of the SP was also affected by extraction condition, since GB2s and GB1 showed lower activity in comparison to the other conditions. In conclusion, the conditions of SP extraction influence their biological activities and chemical composition. The data revealed that NaOH/sonication/proteolysis was the best condition to extract anticoagulant and antioxidant SPs from Gracilaria birdiae.

  3. Asn347 Glycosylation of Corticosteroid-binding Globulin Fine-tunes the Host Immune Response by Modulating Proteolysis by Pseudomonas aeruginosa and Neutrophil Elastase.

    PubMed

    Sumer-Bayraktar, Zeynep; Grant, Oliver C; Venkatakrishnan, Vignesh; Woods, Robert J; Packer, Nicolle H; Thaysen-Andersen, Morten

    2016-08-19

    Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues upon elastase-based proteolysis of the exposed reactive center loop (RCL). However, the molecular mechanisms that regulate the RCL proteolysis by co-existing host and bacterial elastases in inflamed/infected tissues remain unknown. We document that RCL-localized Asn(347) glycosylation fine-tunes the RCL cleavage rate by human neutrophil elastase (NE) and Pseudomonas aeruginosa elastase (PAE) by different mechanisms. NE- and PAE-generated fragments of native and exoglycosidase-treated blood-derived CBG of healthy individuals were monitored by gel electrophoresis and LC-MS/MS to determine the cleavage site(s) and Asn(347) glycosylation as a function of digestion time. The site-specific (Val(344)-Thr(345)) and rapid (seconds to minutes) NE-based RCL proteolysis was significantly antagonized by several volume-enhancing Asn(347) glycan features (i.e. occupancy, triantennary GlcNAc branching, and α1,6-fucosylation) and augmented by Asn(347) NeuAc-type sialylation (all p < 0.05). In contrast, the inefficient (minutes to hours) PAE-based RCL cleavage, which occurred equally well at Thr(345)-Leu(346) and Asn(347)-Leu(348), was abolished by the presence of Asn(347) glycosylation but was enhanced by sialoglycans on neighboring CBG N-sites. Molecular dynamics simulations of various Asn(347) glycoforms of uncleaved CBG indicated that multiple Asn(347) glycan features are modulating the RCL digestion efficiencies by NE/PAE. Finally, high concentrations of cortisol showed weak bacteriostatic effects toward virulent P. aeruginosa, which may explain the low RCL potency of the abundantly secreted PAE during host infection. In conclusion, site-specific CBG N-glycosylation regulates the bioavailability of cortisol in inflamed environments by fine-tuning the RCL proteolysis by endogenous and exogenous elastases. This study offers new molecular insight into host- and pathogen

  4. Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1.

    PubMed

    Gilkerson, Jonathan; Kelley, Dior R; Tam, Raymond; Estelle, Mark; Callis, Judy

    2015-06-01

    Although many ubiquitin-proteasome substrates have been characterized in plants, very little is known about the corresponding ubiquitin attachment(s) underlying regulated proteolysis. Current dogma asserts that ubiquitin is typically covalently attached to a substrate through an isopeptide bond between the ubiquitin carboxy terminus and a substrate lysyl amino group. However, nonlysine (non-Lys) ubiquitin attachment has been observed in other eukaryotes, including the N terminus, cysteine, and serine/threonine modification. Here, we investigate site(s) of ubiquitin attachment on indole-3-acetic acid1 (IAA1), a short-lived Arabidopsis (Arabidopsis thaliana) Auxin/indole-3-acetic acid (Aux/IAA) family member. Most Aux/IAA proteins function as negative regulators of auxin responses and are targeted for degradation after ubiquitination by the ubiquitin ligase SCF(TIR1/AFB) (for S-Phase Kinase-Associated Protein1, Cullin, F-box [SCF] with Transport Inhibitor Response1 [TIR1]/Auxin Signaling F-box [AFB]) by an interaction directly facilitated by auxin. Surprisingly, using a Histidine-Hemaglutinin (HIS(6x)-HA(3x)) epitope-tagged version expressed in vivo, Lys-less IAA1 was ubiquitinated and rapidly degraded in vivo. Lys-substituted versions of IAA1 localized to the nucleus as Yellow Fluorescent Protein fusions and interacted with both TIR1 and IAA7 in yeast (Saccharomyces cerevisiae) two-hybrid experiments, indicating that these proteins were functional. Ubiquitination on both HIS(6x)-HA(3x)-IAA1 and Lys-less HIS(6x)-HA(3x)-IAA1 proteins was sensitive to sodium hydroxide treatment, indicative of ubiquitin oxyester formation on serine or threonine residues. Additionally, base-resistant forms of ubiquitinated IAA1 were observed for HIS(6x)-HA(3x)-IAA1, suggesting additional lysyl-linked ubiquitin on this protein. Characterization of other Aux/IAA proteins showed that they have diverse degradation rates, adding additional complexity to auxin signaling. Altogether, these data

  5. Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1.

    PubMed

    Gilkerson, Jonathan; Kelley, Dior R; Tam, Raymond; Estelle, Mark; Callis, Judy

    2015-06-01

    Although many ubiquitin-proteasome substrates have been characterized in plants, very little is known about the corresponding ubiquitin attachment(s) underlying regulated proteolysis. Current dogma asserts that ubiquitin is typically covalently attached to a substrate through an isopeptide bond between the ubiquitin carboxy terminus and a substrate lysyl amino group. However, nonlysine (non-Lys) ubiquitin attachment has been observed in other eukaryotes, including the N terminus, cysteine, and serine/threonine modification. Here, we investigate site(s) of ubiquitin attachment on indole-3-acetic acid1 (IAA1), a short-lived Arabidopsis (Arabidopsis thaliana) Auxin/indole-3-acetic acid (Aux/IAA) family member. Most Aux/IAA proteins function as negative regulators of auxin responses and are targeted for degradation after ubiquitination by the ubiquitin ligase SCF(TIR1/AFB) (for S-Phase Kinase-Associated Protein1, Cullin, F-box [SCF] with Transport Inhibitor Response1 [TIR1]/Auxin Signaling F-box [AFB]) by an interaction directly facilitated by auxin. Surprisingly, using a Histidine-Hemaglutinin (HIS(6x)-HA(3x)) epitope-tagged version expressed in vivo, Lys-less IAA1 was ubiquitinated and rapidly degraded in vivo. Lys-substituted versions of IAA1 localized to the nucleus as Yellow Fluorescent Protein fusions and interacted with both TIR1 and IAA7 in yeast (Saccharomyces cerevisiae) two-hybrid experiments, indicating that these proteins were functional. Ubiquitination on both HIS(6x)-HA(3x)-IAA1 and Lys-less HIS(6x)-HA(3x)-IAA1 proteins was sensitive to sodium hydroxide treatment, indicative of ubiquitin oxyester formation on serine or threonine residues. Additionally, base-resistant forms of ubiquitinated IAA1 were observed for HIS(6x)-HA(3x)-IAA1, suggesting additional lysyl-linked ubiquitin on this protein. Characterization of other Aux/IAA proteins showed that they have diverse degradation rates, adding additional complexity to auxin signaling. Altogether, these data

  6. Effects of fibre type and structure of longissimus lumborum (Ll), biceps femoris (Bf) and semimembranosus (Sm) deer muscles salting with different Nacl addition on proteolysis index and texture of dry-cured meats.

    PubMed

    Żochowska-Kujawska, J

    2016-11-01

    The aim of the present study was to describe the effect of fibre type and structure as well as NaCl level on the proteolysis index and texture parameters observed in dry-cured meats produced from individual deer muscles. The biceps femoris, semimembranosus and longissimus lumborum muscles were cut from deer main elements, shaped into blocks by trimming off the edges, cured by adding 4, 6 and 8% of salt (w/w) and dried in a ripening chamber for 29days. The results indicated that deer dry-cured muscles with higher percentage of red fibres (type I) showed higher texture parameters, proteolysis index as well as lower moisture losses than muscles with higher amount of white fibres (type IIB). Dry-cured deer muscles with lower NaCl content showed higher values of proteolysis index and lower hardness, cohesiveness, springiness, and chewiness, as well as lower changes in structure elements. PMID:27442183

  7. A helix-to-coil transition at the ε-cut site in the transmembrane dimer of the amyloid precursor protein is required for proteolysis

    PubMed Central

    Sato, Takeshi; Tang, Tzu-chun; Reubins, Gabriella; Fei, Jeffrey Z.; Fujimoto, Taiki; Kienlen-Campard, Pascal; Constantinescu, Stefan N.; Octave, Jean-Noel; Aimoto, Saburo; Smith, Steven O.

    2009-01-01

    Processing of amyloid precursor protein (APP) by γ-secretase is the last step in the formation of the Aβ peptides associated Alzheimer's disease. Solid-state NMR spectroscopy is used to establish the structural features of the transmembrane (TM) and juxtamembrane (JM) domains of APP that facilitate proteolysis. Using peptides corresponding to the APP TM and JM regions (residues 618–660), we show that the TM domain forms an α-helical homodimer mediated by consecutive GxxxG motifs. We find that the APP TM helix is disrupted at the intracellular membrane boundary near the ε-cleavage site. This helix-to-coil transition is required for γ-secretase processing; mutations that extend the TM α-helix inhibit ε cleavage, leading to a low production of Aβ peptides and an accumulation of the α- and β-C-terminal fragments. Our data support a progressive cleavage mechanism for APP proteolysis that depends on the helix-to-coil transition at the TM-JM boundary and unraveling of the TM α-helix. PMID:19164538

  8. Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis.

    PubMed Central

    Hershko, A; Ciechanover, A; Heller, H; Haas, A L; Rose, I A

    1980-01-01

    The heat-stable polypeptide ATP-dependent proteolysis factor 1 (APF-1) of the reticulocyte proteolytic system forms covalent compounds with proteins in an ATP-requiring reaction. APF-1 and lysozyme, a good substrate for ATP-dependent proteolysis, form multiple conjugates, as was shown by comigration of label from each upon gel electrophoresis. Multiple bands were also seen with other substrates of the ATP-dependent proteolytic system, such as globin or alpha-lactalbumin. Analysis of the ratio of APF-1 to lysozyme radioactivities and of the molecular weights of the bands indicated that they consist of increasing numbers of the APF-1 polypeptide bound to one molecule of lysozyme. The covalent linkage is probably of an isopeptide nature, because it is stable to hydroxylamine and alkali, and polylysine is able to give conjugates of APF-1. Removal of ATP after formation of the 125I-labeled APF-1 conjugates with endogenous proteins caused the regeneration of APF-1, indicating presence of an amidase. This reaction is thought to compete with proteases that may act on APF-1-protein conjugates, especially those containing several APF-1 ligands. A sequence of reactions in which the linkage of APF-1 to the substrate is followed by the proteolytic breakdown of the substrate is proposed to explain the role of ATP. Images PMID:6990414

  9. Cell motility and ECM proteolysis regulate tumor growth and tumor relapse by altering the fraction of cancer stem cells and their spatial scattering

    NASA Astrophysics Data System (ADS)

    Kumar, Sandeep; Kulkarni, Rahul; Sen, Shamik

    2016-06-01

    Tumors consist of multiple cell sub-populations including cancer stem cells (CSCs), transiently amplifying cells and terminally differentiated cells (TDCs), with the CSC fraction dictating the aggressiveness of the tumor and drug sensitivity. In epithelial cancers, tumor growth is influenced greatly by properties of the extracellular matrix (ECM), with cancer progression associated with an increase in ECM density. However, the extent to which increased ECM confinement induced by an increase in ECM density influences tumor growth and post treatment relapse dynamics remains incompletely understood. In this study, we use a cellular automata-based discrete modeling approach to study the collective influence of ECM density, cell motility and ECM proteolysis on tumor growth, tumor heterogeneity, and tumor relapse after drug treatment. We show that while increased confinement suppresses tumor growth and the spatial scattering of CSCs, this effect can be reversed when cells become more motile and proteolytically active. Our results further suggest that, in addition to the absolute number of CSCs, their spatial positioning also plays an important role in driving tumor growth. In a nutshell, our study suggests that, in confined environments, cell motility and ECM proteolysis are two key factors that regulate tumor growth and tumor relapse dynamics by altering the number and spatial distribution of CSCs.

  10. Decreased rate of protein synthesis, caspase-3 activity, and ubiquitin-proteasome proteolysis in soleus muscles from growing rats fed a low-protein, high-carbohydrate diet.

    PubMed

    Batistela, Emanuele; Pereira, Mayara Peron; Siqueira, Juliany Torres; Paula-Gomes, Silvia; Zanon, Neusa Maria; Oliveira, Eduardo Brandt; Navegantes, Luiz Carlos Carvalho; Kettelhut, Isis C; Andrade, Claudia Marlise Balbinotti; Kawashita, Nair Honda; Baviera, Amanda Martins

    2014-06-01

    The aim of this study was to investigate the changes in the rates of both protein synthesis and breakdown, and the activation of intracellular effectors that control these processes in soleus muscles from growing rats fed a low-protein, high-carbohydrate (LPHC) diet for 15 days. The mass and the protein content, as well as the rate of protein synthesis, were decreased in the soleus from LPHC-fed rats. The availability of amino acids was diminished, since the levels of various essential amino acids were decreased in the plasma of LPHC-fed rats. Overall rate of proteolysis was also decreased, explained by reductions in the mRNA levels of atrogin-1 and MuRF-1, ubiquitin conjugates, proteasome activity, and in the activity of caspase-3. Soleus muscles from LPHC-fed rats showed increased insulin sensitivity, with increased levels of insulin receptor and phosphorylation levels of AKT, which probably explains the inhibition of both the caspase-3 activity and the ubiquitin-proteasome system. The fall of muscle proteolysis seems to represent an adaptive response that contributes to spare proteins in a condition of diminished availability of dietary amino acids. Furthermore, the decreased rate of protein synthesis may be the driving factor to the lower muscle mass gain in growing rats fed the LPHC diet.

  11. α2β1 integrin affects metastatic potential of ovarian carcinoma spheroids by supporting disaggregation and proteolysis

    PubMed Central

    Shield, Kristy; Riley, Clyde; Quinn, Michael A; Rice, Gregory E; Ackland, Margaret L; Ahmed, Nuzhat

    2007-01-01

    activation of MMP's observed in monolayer cells. Flow cytometric analysis demonstrated enhanced expression of α2 and diminution of α6 integrin subunits in spheroids versus monolayer cells. No change in the expression of α3, αv and β1 subunits was evident. Conversely, except for αv integrin, a 1.5–7.5-fold decrease in α2, α3, α6 and β1 integrin subunit expression was observed in IOSE29 cells within 2 days. Neutralizing antibodies against α2, β1 subunits and α2β1 integrin inhibited disaggregation as well as activation of MMPs in spheroids. Conclusion Our results suggest that enhanced expression of α2β1 integrin may influence spheroid disaggregation and proteolysis responsible for the peritoneal dissemination of ovarian carcinoma. This may indicate a new therapeutic target for the suppression of the peritoneal metastasis associated with advanced ovarian carcinomas. PMID:17567918

  12. Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation

    PubMed Central

    2013-01-01

    Background Cell division is positively regulated by cyclin-dependent kinases (CDKs) partnered with cyclins and negatively regulated by CDK inhibitors. In the frog, Xenopus laevis, three types of CDK inhibitors have been described: p27Xic1 (Xic1) which shares sequence homology with both p21Cip1 and p27Kip1 from mammals, p16Xic2 (Xic2) which shares sequence homology with p21Cip1, and p17Xic3 (Xic3) which shares sequence homology with p27Kip1. While past studies have demonstrated that during DNA polymerase switching, Xic1 is targeted for protein turnover dependent upon DNA, Proliferating Cell Nuclear Antigen (PCNA), and the ubiquitin ligase CRL4Cdt2, little is known about the processes that regulate Xic2 or Xic3. Methods We used the Xenopus interphase egg extract as a model system to examine the regulation of Xic2 by proteolysis and phosphorylation. Results Our studies indicated that following primer synthesis during the initiation of DNA replication, Xic2 is targeted for DNA- and PCNA-dependent ubiquitin-mediated proteolysis and that Cdt2 can promote Xic2 turnover. Additionally, during interphase, Xic2 is phosphorylated by CDK2 at Ser-98 and Ser-131 in a DNA-independent manner, inhibiting Xic2 turnover. In the presence of double-stranded DNA ends, Xic2 is also phosphorylated at Ser-78 and Ser-81 by a caffeine-sensitive kinase, but this phosphorylation does not alter Xic2 turnover. Conversely, in the presence or absence of DNA, Xic3 was stable in the Xenopus interphase egg extract and did not exhibit a shift indicative of phosphorylation. Conclusions During interphase, Xic2 is targeted for DNA- and PCNA-dependent proteolysis that is negatively regulated by CDK2 phosphorylation. During a response to DNA damage, Xic2 may be alternatively regulated by phosphorylation by a caffeine-sensitive kinase. Our studies suggest that the three types of Xenopus CDK inhibitors, Xic1, Xic2, and Xic3 appear to be uniquely regulated which may reflect their specialized roles during cell

  13. Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis Using Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array*

    PubMed Central

    Shinkai-Ouchi, Fumiko; Koyama, Suguru; Ono, Yasuko; Hata, Shoji; Ojima, Koichi; Shindo, Mayumi; duVerle, David; Ueno, Mika; Kitamura, Fujiko; Doi, Naoko; Takigawa, Ichigaku; Mamitsuka, Hiroshi; Sorimachi, Hiroyuki

    2016-01-01

    Calpains are intracellular Ca2+-regulated cysteine proteases that are essential for various cellular functions. Mammalian conventional calpains (calpain-1 and calpain-2) modulate the structure and function of their substrates by limited proteolysis. Thus, it is critically important to determine the site(s) in proteins at which calpains cleave. However, the calpains' substrate specificity remains unclear, because the amino acid (aa) sequences around their cleavage sites are very diverse. To clarify calpains' substrate specificities, 84 20-mer oligopeptides, corresponding to P10-P10′ of reported cleavage site sequences, were proteolyzed by calpains, and the catalytic efficiencies (kcat/Km) were globally determined by LC/MS. This analysis revealed 483 cleavage site sequences, including 360 novel ones. The kcat/Kms for 119 sites ranged from 12.5–1,710 M−1s−1. Although most sites were cleaved by both calpain-1 and −2 with a similar kcat/Km, sequence comparisons revealed distinct aa preferences at P9-P7/P2/P5′. The aa compositions of the novel sites were not statistically different from those of previously reported sites as a whole, suggesting calpains have a strict implicit rule for sequence specificity, and that the limited proteolysis of intact substrates is because of substrates' higher-order structures. Cleavage position frequencies indicated that longer sequences N-terminal to the cleavage site (P-sites) were preferred for proteolysis over C-terminal (P′-sites). Quantitative structure-activity relationship (QSAR) analyses using partial least-squares regression and >1,300 aa descriptors achieved kcat/Km prediction with r = 0.834, and binary-QSAR modeling attained an 87.5% positive prediction value for 132 reported calpain cleavage sites independent of our model construction. These results outperformed previous calpain cleavage predictors, and revealed the importance of the P2, P3′, and P4′ sites, and P1-P2 cooperativity. Furthermore, using our

  14. Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis Using Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array.

    PubMed

    Shinkai-Ouchi, Fumiko; Koyama, Suguru; Ono, Yasuko; Hata, Shoji; Ojima, Koichi; Shindo, Mayumi; duVerle, David; Ueno, Mika; Kitamura, Fujiko; Doi, Naoko; Takigawa, Ichigaku; Mamitsuka, Hiroshi; Sorimachi, Hiroyuki

    2016-04-01

    Calpains are intracellular Ca(2+)-regulated cysteine proteases that are essential for various cellular functions. Mammalian conventional calpains (calpain-1 and calpain-2) modulate the structure and function of their substrates by limited proteolysis. Thus, it is critically important to determine the site(s) in proteins at which calpains cleave. However, the calpains' substrate specificity remains unclear, because the amino acid (aa) sequences around their cleavage sites are very diverse. To clarify calpains' substrate specificities, 84 20-mer oligopeptides, corresponding to P10-P10' of reported cleavage site sequences, were proteolyzed by calpains, and the catalytic efficiencies (kcat/Km) were globally determined by LC/MS. This analysis revealed 483 cleavage site sequences, including 360 novel ones. Thekcat/Kms for 119 sites ranged from 12.5-1,710 M(-1)s(-1) Although most sites were cleaved by both calpain-1 and -2 with a similarkcat/Km, sequence comparisons revealed distinct aa preferences at P9-P7/P2/P5'. The aa compositions of the novel sites were not statistically different from those of previously reported sites as a whole, suggesting calpains have a strict implicit rule for sequence specificity, and that the limited proteolysis of intact substrates is because of substrates' higher-order structures. Cleavage position frequencies indicated that longer sequences N-terminal to the cleavage site (P-sites) were preferred for proteolysis over C-terminal (P'-sites). Quantitative structure-activity relationship (QSAR) analyses using partial least-squares regression and >1,300 aa descriptors achievedkcat/Kmprediction withr= 0.834, and binary-QSAR modeling attained an 87.5% positive prediction value for 132 reported calpain cleavage sites independent of our model construction. These results outperformed previous calpain cleavage predictors, and revealed the importance of the P2, P3', and P4' sites, and P1-P2 cooperativity. Furthermore, using our binary-QSAR model

  15. A Novel Phosphorylation Site, Serine 199, in the C-Terminus of Cardiac Troponin I Regulates Calcium Sensitivity and Susceptibility to Calpain-Induced Proteolysis

    PubMed Central

    Wijnker, Paul J.M.; Li, Yuejin; Zhang, Pingbo; Foster, D. Brian; dos Remedios, Cris; Van Eyk, Jennifer E.; Stienen, Ger J.M.; Murphy, Anne M.; van der Velden, Jolanda

    2015-01-01

    Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium. The functional consequences of cTnI-Ser199 phosphorylation in the heart are unknown. Therefore, we investigated the impact of phosphorylation of cTnI-Ser199 on myofilament function in human cardiac tissue and the susceptibility of cTnI to proteolysis. cTnI-Ser199 was replaced by aspartic acid (199D) or alanine (199A) to mimic phosphorylation and dephosphorylation, respectively, with recombinant wild-type (Wt) cTn as a negative control. Force development was measured at various [Ca2+] and at sarcomere lengths of 1.8 and 2.2 μm in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca2+-sensitivity (pCa50) at 2.2 μm was significantly higher in 199D (pCa50=5.79±0.01) compared to 199A (pCa50=5.65±0.01) and Wt (pCa50=5.66±0.02) at ~63% cTn exchange. Myofilament Ca2+-sensitivity was significantly higher even with only 5.9±2.5% 199D exchange compared to 199A, and saturated at 12.3±2.6% 199D exchange. Ser199 pseudo-phosphorylation decreased cTnI binding to both actin and actin-tropomyosin. Moreover, altered susceptibility of cTnI to proteolysis by calpain I was found when Ser199 was pseudo-phosphorylated. Our data demonstrate that low levels of cTnI-Ser199 pseudo-phosphorylation (~6%) increase myofilament Ca2+-sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudo-phosphorylation or mutation regulates calpain I mediated proteolysis of cTnI. PMID:25771144

  16. Conventional protein kinase Cβ-mediated phosphorylation inhibits collapsin response-mediated protein 2 proteolysis and alleviates ischemic injury in cultured cortical neurons and ischemic stroke-induced mice.

    PubMed

    Yang, Xuan; Zhang, Xinxin; Li, Yun; Han, Song; Howells, David W; Li, Shujuan; Li, Junfa

    2016-05-01

    We previously reported that conventional protein kinase C (cPKC)β participated in hypoxic preconditioning-induced neuroprotection against cerebral ischemic injury, and collapsin response-mediated protein 2 (CRMP2) was identified as a cPKCβ interacting protein. In this study, we explored the regulation of CRMP2 phosphorylation and proteolysis by cPKCβ, and their role in ischemic injury of oxygen-glucose deprivation (OGD)-treated cortical neurons and brains of mice with middle cerebral artery occlusion-induced ischemic stroke. The results demonstrated that cPKCβ-mediated CRMP2 phosphorylation via the cPKCβ-selective activator 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and inhibition of calpain-mediated CRMP2 proteolysis by calpeptin and a fusing peptide containing TAT peptide and the calpain cleavage site of CRMP2 (TAT-CRMP2) protected neurons against OGD-induced cell death through inhibiting CRMP2 proteolysis in cultured cortical neurons. The OGD-induced nuclear translocation of the CRMP2 breakdown product was inhibited by DOPPA, calpeptin, and TAT-CRMP2 in cortical neurons. In addition, both cPKCβ activation and CRMP2 proteolysis inhibition by hypoxic preconditioning and intracerebroventricular injections of DOPPA, calpeptin, and TAT-CRMP2 improved the neurological deficit in addition to reducing the infarct volume and proportions of cells with pyknotic nuclei in the peri-infact region of mice with ischemic stroke. These results suggested that cPKCβ modulates CRMP2 phosphorylation and proteolysis, and cPKCβ activation alleviates ischemic injury in the cultured cortical neurons and brains of mice with ischemic stroke through inhibiting CRMP2 proteolysis by phosphorylation. Focal cerebral ischemia induces a large flux of Ca(2+) to activate calpain which cleaves collapsin response mediator (CRMP) 2 into breakdown product (BDP). Inhibition of CRMP2 cleavage by calpeptin and TAT-CRMP2 alleviates ischemic injury. Conventional protein kinase C (c

  17. Lipolysis, proteolysis and sensory characteristics of a Spanish fermented dry-cured meat product (salchichón) with oregano essential oil used as surface mold inhibitor.

    PubMed

    Martín-Sánchez, Ana María; Chaves-López, Clemencia; Sendra, Esther; Sayas, Estrella; Fenández-López, Juana; Pérez-Álvarez, José Ángel

    2011-09-01

    The superficial antifungal activity of oregano essential oil (OEO) in Spanish fermented dry-cured sausages ("salchichón"), and its effects on lipolysis, proteolysis and sensory characteristics were evaluated. The surface application of OEO reduced mold contamination on the surface, without significantly affecting the drying process. To evaluate the intensity of lipolysis during the ripening process, the profile and content of free fatty acids were determined. The addition of OEO led to a higher amount of unsaturated fatty acids, but lipolysis was not greatly affected. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which was used to qualitatively assess the proteolytic changes in the sarcoplasmic and myofibrillar proteins during the process, pointed to very similar patterns in all the sausages. OEO did not significantly affect the sensory properties, but increased hardness, resulting in a better texture. Therefore, a shorter ripening time may be necessary for sausages treated with OEO. PMID:21497448

  18. Lipolysis, proteolysis and sensory characteristics of a Spanish fermented dry-cured meat product (salchichón) with oregano essential oil used as surface mold inhibitor.

    PubMed

    Martín-Sánchez, Ana María; Chaves-López, Clemencia; Sendra, Esther; Sayas, Estrella; Fenández-López, Juana; Pérez-Álvarez, José Ángel

    2011-09-01

    The superficial antifungal activity of oregano essential oil (OEO) in Spanish fermented dry-cured sausages ("salchichón"), and its effects on lipolysis, proteolysis and sensory characteristics were evaluated. The surface application of OEO reduced mold contamination on the surface, without significantly affecting the drying process. To evaluate the intensity of lipolysis during the ripening process, the profile and content of free fatty acids were determined. The addition of OEO led to a higher amount of unsaturated fatty acids, but lipolysis was not greatly affected. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which was used to qualitatively assess the proteolytic changes in the sarcoplasmic and myofibrillar proteins during the process, pointed to very similar patterns in all the sausages. OEO did not significantly affect the sensory properties, but increased hardness, resulting in a better texture. Therefore, a shorter ripening time may be necessary for sausages treated with OEO.

  19. A single amino acid of a Salmonella virulence protein contributes to pathogenicity by protecting from the FtsH-mediated proteolysis.

    PubMed

    Choi, Eunna; Kwon, Kyoung; Lee, Eun-Jin

    2015-05-22

    FtsH is a membrane-bound ATP-dependent protease in bacteria that is critical for degrading membrane proteins. The MgtC virulence protein from Salmonella enterica is located at the inner membrane and required for survival inside macrophages. Here we report that a single substitution at tryptophan 226 of the MgtC protein to alanine promotes the FtsH-mediated proteolysis. The Trp residue is located at the very C-terminus of the cytoplasmic domain of the MgtC protein and conserved only in intracellular pathogens surviving within a macrophage phagosome, suggesting that Salmonella may acquire the tryptophan residue to prevent MgtC degradation by the FtsH protease. Moreover, the reduced proteolytic activity of the FtsH protease during infection further increases MgtC production, promoting Salmonella's pathogenicity inside phagocytic cells.

  20. Reverse transcriptase of RNA tumor viruses. V. In vitro proteolysis of reverse transcriptase from avian myeloblastosis virus and isolation of a polypeptide manifesting only RNase H activity.

    PubMed Central

    Lai, M H; Verma, I M

    1978-01-01

    Purified avian myeloblastosis virus reverse transcriptase contains two subunits that are structurally related. The large subunit, beta (molecular weight, 95,000), was converted in vitro by chymotrypsin into a polypeptide of molecular weight 63,000. This polypeptide was indistinguishable from the small subunit, alpha (molecular weight, 65,000), in its chromatographic behavior on the phosphocellulose column and its tryptic peptide composition. During this proteolytic conversion, a polypeptide of molecular weight 32,000 (fragment B) was obtained. It was composed of tryptic peptides unique to beta and appeared to be derived from the portion of the beta subunit that was cleaved off during the conversion of beta into alpha. Upon continued proteolysis, a smaller polypeptide of molecular weight 24,000 (fragment A) was generated. This polypeptide manifested only RNase H activity and shared common amino acid sequences with beta and alpha subunits. Fragment A did not share any amino acid sequence homology with fragment B. Images PMID:75271

  1. Influence of pig rennet on proteolysis, organic acids content and microbiota of Pecorino di Farindola, a traditional Italian ewe's raw milk cheese.

    PubMed

    Tofalo, Rosanna; Schirone, Maria; Fasoli, Giuseppe; Perpetuini, Giorgia; Patrignani, Francesca; Manetta, Anna Chiara; Lanciotti, Rosalba; Corsetti, Aldo; Martino, Giuseppe; Suzzi, Giovanna

    2015-05-15

    The use of pig rennet is very ancient and in Italy is only applied in the manufacture of Pecorino di Farindola cheese. In order to evaluate the key role of this rennet in the establishment of peculiar features of Pecorino di Farindola, cheeses made from raw ewes' milk using calf (A) and kid (B) rennets were compared to those produced with pig (C) rennet. The use of pig rennet for Pecorino di Farindola cheese making confers physico-chemical and proteolytic characteristics that differentiate it from cheeses produced with other coagulants. However, no microbiological differences were observed. Chesses made with pig and kid rennets were characterised by higher proteolysis after 7 days of ripening. The content of isovaleric and propionic acids at the end of ripening was correlated with the presence of propionibacteria.

  2. Limited proteolysis and sequence analysis of the 2-oxo acid dehydrogenase complexes from Escherichia coli. Cleavage sites and domains in the dihydrolipoamide acyltransferase components.

