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Sample records for autoinducer-2 processing protein

  1. Auto-inducing media for uniform isotope labeling of proteins with 15N, 13C and 2H

    International Nuclear Information System (INIS)

    Guthertz, Nicolas; Klopp, Julia; Winterhalter, Aurélie; Fernández, César; Gossert, Alvar D.

    2015-01-01

    Auto-inducing media for protein expression offer many advantages like robust reproducibility, high yields of soluble protein and much reduced workload. Here, an auto-inducing medium for uniform isotope labelling of proteins with 15 N, 13 C and/or 2 H in E. coli is presented. So far, auto-inducing media have not found widespread application in the NMR field, because of the prohibitively high cost of labeled lactose, which is an essential ingredient of such media. Here, we propose using lactose that is only selectively labeled on the glucose moiety. It can be synthesized from inexpensive and readily available substrates: labeled glucose and unlabeled activated galactose. With this approach, uniformly isotope labeled proteins were expressed in unattended auto-inducing cultures with incorporation of 13 C, 15 N of 96.6 % and 2 H, 15 N of 98.8 %. With the present protocol, the NMR community could profit from the many advantages that auto-inducing media offer

  2. Auto-inducing media for uniform isotope labeling of proteins with {sup 15}N, {sup 13}C and {sup 2}H

    Energy Technology Data Exchange (ETDEWEB)

    Guthertz, Nicolas [Institute of Cancer Research, Division of Structural Biology (United Kingdom); Klopp, Julia; Winterhalter, Aurélie; Fernández, César; Gossert, Alvar D., E-mail: alvar.gossert@novartis.com [Novartis Institutes for BioMedical Research (Switzerland)

    2015-06-15

    Auto-inducing media for protein expression offer many advantages like robust reproducibility, high yields of soluble protein and much reduced workload. Here, an auto-inducing medium for uniform isotope labelling of proteins with {sup 15}N, {sup 13}C and/or {sup 2}H in E. coli is presented. So far, auto-inducing media have not found widespread application in the NMR field, because of the prohibitively high cost of labeled lactose, which is an essential ingredient of such media. Here, we propose using lactose that is only selectively labeled on the glucose moiety. It can be synthesized from inexpensive and readily available substrates: labeled glucose and unlabeled activated galactose. With this approach, uniformly isotope labeled proteins were expressed in unattended auto-inducing cultures with incorporation of {sup 13}C, {sup 15}N of 96.6 % and {sup 2}H, {sup 15}N of 98.8 %. With the present protocol, the NMR community could profit from the many advantages that auto-inducing media offer.

  3. Bacteroides species produce Vibrio harveyi autoinducer 2-related molecules.

    Science.gov (United States)

    Antunes, Luis Caetano Martha; Ferreira, Lívia Queiroz; Ferreira, Eliane Oliveira; Miranda, Karla Rodrigues; Avelar, Kátia Eliane Santos; Domingues, Regina Maria Cavalcanti Pilotto; Ferreira, Maria Candida de Souza

    2005-10-01

    Quorum sensing is a density-dependent gene regulation mechanism that has been described in many bacterial species in the last decades. Bacteria that use quorum sensing as part of their gene regulation circuits produce molecules called autoinducers that accumulate in the environment and activate target genes in a quorum-dependent way. Some specific clues led us to hypothesize that Bacteroides species can produce autoinducers and possess a quorum sensing system. First, Bacteroides are anaerobic bacteria that are frequently involved in polymicrobial infections. These infections often involve Pseudomonas aeruginosa and Staphylococcus aureus, two of the best understood examples of bacteria that employ quorum sensing systems as part of their pathogenesis. Also, studies have detected the presence of a quorum sensing gene involved in the production of autoinducers in Porphyromonas gingivalis, a species closely related to the Bacteroides genus. These and other evidences prompted us to investigate if Bacteroides strains could produce autoinducer molecules that could be detected by a Vibrio harveyi reporter system. In this paper, we show that supernatants of B. fragilis, B. vulgatus and B. distasonis strains are able to stimulate the V. harveyi quorum sensing system 2. Also, we were able to demonstrate that the stimulation detected is due to the production of autoinducer molecules and not the growth of reporter strains after addition of supernatant. Moreover, the phenomenon observed does not seem to represent the degradation of repressors possibly present in the culture medium used. We could also amplify bands from some of the strains tested using primers designed to the luxS gene of Escherichia coli. Altogether, our results show that B. fragilis, B. vulgatus and B. distasonis (but possibly some other species) can produce V. harveyi autoinducer 2-related molecules. However, the role of such molecules in the biology of these organisms remains unknown.

  4. A Host-Produced Autoinducer-2 Mimic Activates Bacterial Quorum Sensing.

    Science.gov (United States)

    Ismail, Anisa S; Valastyan, Julie S; Bassler, Bonnie L

    2016-04-13

    Host-microbial symbioses are vital to health; nonetheless, little is known about the role crosskingdom signaling plays in these relationships. In a process called quorum sensing, bacteria communicate with one another using extracellular signal molecules called autoinducers. One autoinducer, AI-2, is proposed to promote interspecies bacterial communication, including in the mammalian gut. We show that mammalian epithelia produce an AI-2 mimic activity in response to bacteria or tight-junction disruption. This AI-2 mimic is detected by the bacterial AI-2 receptor, LuxP/LsrB, and can activate quorum-sensing-controlled gene expression, including in the enteric pathogen Salmonella typhimurium. AI-2 mimic activity is induced when epithelia are directly or indirectly exposed to bacteria, suggesting that a secreted bacterial component(s) stimulates its production. Mutagenesis revealed genes required for bacteria to both detect and stimulate production of the AI-2 mimic. These findings uncover a potential role for the mammalian AI-2 mimic in fostering crosskingdom signaling and host-bacterial symbioses. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Autoinducers act as biological timers in Vibrio harveyi.

    Science.gov (United States)

    Anetzberger, Claudia; Reiger, Matthias; Fekete, Agnes; Schell, Ursula; Stambrau, Nina; Plener, Laure; Kopka, Joachim; Schmitt-Kopplin, Phillippe; Hilbi, Hubert; Jung, Kirsten

    2012-01-01

    Quorum sensing regulates cell density-dependent phenotypes and involves the synthesis, excretion and detection of so-called autoinducers. Vibrio harveyi strain ATCC BAA-1116 (recently reclassified as Vibrio campbellii), one of the best-characterized model organisms for the study of quorum sensing, produces and responds to three autoinducers. HAI-1, AI-2 and CAI-1 are recognized by different receptors, but all information is channeled into the same signaling cascade, which controls a specific set of genes. Here we examine temporal variations of availability and concentration of the three autoinducers in V. harveyi, and monitor the phenotypes they regulate, from the early exponential to the stationary growth phase in liquid culture. Specifically, the exponential growth phase is characterized by an increase in AI-2 and the induction of bioluminescence, while HAI-1 and CAI-1 are undetectable prior to the late exponential growth phase. CAI-1 activity reaches its maximum upon entry into stationary phase, while molar concentrations of AI-2 and HAI-1 become approximately equal. Similarly, autoinducer-dependent exoproteolytic activity increases at the transition into stationary phase. These findings are reflected in temporal alterations in expression of the luxR gene that encodes the master regulator LuxR, and of four autoinducer-regulated genes during growth. Moreover, in vitro phosphorylation assays reveal a tight correlation between the HAI-1/AI-2 ratio as input and levels of receptor-mediated phosphorylation of LuxU as output. Our study supports a model in which the combinations of autoinducers available, rather than cell density per se, determine the timing of various processes in V. harveyi populations.

  6. Autoinducers act as biological timers in Vibrio harveyi.

    Directory of Open Access Journals (Sweden)

    Claudia Anetzberger

    Full Text Available Quorum sensing regulates cell density-dependent phenotypes and involves the synthesis, excretion and detection of so-called autoinducers. Vibrio harveyi strain ATCC BAA-1116 (recently reclassified as Vibrio campbellii, one of the best-characterized model organisms for the study of quorum sensing, produces and responds to three autoinducers. HAI-1, AI-2 and CAI-1 are recognized by different receptors, but all information is channeled into the same signaling cascade, which controls a specific set of genes. Here we examine temporal variations of availability and concentration of the three autoinducers in V. harveyi, and monitor the phenotypes they regulate, from the early exponential to the stationary growth phase in liquid culture. Specifically, the exponential growth phase is characterized by an increase in AI-2 and the induction of bioluminescence, while HAI-1 and CAI-1 are undetectable prior to the late exponential growth phase. CAI-1 activity reaches its maximum upon entry into stationary phase, while molar concentrations of AI-2 and HAI-1 become approximately equal. Similarly, autoinducer-dependent exoproteolytic activity increases at the transition into stationary phase. These findings are reflected in temporal alterations in expression of the luxR gene that encodes the master regulator LuxR, and of four autoinducer-regulated genes during growth. Moreover, in vitro phosphorylation assays reveal a tight correlation between the HAI-1/AI-2 ratio as input and levels of receptor-mediated phosphorylation of LuxU as output. Our study supports a model in which the combinations of autoinducers available, rather than cell density per se, determine the timing of various processes in V. harveyi populations.

  7. The Staphylococcus aureus autoinducer-2 synthase LuxS is regulated by Ser/Thr phosphorylation.

    Science.gov (United States)

    Cluzel, Marie-Eve; Zanella-Cléon, Isabelle; Cozzone, Alain J; Fütterer, Klaus; Duclos, Bertrand; Molle, Virginie

    2010-12-01

    The Staphylococcus aureus autoinducer-2 (AI-2) producer protein LuxS is phosphorylated by the Ser/Thr kinase Stk1 at a unique position, Thr14. The enzymatic activity of the phosphorylated isoform of LuxS was abrogated compared to that of nonphosphorylated LuxS, thus providing the first evidence of an AI-2-producing enzyme regulated by phosphorylation and demonstrating that S. aureus possesses an original and specific system for controlling AI-2 synthesis.

  8. The Staphylococcus aureus Autoinducer-2 Synthase LuxS Is Regulated by Ser/Thr Phosphorylation▿

    Science.gov (United States)

    Cluzel, Marie-Eve; Zanella-Cléon, Isabelle; Cozzone, Alain J.; Fütterer, Klaus; Duclos, Bertrand; Molle, Virginie

    2010-01-01

    The Staphylococcus aureus autoinducer-2 (AI-2) producer protein LuxS is phosphorylated by the Ser/Thr kinase Stk1 at a unique position, Thr14. The enzymatic activity of the phosphorylated isoform of LuxS was abrogated compared to that of nonphosphorylated LuxS, thus providing the first evidence of an AI-2-producing enzyme regulated by phosphorylation and demonstrating that S. aureus possesses an original and specific system for controlling AI-2 synthesis. PMID:20870760

  9. Methods for analysis of bacterial autoinducer-2 production.

    Science.gov (United States)

    Taga, Michiko E

    2005-07-01

    The quorum-sensing signal molecule autoinducer-2 (AI-2) is produced by over 50 diverse bacterial species and controls many different processes, including antibiotic production, biofilm formation, and virulence. AI-2 production often varies according to growth phase, media conditions, and the presence of specific factors. This unit describes a biological assay for AI-2 activity produced by a bacterial strain of interest. The assay employs an AI-2 reporter strain, Vibrio harveyi BB170, which produces light in response to AI-2. In the first stage of the assay, culture fluids of the bacterial strain of interest are collected over a time course of growth and filtered to remove cells. In the next stage, these culture fluids are mixed with BB170, and the light produced in response to AI-2 in the culture fluids is measured using a luminometer. BB170 is exquisitely sensitive to AI-2, and therefore, even low amounts of AI-2 can be detected using this bioassay.

  10. Is autoinducer-2 a universal signal for interspecies communication: a comparative genomic and phylogenetic analysis of the synthesis and signal transduction pathways

    Directory of Open Access Journals (Sweden)

    Wagner-Döbler Irene

    2004-09-01

    Full Text Available Abstract Background Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. Recently, it has been proposed that autoinducer-2 (AI-2, a furanosyl borate diester derived from the recycling of S-adenosyl-homocysteine (SAH to homocysteine, serves as a universal signal for interspecies communication. Results In this study, 138 completed genomes were examined for the genes involved in the synthesis and detection of AI-2. Except for some symbionts and parasites, all organisms have a pathway to recycle SAH, either using a two-step enzymatic conversion by the Pfs and LuxS enzymes or a one-step conversion using SAH-hydrolase (SahH. 51 organisms including most Gamma-, Beta-, and Epsilonproteobacteria, and Firmicutes possess the Pfs-LuxS pathway, while Archaea, Eukarya, Alphaproteobacteria, Actinobacteria and Cyanobacteria prefer the SahH pathway. In all 138 organisms, only the three Vibrio strains had strong, bidirectional matches to the periplasmic AI-2 binding protein LuxP and the central signal relay protein LuxU. The initial two-component sensor kinase protein LuxQ, and the terminal response regulator luxO are found in most Proteobacteria, as well as in some Firmicutes, often in several copies. Conclusions The genomic analysis indicates that the LuxS enzyme required for AI-2 synthesis is widespread in bacteria, while the periplasmic binding protein LuxP is only present in Vibrio strains. Thus, other organisms may either use components different from the AI-2 signal transduction system of Vibrio strains to sense the signal of AI-2, or they do not have such a quorum sensing system at all.

  11. Evaluation of two autoinducer-2 quantification methods for application in marine environments

    KAUST Repository

    Wang, Tian-Nyu; Kaksonen, Anna H.; Hong, Pei-Ying

    2018-01-01

    This study evaluated two methods, namely high performance liquid chromatography with fluorescence detection (HPLC-FLD) and Vibrio harveyi BB170 bioassay, for autoinducer-2 (AI-2) quantification in marine samples. Using both methods, the study also

  12. Autoinducers act as biological timers in Vibrio harveyi

    OpenAIRE

    Anetzberger, C.; Reiger, M.; Fekete, A.; Schell, U.; Stambrau, N.; Plener, L.; Kopka, J.; Schmitt-Kopplin, P.; Hilbi, H.; Jung, K.

    2012-01-01

    Quorum sensing regulates cell density-dependent phenotypes and involves the synthesis, excretion and detection of so-called autoinducers. Vibrio harveyi strain ATCC BAA-1116 (recently reclassified as Vibrio campbellii), one of the best-characterized model organisms for the study of quorum sensing, produces and responds to three autoinducers. HAI-1, AI-2 and CAI-1 are recognized by different receptors, but all information is channeled into the same signaling cascade, which controls a specific ...

  13. Detection and quantification of Vibrio fischeri autoinducer from symbiotic squid light organs.

    Science.gov (United States)

    Boettcher, K J; Ruby, E G

    1995-02-01

    Vibrio fischeri is the specific light organ symbiont of the sepiolid squid species Euprymna scolopes and Euprymna morsei. Both species of squid are luminescent by virtue of their bacterial symbionts, but the natural symbionts of E. scolopes do not produce visible luminescence in laboratory culture. The primary cause of this depressed luminescence by E. scolopes symbionts in culture was found to be the production of relatively low levels of V. fischeri autoinducer, a positive transcriptional coregulator of the lux regulon, identified as N-(3-oxohexanoyl) homoserine lactone. Concentrations of autoinducer activity produced by these symbionts in culture were quantified and found to be at least 10-fold lower than those produced by E. morsei isolates (which are visibly luminous outside the association) and perhaps 10,000-fold lower than those of the brightest V. fischeri strains. Despite the differences in their symbiont strains, the intact light organs of the two species of squid contained comparable amounts of extractable autoinducer activity (between 100 and 200 pg per adult animal). The chromatographic behavior of this autoinducer activity on reverse-phase high-performance liquid chromatography was consistent with its presumptive identification as V. fischeri autoinducer. Within the 5-microliter volume of the epithelial core of the light organ in which the symbiotic V. fischeri strains are housed, these amounts would result in an effective autoinducer concentration of at least 100 nM. Because these levels are over 40-fold higher than the concentration needed for the induction of luminescence of bacteria in culture, we conclude that the inherent degree of autoinducer production by strains of V. fischeri may not influence their effectiveness as light organ symbionts. Furthermore, this study provides the first direct evidence that the phenomenon of cell density-dependent autoinduction, discovered and described first for laboratory cultures of V. fischeri but believed to

  14. Autoinducer-2 Quorum Sensing Contributes to Regulation of Microcin PDI in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Shao-Yeh Lu

    2017-12-01

    Full Text Available The Escherichia coli quorum sensing (QS signal molecule, autoinducer-2 (AI-2, reaches its maximum concentration during mid-to-late growth phase after which it quickly degrades during stationary phase. This pattern of AI-2 concentration coincides with the up- then down-regulation of a recently described microcin PDI (mccPDI effector protein (McpM. To determine if there is a functional relationship between these systems, a prototypical mccPDI-expressing strain of E. coli 25 was used to generate ΔluxS, ΔlsrACDBFG (Δlsr, and ΔlsrR mutant strains that are deficient in AI-2 production, transportation, and AI-2 transport regulation, respectively. Trans-complementation, RT-qPCR, and western blot assays were used to detect changes of microcin expression and synthesis under co-culture and monoculture conditions. Compared to the wild-type strain, the AI-2-deficient strain (ΔluxS and -uptake negative strain (Δlsr were >1,000-fold less inhibitory to susceptible bacteria (P < 0.05. With in trans complementation of luxS, the AI-2 deficient mutant reduced the susceptible E. coli population by 4-log, which was within 1-log of the wild-type phenotype. RT-qPCR and western blot results for the AI-2 deficient E. coli 25 showed a 5-fold reduction in mcpM transcription with an average 2-h delay in McpM synthesis. Furthermore, overexpression of sRNA micC and micF (both involved in porin protein regulation was correlated with mcpM regulation, consistent with a possible link between QS and mcpM regulation. This is the direct first evidence that microcin regulation can be linked to quorum sensing in a Gram-negative bacterium.

  15. Establishing bacterial communities by 'word of mouth': LuxS and autoinducer 2 in biofilm development.

    Science.gov (United States)

    Hardie, Kim Rachael; Heurlier, Karin

    2008-08-01

    Multicellular bacterial communities (biofilms) abound in nature, and their successful formation and survival is likely to require cell-cell communication--including quorum sensing--to co-ordinate appropriate gene expression. The only mode of quorum sensing that is shared by both Gram-positive and Gram-negative bacteria involves the production of the signalling molecule autoinducer 2 by LuxS. A survey of the current literature reveals that luxS contributes to biofilm development in some bacteria. However, inconsistencies prevent biofilm development being attributed to the production of AI2 in all cases.

  16. Modeling and validation of autoinducer-mediated bacterial gene expression in microfluidic environments

    Science.gov (United States)

    Austin, Caitlin M.; Stoy, William; Su, Peter; Harber, Marie C.; Bardill, J. Patrick; Hammer, Brian K.; Forest, Craig R.

    2014-01-01

    Biosensors exploiting communication within genetically engineered bacteria are becoming increasingly important for monitoring environmental changes. Currently, there are a variety of mathematical models for understanding and predicting how genetically engineered bacteria respond to molecular stimuli in these environments, but as sensors have miniaturized towards microfluidics and are subjected to complex time-varying inputs, the shortcomings of these models have become apparent. The effects of microfluidic environments such as low oxygen concentration, increased biofilm encapsulation, diffusion limited molecular distribution, and higher population densities strongly affect rate constants for gene expression not accounted for in previous models. We report a mathematical model that accurately predicts the biological response of the autoinducer N-acyl homoserine lactone-mediated green fluorescent protein expression in reporter bacteria in microfluidic environments by accommodating these rate constants. This generalized mass action model considers a chain of biomolecular events from input autoinducer chemical to fluorescent protein expression through a series of six chemical species. We have validated this model against experimental data from our own apparatus as well as prior published experimental results. Results indicate accurate prediction of dynamics (e.g., 14% peak time error from a pulse input) and with reduced mean-squared error with pulse or step inputs for a range of concentrations (10 μM–30 μM). This model can help advance the design of genetically engineered bacteria sensors and molecular communication devices. PMID:25379076

  17. Autoinducer-2-like activity associated with foods and its interaction with food additives.

    Science.gov (United States)

    Lu, Lingeng; Hume, Michael E; Pillai, Suresh D

    2004-07-01

    The autoinducer-2 (AI-2) molecule produced by bacteria as part of quorum sensing is considered to be a universal inducer signal in bacteria because it reportedly influences gene expression in a variety of both gram-negative and gram-positive bacteria. The objective of this study was to determine whether selected fresh produce and processed foods have AI-2-like activity and whether specific food additives can act as AI-2 mimics and result in AI-2-like activity. The luminescence-based response of the reporter strain Vibrio harveyi BB170 was used as the basis for determining AI-2 activity in the selected foods and food ingredients. Maximum AI-2 activity was seen on the frozen fish sample (203-fold, compared with the negative control) followed by tomato, cantaloupe, carrots, tofu, and milk samples. Interestingly, some samples were capable of inhibiting AI-2 activity. Turkey patties showed the highest inhibition (99.8% compared with the positive control) followed by chicken breast (97.5%), homemade cheeses (93.7%), beef steak (90.6%), and beef patties (84.4%). AI-2 activity was almost totally inhibited by sodium propionate, whereas sodium benzoate caused 93.3% inhibition, compared with 75% inhibition by sodium acetate. Sodium nitrate did not have any appreciable effect, even at 200 ppm. Understanding the relationships that exist between AI-2 activity on foods and the ecology of pathogens and food spoilage bacteria on foods could yield clues about factors controlling food spoilage and pathogen virulence.

  18. Determination of autoinducer-2 in biological samples by high-performance liquid chromatography with fluorescence detection using pre-column derivatization.

    Science.gov (United States)

    Song, Xiang-Ning; Qiu, Hai-Bin; Xiao, Xiang; Cheng, Yuan-Yuan; Li, Wen-Wei; Sheng, Guo-Ping; Li, Xiao-Yan; Yu, Han-Qing

    2014-09-26

    Autoinducer-2 (AI-2), as a small-molecular-weight organic molecule secreted and perceived by various bacteria, enables intra- and inter-species communications. Quantitative determination of AI-2 is essential for exploring the bacterial AI-2-related physiological and biochemical processes. However, current strategies for sensitive detection of AI-2 require sophisticated instruments and complicated procedures. In this work, on the basis of the derivatization of AI-2 with 2,3-diaminonaphthalene, a simple, sensitive and cost-effective high-performance liquid chromatography with fluorescence detector (HPLC-FLD) method is developed for the quantitative detection of AI-2. Under the optimized conditions, this method had a broad linear range of 10-14,000 ng/ml (R(2)=0.9999), and a low detection limit of 1.0 ng/ml. Furthermore, the effectiveness of this approach was further validated through measuring the AI-2 concentrations in the cell-free culture supernatants of both Escherichia coli and Vibrio harveyi. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Mapping Stress-Induced Changes in Autoinducer AI-2 Production in Chemostat-Cultivated Escherichia coli K-12

    Science.gov (United States)

    DeLisa, Matthew P.; Valdes, James J.; Bentley, William E.

    2001-01-01

    Numerous gram-negative bacteria employ a cell-to-cell signaling mechanism, termed quorum sensing, for controlling gene expression in response to population density. Recently, this phenomenon has been discovered in Escherichia coli, and while pathogenic E. coli utilize quorum sensing to regulate pathogenesis (i.e., expression of virulence genes), the role of quorum sensing in nonpathogenic E. coli is less clear, and in particular, there is no information regarding the role of quorum sensing during the overexpression of recombinant proteins. The production of autoinducer AI-2, a signaling molecule employed by E. coli for intercellular communication, was studied in E. coli W3110 chemostat cultures using a Vibrio harveyi AI-2 reporter assay (M. G. Surrette and B. L. Bassler, Proc. Natl. Acad. Sci. USA 95:7046–7050, 1998). Chemostat cultures enabled a study of AI-2 regulation through steady-state and transient responses to a variety of environmental stimuli. Results demonstrated that AI-2 levels increased with the steady-state culture growth rate. In addition, AI-2 increased following pulsed addition of glucose, Fe(III), NaCl, and dithiothreitol and decreased following aerobiosis, amino acid starvation, and isopropyl-β-d-thiogalactopyranoside-induced expression of human interleukin-2 (hIL-2). In general, the AI-2 responses to several perturbations were indicative of a shift in metabolic activity or state of the cells induced by the individual stress. Because of our interest in the expression of heterologous proteins in E. coli, the transcription of four quorum-regulated genes and 20 stress genes was mapped during the transient response to induced expression of hIL-2. Significant regulatory overlap was revealed among several stress and starvation genes and known quorum-sensing genes. PMID:11292813

  20. The Vibrio cholerae quorum-sensing autoinducer CAI-1: analysis of the biosynthetic enzyme CqsA

    Energy Technology Data Exchange (ETDEWEB)

    Kelly, R.; Bolitho, M; Higgins, D; Lu, W; Ng, W; Jeffrey, P; Rabinowitz, J; Semmelhack, M; Hughson, F; Bassler, B

    2009-01-01

    Vibrio cholerae, the bacterium that causes the disease cholera, controls virulence factor production and biofilm development in response to two extracellular quorum-sensing molecules, called autoinducers. The strongest autoinducer, called CAI-1 (for cholera autoinducer-1), was previously identified as (S)-3-hydroxytridecan-4-one. Biosynthesis of CAI-1 requires the enzyme CqsA. Here, we determine the CqsA reaction mechanism, identify the CqsA substrates as (S)-2-aminobutyrate and decanoyl coenzyme A, and demonstrate that the product of the reaction is 3-aminotridecan-4-one, dubbed amino-CAI-1. CqsA produces amino-CAI-1 by a pyridoxal phosphate-dependent acyl-CoA transferase reaction. Amino-CAI-1 is converted to CAI-1 in a subsequent step via a CqsA-independent mechanism. Consistent with this, we find cells release {ge}100 times more CAI-1 than amino-CAI-1. Nonetheless, V. cholerae responds to amino-CAI-1 as well as CAI-1, whereas other CAI-1 variants do not elicit a quorum-sensing response. Thus, both CAI-1 and amino-CAI-1 have potential as lead molecules in the development of an anticholera treatment.

  1. Physical and functional maps of the luminescence gene cluster in an autoinducer-deficient Vibrio fischeri strain isolated from a squid light organ.

    Science.gov (United States)

    Gray, K M; Greenberg, E P

    1992-07-01

    Vibrio fischeri ES114 is an isolate representing the specific bacterial light organ symbiont of the squid Euprymna scolopes. An interesting feature of this strain of V. fischeri is that it is visibly luminous within the light organ of the squid host but is nonluminous when grown under standard laboratory conditions. Luminescence can be restored in laboratory culture, however, by the addition of autoinducer, a species-specific inducer of the V. fischeri luminescence (lux) genes. Most other isolates of V. fischeri produce autoinducer in sufficient quantities to induce luminescence in laboratory culture. We have cloned an 8.8-kb DNA fragment from V. fischeri ES114 that encodes all of the functions necessary for luminescence in Escherichia coli in the absence of exogenous autoinducer. This DNA contains both of the recognized V. fischeri lux regulatory genes, one of which (luxI) directs E. coli to synthesize autoinducer. The organization of the individual lux genes within this DNA fragment appears to be the same as that in the other strains of V. fischeri studied; the restriction map of the V. fischeri ES114 lux DNA has diverged substantially, however, from the largely conserved maps of V. fischeri MJ1 and ATCC 7744. Although E. coli containing the V. fischeri ES114 lux DNA synthesizes considerable amounts of autoinducer, V. fischeri ES114 synthesizes autoinducer only in small amounts, even when transcription of the lux genes, including luxI, is activated by the addition of exogenous autoinducer. Nonetheless, transconjugants of V. fischeri ES114 that contain multicopy plasmids bearing the ES114 lux genes synthesize sufficient autoinducer to induce luminescence. These results suggest that V. fischeri ES11r does not lack a functional luxl, nor is it deficient in the ability to synthesize metabolic precursors for autoinducer synthesis.

  2. Esterification of jatropha oil via ultrasonic irradiation with auto-induced temperature-rise effect

    International Nuclear Information System (INIS)

    Andrade-Tacca, Cesar Augusto; Chang, Chia-Chi; Chen, Yi-Hung; Manh, Do-Van; Chang, Ching-Yuan; Ji, Dar-Ren; Tseng, Jyi-Yeong; Shie, Je-Lueng

    2014-01-01

    Auto-induced temperature-rise effects of ultrasonic irradiation (UI) on the esterification performance of jatropha oil (JO) were studied. Comparisons with other methods of mechanical mixing (MM) and hand shaking mixing were made. Major system parameters examined include: esterification time (t E ), settling time (t S ) after esterification and temperature. Properties of acid value (AV), iodine value (IV), kinematic viscosity (KV) and density of JO and ester product were measured. The esterification conversion efficiencies (η) were determined and assessed. Sulfuric acid was used to catalyze the esterification using methyl alcohol. For esterification without temperature control, η at t E  = 10 and 30 min for UI of 56.73 and 83.23% are much higher than those for MM of 36.76 and 42.48%, respectively. At t E  = 10 min, the jatropha oil esters produced via UI and MM respectively possess AV of 15.82 and 23.12 mg KOH/g, IV of 111.49 and 113.22 g I 2 /100 g, KV of 22.41 and 22.51 mm 2 /s and density of 913.8 and 913.58 kg/m 3 , showing that UI is much better than MM in enhancing the reduction of AV. The t E exhibits more vigorous effect on AV for UI than MM. The UI offers auto-induced temperature-rise, improving the mixing and esterification extents. - Highlights: • Esterification of jatropha oil is pronounced under ultrasonic irradiation (UI). • UI can auto-induce temperature rise. • The induced temperature rise assists the mixing of UI in enhancing esterification. • UI offers better esterification than mechanical mixing with external heating. • An 83.23% reduction of FFA in jatropha-ester is achievable via UI in 30 min

  3. Evaluation of two autoinducer-2 quantification methods for application in marine environments

    KAUST Repository

    Wang, Tian-Nyu

    2018-02-11

    This study evaluated two methods, namely high performance liquid chromatography with fluorescence detection (HPLC-FLD) and Vibrio harveyi BB170 bioassay, for autoinducer-2 (AI-2) quantification in marine samples. Using both methods, the study also investigated the stability of AI-2 in varying pH, temperature and media, as well as quantified the amount of AI-2 signals in marine samples.HPLC-FLD method showed a higher level of reproducibility and precision compared to V. harveyi BB170 bioassay. Alkaline pH > 8 and high temperature (> 37°C) increased the instability of AI-2. The AI-2 concentrations in seawater were low, ca. 3.2-27.6 pmol l-1 whereas 8- week old marine biofilm grew on an 18.8 cm2 substratum accumulated ca. 0.207 nmol of AI-2.Both methods have pros and cons for AI-2 quantification in marine samples. Regardless, both methods reported a ubiquitous presence of AI-2 in both planktonic and biomass fractions of seawater, as well as in marine biofilm.In this study, AI-2 signals were for the first time enumerated in marine samples to reveal the ubiquitous presence of AI-2 in this environment. The findings suggest a possible role of AI-2 in biofilm formation in marine environment, and the contribution of AI-2 in biofilm-associated problems such as biofouling and biocorrosion. This article is protected by copyright. All rights reserved.

  4. The new pLAI (lux regulon based auto-inducible expression system for recombinant protein production in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Nocadello Salvatore

    2012-01-01

    Full Text Available Abstract Background After many years of intensive research, it is generally assumed that no universal expression system can exist for high-level production of a given recombinant protein. Among the different expression systems, the inducible systems are the most popular for their tight regulation. However, induction is in many cases less favorable due to the high cost and/or toxicity of inducers, incompatibilities with industrial scale-up or detrimental growth conditions. Expression systems using autoinduction (or self-induction prove to be extremely versatile allowing growth and induction of recombinant proteins without the need to monitor cell density or add inducer. Unfortunately, almost all the actual auto inducible expression systems need endogenous or induced metabolic changes during the growth to trigger induction, both frequently linked to detrimental condition to cell growth. In this context, we use a simple modular approach for a cell density-based genetic regulation in order to assemble an autoinducible recombinant protein expression system in E. coli. Result The newly designed pLAI expression system places the expression of recombinant proteins in Escherichia coli under control of the regulatory genes of the lux regulon of Vibrio fischeri's Quorum Sensing (QS system. The pLAI system allows a tight regulation of the recombinant gene allowing a negligible basal expression and expression only at high cell density. Sequence optimization of regulative genes of QS of V. fischeri for expression in E. coli upgraded the system to high level expression. Moreover, partition of regulative genes between the plasmid and the host genome and introduction of a molecular safety lock permitted tighter control of gene expression. Conclusion Coupling gene expression to cell density using cell-to-cell communication provides a promising approach for recombinant protein production. The system allows the control of expression of the target recombinant gene

  5. Analysing traces of autoinducer-2 requires standardization of the Vibrio harveyi bioassay.

    Science.gov (United States)

    Vilchez, Ramiro; Lemme, André; Thiel, Verena; Schulz, Stefan; Sztajer, Helena; Wagner-Döbler, Irene

    2007-01-01

    Autoinducer-2 (furanosyl borate diester) is a biologically active compound whose role as a universal bacterial signalling molecule is currently under intense investigation. Because of its instability and the low concentrations of it found in biological samples, its detection relies at present on a bioassay that measures the difference in the timing of the luminescence of the Vibrio harveyi BB170 sensor strain with and without externally added AI-2. Here we systematically investigated which parameters affected the fold induction values of luminescence obtained in the bioassay and developed a modified protocol. Our experiments showed that growth and luminescence of V. harveyi BB170 are strongly influenced by trace elements. In particular, addition of Fe(3+) within a certain concentration range to the growth medium of the preinoculum culture improved the reproducibility and reduced the variance of the bioassay. In contrast, trace elements and vitamins introduced directly into the bioassay caused inhibitory effects. The initial density and luminescence of the sensor strain are very important and the values required for these parameters were defined. Borate interferes with the detection of AI-2 by giving false positive results. The response of V. harveyi BB170 to chemically synthesized AI-2 in the bioassay is nonlinear except over a very small concentration range; it is maximum over three orders of magnitude and shows inhibition above 35 microM. Based on the modified protocol, we were able to detect AI-2 in the absence of inhibitors with maximum fold induction values for the positive control (chemically synthesized AI-2) of >120 with a standard deviation of approximately 30% in a reliable and reproducible way.

  6. Short communication: The role of autoinducer 2 (AI-2) on antibiotic resistance regulation in an Escherichia coli strain isolated from a dairy cow with mastitis.

    Science.gov (United States)

    Xue, Ting; Yu, Lumin; Shang, Fei; Li, Wenchang; Zhang, Ming; Ni, Jingtian; Chen, Xiaolin

    2016-06-01

    Extended spectrum β-lactamase (ESBL)-positive Escherichia coli is a major etiological organism responsible for bovine mastitis. The autoinducer 2 (AI-2) quorum sensing system is widely present in many species of gram-negative and gram-positive bacteria and has been proposed to be involved in interspecies communication. In E. coli model strains, the functional mechanisms of AI-2 have been well studied; however, in clinical antibiotic-resistant E. coli strains, whether AI-2 affects the expression of antibiotic resistance genes has not been reported. In this study, we report that exogenous AI-2 increased the antibiotic resistance of a clinical E. coli strain isolated from a dairy cow with mastitis by upregulating the expression of TEM-type enzyme in an LsrR (LuxS regulated repressor)-dependent manner. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  7. Quorum-sensing regulator RhlR but not its autoinducer RhlI enables Pseudomonas to evade opsonization.

    Science.gov (United States)

    Haller, Samantha; Franchet, Adrien; Hakkim, Abdul; Chen, Jing; Drenkard, Eliana; Yu, Shen; Schirmeier, Stefanie; Li, Zi; Martins, Nelson; Ausubel, Frederick M; Liégeois, Samuel; Ferrandon, Dominique

    2018-03-09

    When Drosophila melanogaster feeds on Pseudomonas aeruginosa , some bacteria cross the intestinal barrier and eventually proliferate in the hemocoel. This process is limited by hemocytes through phagocytosis. P. aeruginosa requires the quorum-sensing regulator RhlR to elude the cellular immune response of the fly. RhlI synthesizes the autoinducer signal that activates RhlR. Here, we show that rhlI mutants are unexpectedly more virulent than rhlR mutants, both in fly and in nematode intestinal infection models, suggesting that RhlR has RhlI-independent functions. We also report that RhlR protects P. aeruginosa from opsonization mediated by the Drosophila thioester-containing protein 4 (Tep4). RhlR mutant bacteria show higher levels of Tep4- mediated opsonization, as compared to rhlI mutants, which prevents lethal bacteremia in the Drosophila hemocoel. In contrast, in a septic model of infection, in which bacteria are introduced directly into the hemocoel, Tep4 mutant flies are more resistant to wild-type P. aeruginosa, but not to the rhlR mutant. Thus, depending on the infection route, the Tep4 opsonin can either be protective or detrimental to host defense. © 2018 The Authors.

  8. Exploring the selectivity of auto-inducer complex with LuxR using molecular docking, mutational studies and molecular dynamics simulations

    Science.gov (United States)

    Rajamanikandan, Sundaraj; Srinivasan, Pappu

    2017-03-01

    Bacteria communicate with one another using extracellular signaling molecules called auto-inducers (AHLs), a process termed as quorum sensing. The quorum sensing process allows bacteria to regulate various physiological activities. In this regard, quorum sensing master regulator LuxR from Vibrio harveyi represents an attractive therapeutic target for the development of novel anti-quorum sensing agents. Eventhough the binding of AHL complex with LuxR is evidenced in earlier reports, but their mode of binding is not clearly determined. Therefore, in the present work, molecular docking, in silico mutational studies, molecular dynamics simulations and free energy calculations were performed to understand the selectivity of AHL into the binding site of LuxR. The results revealed that Asn133 and Gln137 residues play a crucial role in recognizing AHL more effectively into the binding site of LuxR with good binding free energy. In addition to that, the carbonyl group presents in the lactone ring and amide group of AHL plays a vital role in the formation of hydrogen bond interactions with the protein. Further, structure based virtual screening was performed using ChemBridge database to screen potent lead molecules against LuxR. 4-benzyl-2-pyrrolidinone and N-[2(1-cyclohexen-1-yl) enthyl]-N'(2-ethoxyphenyl) were selected based on dock score, binding affinity and mode of interactions with the receptor. Furthermore, binding free energy, density functional theory and ADME prediction were performed to rank the lead molecules. Thus, the identified lead molecules can be used for the development of anti-quorum sensing drugs.

  9. Autoinducer-2 plays a crucial role in gut colonization and probiotic functionality of Bifidobacterium breve UCC2003.

    Science.gov (United States)

    Christiaen, Steven E A; O'Connell Motherway, Mary; Bottacini, Francesca; Lanigan, Noreen; Casey, Pat G; Huys, Geert; Nelis, Hans J; van Sinderen, Douwe; Coenye, Tom

    2014-01-01

    In the present study we show that luxS of Bifidobacterium breve UCC2003 is involved in the production of the interspecies signaling molecule autoinducer-2 (AI-2), and that this gene is essential for gastrointestinal colonization of a murine host, while it is also involved in providing protection against Salmonella infection in Caenorhabditis elegans. We demonstrate that a B. breve luxS-insertion mutant is significantly more susceptible to iron chelators than the WT strain and that this sensitivity can be partially reverted in the presence of the AI-2 precursor DPD. Furthermore, we show that several genes of an iron starvation-induced gene cluster, which are downregulated in the luxS-insertion mutant and which encodes a presumed iron-uptake system, are transcriptionally upregulated under in vivo conditions. Mutation of two genes of this cluster in B. breve UCC2003 renders the derived mutant strains sensitive to iron chelators while deficient in their ability to confer gut pathogen protection to Salmonella-infected nematodes. Since a functional luxS gene is present in all tested members of the genus Bifidobacterium, we conclude that bifidobacteria operate a LuxS-mediated system for gut colonization and pathogen protection that is correlated with iron acquisition.

  10. Autoinducer-2 plays a crucial role in gut colonization and probiotic functionality of Bifidobacterium breve UCC2003.

    Directory of Open Access Journals (Sweden)

    Steven E A Christiaen

    Full Text Available In the present study we show that luxS of Bifidobacterium breve UCC2003 is involved in the production of the interspecies signaling molecule autoinducer-2 (AI-2, and that this gene is essential for gastrointestinal colonization of a murine host, while it is also involved in providing protection against Salmonella infection in Caenorhabditis elegans. We demonstrate that a B. breve luxS-insertion mutant is significantly more susceptible to iron chelators than the WT strain and that this sensitivity can be partially reverted in the presence of the AI-2 precursor DPD. Furthermore, we show that several genes of an iron starvation-induced gene cluster, which are downregulated in the luxS-insertion mutant and which encodes a presumed iron-uptake system, are transcriptionally upregulated under in vivo conditions. Mutation of two genes of this cluster in B. breve UCC2003 renders the derived mutant strains sensitive to iron chelators while deficient in their ability to confer gut pathogen protection to Salmonella-infected nematodes. Since a functional luxS gene is present in all tested members of the genus Bifidobacterium, we conclude that bifidobacteria operate a LuxS-mediated system for gut colonization and pathogen protection that is correlated with iron acquisition.

  11. Inactivation of ferric uptake regulator (Fur) attenuates Helicobacter pylori J99 motility by disturbing the flagellar motor switch and autoinducer-2 production.

    Science.gov (United States)

    Lee, Ai-Yun; Kao, Cheng-Yen; Wang, Yao-Kuan; Lin, Ssu-Yuan; Lai, Tze-Ying; Sheu, Bor-Shyang; Lo, Chien-Jung; Wu, Jiunn-Jong

    2017-08-01

    Flagellar motility of Helicobacter pylori has been shown to be important for the bacteria to establish initial colonization. The ferric uptake regulator (Fur) is a global regulator that has been identified in H. pylori which is involved in the processes of iron uptake and establishing colonization. However, the role of Fur in H. pylori motility is still unclear. Motility of the wild-type, fur mutant, and fur revertant J99 were determined by a soft-agar motility assay and direct video observation. The bacterial shape and flagellar structure were evaluated by transmission electron microscopy. Single bacterial motility and flagellar switching were observed by phase-contrast microscopy. Autoinducer-2 (AI-2) production in bacterial culture supernatant was analyzed by a bioluminescence assay. The fur mutant showed impaired motility in the soft-agar assay compared with the wild-type J99 and fur revertant. The numbers and lengths of flagellar filaments on the fur mutant cells were similar to those of the wild-type and revertant cells. Phenotypic characterization showed similar swimming speed but reduction in switching rate in the fur mutant. The AI-2 production of the fur mutant was dramatically reduced compared with wild-type J99 in log-phase culture medium. These results indicate that Fur positively modulates H. pylori J99 motility through interfering with bacterial flagellar switching. © 2017 John Wiley & Sons Ltd.

  12. F-box only protein 2 (Fbxo2) regulates amyloid precursor protein levels and processing.

    Science.gov (United States)

    Atkin, Graham; Hunt, Jack; Minakawa, Eiko; Sharkey, Lisa; Tipper, Nathan; Tennant, William; Paulson, Henry L

    2014-03-07

    The amyloid precursor protein (APP) is an integral membrane glycoprotein whose cleavage products, particularly amyloid-β, accumulate in Alzheimer disease (AD). APP is present at synapses and is thought to play a role in both the formation and plasticity of these critical neuronal structures. Despite the central role suggested for APP in AD pathogenesis, the mechanisms regulating APP in neurons and its processing into cleavage products remain incompletely understood. F-box only protein 2 (Fbxo2), a neuron-enriched ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans on glycoproteins, was previously implicated in APP processing by facilitating the degradation of the APP-cleaving β-secretase, β-site APP-cleaving enzyme. Here, we sought to determine whether Fbxo2 plays a similar role for other glycoproteins in the amyloid processing pathway. We present in vitro and in vivo evidence that APP is itself a substrate for Fbxo2. APP levels were decreased in the presence of Fbxo2 in non-neuronal cells, and increased in both cultured hippocampal neurons and brain tissue from Fbxo2 knock-out mice. The processing of APP into its cleavage products was also increased in hippocampi and cultured hippocampal neurons lacking Fbxo2. In hippocampal slices, this increase in cleavage products was accompanied by a significant reduction in APP at the cell surface. Taken together, these results suggest that Fbxo2 regulates APP levels and processing in the brain and may play a role in modulating AD pathogenesis.

  13. The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA sinI in Sinorhizobium meliloti

    Czech Academy of Sciences Publication Activity Database

    Baumgardt, K.; Šmídová, Klára; Rahn, H.; Lochnit, G.; Robledo, M.; Evguenieva-Hackenberg, E.

    2016-01-01

    Roč. 13, č. 5 (2016), s. 486-499 ISSN 1547-6286 Institutional support: RVO:61388971 Keywords : Agrobacterium * autoinducer synthase * degradosome Subject RIV: EE - Microbiology, Virology Impact factor: 3.900, year: 2016

  14. Streptococcus gordonii LuxS/autoinducer-2 quorum-sensing system modulates the dual-species biofilm formation with Streptococcus mutans.

    Science.gov (United States)

    Wang, Xiao; Li, Xiaolan; Ling, Junqi

    2017-07-01

    Dental plaques are mixed-species biofilms that are related to the development of dental caries. Streptococcus mutans (S. mutans) is an important cariogenic bacterium that forms mixed-species biofilms with Streptococcus gordonii (S. gordonii), an early colonizer of the tooth surface. The LuxS/autoinducer-2(AI-2) quorum sensing system is involved in the regulation of mixed-species biofilms, and AI-2 is proposed as a universal signal for the interaction between bacterial species. In this work, a S. gordonii luxS deficient strain was constructed to investigate the effect of the S. gordonii luxS gene on dual-species biofilm formed by S. mutans and S. gordonii. In addition, AI-2 was synthesized in vitro by incubating recombinant LuxS and Pfs enzymes of S. gordonii together. The effect of AI-2 on S. mutans single-species biofilm formation and cariogenic virulence gene expression were also assessed. The results showed that luxS disruption in S. gordonii altered dual-species biofilm formation, architecture, and composition, as well as the susceptibility to chlorhexidine. And the in vitro synthesized AI-2 had a concentration-dependent effect on S. mutans biofilm formation and virulence gene expression. These findings indicate that LuxS/AI-2 quorum-sensing system of S. gordonii plays a role in regulating the dual-species biofilm formation with S. mutans. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. The small nucleoid protein Fis is involved in Vibrio cholerae quorum sensing.

    Science.gov (United States)

    Lenz, Derrick H; Bassler, Bonnie L

    2007-02-01

    Quorum sensing is a process of cell-cell communication that bacteria use to relay information to one another about the cell density and species composition of the bacterial community. Quorum sensing involves the production, secretion and population-wide detection of small signalling molecules called autoinducers. This process allows bacteria to synchronize group behaviours and act as multicellular units. The human pathogen, Vibrio cholerae, uses quorum sensing to co-ordinate such complex behaviours as pathogenicity and biofilm formation. The quorum-sensing circuit of V. cholerae consists of two autoinducer/sensor systems, CAI-1/CqsS and AI-2/LuxPQ, and the VarS/A-CsrA/BCD growth-phase regulatory system. Genetic analysis suggests that an additional regulatory arm involved in quorum sensing exists in V. cholerae. All of these systems channel information into the histidine phosphotransfer protein, LuxU, and/or the response regulator, LuxO. LuxO, when phosphorylated, activates the expression of four genes encoding the Qrr (quorum regulatory RNAs) small RNAs (sRNAs). The Qrr sRNAs destabilize the hapR transcript encoding the master regulator of quorum sensing, HapR. Here we identify the nucleoid protein Fis as playing a major role in the V. cholerae quorum-sensing circuit. Fis fulfils the predictions required to be the putative additional component that inputs information into the cascade: its expression is regulated in a growth phase-dependent manner; it requires LuxO but acts independently of LuxU, and it regulates all four qrr genes and, in turn, HapR by directly binding to the qrr gene promoters and modulating their expression.

  16. A positive feedback-based gene circuit to increase the production of a membrane protein

    Directory of Open Access Journals (Sweden)

    Gennis Robert B

    2010-05-01

    Full Text Available Abstract Background Membrane proteins are an important class of proteins, playing a key role in many biological processes, and are a promising target in pharmaceutical development. However, membrane proteins are often difficult to produce in large quantities for the purpose of crystallographic or biochemical analyses. Results In this paper, we demonstrate that synthetic gene circuits designed specifically to overexpress certain genes can be applied to manipulate the expression kinetics of a model membrane protein, cytochrome bd quinol oxidase in E. coli, resulting in increased expression rates. The synthetic circuit involved is an engineered, autoinducer-independent variant of the lux operon activator LuxR from V. fischeri in an autoregulatory, positive feedback configuration. Conclusions Our proof-of-concept experiments indicate a statistically significant increase in the rate of production of the bd oxidase membrane protein. Synthetic gene networks provide a feasible solution for the problem of membrane protein production.

  17. A combination of luxR1 and luxR2 genes activates Pr-promoters of psychrophilic Aliivibrio logei lux-operon independently of chaperonin GroEL/ES and protease Lon at high concentrations of autoinducer.

    Science.gov (United States)

    Konopleva, Maria N; Khrulnova, Svetlana A; Baranova, Ancha; Ekimov, Leonid V; Bazhenov, Sergey V; Goryanin, Ignatiy I; Manukhov, Ilya V

    2016-05-13

    Lux-operon of psychrophilic bacteria Aliivibrio logei contains two copies of luxR and is regulated by Type I quorum sensing (QS). Activation of lux-operon of psychrophilic bacteria A. logei by LuxR1 requires about 100 times higher concentrations of autoinducer (AI) than the activation by LuxR2. On the other hand, LuxR1 does not require GroEL/ES chaperonin for its folding and cannot be degraded by protease Lon, while LuxR2 sensitive to Lon and requires GroEL/ES. Here we show that at 10(-5) - 10(-4)М concentrations of AI a combination of luxR1 and luxR2 products is capable of activating the Pr-promoters of A. logei lux-operon in Escherichia coli independently of GroEL/ES and protease Lon. The presence of LuxR1 assists LuxR2 in gro(-) cells when AI was added at high concentration, while at low concentration of AI in a cell LuxR1 decreases the LuxR2 activity. These observations may be explained by the formation of LuxR1/LuxR2 heterodimers that act in complex with AI independently from GroEL/ES and protease Lon. This study expands current understanding of QS regulation in A. logei as it implies cooperative regulation of lux-operon by LuxR1 and LuxR2 proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Potential for luxS related signalling in marine bacteria and production of autoinducer-2 in the genus Shewanella

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    Wagner-Döbler Irene

    2008-01-01

    Full Text Available Abstract Background The autoinducer-2 (AI-2 group of signalling molecules are produced by both Gram positive and Gram negative bacteria as the by-product of a metabolic transformation carried out by the LuxS enzyme. They are the only non species-specific quorum sensing compounds presently known in bacteria. The luxS gene coding for the AI-2 synthase enzyme was found in many important pathogens. Here, we surveyed its occurrence in a collection of 165 marine isolates belonging to abundant marine phyla using conserved degenerated PCR primers and sequencing of selected positive bands to determine if the presence of the luxS gene is phylogenetically conserved or dependent on the habitat. Results The luxS gene was not present in any of the Alphaproteobacteria (n = 71 and Bacteroidetes strains (n = 29 tested; by contrast, these bacteria harboured the sahH gene, coding for an alternative enzyme for the detoxification of S-adenosylhomocysteine (SAH in the activated methyl cycle. Within the Gammaproteobacteria (n = 76, luxS was found in all Shewanella, Vibrio and Alteromonas isolates and some Pseudoalteromonas and Halomonas species, while sahH was detected in Psychrobacter strains. A number of Gammaproteobacteria (n = 27 appeared to have neither the luxS nor the sahH gene. We then studied the production of AI-2 in the genus Shewanella using the Vibrio harveyi bioassay. All ten species of Shewanella tested produced a pronounced peak of AI-2 towards the end of the exponential growth phase in several media investigated. The maximum of AI-2 activity was different in each Shewanella species, ranging from 4% to 46% of the positive control. Conclusion The data are consistent with those of fully sequenced bacterial genomes and show that the potential for luxS related signalling is dependent on phylogenetic affiliation rather than ecological niche and is largest in certain groups of Gammaproteobacteria in the marine environment. This is the first report on AI-2

  19. Phosphorylation of the human respiratory syncytial virus P protein mediates M2-2 regulation of viral RNA synthesis, a process that involves two P proteins.

    Science.gov (United States)

    Asenjo, Ana; Villanueva, Nieves

    2016-01-04

    The M2-2 protein regulates the balance between human respiratory syncytial virus (HRSV) transcription and replication. Here it is shown that M2-2 mediated transcriptional inhibition is managed through P protein phosphorylation. Transcription inhibition by M2-2 of the HRSV based minigenome pRSVluc, required P protein phosphorylation at serines (S) in positions 116, 117, 119 and increased inhibition is observed if S232 or S237 is also phosphorylated. Phosphorylation of these residues is required for viral particle egression from infected cells. Viral RNA synthesis complementation assays between P protein variants, suggest that two types of P proteins participate in the process as components of RNA dependent RNA polymerase (RdRp). Type I is only functional when, as a homotetramer, it is bound to N and L proteins through residues 203-241. Type II is functionally independent of these interactions and binds to N protein at a region outside residues 232-241. P protein type I phosphorylation at S116, S117 and S119, did not affect the activity of RdRp but this phosphorylation in type II avoids its interaction with N protein and impairs RdRp functionality for transcription and replication. Structural changes in the RdRp, mediated by phosphorylation turnover at the indicated residues, in the two types of P proteins, may result in a fine adjustment, late in the infectious cycle, of transcription, replication and progression in the morphogenetic process that ends in egression of the viral particles from infected cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. α-Hydroxyketone Synthesis and Sensing by Legionella and Vibrio

    Directory of Open Access Journals (Sweden)

    Hubert Hilbi

    2012-03-01

    Full Text Available Bacteria synthesize and sense low molecular weight signaling molecules, termed autoinducers, to measure their population density and community complexity. One class of autoinducers, the α-hydroxyketones (AHKs, is produced and detected by the water-borne opportunistic pathogens Legionella pneumophila and Vibrio cholerae, which cause Legionnaires’ disease and cholera, respectively. The “Legionella quorum sensing” (lqs or “cholera quorum sensing” (cqs genes encode enzymes that produce and sense the AHK molecules “Legionella autoinducer-1” (LAI-1; 3-hydroxypentadecane-4-one or cholera autoinducer-1 (CAI-1; 3-hydroxytridecane-4-one. AHK signaling regulates the virulence of L. pneumophila and V. cholerae, pathogen-host cell interactions, formation of biofilms or extracellular filaments, expression of a genomic “fitness island” and competence. Here, we outline the processes, wherein AHK signaling plays a role, and review recent insights into the function of proteins encoded by the lqs and cqs gene clusters. To this end, we will focus on the autoinducer synthases catalysing the biosynthesis of AHKs, on the cognate trans-membrane sensor kinases detecting the signals, and on components of the down-stream phosphorelay cascade that promote the transmission and integration of signaling events regulating gene expression.

  1. Role and regulation of the orphan AphA protein of quorum sensing in pathogenic Vibrios.

    Science.gov (United States)

    Lu, Renfei; Osei-Adjei, George; Huang, Xinxiang; Zhang, Yiquan

    2018-03-01

    Quorum sensing (QS), a cell-to-cell communication process, is widely distributed in the bacterial kingdom. Bacteria use QS to control gene expression in response to cell density by detecting the signal molecules called autoinducers. AphA protein is the master QS regulator of vibrios operating at low cell density. It regulates the expression of a variety of genes, especially those encoding virulence factors, flagella/motility and biofilm formation. The role and regulation of AphA in vibrios, especially in human pathogenic vibrios, are summarized in this review. Clarification of the roles of AphA will help us to understand the pathogenesis of vibrios.

  2. Expression of tropodithietic acid biosynthesis is controlled by a novel autoinducer.

    Science.gov (United States)

    Geng, Haifeng; Belas, Robert

    2010-09-01

    The interactions between marine prokaryotic and eukaryotic microorganisms are crucial to many biological and biogeochemical processes in the oceans. Often the interactions are mutualistic, as in the symbiosis between phytoplankton, e.g., the dinoflagellate Pfiesteria piscicida and Silicibacter sp. TM1040, a member of the Roseobacter taxonomic lineage. It is hypothesized that an important component of this symbiosis is bacterial production of tropodithietic acid (TDA), a biologically active tropolone compound whose synthesis requires the expression of tdaABCDEF (tdaA-F), as well as six additional genes (cysI, malY, paaIJK, and tdaH). The factors controlling tda gene expression are not known, although growth in laboratory standing liquid cultures drastically increases TDA levels. In this report, we measured the transcription of tda genes to gain a greater understanding of the factors controlling their expression. While the expression of tdaAB was constitutive, tdaCDE and tdaF mRNA increased significantly (3.7- and 17.4-fold, respectively) when cells were grown in standing liquid broth compared to their levels with shaking liquid culturing. No transcription of tdaC was detected when a tdaCp::lacZ transcriptional fusion was placed in 11 of the 12 Tda(-) mutant backgrounds, with cysI being the sole exception. The expression of tdaC could be restored to 9 of the remaining 11 Tda(-) mutants-tdaA and tdaH failed to respond-by placing wild-type (Tda(+)) strains in close proximity or by supplying exogenous TDA to the mutant, suggesting that TDA induces tda gene expression. These results indicate that TDA acts as an autoinducer of its own synthesis and suggest that roseobacters may use TDA as a quorum signal.

  3. Protein Molecular Structures, Protein SubFractions, and Protein Availability Affected by Heat Processing: A Review

    International Nuclear Information System (INIS)

    Yu, P.

    2007-01-01

    The utilization and availability of protein depended on the types of protein and their specific susceptibility to enzymatic hydrolysis (inhibitory activities) in the gastrointestine and was highly associated with protein molecular structures. Studying internal protein structure and protein subfraction profiles leaded to an understanding of the components that make up a whole protein. An understanding of the molecular structure of the whole protein was often vital to understanding its digestive behavior and nutritive value in animals. In this review, recently obtained information on protein molecular structural effects of heat processing was reviewed, in relation to protein characteristics affecting digestive behavior and nutrient utilization and availability. The emphasis of this review was on (1) using the newly advanced synchrotron technology (S-FTIR) as a novel approach to reveal protein molecular chemistry affected by heat processing within intact plant tissues; (2) revealing the effects of heat processing on the profile changes of protein subfractions associated with digestive behaviors and kinetics manipulated by heat processing; (3) prediction of the changes of protein availability and supply after heat processing, using the advanced DVE/OEB and NRC-2001 models, and (4) obtaining information on optimal processing conditions of protein as intestinal protein source to achieve target values for potential high net absorbable protein in the small intestine. The information described in this article may give better insight in the mechanisms involved and the intrinsic protein molecular structural changes occurring upon processing.

  4. Chemical Changes in Proteins Produced by Thermal Processing.

    Science.gov (United States)

    Dutson, T. R.; Orcutt, M. W.

    1984-01-01

    Discusses effects of thermal processing on proteins, focusing on (1) the Maillard reaction; (2) heat denaturation of proteins; (3) aggregation, precipitation, gelation, and degradation; and (4) other thermally induced protein reactions. Also discusses effects of thermal processing on muscle foods, egg proteins, fruits and vegetables, and cereal…

  5. High-salt brines compromise autoinducer-mediated bacteriocinogenic Lactobacillus plantarum survival in Spanish-style green olive fermentations.

    Science.gov (United States)

    Caballero-Guerrero, Belén; Lucena-Padrós, Helena; Maldonado-Barragán, Antonio; Ruiz-Barba, José Luis

    2013-02-01

    The effect of NaCl on plantaricin production by five Lactobacillus plantarum strains was investigated. Plantaricin production in these strains was found to be regulated by three-component regulatory systems ruled by two different autoinducer peptides (AIPs), either PLNC8IF or Plantaricin A. Bacteriocin activity exhibited by these strains came to a halt in liquid medium containing NaCl concentrations of or above 2%. In contrast, bacteriocin activity was still observed when the producing strains were growing on solid medium containing up to 4% NaCl. Bacteriocin activity in liquid medium containing up to 2% NaCl could be restored by coculturing the producing strains with a selected plantaricin-production inducing strain of Lactococcus lactis. Growth of these bacteriocinogenic L. plantarum strains was monitored in traditional Spanish-style green olive fermentations. Survival of these strains could not be enhanced when provided with a range of plantaricin-production inducing mechanisms previously described, such as constitutive AIP production or coinoculation with a specific bacteriocin-production inducing strain of L. lactis. Our results suggest that it is advisable the use of constitutive bacteriocin producers, or at least non-AIP-dependant ones, as starters for olive fermentations due to the intrinsic physical characteristics of this food fermentation, especially the high salt concentration of the brines currently used. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Regulation of protein phosphatase 2A during embryonic diapause process in the silkworm, Bombyx mori.

    Science.gov (United States)

    Gu, Shi-Hong; Hsieh, Hsiao-Yen; Lin, Pei-Ling

    2017-11-01

    Regulation of protein phosphorylation requires coordinated interactions between protein kinases and protein phosphatases. In the present study, we investigated regulation of protein phosphatase 2A (PP2A) during the embryonic diapause process of B. mori. An immunoblotting analysis showed that Bombyx eggs contained a catalytic C subunit, a major regulatory B subunit (B55/PR55 subunit), and a structural A subunit, with the A and B subunits undergoing differential changes between diapause and non-diapause eggs during embryonic process. In non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling of diapausing eggs at 5°C for 70days and then were transferred to 25°C, protein levels of the A and B subunits of PP2A gradually increased toward embryonic development. However, protein levels of the A and B subunits in diapause eggs remained at low levels during the first 8days after oviposition. The direct determination of PP2A enzymatic activity showed that the activity remained at low levels in diapause eggs during the first 8days after oviposition. However, in non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling, PP2A enzymatic activity sharply increased during the first several days, reached a peak during the middle embryonic development, and then greatly decreased 3 or 4days before hatching. Examination of temporal changes in mRNA expression levels of the catalytic β subunit and regulatory subunit of PP2A showed high levels in eggs whose diapause initiation was prevented by HCl compared to those in diapause eggs. These results demonstrate that the higher PP2A gene expression and PP2A A and B subunit protein levels and increased enzymatic activity are related to embryonic development of B. mori. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Synthesis and processing of sphingolipid activator protein-2 (SAP-2) in cultured human fibroblasts

    International Nuclear Information System (INIS)

    Fujibayashi, S.; Wenger, D.A.

    1986-01-01

    Sphingolipid activator proteins (SAP) are relatively small molecular weight proteins that stimulate the enzymatic hydrolysis of sphingolipids in the presence of specific lysosomal hydrolases. SAP-2 has previously been demonstrated to activate the hydrolysis of glucosylceramide, galactosylceramide, and, possibly, sphingomyelin. Using monospecific rabbit antibodies against human spleen SAP-2, the synthesis and processing of SAP-2 were studied in cultured human fibroblasts. When [ 35 S]methionine was presented in the medium to control human cells for 4 h, five major areas of radiolabeling were found. These had apparent molecular weights of 73,000, 68,000, 50,000, 12,000, and 9000. Further studies indicated that the major extracellular product in normal cells given NH4Cl along with the [ 35 S]methionine and in medium from cultures from patients with I cell disease had an apparent molecular weight of 73,000. The Mr = 68,000 and 73,000 species can be converted to a species with an apparent molecular weight of 50,000 by the action of endoglycosidase F. After labeling cells for 1 h followed by a 1-h chase, the Mr = 12,000 and 9000 species appear. Treatment of the immunoprecipitated mixture with endoglycosidase F resulted in conversion of these species to one band with an apparent molecular weight of 7600. These studies indicate that this relatively low molecular weight protein is rapidly synthesized from a relatively large molecular weight highly glycosylated precursor

  8. Signaling system in Porphyromonas gingivalis based on a LuxS protein.

    Science.gov (United States)

    Chung, W O; Park, Y; Lamont, R J; McNab, R; Barbieri, B; Demuth, D R

    2001-07-01

    The luxS gene of quorum-sensing Vibrio harveyi is required for type 2 autoinducer production. We identified a Porphyromonas gingivalis open reading frame encoding a predicted peptide of 161 aa that shares 29% identity with the amino acid sequence of the LuxS protein of V. harveyi. Conditioned medium from a late-log-phase P. gingivalis culture induced the luciferase operon of V. harveyi, but that from a luxS insertional mutant did not. In P. gingivalis, the expression of luxS mRNA was environmentally controlled and varied according to the cell density and the osmolarity of the culture medium. In addition, differential display PCR showed that the inactivation of P. gingivalis luxS resulted in up-regulation of a hemin acquisition protein and an arginine-specific protease and reduced expression of a hemin-regulated protein, a TonB homologue, and an excinuclease. The data suggest that the luxS gene in P. gingivalis may function to control the expression of genes involved in the acquisition of hemin.

  9. Processing and fatty acid acylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Fujiyama, A.; Tamanoi, F.

    1986-01-01

    The authors demonstrate the pathway for the biosynthesis of RAS1 and RAS2 gene products of Saccharomyces cerevisiae leading to their localization in membranes. The primary translation products of these genes are detected in a soluble fraction. Shortly after synthesis, these precursor molecules are converted to forms that migrate slightly faster than the precursor forms on a NaDodSO 4 /polyacrylamide gel. These processed proteins are further modified by fatty acid acylation, which is detected by [ 3 H]palmitic acid labeling. The acylated derivatives are found exclusively in cell membranes, indicating the translocation of the RAS proteins from cytosol to membranes during maturation process. The attached fatty acids can be released by mild alkaline hydrolysis, suggesting that the linkage between the fatty acid and the protein is an ester bond. The site of the modification by fatty acid is presumably localized to the COOH-terminal portion of the RAS proteins. Fraction of the membranes by sucrose gradient demonstrates that a majority of the fatty-acylated RAS proteins are localized in plasma membrane

  10. Detection of possible AI-2-mediated quorum sensing system in commensal intestinal bacteria.

    Science.gov (United States)

    Lukás, F; Gorenc, G; Kopecný, J

    2008-01-01

    The Vibrio harveyi strain BB170-autoinducer bioassay was used to detect possible quorum sensing autoinducer-2 molecule (AI-2) in culture fluids of commensal intestinal bacteria. Culture fluids of Bacteroides vulgatus, Clostridium proteoclasticum, Escherichia coli, Eubacterium rectale, Lachnospira multipara, Pseudobutyrivibrio ruminis, Roseburia intestinalis, Ruminococcus albus and Ruminococcus flavefaciens contained AI-2-like molecules. The PCR bands from some of the tested strains could be also amplified using primers designed for the luxS gene. These findings suggest that AI-2 is present in the gastrointestinal tract; however, it has not yet been proved whether it is used for bacterial cell-to-cell communication.

  11. Recent progresses on AI-2 bacterial quorum sensing inhibitors.

    Science.gov (United States)

    Zhu, Peng; Li, Minyong

    2012-01-01

    Quorum sensing (QS) is a communication procedure that predominates gene expression in response to cell density and fluctuations in the neighboring environment as a result of discerning molecules termed autoinducers (AIs). It has been embroiled that QS can govern bacterial behaviors such as the secretion of virulence factors, biofilm formation, bioluminescence production, conjugation, sporulation and swarming motility. Autoinducer 2 (AI-2), a QS signaling molecule brought up to be involved in interspecies communication, exists in both gram-negative and -positive bacteria. Therefore, novel approaches to interrupt AI-2 quorum sensing are being recognized as next generation antimicrobials. In the present review article, we summarized recent progresses on AI-2 bacterial quorum sensing inhibitors and discussed their potential as the antibacterial agents.

  12. Stopping AI-2 chatter by means of an indigenous bacterium ( Acinetobacter sp. DKY-1): A new anti-biofouling strategy in an MBR for wastewater treatment.

    Science.gov (United States)

    Lee, Kibaek; Kim, Yea-Won; Lee, Seonki; Lee, Sang Hyun; Nahm, Chang Hyun; Kwon, Hyeokpil; Park, Pyung-Kyu; Choo, Kwang-Ho; Koyuncu, Ismail; Drews, Anja; Lee, Chung-Hak; Lee, Jung-Kee

    2018-05-01

    Bacterial quorum quenching (QQ) by means of degrading signaling molecules has been applied to anti-biofouling strategy in a membrane bioreactor (MBR) for wastewater treatment. However, the target signaling molecules have been limited to N-acyl homoserine lactones participating in intra-species quorum sensing. Here, an approach to disrupt autoinducer-2 (AI-2) signaling molecules participating in inter-species quorum sensing, was pursued as a next-generation anti-biofouling strategy in an MBR for wastewater treatment. We isolated an indigenous QQ bacterium ( Acinetobacter sp. DKY-1) that can attenuate the expression of quorum sensing (QS) response through inactivation of autoinducer-2 signaling molecule, 4,5-dihydroxy-2,3-pentanedione (DPD) among four kinds of autoinducer-2 QS bacteria. DKY-1 released AI-2 QQ compound(s), which was verified to be hydrophilic with a molecular weight biofouling. This new approach, combining molecular biology with wastewater engineering, could enlarge the range of QQ-MBR for anti-biofouling and energy savings in the field of wastewater treatment.

  13. Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway.

    Science.gov (United States)

    Bassler, B L; Wright, M; Silverman, M R

    1994-07-01

    Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of extracellular signal molecules (autoinducers) in the culture medium. One signal-response system is encoded by the luxL,M,N locus. The luxL and luxM genes are required for the production of an autoinducer (probably beta-hydroxybutyl homoserine lactone), and the luxN gene is required for the response to that autoinducer. Analysis of the phenotypes of LuxL,M and N mutants indicated that an additional signal-response system also controls density sensing. We report here the identification, cloning and analysis of luxP and luxQ, which encode functions required for a second density-sensing system. Mutants with defects in luxP and luxQ are defective in response to a second autoinducer substance. LuxQ, like LuxN, is similar to members of the family of two-component, signal transduction proteins and contains both a histidine protein kinase and a response regulator domain. Analysis of signalling mutant phenotypes indicates that there are at least two separate signal-response pathways which converge to regulate expression of luminescence in V. harveyi.

  14. Food-cooking processes modulate allergenic properties of hen's egg white proteins.

    Science.gov (United States)

    Liu, Xiaoyu; Feng, Bai-Sui; Kong, Xiaoli; Xu, Hong; Li, Xiumin; Yang, Ping-Chang; Liu, Zhigang

    2013-01-01

    Reducing the allergenicity of food allergens can suppress the clinical symptoms of food allergy. The objective of the present study was to investigate the effects of processing on the allergenic properties of hen's egg white proteins. Eggs were processed by traditional Chinese cooking, including steaming, water boiling, frying, spicing and tea boiling. The contents of processed egg protein were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis; the allergenicity was evaluated by Western blotting, enzyme-linked immunosorbent assay and enzyme allergosorbent test inhibition. Circular dichroism spectrum analysis of four major egg allergens from various egg products was performed as well. A mouse model of food allergy was developed to test the allergenicity of processed egg protein in vivo. Protein degradation was significant following tea boiling and spiced-tea boiling. The total allergenic potential of water-boiled egg and fried egg was relatively higher than that of steamed egg, spiced egg and tea-boiled egg. Challenge with proteins from raw egg, water-boiled egg and fried egg induced skewed T-helper 2 pattern responses (Th2 responses) in the intestine of mice sensitized to egg proteins; however, when the mice sensitized to egg proteins were challenged with proteins from steamed egg, spiced egg and tea-boiled egg, respectively, only weak Th2 responses were induced in their intestine. Processing by steaming, spicing, or tea boiling can weaken the allergenicity of egg proteins. Copyright © 2012 S. Karger AG, Basel.

  15. A novel family of katanin-like 2 protein isoforms (KATNAL2), interacting with nucleotide-binding proteins Nubp1 and Nubp2, are key regulators of different MT-based processes in mammalian cells.

    Science.gov (United States)

    Ververis, Antonis; Christodoulou, Andri; Christoforou, Maria; Kamilari, Christina; Lederer, Carsten W; Santama, Niovi

    2016-01-01

    Katanins are microtubule (MT)-severing AAA proteins with high phylogenetic conservation throughout the eukaryotes. They have been functionally implicated in processes requiring MT remodeling, such as spindle assembly in mitosis and meiosis, assembly/disassembly of flagella and cilia and neuronal morphogenesis. Here, we uncover a novel family of katanin-like 2 proteins (KATNAL2) in mouse, consisting of five alternatively spliced isoforms encoded by the Katnal2 genomic locus. We further demonstrate that in vivo these isoforms are able to interact with themselves, with each other and moreover directly and independently with MRP/MinD-type P-loop NTPases Nubp1 and Nubp2, which are integral components of centrioles, negative regulators of ciliogenesis and implicated in centriole duplication in mammalian cells. We find KATNAL2 localized on interphase MTs, centrioles, mitotic spindle, midbody and the axoneme and basal body of sensory cilia in cultured murine cells. shRNAi of Katnal2 results in inefficient cytokinesis and severe phenotypes of enlarged cells and nuclei, increased numbers of centrioles and the manifestation of aberrant multipolar mitotic spindles, mitotic defects, chromosome bridges, multinuclearity, increased MT acetylation and an altered cell cycle pattern. Silencing or stable overexpression of KATNAL2 isoforms drastically reduces ciliogenesis. In conclusion, KATNAL2s are multitasking enzymes involved in the same cell type in critically important processes affecting cytokinesis, MT dynamics, and ciliogenesis and are also implicated in cell cycle progression.

  16. Quorum sensing in Acinetobacter: with special emphasis on antibiotic resistance, biofilm formation and quorum quenching

    Directory of Open Access Journals (Sweden)

    Bindu Subhadra

    2016-02-01

    Full Text Available Acinetobacter is an important nosocomial, opportunistic human pathogen that is gradually gaining more attention as a major health threat worldwide. Quorum sensing (QS is a cell-cell communication system in which specific signaling molecules called autoinducers accumulate in the medium as the population density grows and control various physiological processes including production of virulence factors, biofilm and development of antibiotic resistance. The complex QS machinery in Acinetobacter is mediated by a two-component system which is homologous to the typical LuxI/LuxR system found in Gram-negative bacteria. This cell signaling system comprises of a sensor protein that functions as autoinducer synthase and a receptor protein which binds to the signal molecules, acyl homoserine lactones inducing a cascade of reactions. Lately, disruption of QS has emerged as an anti-virulence strategy with great therapeutic potential. Here, we depict the current understanding of the existing QS network in Acinetobacter and describe important anti-virulent strategies developed in order to effectively tackle this pathogen. In addition, the prospects of quorum quenching to control Acinetobacter infections is also been discussed.

  17. Identification of AI-2 Quorum Sensing Inhibitors in Vibrio harveyi Through Structure-Based Virtual Screening.

    Science.gov (United States)

    Jiang, Tianyu; Zhu, Peng; Du, Lupei; Li, Minyong

    2018-01-01

    Quorum sensing (QS) is a cell-to-cell communication system that regulates gene expression as a result of the production and perception of signal molecules called autoinducers (AIs). AI-2 is a QS autoinducer produced by both Gram-negative and Gram-positive bacteria, in which it regulates intraspecies and interspecies communication. The identification of QS inhibitors is considered a promising strategy for the development of anti-virulence drugs with reduced selective pressure for resistance. Here we describe a high-throughput virtual screening approach to identify AI-2 quorum sensing inhibitors on the basis of Vibrio harveyi LuxPQ crystal structure. Seven potent inhibitors with IC 50 values in the micromolar range were selected with no effect or low effect on V. harveyi growth rate.