    PubMed Central

    Packman, L C; Perham, R N

    1987-01-01

    The structures of the dihydrolipoamide acyltransferase (E2) components of the 2-oxo acid dehydrogenase complexes from Escherichia coli were investigated by limited proteolysis. Trypsin and Staphylococcus aureus V8 proteinase were used to excise the three lipoyl domains from the E2p component of the pyruvate dehydrogenase complex and the single lipoyl domain from the E2o component of the 2-oxoglutarate dehydrogenase complex. The principal sites of action of these enzymes on each E2 chain were determined by sequence analysis of the isolated lipoyl fragments and of the truncated E2p and E2o chains. Each of the numerous cleavage sites (12 in E2p, six in E2o) fell within similar segments of the E2 chains, namely stretches of polypeptide rich in alanine, proline and/or charged amino acids. These regions are clearly accessible to proteinases of Mr 24,000-28,000 and, on the basis of n.m.r. spectroscopy, some of them have previously been implicated in facilitating domain movements by virtue of their conformational flexibility. The limited proteolysis data suggest that E2p and E2o possess closer architectural similarities than would be predicted from inspection of their amino acid sequences. As a result of this work, an error was detected in the sequence of E2o inferred from the previously published sequence of the encoding gene, sucB. The relevant peptides from E2o were purified and sequenced by direct means; an amended sequence is presented. Images Fig. 1. Fig. 2. PMID:3297046

  3. Arsenite reduces insulin secretion in rat pancreatic {beta}-cells by decreasing the calcium-dependent calpain-10 proteolysis of SNAP-25

    SciTech Connect

    Diaz-Villasenor, Andrea; Burns, Anna L.; Salazar, Ana Maria; Sordo, Monserrat; Hiriart, Marcia; Cebrian, Mariano E.; Ostrosky-Wegman, Patricia

    2008-09-15

    An increase in the prevalence of type 2 diabetes has been consistently observed among residents of high arsenic exposure areas. We have previously shown that in rat pancreatic {beta}-cells, low arsenite doses impair the secretion of insulin without altering its synthesis. To further study the mechanism by which arsenite reduces insulin secretion, we evaluated the effects of arsenite on the calcium-calpain pathway that triggers insulin exocytosis in RINm5F cells. Cell cycle and proliferation analysis were also performed to complement the characterization. Free [Ca{sup 2+}]i oscillations needed for glucose-stimulated insulin secretion were abated in the presence of subchronic low arsenite doses (0.5-2 {mu}M). The global activity of calpains increased with 2 {mu}M arsenite. However, during the secretion of insulin stimulated with glucose (15.6 mM), 1 {mu}M arsenite decreased the activity of calpain-10, measured as SNAP-25 proteolysis. Both proteins are needed to fuse insulin granules with the membrane to produce insulin exocytosis. Arsenite also induced a slowdown in the {beta} cell line proliferation in a dose-dependent manner, reflected by a reduction of dividing cells and in their arrest in G2/M. Data obtained showed that one of the mechanisms by which arsenite impairs insulin secretion is by decreasing the oscillations of free [Ca{sup 2+}]i, thus reducing calcium-dependent calpain-10 partial proteolysis of SNAP-25. The effects in cell division and proliferation observed with arsenite exposure can be an indirect consequence of the decrease in insulin secretion.

  4. Resistance to UV-induced apoptosis by β-HPV5 E6 involves targeting of activated BAK for proteolysis by recruitment of the HERC1 ubiquitin ligase.

    PubMed

    Holloway, Amy; Simmonds, Mark; Azad, Abul; Fox, Joanna L; Storey, Alan

    2015-06-15

    UV exposure is the main etiological agent in the development of non-melanoma skin cancer (NMSC), but mounting evidence suggests a co-factorial role for β-genus HPV types early in tumor initiation or progression. UV damage initiates an apoptotic response, driven at the mitochondrial level by BCL-2 family proteins, that eliminates damaged cells that may accumulate deleterious mutations and acquire tumorigenic properties. BAK is a pro-apoptotic BCL-2 protein that functions ultimately to form pores that permeabilize the mitochondrial outer membrane, thereby committing a cell to death, a process involving changes in BAK phosphorylation and conformation. The E6 protein of β-type HPV5 signals BAK for proteasomal degradation, a function that confers protection from UV-induced apoptosis. We find that HPV5 E6 does not constitutively target BAK for proteolysis, but targets the latter stages of BAK activation, following changes in phosphorylation and conformation. A mutational analysis identified the lysine residue on BAK required for proteolysis, and a functional siRNA screen identified the HECT domain E3 ubiquitin ligase HERC1 as being required for E6-mediated BAK degradation. We show that HERC1 interacts with BAK in E6-expressing cells that have been damaged by UV, and provide evidence that the interaction of HERC1 with BAK requires access to a hydrophobic surface on BAK that binds BH3 domains of BCL-2 proteins. We also show that HERC1 contains a putative BH3 domain that can bind to BAK. These findings reveal a specific and unique mechanism used by the HPV5 E6 protein to target BAK.

  5. Regulated intramembrane proteolysis of the virulence activator TcpP in Vibrio cholerae is initiated by the tail-specific protease (Tsp).

    PubMed

    Teoh, Wei Ping; Matson, Jyl S; DiRita, Victor J

    2015-09-01

    Vibrio cholerae uses a multiprotein transcriptional regulatory cascade to control expression of virulence factors cholera toxin and toxin-co-regulated pilus. Two proteins in this cascade are ToxR and TcpP - unusual membrane-localized transcription factors with relatively undefined periplasmic domains and transcription activator cytoplasmic domains. TcpP and ToxR function with each other and two other membrane-localized proteins, TcpH and ToxS, to activate transcription of toxT, encoding the direct activator of toxin and pilus genes. Under some conditions, TcpP is degraded in a two-step proteolytic pathway known as regulated intramembrane proteolysis (RIP), thereby inactivating the cascade. The second step in this proteolytic pathway involves the zinc metalloprotease YaeL; V. cholerae cells lacking YaeL accumulate a truncated yet active form of TcpP termed TcpP*. We hypothesized that a protease acting prior to YaeL degrades TcpP to TcpP*, which is the substrate of YaeL. In this study, we demonstrate that a C-terminal protease called Tsp degrades TcpP to form TcpP*, which is then acted upon by YaeL. We present evidence that TcpH and Tsp serve to protect full-length TcpP from spurious proteolysis by YaeL. Cleavage by Tsp occurs in the periplasmic domain of TcpP and requires residues TcpPA172 and TcpPI174 for wild-type activity.

  6. Increase in ubiquitin-protein conjugates concomitant with the increase in proteolysis in rat skeletal muscle during starvation and atrophy denervation

    NASA Technical Reports Server (NTRS)

    Wing, S. S.; Haas, A. L.; Goldberg, A. L.

    1995-01-01

    The rapid loss of skeletal-muscle protein during starvation and after denervation occurs primarily through increased rates of protein breakdown and activation of a non-lysosomal ATP-dependent proteolytic process. To investigate whether protein flux through the ubiquitin (Ub)-proteasome pathway is enhanced, as was suggested by related studies, we measured, using specific polyclonal antibodies, the levels of Ub-conjugated proteins in normal and atrophying muscles. The content of these critical intermediates had increased 50-250% after food deprivation in the extensor digitorum longus and soleus muscles 2 days after denervation. Like rates of proteolysis, the amount of Ub-protein conjugates and the fraction of Ub conjugated to proteins increased progressively during food deprivation and returned to normal within 1 day of refeeding. During starvation, muscles of adrenalectomized rats failed to increase protein breakdown, and they showed 50% lower levels of Ub-protein conjugates than those of starved control animals. The changes in the pools of Ub-conjugated proteins (the substrates for the 26S proteasome) thus coincided with and can account for the alterations in overall proteolysis. In this pathway, large multiubiquitinated proteins are preferentially degraded, and the Ub-protein conjugates that accumulated in atrophying muscles were of high molecular mass (> 100 kDa). When innervated and denervated gastrocnemius muscles were fractionated, a significant increase in ubiquitinated proteins was found in the myofibrillar fraction, the proteins of which are preferentially degraded on denervation, but not in the soluble fraction. Thus activation of this proteolytic pathway in atrophying muscles probably occurs initially by increasing Ub conjugation to cell proteins. The resulting accumulation of Ub-protein conjugates suggests that their degradation by the 26S proteasome complex subsequently becomes rate-limiting in these catabolic states.

  7. Lipolysis, proteolysis and formation of volatile components during ripening of a fermented sausage with Pediococcus pentosaceus and Staphylococcus xylosus as starter cultures.

    PubMed

    Johansson, G; Berdagué, J L; Larsson, M; Tran, N; Borch, E

    1994-01-01

    Bacterial growth, formation of acids, lipolysis, proteolysis, fat oxidation, formation of volatile compounds and flavour characteristics were followed during ripening and storage of a fermented sausage. The starter culture used was composed of Pediococcus pentosaceus and Staphylococcus xylosus. The number of Pediococcus sp. increased by 1.5 log cfu/g during the first day of processing and remained constant at this level for 3 weeks. The corresponding initial increase in the numbers of Staphylococcus sp., 0.4 log cfu/g, was followed by a rapid decrease in the viable numbers. Lactic acid, mainly d-lactic acid, and acetic acid were formed during ripening. The triglycerides were hydrolysed to 1,2-diglycerides and free fatty acids at the beginning of ripening, followed by the formation of 1,3-diglycerides and monoglycerides, indicating lipolytic activity. Moreover, the nonprotein nitrogen increased during ripening as a result of the proteolytic activity. Most of the changes with respect to pH, formation of d-lactic acid, acetic acid, peroxides and flavour development occurred during the initial 3 days of ripening, when growth of Pediococcus sp. and Staphylococcus sp. occurred. Lipolysis as well as proteolysis continued after this initial period. The volatile compounds identified belonged to several chemical families, viz. aliphatic and aromatic hydrocarbons, aldehydes, ketones, alcohols, phenols, carboxylic acids, esters, nitrogen compounds, sulphur compounds, chloride compounds, terpenes and furans. Many of the volatile compounds probably originated from smoke and seasoning (onion/garlic and pepper), while others were a result of the activities of muscle enzymes and bacteria. PMID:22059658

  8. Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system

    SciTech Connect

    Johnson, Steven; Roversi, Pietro; Espina, Marianela; Deane, Janet E.; Birket, Susan; Picking, William D.; Blocker, Ariel; Picking, Wendy L.; Lea, Susan M.

    2006-09-01

    IpaD, the putative needle-tip protein of the S. flexneri type III secretion system, has been crystallized in a variety of crystal forms using in-drop proteolysis. Native and selenomethionine-labelled data collection and preliminary analyses are reported. IpaD, the putative needle-tip protein of the Shigella flexneri type III secretion system, has been overexpressed and purified. Crystals were grown of the native protein in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 Å, and data were collected to 2.9 Å resolution. Analysis of the native Patterson map revealed a peak at 50% of the origin on the Harker section v = 0.5, suggesting twofold non-crystallographic symmetry parallel to the b crystallographic axis. As attempts to derivatize or grow selenomethionine-labelled protein crystals failed, in-drop proteolysis was used to produce new crystal forms. A trace amount of subtilisin Carlsberg was added to IpaD before sparse-matrix screening, resulting in the production of several new crystal forms. This approach produced SeMet-labelled crystals and diffraction data were collected to 3.2 Å resolution. The SeMet crystals belong to space group C2, with unit-cell parameters a = 139.4, b = 45.0, c = 99.5 Å, β = 107.9°. An anomalous difference Patterson map revealed peaks on the Harker section v = 0, while the self-rotation function indicates the presence of a twofold noncrystallographic symmetry axis, which is consistent with two molecules per asymmetric unit.

  9. Study of the chemical composition, proteolysis, volatile compounds, and textural properties of industrial and traditional Beaten (Bieno sirenje) ewe milk cheese.

    PubMed

    Sulejmani, E; Hayaloglu, A A; Rafajlovska, V

    2014-03-01

    The objective of this study was to determine the gross composition, proteolysis, and volatile and texture profiles during ripening of industrial (IND) and traditional (TRD) Beaten (Bieno sirenje) cheeses made by using ewe milk. In the course of the analyses, statistical differences were determined in some physicochemical parameters, nitrogen fractions, and total free amino acid levels between TRD and IND types of cheese. Higher levels of proteolysis were observed in IND cheeses than in TRD cheeses during ripening. Levels of residual β- and αs-caseins were 72.2 and 48.7%, respectively, in 180-d-old TRD cheeses. However, the residual levels were 52.8% for β-casein and 18% for αs-casein in IND cheeses. Similar differences were noted for the reversed-phase HPLC peptide profiles of 2 types of cheeses. Also, higher concentrations of peptides were eluted in IND cheeses than in TRD cheeses during ripening. A total of 73 volatile compounds, including alcohols (16), esters (17), acids (14), terpenes (7), ketones (5), aldehydes (4), and miscellaneous (10) were identified. The IND cheeses contained higher levels of carboxylic acids, esters, alcohols, and terpenes than the TRD cheeses; however, the same levels of methyl ketones were determined in the 2 types of cheeses at the end of ripening. These may be due to some differences (e.g., pasteurization and scalding temperature, among other factors) in the manufacture of the 2 types of Beaten cheeses. The textural profile of Beaten cheeses showed that TRD production method resulted in firmer, less fracturable, and stiffer cheeses than the IND production method. In conclusion, the results suggest that the use of industrial production method (pasteurization of cheese milk and curd scalding at 70°C) in the manufacture of Beaten ewe milk cheese enriched the volatile profile of the cheese.

  10. The proteolysis adaptor, NblA, is essential for degradation of the core pigment of the cyanobacterial light-harvesting complex.

    PubMed

    Sendersky, Eleonora; Kozer, Noga; Levi, Mali; Moizik, Michael; Garini, Yuval; Shav-Tal, Yaron; Schwarz, Rakefet

    2015-09-01

    The cyanobacterial light-harvesting complex, the phycobilisome, is degraded under nutrient limitation, allowing the cell to adjust light absorbance to its metabolic capacity. This large light-harvesting antenna comprises a core complex of the pigment allophycocyanin, and rod-shaped pigment assemblies emanating from the core. NblA, a low-molecular-weight protein, is essential for degradation of the phycobilisome. NblA mutants exhibit high absorbance of rod pigments under conditions that generally elicit phycobilisome degradation, implicating NblA in degradation of these pigments. However, the vast abundance of rod pigments and the substantial overlap between the absorbance spectra of rod and core pigments has made it difficult to directly associate NblA with proteolysis of the phycobilisome core. Furthermore, lack of allophycocyanin degradation in an NblA mutant may reflect a requirement for rod degradation preceding core degradation, and does not prove direct involvement of NblA in proteolysis of the core pigment. Therefore, in this study, we used a mutant lacking phycocyanin, the rod pigment of Synechococcus elongatusPCC7942, to examine whether NblA is required for allophycocyanin degradation. We demonstrate that NblA is essential for degradation of the core complex of the phycobilisome. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for the interaction of NblA with allophycocyanin, and indicated that NblA interacts with allophycocyanin complexes that are associated with the photosynthetic membranes. Based on these data, as well as previous observations indicating interaction of NblA with phycobilisomes attached to the photosynthetic membranes, we suggest a model for sequential phycobilisome disassembly by NblA. PMID:26173720

  11. Enhanced waterlogging tolerance in barley by manipulation of expression of the N-end rule pathway E3 ligase PROTEOLYSIS6.

    PubMed

    Mendiondo, Guillermina M; Gibbs, Daniel J; Szurman-Zubrzycka, Miriam; Korn, Arnd; Marquez, Julietta; Szarejko, Iwona; Maluszynski, Miroslaw; King, John; Axcell, Barry; Smart, Katherine; Corbineau, Francoise; Holdsworth, Michael J

    2016-01-01

    Increased tolerance of crops to low oxygen (hypoxia) during flooding is a key target for food security. In Arabidopsis thaliana (L.) Heynh., the N-end rule pathway of targeted proteolysis controls plant responses to hypoxia by regulating the stability of group VII ethylene response factor (ERFVII) transcription factors, controlled by the oxidation status of amino terminal (Nt)-cysteine (Cys). Here, we show that the barley (Hordeum vulgare L.) ERFVII BERF1 is a substrate of the N-end rule pathway in vitro. Furthermore, we show that Nt-Cys acts as a sensor for hypoxia in vivo, as the stability of the oxygen-sensor reporter protein MCGGAIL-GUS increased in waterlogged transgenic plants. Transgenic RNAi barley plants, with reduced expression of the N-end rule pathway N-recognin E3 ligase PROTEOLYSIS6 (HvPRT6), showed increased expression of hypoxia-associated genes and altered seed germination phenotypes. In addition, in response to waterlogging, transgenic plants showed sustained biomass, enhanced yield, retention of chlorophyll, and enhanced induction of hypoxia-related genes. HvPRT6 RNAi plants also showed reduced chlorophyll degradation in response to continued darkness, often associated with waterlogged conditions. Barley Targeting Induced Local Lesions IN Genomes (TILLING) lines, containing mutant alleles of HvPRT6, also showed increased expression of hypoxia-related genes and phenotypes similar to RNAi lines. We conclude that the N-end rule pathway represents an important target for plant breeding to enhance tolerance to waterlogging in barley and other cereals. PMID:25657015

  12. CRM1/Ran-Mediated Nuclear Export of p27Kip1 Involves a Nuclear Export Signal and Links p27 Export and Proteolysis

    PubMed Central

    Connor, Michael K.; Kotchetkov, Rouslan; Cariou, Sandrine; Resch, Ansgar; Lupetti, Rafaella; Beniston, Richard G.; Melchior, Frauke; Hengst, Ludger; Slingerland, Joyce M.

    2003-01-01

    We show that p27 localization is cell cycle regulated and we suggest that active CRM1/RanGTP-mediated nuclear export of p27 may be linked to cytoplasmic p27 proteolysis in early G1. p27 is nuclear in G0 and early G1 and appears transiently in the cytoplasm at the G1/S transition. Association of p27 with the exportin CRM1 was minimal in G0 and increased markedly during G1-to-S phase progression. Proteasome inhibition in mid-G1 did not impair nuclear import of p27, but led to accumulation of p27 in the cytoplasm, suggesting that export precedes degradation for at least part of the cellular p27 pool. p27-CRM1 binding and nuclear export were inhibited by S10A mutation but not by T187A mutation. A putative nuclear export sequence in p27 is identified whose mutation reduced p27-CRM1 interaction, nuclear export, and p27 degradation. Leptomycin B (LMB) did not inhibit p27-CRM1 binding, nor did it prevent p27 export in vitro or in heterokaryon assays. Prebinding of CRM1 to the HIV-1 Rev nuclear export sequence did not inhibit p27-CRM1 interaction, suggesting that p27 binds CRM1 at a non-LMB-sensitive motif. LMB increased total cellular p27 and may do so indirectly, through effects on other p27 regulatory proteins. These data suggest a model in which p27 undergoes active, CRM1-dependent nuclear export and cytoplasmic degradation in early G1. This would permit the incremental activation of cyclin E-Cdk2 leading to cyclin E-Cdk2-mediated T187 phosphorylation and p27 proteolysis in late G1 and S phase. PMID:12529437

  13. CRM1/Ran-mediated nuclear export of p27(Kip1) involves a nuclear export signal and links p27 export and proteolysis.

    PubMed

    Connor, Michael K; Kotchetkov, Rouslan; Cariou, Sandrine; Resch, Ansgar; Lupetti, Rafaella; Beniston, Richard G; Melchior, Frauke; Hengst, Ludger; Slingerland, Joyce M

    2003-01-01

    We show that p27 localization is cell cycle regulated and we suggest that active CRM1/RanGTP-mediated nuclear export of p27 may be linked to cytoplasmic p27 proteolysis in early G1. p27 is nuclear in G0 and early G1 and appears transiently in the cytoplasm at the G1/S transition. Association of p27 with the exportin CRM1 was minimal in G0 and increased markedly during G1-to-S phase progression. Proteasome inhibition in mid-G1 did not impair nuclear import of p27, but led to accumulation of p27 in the cytoplasm, suggesting that export precedes degradation for at least part of the cellular p27 pool. p27-CRM1 binding and nuclear export were inhibited by S10A mutation but not by T187A mutation. A putative nuclear export sequence in p27 is identified whose mutation reduced p27-CRM1 interaction, nuclear export, and p27 degradation. Leptomycin B (LMB) did not inhibit p27-CRM1 binding, nor did it prevent p27 export in vitro or in heterokaryon assays. Prebinding of CRM1 to the HIV-1 Rev nuclear export sequence did not inhibit p27-CRM1 interaction, suggesting that p27 binds CRM1 at a non-LMB-sensitive motif. LMB increased total cellular p27 and may do so indirectly, through effects on other p27 regulatory proteins. These data suggest a model in which p27 undergoes active, CRM1-dependent nuclear export and cytoplasmic degradation in early G1. This would permit the incremental activation of cyclin E-Cdk2 leading to cyclin E-Cdk2-mediated T187 phosphorylation and p27 proteolysis in late G1 and S phase.

  14. Genomic and proteomic analyses reveal non-neofunctionalized vitellogenins in a basal clupeocephalan, the Atlantic herring, and point to the origin of maturational yolk proteolysis in marine teleosts.

    PubMed

    Kristoffersen, Børge A; Nerland, Audun; Nilsen, Frank; Kolarevic, Jelena; Finn, Roderick Nigel

    2009-05-01

    Oocyte hydration is a unique event in oviparous marine teleosts that provides the single-celled egg with an essential pool of water for survival during early development in the saline oceanic environment. A conserved mechanism of maturational yolk proteolysis of a neofunctionalized vitellogenin (VtgAa) has been shown to underlie the hydration event in all teleosts that spawn pelagic eggs (pelagophils), and is argued to be a key adaptation for teleost radiation in the oceanic environment 55 Ma. We have recently shown that a small pool of free amino acids (FAAs) significantly contributes to the osmolarity of the ovulated egg in an ancestral marine teleost, the Atlantic herring that spawns benthic eggs (benthophil). To determine whether multiple forms of vtg exist and whether neofunctionalization of the gene products are related to the egg FAA pool in this species, genomic sequences conserved between the exons of Atlantic herring and zebrafish were amplified. This approach identified a small polymorphic intron between exons 9 and 10 in Atlantic herring and demonstrated that two closely related major vtg transcripts (chvtgAc1 and chvtgAc2) are expressed during oogenesis. A separate polymerase chain reaction-based approach identified a more ancestral phosvitinless transcript (chvtgC). Proteomic analyses of the translated products of the major vtg forms demonstrated that the yolk proteins are similarly processed during deposition, and oocyte maturation and reveal that vtgs have duplicated but not neofunctionalized in this species. Phylogenetic analyses consistently clustered the transcripts and proteins as the basal sister group to the Ostariophysi in full congruence with the Clupeocephalan rank, and suggest that expansion of ostariophysan vtgAo1 and vtgAo2 genes occurred in a lineage-specific manner after separation from the Clupeiformes. Three-dimensional modeling of the ChvtgAc1 sequence against the resolved lamprey lipovitellin module revealed that the tertiary

  15. A preliminary study on the effect of adding yeast extract to cheese curd on proteolysis and flavour development of reduced-fat Cheddar.

    PubMed

    Shakeel-Ur-Rehman; Farkye, Nana Y; Vedamuthu, Ebenezer R; Drake, Mary A

    2003-02-01

    Yeast extract was used as a nutrient for growing lactobacilli in reduced-fat Cheddar cheese as early growth of non-starter lactic acid bacteria (NSLAB) in Cheddar cheese is suppressed by pasteurization of milk and the hostile environment of the cheese. Reduced-fat Cheddar cheese was manufactured from 100 kg standardized milk on two occasions. After milling, the curd was divided into two portions, C and E. To control portion, C, salt was added at normal levels. A mixture of salt and yeast extract was added to the experimental, E. The cheeses were ripened for 7 months at 8 degrees C and assessed for proteolysis and NSLAB growth during ripening. Mean % moisture, fat, protein, salt and pH were 40.6, 20.5, 31.1, 1.72 and 5.22 respectively, in E cheeses, and 39.5, 20.5, 30.9, 1.68 and 5.22, respectively, in C cheese. NSLAB counts in E cheeses were 10(1), 10(3), 10(5) cfu/g compared with 0, 10(1), 10(4) cfu/g in C respectively, after 1, 7 and 30 d of ripening. After 60 d, cell densities of NSLAB were similar (approximately 10(6) cfu/g) in C and E cheese. Addition of yeast extract to curd affected neither the electrophoretic patterns of cheese nor its water-soluble N content during ripening. However, the total free amino acids were significantly higher in E cheese than C cheese throughout ripening, suggesting faster secondary proteolysis in the former cheeses. A 6-member trained descriptive panel evaluated the cheese at 7 months and found that the E cheeses had higher intensities of whey, fruity, sulphur, nutty, sweet and sour flavours, but had lower intensities of brothy flavours as compared to C cheeses. Also, the E cheeses were perceived to be more mature than corresponding C cheese. Results show that addition of yeast extract to cheese curd is a promising method of enhancing flavour development in ripened cheeses.

  16. Transformation by E1A Oncoprotein Involves Ubiquitin-Mediated Proteolysis of the Neuronal and Tumor Repressor REST in the Nucleus

    PubMed Central

    Guan, Hancheng

    2012-01-01

    The adenovirus early region 1A (E1A) protein promotes cell immortalization and transformation by mediating the activities of key cellular regulators. The repressor element 1-silencing transcription factor (REST), which is a major neuronal and tumor suppressor, was previously found mainly in the cytoplasm rather than in the nuclei of adenovirus-transformed rodent cells (22). We now demonstrate that the loss of REST in the nucleus is due to its rapid degradation by the ubiquitin-proteasome system. Only nuclear REST, but not its cytoplasmic counterpart, was ubiquitinated and degraded. REST degradation was blocked by the ubiquitination inhibitor PYR-41 and the proteasome inhibitor MG-132 but not by the nuclear export inhibitor leptomycin B. REST degradation required both of its two C-terminal degrons that are recognized by the ubiquitin ligase SCFβ-TrCP, since deletion or mutation of either degron eliminated degradation. Importantly, E1A was shown to mediate REST ubiquitination and degradation by upregulating β-TrCP. Knockdown of E1A in virus-transformed cells reduced both β-TrCP and ubiquitination of nuclear REST. In contrast, when expressed in HeLa cells, E1A enhanced the degradation of nuclear REST. Reconstitution of REST in virus-transformed cells negatively affected E1A-mediated cell proliferation and anchorage-independent growth. These data strongly indicate that E1A stimulates ubiquitination and proteolysis of REST in the nucleus, thereby abolishing the tumor suppressor functions of REST. PMID:22419809

  17. Preparation of graphene oxide-modified affinity capillary monoliths based on three types of amino donor for chiral separation and proteolysis.

    PubMed

    Hong, Tingting; Chen, Xueping; Xu, Yujing; Cui, Xiaoqin; Bai, Ruihan; Jin, Can; Li, Ruijun; Ji, Yibing

    2016-07-22

    Novel graphene oxide (GO)-modified affinity capillary monoliths were developed employing human serum albumin (HSA) or pepsin as chiral selector. Three types of amino donors for GO immobilization, including ammonium hydroxide (NH4OH), ethanediamine (EDA) and polyethyleneimine (PEI), were applied to explore the effect of spacer arm on enantioseparation. It was observed that HSA-GO-EDA-based affinity capillary monoliths exhibited better chiral recognition ability in comparison with the other two spacer-based monoliths. Under the optimized conditions, the obtained columns revealed satisfactory repeatability concerning column-to-column, run-to-run and interday repeatability. In addition, the impact of GO concentration on enantiomeric separation was also investigated. HSA-GO-EDA-based affinity capillary monoliths provided higher chiral selectivity for nine pairs of enantiomers compared to the columns without GO. Furthermore, the influence of amino donors and GO on proteolytic activity of pepsin-based immobilized enzymatic reactor (IMER) was discussed. Unfortunately, pepsin-GO-PEI-based affinity capillary monoliths possessed the highest protein digestion capacity, which was different from the effect of amino donors on enantiorecognition. Moreover, GO presented as a favorable choice to improve the enzymatic activity of IMER. These results proved that GO-functionalized affinity capillary monoliths have promising potential for chiral separation and proteolysis.

  18. A model of low-level primary blast brain trauma results in cytoskeletal proteolysis and chronic functional impairment in the absence of lung barotrauma.

    PubMed

    Park, Eugene; Gottlieb, James J; Cheung, Bob; Shek, Pang N; Baker, Andrew J

    2011-03-01

    Shock-wave exposure from improvised explosive devices (IEDs) has been implicated as a possible contributing factor to neurological impairment reported in combat veterans. However, evidence-based substantiation of this implication, particularly for low-level exposure in the absence of external signs of trauma, remain elusive. Accordingly, we constructed an open-ended shock tube producing a short-duration, low-amplitude shockwave. Low-level (11.5 kPa static overpressure) complex shock-wave exposure in rats resulted in no histological evidence of lung injury. By contrast, delayed cytoskeletal proteolysis of αII-spectrin was detected in the cortex and hippocampus by 12 h post-injury. Cell death was minimal and localized predominantly in the corpus callosum and periventricular regions. These regions, with presumably different density interfaces, exhibit biological responses to shockwaves consistent with interface turbulence described by Richtmyer-Meshkov instability. Evoked compound action potential (CAP) recordings from the corpus callosum showed a significant increase in the duration of CAP responses at 14 and 30 days post-injury, and a gradual depression in the unmyelinated fiber amplitude. Shielding the head attenuated αII-spectrin cytoskeletal breakdown, thus directly implicating low-level shock-wave exposure as a cause of brain injury in the rat. Despite anatomical and scaling differences in rats compared to humans, the results suggest the potential for undiagnosed traumatic brain pathologies occurring in combat veterans following shock-wave exposure.

  19. Soluble Phenolic Compounds in Different Cultivars of Red Clover and Alfalfa, and their Implication for Protection against Proteolysis and Ammonia Production in Ruminants.

    PubMed

    Kagan, Isabelle A; Goff, Ben M; Flythe, Michael D

    2015-07-01

    Red clover (Trifolium pratense) contains soluble phenolic compounds with roles in inhibiting proteolysis and ammonia production. Alfalfa (Medicago sativa) has been found to have a low phenolic content, but few alfalfa and red clover cultivars have been compared for phenolic content. Total soluble phenolics were quantified by a Folin-Ciocalteu colorimetric assay in nine red clover and 27 alfalfa cultivars. Mean total phenolic contents of red clover and alfalfa were 36.5 ± 4.3 mg/gdw and 15.8 ± 1.4 mg/gdw, respectively, with the greater standard deviation of red clover possibly indicating more diversity in phenolic content. Because different phenolic standards had different response factors in the colorimetric assay, the red clover and 11 alfalfa cultivars were analyzed by HPLC to determine if the differences in total soluble phenolics between genera reflected differences in the amounts of phenolics or in the classes of phenolics responding to the colorimetric assay. Two red clover cultivars differed in total phenolics and phaselic acid. Alfalfa produced different phenolic compounds from red clover, at lower concentrations. Extracts of two red clover cultivars were separated by thin-layer chromatography (TLC), and the bands were assayed for activity against Clostridium sticklandii, a bovine ruminal hyper ammonia-producing bacterium (HAB). Only biochanin A had anti-HAB activity. Inhibitory amounts indicated that five red clover cultivars could be suitable sources of anti-HAB activity. PMID:26411026

  20. The Role of G-Protein-Coupled Receptor Proteolysis Site Cleavage of Polycystin-1 in Renal Physiology and Polycystic Kidney Disease

    PubMed Central

    Trudel, Marie; Yao, Qin; Qian, Feng

    2016-01-01

    Polycystin-1 (PC1) plays an essential role in renal tubular morphogenesis, and PC1 dysfunction causes human autosomal dominant polycystic kidney disease. A fundamental characteristic of PC1 is post-translational modification via cleavage at the juxtamembrane GPCR proteolysis site (GPS) motif that is part of the larger GAIN domain. Given the considerable biochemical complexity of PC1 molecules generated in vivo by this process, GPS cleavage has several profound implications on the intracellular trafficking and localization in association with their particular function. The critical nature of GPS cleavage is further emphasized by the increasing numbers of PKD1 mutations that significantly affect this cleavage process. The GAIN domain with the GPS motif therefore represents the key structural element with fundamental importance for PC1 and might be polycystic kidney disease’s (PKD) Achilles’ heel in a large spectrum of PKD1 missense mutations. We highlight the central roles of PC1 cleavage for the regulation of its biogenesis, intracellular trafficking and function, as well as its significance in polycystic kidney disease. PMID:26805887

  1. Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system

    PubMed Central

    Johnson, Steven; Roversi, Pietro; Espina, Marianela; Deane, Janet E.; Birket, Susan; Picking, William D.; Blocker, Ariel; Picking, Wendy L.; Lea, Susan M.

    2006-01-01

    IpaD, the putative needle-tip protein of the Shigella flexneri type III secretion system, has been overexpressed and purified. Crystals were grown of the native protein in space group P212121, with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 Å, and data were collected to 2.9 Å resolution. Analysis of the native Patterson map revealed a peak at 50% of the origin on the Harker section v = 0.5, suggesting twofold non-crystallographic symmetry parallel to the b crystallographic axis. As attempts to derivatize or grow selenomethionine-labelled protein crystals failed, in-drop proteolysis was used to produce new crystal forms. A trace amount of subtilisin Carlsberg was added to IpaD before sparse-matrix screening, resulting in the production of several new crystal forms. This approach produced SeMet-labelled crystals and diffraction data were collected to 3.2 Å resolution. The SeMet crystals belong to space group C2, with unit-cell parameters a = 139.4, b = 45.0, c = 99.5 Å, β = 107.9°. An anomalous difference Patterson map revealed peaks on the Harker section v = 0, while the self-rotation function indicates the presence of a twofold noncrystallographic symmetry axis, which is consistent with two molecules per asymmetric unit. PMID:16946465

  2. The Role of G-Protein-Coupled Receptor Proteolysis Site Cleavage of Polycystin-1 in Renal Physiology and Polycystic Kidney Disease.

    PubMed

    Trudel, Marie; Yao, Qin; Qian, Feng

    2016-01-01

    Polycystin-1 (PC1) plays an essential role in renal tubular morphogenesis, and PC1 dysfunction causes human autosomal dominant polycystic kidney disease. A fundamental characteristic of PC1 is post-translational modification via cleavage at the juxtamembrane GPCR proteolysis site (GPS) motif that is part of the larger GAIN domain. Given the considerable biochemical complexity of PC1 molecules generated in vivo by this process, GPS cleavage has several profound implications on the intracellular trafficking and localization in association with their particular function. The critical nature of GPS cleavage is further emphasized by the increasing numbers of PKD1 mutations that significantly affect this cleavage process. The GAIN domain with the GPS motif therefore represents the key structural element with fundamental importance for PC1 and might be polycystic kidney disease's (PKD) Achilles' heel in a large spectrum of PKD1 missense mutations. We highlight the central roles of PC1 cleavage for the regulation of its biogenesis, intracellular trafficking and function, as well as its significance in polycystic kidney disease. PMID:26805887

  3. Specific proteolysis of native alanine racemases from Salmonella typhimurium: identification of the cleavage site and characterization of the clipped two-domain proteins

    SciTech Connect

    Galakatos, N.G.; Walsh, C.T.

    1987-12-15

    Native DadB and Alr alanine racemases (M/sub r/ 39,000) from Salmonella typhimurium are proteolyzed at homologous positions by ..cap alpha..-chymotrypsin, trypsin, and subtilisin to generate in all cases two nonoverlapping polypeptides of M/sub r/ 28,000 and 11,000. Under nondenaturing conditions, chymotryptic digest results in an associated form of the two fragments which possesses 3% of the original catalytic activity, incorporates 0.76 equiv of the mechanism-based inactivator ..beta..-chloro-(/sup 14/C)-D-alanine, and exhibits a UV circular dichroism profile identical with that of native enzyme. Protein sequence analysis of the denatured chymotryptic fragments indicates the presence of a tetrapeptide interdomain hinge (DadB, residues 254-257; Alr, residues 256-259) that is attacked at both ends during proteolysis. Under the previously employed digest conditions, NaB/sup 3/H/sub 4/-reduced DadB holoenzyme is resistant to ..cap alpha..-chymotrypsin and trypsin and is labile only toward subtilisin. These data suggest that the hinge structure is essential for a catalytically efficient enzyme species and is sensitive to active site geometry. The sequence at the hinge region is also conserved in alanine racemases from Gram-positive bacteria.

  4. A cytoplasmic C-terminal fragment of syndecan-1 is generated by sequential proteolysis and antagonizes syndecan-1 dependent lung tumor cell migration

    PubMed Central

    Pasqualon, Tobias; Pruessmeyer, Jessica; Jankowski, Vera; Babendreyer, Aaron; Groth, Esther; Schumacher, Julian; Koenen, Andrea; Weidenfeld, Sarah; Schwarz, Nicole; Denecke, Bernd; Jahr, Holger; Dreymueller, Daniela; Jankowski, Joachim; Ludwig, Andreas

    2015-01-01

    Syndecan-1 is a surface expressed heparan sulphate proteoglycan, which is upregulated by several tumor types and involved in tumor cell migration and metastasis. Syndecan-1 is shed from the cell surface and the remaining transmembrane fragment undergoes intramembrane proteolysis by γ-secretase. We here show that this generates a cytoplasmic C-terminal fragment (cCTF). In epithelial lung tumor A549 cells the endogenously produced cCTF accumulated when its proteasomal degradation was blocked with bortezomib and this accumulation was prevented by γ-secretase inhibition. Overexpression of the cCTF suppressed migration and invasion of A549 cells. This inhibitory effect was only seen when endogenous syndecan-1 was present, but not in syndecan-1 deficient cells. Further, overexpression of syndecan-1 cCTF increased the basal activation of Src kinase, focal adhesion kinase (FAK) and Rho GTPase. This was associated with increased adhesion to fibronectin and collagen G and an increased recruitment of paxillin to focal adhesions. Moreover, lung tumor formation of A549 cells in mice was reduced by overexpression of syndecan-1 cCTF. Finally, delivery of a synthetic peptide corresponding to the syndecan-1 cCTF suppressed A549 cell migration and increased basal phosphorylation of Src and FAK. Our data indicate that the syndecan-1 cCTF antagonizes syndecan-1 dependent tumor cell migration in vitro and in vivo by dysregulating proadhesive signaling pathways and suggest that the cCTF can be used as an inhibitory peptide. PMID:26378057

  5. N-lactoyl-amino acids are ubiquitous metabolites that originate from CNDP2-mediated reverse proteolysis of lactate and amino acids

    PubMed Central

    Jansen, Robert S.; Addie, Ruben; Merkx, Remco; Fish, Alexander; Mahakena, Sunny; Bleijerveld, Onno B.; Altelaar, Maarten; IJlst, Lodewijk; Wanders, Ronald J.; Borst, P.; van de Wetering, Koen

    2015-01-01

    Despite technological advances in metabolomics, large parts of the human metabolome are still unexplored. In an untargeted metabolomics screen aiming to identify substrates of the orphan transporter ATP-binding cassette subfamily C member 5 (ABCC5), we identified a class of mammalian metabolites, N-lactoyl-amino acids. Using parallel protein fractionation in conjunction with shotgun proteomics on fractions containing N-lactoyl-Phe–forming activity, we unexpectedly found that a protease, cytosolic nonspecific dipeptidase 2 (CNDP2), catalyzes their formation. N-lactoyl-amino acids are ubiquitous pseudodipeptides of lactic acid and amino acids that are rapidly formed by reverse proteolysis, a process previously considered to be negligible in vivo. The plasma levels of these metabolites strongly correlate with plasma levels of lactate and amino acid, as shown by increased levels after physical exercise and in patients with phenylketonuria who suffer from elevated Phe levels. Our approach to identify unknown metabolites and their biosynthesis has general applicability in the further exploration of the human metabolome. PMID:25964343

  6. 7-Dehydrocholesterol-dependent proteolysis of HMG-CoA reductase suppresses sterol biosynthesis in a mouse model of Smith-Lemli-Opitz/RSH syndrome.

    PubMed

    Fitzky, B U; Moebius, F F; Asaoka, H; Waage-Baudet, H; Xu, L; Xu, G; Maeda, N; Kluckman, K; Hiller, S; Yu, H; Batta, A K; Shefer, S; Chen, T; Salen, G; Sulik, K; Simoni, R D; Ness, G C; Glossmann, H; Patel, S B; Tint, G S

    2001-09-01

    Smith-Lemli-Opitz/RSH syndrome (SLOS), a relatively common birth-defect mental-retardation syndrome, is caused by mutations in DHCR7, whose product catalyzes an obligate step in cholesterol biosynthesis, the conversion of 7-dehydrocholesterol to cholesterol. A null mutation in the murine Dhcr7 causes an identical biochemical defect to that seen in SLOS, including markedly reduced tissue cholesterol and total sterol levels, and 30- to 40-fold elevated concentrations of 7-dehydrocholesterol. Prenatal lethality was not noted, but newborn homozygotes breathed with difficulty, did not suckle, and died soon after birth with immature lungs, enlarged bladders, and, frequently, cleft palates. Despite reduced sterol concentrations in Dhcr7(-/-) mice, mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme for sterol biosynthesis, the LDL receptor, and SREBP-2 appeared neither elevated nor repressed. In contrast to mRNA, protein levels and activities of HMG-CoA reductase were markedly reduced. Consistent with this finding, 7-dehydrocholesterol accelerates proteolysis of HMG-CoA reductase while sparing other key proteins. These results demonstrate that in mice without Dhcr7 activity, accumulated 7-dehydrocholesterol suppresses sterol biosynthesis posttranslationally. This effect might exacerbate abnormal development in SLOS by increasing the fetal cholesterol deficiency. PMID:11560960

  7. 7-Dehydrocholesterol–dependent proteolysis of HMG-CoA reductase suppresses sterol biosynthesis in a mouse model of Smith-Lemli-Opitz/RSH syndrome

    PubMed Central

    Fitzky, Barbara U.; Moebius, Fabian F.; Asaoka, Hitoshi; Waage-Baudet, Heather; Xu, Liwen; Xu, Guorong; Maeda, Nobuyo; Kluckman, Kimberly; Hiller, Sylvia; Yu, Hongwei; Batta, Ashok K.; Shefer, Sarah; Chen, Thomas; Salen, Gerald; Sulik, Kathleen; Simoni, Robert D.; Ness, Gene C.; Glossmann, Hartmut; Patel, Shailendra B.; Tint, G.S.

    2001-01-01

    Smith-Lemli-Opitz/RSH syndrome (SLOS), a relatively common birth-defect mental-retardation syndrome, is caused by mutations in DHCR7, whose product catalyzes an obligate step in cholesterol biosynthesis, the conversion of 7-dehydrocholesterol to cholesterol. A null mutation in the murine Dhcr7 causes an identical biochemical defect to that seen in SLOS, including markedly reduced tissue cholesterol and total sterol levels, and 30- to 40-fold elevated concentrations of 7-dehydrocholesterol. Prenatal lethality was not noted, but newborn homozygotes breathed with difficulty, did not suckle, and died soon after birth with immature lungs, enlarged bladders, and, frequently, cleft palates. Despite reduced sterol concentrations in Dhcr7–/– mice, mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme for sterol biosynthesis, the LDL receptor, and SREBP-2 appeared neither elevated nor repressed. In contrast to mRNA, protein levels and activities of HMG-CoA reductase were markedly reduced. Consistent with this finding, 7-dehydrocholesterol accelerates proteolysis of HMG-CoA reductase while sparing other key proteins. These results demonstrate that in mice without Dhcr7 activity, accumulated 7-dehydrocholesterol suppresses sterol biosynthesis posttranslationally. This effect might exacerbate abnormal development in SLOS by increasing the fetal cholesterol deficiency. PMID:11560960

  8. Measurement of proteolysis in natural water as an approach to the study of natural cycling and pollution impact. Technical completion report

    SciTech Connect

    Sjogren, R.E.

    1982-12-01

    Studies were conducted using a spectrophotometric assay that utilizes a particulate chromogenic protein to detect and follow proteolytic activity of natural mixed bacterial populations from lake water. Seasonal changes in proteolytic rates were found to be related to lake turnover, algal blooms, and are dependent on the dominant proteolytic bacterium. In addition, temperature, but not pH changes, leads to changes in proteolytic rates. Addition of 100 micrograms per liter of orthophosphate leads to an increase in proteolytic rates by 54 percent, while the addition of metal cofactors leads to variable increases (about 5 percent) in rates when compared to control flasks. The addition of chemical contaminants generally leads to a reduction in the rate of proteolysis. Toxic heavy metals were also inhibitory. Recognition of the sensitivity of the assay to varying levels of added orthophosphate led to the development of a procedure for assessment of trophic state of freshwater ecosystems. Preliminary data demonstrate a reasonable correlation between biologically available phosphorus and trophic state.

  9. Interaction of factor IXa with factor VIIIa. Effects of protease domain Ca2+ binding site, proteolysis in the autolysis loop, phospholipid, and factor X.

    PubMed

    Mathur, A; Zhong, D; Sabharwal, A K; Smith, K J; Bajaj, S P

    1997-09-12

    We previously identified a high affinity Ca2+ binding site in the protease domain of factor IXa involving Glu235 (Glu70 in chymotrypsinogen numbering; hereafter, the numbers in brackets refer to the chymotrypsin equivalents) and Glu245[80] as putative ligands. To delineate the function of this Ca2+ binding site, we expressed IXwild type (IXWT), IXE235K, and IXE245V in 293 kidney cells and compared their properties with those of factor IX isolated from normal plasma (IXNP); each protein had the same Mr and gamma-carboxyglutamic acid content. Activation of each factor IX protein by factor VIIa.Ca2+.tissue factor was normal as analyzed by sodium dodecyl sulfate-gel electrophoresis. The coagulant activity of IXaWT was approximately 93%, of IXaE235K was approximately 27%, and of IXaE245V was approximately 4% compared with that of IXaNP. In contrast, activation by factor XIa.Ca2+ led to proteolysis at Arg318-Ser319[150-151] in the protease domain autolysis loop of IXaE245V with a concomitant loss of coagulant activity; this proteolysis was moderate in IXaE235K and minimal in IXaWT or IXaNP. Interaction of each activated mutant with an active site probe, p-aminobenzamidine, was also examined; the Kd of interaction in the absence and presence (in parentheses) of Ca2+ was: IXaNP or IXaWT 230 microM (78 microM), IXaE235K 150 microM (145 microM), IXaE245V 225 microM (240 microM), and autolysis loop cleaved IXaE245V 330 microM (350 microM). Next, we evaluated the apparent Kd (Kd,app) of interaction of each activated mutant with factor VIIIa. We first investigated the EC50 of interaction of IXaNP as well as of IXaWT with factor VIIIa in the presence and absence of phospholipid (PL) and varying concentrations of factor X. At each factor X concentration and constant factor VIIIa, EC50 was the free IXaNP or IXaWT concentration that yielded a half-maximal rate of factor Xa generation. EC50 values for IXaNP and IXaWT were similar and are as follows: PL-minus/X-minus (extrapolated

  10. Conditional Proteolysis of the Membrane Protein YfgM by the FtsH Protease Depends on a Novel N-terminal Degron*

    PubMed Central

    Bittner, Lisa-Marie; Westphal, Kai; Narberhaus, Franz

    2015-01-01

    Regulated proteolysis efficiently and rapidly adapts the bacterial proteome to changing environmental conditions. Many protease substrates contain recognition motifs, so-called degrons, that direct them to the appropriate protease. Here we describe an entirely new degron identified in the cytoplasmic N-terminal end of the membrane-anchored protein YfgM of Escherichia coli. YfgM is stable during exponential growth and degraded in stationary phase by the essential FtsH protease. The alarmone (p)ppGpp, but not the previously described YfgM interactors RcsB and PpiD, influence YfgM degradation. By scanning mutagenesis, we define individual amino acids responsible for turnover of YfgM and find that the degron does not at all comply with the known N-end rule pathway. The YfgM degron is a distinct module that facilitates FtsH-mediated degradation when fused to the N terminus of another monotopic membrane protein but not to that of a cytoplasmic protein. Several lines of evidence suggest that stress-induced degradation of YfgM relieves the response regulator RcsB and thereby permits cellular protection by the Rcs phosphorelay system. On the basis of these and other results in the literature, we propose a model for how the membrane-spanning YfgM protein serves as connector between the stress responses in the periplasm and cytoplasm. PMID:26092727

  11. Cysteine and serine protease-mediated proteolysis in body homogenate of a zooplankter, Moina macrocopa, is inhibited by the toxic cyanobacterium, Microcystis aeruginosa PCC7806.

    PubMed

    Agrawal, Manish Kumar; Bagchi, Divya; Bagchi, Suvendra Nath

    2005-05-01

    The paper describes the characterization of proteases in the whole body homogenate of Moina macrocopa, which can possibly be inhibited by the extracts of Microcystis aeruginosa PCC7806. With the use of oligopeptide substrates and specific inhibitors, we detected the activities of trypsin, chymotrypsin, elastase and cysteine protease. Cysteine protease, the predominant enzyme behind proteolysis of a natural substrate, casein, was partially purified by gel filtration. The substrate SDS-polyacrylamide gel electrophoresis of body homogenate revealed the presence of nine bands of proteases (17-72 kDa). The apparent molecular mass of an exclusive cysteine protease was 60 kDa, whereas of trypsin, it was 17-24 kDa. An extract of M. aeruginosa PCC7806 significantly inhibited the activities of trypsin, chymotrypsin and cysteine protease in M. macrocopa body homogenate at estimated IC(50) of 6- to 79-microg dry mass mL(-1). Upon fractionation by C-18 solid-phase extraction, 60% methanolic elute contained all the protease inhibitors, and these metabolites could be further separated by reverse-phase liquid chromatography. The metabolites inhibitory to M. macrocopa proteases also inhibited the corresponding class of proteases of mammalian/plant origin. The study suggests that protease inhibition may contribute to chemical interaction of cyanobacteria and crustacean zooplankton.

  12. tmRNAs that encode proteolysis-inducing tags are found in all known bacterial genomes: A two-piece tmRNA functions in Caulobacter

    PubMed Central

    Keiler, Kenneth C.; Shapiro, Lucy; Williams, Kelly P.

    2000-01-01

    A general mechanism in bacteria to rescue stalled ribosomes and to clear the cell of incomplete polypeptides involves an RNA species, tmRNA (SsrA), which functions as both a tRNA and an mRNA. This RNA encodes a peptide tag that is incorporated at the end of the aberrant polypeptide and targets it for proteolysis. We have identified a circularly permuted version of the tmRNA gene in α-proteobacteria as well as in a lineage of cyanobacteria. The genes in these two groups seem to have arisen from two independent permutation events. As a result of the altered genetic structure, these tmRNAs are composed of two distinct RNA molecules. The mature two-piece tmRNAs are predicted to have a tRNA-like domain and an mRNA-like domain similar to those of standard one-piece tmRNAs, with a break located in the loop containing the tag reading frame. A related sequence was found in the mitochondrial genome of Reclinomonas americana, but only the tRNA-like portion is retained. Although several sequence and structural motifs that are conserved among one-piece tmRNAs have been lost, the α-proteobacterium Caulobacter crescentus produces a functional two-piece tmRNA. PMID:10884408

  13. Preparation of graphene oxide-modified affinity capillary monoliths based on three types of amino donor for chiral separation and proteolysis.

    PubMed

    Hong, Tingting; Chen, Xueping; Xu, Yujing; Cui, Xiaoqin; Bai, Ruihan; Jin, Can; Li, Ruijun; Ji, Yibing

    2016-07-22

    Novel graphene oxide (GO)-modified affinity capillary monoliths were developed employing human serum albumin (HSA) or pepsin as chiral selector. Three types of amino donors for GO immobilization, including ammonium hydroxide (NH4OH), ethanediamine (EDA) and polyethyleneimine (PEI), were applied to explore the effect of spacer arm on enantioseparation. It was observed that HSA-GO-EDA-based affinity capillary monoliths exhibited better chiral recognition ability in comparison with the other two spacer-based monoliths. Under the optimized conditions, the obtained columns revealed satisfactory repeatability concerning column-to-column, run-to-run and interday repeatability. In addition, the impact of GO concentration on enantiomeric separation was also investigated. HSA-GO-EDA-based affinity capillary monoliths provided higher chiral selectivity for nine pairs of enantiomers compared to the columns without GO. Furthermore, the influence of amino donors and GO on proteolytic activity of pepsin-based immobilized enzymatic reactor (IMER) was discussed. Unfortunately, pepsin-GO-PEI-based affinity capillary monoliths possessed the highest protein digestion capacity, which was different from the effect of amino donors on enantiorecognition. Moreover, GO presented as a favorable choice to improve the enzymatic activity of IMER. These results proved that GO-functionalized affinity capillary monoliths have promising potential for chiral separation and proteolysis. PMID:27334417

  14. Topology of mannosidase II in rat liver Golgi membranes and release of the catalytic domain by selective proteolysis.

    PubMed

    Moremen, K W; Touster, O

    1986-08-15

    The orientation of mannosidase II, an integral Golgi membrane protein involved in asparagine-linked oligosaccharide processing, has been examined in rat liver Golgi membranes. Previous studies on mannosidase II purified from Golgi membranes revealed an intact subunit of 124,000 daltons, as well as a catalytically active 110,000-dalton degradation product generated during purification (Moremen, K. W., and Touster, O. (1985) J. Biol. Chem. 260, 6654-6662). In Triton X-100 extracts of Golgi membranes, the intact enzyme was cleaved by a variety of proteases to generate degradation products similar to those observed previously. At appropriate concentrations, chymotrypsin, pronase, and proteinase K generated 110,000-dalton species, while trypsin and Staphylococcus aureus V8 protease generated 115,000-dalton forms. Cleavage by chymotrypsin under mild conditions (10 micrograms/ml, 10 min, 20 degrees C) resulted in a complete conversion to a catalytically active 110,000-dalton form of the enzyme which was extremely resistant to further degradation. Attempts to demonstrate these protease digestions in nonpermeabilized Golgi membranes were unsuccessful, a result suggesting that the protease-sensitive regions are not accessible on the external surface of the membrane. In Golgi membranes permeabilized by treatment with 0.5% saponin, mannosidase II could readily be cleaved to the 110,000-dalton form by digestion with chymotrypsin under conditions similar to those which result in a proteolytic inactivation of galactosyltransferase, a lumenal Golgi membrane marker. Although mannosidase II catalytic activity was not diminished by this chymotrypsin digestion, as much as 90% of the enzyme activity was converted to a nonsedimentable form. To examine the effect of the proteolytic cleavage on the partition behavior of the enzyme, control and chymotrypsin-treated Triton X-114 extracts of Golgi membranes were examined by phase separation at 35 degrees C. The undigested enzyme partitioned

  15. Calpain-mediated proteolysis of polycystin-1 C-terminus induces JAK2 and ERK signal alterations

    SciTech Connect

    Kim, Hyunho; Kang, Ah-Young; Ko, Ah-ra; Park, Hayne Cho; So, Insuk; Park, Jong Hoon; Cheong, Hae Il; Hwang, Young-Hwan; and others

    2014-01-01

    Autosomal dominant polycystic kidney disease (ADPKD), a hereditary renal disease caused by mutations in PKD1 (85%) or PKD2 (15%), is characterized by the development of gradually enlarging multiple renal cysts and progressive renal failure. Polycystin-1 (PC1), PKD1 gene product, is an integral membrane glycoprotein which regulates a number of different biological processes including cell proliferation, apoptosis, cell polarity, and tubulogenesis. PC1 is a target of various proteolytic cleavages and proteosomal degradations, but its role in intracellular signaling pathways remains poorly understood. Herein, we demonstrated that PC1 is a novel substrate for μ- and m-calpains, which are calcium-dependent cysteine proteases. Overexpression of PC1 altered both Janus-activated kinase 2 (JAK2) and extracellular signal-regulated kinase (ERK) signals, which were independently regulated by calpain-mediated PC1 degradation. They suggest that the PC1 function on JAK2 and ERK signaling pathways might be regulated by calpains in response to the changes in intracellular calcium concentration. - Highlights: • Polycystin-1 is a target of ubiquitin-independent degradation by calpains. • The PEST domain is required for calpain-mediated degradation of polycystin-1. • Polycystin-1 may independently regulate JAK2 and ERK signaling pathways.

  16. Oxidative and proteolysis-related parameters of skeletal muscle from hamsters with experimental pulmonary emphysema: a comparison between papain and elastase induction

    PubMed Central

    Brunnquell, Cláudia R; Vieira, Nichelle A; Sábio, Laís R; Sczepanski, Felipe; Cecchini, Alessandra L; Cecchini, Rubens; Guarnier, Flávia A

    2015-01-01

    The objective of this study was to investigate whether emphysema induced by elastase or papain triggers the same effects on skeletal muscle, related to oxidative stress and proteolysis, in hamsters. For this purpose, we evaluated pulmonary lesions, body weight, muscle loss, oxidative stress (thiobarbituric acid-reactive substances, total and oxidized glutathiones, chemiluminescence stimulated by tert-butyl hydroperoxide and carbonyl proteins), chymotrypsin-like and calpain-like proteolytic activities and muscle fibre cross-sectional area in the gastrocnemius muscles of emphysemic hamsters. Two groups of animals received different intratracheal inductions of experimental emphysema: by 40 mg/ml papain (EP) or 5.2 IU/100 g animal (EE) elastase (n = 10 animals/group). The control group received intratracheal instillation of 300 μl sterile NaCl 0.9%. Compared with the control group, the EP group had reduced muscle weight (18.34%) and the EE group had increased muscle weight (8.37%). Additionally, tert-butyl hydroperoxide-initiated chemiluminescence, carbonylated proteins and chymotrypsin-like proteolytic activity were all elevated in the EP group compared to the CS group, while total glutathione was decreased compared to the EE group. The EE group showed more fibres with increased cross-sectional areas and increased calpain-like activity. Together, these data show that elastase and papain, when used to induce experimental models of emphysema, lead to different speeds and types of adaptation. These findings provide more information on choosing a suitable experimental model for studying skeletal muscle adaptations in emphysema. PMID:26102076

  17. Oxidative and proteolysis-related parameters of skeletal muscle from hamsters with experimental pulmonary emphysema: a comparison between papain and elastase induction.

    PubMed

    Brunnquell, Cláudia R; Vieira, Nichelle A; Sábio, Laís R; Sczepanski, Felipe; Cecchini, Alessandra L; Cecchini, Rubens; Guarnier, Flávia A

    2015-06-01

    The objective of this study was to investigate whether emphysema induced by elastase or papain triggers the same effects on skeletal muscle, related to oxidative stress and proteolysis, in hamsters. For this purpose, we evaluated pulmonary lesions, body weight, muscle loss, oxidative stress (thiobarbituric acid-reactive substances, total and oxidized glutathiones, chemiluminescence stimulated by tert-butyl hydroperoxide and carbonyl proteins), chymotrypsin-like and calpain-like proteolytic activities and muscle fibre cross-sectional area in the gastrocnemius muscles of emphysemic hamsters. Two groups of animals received different intratracheal inductions of experimental emphysema: by 40 mg/ml papain (EP) or 5.2 IU/100 g animal (EE) elastase (n = 10 animals/group). The control group received intratracheal instillation of 300 μl sterile NaCl 0.9%. Compared with the control group, the EP group had reduced muscle weight (18.34%) and the EE group had increased muscle weight (8.37%). Additionally, tert-butyl hydroperoxide-initiated chemiluminescence, carbonylated proteins and chymotrypsin-like proteolytic activity were all elevated in the EP group compared to the CS group, while total glutathione was decreased compared to the EE group. The EE group showed more fibres with increased cross-sectional areas and increased calpain-like activity. Together, these data show that elastase and papain, when used to induce experimental models of emphysema, lead to different speeds and types of adaptation. These findings provide more information on choosing a suitable experimental model for studying skeletal muscle adaptations in emphysema.

  18. Aspergillus Oryzae S2 α-Amylase Domain C Involvement in Activity and Specificity: In Vivo Proteolysis, Molecular and Docking Studies

    PubMed Central

    Sahnoun, Mouna; Jemli, Sonia; Trabelsi, Sahar; Ayadi, Leila; Bejar, Samir

    2016-01-01

    We previously reported that Aspergillus oryzae strain S2 had produced two α-amylase isoforms named AmyA and AmyB. The apparent molecular masses revealed by SDS-PAGE were 50 and 42 kDa, respectively. Yet AmyB has a higher catalytic efficiency. Based on a monitoring study of the α-amylase production in both the presence and absence of different protease inhibitors, a chymotrypsin proteolysis process was detected in vivo generating AmyB. A. oryzae S2 α-amylase gene was amplified, cloned and sequenced. The sequence analysis revealed nine exons, eight introns and an encoding open reading frame of 1500 bp corresponding to AmyA isoform. The amino-acid sequence analysis revealed aY371 potential chymotrypsin cleaving site, likely to be the AmyB C-Terminal end and two other potential sites at Y359, and F379. A zymogram with a high acrylamide concentration was used. It highlighted two other closed apparent molecular mass α-amylases termed AmyB1 and AmyB2 reaching40 kDa and 43 kDa. These isoforms could be possibly generated fromY359, and F379secondary cut, respectively. The molecular modeling study showed that AmyB preserved the (β/α)8 barrel domain and the domain B but lacked the C-terminal domain C. The contact map analysis and the docking studies strongly suggested a higher activity and substrate binding affinity for AmyB than AmyA which was previously experimentally exhibited. This could be explained by the easy catalytic cleft accessibility. PMID:27101008

  19. Loss of Peripheral Protection in Pancreatic Islets by Proteolysis-Driven Impairment of VTCN1 (B7-H4) Presentation Is Associated with the Development of Autoimmune Diabetes.

    PubMed

    Radichev, Ilian A; Maneva-Radicheva, Lilia V; Amatya, Christina; Salehi, Maryam; Parker, Camille; Ellefson, Jacob; Burn, Paul; Savinov, Alexei Y

    2016-02-15

    Ag-specific activation of T cells is an essential process in the control of effector immune responses. Defects in T cell activation, particularly in the costimulation step, have been associated with many autoimmune conditions, including type 1 diabetes (T1D). Recently, we demonstrated that the phenotype of impaired negative costimulation, due to reduced levels of V-set domain-containing T cell activation inhibitor 1 (VTCN1) protein on APCs, is shared between diabetes-susceptible NOD mice and human T1D patients. In this study, we show that a similar process takes place in the target organ, as both α and β cells within pancreatic islets gradually lose their VTCN1 protein during autoimmune diabetes development despite upregulation of the VTCN1 gene. Diminishment of functional islet cells' VTCN1 is caused by the active proteolysis by metalloproteinase N-arginine dibasic convertase 1 (NRD1) and leads to the significant induction of proliferation and cytokine production by diabetogenic T cells. Inhibition of NRD1 activity, alternatively, stabilizes VTCN1 and dulls the anti-islet T cell responses. Therefore, we suggest a general endogenous mechanism of defective VTCN1 negative costimulation, which affects both lymphoid and peripheral target tissues during T1D progression and results in aggressive anti-islet T cell responses. This mechanism is tied to upregulation of NRD1 expression and likely acts in two synergistic proteolytic modes: cell-intrinsic intracellular and cell-extrinsic systemic. Our results highlight an importance of VTCN1 stabilization on cell surfaces for the restoration of altered balance of immune control during T1D. PMID:26773144

  20. Multiple vitellogenins and product yolk proteins in European sea bass (Dicentrarchus labrax): Molecular characterization, quantification in plasma, liver and ovary, and maturational proteolysis.