  18. Optimizing Urine Processing Protocols for Protein and Metabolite Detection.

    Science.gov (United States)

    Siddiqui, Nazema Y; DuBois, Laura G; St John-Williams, Lisa; Will, Thompson J; Grenier, Carole; Burke, Emily; Fraser, Matthew O; Amundsen, Cindy L; Murphy, Susan K

    In urine, factors such as timing of voids, and duration at room temperature (RT) may affect the quality of recovered protein and metabolite data. Additives may aid with detection, but can add more complexity in sample collection or analysis. We aimed to identify the optimal urine processing protocol for clinically-obtained urine samples that allows for the highest protein and metabolite yields with minimal degradation. Healthy women provided multiple urine samples during the same day. Women collected their first morning (1 st AM) void and another "random void". Random voids were aliquotted with: 1) no additive; 2) boric acid (BA); 3) protease inhibitor (PI); or 4) both BA + PI. Of these aliquots, some were immediately stored at 4°C, and some were left at RT for 4 hours. Proteins and individual metabolites were quantified, normalized to creatinine concentrations, and compared across processing conditions. Sample pools corresponding to each processing condition were analyzed using mass spectrometry to assess protein degradation. Ten Caucasian women between 35-65 years of age provided paired 1 st morning and random voided urine samples. Normalized protein concentrations were slightly higher in 1 st AM compared to random "spot" voids. The addition of BA did not significantly change proteins, while PI significantly improved normalized protein concentrations, regardless of whether samples were immediately cooled or left at RT for 4 hours. In pooled samples, there were minimal differences in protein degradation under the various conditions we tested. In metabolite analyses, there were significant differences in individual amino acids based on the timing of the void. For comparative translational research using urine, information about void timing should be collected and standardized. For urine samples processed in the same day, BA does not appear to be necessary while the addition of PI enhances protein yields, regardless of 4°C or RT storage temperature.

  19. Effect of Processing Intensity on Immunologically Active Bovine Milk Serum Proteins.

    Science.gov (United States)

    Brick, Tabea; Ege, Markus; Boeren, Sjef; Böck, Andreas; von Mutius, Erika; Vervoort, Jacques; Hettinga, Kasper

    2017-08-31

    Consumption of raw cow's milk instead of industrially processed milk has been reported to protect children from developing asthma, allergies, and respiratory infections. Several heat-sensitive milk serum proteins have been implied in this effect though unbiased assessment of milk proteins in general is missing. The aim of this study was to compare the native milk serum proteome between raw cow's milk and various industrially applied processing methods, i.e., homogenization, fat separation, pasteurization, ultra-heat treatment (UHT), treatment for extended shelf-life (ESL), and conventional boiling. Each processing method was applied to the same three pools of raw milk. Levels of detectable proteins were quantified by liquid chromatography/tandem mass spectrometry following filter aided sample preparation. In total, 364 milk serum proteins were identified. The 140 proteins detectable in 66% of all samples were entered in a hierarchical cluster analysis. The resulting proteomics pattern separated mainly as high (boiling, UHT, ESL) versus no/low heat treatment (raw, skimmed, pasteurized). Comparing these two groups revealed 23 individual proteins significantly reduced by heating, e.g., lactoferrin (log2-fold change = -0.37, p = 0.004), lactoperoxidase (log2-fold change = -0.33, p = 0.001), and lactadherin (log2-fold change = -0.22, p = 0.020). The abundance of these heat sensitive proteins found in higher quantity in native cow's milk compared to heat treated milk, renders them potential candidates for protection from asthma, allergies, and respiratory infections.

  20. Exploring novel food proteins and processing technologies

    NARCIS (Netherlands)

    Avila Ruiz, Geraldine

    2016-01-01

    Foods rich in protein are nowadays high in demand worldwide. To ensure a sustainable supply and a high quality of protein foods, novel food proteins and processing technologies need to be explored to understand whether they can be used for the development of high-quality protein foods. Therefore,

  1. Engineering Synthetic Proteins to Generate Ca2+ Signals in Mammalian Cells.

    Science.gov (United States)

    Qudrat, Anam; Truong, Kevin

    2017-03-17

    The versatility of Ca 2+ signals allows it to regulate diverse cellular processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g., VEGFR2), Ca 2+ signals are generated upon binding their ligand(s) (e.g., VEGF-A). Here, we employed a design strategy to engineer proteins that generate a Ca 2+ signal upon binding various extracellular stimuli by creating fusions of protein domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. To test the strategy, we created chimeric proteins that generate Ca 2+ signals upon stimulation with various extracellular stimuli (e.g., rapamycin, EDTA or extracellular free Ca 2+ ). By coupling these chimeric proteins that generate Ca 2+ signals with proteins that respond to Ca 2+ signals, we rewired, for example, dynamic cellular blebbing to increases in extracellular free Ca 2+ . Thus, using this design strategy, it is possible to engineer proteins to generate a Ca 2+ signal to rewire a wide range of extracellular stimuli to a wide range of Ca 2+ -activated processes.

  2. Self-processing 2A-polyproteins--a system for co-ordinate expression of multiple proteins in transgenic plants.

    Science.gov (United States)

    Halpin, C; Cooke, S E; Barakate, A; El Amrani, A; Ryan, M D

    1999-02-01

    Achieving co-ordinate, high-level and stable expression of multiple transgenes in plants is currently difficult. Expression levels are notoriously variable and influenced by factors that act independently on transgenes at different genetic loci. Instability of expression due to loss, re-arrangement or silencing of transgenes may occur, and is exacerbated by increasing numbers of transgenic loci and repeated use of homologous sequences. Even linking two or more genes within a T-DNA does not necessarily result in co-ordinate expression. Linking proteins in a single open reading frame--a polyprotein--is a strategy for co-ordinate expression used by many viruses. After translation, polyproteins are processed into constituent polypeptides, usually by proteinases encoded within the polyprotein itself. However, in foot-and-mouth disease virus (FMDV), a sequence (2A) of just 16-20 amino acids appears to have the unique capability to mediate cleavage at its own C-terminus by an apparently enzyme-independent, novel type of reaction. This sequence can also mediate cleavage in a heterologous protein context in a range of eukaryotic expression systems. We have constructed a plasmid in which the 2A sequence is inserted between the reporter genes chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS), maintaining a single open reading frame. Here we report that expression of this construct in wheatgerm lysate and transgenic plants results in efficient cleavage of the polyprotein and co-ordinate expression of active CAT and GUS. Self-processing polyproteins using the FMDV 2A sequence could therefore provide a system for ensuring co-ordinated, stable expression of multiple introduced proteins in plant cells.

  3. The calcium-binding protein ALG-2 regulates protein secretion and trafficking via interactions with MISSL and MAP1B proteins.

    Science.gov (United States)

    Takahara, Terunao; Inoue, Kuniko; Arai, Yumika; Kuwata, Keiko; Shibata, Hideki; Maki, Masatoshi

    2017-10-13

    Mobilization of intracellular calcium is essential for a wide range of cellular processes, including signal transduction, apoptosis, and vesicular trafficking. Several lines of evidence have suggested that apoptosis-linked gene 2 (ALG-2, also known as PDCD6 ), a calcium-binding protein, acts as a calcium sensor linking calcium levels with efficient vesicular trafficking, especially at the endoplasmic reticulum (ER)-to-Golgi transport step. However, how ALG-2 regulates these processes remains largely unclear. Here, we report that M APK1- i nteracting and s pindle- s tabilizing (MISS)- l ike (MISSL), a previously uncharacterized protein, interacts with ALG-2 in a calcium-dependent manner. Live-cell imaging revealed that upon a rise in intracellular calcium levels, GFP-tagged MISSL (GFP-MISSL) dynamically relocalizes in a punctate pattern and colocalizes with ALG-2. MISSL knockdown caused disorganization of the components of the ER exit site, the ER-Golgi intermediate compartment, and Golgi. Importantly, knockdown of either MISSL or ALG-2 attenuated the secretion of se creted a lkaline p hosphatase (SEAP), a model secreted cargo protein, with similar reductions in secretion by single- and double-protein knockdowns, suggesting that MISSL and ALG-2 act in the same pathway to regulate the secretion process. Furthermore, ALG-2 or MISSL knockdown delayed ER-to-Golgi transport of procollagen type I. We also found that ALG-2 and MISSL interact with microtubule-associated protein 1B (MAP1B) and that MAP1B knockdown reverts the reduced secretion of SEAP caused by MISSL or ALG-2 depletion. These results suggest that a change in the intracellular calcium level plays a role in regulation of the secretory pathway via interaction of ALG-2 with MISSL and MAP1B. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Optimization of Protein Hydrolysate Production Process from Jatropha curcas Cake

    OpenAIRE

    Waraporn Apiwatanapiwat; Pilanee Vaithanomsat; Phanu Somkliang; Taweesiri Malapant

    2009-01-01

    This was the first document revealing the investigation of protein hydrolysate production optimization from J. curcas cake. Proximate analysis of raw material showed 18.98% protein, 5.31% ash, 8.52% moisture and 12.18% lipid. The appropriate protein hydrolysate production process began with grinding the J. curcas cake into small pieces. Then it was suspended in 2.5% sodium hydroxide solution with ratio between solution/ J. curcas cake at 80:1 (v/w). The hydrolysis reactio...

  5. RECOVERY OF PROTEIN FROM MUNG BEAN STARCH PROCESSING WASTEWATER BY ROTATING ULTRAFILTRATION

    Directory of Open Access Journals (Sweden)

    PENPORN SRINIWORN

    2016-07-01

    Full Text Available Mung bean wastewater containing valuable protein is very potential to be recovered for reuse. In this study, rotary disk ultrafiltration was employed to recover this protein. The effects of transmembrane pressure (TMP and membrane rotational speeds on process efficiency were studied and the optimum condition was chosen based on membrane permeate flux and protein retention. The results suggested that the use of TMP of 1.2 bar and rotating speed of 1,683 rpm under total recycle mode tended to achieve highest permeate flux (43 L/m3h compared to those using lower TMP and rotating speeds. The permeate fluxes under total recycle mode and batch concentration mode tended to increase with processing time, indicating the effectiveness of rotating shear force. In addition, the effect of stabilization technique on process performance under batch concentration mode was also studied. However, the variable did not show positive impacts on permeate flux and protein retention improvement. The optimum condition to achieve volume concentration factor (VCF of 5 was TMP of 1.2 bar and rotating speed of 1,403 rpm without stabilization. Under this condition, the average flux, protein retention and energy consumption were 42 L/m2h, 96% and 81 kWh/m3, respectively.

  6. Type 2 diabetes mellitus with early phase acute inflammatory protein on serum protein electrophoresis

    Directory of Open Access Journals (Sweden)

    ET Tuladhar

    2012-03-01

    Full Text Available Background: The onset of Type 2 diabetes has been associated with low grade systemic inflammation. The inflammatory status has been studied by measuring acute phase reactant proteins like hsCRP, α1- antitrypsin, α1-acid glycoprotein, ceruloplasmin, fibrinogen. Most of these acute phase reactants form α1 and α2 bands on electropherogram of serum proteins. The aim of this study was to evaluate inflammatory status in controlled and uncontrolled type 2 diabetes using cellulose acetate electrophoresis and to find the impact of glycemic status as indicated by HbA1c on inflammation process. Materials and Methods: Serum protein electrophoresis was done on serum samples of 60 cases of Diabetes [controlled and uncontrolled] using cellulose acetate paper technique. The electropherogram obtained was stained with Ponseu S and then quantitated using densitometer. Glycemic status was studied by HbA1c analysis. The density of α1and α2 bands in electropherogram were correlated with HbA1c level. Result: A significant increase in the percentage of α1 and α2 band proteins (0.765 and 0.716, p<0.001 were found with the increasing level of HbA1c. With cutoff of HbA1c 7% (American Diabetic Association recommended, the α1 and α2 serum proteins concentration are significantly higher (p<0.001 in uncontrolled diabetes mellitus compared to controlled diabetes mellitus Conclusion: Cellulose acetate electrophoresis of serum proteins show early phase acute inflammatory status in uncontrolled type 2 diabetes mellitus. The process of systemic inflammation worsens with uncontrolled glycemia as indicated by HbA1c. Inflammatory status should be studied adjunct to glycemic status. DOI: http://dx.doi.org/10.3126/jpn.v2i3.6024 JPN 2012; 2(3: 211-214

  7. Glucagon-Like Peptide 2 Stimulates Postresection Intestinal Adaptation in Preterm Pigs by Affecting Proteins Related to Protein, Carbohydrate, and Sulphur Metabolism

    DEFF Research Database (Denmark)

    Jiang, Pingping; Vegge, Andreas; Thymann, Thomas

    2017-01-01

    cellular structural proteins, while the added GLP-2 treatment affected proteins involved in protein processing and the metabolism of protein, carbohydrate, and sulphur. CONCLUSION: In the first days following resection, proteins affected by resection plus GLP-2 treatment differed markedly from those...

  8. Beyond voltage-gated ion channels: Voltage-operated membrane proteins and cellular processes.

    Science.gov (United States)

    Zhang, Jianping; Chen, Xingjuan; Xue, Yucong; Gamper, Nikita; Zhang, Xuan

    2018-04-18

    Voltage-gated ion channels were believed to be the only voltage-sensitive proteins in excitable (and some non-excitable) cells for a long time. Emerging evidence indicates that the voltage-operated model is shared by some other transmembrane proteins expressed in both excitable and non-excitable cells. In this review, we summarize current knowledge about voltage-operated proteins, which are not classic voltage-gated ion channels as well as the voltage-dependent processes in cells for which single voltage-sensitive proteins have yet to be identified. Particularly, we will focus on the following. (1) Voltage-sensitive phosphoinositide phosphatases (VSP) with four transmembrane segments homologous to the voltage sensor domain (VSD) of voltage-gated ion channels; VSPs are the first family of proteins, other than the voltage-gated ion channels, for which there is sufficient evidence for the existence of the VSD domain; (2) Voltage-gated proton channels comprising of a single voltage-sensing domain and lacking an identified pore domain; (3) G protein coupled receptors (GPCRs) that mediate the depolarization-evoked potentiation of Ca 2+ mobilization; (4) Plasma membrane (PM) depolarization-induced but Ca 2+ -independent exocytosis in neurons. (5) Voltage-dependent metabolism of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P 2 , PIP 2 ) in the PM. These recent discoveries expand our understanding of voltage-operated processes within cellular membranes. © 2018 Wiley Periodicals, Inc.

  9. Effects of grain source, grain processing, and protein degradability on rumen kinetics and microbial protein synthesis in Boer kids.

    Science.gov (United States)

    Brassard, M-E; Chouinard, P Y; Berthiaume, R; Tremblay, G F; Gervais, R; Martineau, R; Cinq-Mars, D

    2015-11-01

    Microbial protein synthesis in the rumen would be optimized when dietary carbohydrates and proteins have synchronized rates and extent of degradation. The aim of this study was to evaluate the effect of varying ruminal degradation rate of energy and nitrogen sources on intake, nitrogen balance, microbial protein yield, and kinetics of nutrients in the rumen of growing kids. Eight Boer goats (38.2 ± 3.0 kg) were used. The treatments were arranged in a split-plot Latin square design with grain sources (barley or corn) forming the main plots (squares). Grain processing methods and levels of protein degradability formed the subplots in a 2 × 2 factorial arrangement for a total of 8 dietary treatments. The grain processing method was rolling for barley and cracking for corn. Levels of protein degradability were obtained by feeding untreated soybean meal (SBM) or heat-treated soybean meal (HSBM). Each experimental period lasted 21 d, consisting of a 10-d adaptation period, a 7-d digestibility determination period, and a 4-d rumen evacuation and sampling period. Kids fed with corn had higher purine derivatives (PD) excretion when coupled with SBM compared with HSBM and the opposite occurred with barley-fed kids ( ≤ 0.01). Unprocessed grain offered with SBM led to higher PD excretion than with HSBM whereas protein degradability had no effect when processed grain was fed ( ≤ 0.03). Results of the current experiment with high-concentrate diets showed that microbial N synthesis could be maximized in goat kids by combining slowly fermented grains (corn or unprocessed grains) with a highly degradable protein supplement (SBM). With barley, a more rapidly fermented grain, a greater microbial N synthesis was observed when supplementing a low-degradable protein (HSBM).

  10. Investigating interactions between phospholipase B-Like 2 and antibodies during Protein A chromatography.

    Science.gov (United States)

    Tran, Benjamin; Grosskopf, Vanessa; Wang, Xiangdan; Yang, Jihong; Walker, Don; Yu, Christopher; McDonald, Paul

    2016-03-18

    Purification processes for therapeutic antibodies typically exploit multiple and orthogonal chromatography steps in order to remove impurities, such as host-cell proteins. While the majority of host-cell proteins are cleared through purification processes, individual host-cell proteins such as Phospholipase B-like 2 (PLBL2) are more challenging to remove and can persist into the final purification pool even after multiple chromatography steps. With packed-bed chromatography runs using host-cell protein ELISAs and mass spectrometry analysis, we demonstrated that different therapeutic antibodies interact to varying degrees with host-cell proteins in general, and PLBL2 specifically. We then used a high-throughput Protein A chromatography method to further examine the interaction between our antibodies and PLBL2. Our results showed that the co-elution of PLBL2 during Protein A chromatography is highly dependent on the individual antibody and PLBL2 concentration in the chromatographic load. Process parameters such as antibody resin load density and pre-elution wash conditions also influence the levels of PLBL2 in the Protein A eluate. Furthermore, using surface plasmon resonance, we demonstrated that there is a preference for PLBL2 to interact with IgG4 subclass antibodies compared to IgG1 antibodies. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. AI-2 does not function as a quorum sensing molecule in Campylobacter jejuni during exponential growth in vitro

    NARCIS (Netherlands)

    Holmes, K.; Tavender, T.J.; Winzer, K.; Wells, J.; Hardie, K.

    2009-01-01

    Background - Campylobacter jejuni contains a homologue of the luxS gene shown to be responsible for the production of the signalling molecule autoinducer-2 (AI-2) in Vibrio harveyi and Vibrio cholerae. The aim of this study was to determine whether AI-2 acted as a diffusible quorum sensing signal

  12. Effects of colonization, luminescence, and autoinducer on host transcription during development of the squid-vibrio association.

    Science.gov (United States)

    Chun, Carlene K; Troll, Joshua V; Koroleva, Irina; Brown, Bartley; Manzella, Liliana; Snir, Einat; Almabrazi, Hakeem; Scheetz, Todd E; Bonaldo, Maria de Fatima; Casavant, Thomas L; Soares, M Bento; Ruby, Edward G; McFall-Ngai, Margaret J

    2008-08-12

    The light-organ symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri offers the opportunity to decipher the hour-by-hour events that occur during the natural colonization of an animal's epithelial surface by its microbial partners. To determine the genetic basis of these events, a glass-slide microarray was used to characterize the light-organ transcriptome of juvenile squid in response to the initiation of symbiosis. Patterns of gene expression were compared between animals not exposed to the symbiont, exposed to the wild-type symbiont, or exposed to a mutant symbiont defective in either of two key characters of this association: bacterial luminescence or autoinducer (AI) production. Hundreds of genes were differentially regulated as a result of symbiosis initiation, and a hierarchy existed in the magnitude of the host's response to three symbiont features: bacterial presence > luminescence > AI production. Putative host receptors for bacterial surface molecules known to induce squid development are up-regulated by symbiont light production, suggesting that bioluminescence plays a key role in preparing the host for bacteria-induced development. Further, because the transcriptional response of tissues exposed to AI in the natural context (i.e., with the symbionts) differed from that to AI alone, the presence of the bacteria potentiates the role of quorum signals in symbiosis. Comparison of these microarray data with those from other symbioses, such as germ-free/conventionalized mice and zebrafish, revealed a set of shared genes that may represent a core set of ancient host responses conserved throughout animal evolution.

  13. Crystallization Process of Protein Rv0731c from Mycobacterium Tuberculosis for a Successful Atomic Resolution Crystal Structure at 1.2 Angstrom

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Liang Cong

    2009-06-08

    Proteins are bio-macromolecules consisting of basic 20 amino acids and have distinct three-dimensional folds. They are essential parts of organisms and participate in every process within cells. Proteins are crucial for human life, and each protein within the body has a specific function, such as antibodies, contractile proteins, enzymes, hormonal proteins, structural proteins, storage proteins and transport proteins. Determining three-dimensional structure of a protein can help researchers discover the remarkable protein folding, binding site, conformation and etc, in order to understand well of protein interaction and aid for possible drug design. The research on protein structure by X-ray protein crystallography carried by Li-Wei Hung's research group in the Physical Bioscience Division at Lawrence Berkeley National Laboratory (LBNL) is focusing on protein crystallography. The research in this lab is in the process of from crystallizing the proteins to determining the three dimensional crystal structures of proteins. Most protein targets are selected from Mycobacterium Tuberculosis. TB (Tuberculosis) is a possible fatal infectious disease. By studying TB target protein can help discover antituberculer drugs, and find treatment for TB. The high-throughput mode of crystallization, crystal harvesting, crystal screening and data collection are applied to the research pipeline (Figure 1). The X-ray diffraction data by protein crystals can be processed and analyzed to result in a three dimensional representation of electron density, producing a detailed model of protein structure. Rv0731c is a conserved hypothetical protein with unknown function from Mycobacterium Tuberculosis. This paper is going to report the crystallization process and brief structure information of Rv0731c.

  14. Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence.

    Science.gov (United States)

    Bassler, B L; Wright, M; Showalter, R E; Silverman, M R

    1993-08-01

    Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of an extracellular signal molecule (autoinducer) in the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode functions necessary for the synthesis of, and response to, a signal molecule. Sequence analysis of the region encoding these functions revealed three open reading frames, two (luxL and luxM) that are required for production of an autoinducer substance and a third (luxN) that is required for response to this signal substance. The LuxL and LuxM proteins are not similar in amino acid sequence to other proteins in the database, but the LuxN protein contains regions of sequence resembling both the histidine protein kinase and the response regulator domains of the family of two-component, signal transduction proteins. The phenotypes of mutants with luxL, luxM and luxN defects indicated that an additional signal-response system controlling density-dependent expression of luminescence remains to be identified.

  15. Myocardial ischemic preconditioning upregulated protein 1(Mipu1):zinc finger protein 667 - a multifunctional KRAB/C{sub 2}H{sub 2} zinc finger protein

    Energy Technology Data Exchange (ETDEWEB)

    Han, D.; Zhang, C. [Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, Post-doctoral Mobile Stations for Basic Medicine, University of South China, Hengyang City, Hunan Province (China); Fan, W.J. [Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, Post-doctoral Mobile Stations for Basic Medicine, University of South China, Hengyang City, Hunan Province (China); The Second Affiliated Hospital, University of South China, Hengyang City, Hunan Province (China); Pan, W.J.; Feng, D.M.; Qu, S.L.; Jiang, Z.S. [Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, Post-doctoral Mobile Stations for Basic Medicine, University of South China, Hengyang City, Hunan Province (China)

    2014-10-31

    Myocardial ischemic preconditioning upregulated protein 1 (Mipu1) is a newly discovered upregulated gene produced in rats during the myocardial ischemic preconditioning process. Mipu1 cDNA contains a 1824-base pair open reading frame and encodes a 608 amino acid protein with an N-terminal Krüppel-associated box (KRAB) domain and classical zinc finger C{sub 2}H{sub 2} motifs in the C-terminus. Mipu1 protein is located in the cell nucleus. Recent studies found that Mipu1 has a protective effect on the ischemia-reperfusion injury of heart, brain, and other organs. As a nuclear factor, Mipu1 may perform its protective function through directly transcribing and repressing the expression of proapoptotic genes to repress cell apoptosis. In addition, Mipu1 also plays an important role in regulating the gene expression of downstream inflammatory mediators by inhibiting the activation of activator protein-1 and serum response element.

  16. Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Tacchi, Jessica L; Raymond, Benjamin B A; Haynes, Paul A; Berry, Iain J; Widjaja, Michael; Bogema, Daniel R; Woolley, Lauren K; Jenkins, Cheryl; Minion, F Chris; Padula, Matthew P; Djordjevic, Steven P

    2016-02-01

    Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC-MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity. © 2016 The Authors.

  17. Foot-and-mouth disease virus capsid proteins; analysis of protein processing, assembly and utility as vaccines

    DEFF Research Database (Denmark)

    Belsham, Graham

    Foot-and-mouth disease (FMD) remains one of the most economically important infectious diseases of production animals globally. The infection is caused by foot-and-mouth disease virus (FMDV), a member of the picornavirus family. The positive sense RNA genome of the virus includes a single, large......, open reading frame that encodes a polyprotein. The intact polyprotein is never observed as it is processed, both during and after translation, to 15 different mature proteins plus a variety of precursors. The FMDV capsid protein precursor, P1-2A, is cleaved by the virus encoded 3C protease (3Cpro......) to generate VP0, VP3, VP1 and the peptide 2A. Sixty copies of each of the capsid proteins “self-assemble” into empty capsid particles or with the RNA genome into infectious viruses. These particles normally lack 2A but it is possible to construct and isolate mutant FMDVs in which the cleavage of the VP1/2A...

  18. Using Förster-Resonance Energy Transfer to Measure Protein Interactions Between Bcl-2 Family Proteins on Mitochondrial Membranes.

    Science.gov (United States)

    Pogmore, Justin P; Pemberton, James M; Chi, Xiaoke; Andrews, David W

    2016-01-01

    The Bcl-2 family of proteins regulates the process of mitochondrial outer membrane permeabilization, causing the release of cytochrome c and committing a cell to apoptosis. The majority of the functional interactions between these proteins occur at, on, or within the mitochondrial outer membrane, complicating structural studies of the proteins and complexes. As a result most in vitro studies of these protein-protein interactions use truncated proteins and/or detergents which can cause artificial interactions. Herein, we describe a detergent-free, fluorescence-based, in vitro technique to study binding between full-length recombinant Bcl-2 family proteins, particularly cleaved BID (cBID) and BCL-XL, on the membranes of purified mitochondria.

  19. Biorefinery process for protein extraction from oriental mustard (Brassica juncea (L.) Czern.) using ethanol stillage.

    Science.gov (United States)

    Ratanapariyanuch, Kornsulee; Tyler, Robert T; Shim, Youn Young; Reaney, Martin Jt

    2012-01-12

    Large volumes of treated process water are required for protein extraction. Evaporation of this water contributes greatly to the energy consumed in enriching protein products. Thin stillage remaining from ethanol production is available in large volumes and may be suitable for extracting protein rich materials. In this work protein was extracted from ground defatted oriental mustard (Brassica juncea (L.) Czern.) meal using thin stillage. Protein extraction efficiency was studied at pHs between 7.6 and 10.4 and salt concentrations between 3.4 × 10-2 and 1.2 M. The optimum extraction efficiency was pH 10.0 and 1.0 M NaCl. Napin and cruciferin were the most prevalent proteins in the isolate. The isolate exhibited high in vitro digestibility (74.9 ± 0.80%) and lysine content (5.2 ± 0.2 g/100 g of protein). No differences in the efficiency of extraction, SDS-PAGE profile, digestibility, lysine availability, or amino acid composition were observed between protein extracted with thin stillage and that extracted with NaCl solution. The use of thin stillage, in lieu of water, for protein extraction would decrease the energy requirements and waste disposal costs of the protein isolation and biofuel production processes.

  20. Utilization of protein-rich residues in biotechnological processes.

    Science.gov (United States)

    Pleissner, Daniel; Venus, Joachim

    2016-03-01

    A drawback of biotechnological processes, where microorganisms convert biomass constituents, such as starch, cellulose, hemicelluloses, lipids, and proteins, into wanted products, is the economic feasibility. Particularly the cost of nitrogen sources in biotechnological processes can make up a large fraction of total process expenses. To further develop the bioeconomy, it is of considerable interest to substitute cost-intensive by inexpensive nitrogen sources. The aim of this mini-review was to provide a comprehensive insight of utilization methods of protein-rich residues, such as fish waste, green biomass, hairs, and food waste. The methods described include (i) production of enzymes, (ii) recovery of bioactive compounds, and/or (iii) usage as nitrogen source for microorganisms in biotechnological processes. In this aspect, the utilization of protein-rich residues, which are conventionally considered as waste, allows the development of value-adding processes for the production of bioactive compounds, biomolecules, chemicals, and materials.

  1. Interferences of Silica Nanoparticles in Green Fluorescent Protein Folding Processes.

    Science.gov (United States)

    Klein, Géraldine; Devineau, Stéphanie; Aude, Jean Christophe; Boulard, Yves; Pasquier, Hélène; Labarre, Jean; Pin, Serge; Renault, Jean Philippe

    2016-01-12

    We investigated the relationship between unfolded proteins, silica nanoparticles and chaperonin to determine whether unfolded proteins could stick to silica surfaces and how this process could impair heat shock protein activity. The HSP60 catalyzed green fluorescent protein (GFP) folding was used as a model system. The adsorption isotherms and adsorption kinetics of denatured GFP were measured, showing that denaturation increases GFP affinity for silica surfaces. This affinity is maintained even if the surfaces are covered by a protein corona and allows silica NPs to interfere directly with GFP folding by trapping it in its unstructured state. We determined also the adsorption isotherms of HSP60 and its chaperonin activity once adsorbed, showing that SiO2 NP can interfere also indirectly with protein folding through chaperonin trapping and inhibition. This inhibition is specifically efficient when NPs are covered first with a layer of unfolded proteins. These results highlight for the first time the antichaperonin activity of silica NPs and ask new questions about the toxicity of such misfolded proteins/nanoparticles assembly toward cells.

  2. Inhibitory function of adapter-related protein complex 2 alpha 1 subunit in the process of nuclear translocation of human immunodeficiency virus type 1 genome

    International Nuclear Information System (INIS)

    Kitagawa, Yukiko; Kameoka, Masanori; Shoji-Kawata, Sanae; Iwabu, Yukie; Mizuta, Hiroyuki; Tokunaga, Kenzo; Fujino, Masato; Natori, Yukikazu; Yura, Yoshiaki; Ikuta, Kazuyoshi

    2008-01-01

    The transfection of human cells with siRNA against adapter-related protein complex 2 alpha 1 subunit (AP2α) was revealed to significantly up-regulate the replication of human immunodeficiency virus type 1 (HIV-1). This effect was confirmed by cell infection with vesicular stomatitis virus G protein-pseudotyped HIV-1 as well as CXCR4-tropic and CCR5-tropic HIV-1. Viral adsorption, viral entry and reverse transcription processes were not affected by cell transfection with siRNA against AP2α. In contrast, viral nuclear translocation as well as the integration process was significantly up-regulated in cells transfected with siRNA against AP2α. Confocal fluorescence microscopy revealed that a subpopulation of AP2α was not only localized in the cytoplasm but was also partly co-localized with lamin B, importin β and Nup153, implying that AP2α negatively regulates HIV-1 replication in the process of nuclear translocation of viral DNA in the cytoplasm or the perinuclear region. We propose that AP2α may be a novel target for disrupting HIV-1 replication in the early stage of the viral life cycle

  3. Dynamical analysis of yeast protein interaction network during the sake brewing process.

    Science.gov (United States)

    Mirzarezaee, Mitra; Sadeghi, Mehdi; Araabi, Babak N

    2011-12-01

    Proteins interact with each other for performing essential functions of an organism. They change partners to get involved in various processes at different times or locations. Studying variations of protein interactions within a specific process would help better understand the dynamic features of the protein interactions and their functions. We studied the protein interaction network of Saccharomyces cerevisiae (yeast) during the brewing of Japanese sake. In this process, yeast cells are exposed to several stresses. Analysis of protein interaction networks of yeast during this process helps to understand how protein interactions of yeast change during the sake brewing process. We used gene expression profiles of yeast cells for this purpose. Results of our experiments revealed some characteristics and behaviors of yeast hubs and non-hubs and their dynamical changes during the brewing process. We found that just a small portion of the proteins (12.8 to 21.6%) is responsible for the functional changes of the proteins in the sake brewing process. The changes in the number of edges and hubs of the yeast protein interaction networks increase in the first stages of the process and it then decreases at the final stages.

  4. Surface dynamics in allosteric regulation of protein-protein interactions: modulation of calmodulin functions by Ca2+.

    Directory of Open Access Journals (Sweden)

    Yosef Y Kuttner

    2013-04-01

    Full Text Available Knowledge of the structural basis of protein-protein interactions (PPI is of fundamental importance for understanding the organization and functioning of biological networks and advancing the design of therapeutics which target PPI. Allosteric modulators play an important role in regulating such interactions by binding at site(s orthogonal to the complex interface and altering the protein's propensity for complex formation. In this work, we apply an approach recently developed by us for analyzing protein surfaces based on steered molecular dynamics simulation (SMD to the study of the dynamic properties of functionally distinct conformations of a model protein, calmodulin (CaM, whose ability to interact with target proteins is regulated by the presence of the allosteric modulator Ca(2+. Calmodulin is a regulatory protein that acts as an intracellular Ca(2+ sensor to control a wide variety of cellular processes. We demonstrate that SMD analysis is capable of pinpointing CaM surfaces implicated in the recognition of both the allosteric modulator Ca(2+ and target proteins. Our analysis of changes in the dynamic properties of the CaM backbone elicited by Ca(2+ binding yielded new insights into the molecular mechanism of allosteric regulation of CaM-target interactions.

  5. Dengue-2 Structural Proteins Associate with Human Proteins to Produce a Coagulation and Innate Immune Response Biased Interactome

    Directory of Open Access Journals (Sweden)

    Soares Luis RB

    2011-01-01

    Full Text Available Abstract Background Dengue virus infection is a public health threat to hundreds of millions of individuals in the tropical regions of the globe. Although Dengue infection usually manifests itself in its mildest, though often debilitating clinical form, dengue fever, life-threatening complications commonly arise in the form of hemorrhagic shock and encephalitis. The etiological basis for the virus-induced pathology in general, and the different clinical manifestations in particular, are not well understood. We reasoned that a detailed knowledge of the global biological processes affected by virus entry into a cell might help shed new light on this long-standing problem. Methods A bacterial two-hybrid screen using DENV2 structural proteins as bait was performed, and the results were used to feed a manually curated, global dengue-human protein interaction network. Gene ontology and pathway enrichment, along with network topology and microarray meta-analysis, were used to generate hypothesis regarding dengue disease biology. Results Combining bioinformatic tools with two-hybrid technology, we screened human cDNA libraries to catalogue proteins physically interacting with the DENV2 virus structural proteins, Env, cap and PrM. We identified 31 interacting human proteins representing distinct biological processes that are closely related to the major clinical diagnostic feature of dengue infection: haemostatic imbalance. In addition, we found dengue-binding human proteins involved with additional key aspects, previously described as fundamental for virus entry into cells and the innate immune response to infection. Construction of a DENV2-human global protein interaction network revealed interesting biological properties suggested by simple network topology analysis. Conclusions Our experimental strategy revealed that dengue structural proteins interact with human protein targets involved in the maintenance of blood coagulation and innate anti

  6. Bioenergy, protein and fibres from grass - biogas process monitoring; Bioenergie, Protein und Fasern aus Gras - Monitoring des Biogasprozesses

    Energy Technology Data Exchange (ETDEWEB)

    Baier, U.; Delavy, P.

    2003-07-01

    Starting in Summer 2001 the first full scale Swiss Bio-refinery for grass processing took up operation in Schaffhausen. Grass processing covers the production of technical fibres and protein concentrate as well as anaerobic digestion of residual slops for the production of biogas and 'green' electricity. The refinery is operated by the company Bioenergie Schaffhausen as a P+D (pilot + demonstration) project of the Swiss Federal Office of Energy. Under full load it will deliver 2,000 MWh of 'green' electricity (10% own needs) and 3,000 MWh heat (50% own needs). Prior to start up the Swiss technology holder 2B Biorefineries AG mandated the University of Applied Sciences HSW with lab scale testing of the mesophilic biogas potential and anaerobic degradability of residual grass processing slops. Nutrient limitations and possible inhibition risks were evaluated. During the initial 8 months of full scale operation of the refinery in Schaffhausen an intensive monitoring of the anaerobic digester's performance was carried out. Carbon and nitrogen mass balances have been set up and the development of the granular EGSB sludge was characterised. From operational data a set of performance values was elaborated. The first year of operation was characterised by only partial exploitation of the refinery's grass processing capacity. Furthermore the protein separation and production unit has not yet been incorporated. Consequently, the EGSB biogas reactor showed a significant hydraulic underload when compared to dimensioning basics. Raw residuals were characterised by a higher particulate protein fraction. Operational conditions for the EGSB reactor were worked out to allow stable operation at elevated load conditions and with protein separation in operation. (author)

  7. Bioenergy, protein and fibres from grass - biogas process monitoring; Bioenergie, Protein und Fasern aus Gras - Monitoring des Biogasprozesses

    Energy Technology Data Exchange (ETDEWEB)

    Baier, U; Delavy, P

    2003-07-01

    Starting in Summer 2001 the first full scale Swiss Bio-refinery for grass processing took up operation in Schaffhausen. Grass processing covers the production of technical fibres and protein concentrate as well as anaerobic digestion of residual slops for the production of biogas and 'green' electricity. The refinery is operated by the company Bioenergie Schaffhausen as a P+D (pilot + demonstration) project of the Swiss Federal Office of Energy. Under full load it will deliver 2,000 MWh of 'green' electricity (10% own needs) and 3,000 MWh heat (50% own needs). Prior to start up the Swiss technology holder 2B Biorefineries AG mandated the University of Applied Sciences HSW with lab scale testing of the mesophilic biogas potential and anaerobic degradability of residual grass processing slops. Nutrient limitations and possible inhibition risks were evaluated. During the initial 8 months of full scale operation of the refinery in Schaffhausen an intensive monitoring of the anaerobic digester's performance was carried out. Carbon and nitrogen mass balances have been set up and the development of the granular EGSB sludge was characterised. From operational data a set of performance values was elaborated. The first year of operation was characterised by only partial exploitation of the refinery's grass processing capacity. Furthermore the protein separation and production unit has not yet been incorporated. Consequently, the EGSB biogas reactor showed a significant hydraulic underload when compared to dimensioning basics. Raw residuals were characterised by a higher particulate protein fraction. Operational conditions for the EGSB reactor were worked out to allow stable operation at elevated load conditions and with protein separation in operation. (author)

  8. Ultra-processed foods, protein leverage and energy intake in the USA.

    Science.gov (United States)

    Martínez Steele, Euridice; Raubenheimer, David; Simpson, Stephen J; Baraldi, Larissa Galastri; Monteiro, Carlos A

    2018-01-01

    Experimental studies have shown that human macronutrient regulation minimizes variation in absolute protein intake and consequently energy intake varies passively with dietary protein density ('protein leverage'). According to the 'protein leverage hypothesis' (PLH), protein leverage interacts with a reduction in dietary protein density to drive energy overconsumption and obesity. Worldwide increase in consumption of ultra-processed foods (UPF) has been hypothesized to be an important determinant of dietary protein dilution, and consequently an ecological driving force of energy overconsumption and the obesity pandemic. The present study examined the relationships between dietary contribution of UPF, dietary proportional protein content and the absolute intakes of protein and energy. National representative cross-sectional study. National Health and Nutrition Examination Survey 2009-2010. Participants (n 9042) aged ≥2 years with at least one day of 24 h dietary recall data. We found a strong inverse relationship between consumption of UPF and dietary protein density, with mean protein content dropping from 18·2 to 13·3 % between the lowest and highest quintiles of dietary contribution of UPF. Consistent with the PLH, increase in the dietary contribution of UPF (previously shown to be inversely associated with protein density) was also associated with a rise in total energy intake, while absolute protein intake remained relatively constant. The protein-diluting effect of UPF might be one mechanism accounting for their association with excess energy intake. Reducing UPF contribution in the US diet may be an effective way to increase its dietary protein concentration and prevent excessive energy intake.

  9. The LIM-only protein FHL2 attenuates lung inflammation during bleomycin-induced fibrosis.

    Directory of Open Access Journals (Sweden)

    Abdulaleem Alnajar

    Full Text Available Fibrogenesis is usually initiated when regenerative processes have failed and/or chronic inflammation occurs. It is characterised by the activation of tissue fibroblasts and dysregulated synthesis of extracellular matrix proteins. FHL2 (four-and-a-half LIM domain protein 2 is a scaffolding protein that interacts with numerous cellular proteins, regulating signalling cascades and gene transcription. It is involved in tissue remodelling and tumour progression. Recent data suggest that FHL2 might support fibrogenesis by maintaining the transcriptional expression of alpha smooth muscle actin and the excessive synthesis and assembly of matrix proteins in activated fibroblasts. Here, we present evidence that FHL2 does not promote bleomycin-induced lung fibrosis, but rather suppresses this process by attenuating lung inflammation. Loss of FHL2 results in increased expression of the pro-inflammatory matrix protein tenascin C and downregulation of the macrophage activating C-type lectin receptor DC-SIGN. Consequently, FHL2 knockout mice developed a severe and long-lasting lung pathology following bleomycin administration due to enhanced expression of tenascin C and impaired activation of inflammation-resolving macrophages.

  10. The 2DX robot: a membrane protein 2D crystallization Swiss Army knife.

    Science.gov (United States)

    Iacovache, Ioan; Biasini, Marco; Kowal, Julia; Kukulski, Wanda; Chami, Mohamed; van der Goot, F Gisou; Engel, Andreas; Rémigy, Hervé-W

    2010-03-01

    Among the state-of-the-art techniques that provide experimental information at atomic scale for membrane proteins, electron crystallography, atomic force microscopy and solid state NMR make use of two-dimensional crystals. We present a cyclodextrin-driven method for detergent removal implemented in a fully automated robot. The kinetics of the reconstitution processes is precisely controlled, because the detergent complexation by cyclodextrin is of stoichiometric nature. The method requires smaller volumes and lower protein concentrations than established 2D crystallization methods, making it possible to explore more conditions with the same amount of protein. The method yielded highly ordered 2D crystals diffracting to high resolution from the pore-forming toxin Aeromonas hydrophila aerolysin (2.9A), the plant aquaporin SoPIP2;1 (3.1A) and the human aquaporin-8 (hAQP8; 3.3A). This new method outperforms traditional 2D crystallization approaches in terms of accuracy, flexibility, throughput, and allows the usage of detergents having low critical micelle concentration (CMC), which stabilize the structure of membrane proteins in solution. (c) 2009 Elsevier Inc. All rights reserved.

  11. The adsorption features between insecticidal crystal protein and nano-Mg(OH)2.

    Science.gov (United States)

    Pan, Xiaohong; Xu, Zhangyan; Zheng, Yilin; Huang, Tengzhou; Li, Lan; Chen, Zhi; Rao, Wenhua; Chen, Saili; Hong, Xianxian; Guan, Xiong

    2017-12-01

    Nano-Mg(OH) 2 , with low biological toxicity, is an ideal nano-carrier for insecticidal protein to improve the bioactivity. In this work, the adsorption features of insecticidal protein by nano-Mg(OH) 2 have been studied. The adsorption capacity could reach as high as 136 mg g -1 , and the adsorption isotherm had been fitted with Langmuir and Freundlich models. Moreover, the adsorption kinetics followed a pseudo-first or -second order rate model, and the adsorption was spontaneous and an exothermic process. However, high temperatures are not suitable for adsorption, which implies that the temperature would be a critical factor during the adsorption process. In addition, FT-IR confirmed that the protein was adsorbed on the nano-Mg(OH) 2 , zeta potential analysis suggested that insecticidal protein was loaded onto the nano-Mg(OH) 2 not by electrostatic adsorption but maybe by intermolecular forces, and circular dichroism spectroscopy of Cry11Aa protein before and after loading with nano-Mg(OH) 2 was changed. The study applied the adsorption information between Cry11Aa and nano-Mg(OH) 2 , which would be useful in the practical application of nano-Mg(OH) 2 as a nano-carrier.

  12. A high-throughput 2D-analytical technique to obtain single protein parameters from complex cell lysates for in silico process development of ion exchange chromatography.

    Science.gov (United States)

    Kröner, Frieder; Elsäßer, Dennis; Hubbuch, Jürgen

    2013-11-29

    The accelerating growth of the market for biopharmaceutical proteins, the market entry of biosimilars and the growing interest in new, more complex molecules constantly pose new challenges for bioseparation process development. In the presented work we demonstrate the application of a multidimensional, analytical separation approach to obtain the relevant physicochemical parameters of single proteins in a complex mixture for in silico chromatographic process development. A complete cell lysate containing a low titre target protein was first fractionated by multiple linear salt gradient anion exchange chromatography (AEC) with varying gradient length. The collected fractions were subsequently analysed by high-throughput capillary gel electrophoresis (HT-CGE) after being desalted and concentrated. From the obtained data of the 2D-separation the retention-volumes and the concentration of the single proteins were determined. The retention-volumes of the single proteins were used to calculate the related steric-mass action model parameters. In a final evaluation experiment the received parameters were successfully applied to predict the retention behaviour of the single proteins in salt gradient AEC. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Thermoplastic processing of proteins for film formation--a review.

    Science.gov (United States)

    Hernandez-Izquierdo, V M; Krochta, J M

    2008-03-01

    Increasing interest in high-quality food products with increased shelf life and reduced environmental impact has encouraged the study and development of edible and/or biodegradable polymer films and coatings. Edible films provide the opportunity to effectively control mass transfer among different components in a food or between the food and its surrounding environment. The diversity of proteins that results from an almost limitless number of side-chain amino-acid sequential arrangements allows for a wide range of interactions and chemical reactions to take place as proteins denature and cross-link during heat processing. Proteins such as wheat gluten, corn zein, soy protein, myofibrillar proteins, and whey proteins have been successfully formed into films using thermoplastic processes such as compression molding and extrusion. Thermoplastic processing can result in a highly efficient manufacturing method with commercial potential for large-scale production of edible films due to the low moisture levels, high temperatures, and short times used. Addition of water, glycerol, sorbitol, sucrose, and other plasticizers allows the proteins to undergo the glass transition and facilitates deformation and processability without thermal degradation. Target film variables, important in predicting biopackage performance under various conditions, include mechanical, thermal, barrier, and microstructural properties. Comparisons of film properties should be made with care since results depend on parameters such as film-forming materials, film formulation, fabrication method, operating conditions, testing equipment, and testing conditions. Film applications include their use as wraps, pouches, bags, casings, and sachets to protect foods, reduce waste, and improve package recyclability.

  14. Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein.

    Directory of Open Access Journals (Sweden)

    Benjamin Dälken

    Full Text Available BACKGROUND: The apoptosis-inducing serine protease granzyme B (GrB is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. METHODS AND FINDINGS: We investigated the influence of bacterial maltose-binding protein (MBP fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal α-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. CONCLUSIONS: Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins.

  15. Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein

    Science.gov (United States)

    Dälken, Benjamin; Jabulowsky, Robert A.; Oberoi, Pranav; Benhar, Itai; Wels, Winfried S.

    2010-01-01

    Background The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. Methods and Findings We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal α-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. Conclusions Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins. PMID:21203542

  16. The Autism Related Protein Contactin-Associated Protein-Like 2 (CNTNAP2 Stabilizes New Spines: An In Vivo Mouse Study.

    Directory of Open Access Journals (Sweden)

    Amos Gdalyahu

    Full Text Available The establishment and maintenance of neuronal circuits depends on tight regulation of synaptic contacts. We hypothesized that CNTNAP2, a protein associated with autism, would play a key role in this process. Indeed, we found that new dendritic spines in mice lacking CNTNAP2 were formed at normal rates, but failed to stabilize. Notably, rates of spine elimination were unaltered, suggesting a specific role for CNTNAP2 in stabilizing new synaptic circuitry.

  17. Gardenia jasminoides Encodes an Inhibitor-2 Protein for Protein Phosphatase Type 1

    Science.gov (United States)

    Gao, Lan; Li, Hao-Ming

    2017-08-01

    Protein phosphatase-1 (PP1) regulates diverse, essential cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling. Inhibitor-2 (I-2) can inhibit the activity of PP1 and has been found in diverse organisms. In this work, a Gardenia jasminoides fruit cDNA library was constructed, and the GjI-2 cDNA was isolated from the cDNA library by sequencing method. The GjI-2 cDNA contains a predicted 543 bp open reading frame that encodes 180 amino acids. The bioinformatics analysis suggested that the GjI-2 has conserved PP1c binding motif, and contains a conserved phosphorylation site, which is important in regulation of its activity. The three-dimensional model structure of GjI-2 was buite, its similar with the structure of I-2 from mouse. The results suggest that GjI-2 has relatively conserved RVxF, FxxR/KxR/K and HYNE motif, and these motifs are involved in interaction with PP1.

  18. A Proteomic Approach for the Identification of Up-Regulated Proteins Involved in the Metabolic Process of the Leiomyoma.

    Science.gov (United States)

    Ura, Blendi; Scrimin, Federica; Arrigoni, Giorgio; Franchin, Cinzia; Monasta, Lorenzo; Ricci, Giuseppe

    2016-04-09

    Uterine leiomyoma is the most common benign smooth muscle cell tumor of the uterus. Proteomics is a powerful tool for the analysis of complex mixtures of proteins. In our study, we focused on proteins that were upregulated in the leiomyoma compared to the myometrium. Paired samples of eight leiomyomas and adjacent myometrium were obtained and submitted to two-dimensional gel electrophoresis (2-DE) and mass spectrometry for protein identification and to Western blotting for 2-DE data validation. The comparison between the patterns revealed 24 significantly upregulated (p leiomyoma and not with the normal myometrium. The overexpression of seven proteins involved in the metabolic processes of the leiomyoma was further validated by Western blotting and 2D Western blotting. Four of these proteins have never been associated with the leiomyoma before. The 2-DE approach coupled with mass spectrometry, which is among the methods of choice for comparative proteomic studies, identified a number of proteins overexpressed in the leiomyoma and involved in several biological processes, including metabolic processes. A better understanding of the mechanism underlying the overexpression of these proteins may be important for therapeutic purposes.

  19. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Ková cs, Krisztiá n A.; Steinmann, Myriam; Halfon, Olivier; Magistretti, Pierre J.; Cardinaux, Jean René

    2015-01-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  20. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Kovács, Krisztián A.

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  1. Reduction of FFA in jatropha curcas oil via sequential direct-ultrasonic irradiation and dosage of methanol/sulfuric acid catalyst mixture on esterification process

    International Nuclear Information System (INIS)

    Andrade-Tacca, Cesar Augusto; Chang, Chia-Chi; Chen, Yi-Hung; Ji, Dar-Ren; Wang, Yi-Yu; Yen, Yue-Quen; Chang, Ching-Yuan

    2014-01-01

    Highlights: • Ultrasonic irradiation (UI) can auto-induce temperature rise. • Esterification at higher temperature (T) by UI offers greater reduction of acid value. • Sequential UI and catalyst dosing enhance esterification conversion efficiency (η). • UR of jatropha oil at higher T results in less water content on ester as product. • A 99.35% of η is achievable via sequential UI and dosing of 5 mL per dose. - Abstract: Production of jatropha-ester (JO-ester) from jatropha oil (JO) under sequential direct-ultrasonic irradiation (UI) with auto-induced temperature rise followed by adding a mixture of methanol/sulfuric-acid catalyst (M/C) dose between high temperature intervals was studied. Comparisons with various doses of 5, 10, 16.6 and 25 mL at different temperature intervals of 108.9–120 °C, 100–120 °C, 85–120 °C and 75–120 °C respectively were performed. System parameters examined include: esterification times (t E ) for UI, settling time (t S ) after esterification and temperature (T). Properties of acid value (AV), iodine value (IV), kinematic viscosity (kV), density (ρ LO ) and water content (m w ) of JO and JO-ester product were measured. The esterification conversion efficiencies (η) were determined and assessed. An η of 99.35% was obtained at temperature interval of 108.9–120 °C with 5 mL per dose for 20 doses and t E of 167.39 min (denoted as Process U 120-5 ), which is slightly higher than η of 98.87% at temperature interval of 75–120 °C with 25 mL per dose for 4 doses and t E of 108.79 min (noted as Process U 120-25 ). The JO-ester obtained via sequential UI with adding doses of 5 mL possess AV of 0.24 mg KOH/g, IV of 124.77 g I 2 /100 g, kV of 9.89 mm 2 /s, ρ LO of 901.73 kg/m 3 and m w of 0.3 wt.% showing that sequential UI and dose at higher temperature interval can give higher reduction of AV compared with 36.56 mg KOH/g of original oil. The effects of t S and t E on AV are of minor and moderate importance

  2. Luminescence, virulence and quorum sensing signal production by pathogenic Vibrio campbellii and Vibrio harveyi isolates.

    Science.gov (United States)

    Defoirdt, T; Verstraete, W; Bossier, P

    2008-05-01

    To study the relationship between luminescence, autoinducer production and virulence of pathogenic vibrios. Luminescence, quorum sensing signal production and virulence towards brine shrimp nauplii of 13 Vibrio campbellii and Vibrio harveyi strains were studied. Although only two of the tested strains were brightly luminescent, all of them were shown to produce the three different types of quorum sensing signals known to be produced by Vibrio harveyi. Cell-free culture fluids of all strains significantly induced bioluminescence in the cholerae autoinducer 1, autoinducer 2 and harveyi autoinducer 1 reporter strains JAF375, JMH597 and JMH612, respectively. There was no relation between luminescence and signal production and virulence towards brine shrimp. There is a large difference between different strains of Vibrio campbellii and Vibrio harveyi with respect to bioluminescence. However, this is not reflected in signal production and virulence towards gnotobiotic brine shrimp. Moreover, there seems to be no relation between quorum sensing signal production and virulence towards brine shrimp. The results presented here indicate that strains that are most brightly luminescent are not necessarily the most virulent ones and that the lower virulence of some of the strains is not due to a lack of autoinducer production.

  3. Effect of Wnt-1 inducible signaling pathway protein-2 (WISP-2/CCN5), a downstream protein of Wnt signaling, on adipocyte differentiation

    International Nuclear Information System (INIS)

    Inadera, Hidekuni; Shimomura, Akiko; Tachibana, Shinjiro

    2009-01-01

    Wnt signaling negatively regulates adipocyte differentiation, and ectopic expression of Wnt-1 in 3T3-L1 cells induces several downstream molecules of Wnt signaling, including Wnt-1 inducible signaling pathway protein (WISP)-2. In this study, we examined the role of WISP-2 in the process of adipocyte differentiation using an in vitro cell culture system. In the differentiation of 3T3-L1 cells, WISP-2 expression was observed in growing cells and declined thereafter. In the mitotic clonal expansion phase of adipocyte differentiation, WISP-2 expression was transiently down-regulated concurrently with up-regulation of CCAAT/enhancer-binding protein δ expression. Treatment of 3T3-L1 cells in the differentiation medium with lithium, an activator of Wnt signaling, inhibited the differentiation process with concomitant induction of WISP-2. Treatment of differentiated cells with lithium induced de-differentiation as evidenced by profound reduction of peroxisome proliferator-activator receptor γ expression and concomitant induction of WISP-2. However, de-differentiation of differentiated cells induced by tumor necrosis factor-α did not induce WISP-2 expression. To directly examine the effect of WISP-2 on adipocyte differentiation, 3T3-L1 cells were infected with a retrovirus carrying WISP-2. Although forced expression of WISP-2 inhibited preadipocyte proliferation, it had no effect on adipocyte differentiation. Thus, although WISP-2 is a downstream protein of Wnt signaling, the role of WISP-2 on adipocyte differentiation may be marginal, at least in this in vitro culture model.

  4. Quorum sensing signaling molecules produced by reference and emerging soft-rot bacteria (Dickeya and Pectobacterium spp..

    Directory of Open Access Journals (Sweden)

    Alexandre Crépin

    Full Text Available Several small diffusible molecules are involved in bacterial quorum sensing and virulence. The production of autoinducers-1 and -2, quinolone, indole and γ-amino butyrate signaling molecules was investigated in a set of soft-rot bacteria belonging to six Dickeya or Pectobacterium species including recent or emerging potato isolates.Using bacterial biosensors, immunoassay, and chromatographic analysis, we showed that soft-rot bacteria have the common ability to produce transiently during their exponential phase of growth the N-3-oxo-hexanoyl- or the N-3-oxo-octanoyl-l-homoserine lactones and a molecule of the autoinducer-2 family. Dickeya spp. produced in addition the indole-3-acetic acid in tryptophan-rich conditions. All these signaling molecules have been identified for the first time in the novel Dickeya solani species. In contrast, quinolone and γ-amino butyrate signals were not identified and the corresponding synthases are not present in the available genomes of soft-rot bacteria. To determine if the variations of signal production according to growth phase could result from expression modifications of the corresponding synthase gene, the respective mRNA levels were estimated by reverse transcriptase-PCR. While the N-acyl-homoserine lactone production is systematically correlated to the synthase expression, that of the autoinducer-2 follows the expression of an enzyme upstream in the activated methyl cycle and providing its precursor, rather than the expression of its own synthase.Despite sharing the S-adenosylmethionine precursor, no strong link was detected between the production kinetics or metabolic pathways of autoinducers-1 and -2. In contrast, the signaling pathway of autoinducer-2 seems to be switched off by the indole-3-acetic acid pathway under tryptophan control. It therefore appears that the two genera of soft-rot bacteria have similarities but also differences in the mechanisms of communication via the diffusible molecules

  5. Quorum Sensing Signaling Molecules Produced by Reference and Emerging Soft-Rot Bacteria (Dickeya and Pectobacterium spp.)

    Science.gov (United States)

    Crépin, Alexandre; Barbey, Corinne; Beury-Cirou, Amélie; Hélias, Valérie; Taupin, Laure; Reverchon, Sylvie; Nasser, William; Faure, Denis; Dufour, Alain; Orange, Nicole; Feuilloley, Marc; Heurlier, Karin; Burini, Jean-François; Latour, Xavier

    2012-01-01

    Background Several small diffusible molecules are involved in bacterial quorum sensing and virulence. The production of autoinducers-1 and -2, quinolone, indole and γ-amino butyrate signaling molecules was investigated in a set of soft-rot bacteria belonging to six Dickeya or Pectobacterium species including recent or emerging potato isolates. Methodology/Principal Findings Using bacterial biosensors, immunoassay, and chromatographic analysis, we showed that soft-rot bacteria have the common ability to produce transiently during their exponential phase of growth the N-3-oxo-hexanoyl- or the N-3-oxo-octanoyl-l-homoserine lactones and a molecule of the autoinducer-2 family. Dickeya spp. produced in addition the indole-3-acetic acid in tryptophan-rich conditions. All these signaling molecules have been identified for the first time in the novel Dickeya solani species. In contrast, quinolone and γ-amino butyrate signals were not identified and the corresponding synthases are not present in the available genomes of soft-rot bacteria. To determine if the variations of signal production according to growth phase could result from expression modifications of the corresponding synthase gene, the respective mRNA levels were estimated by reverse transcriptase-PCR. While the N-acyl-homoserine lactone production is systematically correlated to the synthase expression, that of the autoinducer-2 follows the expression of an enzyme upstream in the activated methyl cycle and providing its precursor, rather than the expression of its own synthase. Conclusions/Significance Despite sharing the S-adenosylmethionine precursor, no strong link was detected between the production kinetics or metabolic pathways of autoinducers-1 and -2. In contrast, the signaling pathway of autoinducer-2 seems to be switched off by the indole-3-acetic acid pathway under tryptophan control. It therefore appears that the two genera of soft-rot bacteria have similarities but also differences in the

  6. Protein phosphatase 2A dysfunction in Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    Jean-Marie eSontag

    2014-03-01

    Full Text Available Protein Phosphatase 2A (PP2A is a large family of enzymes that account for the majority of brain Ser/Thr phosphatase activity. While PP2A enzymes collectively modulate most cellular processes, sophisticated regulatory mechanisms are ultimately responsible for ensuring isoform-specific substrate specificity. Of particular interest to the Alzheimer’s disease (AD field, alterations in PP2A regulators and PP2A catalytic activity, subunit expression, methylation and/or phosphorylation, have been reported in AD-affected brain regions. PP2A dysfunction has been linked to Tau hyperphosphorylation, amyloidogenesis and synaptic deficits that are pathological hallmarks of this neurodegenerative disorder. Deregulation of PP2A enzymes also affects the activity of many Ser/Thr protein kinases implicated in AD. This review will more specifically discuss the role of the PP2A/B holoenzyme and PP2A methylation in AD pathogenesis. The PP2A/B isoform binds to tau and is the primary tau phosphatase. Its deregulation correlates with increased tau phosphorylation in vivo and in AD. Disruption of PP2A/B-Tau protein interactions likely contribute to Tau deregulation in AD. Significantly, alterations in one-carbon metabolism that impair PP2A methylation are associated with increased risk for sporadic AD, and enhanced AD-like pathology in animal models. Experimental studies have linked deregulation of PP2A methylation with down-regulation of PP2A/B, enhanced phosphorylation of Tau and amyloid precursor protein, Tau mislocalization, microtubule destabilization and neuritic defects. While it remains unclear what are the primary events that underlie PP2A dysfunction in AD, deregulation of PP2A enzymes definitely affects key players in the pathogenic process. As such, there is growing interest in developing PP2A-centric therapies for AD, but this may be a daunting task without a better understanding of the regulation and function of specific PP2A enzymes.

  7. Amyloid Precursor Proteins Are Dynamically Trafficked and Processed During Neuronal Development

    Directory of Open Access Journals (Sweden)

    Jenna M. Ramaker

    2016-11-01

    Full Text Available Proteolytic processing of the Amyloid Precursor Protein (APP produces beta-amyloid (Aβ peptide fragments that accumulate in Alzheimer’s Disease (AD, but APP may also regulate multiple aspects of neuronal development, albeit via mechanisms that are not well understood. APP is a member of a family of transmembrane glycoproteins expressed by all higher organisms, including two mammalian orthologs (APLP1 and APLP2 that have complicated investigations into the specific activities of APP. By comparison, insects express only a single APP-related protein (APP-Like, or APPL that contains the same protein interaction domains identified in APP. However, unlike its mammalian orthologs, APPL is only expressed by neurons, greatly simplifying an analysis of its functions in vivo. Like APP, APPL is processed by secretases to generate a similar array of extracellular and intracellular cleavage fragments, as well as an Aβ-like fragment that can induce neurotoxic responses in the brain. Exploiting the complementary advantages of two insect models (Drosophila melanogaster and Manduca sexta, we have investigated the regulation of APPL trafficking and processing with respect to different aspects of neuronal development. By comparing the behavior of endogenously expressed APPL with fluorescently tagged versions of APPL and APP, we have shown that some full-length protein is consistently trafficked into the most motile regions of developing neurons both in vitro and in vivo. Concurrently, much of the holoprotein is rapidly processed into N- and C-terminal fragments that undergo bi-directional transport within distinct vesicle populations. Unexpectedly, we also discovered that APPL can be transiently sequestered into an amphisome-like compartment in developing neurons, while manipulations targeting APPL cleavage altered their motile behavior in cultured embryos. These data suggest that multiple mechanisms restrict the bioavailability of the holoprotein to regulate

  8. Gold nanoparticles enhance the X-ray-induced degradation of human centrin 2 protein

    International Nuclear Information System (INIS)

    Brun, Emilie; Duchambon, Patricia; Blouquit, Yves; Keller, Gerard; Sanche, Leon; Sicard-Roselli, Cecile

    2009-01-01

    In the war against cancer, radiotherapy is a prominent tool but counterbalanced by the fact that it also induces damages in healthy tissues. Nanotechnologies could open a new possibility to decrease these side effects. In particular, gold nanoparticles (GNPs) could be used as radio-sensitizers. As the role of proteins in the processes leading to cell death cannot be neglected, their radio-sensitization by GNPs is of great interest. This is particularly true in the case of the human centrin 2 protein, which has been proposed to be involved in DNA repair processes. To investigate this effect, we quantified for the first time the degradation of this protein in a gold colloidal solution when submitted to X-rays. We showed that the X-ray-induced degradation of the human centrin 2 protein is enhanced 1.5-fold in the presence of GNPs, even though no covalent bond exists between protein and GNPs. Among the conditions tested, the maximum enhancement was found with the higher GNP:protein ratio of 2x10 -4 and with the higher X-ray energy of 49 keV

  9. Synthesis and characterization of novel 2, 2'-bipyrimidine fluorescent derivative for protein binding

    Directory of Open Access Journals (Sweden)

    Padalkar Vikas S

    2011-11-01

    Full Text Available Abstract Background Fluorescent dyes with biocompatible functional group and good fluorescence behavior are used as biosensor for monitoring different biological processes as well as detection of protein assay. All reported fluorophore used as sensors are having high selectivity and sensitivity but till there is more demand to synthesized new fluorophore which have improved fluorescence properties and good biocompatibility. Results Novel 4, 4'-(1, 1'-(5-(2-methoxyphenoxy-[2, 2'-bipyrimidine]-4, 6-diylbis(1H-pyrazol-3, 1-diyl dianiline fluorescent dye was synthesized by multistep synthesis from 2-phenylacetonitrile, 2-chloropyrimidine and 2-methoxyphenol. This dye has absorption at 379 nm with intense single emission at 497 nm having fairly good quantum yield (0.375 and Stokes shift. The intermediates and dye were characterized by FT-IR, 1H NMR, 13C NMR and Mass spectral analysis. The pyrazole bipyrimidine based fluorescent dye possessing two amino groups suitable for binding with protein is reported. Its utility as a biocompatible conjugate was explained by conjugation with bovine serum albumin. The method is based on direct fluorescence detection of fluorophore-labelled protein before and after conjugation. Purified fluorescent conjugate was subsequently analyzed by fluorimetry. The analysis showed that the tested conjugation reaction yielded fluorescent conjugates of the dye through carbodiimide chemistry. Conclusion In summery synthesized fluorophore pyrazole-bipyrimidine has very good interaction towards protein bovine serum albumin and it acts as good candidate for protein assay.

  10. Co-operative intra-protein structural response due to protein-protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding.

    Science.gov (United States)

    Samanta, Sudipta; Mukherjee, Sanchita

    2017-10-01

    The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.

  11. Co-operative intra-protein structural response due to protein-protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding

    Science.gov (United States)

    Samanta, Sudipta; Mukherjee, Sanchita

    2017-10-01

    The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.

  12. Protein: MPB2 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPB2 Ubiquitin ligases WWP1 WWP1 NEDD4-like E3 ubiquitin-protein ligase WWP1 Atrophin-1-interacting pr...otein 5, WW domain-containing protein 1 9606 Homo sapiens Q9H0M0 11059 2OP7, 1ND7 11059 ...

  13. SH2/SH3 signaling proteins.

    Science.gov (United States)

    Schlessinger, J

    1994-02-01

    SH2 and SH3 domains are small protein modules that mediate protein-protein interactions in signal transduction pathways that are activated by protein tyrosine kinases. SH2 domains bind to short phosphotyrosine-containing sequences in growth factor receptors and other phosphoproteins. SH3 domains bind to target proteins through sequences containing proline and hydrophobic amino acids. SH2 and SH3 domain containing proteins, such as Grb2 and phospholipase C gamma, utilize these modules in order to link receptor and cytoplasmic protein tyrosine kinases to the Ras signaling pathway and to phosphatidylinositol hydrolysis, respectively. The three-dimensional structures of several SH2 and SH3 domains have been determined by NMR and X-ray crystallography, and the molecular basis of their specificity is beginning to be unveiled.

  14. Fractionation of whey protein isolate with supercritical carbon dioxide – process modeling and cost estimation

    Science.gov (United States)

    An economical and environmentally friendly whey protein fractionation process was developed using supercritical carbon dioxide (sCO2) as an acid to produce enriched fractions of alpha-lactalbumin (alpha-La) and beta-lactoglobulin (beta-Lg) from a commercial whey protein isolate (WPI) containing 55% ...

  15. IQGAP1 is a novel CXCR2-interacting protein and essential component of the "chemosynapse".

    Directory of Open Access Journals (Sweden)

    Nicole F Neel

    Full Text Available Chemotaxis is essential for a number of physiological processes including leukocyte recruitment. Chemokines initiate intracellular signaling pathways necessary for chemotaxis through binding seven transmembrane G protein-couple receptors. Little is known about the proteins that interact with the intracellular domains of chemokine receptors to initiate cellular signaling upon ligand binding. CXCR2 is a major chemokine receptor expressed on several cell types, including endothelial cells and neutrophils. We hypothesize that multiple proteins interact with the intracellular domains of CXCR2 upon ligand stimulation and these interactions comprise a "chemosynapse", and play important roles in transducing CXCR2 mediated signaling processes.In an effort to define the complex of proteins that assemble upon CXCR2 activation to relay signals from activated chemokine receptors, a proteomics approach was employed to identify proteins that co-associate with CXCR2 with or without ligand stimulation. The components of the CXCR2 "chemosynapse" are involved in processes ranging from intracellular trafficking to cytoskeletal modification. IQ motif containing GTPase activating protein 1 (IQGAP1 was among the novel proteins identified to interact directly with CXCR2. Herein, we demonstrate that CXCR2 co-localizes with IQGAP1 at the leading edge of polarized human neutrophils and CXCR2 expressing differentiated HL-60 cells. Moreover, amino acids 1-160 of IQGAP1 directly interact with the carboxyl-terminal domain of CXCR2 and stimulation with CXCL8 enhances IQGAP1 association with Cdc42.Our studies indicate that IQGAP1 is a novel essential component of the CXCR2 "chemosynapse".

  16. Protein-protein docking with F(2Dock 2.0 and GB-rerank.

    Directory of Open Access Journals (Sweden)

    Rezaul Chowdhury

    Full Text Available Computational simulation of protein-protein docking can expedite the process of molecular modeling and drug discovery. This paper reports on our new F(2 Dock protocol which improves the state of the art in initial stage rigid body exhaustive docking search, scoring and ranking by introducing improvements in the shape-complementarity and electrostatics affinity functions, a new knowledge-based interface propensity term with FFT formulation, a set of novel knowledge-based filters and finally a solvation energy (GBSA based reranking technique. Our algorithms are based on highly efficient data structures including the dynamic packing grids and octrees which significantly speed up the computations and also provide guaranteed bounds on approximation error.The improved affinity functions show superior performance compared to their traditional counterparts in finding correct docking poses at higher ranks. We found that the new filters and the GBSA based reranking individually and in combination significantly improve the accuracy of docking predictions with only minor increase in computation time. We compared F(2 Dock 2.0 with ZDock 3.0.2 and found improvements over it, specifically among 176 complexes in ZLab Benchmark 4.0, F(2 Dock 2.0 finds a near-native solution as the top prediction for 22 complexes; where ZDock 3.0.2 does so for 13 complexes. F(2 Dock 2.0 finds a near-native solution within the top 1000 predictions for 106 complexes as opposed to 104 complexes for ZDock 3.0.2. However, there are 17 and 15 complexes where F(2 Dock 2.0 finds a solution but ZDock 3.0.2 does not and vice versa; which indicates that the two docking protocols can also complement each other.The docking protocol has been implemented as a server with a graphical client (TexMol which allows the user to manage multiple docking jobs, and visualize the docked poses and interfaces. Both the server and client are available for download. Server: http

  17. Protein-Protein Docking with F2Dock 2.0 and GB-Rerank

    Science.gov (United States)

    Chowdhury, Rezaul; Rasheed, Muhibur; Keidel, Donald; Moussalem, Maysam; Olson, Arthur; Sanner, Michel; Bajaj, Chandrajit

    2013-01-01

    Motivation Computational simulation of protein-protein docking can expedite the process of molecular modeling and drug discovery. This paper reports on our new F2 Dock protocol which improves the state of the art in initial stage rigid body exhaustive docking search, scoring and ranking by introducing improvements in the shape-complementarity and electrostatics affinity functions, a new knowledge-based interface propensity term with FFT formulation, a set of novel knowledge-based filters and finally a solvation energy (GBSA) based reranking technique. Our algorithms are based on highly efficient data structures including the dynamic packing grids and octrees which significantly speed up the computations and also provide guaranteed bounds on approximation error. Results The improved affinity functions show superior performance compared to their traditional counterparts in finding correct docking poses at higher ranks. We found that the new filters and the GBSA based reranking individually and in combination significantly improve the accuracy of docking predictions with only minor increase in computation time. We compared F2 Dock 2.0 with ZDock 3.0.2 and found improvements over it, specifically among 176 complexes in ZLab Benchmark 4.0, F2 Dock 2.0 finds a near-native solution as the top prediction for 22 complexes; where ZDock 3.0.2 does so for 13 complexes. F2 Dock 2.0 finds a near-native solution within the top 1000 predictions for 106 complexes as opposed to 104 complexes for ZDock 3.0.2. However, there are 17 and 15 complexes where F2 Dock 2.0 finds a solution but ZDock 3.0.2 does not and vice versa; which indicates that the two docking protocols can also complement each other. Availability The docking protocol has been implemented as a server with a graphical client (TexMol) which allows the user to manage multiple docking jobs, and visualize the docked poses and interfaces. Both the server and client are available for download. Server: http

  18. A Review on Structures and Functions of Bcl-2 Family Proteins from Homo sapiens.

    Science.gov (United States)

    Sivakumar, Dakshinamurthy; Sivaraman, Thirunavukkarasu

    2016-01-01

    Cancer cells evade apoptosis, which is regulated by proteins of Bcl-2 family in the intrinsic pathways. Numerous experimental three-dimensional (3D) structures of the apoptotic proteins and the proteins bound with small chemical molecules/peptides/proteins have been reported in the literature. In this review article, the 3D structures of the Bcl-2 family proteins from Homo sapiens and as well complex structures of the anti-apoptotic proteins bound with small molecular inhibitors reported in the literature to date have been comprehensively listed out and described in detail. Moreover, the molecular mechanisms by which the Bcl-2 family proteins modulate the apoptotic processes and strategies for designing antagonists to anti-apoptotic proteins have been concisely discussed.

  19. Glucohexaose-induced protein phosphatase 2C regulates cell redox ...

    Indian Academy of Sciences (India)

    Q M Chen

    2018-02-13

    Feb 13, 2018 ... glucohexaose, CsPP2C80s play a positive regulatory role in process of ABA combined with ABA receptors ..... protein kinases (SnRKs) involve in the stress responses .... In this work, the endogenous ABA content increased.

  20. Scalable organic solvent free supercritical fluid spray drying process for producing dry protein formulations.

    Science.gov (United States)

    Nuchuchua, O; Every, H A; Hofland, G W; Jiskoot, W

    2014-11-01

    In this study, we evaluated the influence of supercritical carbon dioxide (scCO2) spray drying conditions, in the absence of organic solvent, on the ability to produce dry protein/trehalose formulations at 1:10 and 1:4 (w/w) ratios. When using a 4L drying vessel, we found that decreasing the solution flow rate and solution volume, or increasing the scCO2 flow rate resulted in a significant reduction in the residual water content in dried products (Karl Fischer titration). The best conditions were then used to evaluate the ability to scale the scCO2 spray drying process from 4L to 10L chamber. The ratio of scCO2 and solution flow rate was kept constant. The products on both scales exhibited similar residual moisture contents, particle morphologies (SEM), and glass transition temperatures (DSC). After reconstitution, the lysozyme activity (enzymatic assay) and structure (circular dichroism, HP-SEC) were fully preserved, but the sub-visible particle content was slightly increased (flow imaging microscopy, nanoparticle tracking analysis). Furthermore, the drying condition was applicable to other proteins resulting in products of similar quality as the lysozyme formulations. In conclusion, we established scCO2 spray drying processing conditions for protein formulations without an organic solvent that holds promise for the industrial production of dry protein formulations. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. AI-2 signalling is induced by acidic shock in probiotic strains of Lactobacillus spp

    DEFF Research Database (Denmark)

    Moslehi Jenabian, Saloomeh; Gori, Klaus; Jespersen, Lene

    2009-01-01

    Survival and ability to respond to various environmental stresses such as low pH are important factors for lactobacilli for their function as probiotics. LuxS-mediated quorum sensing mechanism, which is based on the production of universal signal molecule called autoinducer-2 (AI-2), regulates...... important physiological traits and a variety of adaptive processes in different bacteria. The aim of this study was to investigate the effect of acidic stress on LuxS-mediated quorum sensing (AI-2 signalling) in four probiotic strains of different Lactobacillus species. Initially, the production of AI-2...... concomitant with the reduction of pH, reaching maximum at late exponential phase (L. rhamnosus GG) or at stationary phase (L. salivarius UCC118, L. acidophilus NCFM and L. johnsonii NCC533). Acidic shock experiments were conducted on L. rhamnosus GG and L. acidophilus NCFM after exposure to different acidic...

  2. High pressure processing of meat: effects on ultrastructure and protein digestibility.

    Science.gov (United States)

    Kaur, Lovedeep; Astruc, Thierry; Vénien, Annie; Loison, Olivier; Cui, Jian; Irastorza, Marion; Boland, Mike

    2016-05-18

    The effects of high pressure processing (HPP, at 175 and 600 MPa) on the ultrastructure and in vitro protein digestion of bovine longissimus dorsi muscle meat were studied. HPP caused a significant change in the visual appearance and texture of the meat subjected to HPP at 600 MPa so that it appeared similar to cooked meat, unlike the meat subjected to HPP at 175 MPa that showed no significant visible change in the colour and texture compared to the raw meat. The muscles were subjected to digestion under simulated gastric conditions for 1 h and then under simulated small-intestinal conditions for a further 2 h. The digests were analysed using gel electrophoresis (SDS-PAGE) and ninhydrin assay for amino N. The effect of the acid conditions of the stomach alone was also investigated. Reduced SDS-PAGE results showed that pepsin-digested (60 min) HPP meats showed fewer proteins or peptides of high molecular weight than the pepsin-digested untreated meat, suggesting more breakdown of the parent proteins in HPP-treated meats. This effect was more pronounced in the muscles treated at 600 MPa. These results are in accordance with microscopy results, which showed greater changes in the myofibrillar structure after simulated gastric digestion of the sample processed at 600 MPa than at 175 MPa. Transmission electron microscopy also showed the presence of protein aggregates in the former sample, resulting probably from protein denaturation of sarcoplasmic proteins, in the subcellular space and between myofibrils; along with cell contraction (similar to that caused by heating) in the former.

  3. Integrating protein engineering with process design for biocatalysis

    DEFF Research Database (Denmark)

    Woodley, John M.

    2017-01-01

    Biocatalysis uses enzymes for chemical synthesis and production, offering selective, safe and sustainable catalysis. While today the majority of applications are in the pharmaceutical sector, new opportunities are arising every day in other industry sectors, where production costs become a more...... important driver. In the early applications of the technology, it was necessary to design processes to match the properties of the biocatalyst. With the advent of protein engineering, organic chemists started to develop and improve enzymes to suit their needs. Likewise in industry, although not widespread......, a new paradigm was already implemented several years ago to engineer enzymes to suit process needs. Today, a new era is entered, where the effectiveness with which such integrated protein and process engineering is achieved becomes critical to implementation. In this paper, the development of a tool...

  4. Identification of ZASP, a novel protein associated to Zona occludens-2

    Energy Technology Data Exchange (ETDEWEB)

    Lechuga, Susana; Alarcon, Lourdes; Solano, Jesus [Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (Cinvestav), Mexico, D.F. 07360 (Mexico); Huerta, Miriam; Lopez-Bayghen, Esther [Department of Genetics and Molecular Biology, Center for Research and Advanced Studies (Cinvestav), Mexico, D.F. 07360 (Mexico); Gonzalez-Mariscal, Lorenza, E-mail: lorenza@fisio.cinvestav.mx [Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (Cinvestav), Mexico, D.F. 07360 (Mexico)

    2010-11-15

    With the aim of discovering new molecular interactions of the tight junction protein ZO-2, a two-hybrid screen was performed on a human kidney cDNA library using as bait the middle segment of ZO-2. Through this assay we identified a 24-kDa novel protein herein named ZASP for ZO-2 associated speckle protein. ZO-2/ZASP interaction further confirmed by pull down and immunoprecipitation experiments, requires the presence of the intact PDZ binding motif SQV of ZASP and the third PDZ domain of ZO-2. ZASP mRNA and protein are present in the kidney and in several epithelial cell lines. Endogenous ZASP is expressed primarily in nuclear speckles in co-localization with splicing factor SC-35. Nocodazole treatment and wash out reveals that ZASP disappears from the nucleus during mitosis in accordance with speckle disassembly during metaphase. ZASP amino acid sequence exhibits a canonical nuclear exportation signal and in agreement the protein exits the nucleus through a process mediated by exportin/CRM1. ZASP over-expression blocks the inhibitory activity of ZO-2 on cyclin D1 gene transcription and protein expression. The identification of ZASP helps to unfold the complex nuclear molecular arrays that form on ZO-2 scaffolds.

  5. Identification of ZASP, a novel protein associated to Zona occludens-2.

    Science.gov (United States)

    Lechuga, Susana; Alarcón, Lourdes; Solano, Jesús; Huerta, Miriam; Lopez-Bayghen, Esther; González-Mariscal, Lorenza

    2010-11-15

    With the aim of discovering new molecular interactions of the tight junction protein ZO-2, a two-hybrid screen was performed on a human kidney cDNA library using as bait the middle segment of ZO-2. Through this assay we identified a 24-kDa novel protein herein named ZASP for ZO-2 associated speckle protein. ZO-2/ZASP interaction further confirmed by pull down and immunoprecipitation experiments, requires the presence of the intact PDZ binding motif SQV of ZASP and the third PDZ domain of ZO-2. ZASP mRNA and protein are present in the kidney and in several epithelial cell lines. Endogenous ZASP is expressed primarily in nuclear speckles in co-localization with splicing factor SC-35. Nocodazole treatment and wash out reveals that ZASP disappears from the nucleus during mitosis in accordance with speckle disassembly during metaphase. ZASP amino acid sequence exhibits a canonical nuclear exportation signal and in agreement the protein exits the nucleus through a process mediated by exportin/CRM1. ZASP over-expression blocks the inhibitory activity of ZO-2 on cyclin D1 gene transcription and protein expression. The identification of ZASP helps to unfold the complex nuclear molecular arrays that form on ZO-2 scaffolds. Copyright © 2010 Elsevier Inc. All rights reserved.

  6. Identification of ZASP, a novel protein associated to Zona occludens-2

    International Nuclear Information System (INIS)

    Lechuga, Susana; Alarcon, Lourdes; Solano, Jesus; Huerta, Miriam; Lopez-Bayghen, Esther; Gonzalez-Mariscal, Lorenza

    2010-01-01

    With the aim of discovering new molecular interactions of the tight junction protein ZO-2, a two-hybrid screen was performed on a human kidney cDNA library using as bait the middle segment of ZO-2. Through this assay we identified a 24-kDa novel protein herein named ZASP for ZO-2 associated speckle protein. ZO-2/ZASP interaction further confirmed by pull down and immunoprecipitation experiments, requires the presence of the intact PDZ binding motif SQV of ZASP and the third PDZ domain of ZO-2. ZASP mRNA and protein are present in the kidney and in several epithelial cell lines. Endogenous ZASP is expressed primarily in nuclear speckles in co-localization with splicing factor SC-35. Nocodazole treatment and wash out reveals that ZASP disappears from the nucleus during mitosis in accordance with speckle disassembly during metaphase. ZASP amino acid sequence exhibits a canonical nuclear exportation signal and in agreement the protein exits the nucleus through a process mediated by exportin/CRM1. ZASP over-expression blocks the inhibitory activity of ZO-2 on cyclin D1 gene transcription and protein expression. The identification of ZASP helps to unfold the complex nuclear molecular arrays that form on ZO-2 scaffolds.

  7. Gold nanoparticles enhance the X-ray-induced degradation of human centrin 2 protein

    Energy Technology Data Exchange (ETDEWEB)

    Brun, Emilie [Laboratoire de Chimie Physique, CNRS UMR 8000, Universite Paris-Sud 11, Bat. 350, 91405 Orsay Cedex (France); Duchambon, Patricia; Blouquit, Yves [INSERM U759, Imagerie Integrative, Campus Universitaire d' Orsay, Bat. 112, Institut Curie, Centre de Recherche, Laboratoire R. Latarjet, Campus Universitaire d' Orsay, 91405 Orsay Cedex (France); Keller, Gerard [UMR CNRS 8612, Physico-Chimie-Pharmacotechnie-Biopharmacie, Universite Paris 11, Faculte de Pharmacie, 5 rue Jean-Baptiste Clement, 92296 Chatenay-Malabry (France); Sanche, Leon [Groupe en Sciences des Radiations, Departement de Medecine Nucleaire et Radiobiologie, Faculte de Medecine, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4 (Canada); Sicard-Roselli, Cecile [Laboratoire de Chimie Physique, CNRS UMR 8000, Universite Paris-Sud 11, Bat. 350, 91405 Orsay Cedex (France)], E-mail: cecile.sicard@u-psud.fr

    2009-03-15

    In the war against cancer, radiotherapy is a prominent tool but counterbalanced by the fact that it also induces damages in healthy tissues. Nanotechnologies could open a new possibility to decrease these side effects. In particular, gold nanoparticles (GNPs) could be used as radio-sensitizers. As the role of proteins in the processes leading to cell death cannot be neglected, their radio-sensitization by GNPs is of great interest. This is particularly true in the case of the human centrin 2 protein, which has been proposed to be involved in DNA repair processes. To investigate this effect, we quantified for the first time the degradation of this protein in a gold colloidal solution when submitted to X-rays. We showed that the X-ray-induced degradation of the human centrin 2 protein is enhanced 1.5-fold in the presence of GNPs, even though no covalent bond exists between protein and GNPs. Among the conditions tested, the maximum enhancement was found with the higher GNP:protein ratio of 2x10{sup -4} and with the higher X-ray energy of 49 keV.

  8. Depletion of key protein components of the RISC pathway impairs pre-ribosomal RNA processing.

    Science.gov (United States)

    Liang, Xue-Hai; Crooke, Stanley T

    2011-06-01

    Little is known about whether components of the RNA-induced silencing complex (RISC) mediate the biogenesis of RNAs other than miRNA. Here, we show that depletion of key proteins of the RISC pathway by antisense oligonucleotides significantly impairs pre-rRNA processing in human cells. In cells depleted of Drosha or Dicer, different precursors to 5.8S rRNA strongly accumulated, without affecting normal endonucleolytic cleavages. Moderate yet distinct processing defects were also observed in Ago2-depleted cells. Physical links between pre-rRNA and these proteins were identified by co-immunoprecipitation analyses. Interestingly, simultaneous depletion of Dicer and Drosha led to a different processing defect, causing slower production of 28S rRNA and its precursor. Both Dicer and Ago2 were detected in the nuclear fraction, and reduction of Dicer altered the structure of the nucleolus, where pre-rRNA processing occurs. Together, these results suggest that Drosha and Dicer are implicated in rRNA biogenesis.

  9. The cyst nematode SPRYSEC protein RBP-1 elicits Gpa2- and RanGAP2-dependent plant cell death.

    Directory of Open Access Journals (Sweden)

    Melanie Ann Sacco

    2009-08-01

    Full Text Available Plant NB-LRR proteins confer robust protection against microbes and metazoan parasites by recognizing pathogen-derived avirulence (Avr proteins that are delivered to the host cytoplasm. Microbial Avr proteins usually function as virulence factors in compatible interactions; however, little is known about the types of metazoan proteins recognized by NB-LRR proteins and their relationship with virulence. In this report, we demonstrate that the secreted protein RBP-1 from the potato cyst nematode Globodera pallida elicits defense responses, including cell death typical of a hypersensitive response (HR, through the NB-LRR protein Gpa2. Gp-Rbp-1 variants from G. pallida populations both virulent and avirulent to Gpa2 demonstrated a high degree of polymorphism, with positive selection detected at numerous sites. All Gp-RBP-1 protein variants from an avirulent population were recognized by Gpa2, whereas virulent populations possessed Gp-RBP-1 protein variants both recognized and non-recognized by Gpa2. Recognition of Gp-RBP-1 by Gpa2 correlated to a single amino acid polymorphism at position 187 in the Gp-RBP-1 SPRY domain. Gp-RBP-1 expressed from Potato virus X elicited Gpa2-mediated defenses that required Ran GTPase-activating protein 2 (RanGAP2, a protein known to interact with the Gpa2 N terminus. Tethering RanGAP2 and Gp-RBP-1 variants via fusion proteins resulted in an enhancement of Gpa2-mediated responses. However, activation of Gpa2 was still dependent on the recognition specificity conferred by amino acid 187 and the Gpa2 LRR domain. These results suggest a two-tiered process wherein RanGAP2 mediates an initial interaction with pathogen-delivered Gp-RBP-1 proteins but where the Gpa2 LRR determines which of these interactions will be productive.

  10. Genes2Networks: connecting lists of gene symbols using mammalian protein interactions databases

    Directory of Open Access Journals (Sweden)

    Ma'ayan Avi

    2007-10-01

    Full Text Available Abstract Background In recent years, mammalian protein-protein interaction network databases have been developed. The interactions in these databases are either extracted manually from low-throughput experimental biomedical research literature, extracted automatically from literature using techniques such as natural language processing (NLP, generated experimentally using high-throughput methods such as yeast-2-hybrid screens, or interactions are predicted using an assortment of computational approaches. Genes or proteins identified as significantly changing in proteomic experiments, or identified as susceptibility disease genes in genomic studies, can be placed in the context of protein interaction networks in order to assign these genes and proteins to pathways and protein complexes. Results Genes2Networks is a software system that integrates the content of ten mammalian interaction network datasets. Filtering techniques to prune low-confidence interactions were implemented. Genes2Networks is delivered as a web-based service using AJAX. The system can be used to extract relevant subnetworks created from "seed" lists of human Entrez gene symbols. The output includes a dynamic linkable three color web-based network map, with a statistical analysis report that identifies significant intermediate nodes used to connect the seed list. Conclusion Genes2Networks is powerful web-based software that can help experimental biologists to interpret lists of genes and proteins such as those commonly produced through genomic and proteomic experiments, as well as lists of genes and proteins associated with disease processes. This system can be used to find relationships between genes and proteins from seed lists, and predict additional genes or proteins that may play key roles in common pathways or protein complexes.

  11. Small Molecule Inhibitors of AI-2 Signaling in Bacteria: State-of-the-Art and Future Perspectives for Anti-Quorum Sensing Agents

    Science.gov (United States)

    Guo, Min; Gamby, Sonja; Zheng, Yue; Sintim, Herman O.

    2013-01-01

    Bacteria respond to different small molecules that are produced by other neighboring bacteria. These molecules, called autoinducers, are classified as intraspecies (i.e., molecules produced and perceived by the same bacterial species) or interspecies (molecules that are produced and sensed between different bacterial species). AI-2 has been proposed as an interspecies autoinducer and has been shown to regulate different bacterial physiology as well as affect virulence factor production and biofilm formation in some bacteria, including bacteria of clinical relevance. Several groups have embarked on the development of small molecules that could be used to perturb AI-2 signaling in bacteria, with the ultimate goal that these molecules could be used to inhibit bacterial virulence and biofilm formation. Additionally, these molecules have the potential to be used in synthetic biology applications whereby these small molecules are used as inputs to switch on and off AI-2 receptors. In this review, we highlight the state-of-the-art in the development of small molecules that perturb AI-2 signaling in bacteria and offer our perspective on the future development and applications of these classes of molecules. PMID:23994835

  12. Cloning, purification and preliminary crystallographic studies of the 2AB protein from hepatitis A virus

    International Nuclear Information System (INIS)

    Garriga, Damià; Vives-Adrián, Laia; Buxaderas, Mònica; Ferreira-da-Silva, Frederico; Almeida, Bruno; Macedo-Ribeiro, Sandra; Pereira, Pedro José Barbosa; Verdaguer, Núria

    2011-01-01

    The 2AB protein derived from the nonstructural P2 region of hepatitis A virus has been cloned, purified and crystallized. The preliminary characterization of native and selenomethionine-derivative crystals is reported. The Picornaviridae family contains a large number of human pathogens such as rhinovirus, poliovirus and hepatitis A virus (HAV). Hepatitis A is an infectious disease that causes liver inflammation. It is highly endemic in developing countries with poor sanitation, where infections often occur in children. As in other picornaviruses, the genome of HAV contains one open reading frame encoding a single polyprotein that is subsequently processed by viral proteinases to originate mature viral proteins during and after the translation process. In the polyprotein, the N-terminal P1 region generates the four capsid proteins, while the C-terminal P2 and P3 regions contain the enzymes, precursors and accessory proteins essential for polyprotein processing and virus replication. Here, the first crystals of protein 2AB of HAV are reported. The crystals belonged to space group P4 1 or P4 3 , with unit-cell parameters a = b = 90.42, c = 73.43 Å, and contained two molecules in the asymmetric unit. Native and selenomethionine-derivative crystals diffracted to 2.7 and 3.2 Å resolution, respectively

  13. Enhancement of Protein and Pigment Content in Two Chlorella Species Cultivated on Industrial Process Water

    DEFF Research Database (Denmark)

    Safafar, Hamed; Uldall Nørregaard, Patrick; Ljubic, Anita

    2016-01-01

    Chlorella pyrenoidosa and Chlorella vulgaris were cultivated in pre-gasified industrial process water with high concentration of ammonia representing effluent from a local biogas plant. The study aimed to investigate the effects of growth media and cultivation duration on the nutritional...... pyrenoidosa produced the highest concentrations of protein (65.2% ± 1.30% DW) while Chlorella vulgaris accumulated extremely high concentrations of lutein and chlorophylls (7.14 ± 0.66 mg/g DW and 32.4 ± 1.77 mg/g DW, respectively). Cultivation of Chlorella species in industrial process water...... composition of biomass. Variations in proteins, lipid, fatty acid composition, amino acids, tocopherols, and pigments were studied. Both species grew well in industrial process water. The contents of proteins were affected significantly by the growth media and cultivation duration. Microalga Chlorella...

  14. Protein: MPB2 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPB2 Ubiquitin ligases SMURF1 KIAA1625 SMURF1 E3 ubiquitin-protein ligase SMURF1 SM...AD ubiquitination regulatory factor 1, SMAD-specific E3 ubiquitin-protein ligase 1 9606 Homo sapiens Q9HCE7 57154 2LB1, 2LAZ, 2LB0, 3PYC 57154 Q9HCE7 ...

  15. (PS)2: protein structure prediction server version 3.0.

    Science.gov (United States)

    Huang, Tsun-Tsao; Hwang, Jenn-Kang; Chen, Chu-Huang; Chu, Chih-Sheng; Lee, Chi-Wen; Chen, Chih-Chieh

    2015-07-01

    Protein complexes are involved in many biological processes. Examining coupling between subunits of a complex would be useful to understand the molecular basis of protein function. Here, our updated (PS)(2) web server predicts the three-dimensional structures of protein complexes based on comparative modeling; furthermore, this server examines the coupling between subunits of the predicted complex by combining structural and evolutionary considerations. The predicted complex structure could be indicated and visualized by Java-based 3D graphics viewers and the structural and evolutionary profiles are shown and compared chain-by-chain. For each subunit, considerations with or without the packing contribution of other subunits cause the differences in similarities between structural and evolutionary profiles, and these differences imply which form, complex or monomeric, is preferred in the biological condition for the subunit. We believe that the (PS)(2) server would be a useful tool for biologists who are interested not only in the structures of protein complexes but also in the coupling between subunits of the complexes. The (PS)(2) is freely available at http://ps2v3.life.nctu.edu.tw/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Rational and systematic protein purification process development: the next generation.

    Science.gov (United States)

    Nfor, Beckley K; Verhaert, Peter D E M; van der Wielen, Luuk A M; Hubbuch, Jürgen; Ottens, Marcel

    2009-12-01

    Current biopharmaceutical manufacturing strongly relies on using purification platform processes, offering harmonization of practices and speed-to-market. However, the ability of such processes to respond quickly to anticipated higher quality and capacity demands is under question. Here, we describe novel approaches for purification process development that incorporate biothermodynamics, modern high throughput experimentation and simulation tools. Such development leads to production platform-specific databases containing thermodynamic protein descriptors of major host cell proteins over a range of experimental conditions. This will pave the way for in silico purification process development, providing better process understanding and the potential to respond quickly to product quality and market demands. Future efforts will focus on improving this field further and enabling more rationale in process development.

  17. Sequence motif upstream of the Hendra virus fusion protein cleavage site is not sufficient to promote efficient proteolytic processing

    International Nuclear Information System (INIS)

    Craft, Willie Warren; Dutch, Rebecca Ellis

    2005-01-01

    The Hendra virus fusion (HeV F) protein is synthesized as a precursor, F 0 , and proteolytically cleaved into the mature F 1 and F 2 heterodimer, following an HDLVDGVK 109 motif. This cleavage event is required for fusogenic activity. To determine the amino acid requirements for processing of the HeV F protein, we constructed multiple mutants. Individual and simultaneous alanine substitutions of the eight residues immediately upstream of the cleavage site did not eliminate processing. A chimeric SV5 F protein in which the furin site was substituted for the VDGVK 109 motif of the HeV F protein was not processed but was expressed on the cell surface. Another chimeric SV5 F protein containing the HDLVDGVK 109 motif of the HeV F protein underwent partial cleavage. These data indicate that the upstream region can play a role in protease recognition, but is neither absolutely required nor sufficient for efficient processing of the HeV F protein

  18. Identification of Pathways Critical to Quorum Sensing and Virulence Induction

    Energy Technology Data Exchange (ETDEWEB)

    Ognibene, Ted J. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Young, N. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Holtz-Morris, A. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Daley, P. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2009-02-27

    Quorum sensing is a mode of intercellular communication between bacteria that allows them to collectively regulate behavior such as virulence, sporulation, motility and biofilm formation. It is mediated by bacterially synthesized, diffusible, signaling molecules (autoinducers) that increase in concentration as a bacterial population expands until a critical threshold concentration is reached. However, in most bacterial species that produce autoinducer molecules, the physiologic concentration of these molecules is unknown. Moreover, many bacterial species, including Y. pestis, produce an array of quorum sensing molecules and the physiologic concentration of each individual type of autoinducer molecule is not known. There is a need to accurately and precisely quantitate these molecules, as it may be that different types of autoinducer molecules have different effects on virulence in the bacterium. We focused our efforts on the construction of a platform to identify and quantitate autoinducer molecules using FTICR, 14C isotope labeling and accelerator mass spectrometry (AMS). Specifically, we focused on autoinducer-1 type molecules, acylhomoserine lactone (HSL), derived from S-adenosylmethionine (SAM).

  19. Quorum-Quenching Human Designer Cells for Closed-Loop Control of Pseudomonas aeruginosa Biofilms.

    Science.gov (United States)

    Sedlmayer, Ferdinand; Jaeger, Tina; Jenal, Urs; Fussenegger, Martin

    2017-08-09

    Current antibiotics gradually lose their efficacy against chronic Pseudomonas aeruginosa infections due to development of increased resistance mediated by biofilm formation, as well as the large arsenal of microbial virulence factors that are coordinated by the cell density-dependent phenomenon of quorum sensing. Here, we address this issue by using synthetic biology principles to rationally engineer quorum-quencher cells with closed-loop control to autonomously dampen virulence and interfere with biofilm integrity. Pathogen-derived signals dynamically activate a synthetic mammalian autoinducer sensor driving downstream expression of next-generation anti-infectives. Engineered cells were able to sensitively score autoinducer levels from P. aeruginosa clinical isolates and mount a 2-fold defense consisting of an autoinducer-inactivating enzyme to silence bacterial quorum sensing and a bipartite antibiofilm effector to dissolve the biofilm matrix. The self-guided cellular device fully cleared autoinducers, potentiated bacterial antibiotic susceptibility, substantially reduced biofilms, and alleviated cytotoxicity to lung epithelial cells. We believe this strategy of dividing otherwise coordinated pathogens and breaking up their shielded stronghold represents a blueprint for cellular anti-infectives in the postantibiotic era.

  20. Defective Proteasome Delivery of Polyubiquitinated Proteins by Ubiquilin-2 Proteins Containing ALS Mutations.

    Directory of Open Access Journals (Sweden)

    Lydia Chang

    Full Text Available Ubiquilin proteins facilitate delivery of ubiquitinated proteins to the proteasome for degradation. Interest in the proteins has been heightened by the discovery that gene mutations in UBQLN2 cause dominant inheritance of amyotrophic lateral sclerosis (ALS. However, the mechanisms by which the mutations cause ALS are not known. Here we report on the underlying defect of ubiquilin-2 proteins containing ALS-linked mutations in affecting proteasome-mediated degradation. We found that overexpression of ubiquilin-2 proteins containing any one of five different ALS mutations slow degradation of Myc, a prototypic proteasome substrate. Examination of coprecipitating proteins indicated that the mutant proteins are generally capable of binding polyubiquitinated proteins, but defective in binding the proteasome. GST-pulldown studies revealed that many of the mutants bind weaker to the S5a subunit of the proteasome, compared with wild type (WT ubiquilin-2 protein. The results suggest the mutant proteins are unable to deliver their captured cargo to the proteasome for degradation, which presumably leads to toxicity. Quantification of cell death is consistent with this idea. Measurement of protein turnover further indicated the mutant proteins have longer half-lives than WT ubiquilin-2. Our studies provide novel insight into the mechanism by which ALS-linked mutations in UBQLN2 interfere with protein degradation.

  1. Recent evolution of the NF-κB and inflammasome regulating protein POP2 in primates

    Directory of Open Access Journals (Sweden)

    Harton Jonathan A

    2011-03-01

    Full Text Available Abstract Background Pyrin-only protein 2 (POP2 is a small human protein comprised solely of a pyrin domain that inhibits NF-κB p65/RelA and blocks the formation of functional IL-1β processing inflammasomes. Pyrin proteins are abundant in mammals and several, like POP2, have been linked to activation or regulation of inflammatory processes. Because POP2 knockout mice would help probe the biological role of inflammatory regulation, we thus considered whether POP2 is common in the mammalian lineage. Results BLAST searches revealed that POP2 is absent from the available genomes of not only mice and rats, but those of other domestic mammals and New World monkeys as well. POP2 is however present in the genome of the primate species most closely related to humans including Pan troglodytes (chimpanzees, Macaca mulatta (rhesus macaques and others. Interestingly, chimpanzee POP2 is identical to human POP2 (huPOP2 at both the DNA and protein level. Macaque POP2 (mqPOP2, although highly conserved is not identical to the human sequence; however, both functions of the human protein are retained. Further, POP2 appears to have arisen in the mammalian genome relatively recently (~25 mya and likely derived from retrogene insertion of NLRP2. Conclusion Our findings support the hypothesis that the NLR loci of mammals, encoding proteins involved in innate and adaptive immunity as well as mammalian development, have been subject to recent and strong selective pressures. Since POP2 is capable of regulating signaling events and processes linked to innate immunity and inflammation, its presence in the genomes of hominids and Old World primates further suggests that additional regulation of these signals is important in these species.

  2. Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding protein.

    Science.gov (United States)

    Anjos, Liliana; Gomes, Ana S; Melo, Eduardo P; Canário, Adelino V; Power, Deborah M

    2013-03-01

    Cartilage Acidic Protein 2 (CRTAC2) is a novel protein present from prokaryotes to vertebrates with abundant expression in the teleost fish pituitary gland and an isoform of CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containing N-terminal integrin-like Ca(2+)-binding motifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca(2+) with high affinity (K(d)=1.46nM) and favourable Gibbs free energy (∆G=-12.4kcal/mol). The stoichiometry for Ca(2+) bound to sbCRTAC2 at saturation indicated six Ca(2+) ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca(2+). Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25°C and 95°C and the fully unfolded state is only induced by chemical denaturing (4M GndCl). sbCRTAC has a widespread tissue distribution and is present as high molecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell-cell and cell-matrix interactions. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Polycomb protein SCML2 associates with USP7 and counteracts histone H2A ubiquitination in the XY chromatin during male meiosis

    NARCIS (Netherlands)

    Luo, Mengcheng; Zhou, Jian; Leu, N. Adrian; Abreu, Carla M.; Wang, Jianle; Anguera, Montserrat C.; de Rooij, Dirk G.; Jasin, Maria; Wang, P. Jeremy

    2015-01-01

    Polycomb group proteins mediate transcriptional silencing in diverse developmental processes. Sex chromosomes undergo chromosome-wide transcription silencing during male meiosis. Here we report that mouse SCML2 (Sex comb on midleg-like 2), an X chromosome-encoded polycomb protein, is specifically

  4. Enhancement of Protein and Pigment Content in Two Chlorella Species Cultivated on Industrial Process Water

    Directory of Open Access Journals (Sweden)

    Hamed Safafar

    2016-12-01

    Full Text Available Chlorella pyrenoidosa and Chlorella vulgaris were cultivated in pre-gasified industrial process water with high concentration of ammonia representing effluent from a local biogas plant. The study aimed to investigate the effects of growth media and cultivation duration on the nutritional composition of biomass. Variations in proteins, lipid, fatty acid composition, amino acids, tocopherols, and pigments were studied. Both species grew well in industrial process water. The contents of proteins were affected significantly by the growth media and cultivation duration. Microalga Chlorella pyrenoidosa produced the highest concentrations of protein (65.2% ± 1.30% DW while Chlorella vulgaris accumulated extremely high concentrations of lutein and chlorophylls (7.14 ± 0.66 mg/g DW and 32.4 ± 1.77 mg/g DW, respectively. Cultivation of Chlorella species in industrial process water is an environmentally friendly, sustainable bioremediation method with added value biomass production and resource valorization, since the resulting biomass also presented a good source of proteins, amino acids, and carotenoids for potential use in aquaculture feed industry.

  5. A new theoretical approach to analyze complex processes in cytoskeleton proteins.

    Science.gov (United States)

    Li, Xin; Kolomeisky, Anatoly B

    2014-03-20

    Cytoskeleton proteins are filament structures that support a large number of important biological processes. These dynamic biopolymers exist in nonequilibrium conditions stimulated by hydrolysis chemical reactions in their monomers. Current theoretical methods provide a comprehensive picture of biochemical and biophysical processes in cytoskeleton proteins. However, the description is only qualitative under biologically relevant conditions because utilized theoretical mean-field models neglect correlations. We develop a new theoretical method to describe dynamic processes in cytoskeleton proteins that takes into account spatial correlations in the chemical composition of these biopolymers. Our approach is based on analysis of probabilities of different clusters of subunits. It allows us to obtain exact analytical expressions for a variety of dynamic properties of cytoskeleton filaments. By comparing theoretical predictions with Monte Carlo computer simulations, it is shown that our method provides a fully quantitative description of complex dynamic phenomena in cytoskeleton proteins under all conditions.

  6. UO{sub 2}{sup 2+}/protein complexation sites screening

    Energy Technology Data Exchange (ETDEWEB)

    Guilbaud, P.; Pible, O

    2004-07-01

    Uranium(VI) is likely to make strong coordination with some proteins in the plasma and in targeted cells. In the frame of a nuclear toxicology program, a biochemical strategy has been developed to identify these targets in complex biological media. The present work focuses on an approach based on the screening of 3D protein structures in order to identify proteins able to bind UO{sub 2}{sup 2+} and the corresponding complexation sites in these proteins. Our preliminary results show that indeed a few proteins display a high affinity to uranyl salt. The site of interaction may be mapped using molecular modeling, providing coherent results with the biochemical data. (authors)

  7. In-line Fourier-transform infrared spectroscopy as a versatile process analytical technology for preparative protein chromatography.

    Science.gov (United States)

    Großhans, Steffen; Rüdt, Matthias; Sanden, Adrian; Brestrich, Nina; Morgenstern, Josefine; Heissler, Stefan; Hubbuch, Jürgen

    2018-04-27

    Fourier-transform infrared spectroscopy (FTIR) is a well-established spectroscopic method in the analysis of small molecules and protein secondary structure. However, FTIR is not commonly applied for in-line monitoring of protein chromatography. Here, the potential of in-line FTIR as a process analytical technology (PAT) in downstream processing was investigated in three case studies addressing the limits of currently applied spectroscopic PAT methods. A first case study exploited the secondary structural differences of monoclonal antibodies (mAbs) and lysozyme to selectively quantify the two proteins with partial least squares regression (PLS) giving root mean square errors of cross validation (RMSECV) of 2.42 g/l and 1.67 g/l, respectively. The corresponding Q 2 values are 0.92 and, respectively, 0.99, indicating robust models in the calibration range. Second, a process separating lysozyme and PEGylated lysozyme species was monitored giving an estimate of the PEGylation degree of currently eluting species with RMSECV of 2.35 g/l for lysozyme and 1.24 g/l for PEG with Q 2 of 0.96 and 0.94, respectively. Finally, Triton X-100 was added to a feed of lysozyme as a typical process-related impurity. It was shown that the species could be selectively quantified from the FTIR 3D field without PLS calibration. In summary, the proposed PAT tool has the potential to be used as a versatile option for monitoring protein chromatography. It may help to achieve a more complete implementation of the PAT initiative by mitigating limitations of currently used techniques. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  8. Structural changes induced by high-pressure processing in micellar casein and milk protein concentrates.

    Science.gov (United States)

    Cadesky, Lee; Walkling-Ribeiro, Markus; Kriner, Kyle T; Karwe, Mukund V; Moraru, Carmen I

    2017-09-01

    Reconstituted micellar casein concentrates and milk protein concentrates of 2.5 and 10% (wt/vol) protein concentration were subjected to high-pressure processing at pressures from 150 to 450 MPa, for 15 min, at ambient temperature. The structural changes induced in milk proteins by high-pressure processing were investigated using a range of physical, physicochemical, and chemical methods, including dynamic light scattering, rheology, mid-infrared spectroscopy, scanning electron microscopy, proteomics, and soluble mineral analyses. The experimental data clearly indicate pressure-induced changes of casein micelles, as well as denaturation of serum proteins. Calcium-binding α S1 - and α S2 -casein levels increased in the soluble phase after all pressure treatments. Pressurization up to 350 MPa also increased levels of soluble calcium and phosphorus, in all samples and concentrations, whereas treatment at 450 MPa reduced the levels of soluble Ca and P. Experimental data suggest dissociation of calcium phosphate and subsequent casein micelle destabilization as a result of pressure treatment. Treatment of 10% micellar casein concentrate and 10% milk protein concentrate samples at 450 MPa resulted in weak, physical gels, which featured aggregates of uniformly distributed, casein substructures of 15 to 20 nm in diameter. Serum proteins were significantly denatured by pressures above 250 MPa. These results provide information on pressure-induced changes in high-concentration protein systems, and may inform the development on new milk protein-based foods with novel textures and potentially high nutritional quality, of particular interest being the soft gel structures formed at high pressure levels. The Authors. Published by the Federation of Animal Science Societies and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

  9. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process.

    Science.gov (United States)

    Singh, Anupam; Upadhyay, Vaibhav; Upadhyay, Arun Kumar; Singh, Surinder Mohan; Panda, Amulya Kumar

    2015-03-25

    Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from inclusion bodies is labor intensive and the yields of recombinant protein are often low. Here we review the developments in the field that are targeted at improving the yield, as well as quality of the recombinant protein by optimizing the individual steps of the process, especially solubilization of the inclusion bodies and refolding of the solubilized protein. Mild solubilization methods have been discussed which are based on the understanding of the fact that protein molecules in inclusion body aggregates have native-like structure. These methods solubilize the inclusion body aggregates while preserving the native-like protein structure. Subsequent protein refolding and purification results in high recovery of bioactive protein. Other parameters which influence the overall recovery of bioactive protein from inclusion bodies have also been discussed. A schematic model describing the utility of mild solubilization methods for high throughput recovery of bioactive protein has also been presented.

  10. Inhibition of tumor metastasis by a growth factor receptor bound protein 2 Src homology 2 domain-binding antagonist.

    Science.gov (United States)

    Giubellino, Alessio; Gao, Yang; Lee, Sunmin; Lee, Min-Jung; Vasselli, James R; Medepalli, Sampath; Trepel, Jane B; Burke, Terrence R; Bottaro, Donald P

    2007-07-01

    Metastasis, the primary cause of death in most forms of cancer, is a multistep process whereby cells from the primary tumor spread systemically and colonize distant new sites. Blocking critical steps in this process could potentially inhibit tumor metastasis and dramatically improve cancer survival rates; however, our understanding of metastasis at the molecular level is still rudimentary. Growth factor receptor binding protein 2 (Grb2) is a widely expressed adapter protein with roles in epithelial cell growth and morphogenesis, as well as angiogenesis, making it a logical target for anticancer drug development. We have previously shown that a potent antagonist of Grb2 Src homology-2 domain-binding, C90, blocks growth factor-driven cell motility in vitro and angiogenesis in vivo. We now report that C90 inhibits metastasis in vivo in two aggressive tumor models, without affecting primary tumor growth rate. These results support the potential efficacy of this compound in reducing the metastatic spread of primary solid tumors and establish a critical role for Grb2 Src homology-2 domain-mediated interactions in this process.

  11. Oxidation of lipid and protein in horse mackerel (Trachurus trachurus) mince and washed minces during processing and storage

    DEFF Research Database (Denmark)

    Eymard, Sylvie; Baron, Caroline; Jacobsen, Charlotte

    2009-01-01

    : M1, M2 and M3, with one, two and three washing steps, respectively. The different products were characterised (i.e. lipid content, protein, water, iron, fatty acid profile and tocopherol content) and analysed for protein and lipid oxidation in order to investigate the impact of the washing steps...... was followed by determination of protein solubility, protein thiol groups and protein carbonyl groups using colorimetric methods as well as western blotting for protein carbonyl groups. Lipid and protein oxidation markers indicated that both lipid and protein oxidation took place during processing...

  12. A Signal Processing Method to Explore Similarity in Protein Flexibility

    Directory of Open Access Journals (Sweden)

    Simina Vasilache

    2010-01-01

    Full Text Available Understanding mechanisms of protein flexibility is of great importance to structural biology. The ability to detect similarities between proteins and their patterns is vital in discovering new information about unknown protein functions. A Distance Constraint Model (DCM provides a means to generate a variety of flexibility measures based on a given protein structure. Although information about mechanical properties of flexibility is critical for understanding protein function for a given protein, the question of whether certain characteristics are shared across homologous proteins is difficult to assess. For a proper assessment, a quantified measure of similarity is necessary. This paper begins to explore image processing techniques to quantify similarities in signals and images that characterize protein flexibility. The dataset considered here consists of three different families of proteins, with three proteins in each family. The similarities and differences found within flexibility measures across homologous proteins do not align with sequence-based evolutionary methods.

  13. @TOME-2: a new pipeline for comparative modeling of protein-ligand complexes.

    Science.gov (United States)

    Pons, Jean-Luc; Labesse, Gilles

    2009-07-01

    @TOME 2.0 is new web pipeline dedicated to protein structure modeling and small ligand docking based on comparative analyses. @TOME 2.0 allows fold recognition, template selection, structural alignment editing, structure comparisons, 3D-model building and evaluation. These tasks are routinely used in sequence analyses for structure prediction. In our pipeline the necessary software is efficiently interconnected in an original manner to accelerate all the processes. Furthermore, we have also connected comparative docking of small ligands that is performed using protein-protein superposition. The input is a simple protein sequence in one-letter code with no comment. The resulting 3D model, protein-ligand complexes and structural alignments can be visualized through dedicated Web interfaces or can be downloaded for further studies. These original features will aid in the functional annotation of proteins and the selection of templates for molecular modeling and virtual screening. Several examples are described to highlight some of the new functionalities provided by this pipeline. The server and its documentation are freely available at http://abcis.cbs.cnrs.fr/AT2/

  14. An Improved 2-Dimensional Gel Electrophoresis Method for Resolving Human Erythrocyte Membrane Proteins.

    Science.gov (United States)

    Kumar, Manoj; Singh, Rajendra; Meena, Anil; Patidar, Bhagwan S; Prasad, Rajendra; Chhabra, Sunil K; Bansal, Surendra K

    2017-01-01

    The 2-dimensional gel electrophoresis (2-DE) technique is widely used for the analysis of complex protein mixtures extracted from biological samples. It is one of the most commonly used analytical techniques in proteomics to study qualitative and quantitative protein changes between different states of a cell or an organism (eg, healthy and diseased), conditionally expressed proteins, posttranslational modifications, and so on. The 2-DE technique is used for its unparalleled ability to separate thousands of proteins simultaneously. The resolution of the proteins by 2-DE largely depends on the quality of sample prepared during protein extraction which increases results in terms of reproducibility and minimizes protein modifications that may result in artifactual spots on 2-DE gels. The buffer used for the extraction and solubilization of proteins influences the quality and reproducibility of the resolution of proteins on 2-DE gel. The purification by cleanup kit is another powerful process to prevent horizontal streaking which occurs during isoelectric focusing due to the presence of contaminants such as salts, lipids, nucleic acids, and detergents. Erythrocyte membrane proteins serve as prototypes for multifunctional proteins in various erythroid and nonerythroid cells. In this study, we therefore optimized the selected major conditions of 2-DE for resolving various proteins of human erythrocyte membrane. The modification included the optimization of conditions for sample preparation, cleanup of protein sample, isoelectric focusing, equilibration, and storage of immobilized pH gradient strips, which were further carefully examined to achieve optimum conditions for improving the quality of protein spots on 2-DE gels. The present improved 2-DE analysis method enabled better detection of protein spots with higher quality and reproducibility. Therefore, the conditions established in this study may be used for the 2-DE analysis of erythrocyte membrane proteins for

  15. Regulation of amyloid precursor protein processing by its KFERQ motif.

    Science.gov (United States)

    Park, Ji-Seon; Kim, Dong-Hou; Yoon, Seung-Yong

    2016-06-01

    Understanding of trafficking, processing, and degradation mechanisms of amyloid precursor protein (APP) is important because APP can be processed to produce β-amyloid (Aβ), a key pathogenic molecule in Alzheimer's disease (AD). Here, we found that APP contains KFERQ motif at its C-terminus, a consensus sequence for chaperone-mediated autophagy (CMA) or microautophagy which are another types of autophagy for degradation of pathogenic molecules in neurodegenerative diseases. Deletion of KFERQ in APP increased C-terminal fragments (CTFs) and secreted N-terminal fragments of APP and kept it away from lysosomes. KFERQ deletion did not abolish the interaction of APP or its cleaved products with heat shock cognate protein 70 (Hsc70), a protein necessary for CMA or microautophagy. These findings suggest that KFERQ motif is important for normal processing and degradation of APP to preclude the accumulation of APP-CTFs although it may not be important for CMA or microautophagy. [BMB Reports 2016; 49(6): 337-342].

  16. Protein phosphatase 2A mediates JS-K-induced apoptosis by affecting Bcl-2 family proteins in human hepatocellular carcinoma HepG2 cells.

    Science.gov (United States)

    Liu, Ling; Huang, Zile; Chen, Jingjing; Wang, Jiangang; Wang, Shuying

    2018-04-25

    Protein phosphatase 2A (PP2A) is an important enzyme within various signal transduction pathways. The present study was investigated PP2A mediates JS-K-induced apoptosis by affecting Bcl-2 family protein. JS-K showed diverse inhibitory effects in five HCC cell lines, especially HepG2 cells. JS-K caused a dose- and time-dependent reduction in cell viability and increased in levels of LDH release. Meanwhile, JS-K- induced apoptosis was characterized by mitochondrial membrane potential reduction, Hoechst 33342 + /PI + dual staining, release of cytochrome c (Cyt c), and activation of cleaved caspase-9/3. Moreover, JS-K-treatment could lead to the activation of protein phosphatase 2A-C (PP2A-C), decrease of anti-apoptotic Bcl-2 family-protein expression including p-Bcl-2 (Ser70), Bcl-2, Bcl-xL, and Mcl-1 as well as the increase of pro-apoptosis Bcl-2 family-protein including Bim, Bad, Bax, and Bak. Furthermore, JS-K caused a marked increase of intracellular NO levels while pre-treatment with Carboxy-PTIO (a NO scavenger) reduced the cytotoxicity effects and the apoptosis rate. Meanwhile, pre-treatment with Carboxy-PTIO attenuated the JS-K-induced up-regulation of PP2A, Cyt c, and cleaved-caspase-9/3 activation. The silencing PP2A-C by siRNA could abolish the activation of PP2A-C, down-regulation of anti-apoptotic Bcl-2 family-protein (p-Bcl-2, Bcl-2, Bcl-xL, and Mcl-1), increase of pro-apoptosis Bcl-2 family-protein (Bim, Bad, Bax, and Bak) and apoptotic-related protein (Cyt c, cleaved caspase-9/3) that were caused by JS-K in HepG2 cells. In addition, pre-treatment with OA (a PP2A inhibitor) also attenuated the above effects induced by JS-K. In summary, NO release from JS-K induces apoptosis through PP2A activation, which contributed to the regulation of Bcl-2 family proteins. © 2018 Wiley Periodicals, Inc.

  17. Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

    Science.gov (United States)

    Bauß, Katharina; Knapp, Barbara; Jores, Pia; Roepman, Ronald; Kremer, Hannie; Wijk, Erwin V; Märker, Tina; Wolfrum, Uwe

    2014-08-01

    The human Usher syndrome (USH) is a complex ciliopathy with at least 12 chromosomal loci assigned to three clinical subtypes, USH1-3. The heterogeneous USH proteins are organized into protein networks. Here, we identified Magi2 (membrane-associated guanylate kinase inverted-2) as a new component of the USH protein interactome, binding to the multifunctional scaffold protein SANS (USH1G). We showed that the SANS-Magi2 complex assembly is regulated by the phosphorylation of an internal PDZ-binding motif in the sterile alpha motif domain of SANS by the protein kinase CK2. We affirmed Magi2's role in receptor-mediated, clathrin-dependent endocytosis and showed that phosphorylated SANS tightly regulates Magi2-mediated endocytosis. Specific depletions by RNAi revealed that SANS and Magi2-mediated endocytosis regulates aspects of ciliogenesis. Furthermore, we demonstrated the localization of the SANS-Magi2 complex in the periciliary membrane complex facing the ciliary pocket of retinal photoreceptor cells in situ. Our data suggest that endocytotic processes may not only contribute to photoreceptor cell homeostasis but also counterbalance the periciliary membrane delivery accompanying the exocytosis processes for the cargo vesicle delivery. In USH1G patients, mutations in SANS eliminate Magi2 binding and thereby deregulate endocytosis, lead to defective ciliary transport modules and ultimately disrupt photoreceptor cell function inducing retinal degeneration. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Piezo Proteins: Regulators of Mechanosensation and Other Cellular Processes*

    OpenAIRE

    Bagriantsev, Sviatoslav N.; Gracheva, Elena O.; Gallagher, Patrick G.

    2014-01-01

    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular deve...

  19. Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes

    DEFF Research Database (Denmark)

    Gannagé, Monique; Dormann, Dorothee; Albrecht, Randy

    2009-01-01

    Influenza A virus is an important human pathogen causing significant morbidity and mortality every year and threatening the human population with epidemics and pandemics. Therefore, it is important to understand the biology of this virus to develop strategies to control its pathogenicity. Here, we...... demonstrate that influenza A virus inhibits macroautophagy, a cellular process known to be manipulated by diverse pathogens. Influenza A virus infection causes accumulation of autophagosomes by blocking their fusion with lysosomes, and one viral protein, matrix protein 2, is necessary and sufficient...... for this inhibition of autophagosome degradation. Macroautophagy inhibition by matrix protein 2 compromises survival of influenza virus-infected cells but does not influence viral replication. We propose that influenza A virus, which also encodes proapoptotic proteins, is able to determine the death of its host cell...

  20. PENGARUH PENAMBAHAN MAGNESIUM STEARAT DAN JENIS PROTEIN PADA PEMBUATAN BIODEGRADABLE FOAM DENGAN METODE BAKING PROCESS

    Directory of Open Access Journals (Sweden)

    Nanik hendrawati

    2015-12-01

    Full Text Available Biodegradable foam with cassava starch, protein and chitosan as the basic ingredients can be produced by using baking process method. Variation on magnesium stearate amount and protein types gave different effect on the biodegradable foam quality. The amount of magnesium stearate was varied as 1; 1.6; 2.2; 2.8; 3.4 and 4 % w/w and the sources of protein used in this research were taken from soy bean, peanut and egg white. The foam produced in this research was then tested for its mechanical properties, water resistance and biodegradability. It was found that addition of magnesium stearate as much as 4% w/w reduced water adsorption and biodegradability of foam. Magnesium stearate affected the ability of absorption of water and foam degradation, but did not influence on tensile strength. Different types of protein also gave influence on water absorption, biodegradability and tensile strength. The best improvement of tensile strenght among the compounds tested was shown by soy bean based foam.

  1. The RB/E2F pathway and regulation of RNA processing

    Energy Technology Data Exchange (ETDEWEB)

    Ahlander, Joseph [Department of Molecular and Cellular Biology, 1007 East Lowell Street, University of Arizona, Tucson, AZ 85721 (United States); Bosco, Giovanni, E-mail: gbosco@email.arizona.edu [Department of Molecular and Cellular Biology, 1007 East Lowell Street, University of Arizona, Tucson, AZ 85721 (United States)

    2009-07-03

    The retinoblastoma tumor suppressor protein (RB) is inactivated in a majority of cancers. RB restricts cell proliferation by inhibiting the E2F family of transcription factors. The current model for RB/E2F function describes its role in regulating transcription at gene promoters. Whether the RB or E2F proteins might play a role in gene expression beyond transcription initiation is not well known. This review describes evidence that points to a novel role for the RB/E2F network in the regulation of RNA processing, and we propose a model as a framework for future research. The elucidation of a novel role of RB in RNA processing will have a profound impact on our understanding of the role of this tumor suppressor family in cell and developmental biology.

  2. Piezo Proteins: Regulators of Mechanosensation and Other Cellular Processes*

    Science.gov (United States)

    Bagriantsev, Sviatoslav N.; Gracheva, Elena O.; Gallagher, Patrick G.

    2014-01-01

    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature. PMID:25305018

  3. Regulation of Long-Chain N-Acyl-Homoserine Lactones in Agrobacterium vitis

    OpenAIRE

    Hao, Guixia; Burr, Thomas J.

    2006-01-01

    Homologs of quorum-sensing luxR and luxI regulatory genes, avsR and avsI, were identified in Agrobacterium vitis strain F2/5. Compared to other LuxI proteins from related species, the deduced AvsI shows the greatest identity to SinI (71%) from Sinorhizobium meliloti Rm1021. AvsR possesses characteristic autoinducer binding and helix-turn-helix DNA binding domains and shares a high level of identity with SinR (38%) from Rm1021. Site-directed mutagenesis of avsR and avsI was performed, and both...

  4. The conserved, disease-associated RNA binding protein dNab2 interacts with the Fragile-X protein ortholog in Drosophila neurons

    Science.gov (United States)

    Bienkowski, Rick S.; Banerjee, Ayan; Rounds, J. Christopher; Rha, Jennifer; Omotade, Omotola F.; Gross, Christina; Morris, Kevin J.; Leung, Sara W.; Pak, ChangHui; Jones, Stephanie K.; Santoro, Michael R.; Warren, Stephen T.; Zheng, James Q.; Bassell, Gary J.; Corbett, Anita H.; Moberg, Kenneth H.

    2017-01-01

    Summary The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA-binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. Here we present a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with and the Fragile-X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII but not futsch mRNA, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A)-tail length similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. In sum these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-specific defects in patients lacking ZC3H14. PMID:28793261

  5. The Conserved, Disease-Associated RNA Binding Protein dNab2 Interacts with the Fragile X Protein Ortholog in Drosophila Neurons

    Directory of Open Access Journals (Sweden)

    Rick S. Bienkowski

    2017-08-01

    Full Text Available The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A RNA binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. Here, we present a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory, and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII, but not futsch, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A tail length, similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. Altogether, these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-specific defects in patients lacking ZC3H14.

  6. Microchidia protein 2, MORC2, downregulates the cytoskeleton adapter protein, ArgBP2, via histone methylation in gastric cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Tong, Yuxin; Li, Yan [Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, Liaoning 110122 (China); Gu, Hui [Department of Key Laboratory of Health Ministry for Congenital Malformation Shengjing Hospital, China Medical University, Shenyang, Liaoning 110004 (China); Wang, Chunyu [Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, Liaoning 110122 (China); Liu, Funan [Department of Surgical Oncology, The First Hospital of China Medical University, Shenyang, Liaoning 110001 (China); Shao, Yangguang; Li, Jiabin; Cao, Liu [Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, Liaoning 110122 (China); Li, Feng, E-mail: fli@mail.cmu.edu.cn [Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, Liaoning 110122 (China)

    2015-11-27

    ArgBP2 is an adapter protein that plays an important role in actin-dependent processes such as cell adhesion and migration. However, its function and regulation mechanisms in gastric cancer have not yet been investigated. Here, we showed the low expression of ArgBP2 mRNA level in gastric tumor samples and its repressive function in the proliferation, migration, and invasion of gastric cancer cells. Then, we cloned and identified ArgBP2 promoter and verified that MORC2 bound to the promoter. Moreover, we demonstrated that MORC2 enhanced the recruitment of EZH2, which promoted the tri-methylation of H3K27, leading to the transcriptional repression of ArgBP2. Our results might thus contribute to understanding the molecular mechanisms of ArgBP2 regulation and suggesting ArgBP2 as a potential therapeutic target for gastric cancer. - Highlights: • ArgBP2 inhibits proliferation, migration, and invasion of gastric cancer cells. • Identification of ArgBP2 promoter and its transcription factor MORC2. • EZH2 is required in MORC2 down-regulating ArgBP2 via histone methylation.

  7. Microchidia protein 2, MORC2, downregulates the cytoskeleton adapter protein, ArgBP2, via histone methylation in gastric cancer cells

    International Nuclear Information System (INIS)

    Tong, Yuxin; Li, Yan; Gu, Hui; Wang, Chunyu; Liu, Funan; Shao, Yangguang; Li, Jiabin; Cao, Liu; Li, Feng

    2015-01-01

    ArgBP2 is an adapter protein that plays an important role in actin-dependent processes such as cell adhesion and migration. However, its function and regulation mechanisms in gastric cancer have not yet been investigated. Here, we showed the low expression of ArgBP2 mRNA level in gastric tumor samples and its repressive function in the proliferation, migration, and invasion of gastric cancer cells. Then, we cloned and identified ArgBP2 promoter and verified that MORC2 bound to the promoter. Moreover, we demonstrated that MORC2 enhanced the recruitment of EZH2, which promoted the tri-methylation of H3K27, leading to the transcriptional repression of ArgBP2. Our results might thus contribute to understanding the molecular mechanisms of ArgBP2 regulation and suggesting ArgBP2 as a potential therapeutic target for gastric cancer. - Highlights: • ArgBP2 inhibits proliferation, migration, and invasion of gastric cancer cells. • Identification of ArgBP2 promoter and its transcription factor MORC2. • EZH2 is required in MORC2 down-regulating ArgBP2 via histone methylation.

  8. Covariance NMR Processing and Analysis for Protein Assignment.

    Science.gov (United States)

    Harden, Bradley J; Frueh, Dominique P

    2018-01-01

    During NMR resonance assignment it is often necessary to relate nuclei to one another indirectly, through their common correlations to other nuclei. Covariance NMR has emerged as a powerful technique to correlate such nuclei without relying on error-prone peak peaking. However, false-positive artifacts in covariance spectra have impeded a general application to proteins. We recently introduced pre- and postprocessing steps to reduce the prevalence of artifacts in covariance spectra, allowing for the calculation of a variety of 4D covariance maps obtained from diverse combinations of pairs of 3D spectra, and we have employed them to assign backbone and sidechain resonances in two large and challenging proteins. In this chapter, we present a detailed protocol describing how to (1) properly prepare existing 3D spectra for covariance, (2) understand and apply our processing script, and (3) navigate and interpret the resulting 4D spectra. We also provide solutions to a number of errors that may occur when using our script, and we offer practical advice when assigning difficult signals. We believe such 4D spectra, and covariance NMR in general, can play an integral role in the assignment of NMR signals.

  9. Identification of poly(rC) binding protein 2 (PCBP2) as a target protein of immunosuppressive agent 15-deoxyspergualin

    Energy Technology Data Exchange (ETDEWEB)

    Murahashi, Masataka; Simizu, Siro; Morioka, Masahiko [Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522 (Japan); Umezawa, Kazuo, E-mail: umezawa@aichi-med-u.ac.jp [Department of Molecular Target Medicine, Aichi Medical University School of Medicine, 1-1 Yazako-Karimata, Nagakute 480-1195 (Japan)

    2016-08-05

    15-Deoxyspergualin (DSG) is an immunosuppressive agent being clinically used. Unlike tacrolimus and cyclosporine A, it does not inhibit the calcineurin pathway, and its mechanism of action and target molecule have not been elucidated. Therefore, we previously prepared biotinylated derivative of DSG (BDSG) to fish up the target protein. In the present research, we identified poly(rC) binding protein 2 (PCBP2) as a DSG-binding protein using this probe. DSG was confirmed to bind to PCBP2 by pull-down assay. Intracellular localization of PCBP2 was changed from the nucleus to the cytoplasm by DSG treatment. DSG inhibited the cell growth, and over-expression of PCBP2 reduced the anti-proliferative activity of DSG. PCBP2 is known to regulate various proteins including STAT1/2. Thus, we found PCBP2 as the first target protein of DSG that can explain the immunosuppressive activity. -- Highlights: •Fifteen-deoxyspergualin (DSG) is an immunosuppressive agent clinically used. •We have identified PCBP2, an RNA-binding protein, as a molecular target of DSG. •Alteration of PCBP2 activity may explain the immunosuppressive activity of DSG.

  10. High-Pressure-High-Temperature Processing Reduces Maillard Reaction and Viscosity in Whey Protein-Sugar Solutions.

    Science.gov (United States)

    Avila Ruiz, Geraldine; Xi, Bingyan; Minor, Marcel; Sala, Guido; van Boekel, Martinus; Fogliano, Vincenzo; Stieger, Markus

    2016-09-28

    The aim of the study was to determine the influence of pressure in high-pressure-high-temperature (HPHT) processing on Maillard reactions and protein aggregation of whey protein-sugar solutions. Solutions of whey protein isolate containing either glucose or trehalose at pH 6, 7, and 9 were treated by HPHT processing or conventional high-temperature (HT) treatments. Browning was reduced, and early and advanced Maillard reactions were retarded under HPHT processing at all pH values compared to HT treatment. HPHT induced a larger pH drop than HT treatments, especially at pH 9, which was not associated with Maillard reactions. After HPHT processing at pH 7, protein aggregation and viscosity of whey protein isolate-glucose/trehalose solutions remained unchanged. It was concluded that HPHT processing can potentially improve the quality of protein-sugar-containing foods, for which browning and high viscosities are undesired, such as high-protein beverages.

  11. Protein-induced changes during the maturation process of human dendritic cells: A 2-D DIGE approach

    DEFF Research Database (Denmark)

    Ferreira, Gb; Overbergh, L; Hansen, Kasper Lage

    2008-01-01

    Dendritic cells (DCs) are unique antigen presenting cells, which upon maturation change from a specialized antigen-capturing cell towards a professional antigen presenting cells. In this study, a 2-D DIGE analysis of immature and mature DCs was performed, to identify proteins changing in expression...... upon maturation. The protein expression profile of immature and mature DCs, derived from CD14+ peripheral blood monocytes was investigated using two pH ranges (pH 4-7 and 6-9) (n = 4). Ninety one differentially expressed spots (p...

  12. Protein Phosphatase 2A in the Regulation of Wnt Signaling, Stem Cells, and Cancer.

    Science.gov (United States)

    Thompson, Joshua J; Williams, Christopher S

    2018-02-26

    Protein phosphorylation is a ubiquitous cellular process that allows for the nuanced and reversible regulation of protein activity. Protein phosphatase 2A (PP2A) is a heterotrimeric serine-threonine phosphatase-composed of a structural, regulatory, and catalytic subunit-that controls a variety of cellular events via protein dephosphorylation. While much is known about PP2A and its basic biochemistry, the diversity of its components-especially the multitude of regulatory subunits-has impeded the determination of PP2A function. As a consequence of this complexity, PP2A has been shown to both positively and negatively regulate signaling networks such as the Wnt pathway. Wnt signaling modulates major developmental processes, and is a dominant mediator of stem cell self-renewal, cell fate, and cancer stem cells. Because PP2A affects Wnt signaling both positively and negatively and at multiple levels, further understanding of this complex dynamic may ultimately provide insight into stem cell biology and how to better treat cancers that result from alterations in Wnt signaling. This review will summarize literature that implicates PP2A as a tumor suppressor, explore PP2A mutations identified in human malignancy, and focus on PP2A in the regulation of Wnt signaling and stem cells so as to better understand how aberrancy in this pathway can contribute to tumorigenesis.

  13. SH2 domain-containing protein tyrosine phosphatase 2 and focal adhesion kinase protein interactions regulate pulmonary endothelium barrier function.

    Science.gov (United States)

    Chichger, Havovi; Braza, Julie; Duong, Huetran; Harrington, Elizabeth O

    2015-06-01

    Enhanced protein tyrosine phosphorylation is associated with changes in vascular permeability through formation and dissolution of adherens junctions and regulation of stress fiber formation. Inhibition of the protein tyrosine phosphorylase SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) increases tyrosine phosphorylation of vascular endothelial cadherin and β-catenin, resulting in disruption of the endothelial monolayer and edema formation in the pulmonary endothelium. Vascular permeability is a hallmark of acute lung injury (ALI); thus, enhanced SHP2 activity offers potential therapeutic value for the pulmonary vasculature in diseases such as ALI, but this has not been characterized. To assess whether SHP2 activity mediates protection against edema in the endothelium, we assessed the effect of molecular activation of SHP2 on lung endothelial barrier function in response to the edemagenic agents LPS and thrombin. Both LPS and thrombin reduced SHP2 activity, correlated with decreased focal adhesion kinase (FAK) phosphorylation (Y(397) and Y(925)) and diminished SHP2 protein-protein associations with FAK. Overexpression of constitutively active SHP2 (SHP2(D61A)) enhanced baseline endothelial monolayer resistance and completely blocked LPS- and thrombin-induced permeability in vitro and significantly blunted pulmonary edema formation induced by either endotoxin (LPS) or Pseudomonas aeruginosa exposure in vivo. Chemical inhibition of FAK decreased SHP2 protein-protein interactions with FAK concomitant with increased permeability; however, overexpression of SHP2(D61A) rescued the endothelium and maintained FAK activity and FAK-SHP2 protein interactions. Our data suggest that SHP2 activation offers the pulmonary endothelium protection against barrier permeability mediators downstream of the FAK signaling pathway. We postulate that further studies into the promotion of SHP2 activation in the pulmonary endothelium may offer a therapeutic approach for patients

  14. L2, the minor capsid protein of papillomavirus

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Joshua W. [Department of Pathology, The Johns Hopkins University, Baltimore, MD 21287 (United States); Roden, Richard B.S., E-mail: roden@jhmi.edu [Department of Pathology, The Johns Hopkins University, Baltimore, MD 21287 (United States); Department of Oncology, The Johns Hopkins University, Baltimore, MD 21287 (United States); Department of Gynecology and Obstetrics, The Johns Hopkins University, Baltimore, MD 21287 (United States)

    2013-10-15

    The capsid protein L2 plays major roles in both papillomavirus assembly and the infectious process. While L1 forms the majority of the capsid and can self-assemble into empty virus-like particles (VLPs), L2 is a minor capsid component and lacks the capacity to form VLPs. However, L2 co-assembles with L1 into VLPs, enhancing their assembly. L2 also facilitates encapsidation of the ∼8 kbp circular and nucleosome-bound viral genome during assembly of the non-enveloped T=7d virions in the nucleus of terminally differentiated epithelial cells, although, like L1, L2 is not detectably expressed in infected basal cells. With respect to infection, L2 is not required for particles to bind to and enter cells. However L2 must be cleaved by furin for endosome escape. L2 then travels with the viral genome to the nucleus, wherein it accumulates at ND-10 domains. Here, we provide an overview of the biology of L2. - Highlights: • L2 is the minor antigen of the non-enveloped T=7d icosahedral Papillomavirus capsid. • L2 is a nuclear protein that can traffic to ND-10 and facilitate genome encapsidation. • L2 is critical for infection and must be cleaved by furin. • L2 is a broadly protective vaccine antigen recognized by neutralizing antibodies.

  15. L2, the minor capsid protein of papillomavirus

    International Nuclear Information System (INIS)

    Wang, Joshua W.; Roden, Richard B.S.

    2013-01-01

    The capsid protein L2 plays major roles in both papillomavirus assembly and the infectious process. While L1 forms the majority of the capsid and can self-assemble into empty virus-like particles (VLPs), L2 is a minor capsid component and lacks the capacity to form VLPs. However, L2 co-assembles with L1 into VLPs, enhancing their assembly. L2 also facilitates encapsidation of the ∼8 kbp circular and nucleosome-bound viral genome during assembly of the non-enveloped T=7d virions in the nucleus of terminally differentiated epithelial cells, although, like L1, L2 is not detectably expressed in infected basal cells. With respect to infection, L2 is not required for particles to bind to and enter cells. However L2 must be cleaved by furin for endosome escape. L2 then travels with the viral genome to the nucleus, wherein it accumulates at ND-10 domains. Here, we provide an overview of the biology of L2. - Highlights: • L2 is the minor antigen of the non-enveloped T=7d icosahedral Papillomavirus capsid. • L2 is a nuclear protein that can traffic to ND-10 and facilitate genome encapsidation. • L2 is critical for infection and must be cleaved by furin. • L2 is a broadly protective vaccine antigen recognized by neutralizing antibodies

  16. Cross-talk between amyloidogenic proteins in type-2 diabetes and Parkinson’s disease

    OpenAIRE

    Horvath, Istvan; Wittung-Stafshede, Pernilla

    2016-01-01

    Protein assembly into ordered so-called amyloid fibers is a process that promotes several neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease (PD). Also type-2 diabetes (T2D) is a disease involving amyloid formation, although it occurs in the pancreas. Since the protein that forms amyloids in PD, α-synuclein (aS), is also expressed in the pancreas, we investigated whether it could affect aggregation of the peptide involved in T2D, and vice versa. Using in vitro methods an...

  17. Protein: MPB2 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPB2 Ubiquitin ligases STUB1 CHIP STUB1 E3 ubiquitin-protein ligase CHIP Antigen NY...-CO-7, CLL-associated antigen KW-8, Carboxy terminus of Hsp70-interacting protein, STIP1 homology and U box-containing pr

  18. Effect of thermal processing on estimated metabolizable protein supply to dairy cattle from camelina seeds: relationship with protein molecular structural changes.

    Science.gov (United States)

    Peng, Quanhui; Khan, Nazir A; Wang, Zhisheng; Zhang, Xuewei; Yu, Peiqiang

    2014-08-20

    This study evaluated the effect of thermal processing on the estimated metabolizable protein (MP) supply to dairy cattle from camelina seeds (Camelina sativa L. Crantz) and determined the relationship between heat-induced changes in protein molecular structural characteristics and the MP supply. Seeds from two camelina varieties were sampled in two consecutive years and were either kept raw or were heated in an autoclave (moist heating) or in an air-draft oven (dry heating) at 120 °C for 1 h. The MP supply to dairy cattle was modeled by three commonly used protein evaluation systems. The protein molecular structures were analyzed by Fourier transform/infrared-attenuated total reflectance molecular spectroscopy. The results showed that both the dry and moist heating increased the contents of truly absorbable rumen-undegraded protein (ARUP) and total MP and decreased the degraded protein balance (DPB). However, the moist-heated camelina seeds had a significantly higher (P seeds. The regression equations showed that intensities of the protein molecular structural bands can be used to estimate the contents of ARUP, MP, and DPB with high accuracy (R(2) > 0.70). These results show that protein molecular structural characteristics can be used to rapidly assess the MP supply to dairy cattle from raw and heat-treated camelina seeds.

  19. Tobacco BY-2 Media Component Optimization for a Cost-Efficient Recombinant Protein Production.

    Science.gov (United States)

    Häkkinen, Suvi T; Reuter, Lauri; Nuorti, Ninni; Joensuu, Jussi J; Rischer, Heiko; Ritala, Anneli

    2018-01-01

    Plant cells constitute an attractive platform for production of recombinant proteins as more and more animal-free products and processes are desired. One of the challenges in using plant cells as production hosts has been the costs deriving from expensive culture medium components. In this work, the aim was to optimize the levels of most expensive components in the nutrient medium without compromising the accumulation of biomass and recombinant protein yields. Wild-type BY-2 culture and transgenic tobacco BY-2 expressing green fluorescent protein-Hydrophobin I (GFP-HFBI) fusion protein were used to determine the most inexpensive medium composition. One particularly high-accumulating BY-2 clone, named 'Hulk,' produced 1.1 ± 0.2 g/l GFP-HFBI in suspension and kept its high performance during prolonged subculturing. In addition, both cultures were successfully cryopreserved enabling truly industrial application of this plant cell host. With the optimized culture medium, 43-55% cost reduction with regard to biomass and up to 69% reduction with regard to recombinant protein production was achieved.

  20. Interaction of a non-histone chromatin protein (high-mobility group protein 2) with DNA

    International Nuclear Information System (INIS)

    Goodwin, G.H.; Shooter, K.V.; Johns, E.W.

    1975-01-01

    The interaction with DNA of the calf thymus chromatin non-histone protein termed the high-mobility group protein 2 has been studied by sedimentation analysis in the ultracentrifuge and by measuring the binding of the 125 I-labelled protein to DNA. The results have been compared with those obtained previously by us [Eur. J. Biochem. (1974) 47, 263-270] for the interaction of high-mobility group protein 1 with DNA. Although the binding parameters are similar for these two proteins, high-mobility group protein 2 differs from high-mobility group protein 1 in that the former appears to change the shape of the DNA to a more compact form. The molecular weight of high-mobility group protein 2 has been determined by equilibrium sedimentation and a mean value of 26,000 was obtained. A low level of nuclease activity detected in one preparation of high-mobility group protein 2 has been investigated. (orig.) [de

  1. Dephosphorylation of chicken cardiac myofibril C-protein by protein phosphatases 1 and 2A

    International Nuclear Information System (INIS)

    Thysseril, T.J.; Hegazy, M.G.; Schlender, K.K.

    1987-01-01

    C-Protein, which is a regulatory component of cardiac muscle myofibrils, is phosphorylated in response to β-adrenergic agonists by a cAMP-dependent mechanism and dephosphorylated in response to cholinergic agonists. It is believed that the cAMP-dependent phosphorylation is due to cAMP-dependent protein kinase. The protein phosphatase(s) involved in the dephosphorylation of C-protein has not been determined. In this study, chicken cardiac C-protein was phosphorylated with the cAMP-dependent protein kinase to about 3 mol phosphate/mol C-protein. Incubation of [ 32 P]C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of 32 [P]. Phosphopeptide maps and phosphoamino acid analysis revealed that the major site(s) dephosphorylated by either phosphatase was a phosphothreonine residue(s) located on the same tryptic peptide and on the same CNBr fragment. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A completely dephosphorylated C-protein. Preliminary studies showed that the major protein phosphatase associated with the myofibril was phosphatase 2A. These results indicate the phosphatase 2A may be important in the regulation of the phosphorylation state of C-protein

  2. Reprint of Design of synthetic microbial communities for biotechnological production processes.

    Science.gov (United States)

    Jagmann, Nina; Philipp, Bodo

    2014-12-20

    In their natural habitats microorganisms live in multi-species communities, in which the community members exhibit complex metabolic interactions. In contrast, biotechnological production processes catalyzed by microorganisms are usually carried out with single strains in pure cultures. A number of production processes, however, may be more efficiently catalyzed by the concerted action of microbial communities. This review will give an overview of organismic interactions between microbial cells and of biotechnological applications of microbial communities. It focuses on synthetic microbial communities that consist of microorganisms that have been genetically engineered. Design principles for such synthetic communities will be exemplified based on plausible scenarios for biotechnological production processes. These design principles comprise interspecific metabolic interactions via cross-feeding, regulation by interspecific signaling processes via metabolites and autoinducing signal molecules, and spatial structuring of synthetic microbial communities. In particular, the implementation of metabolic interdependencies, of positive feedback regulation and of inducible cell aggregation and biofilm formation will be outlined. Synthetic microbial communities constitute a viable extension of the biotechnological application of metabolically engineered single strains and enlarge the scope of microbial production processes. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Effects of rumen-degradable protein:rumen-undegradable protein ratio and corn processing on production performance, nitrogen efficiency, and feeding behavior of Holstein dairy cows.

    Science.gov (United States)

    Savari, M; Khorvash, M; Amanlou, H; Ghorbani, G R; Ghasemi, E; Mirzaei, M

    2018-02-01

    This study was conducted to investigate the effects of the ratio of rumen-degradable protein (RDP) to rumen-undegradable protein (RUP) and corn processing method on production performance, nitrogen (N) efficiency, and feeding behavior of high-producing Holstein dairy cows. Twelve multiparous Holstein cows (second parity; milk yield = 48 ± 3 kg/d) were assigned to a replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments. Factor 1 was corn processing method [ground corn (GC) or steam flaked corn (SFC) with a flake density of about 390 g/L], and factor 2 was RDP:RUP ratio [low ratio (LR) = 60:40; high ratio (HR) = 65:35] based on crude protein (%). The crude protein concentrations were kept constant across the treatments (16.7% of DM). No significant interactions of main treatment effects occurred for lactation performance data. Cows fed 2 different RDP:RUP ratios exhibited similar dry matter intake (DMI), but those fed SFC showed decreased feed intake compared with those receiving GC (25.1 ± 0.48 vs. 26.2 ± 0.47 kg/d, respectively). Cows fed HR diets produced more milk than did those fed LR diets (44.4 ± 1.05 vs. 43.2 ± 1.05 kg/d, respectively). Milk fat content decreased but milk protein content increased in cows fed SFC compared with those fed GC. Feed efficiency (i.e., milk yield/DMI) was enhanced with increasing ratio of RDP:RUP (1.68 ± 0.04 vs. 1.74 ± 0.04 for LR and HR, respectively). Apparent N efficiency was higher in cows fed HR than in those fed LR (30.4 ± 0.61 vs. 29.2 ± 0.62, respectively). Compared with cows fed the GC-based diet, those receiving SFC exhibited lower values of N intake, N-NH 3 concentration, and fecal N excretion. Cows receiving SFC-based diets spent more time ruminating (min/kg of DMI) than did those fed GC. Although these results showed no interaction effects of RDP:RUP ratio and corn processing method on performance, higher RDP:RUP ratios and ground corn can be effective feeding strategies for

  4. Protein Solubility as Quality Index for Processed Soybean

    Directory of Open Access Journals (Sweden)

    Rodica Căpriţă

    2010-05-01

    Full Text Available Protein quality of soybean meal (SBM is linked to both the reduction of antinutritional factors (ANFs, and the optimization of protein digestibility. Both insufficient- and over-heating result in poor quality SBM. Inadequate heating fails to completely destroy the ANFs, which may have a detrimental impact on animal performance, while excessive heating reduces the availability of lysine via the Maillard reaction and possibly, to a lesser extent, of other amino acids. The objective of our study was to compare some biochemical laboratory procedures for assessing quality of SBM: urease index (UI, protein dispersibility index (PDI, KOH protein solubility (PS, and nitrogen solubility index (NSI. The experimental data reveal that UI is not useful to determine excessive heat treatment since additional heating has no effect on the urease index. KOH protein solubility remains high, during initial heat treatment. In marked contrast, the PDI and NSI decreased incrementally from 78% to 20% and from 97% to 60%, respectively, when heating 0 to 30 minutes. Combing the PDI test with the urease test could be useful to monitor soybean quality. SBM containing low UI (0.3 or below and high PDI (40 to 45% may indicate that the sample is definitely high quality because it has been adequately heat processed, but not overprocessed.