    PubMed

    Yilmaz, Ozlem; Prat, Francisco; Ibáñez, A Jose; Köksoy, Sadi; Amano, Haruna; Sullivan, Craig V

    2016-01-01

    Three complete vitellogenin (Vtg) polypeptides of European sea bass (Dicentrarchus labrax), an acanthomorph teleost spawning pelagic eggs in seawater, were deduced from cDNA and identified as VtgAa, VtgAb and VtgC based on current Vtg nomenclature and phylogeny. Label free quantitative mass spectrometry verified the presence of the three sea bass Vtgs or their product yolk proteins (YPs) in liver, plasma and ovary of postvitellogenic females. As evidenced by normalized spectral counts, VtgAb-derived protein was 2- to 5-fold more abundant, depending on sample type, than for VtgAa, while VtgC-derived protein was less abundant, albeit only 3-fold lower than for VtgAb in the ovary. Western blotting with Vtg type-specific antisera raised against corresponding gray mullet (Mugil cephalus) lipovitellins (Lvs) detected all three types of sea bass Vtg in the blood plasma of gravid females and/or estrogenized males and showed that all three forms of sea bass Lv undergo limited partial degradation during oocyte maturation. The comparatively high levels of VtgC-derived YPs in fully-grown oocytes and the maturational proteolysis of all three types of Lv differ from what has been reported for other teleosts spawning pelagic eggs in seawater but are similar to recent findings for two species of North American Moronidae, the striped bass (Morone saxatilis) and white perch (Morone americana), which spawn pelagic and demersal eggs, respectively in fresh water. Together with the high Vtg sequence homologies and virtually identical structural features of each type of Vtg between species, these findings indicate that the moronid multiple Vtg systems do not substantially vary with reproductive environment. PMID:26643259

  1. Impact of polymorphism of the regulatory subunit of the μ-calpain (CAPN1S) on the proteolysis process and meat tenderness of young cattle.

    PubMed

    Iwanowska, Agnieszka; Grześ, Bożena; Mikołajczak, Beata; Iwańska, Ewa; Juszczuk-Kubiak, Edyta; Rosochacki, Stanisław J; Pospiech, Edward

    2011-02-01

    The objective of this study was to estimate the impact of the polymorphism of μ-calpain (CAPN1S) gene on protein changes of the cattle muscle tissue and its tenderness during 10-day cold storage. The analysis was performed on the longest dorsal and lumbar muscles collected from 76 bulls 6 to 12 months of age. Polymorphism identification of the above-mentioned gene was conducted using the PCR-RFLP technique. Its effect on the course of the proteolysis process was assessed by monitoring changes in proportions of tissue proteins during 10-day process of meat ageing. Special attention was focused on changes in native titin (T1) share and products of its degradation (proteins of molecular weight (m.w.) of 2400 and 200 kDa), α-actinin and protein of 37 kDa as well as myosin heavy chains (MHC). In the case of the last proteins, their polymorphism was evaluated as well. Meat tenderness was estimated measuring the value of shear force and sensorially. The highest tenderness was ascertained for the heterozygote. Its improvement was associated with a significant decrease in proportions of proteins of molecular weight of approximately 37 kDa accompanied by an increase of those with 200 kDa molecular weight. Muscles derived from cattle of CT genotype were characterised by the highest proportions of type 2a MHC isoform. Value differences between proportions determined for the heterozygote and CC and TT homozygotes of the CAPN1S gene were statistically significant. Therefore, it can be presumed that the process of meat tenderisation was especially connected with MHC polymorphism.

  2. Aspergillus Oryzae S2 α-Amylase Domain C Involvement in Activity and Specificity: In Vivo Proteolysis, Molecular and Docking Studies.

    PubMed

    Sahnoun, Mouna; Jemli, Sonia; Trabelsi, Sahar; Ayadi, Leila; Bejar, Samir

    2016-01-01

    We previously reported that Aspergillus oryzae strain S2 had produced two α-amylase isoforms named AmyA and AmyB. The apparent molecular masses revealed by SDS-PAGE were 50 and 42 kDa, respectively. Yet AmyB has a higher catalytic efficiency. Based on a monitoring study of the α-amylase production in both the presence and absence of different protease inhibitors, a chymotrypsin proteolysis process was detected in vivo generating AmyB. A. oryzae S2 α-amylase gene was amplified, cloned and sequenced. The sequence analysis revealed nine exons, eight introns and an encoding open reading frame of 1500 bp corresponding to AmyA isoform. The amino-acid sequence analysis revealed aY371 potential chymotrypsin cleaving site, likely to be the AmyB C-Terminal end and two other potential sites at Y359, and F379. A zymogram with a high acrylamide concentration was used. It highlighted two other closed apparent molecular mass α-amylases termed AmyB1 and AmyB2 reaching40 kDa and 43 kDa. These isoforms could be possibly generated fromY359, and F379secondary cut, respectively. The molecular modeling study showed that AmyB preserved the (β/α)8 barrel domain and the domain B but lacked the C-terminal domain C. The contact map analysis and the docking studies strongly suggested a higher activity and substrate binding affinity for AmyB than AmyA which was previously experimentally exhibited. This could be explained by the easy catalytic cleft accessibility. PMID:27101008

  3. Manganese Upregulates Cellular Prion Protein and Contributes to Altered Stabilization and Proteolysis: Relevance to Role of Metals in Pathogenesis of Prion Disease

    PubMed Central

    Choi, Christopher J.; Anantharam, Vellareddy; Martin, Dustin P.; Nicholson, Eric M.; Richt, Jürgen A.; Kanthasamy, Arthi; Kanthasamy, Anumantha G.

    2010-01-01

    Prion diseases are fatal neurodegenerative diseases resulting from misfolding of normal cellular prion (PrPC) into an abnormal form of scrapie prion (PrPSc). The cellular mechanisms underlying the misfolding of PrPC are not well understood. Since cellular prion proteins harbor divalent metal-binding sites in the N-terminal region, we examined the effect of manganese on PrPC processing in in vitro models of prion disease. Exposure to manganese significantly increased PrPC levels both in cytosolic and in membrane-rich fractions in a time-dependent manner. Manganese-induced PrPC upregulation was independent of messenger RNA transcription or stability. Additionally, manganese treatment did not alter the PrPC degradation by either proteasomal or lysosomal pathways. Interestingly, pulse-chase analysis showed that the PrPC turnover rate was significantly altered with manganese treatment, indicating increased stability of PrPC with the metal exposure. Limited proteolysis studies with proteinase-K further supported that manganese increases the stability of PrPC. Incubation of mouse brain slice cultures with manganese also resulted in increased prion protein levels and higher intracellular manganese accumulation. Furthermore, exposure of manganese to an infectious prion cell model, mouse Rocky Mountain Laboratory–infected CAD5 cells, significantly increased prion protein levels. Collectively, our results demonstrate for the first time that divalent metal manganese can alter the stability of prion proteins and suggest that manganese-induced stabilization of prion protein may play a role in prion protein misfolding and prion disease pathogenesis. PMID:20176619

  4. Comparative Transcriptome Analysis Reveals that a Ubiquitin-Mediated Proteolysis Pathway Is Important for Primary and Secondary Hair Follicle Development in Cashmere Goats

    PubMed Central

    Zheng, Zhu-qing; Fu, Shao-yin; Tarekegn, Getinet Mekuriaw; Bai, Xue; Bai, Yong-sheng; Li, Heng; Zhang, Wen-guang

    2016-01-01

    Background The fleece of cashmere goats contains two distinct populations of fibers, a short and fine non-medullated insulating cashmere fiber and a long and coarse medullated guard hair. The former is produced by secondary follicles (SFs) and the later by primary follicles (PFs). Evidence suggests that the induction of PFs and SFs may require different signaling pathways. The regulation of BMP2/4 signaling by noggin and Edar signaling via Downless genes are essential for the induction of SFs and PFs, respectively. However, these differently expressed genes of the signaling pathway cannot directly distinguish between the PFs and SFs. Results In this study, we selected RNA samples from 11 PFs and 7 SFs that included 145,525 exons. The pathway analysis of 4512 differentially expressed exons revealed that the most statistically significant metabolic pathway was related to the ubiquitin–mediated proteolysis pathway (UMPP) (P<3.32x 10−7). In addition, the 51 exons of the UMPP that were differentially expressed between the different types of hair follicle (HFs) were compared by cluster analysis. This resulted in the PFs and SFs being divided into two classes. The expression level of two selected exons was analyzed by qRT-PCR, and the results indicated that the expression patterns were consistent with the deep sequencing results obtained by RNA-Seq. Conclusions Based on the comparative transcriptome analysis of 18 HFs from cashmere goats, a large number of differentially expressed exons were identified using a high-throughput sequencing approach. This study suggests that UMPP activation is a prominent signaling pathway for distinguishing the PFs and SFs of cashmere goats. It is also a meaningful contribution to the theoretical basis of the biological study of the HFs of cashmere goats and other mammals. PMID:27695037

  5. SILAC-Pulse Proteolysis: A Mass Spectrometry-Based Method for Discovery and Cross-Validation in Proteome-Wide Studies of Ligand Binding

    NASA Astrophysics Data System (ADS)

    Adhikari, Jagat; Fitzgerald, Michael C.

    2014-12-01

    Reported here is the use of stable isotope labeling with amino acids in cell culture (SILAC) and pulse proteolysis (PP) for detection and quantitation of protein-ligand binding interactions on the proteomic scale. The incorporation of SILAC into PP enables the PP technique to be used for the unbiased detection and quantitation of protein-ligand binding interactions in complex biological mixtures (e.g., cell lysates) without the need for prefractionation. The SILAC-PP technique is demonstrated in two proof-of-principle experiments using proteins in a yeast cell lysate and two test ligands including a well-characterized drug, cyclosporine A (CsA), and a non-hydrolyzable adenosine triphosphate (ATP) analogue, adenylyl imidodiphosphate (AMP-PNP). The well-known tight-binding interaction between CsA and cyclophilin A was successfully detected and quantified in replicate analyses, and a total of 33 proteins from a yeast cell lysate were found to have AMP-PNP-induced stability changes. In control experiments, the method's false positive rate of protein target discovery was found to be in the range of 2.1% to 3.6%. SILAC-PP and the previously reported stability of protein from rates of oxidation (SPROX) technique both report on the same thermodynamic properties of proteins and protein-ligand complexes. However, they employ different probes and mass spectrometry-based readouts. This creates the opportunity to cross-validate SPROX results with SILAC-PP results, and vice-versa. As part of this work, the SILAC-PP results obtained here were cross-validated with previously reported SPROX results on the same model systems to help differentiate true positives from false positives in the two experiments.

  6. Multiple vitellogenins and product yolk proteins in European sea bass (Dicentrarchus labrax): Molecular characterization, quantification in plasma, liver and ovary, and maturational proteolysis.

    PubMed

    Yilmaz, Ozlem; Prat, Francisco; Ibáñez, A Jose; Köksoy, Sadi; Amano, Haruna; Sullivan, Craig V

    2016-01-01

    Three complete vitellogenin (Vtg) polypeptides of European sea bass (Dicentrarchus labrax), an acanthomorph teleost spawning pelagic eggs in seawater, were deduced from cDNA and identified as VtgAa, VtgAb and VtgC based on current Vtg nomenclature and phylogeny. Label free quantitative mass spectrometry verified the presence of the three sea bass Vtgs or their product yolk proteins (YPs) in liver, plasma and ovary of postvitellogenic females. As evidenced by normalized spectral counts, VtgAb-derived protein was 2- to 5-fold more abundant, depending on sample type, than for VtgAa, while VtgC-derived protein was less abundant, albeit only 3-fold lower than for VtgAb in the ovary. Western blotting with Vtg type-specific antisera raised against corresponding gray mullet (Mugil cephalus) lipovitellins (Lvs) detected all three types of sea bass Vtg in the blood plasma of gravid females and/or estrogenized males and showed that all three forms of sea bass Lv undergo limited partial degradation during oocyte maturation. The comparatively high levels of VtgC-derived YPs in fully-grown oocytes and the maturational proteolysis of all three types of Lv differ from what has been reported for other teleosts spawning pelagic eggs in seawater but are similar to recent findings for two species of North American Moronidae, the striped bass (Morone saxatilis) and white perch (Morone americana), which spawn pelagic and demersal eggs, respectively in fresh water. Together with the high Vtg sequence homologies and virtually identical structural features of each type of Vtg between species, these findings indicate that the moronid multiple Vtg systems do not substantially vary with reproductive environment.

  7. Limited Proteolysis Reveals That Amyloids from the 3D Domain-Swapping Cystatin B Have a Non-Native β-Sheet Topology.

    PubMed

    Davis, Peter J; Holmes, David; Waltho, Jonathan P; Staniforth, Rosemary A

    2015-07-31

    3D domain-swapping proteins form multimers by unfolding and then sharing of secondary structure elements, often with native-like interactions. Runaway domain swapping is proposed as a mechanism for folded proteins to form amyloid fibres, with examples including serpins and cystatins. Cystatin C amyloids cause a hereditary form of cerebral amyloid angiopathy whilst cystatin B aggregates are found in cases of Unverricht-Lundborg Syndrome, a progressive form of myoclonic epilepsy. Under conditions that favour fibrillisation, cystatins populate stable 3D domain-swapped dimers both in vitro and in vivo that represent intermediates on route to the formation of fibrils. Previous work on cystatin B amyloid fibrils revealed that the α-helical region of the protein becomes disordered and identified the conservation of a continuous 20-residue elongated β-strand (residues 39-58), the latter being a salient feature of the dimeric 3D domain-swapped structure. Here we apply limited proteolysis to cystatin B amyloid fibrils and show that not only the α-helical N-terminal of the protein (residues 1-35) but also the C-terminal of the protein (residues 80-98) can be removed without disturbing the underlying fibril structure. This observation is incompatible with previous models of cystatin amyloid fibrils where the β-sheet is assumed to retain its native antiparallel arrangement. We conclude that our data favour a more generic, at least partially parallel, arrangement for cystatin β-sheet structure in mature amyloids and propose a model that remains consistent with available data for amyloids from either cystatin B or cystatin C.

  8. Evaluating the role of conserved amino acids in bacterial O-oligosaccharyltransferases by in vivo, in vitro and limited proteolysis assays.

    PubMed

    Musumeci, Matias A; Faridmoayer, Amirreza; Watanabe, Yasuharu; Feldman, Mario F

    2014-01-01

    Bacterial O-Oligosaccharyltransferases (O-OTases) constitute a growing family of enzymes that catalyze the transfer of a glycan from a lipid carrier to protein acceptors. O-OTases are inner membrane proteins that display limited sequence similarity, except for the Wzy_C signature domain also present in a predicted periplasmic loop of the WaaL ligase, the enzyme responsible for transferring the O antigen to the lipid A core. The mechanism of O-OTase-dependent glycosylation is poorly understood. In this work, conserved amino acid residues in the O-OTases were replaced with alanine in PglL, the O-OTase of Neisseria meningitidis. The activity of wild-type PglL and its mutant derivatives were analyzed in vivo in engineered Escherichia coli cells, and in in vitro assays. We identified two additional sites of pilin glycosylated exclusively by PglL in E. coli. Both sites are modified with phosphoglycerol (PG) by different enzymes in Neisseria gonorrhoeae and Neisseria meningitidis. Limited proteolysis experiments revealed a conformational change that is triggered upon interaction of the C-terminal region of PglL with the lipid-linked oligosaccharide (LLO) substrate. These experiments showed that Q178 and Y405 are required for optimal function, whereas H349 is essential for activity and plays a critical role in the interaction with LLO. The equivalent His residue is also essential for WaaL activity, which suggests a common mechanism for both enzymes, and supports the hypothesis that O-glycosylation and lipopolysaccharide (LPS) synthesis are evolutionarily related. These results contribute to the elucidation of the mechanism of O-OTases, which are promising targets for novel antibiotics and present an enormous potential for glycoengineering novel vaccines and therapeutics. PMID:24092836

  9. Age-related differences in lean mass, protein synthesis and skeletal muscle markers of proteolysis after bed rest and exercise rehabilitation.

    PubMed

    Tanner, Ruth E; Brunker, Lucille B; Agergaard, Jakob; Barrows, Katherine M; Briggs, Robert A; Kwon, Oh Sung; Young, Laura M; Hopkins, Paul N; Volpi, Elena; Marcus, Robin L; LaStayo, Paul C; Drummond, Micah J

    2015-09-15

    Bed rest-induced muscle loss and impaired muscle recovery may contribute to age-related sarcopenia. It is unknown if there are age-related differences in muscle mass and muscle anabolic and catabolic responses to bed rest. A secondary objective was to determine if rehabilitation could reverse bed rest responses. Nine older and fourteen young adults participated in a 5-day bed rest challenge (BED REST). This was followed by 8 weeks of high intensity resistance exercise (REHAB). Leg lean mass (via dual-energy X-ray absorptiometry; DXA) and strength were determined. Muscle biopsies were collected during a constant stable isotope infusion in the postabsorptive state and after essential amino acid (EAA) ingestion on three occasions: before (PRE), after bed rest and after rehabilitation. Samples were assessed for protein synthesis, mTORC1 signalling, REDD1/2 expression and molecular markers related to muscle proteolysis (MURF1, MAFBX, AMPKα, LC3II/I, Beclin1). We found that leg lean mass and strength decreased in older but not younger adults after bedrest (P < 0.05) and was restored after rehabilitation. EAA-induced mTORC1 signalling and protein synthesis increased before bed rest in both age groups (P < 0.05). Although both groups had blunted mTORC1 signalling, increased REDD2 and MURF1 mRNA after bedrest, only older adults had reduced EAA-induced protein synthesis rates and increased MAFBX mRNA, p-AMPKα and the LC3II/I ratio (P < 0.05). We conclude that older adults are more susceptible than young persons to muscle loss after short-term bed rest. This may be partially explained by a combined suppression of protein synthesis and a marginal increase in proteolytic markers. Finally, rehabilitation restored bed rest-induced deficits in lean mass and strength in older adults.

  10. Integrated Means Integrity

    ERIC Educational Resources Information Center

    Odegard, John D.

    1978-01-01

    Describes the operation of the Cessna Pilot Center (CPC) flight training systems. The program is based on a series of integrated activities involving stimulus, response, reinforcement and association components. Results show that the program can significantly reduce in-flight training time. (CP)

  11. Integrating Art.

    ERIC Educational Resources Information Center

    BCATA Journal for Art Teachers, 1991

    1991-01-01

    These articles focus on art as a component of interdisciplinary integration. (1) "Integrated Curriculum and the Visual Arts" (Anna Kindler) considers various aspects of integration and implications for art education. (2) "Integration: The New Literacy" (Tim Varro) illustrates how the use of technology can facilitate cross-curricular integration.…

  12. Effect of sodium, potassium, magnesium, and calcium salt cations on pH, proteolysis, organic acids, and microbial populations during storage of full-fat Cheddar cheese.

    PubMed

    McMahon, D J; Oberg, C J; Drake, M A; Farkye, N; Moyes, L V; Arnold, M R; Ganesan, B; Steele, J; Broadbent, J R

    2014-01-01

    Sodium reduction in cheese can assist in reducing overall dietary Na intake, yet saltiness is an important aspect of cheese flavor. Our objective was to evaluate the effect of partial substitution of Na with K on survival of lactic acid bacteria (LAB) and nonstarter LAB (NSLAB), pH, organic acid production, and extent of proteolysis as water-soluble nitrogen (WSN) and protein profiles using urea-PAGE, in Cheddar cheese during 9mo of storage. Seven Cheddar cheeses with molar salt contents equivalent to 1.7% salt but with different ratios of Na, K, Ca, and Mg cations were manufactured as well as a low-salt cheese with 0.7% salt. The 1.7% salt cheeses had a mean composition of 352g of moisture/kg, 259g of protein/kg and 50% fat-on-dry-basis, and 17.5g of salt/kg (measured as Cl(-)). After salting, a faster initial decrease in cheese pH occurred with low salt or K substitution and it remained lower throughout storage. No difference in intact casein levels or percentage WSN levels between the various cheeses was observed, with the percentage WSN increasing from 5% at d 1 to 25% at 9mo. A greater decrease in intact αs1-casein than β-casein was detected, and the ratio of αs1-casein (f121-199) to αs1-casein could be used as an index of ripening. Typical changes in bacteria microflora occurred during storage, with lactococci decreasing gradually and NSLAB increasing. Lowering the Na content, even with K replacement, extended the crossover time when NSLAB became dominant. The crossover time was 4.5mo for the control cheese and was delayed to 5.2, 6.0, 6.1, and 6.2mo for cheeses with 10, 25, 50, and 75% K substitution. Including 10% Mg or Ca, along with 40% K, further increased crossover time, whereas the longest crossover time (7.3mo) was for low-salt cheese. By 9mo, NSLAB levels in all cheeses had increased from initial levels of ≤10(2) to approximately 10(6)cfu/g. Lactococci remained at 10(6) cfu/g in the low-salt cheese even after 9mo of storage. The propionic acid

  13. Effect of heat treatment on true digestibility in the rat, in vitro proteolysis and available lysine content of cottonseed meal protein.

    PubMed

    Craig, W M; Broderick, G A

    1981-02-01

    Effects of heating cottonseed meal (CSM) protein were quantitatively assessed by determination of true digestibility (TD), in vitro proteolysis, N solubility and fluorodinitrobenzene (FDNB) available lysine. Flaked, dehulled cottonseed was extracted with hexane and desolventized at 25 C, then autoclaved (121 C, 1.1 kg/cm(2)) for 0, 15, 30, 60, 90 or 120 minutes. Free gossypol was subsequently extracted, and TD was determined in weanling rats. Metabolic fecal N (the fecal N excreted by rats fed a basal diet containing 4% casein protein) was 1.84 +/- .10 mg N/g dry matter intake. TD and FDNB-available lysine (percentage of total) were 91 and 89%, respectively, in the unheated meal. TD and FDNB-available lysine were reduced to 84 and 78% after 60 min of autoclaving, and to 71 and 44% after 120 min of autoclaving. The effect of heat treatment on TD was described by the equation: % TD = 100 - 9.28e(.0096t) (r = .998), where t = minutes of autoclaving. This indicated an accelerated decline in TD as heating time increased. No more than 40% of the loss in FDNB-available lysine was attributable to gossypol binding. In vitro release of total amino acids from autoclaved CSM samples during pepsin-pancreatin incubations was highly correlated to TD (r = .996), but N solubility in .02 N NaOH was poorly correlated to TD. In samples of solvent-extracted and screw-pressed CSM, TD (estimated from pepsin-pancreatin incubations) ranged from 80 to 85% and FDNB-available lysine ranged from 73 to 85%, and both were only slightly lower in screw-pressed than in solvent-extracted meals. Intake of FDNB-available lysine was correlated (r = .902) to weight gain in rats fed diets containing the CSM that were more severely autoclaved. Results suggest that heat treatment must be more severe than that which normally occurs in commercial CSM processing to cause substantial, selective loss in lysine availability.

  14. Effect of sodium, potassium, magnesium, and calcium salt cations on pH, proteolysis, organic acids, and microbial populations during storage of full-fat Cheddar cheese.

    PubMed

    McMahon, D J; Oberg, C J; Drake, M A; Farkye, N; Moyes, L V; Arnold, M R; Ganesan, B; Steele, J; Broadbent, J R

    2014-01-01

    Sodium reduction in cheese can assist in reducing overall dietary Na intake, yet saltiness is an important aspect of cheese flavor. Our objective was to evaluate the effect of partial substitution of Na with K on survival of lactic acid bacteria (LAB) and nonstarter LAB (NSLAB), pH, organic acid production, and extent of proteolysis as water-soluble nitrogen (WSN) and protein profiles using urea-PAGE, in Cheddar cheese during 9mo of storage. Seven Cheddar cheeses with molar salt contents equivalent to 1.7% salt but with different ratios of Na, K, Ca, and Mg cations were manufactured as well as a low-salt cheese with 0.7% salt. The 1.7% salt cheeses had a mean composition of 352g of moisture/kg, 259g of protein/kg and 50% fat-on-dry-basis, and 17.5g of salt/kg (measured as Cl(-)). After salting, a faster initial decrease in cheese pH occurred with low salt or K substitution and it remained lower throughout storage. No difference in intact casein levels or percentage WSN levels between the various cheeses was observed, with the percentage WSN increasing from 5% at d 1 to 25% at 9mo. A greater decrease in intact αs1-casein than β-casein was detected, and the ratio of αs1-casein (f121-199) to αs1-casein could be used as an index of ripening. Typical changes in bacteria microflora occurred during storage, with lactococci decreasing gradually and NSLAB increasing. Lowering the Na content, even with K replacement, extended the crossover time when NSLAB became dominant. The crossover time was 4.5mo for the control cheese and was delayed to 5.2, 6.0, 6.1, and 6.2mo for cheeses with 10, 25, 50, and 75% K substitution. Including 10% Mg or Ca, along with 40% K, further increased crossover time, whereas the longest crossover time (7.3mo) was for low-salt cheese. By 9mo, NSLAB levels in all cheeses had increased from initial levels of ≤10(2) to approximately 10(6)cfu/g. Lactococci remained at 10(6) cfu/g in the low-salt cheese even after 9mo of storage. The propionic acid

  15. Aspartate mutations in presenilin and gamma-secretase inhibitors both impair notch1 proteolysis and nuclear translocation with relative preservation of notch1 signaling.

    PubMed

    Berezovska, O; Jack, C; McLean, P; Aster, J C; Hicks, C; Xia, W; Wolfe, M S; Kimberly, W T; Weinmaster, G; Selkoe, D J; Hyman, B T

    2000-08-01

    It has been hypothesized that a presenilin 1 (PS1)-related enzymatic activity is responsible for proteolytic cleavage of the C-terminal intracellular protein of Notch1, in addition to its role in beta-amyloid protein (Abeta) formation from the amyloid precursor protein (APP). We developed an assay to monitor ligand-induced Notch1 proteolysis and nuclear translocation in individual cells : Treatment of full-length Notch1-enhanced green fluorescent protein-transfected Chinese hamster ovary (CHO) cells with a soluble preclustered form of the physiologic ligand Delta leads to rapid accumulation of the C terminus of Notch1 in the nucleus and to transcriptional activation of a C-promoter binding factor 1 (CBF1) reporter construct. Nuclear translocation was blocked by cotransfection with Notch's physiologic inhibitor Numb. Using this assay, we now confirm and extend the observation that PS1 is involved in Notch1 nuclear translocation and signaling in mammalian cells. We demonstrate that the D257A and the D385A PS1 mutations, which had been shown previously to block APP gamma-secretase activity, also prevent Notch1 cleavage and translocation to the nucleus but do not alter Notch1 trafficking to the cell surface. We also show that two APP gamma-secretase inhibitors block Notch1 nuclear translocation with an IC(50) similar to that reported for APP gamma-secretase. Notch1 signaling, assessed by measuring the activity of CBF1, a downstream transcription factor, was impaired but not abolished by the PS1 aspartate mutations or gamma-secretase inhibitors. Our results support the hypotheses that (a) PS1-dependent APP gamma-secretase-like enzymatic activity is critical for both APP and Notch processing and (b) the Notch1 signaling pathway remains partially activated even when Notch1 proteolytic processing and nuclear translocation are markedly inhibited. The latter is an important finding from the perspective of therapeutic treatment of Alzheimer's disease by targeting gamma

  16. Pulmonary emphysema and proteolysis: 1986

    SciTech Connect

    Taylor, J.C.; Mittman, C. )

    1987-01-01

    This book deals with the topic of pulmonary emphysema. Included are the following chapters: Abnormality of secretion of Z Alpha-1-antitrypsin, Proteases, antiproteases, and oxidants in the pathogenesis of pulmonary emphysema, Alveolar Leukocytes and protease responses with continuous vs. intermittent exposures to NO{sub 2}.

  17. Pulmonary emphysema and proteolysis. 1986

    SciTech Connect

    Taylor, J.C.; Mittman, C. )

    1987-01-01

    This book contains over 50 selections. Some of the titles are: Evaluation of Parenteral Administration of Recombinant DNA Produced Alpha-1-Antitrypsin to Primates; Properties of Mutant Forms of Alpha-1-Proteinase Inhibitor Prepared by Recombinant DNA Technology; Natural and Genetically Engineered Proteinase Inhibitors as Protective Agents against Connective Tissue Damage in an in Vitro System; and Structure, Genomic Organization and Tissue Distribution of Human Secretary Leukocyte-Protease Inhibitor (SLPI): A Potent Inhibitor of Neutrophil Elastase.

  18. Proteolysis in Plastids of Arabidopsis Thaliana: Functional Analysis of ClpS1,2,T and their Physical and Genetic Interactions with the ClpPR Protease Core Complex and Clp Chaperones

    SciTech Connect

    van Wijk, Klaas

    2009-01-12

    Chloroplasts are essential organelles required for plant growth and biomass production. They synthesize many essential secondary metabolites (e.g. hormones, isoprenoids, amino acids, etc.) and house the photosynthetic apparatus needed for conversion of light energy and CO2 into chemical energy [in the form of reduced carbohydrates, ATP and NADPH]. Thus chloroplasts are essential for life on earth and essential for production of bioenergy. Formation and maintenance of a functional chloroplast requires an extensive investment in the biogenesis and homeostasis apparatus. Protease and proteolysis play a critical role in these processes, with the Clp gene family being particularly central. Proteolysis of proteins and protein complexes in plastids is poorly understood, and is not only critical for biogenesis, adaptation and maintenance but is also important for plant development. Several years ago, the vanWijk lab identified a large and relatively abundant ClpP/R/S complex, along with ClpC1,C2 and ClpD chaperones and a putative Clp affinity modulator in plastids. So far, no substrate recognition mechanism has been determined for any Clp complex in plants. The purpose of this grant was to initiate functional analysis of three members of the Clp family.

  19. Teaching Integrity

    ERIC Educational Resources Information Center

    Saunders, Sue; Butts, Jennifer Lease

    2011-01-01

    Integrity is one of those essential yet highly ambiguous concepts. For the purpose of this chapter, integrity is defined as that combination of both attributes and actions that makes entities appear to be whole and ethical, as well as consistent. Like the concepts of leadership or wisdom or community or collaboration, integrity is a key element of…

  20. The anaphase-promoting complex works together with the SCF complex for proteolysis of the S-phase cyclin Clb6 during the transition from G1 to S phase.

    PubMed

    Wu, Shiao-Yii; Kuan, Vivian Jen-Wei; Tzeng, Yao-Wei; Schuyler, Scott C; Juang, Yue-Li

    2016-06-01

    In Saccharomyces cerevisiae, the S-phase cyclin Clb6 is expressed shortly before the G1/S transition. It has been shown that in S phase the SCF(Cdc4) ubiquitin ligase controls Clb6 proteolysis, which requires cyclin-dependent kinases activity. A Clb6-3A mutant, bearing non-phosphorylatable mutations at S6A, T39A, and S147A, was observed to be hyperstabilized in S-phase but was unstable in mitosis. In this study, we found that the APC(Cdh1) form of the Anaphase-Promoting Complex (APC) was required for Clb6 proteolysis in both early and late G1. An in vitro ubiquitination assay confirmed that Clb6 is a substrate for APC(Cdh1). A KEN box and a destruction box in the Clb6N-terminus were identified. Mutations in the KEN box (mkb) and/or the destruction box (mdb) enhanced Clb6 stability in G1. Expression of Clb6mkd, bearing both mutations in the mkb and mdb, allowed cells to bypass the late G1 arrest caused by cdc4-1. This bypass phenotype was observed to depend upon CDK phosphorylation at residues S6, T39 and S147. Compared to Clb6, overexpression of Clb6ST, bearing all five mutations of S6A, T39A, S147A, mkb and mdb in combination, had a greater effect on promoting expression of Clb2 and S-phase entry, caused a greater G2 delay and a greater defect in cell division. Swe1 was also required for bud emergence when Clb6ST was overexpressed. Our observations suggest that both APC(Cdh1) and SCF(Cdc4)-dependent proteolysis of Clb6 at the G1/S border are crucial for multiple cell cycle regulated events including proper expression of Clb2, the G1/S and G2/M cell cycle transitions and for proper completion of cell division at mitotic exit.

  1. Ablation of Prion Protein in Wild Type Human Amyloid Precursor Protein (APP) Transgenic Mice Does Not Alter The Proteolysis of APP, Levels of Amyloid-β or Pathologic Phenotype

    PubMed Central

    Baybutt, Herbert; Diack, Abigail B.; Kellett, Katherine A. B.; Piccardo, Pedro; Manson, Jean C.

    2016-01-01

    The cellular prion protein (PrPC) has been proposed to play an important role in the pathogenesis of Alzheimer’s disease. In cellular models PrPC inhibited the action of the β-secretase BACE1 on wild type amyloid precursor protein resulting in a reduction in amyloid-β (Aβ) peptides. Here we have assessed the effect of genetic ablation of PrPC in transgenic mice expressing human wild type amyloid precursor protein (line I5). Deletion of PrPC had no effect on the α- and β-secretase proteolysis of the amyloid precursor protein (APP) nor on the amount of Aβ38, Aβ40 or Aβ42 in the brains of the mice. In addition, ablation of PrPC did not alter Aβ deposition or histopathology phenotype in this transgenic model. Thus using this transgenic model we could not provide evidence to support the hypothesis that PrPC regulates Aβ production. PMID:27447728

  2. Ablation of Prion Protein in Wild Type Human Amyloid Precursor Protein (APP) Transgenic Mice Does Not Alter The Proteolysis of APP, Levels of Amyloid-β or Pathologic Phenotype.

    PubMed

    Whitehouse, Isobel J; Brown, Deborah; Baybutt, Herbert; Diack, Abigail B; Kellett, Katherine A B; Piccardo, Pedro; Manson, Jean C; Hooper, Nigel M

    2016-01-01

    The cellular prion protein (PrPC) has been proposed to play an important role in the pathogenesis of Alzheimer's disease. In cellular models PrPC inhibited the action of the β-secretase BACE1 on wild type amyloid precursor protein resulting in a reduction in amyloid-β (Aβ) peptides. Here we have assessed the effect of genetic ablation of PrPC in transgenic mice expressing human wild type amyloid precursor protein (line I5). Deletion of PrPC had no effect on the α- and β-secretase proteolysis of the amyloid precursor protein (APP) nor on the amount of Aβ38, Aβ40 or Aβ42 in the brains of the mice. In addition, ablation of PrPC did not alter Aβ deposition or histopathology phenotype in this transgenic model. Thus using this transgenic model we could not provide evidence to support the hypothesis that PrPC regulates Aβ production. PMID:27447728

  3. Integration-independent Transgenic Huntington Disease Fragment Mouse Models Reveal Distinct Phenotypes and Life Span in Vivo*

    PubMed Central

    O'Brien, Robert; DeGiacomo, Francesco; Holcomb, Jennifer; Bonner, Akilah; Ring, Karen L.; Zhang, Ningzhe; Zafar, Khan; Weiss, Andreas; Lager, Brenda; Schilling, Birgit; Gibson, Bradford W.; Chen, Sylvia; Kwak, Seung; Ellerby, Lisa M.

    2015-01-01

    The cascade of events that lead to cognitive decline, motor deficits, and psychiatric symptoms in patients with Huntington disease (HD) is triggered by a polyglutamine expansion in the N-terminal region of the huntingtin (HTT) protein. A significant mechanism in HD is the generation of mutant HTT fragments, which are generally more toxic than the full-length HTT. The protein fragments observed in human HD tissue and mouse models of HD are formed by proteolysis or aberrant splicing of HTT. To systematically investigate the relative contribution of the various HTT protein proteolysis events observed in vivo, we generated transgenic mouse models of HD representing five distinct proteolysis fragments ending at amino acids 171, 463, 536, 552, and 586 with a polyglutamine length of 148. All lines contain a single integration at the ROSA26 locus, with expression of the fragments driven by the chicken β-actin promoter at nearly identical levels. The transgenic mice N171-Q148 and N552-Q148 display significantly accelerated phenotypes and a shortened life span when compared with N463-Q148, N536-Q148, and N586-Q148 transgenic mice. We hypothesized that the accelerated phenotype was due to altered HTT protein interactions/complexes that accumulate with age. We found evidence for altered HTT complexes in caspase-2 fragment transgenic mice (N552-Q148) and a stronger interaction with the endogenous HTT protein. These findings correlate with an altered HTT molecular complex and distinct proteins in the HTT interactome set identified by mass spectrometry. In particular, we identified HSP90AA1 (HSP86) as a potential modulator of the distinct neurotoxicity of the caspase-2 fragment mice (N552-Q148) when compared with the caspase-6 transgenic mice (N586-Q148). PMID:26025364

  4. Integrated Learning

    ERIC Educational Resources Information Center

    Gnanakan, Ken

    2012-01-01

    This book upholds the idea of learning and education as a means to individual development and social empowerment. It presents a holistic picture, looking at learning as an integral part of one's social and physical life. Strongly differing from existing classroom perspectives, the book analyses integrated learning at its broadest possible…

  5. Integrated Science.

    ERIC Educational Resources Information Center

    Rainey, Larry; Miller, Roxanne Greitz

    1997-01-01

    Describes the Integrated Science program that integrates biology, earth/space science, chemistry, and physics over a three-year, spiraling sequence arranged around broad themes such as cycles, changes, patterns, and waves. Includes weekly telecasts via public television and satellite, teacher manuals, student handbooks, e-mail connections, staff…

  6. Integrative Education.

    ERIC Educational Resources Information Center

    Walker, Dean

    1995-01-01

    In contrast with subject-bound education, integrative education promotes the construction of broad "mental programs" that require students to use skills and information in new, realistic contexts. Early childhood education has long been a model of integrative education, emphasizing the whole child and offering a wide range of experiences that…

  7. Integrated care

    PubMed Central

    Gröne, Oliver; Garcia-Barbero, Mila

    2001-01-01

    Abstract The WHO European Office for Integrated Health Care Services in Barcelona is an integral part of the World Health Organizations' Regional Office for Europe. The main purpose of the Barcelona office is within the integration of services to encourage and facilitate changes in health care services in order to promote health and improve management and patient satisfaction by working for quality, accessibility, cost-effectiveness and participation. This position paper outlines the need for Integrated Care from a European perspective, provides a theoretical framework for the meaning of Integrated Care and its strategies and summarizes the programmes of the office that will support countries in the WHO European Region to improve health services. PMID:16896400

  8. Integrative psychotherapy.

    PubMed

    Kozarić-Kovacić, Dragica

    2008-09-01

    The main purposes of the article are to present the history of integration in psychotherapy, the reasons of the development integrative approaches, and the approaches to integration in psychotherapy. Three approaches to integration in psychotherapy exist: theoretical integration, theoretical eclecticism, and common factors in different psychotherapeutic trends. In integrative psychotherapy, the basic epistemology, theory, and clinical practice are based on the phenomenology, field theory, holism, dialogue, and co-creation of dialogue in the therapeutic relationship. The main criticism is that integrative psychotherapy suffers from confusion and many unresolved controversies. It is difficult to theoretically and methodologically define the clinically applied model that is based on such a different epistemological and theoretical presumptions. Integrative psychotherapy is a synthesis of humanistic psychotherapy, object relations theory, and psychoanalytical self psychology. It focuses on the dynamics and potentials of human relationships, with a goal of changing the relations and understanding internal and external resistances. The process of integrative psychotherapy is primarily focused on the developmental-relational model and co-creation of psychotherapeutic relationship as a single interactive event, which is not unilateral, but rather a joint endeavor by both the therapist and the patient/client. The need for a relationship is an important human need and represents a process of attunement that occurs as a response to the need for a relationship, a unique interpersonal contact between two people. If this need is not met, it manifests with the different feelings and various defenses. To meet this need, we need to have another person with whom we can establish a sensitive, attuned relationship. Thus, the therapist becomes this person who tries to supplement what the person did not receive. Neuroscience can be a source of integration through different therapies. We

  9. Osteoconductive carriers for integrated bone repair

    PubMed Central

    Ganey, Timothy; Hutton, William; Meisel, Hans Jörg

    2009-01-01

    Successful bone repair is judged in achieving restitution of space and mechanical integrity, and in regaining function. When the biology or anatomy are insufficient to attain a full repair, therapeutic use of graft material has been used to omit compliance features such as strain tolerance, reduced stiffness, and attenuated strength, and instead promote primary or membranous-type bone formation within the physical approximation of a graft material. The challenge of most conductive materials is that they emerge from a static platform and in placement force the living system to adapt to placement, dimension, different properties, and eventually are only successful in degradation and replacement, or in integration. The synergy and interdependency between adhesion, ECM, and proteolysis are important concepts that must be understood to engineer scaffolds capable of holding up to standards which are more than cell decoration. Moreover, the reactive specificity to loading, degradation, therapeutic delivery during absorption remains a key aim of both academic and industrial designs. Achieving conductivity comes with challenges of best fit integration, delivery, and in integrated modeling. The more liquid is the delivery, the more modular the components, and adaptive the matrix to meeting the intended application, the more likely that the conductivity will not be excluded by the morphology of the injury site. Considerations for osteoconductive materials for bone repair and replacement have developed conceptually and advanced parallel with a better understanding of not only bone biology but of materials science. First models of material replacements utilized a reductionist-constructionist logic; define the constituents of the material in terms of its morphology and chemical composition, and then engineer material with similar content and properties as a means of accommodating a replacement. Unfortunately for biologic systems, empiric formulation is insufficient to promote

  10. Functional Integration

    NASA Astrophysics Data System (ADS)

    Cartier, Pierre; DeWitt-Morette, Cecile

    2010-06-01

    Acknowledgements; List symbols, conventions, and formulary; Part I. The Physical and Mathematical Environment: 1. The physical and mathematical environment; Part II. Quantum Mechanics: 2. First lesson: gaussian integrals; 3. Selected examples; 4. Semiclassical expansion: WKB; 5. Semiclassical expansion: beyond WKB; 6. Quantum dynamics: path integrals and operator formalism; Part III. Methods from Differential Geometry: 7. Symmetries; 8. Homotopy; 9. Grassmann analysis: basics; 10. Grassmann analysis: applications; 11. Volume elements, divergences, gradients; Part IV. Non-Gaussian Applications: 12. Poisson processes in physics; 13. A mathematical theory of Poisson processes; 14. First exit time: energy problems; Part V. Problems in Quantum Field Theory: 15. Renormalization 1: an introduction; 16. Renormalization 2: scaling; 17. Renormalization 3: combinatorics; 18. Volume elements in quantum field theory Bryce DeWitt; Part VI. Projects: 19. Projects; Appendix A. Forward and backward integrals: spaces of pointed paths; Appendix B. Product integrals; Appendix C. A compendium of gaussian integrals; Appendix D. Wick calculus Alexander Wurm; Appendix E. The Jacobi operator; Appendix F. Change of variables of integration; Appendix G. Analytic properties of covariances; Appendix H. Feynman's checkerboard; Bibliography; Index.

  11. Functional Integration

    NASA Astrophysics Data System (ADS)

    Cartier, Pierre; DeWitt-Morette, Cecile

    2006-11-01

    Acknowledgements; List symbols, conventions, and formulary; Part I. The Physical and Mathematical Environment: 1. The physical and mathematical environment; Part II. Quantum Mechanics: 2. First lesson: gaussian integrals; 3. Selected examples; 4. Semiclassical expansion: WKB; 5. Semiclassical expansion: beyond WKB; 6. Quantum dynamics: path integrals and operator formalism; Part III. Methods from Differential Geometry: 7. Symmetries; 8. Homotopy; 9. Grassmann analysis: basics; 10. Grassmann analysis: applications; 11. Volume elements, divergences, gradients; Part IV. Non-Gaussian Applications: 12. Poisson processes in physics; 13. A mathematical theory of Poisson processes; 14. First exit time: energy problems; Part V. Problems in Quantum Field Theory: 15. Renormalization 1: an introduction; 16. Renormalization 2: scaling; 17. Renormalization 3: combinatorics; 18. Volume elements in quantum field theory Bryce DeWitt; Part VI. Projects: 19. Projects; Appendix A. Forward and backward integrals: spaces of pointed paths; Appendix B. Product integrals; Appendix C. A compendium of gaussian integrals; Appendix D. Wick calculus Alexander Wurm; Appendix E. The Jacobi operator; Appendix F. Change of variables of integration; Appendix G. Analytic properties of covariances; Appendix H. Feynman's checkerboard; Bibliography; Index.

  12. Integrative neuroscience.

    PubMed

    Gordon, Evian

    2003-07-01

    A fundamental impediment to an "Integrative Neuroscience" is the sense that scientists building models at one particular scale often see that scale as the epicentre of all brain function. This fragmentation has begun to change in a very distinctive way. Multidisciplinary efforts have provided the impetus to break down the boundaries and encourage a freer exchange of information across disciplines and scales. Despite huge deficits of knowledge, sufficient facts about the brain already exist, for an Integrative Neuroscience to begin to lift us clear of the jungle of detail, and shed light upon the workings of the brain as a system. Integrations of brain theory can be tested using judicious paradigm designs and measurement of temporospatial activity reflected in brain imaging technologies. However, to test realistically these new hypotheses requires consistent findings of the normative variability in very large numbers of control subjects, coupled with high sensitivity and specificity of findings in psychiatric disorders. Most importantly, these findings need to be analyzed and modeled with respect to the fundamental mechanisms underlying these measures. Without this convergence of theory, databases, and methodology (including across scale physiologically realistic numerical models), the clinical utility of brain imaging technologies in psychiatry will be significantly impeded. The examples provided in this paper of integration of theory, temporospatial integration of neuroimaging technologies, and a numerical simulation of brain function, bear testimony to the ongoing conversion of an Integrative Neuroscience from an exemplar status into reality.

  13. Classical integrability

    NASA Astrophysics Data System (ADS)

    Torrielli, Alessandro

    2016-08-01

    We review some essential aspects of classically integrable systems. The detailed outline of the sections consists of: 1. Introduction and motivation, with historical remarks; 2. Liouville theorem and action-angle variables, with examples (harmonic oscillator, Kepler problem); 3. Algebraic tools: Lax pairs, monodromy and transfer matrices, classical r-matrices and exchange relations, non-ultralocal Poisson brackets, with examples (non-linear Schrödinger model, principal chiral field); 4. Features of classical r-matrices: Belavin–Drinfeld theorems, analyticity properties, and lift of the classical structures to quantum groups; 5. Classical inverse scattering method to solve integrable differential equations: soliton solutions, spectral properties and the Gel’fand–Levitan–Marchenko equation, with examples (KdV equation, Sine-Gordon model). Prepared for the Durham Young Researchers Integrability School, organised by the GATIS network. This is part of a collection of lecture notes.

  14. Classical integrability

    NASA Astrophysics Data System (ADS)

    Torrielli, Alessandro

    2016-08-01

    We review some essential aspects of classically integrable systems. The detailed outline of the sections consists of: 1. Introduction and motivation, with historical remarks; 2. Liouville theorem and action-angle variables, with examples (harmonic oscillator, Kepler problem); 3. Algebraic tools: Lax pairs, monodromy and transfer matrices, classical r-matrices and exchange relations, non-ultralocal Poisson brackets, with examples (non-linear Schrödinger model, principal chiral field); 4. Features of classical r-matrices: Belavin-Drinfeld theorems, analyticity properties, and lift of the classical structures to quantum groups; 5. Classical inverse scattering method to solve integrable differential equations: soliton solutions, spectral properties and the Gel’fand-Levitan-Marchenko equation, with examples (KdV equation, Sine-Gordon model). Prepared for the Durham Young Researchers Integrability School, organised by the GATIS network. This is part of a collection of lecture notes.

  15. The Integrated Hazard Analysis Integrator

    NASA Technical Reports Server (NTRS)

    Morris, A. Terry; Massie, Michael J.

    2009-01-01

    Hazard analysis addresses hazards that arise in the design, development, manufacturing, construction, facilities, transportation, operations and disposal activities associated with hardware, software, maintenance, operations and environments. An integrated hazard is an event or condition that is caused by or controlled by multiple systems, elements, or subsystems. Integrated hazard analysis (IHA) is especially daunting and ambitious for large, complex systems such as NASA s Constellation program which incorporates program, systems and element components that impact others (International Space Station, public, International Partners, etc.). An appropriate IHA should identify all hazards, causes, controls and verifications used to mitigate the risk of catastrophic loss of crew, vehicle and/or mission. Unfortunately, in the current age of increased technology dependence, there is the tendency to sometimes overlook the necessary and sufficient qualifications of the integrator, that is, the person/team that identifies the parts, analyzes the architectural structure, aligns the analysis with the program plan and then communicates/coordinates with large and small components, each contributing necessary hardware, software and/or information to prevent catastrophic loss. As viewed from both Challenger and Columbia accidents, lack of appropriate communication, management errors and lack of resources dedicated to safety were cited as major contributors to these fatalities. From the accident reports, it would appear that the organizational impact of managers, integrators and safety personnel contributes more significantly to mission success and mission failure than purely technological components. If this is so, then organizations who sincerely desire mission success must put as much effort in selecting managers and integrators as they do when designing the hardware, writing the software code and analyzing competitive proposals. This paper will discuss the necessary and

  16. Information Integrity

    ERIC Educational Resources Information Center

    Graves, Eric

    2013-01-01

    This dissertation introduces the concept of Information Integrity, which is the detection and possible correction of information manipulation by any intermediary node in a communication system. As networks continue to grow in complexity, information theoretic security has failed to keep pace. As a result many parties whom want to communicate,…

  17. Numerical Integration

    ERIC Educational Resources Information Center

    Sozio, Gerry

    2009-01-01

    Senior secondary students cover numerical integration techniques in their mathematics courses. In particular, students would be familiar with the "midpoint rule," the elementary "trapezoidal rule" and "Simpson's rule." This article derives these techniques by methods which secondary students may not be familiar with and an approach that…

  18. Regulation of haemolysin (VvhA) production by ferric uptake regulator (Fur) in Vibrio vulnificus: repression of vvhA transcription by Fur and proteolysis of VvhA by Fur-repressive exoproteases.

    PubMed

    Lee, Hyun-Jung; Kim, Jeong-A; Lee, Mi-Ae; Park, Soon-Jung; Lee, Kyu-Ho

    2013-05-01

    VvhA produced by Vibrio vulnificus exhibits cytolytic activity to human cells including erythrocytes. Since haemolysis by VvhA may provide iron for bacterial growth and pathogenicity, we investigated the expression of VvhA to elucidate the regulatory roles of Fur, a major transcription factor controlling iron-homeostasis. Fur repressed the transcription of vvhBA operon via binding to the promoter region. However, haemolysin content and haemolytic activity were lowered in cell-free supernatant of fur mutant. This discrepancy between the levels of vvhA transcript and VvhA protein in fur mutant was caused by exoproteolytic activities of the elastase VvpE and another metalloprotease VvpM, which were also regulated by Fur. vvpE gene expression was repressed by Fur via binding to the Fur-box homologous region. Regulation of VvpM expression by Fur did not occur at the level of vvpM transcription. In vitro proteolysis assays showed that both proteases efficiently degraded VvhA. In addition, the extracellular levels of VvhA were higher in culture supernatants of vvpE or vvpM mutants than in the wild type. Thus this study demonstrates that Fur regulates haemolysin production at the transcription level of the vvhBA operon and at the post-translation level by regulating the expressions of two VvhA-degrading exoproteases, VvpE and VvpM.

  19. Optimized verification method for detection of an albumin-sulfur mustard adduct at Cys(34) using a hybrid quadrupole time-of-flight tandem mass spectrometer after direct plasma proteolysis.

    PubMed

    John, Harald; Siegert, Markus; Gandor, Felix; Gawlik, Michael; Kranawetvogl, Andreas; Karaghiosoff, Konstantin; Thiermann, Horst

    2016-02-26

    The vesicant sulfur mustard (SM) is a banned chemical warfare agent that is controlled by the Organisation for the Prohibition of Chemical Weapons (OPCW). Bioanalytical procedures are mandatory for proving an alleged use and incorporation of SM into the body. We herein present the development and application of a novel optimized procedure suitable for qualitative verification analysis of plasma targeting the SM-adduct of human serum albumin (HSA) alkylated at the cysteine(34) residue. Diluted human plasma is directly mixed with pronase in an ultrafiltration device (10kDa cut-off) for proteolysis (4h, 37°C). Following ultrafiltration the filtrate is diluted and analyzed by microbore liquid chromatography-electrospray ionization high resolution tandem-mass spectrometry (μLC-ESI HR MS/MS) targeting the alkylated dipeptide hydroxyethylthioethyl-CysPro (HETE-CP). A hybrid quadrupole time-of-flight mass spectrometer provided high mass spectrometric resolution in the MS/MS mode enabling highest selectivity and sensitivity (lower limit of detection corresponding to 9.8nM SM in plasma). Kinetics of HETE-CP formation from heparin-, citrate-, and EDTA-plasma as well as serum are presented and the influence of different EDTA and pronase concentrations was characterized. The novel procedure was applied to plasma samples provided by the OPCW as well as to patientś plasma derived from real cases of SM-poisoning.

  20. Optimized verification method for detection of an albumin-sulfur mustard adduct at Cys(34) using a hybrid quadrupole time-of-flight tandem mass spectrometer after direct plasma proteolysis.

    PubMed

    John, Harald; Siegert, Markus; Gandor, Felix; Gawlik, Michael; Kranawetvogl, Andreas; Karaghiosoff, Konstantin; Thiermann, Horst

    2016-02-26

    The vesicant sulfur mustard (SM) is a banned chemical warfare agent that is controlled by the Organisation for the Prohibition of Chemical Weapons (OPCW). Bioanalytical procedures are mandatory for proving an alleged use and incorporation of SM into the body. We herein present the development and application of a novel optimized procedure suitable for qualitative verification analysis of plasma targeting the SM-adduct of human serum albumin (HSA) alkylated at the cysteine(34) residue. Diluted human plasma is directly mixed with pronase in an ultrafiltration device (10kDa cut-off) for proteolysis (4h, 37°C). Following ultrafiltration the filtrate is diluted and analyzed by microbore liquid chromatography-electrospray ionization high resolution tandem-mass spectrometry (μLC-ESI HR MS/MS) targeting the alkylated dipeptide hydroxyethylthioethyl-CysPro (HETE-CP). A hybrid quadrupole time-of-flight mass spectrometer provided high mass spectrometric resolution in the MS/MS mode enabling highest selectivity and sensitivity (lower limit of detection corresponding to 9.8nM SM in plasma). Kinetics of HETE-CP formation from heparin-, citrate-, and EDTA-plasma as well as serum are presented and the influence of different EDTA and pronase concentrations was characterized. The novel procedure was applied to plasma samples provided by the OPCW as well as to patientś plasma derived from real cases of SM-poisoning. PMID:26449527

  1. Oral Administration of Aloe vera (L.) Burm. f. (Xanthorrhoeaceae) and Honey Improves the Host Body Composition and Modulates Proteolysis Through Reduction of Tumor Progression and Oxidative Stress in Rats.

    PubMed

    Tomasin, Rebeka; de Andrade, Rafael Siqueira; Gomes-Marcondes, Maria Cristina Cintra

    2015-10-01

    Oxidative stress has a dual role in cancer; it is linked with tumorigenic events and host wasting, as well as senescence and apoptosis. Researchers have demonstrated the importance of coadjuvant therapies in cancer treatment, and Aloe vera and honey have immunomodulatory, anticancer, and antioxidant properties. The preventive and therapeutic effects of Aloe vera (L.) Burm. f. (Xanthorrhoeaceae) and honey in tumor progression and host wasting were analyzed in Walker 256 carcinoma-bearing rats. The animals were distributed into the following groups: C=control-untreated, W=tumor-untreated, WA=treated after tumor induction, A=control-treated, AW=treated before tumor induction, and AWA=treated before and after tumor induction. Proteolysis and oxidative stress were analyzed in the tumor, liver, muscle, and myocardial tissues. The results suggest that the Aloe vera and honey treatment affect the tumor and host by different mechanisms; the treatment-modulated host wasting and cachexia, whereas it promoted oxidative stress and damage in tumor tissues, particularly in a therapeutic context (WA).

  2. Integrating maps requires integrated data

    SciTech Connect

    Weissenbach, J.; Bentolila, S.

    1996-06-01

    The molecular genetics movement has proceeded admirably thanks to new technologies such as polymerase chain reaction (PCR) sequencing. However, the integration of mapping data has not yet been achieved. This article discussing several problem areas and serves as a reminder of the urgency of such concerns.

  3. RADIATION INTEGRATOR

    DOEpatents

    Glass, F.M.; Wilson, H.N.

    1959-02-17

    Radiation detecting and measuring systems, particularly a compact, integrating, background monitor, are discussed. One of the principal features of the system is the use of an electrometer tube where the input of the tube is directly connected to an electrode of the radiation detector and a capacitor is coupled to the tube input. When a predetermined quantity of radiation has been integrated, a trigger signal is fed to a recorder and a charge is delivered to the capacitor to render the tube inoperative. The capacitor is then recharged for the next period of operation. With this arrangement there is a substantial reduction in lead lengths and the principal components may be enclosed and hermetically sealed to insure low leakage.

  4. Integrated Fluorescence

    NASA Technical Reports Server (NTRS)

    Tuma, Margaret (Inventor); Gruhlke, Russell W. (Inventor)

    1998-01-01

    A detection method is integrated with a filtering method and an enhancement method to create a fluorescence sensor that can be miniaturized. The fluorescence sensor comprises a thin film geometry including a waveguide layer, a metal film layer and sensor layer. The thin film geometry of the fluorescence sensor allows the detection of fluorescent radiation over a narrow wavelength interval. This enables wavelength discrimination and eliminates the detection of unwanted light from unknown or spurious sources.

  5. Synaptobrevin/vesicle-associated membrane protein (VAMP) of Aplysia californica: structure and proteolysis by tetanus toxin and botulinal neurotoxins type D and F.

    PubMed Central

    Yamasaki, S; Hu, Y; Binz, T; Kalkuhl, A; Kurazono, H; Tamura, T; Jahn, R; Kandel, E; Niemann, H

    1994-01-01

    Synaptobrevin/vesicle-associated membrane protein (VAMP) and syntaxin are potential vesicle donor and target membrane receptors of a docking complex that requires N-ethylmaleimide-sensitive factor (NSF) and soluble NSF-attachment proteins as soluble factors for vesicle fusion with target membranes. Members of this docking complex are the target of clostridial neurotoxins that act as zinc-dependent proteases. Molecular cloning of the Aplysia californica synaptobrevin cDNA revealed a 180-residue polypeptide (M(r), 19,745) with a central transmembrane region and an atypically large C-terminal intravesicular domain. This polypeptide integrates into membranes at both the co- and posttranslational level, as shown by modification of an artificially introduced N-glycosylation site. The soluble and membrane-anchored forms of synaptobrevin are cleaved by the light chains of the botulinal toxins type D and F and by tetanus toxin involving the peptide bonds Lys49-Ile50, Gln48-Lys49, and Gln66-Phe67, respectively. The active center of teh tetanus toxin light chain was identified by site-specific mutagenesis. His233, His237, Glu234, and Glu270/271 are essential to this proteolytic activity. Modification of histidine residues resulted in loss of zinc binding, whereas a replacement of Glu234 only slightly reduced the zinc content. Images PMID:8197120

  6. STRUCTURE OF THE EGF RECEPTOR TRANSACTIVATION CIRCUIT INTEGRATES MULTIPLE SIGNALS WITH CELL CONTEXT

    PubMed Central

    Joslin, Elizabeth J.; Shankaran, Harish; Opresko, Lee K.; Bollinger, Nikki; Lauffenburger, Douglas A.; Wiley, H. Steven

    2012-01-01

    Summary Transactivation of the epidermal growth factor receptor (EGFR) is thought to be a process by which a variety of cellular inputs can be integrated into a single signaling pathway through either stimulated proteolysis (shedding) of membrane-anchored EGFR ligands or by modification of the activity of the EGFR. As a first step towards building a predictive model of the EGFR transactivation circuit, we quantitatively defined how signals from multiple agonists were integrated both upstream and downstream of the EGFR to regulate extracellular signal regulated kinase (ERK) activity in human mammary epithelial cells. By using a “non-binding” reporter of ligand shedding, we found that transactivation triggers a positive feedback loop from ERK back to the EGFR such that ligand shedding drives EGFR-stimulated ERK that in turn drives further ligand shedding. Importantly, activated Ras and ERK levels were nearly linear functions of ligand shedding and the effect of multiple, sub-saturating inputs was additive. Simulations showed that ERK-mediated feedback through ligand shedding resulted in a stable steady-state level of activated ERK, but also showed that the extracellular environment can modulate the level of feedback. Our results suggest that the transactivation circuit acts as a context-dependent integrator and amplifier of multiple extracellular signals and that signal integration can effectively occur at multiple points in the EGFR pathway. PMID:20458382

  7. ACCELERATION INTEGRATOR

    DOEpatents

    Pope, K.E.

    1958-01-01

    This patent relates to an improved acceleration integrator and more particularly to apparatus of this nature which is gyrostabilized. The device may be used to sense the attainment by an airborne vehicle of a predetermined velocitv or distance along a given vector path. In its broad aspects, the acceleration integrator utilizes a magnetized element rotatable driven by a synchronous motor and having a cylin drical flux gap and a restrained eddy- current drag cap deposed to move into the gap. The angular velocity imparted to the rotatable cap shaft is transmitted in a positive manner to the magnetized element through a servo feedback loop. The resultant angular velocity of tae cap is proportional to the acceleration of the housing in this manner and means may be used to measure the velocity and operate switches at a pre-set magnitude. To make the above-described dcvice sensitive to acceleration in only one direction the magnetized element forms the spinning inertia element of a free gyroscope, and the outer housing functions as a gimbal of a gyroscope.

  8. Mitotic lifecycle of chromosomal 3xHMG-box proteins and the role of their N-terminal domain in the association with rDNA loci and proteolysis.

    PubMed

    Antosch, Martin; Schubert, Veit; Holzinger, Philipp; Houben, Andreas; Grasser, Klaus D

    2015-12-01

    The high mobility group (HMG)-box is a DNA-binding domain characteristic of various eukaryotic DNA-binding proteins. 3xHMG-box proteins (containing three copies of the HMG-box domain and a unique basic N-terminal domain) are specific for plants and the Arabidopsis genome encodes two versions termed 3xHMG-box1 and 3xHMG-box2, whose expression is cell cycle-dependent, peaking during mitosis. Here, we analysed in detail the spatiotemporal expression, subcellular localisation and chromosome association of the Arabidopsis thaliana 3xHMG-box proteins. Live cell imaging and structured illumination microscopy revealed that the expression of the 3xHMG-box proteins is induced in late G2 phase of the cell cycle and upon nuclear envelope breakdown in prophase they rapidly associate with the chromosomes. 3xHMG-box1 associates preferentially with 45S rDNA loci and the basic N-terminal domain is involved in the targeting of rDNA loci. Shortly after mitosis the 3xHMG-box proteins are degraded and an N-terminal destruction-box mediates the proteolysis. Ectopic expression/localisation of 3xHMG-box1 in interphase nuclei results in reduced plant growth and various developmental defects including early bolting and abnormal flower morphology. The remarkable conservation of 3xHMG-box proteins within the plant kingdom, their characteristic expression during mitosis, and their striking association with chromosomes, suggest that they play a role in the organisation of plant mitotic chromosomes. PMID:26213803

  9. Ubiquitin-specific Protease 7 Regulates Nucleotide Excision Repair through Deubiquitinating XPC Protein and Preventing XPC Protein from Undergoing Ultraviolet Light-induced and VCP/p97 Protein-regulated Proteolysis*

    PubMed Central

    He, Jinshan; Zhu, Qianzheng; Wani, Gulzar; Sharma, Nidhi; Han, Chunhua; Qian, Jiang; Pentz, Kyle; Wang, Qi-en; Wani, Altaf A.