  5. Protein kinase CK2 in health and disease: Protein kinase CK2: from structures to insights

    DEFF Research Database (Denmark)

    Niefind, K; Raaf, J; Issinger, Olaf-Georg

    2009-01-01

    the critical region of CK2alpha recruitment is pre-formed in the unbound state. In CK2alpha the activation segment - a key element of protein kinase regulation - adapts invariably the typical conformation of the active enzymes. Recent structures of human CK2alpha revealed a surprising plasticity in the ATP......Within the last decade, 40 crystal structures corresponding to protein kinase CK2 (former name 'casein kinase 2'), to its catalytic subunit CK2alpha and to its regulatory subunit CK2beta were published. Together they provide a valuable, yet by far not complete basis to rationalize the biochemical...

  6. Protein Corona Prevents TiO2 Phototoxicity.

    Directory of Open Access Journals (Sweden)

    Maja Garvas

    Full Text Available TiO2 nanoparticles have generally low toxicity in the in vitro systems although some toxicity is expected to originate in the TiO2-associated photo-generated radical production, which can however be modulated by the radical trapping ability of the serum proteins. To explore the role of serum proteins in the phototoxicity of the TiO2 nanoparticles we measure viability of the exposed cells depending on the nanoparticle and serum protein concentrations.Fluorescence and spin trapping EPR spectroscopy reveal that the ratio between the nanoparticle and protein concentrations determines the amount of the nanoparticles' surface which is not covered by the serum proteins and is proportional to the amount of photo-induced radicals. Phototoxicity thus becomes substantial only at the protein concentration being too low to completely coat the nanotubes' surface.These results imply that TiO2 nanoparticles should be applied with ligands such as proteins when phototoxic effects are not desired - for example in cosmetics industry. On the other hand, the nanoparticles should be used in serum free medium or any other ligand free medium, when phototoxic effects are desired - as for efficient photodynamic cancer therapy.

  7. Effect of processing intensity on immunologically active bovine milk serum proteins

    NARCIS (Netherlands)

    Brick, Tabea; Ege, Markus; Boeren, Sjef; Böck, Andreas; Mutius, Von Erika; Vervoort, Jacques; Hettinga, Kasper

    2017-01-01

    Consumption of raw cow’s milk instead of industrially processed milk has been reported to protect children from developing asthma, allergies, and respiratory infections. Several heat-sensitive milk serum proteins have been implied in this effect though unbiased assessment of milk proteins in general

  8. Piezo proteins: regulators of mechanosensation and other cellular processes.

    Science.gov (United States)

    Bagriantsev, Sviatoslav N; Gracheva, Elena O; Gallagher, Patrick G

    2014-11-14

    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Peculiarities of the free radical processes in rat liver mitochondria under toxic hepatitis on the background of alimentary protein deficiency

    Directory of Open Access Journals (Sweden)

    G. P. Kopylchuk

    2016-04-01

    Full Text Available The rate of superoxide anion radical, hydroxyl radical and hydrogen peroxide generation, the level of oxidative modification of mitochondrial proteins in the liver of rats with toxic hepatitis was investigated on the background of alimentary protein deficiency. We did not find significant increases of the intensity of free radical processes in liver mitochondria of rats maintained on the protein-deficient ration. The most significant intensification of free radical processes in liver mitochondria is observed under the conditions of toxic hepatitis, induced on the background of alimentary protein deprivation. Under these conditions the aggravation of all studied forms of reactive oxygen species generation was observed in liver mitochondria. The generation rates were increased as follows: O2 – by 1.7 times, Н2О2 – by 1.5 times, •ОН – practically double on the background of accumulation of oxidized mitochondria-derived proteins. The established changes in thiol groups’ redox status of respiratory chain proteins insoluble in 0.05 M sodium-phosphate buffer (pH 11.5, and changes of their carbonyl derivatives content may be considered as one of the regulatory factors of mitochondrial energy-generating function.

  10. Interaction of the amyloid precursor protein-like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

    Energy Technology Data Exchange (ETDEWEB)

    Dahms, Sven O., E-mail: sdahms@fli-leibniz.de [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany); Mayer, Magnus C. [Freie Universität Berlin, Thielallee 63, 14195 Berlin (Germany); Miltenyi Biotec GmbH, Robert-Koch-Strasse 1, 17166 Teterow (Germany); Roeser, Dirk [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany); Multhaup, Gerd [McGill University Montreal, Montreal, Quebec H3G 1Y6 (Canada); Than, Manuel E., E-mail: sdahms@fli-leibniz.de [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany)

    2015-03-01

    Two X-ray structures of APLP1 E2 with and without a heparin dodecasaccharide are presented, revealing two distinct binding modes of the protein to heparan sulfate. The data provide a mechanistic explanation of how APP-like proteins bind to heparan sulfates and how they specifically recognize nonreducing structures of heparan sulfates. Beyond the pathology of Alzheimer’s disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects various (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Å resolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2){sub 2}–(heparin){sub 2} complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins.

  11. Adaptor proteins intersectin 1 and 2 bind similar proline-rich ligands but are differentially recognized by SH2 domain-containing proteins.

    Directory of Open Access Journals (Sweden)

    Olga Novokhatska

    Full Text Available BACKGROUND: Scaffolding proteins of the intersectin (ITSN family, ITSN1 and ITSN2, are crucial for the initiation stage of clathrin-mediated endocytosis. These proteins are closely related but have implications in distinct pathologies. To determine how these proteins could be separated in certain cell pathways we performed a comparative study of ITSNs. METHODOLOGY/PRINCIPAL FINDINGS: We have shown that endogenous ITSN1 and ITSN2 colocalize and form a complex in cells. A structural comparison of five SH3 domains, which mediated most ITSNs protein-protein interactions, demonstrated a similarity of their ligand-binding sites. We showed that the SH3 domains of ITSN2 bound well-established interactors of ITSN1 as well as newly identified ITSNs protein partners. A search for a novel interacting interface revealed multiple tyrosines that could be phosphorylated in ITSN2. Phosphorylation of ITSN2 isoforms but not ITSN1 short isoform was observed in various cell lines. EGF stimulation of HeLa cells enhanced tyrosine phosphorylation of ITSN2 isoforms and enabled their recognition by the SH2 domains of the Fyn, Fgr and Abl1 kinases, the regulatory subunit of PI3K, the adaptor proteins Grb2 and Crk, and phospholipase C gamma. The SH2 domains mentioned were unable to bind ITSN1 short isoform. CONCLUSIONS/SIGNIFICANCE: Our results indicate that during evolution of vertebrates ITSN2 acquired a novel protein-interaction interface that allows its specific recognition by the SH2 domains of signaling proteins. We propose that these data could be important to understand the functional diversity of paralogous ITSN proteins.

  12. The protein network surrounding the human telomere repeat binding factors TRF1, TRF2, and POT1.

    Directory of Open Access Journals (Sweden)

    Richard J Giannone

    2010-08-01

    Full Text Available Telomere integrity (including telomere length and capping is critical in overall genomic stability. Telomere repeat binding factors and their associated proteins play vital roles in telomere length regulation and end protection. In this study, we explore the protein network surrounding telomere repeat binding factors, TRF1, TRF2, and POT1 using dual-tag affinity purification in combination with multidimensional protein identification technology liquid chromatography--tandem mass spectrometry (MudPIT LC-MS/MS. After control subtraction and data filtering, we found that TRF2 and POT1 co-purified all six members of the telomere protein complex, while TRF1 identified five of six components at frequencies that lend evidence towards the currently accepted telomere architecture. Many of the known TRF1 or TRF2 interacting proteins were also identified. Moreover, putative associating partners identified for each of the three core components fell into functional categories such as DNA damage repair, ubiquitination, chromosome cohesion, chromatin modification/remodeling, DNA replication, cell cycle and transcription regulation, nucleotide metabolism, RNA processing, and nuclear transport. These putative protein-protein associations may participate in different biological processes at telomeres or, intriguingly, outside telomeres.

  13. Aggregation of egg white proteins with pulsed electric fields and thermal processes.

    Science.gov (United States)

    Wu, Li; Zhao, Wei; Yang, Ruijin; Yan, Wenxu; Sun, Qianyan

    2016-08-01

    Pulsed electric field (PEF) processing is progressing towards application for liquid egg to ensure microbial safety. However, it usually causes protein aggregation, and the mechanism is still unclear. In this study, egg white protein was applied to investigate the changes in protein structure and mechanism of aggregates formation and a comparison was made with thermal treatment. Soluble protein content decreased with the increase of turbidity after both treatments. Fluorescence intensity and free sulfhydryl content were increased after being treated at 70 °C for 4 min. Less-remarkable changes of hydrophobicity were observed after PEF treatments (30 kV cm(-1) , 800 µs). Soluble and insoluble aggregates were observed by thermal treatment, and disulfide bonds were the main binding forces. The main components of insoluble aggregates formed by thermal treatment were ovotransferrin (30.58%), lysozyme (18.47%) and ovalbumin (14.20%). While only insoluble aggregates were detected during PEF processes, which consists of ovotransferrin (11.86%), lysozyme (21.11%) and ovalbumin (31.07%). Electrostatic interaction played a very important role in the aggregates formation. PEF had a minor impact on the structure of egg white protein. PEF had insignificant influence on heat-sensitive protein, indicating that PEF has potential in processing food with high biological activity and heat sensitive properties. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  14. Shoc2/Sur8 protein regulates neurite outgrowth.

    Directory of Open Access Journals (Sweden)

    Gonzalo Leon

    Full Text Available The Shoc2 protein has been implicated in the positive regulation of the Ras-ERK pathway by increasing the functional binding interaction between Ras and Raf, leading to increased ERK activity. Here we found that Shoc2 overexpression induced sustained ERK phosphorylation, notably in the case of EGF stimulation, and Shoc2 knockdown inhibited ERK activation. We demonstrate that ectopic overexpression of human Shoc2 in PC12 cells significantly promotes neurite extension in the presence of EGF, a stimulus that induces proliferation rather than differentiation in these cells. Finally, Shoc2 depletion reduces both NGF-induced neurite outgrowth and ERK activation in PC12 cells. Our data indicate that Shoc2 is essential to modulate the Ras-ERK signaling outcome in cell differentiation processes involved in neurite outgrowth.

  15. Pdsg1 and Pdsg2, novel proteins involved in developmental genome remodelling in Paramecium.

    Directory of Open Access Journals (Sweden)

    Miroslav Arambasic

    Full Text Available The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2, involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization.

  16. Pdsg1 and Pdsg2, novel proteins involved in developmental genome remodelling in Paramecium.

    Science.gov (United States)

    Arambasic, Miroslav; Sandoval, Pamela Y; Hoehener, Cristina; Singh, Aditi; Swart, Estienne C; Nowacki, Mariusz

    2014-01-01

    The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2), involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization.

  17. Pico- and femtosecond laser-induced crosslinking of protein microstructures: evaluation of processability and bioactivity

    Energy Technology Data Exchange (ETDEWEB)

    Turunen, S; Kaepylae, E; Kellomaeki, M [Tampere University of Technology, Department of Biomedical Engineering, PO Box 692, 33101 Tampere (Finland); Terzaki, K; Fotakis, C; Farsari, M [Institute of Electronic Structure and Laser (IESL), Foundation for Research and Technology Hellas (FORTH), N. Plastira 100, 70013, Heraklion, Crete (Greece); Viitanen, J, E-mail: elli.kapyla@tut.fi [VTT Technical Research Centre of Finland, PO Box 1300, 33101 Tampere (Finland)

    2011-12-15

    This study reports the pico- and femtosecond laser-induced photocrosslinking of protein microstructures. The capabilities of a picosecond Nd:YAG laser to promote multiphoton excited crosslinking of proteins were evaluated by fabricating 2D and 3D microstructures of avidin, bovine serum albumin (BSA) and biotinylated bovine serum albumin (bBSA). The multiphoton absorption-induced photocrosslinking of proteins was demonstrated here for the first time with a non-toxic biomolecule flavin mononucleotide (FMN) as the photosensitizer. Sub-micrometer and micrometer scale structures were fabricated from several different compositions of protein and photosensitizer by varying the average laser power and scanning speed in order to determine the optimal process parameters for efficient photocrosslinking. In addition, the retention of ligand-binding ability of the crosslinked protein structures was shown by fluorescence imaging of immobilized biotin or streptavidin conjugated fluorescence labels. The surface topography and the resolution of the protein patterns fabricated with the Nd:YAG laser were compared to the results obtained with a femtosecond Ti:Sapphire laser. Quite similar grain characteristics and comparable feature sizes were achieved with both laser sources, which demonstrates the utility of the low-cost Nd:YAG microlaser for direct laser writing of protein microstructures.

  18. Pico- and femtosecond laser-induced crosslinking of protein microstructures: evaluation of processability and bioactivity

    International Nuclear Information System (INIS)

    Turunen, S; Kaepylae, E; Kellomaeki, M; Terzaki, K; Fotakis, C; Farsari, M; Viitanen, J

    2011-01-01

    This study reports the pico- and femtosecond laser-induced photocrosslinking of protein microstructures. The capabilities of a picosecond Nd:YAG laser to promote multiphoton excited crosslinking of proteins were evaluated by fabricating 2D and 3D microstructures of avidin, bovine serum albumin (BSA) and biotinylated bovine serum albumin (bBSA). The multiphoton absorption-induced photocrosslinking of proteins was demonstrated here for the first time with a non-toxic biomolecule flavin mononucleotide (FMN) as the photosensitizer. Sub-micrometer and micrometer scale structures were fabricated from several different compositions of protein and photosensitizer by varying the average laser power and scanning speed in order to determine the optimal process parameters for efficient photocrosslinking. In addition, the retention of ligand-binding ability of the crosslinked protein structures was shown by fluorescence imaging of immobilized biotin or streptavidin conjugated fluorescence labels. The surface topography and the resolution of the protein patterns fabricated with the Nd:YAG laser were compared to the results obtained with a femtosecond Ti:Sapphire laser. Quite similar grain characteristics and comparable feature sizes were achieved with both laser sources, which demonstrates the utility of the low-cost Nd:YAG microlaser for direct laser writing of protein microstructures.

  19. Effect of the synthetic cannabinoid HU-210 on quorum sensing and on the production of quorum sensing-mediated virulence factors by Vibrio harveyi

    OpenAIRE

    Soni, Divya; Smoum, Reem; Breuer, Aviva; Mechoulam, Raphael; Steinberg, Doron

    2015-01-01

    Background Bacterial populations communicate through the cell density-dependent mechanism of quorum sensing (QS). Vibrio harveyi, one of the best studied model organisms for QS, was used to explore effects of the synthetic cannabinoid HU-210 on QS and different QS-regulated physiological processes in bacteria. Results Analysis of QS-regulated bioluminescence in wild-type and mutant strains of V. harveyi revealed that HU-210 affects the autoinducer-2 (AI-2) pathway, one of three known QS casca...

  20. Topological analysis of Chlamydia trachomatis L2 outer membrane protein 2

    DEFF Research Database (Denmark)

    Mygind, P; Christiansen, Gunna; Birkelund, Svend

    1998-01-01

    Using monospecific polyclonal antisera to different parts of Chlamydia trachomatis L2 outer membrane protein 2 (Omp2), we show that the protein is localized at the inner surface of the outer membrane. Omp2 becomes immunoaccessible when Chlamydia elementary bodies are treated with dithiothreitol...

  1. Processing of metacaspase 2 from Trypanosoma brucei (TbMCA2) broadens its substrate specificity.

    Science.gov (United States)

    Gilio, Joyce M; Marcondes, Marcelo F; Ferrari, Débora; Juliano, Maria A; Juliano, Luiz; Oliveira, Vitor; Machado, Maurício F M

    2017-04-01

    Metacaspases are members of the cysteine peptidase family and may be implicated in programmed cell death in plants and lower eukaryotes. These proteases exhibit calcium-dependent activity and specificity for arginine residues at P 1 . In contrast to caspases, they do not require processing or dimerization for activity. Indeed, unprocessed metacaspase-2 of Trypanosoma brucei (TbMCA2) is active; however, it has been shown that cleavages at Lys 55 and Lys 268 increase TbMCA2 hydrolytic activity on synthetic substrates. The processed TbMCA2 comprises 3 polypeptide chains that remain attached by non-covalent bonds. Replacement of Lys 55 and Lys 268 with Gly via site-directed mutagenesis results in non-processed but enzymatically active mutant, TbMCA2 K55/268G. To investigate the importance of this processing for the activity and specificity of TbMCA2, we performed activity assays comparing the non-processed mutant (TbMCA2 K55/268G) with the processed TbMCA2 form. Significant differences between TbMCA2 WT (processed form) and TbMCA2 K55/268G (non-processed form) were observed. Specifically, we verified that although non-processed TbMCA2 is active when assayed with small synthetic substrates, the TbMCA2 form does not exhibit hydrolytic activity on large substrates such as azocasein, while processed TbMCA2 is able to readily digest this protein. Such differences can be relevant for understanding the physiological regulation and function of TbMCA2. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Cellular Reprogramming Employing Recombinant Sox2 Protein

    Directory of Open Access Journals (Sweden)

    Marc Thier

    2012-01-01

    Full Text Available Induced pluripotent stem (iPS cells represent an attractive option for the derivation of patient-specific pluripotent cells for cell replacement therapies as well as disease modeling. To become clinically meaningful, safe iPS cells need to be generated exhibiting no permanent genetic modifications that are caused by viral integrations of the reprogramming transgenes. Recently, various experimental strategies have been applied to accomplish transgene-free derivation of iPS cells, including the use of nonintegrating viruses, episomal expression, or excision of transgenes after reprogramming by site-specific recombinases or transposases. A straightforward approach to induce reprogramming factors is the direct delivery of either synthetic mRNA or biologically active proteins. We previously reported the generation of cell-permeant versions of Oct4 (Oct4-TAT and Sox2 (Sox2-TAT proteins and showed that Oct4-TAT is reprogramming-competent, that is, it can substitute for Oct4-encoding virus. Here, we explore conditions for enhanced Sox2-TAT protein stabilization and functional delivery into somatic cells. We show that cell-permeant Sox2 protein can be stabilized by lipid-rich albumin supplements in serum replacement or low-serum-supplemented media. Employing optimized conditions for protein delivery, we demonstrate that Sox2-TAT protein is able to substitute for viral Sox2. Sox2-piPS cells express pluripotency-associated markers and differentiate into all three germ layers.

  3. Karyopherin alpha2 is essential for rRNA transcription and protein synthesis in proliferative keratinocytes.

    Directory of Open Access Journals (Sweden)

    Noriko Umegaki-Arao

    Full Text Available Karyopherin proteins mediate nucleocytoplasmic trafficking and are critical for protein and RNA subcellular localization. Recent studies suggest KPNA2 expression is induced in tumor cells and is strongly associated with prognosis, although the precise roles and mechanisms of KPNA2 overexpression in proliferative disorders have not been defined. We found that KPNA2 expression is induced in various proliferative disorders of the skin such as psoriasis, Bowen's disease, actinic keratosis, squamous cell carcinoma, Paget's disease, Merkel cell carcinoma, and mycosis fungoides. siRNA-mediated KPNA suppression revealed that KPNA2 is essential for significant suppression of HaCaT proliferation under starvation conditions. Ribosomal RNA transcription and protein synthesis were suppressed by starvation combined with knockdown of KPNA (including KPNA2 expression. KPNA2 localized to the nucleolus and interacted with proteins associated with mRNA processing, ribonucleoprotein complex biogenesis, chromatin modification, and transcription, as demonstrated by tandem affinity purification and mass spectrometry. KPNA2 may be an important promoter of ribosomal RNA and protein synthesis in tumor cells.

  4. Autoinducer-2 activity produced by bacteria found in smear of surface ripened cheeses

    DEFF Research Database (Denmark)

    Gori, Klaus; Moslehi Jenabian, Saloomeh; Purrotti, Micol

    2011-01-01

    -2) activity using the Vibrio harveyi (BB170) bioluminescence assay. In contrast, Brevibacterium casei and Brevibacterium linens strains were not found to have AI-2 activity. When exposed to low pH and high NaCl concentrations, AI-2 activities increased between 5.0 and 11.6× for C. casei 44701, M...

  5. Preparation and characterization of protein isolate from Yellowfin tuna Thunnus albacares roe by isoelectric solubilization/precipitation process

    Directory of Open Access Journals (Sweden)

    Hyun Ji Lee

    2016-05-01

    Full Text Available Abstract Isoelectric solubilization/precipitation (ISP processing allows selective, pH-induced water solubility of proteins with concurrent separation of lipids and removal of materials not intended for human consumption such as bone, scales, skin, etc. Recovered proteins retain functional properties and nutritional value. Four roe protein isolates (RPIs from yellowfin tuna roe were prepared under different solubilization and precipitation condition (pH 11/4.5, pH 11/5.5, pH 12/4.5 and pH 12/5.5. RPIs contained 2.3–5.0 % moisture, 79.1–87.8 % protein, 5.6–7.4 % lipid and 3.0–3.8 % ash. Protein content of RPI-1 and RPI-2 precipitated at pH 4.5 and 5.5 after alkaline solubilization at pH 11, was higher than those of RPI-3 and RPI-4 after alkaline solubilization at pH 12 (P < 0.05. Lipid content (5.6–7.4 % of RPIs was lower than that of freeze-dried concentrate (10.6 %. And leucine and lysine of RPIs were the most abundant amino acids (8.8–9.4 and 8.5–8.9 g/100 g protein, respectively. S, Na, P, K as minerals were the major elements in RPIs. SDS-PAGE of RPIs showed bands at 100, 45, 25 and 15 K. Moisture and protein contents of process water as a 2’nd byproduct were 98.9–99.0 and 1.3–1.8 %, respectively. Therefore, yellowfin tuna roe isolate could be a promising source of valuable nutrients for human food and animal feeds.

  6. Disparate effects of p24alpha and p24delta on secretory protein transport and processing.

    Directory of Open Access Journals (Sweden)

    Jeroen R P M Strating

    Full Text Available BACKGROUND: The p24 family is thought to be somehow involved in endoplasmic reticulum (ER-to-Golgi protein transport. A subset of the p24 proteins (p24alpha(3, -beta(1, -gamma(3 and -delta(2 is upregulated when Xenopus laevis intermediate pituitary melanotrope cells are physiologically activated to produce vast amounts of their major secretory cargo, the prohormone proopiomelanocortin (POMC. METHODOLOGY/PRINCIPAL FINDINGS: Here we find that transgene expression of p24alpha(3 or p24delta(2 specifically in the Xenopus melanotrope cells in both cases causes an effective displacement of the endogenous p24 proteins, resulting in severely distorted p24 systems and disparate melanotrope cell phenotypes. Transgene expression of p24alpha(3 greatly reduces POMC transport and leads to accumulation of the prohormone in large, ER-localized electron-dense structures, whereas p24delta(2-transgenesis does not influence the overall ultrastructure of the cells nor POMC transport and cleavage, but affects the Golgi-based processes of POMC glycomaturation and sulfation. CONCLUSIONS/SIGNIFICANCE: Transgenic expression of two distinct p24 family members has disparate effects on secretory pathway functioning, illustrating the specificity and non-redundancy of our transgenic approach. We conclude that members of the p24 family furnish subcompartments of the secretory pathway with specific sets of machinery cargo to provide the proper microenvironments for efficient and correct secretory protein transport and processing.

  7. Identification of brain-specific angiogenesis inhibitor 2 as an interaction partner of glutaminase interacting protein

    International Nuclear Information System (INIS)

    Zencir, Sevil; Ovee, Mohiuddin; Dobson, Melanie J.; Banerjee, Monimoy; Topcu, Zeki; Mohanty, Smita

    2011-01-01

    Highlights: → Brain-specific angiogenesis inhibitor 2 (BAI2) is a new partner protein for GIP. → BAI2 interaction with GIP was revealed by yeast two-hybrid assay. → Binding of BAI2 to GIP was characterized by NMR, CD and fluorescence. → BAI2 and GIP binding was mediated through the C-terminus of BAI2. -- Abstract: The vast majority of physiological processes in living cells are mediated by protein-protein interactions often specified by particular protein sequence motifs. PDZ domains, composed of 80-100 amino acid residues, are an important class of interaction motif. Among the PDZ-containing proteins, glutaminase interacting protein (GIP), also known as Tax Interacting Protein TIP-1, is unique in being composed almost exclusively of a single PDZ domain. GIP has important roles in cellular signaling, protein scaffolding and modulation of tumor growth and interacts with a number of physiological partner proteins, including Glutaminase L, β-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kir 2.3. To identify the network of proteins that interact with GIP, a human fetal brain cDNA library was screened using a yeast two-hybrid assay with GIP as bait. We identified brain-specific angiogenesis inhibitor 2 (BAI2), a member of the adhesion-G protein-coupled receptors (GPCRs), as a new partner of GIP. BAI2 is expressed primarily in neurons, further expanding GIP cellular functions. The interaction between GIP and the carboxy-terminus of BAI2 was characterized using fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy assays. These biophysical analyses support the interaction identified in the yeast two-hybrid assay. This is the first study reporting BAI2 as an interaction partner of GIP.

  8. Myeloperoxidase-mediated protein lysine oxidation generates 2-aminoadipic acid and lysine nitrile in vivo.

    Science.gov (United States)

    Lin, Hongqiao; Levison, Bruce S; Buffa, Jennifer A; Huang, Ying; Fu, Xiaoming; Wang, Zeneng; Gogonea, Valentin; DiDonato, Joseph A; Hazen, Stanley L

    2017-03-01

    Recent studies reveal 2-aminoadipic acid (2-AAA) is both elevated in subjects at risk for diabetes and mechanistically linked to glucose homeostasis. Prior studies also suggest enrichment of protein-bound 2-AAA as an oxidative post-translational modification of lysyl residues in tissues associated with degenerative diseases of aging. While in vitro studies suggest redox active transition metals or myeloperoxidase (MPO) generated hypochlorous acid (HOCl) may produce protein-bound 2-AAA, the mechanism(s) responsible for generation of 2-AAA during inflammatory diseases are unknown. In initial studies we observed that traditional acid- or base-catalyzed protein hydrolysis methods previously employed to measure tissue 2-AAA can artificially generate protein-bound 2-AAA from an alternative potential lysine oxidative product, lysine nitrile (LysCN). Using a validated protease-based digestion method coupled with stable isotope dilution LC/MS/MS, we now report protein bound 2-AAA and LysCN are both formed by hypochlorous acid (HOCl) and the MPO/H 2 O 2 /Cl - system of leukocytes. At low molar ratio of oxidant to target protein N ε -lysine moiety, 2-AAA is formed via an initial N ε -monochloramine intermediate, which ultimately produces the more stable 2-AAA end-product via sequential generation of transient imine and semialdehyde intermediates. At higher oxidant to target protein N ε -lysine amine ratios, protein-bound LysCN is formed via initial generation of a lysine N ε -dichloramine intermediate. In studies employing MPO knockout mice and an acute inflammation model, we show that both free and protein-bound 2-AAA, and in lower yield, protein-bound LysCN, are formed by MPO in vivo during inflammation. Finally, both 2-AAA and to lesser extent LysCN are shown to be enriched in human aortic atherosclerotic plaque, a tissue known to harbor multiple MPO-catalyzed protein oxidation products. Collectively, these results show that MPO-mediated oxidation of protein lysyl

  9. HPDB-Haskell library for processing atomic biomolecular structures in Protein Data Bank format.

    Science.gov (United States)

    Gajda, Michał Jan

    2013-11-23

    Protein DataBank file format is used for the majority of biomolecular data available today. Haskell is a lazy functional language that enjoys a high-level class-based type system, a growing collection of useful libraries and a reputation for efficiency. I present a fast library for processing biomolecular data in the Protein Data Bank format. I present benchmarks indicating that this library is faster than other frequently used Protein Data Bank parsing programs. The proposed library also features a convenient iterator mechanism, and a simple API modeled after BioPython. I set a new standard for convenience and efficiency of Protein Data Bank processing in a Haskell library, and release it to open source.

  10. Influences of different thermal processings in milk, bovine meat and frog protein structure.

    Science.gov (United States)

    Coura Oliveira, Tatiana; Lopes Lima, Samuel; Bressan, Josefina

    2013-01-01

    Several studies have associated the digestibility of proteins to its imunogenic potential. Though, it was objectified to evaluate the impact of the thermal processing with high and low temperatures on the proteins structure of three types of foods, by means of the digestibility in vitro and electroforesis en gel de poliacrilamida. The pasteurize was observed in such a way, firing 95 ºC during 15 minutes, how much freeze dried causes qualitative and quantitative modifications of constituent proteins of the food. The most sensible proteins to the increasing thermal processing order were beef, frog meat, and the last, cow milk. Copyright © AULA MEDICA EDICIONES 2013. Published by AULA MEDICA. All rights reserved.

  11. Improved protein hydrogen/deuterium exchange mass spectrometry platform with fully automated data processing.

    Science.gov (United States)

    Zhang, Zhongqi; Zhang, Aming; Xiao, Gang

    2012-06-05

    Protein hydrogen/deuterium exchange (HDX) followed by protease digestion and mass spectrometric (MS) analysis is accepted as a standard method for studying protein conformation and conformational dynamics. In this article, an improved HDX MS platform with fully automated data processing is described. The platform significantly reduces systematic and random errors in the measurement by introducing two types of corrections in HDX data analysis. First, a mixture of short peptides with fast HDX rates is introduced as internal standards to adjust the variations in the extent of back exchange from run to run. Second, a designed unique peptide (PPPI) with slow intrinsic HDX rate is employed as another internal standard to reflect the possible differences in protein intrinsic HDX rates when protein conformations at different solution conditions are compared. HDX data processing is achieved with a comprehensive HDX model to simulate the deuterium labeling and back exchange process. The HDX model is implemented into the in-house developed software MassAnalyzer and enables fully unattended analysis of the entire protein HDX MS data set starting from ion detection and peptide identification to final processed HDX output, typically within 1 day. The final output of the automated data processing is a set (or the average) of the most possible protection factors for each backbone amide hydrogen. The utility of the HDX MS platform is demonstrated by exploring the conformational transition of a monoclonal antibody by increasing concentrations of guanidine.

  12. Effect of Processing on the In Vitro and In Vivo Protein Quality of Beans (Phaseolus vulgaris and Vicia Faba

    Directory of Open Access Journals (Sweden)

    Matthew G. Nosworthy

    2018-05-01

    Full Text Available In this work, the protein quality of different bean types after undergoing the preparatory methods of baking, cooking and extrusion was assayed. Protein quality was assessed using a rodent bioassay to evaluate growth and protein digestibility while amino acid composition was determined via HPLC. In vivo protein digestibility was compared to an in vitro assessment method. The average protein digestibility corrected amino acid score (PDCAAS for processed beans was higher than the digestible indispensable amino acid score (DIAAS (61% vs. 45%. Extrusion/cooking of Phaseolus varieties resulted in higher PDCAAS (66% on average and DIAAS values (61% on average than baked (52% and 48% while baked faba beans had higher PDCAAS (66% and DIAAS (61% values. A significant correlation was found between PDCAAS and in vitro PDCAAS (R2 = 0.7497. This demonstrates which bean processing method will generate the optimal protein quality, which has benefits for both industrial production and individual domestic preparation.

  13. [A study of recombinant human sestrin 1 and sestrin 2 proteins produced in a prokaryotic system].

    Science.gov (United States)

    Rai, N; Kumar, R; Haque, Md A; Hassan, Md I; Dey, S

    2017-01-01

    Sestrins are highly conserved stress-inducible proteins capable of suppressing the production of ROS and signalling through mTORC1. Here we report a study of human sestrin1 (sesn1) and sestrin2 (sesn2) proteins produced in a pET28^(+) vector based prokaryotic system. Mass spectrometry analysis, western blot and surface plasmon resonance (SPR) of affinity purified sesn1 and sesn2 proteins confirmed their identity; biophysical characteristics were observed using circular dichroism (CD) showing that sesn1 and sesn2 have a predominant α-helical structure. Here we describe a simple, one step purification process to purify a large amount of sestrin proteins with significant yield. Further study of recombinant human sestrins may further facilitate the understanding of their roles in eukaryotic cells.

  14. Processing and characteristics of canola protein-based biodegradable packaging: A review.

    Science.gov (United States)

    Zhang, Yachuan; Liu, Qiang; Rempel, Curtis

    2018-02-11

    Interest increased recently in manufacturing food packaging, such as films and coatings, from protein-based biopolymers. Among various protein sources, canola protein is a novel source for manufacturing polymer films. It can be concentrated or isolated by aqueous extraction technology followed by protein precipitation. Using this procedure, it was claimed that more than 99% of protein was extracted from the defatted canola meal, and protein recovery was 87.5%. Canola protein exhibits thermoplastic properties when plasticizers are present, including water, glycerol, polyethylene glycol, and sorbitol. Addition of these plasticizers allows the canola protein to undergo glass transition and facilitates deformation and processability. Normally, canola protein-based bioplastics showed low mechanical properties, which had tensile strength (TS) of 1.19 to 4.31 MPa. So, various factors were explored to improve it, including blending with synthetic polymers, modifying protein functionality through controlled denaturation, and adding cross-linking agents. Canola protein-based bioplastics were reported to have glass transition temperature, T g , below -50°C but it highly depends on the plasticizer content. Canola protein-based bioplastics have demonstrated comparable mechanical and moisture barrier properties compared with other plant protein-based bioplastics. They have great potential in food packaging applications, including their use as wraps, sacks, sachets, or pouches.

  15. Heterotrimeric G protein beta1gamma2 subunits change orientation upon complex formation with G protein-coupled receptor kinase 2 (GRK2) on a model membrane.

    Science.gov (United States)

    Boughton, Andrew P; Yang, Pei; Tesmer, Valerie M; Ding, Bei; Tesmer, John J G; Chen, Zhan

    2011-09-13

    Few experimental techniques can assess the orientation of peripheral membrane proteins in their native environment. Sum Frequency Generation (SFG) vibrational spectroscopy was applied to study the formation of the complex between G protein-coupled receptor (GPCR) kinase 2 (GRK2) and heterotrimeric G protein β(1)γ(2) subunits (Gβγ) at a lipid bilayer, without any exogenous labels. The most likely membrane orientation of the GRK2-Gβγ complex differs from that predicted from the known protein crystal structure, and positions the predicted receptor docking site of GRK2 such that it would more optimally interact with GPCRs. Gβγ also appears to change its orientation after binding to GRK2. The developed methodology is widely applicable for the study of other membrane proteins in situ.

  16. C2 Domains as Protein-Protein Interaction Modules in the Ciliary Transition Zone

    Directory of Open Access Journals (Sweden)

    Kim Remans

    2014-07-01

    Full Text Available RPGR-interacting protein 1 (RPGRIP1 is mutated in the eye disease Leber congenital amaurosis (LCA and its structural homolog, RPGRIP1-like (RPGRIP1L, is mutated in many different ciliopathies. Both are multidomain proteins that are predicted to interact with retinitis pigmentosa G-protein regulator (RPGR. RPGR is mutated in X-linked retinitis pigmentosa and is located in photoreceptors and primary cilia. We solved the crystal structure of the complex between the RPGR-interacting domain (RID of RPGRIP1 and RPGR and demonstrate that RPGRIP1L binds to RPGR similarly. RPGRIP1 binding to RPGR affects the interaction with PDEδ, the cargo shuttling factor for prenylated ciliary proteins. RPGRIP1-RID is a C2 domain with a canonical β sandwich structure that does not bind Ca2+ and/or phospholipids and thus constitutes a unique type of protein-protein interaction module. Judging from the large number of C2 domains in most of the ciliary transition zone proteins identified thus far, the structure presented here seems to constitute a cilia-specific module that is present in multiprotein transition zone complexes.

  17. Microbial Protein Production from Candida tropicalis ATCC13803 in a Submerged Batch Fermentation Process

    Directory of Open Access Journals (Sweden)

    Sahar Golaghaiee

    2017-01-01

    Full Text Available Background and Objective: Microbial protein production can resolve one of the major world challenges, i.e. lack of protein sources. Candida tropicalis growth was investigated to specify a medium to reach the highest cell proliferation and protein production.Material and Methods: Fractional factorial design and the index of signal to noise ratio were applied for optimization of microbial protein production. Optimization process was conducted based on the experimental results of Taguchi approach designs. Fermentationwas performed at 25oC and the agitation speed of 300 rpm for 70 h. Ammonium sulfate, iron sulfate, glycine and glucose concentrations were considered as process variables. Optimization of the culture medium composition was conducted in order to obtain the highest cell biomass concentration and protein content. Experiment design was performed based on the Taguchi approach and L-16 orthogonal arrays using Qualitek-4 software.Results and Conclusion: Maximum biomass of 8.72 log (CFU ml-1 was obtained using the optimized medium with 0.3, 0.15, 2 and 80 g l-1 of ammonium sulfate, iron sulfate, glycine and glucose, respectively. Iron sulfate and ammonium sulfate with 41.76% (w w-1 and 35.27% (w w-1 contributions, respectively, were recognized as the main components for cell growth. Glucose and glycine with 17.12% and 5.86% (w w-1 contributions,respectively, also affected cell production. The highest interaction severity index of +54.16% was observed between glycine and glucose while the least one of +0.43% was recorded for ammonium sulfate and glycine. A deviation of 7% between the highestpredicted cell numbers and the experimented count confirms the suitability of the applied statistical method. High protein content of 52.16% (w w-1 as well as low fat and nucleic acids content suggest that Candida tropicalis is a suitable case for commercial processes.Conflict of interest: The authors declare that there is no conflict of interest.

  18. Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

    International Nuclear Information System (INIS)

    Rodriguez, J.B.R.; Muzi-Filho, H.; Valverde, R.H.F.; Quintas, L.E.M.; Noel, F.; Einicker-Lamas, M.; Cunha, V.M.N.

    2013-01-01

    Ca 2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca 2+ -ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca 2+ -ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca 2+ (Ca 0.5 = 780 nM) and a low sensitivity to vanadate (IC 50 = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca 2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca 2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca 2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca 2+ pumping activity

  19. Single cell analysis of Vibrio harveyi uncovers functional heterogeneity in response to quorum sensing signals

    Directory of Open Access Journals (Sweden)

    Anetzberger Claudia

    2012-09-01

    Full Text Available Abstract Background Vibrio harveyi and closely related species are important pathogens in aquaculture. A complex quorum sensing cascade involving three autoinducers controls bioluminescence and several genes encoding virulence factors. Single cell analysis of a V. harveyi population has already indicated intercellular heterogeneity in the production of bioluminescence. This study was undertaken to analyze the expression of various autoinducer-dependent genes in individual cells. Results Here we used reporter strains bearing promoter::gfp fusions to monitor the induction/repression of three autoinducer-regulated genes in wild type conjugates at the single cell level. Two genes involved in pathogenesis - vhp and vscP, which code for an exoprotease and a component of the type III secretion system, respectively, and luxC (the first gene in the lux operon were chosen for analysis. The lux operon and the exoprotease gene are induced, while vscP is repressed at high cell density. As controls luxS and recA, whose expression is not dependent on autoinducers, were examined. The responses of the promoter::gfp fusions in individual cells from the same culture ranged from no to high induction. Importantly, simultaneous analysis of two autoinducer induced phenotypes, bioluminescence (light detection and exoproteolytic activity (fluorescence of a promoter::gfp fusion, in single cells provided evidence for functional heterogeneity within a V. harveyi population. Conclusions Autoinducers are not only an indicator for cell density, but play a pivotal role in the coordination of physiological activities within the population.

  20. Single cell analysis of Vibrio harveyi uncovers functional heterogeneity in response to quorum sensing signals.

    Science.gov (United States)

    Anetzberger, Claudia; Schell, Ursula; Jung, Kirsten

    2012-09-18

    Vibrio harveyi and closely related species are important pathogens in aquaculture. A complex quorum sensing cascade involving three autoinducers controls bioluminescence and several genes encoding virulence factors. Single cell analysis of a V. harveyi population has already indicated intercellular heterogeneity in the production of bioluminescence. This study was undertaken to analyze the expression of various autoinducer-dependent genes in individual cells. Here we used reporter strains bearing promoter::gfp fusions to monitor the induction/repression of three autoinducer-regulated genes in wild type conjugates at the single cell level. Two genes involved in pathogenesis - vhp and vscP, which code for an exoprotease and a component of the type III secretion system, respectively, and luxC (the first gene in the lux operon) were chosen for analysis. The lux operon and the exoprotease gene are induced, while vscP is repressed at high cell density. As controls luxS and recA, whose expression is not dependent on autoinducers, were examined. The responses of the promoter::gfp fusions in individual cells from the same culture ranged from no to high induction. Importantly, simultaneous analysis of two autoinducer induced phenotypes, bioluminescence (light detection) and exoproteolytic activity (fluorescence of a promoter::gfp fusion), in single cells provided evidence for functional heterogeneity within a V. harveyi population. Autoinducers are not only an indicator for cell density, but play a pivotal role in the coordination of physiological activities within the population.

  1. The physical basis of bacterial quorum communication

    CERN Document Server

    2015-01-01

    This book aims to educate physical scientists and quantitatively-oriented biologists on the application of physical experimentation and analysis, together with appropriate modeling, to understanding and interpreting microbial chemical communication and especially quorum sensing (QS). Quorum sensing describes a chemical communication behavior that is nearly universal among bacteria. Individual cells release a diffusible small molecule (an autoinducer) into their environment. A high concentration of this autoinducer serves as a signal of high population density, triggering new patterns of gene expression throughout the population. However QS is often much more complex than simple census-taking. Many QS bacteria produce and detect multiple autoinducers, which generate quorum signal cross talk with each other and with other bacterial species. QS gene regulatory networks operate in physically complex environments and respond to a range of inputs in addition to autoinducer signals. While many individual QS systems ...

  2. Loss of Niemann-Pick C1 or C2 protein results in similar biochemical changes suggesting that these proteins function in a common lysosomal pathway.

    Directory of Open Access Journals (Sweden)

    Sayali S Dixit

    Full Text Available Niemann-Pick Type C (NPC disease is a lysosomal storage disorder characterized by accumulation of unesterified cholesterol and other lipids in the endolysosomal system. NPC disease results from a defect in either of two distinct cholesterol-binding proteins: a transmembrane protein, NPC1, and a small soluble protein, NPC2. NPC1 and NPC2 are thought to function closely in the export of lysosomal cholesterol with both proteins binding cholesterol in vitro but they may have unrelated lysosomal roles. To investigate this possibility, we compared biochemical consequences of the loss of either protein. Analyses of lysosome-enriched subcellular fractions from brain and liver revealed similar decreases in buoyant densities of lysosomes from NPC1 or NPC2 deficient mice compared to controls. The subcellular distribution of both proteins was similar and paralleled a lysosomal marker. In liver, absence of either NPC1 or NPC2 resulted in similar alterations in the carbohydrate processing of the lysosomal protease, tripeptidyl peptidase I. These results highlight biochemical alterations in the lysosomal system of the NPC-mutant mice that appear secondary to lipid storage. In addition, the similarity in biochemical phenotypes resulting from either NPC1 or NPC2 deficiency supports models in which the function of these two proteins within lysosomes are linked closely.

  3. Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses1[OPEN

    Science.gov (United States)

    Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A.; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A.; Kay, Steve A.; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R.; Schroeder, Julian I.

    2015-01-01

    The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

  4. Molecular characterization of a new Babesia bovis thrombospondin-related anonymous protein (BbTRAP2.

    Directory of Open Access Journals (Sweden)

    Mohamad Alaa Terkawi

    Full Text Available A gene encoding a Babesia bovis protein that shares significant degree of similarity to other apicomplexan thrombospondin-related anonymous proteins (TRAPs was found in the genomic database and designated as BbTRAP2. Recombinant protein containing a conserved region of BbTRAP2 was produced in E. coli. A high antigenicity of recombinant BbTRAP2 (rBbTRAP2 was observed with field B. bovis-infected bovine sera collected from geographically different regions of the world. Moreover, antiserum against rBbTRAP2 specifically reacted with the authentic protein by Western blot analysis and an indirect fluorescent antibody test. Three bands corresponding to 104-, 76-, and 44-kDa proteins were identified in the parasite lysates and two bands of 76- and 44-kDa proteins were detected in the supernatant of cultivated parasites, indicating that BbTRAP2 was proteolytically processed and shed into the culture. Apical and surface localizations of BbTRAP2 were observed in the intracellular and extracellular parasites, respectively, by confocal laser microscopic examination. Moreover, native BbTRAP2 was precipitated by bovine erythrocytes, suggesting its role in the attachment to erythrocytes. Furthermore, the specific antibody to rBbTRAP2 inhibited the growth of B. bovis in a concentration-dependent manner. Consistently, pre-incubation of the free merozoites with the antibody to rBbTRAP2 resulted in an inhibition of the parasite invasion into host erythrocytes. Interestingly, the antibody to rBbTRAP2 was the most inhibitive for the parasite's growth as compared to those of a set of antisera produced against different recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c, rhoptry-associated protein 1 C-terminal (BbRAP-1CT, and spherical body protein 1 (BbSBP-1. These results suggest that BbTRAP2 might be a potential candidate for development of a subunit vaccine against B. bovis infection.

  5. Production of solid lipid submicron particles for protein delivery using a novel supercritical gas-assisted melting atomization process.

    Science.gov (United States)

    Salmaso, Stefano; Elvassore, Nicola; Bertucco, Alberto; Caliceti, Paolo

    2009-02-01

    A supercritical carbon dioxide micronization technique based on gas-assisted melting atomization has been designed to prepare protein-loaded solid lipid submicron particles. The supercritical process was applied to homogeneous dispersions of insulin in lipid mixtures: (1) tristearin, Tween-80, phosphatidylcholine and 5 kDa PEG (1:0.1:0.9:1 and 1:0.1:0.9:2 weight ratio); and (2) tristearin, dioctyl sulfosuccinate and phosphatidylcholine (1:1:0.5 weight ratio). Optimized process conditions yielded dry nonagglomerated powders with high product recovery (70%, w/w). Dynamic light scattering and transmission electron microscopy showed that two size fractions of particles, with 80-120 and 200-400 nm diameters, were produced. In all final products, dimethylsulfoxide used to prepare the insulin/lipid mixture was below 20 ppm. Protein encapsulation efficiency increased up to 80% as the DMSO content in the insulin/lipid mixture increased. Compared to the particles without PEG, the polymer-containing particles dispersed rapidly in water, and the dispersions were more stable under centrifugation as less than 20% of suspended particles precipitated after extensive centrifugation. In vitro, the protein was slowly released from the formulation without PEG, while a burst and faster release were obtained from the formulations containing PEG. Subcutaneous injection to diabetic mice of insulin extracted from the particles showed that the supercritical process did not impair the protein hypoglycemic activity.

  6. HCV E2 glycoprotein: mutagenesis of N-linked glycosylation sites and its effects on E2 expression and processing

    International Nuclear Information System (INIS)

    Slater-Handshy, Tiffany; Droll, Deborah A.; Fan Xiaofeng; Di Bisceglie, Adrian M.; Chambers, Thomas J.

    2004-01-01

    An expression system for analysis of the synthesis and processing of the E2 glycoprotein of a hepatitis C virus (HCV) genotype 1a strain was developed in transiently transfected cells. E2 proteins representing the entire length of the protein, including the transmembrane segment (E2) as well as two truncated versions (E2 660 and E2 715 ), were characterized for acquisition of N-linked glycans and transport to the media of transfected cells. To investigate the utilization of the 10 potential N-linked glycosylation sites on this E2 protein, a series of mutations consisting of single or multiple (two, three, four or eight) ablations of asparagine residues in the background of the E2 660 construct were analyzed. E2 660 proteins harboring single or multiple site mutations were produced at levels similar to that of wild-type protein, but secretion of the single mutants was mildly diminished, and elimination of two or more sites dramatically reduced delivery of the protein to the media. Similar results were obtained in Huh-7 cells with respect to intracellular synthesis and secretion of the mutant proteins. Analysis of oligosaccharide composition using endoglycosidase digestion revealed that all of the glycan residues on the intracellular forms of E2 660 , E2 715 , and E2 contained N-linked glycans modified into high-mannose carbohydrates, in contrast to the secreted forms, which were endo H resistant. The parental E2 660 protein could be readily detected in Huh-7 cells using anti-polyhistidine or antibody to recombinant E2. In contrast, E2 660 lacking the eight N-linked glycans was expressed but not detectable with anti-E2 antibody, and proteins lacking four glycans exhibited reduced reactivity. These experiments provide direct evidence that the presence of multiple N-linked glycans is required for the proper folding of the E2 protein in the ER and secretory pathway as well as for formation of its antigenic structure

  7. N-acyl homoserine lactone-degrading microbial enrichment cultures isolated from Penaeus vannamei shrimp gut and their probiotic properties in Brachionus plicatilis cultures.

    Science.gov (United States)

    Tinh, Nguyen Thi Ngoc; Asanka Gunasekara, R A Y S; Boon, Nico; Dierckens, Kristof; Sorgeloos, Patrick; Bossier, Peter

    2007-10-01

    Three bacterial enrichment cultures (ECs) were isolated from the digestive tract of Pacific white shrimp Penaeus vannamei, by growing the shrimp microbial communities in a mixture of N-acyl homoserine lactone (AHL) molecules. The ECs, characterized by denaturing gradient gel electrophoresis analysis and subsequent rRNA sequencing, degraded AHL molecules in the degradation assays. Apparently, the resting cells of the ECs also degraded one of the three types of quorum-sensing signal molecules produced by Vibrio harveyi in vitro [i.e. harveyi autoinducer 1 (HAI-1)]. The most efficient AHL-degrading ECs, EC5, was tested in Brachionus experiments. EC5 degraded the V. harveyi HAI-1 autoinducer in vivo, neutralizing the negative effect of V. harveyi autoinducer 2 (AI-2) mutant, in which only the HAI-1- and CAI-1-mediated components of the quorum-sensing system are functional on the growth of Brachionus. This suggests that EC5 interferes with HAI-1-regulated metabolism in V. harveyi. These AHL-degrading ECs need to be tested in other aquatic systems for their probiotic properties, preferably in combination with specific AI-2-degrading bacteria.

  8. A novel processing system of sterol regulatory element-binding protein-1c regulated by polyunsaturated fatty acid.

    Science.gov (United States)

    Nakakuki, Masanori; Kawano, Hiroyuki; Notsu, Tatsuto; Imada, Kazunori; Mizuguchi, Kiyoshi; Shimano, Hitoshi

    2014-05-01

    The proteolytic cascade is the key step in transactivation of sterol regulatory element-binding proteins (SREBPs), a transcriptional factor of lipid synthesis. Proteolysis of SREBP-2 is strictly regulated by sterols, but that of SREBP-1c was not strongly sterol-regulated, but inhibited by polyunsaturated fatty acids (PUFAs). In this study, the proteolytic processing of SREBP-1 and -2 was examined by transfection studies of cDNA-encoding mutants in which all the known cleavage sites were disrupted. In cultured cells, sterol-regulated SREBP-2 processing was completely eliminated by mutation of cleavage sites. In contrast, the corresponding SREBP-1c mutants as well as wild type exhibited large amounts of cleaved products in the nuclear extracts from culture cells and murine liver in vivo. The nuclear form of the mutant SREBP-1c was induced by delipidated condition and suppressed by eicosapentaenoic acid, an n-3 PUFA, but not by sterols. This novel processing mechanism was affected by neither SREBP cleavage-activating protein (SCAP) nor insulin-induced gene (Insig)-1, unlike SREBP-2, but abolished by a serine protease inhibitor. Through analysis of deletion mutant, a site-2 protease recognition sequence (DRSR) was identified to be involved in this novel processing. These findings suggest that SREBP-1c cleavage could be subjected to a novel PUFA-regulated cleavage system in addition to the sterol-regulatory SCAP/Insig system.

  9. Activity of autoinducer two (AI-2) in bacteria isolated from surface ripened cheeses

    DEFF Research Database (Denmark)

    Gori, Klaus; Jespersen, Lene

    2007-01-01

    ). Corynebacterium casei, Microbacterium barkeri, Microbacterium gubbeenense and S. equorum subsp. linens (all isolated from the smear of surface ripened cheeses) using the AI-2 bioluminescence assay. This indicates that AI-2 signaling could take place between bacteria found in the smear of surface ripened cheeses....

  10. The unique fold and lability of the [2Fe-2S] clusters of NEET proteins mediate their key functions in health and disease.

    Science.gov (United States)

    Karmi, Ola; Marjault, Henri-Baptiste; Pesce, Luca; Carloni, Paolo; Onuchic, Jose' N; Jennings, Patricia A; Mittler, Ron; Nechushtai, Rachel

    2018-02-12

    NEET proteins comprise a new class of [2Fe-2S] cluster proteins. In human, three genes encode for NEET proteins: cisd1 encodes mitoNEET (mNT), cisd2 encodes the Nutrient-deprivation autophagy factor-1 (NAF-1) and cisd3 encodes MiNT (Miner2). These recently discovered proteins play key roles in many processes related to normal metabolism and disease. Indeed, NEET proteins are involved in iron, Fe-S, and reactive oxygen homeostasis in cells and play an important role in regulating apoptosis and autophagy. mNT and NAF-1 are homodimeric and reside on the outer mitochondrial membrane. NAF-1 also resides in the membranes of the ER associated mitochondrial membranes (MAM) and the ER. MiNT is a monomer with distinct asymmetry in the molecular surfaces surrounding the clusters. Unlike its paralogs mNT and NAF-1, it resides within the mitochondria. NAF-1 and mNT share similar backbone folds to the plant homodimeric NEET protein (At-NEET), while MiNT's backbone fold resembles a bacterial MiNT protein. Despite the variation of amino acid composition among these proteins, all NEET proteins retained their unique CDGSH domain harboring their unique 3Cys:1His [2Fe-2S] cluster coordination through evolution. The coordinating exposed His was shown to convey the lability to the NEET proteins' [2Fe-2S] clusters. In this minireview, we discuss the NEET fold and its structural elements. Special attention is given to the unique lability of the NEETs' [2Fe-2S] cluster and the implication of the latter to the NEET proteins' cellular and systemic function in health and disease.

  11. CCS mRNA transcripts and serum CCS protein as copper marker in adults suffering inflammatory processes.

    Science.gov (United States)

    Araya, Magdalena; Gutiérrez, Ricardo; Arredondo, Miguel

    2014-08-01

    The chaperone to Zn-Cu superoxide dismutase (CCS) has been postulated as a candidate copper indicator, changing in a consistent manner in induced and recovered copper deficiency, in experimental cell and animal models. In real life people have various conditions that may modify molecules acting as acute phase proteins, such as serum ceruloplasmin and copper concentration and could alter CCS responses. With the hypothesis that CCS mRNA transcripts and protein would be different in individuals suffering inflammatory processes in comparison to healthy individuals, we assessed adult individuals who, although not ill had conditions known to induce variable degrees of inflammation. Screening of 600 adults resulted in two study groups, formed on the basis of their clinical history and levels of serum C reactive protein (CRP): Group 1 (n = 61, mean (range) CRP = 0.9 (0.3-2.0 mg/dL) and Group 2 (n = 150, mean (range) CRP = 6.1 (4.3-8.7 mg/dL). Results showed that mRNA transcripts relative abundance was not different for CCS, MTIIA, TNF-alpha and Cu-Zn-SOD by group (p > 0.05, one way Anova), nor between sexes (p > 0.05, one way Anova). Distribution of CCS mRNA transcripts and CCS protein in serum did not show any differences or trends. Results disproved our hypothesis that CCS abundance of transcripts and CCS protein would be different in individuals suffering inflammatory processes, adding further support to the idea that CCS may be a copper marker.

  12. Biosensors for the Detection and Quantification of AI-2 Class Quorum-Sensing Compounds.

    Science.gov (United States)

    Rajamani, Sathish; Sayre, Richard

    2018-01-01

    Intercellular small-molecular-weight signaling molecules modulate a variety of biological functions in bacteria. One of the more complex behaviors mediated by intercellular signaling molecules is the suite of activities regulated by quorum-sensing molecules. These molecules mediate a variety of population-dependent responses including the expression of genes that regulate bioluminescence, type III secretion, siderophore production, colony morphology, biofilm formation, and metalloprotease production. Given their central role in regulating these responses, the detection and quantification of QS molecules have important practical implications. Until recently, the detection of QS molecules from Gram-negative bacteria has relied primarily on bacterial reporter systems. These bioassays though immensely useful are subject to interference by compounds that affect bacterial growth and metabolism. In addition, the reporter response is highly dependent on culture age and cell population density. To overcome such limitations, we developed an in vitro protein-based assay system for the rapid detection and quantification of the furanosyl borate diester (BAI-2) subclass of autoinducer-2 (AI-2) QS molecules. The biosensor is based on the interaction of BAI-2 with the Vibrio harveyi QS receptor LuxP. Conformation changes associated with BAI-2 binding to the LuxP receptor change the orientation of cyan and yellow variants of GFP (CFP and YFP) fused to the N- and C-termini, respectively, of the LuxP receptor. LuxP-BAI2 binding induces changes in fluorescence resonance energy transfer (FRET) between CFP and YFP, whose magnitude of change is ligand concentration dependent. Ligand-insensitive LuxP mutant FRET protein sensors were also developed for use as control biosensors. The FRET-based BAI-2 biosensor responds selectively to both synthetic and biologically derived BAI-2 compounds. This report describes the use of the LuxP-FRET biosensor for the detection and quantification of BAI-2.

  13. Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

    Directory of Open Access Journals (Sweden)

    Yuping Ren

    2017-12-01

    Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in pediatric populations globally. This study examined proteomic profile changes in A549 cells infected with hMPV and two attenuated mutants with deleted PDZ domain-binding motif(s in the M2-2 protein. These motifs are involved in the interruption of antiviral signaling, namely the interaction between the TNF receptor associated factor (TRAF and mitochondrial antiviral-signaling (MAVS proteins. The aim of this study was to provide insight into the overall and novel impact of M2-2 motifs on cellular responses via an unbiased comparison. Tandem mass tagging, stable isotope labeling, and high-resolution mass spectrometry were used for quantitative proteomic analysis. Using quantitative proteomics and Venn analysis, 1248 common proteins were detected in all infected samples of both technical sets. Hierarchical clustering of the differentiated proteome displayed distinct proteomic signatures that were controlled by the motif(s. Bioinformatics and experimental analysis confirmed the differentiated proteomes, revealed novel cellular biological events, and implicated key pathways controlled by hMPV M2-2 PDZ domain-binding motif(s. This provides further insight for evaluating M2-2 mutants as potent vaccine candidates.

  14. Incorporation of radiolabeled whey proteins into casein micelles by heat processing

    International Nuclear Information System (INIS)

    Noh, B.; Richardson, T.

    1989-01-01

    Skim milk was heated at .70, 95, and 140 degree C to simulate the processes of pasteurization, forewarming, and UHT sterilization, and the specific interactions between α-lactalbumin or β-lactoglobulin and the caseins studied using tracer amounts of added 14 C-labeled whey protein. Radioactivities of the whey and of the washed casein pellets from renneted skim milk were measured and the extent of the interaction estimated. Upon heating skim milk at 70 degree C for 45 s, less than 2% β-lactoglobulin and less than .3% α-lactalbumin were incorporated into the curd. Heating at 95 degree C for .5 to 20 min resulted in 58 to 85% of the β-lactoglobulin and 8 to 55% of the α-lactalbumin becoming associated with the curd. Heating at 140 degree C for 2 and 4 s caused 43 and 54% of the β-lactoglobulin and 9 and 12% of the α-lactalbumin, respectively, to be bound to the curd fraction. The radiolabeling technique is very sensitive and useful for tracing low levels of interaction between whey proteins and casein in heated milk systems

  15. Processed Meat Protein and Heat-Stable Peptide Marker Identification Using Microwave-Assisted Tryptic Digestion

    Directory of Open Access Journals (Sweden)

    Magdalena Montowska

    2016-01-01

    Full Text Available New approaches to rapid examination of proteins and peptides in complex food matrices are of great interest to the community of food scientists. The aim of the study is to examine the influence of microwave irradiation on the acceleration of enzymatic cleavage and enzymatic digestion of denatured proteins in cooked meat of five species (cattle, horse, pig, chicken and turkey and processed meat products (coarsely minced, smoked, cooked and semi-dried sausages. Severe protein aggregation occurred not only in heated meat under harsh treatment at 190 °C but also in processed meat products. All the protein aggregates were thoroughly hydrolyzed aft er 1 h of trypsin treatment with short exposure times of 40 and 20 s to microwave irradiation at 138 and 303 W. There were much more missed cleavage sites observed in all microwave-assisted digestions. Despite the incompleteness of microwave-assisted digestion, six unique peptide markers were detected, which allowed unambiguous identification of processed meat derived from the examined species. Although the microwave-assisted tryptic digestion can serve as a tool for rapid and high-throughput protein identification, great caution and pre-evaluation of individual samples is recommended in protein quantitation.

  16. Functional Regulation of the Plasma Protein Histidine-Rich Glycoprotein by Zn2+ in Settings of Tissue Injury

    Directory of Open Access Journals (Sweden)

    Kristin M. Priebatsch

    2017-03-01

    Full Text Available Divalent metal ions are essential nutrients for all living organisms and are commonly protein-bound where they perform important roles in protein structure and function. This regulatory control from metals is observed in the relatively abundant plasma protein histidine-rich glycoprotein (HRG, which displays preferential binding to the second most abundant transition element in human systems, Zinc (Zn2+. HRG has been proposed to interact with a large number of protein ligands and has been implicated in the regulation of various physiological and pathological processes including the formation of immune complexes, apoptotic/necrotic and pathogen clearance, cell adhesion, antimicrobial activity, angiogenesis, coagulation and fibrinolysis. Interestingly, these processes are often associated with sites of tissue injury or tumour growth, where the concentration and distribution of Zn2+ is known to vary. Changes in Zn2+ levels have been shown to modify HRG function by altering its affinity for certain ligands and/or providing protection against proteolytic disassembly by serine proteases. This review focuses on the molecular interplay between HRG and Zn2+, and how Zn2+ binding modifies HRG-ligand interactions to regulate function in different settings of tissue injury.

  17. Protein kinase CK2 in human diseases

    DEFF Research Database (Denmark)

    Guerra, Barbara; Issinger, Olaf-Georg

    2008-01-01

    Protein kinase CK2 (formerly referred to as casein kinase II) is an evolutionary conserved, ubiquitous protein kinase. There are two paralog catalytic subunits, i.e. alpha (A1) and alpha' (A2). The alpha and alpha' subunits are linked to two beta subunits to produce a heterotetrameric structure...

  18. COPS5 (Jab1) protein increases β site processing of amyloid precursor protein and amyloid β peptide generation by stabilizing RanBP9 protein levels.

    Science.gov (United States)

    Wang, Hongjie; Dey, Debleena; Carrera, Ivan; Minond, Dmitriy; Bianchi, Elisabetta; Xu, Shaohua; Lakshmana, Madepalli K

    2013-09-13

    Increased processing of amyloid precursor protein (APP) and accumulation of neurotoxic amyloid β peptide (Aβ) in the brain is central to the pathogenesis of Alzheimer's disease (AD). Therefore, the identification of molecules that regulate Aβ generation is crucial for future therapeutic approaches for AD. We demonstrated previously that RanBP9 regulates Aβ generation in a number of cell lines and primary neuronal cultures by forming tripartite protein complexes with APP, low-density lipoprotein-related protein, and BACE1, consequently leading to increased amyloid plaque burden in the brain. RanBP9 is a scaffold protein that exists and functions in multiprotein complexes. To identify other proteins that may bind RanBP9 and regulate Aβ levels, we used a two-hybrid analysis against a human brain cDNA library and identified COPS5 as a novel RanBP9-interacting protein. This interaction was confirmed by coimmunoprecipitation experiments in both neuronal and non-neuronal cells and mouse brain. Colocalization of COPS5 and RanBP9 in the same subcellular compartments further supported the interaction of both proteins. Furthermore, like RanBP9, COPS5 robustly increased Aβ generation, followed by increased soluble APP-β (sAPP-β) and decreased soluble-APP-α (sAPP-α) levels. Most importantly, down-regulation of COPS5 by siRNAs reduced Aβ generation, implying that endogenous COPS5 regulates Aβ generation. Finally, COPS5 levels were increased significantly in AD brains and APΔE9 transgenic mice, and overexpression of COPS5 strongly increased RanBP9 protein levels by increasing its half-life. Taken together, these results suggest that COPS5 increases Aβ generation by increasing RanBP9 levels. Thus, COPS5 is a novel RanBP9-binding protein that increases APP processing and Aβ generation by stabilizing RanBP9 protein levels.

  19. Malting process optimization for protein digestibility enhancement in finger millet grain.

    Science.gov (United States)

    Hejazi, Sara Najdi; Orsat, Valérie

    2016-04-01

    Finger millet (Eleusine coracana) is a nutritious, gluten-free, and drought resistant cereal containing high amounts of protein, carbohydrate, and minerals. However, bio-availability of these nutrients is restricted due to the presence of an excessive level of anti-nutrient components, mainly phytic acid, tannin, and oxalate. It has been shown that a well-designed malting/germination process can significantly reduce these anti-nutrients and consequently enhance the nutrient availability. In the present study, the effects of two important germination factors, duration and temperature, on the enhancement of in-vitro protein digestibility of finger millet were thoroughly investigated and optimized. Based on a central composite design, the grains were germinated for 24, 36, and 48 h at 22, 26, and 30 °C. For all factor combinations, protein, peptide, phytic acid, tannin, and oxalate contents were evaluated and digestibility was assessed. It was shown that during the malting/germinating process, both temperature and duration factors significantly influenced the investigated quantities. Germination of finger millet for 48 h at 30 °C increased protein digestibility from 74 % (for native grain) up to 91 %. Besides, it notably decreased phytic acid, tannin, and oxalate contents by 45 %, 46 %, and 29 %, respectively. Linear correlations between protein digestibility and these anti-nutrients were observed.

  20. Effect of the synthetic cannabinoid HU-210 on quorum sensing and on the production of quorum sensing-mediated virulence factors by Vibrio harveyi.

    Science.gov (United States)

    Soni, Divya; Smoum, Reem; Breuer, Aviva; Mechoulam, Raphael; Steinberg, Doron

    2015-08-12

    Bacterial populations communicate through the cell density-dependent mechanism of quorum sensing (QS). Vibrio harveyi, one of the best studied model organisms for QS, was used to explore effects of the synthetic cannabinoid HU-210 on QS and different QS-regulated physiological processes in bacteria. Analysis of QS-regulated bioluminescence in wild-type and mutant strains of V. harveyi revealed that HU-210 affects the autoinducer-2 (AI-2) pathway, one of three known QS cascades of V. harveyi. Furthermore, QS-mediated biofilm formation and swimming motility in the mutant strain BB152 (AI-1(-), AI-2(+)) were significantly reduced in the presence of HU-210. HU-210 inhibited QS-mediated virulence factor production without any inhibitory effect on bacterial growth. It also alters the expression of several genes, which are regulated by QS, specifically downregulating the genes of the AI-2 QS cascade. First evidence is being provided for interference of bacterial signal-transduction systems by a synthetic cannabinoid. The effect of HU-210 was specific to the AI-2 cascade in V. harveyi. AI-2 is known as a "universal autoinducer" and interference with its activity opens a broad spectrum of applications for synthetic cannabinoids in future research as a potential anti-QS agent.

  1. Influence of endurance training on skeletal muscle mitophagy regulatory proteins in type 2 diabetic men.

    Science.gov (United States)

    Brinkmann, Christian; Przyklenk, Axel; Metten, Alexander; Schiffer, Thorsten; Bloch, Wilhelm; Brixius, Klara; Gehlert, Sebastian

    2017-11-01

    Mitophagy is a form of autophagy for the elimination of mitochondria. Mitochondrial content and function are reduced in the skeletal muscle of patients with type 2 diabetes mellitus (T2DM). Physical training has been shown to restore mitochondrial capacity in T2DM patients, but the role of mitophagy has not been examined in this context. This study analyzes the impact of a 3-month endurance training on important skeletal muscle mitophagy regulatory proteins and oxidative phosphorylation (OXPHOS) complexes in T2DM patients. Muscle biopsies were obtained from eight overweight/obese T2DM men (61±10 years) at T1 (6 weeks pre-training), T2 (1 week pre-training), and T3 (3 to 4 days post-training). Protein contents were determined by Western blotting. The training increased mitochondrial complex II significantly (T2-T3: +29%, p = 0.037). The protein contents of mitophagy regulatory proteins (phosphorylated form of forkhead box O3A (pFOXO3A), mitochondrial E3 ubiquitin protein ligase-1 (MUL1), Bcl-2/adenovirus E1B 19-kD interacting protein-3 (BNIP3), microtubule-associated protein 1 light chain-3B (the ratio LC3B-II/LC3B-I was determined)) did not differ significantly between T1, T2, and T3. The results imply that training-induced changes in OXPHOS subunits (significant increase in complex II) are not accompanied by changes in mitophagy regulatory proteins in T2DM men. Future studies should elucidate whether acute exercise might affect mitophagic processes in T2DM patients (and whether a transient regulation of mitophagy regulatory proteins is evident) to fully clarify the role of physical activity and mitophagy for mitochondrial health in this particular patient group.

  2. Conservation, diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome

    Directory of Open Access Journals (Sweden)

    Böhm Siegfried

    2004-07-01

    Full Text Available Background The classical C2H2 zinc finger domain is involved in a wide range of functions and can bind to DNA, RNA and proteins. The comparison of zinc finger proteins in several eukaryotes has shown that there is a lot of lineage specific diversification and expansion. Although the number of characterized plant proteins that carry the classical C2H2 zinc finger motifs is growing, a systematic classification and analysis of a plant genome zinc finger gene set is lacking. Results We found through in silico analysis 176 zinc finger proteins in Arabidopsis thaliana that hence constitute the most abundant family of putative transcriptional regulators in this plant. Only a minority of 33 A. thaliana zinc finger proteins are conserved in other eukaryotes. In contrast, the majority of these proteins (81% are plant specific. They are derived from extensive duplication events and form expanded families. We assigned the proteins to different subgroups and families and focused specifically on the two largest and evolutionarily youngest families (A1 and C1 that are suggested to be primarily involved in transcriptional regulation. The newly defined family A1 (24 members comprises proteins with tandemly arranged zinc finger domains. Family C1 (64 members, earlier described as the EPF-family in Petunia, comprises proteins with one isolated or two to five dispersed fingers and a mostly invariant QALGGH motif in the zinc finger helices. Based on the amino acid pattern in these helices we could describe five different signature sequences prevalent in C1 zinc finger domains. We also found a number of non-finger domains that are conserved in these families. Conclusions Our analysis of the few evolutionarily conserved zinc finger proteins of A. thaliana suggests that most of them could be involved in ancient biological processes like RNA metabolism and chromatin-remodeling. In contrast, the majority of the unique A. thaliana zinc finger proteins are known or

  3. Endoplasmic reticulum proteins SDF2 and SDF2L1 act as components of the BiP chaperone cycle to prevent protein aggregation.

    Science.gov (United States)

    Fujimori, Tsutomu; Suno, Ryoji; Iemura, Shun-Ichiro; Natsume, Tohru; Wada, Ikuo; Hosokawa, Nobuko

    2017-08-01

    The folding of newly synthesized proteins in the endoplasmic reticulum (ER) is assisted by ER-resident chaperone proteins. BiP (immunoglobulin heavy-chain-binding protein), a member of the HSP70 family, plays a central role in protein quality control. The chaperone function of BiP is regulated by its intrinsic ATPase activity, which is stimulated by ER-resident proteins of the HSP40/DnaJ family, including ERdj3. Here, we report that two closely related proteins, SDF2 and SDF2L1, regulate the BiP chaperone cycle. Both are ER-resident, but SDF2 is constitutively expressed, whereas SDF2L1 expression is induced by ER stress. Both luminal proteins formed a stable complex with ERdj3 and potently inhibited the aggregation of different types of misfolded ER cargo. These proteins associated with non-native proteins, thus promoting the BiP-substrate interaction cycle. A dominant-negative ERdj3 mutant that inhibits the interaction between ERdj3 and BiP prevented the dissociation of misfolded cargo from the ERdj3-SDF2L1 complex. Our findings indicate that SDF2 and SDF2L1 associate with ERdj3 and act as components in the BiP chaperone cycle to prevent the aggregation of misfolded proteins, partly explaining the broad folding capabilities of the ER under various physiological conditions. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  4. Respiratory syncytial virus M2-1 protein induces the activation of nuclear factor kappa B

    Energy Technology Data Exchange (ETDEWEB)

    Reimers, Kerstin [Klinik fuer Plastische, Hand-und Wiederherstellungschirurgie, Podbielskistrasse 380, D-30659 Hannover (Germany); Buchholz, Katja [Institut fuer Medizinische Mikrobiologie, Otto-von-Guericke-Universitaet Magdeburg, Leipzigerstrasse 44, D-39120 Magdeburg (Germany); Werchau, Hermann [Institut fuer Medizinische Mikrobiologie, Otto-von-Guericke-Universitaet Magdeburg, Leipzigerstrasse 44, D-39120 Magdeburg (Germany)

    2005-01-20

    Respiratory syncytial virus (RSV) induces the production of a number of cytokines and chemokines by activation of nuclear factor kappa B (NF-{kappa}B). The activation of NF-{kappa}B has been shown to depend on viral replication in the infected cells. In this study, we demonstrate that expression of RSV M2-1 protein, a transcriptional processivity and anti-termination factor, is sufficient to activate NF-{kappa}B in A549 cells. Electromobility shift assays show increased NF-{kappa}B complexes in the nuclei of M2-1-expressing cells. M2-1 protein is found in nuclei of M2-1-expressing cells and in RSV-infected cells. Co-immunoprecipitations of nuclear extracts of M2-1-expressing cells and of RSV-infected cells revealed an association of M2-1 with Rel A protein. Furthermore, the activation of NF-{kappa}B depends on the C-terminus of the RSV M2-1 protein, as shown by NF-{kappa}B-induced gene expression of a reporter gene construct.