    2014-01-01

    Ubiquitin specific protease 7 (USP7) is a known deubiquitinating enzyme for tumor suppressor p53 and its downstream regulator, E3 ubiquitin ligase Mdm2. Here we report that USP7 regulates nucleotide excision repair (NER) via deubiquitinating xeroderma pigmentosum complementation group C (XPC) protein, a critical damage recognition factor that binds to helix-distorting DNA lesions and initiates NER. XPC is ubiquitinated during the early stage of NER of UV light-induced DNA lesions. We demonstrate that transiently compromising cellular USP7 by siRNA and chemical inhibition leads to accumulation of ubiquitinated forms of XPC, whereas complete USP7 deficiency leads to rapid ubiquitin-mediated XPC degradation upon UV irradiation. We show that USP7 physically interacts with XPC in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytically inactive or interaction-defective mutants, reduces the ubiquitinated forms of XPC. Importantly, USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination assays in vitro. We further show that valosin-containing protein (VCP)/p97 is involved in UV light-induced XPC degradation in USP7-deficient cells. VCP/p97 is readily recruited to DNA damage sites and colocalizes with XPC. Chemical inhibition of the activity of VCP/p97 ATPase causes an increase in ubiquitinated XPC on DNA-damaged chromatin. Moreover, USP7 deficiency severely impairs the repair of cyclobutane pyrimidine dimers and, to a lesser extent, affects the repair of 6-4 photoproducts. Taken together, our findings uncovered an important role of USP7 in regulating NER via deubiquitinating XPC and by preventing its VCP/p97-regulated proteolysis. PMID:25118285

  10. Nonplanar integrability

    NASA Astrophysics Data System (ADS)

    Carlson, Warren; de Mello Koch, Robert; Lin, Hai

    2011-03-01

    In this article we study operators with a dimension Δ ˜ O( N) and show that simple analytic expressions for the action of the dilatation operator can be found. The operators we consider are restricted Schur polynomials. There are two distinct classes of operators that we consider: operators labeled by Young diagrams with two long columns or two long rows. The main complication in working with restricted Schur polynomials is in building a projector from a given S n+ m irreducible representation to an S n × S m irreducible representation (both specified by the labels of the restricted Schur polynomial). We give an explicit construction of these projectors by reducing it to the simple problem of addition of angular momentum in ordinary non-relativistic quantum mechanics. The diagonalization of the dilatation operator reduces to solving three term recursion relations. The fact that the recursion relations have only three terms is a direct consequence of the weak mixing at one loop of the restricted Schur polynomials. The recursion relations can be solved exactly in terms of symmetric Kravchuk polynomials or in terms of Clebsch-Gordan coefficients. This proves that the dilatation operator reduces to a decoupled set of harmonic oscillators and therefore it is integrable.

  11. (67/68)Ga-labeling agent that liberates (67/68)Ga-NOTA-methionine by lysosomal proteolysis of parental low molecular weight polypeptides to reduce renal radioactivity levels.

    PubMed

    Uehara, Tomoya; Rokugawa, Takemi; Kinoshita, Mai; Nemoto, Souki; Fransisco Lazaro, Guerra Gomez; Hanaoka, Hirofumi; Arano, Yasushi

    2014-11-19

    The renal localization of gallium-67 or gallium-68 ((67/68)Ga)-labeled low molecular weight (LMW) probes such as peptides and antibody fragments constitutes a problem in targeted imaging. Wu et al. previously showed that (67)Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bz-NOTA)-conjugated methionine ((67)Ga-NOTA-Met) was rapidly excreted from the kidney in urine following lysosomal proteolysis of the parental (67)Ga-NOTA-Bz-SCN-disulfide-stabilized Fv fragment (Bioconjugate Chem., (1997) 8, 365-369). In the present study, a new (67/68)Ga-labeling reagent for LMW probes that liberates (67/68)Ga-NOTA-Met was designed, synthesized, and evaluated using longer-lived (67)Ga in order to reduce renal radioactivity levels. We employed a methionine-isoleucine (MI) dipeptide bond as the cleavable linkage. The amine residue of MI was coupled with SCN-Bz-NOTA for (67)Ga-labeling, while the carboxylic acid residue of MI was derivatized to maleimide for antibody conjugation in order to synthesize NOTA-MI-Mal. A Fab fragment of the anti-Her2 antibody was thiolated with iminothiolane, and NOTA-MI-Mal was conjugated with the antibody fragment by maleimide-thiol chemistry. The Fab fragment was also conjugated with SCN-Bz-NOTA (NOTA-Fab) for comparison. (67)Ga-NOTA-MI-Fab was obtained at radiochemical yields of over 95% and was stable in murine serum for 24 h. In the biodistribution study using normal mice, (67)Ga-NOTA-MI-Fab registered significantly lower renal radioactivity levels from 1 to 6 h postinjection than those of (67)Ga-NOTA-Fab. An analysis of urine samples obtained 6 h after the injection of (67)Ga-NOTA-MI-Fab showed that the majority of radioactivity was excreted as (67)Ga-NOTA-Met. In the biodistribution study using tumor-bearing mice, the tumor to kidney ratios of (67)Ga-NOTA-MI-Fab were 4 times higher (6 h postinjection) than those of (67)Ga-NOTA-Fab. Although further studies including the structure of radiometabolites and

  12. Defective lysosomal proteolysis and axonal transport are early pathogenic events that worsen with age leading to increased APP metabolism and synaptic Abeta in transgenic APP/PS1 hippocampus

    PubMed Central

    2012-01-01

    Background Axonal pathology might constitute one of the earliest manifestations of Alzheimer disease. Axonal dystrophies were observed in Alzheimer’s patients and transgenic models at early ages. These axonal dystrophies could reflect the disruption of axonal transport and the accumulation of multiple vesicles at local points. It has been also proposed that dystrophies might interfere with normal intracellular proteolysis. In this work, we have investigated the progression of the hippocampal pathology and the possible implication in Abeta production in young (6 months) and aged (18 months) PS1(M146L)/APP(751sl) transgenic mice. Results Our data demonstrated the existence of a progressive, age-dependent, formation of axonal dystrophies, mainly located in contact with congophilic Abeta deposition, which exhibited tau and neurofilament hyperphosphorylation. This progressive pathology was paralleled with decreased expression of the motor proteins kinesin and dynein. Furthermore, we also observed an early decrease in the activity of cathepsins B and D, progressing to a deep inhibition of these lysosomal proteases at late ages. This lysosomal impairment could be responsible for the accumulation of LC3-II and ubiquitinated proteins within axonal dystrophies. We have also investigated the repercussion of these deficiencies on the APP metabolism. Our data demonstrated the existence of an increase in the amyloidogenic pathway, which was reflected by the accumulation of hAPPfl, C99 fragment, intracellular Abeta in parallel with an increase in BACE and gamma-secretase activities. In vitro experiments, using APPswe transfected N2a cells, demonstrated that any imbalance on the proteolytic systems reproduced the in vivo alterations in APP metabolism. Finally, our data also demonstrated that Abeta peptides were preferentially accumulated in isolated synaptosomes. Conclusion A progressive age-dependent cytoskeletal pathology along with a reduction of lysosomal and, in minor

  13. Elliptic integrals: Symmetry and symbolic integration

    SciTech Connect

    Carlson, B.C. |

    1997-12-31

    Computation of elliptic integrals, whether numerical or symbolic, has been aided by the contributions of Italian mathematicians. Tricomi had a strong interest in iterative algorithms for computing elliptic integrals and other special functions, and his writings on elliptic functions and elliptic integrals have taught these subjects to many modern readers (including the author). The theory of elliptic integrals began with Fagnano`s duplication theorem, a generalization of which is now used iteratively for numerical computation in major software libraries. One of Lauricella`s multivariate hypergeometric functions has been found to contain all elliptic integrals as special cases and has led to the introduction of symmetric canonical forms. These forms provide major economies in new integral tables and offer a significant advantage also for symbolic integration of elliptic integrals. Although partly expository the present paper includes some new proofs and proposes a new procedure for symbolic integration.

  14. Impact of Silk Biomaterial Structure on Proteolysis

    PubMed Central

    Brown, Joseph; Lu, Chia-Li; Coburn, Jeannine; Kaplan, David L.

    2014-01-01

    The goal of this study was to determine the impact of silk biomaterial structure (e.g., solution, hydrogel, film) on proteolytic susceptibility. In vitro enzymatic degradation of silk fibroin hydrogels and films was studied using a variety of proteases, including proteinase K, protease XIV, α-chymotrypsin, collagenase, matrix metalloproteinase-1 (MMP-1) and MMP-2. Hydrogels were used to assess bulk degradation while films were used to assess surface degradation. Weight loss, secondary structure determined by Fourier Transform Infrared (FT-IR) spectroscopy and degradation products analyzed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to evaluate degradation through five days. Silk films were significantly degraded by proteinase K, while silk hydrogels were degraded more extensively by protease XIV and proteinase K. Collagenase preferentially degraded the β-sheet content in hydrogels while protease XIV and α-chymotrypsin degraded the amorphous structures. MMP-1 and MMP-2 degraded silk fibroin in solution resulting in a decrease in peptide fragment sizes over time. The link between primary sequence mapping with protease susceptibility provides insight into the role of secondary structure in impacting proteolytic access by comparing solution vs. solid state proteolytic susceptibility. PMID:25240984

  15. [Proteolysis of milk proteins in young children].

    PubMed

    Nikolaevskaia, V R; Chernikov, M P

    1984-01-01

    Distinct pancreatic activity was observed in duodenal chyme of newly born children. Rapid evacuation of protein from stomach was noted in artificial feeding. The tryptic activity was increased in duodenal contents after the introduction of artificial feeding. Amino acids were mainly liberated in the course of cavitary digestion of proteins. Some of the amino acids (cysteine, phenylalanine) were absorbed in a bound form. PMID:6528540

  16. Evaluating Simultaneous Integrals

    ERIC Educational Resources Information Center

    Kwong, Harris

    2012-01-01

    Many integrals require two successive applications of integration by parts. During the process, another integral of similar type is often invoked. We propose a method which can integrate these two integrals simultaneously. All we need is to solve a linear system of equations.

  17. Lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) is a substrate of cathepsin-F, a cysteine protease mutated in type-B-Kufs-disease.

    PubMed

    Peters, Judith; Rittger, Andrea; Weisner, Rebecca; Knabbe, Johannes; Zunke, Friederike; Rothaug, Michelle; Damme, Markus; Berkovic, Samuel F; Blanz, Judith; Saftig, Paul; Schwake, Michael

    2015-02-13

    The lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) has been identified as a receptor for enterovirus 71 uptake and mannose-6-phosphate-independent lysosomal trafficking of the acid hydrolase β-glucocerebrosidase. Here we show that LIMP-2 undergoes proteolytic cleavage mediated by lysosomal cysteine proteases. Heterologous expression and in vitro studies suggest that cathepsin-F is mainly responsible for the lysosomal processing of wild-type LIMP-2. Furthermore, examination of purified lysosomes revealed that LIMP-2 undergoes proteolysis in vivo. Mutations in the gene encoding cathepsin-F (CTSF) have recently been associated with type-B-Kufs-disease, an adult form of neuronal ceroid-lipofuscinosis. In this study we show that disease-causing cathepsin-F mutants fail to cleave LIMP-2. Our findings provide evidence that LIMP-2 represents an in vivo substrate of cathepsin-F with relevance for understanding the pathophysiology of type-B-Kufs-disease.

  18. TIPs for Technology Integration.

    ERIC Educational Resources Information Center

    Mandell, Susan; Sorge, Dennis H.; Russell, James D.

    2002-01-01

    Discusses the role of the teacher in effectively using technology in education based on the Technology Integration Project (TIP). Topics include why use technology; types of computer software; how to select software; software integration strategies; and effectively planning lessons that integrate the chosen software and integration strategy. (LRW)

  19. Slimplectic Integrators: Variational Integrators for Nonconservative systems

    NASA Astrophysics Data System (ADS)

    Tsang, David

    2016-05-01

    Symplectic integrators are widely used for long-term integration of conservative astrophysical problems due to their ability to preserve the constants of motion; however, they cannot in general be applied in the presence of nonconservative interactions. Here we present the “slimplectic” integrator, a new type of numerical integrator that shares many of the benefits of traditional symplectic integrators yet is applicable to general nonconservative systems. We utilize a fixed-time-step variational integrator formalism applied to a newly developed principle of stationary nonconservative action (Galley, 2013, Galley et al 2014). As a result, the generalized momenta and energy (Noether current) evolutions are well-tracked. We discuss several example systems, including damped harmonic oscillators, Poynting–Robertson drag, and gravitational radiation reaction, by utilizing our new publicly available code to demonstrate the slimplectic integrator algorithm. Slimplectic integrators are well-suited for integrations of systems where nonconservative effects play an important role in the long-term dynamical evolution. As such they are particularly appropriate for cosmological or celestial N-body dynamics problems where nonconservative interactions, e.g., gas interactions or dissipative tides, can play an important role.

  20. Slimplectic Integrators: Variational Integrators for Nonconservative systems

    NASA Astrophysics Data System (ADS)

    Tsang, David

    2016-01-01

    Symplectic integrators are widely used for long-term integration of conservative astrophysical problems due to their ability to preserve the constants of motion; however, they cannot in general be applied in the presence of nonconservative interactions. In this Letter, we develop the "slimplectic" integrator, a new type of numerical integrator that shares many of the benefits of traditional symplectic integrators yet is applicable to general nonconservative systems. We utilize a fixed-time-step variational integrator formalism applied to the principle of stationary nonconservative action developed in Galley et al. As a result, the generalized momenta and energy (Noether current) evolutions are well-tracked. We discuss several example systems, including damped harmonic oscillators, Poynting-Robertson drag, and gravitational radiation reaction, by utilizing our new publicly available code to demonstrate the slimplectic integrator algorithm. Slimplectic integrators are well-suited for integrations of systems where nonconservative effects play an important role in the long-term dynamical evolution. As such they are particularly appropriate for cosmological or celestial N-body dynamics problems where nonconservative interactions, e.g., gas interactions or dissipative tides, can play an important role.

  1. Two Related Parametric Integrals

    ERIC Educational Resources Information Center

    Dana-Picard, T.

    2007-01-01

    Two related sequences of definite integrals are considered. By mixing hand-work, computer algebra system assistance and websurfing, fine connections can be studied between integrals and a couple of interesting sequences of integers. (Contains 4 tables.)

  2. Integration in action.

    PubMed

    Green, Sara; Wolkenhauer, Olaf

    2012-09-01

    The workshop on 'Integration in Biology and Biomedicine' was held in May 2012 at the University of Sydney. It brought together scientists and philosophers to discuss the need for, and practice of, integration in the life sciences.

  3. Quality Integrated Education

    ERIC Educational Resources Information Center

    Brazziel, William

    1978-01-01

    Integrated education as it exists in many communities is actually harmful to black children and works to devastate rather than develop black talent; proponents of school integration must put effort into improving the quality of all schools. (JD)

  4. The universal path integral

    NASA Astrophysics Data System (ADS)

    Lloyd, Seth; Dreyer, Olaf

    2016-02-01

    Path integrals calculate probabilities by summing over classical configurations of variables such as fields, assigning each configuration a phase equal to the action of that configuration. This paper defines a universal path integral, which sums over all computable structures. This path integral contains as sub-integrals all possible computable path integrals, including those of field theory, the standard model of elementary particles, discrete models of quantum gravity, string theory, etc. The universal path integral possesses a well-defined measure that guarantees its finiteness. The probabilities for events corresponding to sub-integrals can be calculated using the method of decoherent histories. The universal path integral supports a quantum theory of the universe in which the world that we see around us arises out of the interference between all computable structures.

  5. Integrated optics technology study

    NASA Technical Reports Server (NTRS)

    Chen, B.; Findakly, T.; Innarella, R.

    1982-01-01

    The status and near term potential of materials and processes available for the fabrication of single mode integrated electro-optical components are discussed. Issues discussed are host material and orientation, waveguide formation, optical loss mechanisms, wavelength selection, polarization effects and control, laser to integrated optics coupling fiber optic waveguides to integrated optics coupling, sources, and detectors. Recommendations of the best materials, technology, and processes for fabrication of integrated optical components for communications and fiber gyro applications are given.

  6. Integrated optics technology study

    NASA Technical Reports Server (NTRS)

    Chen, B.

    1982-01-01

    The materials and processes available for the fabrication of single mode integrated electrooptical components are described. Issues included in the study are: (1) host material and orientation, (2) waveguide formation, (3) optical loss mechanisms, (4) wavelength selection, (5) polarization effects and control, (6) laser to integrated optics coupling,(7) fiber optic waveguides to integrated optics coupling, (8) souces, (9) detectors. The best materials, technology and processes for fabrication of integrated optical components for communications and fiber gyro applications are recommended.

  7. Boolean integral calculus

    NASA Technical Reports Server (NTRS)

    Tucker, Jerry H.; Tapia, Moiez A.; Bennett, A. Wayne

    1988-01-01

    The concept of Boolean integration is developed, and different Boolean integral operators are introduced. Given the changes in a desired function in terms of the changes in its arguments, the ways of 'integrating' (i.e. realizing) such a function, if it exists, are presented. The necessary and sufficient conditions for integrating, in different senses, the expression specifying the changes are obtained. Boolean calculus has applications in the design of logic circuits and in fault analysis.

  8. Effect of recombinant prophenin 2 on the integrity and viability of Trichomonas vaginalis.

    PubMed

    Hernandez-Flores, J L; Rodriguez, M C; Gastelum Arellanez, A; Alvarez-Morales, A; Avila, E E

    2015-01-01

    Trichomonas vaginalis is the causal agent of trichomoniasis, which is associated with preterm child delivery, low birth weight, and an increased risk of infection by human papilloma virus and human immunodeficiency virus following exposure. Several reports have established increasing numbers of trichomoniasis cases resistant to metronidazole, the agent used for treatment, and it is therefore important to identify new therapeutic alternatives. Previously, our group reported the effect of tritrpticin, a synthetic peptide derived from porcine prophenin, on T. vaginalis; however, the hemolytic activity of this small peptide complicates its possible use as a therapeutic agent. In this study, we report that the propeptide and the processed peptide of prophenin 2 (cleaved with hydroxylamine) affected the integrity and growth of T. vaginalis and that pro-prophenin 2 displays some resistance to proteolysis by T. vaginalis proteinases at 1 h. Its effect on T. vaginalis as well as its low hemolytic activity and short-time stability to parasite proteinases makes prophenin 2 an interesting candidate for synergistic or alternative treatment against T. vaginalis.

  9. Effect of Recombinant Prophenin 2 on the Integrity and Viability of Trichomonas vaginalis

    PubMed Central

    Hernandez-Flores, J. L.; Rodriguez, M. C.; Gastelum Arellanez, A.; Alvarez-Morales, A.; Avila, E. E.

    2015-01-01

    Trichomonas vaginalis is the causal agent of trichomoniasis, which is associated with preterm child delivery, low birth weight, and an increased risk of infection by human papilloma virus and human immunodeficiency virus following exposure. Several reports have established increasing numbers of trichomoniasis cases resistant to metronidazole, the agent used for treatment, and it is therefore important to identify new therapeutic alternatives. Previously, our group reported the effect of tritrpticin, a synthetic peptide derived from porcine prophenin, on T. vaginalis; however, the hemolytic activity of this small peptide complicates its possible use as a therapeutic agent. In this study, we report that the propeptide and the processed peptide of prophenin 2 (cleaved with hydroxylamine) affected the integrity and growth of T. vaginalis and that pro-prophenin 2 displays some resistance to proteolysis by T. vaginalis proteinases at 1 h. Its effect on T. vaginalis as well as its low hemolytic activity and short-time stability to parasite proteinases makes prophenin 2 an interesting candidate for synergistic or alternative treatment against T. vaginalis. PMID:25815316

  10. Connective tissue integrity is lost in vitamin B-6-deficient chicks

    NASA Technical Reports Server (NTRS)

    Masse, P. G.; Yamauchi, M.; Mahuren, J. D.; Coburn, S. P.; Muniz, O. E.; Howell, D. S.

    1995-01-01

    The objective of the present investigation was to characterize further the connective tissue disorder produced by pyridoxine (vitamin B-6) deficiency, as previously evidenced by electron microscopy. Following the second post-natal week, fast growing male chicks were deprived of pyridoxine for a 1-mo period. Six weeks post-natally, blood concentrations in the experimental deficiency group had declined to deficiency levels as registered by low concentrations of pyridoxal phosphate (coenzyme form) in erythrocytes, but did not reach levels associated with neurological symptoms. Light microscopic study showed abnormalities in the extracellular matrix of the connective tissues. Collagen cross-links and the aldehyde contents were not significantly lower in cartilage and tendon collagens of vitamin B-6-deficient animals than in age-matched controls; also, their proteoglycan degrading protease and collagenase activities measured in articular cartilages were not greater. Thus, proteolysis was an unlikely alternative mechanism to account for the loss of connective tissue integrity. These results point to the need for further investigation into adhesive properties of collagen associated proteoglycans or other proteins in vitamin B-6-deficient connective tissue.

  11. Integrated Education. Feature Issue.

    ERIC Educational Resources Information Center

    York, Jennifer, Ed.; Vandercook, Terri, Ed.

    1988-01-01

    This "feature issue" provides various perspectives on a number of integrated education topics, including successful integration practices and strategies, the changing roles of teachers, the appropriate role of research, the history and future of integrated education, and the realization of dreams of life in the mainstream for children with severe…

  12. Sledge-Hammer Integration

    ERIC Educational Resources Information Center

    Ahner, Henry

    2009-01-01

    Integration (here visualized as a pounding process) is mathematically realized by simple transformations, successively smoothing the bounding curve into a straight line and the region-to-be-integrated into an area-equivalent rectangle. The relationship to Riemann sums, and to the trapezoid and midpoint methods of numerical integration, is…

  13. Social Integration and Divorce.

    ERIC Educational Resources Information Center

    Booth, Alan; And Others

    1991-01-01

    Longitudinal data on over 1,300 married persons suggest that divorce was deterred by absence of divorce in reference group (normative integration) and was deterred for shorter marriages by more friends and organizational affiliations (communicative integration). Sharing friends and organization affiliations with spouse (functional integration) may…

  14. Photonic Integrated Circuits

    NASA Technical Reports Server (NTRS)

    Merritt, Scott; Krainak, Michael

    2016-01-01

    Integrated photonics generally is the integration of multiple lithographically defined photonic and electronic components and devices (e.g. lasers, detectors, waveguides passive structures, modulators, electronic control and optical interconnects) on a single platform with nanometer-scale feature sizes. The development of photonic integrated circuits permits size, weight, power and cost reductions for spacecraft microprocessors, optical communication, processor buses, advanced data processing, and integrated optic science instrument optical systems, subsystems and components. This is particularly critical for small spacecraft platforms. We will give an overview of some NASA applications for integrated photonics.

  15. The Chlamydia protease CPAF regulates host and bacterial proteins to maintain pathogen vacuole integrity and promote virulence

    PubMed Central

    Jorgensen, Ine; Bednar, Maria; Amin, Vishar; Davies, Beckley K.; Ting, Jenny P.Y.; McCafferty, Dewey; Valdivia, Raphael H.

    2011-01-01

    SUMMARY The obligate intracellular bacterial pathogen Chlamydia trachomatis injects numerous effector proteins into the epithelial cell cytoplasm to manipulate host functions important for bacterial survival. In addition, the bacterium secretes a serine protease, chlamydial protease-like activity factor (CPAF). Although several CPAF targets are reported, the significance of CPAF-mediated proteolysis is unclear due to the lack of specific CPAF inhibitors and the diversity of host targets. We report that CPAF also targets chlamydial effectors secreted early during the establishment of the pathogen-containing vacuole (“inclusion”). We designed a cell-permeable CPAF-specific inhibitory peptide and used it to determine that CPAF prevents superinfection by degrading early Chlamydia effectors translocated during entry into a pre-infected cell. Prolonged CPAF inhibition leads to loss of inclusion integrity and caspase-1-dependent death of infected epithelial cells. Thus, CPAF functions in niche protection, inclusion integrity and pathogen survival, making the development of CPAF-specific protease inhibitors an attractive anti-chlamydial therapeutic strategy. PMID:21767809

  16. Misconceptions and Integration

    PubMed Central

    MORTAZ HEJRI, SARA; MIRZAZADEH, AZIM; JALILI, MOHAMMAD

    2015-01-01

    Introduction Pervasive beliefs regarding curricular reform and integration have flourished among medical students, faculty members and medical school administrators. These concepts have extensively impacted the reform process, sometimes by resisting the reforms and sometimes by diverting the curriculum from its planned objectives. In the current paper, we have tried to address the challenges of integration in MD program by looking at the existing literature and the experience of the international universities. Methods We collected the questions frequently asked during the curricular reform process. We, then, evaluated them, and selected 5 main ideas. In order to find their answers, we searched the literature using these keywords: integration, reform, and undergraduate medical curriculum. Results The findings are discussed in five sections: 1) Reform is not equivalent to integration, 2) Integration can be implemented in both high school and graduate programs, 3) Organ-system based integration is not the only method available for integration, 4) Integration of two phases (basic sciences and physiopathology) can be considered but it is not mandatory, 5) Integration does not fade basic sciences in favor of clinical courses. Conclusion It seems that medical education literature and prior experience of the leading universities do not support most of the usual concepts about integration. Therefore, it is important to consider informed decision making based on best evidence rather than personal opinions during the curricular reform process. PMID:26457318

  17. Faces of integration

    PubMed Central

    Williams, Paul; Sullivan, Helen

    2009-01-01

    Theme Two central themes permeate this paper—the interplay between structure and agency in integration processes and the extent to which this is mediated through sensemaking by individual actors. Case study The empirical base for the paper is provided by case study research from Wales which draws on examples of different types of integration in health and social care. The individual case studies highlight different interpretations of integration set against a background of the resources involved, processes employed and outcomes achieved. Discussion A wide ranging discussion exposes the complex interplay and dynamics between structural factors and the manner in which they enable or constrain integration, and individual actors realising their potential agency through leadership, professionalism and boundary spanning to influence outcomes. The importance of structure and agency complementing each other to determine effective integration is emphasised, together with the scope that is available for interpretation and meaning by individual actors within the contested discourse of integration. PMID:20087420

  18. Integrated Marketing Communications

    ERIC Educational Resources Information Center

    Black, Jim

    2004-01-01

    Integration has become a cliche in enrollment management and student services circles. The term is used to describe everything from integrated marketing to seamless services. Often, it defines organizational structures, processes, student information systems, and even communities. In Robert Sevier's article in this issue of "College and…

  19. Elementary Integrated Curriculum Framework

    ERIC Educational Resources Information Center

    Montgomery County Public Schools, 2010

    2010-01-01

    The Elementary Integrated Curriculum (EIC) Framework is the guiding curriculum document for the Elementary Integrated Curriculum and represents the elementary portion of the Montgomery County (Maryland) Public Schools (MCPS) Pre-K-12 Curriculum Frameworks. The EIC Framework contains the detailed indicators and objectives that describe what…

  20. System integration report

    NASA Technical Reports Server (NTRS)

    Badler, N. I.; Korein, J. D.; Meyer, C.; Manoochehri, K.; Rovins, J.; Beale, J.; Barr, B.

    1985-01-01

    Several areas that arise from the system integration issue were examined. Intersystem analysis is discussed as it relates to software development, shared data bases and interfaces between TEMPUS and PLAID, shaded graphics rendering systems, object design (BUILD), the TEMPUS animation system, anthropometric lab integration, ongoing TEMPUS support and maintenance, and the impact of UNIX and local workstations on the OSDS environment.

  1. Integration in Hydraulics.

    ERIC Educational Resources Information Center

    Sworder, Steven C.

    This paper presents an application of integration to the field of hydraulics. An integral relation for the time required to drop the fluid contained in a cylindrical tank from one level to another using a hole in the tank wall is derived. Procedures for constructing the experimental equipment and procedures for determining the coefficient of…

  2. Academic Research Integration System

    ERIC Educational Resources Information Center

    Surugiu, Iula; Velicano, Manole

    2008-01-01

    This paper comprises results concluding the research activity done so far regarding enhanced web services and system integration. The objective of the paper is to define the software architecture for a coherent framework and methodology for enhancing existing web services into an integrated system. This document presents the research work that has…

  3. Integrated Assessments for ELL

    ERIC Educational Resources Information Center

    Armon, Joan; Morris, Linda J.

    2008-01-01

    Despite the challenges posed by increased time, specialized vocabularies, and balance, integrating writing and drawing with science investigations is beneficial for teachers and students. This month's column explains why this integrated approach is beneficial, and illustrates how teachers can use it to meet the needs of students learning English…

  4. An Integrated Teaching Module.

    ERIC Educational Resources Information Center

    Samuel, Marie R.; Seiferth, Berniece B.

    This integrated teaching module provides elementary and junior high school teachers with a "hands-on" approach to studying the Anasazi Indian. Emphasis is on creative exploration that focuses on integrating art, music, poetry, writing, geography, dance, history, anthropology, sociology, and archaeology. Replicas of artifacts, contemporary Indian…

  5. The Fresnel Integrals Revisited

    ERIC Educational Resources Information Center

    Chen, Hongwei

    2009-01-01

    This note presents another elementary method to evaluate the Fresnel integrals. It is interesting to see that this technique is also strong enough to capture a number of pairs of parameter integrals. The main ingredients of the method are the consideration of some related derivatives and linear differential equations.

  6. Undulator Field Integral Measurements

    SciTech Connect

    Wolf, Zachary

    2010-12-07

    The LCLS undulator field integrals must be very small so that the beam trajectory slope and offset stay within tolerance. In order to make accurate measurements of the small field integrals, a long coil will be used. This note describes the design of the coil measurement system.

  7. Integrity in Student Development

    ERIC Educational Resources Information Center

    Roberts, Dennis C.; Banta, Trudy W.

    2011-01-01

    The quest for integrity in practice and theory has been part of the evolution of student personnel work all the way back to the turn of the 20th century. This chapter seeks to take stock of the question of integrity in relation to one of the core knowledge bases used by those engaged in student affairs work today--student development. The authors…

  8. Hermeneutics of Integrative Knowledge.

    ERIC Educational Resources Information Center

    Shin, Un-chol

    This paper examines and compares the formation processes and structures of three types of integrative knowledge that in general represent natural sciences, social sciences, and humanities. These three types can be observed, respectively, in the philosophies of Michael Polanyi, Jurgen Habermas, and Paul Ricoeur. These types of integrative knowledge…

  9. Integration by Hyperbolic Substitution

    ERIC Educational Resources Information Center

    Price, David

    2012-01-01

    Mathematics teachers constantly encourage their students to think independently. The study of integration in calculus provides an excellent opportunity to encourage inventive investigation. In contrast to differentiation, which is predominately mechanical, integration is a more creative process. One such possibility is offered by the study of the…

  10. Integrity in Transactional Leadership

    ERIC Educational Resources Information Center

    Miller, Thomas

    2011-01-01

    This chapter begins with a discussion of the impact of limited resources on the integrity of transactions between students and student affairs administrators. A framework and guiding principles for maintaining integrity are offered, and then some general principles for transactions with students are presented. Next, the chapter involves integrity…

  11. Integrating Module - NEMS Documentation

    EIA Publications

    2014-01-01

    Provides an overview of the complete National Energy Modeling System (NEMS) model, and includes brief descriptions of the modules with which the Integrating Module interacts. The emphasis and focus, however, is on the structure and function of the Integrating Module of NEMS.

  12. Ready, Set, Integrate!

    ERIC Educational Resources Information Center

    McCombs, John

    2003-01-01

    Describes how the American Embassy School (AES) in New Delhi, India achieved school-wide technology integration. Discusses development of a new network; beginning to mentor; organizing the Technology Integration Plan (TIP) by software application; implementing the plan; assessing progress; and results, which overall, were positive. (AEF)

  13. Systems Integration (Fact Sheet)

    SciTech Connect

    Not Available

    2011-10-01

    The Systems Integration (SI) subprogram works closely with industry, universities, and the national laboratories to overcome technical barriers to the large-scale deployment of solar technologies. To support these goals, the subprogram invests primarily in four areas: grid integration, technology validation, solar resource assessment, and balance of system development.

  14. Systems Integration (Fact Sheet)

    SciTech Connect

    DOE Solar Energy Technologies Program

    2011-10-13

    The Systems Integration (SI) subprogram works closely with industry, universities, and the national laboratories to overcome technical barriers to the large-scale deployment of solar technologies. To support these goals, the subprogram invests primarily in four areas: grid integration, technology validation, solar resource assessment, and balance of system development.

  15. What Is Integral Theory?

    ERIC Educational Resources Information Center

    Marquis, Andre

    2007-01-01

    Integral theory is a way of knowing that helps foster the recognition that disparate aspects of reality--such as biological constitution, cultural worldviews, felt-sense of selfhood, and social systems--are all critically important to any knowledge quest. Integral theory provides an "all quadrants, all levels" (K. Wilber, 2006, p. 26)…

  16. Designing for STEM Integration

    ERIC Educational Resources Information Center

    Berland, Leema K.

    2013-01-01

    We are increasingly seeing an emphasis on STEM integration in high school classrooms such that students will learn and apply relevant math and science content while simultaneously developing engineering habits of mind. However, research in both science education and engineering education suggests that this goal of truly integrating STEM is rife…

  17. Retroviral integration: Site matters

    PubMed Central

    Demeulemeester, Jonas; De Rijck, Jan

    2015-01-01

    Here, we review genomic target site selection during retroviral integration as a multistep process in which specific biases are introduced at each level. The first asymmetries are introduced when the virus takes a specific route into the nucleus. Next, by co‐opting distinct host cofactors, the integration machinery is guided to particular chromatin contexts. As the viral integrase captures a local target nucleosome, specific contacts introduce fine‐grained biases in the integration site distribution. In vivo, the established population of proviruses is subject to both positive and negative selection, thereby continuously reshaping the integration site distribution. By affecting stochastic proviral expression as well as the mutagenic potential of the virus, integration site choice may be an inherent part of the evolutionary strategies used by different retroviruses to maximise reproductive success. PMID:26293289

  18. Algebraic integrability: a survey.

    PubMed

    Vanhaecke, Pol

    2008-03-28

    We give a concise introduction to the notion of algebraic integrability. Our exposition is based on examples and phenomena, rather than on detailed proofs of abstract theorems. We mainly focus on algebraic integrability in the sense of Adler-van Moerbeke, where the fibres of the momentum map are affine parts of Abelian varieties; as it turns out, most examples from classical mechanics are of this form. Two criteria are given for such systems (Kowalevski-Painlevé and Lyapunov) and each is illustrated in one example. We show in the case of a relatively simple example how one proves algebraic integrability, starting from the differential equations for the integrable vector field. For Hamiltonian systems that are algebraically integrable in the generalized sense, two examples are given, which illustrate the non-compact analogues of Abelian varieties which typically appear in such systems. PMID:17588863

  19. Integrated assessment briefs

    SciTech Connect

    1995-04-01

    Integrated assessment can be used to evaluate and clarify resource management policy options and outcomes for decision makers. The defining characteristics of integrated assessment are (1) focus on providing information and analysis that can be understood and used by decision makers rather than for merely advancing understanding and (2) its multidisciplinary approach, using methods, styles of study, and considerations from a broader variety of technical areas than would typically characterize studies produced from a single disciplinary standpoint. Integrated assessment may combine scientific, social, economic, health, and environmental data and models. Integrated assessment requires bridging the gap between science and policy considerations. Because not everything can be valued using a single metric, such as a dollar value, the integrated assessment process also involves evaluating trade-offs among dissimilar attributes. Scientists at Oak Ridge National Laboratory (ORNL) recognized the importance and value of multidisciplinary approaches to solving environmental problems early on and have pioneered the development of tools and methods for integrated assessment over the past three decades. Major examples of ORNL`s experience in the development of its capabilities for integrated assessment are given.

  20. Human-technology Integration

    NASA Astrophysics Data System (ADS)

    Mullen, Katharine M.

    Human-technology integration is the replacement of human parts and extension of human capabilities with engineered devices and substrates. Its result is hybrid biological-artificial systems. We discuss here four categories of products furthering human-technology integration: wearable computers, pervasive computing environments, engineered tissues and organs, and prosthetics, and introduce examples of currently realized systems in each category. We then note that realization of a completely artificial sytem via the path of human-technology integration presents the prospect of empirical confirmation of an aware artificially embodied system.

  1. Ethics and academic integrity.

    PubMed

    Milton, Constance L

    2015-01-01

    Academics from across the globe must navigate ever-increasing demands for research, practice, and educational productivity. With the increased demands, nurse faculty must choose value priorities and actions that reflect academic integrity. What does it mean to choose actions that reflect personal integrity in the academic arena? This article begins an important nursing philosophical and theoretical discussion that members and future members of the discipline of nursing must reflect upon and grapple with as they consider what it potentially means to act with straight thinking and integrity in academics. PMID:25520458

  2. GMRES and integral operators

    SciTech Connect

    Kelley, C.T.; Xue, Z.Q.

    1994-12-31

    Many discretizations of integral equations and compact fixed point problems are collectively compact and strongly convergent in spaces of continuous functions. These properties not only lead to stable and convergent approximations but also can be used in the construction of fast multilevel algorithms. Recently the GMRES algorithm has become a standard coarse mesh solver. The purpose of this paper is to show how the special properties of integral operators and their approximations are reflected in the performance of the GMRES iteration and how these properties can be used to strengthen the norm in which convergence takes place. The authors illustrate these ideas with composite Gauss rules for integral equations on the unit interval.

  3. The Integration Paradox

    PubMed Central

    Verkuyten, Maykel

    2016-01-01

    The integration paradox refers to the phenomenon of the more highly educated and structurally integrated immigrants turning away from the host society, rather than becoming more oriented toward it. This article provides an overview of the empirical evidence documenting this paradox in the Netherlands. In addition, the theoretical arguments and the available findings about the social psychological processes involved in this paradox are considered. The existing evidence for the integration paradox and what might explain it form the basis for making suggestion for future theoretical work and empirical research, and for discussing possible policy implications. PMID:27152028

  4. Candidate gene networks and blood biomarkers of methamphetamine-associated psychosis: an integrative RNA-sequencing report.

    PubMed

    Breen, M S; Uhlmann, A; Nday, C M; Glatt, S J; Mitt, M; Metsalpu, A; Stein, D J; Illing, N

    2016-01-01

    The clinical presentation, course and treatment of methamphetamine (METH)-associated psychosis (MAP) are similar to that observed in schizophrenia (SCZ) and subsequently MAP has been hypothesized as a pharmacological and environmental model of SCZ. However, several challenges currently exist in diagnosing MAP accurately at the molecular and neurocognitive level before the MAP model can contribute to the discovery of SCZ biomarkers. We directly assessed subcortical brain structural volumes and clinical parameters of MAP within the framework of an integrative genome-wide RNA-Seq blood transcriptome analysis of subjects diagnosed with MAP (N=10), METH dependency without psychosis (MA; N=10) and healthy controls (N=10). First, we identified discrete groups of co-expressed genes (that is, modules) and tested them for functional annotation and phenotypic relationships to brain structure volumes, life events and psychometric measurements. We discovered one MAP-associated module involved in ubiquitin-mediated proteolysis downregulation, enriched with 61 genes previously found implicated in psychosis and SCZ across independent blood and post-mortem brain studies using convergent functional genomic (CFG) evidence. This module demonstrated significant relationships with brain structure volumes including the anterior corpus callosum (CC) and the nucleus accumbens. Furthermore, a second MAP and psychoticism-associated module involved in circadian clock upregulation was also enriched with 39 CFG genes, further associated with the CC. Subsequently, a machine-learning analysis of differentially expressed genes identified single blood-based biomarkers able to differentiate controls from methamphetamine dependents with 87% accuracy and MAP from MA subjects with 95% accuracy. CFG evidence validated a significant proportion of these putative MAP biomarkers in independent studies including CLN3, FBP1, TBC1D2 and ZNF821 (RNA degradation), ELK3 and SINA3 (circadian clock) and PIGF and

  5. Candidate gene networks and blood biomarkers of methamphetamine-associated psychosis: an integrative RNA-sequencing report

    PubMed Central

    Breen, M S; Uhlmann, A; Nday, C M; Glatt, S J; Mitt, M; Metsalpu, A; Stein, D J; Illing, N

    2016-01-01

    The clinical presentation, course and treatment of methamphetamine (METH)-associated psychosis (MAP) are similar to that observed in schizophrenia (SCZ) and subsequently MAP has been hypothesized as a pharmacological and environmental model of SCZ. However, several challenges currently exist in diagnosing MAP accurately at the molecular and neurocognitive level before the MAP model can contribute to the discovery of SCZ biomarkers. We directly assessed subcortical brain structural volumes and clinical parameters of MAP within the framework of an integrative genome-wide RNA-Seq blood transcriptome analysis of subjects diagnosed with MAP (N=10), METH dependency without psychosis (MA; N=10) and healthy controls (N=10). First, we identified discrete groups of co-expressed genes (that is, modules) and tested them for functional annotation and phenotypic relationships to brain structure volumes, life events and psychometric measurements. We discovered one MAP-associated module involved in ubiquitin-mediated proteolysis downregulation, enriched with 61 genes previously found implicated in psychosis and SCZ across independent blood and post-mortem brain studies using convergent functional genomic (CFG) evidence. This module demonstrated significant relationships with brain structure volumes including the anterior corpus callosum (CC) and the nucleus accumbens. Furthermore, a second MAP and psychoticism-associated module involved in circadian clock upregulation was also enriched with 39 CFG genes, further associated with the CC. Subsequently, a machine-learning analysis of differentially expressed genes identified single blood-based biomarkers able to differentiate controls from methamphetamine dependents with 87% accuracy and MAP from MA subjects with 95% accuracy. CFG evidence validated a significant proportion of these putative MAP biomarkers in independent studies including CLN3, FBP1, TBC1D2 and ZNF821 (RNA degradation), ELK3 and SINA3 (circadian clock) and PIGF and

  6. ELECTRONIC INTEGRATING CIRCUIT

    DOEpatents

    Englemann, R.H.

    1963-08-20

    An electronic integrating circuit using a transistor with a capacitor connected between the emitter and collector through which the capacitor discharges at a rate proportional to the input current at the base is described. Means are provided for biasing the base with an operating bias and for applying a voltage pulse to the capacitor for charging to an initial voltage. A current dividing diode is connected between the base and emitter of the transistor, and signal input terminal means are coupled to the juncture of the capacitor and emitter and to the base of the transistor. At the end of the integration period, the residual voltage on said capacitor is less by an amount proportional to the integral of the input signal. Either continuous or intermittent periods of integration are provided. (AEC)

  7. INTEGRAL and Cataclysmic Variables

    SciTech Connect

    Hudec, R.; Blazek, M.; Galis, R.; Kocka, M.

    2010-07-15

    The results of investigations of cataclysmic variables (CVs) with the ESA INTEGRAL satellite are briefly presented and discussed. It is evident that the satellite serves as an efficient tool to study some of these objects.

  8. Integrated Flywheel Technology, 1983

    NASA Technical Reports Server (NTRS)

    Keckler, C. R. (Editor); Rodriguez, G. E. (Editor); Groom, N. J. (Editor)

    1983-01-01

    Topics of discussion included: technology assessment of the integrated flywheel systems, potential of system concepts, identification of critical areas needing development and, to scope and define an appropriate program for coordinated activity.

  9. Retroviral DNA Integration

    PubMed Central

    2016-01-01

    The integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral integration proceeds via two integrase activities: 3′-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications. PMID:27198982

  10. Payload missions integration

    NASA Technical Reports Server (NTRS)

    Mitchell, R. A. K.

    1983-01-01

    Highlights of the Payload Missions Integration Contract (PMIC) are summarized. Spacelab Missions no. 1 to 3, OSTA partial payloads, Astro-1 Mission, premission definition, and mission peculiar equipment support structure are addressed.

  11. Managing for Organizational Integrity.

    ERIC Educational Resources Information Center

    Paine, Lynn Sharp

    1994-01-01

    Compliance-based ethics programs focus on prevention, detection, and punishment. Companies should adopt an integrity-based approach to ethics management that combines a concern for the law with an emphasis on managerial responsibility for ethical behavior. (JOW)

  12. Aviation Data Integration System

    NASA Technical Reports Server (NTRS)

    Kulkarni, Deepak; Wang, Yao; Windrem, May; Patel, Hemil; Keller, Richard

    2003-01-01

    During the analysis of flight data and safety reports done in ASAP and FOQA programs, airline personnel are not able to access relevant aviation data for a variety of reasons. We have developed the Aviation Data Integration System (ADIS), a software system that provides integrated heterogeneous data to support safety analysis. Types of data available in ADIS include weather, D-ATIS, RVR, radar data, and Jeppesen charts, and flight data. We developed three versions of ADIS to support airlines. The first version has been developed to support ASAP teams. A second version supports FOQA teams, and it integrates aviation data with flight data while keeping identification information inaccessible. Finally, we developed a prototype that demonstrates the integration of aviation data into flight data analysis programs. The initial feedback from airlines is that ADIS is very useful in FOQA and ASAP analysis.

  13. Wellbore Integrity Network

    SciTech Connect

    Carey, James W.; Bachu, Stefan

    2012-06-21

    In this presentation, we review the current state of knowledge on wellbore integrity as developed in the IEA Greenhouse Gas Programme's Wellbore Integrity Network. Wells are one of the primary risks to the successful implementation of CO{sub 2} storage programs. Experimental studies show that wellbore materials react with CO{sub 2} (carbonation of cement and corrosion of steel) but the impact on zonal isolation is unclear. Field studies of wells in CO{sub 2}-bearing fields show that CO{sub 2} does migrate external to casing. However, rates and amounts of CO{sub 2} have not been quantified. At the decade time scale, wellbore integrity is driven by construction quality and geomechanical processes. Over longer time-scales (> 100 years), chemical processes (cement degradation and corrosion) become more important, but competing geomechanical processes may preserve wellbore integrity.

  14. Integrated Programs Bibliography.

    ERIC Educational Resources Information Center

    Russell, Connie; Klassen, Aynsley

    2002-01-01

    Lists 58 journal articles and books, published 1989-2002, on integrated programs at the secondary level. Categories are: updates on the Ontario scene, suggestions for teachers, assessment, research, and program descriptions. (SV)

  15. Building an Integrated Study

    ERIC Educational Resources Information Center

    Collins, Myrtle T.; Greenberg, Marvin

    1974-01-01

    Article described a plan to develop integrated study through music activities. Students learned to become more independent learners while concentrating on more complex and creative activities. (Author/RK)

  16. Silicon-electroceramics integration

    SciTech Connect

    Tuller, H.L.

    1996-12-31

    Electroceramics provides unique functionality as sensors, transducers, and non-volatile storage media while silicon is nearly unique in its signal processing and micromechanical properties. We discuss how these two materials technologies, when integrated, provide unique devices and structures.

  17. Human Systems Integration Introduction

    NASA Video Gallery

    This lecture provides an overview of Human Systems Integration (HSI), its implementation cost and return on investment, HSI domains, how HSI fits into the NASA organization structure, HSI roles and...

  18. ALHAT Integration and Testing

    NASA Video Gallery

    Autonomous Landing and Hazard Avoidance Technology (ALHAT) project integration and testing takes place at NASA Langley Research Center. The ALHAT project is a multi-center initiative to enable vari...

  19. Complementary and Integrative Medicine

    MedlinePlus

    ... medical treatments that are not part of mainstream medicine. When you are using these types of care, it may be called complementary, integrative, or alternative medicine. Complementary medicine is used together with mainstream medical ...

  20. Acoustic integrated extinction

    PubMed Central

    Norris, Andrew N.

    2015-01-01

    The integrated extinction (IE) is defined as the integral of the scattering cross section as a function of wavelength. Sohl et al. (2007 J. Acoust. Soc. Am. 122, 3206–3210. (doi:10.1121/1.2801546)) derived an IE expression for acoustic scattering that is causal, i.e. the scattered wavefront in the forward direction arrives later than the incident plane wave in the background medium. The IE formula was based on electromagnetic results, for which scattering is causal by default. Here, we derive a formula for the acoustic IE that is valid for causal and non-causal scattering. The general result is expressed as an integral of the time-dependent forward scattering function. The IE reduces to a finite integral for scatterers with zero long-wavelength monopole and dipole amplitudes. Implications for acoustic cloaking are discussed and a new metric is proposed for broadband acoustic transparency. PMID:27547100

  1. Integrating Forensic Science.

    ERIC Educational Resources Information Center

    Funkhouser, John; Deslich, Barbara J.

    2000-01-01

    Explains the implementation of forensic science in an integrated curriculum and discusses the advantages of this approach. Lists the forensic science course syllabi studied in three high schools. Discusses the unit on polymers in detail. (YDS)

  2. PEV Integration with Renewables (Presentation)

    SciTech Connect

    Markel, T.

    2014-06-18

    This presentation discusses current research at NREL on integrating plug-in electric vehicles with the grid and using renewable energy to charge the grid. The Electric Vehicle Grid Integration (EVGI) and Integrated Network Testbed for Energy Grid Research and Technology Experimentation (INTEGRATE) are addressing the opportunities and technical requirements for vehicle grid integration that will increase marketability and lead to greater petroleum reduction.

  3. Streamlining Payload Integration

    NASA Technical Reports Server (NTRS)

    Lufkin, Susan N.

    2010-01-01

    Payload integration onto space transport vehicles and the International Space Station (ISS) is a complex process. Yet, cargo transport is the sole reason for any space mission, be it for ferrying humans, science, or hardware. As the largest such effort in history, the ISS offers a wide variety of payload experience. However, for any payload to reach the Space Station under the current process, Payload Developers face a list of daunting tasks that go well beyond just designing the payload to the constraints of the transport vehicle and its stowage topology. Payload customers are required to prove their payload s functionality, structural integrity, and safe integration - including under less than nominal situations. They must also plan for or provide training, procedures, hardware labeling, ground support, and communications. In addition, they must deal with negotiating shared consumables, integrating software, obtaining video, and coordinating the return of data and hardware. All the while, they must meet export laws, launch schedules, budget limits, and the consensus of more than 12 panel and board reviews. Despite the cost and infrastructure overhead, payload proposals have increased. Just in the span from FY08 to FY09, the NASA Payload Space Station Support Office budget rose from $78M to $96M in attempt to manage the growing manifest, but the potential number of payloads still exceeds available Payload Integration Management manpower. The growth has also increased management difficulties due to the fact that payloads are more frequently added to a flight schedule late in the flow. The current standard ISS template for payload integration from concept to payload turn-over is 36 months, or 18 months if the payload already has a preliminary design. Customers are increasingly requiring a turn-around of 3 to 6-months to meet market needs. The following paper suggests options for streamlining the current payload integration process in order to meet customer schedule

  4. 2014 SRP Integration Transcript

    NASA Technical Reports Server (NTRS)

    Steinberg, Susan

    2014-01-01

    HRP's mission is to reduce the risks to human health and performance during long-duration spaceflight. The HRP Integrated Research Plan (IRP) contains the research plans for the 32 risks that require research to characterize and mitigate. From its inception the "integrate" aspect of the IRP has denoted the integrated nature of risks to human health and performance. Even though each risk in the IRP has its own research plan and is tracked separately, the interrelated nature of health and performance requires that they be addressed in an integrative or holistic fashion so that the connectedness of physiological systems within the human body and the integrated response to spaceflight can be addressed. Common characteristics of the spaceflight environment include altered gravity, atmospheres, and light/dark cycles; space radiation; isolation; noise; and periods of high or low workload. Long-term exposure to this unique environment produces a suite of physiological effects such as stress; vision, neurocognitive, and anthropometric changes; circadian misalignment; fluid shifts; cardiovascular deconditioning; immune dysregulation; and altered nutritional requirements. Expanding cross-disciplinary integrative approaches that synthesize concepts or data from two or more disciplines would improve the identification and characterization risk factors, and enable the development of countermeasures relevant to multiple risks. Cross-disciplinary approaches might also help to illuminate problem areas that may arise when a countermeasure adversely impacts risks other than those which it was developed to mitigate, or to identify groupings of physiological changes that are likely to occur that may impact the overall risk posture. In 2014 HRP embarked on a pilot study that combined four SRPs (and 12 HRP risks) - Behavioral Health, Sensorimotor, Cardiovascular, and Bone/Muscle - specifically to discuss cross-disciplinary integration. The points outlined below were suggested to seed the

  5. Integrated spatial light modulator

    NASA Astrophysics Data System (ADS)

    Yu, Dong X.; Storti, George M.; Wrigley, Charles Y.

    1992-08-01

    We described here an integrated spatial light modulator (SLM) employing Quantex electron trapping (ET) materials. The light modulation is accomplished by emission of ET material, upon incident coherent infrared light, where a pattern is written to by previous visible light excitation. The ET based spatial light modulators offer unique advantages over other SLM devices, such as capability of converting incoherent input to coherent light output and of integrating the modulator, the photodetector, and the memory into a single, rugged unit.

  6. Integrated infrared array technology

    NASA Technical Reports Server (NTRS)

    Goebel, J. H.; Mccreight, C. R.

    1986-01-01

    An overview of integrated infrared (IR) array technology is presented. Although the array pixel formats are smaller, and the readout noise of IR arrays is larger, than the corresponding values achieved with optical charge-coupled-device silicon technology, substantial progress is being made in IR technology. Both existing IR arrays and those being developed are described. Examples of astronomical images are given which illustrate the potential of integrated IR arrays for scientific investigations.

  7. Integrated support structure

    NASA Technical Reports Server (NTRS)

    Bruneau, Stephen D.; Campbell, John T.; Struven, Christopher A.

    1990-01-01

    This Major Qualifying Project is part of the Advanced Space Design Program at WPI. The goal is to design a support structure for a NASA GetAway Special experimental canister. The payload integration, weight, volume, and structural integrity of the canister as specified by NASA guidelines were studied. The end result is a complete set of design drawings with interface drawings and data to specify the design and leave a base on which the next group can concentrate.

  8. The Integrated Medical Model

    NASA Technical Reports Server (NTRS)

    Kerstman, Eric; Minard, Charles; Saile, Lynn; Freiere deCarvalho, Mary; Myers, Jerry; Walton, Marlei; Butler, Douglas; Iyengar, Sriram; Johnson-Throop, Kathy; Baumann, David

    2010-01-01

    The goals of the Integrated Medical Model (IMM) are to develop an integrated, quantified, evidence-based decision support tool useful to crew health and mission planners and to help align science, technology, and operational activities intended to optimize crew health, safety, and mission success. Presentation slides address scope and approach, beneficiaries of IMM capabilities, history, risk components, conceptual models, development steps, and the evidence base. Space adaptation syndrome is used to demonstrate the model's capabilities.

  9. IDC Integrated Master Plan.

    SciTech Connect

    Clifford, David J.; Harris, James M.

    2014-12-01

    This is the IDC Re-Engineering Phase 2 project Integrated Master Plan (IMP). The IMP presents the major accomplishments planned over time to re-engineer the IDC system. The IMP and the associate Integrated Master Schedule (IMS) are used for planning, scheduling, executing, and tracking the project technical work efforts. REVISIONS Version Date Author/Team Revision Description Authorized by V1.0 12/2014 IDC Re- engineering Project Team Initial delivery M. Harris

  10. Astrophysical integrated research environment

    NASA Astrophysics Data System (ADS)

    Zhou, Jianfeng; Yang, Yang

    2007-08-01

    Astrophysical Integrated Research Environment (AIRE), aims to integrate astrophysical data, analysis software and astrophysical knowledge into an easy-to-use Internet based environment. Therefore, astrophysicists from different institutes can constitute virtual research groups which are favorable to study some complex multi-band astrophysical phenomena. The AIRE was put into use in Center for Astrophysics, Tsinghua university in 2003. Up to now, there are 219 advanced users in this environment. Several astrophysical researches base on AIRE have generated some important published results.

  11. Imagery Integration Team

    NASA Technical Reports Server (NTRS)

    Calhoun, Tracy; Melendrez, Dave

    2014-01-01

    The Human Exploration Science Office (KX) provides leadership for NASA's Imagery Integration (Integration 2) Team, an affiliation of experts in the use of engineering-class imagery intended to monitor the performance of launch vehicles and crewed spacecraft in flight. Typical engineering imagery assessments include studying and characterizing the liftoff and ascent debris environments; launch vehicle and propulsion element performance; in-flight activities; and entry, landing, and recovery operations. Integration 2 support has been provided not only for U.S. Government spaceflight (e.g., Space Shuttle, Ares I-X) but also for commercial launch providers, such as Space Exploration Technologies Corporation (SpaceX) and Orbital Sciences Corporation, servicing the International Space Station. The NASA Integration 2 Team is composed of imagery integration specialists from JSC, the Marshall Space Flight Center (MSFC), and the Kennedy Space Center (KSC), who have access to a vast pool of experience and capabilities related to program integration, deployment and management of imagery assets, imagery data management, and photogrammetric analysis. The Integration 2 team is currently providing integration services to commercial demonstration flights, Exploration Flight Test-1 (EFT-1), and the Space Launch System (SLS)-based Exploration Missions (EM)-1 and EM-2. EM-2 will be the first attempt to fly a piloted mission with the Orion spacecraft. The Integration 2 Team provides the customer (both commercial and Government) with access to a wide array of imagery options - ground-based, airborne, seaborne, or vehicle-based - that are available through the Government and commercial vendors. The team guides the customer in assembling the appropriate complement of imagery acquisition assets at the customer's facilities, minimizing costs associated with market research and the risk of purchasing inadequate assets. The NASA Integration 2 capability simplifies the process of securing one

  12. UCSC Data Integrator and Variant Annotation Integrator

    PubMed Central

    Hinrichs, Angie S.; Raney, Brian J.; Speir, Matthew L.; Rhead, Brooke; Casper, Jonathan; Karolchik, Donna; Kuhn, Robert M.; Rosenbloom, Kate R.; Zweig, Ann S.; Haussler, David; Kent, W. James

    2016-01-01

    Summary: Two new tools on the UCSC Genome Browser web site provide improved ways of combining information from multiple datasets, optionally including the user's own custom track data and/or data from track hubs. The Data Integrator combines columns from multiple data tracks, showing all items from the first track along with overlapping items from the other tracks. The Variant Annotation Integrator is tailored to adding functional annotations to variant calls; it offers a more restricted set of underlying data tracks but adds predictions of each variant's consequences for any overlapping or nearby gene transcript. When available, it optionally adds additional annotations including effect prediction scores from dbNSFP for missense mutations, ENCODE regulatory summary tracks and conservation scores. Availability and implementation: The web tools are freely available at http://genome.ucsc.edu/ and the underlying database is available for download at http://hgdownload.cse.ucsc.edu/. The software (written in C and Javascript) is available from https://genome-store.ucsc.edu/ and is freely available for academic and non-profit usage; commercial users must obtain a license. Contact: angie@soe.ucsc.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26740527

  13. Integrated work management system.

    SciTech Connect

    Williams, Edward J., Jr.; Henry, Karen Lynne

    2010-06-01

    Sandia National Laboratories develops technologies to: (1) sustain, modernize, and protect our nuclear arsenal (2) Prevent the spread of weapons of mass destruction; (3) Provide new capabilities to our armed forces; (4) Protect our national infrastructure; (5) Ensure the stability of our nation's energy and water supplies; and (6) Defend our nation against terrorist threats. We identified the need for a single overarching Integrated Workplace Management System (IWMS) that would enable us to focus on customer missions and improve FMOC processes. Our team selected highly configurable commercial-off-the-shelf (COTS) software with out-of-the-box workflow processes that integrate strategic planning, project management, facility assessments, and space management, and can interface with existing systems, such as Oracle, PeopleSoft, Maximo, Bentley, and FileNet. We selected the Integrated Workplace Management System (IWMS) from Tririga, Inc. Facility Management System (FMS) Benefits are: (1) Create a single reliable source for facility data; (2) Improve transparency with oversight organizations; (3) Streamline FMOC business processes with a single, integrated facility-management tool; (4) Give customers simple tools and real-time information; (5) Reduce indirect costs; (6) Replace approximately 30 FMOC systems and 60 homegrown tools (such as Microsoft Access databases); and (7) Integrate with FIMS.

  14. HIV DNA Integration

    PubMed Central

    Craigie, Robert; Bushman, Frederic D.

    2012-01-01

    Retroviruses are distinguished from other viruses by two characteristic steps in the viral replication cycle. The first is reverse transcription, which results in the production of a double-stranded DNA copy of the viral RNA genome, and the second is integration, which results in covalent attachment of the DNA copy to host cell DNA. The initial catalytic steps of the integration reaction are performed by the virus-encoded integrase (IN) protein. The chemistry of the IN-mediated DNA breaking and joining steps is well worked out, and structures of IN-DNA complexes have now clarified how the overall complex assembles. Methods developed during these studies were adapted for identification of IN inhibitors, which received FDA approval for use in patients in 2007. At the chromosomal level, HIV integration is strongly favored in active transcription units, which may promote efficient viral gene expression after integration. HIV IN binds to the cellular factor LEDGF/p75, which promotes efficient infection and tethers IN to favored target sites. The HIV integration machinery must also interact with many additional host factors during infection, including nuclear trafficking and pore proteins during nuclear entry, histones during initial target capture, and DNA repair proteins during completion of the DNA joining steps. Models for some of the molecular mechanisms involved have been proposed, but important details remain to be clarified. PMID:22762018

  15. Integrated Budget Office Toolbox

    NASA Technical Reports Server (NTRS)

    Rushing, Douglas A.; Blakeley, Chris; Chapman, Gerry; Robertson, Bill; Horton, Allison; Besser, Thomas; McCarthy, Debbie

    2010-01-01

    The Integrated Budget Office Toolbox (IBOT) combines budgeting, resource allocation, organizational funding, and reporting features in an automated, integrated tool that provides data from a single source for Johnson Space Center (JSC) personnel. Using a common interface, concurrent users can utilize the data without compromising its integrity. IBOT tracks planning changes and updates throughout the year using both phasing and POP-related (program-operating-plan-related) budget information for the current year, and up to six years out. Separating lump-sum funds received from HQ (Headquarters) into separate labor, travel, procurement, Center G&A (general & administrative), and servicepool categories, IBOT creates a script that significantly reduces manual input time. IBOT also manages the movement of travel and procurement funds down to the organizational level and, using its integrated funds management feature, helps better track funding at lower levels. Third-party software is used to create integrated reports in IBOT that can be generated for plans, actuals, funds received, and other combinations of data that are currently maintained in the centralized format. Based on Microsoft SQL, IBOT incorporates generic budget processes, is transportable, and is economical to deploy and support.