  5. A plastome mutation affects processing of both chloroplast and nuclear DNA-encoded plastid proteins.

    Science.gov (United States)

    Johnson, E M; Schnabelrauch, L S; Sears, B B

    1991-01-01

    Immunoblotting of a chloroplast mutant (pm7) of Oenothera showed that three proteins, cytochrome f and the 23 kDa and 16 kDa subunits of the oxygen-evolving subcomplex of photosystem II, were larger than the corresponding mature proteins of the wild type and, thus, appear to be improperly processed in pm7. The mutant is also chlorotic and has little or no internal membrane development in the plastids. The improperly processed proteins, and other proteins that are completely missing, represent products of both the plastid and nuclear genomes. To test for linkage of these defects, a green revertant of pm7 was isolated from cultures in which the mutant plastids were maintained in a nuclear background homozygous for the plastome mutator (pm) gene. In this revertant, all proteins analyzed co-reverted to the wild-type condition, indicating that a single mutation in a plastome gene is responsible for the complex phenotype of pm7. These results suggest that the defect in pm7 lies in a gene that affects a processing protease encoded in the chloroplast genome.

  6. Identification of interacting proteins of retinoid-related orphan nuclear receptor gamma in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Ze-Min Huang1,#, Jun Wu2,#, Zheng-Cai Jia1, Yi Tian1, Jun Tang3, Yan Tang1, Ying Wang2, Yu-Zhang Wu1,* & Bing Ni1,*

    2012-06-01

    Full Text Available The retinoid-related orphan nuclear receptor gamma (RORγplays critical roles in regulation of development, immunity andmetabolism. As transcription factor usually forms a proteincomplex to function, thus capturing and dissecting of theRORγ protein complex will be helpful for exploring themechanisms underlying those functions. After construction ofthe recombinant tandem affinity purification (TAP plasmid,pMSCVpuro RORγ-CTAP(SG, the nuclear localization ofRORγ-CTAP(SG fusion protein was verified. Followingisolation of RORγ protein complex by TAP strategy, sevencandidate interacting proteins were identified. Finally, the heatshock protein 90 (HSP90 and receptor-interacting protein 140(RIP140 were confirmed to interplay with RORγ byco-immunoprecipitation. Interference of HSP90 or/and RIP140genes resulted in dramatically decreased expression ofCYP2C8 gene, the RORγ target gene. Data from this studydemonstrate that HSP90 and RIP140 proteins interact withRORγ protein in a complex format and function asco-activators in the RORγ-mediated regulatory processes ofHepG2 cells.

  7. Non-conventional approaches to food processing in CELSS. I - Algal proteins: Characterization and process optimization

    Science.gov (United States)

    Nakhost, Z.; Karel, M.; Krukonis, V. J.

    1987-01-01

    Protein isolate obtained from green algae (Scenedesmus obliquus) cultivated under controlled conditions was characterized. Molecular weight determination of fractionated algal proteins using SDS-polyacrylamide gel electrophoresis revealed a wide spectrum of molecular weights ranging from 15,000 to 220,000. Isoelectric points of dissociated proteins were in the range of 3.95 to 6.20. Amino acid composition of protein isolate compared favorably with FAO standards. High content of essential amino acids leucine, valine, phenylalanine and lysine makes algal protein isolate a high quality component of CELSS diets. To optimize the removal of algal lipids and pigments supercritical carbon dioxide extraction (with and without ethanol as a co-solvent) was used. Addition of ethanol to supercritical CO2 resulted in more efficient removal of algal lipids and produced protein isolate with a good yield and protein recovery. The protein isolate extracted by the above mixture had an improved water solubility.

  8. Label-free SnO2 nanowire FET biosensor for protein detection

    Science.gov (United States)

    Jakob, Markus H.; Dong, Bo; Gutsch, Sebastian; Chatelle, Claire; Krishnaraja, Abinaya; Weber, Wilfried; Zacharias, Margit

    2017-06-01

    Novel tin oxide field-effect-transistors (SnO2 NW-FET) for pH and protein detection applicable in the healthcare sector are reported. With a SnO2 NW-FET the proof-of-concept of a bio-sensing device is demonstrated using the carrier transport control of the FET channel by a (bio-) liquid modulated gate. Ultra-thin Al2O3 fabricated by a low temperature atomic layer deposition (ALD) process represents a sensitive layer to H+ ions safeguarding the nanowire at the same time. Successful pH sensitivity is demonstrated for pH ranging from 3 to 10. For protein detection, the SnO2 NW-FET is functionalized with a receptor molecule which specifically interacts with the protein of interest to be detected. The feasibility of this approach is demonstrated via the detection of a biotinylated protein using a NW-FET functionalized with streptavidin. An immediate label-free electronic read-out of the signal is shown. The well-established Enzyme-Linked Immunosorbent Assay (ELISA) method is used to determine the optimal experimental procedure which would enable molecular binding events to occur while being compatible with a final label-free electronic read-out on a NW-FET. Integration of the bottom-up fabricated SnO2 NW-FET pH- and biosensor into a microfluidic system (lab-on-a-chip) allows the automated analysis of small volumes in the 400 μl range as would be desired in portable on-site point-of-care (POC) devices for medical diagnosis.

  9. Fabrication of 2D protein microstructures and 3D polymer-protein hybrid microstructures by two-photon polymerization

    Energy Technology Data Exchange (ETDEWEB)

    Engelhardt, Sascha [Lehrstuhl fuer Lasertechnik, RWTH Aachen, Steinbachstrasse 15, Aachen (Germany); Hoch, Eva; Tovar, Guenter E M [Institut fuer Grenzflaechenverfahrenstechnik, Universitaet Stuttgart, Nobelstrasse 12, Stuttgart (Germany); Borchers, Kirsten [Fraunhofer-Institut fuer Grenzflaechen- und Bioverfahrenstechnik, Nobelstrasse 12, Stuttgart (Germany); Meyer, Wolfdietrich; Krueger, Hartmut [Fraunhofer-Institut fuer Angewandte Polymerforschung, Geiselbergstrasse 69, Potsdam (Germany); Gillner, Arnold, E-mail: sascha.engelhardt@ilt.fraunhofer.de [Fraunhofer-Institut fuer Lasertechnik, Steinbachstrasse 15, Aachen (Germany)

    2011-06-15

    Two-photon polymerization (TPP) offers the possibility of creating artificial cell scaffolds composed of micro- and nanostructures with spatial resolutions of less than 1 {mu}m. For use in tissue engineering, the identification of a TPP-processable polymer that provides biocompatibility, biofunctionality and appropriate mechanical properties is a difficult task. ECM proteins such as collagen or fibronectin, which could mimic native tissues best, often lack the mechanical stability. Hence, by generating polymer-protein hybrid structures, the beneficial properties of proteins can be combined with the advantageous characteristics of polymers, such as sufficient mechanical stability. This study describes three steps toward facilitated application of TPP for biomaterial generation. (1) The efficiency of a low-cost ps-laser source is compared to a fs-laser source by testing several materials. A novel photoinitiator for polymerization with a ps-laser source is synthesized and proved to enable increased fabrication throughput. (2) The fabrication of 3D-microstructures with both systems and the fabrication of polymer-protein hybrid structures are demonstrated. (3) The tissue engineering capabilities of TPP are demonstrated by creating cross-linked gelatin microstructures, which clearly forced porcine chondrocytes to adapt their cell morphology.

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

    Science.gov (United States)

    Deng, Jingren; Lazar, Iulia M.

    2016-04-01

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

  11. Reconstitution of the anti-apoptotic Bcl-2 protein into lipid membranes and biophysical evidence for its detergent-driven association with the pro-apoptotic Bax protein.

    Directory of Open Access Journals (Sweden)

    Marcus Wallgren

    Full Text Available The anti-apoptotic B-cell CLL/lymphoma-2 (Bcl-2 protein and its counterpart, the pro-apoptotic Bcl-2-associated X protein (Bax, are key players in the regulation of the mitochondrial pathway of apoptosis. However, how they interact at the mitochondrial outer membrane (MOM and there determine whether the cell will live or be sentenced to death remains unknown. Competing models have been presented that describe how Bcl-2 inhibits the cell-killing activity of Bax, which is common in treatment-resistant tumors where Bcl-2 is overexpressed. Some studies suggest that Bcl-2 binds directly to and sequesters Bax, while others suggest an indirect process whereby Bcl-2 blocks BH3-only proteins and prevents them from activating Bax. Here we present the results of a biophysical study in which we investigated the putative interaction of solubilized full-length human Bcl-2 with Bax and the scope for incorporating the former into a native-like lipid environment. Far-UV circular dichroism (CD spectroscopy was used to detect direct Bcl-2-Bax-interactions in the presence of polyoxyethylene-(23-lauryl-ether (Brij-35 detergent at a level below its critical micelle concentration (CMC. Additional surface plasmon resonance (SPR measurements confirmed this observation and revealed a high affinity between the Bax and Bcl-2 proteins. Upon formation of this protein-protein complex, Bax also prevented the binding of antimycin A2 (a known inhibitory ligand of Bcl-2 to the Bcl-2 protein, as fluorescence spectroscopy experiments showed. In addition, Bcl-2 was able to form mixed micelles with Triton X-100 solubilized neutral phospholipids in the presence of high concentrations of Brij-35 (above its CMC. Following detergent removal, the integral membrane protein was found to have been fully reconstituted into a native-like membrane environment, as confirmed by ultracentrifugation and subsequent SDS-PAGE experiments.

  12. G2E3 is a nucleo-cytoplasmic shuttling protein with DNA damage responsive localization

    International Nuclear Information System (INIS)

    Brooks, William S.; Banerjee, Sami; Crawford, David F.

    2007-01-01

    G2E3 was originally described as a G2/M-specific gene with DNA damage responsive expression. The presence of a conserved HECT domain within the carboxy-terminus of the protein indicated that it likely functions as a ubiquitin ligase or E3. Although HECT domains are known to function in this capacity for many proteins, we demonstrate that a portion of the HECT domain from G2E3 plays an important role in the dynamic subcellular localization of the protein. We have shown that G2E3 is a nucleo-cytoplasmic shuttling protein with nuclear export mediated by a novel nuclear export domain that functions independently of CRM1. In full-length G2E3, a separate region of the HECT domain suppresses the function of the NES. Additionally, G2E3 contains a nucleolar localization signal (NoLS) in its amino terminus. Localization of G2E3 to the nucleolus is a dynamic process, and the protein delocalizes from the nucleolus rapidly after DNA damage. Cell cycle phase-specific expression and highly regulated subcellular localization of G2E3 suggest a possible role in cell cycle regulation and the cellular response to DNA damage

  13. Purification of bone morphogenetic protein-2 from refolding mixtures using mixed-mode membrane chromatography.

    Science.gov (United States)

    Gieseler, Gesa; Pepelanova, Iliyana; Stuckenberg, Lena; Villain, Louis; Nölle, Volker; Odenthal, Uwe; Beutel, Sascha; Rinas, Ursula; Scheper, Thomas

    2017-01-01

    In this study, we present the development of a process for the purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) using mixed-mode membrane chromatography. RhBMP-2 was produced as inclusion bodies in Escherichia coli. In vitro refolding using rapid dilution was carried out according to a previously established protocol. Different membrane chromatography phases were analyzed for their ability to purify BMP-2. A membrane phase with salt-tolerant properties resulting from mixed-mode ligand chemistry was able to selectively purify BMP-2 dimer from refolding mixtures. No further purification or polishing steps were necessary and high product purity was obtained. The produced BMP-2 exhibited a biological activity of 7.4 × 10 5  U/mg, comparable to commercial preparations. Mixed-mode membrane chromatography can be a valuable tool for the direct purification of proteins from solutions with high-conductivity, for example refolding buffers. In addition, in this particular case, it allowed us to circumvent the use of heparin-affinity chromatography, thus allowing the design of an animal-component-free process.

  14. Polycomb-like proteins link the PRC2 complex to CpG islands

    Energy Technology Data Exchange (ETDEWEB)

    Li, Haojie; Liefke, Robert; Jiang, Junyi; Kurland, Jesse Vigoda; Tian, Wei; Deng, Pujuan; Zhang, Weidi; He, Qian; Patel, Dinshaw J.; Bulyk, Martha L.; Shi, Yang; Wang, Zhanxin

    2017-09-06

    The Polycomb repressive complex 2 (PRC2) mainly mediates transcriptional repression1,2 and has essential roles in various biological processes including the maintenance of cell identity and proper differentiation. Polycomb-like (PCL) proteins, such as PHF1, MTF2 and PHF19, are PRC2-associated factors that form sub-complexes with PRC2 core components3, and have been proposed to modulate the enzymatic activity of PRC2 or the recruitment of PRC2 to specific genomic loci4,5,6,7,8,9,10,11,12,13. Mammalian PRC2-binding sites are enriched in CG content, which correlates with CpG islands that display a low level of DNA methylation14. However, the mechanism of PRC2 recruitment to CpG islands is not fully understood. Here we solve the crystal structures of the N-terminal domains of PHF1 and MTF2 with bound CpG-containing DNAs in the presence of H3K36me3-containing histone peptides. We show that the extended homologous regions of both proteins fold into a winged-helix structure, which specifically binds to the unmethylated CpG motif but in a completely different manner from the canonical winged-helix DNA recognition motif. We also show that the PCL extended homologous domains are required for efficient recruitment of PRC2 to CpG island-containing promoters in mouse embryonic stem cells. Our research provides the first, to our knowledge, direct evidence to demonstrate that PCL proteins are crucial for PRC2 recruitment to CpG islands, and further clarifies the roles of these proteins in transcriptional regulation in vivo.

  15. DEVELOPMENT OF TECHNOLOGY FOR WHEAT PROCESSING INTO ALCOHOL AND PROTEIN PRODUCT

    Directory of Open Access Journals (Sweden)

    T. I. Romanyuk

    2015-01-01

    Full Text Available In the alcohol industry it is important to create non-waste technology for grain processing into alcohol. The aim of research was the development of technology for wheat processing into ethanol and protein product. We studied the process of enzymatic hydrolysis of starch with glucoamylase of Glucogam preparation. We determined the optimal dosage of the enzyme 8 units. GlA/g of starch, and the temperature of 55°C. In the study of protein hydrolysis by the concomitant to glucoamylase protease of enzyme Glucogam preparation accumulation of amino nitrogen of 4.5 mg / cm 3 in 7 hours of bioconversion takes place. Separation of the resulting saccharified mass was carried out by centrifugation into the filtrate and protein mass. Centrifugation was carried out at a rotational speed of 2500 rev / min for 8 minutes. Protein was dried to 5% moisture content at temperatures not exceeding 35°C, milled, and examined its properties in comparison with native wheat gluten. The resulting product had the following characteristics: the solubility of 10%, water-holding capacity of 1.53 g / g, and fat binding capacity of 1.9 g /g. We investigated the process of fermentation of clarified wort with the dry solids concentration of 14%. We used the yeast Saccharomyces cerevisiae of race XII and Saccharomyces cerevisiae of race IMB Y-5007 in the dose of 120 million cells per 1 cm3 of wort. Optimum composition of mineral salts was determined. For the yeasts of race XII and IMB Y-5007 fertilizing with diammonium phosphate in a dosage of 1.5 g / dm3 is necessary. The alcohol yield when using the yeasts of race IMB Y-5007 was 60.7 dal/ ton of conditional starch, when using yeasts of race XII it accounts 60,6 dal / ton of conditional starch.

  16. Rat vas deferens SERCA2 is modulated by Ca{sup 2+}/calmodulin protein kinase II-mediated phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez, J.B.R.; Muzi-Filho, H. [Programa de Farmacologia e Inflamação, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Valverde, R.H.F. [Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Quintas, L.E.M. [Programa de Farmacologia e Inflamação, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Noel, F. [Programa de Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Einicker-Lamas, M. [Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Rio de Janeiro, RJ (Brazil); Cunha, V.M.N. [Programa de Farmacologia e Inflamação, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil)

    2013-03-19

    Ca{sup 2+} pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca{sup 2+}-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca{sup 2+} (Ca{sub 0.5} = 780 nM) and a low sensitivity to vanadate (IC{sub 50} = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca{sup 2+} and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca{sup 2+} accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca{sup 2+} and CaM, possibly via CaMKII, in a process that results in stimulation of Ca{sup 2+} pumping activity.

  17. Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis.

    Science.gov (United States)

    Oldereid, N B; Angelis, P D; Wiger, R; Clausen, O P

    2001-05-01

    We investigated the frequency of spontaneous apoptosis and expression of the Bcl-2 family of proteins during normal spermatogenesis in man. Testicular tissue with both normal morphology and DNA content was obtained from necro-donors and fixed in Bouin's solution. A TdT-mediated dUTP end-labelling method (TUNEL) was used for the detection of apoptotic cells. Expression of apoptosis regulatory Bcl-2 family proteins and of p53 and p21(Waf1) was assessed by immunohistochemistry. Germ cell apoptosis was detected in all testes and was mainly seen in primary spermatocytes and spermatids and in a few spermatogonia. Bcl-2 and Bak were preferentially expressed in the compartments of spermatocytes and differentiating spermatids, while Bcl-x was preferentially expressed in spermatogonia. Bax showed a preferential expression in nuclei of round spermatids, whereas Bad was only seen in the acrosome region of various stages of spermatids. Mcl-1 staining was weak without a particular pattern, whereas expression of Bcl-w, p53 and p21(Waf1) proteins was not detected by immunohistochemistry. The results show that spontaneous apoptosis occurs in all male germ cell compartments in humans. Bcl-2 family proteins are distributed preferentially within distinct germ cell compartments suggesting a specific role for these proteins in the processes of differentiation and maturation during human spermatogenesis.

  18. Chemical composition of protein concentrate prepared from Yellowfin tuna Thunnus albacares roe by cook-dried process

    Directory of Open Access Journals (Sweden)

    Hyun Ji Lee

    2016-05-01

    Full Text Available Abstract Roe is the term used to describe fish eggs (oocytes gathered in skeins and is one of the most valuable food products from fishery sources. Thus, means of processing are required to convert the underutilized yellowfin tuna roes (YTR into more marketable and acceptable forms as protein concentrate. Roe protein concentrates (RPCs were prepared by cooking condition (boil-dried concentrate, BDC and steam-dried concentrate, SDC, respectively and un-cooking condition (freeze-dried concentrate, FDC from yellowfin tuna roe. The yield of RPCs was in the range from 22.2 to 25.3 g/100 g of roe. RPCs contained protein (72.3–77.3 %, moisture (4.3–5.6 %, lipid (10.6–11.3 % and ash (4.3–5.7 % as the major constituents. The prominent amino acids of RPCs were aspartic acid, 8.7–9.2, glutamic acid, 13.1–13.2, and leucine, 8.5–8.6 g/100 g of protein. Major differences were not observed in each of the amino acid. K, S, Na, and P as minerals were the major elements in RPCs. No difference noted in sodium dodecyl sulfate polyacrylamide gel electrophoresis protein band (15–100 K possibly representing partial hydrolysis of myosin. Therefore, RPCs from YTR could be use potential protein ingredient for human food and animal feeds.

  19. Protein profiling as early detection biomarkers for TiO2 nanoparticle toxicity in Daphnia magna.

    Science.gov (United States)

    Sá-Pereira, Paula; Diniz, Mário S; Moita, Liliana; Pinheiro, Teresa; Mendonça, Elsa; Paixão, Susana M; Picado, Ana

    2018-05-01

    The mode of action for nanoparticle (NP) toxicity in aquatic organisms is not yet fully understood. In this work, a strategy other than toxicity testing was applied to Daphnia magna exposed to TiO 2 -NPs: the use of nuclear microscopy and the assessment of protein profile. D. magna is a keystone species broadly used as a model system in ecotoxicology. Titanium (Ti) was found in the D. magna digestive tract, mainly in the gut. The penetration of Ti into the epithelial region was greater at higher exposure levels and also observed in eggs in the brood pouch. The protein profile of individuals exposed to different concentrations showed that 2.8 and 5.6 mg/L TiO 2 -NP concentrations induced an over-expression of the majority of proteins, in particular proteins with molecular weight of ∼120, 85 and 15 kDa, while 11.2 mg/L TiO 2 -NP had an inhibitory effect on protein expression. The Matrix-assisted laser desorption ionization with tandem time of flight mass spectrometry (MALDI-TOF/TOF MS) analysis of these proteins consistently identified them as vitellogenin (Vtg)-like proteins, associated with enzymes involved in redox balance. These results indicate that Vtg-like proteins are up-regulated in D. magna exposed to TiO 2 -NPs. Vitellogenesis is associated with the reproduction system, suggesting that TiO 2 -NP exposure can impair reproduction by affecting this process. The precise mode of action of TiO 2 -NPs is still unclear and the results from this study are a first attempt to identify specific proteins as potential markers of TiO 2 -NP toxicity in D. magna, providing useful information for future research.

  20. Analysis of SiO2 nanoparticles binding proteins in rat blood and brain homogenate

    Directory of Open Access Journals (Sweden)

    Shim KH

    2014-12-01

    found for arginine-coated positively charged SiO2 nanoparticles than for the negatively charged nanoparticles. The proteins identified as bound in the corona from SiO2 nanoparticles were further analyzed with ClueGO, a Cytoscape plugin used in protein ontology and for identifying biological interaction pathways. Proteins bound on the surface of nanoparticles may affect functional and conformational properties and distributions in complicated biological processes. Keywords: silica, nanoparticles, protein corona, plasma, brain homogenate, nanotoxicity

  1. Effect of four processed animal proteins in the diet on behavior in laying hens

    NARCIS (Netherlands)

    Krimpen, van M.M.; Veldkamp, T.; Binnendijk, G.P.; Veer, de R.

    2011-01-01

    An experiment was performed to investigate the effect of animal versus vegetable protein sources in the diet on the development of behavior in laying hens. A diet containing protein sources of only vegetable origin was compared with four diets, each containing one of four processed animal proteins

  2. Spatial control of protein phosphatase 2A (de)methylation

    International Nuclear Information System (INIS)

    Longin, Sari; Zwaenepoel, Karen; Martens, Ellen; Louis, Justin V.; Rondelez, Evelien; Goris, Jozef; Janssens, Veerle

    2008-01-01

    Reversible methylation of the protein phosphatase 2A catalytic subunit (PP2A C ) is an important regulatory mechanism playing a crucial role in the selective recruitment of regulatory B subunits. Here, we investigated the subcellular localization of leucine carboxyl methyltransferase (LCMT1) and protein phosphatase methylesterase (PME-1), the two enzymes catalyzing this process. The results show that PME-1 is predominantly localized in the nucleus and harbors a functional nuclear localization signal, whereas LCMT1 is underrepresented in the nucleus and mainly localizes to the cytoplasm, Golgi region and late endosomes. Indirect immunofluorescence with methylation-sensitive anti-PP2A C antibodies revealed a good correlation with the methylation status of PP2A C , demethylated PP2A C being substantially nuclear. Throughout mitosis, demethylated PP2A C is associated with the mitotic spindle and during cytokinesis with the cleavage furrow. Overexpression of PME-1, but not of an inactive mutant, results in increased demethylation of PP2A C in the nucleus, whereas overexpression of a cytoplasmic PME-1 mutant lacking the NLS results in increased demethylation in the cytoplasm-in all cases, however, without any obvious functional consequences. PME-1 associates with an inactive PP2A population, regardless of its esterase activity or localization. We propose that stabilization of this inactive, nuclear PP2A pool is a major in vivo function of PME-1

  3. Rapid and accurate processing method for amide proton exchange rate measurement in proteins

    International Nuclear Information System (INIS)

    Koskela, Harri; Heikkinen, Outi; Kilpelaeinen, Ilkka; Heikkinen, Sami

    2007-01-01

    Exchange between protein backbone amide hydrogen and water gives relevant information about solvent accessibility and protein secondary structure stability. NMR spectroscopy provides a convenient tool to study these dynamic processes with saturation transfer experiments. Processing of this type of NMR spectra has traditionally required peak integration followed by exponential fitting, which can be tedious with large data sets. We propose here a computer-aided method that applies inverse Laplace transform in the exchange rate measurement. With this approach, the determination of exchange rates can be automated, and reliable results can be acquired rapidly without a need for manual processing

  4. The hybrid-cluster protein ('prismane protein') from Escherichia coli. Characterization of the hybrid-cluster protein, redox properties of the [2Fe-2S] and [4Fe-2S-2O] clusters and identification of an associated NADH oxidoreductase containing FAD and[2Fe-2S

    NARCIS (Netherlands)

    Berg, van den W.A.M.; Hagen, W.R.; Dongen, van W.M.A.M.

    2000-01-01

    Hybrid-cluster proteins ('prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe-4S] cubane cluster with spin-admixed S = 3/2 ground-state

  5. Phosphorylation and mRNA splicing of collapsin response mediator protein-2 determine inhibition of rho-associated protein kinase (ROCK) II function in carcinoma cell migration and invasion

    DEFF Research Database (Denmark)

    Morgan-Fisher, Marie; Couchman, John R; Yoneda, Atsuko

    2013-01-01

    The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II......, the presented data show that CRMP-2-dependent regulation of ROCK II activity is mediated through interaction of the CRMP-2L N terminus with the ROCK II catalytic domain as well as by GSK3-dependent phosphorylation of CRMP-2....

  6. ARCPHdb: A comprehensive protein database for SF1 and SF2 helicase from archaea.

    Science.gov (United States)

    Moukhtar, Mirna; Chaar, Wafi; Abdel-Razzak, Ziad; Khalil, Mohamad; Taha, Samir; Chamieh, Hala

    2017-01-01

    Superfamily 1 and Superfamily 2 helicases, two of the largest helicase protein families, play vital roles in many biological processes including replication, transcription and translation. Study of helicase proteins in the model microorganisms of archaea have largely contributed to the understanding of their function, architecture and assembly. Based on a large phylogenomics approach, we have identified and classified all SF1 and SF2 protein families in ninety five sequenced archaea genomes. Here we developed an online webserver linked to a specialized protein database named ARCPHdb to provide access for SF1 and SF2 helicase families from archaea. ARCPHdb was implemented using MySQL relational database. Web interfaces were developed using Netbeans. Data were stored according to UniProt accession numbers, NCBI Ref Seq ID, PDB IDs and Entrez Databases. A user-friendly interactive web interface has been developed to browse, search and download archaeal helicase protein sequences, their available 3D structure models, and related documentation available in the literature provided by ARCPHdb. The database provides direct links to matching external databases. The ARCPHdb is the first online database to compile all protein information on SF1 and SF2 helicase from archaea in one platform. This database provides essential resource information for all researchers interested in the field. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Stability of globular proteins in H2O and D2O

    NARCIS (Netherlands)

    Efimova, Y. M.; Haemers, S.; Wierczinski, B.; Norde, W.; van Well, A. A.

    2007-01-01

    In several experimental techniques D2O rather then H2O is often used as a solvent for proteins. Concerning the influence of the solvent on the stability of the proteins, contradicting results have been reported in literature. In this paper the influence of H2O-D2O solvent substitution on the

  8. Nixtamalized flour from quality protein maize (Zea mays L). optimization of alkaline processing.

    Science.gov (United States)

    Milán-Carrillo, J; Gutiérrez-Dorado, R; Cuevas-Rodríguez, E O; Garzón-Tiznado, J A; Reyes-Moreno, C

    2004-01-01

    Quality of maize proteins is poor, they are deficient in the essential amino acids lysine and tryptophan. Recently, in Mexico were successfully developed nutritionally improved 26 new hybrids and cultivars called quality protein maize (QPM) which contain greater amounts of lysine and tryptophan. Alkaline cooking of maize with lime (nixtamalization) is the first step for producing several maize products (masa, tortillas, flours, snacks). Processors adjust nixtamalization variables based on experience. The objective of this work was to determine the best combination of nixtamalization process variables for producing nixtamalized maize flour (NMF) from QPM V-537 variety. Nixtamalization conditions were selected from factorial combinations of process variables: nixtamalization time (NT, 20-85 min), lime concentration (LC, 3.3-6.7 g Ca(OH)2/l, in distilled water), and steep time (ST, 8-16 hours). Nixtamalization temperature and ratio of grain to cooking medium were 85 degrees C and 1:3 (w/v), respectively. At the end of each cooking treatment the steeping started for the required time. Steeping was finished by draining the cooking liquor (nejayote). Nixtamal (alkaline-cooked maize kernels) was washed with running tap water. Wet nixtamal was dried (24 hours, 55 degrees C) and milled to pass through 80-US mesh screen to obtain NMF. Response surface methodology (RSM) was applied as optimization technique, over four response variables: In vitro protein digestibility (PD), total color difference (deltaE), water absorption index (WAI), and pH. Predictive models for response variables were developed as a function of process variables. Conventional graphical method was applied to obtain maximum PD, WAI and minimum deltaE, pH. Contour plots of each of the response variables were utilized applying superposition surface methodology, to obtain three contour plots for observation and selection of best combination of NT (31 min), LC (5.4 g Ca(OH)2/l), and ST (8.1 hours) for producing

  9. Expression and Production of SH2 Domain Proteins.

    Science.gov (United States)

    Liu, Bernard A; Ogiue-Ikeda, Mari; Machida, Kazuya

    2017-01-01

    The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.

  10. Protein: FBA2 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FBA2 19S regulatory particles(RP) Rpn11 yip5 26S proteasome non-ATPase regulatory subunit 14 26S pr...oteasome regulatory complex subunit p37B, 26S proteasome regulatory subunit rpn11, Yippee-interacting protein 5 7227 Drosophila melanogaster Q9V3H2 Q9V3H2 19075009 ...

  11. Programmed cell death 4 protein (Pdcd4) and homeodomain-interacting protein kinase 2 (Hipk2) antagonistically control translation of Hipk2 mRNA.

    Science.gov (United States)

    Ohnheiser, Johanna; Ferlemann, Eva; Haas, Astrid; Müller, Jan P; Werwein, Eugen; Fehler, Olesja; Biyanee, Abhiruchi; Klempnauer, Karl-Heinz

    2015-07-01

    The tumor suppressor protein programmed cell death 4 (Pdcd4) is a highly conserved RNA-binding protein that inhibits the translation of specific mRNAs. Here, we have identified the homeobox-interacting protein kinase-2 (Hipk2) mRNA as a novel translational target of Pdcd4. Unlike most other protein kinases Hipk2 is constitutively active after being synthesized by the ribosome and its expression and activity are thought to be mainly controlled by modulation of the half-life of the kinase. Our work provides the first evidence that Hipk2 expression is also controlled on the level of translation. We show that Hipk2 stimulates the translation of its own mRNA and that Pdcd4 suppresses the translation of Hipk2 mRNA by interfering with this auto-regulatory feedback mechanism. We also show that the translation of the related kinase Hipk1 is controlled by a similar feedback loop and that Hipk2 also stimulates the translation of Hipk1 mRNA. Taken together, our work describes a novel mechanism of translational suppression by Pdcd4 and shows for the first time that Hipk2 controls its own synthesis by an auto-regulatory feedback mechanism. Furthermore, the effect of Hipk2 on the translation of Hipk1 RNA suggests that Hipk2 and Pdcd4 can act in similar manner to control the translation of other mRNAs. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Genome-wide association study of CSF levels of 59 alzheimer's disease candidate proteins: significant associations with proteins involved in amyloid processing and inflammation.

    Science.gov (United States)

    Kauwe, John S K; Bailey, Matthew H; Ridge, Perry G; Perry, Rachel; Wadsworth, Mark E; Hoyt, Kaitlyn L; Staley, Lyndsay A; Karch, Celeste M; Harari, Oscar; Cruchaga, Carlos; Ainscough, Benjamin J; Bales, Kelly; Pickering, Eve H; Bertelsen, Sarah; Fagan, Anne M; Holtzman, David M; Morris, John C; Goate, Alison M

    2014-10-01

    Cerebrospinal fluid (CSF) 42 amino acid species of amyloid beta (Aβ42) and tau levels are strongly correlated with the presence of Alzheimer's disease (AD) neuropathology including amyloid plaques and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer's Disease Research Center (ADRC) and Alzheimer's Disease Neuroimaging Initiative (ADNI), were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE), Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 4 (CCL4), Interleukin 6 receptor (IL6R) and Matrix metalloproteinase-3 (MMP3)) with study-wide significant p-values (pprocessing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal individuals suggest that these SNPs also influence regulation of these proteins more generally and may therefore be relevant to other diseases.

  13. Fusion protein based on Grb2-SH2 domain for cancer therapy

    International Nuclear Information System (INIS)

    Saito, Yuriko; Furukawa, Takako; Arano, Yasushi; Fujibayashi, Yasuhisa; Saga, Tsuneo

    2010-01-01

    Research highlights: → Grb2 mediates EGFR signaling through binding to phosphorylate EGFR with SH2 domain. → We generated fusion proteins containing 1 or 2 SH2 domains of Grb2 added with TAT. → The one with 2 SH2 domains (TSSF) interfered ERK phosphorylation. → TSSF significantly delayed the growth of EGFR overexpressing tumor in a mouse model. -- Abstract: Epidermal growth factor receptor (EGFR) is one of the very attractive targets for cancer therapy. In this study, we generated fusion proteins containing one or two Src-homology 2 (SH2) domains of growth factor receptor bound protein 2 (Grb2), which bind to phosphorylated EGFR, added with HIV-1 transactivating transcription for cell membrane penetration (termed TSF and TSSF, respectively). We examined if they can interfere Grb2-mediated signaling pathway and suppress tumor growth as expected from the lack of SH3 domain, which is necessary to intermediate EGFR-Grb2 cell signaling, in the fusion proteins. The transduction efficiency of TSSF was similar to that of TSF, but the binding activity of TSSF to EGFR was higher than that of TSF. Treatment of EGFR-overexpressing cells showed that TSSF decreased p42-ERK phosphorylation, while TSF did not. Both the proteins delayed cell growth but did not induce cell death in culture. TSSF also significantly suppressed tumor growth in vivo under consecutive administration. In conclusion, TSSF showed an ability to inhibit EGFR-Grb2 signaling and could have a potential to treat EGFR-activated cancer.

  14. Processing and turnover of the Hedgehog protein in the endoplasmic reticulum.

    Science.gov (United States)

    Chen, Xin; Tukachinsky, Hanna; Huang, Chih-Hsiang; Jao, Cindy; Chu, Yue-Ru; Tang, Hsiang-Yun; Mueller, Britta; Schulman, Sol; Rapoport, Tom A; Salic, Adrian

    2011-03-07

    The Hedgehog (Hh) signaling pathway has important functions during metazoan development. The Hh ligand is generated from a precursor by self-cleavage, which requires a free cysteine in the C-terminal part of the protein and results in the production of the cholesterol-modified ligand and a C-terminal fragment. In this paper, we demonstrate that these reactions occur in the endoplasmic reticulum (ER). The catalytic cysteine needs to form a disulfide bridge with a conserved cysteine, which is subsequently reduced by protein disulfide isomerase. Generation of the C-terminal fragment is followed by its ER-associated degradation (ERAD), providing the first example of an endogenous luminal ERAD substrate that is constitutively degraded. This process requires the ubiquitin ligase Hrd1, its partner Sel1, the cytosolic adenosine triphosphatase p97, and degradation by the proteasome. Processing-defective mutants of Hh are degraded by the same ERAD components. Thus, processing of the Hh precursor competes with its rapid degradation, explaining the impaired Hh signaling of processing-defective mutants, such as those causing human holoprosencephaly.

  15. EST2Prot: Mapping EST sequences to proteins

    Directory of Open Access Journals (Sweden)

    Lin David M

    2006-03-01

    Full Text Available Abstract Background EST libraries are used in various biological studies, from microarray experiments to proteomic and genetic screens. These libraries usually contain many uncharacterized ESTs that are typically ignored since they cannot be mapped to known genes. Consequently, new discoveries are possibly overlooked. Results We describe a system (EST2Prot that uses multiple elements to map EST sequences to their corresponding protein products. EST2Prot uses UniGene clusters, substring analysis, information about protein coding regions in existing DNA sequences and protein database searches to detect protein products related to a query EST sequence. Gene Ontology terms, Swiss-Prot keywords, and protein similarity data are used to map the ESTs to functional descriptors. Conclusion EST2Prot extends and significantly enriches the popular UniGene mapping by utilizing multiple relations between known biological entities. It produces a mapping between ESTs and proteins in real-time through a simple web-interface. The system is part of the Biozon database and is accessible at http://biozon.org/tools/est/.

  16. Markers of protein oxidation

    DEFF Research Database (Denmark)

    Headlam, Henrietta A; Davies, Michael Jonathan

    2004-01-01

    Exposure of proteins to radicals in the presence of O2 gives both side-chain oxidation and backbone fragmentation. These processes can be interrelated, with initial side-chain oxidation giving rise to backbone damage via transfer reactions. We have shown previously that alkoxyl radicals formed...... of this process depends on the extent of oxidation at C-3 compared with other sites. HO*, generated by gamma radiolysis, gave the highest total carbonyl yield, with protein-bound carbonyls predominating over released. In contrast, metal ion/H2O2 systems, gave more released than bound carbonyls, with this ratio...... modulated by EDTA. This is ascribed to metal ion-protein interactions affecting the sites of initial oxidation. Hypochlorous acid gave low concentrations of released carbonyls, but high yields of protein-bound material. The peroxyl radical generator 2,2'-azobis(2-amidinopropane) hydrochloride...

  17. Integration of AI-2 Based Cell-Cell Signaling with Metabolic Cues in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Arindam Mitra

    Full Text Available The quorum sensing molecule Autoinducer-2 (AI-2 is generated as a byproduct of activated methyl cycle by the action of LuxS in Escherichia coli. AI-2 is synthesized, released and later internalized in a cell-density dependent manner. Here, by mutational analysis of the genes, uvrY and csrA, we describe a regulatory circuit of accumulation and uptake of AI-2. We constructed a single-copy chromosomal luxS-lacZ fusion in a luxS + merodiploid strain and evaluated its relative expression in uvrY and csrA mutants. At the entry of stationary phase, the expression of the fusion and AI-2 accumulation was positively regulated by uvrY and negatively regulated by csrA respectively. A deletion of csrA altered message stability of the luxS transcript and CsrA protein exhibited weak binding to 5' luxS regulatory region. DNA protein interaction and chromatin immunoprecipitation analysis confirmed direct interaction of UvrY with the luxS promoter. Additionally, reduced expression of the fusion in hfq deletion mutant suggested involvement of small RNA interactions in luxS regulation. In contrast, the expression of lsrA operon involved in AI-2 uptake, is negatively regulated by uvrY and positively by csrA in a cell-density dependent manner. The dual role of csrA in AI-2 synthesis and uptake suggested a regulatory crosstalk of cell signaling with carbon regulation in Escherichia coli. We found that the cAMP-CRP mediated catabolite repression of luxS expression was uvrY dependent. This study suggests that luxS expression is complex and regulated at the level of transcription and translation. The multifactorial regulation supports the notion that cell-cell communication requires interaction and integration of multiple metabolic signals.

  18. Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

    International Nuclear Information System (INIS)

    Paulin, Sarah; Rosado, Helena; Taylor, Peter W; Jamshad, Mohammed; Dafforn, Timothy R; Garcia-Lara, Jorge; Foster, Simon J; Galley, Nicola F; Roper, David I

    2014-01-01

    Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function. (paper)

  19. Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

    Science.gov (United States)

    Paulin, Sarah; Jamshad, Mohammed; Dafforn, Timothy R.; Garcia-Lara, Jorge; Foster, Simon J.; Galley, Nicola F.; Roper, David I.; Rosado, Helena; Taylor, Peter W.

    2014-07-01

    Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function.

  20. The human nucleolar protein FTSJ3 associates with NIP7 and functions in pre-rRNA processing.

    Directory of Open Access Journals (Sweden)

    Luis G Morello

    Full Text Available NIP7 is one of the many trans-acting factors required for eukaryotic ribosome biogenesis, which interacts with nascent pre-ribosomal particles and dissociates as they complete maturation and are exported to the cytoplasm. By using conditional knockdown, we have shown previously that yeast Nip7p is required primarily for 60S subunit synthesis while human NIP7 is involved in the biogenesis of 40S subunit. This raised the possibility that human NIP7 interacts with a different set of proteins as compared to the yeast protein. By using the yeast two-hybrid system we identified FTSJ3, a putative ortholog of yeast Spb1p, as a human NIP7-interacting protein. A functional association between NIP7 and FTSJ3 is further supported by colocalization and coimmunoprecipitation analyses. Conditional knockdown revealed that depletion of FTSJ3 affects cell proliferation and causes pre-rRNA processing defects. The major pre-rRNA processing defect involves accumulation of the 34S pre-rRNA encompassing from site A' to site 2b. Accumulation of this pre-rRNA indicates that processing of sites A0, 1 and 2 are slower in cells depleted of FTSJ3 and implicates FTSJ3 in the pathway leading to 18S rRNA maturation as observed previously for NIP7. The results presented in this work indicate a close functional interaction between NIP7 and FTSJ3 during pre-rRNA processing and show that FTSJ3 participates in ribosome synthesis in human cells.

  1. Protein carbonylation sites in bovine raw milk and processed milk products.

    Science.gov (United States)

    Milkovska-Stamenova, Sanja; Mnatsakanyan, Ruzanna; Hoffmann, Ralf

    2017-08-15

    During thermal treatment of milk, proteins are oxidized, which may reduce the nutritional value of milk, abolish protein functions supporting human health, especially important for newborns, and yield potentially harmful products. The side chains of several amino acids can be oxidized to reactive carbonyls, which are often used to monitor oxidative stress in organisms. Here we mapped protein carbonylation sites in raw milk and different brands of pasteurized, ultra high temperature (UHT) treated milk, and infant formulas (IFs) after digesting the precipitated proteins with trypsin. Reactive carbonyls were derivatized with O-(biotinylcarbazoylmethyl)hydroxylamine to enrich the modified peptides by avidin-biotin affinity chromatography and analyze them by nanoRP-UPLC-ESI-MS. Overall, 53 unique carbonylated peptides (37 carbonylation sites, 15 proteins) were identified. Most carbonyls were derived from dicarbonyls (mainly glyoxal). The number of carbonylation sites increased with the harsher processing from raw milk (4) to pasteurized (16) and UHT milk (16) and to IF (24). Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris.

    Science.gov (United States)

    Prabhu, Ashish A; Boro, Bibari; Bharali, Biju; Chakraborty, Shuchishloka; Dasu, V Venkata

    2018-03-28

    Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Despite of producing high protein titers, various cellular and process level bottlenecks hinders the expression of recombinant proteins in P. pastoris. In the present review, we have summarized the recent developments in the expression of foreign proteins in P. pastoris. Further, we have discussed various cellular engineering strategies which include codon optimization, pathway engineering, signal peptide processing, development of protease deficient strain and glyco-engineered strains for the high yield protein secretion of recombinant protein. Bioprocess development of recombinant proteins in large scale bioreactor including medium optimization, optimum feeding strategy and co-substrate feeding in fed batch as well as continuous cultivation have been described. The recent advances in system and synthetic biology studies including metabolic flux analysis in understanding the phenotypic characteristics of recombinant Pichia and genome editing with CRISPR-CAS system have also been summarized. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. IGF2 mRNA-binding protein 2: biological function and putative role in type 2 diabetes

    DEFF Research Database (Denmark)

    Christiansen, J.; Kolte, A.M.; Hansen, T.O.

    2009-01-01

    Recent genome-wide association (GWA) studies of type 2 diabetes (T2D) have implicated IGF2 mRNA-binding protein 2 (IMP2/IGF2BP2) as one of the several factors in the etiology of late onset diabetes. IMP2 belongs to a family of oncofetal mRNA-binding proteins implicated in RNA localization...... and T2D Udgivelsesdato: 2009/11......, stability, and translation that are essential for normal embryonic growth and development. This review provides a background to the IMP protein family with an emphasis on human IMP2, followed by a closer look at the GWA studies to evaluate the significance, if any, of the proposed correlation between IMP2...

  4. Rapid microscale in-gel processing and digestion of proteins using surface acoustic waves.

    Science.gov (United States)

    Kulkarni, Ketav P; Ramarathinam, Sri H; Friend, James; Yeo, Leslie; Purcell, Anthony W; Perlmutter, Patrick

    2010-06-21

    A new method for in-gel sample processing and tryptic digestion of proteins is described. Sample preparation, rehydration, in situ digestion and peptide extraction from gel slices are dramatically accelerated by treating the gel slice with surface acoustic waves (SAWs). Only 30 minutes total workflow time is required for this new method to produce base peak chromatograms (BPCs) of similar coverage and intensity to those observed for traditional processing and overnight digestion. Simple set up, good reproducibility, excellent peptide recoveries, rapid turnover of samples and high confidence protein identifications put this technology at the fore-front of the next generation of proteomics sample processing tools.

  5. The TRPC2 channel forms protein-protein interactions with Homer and RTP in the rat vomeronasal organ

    Directory of Open Access Journals (Sweden)

    Brann Jessica H

    2010-05-01

    Full Text Available Abstract Background The signal transduction cascade operational in the vomeronasal organ (VNO of the olfactory system detects odorants important for prey localization, mating, and social recognition. While the protein machinery transducing these external cues has been individually well characterized, little attention has been paid to the role of protein-protein interactions among these molecules. Development of an in vitro expression system for the transient receptor potential 2 channel (TRPC2, which establishes the first electrical signal in the pheromone transduction pathway, led to the discovery of two protein partners that couple with the channel in the native VNO. Results Homer family proteins were expressed in both male and female adult VNO, particularly Homer 1b/c and Homer 3. In addition to this family of scaffolding proteins, the chaperones receptor transporting protein 1 (RTP1 and receptor expression enhancing protein 1 (REEP1 were also expressed. RTP1 was localized broadly across the VNO sensory epithelium, goblet cells, and the soft palate. Both Homer and RTP1 formed protein-protein interactions with TRPC2 in native reciprocal pull-down assays and RTP1 increased surface expression of TRPC2 in in vitro assays. The RTP1-dependent TRPC2 surface expression was paralleled with an increase in ATP-stimulated whole-cell current in an in vitro patch-clamp electrophysiological assay. Conclusions TRPC2 expression and channel activity is regulated by chaperone- and scaffolding-associated proteins, which could modulate the transduction of chemosignals. The developed in vitro expression system, as described here, will be advantageous for detailed investigations into TRPC2 channel activity and cell signalling, for a channel protein that was traditionally difficult to physiologically assess.

  6. The Reg1-interacting proteins, Bmh1, Bmh2, Ssb1, and Ssb2, have roles in maintaining glucose repression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Dombek, Kenneth M; Kacherovsky, Nataly; Young, Elton T

    2004-09-10

    In Saccharomyces cerevisiae, a type 1 protein phosphatase complex composed of the Glc7 catalytic subunit and the Reg1 regulatory subunit represses expression of many glucose-regulated genes. Here we show that the Reg1-interacting proteins Bmh1, Bmh2, Ssb1, and Ssb2 have roles in glucose repression. Deleting both BMH genes causes partially constitutive ADH2 expression without significantly increasing the level of Adr1 protein, the major activator of ADH2 expression. Adr1 and Bcy1, the regulatory subunit of cAMP-dependent protein kinase, are both required for this effect indicating that constitutive expression in Deltabmh1Deltabmh2 cells uses the same activation pathway that operates in Deltareg1 cells. Deletion of both BMH genes and REG1 causes a synergistic relief from repression, suggesting that Bmh proteins also act independently of Reg1 during glucose repression. A two-hybrid interaction with the Bmh proteins was mapped to amino acids 187-232, a region of Reg1 that is conserved in different classes of fungi. Deleting this region partially releases SUC2 from glucose repression. This indicates a role for the Reg1-Bmh interaction in glucose repression and also suggests a broad role for Bmh proteins in this process. An in vivo Reg1-Bmh interaction was confirmed by copurification of Bmh proteins with HA(3)-TAP-tagged Reg1. The nonconventional heat shock proteins Ssb1 and Ssb2 are also copurified with HA(3)-TAP-tagged Reg1. Deletion of both SSB genes modestly decreases repression of ADH2 expression in the presence of glucose, suggesting that Ssb proteins, perhaps through their interaction with Reg1, play a minor role in glucose repression.

  7. High-yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development.

    Science.gov (United States)

    Zhang, Ningning; Gonzalez, Maria; Savary, Brett; Xu, Jianfeng

    2016-03-01

    Low-yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline-O-glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY-2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of "Ser-Pro" dipeptide, or (SP)32 , to study cell growth and protein secretion, culture scale-up, and establishment of perfusion cultures for continuous production. The BY-2 cells accumulated low levels of cell biomass (~7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)32 -tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY-2 cells cultured in a 5-L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day(-1) , generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Detection of the Lux S-mediated quorum-sensing system signal autoinducer-2 of Enterococcus durans SQ-3-2 and optimize the methods%坚强肠球菌SQ-3-2基于Lux S群体感应系统信号分子AI-2的检测及方法优化

    Institute of Scientific and Technical Information of China (English)

    张腾; 贺银凤

    2013-01-01

    To explore that if the Enterococcus durans SQ-3-2 produces the quorum-sensing signal autoinducer-2 by utilizing a biological assay and optimize the test condition at the same time.The experiment employed a biolumi-nescent bacterial reporter strain called Vibrio harveyi BB170, which produces light in response to AI-2.We collected the cell-free culture fluids and added them to the V.harveyi BB170 reporter strain.The resulting light production was measured using a luminometer or scintillation counter.AI-2 activity was deduced according to the induction of luminescence of V.harveyi BB170.The fluorescence of the V.harveyi increased after adding the cell-free culture fluids of E.durans SQ-3-2 and based on the fluorescence intensity of different sample experiment condition was optimized.We found that the E.durans SQ-3-2 produces the quorum-sensing signal AI-2.With the increase of cell density, concentration of signaling molecule AI-2 increased and the maximum value was reached in logarithmic period.Optimal testing condition was sample pH =7 and sample ratio of 1: 100.Experiment lays the foundation to further study of function of AI-2 from E.durans SQ-3-2.At the same time, the optimized experimental conditions for detection of quorum-sensing signal AI-2 of various kinds of lactic acid bacteria is the experimental foundation.%通过生物学方法检测坚强肠球菌SQ-3-2是否产生群体感应信号分子AI-2,并对检测条件进行优化.将坚强肠球菌SQ-3-2培养上清液加入到由哈维氏弧菌BB170构成的特异性报告系统中,使用多功能酶标仪化学发光模式检测荧光强度,通过与AB培养基空白对照进行荧光强度的比较得出坚强肠球菌SQ-3-2是否产生具有活性的AI-2信号分子,同时依据荧光强度的大小对加样比和酸碱度两个条件进行优化.研究结果表明,坚强肠球菌SQ-3-2培养上清液中含有AI-2信号分子,随着菌体密度的增加信号分子AI-2

  9. Evidence for high-pressure-induced rupture of hydrogen bonds in LH2 photosynthetic antenna pigment-protein complexes

    International Nuclear Information System (INIS)

    Kangur, L; Leiger, K; Freiberg, A

    2008-01-01

    The bacteriochlorophyll a-containing LH2 light harvesting complex is an integral membrane protein that catalyzes the photosynthetic process in purple photosynthetic bacteria. The LH2 complexes from Rhodobacter sphaeroides show characteristic strong absorbance at 800 and 850 nm due to the bacteriochlorophyll a molecules confined in two separate areas of the protein. Using these cofactors as intrinsic probes to monitor changes in membrane protein structure, we investigate the response to high hydrostatic pressure up to 2.1 GPa of LH2 complexes embedded into natural membrane environment or extracted with detergent. We demonstrate that high pressure does induce significant alterations to the tertiary structure of the protein in proximity of the protein-bound bacteriochlorophyll a molecules, including breakage of the hydrogen bond they are involved in. The membrane-embedded complexes appear more resilient to damaging effects of the compression than the complexes extracted into detergent environment. This difference has tentatively been explained by more compact structure of the membrane-embedded complexes

  10. Evidence for high-pressure-induced rupture of hydrogen bonds in LH2 photosynthetic antenna pigment-protein complexes

    Energy Technology Data Exchange (ETDEWEB)

    Kangur, L; Leiger, K; Freiberg, A [Institute of Physics, University of Tartu, Riia 142, Tartu 51014 (Estonia)

    2008-07-15

    The bacteriochlorophyll a-containing LH2 light harvesting complex is an integral membrane protein that catalyzes the photosynthetic process in purple photosynthetic bacteria. The LH2 complexes from Rhodobacter sphaeroides show characteristic strong absorbance at 800 and 850 nm due to the bacteriochlorophyll a molecules confined in two separate areas of the protein. Using these cofactors as intrinsic probes to monitor changes in membrane protein structure, we investigate the response to high hydrostatic pressure up to 2.1 GPa of LH2 complexes embedded into natural membrane environment or extracted with detergent. We demonstrate that high pressure does induce significant alterations to the tertiary structure of the protein in proximity of the protein-bound bacteriochlorophyll a molecules, including breakage of the hydrogen bond they are involved in. The membrane-embedded complexes appear more resilient to damaging effects of the compression than the complexes extracted into detergent environment. This difference has tentatively been explained by more compact structure of the membrane-embedded complexes.

  11. Polycomb Protein OsFIE2 Affects Plant Height and Grain Yield in Rice.

    Directory of Open Access Journals (Sweden)

    Xianbo Liu

    Full Text Available Polycomb group (PcG proteins have been shown to affect growth and development in plants. To further elucidate their role in these processes in rice, we isolated and characterized a rice mutant which exhibits dwarfism, reduced seed setting rate, defective floral organ, and small grains. Map-based cloning revealed that abnormal phenotypes were attributed to a mutation of the Fertilization Independent Endosperm 2 (OsFIE2 protein, which belongs to the PcG protein family. So we named the mutant as osfie2-1. Histological analysis revealed that the number of longitudinal cells in the internodes decreased in osfie2-1, and that lateral cell layer of the internodes was markedly thinner than wild-type. In addition, compared to wild-type, the number of large and small vascular bundles decreased in osfie2-1, as well as cell number and cell size in spikelet hulls. OsFIE2 is expressed in most tissues and the coded protein localizes in both nucleus and cytoplasm. Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that OsFIE2 interacts with OsiEZ1 which encodes an enhancer of zeste protein previously identified as a histone methylation enzyme. RNA sequencing-based transcriptome profiling and qRT-PCR analysis revealed that some homeotic genes and genes involved in endosperm starch synthesis, cell division/expansion and hormone synthesis and signaling are differentially expressed between osfie2-1 and wild-type. In addition, the contents of IAA, GA3, ABA, JA and SA in osfie2-1 are significantly different from those in wild-type. Taken together, these results indicate that OsFIE2 plays an important role in the regulation of plant height and grain yield in rice.

  12. Synthesis and processing of structural and intracellular proteins of two enteric coronaviruses

    International Nuclear Information System (INIS)

    Sardinia, L.M.

    1985-01-01

    The synthesis and processing of virus-specific proteins of two economically important enteric coronaviruses, bovine enteric coronavirus (BCV) and transmissible gastroenteritis virus (TGEV), were studied at the molecular level. To determine the time of appearance of virus-specific proteins, virus-infected cells were labeled with 35 S-methionine at various times during infection, immunoprecipitated with specific hyperimmune ascitic fluid, and analyzed by SDS-polyacrylamide gel electrophoresis. The peak of BCV protein synthesis was found to be at 12 hours postinfection (hpi). The appearance of all virus-specific protein was coordinated. In contrast, the peak of TGEV protein synthesis was at 8 hpi, but the nucleocapsid proteins was present as early as 4 hpi. Virus-infected cells were treated with tunicamycin to ascertain the types of glycosidic linkages of the glycoproteins. The peplomer proteins of both viruses were sensitive to inhibition by tunicamycin indicating that they possessed N-linked carbohydrates. The matrix protein of TGEV was similarly affected. The matrix protein of BCV, however, was resistant to tunicamycin treatment and, therefore, has O-linked carbohydrates. Only the nucleocapsid protein of both viruses is phosphorylated as detected by radiolabeling with 32 P-orthophosphate. Pulse-chase studies and comparison of intracellular and virion proteins were done to detect precursor-product relationships

  13. Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein

    DEFF Research Database (Denmark)

    Sørensen, Rikke Kruse; Krantz, James; Barker, Natalie

    2017-01-01

    . The GTPase-activating proteins AGAP1 and AGAP3 were also enriched in the CLASP2 interactome, although subsequent AGAP3 and CLIP2 interactome analysis suggests a preference of AGAP3 for CLIP2. Follow-up MARK2 interactome analysis confirmed reciprocal co-IP of CLASP2 and also revealed MARK2 can co-IP SOGA1......, glycogen synthase, and glycogenin. Investigating the SOGA1 interactome confirmed SOGA1 can reciprocal co-IP both CLASP2 and MARK2 as well as glycogen synthase and glycogenin. SOGA1 was confirmed to colocalize with CLASP2 and also with tubulin, which identifies SOGA1 as a new microtubule-associated protein....... These results introduce the metabolic function of these proposed novel protein networks and their relationship with microtubules as new fields of cytoskeleton-associated protein biology....

  14. GAS2L1 Is a Centriole-Associated Protein Required for Centrosome Dynamics and Disjunction.

    NARCIS (Netherlands)

    Au, F.K.; Jia, Y.; Jiang, K.; Grigoriev, I.S.; Hau, B.K.; Shen, Y.; Du, S.; Akhmanova, A.S.; Qi, R.Z.

    2017-01-01

    Mitotic spindle formation and chromosome segregation require timely separation of the two duplicated centrosomes, and this process is initiated in late G2 by centrosome disjunction. Here we report that GAS2L1, a microtubule- and actin-binding protein, associates with the proximal end of mature

  15. A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L

    International Nuclear Information System (INIS)

    Pager, Cara Theresia; Craft, Willie Warren; Patch, Jared; Dutch, Rebecca Ellis

    2006-01-01

    The Nipah virus fusion (F) protein is proteolytically processed to F 1 + F 2 subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form

  16. Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

    Science.gov (United States)

    Cai, X.; Chang, D.; Rottinghaus, S.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

  17. Overproduction, purification, crystallization and preliminary X-ray analysis of human Fe65-PTB2 in complex with the amyloid precursor protein intracellular domain

    Energy Technology Data Exchange (ETDEWEB)

    Radzimanowski, Jens [Heidelberg University Biochemistry Center, INF328, D-69120 Heidelberg (Germany); Beyreuther, Konrad [Center for Molecular Biology, University Heidelberg, INF282, D-69120 Heidelberg (Germany); Sinning, Irmgard; Wild, Klemens, E-mail: klemens.wild@bzh.uni-heidelberg.de [Heidelberg University Biochemistry Center, INF328, D-69120 Heidelberg (Germany)

    2008-05-01

    Alzheimer’s disease is characterized by proteolytic processing of the amyloid precursor protein (APP), which releases the aggregation-prone amyloid-β (Aβ) peptide and liberates the intracellular domain (AICD) that interacts with various adaptor proteins. The crystallized AICD–Fe65-PTB2 complex is of central importance for APP translocation, nuclear signalling, processing and Aβ generation. Alzheimer’s disease is associated with typical brain deposits (senile plaques) that mainly contain the neurotoxic amyloid β peptide. This peptide results from proteolytic processing of the type I transmembrane protein amyloid precursor protein (APP). During this proteolytic pathway the APP intracellular domain (AICD) is released into the cytosol, where it associates with various adaptor proteins. The interaction of the AICD with the C-terminal phosphotyrosine-binding domain of Fe65 (Fe65-PTB2) regulates APP translocation, signalling and processing. Human AICD and Fe65-PTB2 have been cloned, overproduced and purified in large amounts in Escherichia coli. A complex of Fe65-PTB2 with the C-terminal 32 amino acids of the AICD gave well diffracting hexagonal crystals and data have been collected to 2.1 Å resolution. Initial phases obtained by the molecular-replacement method are of good quality and revealed well defined electron density for the substrate peptide.

  18. Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane.

    Science.gov (United States)

    Koga, Ritsuko; Kubota, Marie; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

    2018-02-28

    MV within the cell. Further studies demonstrated that annexin A2 interacts with the MV matrix (M) protein and mediates the localization of the M protein at the plasma membrane where MV particles are formed. The M protein lines the inner surface of the MV envelope membrane and plays a role in MV particle formation. Our results provide useful information for the understanding of the MV replication process and potential development of anti-viral agents. Copyright © 2018 American Society for Microbiology.

  19. Yeast polypeptide exit tunnel ribosomal proteins L17, L35 and L37 are necessary to recruit late-assembling factors required for 27SB pre-rRNA processing.

    Science.gov (United States)

    Gamalinda, Michael; Jakovljevic, Jelena; Babiano, Reyes; Talkish, Jason; de la Cruz, Jesús; Woolford, John L

    2013-02-01

    Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2.

  20. FRET-based biosensors for the detection and quantification of AI-2 class of quorum sensing compounds.

    Science.gov (United States)

    Rajamani, Sathish; Sayre, Richard

    2011-01-01

    Intercellular small molecular weight signaling molecules modulate a variety of biological functions in bacteria. One of the more complex behaviors mediated by intercellular signaling molecules is the suite of activities regulated by quorum sensing molecules. These molecules mediate a variety of population-dependent responses, including the expression of genes that regulate bioluminescence, type III secretion, siderophore production, colony morphology, biofilm formation, and metalloprotease production. Given their central role in regulating these responses, the detection and quantification of QS molecules has important practical implications. Until recently, the detection of QS molecules from Gram-negative bacteria has relied primarily on bacterial reporter systems. These bioassays though immensely useful are subject to interference by compounds that affect bacterial growth and metabolism. In addition, the reporter response is highly dependent on culture age and cell population density. To overcome such limitations, we developed an in vitro protein-based assay system for the rapid detection and quantification of the furanosyl borate diester (BAI-2) subclass of autoinducer-2 (AI-2) QS molecules. The biosensor is based on the interaction of BAI-2 with the Vibrio harveyi QS receptor LuxP. Conformation changes associated with BAI-2 binding to the LuxP receptor change the orientation of cyan and yellow variants of GFP (CFP and YFP) fused the N- and C-termini, respectively, of the LuxP receptor. LuxP-BAI2 binding induces changes in fluorescence resonance energy transfer (FRET) between CFP and YFP, whose magnitude of change is ligand concentration dependent. A set of ligand-insensitive LuxP-mutant FRET protein sensor was also developed for use as control biosensors. The FRET-based BAI-2 biosensor responds selectively to both synthetic and biologically derived BAI-2compounds. This report describes the use of the LuxP-FRET biosensor for the detection and quantification of

  1. Detect the sensitivity and response of protein molecular structure of whole canola seed (yellow and brown) to different heat processing methods and relation to protein utilization and availability using ATR-FT/IR molecular spectroscopy with chemometrics.

    Science.gov (United States)

    Samadi; Theodoridou, Katerina; Yu, Peiqiang

    2013-03-15

    The objectives of this experiment were to detect the sensitivity and response of protein molecular structure of whole canola seed to different heat processing [moisture (autoclaving) vs. dry (roasting) heating] and quantify heat-induced protein molecular structure changes in relation to protein utilization and availability. In this study, whole canola seeds were autoclaved (moisture heating) and dry (roasting) heated at 120 °C for 1h, respectively. The parameters assessed included changes in (1) chemical composition profile, (2) CNCPS protein subfractions (PA, PB1, PB2, PB3, PC), (3) intestinal absorbed true protein supply, (4) energy values, and (5) protein molecular structures (amide I, amide II, ratio of amide I to II, α-helix, β-sheet, ratio of α-helix to β-sheet). The results showed that autoclave heating significantly decreased (Pseed. Future study is needed to study response and impact of heat processing to each inherent layer of canola seed from outside to inside tissues and between yellow canola and brown canola. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Impact of the lipid bilayer on energy transfer kinetics in the photosynthetic protein LH2.

    Science.gov (United States)

    Ogren, John I; Tong, Ashley L; Gordon, Samuel C; Chenu, Aurélia; Lu, Yue; Blankenship, Robert E; Cao, Jianshu; Schlau-Cohen, Gabriela S

    2018-03-28

    Photosynthetic purple bacteria convert solar energy to chemical energy with near unity quantum efficiency. The light-harvesting process begins with absorption of solar energy by an antenna protein called Light-Harvesting Complex 2 (LH2). Energy is subsequently transferred within LH2 and then through a network of additional light-harvesting proteins to a central location, termed the reaction center, where charge separation occurs. The energy transfer dynamics of LH2 are highly sensitive to intermolecular distances and relative organizations. As a result, minor structural perturbations can cause significant changes in these dynamics. Previous experiments have primarily been performed in two ways. One uses non-native samples where LH2 is solubilized in detergent, which can alter protein structure. The other uses complex membranes that contain multiple proteins within a large lipid area, which make it difficult to identify and distinguish perturbations caused by protein-protein interactions and lipid-protein interactions. Here, we introduce the use of the biochemical platform of model membrane discs to study the energy transfer dynamics of photosynthetic light-harvesting complexes in a near-native environment. We incorporate a single LH2 from Rhodobacter sphaeroides into membrane discs that provide a spectroscopically amenable sample in an environment more physiological than detergent but less complex than traditional membranes. This provides a simplified system to understand an individual protein and how the lipid-protein interaction affects energy transfer dynamics. We compare the energy transfer rates of detergent-solubilized LH2 with those of LH2 in membrane discs using transient absorption spectroscopy and transient absorption anisotropy. For one key energy transfer step in LH2, we observe a 30% enhancement of the rate for LH2 in membrane discs compared to that in detergent. Based on experimental results and theoretical modeling, we attribute this difference to

  3. Processing of Chlamydia abortus polymorphic membrane protein 18D during the chlamydial developmental cycle.

    Directory of Open Access Journals (Sweden)

    Nick M Wheelhouse

    Full Text Available Chlamydia possess a unique family of autotransporter proteins known as the Polymorphic membrane proteins (Pmps. While the total number of pmp genes varies between Chlamydia species, all encode a single pmpD gene. In both Chlamydia trachomatis (C. trachomatis and C. pneumoniae, the PmpD protein is proteolytically cleaved on the cell surface. The current study was carried out to determine the cleavage patterns of the PmpD protein in the animal pathogen C. abortus (termed Pmp18D.Using antibodies directed against different regions of Pmp18D, proteomic techniques revealed that the mature protein was cleaved on the cell surface, resulting in a100 kDa N-terminal product and a 60 kDa carboxy-terminal protein. The N-terminal protein was further processed into 84, 76 and 73 kDa products. Clustering analysis resolved PmpD proteins into three distinct clades with C. abortus Pmp18D, being most similar to those originating from C. psittaci, C. felis and C. caviae.This study indicates that C. abortus Pmp18D is proteolytically processed at the cell surface similar to the proteins of C. trachomatis and C. pneumoniae. However, patterns of cleavage are species-specific, with low sequence conservation of PmpD across the genus. The absence of conserved domains indicates that the function of the PmpD molecule in chlamydia remains to be elucidated.

  4. Processing of Chlamydia abortus polymorphic membrane protein 18D during the chlamydial developmental cycle.

    Science.gov (United States)

    Wheelhouse, Nick M; Sait, Michelle; Aitchison, Kevin; Livingstone, Morag; Wright, Frank; McLean, Kevin; Inglis, Neil F; Smith, David G E; Longbottom, David

    2012-01-01

    Chlamydia possess a unique family of autotransporter proteins known as the Polymorphic membrane proteins (Pmps). While the total number of pmp genes varies between Chlamydia species, all encode a single pmpD gene. In both Chlamydia trachomatis (C. trachomatis) and C. pneumoniae, the PmpD protein is proteolytically cleaved on the cell surface. The current study was carried out to determine the cleavage patterns of the PmpD protein in the animal pathogen C. abortus (termed Pmp18D). Using antibodies directed against different regions of Pmp18D, proteomic techniques revealed that the mature protein was cleaved on the cell surface, resulting in a100 kDa N-terminal product and a 60 kDa carboxy-terminal protein. The N-terminal protein was further processed into 84, 76 and 73 kDa products. Clustering analysis resolved PmpD proteins into three distinct clades with C. abortus Pmp18D, being most similar to those originating from C. psittaci, C. felis and C. caviae. This study indicates that C. abortus Pmp18D is proteolytically processed at the cell surface similar to the proteins of C. trachomatis and C. pneumoniae. However, patterns of cleavage are species-specific, with low sequence conservation of PmpD across the genus. The absence of conserved domains indicates that the function of the PmpD molecule in chlamydia remains to be elucidated.

  5. A sensitive enzyme-linked immunosorbent assay for the determination of fish protein in processed foods.

    Science.gov (United States)

    Shibahara, Yusuke; Uesaka, Yoshihiko; Wang, Jun; Yamada, Shoichi; Shiomi, Kazuo

    2013-01-15

    Fish is one of the most common causes of food allergy and its major allergen is parvalbumin, a 12 kDa muscular protein. In this study, a sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of fish protein in processed foods was developed using a polyclonal antibody raised against Pacific mackerel parvalbumin. The developed sandwich ELISA showed 22.6-99.0% reactivity (based on the reactivity to Pacific mackerel parvalbumin) to parvalbumins from various species of fish. The limits of detection and quantitation were estimated to be 0.23 and 0.70 μg protein per g of food, respectively. When the sandwich ELISA was subjected to inter-laboratory validation, spiked fish protein was recovered from five model processed foods in the range of 69.4-84.8% and the repeatability and reproducibility relative standard deviations were satisfactorily low (≤ 10.5%). Thus, the sandwich ELISA was judged to be a useful tool to determine fish protein in processed foods. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Accesion number Protein name ENOA_MOUSE Alpha-enolase ...

    Indian Academy of Sciences (India)

    Sandra Feijoo Bandin

    Mitochondrial inner membrane protein. CMC1_MOUSE. Calcium-binding mitochondrial carrier protein Aralar1. CMC2_MOUSE. Calcium-binding mitochondrial carrier protein Aralar2. Biological process. Metabolic process. Glycolysis. Lipid metabolism. Respiratory electron transport chain. Others. Calcium ion homeostasis.

  7. Finding the "bio" in biobased products: electrophoretic identification of wheat proteins in processed products.

    Science.gov (United States)

    Robertson, George H; Hurkman, William J; Cao, Trung K; Tanaka, Charlene K; Orts, William J

    2010-04-14

    Verification of the biocontent in biobased or "green" products identifies genuine products, exposes counterfeit copies, supports or refutes content claims, and ensures consumer confidence. When the biocontent includes protein, elemental nitrogen analysis is insufficient for verification since non-protein, but nitrogen-rich, content also may be present. However, the proteins can be extracted, separated by electrophoretic methods, and detected by UV absorption, protein stain, or immunoblotting. We utilized capillary zone electrophoresis (CZE) to separate proteins in a gliadin fraction that had been dissolved in aqueous ethanol (70%) and polyacrylamide gel electrophoresis (PAGE) to separate proteins in a gliadin-plus-glutenin fraction that had been dissolved in water containing both sodium dodecyl sulfate (SDS) and a reducing agent, dithiothreitol (DTT). We sought to verify the presence of these wheat grain proteins in wheat bread, a wheat flake cereal, wheat beer, and an enclosure for an antique automobile ignition coil reputed to contain wheat gluten. Proteins extracted from commercial wheat, corn, and soy flours served as standards, and proteins from heat-altered wheat served as process condition references. This approach successfully identified wheat proteins in these products especially if the process temperature did not exceed 120 degrees C. Above this temperature attenuation was nearly complete for proteins analyzed by CZE, but wheat-like patterns could still be recognized by one- and two-dimensional PAGE. Immunoblots reacted with grain-specific antibodies confirmed the identities of the cereal component especially when the protein pattern was greatly altered by thermal modification, specific protein adsorption, or protein digestion. In addition to verifying that wheat proteins are present, the complementary use of these methods can reveal whether whole wheat gluten or merely an alcohol-soluble fraction had been used in the specific product and indicate the

  8. Neutron scattering studies on protein dynamics using the human myelin peripheral membrane protein P2

    Directory of Open Access Journals (Sweden)

    Laulumaa Saara

    2015-01-01

    Full Text Available Myelin is a multilayered proteolipid membrane structure surrounding selected axons in the vertebrate nervous system, which allows the rapid saltatory conduction of nerve impulses. Deficits in myelin formation and maintenance may lead to chronic neurological disease. P2 is an abundant myelin protein from peripheral nerves, binding between two apposing lipid bilayers. We studied the dynamics of the human myelin protein P2 and its mutated P38G variant in hydrated powders using elastic incoherent neutron scattering. The local harmonic vibrations at low temperatures were very similar for both samples, but the mutant protein had increased flexibility and softness close to physiological temperatures. The results indicate that a drastic mutation of proline to glycine at a functional site can affect protein dynamics, and in the case of P2, they may explain functional differences between the two proteins.

  9. Neutron scattering studies on protein dynamics using the human myelin peripheral membrane protein P2

    Science.gov (United States)

    Laulumaa, Saara; Kursula, Petri; Natali, Francesca

    2015-01-01

    Myelin is a multilayered proteolipid membrane structure surrounding selected axons in the vertebrate nervous system, which allows the rapid saltatory conduction of nerve impulses. Deficits in myelin formation and maintenance may lead to chronic neurological disease. P2 is an abundant myelin protein from peripheral nerves, binding between two apposing lipid bilayers. We studied the dynamics of the human myelin protein P2 and its mutated P38G variant in hydrated powders using elastic incoherent neutron scattering. The local harmonic vibrations at low temperatures were very similar for both samples, but the mutant protein had increased flexibility and softness close to physiological temperatures. The results indicate that a drastic mutation of proline to glycine at a functional site can affect protein dynamics, and in the case of P2, they may explain functional differences between the two proteins.

  10. Neuronal process structure and growth proteins are targets of heavy PTM regulation during brain development

    DEFF Research Database (Denmark)

    Edwards, Alistair V G; Schwämmle, Veit; Larsen, Martin Røssel

    2014-01-01

    UNLABELLED: Brain development is a process requiring precise control of many different cell types. One method to achieve this is through specific and temporally regulated modification of proteins in order to alter structure and function. Post-translational modification (PTM) of proteins is known...... on protein-level events, this study also provides significant insight into detailed roles for individual modified proteins in the developing brain, helping to advance the understanding of the complex protein-driven processes that underlie development. Finally, the use of a novel bioinformatic analytical tool...... provides one of the most comprehensive sets of individual PTM site regulation data for mammalian brain tissue. This will provide a valuable resource for those wishing to perform comparisons or meta-analyses of large scale PTMomic data, as are becoming increasingly common. Furthermore, being focussed...

  11. Dopamine D2L receptor-interacting proteins regulate dopaminergic signaling

    Directory of Open Access Journals (Sweden)

    Norifumi Shioda

    2017-10-01

    Full Text Available Dopamine receptor family proteins include seven transmembrane and trimeric GTP-binding protein-coupled receptors (GPCRs. Among them, the dopamine D2 receptor (D2R is most extensively studied. All clinically used antipsychotic drugs serve as D2R antagonists in the mesolimbic dopamine system, and their ability to block D2R signaling is positively correlated with antipsychotic efficiency. Human genetic studies also show a significant association of DRD2 polymorphisms with disorders including schizophrenia and Parkinson's disease. D2R exists as two alternatively spliced isoforms, the long isoform (D2LR and the short isoform (D2SR, which differ in a 29-amino acid (AA insert in the third cytoplasmic loop. Importantly, previous reports demonstrate functional diversity between the two isoforms in humans. In this review, we focus on binding proteins that specifically interact with the D2LR 29AA insert. We discuss how D2R activities are mediated not only by heterotrimeric G proteins but by D2LR-interacting proteins, which in part regulate diverse D2R activities. Keywords: Dopamine D2L receptor, Antipsychotic drugs, DRD2 polymorphisms, Alternatively spliced isoforms, D2LR-interacting proteins

  12. Using the 2A Protein Coexpression System: Multicistronic 2A Vectors Expressing Gene(s) of Interest and Reporter Proteins.

    Science.gov (United States)

    Luke, Garry A; Ryan, Martin D

    2018-01-01

    To date, a huge range of different proteins-many with cotranslational and posttranslational subcellular localization signals-have been coexpressed together with various reporter proteins in vitro and in vivo using 2A peptides. The pros and cons of 2A co-expression technology are considered below, followed by a simple example of a "how to" protocol to concatenate multiple genes of interest, together with a reporter gene, into a single gene linked via 2As for easy identification or selection of transduced cells.

  13. Myostatin inhibits eEF2K-eEF2 by regulating AMPK to suppress protein synthesis.

    Science.gov (United States)

    Deng, Zhao; Luo, Pei; Lai, Wen; Song, Tongxing; Peng, Jian; Wei, Hong-Kui

    2017-12-09

    Growth of skeletal muscle is dependent on the protein synthesis, and the rate of protein synthesis is mainly regulated in the stage of translation initiation and elongation. Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily, is a negative regulator of protein synthesis. C2C12 myotubes was incubated with 0, 0.01, 0.1, 1, 2, 3 μg/mL myostatin recombinant protein, and then we detected the rates of protein synthesis by the method of SUnSET. We found that high concentrations of myostatin (2 and 3 μg/mL) inhibited protein synthesis by blocking mTOR and eEF2K-eEF2 pathway, while low concentration of myostatin (0.01, 0.1 and 1 μg/mL) regulated eEF2K-eEF2 pathway activity to block protein synthesis without affected mTOR pathway, and myostatin inhibited eEF2K-eEF2 pathway through regulating AMPK pathway to suppress protein synthesis. It provided a new mechanism for myostatin regulating protein synthesis and treating muscle atrophy. Copyright © 2017. Published by Elsevier Inc.

  14. Process for the separation of proteins from acid whey

    Energy Technology Data Exchange (ETDEWEB)

    Mirabel, B

    1980-01-01

    Acid whey from cheese or casein manufacture (pH less than 4.6) and containing about 5.2 g protein/l is passed through a cation exchange resin (of silica coated with a copolymer of styrene/vinyltriethoxysilane carrying SO/sub 3/H functional groups). The proteins adsorbed on the resin (alpha-lactalbumin, beta-lactoglobulin, serum albumin and immunoglobulins) are eluated with an 0.1 M ammonia solution, concentrated under vacuum and freeze-dried, obtaining a final product with 88% undenatured protein. The products are for use in the food and pharmaceutical industries and for dietetic and veterinary purposes.

  15. Dietary protein and fat emulsions, processed by ultrasound and pulsed magnetic field

    Directory of Open Access Journals (Sweden)

    E. I. Verboloz

    2017-01-01

    Full Text Available For the baking of baked goods in order to save fats, different types of endorsement and protein-fatty emulsions which are used as ingredients in goods and for the protection of metal moulds from burning. Usually emulsion is prepared on bakery enterprises by National State Standard Р 51785–2001, involving mechanical beating up of ingredients. The authors suggested and studied the way of manufacturing of more stable food protein-fatty emulsions using ultrasonic transmitter with rigid neodymium magnets on its thickener. As ingredients, there were applied curd whey diluted with water, unpurified sunflower oil and sunflower phosphatides. Ratio of whey and water is 1:7. Physical effects of ultrasound and field of magnets in contact layer of liquid ingredients being dispersed have increased the viscosity and dispersion of protein-fatty emulsions. Hypothesis of increase of stability and sterility of protein-fatty emulsion by the selection of parameters of magnetic field and power of ultrasound transmitter is confirmed experimentally. Microscopic analysis shows high degree of homogeneity of emulsion under the time of processing 3-4 minutes and intensity of ultrasound 2 W/cm2, that is energetically profitable. There was revealed synergism of influence of physical effects of ultrasound and magnetic field on the durability and steadiness of emulsion to mechanical and temperature effect and also cidal effect, prolonging terms of product using. Manufacture of emulsions by the declared way using the ultrasound and magnetic field of constant neodymium magnets decreases number of injected elements-emulsifiers by 3-4 times or excludes their use at all. Existing piezoelectric ultrasound units as well as neodymium magnets have small sizes and low energy consumption, easily built into the line of continuous manufacture of emulsion for the bread production. Such emulsions are less demanding to the storage and transportation.

  16. Crystal structure of human protein kinase CK2

    DEFF Research Database (Denmark)

    Niefind, K; Guerra, B; Ermakowa, I

    2001-01-01

    The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalyt...... as a docking partner for various protein kinases. Furthermore it shows an inter-domain mobility in the catalytic subunit known to be functionally important in protein kinases and detected here for the first time directly within one crystal structure.......The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalytic...... subunits, which make no direct contact with one another. Each catalytic subunit interacts with both regulatory chains, predominantly via an extended C-terminal tail of the regulatory subunit. The CK2 structure is consistent with its constitutive activity and with a flexible role of the regulatory subunit...

  17. N-terminal protein processing: A comparative proteogenomic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Bonissone, Stefano; Gupta, Nitin; Romine, Margaret F.; Bradshaw, Ralph A.; Pevzner, Pavel A.

    2013-01-01

    N-Terminal Methionine Excision (NME) is a universally conserved mechanism with the same specificity across all life forms that removes the first Methionine in proteins when the second residue is Gly, Ala, Ser, Cys, Thr, Pro, or Val. In spite of its necessity for proper cell functioning, the functional role of NME remains unclear. In 1988, Arfin and Bradshaw connected NME with the N-end protein degradation rule and postulated that the role of NME is to expose the stabilizing residues with the goal to resist protein degradation. While this explanation (that treats 7 stabilizing residues in the same manner) has become the de facto dogma of NME, comparative proteogenomics analysis of NME tells a different story. We suggest that the primary role of NME is to expose only two (rather than seven) amino acids Ala and Ser for post-translational modifications (e.g., acetylation) rather than to regulate protein degradation. We argue that, contrary to the existing view, NME is not crucially important for proteins with 5 other stabilizing residue at the 2nd positions that are merely bystanders (their function is not affected by NME) that become exposed to NME because their sizes are comparable or smaller than the size of Ala and Ser.

  18. Phosphorylation Regulates the Bound Structure of an Intrinsically Disordered Protein: The p53-TAZ2 Case.

    Directory of Open Access Journals (Sweden)

    Raúl Esteban Ithuralde

    Full Text Available Disordered regions and Intrinsically Disordered Proteins (IDPs are involved in critical cellular processes and may acquire a stable three-dimensional structure only upon binding to their partners. IDPs may follow a folding-after-binding process, known as induced folding, or a folding-before-binding process, known as conformational selection. The transcription factor p53 is involved in the regulation of cellular events that arise upon stress or DNA damage. The p53 domain structure is composed of an N-terminal transactivation domain (p53TAD, a DNA Binding Domain and a tetramerization domain. The activity of TAD is tightly regulated by interactions with cofactors, inhibitors and phosphorylation. To initiate transcription, p53TAD binds to the TAZ2 domain of CBP, a co-transcription factor, and undergoes a folding and binding process, as revealed by the recent NMR structure of the complex. The activity of p53 is regulated by phosphorylation at multiple sites on the TAD domain and recent studies have shown that modifications at three residues affect the binding towards TAZ2. However, we still do not know how these phosphorylations affect the structure of the bound state and, therefore, how they regulate the p53 function. In this work, we have used computational simulations to understand how phosphorylation affects the structure of the p53TAD:TAZ2 complex and regulates the recognition mechanism. Phosphorylation has been proposed to enhance binding by direct interaction with the folded protein or by changing the unbound conformation of IDPs, for example by pre-folding the protein favoring the recognition mechanism. Here, we show an interesting turn in the p53 case: phosphorylation mainly affects the bound structure of p53TAD, highlighting the complexity of IDP protein-protein interactions. Our results are in agreement with previous experimental studies, allowing a clear picture of how p53 is regulated by phosphorylation and giving new insights into how

  19. Leaching and heating process as alternative to produce fish protein powder from Kilka (Clupeonella cultiventris caspia

    Directory of Open Access Journals (Sweden)

    KAVEH RAHMANIFARAH

    2014-05-01

    Full Text Available Rahmanifarah K, Shabanpour B, Shaviklo AR, Aalami M. 2014. Leaching and heating process as alternative to produce fish protein powder from Kilka (Clupeonella cultiventris caspia. Nusantara Bioscience 6: 1-6. The effect of protein extraction procedures (leached mince and heated suspension on selected properties of fish protein powder (proximate composition, pH, color, density, viscosity, fat adsorption, emulsifying capacity, emulsifying stability, foaming capacity, foaming stability, WBC, protein solubility in water, hygroscopicity, Trichloroacetic acid (TCA-soluble peptides and free sulfhydryl groups was investigated. Results showed that Fish protein powder (FPP produced by leaching mince (LM have higher protein, moisture, ash, pH, L*, viscosity, emulsion capacity, emulsion stability, foam capacity, foam stability, water binding capacity (WBC, protein solubility, hygroscopicity, TCA soluble peptides and free sulfhydryl group content than heated suspension (HS (P0.05. Overall, it was observed that high temperature during heating of suspension in HS method makes possible protein denaturation and aggregation. Consequently, based on functional, chemical and physical properties, extraction of fish protein by leaching process was found to be suitable for the production of fish protein powder.

  20. A protein-tyrosine phosphatase with sequence similarity to the SH2 domain of the protein-tyrosine kinases.

    Science.gov (United States)

    Shen, S H; Bastien, L; Posner, B I; Chrétien, P

    1991-08-22

    The phosphorylation of proteins at tyrosine residues is critical in cellular signal transduction, neoplastic transformation and control of the mitotic cycle. These mechanisms are regulated by the activities of both protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPases). As in the PTKs, there are two classes of PTPases: membrane associated, receptor-like enzymes and soluble proteins. Here we report the isolation of a complementary DNA clone encoding a new form of soluble PTPase, PTP1C. The enzyme possesses a large noncatalytic region at the N terminus which unexpectedly contains two adjacent copies of the Src homology region 2 (the SH2 domain) found in various nonreceptor PTKs and other cytoplasmic signalling proteins. As with other SH2 sequences, the SH2 domains of PTP1C formed high-affinity complexes with the activated epidermal growth factor receptor and other phosphotyrosine-containing proteins. These results suggest that the SH2 regions in PTP1C may interact with other cellular components to modulate its own phosphatase activity against interacting substrates. PTPase activity may thus directly link growth factor receptors and other signalling proteins through protein-tyrosine phosphorylation.

  1. Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity.

    Science.gov (United States)

    Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong

    2011-09-02

    Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca(2+) was located at strategic positions in the oligomerization interface. We further showed that removal of Ca(2+) ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca(2+) ions.

  2. The Human dsRNA binding protein PACT is unable to functionally substitute for the Drosophila dsRNA binding protein R2D2 [v1; ref status: indexed, http://f1000r.es/201

    Directory of Open Access Journals (Sweden)

    Benjamin K Dickerman

    2013-10-01

    Full Text Available The primary function of the dsRNA binding protein (dsRBP PACT/RAX is to activate the dsRNA dependent protein kinase PKR in response to stress signals.  Additionally, it has been identified as a component of the small RNA processing pathway.  A role for PACT/RAX in this pathway represents an important interplay between two modes of post-transcriptional gene regulation.  The function of PACT/RAX in this context is poorly understood.  Thus, additional models are required to clarify the mechanism by which PACT/RAX functions.  In this study, Drosophila melanogaster was employed to identify functionally orthologous dsRNA-binding proteins.  Transgenic Drosophila expressing human PACT were generated to determine whether PACT is capable of functionally substituting for the Drosophila dsRBP R2D2, which has a well-defined role in small RNA biogenesis.  Results presented here indicate that PACT is unable to substitute for R2D2 at the whole organism level.

  3. Ca(2+)-calmodulin-dependent phosphorylation of islet secretory granule proteins

    International Nuclear Information System (INIS)

    Watkins, D.T.

    1991-01-01

    The effect of Ca2+ and calmodulin on phosphorylation of islet secretory granule proteins was studied. Secretory granules were incubated in a phosphorylation reaction mixture containing [32P]ATP and test reagents. The 32P-labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 32P content was visualized by autoradiography, and the relative intensities of specific bands were quantitated. When the reaction mixture contained EGTA and no added Ca2+, 32P was incorporated into two proteins with molecular weights of 45,000 and 13,000. When 10(-4) M Ca2+ was added without EGTA, two additional proteins (58,000 and 48,000 Mr) were phosphorylated, and the 13,000-Mr protein was absent. The addition of 2.4 microM calmodulin markedly enhanced the phosphorylation of the 58,000- and 48,000-Mr proteins and resulted in the phosphorylation of a major protein whose molecular weight (64,000 Mr) is identical to that of one of the calmodulin binding proteins located on the granule surface. Calmodulin had no effect on phosphorylation in the absence of Ca2+ but was effective in the presence of calcium between 10 nM and 50 microM. Trifluoperazine and calmidazolium, calmodulin antagonists, produced a dose-dependent inhibition of the calmodulin effect. 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester that activates protein kinase C, produced no increase in phosphorylation, and 1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride, an inhibitor of protein kinase C, had no effect. These results indicate that Ca(2+)-calmodulin-dependent protein kinases and endogenous substrates are present in islet secretory granules

  4. 2-Aminobenzamide and 2-Aminobenzoic Acid as New MALDI Matrices Inducing Radical Mediated In-Source Decay of Peptides and Proteins

    Science.gov (United States)

    Smargiasso, Nicolas; Quinton, Loic; de Pauw, Edwin

    2012-03-01

    One of the mechanisms leading to MALDI in-source decay (MALDI ISD) is the transfer of hydrogen radicals to analytes upon laser irradiation. Analytes such as peptides or proteins may undergo ISD and this method can therefore be exploited for top-down sequencing. When performed on peptides, radical-induced ISD results in production of c- and z-ions, as also found in ETD and ECD activation. Here, we describe two new compounds which, when used as MALDI matrices, are able to efficiently induce ISD of peptides and proteins: 2-aminobenzamide and 2-aminobenzoic acid. In-source reduction of the disulfide bridge containing peptide Calcitonin further confirmed the radicalar mechanism of the ISD process. ISD of peptides led, in addition to c- and z-ions, to the generation of a-, x-, and y-ions both in positive and in negative ion modes. Finally, good sequence coverage was obtained for the sequencing of myoglobin (17 kDa protein), confirming the effectiveness of both 2-aminobenzamide and 2-aminobenzoic acid as MALDI ISD matrices.

  5. Optimization of the protein concentration process from residual peanut oil-cake

    Directory of Open Access Journals (Sweden)

    Gayol, M. F.

    2013-12-01

    Full Text Available The objective of this study was to find the best process conditions for preparing protein concentrate from residual peanut oil-cake (POC. The study was carried out on POC from industrial peanut oil extraction. Different protein extraction and precipitation conditions were used: water/ flour ratio (10:1, 20:1 and 30:1, pH (8, 9 and 10, NaCl concentration (0 and 0.5 M, extraction time (30, 60 and 120 min, temperature (25, 40 and 60 °C, extraction stages (1, 2 and 3, and precipitation pH (4, 4.5 and 5. The extraction and precipitation conditions which showed the highest protein yield were 10:1 water/flour ratio, extraction at pH 9, no NaCl, 2 extraction stages of 30 min at 40 °C and precipitation at pH 4.5. Under these conditions, the peanut protein concentrate (PC contained 86.22% protein, while the initial POC had 38.04% . POC is an alternative source of protein that can be used for human consumption or animal nutrition. Therefore, it adds value to an industry residue.El objetivo de este trabajo fue encontrar las mejores condiciones para obtener un concentrado de proteínas a partir de la torta residual de maní (POC. El estudio se llevó a cabo en POC provenientes de la extracción industrial de aceite de maní. Se utilizaron distintas condiciones para la extracción y precipitación de proteínas: relación agua / harina (10:1, 20:1 y 30:1, pH de extracción (8, 9 y 10, concentración de NaCl (0 y 0,5 M, tiempo de extracción (30, 60 y 120 min, temperatura (25, 40 y 60 °C, número de etapas de extracción (1, 2 y 3, y el pH de precipitación (4, 4,5 y 5. Las condiciones de extracción y de precipitación que mostraron mayor rendimiento de proteína fueron: relación de 10:1 en agua / harina, pH de extracción de 9, en ausencia de NaCl, 2 etapas de extracción de 30 min cada una a 40 °C y el pH de precipitación de 4,5. En estas condiciones, el concentrado de proteína de maní (PC fue de 86,22%, mientras que el porcentaje de proteínas de

  6. Interactions between milk protein ingredients and other milk components during processing

    DEFF Research Database (Denmark)

    Liu, Guanchen

    research in our group shown that, both MWP and NWP can give a higher viscosity and denser microstructure compared to WPC when used as fat replacer in low-fat yoghurt. In the thesis, we investigated how these two types of commercial whey protein particles interact with other milk components and how...... these interactions affect final acidified milk products. By detecting the properties of the whey protein aggregates, MWP and NWP showed low native whey protein content, low free thiol content and high surface hydrophobicity and were relatively stable at high temperature in the 5 % pure dispersions. When MWP and NWP...... were added to non-fat milk model systems (5% protein in total) and processed into chemically (glucono-delta-lactone) acidified milk gels, the formation of disulfide-linked structures was closely related to the increased particle size of heated milk model systems and the rheological behavior...

  7. Identification of novel NPRAP/δ-catenin-interacting proteins and the direct association of NPRAP with dynamin 2.

    Directory of Open Access Journals (Sweden)

    Carolina Koutras

    Full Text Available Neural plakophilin-related armadillo protein (NPRAP or δ-catenin is a neuronal-specific protein that is best known for its interaction with presenilin 1 (PS1. Interestingly, the hemizygous loss of NPRAP is associated with severe mental retardation in cri du chat syndrome (CDCS, and mutations in PS1 cause an aggressive, early-onset form of Alzheimer's disease. Until recently, studies on the function of NPRAP have focused on its ability to modulate dendritic protrusion elaboration through its binding to cell adhesion and scaffolding molecules. However, mounting evidence indicates that NPRAP participates in intracellular signaling and exists in the nucleus, where it modulates gene expression. This apparent bifunctional nature suggests an elaborate neuronal role, but how NPRAP came to participate in such distinct subcellular events remains a mystery. To gain insight into this pathway, we immunoprecipitated NPRAP from human SH SY5Y cells and identified several novel interacting proteins by mass spectrometry. These included neurofilament alpha-internexin, interferon regulatory protein 2 binding factors, and dynamins 1 and 2. We further validated dynamin 2/NPRAP colocalization and direct interaction in vivo, confirming their bona fide partnership. Interestingly, dynamin 2 has established roles in endocytosis and actin assembly, and both of these processes have the potential to interface with the cell adhesion and intracellular signaling processes that involve NPRAP. Our data provide new avenues for approaching NPRAP biology and suggest a broader role for this protein than previously thought.

  8. Stability of globular proteins in H2O and in D2O

    NARCIS (Netherlands)

    Efimova, Y.M.; Haemers, S.; Wierczinsky, B.; Norde, W.; Well, van A.A.

    2007-01-01

    In several experimental techniques D2O rather then H2O is often used as a solvent for proteins. Concerning the influence of the solvent on the stability of the proteins, contradicting results have been reported in literature. In this paper the influence of H2O-D2O solvent substitution on the

  9. Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2)

    DEFF Research Database (Denmark)

    Theilade, Juliane; Lerche Hansen, Jakob; Haunsø, Stig

    2002-01-01

    G protein-coupled receptor kinase 2 (GRK2) phosphorylates G protein-coupled receptors resulting in uncoupling from G proteins. Receptors modulate GRK2 expression, however the mechanistic basis for this effect is largely unknown. Here we report a novel mechanism by which receptors use...

  10. The dopamine D2 receptor can directly recruit and activate GRK2 without G protein activation.

    Science.gov (United States)

    Pack, Thomas F; Orlen, Margo I; Ray, Caroline; Peterson, Sean M; Caron, Marc G

    2018-04-20

    The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that is critical for many central nervous system functions. The D2R carries out these functions by signaling through two transducers: G proteins and β-arrestins (βarrs). Selectively engaging either the G protein or βarr pathway may be a way to improve drugs targeting GPCRs. The current model of GPCR signal transduction posits a chain of events where G protein activation ultimately leads to βarr recruitment. GPCR kinases (GRKs), which are regulated by G proteins and whose kinase action facilitates βarr recruitment, bridge these pathways. Therefore, βarr recruitment appears to be intimately tied to G protein activation via GRKs. Here we sought to understand how GRK2 action at the D2R would be disrupted when G protein activation is eliminated and the effect of this on βarr recruitment. We used two recently developed biased D2R mutants that can preferentially interact either with G proteins or βarrs as well as a βarr-biased D2R ligand, UNC9994. With these functionally selective tools, we investigated the mechanism whereby the βarr-preferring D2R achieves βarr pathway activation in the complete absence of G protein activation. We describe how direct, G protein-independent recruitment of GRK2 drives interactions at the βarr-preferring D2R and also contributes to βarr recruitment at the WT D2R. Additionally, we found an additive interaction between the βarr-preferring D2R mutant and UNC9994. These results reveal that the D2R can directly recruit GRK2 without G protein activation and that this mechanism may have relevance to achieving βarr-biased signaling. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Matrilin-2, an extracellular adaptor protein, is needed for the regeneration of muscle, nerve and other tissues

    Directory of Open Access Journals (Sweden)

    Éva Korpos

    2015-01-01

    Full Text Available The extracellular matrix (ECM performs essential functions in the differentiation, maintenance and remodeling of tissues during development and regeneration, and it undergoes dynamic changes during remodeling concomitant to alterations in the cell-ECM interactions. Here we discuss recent data addressing the critical role of the widely expressed ECM protein, matrilin-2 (Matn2 in the timely onset of differentiation and regeneration processes in myogenic, neural and other tissues and in tumorigenesis. As a multiadhesion adaptor protein, it interacts with other ECM proteins and integrins. Matn2 promotes neurite outgrowth, Schwann cell migration, neuromuscular junction formation, skeletal muscle and liver regeneration and skin wound healing. Matn2 deposition by myoblasts is crucial for the timely induction of the global switch toward terminal myogenic differentiation during muscle regeneration by affecting transforming growth factor beta/bone morphogenetic protein 7/Smad and other signal transduction pathways. Depending on the type of tissue and the pathomechanism, Matn2 can also promote or suppress tumor growth.

  12. High-Pressure-High-Temperature Processing Reduces Maillard Reaction and Viscosity in Whey Protein-Sugar Solutions

    NARCIS (Netherlands)

    Avila Ruiz, Geraldine; Xi, Bingyan; Minor, Marcel; Sala, Guido; Boekel, van Tiny; Fogliano, Vincenzo; Stieger, Markus

    2016-01-01

    The aim of the study was to determine the influence of pressure in high-pressure-high-temperature (HPHT) processing on Maillard reactions and protein aggregation of whey protein-sugar solutions. Solutions of whey protein isolate containing either glucose or trehalose at pH 6, 7, and 9 were

  13. 2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation.

    Science.gov (United States)

    Lai, Jeffrey K F; Sam, I-Ching; Verlhac, Pauline; Baguet, Joël; Eskelinen, Eeva-Liisa; Faure, Mathias; Chan, Yoke Fun

    2017-07-04

    Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N -ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection.

  14. Increased Protein Stability and Decreased Protein Turnover in the Caenorhabditis elegans Ins/IGF-1 daf-2 Mutant.

    Science.gov (United States)

    Depuydt, Geert; Shanmugam, Nilesh; Rasulova, Madina; Dhondt, Ineke; Braeckman, Bart P

    2016-12-01

    In Caenorhabditis elegans, cellular proteostasis is likely essential for longevity. Autophagy has been shown to be essential for lifespan extension of daf-2 insulin/IGF mutants. Therefore, it can be hypothesized that daf-2 mutants achieve this phenotype by increasing protein turnover. However, such a mechanism would exert a substantial energy cost. By using classical 35 S pulse-chase labeling, we observed that protein synthesis and degradation rates are decreased in young adults of the daf-2 insulin/IGF mutants. Although reduction of protein turnover may be energetically favorable, it may lead to accumulation and aggregation of damaged proteins. As this has been shown not to be the case in daf-2 mutants, another mechanism must exist to maintain proteostasis in this strain. We observed that proteins isolated from daf-2 mutants are more soluble in acidic conditions due to increased levels of trehalose. This suggests that trehalose may decrease the potential for protein aggregation and increases proteostasis in the daf-2 mutants. We postulate that daf-2 mutants save energy by decreasing protein turnover rates and instead stabilize their proteome by trehalose. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America.

  15. Whey protein: The “whey” forward for treatment of type 2 diabetes?

    Science.gov (United States)

    Mignone, Linda E; Wu, Tongzhi; Horowitz, Michael; Rayner, Christopher K

    2015-01-01

    A cost-effective nutritional approach to improve postprandial glycaemia is attractive considering the rising burden of diabetes throughout the world. Whey protein, a by-product of the cheese-making process, can be used to manipulate gut function in order to slow gastric emptying and stimulate incretin hormone secretion, thereby attenuating postprandial glycaemic excursions. The function of the gastrointestinal tract plays a pivotal role in glucose homeostasis, particularly during the postprandial period, and this review will discuss the mechanisms by which whey protein slows gastric emptying and stimulates release of gut peptides, including the incretins. Whey protein is also a rich source of amino acids, and these can directly stimulate beta cells to secrete insulin, which contributes to the reduction in postprandial glycaemia. Appetite is suppressed with consumption of whey, due to its effects on the gut-brain axis and the hypothalamus. These properties of whey protein suggest its potential in the management of type 2 diabetes. However, the optimal dose and timing of whey protein ingestion are yet to be defined, and studies are required to examine the long-term benefits of whey consumption for overall glycaemic control. PMID:26516411

  16. Identification of Tyrosine Phosphorylated Proteins by SH2 Domain Affinity Purification and Mass Spectrometry.

    Science.gov (United States)

    Buhs, Sophia; Gerull, Helwe; Nollau, Peter

    2017-01-01

    Phosphotyrosine signaling plays a major role in the control of many important biological functions such as cell proliferation and apoptosis. Deciphering of phosphotyrosine-dependent signaling is therefore of great interest paving the way for the understanding of physiological and pathological processes of signal transduction. On the basis of the specific binding of SH2 domains to phosphotyrosine residues, we here present an experimental workflow for affinity purification and subsequent identification of tyrosine phosphorylated proteins by mass spectrometry. In combination with SH2 profiling, a broadly applicable platform for the characterization of phosphotyrosine profiles in cell extracts, our pull down strategy enables researchers by now to identify proteins in signaling cascades which are differentially phosphorylated and selectively recognized by distinct SH2 domains.

  17. Single cell protein production of Chlorella sp. using food processing waste as a cultivation medium

    Science.gov (United States)

    Putri, D.; Ulhidayati, A.; Musthofa, I. A.; Wardani, A. K.

    2018-03-01

    The aim of this study was to investigate the effect of various food processing wastes on the production of single cell protein by Chlorella sp. Three various food processing wastes i.e. tofu waste, tempeh waste and cheese whey waste were used as cultivation medium for Chlorella sp. growth. Sea water was used as a control of cultivation medium. The addition of waste into cultivation medium was 10%, 20%, 30%, 40%, and 50%. The result showed that the highest yield of cell mass and protein content was found in 50% tofu waste cultivation medium was 47.8 × 106 cell/ml with protein content was 52.24%. The 50% tofu waste medium showed improved cell yield as nearly as 30% than tempeh waste medium. The yield of biomass and protein content when 30% tempeh waste was used as cultivation medium was 37.1 × 106 cell/ml and 52%, respectively. Thus, food processing waste especially tofu waste would be a promising candidate for cultivation medium for single cell production from Chlorella sp. Moreover, the utilization of waste can reduce environmental pollution and increase protein supply for food supplement or animal feed.

  18. Transient phosphorylation of tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) at Thr-596 during early phases of mitosis

    OpenAIRE

    Hong, Kyung Uk; Choi, Yong-Bock; Lee, Jung-Hwa; Kim, Hyun-Jun; Kwon, Hye-Rim; Seong, Yeon-Sun; Kim, Heung Tae; Park, Joobae; Bae, Chang-Dae; Hong, Kyeong-Man

    2008-01-01

    Tumor associated microtubule associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2) is a mitotic spindle-associated protein whose expression is cell cycle-regulated and also frequently deregulated in cancer cells. Two monoclonal antibodies (mAbs) against TMAP/CKAP2 were produced: B-1-13 and D-12-3. Interestingly, the reactivity of mAb D-12-3 to TMAP/CKAP2 was markedly decreased specifically in mitotic cell lysate. The epitope mapping study showed that mAb D-12-3 re...

  19. [Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

    Science.gov (United States)

    Sheremet, Ia A; Emets, A I; Azmi, A; Vissenberg, K; Verbelen, J-P; Blium, Ia B

    2012-01-01

    In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

  20. Fabrication of protein microarrays for alpha fetoprotein detection by using a rapid photo-immobilization process

    Directory of Open Access Journals (Sweden)

    Sirasa Yodmongkol

    2016-03-01

    Full Text Available In this study, protein microarrays based on sandwich immunoassays are generated to quantify the amount of alpha fetoprotein (AFP in blood serum. For chip generation a mixture of capture antibody and a photoactive copolymer consisting of N,N-dimethylacrylamide (DMAA, methacryloyloxy benzophenone (MaBP, and Na-4-styrenesulfonate (SSNa was spotted onto unmodified polymethyl methacrylate (PMMA substrates. Subsequently to printing of the microarray, the polymer and protein were photochemically cross-linked and the forming, biofunctionalized hydrogels simultaneously bound to the chip surface by short UV- irradiation. The obtained biochip was incubated with AFP antigen, followed by biotinylated AFP antibody and streptavidin-Cy5 and the fluorescence signal read-out. The developed microarray biochip covers the range of AFP in serum samples such as maternal serum in the range of 5 and 100 ng/ml. The chip production process is based on a fast and simple immobilization process, which can be applied to conventional plastic surfaces. Therefore, this protein microarray production process is a promising method to fabricate biochips for AFP screening processes. Keywords: Photo-immobilization, Protein microarray, Alpha fetoprotein, Hydrogel, 3D surface, Down syndrome

  1. A membrane protein / signaling protein interaction network for Arabidopsis version AMPv2

    Directory of Open Access Journals (Sweden)

    Sylvie Lalonde

    2010-09-01

    Full Text Available Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway compatible vector. The mating-based split-ubiquitin system was used to screen for potential protein-protein interactions (pPPIs among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases, 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 387 pPPIs between 179 proteins, yielding a scale-free network (r2=0.863. Eighty of 142 transmembrane receptor-like kinases (RLK tested positive, identifying three homomers, 63 heteromers and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

  2. Inhibition of Poliovirus-Induced Cleavage of Cellular Protein PCBP2 Reduces the Levels of Viral RNA Replication

    Science.gov (United States)

    Chase, Amanda J.; Daijogo, Sarah

    2014-01-01

    ABSTRACT Due to their small genome size, picornaviruses must utilize host proteins to mediate cap-independent translation and viral RNA replication. The host RNA-binding protein poly(rC) binding protein 2 (PCBP2) is involved in both processes in poliovirus infected cells. It has been shown that the viral proteinase 3CD cleaves PCBP2 and contributes to viral translation inhibition. However, cleaved PCBP2 remains active in viral RNA replication. This would suggest that both cleaved and intact forms of PCBP2 have a role in the viral RNA replication cycle. The picornavirus genome must act as a template for both translation and RNA replication. However, a template that is actively being translated cannot function as a template for RNA replication, suggesting that there is a switch in template usage from translation to RNA replication. We demonstrate that the cleavage of PCBP2 by the poliovirus 3CD proteinase is a necessary step for efficient viral RNA replication and, as such, may be important for mediating a switch in template usage from translation to RNA replication. IMPORTANCE Poliovirus, like all positive-strand RNA viruses that replicate in the cytoplasm of eukaryotic cells, uses its genomic RNA as a template for both viral protein synthesis and RNA replication. Given that these processes cannot occur simultaneously on the same template, poliovirus has evolved a mechanism(s) to facilitate the switch from using templates for translation to using them for RNA synthesis. This study explores one possible scenario for how the virus alters the functions of a host cell RNA binding protein to mediate, in part, this important transition. PMID:24371074

  3. Model-based Rational and Systematic Protein Purification Process Development : A Knowledge-based Approach

    NARCIS (Netherlands)

    Kungah Nfor, B.

    2011-01-01

    The increasing market and regulatory (quality and safety) demands on therapeutic proteins calls for radical improvement in their manufacturing processes. Addressing these challenges requires the adoption of strategies and tools that enable faster and more efficient process development. This thesis

  4. Nano titanium dioxide photocatalytic protein tyrosine nitration: A potential hazard of TiO2 on skin

    International Nuclear Information System (INIS)

    Lu, Naihao; Zhu Zhening; Zhao Xuqi; Tao Ran; Yang Xiangliang; Gao Zhonghong

    2008-01-01

    Protein tyrosine nitration is a prevalent post-translational modification which occurs as a result of oxidative and nitrative stress, it may be directly involved in the onset and/or progression of diseases. Considering the existence of nano titanium dioxide (TiO 2 ) in environment and sunscreen products along with the high content of nitrite in sweat, the UV-exposed skin may be a significant target for the photosensitized damage. In this paper, tyrosine nitration of bovine serum albumin (BSA) was initiated in the UV-irradiated reaction mixture containing 0.2-3.0 mg/ml of three commercially nano TiO 2 products and 0.25-1.0 mM NO 2 - . It was found that anatase TiO 2 and Degussa P25 TiO 2 showed prominent photocatalytic activity on promoting the formation of protein tyrosine nitration, and the optimum condition for the reaction was around physiological pH. Meanwhile, the photocatalytic effect of rutile on protein tyrosine nitration was subtle. The potential physiological significance of nano TiO 2 -photocatalytic protein nitration was also demonstrated in mouse skin homogenate. Although the relationship between photocatalytic protein tyrosine nitration and chronic cutaneous diseases needs further study, the toxicity of nano TiO 2 to the skin disease should be paid more attention in the production and utilization process

  5. Cancer associated aberrant protein o-glycosylation can modify antigen processing and immune response

    DEFF Research Database (Denmark)

    Madsen, Caroline B; Petersen, Cecilie; Lavrsen, Kirstine

    2012-01-01

    Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing......, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/- glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo...

  6. Stability of integral membrane proteins under high hydrostatic pressure: the LH2 and LH3 antenna pigment-protein complexes from photosynthetic bacteria.

    Science.gov (United States)

    Kangur, Liina; Timpmann, Kõu; Freiberg, Arvi

    2008-07-03

    The bacteriochlorophyll a-containing LH2 and LH3 antenna complexes are the integral membrane proteins that catalyze the photosynthetic process in purple photosynthetic bacteria. The LH2 complex from Rhodobacter sphaeroides shows characteristic strong absorbance at 800 and 850 nm due to the pigment molecules confined in two separate areas of the protein. In the LH3 complex from Rhodopesudomonas acidophila the corresponding bands peak at 800 and 820 nm. Using the bacteriochlorophyll a cofactors as intrinsic probes to monitor local changes in the protein structure, we investigate spectral responses of the antenna complexes to very high hydrostatic pressures up to 2.5 GPa when embedded into natural membrane environment or extracted with detergent. We first demonstrate that high pressure does induce significant alterations to the tertiary structure of the proteins not only in proximity of the 800 nm-absorbing bacteriochlorophyll a molecules known previously (Gall, A.; et al. Biochemistry 2003, 42, 13019) but also of the 850 nm- and 820 nm-absorbing molecules, including breakage of the hydrogen bond they are involved in. The membrane-protected complexes appear more resilient to damaging effects of the compression compared with the complexes extracted into mixed detergent-buffer environment. Increased resistance of the isolated complexes is observed at high protein concentration resulting aggregation as well as when cosolvent (glycerol) is added into the solution. These stability variations correlate with ability of penetration of the surrounding polar solvent (water) into the hydrophobic protein interiors, being thus the principal reason of the pressure-induced denaturation of the proteins. Considerable variability of elastic properties of the isolated complexes was also observed, tentatively assigned to heterogeneous protein packing in detergent micelles. While a number of the isolated complexes release most of their bacteriochlorophyll a content under high pressure

  7. Effect of Radiation Processing on Protein Quality of Certain Legumes

    International Nuclear Information System (INIS)

    El-Niely, H.F.G

    2007-01-01

    The Effects of irradiation (dose levels of 5, 7.5 and 10 kGy) on nutritive characteristics of peas (Pisum satinum L), cow peas (Vigna unguiculata L.Walp), lentils (Lens culinaris Med), kidney beans (Phaseolus vulgaris L), and chickpeas (Cicer arietinurn L) were examined. Analyses included proximate composition, levels of anti-nutrients (phytic acid, tannins), available lysine (AL), in vitro protein digestibility (IVPD), and protein efficiency ratio (PER) in the growing rat. The results showed that moisture, crude protein, crude fat, crude fiber, and ash were unchanged by the irradiation. Radiation processing significantly (p<0.05) reduced the levels of phytic acid (PA), tannins (TN), and available lysine (AE). IVPD and PER were significantly enhanced in a dose-dependent manner, relative to unirradiated control samples, for all legumes. The data sets for each legume exhibited high correlation coefficients between radiation dose and PA, TN, AE, IVPD, and PER. These results demonstrate the benefits of irradiation on the nutritional properties of these legumes

  8. Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells.

    Science.gov (United States)

    Reuter, Lauri J; Bailey, Michael J; Joensuu, Jussi J; Ritala, Anneli

    2014-05-01

    Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low-cost purification by surfactant-based aqueous two-phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY-2) suspension cell platform and the establishment of pilot-scale propagation and downstream processing including first-step purification by ATPS. Green fluorescent protein-hydrophobin fusion (GFP-HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY-2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP-HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large-scale hydrophobin-assisted production of recombinant proteins in tobacco BY-2 cell suspensions. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  9. Quorum Sensing and the Use of Quorum Quenchers as Natural Biocides to Inhibit Sulfate-Reducing Bacteria

    KAUST Repository

    Scarascia, Giantommaso; Wang, Tiannyu; Hong, Pei-Ying

    2016-01-01

    Sulfate-reducing bacteria (SRB) are one of the main protagonist groups of biocorrosion in the seawater environment. Given their principal role in biocorrosion, it remains a crucial task to develop strategies to reduce the abundance of SRBs. Conventional approaches include the use of biocides and antibiotics, which can impose health, safety, and environmental concerns. This review examines an alternative approach to this problem. This is achieved by reviewing the role of quorum sensing (QS) in SRB populations and its impact on the biofilm formation process. Genome databases of SRBs are mined to look for putative QS systems and homologous protein sequences representative of autoinducer receptors or synthases. Subsequently, this review puts forward the potential use of quorum quenchers as natural biocides against SRBs and outlines the potential strategies for the implementation of this approach.

  10. Quorum Sensing and the Use of Quorum Quenchers as Natural Biocides to Inhibit Sulfate-Reducing Bacteria

    KAUST Repository

    Scarascia, Giantommaso

    2016-12-15

    Sulfate-reducing bacteria (SRB) are one of the main protagonist groups of biocorrosion in the seawater environment. Given their principal role in biocorrosion, it remains a crucial task to develop strategies to reduce the abundance of SRBs. Conventional approaches include the use of biocides and antibiotics, which can impose health, safety, and environmental concerns. This review examines an alternative approach to this problem. This is achieved by reviewing the role of quorum sensing (QS) in SRB populations and its impact on the biofilm formation process. Genome databases of SRBs are mined to look for putative QS systems and homologous protein sequences representative of autoinducer receptors or synthases. Subsequently, this review puts forward the potential use of quorum quenchers as natural biocides against SRBs and outlines the potential strategies for the implementation of this approach.

  11. A novel one-pot process for near-net-shape fabrication of open-porous resorbable hydroxyapatite/protein composites and in vivo assessment

    International Nuclear Information System (INIS)

    Mueller, Berit; Koch, Dietmar; Lutz, Rainer; Schlegel, Karl A.; Treccani, Laura; Rezwan, Kurosch

    2014-01-01

    We present a mild one-pot freeze gelation process for fabricating near-net, complex-shaped hydroxyapatite scaffolds and to directly incorporate active proteins during scaffold processing. In particular, the direct protein incorporation enables a simultaneous adjustment and control of scaffold microstructure, porosity, resorbability and enhancement of initial mechanical and handling stability. Two proteins, serum albumin and lysozyme, are selected and their effect on scaffold stability and microstructure investigated by biaxial strength tests, electron microscopy, and mercury intrusion porosimetry. The resulting hydroxyapatite/protein composites feature adjustable porosities from 50% to 70% and a mechanical strength ranging from 2 to 6 MPa comparable to that of human spongiosa without any sintering step. Scaffold degradation behaviour and protein release are assessed by in vitro studies. A preliminary in vivo assessment of scaffold biocompatibility and resorption behaviour in adult domestic pigs is discussed. After implantation, composites were resorbed up to 50% after only 4 weeks and up to 65% after 8 weeks. In addition, 14% new bone formation after 4 weeks and 37% after 8 weeks were detected. All these investigations demonstrate the outstanding suitability of the one-pot-process to create, in a customisable and reliable way, biocompatible scaffolds with sufficient mechanical strength for handling and surgical insertion, and for potential use as biodegradable bone substitutes and versatile platform for local drug delivery. - Highlights: • We present a one-pot process for directly incorporating protein into HAp scaffolds. • The effect of two model proteins, BSA and LSZ, on scaffold properties is analysed. • HAp/protein scaffolds feature a mechanical strength comparable to human spongiosa. • BSA incorporation in scaffolds leads to strength increase despite porosity increment. • New bone formation in-vivo exceeds established xenograft bone substitutes

  12. Ablation of whirlin long isoform disrupts the USH2 protein complex and causes vision and hearing loss.

    Directory of Open Access Journals (Sweden)

    Jun Yang

    2010-05-01

    Full Text Available Mutations in whirlin cause either Usher syndrome type II (USH2, a deafness-blindness disorder, or nonsyndromic deafness. The molecular basis for the variable disease expression is unknown. We show here that only the whirlin long isoform, distinct from a short isoform by virtue of having two N-terminal PDZ domains, is expressed in the retina. Both long and short isoforms are expressed in the inner ear. The N-terminal PDZ domains of the long whirlin isoform mediates the formation of a multi-protein complex that includes usherin and VLGR1, both of which are also implicated in USH2. We localized this USH2 protein complex to the periciliary membrane complex (PMC in mouse photoreceptors that appears analogous to the frog periciliary ridge complex. The latter is proposed to play a role in photoreceptor protein trafficking through the connecting cilium. Mice carrying a targeted disruption near the N-terminus of whirlin manifest retinal and inner ear defects, reproducing the clinical features of human USH2 disease. This is in contrast to mice with mutations affecting the C-terminal portion of whirlin in which the phenotype is restricted to the inner ear. In mice lacking any one of the USH2 proteins, the normal localization of all USH2 proteins is disrupted, and there is evidence of protein destabilization. Taken together, our findings provide new insights into the pathogenic mechanism of Usher syndrome. First, the three USH2 proteins exist as an obligatory functional complex in vivo, and loss of one USH2 protein is functionally close to loss of all three. Second, defects in the three USH2 proteins share a common pathogenic process, i.e., disruption of the PMC. Third, whirlin mutations that ablate the N-terminal PDZ domains lead to Usher syndrome, but non-syndromic hearing loss will result if they are spared.

  13. Fanconi anemia FANCD2 and FANCI proteins regulate the nuclear dynamics of splicing factors.

    Science.gov (United States)

    Moriel-Carretero, María; Ovejero, Sara; Gérus-Durand, Marie; Vryzas, Dimos; Constantinou, Angelos

    2017-12-04

    Proteins disabled in the cancer-prone disorder Fanconi anemia (FA) ensure the maintenance of chromosomal stability during DNA replication. FA proteins regulate replication dynamics, coordinate replication-coupled repair of interstrand DNA cross-links, and mitigate conflicts between replication and transcription. Here we show that FANCI and FANCD2 associate with splicing factor 3B1 (SF3B1), a key spliceosomal protein of the U2 small nuclear ribonucleoprotein (U2 snRNP). FANCI is in close proximity to SF3B1 in the nucleoplasm of interphase and mitotic cells. Furthermore, we find that DNA replication stress induces the release of SF3B1 from nuclear speckles in a manner that depends on FANCI and on the activity of the checkpoint kinase ATR. In chromatin, both FANCD2 and FANCI associate with SF3B1, prevent accumulation of postcatalytic intron lariats, and contribute to the timely eviction of splicing factors. We propose that FANCD2 and FANCI contribute to the organization of functional domains in chromatin, ensuring the coordination of DNA replication and cotranscriptional processes. © 2017 Moriel-Carretero et al.

  14. Structural basis of G protein-coupled receptor-Gi protein interaction: formation of the cannabinoid CB2 receptor-Gi protein complex.

    Science.gov (United States)

    Mnpotra, Jagjeet S; Qiao, Zhuanhong; Cai, Jian; Lynch, Diane L; Grossfield, Alan; Leioatts, Nicholas; Hurst, Dow P; Pitman, Michael C; Song, Zhao-Hui; Reggio, Patricia H

    2014-07-18

    In this study, we applied a comprehensive G protein-coupled receptor-Gαi protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB2)-Gαi interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gαi C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gαi C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gαi α4β6 loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB2·Gi complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB2 R*·Gαi1β1γ2 complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the β2-AR*·Gαsβ1γ2 complex crystal structure, the Gαi1β1γ2 protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gαi1 α5 helix tilt changed due to the outward movement of TMH5 in CB2 R*. 2) A 25° clockwise rotation of Gαi1β1γ2 underneath CB2 R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gαi1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to Gi proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Influence of Prolonged Storage Process, Pasteurization, and Heat Treatment on Biologically-active Human Milk Proteins

    Directory of Open Access Journals (Sweden)

    Jih-Chin Chang

    2013-12-01

    Conclusion: Various freezing/heating/pasteurization processes applied to human milk prior to delivery to neonates could affect the concentration of immunomodulatory proteins, especially lactoferrin, secretory immunoglobulin A, and lysozyme. Leptin was unaffected by the various handling processes tested. Fresh milk was found to be the best food for neonates. Further studies are warranted to evaluate the functional activity of these proteins and their effects on infants' immunological status.

  16. Whey protein processing influences formula-induced gut maturation in preterm pigs.

    Science.gov (United States)

    Li, Yanqi; Østergaard, Mette V; Jiang, Pingping; Chatterton, Dereck E W; Thymann, Thomas; Kvistgaard, Anne S; Sangild, Per T

    2013-12-01

    Immaturity of the gut predisposes preterm infants to nutritional challenges potentially leading to clinical complications such as necrotizing enterocolitis. Feeding milk formulas is associated with greater risk than fresh colostrum or milk, probably due to loss of bioactive proteins (e.g., immunoglobulins, lactoferrin, insulin-like growth factor, transforming growth factor-β) during industrial processing (e.g., pasteurization, filtration, spray-drying). We hypothesized that the processing method for whey protein concentrate (WPC) would affect gut maturation in formula-fed preterm pigs used as a model for preterm infants. Fifty-five caesarean-delivered preterm pigs were distributed into 4 groups given 1 of 4 isoenergetic diets: formula containing conventional WPC (filtration, multi-pasteurization, standard spray-drying) (CF); formula containing gently treated WPC (reduced filtration and pasteurization, gentle spray-drying) (GF); formula containing minimally treated WPC (rennet precipitation, reduced filtration, heat treatment preserve the bioactivity and nutritional value of formulas for sensitive newborns.

  17. Effect of drying process assisted by high-pressure impregnation on protein quality and digestibility in red abalone (Haliotis rufescens).

    Science.gov (United States)

    Cepero-Betancourt, Yamira; Oliva-Moresco, Patricio; Pasten-Contreras, Alexis; Tabilo-Munizaga, Gipsy; Pérez-Won, Mario; Moreno-Osorio, Luis; Lemus-Mondaca, Roberto

    2017-10-01

    Abalone (Haliotis spp.) is an exotic seafood product recognized as a protein source of high biological value. Traditional methods used to preserve foods such as drying technology can affect their nutritional quality (protein quality and digestibility). A 28-day rat feeding study was conducted to evaluate the effects of the drying process assisted by high-pressure impregnation (HPI) (350, 450, and 500 MPa × 5 min) on chemical proximate and amino acid compositions and nutritional parameters, such as protein efficiency ratio (PER), true digestibility (TD), net protein ratio, and protein digestibility corrected amino acid score (PDCAAS) of dried abalone. The HPI-assisted drying process ensured excellent protein quality based on PER values, regardless of the pressure level. At 350 and 500 MPa, the HPI-assisted drying process had no negative effect on TD and PDCAAS then, based on nutritional parameters analysed, we recommend HPI-assisted drying process at 350 MPa × 5 min as the best process condition to dry abalone. Variations in nutritional parameters compared to casein protein were observed; nevertheless, the high protein quality and digestibility of HPI-assisted dried abalones were maintained to satisfy the metabolic demands of human beings.

  18. Arabidopsis COLD SHOCK DOMAIN PROTEIN2 is a RNA chaperone that is regulated by cold and developmental signals

    International Nuclear Information System (INIS)

    Sasaki, Kentaro; Kim, Myung-Hee; Imai, Ryozo

    2007-01-01

    Bacterial cold shock proteins (CSPs) are RNA chaperones that unwind RNA secondary structures. Arabidopsis COLD SHOCK DOMAIN PROTEIN2 (AtCSP2) contains a domain that is shared with bacterial CSPs. Here we showed that AtCSP2 binds to RNA and unwinds nucleic acid duplex. Heterologous expression of AtCSP2 complemented cold sensitivity of an Escherichia coli csp quadruple mutant, indicating that AtCSP2 function as a RNA chaperone in E. coli. AtCSP2 mRNA and protein levels increased during cold acclimation, but the protein accumulation was most prominent after 10 days of cold treatment. AtCSP2 promoter::GUS transgenic plants revealed that AtCSP2 is expressed only in root and shoot apical regions during vegetative growth but is expressed in reproductive organs such as pollens, ovules and embryos. These data indicated that AtCSP2 is involved in developmental processes as well as cold adaptation. Localization of AtCSP2::GFP in nucleolus and cytoplasm suggested different nuclear and cytosolic RNA targets

  19. Localization of Usher 1 proteins to the photoreceptor calyceal processes, which are absent from mice

    Science.gov (United States)

    Sahly, Iman; Dufour, Eric; Schietroma, Cataldo; Michel, Vincent; Bahloul, Amel; Perfettini, Isabelle; Pepermans, Elise; Estivalet, Amrit; Carette, Diane; Aghaie, Asadollah; Ebermann, Inga; Lelli, Andrea; Iribarne, Maria; Hardelin, Jean-Pierre; Weil, Dominique; Sahel, José-Alain

    2012-01-01

    The mechanisms underlying retinal dystrophy in Usher syndrome type I (USH1) remain unknown because mutant mice lacking any of the USH1 proteins—myosin VIIa, harmonin, cadherin-23, protocadherin-15, sans—do not display retinal degeneration. We found here that, in macaque photoreceptor cells, all USH1 proteins colocalized at membrane interfaces (i) between the inner and outer segments in rods and (ii) between the microvillus-like calyceal processes and the outer segment basolateral region in rods and cones. This pattern, conserved in humans and frogs, was mediated by the formation of an USH1 protein network, which was associated with the calyceal processes from the early embryonic stages of outer segment growth onwards. By contrast, mouse photoreceptors lacked calyceal processes and had no USH1 proteins at the inner–outer segment interface. We suggest that USH1 proteins form an adhesion belt around the basolateral region of the photoreceptor outer segment in humans, and that defects in this structure cause the retinal degeneration in USH1 patients. PMID:23045546

  20. Virus-specific proteins in cells infected with tomato black ring nepovirus: evidence for proteolytic processing in vivo.

    Science.gov (United States)

    Demangeat, G; Hemmer, O; Reinbolt, J; Mayo, M A; Fritsch, C

    1992-07-01

    The synthesis of proteins encoded by the RNA of tomato black ring virus (TBRV) in vivo was studied in protoplasts by direct labelling with [35S]methionine, and in protoplasts and plants by immunoblotting experiments with specific antisera. Comparison of the proteins synthesized in infected and mock-inoculated protoplasts suggested that proteins of M(r) 120K, 90K, 80K, 57K and 46K were virus-specific. The proteins derived from the RNA-1-encoded polyprotein detected by immunoblotting were a stable 120K protein and, only in protoplasts, small amounts of a 90K protein which contains the C-terminal part of the 120K protein and the polymerase domain. The results suggest that the polymerase and the adjacent protease function in vivo largely or solely when combined in a 120K protein. The proteins derived from the RNA-2-encoded polyprotein detected by immunoblotting were 59K and 57K proteins, which reacted with antiserum to TBRV particles, and a 46K protein. In extracts of infected Nicotiana clevelandii and Chenopodium quinoa made soon after inoculation, the 59K protein was more abundant than the 57K protein; later samples contained similar quantities of each protein. The 57K protein comigrated with protein extracted from virus particles. The results of amino acid sequencing suggested that the 57K protein is derived from the 59K protein by the loss of nine C-terminal amino acids. Antiserum to a peptide adjacent to the 57K protein in the 150K polyprotein detected a 46K protein in protoplasts and plant tissue. The results support the processing scheme for TBRV polyproteins proposed after analysis of the products of in vitro translation.

  1. WW domain-binding protein 2: an adaptor protein closely linked to the development of breast cancer.

    Science.gov (United States)

    Chen, Shuai; Wang, Han; Huang, Yu-Fan; Li, Ming-Li; Cheng, Jiang-Hong; Hu, Peng; Lu, Chuan-Hui; Zhang, Ya; Liu, Na; Tzeng, Chi-Meng; Zhang, Zhi-Ming

    2017-07-19

    The WW domain is composed of 38 to 40 semi-conserved amino acids shared with structural, regulatory, and signaling proteins. WW domain-binding protein 2 (WBP2), as a binding partner of WW domain protein, interacts with several WW-domain-containing proteins, such as Yes kinase-associated protein (Yap), paired box gene 8 (Pax8), WW-domain-containing transcription regulator protein 1 (TAZ), and WW-domain-containing oxidoreductase (WWOX) through its PPxY motifs within C-terminal region, and further triggers the downstream signaling pathway in vitro and in vivo. Studies have confirmed that phosphorylated form of WBP2 can move into nuclei and activate the transcription of estrogen receptor (ER) and progesterone receptor (PR), whose expression were the indicators of breast cancer development, indicating that WBP2 may participate in the progression of breast cancer. Both overexpression of WBP2 and activation of tyrosine phosphorylation upregulate the signal cascades in the cross-regulation of the Wnt and ER signaling pathways in breast cancer. Following the binding of WBP2 to the WW domain region of TAZ which can accelerate migration, invasion and is required for the transformed phenotypes of breast cancer cells, the transformation of epithelial to mesenchymal of MCF10A is activated, suggesting that WBP2 is a key player in regulating cell migration. When WBP2 binds with WWOX, a tumor suppressor, ER transactivation and tumor growth can be suppressed. Thus, WBP2 may serve as a molecular on/off switch that controls the crosstalk between E2, WWOX, Wnt, TAZ, and other oncogenic signaling pathways. This review interprets the relationship between WBP2 and breast cancer, and provides comprehensive views about the function of WBP2 in the regulation of the pathogenesis of breast cancer and endocrine therapy in breast cancer treatment.

  2. The point mutation process in proteins

    Science.gov (United States)

    Schwartz, R. M.; Dayhoff, M. O.

    1978-01-01

    An optimized scoring matrix for residue-by-residue comparisons of distantly related protein sequences has been developed. The scoring matrix is based on observed exchanges and mutabilities of amino acids in 1572 closely related sequences derived from a cross-section of protein groups. Very few superimposed or parallel mutations are included in the data. The scoring matrix is most useful for demonstrating the relatedness of proteins between 65 and 85% different.

  3. Uncoupling protein 2 G(-866A polymorphism: a new gene polymorphism associated with C-reactive protein in type 2 diabetic patients C-reactive protein in type 2 diabetic patients

    Directory of Open Access Journals (Sweden)

    Cocozza Sergio

    2010-10-01

    Full Text Available Abstract Background This study evaluated the relationship between the G(-866A polymorphism of the uncoupling protein 2 (UCP2 gene and high-sensitivity C reactive protein (hs-CRP plasma levels in diabetic patients. Methods We studied 383 unrelated people with type 2 diabetes aged 40-70 years. Anthropometry, fasting lipids, glucose, HbA1c, and hs-CRP were measured. Participants were genotyped for the G (-866A polymorphism of the uncoupling protein 2 gene. Results Hs-CRP (mg/L increased progressively across the three genotype groups AA, AG, or GG, being respectively 3.0 ± 3.2, 3.6 ± 5.0, and 4.8 ± 5.3 (p for trend = 0.03. Since hs-CRP values were not significantly different between AA and AG genotype, these two groups were pooled for further analyses. Compared to participants with the AA/AG genotypes, homozygotes for the G allele (GG genotype had significantly higher hs-CRP levels (4.8 ± 5.3 vs 3.5 ± 4.7 mg/L, p = 0.01 and a larger proportion (53.9% vs 46.1%, p = 0.013 of elevated hs-CRP (> 2 mg/L. This was not explained by major confounders such as age, gender, BMI, waist circumference, HbA1c, smoking, or medications use which were comparable in the two genotype groups. Conclusions The study shows for the first time, in type 2 diabetic patients, a significant association of hs-CRP levels with the G(-866A polymorphism of UCP2 beyond the effect of major confounders.

  4. Calcium phosphate–gold nanoparticles nanocomposite for protein adsorption and mediator-free H2O2 biosensor construction

    International Nuclear Information System (INIS)

    Xu Qin; Lu Guiju; Bian XiaoJun; Jin Gendi; Wang Wei; Hu Xiaoya; Wang Yang; Yang Zhanjun

    2012-01-01

    This work reports a new method for the preparation and application of a kind of biocompatible calcium phosphate–gold nanoparticles (Ca 3 (PO 4 ) 2 –AuNPs) nanocomposite. UV–vis spectroscopy and transmittance electron microscopy (TEM) have been used to monitor the formation process of the nanocomposite and to examine the interaction between calcium phosphate and gold nanoparticles (AuNPs). The nanocomposite has multiple sites and improved conductivity which make it suitable for the binding of proteins to construct electrochemical sensors. Myoglobin (Mb) adsorbed on the nanocomposite retained its native structure which was proved by Fourier transform infrared spectroscopy (FTIR). Direct electron transfer between the adsorbed Mb and the electrode was observed. Further results demonstrated that the adsorbed Mb has good electrocatalytic activity towards the reduction of H 2 O 2 in the absence of any mediator. Highlights: ► Using gelatin modified gold nanoparticles to prepare needle-like calcium phosphate. ► Calcium phosphate provides multiple sites for protein adsorption. ► Gold nanoparticles act as electron tunneling. ► Myoglobin adsorbed on the material showed direct electrochemistry and good catalysis.

  5. α-Actinin-2, a cytoskeletal protein, binds to angiogenin

    International Nuclear Information System (INIS)

    Hu Huajun; Gao Xiangwei; Sun Yishan; Zhou Jiliang; Yang Min; Xu Zhengping

    2005-01-01

    Angiogenin is an angiogenic factor which is involved in tumorigenesis. However, no particular intracellular protein is known to interact directly with angiogenin. In the present study, we reported the identification of α-actinin-2, an actin-crosslinking protein, as a potential angiogenin-interacting partner by yeast two-hybrid screening. This interaction was confirmed by different approaches. First, angiogenin was pulled down together with His-tagged α-actinin-2 by Ni 2+ -agarose resins. Second, α-actinin-2 was coimmunoprecipitated with angiogenin by anti-angiogenin monoclonal antibody. Third, the in vivo interaction of these two proteins was revealed by fluorescence resonance energy transfer analysis. Since members of α-actinin family play pivotal roles in cell proliferation, migration, and invasion, the interaction between α-actinin-2 and angiogenin may underline one possible mechanism of angiogenin in angiogenesis. Our finding presents the first evidence of an interaction of a cytosolic protein with angiogenin, which might be a novel interference target for anti-angiogenesis and anti-tumor therapy

  6. Degradability of dry matter and crude protein of dry grains and wet grain silages from different processing corn hybrids (Zea mays)

    OpenAIRE

    Wagner dos Reis; Ciniro Costa; Paulo Roberto de Lima Meirelles; Marina Gabriela Berchiol da Silva; Marco Aurélio Factori; Janaína Conte Hadlich; Kátia de Oliveira; Erikelly Aline Ribeiro de Santana; Cristiano Magalhães Pariz; Josineudson Augusto II de Vasconcelos Silva

    2013-01-01

    The objective of this work was to evaluate the effect of processing two corn hybrids conserved, dry and humid grains, the dry matter (DM) and crude protein (CP) degradability in situ. The particle size was determined and difference was verified in MGD (Medium Geometric Diameter) of processed ingredients. Three sheep were used with rumen canulated, in a completely randomized design, using a factorial outline 2 x 2 x 3, being two corn hybrid, two conservation methods and three processing forms ...

  7. Arabidopsis Intracellular NHX-Type Sodium-Proton Antiporters are Required for Seed Storage Protein Processing.

    Science.gov (United States)

    Ashnest, Joanne R; Huynh, Dung L; Dragwidge, Jonathan M; Ford, Brett A; Gendall, Anthony R

    2015-11-01

    The Arabidopsis intracellular sodium-proton exchanger (NHX) proteins AtNHX5 and AtNHX6 have a well-documented role in plant development, and have been used to improve salt tolerance in a variety of species. Despite evidence that intracellular NHX proteins are important in vacuolar trafficking, the mechanism of this role is poorly understood. Here we show that NHX5 and NHX6 are necessary for processing of the predominant seed storage proteins, and also influence the processing and activity of a vacuolar processing enzyme. Furthermore, we show by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) technology that the C-terminal tail of NHX6 interacts with a component of Retromer, another component of the cell sorting machinery, and that this tail is critical for NHX6 activity. These findings demonstrate that NHX5 and NHX6 are important in processing and activity of vacuolar cargo, and suggest a mechanism by which NHX intracellular (IC)-II antiporters may be involved in subcellular trafficking. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  8. Time-sequential changes of differentially expressed miRNAs during the process of anterior lumbar interbody fusion using equine bone protein extract, rhBMP-2 and autograft

    Science.gov (United States)

    Chen, Da-Fu; Zhou, Zhi-Yu; Dai, Xue-Jun; Gao, Man-Man; Huang, Bao-Ding; Liang, Tang-Zhao; Shi, Rui; Zou, Li-Jin; Li, Hai-Sheng; Bünger, Cody; Tian, Wei; Zou, Xue-Nong

    2014-03-01

    The precise mechanism of bone regeneration in different bone graft substitutes has been well studied in recent researches. However, miRNAs regulation of the bone formation has been always mysterious. We developed the anterior lumbar interbody fusion (ALIF) model in pigs using equine bone protein extract (BPE), recombinant human bone morphogenetic protein-2 (rhBMP-2) on an absorbable collagen sponge (ACS), and autograft as bone graft substitute, respectively. The miRNA and gene expression profiles of different bone graft materials were examined using microarray technology and data analysis, including self-organizing maps, KEGG pathway and Biological process GO analyses. We then jointly analyzed miRNA and mRNA profiles of the bone fusion tissue at different time points respectively. Results showed that miRNAs, including let-7, miR-129, miR-21, miR-133, miR-140, miR-146, miR-184, and miR-224, were involved in the regulation of the immune and inflammation response, which provided suitable inflammatory microenvironment for bone formation. At late stage, several miRNAs directly regulate SMAD4, Estrogen receptor 1 and 5-hydroxytryptamine (serotonin) receptor 2C for bone formation. It can be concluded that miRNAs play important roles in balancing the inflammation and bone formation.

  9. Porcine circovirus type 2 ORF4 protein binds heavy chain ferritin

    Indian Academy of Sciences (India)

    Porcine circovirus type 2 ORF4 protein binds heavy chain ferritin. Qizhuang Lv Kangkang Guo Tao Wang ... Keywords. Cellular protein; FHC; ORF4 protein; porcine circovirus type 2 (PCV2); yeast two-hybrid ... Journal of Biosciences | News ...

  10. [A case of IgA2-lambda type M-protein that IgA concentration differs from the values of M-protein by serum protein electrophoresis].

    Science.gov (United States)

    Fukushima, M; Sugano, M; Ichikawa, T; Honda, T; Totsuka, M; Katsuyama, T; Fujita, K

    2001-07-01

    We report an IgA-lambda type M-protein in which the IgA concentration differed from the values of M-protein by serum protein electrophoresis found in a 53-year-old man with multiple myeloma. The M-protein value as determined by serum protein electrophoresis was 6,170 mg/dl. However, the serum IgA concentration was 3,052 mg/dl by turbidimetric immunoassay. Immuno-fixation electrophoresis using IgA subclass antisera revealed that this M-protein was the IgA2-lambda type. Western blotting analysis showed that the IgA2 molecules were composed of two approximately 68 kDa alpha 2 chains and two 28 kDa lambda chains. In addition the free lambda chain band was detected at the position of 28 kDa without 2-mercaptoethanol(2-ME) even though the patient IgA was purified. Since it is known that IgA2m(1) allotype easily release light chains from the IgA molecules in SDS-PAGE without 2-ME, we speculated that in this patient the IgA was the IgA2m(1) allotype. After peripheral blood stem cell transplantation(PBSCT), immunofixation electrophoresis of the patient serum revealed not only the bands of IgA2-lambda type M-protein, but also three bands of IgG1-kappa type M-protein in the gamma region.

  11. Melanoma cells present high levels of HLA-A2-tyrosinase in association with instability and aberrant intracellular processing of tyrosinase.

    Science.gov (United States)

    Michaeli, Yael; Sinik, Keren; Haus-Cohen, Maya; Reiter, Yoram

    2012-04-01

    Short-lived protein translation products are proposed to be a major source of substrates for major histocompatibility complex (MHC) class I antigen processing and presentation; however, a direct link between protein stability and the presentation level of MHC class I-peptide complexes has not been made. We have recently discovered that the peptide Tyr((369-377)) , derived from the tyrosinase protein is highly presented by HLA-A2 on the surface of melanoma cells. To examine the molecular mechanisms responsible for this presentation, we compared characteristics of tyrosinase in melanoma cells lines that present high or low levels of HLA-A2-Tyr((369-377)) complexes. We found no correlation between mRNA levels and the levels of HLA-A2-Tyr((369-377)) presentation. Co-localization experiments revealed that, in cell lines presenting low levels of HLA-A2-Tyr((369-377)) complexes, tyrosinase co-localizes with LAMP-1, a melanosome marker, whereas in cell lines presenting high HLA-A2-Tyr((369-377)) levels, tyrosinase localizes to the endoplasmic reticulum. We also observed differences in tyrosinase molecular weight and glycosylation composition as well as major differences in protein stability (t(1/2) ). By stabilizing the tyrosinase protein, we observed a dramatic decrease in HLA-A2-tyrosinase presentation. Our findings suggest that aberrant processing and instability of tyrosinase are responsible for the high presentation of HLA-A2-Tyr((369-377)) complexes and thus shed new light on the relationship between intracellular processing, stability of proteins, and MHC-restricted peptide presentation. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. The missing piece in the puzzle: Prediction of aggregation via the protein-protein interaction parameter A∗2.

    Science.gov (United States)

    Koepf, Ellen; Schroeder, Rudolf; Brezesinski, Gerald; Friess, Wolfgang

    2018-07-01

    The tendency of protein pharmaceuticals to form aggregates is a major challenge during formulation development, as aggregation affects quality and safety of the product. In particular, the formation of large native-like particles in the context of liquid-air interfacial stress is a well-known but not fully understood problem. Focusing on the two most fundamental criteria of protein formulation affecting protein-protein interaction, the impact of pH and ionic strength on the interaction parameter A ∗ 2 and its link to aggregation upon mechanical stress was investigated. A ∗ 2 of two monoclonal antibodies (mABs) and a polyclonal IgG was determined using dynamic light scattering and was correlated to the number of particles formed upon shaking in vials analyzed by visual inspection, turbidity analysis, light obscuration and micro-flow imaging. A good correlation between aggregation induced by interfacial stress and formulation pH was given. It could be shown that A ∗ 2 was highest for mAB 1 and lowest for IgG, what was in good accordance with the number of particles formed. Shaking of IgG resulted in overall higher numbers of particles compared to the two mABs. A ∗ 2 decreased and particle numbers increased with increasing pH. Different to pH, ionic strength only slightly affected A ∗ 2 . Nevertheless, at high ionic (100 mM) strength the samples exhibited more pronounced particle formation, particularly of large particles >25 µm, which was most pronounced at high pH. Protein solutions were identified to form continuous films with an inhomogeneous protein distribution at the liquid-air interface. These areas of agglomerated, native-like protein material can be transferred into the bulk solution by compression-decompression of the interface. Whether or not those clusters lead to the appearance of large protein aggregates or fall apart depends on the attractive or repulsive forces between protein molecules. Thus, protein aggregation due to interfacial

  13. Accelerating large-scale protein structure alignments with graphics processing units

    Directory of Open Access Journals (Sweden)

    Pang Bin

    2012-02-01

    Full Text Available Abstract Background Large-scale protein structure alignment, an indispensable tool to structural bioinformatics, poses a tremendous challenge on computational resources. To ensure structure alignment accuracy and efficiency, efforts have been made to parallelize traditional alignment algorithms in grid environments. However, these solutions are costly and of limited accessibility. Others trade alignment quality for speedup by using high-level characteristics of structure fragments for structure comparisons. Findings We present ppsAlign, a parallel protein structure Alignment framework designed and optimized to exploit the parallelism of Graphics Processing Units (GPUs. As a general-purpose GPU platform, ppsAlign could take many concurrent methods, such as TM-align and Fr-TM-align, into the parallelized algorithm design. We evaluated ppsAlign on an NVIDIA Tesla C2050 GPU card, and compared it with existing software solutions running on an AMD dual-core CPU. We observed a 36-fold speedup over TM-align, a 65-fold speedup over Fr-TM-align, and a 40-fold speedup over MAMMOTH. Conclusions ppsAlign is a high-performance protein structure alignment tool designed to tackle the computational complexity issues from protein structural data. The solution presented in this paper allows large-scale structure comparisons to be performed using massive parallel computing power of GPU.

  14. Aquaporin Protein-Protein Interactions

    Directory of Open Access Journals (Sweden)

    Jennifer Virginia Roche

    2017-10-01

    Full Text Available Aquaporins are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across cell membranes. In eukaryotes, they are frequently regulated by gating or trafficking, allowing for the cell to control membrane permeability in a specific manner. Protein–protein interactions play crucial roles in both regulatory processes and also mediate alternative functions such as cell adhesion. In this review, we summarize recent knowledge about aquaporin protein–protein interactions; dividing the interactions into three types: (1 interactions between aquaporin tetramers; (2 interactions between aquaporin monomers within a tetramer (hetero-tetramerization; and (3 transient interactions with regulatory proteins. We particularly focus on the structural aspects of the interactions, discussing the small differences within a conserved overall fold that allow for aquaporins to be differentially regulated in an organism-, tissue- and trigger-specific manner. A deep knowledge about these differences is needed to fully understand aquaporin function and regulation in many physiological processes, and may enable design of compounds targeting specific aquaporins for treatment of human disease.

  15. Differential protein expression using proteomics from a crustacean brine shrimp (Artemia sinica) under CO2-driven seawater acidification.

    Science.gov (United States)

    Chang, Xue-Jiao; Zheng, Chao-Qun; Wang, Yu-Wei; Meng, Chuang; Xie, Xiao-Lu; Liu, Hai-Peng

    2016-11-01

    Gradually increasing atmospheric CO 2 partial pressure (pCO 2 ) has caused an imbalance in carbonate chemistry and resulted in decreased seawater pH in marine ecosystems, termed seawater acidification. Anthropogenic seawater acidification is postulated to affect the physiology of many marine calcifying organisms. To understand the possible effects of seawater acidification on the proteomic responses of a marine crustacean brine shrimp (Artemia sinica) three groups of cysts were hatched and further raised in seawater at different pH levels (8.2 as control and 7.8 and 7.6 as acidification stress levels according to the predicted levels at the end of this century and next century, respectively) for 1, 7 and 14 days followed by examination of the protein expression changes via two-dimensional gel electrophoresis. Searches of protein databases revealed that 67 differential protein spots were altered due to lower pH level (7.6 and 7.8) stress in comparison to control groups (pH 8.2) by mass spectrometry. Generally, these differentially expressed proteins included the following: 1) metabolic process-related proteins involved in glycolysis and glucogenesis, nucleotide/amino acid/fatty acid metabolism, protein biosynthesis, DNA replication and apoptosis; 2) stress response-related proteins, such as peroxiredoxin, thioredoxin peroxidase, 70-kDa heat shock protein, Na/K ATPase, and ubiquinol-cytochrome c reductase; 3) immune defence-related proteins, such as prophenoloxidase and ferritin; 4) cytoskeletal-related proteins, such as myosin light chain, TCP1 subunit 2, tropomyosin and tubulin alpha chain; and 5) signal transduction-related proteins, such as phospholipase C-like protein, 14-3-3 zeta, translationally controlled tumour protein and RNA binding motif protein. Taken together, these data support the idea that CO 2 -driven seawater acidification may affect protein expression in the crustacean A. sinica and possibly also in other species that feed on brine shrimp in the

  16. Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins.

    Science.gov (United States)

    Hyun, Seong-In; Weisberg, Andrea; Moss, Bernard

    2017-08-01

    The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vΔI2. As anticipated, vΔI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vΔI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes. IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights into

  17. Effect of four processed animal proteins in the diet on digestibility and performance in laying hens.

    NARCIS (Netherlands)

    Krimpen, van M.M.; Veldkamp, T.; Binnendijk, G.P.; Veer, de R.

    2010-01-01

    An experiment was performed to investigate the effect of animal vs. vegetable protein sources in the diet of laying hens on the development of hen performance. A diet containing protein sources of only vegetable origin was compared with 4 diets, each containing 1 of 4 processed animal proteins

  18. Use of a protein engineering strategy to overcome limitations in the production of "Difficult to Express" recombinant proteins.

    Science.gov (United States)

    Hussain, Hirra; Fisher, David I; Abbott, W Mark; Roth, Robert G; Dickson, Alan J

    2017-10-01

    Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant

  19. Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca[superscript 2+]-dependent Double-stranded DNA Binding Activity

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong (Cornell); (NWU)

    2012-05-22

    Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 {angstrom} tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is {approx}26 {angstrom} wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an {alpha}/{beta} domain and an {alpha}-helical domain; significant hinge motion was observed between these two domains. Ca{sup 2+} was located at strategic positions in the oligomerization interface. We further showed that removal of Ca{sup 2+} ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca{sup 2+} ions.