  16. Ideal Integrating Bolometer

    NASA Technical Reports Server (NTRS)

    Kogut, A.; DiPirro, M.; Moseley, S. H.

    2004-01-01

    We describe a new "ideal integrator" bolometer as a prototype for a new generation of sensitive, flexible far-IR detectors suitable for use in large arrays. The combination of a non-dissipative sensor coupled with a fast heat switch provides breakthrough capabilities in both sensitivity and operation. The bolometer temperature varies linearly with the integrated infrared power incident on the detector, and may be sampled intermittently without loss of information between samples. The sample speed and consequent dynamic range depend only on the heat switch reset cycle and can be selected in software. Between samples, the device acts as an ideal integrator with noise significantly lower than resistive bolometers. Since there is no loss of information between samples, the device is well-suited for large arrays. A single SQUID readout could process an entire column of detectors, greatly reducing the complexity, power requirements, and cost of readout electronics for large pixel arrays.

  17. Chewing over physiology integration.

    PubMed

    Abdulkader, Fernando; Azevedo-Martins, Anna Karenina; Miranda, Manoel de Arcisio; Brunaldi, Kellen

    2005-03-01

    An important challenge for both students and teachers of physiology is to integrate the different areas in which physiological knowledge is didactically divided. In developing countries, such an issue is even more demanding, because budget restrictions often affect the physiology program with laboratory classes being the first on the list when it comes to cuts in expenses. With the aim of addressing this kind of problem, the graduate students of our department organized a physiology summer course offered to undergraduate students. The objective was to present the different physiological systems in an integrated fashion. The strategy pursued was to plan laboratory classes whose experimental results were the basis for the relevant theoretical discussions. The subject we developed to illustrate physiology integration was the study of factors influencing salivary secretion.

  18. Partially integrated exhaust manifold

    SciTech Connect

    Hayman, Alan W; Baker, Rodney E

    2015-01-20

    A partially integrated manifold assembly is disclosed which improves performance, reduces cost and provides efficient packaging of engine components. The partially integrated manifold assembly includes a first leg extending from a first port and terminating at a mounting flange for an exhaust gas control valve. Multiple additional legs (depending on the total number of cylinders) are integrally formed with the cylinder head assembly and extend from the ports of the associated cylinder and terminate at an exit port flange. These additional legs are longer than the first leg such that the exit port flange is spaced apart from the mounting flange. This configuration provides increased packaging space adjacent the first leg for any valving that may be required to control the direction and destination of exhaust flow in recirculation to an EGR valve or downstream to a catalytic converter.

  19. Integrated digital networks

    NASA Astrophysics Data System (ADS)

    Schwartz, M.; Stern, T.; Lazar, A.

    1984-09-01

    The basic problem described in this document is that of transmitting mixtures of traffic of disparate types over a variety of communication networks. Typical examples include the transmission of interactive data, long streams of data (e.g., file transfers), voice, video, and facsimile in an integrated fashion. Network types include local area networks, metropolitan area networks, large geographically-dispersed terrestrial networks, and satellite networks. Ongoing standards work in the C CITT, supported by telephone administrations worldwide, has focused on the concept of Integrated Services Digital Networks (ISDN) toward which worldwide telecommunications will be moving. Computer manufacturers with a great deal of interest in communications (IBM is a prominent example) have begun to devote considerable effort as well to the concept of traffic integration over networks.

  20. Integrated building design

    NASA Astrophysics Data System (ADS)

    Sanguinetti, Jennifer

    2005-04-01

    For many years, building design has been a very linear process with owners speaking to architects who then design building shells that they pass along to sub-consultants who must fit their systems into the allotted spaces. While this process has some advantages, it provides little opportunity to optimize systems based on such factors as energy use or occupant comfort. This presentation will focus on the evolution and implications of integrated building design, a method that has provided greater opportunities for interaction between design disciplines and with building users early on in the design process. Integration has resulted in buildings that are more sustainable than typical buildings and that can respond better to the needs of the owner and users. Examples of the application of the process and the resulting buildings will be presented from the view of a design engineer with experience of both processes. Specifically, the potential contribution of an acoustical consultant in the integrated process will be explored.

  1. Integral diode solar cells

    SciTech Connect

    Mardesich, W.; Gillanders, M.S.

    1984-05-01

    To achieve high power at minimum weight, innovative array designs are needed. In the case where shadows fall across a series element in a simple circuit, the effective power will be reduced or eliminated. The conventional method of eliminating this loss is the introduction of bypass diodes. This method increases cost and weight and reduces available surface area. An alternative solution to the shadowing problem is to use integral diode solar cells. The integral diode cell has a built-in diode on the back that protects the adjacent cell and passes the current if it is shadowed. This paper will describe the effort to produce the integral diode cells in a production facility with a minimum cost impact. The electrical characterization of the cell as well as the diode will be presented. These cells can be readily manufactured in a production facility using photoresist defined contacting process.

  2. Chewing over physiology integration.

    PubMed

    Abdulkader, Fernando; Azevedo-Martins, Anna Karenina; Miranda, Manoel de Arcisio; Brunaldi, Kellen

    2005-03-01

    An important challenge for both students and teachers of physiology is to integrate the different areas in which physiological knowledge is didactically divided. In developing countries, such an issue is even more demanding, because budget restrictions often affect the physiology program with laboratory classes being the first on the list when it comes to cuts in expenses. With the aim of addressing this kind of problem, the graduate students of our department organized a physiology summer course offered to undergraduate students. The objective was to present the different physiological systems in an integrated fashion. The strategy pursued was to plan laboratory classes whose experimental results were the basis for the relevant theoretical discussions. The subject we developed to illustrate physiology integration was the study of factors influencing salivary secretion. PMID:15718383

  3. Integrated Avionics System (IAS)

    NASA Technical Reports Server (NTRS)

    Hunter, D. J.

    2001-01-01

    As spacecraft designs converge toward miniaturization and with the volumetric and mass constraints placed on avionics, programs will continue to advance the 'state of the art' in spacecraft systems development with new challenges to reduce power, mass, and volume. Although new technologies have improved packaging densities, a total system packaging architecture is required that not only reduces spacecraft volume and mass budgets, but increase integration efficiencies, provide modularity and scalability to accommodate multiple missions. With these challenges in mind, a novel packaging approach incorporates solutions that provide broader environmental applications, more flexible system interconnectivity, scalability, and simplified assembly test and integration schemes. This paper will describe the fundamental elements of the Integrated Avionics System (IAS), Horizontally Mounted Cube (HMC) hardware design, system and environmental test results. Additional information is contained in the original extended abstract.

  4. Integration and Beyond

    PubMed Central

    Stead, William W.; Miller, Randolph A.; Musen, Mark A.; Hersh, William R.

    2000-01-01

    The vision of integrating information—from a variety of sources, into the way people work, to improve decisions and process—is one of the cornerstones of biomedical informatics. Thoughts on how this vision might be realized have evolved as improvements in information and communication technologies, together with discoveries in biomedical informatics, and have changed the art of the possible. This review identified three distinct generations of “integration” projects. First-generation projects create a database and use it for multiple purposes. Second-generation projects integrate by bringing information from various sources together through enterprise information architecture. Third-generation projects inter-relate disparate but accessible information sources to provide the appearance of integration. The review suggests that the ideas developed in the earlier generations have not been supplanted by ideas from subsequent generations. Instead, the ideas represent a continuum of progress along the three dimensions of workflow, structure, and extraction. PMID:10730596

  5. Reflections on 'autistic integrity'.

    PubMed

    Russell, Barbara

    2012-03-01

    Autism, particularly its moderate to severe forms, has prompted considerable scientific study and clinical involvement because the associated behaviours imply disconnections with valued features of a 'good' life, such as close relationships, enjoyment, and adaptability. Proposed causes of autism involve potent philosophical concepts including consciousness, identity, mind, and relationality. The concept of autistic integrity is used by Barnbaum in The Ethics of Autism: Among Them, But Not of Them to help provide moral justification to stop efforts to cure adults with autism, especially if the cause is presumed to be a lack of a theory of mind.(1) This article has two goals: (1) to apply four familiar definitions or characterizations of integrity to the case of moderate to severe autism, and (2) to examine whether autistic integrity does provide the moral justification Barnbaum seeks.

  6. Integrative oncology: an overview.

    PubMed

    Deng, Gary; Cassileth, Barrie

    2014-01-01

    Integrative oncology, the diagnosis-specific field of integrative medicine, addresses symptom control with nonpharmacologic therapies. Known commonly as "complementary therapies" these are evidence-based adjuncts to mainstream care that effectively control physical and emotional symptoms, enhance physical and emotional strength, and provide patients with skills enabling them to help themselves throughout and following mainstream cancer treatment. Integrative or complementary therapies are rational and noninvasive. They have been subjected to study to determine their value, to document the problems they ameliorate, and to define the circumstances under which such therapies are beneficial. Conversely, "alternative" therapies typically are promoted literally as such; as actual antitumor treatments. They lack biologic plausibility and scientific evidence of safety and efficacy. Many are outright fraudulent. Conflating these two very different categories by use of the convenient acronym "CAM," for "complementary and alternative therapies," confuses the issue and does a substantial disservice to patients and medical professionals. Complementary and integrative modalities have demonstrated safety value and benefits. If the same were true for "alternatives," they would not be "alternatives." Rather, they would become part of mainstream cancer care. This manuscript explores the medical and sociocultural context of interest in integrative oncology as well as in "alternative" therapies, reviews commonly-asked patient questions, summarizes research results in both categories, and offers recommendations to help guide patients and family members through what is often a difficult maze. Combining complementary therapies with mainstream oncology care to address patients' physical, psychologic and spiritual needs constitutes the practice of integrative oncology. By recommending nonpharmacologic modalities that reduce symptom burden and improve quality of life, physicians also enable

  7. NASA Integrated Network COOP

    NASA Technical Reports Server (NTRS)

    Anderson, Michael L.; Wright, Nathaniel; Tai, Wallace

    2012-01-01

    Natural disasters, terrorist attacks, civil unrest, and other events have the potential of disrupting mission-essential operations in any space communications network. NASA's Space Communications and Navigation office (SCaN) is in the process of studying options for integrating the three existing NASA network elements, the Deep Space Network, the Near Earth Network, and the Space Network, into a single integrated network with common services and interfaces. The need to maintain Continuity of Operations (COOP) after a disastrous event has a direct impact on the future network design and operations concepts. The SCaN Integrated Network will provide support to a variety of user missions. The missions have diverse requirements and include anything from earth based platforms to planetary missions and rovers. It is presumed that an integrated network, with common interfaces and processes, provides an inherent advantage to COOP in that multiple elements and networks can provide cross-support in a seamless manner. The results of trade studies support this assumption but also show that centralization as a means of achieving integration can result in single points of failure that must be mitigated. The cost to provide this mitigation can be substantial. In support of this effort, the team evaluated the current approaches to COOP, developed multiple potential approaches to COOP in a future integrated network, evaluated the interdependencies of the various approaches to the various network control and operations options, and did a best value assessment of the options. The paper will describe the trade space, the study methods, and results of the study.

  8. Integrated Functionality: Nanosensors

    NASA Astrophysics Data System (ADS)

    Devreese, Jozef T.

    2008-03-01

    Integrated nanosensors are nanostructured systems in which several sensors of different types have been integrated on a single platform including those sensitive to optical, magnetic, chemical, or biological stimuli [1,2]. Nanoparticle-based detector systems rely on the development of nanoparticles as sensing species. I discuss state-of-the-art nanosensors which are based on various advanced materials: nanoshells and nanorice, gold nanoparticles with attached fluorescent dyes, nanopores, carbon nanotubes, neuroelectronic hybrid systems, semiconductor nanocrystals and quantum-dot quantum wells (QDQW's). Phonon-assisted optical processes in semiconductor nanocrystals and QDQW's are highly sensitive to their shape and geometry i. a. due to the non-adiabaticity of the exciton-phonon systems. This sensitivity opens new perspectives for applications of quantum dots in optical sensing. For example, the simultaneous consideration of the tetrahedral shape of a CdS/HgS/CdS QDQW, interface optical phonons, and non-adiabatic phonon-assisted transitions allows for control of the photoluminescence of a QDQW [3]. Quantum-dot-based systems are also considered as examples of communication with nanodevices, which is a prerequisite of their integration. Nanosensors allow for building a new class of integrated devices, which provide the elemental base for ``intelligent sensors'' capable of data processing, storage and analysis. The development of integrated nanosensors could have many applications in several fields such as process monitoring, robotics, environmental, medical, consumer, homeland security [1] I. K. Schuller, http://nanosensors.ucsd.edu/Introduction.htm. [2] J. T. Devreese and Y. Bruynseraede, Integrated Nanosensors. McGraw-Hill 2008 Yearbook of Science & Technology, McGraw-Hill, 2008. [3] V. A. Fonoberov, E. P. Pokatilov, V. M. Fomin, and J. T. Devreese, Phys. Rev. Lett. 92, 127402 (2004).

  9. Embracing Integrative Multiomics Approaches

    PubMed Central

    2016-01-01

    As “-omics” data technology advances and becomes more readily accessible to address complex biological questions, increasing amount of cross “-omics” dataset is inspiring the use and development of integrative bioinformatics analysis. In the current review, we discuss multiple options for integrating data across “-omes” for a range of study designs. We discuss established methods for such analysis and point the reader to in-depth discussions for the various topics. Additionally, we discuss challenges and new directions in the area. PMID:27689071

  10. Integrating with integrins

    NASA Technical Reports Server (NTRS)

    Schwartz, M. A.; Ingber, D. E.

    1994-01-01

    Our central claim is that signaling by integrins provides a mechanism by which signals generated in response to adhesion, soluble hormones, and mechanical forces can interact. Such interactions permit cells to integrate these different classes of external stimuli and hence to orchestrate an efficient response. This integrating function of integrins is likely to be essential for much of development and physiology, as well as complex pathologies such as cancer. Understanding in detail how these signals are transduced and processed is likely to be an important area of research in the near future.

  11. Smart Grid Integration Laboratory

    SciTech Connect

    Troxell, Wade

    2011-12-22

    The initial federal funding for the Colorado State University Smart Grid Integration Laboratory is through a Congressionally Directed Project (CDP), DE-OE0000070 Smart Grid Integration Laboratory. The original program requested in three one-year increments for staff acquisition, curriculum development, and instrumentation all which will benefit the Laboratory. This report focuses on the initial phase of staff acquisition which was directed and administered by DOE NETL/ West Virginia under Project Officer Tom George. Using this CDP funding, we have developed the leadership and intellectual capacity for the SGIC. This was accomplished by investing (hiring) a core team of Smart Grid Systems engineering faculty focused on education, research, and innovation of a secure and smart grid infrastructure. The Smart Grid Integration Laboratory will be housed with the separately funded Integrid Laboratory as part of CSU's overall Smart Grid Integration Center (SGIC). The period of performance of this grant was 10/1/2009 to 9/30/2011 which included one no cost extension due to time delays in faculty hiring. The Smart Grid Integration Laboratory's focus is to build foundations to help graduate and undergraduates acquire systems engineering knowledge; conduct innovative research; and team externally with grid smart organizations. Using the results of the separately funded Smart Grid Workforce Education Workshop (May 2009) sponsored by the City of Fort Collins, Northern Colorado Clean Energy Cluster, Colorado State University Continuing Education, Spirae, and Siemens has been used to guide the hiring of faculty, program curriculum and education plan. This project develops faculty leaders with the intellectual capacity to inspire its students to become leaders that substantially contribute to the development and maintenance of Smart Grid infrastructure through topics such as: (1) Distributed energy systems modeling and control; (2) Energy and power conversion; (3) Simulation of

  12. Integral rocket ramjets

    NASA Astrophysics Data System (ADS)

    Calzone, R. F.

    1994-03-01

    A rough overview of the important aspects and problem areas associated with the development of Integral Rocket Ramjet (IRR) technology is given in this report. The IRR is a supersonic air-breathing concept in which the gas generator produces fuel-rich gases. These fuel-rich gases are burnt in the secondary combustion chamber with ambient air captured and decelerated in the inlet. During the boost phase, a solid propellant booster provides the thrust necessary to achieve the velocity at which the ramjet may be operated (about M = 2). The booster is integrated in the secondary combustion chamber.

  13. Integrated Safety Analysis Tiers

    NASA Technical Reports Server (NTRS)

    Shackelford, Carla; McNairy, Lisa; Wetherholt, Jon

    2009-01-01

    Commercial partnerships and organizational constraints, combined with complex systems, may lead to division of hazard analysis across organizations. This division could cause important hazards to be overlooked, causes to be missed, controls for a hazard to be incomplete, or verifications to be inefficient. Each organization s team must understand at least one level beyond the interface sufficiently enough to comprehend integrated hazards. This paper will discuss various ways to properly divide analysis among organizations. The Ares I launch vehicle integrated safety analyses effort will be utilized to illustrate an approach that addresses the key issues and concerns arising from multiple analysis responsibilities.

  14. On integrable conservation laws

    PubMed Central

    Arsie, Alessandro; Lorenzoni, Paolo; Moro, Antonio

    2015-01-01

    We study normal forms of scalar integrable dispersive (not necessarily Hamiltonian) conservation laws, via the Dubrovin–Zhang perturbative scheme. Our computations support the conjecture that such normal forms are parametrized by infinitely many arbitrary functions that can be identified with the coefficients of the quasi-linear part of the equation. Moreover, in general, we conjecture that two scalar integrable evolutionary partial differential equations having the same quasi-linear part are Miura equivalent. This conjecture is also consistent with the tensorial behaviour of these coefficients under general Miura transformations. PMID:25568614

  15. Environmental integrity, racism, health.

    PubMed

    Westra, L

    1996-05-17

    Environmental degradation seriously affects human health. Thus, a close relationship exists between the protection of ecosystem integrity and wilderness on one hand, and human health on the other. However, there is an overarching, holistic perspective in laws and regulations--as well as morality--to to maintain a healthy relationship between the two. Problem areas focused on in this paper are: (a) climate change and global warming; (b) food production; and (c) global equity. This paper argues for the principle of integrity, which provides an holistic perspective, suggested as a better approach than that of current regulations to mitigate against associated threats to human health.

  16. Finnish care integrated?

    PubMed Central

    Niskanen, J. Jouni

    2002-01-01

    Abstract The public Finnish social and health care system has been challenged by the economic crisis, administrative reforms and increased demands. Better integration as a solution includes many examples, which have been taken to use. The most important are the rewritten national and municipals strategies and quality recommendations, where the different sectors and the levels of care are seen as one entity. Many reorganisations have taken place, both nationally and locally, and welfare clusters have been established. The best examples of integrated care are the forms of teamwork, care management, emphasis on non-institutional care and the information technology. PMID:16896395

  17. Integrated biogas systems

    NASA Astrophysics Data System (ADS)

    Amaratunga, M.

    1980-01-01

    Integrated biogas systems as alternatives to fossil fuels in Sri Lanka are considered from standpoints of population growth, land availability, and employment opportunities. Agricultural practices would be improved by use of chemical fertilizers, and health/nutrition problems be alleviated by using biogas systems. Fuel for cooking and rural industries will become more easily available; water weeds, such as water hyacinth and salvinia which pose a threat to waterways and rice paddy lands could be used for the production of biogas and fertilizers. A concept of an integrated biogas system comprising photosynthesis and anaerobic degradation processes to produce food and energy is presented.

  18. The benefits of integration.

    PubMed

    Ciuffi, A

    2016-04-01

    Retroviruses, including the human immunodeficiency virus (HIV), are notorious for two essential steps of their viral replication: reverse transcription and integration. This latter property is considered to be essential for productive replication and ensures the stable long-term insertion of the viral genome sequence in the host chromatin, thereby leading to the life-long association of the virus with the infected cell. Using HIV as a prototypic example, the present review aims to provide an overview of how and where integration occurs, as well as presenting general consequences for both the virus and the infected host. PMID:27107301

  19. Integrated heterodyne terahertz transceiver

    DOEpatents

    Lee, Mark; Wanke, Michael C.

    2009-06-23

    A heterodyne terahertz transceiver comprises a quantum cascade laser that is integrated on-chip with a Schottky diode mixer. An antenna connected to the Schottky diode receives a terahertz signal. The quantum cascade laser couples terahertz local oscillator power to the Schottky diode to mix with the received terahertz signal to provide an intermediate frequency output signal. The fully integrated transceiver optimizes power efficiency, sensitivity, compactness, and reliability. The transceiver can be used in compact, fieldable systems covering a wide variety of deployable applications not possible with existing technology.

  20. Integrated heterodyne terahertz transceiver

    DOEpatents

    Wanke, Michael C.; Lee, Mark; Nordquist, Christopher D.; Cich, Michael J.

    2012-09-25

    A heterodyne terahertz transceiver comprises a quantum cascade laser that is integrated on-chip with a Schottky diode mixer. A terahertz signal can be received by an antenna connected to the mixer, an end facet or sidewall of the laser, or through a separate active section that can amplify the incident signal. The quantum cascade laser couples terahertz local oscillator power to the Schottky diode to mix with the received terahertz signal to provide an intermediate frequency output signal. The fully integrated transceiver optimizes power efficiency, sensitivity, compactness, and reliability. The transceiver can be used in compact, fieldable systems covering a wide variety of deployable applications not possible with existing technology.

  1. Bayesian Integrated Microbial Forensics

    SciTech Connect

    Jarman, Kristin H.; Kreuzer-Martin, Helen W.; Wunschel, David S.; Valentine, Nancy B.; Cliff, John B.; Petersen, Catherine E.; Colburn, Heather A.; Wahl, Karen L.

    2008-06-01

    In the aftermath of the 2001 anthrax letters, researchers have been exploring ways to predict the production environment of unknown source microorganisms. Different mass spectral techniques are being developed to characterize components of a microbe’s culture medium including water, carbon and nitrogen sources, metal ions added, and the presence of agar. Individually, each technique has the potential to identify one or two ingredients in a culture medium recipe. However, by integrating data from multiple mass spectral techniques, a more complete characterization is possible. We present a Bayesian statistical approach to integrated microbial forensics and illustrate its application on spores grown in different culture media.

  2. Integrated traditional Chinese medicine.

    PubMed

    Robinson, Nicola

    2006-05-01

    To experience the integration of traditional Chinese medicine (TCM) in China was 'the chance of a lifetime; thanks to the support of the Winston Churchill Memorial Trust. The scale and range of TCM available in terms of health care provision, education and research is unique in the world. This holistic integrative medicine is part of Chinese culture. Regulation and training of practitioners has similarities with current structures emerging in the UK in preparation for the statutory regulation for acupuncture and herbal medicine. China's research activity is a critical component of informing the debate on evidence-based practice and now real opportunities for collaboration and dissemination are beginning to emerge. PMID:16648091

  3. Embracing Integrative Multiomics Approaches

    PubMed Central

    2016-01-01

    As “-omics” data technology advances and becomes more readily accessible to address complex biological questions, increasing amount of cross “-omics” dataset is inspiring the use and development of integrative bioinformatics analysis. In the current review, we discuss multiple options for integrating data across “-omes” for a range of study designs. We discuss established methods for such analysis and point the reader to in-depth discussions for the various topics. Additionally, we discuss challenges and new directions in the area.

  4. Two integrable systems with integrals of motion of degree four

    NASA Astrophysics Data System (ADS)

    Tsiganov, A. V.

    2016-03-01

    We discuss the possibility of using second-order Killing tensors to construct Liouville-integrable Hamiltonian systems that are not Nijenhuis integrable. As an example, we consider two Killing tensors with a nonzero Haantjes torsion that satisfy weaker geometric conditions and also three-dimensional systems corresponding to them that are integrable in Euclidean space and have two quadratic integrals of motion and one fourth-order integral in momenta.

  5. Measuring integrated care.

    PubMed

    Strandberg-Larsen, Martin

    2011-02-01

    The positive outcomes of coordination of healthcare services are to an increasing extent becoming clear. However the complexity of the field is an inhibiting factor for vigorously designed trial studies. Conceptual clarity and a consistent theoretical frame-work are thus needed. While researchers respond to these needs, patients and providers face the multiple challenges of today's healthcare environment. Decision makers, planners and managers need evidence based policy options and information on the scope of the integrated care challenges they are facing. The US managed care organization Kaiser Permanente has been put forward as an example for European healthcare systems to follow, although the evidence base is far from conclusive. The thesis has five objectives: 1) To contribute to the understanding of the concept of integration in healthcare systems and to identify measurement methods to capture the multi-dimensional aspects of integrated healthcare delivery. 2) To assess the level of integration of the Danish healthcare system. 3) To assess the use of joint health plans as a tool for coordination between the regional and local level in the Danish healthcare system. 4) To compare the inputs and performance of the Danish healthcare system and the managed care organization Kaiser Permanente, California, US. 5) To compare primary care clinicians' perception of clinical integration in two healthcare systems: Kaiser Permanente, Northern California and the Danish healthcare system. Further to examine the associations between specific organizational factors and clinical integration within each system. The literature was systematically searched to identify methods for measurement of integrated healthcare delivery. A national cross-sectional survey was conducted among major professional stake-holders at five different levels of the Danish healthcare system. The survey data were used to allow for analysis of the level of integration achieved. Data from the survey were

  6. Integrity in Student Affairs Organizations

    ERIC Educational Resources Information Center

    Baird, Leonard

    2011-01-01

    "Integrity" is a term that is intuitively appealing, but hard to define and implement. This chapter discusses those conceptual complexities as well as an ideal portrait of organizations with integrity, a description of the challenges to the integrity of organizations, a discussion of the enhancement of integrity through compliance programs and…

  7. An Integrated Model Recontextualized

    ERIC Educational Resources Information Center

    O'Meara, KerryAnn; Saltmarsh, John

    2016-01-01

    In this commentary, authors KerryAnn O'Meara and John Saltmarsh reflect on their 2008 "Journal of Higher Education Outreach and Engagement" article "An Integrated Model for Advancing the Scholarship of Engagement: Creating Academic Homes for the Engaged Scholar," reprinted in this 20th anniversary issue of "Journal of…

  8. The New Integration

    ERIC Educational Resources Information Center

    Kahlenberg, Richard D.

    2006-01-01

    The goal of closing achievement gaps between students of different socioeconomic status and race has eluded public schools for decades. Facing increased pressure from NCLB to reach this goal, some school districts have turned to a new experiment based on an old-fashioned vision: integrating students by socioeconomic income. Kahlenberg reviews the…

  9. Visual integration in autism

    PubMed Central

    Smith, Danielle; Ropar, Danielle; Allen, Harriet A.

    2015-01-01

    Atypical integration is a topic of debate in the autism literature. Some theories suggest that altered perception in autism spectrum disorder (ASD) is due to a failure to integrate information from meaningful context into the final percept, whereas others suggest that integration of low-level features is impaired. Empirical research which forms the basis for these theories has failed to account for higher-level influences not inherent in the stimuli (i.e., instructions and goals) and assess integration at both lower and higher perceptual levels within the same task. Here, we describe how perceived expectations and goals of a task can modulate the processing of low-level visual input via the medial prefrontal cortex (mPFC). We then go on to illustrate how future research might assess the relative contribution of both low and high-level processes using the same paradigm. We conclude by recommending that when results appear conflicting, consideration of the relative strength of low-level input vs. feedback or high-level processes may prove helpful. Importantly, research in this area needs to more broadly consider the various influences on perception, and find better ways to assess the contributions of early and later visual processes. PMID:26190994

  10. External Cargo Integration Overview

    NASA Technical Reports Server (NTRS)

    Gueera, Alan

    2005-01-01

    This viewgraph presentation reviews the system integration efforts for external cargo for the International Space Station (ISS). The role and responsibility of the External Carriers Ofice is reviewed. The presentation also reviews the application of the office to the Commercial Cargo Services contract.

  11. Integration: Reversing Traditional Pedagogy

    ERIC Educational Resources Information Center

    Yost, David

    2008-01-01

    A derivative is the limit of a quotient. It is an abstraction of division. Since division is harder to understand than multiplication, teachers teach it later, hopefully only after a sound understanding of multiplication has been attained. For the same reason, it may make sense to teach integration first, and move on to differential calculus only…

  12. Integrating Refugee Children.

    ERIC Educational Resources Information Center

    Vershok, Anna

    2003-01-01

    Notes that the Moscow Center for the Integration and Education of Refugee Children attempts to provide real schooling for refugee children displaced from the Chechen Republic. Suggests main task was to prepare the children for regular school, and to help them adapt to both their new school and their new home, Moscow. Hopes this experience may help…

  13. Integrated Pest Management.

    ERIC Educational Resources Information Center

    Council on Environmental Quality, Washington, DC.

    After a brief discussion of the problems of pesticide use and the status of current pest control practices, a definition of integrated pest management is given along with some examples of its successful application, and a description of some of the reasons why the concept has not been applied more widely. The major techniques which can be used as…

  14. Education and European Integration.

    ERIC Educational Resources Information Center

    Lowe, John

    1992-01-01

    Reviews implications for education and training of the movement toward integration among European Community nations and the end of Communist governments. Discusses common concerns for new Europe, including data sharing, teacher training, educational quality, disadvantaged learners, demographic and employment trends, European Studies curricula, and…

  15. SALAD helicopter integrated sensor

    SciTech Connect

    Soo Hoo, M.S.

    1988-08-01

    The theory and operation of an integrated acoustic and seismic sensor for use with the SALAD helicopter detection system is presented. This sensor incorporates a microphone, geophone, acoustic preamplifier, and tamper indicating features in a buryable, compact aluminum package. This sensor is intended for deployment within a pre-selected, controlled media.

  16. Explorations in Integrated Science

    ERIC Educational Resources Information Center

    Lega, Joceline C.; Buxner, Sanlyn; Blonder, Benjamin; Tama, Florence

    2014-01-01

    We describe a third-year undergraduate course that focuses on multiscale modeling and protein folding and has as its primary goal the encouragement of students to integrate thinking across and beyond disciplinary boundaries. The ability to perform innovative and successful research work in STEM (science, technology, engineering, and mathematics)…

  17. Integrated Learning Management Systems

    ERIC Educational Resources Information Center

    Clark, Sharon; Cossarin, Mary; Doxsee, Harry; Schwartz, Linda

    2004-01-01

    Four integrated learning management packages were reviewed: "CentraOne", "IntraLearn", "Lyceum", and "Silicon Chalk". These products provide different combinations of synchronous and asynchronous tools. The current report examines the products in relation to their specific value for distance educators and students.

  18. Integrated Leisure and Recreation.

    ERIC Educational Resources Information Center

    Schleien, Stuart, Ed.; Rynders, John, Ed.

    1989-01-01

    This "feature issue" focuses on integrated leisure and recreation for developmentally disabled persons and includes descriptions of innovative leisure/recreation programs which allow the realization of the concepts of normalization and least restrictive environment. Brief articles include the following titles and authors: "Challenging the…

  19. The Integral Fast Reactor

    SciTech Connect

    Till, C.E.; Chang, Y.I. ); Lineberry, M.J. )

    1990-01-01

    Argonne National Laboratory, since 1984, has been developing the Integral Fast Reactor (IFR). This paper will describe the way in which this new reactor concept came about; the technical, public acceptance, and environmental issues that are addressed by the IFR; the technical progress that has been made; and our expectations for this program in the near term. 5 refs., 3 figs.

  20. 3 CFR - Scientific Integrity

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Departments and Agencies Science and the scientific process must inform and guide decisions of my..., and protection of national security. The public must be able to trust the science and scientific..., and integrity. By this memorandum, I assign to the Director of the Office of Science and...