  20. Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and VDAC1.

    Directory of Open Access Journals (Sweden)

    Dmitriy Zamarin

    2005-09-01

    Full Text Available The influenza virus PB1-F2 is an 87-amino acid mitochondrial protein that previously has been shown to induce cell death, although the mechanism of apoptosis induction has remained unclear. In the process of characterizing its mechanism of action we found that the viral PB1-F2 protein sensitizes cells to apoptotic stimuli such as tumor necrosis factor alpha, as demonstrated by increased cleavage of caspase 3 substrates in PB1-F2-expressing cells. Moreover, treatment of purified mouse liver mitochondria with recombinant PB1-F2 protein resulted in cytochrome c release, loss of the mitochondrial membrane potential, and enhancement of tBid-induced mitochondrial permeabilization, suggesting a possible mechanism for the observed cellular sensitization to apoptosis. Using glutathione-S-transferase pulldowns with subsequent mass spectrometric analysis, we identified the mitochondrial interactors of the PB1-F2 protein and showed that the viral protein uniquely interacts with the inner mitochondrial membrane adenine nucleotide translocator 3 and the outer mitochondrial membrane voltage-dependent anion channel 1, both of which are implicated in the mitochondrial permeability transition during apoptosis. Consistent with this interaction, blockers of the permeability transition pore complex (PTPC inhibited PB1-F2-induced mitochondrial permeabilization. Based on our findings, we propose a model whereby the proapoptotic PB1-F2 protein acts through the mitochondrial PTPC and may play a role in the down-regulation of the host immune response to infection.

  1. Direct protein-protein interaction between PLCγ1 and the bradykinin B2 receptor-Importance of growth conditions

    International Nuclear Information System (INIS)

    Duchene, Johan; Chauhan, Sharmila D.; Lopez, Frederic; Pecher, Christiane; Esteve, Jean-Pierre; Girolami, Jean-Pierre; Bascands, Jean-Loup; Schanstra, Joost P.

    2005-01-01

    Recently, we have described a novel protein-protein interaction between the G-protein coupled bradykinin B2 receptor and tyrosine phosphatase SHP-2 via an immunoreceptor tyrosine-based inhibition motif (ITIM) sequence located in the C-terminal part of the B2 receptor and the Src homology (SH2) domains of SHP-2. Here we show that phospholipase C (PLC)γ1, another SH2 domain containing protein, can also interact with this ITIM sequence. Using surface plasmon resonance analysis, we observed that PLCγ1 interacted with a peptide containing the phosphorylated form of the bradykinin B2 receptor ITIM sequence. In CHO cells expressing the wild-type B2 receptor, bradykinin-induced transient recruitment and activation of PLCγ1. Interestingly, this interaction was only observed in quiescent and not in proliferating cells. Mutation of the key ITIM residue abolished this interaction with and activation of PLCγ1. Finally we also identified bradykinin-induced PLCγ1 recruitment and activation in primary culture renal mesangial cells

  2. Bcl-2 Protein Expression in Egyptian Acute Myeloid Leukemia

    International Nuclear Information System (INIS)

    El-Shakankiry, N.; El-Sayed, Gh.M.M.; El-Maghraby, Sh.; Moneer, M.M.

    2009-01-01

    Objective: The primary cause of treatment failure in acute myeloid leukemia (AML) is the emergence of both resistant disease and early relapse. The bcl-2 gene encodes a 26-kDa protein that promotes cell survival by blocking programmed cell death (apoptosis). In the present study, bcl-2 protein expression was evaluated in newly diagnosed AML patients and correlated with the induction of remission and overall survival (OS), in an attempt to define patients who might benefit from modified therapeutic strategies. Patients and methods: Pretreatment cellular bcl-2 protein expression was measured in bone marrow samples obtained from 68 patients of newly diagnosed acute myeloid leukemia and 10 healthy controls by western blotting. Results: The mean bcl-2 protein expression was significantly higher in patients (0.68610.592) compared to controls (0.313±0.016) (p=0.002). The overall survival for patients with mean bcl-2 expression of less, and more than or equal to 0.315, was 67% and 56%, respectively, with no significant difference between the two groups 0»=0.86). Conclusion: Even though we did not observe a significant difference in overall survival between patients with high and low levels of bcl-2, modulation of this protein might still be considered as an option for enhancing the effectiveness of conventional chemotherapy.

  3. Recent insights into Protein Phosphatase 2A structure and regulation: the reasons why PP2A is no longer considered as a lazy passive housekeeping enzyme

    Directory of Open Access Journals (Sweden)

    Martin, M.

    2010-01-01

    Full Text Available Although intracellular signal transduction is often portrayed as a protein kinase "domino effect", the counterbalancing function of phosphatases, and thus the control of phosphatase activity, is equally relevant to proper regulation of cellular function. Protein Phosphatase 2A (PP2A is a widely expressed family of protein phosphatases made of a core dimer, composed of a catalytic (C subunit and a structural (A subunit, in association with a third variable regulatory (B subunit. Although viewed as a constitutive housekeeping enzyme in the past, PP2A is a highly regulated phosphatase and is emerging as an important regulator of multiple cellular processes involving protein phosphorylation. The regulation of PP2A is mainly accomplished by the identity of the regulatory B-type subunit, which determines substrate specificity, subcellular localization and catalytic activity of the PP2A holoenzyme. In agreement with this, recent findings on the structure and post-translational modifications of PP2A emphasize the importance of PP2A holoenzyme composition in its regulation and pleiotropic activities.

  4. Direct inhibition of RNAse T2 expression by the HTLV-1 viral protein Tax.

    Science.gov (United States)

    Polakowski, Nicholas; Han, Hongjin; Lemasson, Isabelle

    2011-08-01

    Adult T-cell leukemia (ATL) is one of the primary diseases caused by Human T-cell Leukemia Virus type 1 (HTLV-1) infection. The virally-encoded Tax protein is believed to initiate early events in the development of this disease, as it is able to promote immortalization of T-cells and transformation of other cell types. These processes may be aided by the ability of the viral protein to directly deregulate expression of specific cellular genes through interactions with numerous transcriptional regulators. To identify gene promoters where Tax is localized, we isolated Tax-DNA complexes from an HTLV-1-infected T-cell line through a chromatin immunoprecipitation (ChIP) assay and used the DNA to probe a CpG island microarray. A site within the RNASET2 gene was found to be occupied by Tax. Real-time PCR analysis confirmed this result, and transient expression of Tax in uninfected cells led to the recruitment of the viral protein to the promoter. This event correlated with a decrease in the level of RNase T2 mRNA and protein, suggesting that Tax represses expression of this gene. Loss of RNase T2 expression occurs in certain hematological malignancies and other forms of cancer, and RNase T2 was recently reported to function as a tumor suppressor. Consequently, a reduction in the level of RNase T2 by Tax may play a role in ATL development.

  5. Direct Inhibition of RNAse T2 Expression by the HTLV-1 Viral Protein Tax

    Directory of Open Access Journals (Sweden)

    Isabelle Lemasson

    2011-08-01

    Full Text Available Adult T-cell leukemia (ATL is one of the primary diseases caused by Human T-cell Leukemia Virus type 1 (HTLV-1 infection. The virally-encoded Tax protein is believed to initiate early events in the development of this disease, as it is able to promote immortalization of T-cells and transformation of other cell types. These processes may be aided by the ability of the viral protein to directly deregulate expression of specific cellular genes through interactions with numerous transcriptional regulators. To identify gene promoters where Tax is localized, we isolated Tax-DNA complexes from an HTLV-1-infected T-cell line through a chromatin immunoprecipitation (ChIP assay and used the DNA to probe a CpG island microarray. A site within the RNASET2 gene was found to be occupied by Tax. Real-time PCR analysis confirmed this result, and transient expression of Tax in uninfected cells led to the recruitment of the viral protein to the promoter. This event correlated with a decrease in the level of RNase T2 mRNA and protein, suggesting that Tax represses expression of this gene. Loss of RNase T2 expression occurs in certain hematological malignancies and other forms of cancer, and RNase T2 was recently reported to function as a tumor suppressor. Consequently, a reduction in the level of RNase T2 by Tax may play a role in ATL development.

  6. SGLT2 Protein Expression Is Increased in Human Diabetic Nephropathy

    Science.gov (United States)

    Wang, Xiaoxin X.; Levi, Jonathan; Luo, Yuhuan; Myakala, Komuraiah; Herman-Edelstein, Michal; Qiu, Liru; Wang, Dong; Peng, Yingqiong; Grenz, Almut; Lucia, Scott; Dobrinskikh, Evgenia; D'Agati, Vivette D.; Koepsell, Hermann; Kopp, Jeffrey B.; Rosenberg, Avi Z.; Levi, Moshe

    2017-01-01

    There is very limited human renal sodium gradient-dependent glucose transporter protein (SGLT2) mRNA and protein expression data reported in the literature. The first aim of this study was to determine SGLT2 mRNA and protein levels in human and animal models of diabetic nephropathy. We have found that the expression of SGLT2 mRNA and protein is increased in renal biopsies from human subjects with diabetic nephropathy. This is in contrast to db-db mice that had no changes in renal SGLT2 protein expression. Furthermore, the effect of SGLT2 inhibition on renal lipid content and inflammation is not known. The second aim of this study was to determine the potential mechanisms of beneficial effects of SGLT2 inhibition in the progression of diabetic renal disease. We treated db/db mice with a selective SGLT2 inhibitor JNJ 39933673. We found that SGLT2 inhibition caused marked decreases in systolic blood pressure, kidney weight/body weight ratio, urinary albumin, and urinary thiobarbituric acid-reacting substances. SGLT2 inhibition prevented renal lipid accumulation via inhibition of carbohydrate-responsive element-binding protein-β, pyruvate kinase L, SCD-1, and DGAT1, key transcriptional factors and enzymes that mediate fatty acid and triglyceride synthesis. SGLT2 inhibition also prevented inflammation via inhibition of CD68 macrophage accumulation and expression of p65, TLR4, MCP-1, and osteopontin. These effects were associated with reduced mesangial expansion, accumulation of the extracellular matrix proteins fibronectin and type IV collagen, and loss of podocyte markers WT1 and synaptopodin, as determined by immunofluorescence microscopy. In summary, our study showed that SGLT2 inhibition modulates renal lipid metabolism and inflammation and prevents the development of nephropathy in db/db mice. PMID:28196866

  7. Cyclooxygenase-2 and hypoxia-regulated proteins are modulated by basic fibroblast growth factor in acute renal failure

    Directory of Open Access Journals (Sweden)

    Sandra Villanueva

    2012-01-01

    Full Text Available Acute renal failure (ARF can be caused by injuries that induce tissue hypoxia, which in turn can trigger adaptive or inflammatory responses. We previously showed the participation of basic fibroblast growth factor (FGF-2 in renal repair. Based on this, the aim of this study was to analyze the effect of FGF-2 signaling pathway manipulation at hypoxia-induced protein levels, as well as in key proteins from the vasoactive systems of the kidney. We injected rat kidneys with FGF-2 recombinant protein (r-FGF or FGF-2 receptor antisense oligonucleotide (FGFR2-ASO after bilateral ischemia, and evaluated the presence of iNOS, EPO and HO-1, in representation of hypoxia-induced proteins, as well as COX-2, renin, kallikrein, and B2KR, in representation of the vasoactive systems of the kidney. A reduction in iNOS, HO-1, EPO, renin, kallikrein, B2KR, and in renal damage was observed in animals treated with r-FGF. The opposite effect was found with FGF-2 receptor down-regulation. In contrast, COX-2 protein levels were higher in kidneys treated with r-FGF and lower in those that received FGFR2-ASO, as compared to saline treated kidneys. These results suggest that the protective role of FGF-2 in the pathogenesis of ARF induced by I/R is a complex process, through which a differential regulation of metabolic pathways takes place.

  8. Piper betle Induced Cytoprotective Genes and Proteins via the Nrf2/ARE Pathway in Aging Mice.

    Science.gov (United States)

    Aliahmat, Nor Syahida; Abdul Sani, Nur Fathiah; Wan Hasan, Wan Nuraini; Makpol, Suzana; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum

    2016-01-01

    The objective of this study was to elucidate the underlying antioxidant mechanism of aqueous extract of Piper betle (PB) in aging rats. The nuclear factor erythroid 2-related factor 2 (Nrf2)/ARE pathway involving phase II detoxifying and antioxidant enzymes plays an important role in the antioxidant system by reducing electrophiles and reactive oxygen species through induction of phase II enzymes and proteins. Genes and proteins of phase II detoxifying antioxidant enzymes were analyzed by QuantiGenePlex 2.0 Assay and Western blot analysis. PB significantly induced genes and proteins of phase II and antioxidant enzymes, NAD(P)H quinone oxidoreductase 1, and catalase in aging mice (p < 0.05). The expression of these enzymes were stimulated via translocation of Nrf2 into the nucleus, indicating the involvement of ARE, a cis-acting motif located in the promoter region of nearly all phase II genes. PB was testified for the first time to induce cytoprotective genes through the Nrf2/ARE signaling pathway, thus unraveling the antioxidant mechanism of PB during the aging process. © 2016 S. Karger AG, Basel.

  9. Engineered Trx2p industrial yeast strain protects glycolysis and fermentation proteins from oxidative carbonylation during biomass propagation

    Directory of Open Access Journals (Sweden)

    Gómez-Pastor Rocío

    2012-01-01

    Full Text Available Abstract Background In the yeast biomass production process, protein carbonylation has severe adverse effects since it diminishes biomass yield and profitability of industrial production plants. However, this significant detriment of yeast performance can be alleviated by increasing thioredoxins levels. Thioredoxins are important antioxidant defenses implicated in many functions in cells, and their primordial functions include scavenging of reactive oxygen species that produce dramatic and irreversible alterations such as protein carbonylation. Results In this work we have found several proteins specifically protected by yeast Thioredoxin 2 (Trx2p. Bidimensional electrophoresis and carbonylated protein identification from TRX-deficient and TRX-overexpressing cells revealed that glycolysis and fermentation-related proteins are specific targets of Trx2p protection. Indeed, the TRX2 overexpressing strain presented increased activity of the central carbon metabolism enzymes. Interestingly, Trx2p specifically preserved alcohol dehydrogenase I (Adh1p from carbonylation, decreased oligomer aggregates and increased its enzymatic activity. Conclusions The identified proteins suggest that the fermentative capacity detriment observed under industrial conditions in T73 wine commercial strain results from the oxidative carbonylation of specific glycolytic and fermentation enzymes. Indeed, increased thioredoxin levels enhance the performance of key fermentation enzymes such as Adh1p, which consequently increases fermentative capacity.

  10. Continuous desalting of refolded protein solution improves capturing in ion exchange chromatography: A seamless process.

    Science.gov (United States)

    Walch, Nicole; Jungbauer, Alois

    2017-06-01

    Truly continuous biomanufacturing processes enable an uninterrupted feed stream throughout the whole production without the need for holding tanks. We have utilized microporous anion and cation exchangers into which only salts, but not proteins, can penetrate into the pores for desalting of protein solutions, while diafiltration or dilution is usually employed for feed adjustments. Anion exchange and cation exchange chromatography columns were connected in series to remove both anions and cations. To increase operation performance, a continuous process was developed comprised of four columns. Continuous mode was achieved by staggered cycle operation, where one set of columns, consisting of one anion exchange and one cation exchange column, was loaded during the regeneration of the second set. Refolding, desalting and subsequent ion exchange capturing with a scFv as the model protein was demonstrated. The refolding solution was successfully desalted resulting in a consistent conductivity below 0.5 mS/cm from initial values of 10 to 11 mS/cm. With continuous operation process time could be reduced by 39% while productivity was increased to 163% compared to batch operation. Desalting of the protein solution resulted in up to 7-fold higher binding capacities in the subsequent ion exchange capture step with conventional protein binding resins. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. The removal process of 2,2-dichloroacetamide (DCAcAm), a new disinfection by-product, in drinking water treatment process and its toxicity on zebrafish.

    Science.gov (United States)

    Lin, Tao; Zhou, Dongju; Yu, Shilin; Chen, Wei

    2016-09-01

    The removal process of 2,2-dichloroacetamide (DCAcAm), a new disinfection by-product (DBP) in conventional drinking water treatment plant (C-DWTP) and advanced DWTP (ADWTP) was studied with newly maximum formation potential (MFP) process. It was demonstrated that the advanced treatment displayed greater removal efficiency towards DCAcAm formation potential (MFP) than the conventional treatment. The hydrophilic natural organic matter and natural organic matter with molecular weight 10 kDa leaded to more DCAcAm formation, and the aromatic protein was inferred as one part of DCAcAm precursor. DCAcAm was found to cause delayed development and malformation to zebrafish embryos at embryonic growth stage. Compared with heart toxicity, it caused a significant neuron toxicity. It also could cause the acute DNA damage to adult zebrafish, which should be extremely cautioned. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Protective immunity provided by HLA-A2 epitopes for fusion and hemagglutinin proteins of measles virus

    International Nuclear Information System (INIS)

    Oh, Sang Kon; Stegman, Brian; Pendleton, C. David; Ota, Martin O.; Pan, C.-H.; Griffin, Diane E.; Burke, Donald S.; Berzofsky, Jay A.

    2006-01-01

    Natural infection and vaccination with a live-attenuated measles virus (MV) induce CD8 + T-cell-mediated immune responses that may play a central role in controlling MV infection. In this study, we show that newly identified human HLA-A2 epitopes from MV hemagglutinin (H) and fusion (F) proteins induced protective immunity in HLA-A2 transgenic mice challenged with recombinant vaccinia viruses expressing F or H protein. HLA-A2 epitopes were predicted and synthesized. Five and four peptides from H and F, respectively, bound to HLA-A2 molecules in a T2-binding assay, and four from H and two from F could induce peptide-specific CD8 + T cell responses in HLA-A2 transgenic mice. Further experiments proved that three peptides from H (H9-567, H10-250, and H10-516) and one from F protein (F9-57) were endogenously processed and presented on HLA-A2 molecules. All peptides tested in this study are common to 5 different strains of MV including Edmonston. In both A2K b and HHD-2 mice, the identified peptide epitopes induced protective immunity against recombinant vaccinia viruses expressing H or F. Because F and H proteins induce neutralizing antibodies, they are major components of new vaccine strategies, and therefore data from this study will contribute to the development of new vaccines against MV infection

  13. Human metapneumovirus M2-2 protein inhibits innate immune response in monocyte-derived dendritic cells.

    Directory of Open Access Journals (Sweden)

    Junping Ren

    Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in young children, the elderly and immunocompromised patients. Repeated hMPV infections occur throughout life. However, immune evasion mechanisms of hMPV infection are largely unknown. Recently, our group has demonstrated that hMPV M2-2 protein, an important virulence factor, contributes to immune evasion in airway epithelial cells by targeting the mitochondrial antiviral-signaling protein (MAVS. Whether M2-2 regulates the innate immunity in human dendritic cells (DC, an important family of immune cells controlling antigen presenting, is currently unknown. We found that human DC infected with a virus lacking M2-2 protein expression (rhMPV-ΔM2-2 produced higher levels of cytokines, chemokines and IFNs, compared to cells infected with wild-type virus (rhMPV-WT, suggesting that M2-2 protein inhibits innate immunity in human DC. In parallel, we found that myeloid differentiation primary response gene 88 (MyD88, an essential adaptor for Toll-like receptors (TLRs, plays a critical role in inducing immune response of human DC, as downregulation of MyD88 by siRNA blocked the induction of immune regulatory molecules by hMPV. Since M2-2 is a cytoplasmic protein, we investigated whether M2-2 interferes with MyD88-mediated antiviral signaling. We found that indeed M2-2 protein associated with MyD88 and inhibited MyD88-dependent gene transcription. In this study, we also identified the domains of M2-2 responsible for its immune inhibitory function in human DC. In summary, our results demonstrate that M2-2 contributes to hMPV immune evasion by inhibiting MyD88-dependent cellular responses in human DC.

  14. Mannan-binding protein forms complexes with alpha-2-macroglobulin. A protein model for the interaction

    DEFF Research Database (Denmark)

    Storgaard, P; Holm Nielsen, E; Skriver, E

    1995-01-01

    We report that alpha-2-macroglobulin (alpha 2M) can form complexes with a high molecular weight porcine mannan-binding protein (pMBP-28). The alpha 2M/pMBP-28 complexes was isolated by PEG-precipitation and affinity chromatography on mannan-Sepharose, protein A-Sepharose and anti-IgM Sepharose......-PAGE, which reacted with antibodies against alpha 2M and pMBP-28, respectively, in Western blotting. Furthermore, alpha 2M/pMBP-28 complexes were demonstrated by electron microscopy. Fractionation of pMBP-containing D-mannose eluate from mannan-Sepharose on Superose 6 showed two protein peaks which reacted...... with anti-C1 s antibodies in ELISA, one of about 650-800 kDa, which in addition contained pMBP-28 and anti-alpha 2M reactive material, the other with an M(r) of 100-150 kDa. The latter peak revealed rhomboid molecules (7 x 15 nm) in the electron microscope and a 67 kDa band in SDS-PAGE under reducing...

  15. Coffee induces breast cancer resistance protein expression in Caco-2 cells.

    Science.gov (United States)

    Isshiki, Marina; Umezawa, Kazuo; Tamura, Hiroomi

    2011-01-01

    Coffee is a beverage that is consumed world-wide on a daily basis and is known to induce a series of metabolic and pharmacological effects, especially in the digestive tract. However, little is known concerning the effects of coffee on transporters in the gastrointestinal tract. To elucidate the effect of coffee on intestinal transporters, we investigated its effect on expression of the breast cancer resistance protein (BCRP/ABCG2) in a human colorectal cancer cell line, Caco-2. Coffee induced BCRP gene expression in Caco-2 cells in a coffee-dose dependent manner. Coffee treatment of Caco-2 cells also increased the level of BCRP protein, which corresponded to induction of gene expression, and also increased cellular efflux activity, as judged by Hoechst33342 accumulation. None of the major constituents of coffee tested could induce BCRP gene expression. The constituent of coffee that mediated this induction was extractable with ethyl acetate and was produced during the roasting process. Dehydromethylepoxyquinomicin (DHMEQ), an inhibitor of nuclear factor (NF)-κB, inhibited coffee-mediated induction of BCRP gene expression, suggesting involvement of NF-κB in this induction. Our data suggest that daily consumption of coffee might induce BCRP expression in the gastrointestinal tract and may affect the bioavailability of BCRP substrates.

  16. HLA class I is most tightly linked to levels of tapasin compared with other antigen-processing proteins in glioblastoma.

    Science.gov (United States)

    Thuring, Camilla; Follin, Elna; Geironson, Linda; Freyhult, Eva; Junghans, Victoria; Harndahl, Mikkel; Buus, Søren; Paulsson, Kajsa M

    2015-09-15

    Tumour cells can evade the immune system by dysregulation of human leukocyte antigens (HLA-I). Low quantity and/or altered quality of HLA-I cell surface expression is the result of either HLA-I alterations or dysregulations of proteins of the antigen-processing machinery (APM). Tapasin is an APM protein dedicated to the maturation of HLA-I and dysregulation of tapasin has been linked to higher malignancy in several different tumours. We studied the expression of APM components and HLA-I, as well as HLA-I tapasin-dependency profiles in glioblastoma tissues and corresponding cell lines. Tapasin displayed the strongest correlation to HLA-I heavy chain but also clustered with β2-microglobulin, transporter associated with antigen processing (TAP) and LMP. Moreover, tapasin also correlated to survival of glioblastoma patients. Some APM components, for example, TAP1/TAP2 and LMP2/LMP7, showed variable but coordinated expression, whereas ERAP1/ERAP2 displayed an imbalanced expression pattern. Furthermore, analysis of HLA-I profiles revealed variable tapasin dependence of HLA-I allomorphs in glioblastoma patients. Expression of APM proteins is highly variable between glioblastomas. Tapasin stands out as the APM component strongest correlated to HLA-I expression and we proved that HLA-I profiles in glioblastoma patients include tapasin-dependent allomorphs. The level of tapasin was also correlated with patient survival time. Our results support the need for individualisation of immunotherapy protocols.

  17. Chinese mitten crab (Eriocheir sinensis) iron-sulphur cluster assembly protein 2 (EsIscA2) is differentially regulated after immune and oxidative stress challenges.

    Science.gov (United States)

    Zhang, Peng; Liu, Yu; Wang, Min; Dong, Miren; Liu, Zhaoqun; Jia, Zhihao; Wang, Weilin; Zhang, Anguo; Wang, Lingling; Song, Linsheng

    2018-07-01

    Iron-sulphur clusters (ISCs), one of the oldest and most versatile cofactors of proteins, are involved in catalysis reactions, electron transport reactions, regulation processes as well as sensing of ambient conditions. Iron-sulphur cluster assembly protein (IscA) is a scaffold protein member of ISC formation system, which plays a significant role in the assembly and maturation process of ISC proteins. In the present study, the cDNA sequence of iron-sulphur cluster assembly protein 2 (designated as EsIscA2) was cloned from Eriocheir sinensis. The open reading frame (ORF) of EsIscA2 was of 507 bp, encoding a peptide of 168 amino acids with a typically conserved Fe-S domain. A tetrameric form was predicated by the SWISS-MODEL prediction algorithm, and three conserved cysteine residues (Cys-93, Cys-158, Cys-160) from each IscA monomer were predicted to form a 'cysteine pocket'. The deduced amino acid sequence of EsIscA2 shared over 50% similarity with that of other IscAs. EsIscA2 was clustered with IscA2 proteins from invertebrates and vertebrates, indicating that the protein was highly conservative in the evolution. rEsIscA2 exhibited a high iron binding affinity in the concentration ranging from 2 to 200 μM. EsIscA2 transcripts were detected in all the tested tissues including gonad, hemocytes, gill, muscle, heart, hepatopancreas and eyestalk, and EsIscA2 protein was detected in the mitochondria of hemocytes. The highest mRNA expression level of EsIscA2 was detected in muscle and hepatopancreas, which was about 34.66-fold (p < 0.05) and 27.07-fold (p < 0.05) of that in hemocytes, respectively. After Aeromonas hydrophila and lipopolysaccharide (LPS) stimulations, the mRNA expression of EsIscA2 in hemocytes was down-regulated and reached the lowest level at 24 h (0.31-fold, p < 0.05) and 48 h (0.29-fold, p < 0.05) compared to control group, respectively. And the expression of EsIscA2 mRNA in hepatopancreas was repressed from 6 h to 48 h post

  18. Protein phosphatase 2A regulates central sensitization in the spinal cord of rats following intradermal injection of capsaicin

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    Fang Li

    2006-03-01

    Full Text Available Abstract Background Intradermal injection of capsaicin into the hind paw of rats induces spinal cord central sensititzation, a process in which the responsiveness of central nociceptive neurons is amplified. In central sensitization, many signal transduction pathways composed of several cascades of intracellular enzymes are involved. As the phosphorylation state of neuronal proteins is strictly controlled and balanced by the opposing activities of protein kinases and phosphatases, the involvement of phosphatases in these events needs to be investigated. This study is designed to determine the influence of serine/threonine protein phosphatase type 2A (PP2A on the central nociceptive amplification process, which is induced by intradermal injection of capsaicin in rats. Results In experiment 1, the expression of PP2A protein in rat spinal cord at different time points following capsaicin or vehicle injection was examined using the Western blot method. In experiment 2, an inhibitor of PP2A (okadaic acid, 20 nM or fostriecin, 30 nM was injected into the subarachnoid space of the spinal cord, and the spontaneous exploratory activity of the rats before and after capsaicin injection was recorded with an automated photobeam activity system. The results showed that PP2A protein expression in the spinal cord was significantly upregulated following intradermal injection of capsaicin in rats. Capsaicin injection caused a significant decrease in exploratory activity of the rats. Thirty minutes after the injection, this decrease in activity had partly recovered. Infusion of a phosphatase inhibitor into the spinal cord intrathecal space enhanced the central sensitization induced by capsaicin by making the decrease in movement last longer. Conclusion These findings indicate that PP2A plays an important role in the cellular mechanisms of spinal cord central sensitization induced by intradermal injection of capsaicin in rats, which may have implications in

  19. Casein phosphopeptides and CaCl2 increase penicillin production and cause an increment in microbody/peroxisome proteins in Penicillium chrysogenum.

    Science.gov (United States)

    Domínguez-Santos, Rebeca; Kosalková, Katarina; García-Estrada, Carlos; Barreiro, Carlos; Ibáñez, Ana; Morales, Alejandro; Martín, Juan-Francisco

    2017-03-06

    Transport of penicillin intermediates and penicillin secretion are still poorly characterized in Penicillium chrysogenum (re-identified as Penicillium rubens). Calcium (Ca 2+ ) plays an important role in the metabolism of filamentous fungi, and casein phosphopeptides (CPP) are involved in Ca 2+ internalization. In this study we observe that the effect of CaCl 2 and CPP is additive and promotes an increase in penicillin production of up to 10-12 fold. Combination of CaCl 2 and CPP greatly promotes expression of the three penicillin biosynthetic genes. Comparative proteomic analysis by 2D-DIGE, identified 39 proteins differentially represented in P. chrysogenum Wisconsin 54-1255 after CPP/CaCl 2 addition. The most interesting group of overrepresented proteins were a peroxisomal catalase, three proteins of the methylcitrate cycle, two aminotransferases and cystationine β-synthase, which are directly or indirectly related to the formation of penicillin amino acid precursors. Importantly, two of the enzymes of the penicillin pathway (isopenicillin N synthase and isopenicillin N acyltransferase) are clearly induced after CPP/CaCl 2 addition. Most of these overrepresented proteins are either authentic peroxisomal proteins or microbody-associated proteins. This evidence suggests that addition of CPP/CaCl 2 promotes the formation of penicillin precursors and the penicillin biosynthetic enzymes in peroxisomes and vesicles, which may be involved in transport and secretion of penicillin. Penicillin biosynthesis in Penicillium chrysogenum is one of the best characterized secondary metabolism processes. However, the mechanism by which penicillin is secreted still remains to be elucidated. Taking into account the role played by Ca 2+ and CPP in the secretory pathway and considering the positive effect that Ca 2+ exerts on penicillin production, the analysis of global protein changes produced after CPP/CaCl 2 addition is very helpful to decipher the processes related to the

  20. TaPP2C1, a Group F2 Protein Phosphatase 2C Gene, Confers Resistance to Salt Stress in Transgenic Tobacco.

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    Wei Hu

    Full Text Available Group A protein phosphatases 2Cs (PP2Cs are essential components of abscisic acid (ABA signaling in Arabidopsis; however, the function of group F2 subfamily PP2Cs is currently less known. In this study, TaPP2C1 which belongs to group F2 was isolated and characterized from wheat. Expression of the TaPP2C1-GFP fusion protein suggested its ubiquitous localization within a cell. TaPP2C1 expression was downregulated by abscisic acid (ABA and NaCl treatments, but upregulated by H2O2 treatment. Overexpression of TaPP2C1 in tobacco resulted in reduced ABA sensitivity and increased salt resistance of transgenic seedlings. Additionally, physiological analyses showed that improved resistance to salt stress conferred by TaPP2C1 is due to the reduced reactive oxygen species (ROS accumulation, the improved antioxidant system, and the increased transcription of genes in the ABA-independent pathway. Finally, transgenic tobacco showed increased resistance to oxidative stress by maintaining a more effective antioxidant system. Taken together, these results demonstrated that TaPP2C1 negatively regulates ABA signaling, but positively regulates salt resistance. TaPP2C1 confers salt resistance through activating the antioxidant system and ABA-independent gene transcription process.

  1. Bcl-2 protein expression is associated with p27 and p53 protein expressions and MIB-1 counts in breast cancer

    International Nuclear Information System (INIS)

    Tsutsui, Shinichi; Yasuda, Kazuhiro; Suzuki, Kosuke; Takeuchi, Hideya; Nishizaki, Takashi; Higashi, Hidefumi; Era, Shoichi

    2006-01-01

    Recent experimental studies have shown that Bcl-2, which has been established as a key player in the control of apoptosis, plays a role in regulating the cell cycle and proliferation. The aim of this study was to investigate the relationship between Bcl-2 and p27 protein expression, p53 protein expression and the proliferation activity as defined by the MIB-1 counts. The prognostic implication of Bcl-2 protein expression in relation to p27 and p53 protein expressions and MIB-1 counts for breast cancer was also evaluated. The immunohistochemical expression of Bcl-2 protein was evaluated in a series of 249 invasive ductal carcinomas of the breast, in which p27 and p53 protein expressions and MIB-1 counts had been determined previously. The Bcl-2 protein expression was found to be decreased in 105 (42%) cases. A decreased Bcl-2 protein expression was significantly correlated with a nuclear grade of III, a negative estrogen receptor, a decreased p27 protein expression, a positive p53 protein expression, positive MIB-1 counts and a positive HER2 protein expression. The incidence of a nuclear grade of III and positive MIB-1 counts increased as the number of abnormal findings of Bcl-2, p27 and p53 protein expressions increased. A univariate analysis indicated a decreased Bcl-2 protein expression to be significantly (p = 0.0089) associated with a worse disease free survival (DFS), while a multivariate analysis indicated the lymph node status and MIB-1 counts to be independently significant prognostic factors for the DFS. The Bcl-2 protein expression has a close correlation with p27 and p53 protein expressions and the proliferation activity determined by MIB-1 counts in invasive ductal carcinoma of the breast. The prognostic value of Bcl-2 as well as p27 and p53 protein expressions was dependent on the proliferation activity in breast cancer

  2. Bcl-2 protein expression is associated with p27 and p53 protein expressions and MIB-1 counts in breast cancer

    Directory of Open Access Journals (Sweden)

    Nishizaki Takashi

    2006-07-01

    Full Text Available Abstract Background Recent experimental studies have shown that Bcl-2, which has been established as a key player in the control of apoptosis, plays a role in regulating the cell cycle and proliferation. The aim of this study was to investigate the relationship between Bcl-2 and p27 protein expression, p53 protein expression and the proliferation activity as defined by the MIB-1 counts. The prognostic implication of Bcl-2 protein expression in relation to p27 and p53 protein expressions and MIB-1 counts for breast cancer was also evaluated. Methods The immunohistochemical expression of Bcl-2 protein was evaluated in a series of 249 invasive ductal carcinomas of the breast, in which p27 and p53 protein expressions and MIB-1 counts had been determined previously. Results The Bcl-2 protein expression was found to be decreased in 105 (42% cases. A decreased Bcl-2 protein expression was significantly correlated with a nuclear grade of III, a negative estrogen receptor, a decreased p27 protein expression, a positive p53 protein expression, positive MIB-1 counts and a positive HER2 protein expression. The incidence of a nuclear grade of III and positive MIB-1 counts increased as the number of abnormal findings of Bcl-2, p27 and p53 protein expressions increased. A univariate analysis indicated a decreased Bcl-2 protein expression to be significantly (p = 0.0089 associated with a worse disease free survival (DFS, while a multivariate analysis indicated the lymph node status and MIB-1 counts to be independently significant prognostic factors for the DFS. Conclusion The Bcl-2 protein expression has a close correlation with p27 and p53 protein expressions and the proliferation activity determined by MIB-1 counts in invasive ductal carcinoma of the breast. The prognostic value of Bcl-2 as well as p27 and p53 protein expressions was dependent on the proliferation activity in breast cancer.

  3. Degradability of dry matter and crude protein of dry grains and wet grain silages from different processing corn hybrids (Zea mays

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    Wagner dos Reis

    2013-03-01

    Full Text Available The objective of this work was to evaluate the effect of processing two corn hybrids conserved, dry and humid grains, the dry matter (DM and crude protein (CP degradability in situ. The particle size was determined and difference was verified in MGD (Medium Geometric Diameter of processed ingredients. Three sheep were used with rumen canulated, in a completely randomized design, using a factorial outline 2 x 2 x 3, being two corn hybrid, two conservation methods and three processing forms (whole, coarsely and finely ground, with five times of incubation (3, 6, 12, 24 and 48 hours. The fraction A in SDC (silage of dent corn of DM was superior to GDC (grain of dent corn in all of the particles size. The ensiling process increased the DM solubility, reducing the fraction B in comparison to dry grain. The values regarding the fractions DP and DE the 5% per hour of the protein, were larger for SDC and GDC, it presents a decreasing when the incubation time advances. The fermentation rate was superior for SDC and GDC. The ensiling process has positive effect in the decreasing of DM and CP in comparison to GDC.

  4. Development of a Model Protein Interaction Pair as a Benchmarking Tool for the Quantitative Analysis of 2-Site Protein-Protein Interactions.

    Science.gov (United States)

    Yamniuk, Aaron P; Newitt, John A; Doyle, Michael L; Arisaka, Fumio; Giannetti, Anthony M; Hensley, Preston; Myszka, David G; Schwarz, Fred P; Thomson, James A; Eisenstein, Edward

    2015-12-01

    A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar. It is demonstrated that this system can serve as a benchmarking tool for the quantitative analysis of 2-site protein-protein interactions. The protein interaction pair enables determination of precise binding constants for the barstar protein binding to 2 distinct sites on the bivalent barnase binding partner (termed binase), where the 2 binding sites were engineered to possess affinities that differed by 2 orders of magnitude. Multiple MIRG laboratories characterized the interaction using isothermal titration calorimetry (ITC), AUC, and surface plasmon resonance (SPR) methods to evaluate the feasibility of the system as a benchmarking model. Although general agreement was seen for the binding constants measured using solution-based ITC and AUC approaches, weaker affinity was seen for surface-based method SPR, with protein immobilization likely affecting affinity. An analysis of the results from multiple MIRG laboratories suggests that the bivalent barnase-barstar system is a suitable model for benchmarking new approaches for the quantitative characterization of complex biomolecular interactions.

  5. Rice Dwarf Virus P2 Protein Hijacks Auxin Signaling by Directly Targeting the Rice OsIAA10 Protein, Enhancing Viral Infection and Disease Development.

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    Lian Jin

    2016-09-01

    Full Text Available The phytohormone auxin plays critical roles in regulating myriads of plant growth and developmental processes. Microbe infection can disturb auxin signaling resulting in defects in these processes, but the underlying mechanisms are poorly understood. Auxin signaling begins with perception of auxin by a transient co-receptor complex consisting of an F-box transport inhibitor response 1/auxin signaling F-box (TIR1/AFB protein and an auxin/indole-3-acetic acid (Aux/IAA protein. Auxin binding to the co-receptor triggers ubiquitination and 26S proteasome degradation of the Aux/IAA proteins, leading to subsequent events, including expression of auxin-responsive genes. Here we report that Rice dwarf virus (RDV, a devastating pathogen of rice, causes disease symptoms including dwarfing, increased tiller number and short crown roots in infected rice as a result of reduced sensitivity to auxin signaling. The RDV capsid protein P2 binds OsIAA10, blocking the interaction between OsIAA10 and OsTIR1 and inhibiting 26S proteasome-mediated OsIAA10 degradation. Transgenic rice plants overexpressing wild-type or a dominant-negative (degradation-resistant mutant of OsIAA10 phenocopy RDV symptoms are more susceptible to RDV infection; however, knockdown of OsIAA10 enhances the resistance of rice to RDV infection. Our findings reveal a previously unknown mechanism of viral protein reprogramming of a key step in auxin signaling initiation that enhances viral infection and pathogenesis.

  6. Myb-binding protein 1a (Mybbp1a) regulates levels and processing of pre-ribosomal RNA.

    Science.gov (United States)

    Hochstatter, Julia; Hölzel, Michael; Rohrmoser, Michaela; Schermelleh, Lothar; Leonhardt, Heinrich; Keough, Rebecca; Gonda, Thomas J; Imhof, Axel; Eick, Dirk; Längst, Gernot; Németh, Attila

    2012-07-13

    Ribosomal RNA gene transcription, co-transcriptional processing, and ribosome biogenesis are highly coordinated processes that are tightly regulated during cell growth. In this study we discovered that Mybbp1a is associated with both the RNA polymerase I complex and the ribosome biogenesis machinery. Using a reporter assay that uncouples transcription and RNA processing, we show that Mybbp1a represses rRNA gene transcription. In addition, overexpression of the protein reduces RNA polymerase I loading on endogenous rRNA genes as revealed by chromatin immunoprecipitation experiments. Accordingly, depletion of Mybbp1a results in an accumulation of the rRNA precursor in vivo but surprisingly also causes growth arrest of the cells. This effect can be explained by the observation that the modulation of Mybbp1a protein levels results in defects in pre-rRNA processing within the cell. Therefore, the protein may play a dual role in the rRNA metabolism, potentially linking and coordinating ribosomal DNA transcription and pre-rRNA processing to allow for the efficient synthesis of ribosomes.

  7. Modulation of the cytochrome P450-mediated metabolism of ifosfamide by ketoconazole and rifampin

    NARCIS (Netherlands)

    Kerbusch, T.; Jansen, R. L.; Mathôt, R. A.; Huitema, A. D.; Jansen, M.; van Rijswijk, R. E.; Beijnen, J. H.

    2001-01-01

    The autoinducible metabolic transformation of the anticancer agent ifosfamide involves activation through 4-hydroxyifosfamide to the ultimate cytotoxic ifosforamide mustard and deactivation to 2- and 3-dechloroethylifosfamide with concomitant release of the neurotoxic chloroacetaldehyde. Activation

  8. Reverse engineering of an affinity-switchable molecular interaction characterized by atomic force microscopy single-molecule force spectroscopy.

    Science.gov (United States)

    Anselmetti, Dario; Bartels, Frank Wilco; Becker, Anke; Decker, Björn; Eckel, Rainer; McIntosh, Matthew; Mattay, Jochen; Plattner, Patrik; Ros, Robert; Schäfer, Christian; Sewald, Norbert

    2008-02-19

    Tunable and switchable interaction between molecules is a key for regulation and control of cellular processes. The translation of the underlying physicochemical principles to synthetic and switchable functional entities and molecules that can mimic the corresponding molecular functions is called reverse molecular engineering. We quantitatively investigated autoinducer-regulated DNA-protein interaction in bacterial gene regulation processes with single atomic force microscopy (AFM) molecule force spectroscopy in vitro, and developed an artificial bistable molecular host-guest system that can be controlled and regulated by external signals (UV light exposure and thermal energy). The intermolecular binding functionality (affinity) and its reproducible and reversible switching has been proven by AFM force spectroscopy at the single-molecule level. This affinity-tunable optomechanical switch will allow novel applications with respect to molecular manipulation, nanoscale rewritable molecular memories, and/or artificial ion channels, which will serve for the controlled transport and release of ions and neutral compounds in the future.

  9. A gene-protein assay for human epidermal growth factor receptor 2 (HER2: brightfield tricolor visualization of HER2 protein, the HER2 gene, and chromosome 17 centromere (CEN17 in formalin-fixed, paraffin-embedded breast cancer tissue sections

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    Nitta Hiroaki

    2012-05-01

    Full Text Available Abstract Background The eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH or immunohistochemistry (IHC, respectively. Our objective was to combine the US Food and Drug Administration (FDA-approved HER2 & chromosome 17 centromere (CEN17 brightfield ISH (BISH and HER2 IHC assays into a single automated HER2 gene-protein assay allowing simultaneous detection of all three targets in a single tissue section. Methods The HER2 gene-protein assay was optimized using formalin-fixed, paraffin-embedded (FFPE samples of the xenograft tumors MCF7 [HER2 negative (non-amplified gene, protein negative] and Calu-3 [HER2 positive (amplified gene, protein positive]. HER2 IHC was performed using a rabbit monoclonal anti-HER2 antibody (clone 4B5 and a conventional 3,3'-diaminobenzidine IHC detection. The HER2 & CEN17 BISH signals were visualized using horseradish peroxidase-based silver and alkaline phosphatase-based red detection systems, respectively with a cocktail of 2,4-dinitrophenyl-labeled HER2 and digoxigenin-labeled CEN17 probes. The performance of the gene-protein assay on tissue microarray slides containing 189 randomly selected FFPE clinical breast cancer tissue cores was compared to that of the separate HER2 IHC and HER2 & CEN17 BISH assays. Results HER2 protein detection was optimal when the HER2 IHC protocol was used before (rather than after the BISH protocol. The sequential use of HER2 IHC and HER2 & CEN17 BISH detection steps on FFPE xenograft tumor sections appropriately co-localized the HER2 protein, HER2 gene, and CEN17 signals after mitigating the silver background staining by using a naphthol phosphate-containing hybridization buffer for the hybridization step. The HER2 protein and HER2 gene status obtained using the multiplex HER2 gene-protein

  10. Effects of annealing on the physical properties of therapeutic proteins during freeze drying process.

    Science.gov (United States)

    Lim, Jun Yeul; Lim, Dae Gon; Kim, Ki Hyun; Park, Sang-Koo; Jeong, Seong Hoon

    2018-02-01

    Effects of annealing steps during the freeze drying process on etanercept, model protein, were evaluated using various analytical methods. The annealing was introduced in three different ways depending on time and temperature. Residual water contents of dried cakes varied from 2.91% to 6.39% and decreased when the annealing step was adopted, suggesting that they are directly affected by the freeze drying methods Moreover, the samples were more homogenous when annealing was adopted. Transition temperatures of the excipients (sucrose, mannitol, and glycine) were dependent on the freeze drying steps. Size exclusion chromatography showed that monomer contents were high when annealing was adopted and also they decreased less after thermal storage at 60°C. Dynamic light scattering results exhibited that annealing can be helpful in inhibiting aggregation and that thermal storage of freeze-dried samples preferably induced fragmentation over aggregation. Shift of circular dichroism spectrum and of the contents of etanercept secondary structure was observed with different freeze drying steps and thermal storage conditions. All analytical results suggest that the physicochemical properties of etanercept formulation can differ in response to different freeze drying steps and that annealing is beneficial for maintaining stability of protein and reducing the time of freeze drying process. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. CXCL1 and CXCL2 Regulate NLRP3 Inflammasome Activation via G-Protein-Coupled Receptor CXCR2.

    Science.gov (United States)

    Boro, Monoranjan; Balaji, Kithiganahalli Narayanaswamy

    2017-09-01

    Inflammation is an extensively concerted process that confers protection to the host encountering immune insult. The major inflammatory mediators include IL-1 family members, such as IL-1β, and the functional activation of such molecules is arbitrated by their regulated cleavage brought about by components of a multiprotein complex called inflammasome. In this context, NLR family pyrin domain containing 3 (NLRP3) inflammasome activation often acts as a rate-limiting step in regulating critical cell-fate decisions in various inflammatory scenarios. In this study, we identify the G-protein-coupled receptor CXCR2 (recognizing chemokines CXCL1 and CXCL2) as another arm feeding into the regulated activation of NLRP3 inflammasome in macrophages. We demonstrate that in vivo blocking of CXCL1 and CXCL2 can significantly reduce the Mycobacterium tuberculosis -induced bioactive IL-1β production. Further, CXCL1 could amplify the inflammasome activation in in vivo mouse models of carrageenan-induced inflammation in footpads and air pouches. The mechanistic insights revealed CXCR2-driven protein kinase C μ-dependent integrin-linked kinase to be essential for CXCL1-mediated activation of NLRP3 inflammasome. Blocking the activity of integrin-linked kinase or protein kinase C μ either by small interfering RNA-mediated knockdown or pharmacological inhibitor compromised inflammasome activation and subsequent production of bioactive IL-1β. Taken together, our study demonstrates CXCR2-driven activation of NLRP3 inflammasome in macrophages and indicates a potential host-directed therapeutic target to limit the damaging inflammation associated with overt production of proinflammatory IL-1β. Copyright © 2017 by The American Association of Immunologists, Inc.

  12. LuxS/AI-2 system is involved in antibiotic susceptibility and autolysis in Staphylococcus aureus NCTC 8325.

    Science.gov (United States)

    Xue, Ting; Zhao, Liping; Sun, Baolin

    2013-01-01

    Current treatment for Staphylococcus aureus infections relies heavily upon the cell wall synthesis inhibitor antibiotics such as penicillin, oxacillin, vancomycin and teicoplanin. Increasing antibiotic resistance requires the development of new approaches to combating infection. Autoinducer-2 (AI-2) exists widely both in Gram-negative and Gram-positive pathogens and is suggested as a universal language for intraspecies and interspecies communication. This study demonstrates the association between AI-2 signalling and cell wall synthesis inhibitor antibiotic susceptibility in S. aureus. In addition, a luxS mutant exhibited decreased autolysis and upregulated vancomycin resistance-associated VraRS two-component regulatory system. This finding may provide novel clues for antimicrobial therapy in S. aureus infection. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  13. Arabidopsis ASYMMETRIC LEAVES2 protein required for leaf morphogenesis consistently forms speckles during mitosis of tobacco BY-2 cells via signals in its specific sequence.

    Science.gov (United States)

    Luo, Lilan; Ando, Sayuri; Sasabe, Michiko; Machida, Chiyoko; Kurihara, Daisuke; Higashiyama, Tetsuya; Machida, Yasunori

    2012-09-01

    Leaf primordia with high division and developmental competencies are generated around the periphery of stem cells at the shoot apex. Arabidopsis ASYMMETRIC-LEAVES2 (AS2) protein plays a key role in the regulation of many genes responsible for flat symmetric leaf formation. The AS2 gene, expressed in leaf primordia, encodes a plant-specific nuclear protein containing an AS2/LOB domain with cysteine repeats (C-motif). AS2 proteins are present in speckles in and around the nucleoli, and in the nucleoplasm of some leaf epidermal cells. We used the tobacco cultured cell line BY-2 expressing the AS2-fused yellow fluorescent protein to examine subnuclear localization of AS2 in dividing cells. AS2 mainly localized to speckles (designated AS2 bodies) in cells undergoing mitosis and distributed in a pairwise manner during the separation of sets of daughter chromosomes. Few interphase cells contained AS2 bodies. Deletion analyses showed that a short stretch of the AS2 amino-terminal sequence and the C-motif play negative and positive roles, respectively, in localizing AS2 to the bodies. These results suggest that AS2 bodies function to properly distribute AS2 to daughter cells during cell division in leaf primordia; and this process is controlled at least partially by signals encoded by the AS2 sequence itself.

  14. The evaluation of lyophilized polymer matrices for administering recombinant human bone morphogenetic protein-2.

    Science.gov (United States)

    Duggirala, S S; Rodgers, J B; DeLuca, P P

    1996-07-01

    Novel unitary devices, prepared by lyophilization of viscous solutions of sodium carboxymethylcellulose (CMC) and methylcellulose (MC), were evaluated as sustained-release delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2). In vitro characterization of the unitary devices, which contained rhBMP-2-loaded poly (d,l lactide-co-glycolide) (PLGA) bioerodible particles (BEPs), was conducted over a 2-month period. Determinations included buffer uptake, mass and molecular weight loss and rhBMP-2 release from the unitary devices. CMC devices imbibed approximately 16 times their weight of buffer, while with MC, equilibrium uptake was approximately 6 times the dry weight of the devices. Overall mass loss percentages were approximately 55 and 35%, respectively, for CMC and MC devices. rhBMP-2 release from the devices was essentially a triphasic process: an initial phase during which "free" protein (rhBMP-2 present on the surface and within the pores of the PLGA BEPs) was released, a lag period during which no release was discerned, and then release of "bound" rhBMP-2 (protein adsorbed to the BEPs). The release of bound protein correlated with the mass loss of the polymer which began after 3 weeks. Release from the unitary devices was lower than that from the BEPs alone, due to a retardation effect of the gelled CMC/MC polymers. In rabbits in which full-thickness cranial bone defects were created, the implants were well tolerated and induced significant new bone growth during an 8-week evaluation period. The CMC devices appear to have induced bone earlier (at 2 weeks), but this did not affect eventual 8-week results. CMC devices without rhBMP-2 appeared to provide some bone conduction, in contrast to the blank MC devices.

  15. CONFOLD2: improved contact-driven ab initio protein structure modeling.

    Science.gov (United States)

    Adhikari, Badri; Cheng, Jianlin

    2018-01-25

    Contact-guided protein structure prediction methods are becoming more and more successful because of the latest advances in residue-residue contact prediction. To support contact-driven structure prediction, effective tools that can quickly build tertiary structural models of good quality from predicted contacts need to be developed. We develop an improved contact-driven protein modelling method, CONFOLD2, and study how it may be effectively used for ab initio protein structure prediction with predicted contacts as input. It builds models using various subsets of input contacts to explore the fold space under the guidance of a soft square energy function, and then clusters the models to obtain the top five models. CONFOLD2 obtains an average reconstruction accuracy of 0.57 TM-score for the 150 proteins in the PSICOV contact prediction dataset. When benchmarked on the CASP11 contacts predicted using CONSIP2 and CASP12 contacts predicted using Raptor-X, CONFOLD2 achieves a mean TM-score of 0.41 on both datasets. CONFOLD2 allows to quickly generate top five structural models for a protein sequence when its secondary structures and contacts predictions at hand. The source code of CONFOLD2 is publicly available at https://github.com/multicom-toolbox/CONFOLD2/ .

  16. Toponomics analysis of functional interactions of the ubiquitin ligase PAM (Protein Associated with Myc) during spinal nociceptive processing.

    Science.gov (United States)

    Pierre, Sandra; Maeurer, Christian; Coste, Ovidiu; Becker, Wiebke; Schmidtko, Achim; Holland, Sabrina; Wittpoth, Claus; Geisslinger, Gerd; Scholich, Klaus

    2008-12-01

    Protein associated with Myc (PAM) is a giant E3 ubiquitin ligase of 510 kDa. Although the role of PAM during neuronal development is well established, very little is known about its function in the regulation of synaptic strength. Here we used multiepitope ligand cartography (MELC) to study protein network profiles associated with PAM during the modulation of synaptic strength. MELC is a novel imaging technology that utilizes biomathematical tools to describe protein networks after consecutive immunohistochemical visualization of up to 100 proteins on the same sample. As an in vivo model to modulate synaptic strength we used the formalin test, a common model for acute and inflammatory pain. MELC analysis was performed with 37 different antibodies or fluorescence tags on spinal cord slices and led to the identification of 1390 PAM-related motifs that distinguish untreated and formalin-treated spinal cords. The majority of these motifs related to ubiquitin-dependent processes and/or the actin cytoskeleton. We detected an intermittent colocalization of PAM and ubiquitin with TSC2, a known substrate of PAM, and the glutamate receptors mGluR5 and GLUR1. Importantly these complexes were detected exclusively in the presence of F-actin. A direct PAM/F-actin interaction was confirmed by colocalization and cosedimentation. The binding of PAM toward F-actin varied strongly between the PAM splice forms found in rat spinal cords. PAM did not ubiquitylate actin or alter actin polymerization and depolymerization. However, F-actin decreased the ubiquitin ligase activity of purified PAM. Because PAM activation is known to involve its translocation, the binding of PAM to F-actin may serve to control its subcellular localization as well as its activity. Taken together we show that defining protein network profiles by topological proteomics analysis is a useful tool to identify previously unknown protein/protein interactions that underlie synaptic processes.

  17. Effect of high hydrostatic pressure processing on in vitro digestion of milk proteins and fats

    Science.gov (United States)

    The use of high hydrostatic pressure processing (HPP) is increasing in popularity in the food industry. Its ability to modify milk proteins and fats suggests that it may be useful in creating foods that suppress appetite; however, its effect on the digestibility of proteins and fats is unclear. The...

  18. Mental retardation-related protease, motopsin (prss12), binds to the BRICHOS domain of the integral membrane protein 2a.

    Science.gov (United States)

    Mitsui, Shinichi; Osako, Yoji; Yuri, Kazunari

    2014-01-01

    Motopsin (prss12), a mosaic serine protease secreted by neuronal cells, is believed to be important for cognitive function, as the loss of its function causes severe nonsyndromic mental retardation. To understand the molecular role of motopsin, we identified the integral membrane protein 2a (Itm2a) as a motopsin-interacting protein using a yeast two-hybrid system. A pull-down assay showed that the BRICHOS domain of Itm2a was essential for this interaction. Motopsin and Itm2a co-localized in COS cells and in cultured neurons when transiently expressed in these cells. Both proteins were co-immunoprecipitated from lysates of these transfected COS cells. Itm2a was strongly detected in a brain lysate prepared between postnatal day 0 and 10, during which period motopsin protein was also enriched in the brain. Immunohistochemistry detected Itm2a as patchy spots along endothelial cells of brain capillaries (which also expressed myosin II regulatory light chain [RLC]), and on glial fibrillary acidic protein (GFAP)-positive processes in the developing cerebral cortex. The data raise the possibility that secreted motopsin interacts with endothelial cells in the developing brain. © 2013 International Federation for Cell Biology.

  19. Porcine circovirus-2 capsid protein induces cell death in PK15 cells

    Energy Technology Data Exchange (ETDEWEB)

    Walia, Rupali; Dardari, Rkia, E-mail: rdardari@ucalgary.ca; Chaiyakul, Mark; Czub, Markus

    2014-11-15

    Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathways involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis. - Highlights: • IFN-γ enhances PCV2 replication that leads to cell death in PK15 cells. • IFN-γ enhances nuclear localization of the PCV2 Capsid protein. • Transient PCV2a and 2b-Capsid protein expression induces cell death. • Cell death is not dictated by specific Capsid protein sub-localization.

  20. Effect of radiation processing on antinutrients, in-vitro protein digestibility and protein efficiency ratio bioassay of legume seeds

    Energy Technology Data Exchange (ETDEWEB)

    El-Niely, Hania F.G. [Food Irradiation Research Department, National Center for Radiation Research and Technology, Atomic Energy Authority, P.O. Box 29, Nasr City, Cairo (Egypt)]. E-mail: elniely@hotmail.com

    2007-06-15

    The effects of irradiation (dose levels of 5, 7.5 and 10 kGy) on nutritive characteristics of peas (Pisum satinum L), cowpeas (Vigna unguiculata L.Walp), lentils (Lens culinaris Med), kidneybeans (Phaseolus vulgaris L), and chickpeas (Cicer arietinum L) were examined. Analyses included proximate composition, levels of anti-nutrients (phytic acid, tannins), available lysine (AL), in vitro protein digestibility (IVPD), and protein efficiency ratio (PER) in the growing rat. The results showed that moisture, crude protein, crude fat, crude fiber, and ash were unchanged by the irradiation. Radiation processing significantly (p<0.05) reduced the levels of phytic acid (PA), tannins (TN), and AL. IVPD and PER were significantly enhanced in a dose-dependent manner, relative to unirradiated control samples, for all legumes. The data sets for each legume exhibited high correlation coefficients between radiation dose and PA, TN, AL, IVPD, and PER. These results demonstrate the benefits of irradiation on the nutritional properties of these legumes.

  1. Effect of radiation processing on antinutrients, in-vitro protein digestibility and protein efficiency ratio bioassay of legume seeds

    International Nuclear Information System (INIS)

    El-Niely, Hania F.G.

    2007-01-01

    The effects of irradiation (dose levels of 5, 7.5 and 10 kGy) on nutritive characteristics of peas (Pisum satinum L), cowpeas (Vigna unguiculata L.Walp), lentils (Lens culinaris Med), kidneybeans (Phaseolus vulgaris L), and chickpeas (Cicer arietinum L) were examined. Analyses included proximate composition, levels of anti-nutrients (phytic acid, tannins), available lysine (AL), in vitro protein digestibility (IVPD), and protein efficiency ratio (PER) in the growing rat. The results showed that moisture, crude protein, crude fat, crude fiber, and ash were unchanged by the irradiation. Radiation processing significantly (p<0.05) reduced the levels of phytic acid (PA), tannins (TN), and AL. IVPD and PER were significantly enhanced in a dose-dependent manner, relative to unirradiated control samples, for all legumes. The data sets for each legume exhibited high correlation coefficients between radiation dose and PA, TN, AL, IVPD, and PER. These results demonstrate the benefits of irradiation on the nutritional properties of these legumes

  2. Protein Coexpression Using FMDV 2A: Effect of “Linker” Residues

    Directory of Open Access Journals (Sweden)

    Ekaterina Minskaia

    2013-01-01

    Full Text Available Many biomedical applications absolutely require, or are substantially enhanced by, coexpression of multiple proteins from a single vector. Foot-and-mouth disease virus 2A (F2A and “2A-like” sequences (e.g., Thosea asigna virus 2A; T2A are used widely for this purpose since multiple proteins can be coexpressed by linking open reading frames (ORFs to form a single cistron. The activity of F2A “cleavage” may, however, be compromised by both the use of shorter versions of F2A and the sequences (derived from multiple-purpose cloning sites used to link F2A to the upstream protein. To characterise these effects, different lengths of F2A and T2A were inserted between green and cherry fluorescent proteins. Mutations were introduced in the linker region immediately upstream of both F2A- and T2A-based constructs and activities determined using both cell-free translation systems and transfected cells. In shorter versions of F2A, activity may be affected by both the C-terminal sequence of the protein upstream and, equally strikingly, the residues immediately upstream introduced during cloning. Mutations significantly improved activity for shorter versions of F2A but could decrease activity in the case of T2A. These data will aid the design of cloning strategies for the co-expression of multiple proteins in biomedical/biotechnological applications.

  3. Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

    Science.gov (United States)

    Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

    2014-01-17

    The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

  4. 2-Oxoglutarate levels control adenosine nucleotide binding by Herbaspirillum seropedicae PII proteins.

    Science.gov (United States)

    Oliveira, Marco A S; Gerhardt, Edileusa C M; Huergo, Luciano F; Souza, Emanuel M; Pedrosa, Fábio O; Chubatsu, Leda S

    2015-12-01

    Nitrogen metabolism in Proteobacteria is controlled by the Ntr system, in which PII proteins play a pivotal role, controlling the activity of target proteins in response to the metabolic state of the cell. Characterization of the binding of molecular effectors to these proteins can provide information about their regulation. Here, the binding of ATP, ADP and 2-oxoglutarate (2-OG) to the Herbaspirillum seropedicae PII proteins, GlnB and GlnK, was characterized using isothermal titration calorimetry. Results show that these proteins can bind three molecules of ATP, ADP and 2-OG with homotropic negative cooperativity, and 2-OG binding stabilizes the binding of ATP. Results also show that the affinity of uridylylated forms of GlnB and GlnK for nucleotides is significantly lower than that of the nonuridylylated proteins. Furthermore, fluctuations in the intracellular concentration of 2-OG in response to nitrogen availability are shown. Results suggest that under nitrogen-limiting conditions, PII proteins tend to bind ATP and 2-OG. By contrast, after an ammonium shock, a decrease in the 2-OG concentration is observed causing a decrease in the affinity of PII proteins for ATP. This phenomenon may facilitate the exchange of ATP for ADP on the ligand-binding pocket of PII proteins, thus it is likely that under low ammonium, low 2-OG levels would favor the ADP-bound state. © 2015 FEBS.

  5. Targeted mass spectrometry analysis of the proteins IGF1, IGF2, IBP2, IBP3 and A2GL by blood protein precipitation

    DEFF Research Database (Denmark)

    Such-Sanmartín, Gerard; Bache, Nicolai; Callesen, Anne K

    2015-01-01

    aggravated when using fast high-throughput methods, which are necessary for analysis of hundreds and thousands of samples in clinical laboratories. The blood proteins IGF1, IGF2, IBP2, IBP3 and A2GL have been proposed as indirect biomarkers for detection of GH administration and as putative biomarkers...

  6. Influences of different thermal processings in milk, bovine meat and frog protein structure

    OpenAIRE

    Tatiana Coura Oliveira; Samuel Lopes Lima; Josefina Bressan

    2013-01-01

    Several studies have associated the digestibility of proteins to its imunogenic potential. Though, it was objectified to evaluate the impact of the thermal processing with high and low temperatures on the proteins structure of three types of foods, by means of the digestibility in vitro and electroforesis en gel de poliacrilamida. The pasteurize was observed in such a way, firing 95 ºC during 15 minutes, how much freeze dried causes qualitative and quantitative modifications of constituent pr...

  7. Using Simple Manipulatives to Improve Student Comprehension of a Complex Biological Process: Protein Synthesis

    Science.gov (United States)

    Guzman, Karen; Bartlett, John

    2012-01-01

    Biological systems and living processes involve a complex interplay of biochemicals and macromolecular structures that can be challenging for undergraduate students to comprehend and, thus, misconceptions abound. Protein synthesis, or translation, is an example of a biological process for which students often hold many misconceptions. This article…

  8. Quorum Sensing and the Use of Quorum Quenchers as Natural Biocides to Inhibit Sulfate-Reducing Bacteria

    Directory of Open Access Journals (Sweden)

    Giantommaso Scarascia

    2016-12-01

    Full Text Available Sulfate-reducing bacteria (SRB are one of the main protagonist groups of biocorrosion in the seawater environment. Given their principal role in biocorrosion, it remains a crucial task to develop strategies to reduce the abundance of SRBs. Conventional approaches include the use of biocides and antibiotics, which can impose health, safety, and environmental concerns. This review examines an alternative approach to this problem. This is achieved by reviewing the role of quorum sensing (QS in SRB populations and its impact on the biofilm formation process. Genome databases of SRBs are mined to look for putative QS systems and homologous protein sequences representative of autoinducer receptors or synthases. Subsequently, this review puts forward the potential use of quorum quenchers as natural biocides against SRBs and outlines the potential strategies for the implementation of this approach.

  9. Inhibition of the Secretory pathway by Foot-and-Mouth disease virus 2BC protein is reproduced by co-expression of 2B with 2C, and the site of inhibition is determined by the subcellular location of 2C

    DEFF Research Database (Denmark)

    Moffat, Katy; Knox, Caroline; Howell, Gareth

    2007-01-01

    immune responses in vivo. Foot-and-mouth disease virus (FMDV), another picornavirus, can cause persistent infection of ruminants, suggesting it too may inhibit immune responses. Endoplasmic reticulum (ER)-to-Golgi apparatus transport of proteins is blocked by the FMDV 2BC protein. The observation that 2...... blocked in FMDV-infected cells. The block could be reconstituted by coexpression of 2B and 2C, showing that processing of 2BC did not compromise the ability of FMDV to slow secretion. Under these conditions, 2C was located to the Golgi apparatus, and the block in transport also occurred in the Golgi...... apparatus. Interestingly, the block in transport could be redirected to the ER when 2B was coexpressed with a 2C protein fused to an ER retention element. Thus, for FMDV a block in secretion is dependent on both 2B and 2C, with the latter determining the site of the block....

  10. Rhythmic expression of DEC2 protein in vitro and in vivo.

    Science.gov (United States)

    Sato, Fuyuki; Muragaki, Yasuteru; Kawamoto, Takeshi; Fujimoto, Katsumi; Kato, Yukio; Zhang, Yanping

    2016-06-01

    Basic helix-loop-helix (bHLH) transcription factor DEC2 (bHLHE41/Sharp1) is one of the clock genes that show a circadian rhythm in various tissues. DEC2 regulates differentiation, sleep length, tumor cell invasion and apoptosis. Although studies have been conducted on the rhythmic expression of DEC2 mRNA in various tissues, the precise molecular mechanism of DEC2 expression is poorly understood. In the present study, we examined whether DEC2 protein had a rhythmic expression. Western blot analysis for DEC2 protein revealed a rhythmic expression in mouse liver, lung and muscle and in MCF-7 and U2OS cells. In addition, AMP-activated protein kinase (AMPK) activity (phosphorylation of AMPK) in mouse embryonic fibroblasts (MEFs) exhibited a rhythmic expression under the condition of medium change or glucose-depleted medium. However, the rhythmic expression of DEC2 in MEF gradually decreased in time under these conditions. The medium change affected the levels of DEC2 protein and phosphorylation of AMPK. In addition, the levels of DEC2 protein showed a rhythmic expression in vivo and in MCF-7 and U2OS cells. The results showed that the phosphorylation of AMPK immunoreactivity was strongly detected in the liver and lung of DEC2 knockout mice compared with that of wild-type mice. These results may provide new insights into rhythmic expression and the regulation between DEC2 protein and AMPK activity.

  11. Karakteristik Protein dan Nitrogen Non Protein Daging Ikan Cucut Lanyam (Charcharhinus limbatus (Characteristics of Protein and Non Protein Nitrogen in Lanyam Shark Muscle

    Directory of Open Access Journals (Sweden)

    Yuspihana Fitrial

    2017-02-01

    Based on protein solubility of Lanyam muscle at pH 1.5 to 12 obtained two points which is minimum solubility at pH 4.5 and pH 9. Based on the classification Osborn, Lanyam muscle contained albumin (28.64%, globulin (13:44%, prolamin (03.29%, glutelin (33.70%. Observation of non-protein nitrogen levels indicated that the washing process was very effective to reduce non-protein nitrogen levels up to 62.34% and urea levels up to 58% . Differential Scanning Calorimetry Study of Lanyam mince showed two types of protein that has a different stability to heat and after added 2.5% NaCl formed a peak which is a fusion of both these proteins

  12. Regulation of the interaction between protein kinase C-related protein kinase 2 (PRK2) and its upstream kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1)

    DEFF Research Database (Denmark)

    Dettori, Rosalia; Sonzogni, Silvina; Meyer, Lucas

    2009-01-01

    of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by (32)P(i) revealed phosphorylation at two sites......, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found...... that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do...

  13. G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits

    NARCIS (Netherlands)

    Jimenez-Sainz, MC; Murga, C; Kavelaars, A; Jurado-Pueyo, M; Krakstad, BF; Heijnen, CJ; Mayor, F; Aragay, AM

    The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupled-receptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells

  14. Characterization of the aroma of a meatlike process flavoring from soybean-based enzyme-hydrolyzed vegetable protein.

    Science.gov (United States)

    Wu, Yi-Fang G; Cadwallader, Keith R

    2002-05-08

    Defatted soybean meal was converted into enzyme-hydrolyzed vegetable protein (E-HVP) using the proteolytic enzyme Flavorzyme. Total free amino acids increased by 40-fold after enzyme hydrolysis, with leucine being the most abundant, followed by phenylalanine, lysine, glutamine/glutamic acid, and alanine. Volatile components from a meatlike process flavoring made from E-HVP were isolated by direct solvent extraction (DSE)-high vacuum transfer (HVT), dynamic headspace sampling and static headspace sampling and analyzed by gas chromatography (GC)-mass spectrometry and GC-olfactometry. Aroma extract dilution analysis was used to establish a flavor dilution chromatogram of the DSE-HVT extract. Results of these complementary techniques indicated the importance of odorants of high (hydrogen sulfide and methanethiol), intermediate (2-methyl-3-furanthiol, 3-mercapto-2-pentanone, 2-furanmethanethiol, and 3-(methylthiol)propanal) and low volatility (maltol and Furaneol) in the overall aroma of the meatlike process flavoring.

  15. hPDB – Haskell library for processing atomic biomolecular structures in protein data bank format

    OpenAIRE

    Gajda, Michał Jan

    2013-01-01

    Background Protein DataBank file format is used for the majority of biomolecular data available today. Haskell is a lazy functional language that enjoys a high-level class-based type system, a growing collection of useful libraries and a reputation for efficiency. Findings I present a fast library for processing biomolecular data in the Protein Data Bank format. I present benchmarks indicating that this library is faster than other frequently used Protein Data Bank parsing programs. The propo...

  16. Cloning, expression, purification and characterization of Leishmania tropica PDI-2 protein

    Directory of Open Access Journals (Sweden)

    Ali Dina

    2016-01-01

    Full Text Available In Leishmania species, protein disulfide isomerase (PDI is an essential enzyme that catalyzes thiol-disulfide interchange. The present work describes the isolation, cloning, sequencing and expression of the pdI-2 gene. Initially, the gene was amplified from L. tropica genomic DNA by PCR using specific primers before cloning into the expression vector pET-15b. The construct pET/pdI-2 was transformed into BL21(DE3 cells and induced for the protein expression. SDS-PAGE and western blot analysis showed that the expressed protein is about 51 kDa. Cloned gene sequence analysis revealed that the deduced amino acid sequence showed significant homology with those of several parasites PDIs. Finally, recombinant protein was purified with a metal-chelating affinity column. The putative protein was confirmed as a thiol - disulfide oxidoreductase by detecting its activity in an oxidoreductase assay. Assay result of assay suggested that the PDI-2 protein is required for both oxidation and reduction of disulfide bonds in vitro. Antibodies reactive with this 51 kDa protein were detected by Western blot analysis in sera from human infected with L. tropica. This work describes for the first time the enzymatic activity of recombinant L. tropica PDI-2 protein and suggests a role for this protein as an antigen for the detection of leishmaniasis infection.

  17. Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes.

    Science.gov (United States)

    Aizawa, Shu; Contu, Viorica Raluca; Fujiwara, Yuuki; Hase, Katsunori; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji; Kabuta, Tomohiro

    2017-01-02

    Lysosomes degrade macromolecules such as proteins and nucleic acids. We previously identified 2 novel types of autophagy, RNautophagy and DNautophagy, where lysosomes directly take up RNA and DNA, in an ATP-dependent manner, for degradation. We have also reported that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference defective-1), mediates RNA translocation during RNautophagy. In this addendum, we report that SIDT2 also mediates DNA translocation in the process of DNautophagy. These findings help elucidate the mechanisms underlying the direct uptake of nucleic acids by lysosomes and the physiological functions of DNautophagy.

  18. Human surfactant protein A2 gene mutations impair dimmer/trimer assembly leading to deficiency in protein sialylation and secretion.

    Directory of Open Access Journals (Sweden)

    Yi Song

    Full Text Available Surfactant protein A2 (SP-A2 plays an essential role in surfactant metabolism and lung host defense. SP-A2 mutations in the carbohydrate recognition domain have been related to familial pulmonary fibrosis and can lead to a recombinant protein secretion deficiency in vitro. In this study, we explored the molecular mechanism of protein secretion deficiency and the subsequent biological effects in CHO-K1 cells expressing both wild-type and several different mutant forms of SP-A2. We demonstrate that the SP-A2 G231V and F198S mutants impair the formation of dimmer/trimer SP-A2 which contributes to the protein secretion defect. A deficiency in sialylation, but not N-linked glycosylation, is critical to the observed dimmer/trimer impairment-induced secretion defect. Furthermore, both mutant forms accumulate in the ER and form NP-40-insoluble aggregates. In addition, the soluble mutant SP-A2 could be partially degraded through the proteasome pathway but not the lysosome or autophagy pathway. Intriguingly, 4-phenylbutyrate acid (4-PBA, a chemical chaperone, alleviates aggregate formation and partially rescued the protein secretion of SP-A2 mutants. In conclusion, SP-A2 G231V and F198S mutants impair the dimmer/trimer assembly, which contributes to the protein sialylation and secretion deficiency. The intracellular protein mutants could be partially degraded through the proteasome pathway and also formed aggregates. The treatment of the cells with 4-PBA resulted in reduced aggregation and rescued the secretion of mutant SP-A2.

  19. Construction and analysis of protein-protein interaction networks based on proteomics data of prostate cancer

    Science.gov (United States)

    CHEN, CHEN; SHEN, HONG; ZHANG, LI-GUO; LIU, JIAN; CAO, XIAO-GE; YAO, AN-LIANG; KANG, SHAO-SAN; GAO, WEI-XING; HAN, HUI; CAO, FENG-HONG; LI, ZHI-GUO

    2016-01-01

    Currently, using human prostate cancer (PCa) tissue samples to conduct proteomics research has generated a large amount of data; however, only a very small amount has been thoroughly investigated. In this study, we manually carried out the mining of the full text of proteomics literature that involved comparisons between PCa and normal or benign tissue and identified 41 differentially expressed proteins verified or reported more than 2 times from different research studies. We regarded these proteins as seed proteins to construct a protein-protein interaction (PPI) network. The extended network included one giant network, which consisted of 1,264 nodes connected via 1,744 edges, and 3 small separate components. The backbone network was then constructed, which was derived from key nodes and the subnetwork consisting of the shortest path between seed proteins. Topological analyses of these networks were conducted to identify proteins essential for the genesis of PCa. Solute carrier family 2 (facilitated glucose transporter), member 4 (SLC2A4) had the highest closeness centrality located in the center of each network, and the highest betweenness centrality and largest degree in the backbone network. Tubulin, beta 2C (TUBB2C) had the largest degree in the giant network and subnetwork. In addition, using module analysis of the whole PPI network, we obtained a densely connected region. Functional annotation indicated that the Ras protein signal transduction biological process, mitogen-activated protein kinase (MAPK), neurotrophin and the gonadotropin-releasing hormone (GnRH) signaling pathway may play an important role in the genesis and development of PCa. Further investigation of the SLC2A4, TUBB2C proteins, and these biological processes and pathways may therefore provide a potential target for the diagnosis and treatment of PCa. PMID:27121963

  20. Effect of food processing organic matter on photocatalytic bactericidal activity of titanium dioxide (TiO2).

    Science.gov (United States)

    Yemmireddy, Veerachandra K; Hung, Yen-Con

    2015-07-02

    The purpose of this study was to determine the effect of food processing organic matter on photocatalytic bactericidal activity of titanium dioxide (TiO2) nanoparticles (NPs). Produce and meat processing wash solutions were prepared using romaine lettuce and ground beef samples. Physico-chemical properties such as pH, turbidity, chemical oxygen demand (COD), total phenolics (for produce) and protein (for meat) content of the extracts were determined using standard procedures. The photocatalytic bactericidal activity of TiO2 (1 mg/mL) in suspension with or without organic matter against Escherichia coli O157:H7 (5-strain) was determined over a period of 3h. Increasing the concentration of organic matter (either produce or meat) from 0% to 100% resulted in 85% decrease in TiO2 microbicidal efficacy. 'Turbidity, total phenolics, and protein contents in wash solutions had significant effect on the log reduction. Increasing the total phenolics content in produce washes from 20 to 114 mg/L decreased the log reduction from 2.7 to 0.38 CFU/mL, whereas increasing the protein content in meat washes from 0.12 to 1.61 mg/L decreased the log reduction from and 5.74 to 0.87 CFU/mL. Also, a linear correlation was observed between COD and total phenolics as well as COD and protein contents. While classical disinfection kinetic models failed to predict, an empirical equation in the form of "Y=me(nX)" (where Y is log reduction, X is COD, and m and n are reaction rate constants) predicted the disinfection kinetics of TiO2 in the presence of organic matter (R(2)=94.4). This study successfully identified an empirical model with COD as a predictor variable to predict the bactericidal efficacy of TiO2 when used in food processing environment. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Dissociation of SERPINE1 mRNA from the translational repressor proteins Ago2 and TIA-1 upon platelet activation.

    Science.gov (United States)

    Corduan, Aurélie; Plé, Hélène; Laffont, Benoit; Wallon, Thérèse; Plante, Isabelle; Landry, Patricia; Provost, Patrick

    2015-05-01

    Platelets play an important role in haemostasis, as well as in thrombosis and coagulation processes. They harbour a wide variety of messenger RNAs (mRNAs), that can template de novo protein synthesis, and an abundant array of microRNAs, which are known to mediate mRNA translational repression through proteins of the Argonaute (Ago) family. The relationship between platelet microRNAs and proteins capable of mediating translational repression, however, remains unclear. Here, we report that half of platelet microRNAs is associated to mRNA-regulatory Ago2 protein complexes, in various proportions. Associated to these Ago2 complexes are platelet mRNAs known to support de novo protein synthesis. Reporter gene activity assays confirmed the capacity of the platelet microRNAs, found to be associated to Ago2 complexes, to regulate translation of these platelet mRNAs through their 3'UTR. Neither the microRNA repertoire nor the microRNA composition of Ago2 complexes of human platelets changed upon activation with thrombin. However, under conditions favoring de novo synthesis of Plasminogen Activator Inhibitor-1 (PAI-1) protein, we documented a rapid dissociation of the encoding platelet SERPINE1 mRNA from Ago2 protein complexes as well as from the translational repressor protein T-cell-restricted intracellular antigen-1 (TIA-1). These findings are consistent with a scenario by which lifting of the repressive effects of Ago2 and TIA-1 protein complexes, involving a rearrangement of proteinmRNA complexes rather than disassembly of Ago2microRNA complexes, would allow translation of SERPINE1 mRNA into PAI-1 in response to platelet activation.

  2. The carboxy terminus of p53 mimics the polylysine effect of protein kinase CK2-catalyzed MDM2 phosphorylation

    DEFF Research Database (Denmark)

    Guerra, B; Götz, C; Wagner, P

    1997-01-01

    The oncogene product MDM2 can be phosphorylated by protein kinase CK2 in vitro 0.5-1 mol of phosphate were incorporated per mol MDM2 protein. The catalytic subunit of protein kinase CK2 (alpha-subunit) catalyzed the incorporation of twice as much phosphate into the MDM2 protein as it was obtained...

  3. PDB2CD visualises dynamics within protein structures.

    Science.gov (United States)

    Janes, Robert W

    2017-10-01

    Proteins tend to have defined conformations, a key factor in enabling their function. Atomic resolution structures of proteins are predominantly obtained by either solution nuclear magnetic resonance (NMR) or crystal structure methods. However, when considering a protein whose structure has been determined by both these approaches, on many occasions, the resultant conformations are subtly different, as illustrated by the examples in this study. The solution NMR approach invariably results in a cluster of structures whose conformations satisfy the distance boundaries imposed by the data collected; it might be argued that this is evidence of the dynamics of proteins when in solution. In crystal structures, the proteins are often in an energy minimum state which can result in an increase in the extent of regular secondary structure present relative to the solution state depicted by NMR, because the more dynamic ends of alpha helices and beta strands can become ordered at the lower temperatures. This study examines a novel way to display the differences in conformations within an NMR ensemble and between these and a crystal structure of a protein. Circular dichroism (CD) spectroscopy can be used to characterise protein structures in solution. Using the new bioinformatics tool, PDB2CD, which generates CD spectra from atomic resolution protein structures, the differences between, and possible dynamic range of, conformations adopted by a protein can be visualised.

  4. The roles of USH1 proteins and PDZ domain-containing USH proteins in USH2 complex integrity in cochlear hair cells.

    Science.gov (United States)

    Zou, Junhuang; Chen, Qian; Almishaal, Ali; Mathur, Pranav Dinesh; Zheng, Tihua; Tian, Cong; Zheng, Qing Y; Yang, Jun

    2017-02-01

    Usher syndrome (USH) is the most common cause of inherited deaf-blindness, manifested as USH1, USH2 and USH3 clinical types. The protein products of USH2 causative and modifier genes, USH2A, ADGRV1, WHRN and PDZD7, interact to assemble a multiprotein complex at the ankle link region of the mechanosensitive stereociliary bundle in hair cells. Defects in this complex cause stereociliary bundle disorganization and hearing loss. The four USH2 proteins also interact in vitro with USH1 proteins including myosin VIIa, USH1G (SANS), CIB2 and harmonin. However, it is unclear whether the interactions between USH1 and USH2 proteins occur in vivo and whether USH1 proteins play a role in USH2 complex assembly in hair cells. In this study, we identified a novel interaction between myosin VIIa and PDZD7 by FLAG pull-down assay. We further investigated the role of the above-mentioned four USH1 proteins in the cochlear USH2 complex assembly using USH1 mutant mice. We showed that only myosin VIIa is indispensable for USH2 complex assembly at ankle links, indicating the potential transport and/or anchoring role of myosin VIIa for USH2 proteins in hair cells. However, myosin VIIa is not required for USH2 complex assembly in photoreceptors. We further showed that, while PDZ protein harmonin is not involved, its paralogous USH2 proteins, PDZD7 and whirlin, function synergistically in USH2 complex assembly in cochlear hair cells. In summary, our studies provide novel insight into the functional relationship between USH1 and USH2 proteins in the cochlea and the retina as well as the disease mechanisms underlying USH1 and USH2. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  5. Targeting the middle region of CP4-EPSPS protein for its traceability in highly processed soy-related products.

    Science.gov (United States)

    Wu, Honghong; Wang, Xiaofu; Zhou, Xinghu; Zhang, Yihua; Huang, Ming; He, Jian; Shen, Wenbiao

    2017-09-01

    Transgenic components in genetically modified organisms consist not only of the transgenic genes, but also the transgenic protein. However, compared with transgenic DNA, less attention has been paid to the detection of expressed protein, especially those degraded from genetically modified soybean after food processing. In this study, the full length 5-enolpyruvyl-shikimate-3-phosphate synthase (CP4-EPSPS, 47.6 kD) protein was probed with the SC-16 (S19-R33) and the DC-16 (D219-K233) polyclonal antibodies in immunoblots. Both antibodies were able to detect the full length CP4-EPSPS and its residues in soy powder made from Roundup-Ready soybeans after heating and microwaving treatments which also reduced the molecular weight of the protein to 45.8 and 38.7 kD, respectively. Taken together the immunoblot results suggest that the middle region of the CP4-EPSPS protein possessed better stability than its N-terminal during thermal processing. This deduction was further validated by autoclave treatment, where a 37.4 kD residue of the protein was recognized by DC-16. A similar result was obtained in processed smoked sausage containing Roundup Ready soybean protein isolate (as an extender). The additional use of a further polyclonal antibody CK-17 (C372-K388), showed that compared with only the one signal for CP4-EPSPS detected by the SC-16 and CK-17 antibodies, the DC-16 middle region antibody detected four signals for CP4-EPSPS from five market sourced soy protein concentrates. Taken together, the study suggested that the middle region of CP4-EPSPS was more useful than the N- and C-terminal for tracing transgenic CP4-EPSPS protein and its remnants in highly processed soy-related products.

  6. Functional characterization of JMJD2A, a histone deacetylase- and retinoblastoma-binding protein.

    Science.gov (United States)

    Gray, Steven G; Iglesias, Antonio H; Lizcano, Fernando; Villanueva, Raul; Camelo, Sandra; Jingu, Hisaka; Teh, Bin T; Koibuchi, Noriyuki; Chin, William W; Kokkotou, Efi; Dangond, Fernando

    2005-08-05

    To effectively direct targeted repression, the class I histone deacetylases (HDACs) associate with many important regulatory proteins. In this paper we describe the molecular characterization of a member of the Jumonji domain 2 (JMJD2) family of proteins, and demonstrate its binding to both class I HDACs and the retinoblastoma protein (pRb). JMJD2 proteins are characterized by the presence of two leukemia-associated protein/plant homeodomain (LAP/PHD) zinc fingers, one JmjN, one JmjC (containing an internal retinoblastoma-binding protein 2 (RBBP2)-like sequence), and two Tudor domains. The first member of this group, JMJD2A, is widely expressed in human tissues and cell lines, and high endogenous expression of JMJD2A mRNA was found in several cell types, including human T-cell lymphotropic virus 1 (HTLV-1)-infected cell lines. JMJD2A and JMJD2B exhibit cell type-specific responses to the HDAC inhibitor trichostatin A. We show that the JMJD2A protein associates in vivo with pRb and class I HDACs, and mediates repression of E2F-regulated promoters. In HTLV-1 virus-infected cells, we find that JMJD2A binds to the viral Tax protein. Antibodies to JMJD2A recognize the native protein but also a half-sized protein fragment, the latter up-regulated in THP-1 cells during the G(2)/M phase of the cell cycle. The ability of JMJD2A to associate with pRb and HDACs and potentiate pRb-mediated repression of E2F-regulated promoters implies an important role for this protein in cell proliferation and oncogenesis.

  7. Direct interaction between two viral proteins, the nonstructural protein 2C and the capsid protein VP3, is required for enterovirus morphogenesis.

    Directory of Open Access Journals (Sweden)

    Ying Liu

    2010-08-01

    Full Text Available In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV, is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2C(ATPase in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel "reporter virus", we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20 and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2C(ATPase of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2C(ATPase and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20 were blocked in encapsidation (no virus after blind passages but could be rescued if the capsid and 2C(ATPase coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i genome replication is known to be stringently linked to translation, (ii morphogenesis is known to be stringently linked to genome replication, (iii newly synthesized 2C(ATPase is an essential component of the replication complex, and (iv 2C(ATPase has specific affinity to capsid protein(s. These conditions lead to morphogenesis at the site where newly

  8. Effect of dietary protein on the excretion of. cap alpha. /sub 2u/, the sex-dependent protein of the adult male rat

    Energy Technology Data Exchange (ETDEWEB)

    Neuhaus, O W; Flory, W

    1975-01-01

    Adult male rates were maintained on normal (20 percent casein), protein-free (0 percent casein), high protein (50 percent casein), deficient protein (20 percent zein), and a supplemented, deficient protein (20 percent zein plus L-lysine and L-tryptophan) diets. Rats on a protein-free diet excreted approximately 1 mg ..cap alpha../sub 2u//24 h compared with a normal of 10-15 mg/24 h. Depleted rats placed on the normal diet showed a rapid restoration of the normal ..cap alpha../sub 2u/ excretion as well as total urinary proteins. Accumulation of ..cap alpha../sub 2u/ in the blood serum was measured in nephrectomized rats. Rats on the protein free diet accumulated only 30 percent of the ..cap alpha../sub 2u/ compared to normals. On a 50 precent casein diet, rats excreted 30-50 mg ..cap alpha../sub 2u//24 h. However, the accumulation was normal in the serum of nephrectomized rats. A high protein diet did not stimulate ..cap alpha../sub 2u/ synthesis but probably increased the renal loss of all urinary proteins. The excretion of ..cap alpha../sub 2u/ on a zein diet was reduced to the same degree as with the protein-free diet. Supplementation with lysine and tryptophan restored the capacity to eliminate ..cap alpha../sub 2u/ to near normal levels. Accumulation of ..cap alpha../sub 2u/ in the serum of nephrectomized rats kept on the zein diets showed that the effect was to suppress the synthesis of the ..cap alpha../sub 2u/. Supplementation restored the biosynthesis of ..cap alpha../sub 2u/. It is concluded that the effect of dietary protein on the excretion of urinary proteins in the adult male rat is caused in a large part by an influence on the hepatic biosynthesis of ..cap alpha../sub 2u/. The biosynthesis of this protein, which represents approximately 30 percent of the total urinary proteins, is dependent on an adequate supply of dietary protein.

  9. Integrated continuous processing of proteins expressed as inclusion bodies: GCSF as a case study.

    Science.gov (United States)

    Kateja, Nikhil; Agarwal, Harshit; Hebbi, Vishwanath; Rathore, Anurag S

    2017-07-01

    Affordability of biopharmaceuticals continues to be a challenge, particularly in developing economies. This has fuelled advancements in manufacturing that can offer higher productivity and better economics without sacrificing product quality in the form of an integrated continuous manufacturing platform. While platform processes for monoclonal antibodies have existed for more than a decade, development of an integrated continuous manufacturing process for bacterial proteins has received relatively scant attention. In this study, we propose an end-to-end integrated continuous downstream process (from inclusion bodies to unformulated drug substance) for a therapeutic protein expressed in Escherichia coli as inclusion body. The final process consisted of a continuous refolding in a coiled flow inverter reactor directly coupled to a three-column periodic counter-current chromatography for capture of the product followed by a three-column con-current chromatography for polishing. The continuous bioprocessing train was run uninterrupted for 26 h to demonstrate its capability and the resulting output was analyzed for the various critical quality attributes, namely product purity (>99%), high molecular weight impurities (<0.5%), host cell proteins (<100 ppm), and host cell DNA (<10 ppb). All attributes were found to be consistent over the period of operation. The developed assembly offers smaller facility footprint, higher productivity, fewer hold steps, and significantly higher equipment and resin utilization. The complexities of process integration in the context of continuous processing have been highlighted. We hope that the study presented here will promote development of highly efficient, universal, end-to-end, fully continuous platforms for manufacturing of biotherapeutics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:998-1009, 2017. © 2016 American Institute of Chemical Engineers.

  10. The Tp0684 (MglB-2 Lipoprotein of Treponema pallidum: A Glucose-Binding Protein with Divergent Topology.

    Directory of Open Access Journals (Sweden)

    Chad A Brautigam

    Full Text Available Treponema pallidum, the bacterium that causes syphilis, is an obligate human parasite. As such, it must acquire energy, in the form of carbon sources, from the host. There is ample evidence that the principal source of energy for this spirochete is D-glucose acquired from its environment, likely via an ABC transporter. Further, there is genetic evidence of a D-glucose chemotaxis system in T. pallidum. Both of these processes may be dependent on a single lipidated chemoreceptor: Tp0684, also called TpMglB-2 for its sequence homology to MglB of Escherichia coli. To broaden our understanding of this potentially vital protein, we determined a 2.05-Å X-ray crystal structure of a soluble form of the recombinant protein. Like its namesake, TpMglB-2 adopts a bilobed fold that is similar to that of the ligand-binding proteins (LBPs of other ABC transporters. However, the protein has an unusual, circularly permuted topology. This feature prompted a series of biophysical studies that examined whether the protein's topological distinctiveness affected its putative chemoreceptor functions. Differential scanning fluorimetry and isothermal titration calorimetry were used to confirm that the protein bound D-glucose in a cleft between its two lobes. Additionally, analytical ultracentrifugation was employed to reveal that D-glucose binding is accompanied by a significant conformational change. TpMglB-2 thus appears to be fully functional in vitro, and given the probable central importance of the protein to T. pallidum's physiology, our results have implications for the viability and pathogenicity of this obligate human pathogen.

  11. The Tp0684 (MglB-2) Lipoprotein of Treponema pallidum: A Glucose-Binding Protein with Divergent Topology.

    Science.gov (United States)

    Brautigam, Chad A; Deka, Ranjit K; Liu, Wei Z; Norgard, Michael V

    2016-01-01

    Treponema pallidum, the bacterium that causes syphilis, is an obligate human parasite. As such, it must acquire energy, in the form of carbon sources, from the host. There is ample evidence that the principal source of energy for this spirochete is D-glucose acquired from its environment, likely via an ABC transporter. Further, there is genetic evidence of a D-glucose chemotaxis system in T. pallidum. Both of these processes may be dependent on a single lipidated chemoreceptor: Tp0684, also called TpMglB-2 for its sequence homology to MglB of Escherichia coli. To broaden our understanding of this potentially vital protein, we determined a 2.05-Å X-ray crystal structure of a soluble form of the recombinant protein. Like its namesake, TpMglB-2 adopts a bilobed fold that is similar to that of the ligand-binding proteins (LBPs) of other ABC transporters. However, the protein has an unusual, circularly permuted topology. This feature prompted a series of biophysical studies that examined whether the protein's topological distinctiveness affected its putative chemoreceptor functions. Differential scanning fluorimetry and isothermal titration calorimetry were used to confirm that the protein bound D-glucose in a cleft between its two lobes. Additionally, analytical ultracentrifugation was employed to reveal that D-glucose binding is accompanied by a significant conformational change. TpMglB-2 thus appears to be fully functional in vitro, and given the probable central importance of the protein to T. pallidum's physiology, our results have implications for the viability and pathogenicity of this obligate human pathogen.

  12. An effective placental cotyledons proteins extraction method for 2D gel electrophoresis.

    Science.gov (United States)

    Tan, Niu J; Daim, Leona D J; Jamil, Amilia A M; Mohtarrudin, Norhafizah; Thilakavathy, Karuppiah

    2017-03-01

    Effective protein extraction is essential especially in producing a well-resolved proteome on 2D gels. A well-resolved placental cotyledon proteome, with good reproducibility, have allowed researchers to study the proteins underlying the physiology and pathophysiology of pregnancy. The aim of this study is to determine the best protein extraction protocol for the extraction of protein from placental cotyledons tissues for a two-dimensional gel electrophoresis (2D-GE). Based on widely used protein extraction strategies, 12 different extraction methodologies were carefully selected, which included one chemical extraction, two mechanical extraction coupled protein precipitations, and nine chemical extraction coupled protein precipitations. Extracted proteins were resolved in a one-dimensional gel electrophoresis and 2D-GE; then, it was compared with set criteria: extraction efficacy, protein resolution, reproducibility, and recovery efficiency. Our results revealed that a better profile was obtained by chemical extraction in comparison to mechanical extraction. We further compared chemical extraction coupled protein precipitation methodologies, where the DNase/lithium chloride-dense sucrose homogenization coupled dichloromethane-methanol precipitation (DNase/LiCl-DSH-D/MPE) method showed good protein extraction efficiency. This, however, was carried out with the best protein resolution and proteome reproducibility on 2D-gels. DNase/LiCl-DSH-D/MPE was efficient in the extraction of proteins from placental cotyledons tissues. In addition, this methodology could hypothetically allow the protein extraction of any tissue that contains highly abundant lipid and glycogen. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Identification and function of leucine-rich repeat flightless-I-interacting protein 2 (LRRFIP2 in Litopenaeus vannamei.

    Directory of Open Access Journals (Sweden)

    Shuang Zhang

    Full Text Available Leucine-rich repeat flightless-I-interacting protein 2 (LRRFIP2 is a myeloid differentiation factor 88-interacting protein with a positive regulatory function in toll-like receptor signaling. In this study, seven LRRFIP2 protein variants (LvLRRFIP2A-G were identified in Litopenaeus vannamei. All the seven LvLRRFIP2 protein variants encode proteins with a DUF2051 domain. LvLRRFIP2s were upregulated in hemocytes after challenged with lipopolysaccharide, poly I:C, CpG-ODN2006, Vibrio parahaemolyticus, Staphylococcus aureus, and white spot syndrome virus (WSSV. Dual-luciferase reporter assays in Drosophila Schneider 2 cells revealed that LvLRRFIP2 activates the promoters of Drosophila and shrimp AMP genes. The knockdown of LvLRRFIP2 by RNA interference resulted in higher cumulative mortality of L. vannamei upon V. parahaemolyticus but not S. aureus and WSSV infections. The expression of L. vannamei AMP genes were reduced by dsLvLRRFIP2 interference. These results indicate that LvLRRFIP2 has an important function in antibacterials via the regulation of AMP gene expression.

  14. SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1.

    Science.gov (United States)

    Anafi, M; Kiefer, F; Gish, G D; Mbamalu, G; Iscove, N N; Pawson, T

    1997-10-31

    Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases.

  15. The mitochondrial SIR2 related protein 2 (SIR2RP2 impacts Leishmania donovani growth and infectivity.

    Directory of Open Access Journals (Sweden)

    Nimisha Mittal

    2017-05-01

    Full Text Available Leishmania donovani, a protozoan parasite is the major causative agent of visceral leishmaniasis. Increased toxicity and resistance to the existing repertoire of drugs has been reported. Hence, an urgent need exists for identifying newer drugs and drug targets. Previous reports have shown sirtuins (Silent Information Regulator from kinetoplastids as promising drug targets. Leishmania species code for three SIR2 (Silent Information Regulator related proteins. Here, we for the first time report the functional characterization of SIR2 related protein 2 (SIR2RP2 of L. donovani.Recombinant L. donovani SIR2RP2 was expressed in E. coli and purified. The enzymatic functions of SIR2RP2 were determined. The subcellular localization of LdSIR2RP2 was done by constructing C-terminal GFP-tagged full-length LdSIR2RP2. Deletion mutants of LdSIR2RP2 were generated in Leishmania by double targeted gene replacement methodology. These null mutants were tested for their proliferation, virulence, cell cycle defects, mitochondrial functioning and sensitivity to known SIR2 inhibitors.Our data suggests that LdSIR2RP2 possesses NAD+-dependent ADP-ribosyltransferase activity. However, NAD+-dependent deacetylase and desuccinylase activities were not detected. The protein localises to the mitochondrion of the promastigotes. Gene deletion studies showed that ΔLdSIR2RP2 null mutants had restrictive growth phenotype associated with accumulation of cells in the G2/M phase and compromised mitochondrial functioning. The null mutants had attenuated infectivity. Deletion of LdSIR2RP2 resulted in increased sensitivity of the parasites to the known SIR2 inhibitors. The sirtuin inhibitors inhibited the ADP-ribosyltransferase activity of recombinant LdSIR2RP2. In conclusion, sirtuins could be used as potential new drug targets for visceral leishmaniasis.

  16. Xenopus LAP2β protein knockdown affects location of lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability.

    Science.gov (United States)

    Dubińska-Magiera, Magda; Chmielewska, Magdalena; Kozioł, Katarzyna; Machowska, Magdalena; Hutchison, Christopher J; Goldberg, Martin W; Rzepecki, Ryszard

    2016-05-01

    Xenopus LAP2β protein is the single isoform expressed in XTC cells. The protein localizes on heterochromatin clusters both at the nuclear envelope and inside a cell nucleus. The majority of XLAP2β fraction neither colocalizes with TPX2 protein during interphase nor can be immunoprecipitated with XLAP2β antibody. Knockdown of the XLAP2β protein expression in XTC cells by synthetic siRNA and plasmid encoded siRNA resulted in nuclear abnormalities including changes in shape of nuclei, abnormal chromatin structure, loss of nuclear envelope, mislocalization of integral membrane proteins of INM such as lamin B2, mislocalization of nucleoporins, and cell death. Based on timing of cell death, we suggest mechanism associated with nucleus reassembly or with entry into mitosis. This confirms that Xenopus LAP2 protein is essential for the maintenance of cell nucleus integrity and the process of its reassembly after mitosis.

  17. 2A and the auxin-based degron system facilitate control of protein levels in Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Andrea Kreidenweiss

    Full Text Available Analysis of gene function in Plasmodium falciparum, the most important human malaria parasite, is restricted by the lack of robust and simple reverse genetic tools. Approaches to manipulate protein levels post-translationally are powerful tools to study protein-off effects especially in the haploid malaria parasite where genetic knockouts of essential genes are lethal. We investigated if the auxin-inducible degron system is functional in P. falciparum and found that degron-tagged yellow fluorescent protein levels were efficiently reduced upon addition of auxin which otherwise had no effect on parasite viability. The genetic components required in this conditional approach were co-expressed in P. falciparum by applying the small peptide 2A. 2A is a self-processing peptide from Foot-And-Mouth Disease virus that allows the whole conditional system to be accommodated on a single plasmid vector and ensures stoichiometric expression levels.

  18. ProteinShop: A tool for interactive protein manipulation and steering

    Energy Technology Data Exchange (ETDEWEB)

    Crivelli, Silvia; Kreylos, Oliver; Max, Nelson; Hamann, Bernd; Bethel, Wes

    2004-05-25

    We describe ProteinShop, a new visualization tool that streamlines and simplifies the process of determining optimal protein folds. ProteinShop may be used at different stages of a protein structure prediction process. First, it can create protein configurations containing secondary structures specified by the user. Second, it can interactively manipulate protein fragments to achieve desired folds by adjusting the dihedral angles of selected coil regions using an Inverse Kinematics method. Last, it serves as a visual framework to monitor and steer a protein structure prediction process that may be running on a remote machine. ProteinShop was used to create initial configurations for a protein structure prediction method developed by a team that competed in CASP5. ProteinShop's use accelerated the process of generating initial configurations, reducing the time required from days to hours. This paper describes the structure of ProteinShop and discusses its main features.

  19. The small serine-threonine protein SIP2 interacts with STE12 and is involved in ascospore germination in Sordaria macrospora.

    Science.gov (United States)

    Elleuche, Skander; Bernhards, Yasmine; Schäfers, Christian; Varghese, Jans Manjali; Nolting, Nicole; Pöggeler, Stefanie

    2010-12-01

    In fungi, the homoeodomain protein STE12 controls diverse developmental processes, and derives its regulatory specificity from different protein interactions. We recently showed that in the homothallic ascomycete Sordaria macrospora, STE12 is essential for ascospore development, and is able to interact with the alpha-domain mating-type protein SMTA-1 and the MADS box protein MCM1. To further evaluate the functional roles of STE12, we used the yeast two-hybrid approach to identify new STE12-interacting partners. Using STE12 as bait, a small, serine-threonine-rich protein (designated STE12-interacting protein 2, SIP2) was identified. SIP2 is conserved among members of the fungal class Sordariomycetes. In vivo localization studies revealed that SIP2 was targeted to the nucleus and cytoplasm. The STE12/SIP2 interaction was further confirmed in vivo by bimolecular fluorescence complementation. Nuclear localization of SIP2 was apparently mediated by STE12. Unlike deletion of ste12, deletion of sip2 in S. macrospora led to only a slight decrease in ascospore germination, and no other obvious morphological phenotype. In comparison to the Δste12 single knockout strain, ascospore germination was significantly increased in a Δsip2/ste12 double knockout strain. Our data provide evidence for a regulatory role of the novel fungal protein SIP2 in ascospore germination. Copyright © 2010 Elsevier GmbH. All rights reserved.

  20. LC-MS/MS Identification of Species-Specific Muscle Peptides in Processed Animal Proteins.

    Science.gov (United States)

    Marchis, Daniela; Altomare, Alessandra; Gili, Marilena; Ostorero, Federica; Khadjavi, Amina; Corona, Cristiano; Ru, Giuseppe; Cappelletti, Benedetta; Gianelli, Silvia; Amadeo, Francesca; Rumio, Cristiano; Carini, Marina; Aldini, Giancarlo; Casalone, Cristina

    2017-12-06

    An innovative analytical strategy has been applied to identify signature peptides able to distinguish among processed animal proteins (PAPs) derived from bovine, pig, fish, and milk products. Proteomics was first used to elucidate the proteome of each source. Starting from the identified proteins and using a funnel based approach, a set of abundant and well characterized peptides with suitable physical-chemical properties (signature peptides) and specific for each source was selected. An on-target LC-ESI-MS/MS method (MRM mode) was set up using standard peptides and was then applied to selectively identify the PAP source and also to distinguish proteins from bovine carcass and milk proteins. We believe that the method described meets the request of the European Commission which has developed a strategy for gradually lifting the "total ban" toward "species to species ban", therefore requiring official methods for species-specific discrimination in feed.

  1. Amino Acid and Mineral Supplementation in Fermentation Process of Concentrate Protein of Jatropha Seed Cake (Jatropha curcas L.

    Directory of Open Access Journals (Sweden)

    Titin Widiyastuti

    2016-09-01

    Full Text Available The purpose of this study is to assess the optimization of fermentation process by adding a minerals and amino acids so that the potential of protein  of Concentrate Protein-Jatropha seed cake (CP-JSC can be optimally used as a substitute for soybean meal. The method used was completely randomized design. The treatment consisted of F1: Fermentation CP-BBJ + methionine-lysine (0.25%: 0.25%, F2: Fermentation CP-JSC + methionine-lysine (0.5%: 0.5%, F3: F1 + 0.45% Dicalsium Phosphate, F4: F2 + 0.45% Dicalsium Phosphate. Each treatment was repeated four times, When treatment significantly continued by Least Significant Difference (LSD, variables observed are the levels of antinutrients (phorbolester, antitrypsin, the levels of nutrients (fat, protein, crude fiber, Ca, P and gross energy and amino acid. Results of analysis of variance showed that the addition of amino acids and minerals Ca, P in the fermentation process was highly significant effect on the levels of crude fiber and phosphorus (P 0.05. While the levels obtained phorbolester range of 0.055% - 0.08%. It was concluded that the optimization of fermentation can be done without adding the amino acid supplementation of minerals calcium and phosphorus. Supplementation significantly affect a significant increase or decrease in some nutrients (crude fiber, gross energy, phosphor and capable of suppressing a decrease in amino acids. Supplementation of amino acids Lysine and Methionin 0.05% is the best treatment.

  2. Efficient biological process characterization by definitive-screening designs: the formaldehyde treatment of a therapeutic protein as a case study.

    Science.gov (United States)

    Erler, Axel; de Mas, Nuria; Ramsey, Philip; Henderson, Grant

    2013-03-01

    As part of the process-characterization campaign of a candidate vaccine product, a recently developed class of three-level designs-definitive-screening designs-was employed to select a quadratic model that describes the effect of six input process parameters, including protein concentration, formaldehyde-to-protein ratio, lysine concentration, reaction duration, pH, and reaction temperature, on a formylation protein-crosslinking reaction. This design requires only 17 experimental runs. The resulting model was then used to simulate 10,000 runs that account for the variability in the inputs expected on manufacturing scale. The extent of protein polymerization was predicted to be within specifications for all simulated runs, demonstrating the robustness of the unit operation for subsequent process validation and future commercial manufacturing.

  3. Protein surface shielding agents in protein crystallization

    International Nuclear Information System (INIS)

    Hašek, J.

    2011-01-01

    The crystallization process can be controlled by protein surface shielding agents blocking undesirable competitive adhesion modes during non-equilibrium processes of deposition of protein molecules on the surface of growing crystalline blocks. The hypothesis is based on a number of experimental proofs from diffraction experiments and also retrieved from the Protein Data Bank. The molecules adhering temporarily on the surface of protein molecules change the propensity of protein molecules to deposit on the crystal surface in a definite position and orientation. The concepts of competitive adhesion modes and protein surface shielding agents acting on the surface of molecules in a non-equilibrium process of protein crystallization provide a useful platform for the control of crystallization. The desirable goal, i.e. a transient preference of a single dominating adhesion mode between protein molecules during crystallization, leads to uniform deposition of proteins in a crystal. This condition is the most important factor for diffraction quality and thus also for the accuracy of protein structure determination. The presented hypothesis is a generalization of the experimentally well proven behaviour of hydrophilic polymers on the surface of protein molecules of other compounds

  4. Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O2.

    Science.gov (United States)

    Ibrahim, Susan A; Crack, Jason C; Rolfe, Matthew D; Borrero-de Acuña, José Manuel; Thomson, Andrew J; Le Brun, Nick E; Schobert, Max; Stapleton, Melanie R; Green, Jeffrey

    2015-07-03

    The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Genome-wide mRNA processing in methanogenic archaea reveals post-transcriptional regulation of ribosomal protein synthesis.

    Science.gov (United States)

    Qi, Lei; Yue, Lei; Feng, Deqin; Qi, Fengxia; Li, Jie; Dong, Xiuzhu

    2017-07-07

    Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an 'all-or-none' pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. The Phyre2 web portal for protein modeling, prediction and analysis.

    Science.gov (United States)

    Kelley, Lawrence A; Mezulis, Stefans; Yates, Christopher M; Wass, Mark N; Sternberg, Michael J E

    2015-06-01

    Phyre2 is a suite of tools available on the web to predict and analyze protein structure, function and mutations. The focus of Phyre2 is to provide biologists with a simple and intuitive interface to state-of-the-art protein bioinformatics tools. Phyre2 replaces Phyre, the original version of the server for which we previously published a paper in Nature Protocols. In this updated protocol, we describe Phyre2, which uses advanced remote homology detection methods to build 3D models, predict ligand binding sites and analyze the effect of amino acid variants (e.g., nonsynonymous SNPs (nsSNPs)) for a user's protein sequence. Users are guided through results by a simple interface at a level of detail they determine. This protocol will guide users from submitting a protein sequence to interpreting the secondary and tertiary structure of their models, their domain composition and model quality. A range of additional available tools is described to find a protein structure in a genome, to submit large number of sequences at once and to automatically run weekly searches for proteins that are difficult to model. The server is available at http://www.sbg.bio.ic.ac.uk/phyre2. A typical structure prediction will be returned between 30 min and 2 h after submission.

  7. Role of nonstructural protein NS2A in flavivirus assembly

    NARCIS (Netherlands)

    Leung, J.Y.; Pijlman, G.P.; Kondratieva, N.; Hyde, J.; Mackenzie, J.M.; Khromykh, A.A.

    2008-01-01

    Flavivirus nonstructural (NS) proteins are involved in RNA replication and modulation of the host antiviral response; however, evidence is mounting that some NS proteins also have essential roles in virus assembly. Kunjin virus (KUN) NS2A is a small, hydrophobic, transmembrane protein that is part

  8. Asymmetric expression of protein kinase CK2 subunits in human kidney tumors

    DEFF Research Database (Denmark)

    Stalter, G; Siemer, S; Becht, E

    1994-01-01

    of protein kinase CK2 alpha in tumors/normal tissue (T/N) was 1.58 and that of the protein kinase CK2 beta (T/N) was 2.65. The data suggest that the generally described increase in protein kinase CK2 activity in tumor cells may to some extent result from a deregulation in subunit biosynthesis or degradation...

  9. Xylosylation of proteins by expression of human xylosyltransferase 2 in plants.

    Science.gov (United States)

    Matsuo, Kouki; Atsumi, Go

    2018-04-12

    Through the years, the post-translational modification of plant-made recombinant proteins has been a considerable problem. Protein glycosylation is arguably the most important post-translational modification; thus, for the humanization of protein glycosylation in plants, the introduction, repression, and knockout of many glycosylation-related genes has been carried out. In addition, plants lack mammalian-type protein O-glycosylation pathways; thus, for the synthesis of mammalian O-glycans in plants, the construction of these pathways is necessary. In this study, we successfully xylosylated the recombinant human proteoglycan core protein, serglycin, by transient expression of human xylosyltransferase 2 in Nicotiana benthamiana plants. When human serglycin was co-expressed with human xylosyltransferase 2 in plants, multiple serine residues of eight xylosylation candidates were xylosylated. From the results of carbohydrate assays for total soluble proteins, some endogenous plant proteins also appeared to be xylosylated, likely through the actions of xylosyltransferase 2. The xylosylation of core proteins is the initial step of the glycosaminoglycan part of the synthesis of proteoglycans. In the future, these novel findings may lead to whole mammalian proteoglycan synthesis in plants. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  10. Change of the structure and the digestibility of myofibrillar proteins in Nanjing dry-cured duck during processing.

    Science.gov (United States)

    Du, Xiaojing; Sun, Yangying; Pan, Daodong; Wang, Ying; Ou, Changrong; Cao, Jinxuan

    2018-06-01

    To investigate the change of bioavailability and structure of myofibrillar proteins during Nanjing dry-cured duck processing, carbonyl content, sulfhydryl (SH) group, disulfide (SS) group, sodium dodecyl sulfate polyacrylamide gel electrophoresis, surface hydrophobicity, secondary structures and in vitro digestibility were determined. During processing, carbonyl content and surface hydrophobicity increased; SH turned into SS group; α-helix turned into β-sheet and random coil fractions. Protein degradation occurred during dry-curing and drying-ripening stages. The in vitro digestibility of pepsin and pancreatic proteases increased during the salt curing stage and decreased during the drying-ripening stage. The increase of digestibility could be attributed to the mild oxidation, degradation and unfolding of proteins while the decrease of digestibility was related to the intensive oxidation and aggregation of proteins. Protein degradation was not a main factor of digestibility during the drying-ripening stage. Results demonstrated that the bioavailability loss of myofibrillar proteins in Nanjing dry-cured duck occurred during the stage of drying-ripening instead of curing. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  11. Adaptor protein complex 2-mediated endocytosis is crucial for male reproductive organ development in Arabidopsis.

    Science.gov (United States)

    Kim, Soo Youn; Xu, Zheng-Yi; Song, Kyungyoung; Kim, Dae Heon; Kang, Hyangju; Reichardt, Ilka; Sohn, Eun Ju; Friml, Jirí; Juergens, Gerd; Hwang, Inhwan

    2013-08-01

    Fertilization in flowering plants requires the temporal and spatial coordination of many developmental processes, including pollen production, anther dehiscence, ovule production, and pollen tube elongation. However, it remains elusive as to how this coordination occurs during reproduction. Here, we present evidence that endocytosis, involving heterotetrameric adaptor protein complex 2 (AP-2), plays a crucial role in fertilization. An Arabidopsis thaliana mutant ap2m displays multiple defects in pollen production and viability, as well as elongation of staminal filaments and pollen tubes, all of which are pivotal processes needed for fertilization. Of these abnormalities, the defects in elongation of staminal filaments and pollen tubes were partially rescued by exogenous auxin. Moreover, DR5rev:GFP (for green fluorescent protein) expression was greatly reduced in filaments and anthers in ap2m mutant plants. At the cellular level, ap2m mutants displayed defects in both endocytosis of N-(3-triethylammonium-propyl)-4-(4-diethylaminophenylhexatrienyl) pyridinium dibromide, a lypophilic dye used as an endocytosis marker, and polar localization of auxin-efflux carrier PIN FORMED2 (PIN2) in the stamen filaments. Moreover, these defects were phenocopied by treatment with Tyrphostin A23, an inhibitor of endocytosis. Based on these results, we propose that AP-2-dependent endocytosis plays a crucial role in coordinating the multiple developmental aspects of male reproductive organs by modulating cellular auxin level through the regulation of the amount and polarity of PINs.

  12. Effects of 1,2,4-Trichlorobenzene and Mercury Ion Stress on Ca2+ Fluxion and Protein Phosphorylation in Rice

    Directory of Open Access Journals (Sweden)

    Cai-lin GE

    2007-12-01

    Full Text Available The effects of 5 mg/L 1,2,4-trichlorobenzene (TCB and 0.1 mmol/L mercury ion (Hg2+ stresses on Ca2+ fluxion and protein phosphorylation in rice seedlings were investigated by isotope exchange kinetics and in vitro phosphorylation assay. The Ca2+ absorption in rice leaves and Ca2+ transportation from roots to leaves were promoted significantly in response to Hg2+ and TCB treatments for 4-48 h. The Ca2+ absorption peaks presented in the leaves when the rice seedlings were exposed to Hg2+ for 8-12 h or to TCB for 12-24 h. Several Ca2+ absorption peaks presented in the roots during rice seedlings being exposed to Hg2+ and TCB, and the first Ca2+ absorption peak was at 8 h after being exposed to Hg2+ and TCB. The result of isotope exchange kinetic analysis confirmed that short-term (8 h Hg2+ and TCB stresses caused Ca2+ channels or pumps located on plasmalemma to open transiently. The phosphorylation assay showed that short-term TCB stress enhanced protein phosphorylation in rice roots (TCB treatment for 4-8 h and leaves (TCB treatment for 4-24 h, and short-term (4-8 h Hg2+ stress also enhanced protein phosphorylation in rice leaves. The enhancement of protein phosphorylation in both roots and leaves corresponded with the first Ca2+ absorption peak, which confirmed that the enhancement of protein phosphorylation caused by TCB or Hg2+ stress might be partly triggered by the increases of cytosolic calcium. TCB treatment over 12 h inhibited protein phosphorylation in rice roots, which might be partly due to that TCB stress suppressed the protein kinase activity. Whereas, Hg2+ treatment inhibited protein phosphorylation in rice roots, and Hg2+ treatment over 12 h inhibited protein phosphorylation in rice leaves. This might be attributed to that not only the protein kinase activity, but also the expressions of phosphorylation proteins were restrained by Hg2+ stress.

  13. Pichia pastoris-expressed dengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Shailendra Mani

    Full Text Available Dengue is a mosquito-borne viral disease with a global prevalence. It is caused by four closely-related dengue viruses (DENVs 1-4. A dengue vaccine that can protect against all four viruses is an unmet public health need. Live attenuated vaccine development efforts have encountered unexpected interactions between the vaccine viruses, raising safety concerns. This has emphasized the need to explore non-replicating dengue vaccine options. Virus-like particles (VLPs which can elicit robust immunity in the absence of infection offer potential promise for the development of non-replicating dengue vaccine alternatives. We have used the methylotrophic yeast Pichia pastoris to develop DENV envelope (E protein-based VLPs. We designed a synthetic codon-optimized gene, encoding the N-terminal 395 amino acid residues of the DENV-2 E protein. It also included 5' pre-membrane-derived signal peptide-encoding sequences to ensure proper translational processing, and 3' 6× His tag-encoding sequences to facilitate purification of the expressed protein. This gene was integrated into the genome of P. pastoris host and expressed under the alcohol oxidase 1 promoter by methanol induction. Recombinant DENV-2 protein, which was present in the insoluble membrane fraction, was extracted and purified using Ni(2+-affinity chromatography under denaturing conditions. Amino terminal sequencing and detection of glycosylation indicated that DENV-2 E had undergone proper post-translational processing. Electron microscopy revealed the presence of discrete VLPs in the purified protein preparation after dialysis. The E protein present in these VLPs was recognized by two different conformation-sensitive monoclonal antibodies. Low doses of DENV-2 E VLPs formulated in alum were immunogenic in inbred and outbred mice eliciting virus neutralizing titers >1,1200 in flow cytometry based assays and protected AG129 mice against lethal challenge (p<0.05. The formation of immunogenic DENV-2 E

  14. [Effect of protein intervention on amino acid metabolism spectrum of Qi and Yin deficiency type 2 diabetic rats].

    Science.gov (United States)

    Ma, Li-Na; Mao, Xin-Min; Ma, Xiao-Li; Li, Lin-Lin; Wang, Ye; Tao, Yi-Cun; Wang, Jing-Wei; Guo, Jia-Jia; Lan, Yi

    2016-11-01

    To study the effect of plant protein and animal protein on amino acid metabolism spectrum of Qi and Yin deficiency type 2 diabetic rats. 110 male SD rats were randomly divided into blank group (n=10), diabetic model group (n=20), disease-symptoms group (n=80). The rats of blank group received ordinary feeding, while other groups were fed with high sugar and fat diets. During the whole process of feeding, rats of disease-symptoms group were given with Qingpi-Fuzi (15.75 g•kg⁻¹) once a day through oral administration. Five weeks later, the rats were given with a low dose of STZ (40 mg•kg⁻¹) by intraperitoneal injection to establish experimental diabetic models. Then the models were randomly divided into disease-symptoms group 1 (Qi and Yin deficiency diabetic group, 15.75 g•kg⁻¹), disease-symptoms group 2 (plant protein group, 0.5 g•kg⁻¹), disease-symptoms group 3 (animal protein group, 0.5 g•kg⁻¹), disease-symptoms group 4 (berberine group, 0.1 g•kg⁻¹). The drugs were given for 4 weeks by gavage administration. After 4 weeks of protein intervention, the abdominal aortic blood was collected and serum was isolated to analyze its free amino acid by using AQC pre-column derivatization HPLC and fluorescence detector. Four weeks after the protein intervention, plant protein, animal protein and berberine had no obvious effect on body weight and blood sugar in type 2 diabetic rats. As compared with animal protein group, histidine and proline(PYin deficiency type 2 diabetic SD rats. Symbolic differential compounds could be found through metabonomics technology, providing experimental basis for early warning of type 2 diabetes and diagnosis of Qi and Yin deficiency syndrome. Copyright© by the Chinese Pharmaceutical Association.

  15. Lactic acid induces aberrant amyloid precursor protein processing by promoting its interaction with endoplasmic reticulum chaperone proteins.

    Directory of Open Access Journals (Sweden)

    Yiwen Xiang

    Full Text Available BACKGROUND: Lactic acid, a natural by-product of glycolysis, is produced at excess levels in response to impaired mitochondrial function, high-energy demand, and low oxygen availability. The enzyme involved in the production of β-amyloid peptide (Aβ of Alzheimer's disease, BACE1, functions optimally at lower pH, which led us to investigate a potential role of lactic acid in the processing of amyloid precursor protein (APP. METHODOLOGY/PRINCIPAL FINDINGS: Lactic acid increased levels of Aβ40 and 42, as measured by ELISA, in culture medium of human neuroblastoma cells (SH-SY5Y, whereas it decreased APP metabolites, such as sAPPα. In cell lysates, APP levels were increased and APP was found to interact with ER-chaperones in a perinuclear region, as determined by co-immunoprecipitation and fluorescence microscopy studies. Lactic acid had only a very modest effect on cellular pH, did increase the levels of ER chaperones Grp78 and Grp94 and led to APP aggregate formation reminiscent of aggresomes. CONCLUSIONS/SIGNIFICANCE: These findings suggest that sustained elevations in lactic acid levels could be a risk factor in amyloidogenesis related to Alzheimer's disease through enhanced APP interaction with ER chaperone proteins and aberrant APP processing leading to increased generation of amyloid peptides and APP aggregates.

  16. Self-packed filter plates: a good alternative for pre-packed filter plates for developing purification processes of therapeutic proteins

    NARCIS (Netherlands)

    Li, X.; Roo, de G.; Burgers, K.; Ottens, M.; Eppink, M.H.M.

    2012-01-01

    The use of high throughput screening (HTS) has successfully been applied in the past years in downstream process development of therapeutic proteins. Different HTS applications were introduced to speed up the purification process development of these proteins. In the light of these findings, studies

  17. Characterization of the ternary Usher syndrome SANS/ush2a/whirlin protein complex.

    Science.gov (United States)

    Sorusch, Nasrin; Bauß, Katharina; Plutniok, Janet; Samanta, Ananya; Knapp, Barbara; Nagel-Wolfrum, Kerstin; Wolfrum, Uwe

    2017-03-15

    The Usher syndrome (USH) is the most common form of inherited deaf-blindness, accompanied by vestibular dysfunction. Due to the heterogeneous manifestation of the clinical symptoms, three USH types (USH1-3) and additional atypical forms are distinguished. USH1 and USH2 proteins have been shown to function together in multiprotein networks in photoreceptor cells and hair cells. Mutations in USH proteins are considered to disrupt distinct USH protein networks and finally lead to the development of USH.To get novel insights into the molecular pathomechanisms underlying USH, we further characterize the periciliary USH protein network in photoreceptor cells. We show the direct interaction between the scaffold protein SANS (USH1G) and the transmembrane adhesion protein ush2a and that both assemble into a ternary USH1/USH2 complex together with the PDZ-domain protein whirlin (USH2D) via mutual interactions. Immunohistochemistry and proximity ligation assays demonstrate co-localization of complex partners and complex formation, respectively, in the periciliary region, the inner segment and at the synapses of rodent and human photoreceptor cells. Protein-protein interaction assays and co-expression of complex partners reveal that pathogenic mutations in USH1G severely affect formation of the SANS/ush2a/whirlin complex. Translational read-through drug treatment, targeting the c.728C > A (p.S243X) nonsense mutation, restored SANS scaffold function. We conclude that USH1 and USH2 proteins function together in higher order protein complexes. The maintenance of USH1/USH2 protein complexes depends on multiple USH1/USH2 protein interactions, which are disrupted by pathogenic mutations in USH1G protein SANS. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Influence of sodium nitrite on protein oxidation and nitrosation of sausages subjected to processing and storage.

    Science.gov (United States)

    Feng, Xianchao; Li, Chenyi; Jia, Xu; Guo, Yan; Lei, Na; Hackman, Robert M; Chen, Lin; Zhou, Guanghong

    2016-06-01

    The influence of NaNO2 content on protein oxidation and nitrosation was investigated in cooked sausages at different concentrations (0, 50, 100, 200 and 400 mg NaNO2/kg). Dependent on concentration, NaNO2 had both anti- and pro-oxidant effects on protein oxidation. The antioxidant effects of NaNO2 on the protein oxidation were evidenced by significantly lower carbonyl contents, higher free amines and lower surface hydrophobicities. The pro-oxidant effects of NaNO2 on protein oxidation resulted in a decrease of sulfhydryls and an increase of disulfide bonds. NaNO2 also improved the protein nitrosation inducing the formation of 3-nitrotyrosine (3-NT). Moreover, 3-NT had significant correlations with parameters of protein oxidation, indicating that 3-NT may be a possible marker for protein oxidation. Results of this study contribute to an understanding of the impact of NaNO2 on food quality and help to identify optimal formulations of cured meat products. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Fluorescence detection of a protein-bound 2Fe2S cluster.

    Science.gov (United States)

    Hoff, Kevin G; Goodlitt, Rochelle; Li, Rui; Smolke, Christina D; Silberg, Jonathan J

    2009-03-02

    A fluorescent biosensor is described for 2Fe2S clusters that is composed of green fluorescent protein (GFP) fused to glutaredoxin 2 (Grx2), as illustrated here. 2Fe2S detection is based on the reduction of GFP fluorescence upon the 2Fe2S-induced dimerization of GFP-Grx2. This assay is sufficiently sensitive to detect submicromolar changes in 2Fe2S levels, thus making it suitable for high-throughput measurements of metallocluster degradation and synthesis reactions.

  20. Detection of the argonaute protein Ago2 and microRNAs in the RNA induced silencing complex (RISC) using a monoclonal antibody.

    Science.gov (United States)

    Ikeda, Keigo; Satoh, Minoru; Pauley, Kaleb M; Fritzler, Marvin J; Reeves, Westley H; Chan, Edward K L

    2006-12-20

    MicroRNAs (miRNAs) are short RNA molecules responsible for post-transcriptional gene silencing by the degradation or translational inhibition of their target messenger RNAs (mRNAs). This process of gene silencing, known as RNA interference (RNAi), is mediated by highly conserved Argonaute (Ago) proteins which are the key components of the RNA induced silencing complex (RISC). In humans, Ago2 is responsible for the endonuclease cleavage of targeted mRNA and it interacts with the mRNA-binding protein GW182, which is a marker for cytoplasmic foci referred to as GW bodies (GWBs). We demonstrated that the anti-Ago2 monoclonal antibody 4F9 recognized GWBs in a cell cycle dependent manner and was capable of capturing miRNAs associated with Ago2. Since Ago2 protein is the effector protein of RNAi, anti-Ago2 monoclonal antibody may be useful in capturing functional miRNAs.

  1. Phthalic Acid Chemical Probes Synthesized for Protein-Protein Interaction Analysis

    Directory of Open Access Journals (Sweden)

    Chin-Jen Wu

    2013-06-01

    Full Text Available Plasticizers are additives that are used to increase the flexibility of plastic during manufacturing. However, in injection molding processes, plasticizers cannot be generated with monomers because they can peel off from the plastics into the surrounding environment, water, or food, or become attached to skin. Among the various plasticizers that are used, 1,2-benzenedicarboxylic acid (phthalic acid is a typical precursor to generate phthalates. In addition, phthalic acid is a metabolite of diethylhexyl phthalate (DEHP. According to Gene_Ontology gene/protein database, phthalates can cause genital diseases, cardiotoxicity, hepatotoxicity, nephrotoxicity, etc. In this study, a silanized linker (3-aminopropyl triethoxyslane, APTES was deposited on silicon dioxides (SiO2 particles and phthalate chemical probes were manufactured from phthalic acid and APTES–SiO2. These probes could be used for detecting proteins that targeted phthalic acid and for protein-protein interactions. The phthalic acid chemical probes we produced were incubated with epithelioid cell lysates of normal rat kidney (NRK-52E cells to detect the interactions between phthalic acid and NRK-52E extracted proteins. These chemical probes interacted with a number of chaperones such as protein disulfide-isomerase A6, heat shock proteins, and Serpin H1. Ingenuity Pathways Analysis (IPA software showed that these chemical probes were a practical technique for protein-protein interaction analysis.

  2. Phosphorylation of protein synthesis initiation factor 2 (elF-2) in the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Romero, D.P.

    1986-01-01

    Initiation Factor 2 (elF-2) in the yeast Saccharomyces cerevisiae is comprised of 3 subunits. The control of protein synthesis in mammalian cells have been shown to involve the phosphorylation of the small (alpha) subunit by a specific protein kinase. Phosphorylation results in an inhibition of protein synthesis. In order to determine whether or not an analogous system is operative in yeast, the phosphorylation state of the alpha subunit of elF-2 in Saccharomyces was determined during various growth and nongrowth conditions. Cells were radiolabelled with 32 P and 35 S, and the whole cell lysates were analyzed by two dimensional gel electrophoresis. These experiments revealed that the smallest subunit (alpha, M/sub r/ = 31,000) is a phosphoprotein in vivo under a variety of growth and nongrowth conditions. This is in direct contrast to the pattern exhibited in mammalian cells. The fact that the small subunit of elF-2 in yeast is phosphorylated under a variety of physiological conditions indicates that such a covalent modification is important for some aspects of elF-2 function. In order to investigate this problem further, a protein kinase that specifically labels the alpha subunit of elF-2 in vitro was isolated. The kinase is not autophosphorylating, utilizes ATP as a phosphate donor, phosphorylates an exogenous protein, casein, modifies serine residues in elF-2, is cyclic nucleotide-independent, and is strongly inhibited by heparin

  3. N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

    Science.gov (United States)

    Mooney, Jane T; Fredericks, Dale P; Christensen, Thorkild; Bruun Schiødt, Christine; Hearn, Milton T W

    2015-07-01

    The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. Copyright © 2015 John Wiley & Sons, Ltd.

  4. Proteomic study on the stability of proteins in bovine, camel, and caprine milk sera after processing

    NARCIS (Netherlands)

    Zhang, Lina; Boeren, Sjef; Smits, Marcel; Hooijdonk, van Toon; Vervoort, Jacques; Hettinga, Kasper

    2016-01-01

    Milk proteins have been shown to be very sensitive to processing. This study aims to investigate the changes of the bovine, camel, and caprine milk proteins after freezing, pasteurization (62 °C, 30 min), and spray drying by proteomic techniques, filter-aided sample preparation (FASP) and

  5. Characterization of fatty acid binding by the P2 myelin protein

    International Nuclear Information System (INIS)

    Gudaitis, P.G.; Weise, M.J.

    1987-01-01

    In recent years, significant sequence homology has been found between the P2 protein of peripheral myelin and intracellular retinoid- and fatty acid-binding proteins. They have found that salt extracts of bovine intradural nerve roots contain the P2 basic protein in association with free fatty acid. Preliminary results from quantitative analyses showed a ratio of 0.4-1.1 fatty acid (mainly oleate and palmitate) per P2 molecule. P2/ligand interactions were partially characterized using ( 3 H)-oleate in gel permeation assays and binding studies using lipidex to separated bound and free fatty acid. Methyloleate was found to displace ( 3 H)-oleate from P2, indicating that ligand binding interactions are predominantly hydrophobic in nature. On the other hand, myristic acid and retinol did not inhibit the binding of oleate to the protein, results consistent with a decided affinity for long chain fatty acids but not for the retinoids. The binding between P2 and oleic acid showed an apparent Kd in the micromolar range, a value comparable to those found for other fatty acid-binding proteins. From these results they conclude that P2 shares not only structural homology with certain fatty acid binding proteins but also an ability to bind long chain fatty acids. Although the significance of these similarities is not yet clear, they may, by analogy, expect P2 to have a role in PNS lipid metabolism

  6. Effects of processing moisture on the physical properties and in vitro digestibility of starch and protein in extruded brown rice and pinto bean composite flours.

    Science.gov (United States)

    Sumargo, Franklin; Gulati, Paridhi; Weier, Steven A; Clarke, Jennifer; Rose, Devin J

    2016-11-15

    The influence of pinto bean flour and processing moisture on the physical properties and in vitro digestibility of rice-bean extrudates has been investigated. Brown rice: pinto bean flour (0%, 15%, 30%, and 45% bean flour) were extruded under 5 moisture conditions (17.2%, 18.1%, 18.3%, 19.5%, and 20.1%). Physical properties [bulk density, unit density, radial expansion, axial expansion, overall expansion, specific volume, hardness, color, water solubility index, and water absorption index] and in vitro starch and protein digestibilities were determined. Increasing bean flour and processing moisture increased density and hardness while decreasing expansion. Rapidly digestible starch decreased and resistant starch increased as bean substitution and processing moisture increased. In vitro protein digestibility increased with increasing bean flour or with decreasing processing moisture. Incorporating bean flour into extruded snacks can negatively affect physical attributes (hardness, density, and expansion) while positively affecting in vitro starch (decrease) and protein (increase) digestibilities. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Structures and Corresponding Functions of Five Types of Picornaviral 2A Proteins

    Directory of Open Access Journals (Sweden)

    Xiaoyao Yang

    2017-07-01

    Full Text Available Among the few non-structural proteins encoded by the picornaviral genome, the 2A protein is particularly special, irrespective of structure or function. During the evolution of the Picornaviridae family, the 2A protein has been highly non-conserved. We believe that the 2A protein in this family can be classified into at least five distinct types according to previous studies. These five types are (A chymotrypsin-like 2A, (B Parechovirus-like 2A, (C hepatitis-A-virus-like 2A, (D Aphthovirus-like 2A, and (E 2A sequence of the genus Cardiovirus. We carried out a phylogenetic analysis and found that there was almost no homology between each type. Subsequently, we aligned the sequences within each type and found that the functional motifs in each type are highly conserved. These different motifs perform different functions. Therefore, in this review, we introduce the structures and functions of these five types of 2As separately. Based on the structures and functions, we provide suggestions to combat picornaviruses. The complexity and diversity of the 2A protein has caused great difficulties in functional and antiviral research. In this review, researchers can find useful information on the 2A protein and thus conduct improved antiviral research.

  8. The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3

    OpenAIRE

    Halfter, Ursula; Ishitani, Manabu; Zhu, Jian-Kang

    2000-01-01

    The Arabidopsis thaliana SOS2 and SOS3 genes are required for intracellular Na+ and K+ homeostasis and plant tolerance to high Na+ and low K+ environments. SOS3 is an EF hand type calcium-binding protein having sequence similarities with animal neuronal calcium sensors and the yeast calcineurin B. SOS2 is a serine/threonine protein kinase in the SNF1/AMPK family. We report here that SOS3 physically interacts with and activates SOS2 protein kinase. Genetically, sos2sos3 double mutant analysis ...

  9. Tsetse salivary gland proteins 1 and 2 are high affinity nucleic acid binding proteins with residual nuclease activity.

    Directory of Open Access Journals (Sweden)

    Guy Caljon

    Full Text Available Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2 display DNA/RNA non-specific, high affinity nucleic acid binding with K(D values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.

  10. Specific binding of tubeimoside-2 with proteins in hepatocarcinoma HepG2 cells: investigation by molecular spectroscopy

    Science.gov (United States)

    Yang, Sun; Shi-Sheng, Sun; Ying-Yong, Zhao; Jun, Fan

    2012-07-01

    In this study, we compared different binding interactions of TBMS2 with proteins both in hepatocarcinoma HepG2 cells and in normal embryo hepatic L02 cells by using fluorescence, absorption, and CD spectroscopy. The fluorescence data revealed that the fluorescence intensity of proteins in the HepG2 and L02 cells decreased in the presence of TBMS2 by 30.79% and 12.01%, respectively. Binding constants and thermodynamic parameters were obtained for systems of TBMS2 with the two kinds of cell proteins. The results indicated that HepG2 cell proteins had a higher TBMS2 binding activity than those in the L02 cells. Analysis of the TBMS2 cytotoxic activities showed that TBMS2 could selectively induce apoptosis of HepG2 cells by binding to them, while its apoptotic effect on L02 cells was relatively weaker.

  11. Structure and DNA-binding of meiosis-specific protein Hop2

    Science.gov (United States)

    Zhou, Donghua; Moktan, Hem; Pezza, Roberto

    2014-03-01

    Here we report structure elucidation of the DNA binding domain of homologous pairing protein 2 (Hop2), which is important to gene diversity when sperms and eggs are produced. Together with another protein Mnd1, Hop2 enhances the strand invasion activity of recombinase Dmc1 by over 30 times, facilitating proper synapsis of homologous chromosomes. However, the structural and biochemical bases for the function of Hop2 and Mnd1 have not been well understood. As a first step toward such understanding, we recently solved the structure for the N-terminus of Hop2 (1-84) using solution NMR. This fragment shows a typical winged-head conformation with recognized DNA binding activity. DNA interacting sites were then investigated by chemical shift perturbations in a titration experiment. Information of these sites was used to guide protein-DNA docking with MD simulation, revealing that helix 3 is stably lodged in the DNA major groove and that wing 1 (connecting strands 2 and 3) transiently comes in contact with the minor groove in nanosecond time scale. Mutagenesis analysis further confirmed the DNA binding sites in this fragment of the protein.

  12. Influenza A H3N2 subtype virus NS1 protein targets into the nucleus and binds primarily via its C-terminal NLS2/NoLS to nucleolin and fibrillarin

    Science.gov (United States)

    2012-01-01

    Background Influenza A virus non-structural protein 1 (NS1) is a virulence factor, which is targeted into the cell cytoplasm, nucleus and nucleolus. NS1 is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. Previously, we have shown that the NS1 protein of the H3N2 subtype influenza viruses possesses a C-terminal nuclear localization signal (NLS) that also functions as a nucleolar localization signal (NoLS) and targets the protein into the nucleolus. Results Here, we show that the NS1 protein of the human H3N2 virus subtype interacts in vitro primarily via its C-terminal NLS2/NoLS and to a minor extent via its N-terminal NLS1 with the nucleolar proteins, nucleolin and fibrillarin. Using chimeric green fluorescence protein (GFP)-NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions. Conclusion NS1 protein of the human H3N2 virus interacts primarily via the C-terminal NLS2/NoLS and to a minor extent via the N-terminal NLS1 with the main nucleolar proteins, nucleolin, B23 and fibrillarin. PMID:22909121

  13. MERTK interactions with SH2-domain proteins in the retinal pigment epithelium.

    Science.gov (United States)

    Shelby, Shameka J; Colwill, Karen; Dhe-Paganon, Sirano; Pawson, Tony; Thompson, Debra A

    2013-01-01

    The receptor tyrosine kinase MERTK plays an essential role in the phagocytic uptake of shed photoreceptor membranes by the retinal pigment epithelium (RPE). A fundamental aspect of signal transduction by receptor tyrosine kinases involves autophosphorylation of tyrosine residues that recruit Src-homology 2 (SH2)-domain proteins to the receptor intracellular domain. The goal of the current study was to evaluate the interactions of human MERTK with SH2-domain proteins present in the RPE. The MERTK intracellular domain was expressed as a 6xHis-fusion protein (6xHis-rMERTK(571-999)), purified and phosphorylated. Ni(2+)-NTA pull downs were performed using 6xHis-rMERTK(571-999) in incubations with recombinant phosphotyrosine-recognition sequences expressed as GST-fusion proteins. In addition, pull downs of native SH2-domain proteins were performed using 6xHis-rMERTK(571-999) and protein homogenates from rat RPE/choroid. For both recombinant and native proteins, western analysis detected MERTK interactions with GRB2, PIK3R1 (P85α), VAV3, and SRC. Immunohistochemical analysis localized each protein to mouse RPE. In cultured RPE-J cells incubated with rod outer segments (OS), siRNA knockdown of Grb2 had no effect on OS binding, but significantly reduced OS uptake. Pik3r1 localized to early phagosomes along with Rab5 and Eea1. Phosphorylation and activation of Src was detected downstream of phagocytosis and Mertk activation. These findings suggest that MERTK signaling in the RPE involves a cohort of SH2-domain proteins with the potential to regulate both cytoskeletal rearrangement and membrane movement. Identification of the SH2-domain signaling partners of MERTK is an important step toward further defining the mechanism of RPE phagocytosis that is central to the function and survival of the retina.

  14. MERTK interactions with SH2-domain proteins in the retinal pigment epithelium.

    Directory of Open Access Journals (Sweden)

    Shameka J Shelby

    Full Text Available The receptor tyrosine kinase MERTK plays an essential role in the phagocytic uptake of shed photoreceptor membranes by the retinal pigment epithelium (RPE. A fundamental aspect of signal transduction by receptor tyrosine kinases involves autophosphorylation of tyrosine residues that recruit Src-homology 2 (SH2-domain proteins to the receptor intracellular domain. The goal of the current study was to evaluate the interactions of human MERTK with SH2-domain proteins present in the RPE. The MERTK intracellular domain was expressed as a 6xHis-fusion protein (6xHis-rMERTK(571-999, purified and phosphorylated. Ni(2+-NTA pull downs were performed using 6xHis-rMERTK(571-999 in incubations with recombinant phosphotyrosine-recognition sequences expressed as GST-fusion proteins. In addition, pull downs of native SH2-domain proteins were performed using 6xHis-rMERTK(571-999 and protein homogenates from rat RPE/choroid. For both recombinant and native proteins, western analysis detected MERTK interactions with GRB2, PIK3R1 (P85α, VAV3, and SRC. Immunohistochemical analysis localized each protein to mouse RPE. In cultured RPE-J cells incubated with rod outer segments (OS, siRNA knockdown of Grb2 had no effect on OS binding, but significantly reduced OS uptake. Pik3r1 localized to early phagosomes along with Rab5 and Eea1. Phosphorylation and activation of Src was detected downstream of phagocytosis and Mertk activation. These findings suggest that MERTK signaling in the RPE involves a cohort of SH2-domain proteins with the potential to regulate both cytoskeletal rearrangement and membrane movement. Identification of the SH2-domain signaling partners of MERTK is an important step toward further defining the mechanism of RPE phagocytosis that is central to the function and survival of the retina.

  15. The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA Proteins Alter Amyloid-β Precursor Protein (APP Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1.

    Directory of Open Access Journals (Sweden)

    Bjoern von Einem

    Full Text Available Proteolytic processing of amyloid-β precursor protein (APP by beta-site APP cleaving enzyme 1 (BACE1 is the initial step in the production of amyloid beta (Aβ, which accumulates in senile plaques in Alzheimer's disease (AD. Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα, sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.

  16. Partial purification and identification of the thrombozane A2/prostaglandin H2 receptor protein in human platelets

    International Nuclear Information System (INIS)

    Lim, C.T.; Kattelman, E.J.; Arora, S.K.; Venton, D.L.; Le Breton, G.C.

    1986-01-01

    The thromboxane A 2 /prostaglandin H 2 (TXA 2 /PGH 2 ) receptor antagonist [ 3 H]-13-azaprostanoic acid (13-APA) was used to identify and purify the platelet TXA 2 /PGH 2 receptor protein. Optimal solubilization of the 13-APA binding protein was achieved by extraction with 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS) detergent. Preliminary purification of the crude solubilized membrane fraction was performed by gel filtration chromatography using a Sepharose 4B column. Further purification was accomplished by high performance liquid chromatography (HPLC) using a Synchropak GPC-500 column. The HPLC protein profile revealed two protein peaks, only one of which was enriched in [ 3 H]-13-APA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of this peak revealed two bands with molecular weights of 65,000 and 60,000 daltons. In binding studies using the 60,000 dalton-enriched subfraction, unlabelled 13-APA, the TXA 2 /PGH 2 mimetic U46619 and the TXA 2 /PGH 2 antagonist SQ 29,548 all competed for [ 3 H]-13-APA binding whereas TXB 2 did not compete for binding. Heat denaturation of this subfraction resulted in a complete loss of binding activity. These findings indicate that a protein of approximately 60,000 daltons represents the human platelet TXA 2 /PGH 2 receptor

  17. Pancreatic Islet Protein Complexes and Their Dysregulation in Type 2 Diabetes

    DEFF Research Database (Denmark)

    Pedersen, Helle Krogh; Gudmundsdottir, Valborg; Brunak, Søren

    2017-01-01

    Type 2 diabetes (T2D) is a complex disease that involves multiple genes. Numerous risk loci have already been associated with T2D, although many susceptibility genes remain to be identified given heritability estimates. Systems biology approaches hold potential for discovering novel T2D genes...... by considering their biological context, such as tissue-specific protein interaction partners. Pancreatic islets are a key T2D tissue and many of the known genetic risk variants lead to impaired islet function, hence a better understanding of the islet-specific dysregulation in the disease-state is essential...... to unveil the full potential of person-specific profiles. Here we identify 3,692 overlapping pancreatic islet protein complexes (containing 10,805 genes) by integrating islet gene and protein expression data with protein interactions. We found 24 of these complexes to be significantly enriched for genes...

  18. The PTK7-related transmembrane proteins off-track and off-track 2 are co-receptors for Drosophila Wnt2 required for male fertility.

    Science.gov (United States)

    Linnemannstöns, Karen; Ripp, Caroline; Honemann-Capito, Mona; Brechtel-Curth, Katja; Hedderich, Marie; Wodarz, Andreas

    2014-07-01

    Wnt proteins regulate many developmental processes and are required for tissue homeostasis in adult animals. The cellular responses to Wnts are manifold and are determined by the respective Wnt ligand and its specific receptor complex in the plasma membrane. Wnt receptor complexes contain a member of the Frizzled family of serpentine receptors and a co-receptor, which commonly is a single-pass transmembrane protein. Vertebrate protein tyrosine kinase 7 (PTK7) was identified as a Wnt co-receptor required for control of planar cell polarity (PCP) in frogs and mice. We found that flies homozygous for a complete knock-out of the Drosophila PTK7 homolog off track (otk) are viable and fertile and do not show PCP phenotypes. We discovered an otk paralog (otk2, CG8964), which is co-expressed with otk throughout embryonic and larval development. Otk and Otk2 bind to each other and form complexes with Frizzled, Frizzled2 and Wnt2, pointing to a function as Wnt co-receptors. Flies lacking both otk and otk2 are viable but male sterile due to defective morphogenesis of the ejaculatory duct. Overexpression of Otk causes female sterility due to malformation of the oviduct, indicating that Otk and Otk2 are specifically involved in the sexually dimorphic development of the genital tract.

  19. Influence of prolonged storage process, pasteurization, and heat treatment on biologically-active human milk proteins.

    Science.gov (United States)

    Chang, Jih-Chin; Chen, Chao-Huei; Fang, Li-Jung; Tsai, Chi-Ren; Chang, Yu-Chuan; Wang, Teh-Ming

    2013-12-01

    The bioactive proteins in human milk may be influenced by prolonged storage process, pasteurization, and heat treatment. This study was conducted to evaluate the effects of these procedures. Three forms of human milk - freshly expressed, frozen at -20°C for a prolonged duration, and pasteurized milk - were collected from 14 healthy lactating mothers and a milk bank. The concentrations of major bioactive proteins (secretory immunoglobulin A, lactoferrin, lysozyme, and leptin) were quantified using enzyme-linked immunosorbent assay kits. Changes in these proteins by heat treatment at 40°C or 60°C for 30 minutes were further evaluated. The mean concentrations of lactoferrin and secretory immunoglobulin A were significantly reduced by 66% and 25.9%, respectively, in pasteurized milk compared with those in freshly-expressed milk. Heat treatment at 40°C or 60°C did not cause significant changes in lactoferrin and secretory immunoglobulin A, but there was an apparent increase in lysozyme (p = 0.016). There were no significant differences in leptin level among these three forms of milk prior to (p = 0.153) or after heat treatment (p = 0.053). Various freezing/heating/pasteurization processes applied to human milk prior to delivery to neonates could affect the concentration of immunomodulatory proteins, especially lactoferrin, secretory immunoglobulin A, and lysozyme. Leptin was unaffected by the various handling processes tested. Fresh milk was found to be the best food for neonates. Further studies are warranted to evaluate the functional activity of these proteins and their effects on infants' immunological status. Copyright © 2013. Published by Elsevier B.V.

  20. Regulator of G protein signaling 2 (RGS2 and RGS4 form distinct G protein-dependent complexes with protease activated-receptor 1 (PAR1 in live cells.

    Directory of Open Access Journals (Sweden)

    Sungho Ghil

    Full Text Available Protease-activated receptor 1 (PAR1 is a G-protein coupled receptor (GPCR that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to Gi, Gq and G12 to activate linked signaling pathways. Regulators of G protein signaling (RGS proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET, we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven and either RGS2-Luciferase (RGS2-Luc or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gαq/11, and PAR1-RGS4 by Gαo, but not by other Gα subunits. Gαq/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A that eliminates PAR1-Gq/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.