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Sample records for protein stability contribute

  1. Positively selected sites in cetacean myoglobins contribute to protein stability

    DEFF Research Database (Denmark)

    Dasmeh, Pouria; Serohijos, Adrian W R; Kepp, Kasper P

    2013-01-01

    Since divergence ∼50 Ma ago from their terrestrial ancestors, cetaceans underwent a series of adaptations such as a ∼10-20 fold increase in myoglobin (Mb) concentration in skeletal muscle, critical for increasing oxygen storage capacity and prolonging dive time. Whereas the O2-binding affinity...... between Mb folding stability and protein abundance, suggesting that a selection pressure for stability acts proportionally to higher expression. We also identify a major divergence event leading to the common ancestor of whales, during which major stabilization occurred. Most of the positively selected...

  2. Contribution of buried aspartic acid to the stability of the PDZ2 protein

    International Nuclear Information System (INIS)

    Jayasimha, Pruthvi; Shanmuganathan, Aranganathan; Suladze, Saba; Makhatadze, George I.

    2012-01-01

    Highlights: ► Buried Asp residues on average form 2.5 to 3 hydrogen bonds and/or 0.8 salt bridges. ► Contribution of buried Asp to stability was estimated using model protein PDZ2. ► The energetic contribution of Asp56 to PDZ2 stability estimated to be 18 kJ · mol −1 . ► Findings are discussed in terms of contribution of Asp residues to protein stability. - Abstract: Statistical analysis of protein structures shows that buried aspartic acid residues on average form 2.5 to 3 hydrogen bonds and/or 0.8 potential ionic interactions with other protein groups. To estimate the energetic contribution of such buried groups to the Gibbs free energy of proteins, we measured the effects of amino acid substitutions of D56 in a model protein PDZ2 on its stability. We used temperature-induced unfolding monitored by DSC and denaturant-induced unfolding monitored by the changes in fluorescence intensity. We find that all substitutions of D56 lead to protein unfolding, thus suggesting that this buried hydrogen bonded aspartic acid has a significant contribution to the stability. To quantify the changes in the Gibbs free energy, one of the variants, D56N was stabilized by addition of the protective osmolyte TMAO. Comparison of the stability of the D56N variant with the wild-type PDZ2 in the presence and absence of TMAO allowed us to estimate the contribution of D56 to the protein stability to be 18 kJ · mol −1 . These findings are discussed in terms of contribution of buried ionizable groups to protein stability.

  3. Model for calculation of electrostatic contribution into protein stability

    Science.gov (United States)

    Kundrotas, Petras; Karshikoff, Andrey

    2003-03-01

    Existing models of the denatured state of proteins consider only one possible spatial distribution of protein charges and therefore are applicable to a limited number of cases. In this presentation a more general framework for the modeling of the denatured state is proposed. It is based on the assumption that the titratable groups of an unfolded protein can adopt a quasi-random distribution, restricted by the protein sequence. The model was tested on two proteins, barnase and N-terminal domain of the ribosomal protein L9. The calculated free energy of denaturation, Δ G( pH), reproduces the experimental data essentially better than the commonly used null approximation (NA). It was demonstrated that the seemingly good agreement with experimental data obtained by NA originates from the compensatory effect between the pair-wise electrostatic interactions and the desolvation energy of the individual sites. It was also found that the ionization properties of denatured proteins are influenced by the protein sequence.

  4. Long Non-coding RNA, PANDA, Contributes to the Stabilization of p53 Tumor Suppressor Protein.

    Science.gov (United States)

    Kotake, Yojiro; Kitagawa, Kyoko; Ohhata, Tatsuya; Sakai, Satoshi; Uchida, Chiharu; Niida, Hiroyuki; Naemura, Madoka; Kitagawa, Masatoshi

    2016-04-01

    P21-associated noncoding RNA DNA damage-activated (PANDA) is induced in response to DNA damage and represses apoptosis by inhibiting the function of nuclear transcription factor Y subunit alpha (NF-YA) transcription factor. Herein, we report that PANDA affects regulation of p53 tumor-suppressor protein. U2OS cells were transfected with PANDA siRNAs. At 72 h post-transfection, cells were subjected to immunoblotting and quantitative reverse transcription-polymerase chain reaction. Depletion of PANDA was associated with decreased levels of p53 protein, but not p53 mRNA. The stability of p53 protein was markedly reduced by PANDA silencing. Degradation of p53 protein by silencing PANDA was prevented by treatment of MG132, a proteasome inhibitor. Moreover, depletion of PANDA prevented accumulation of p53 protein, as a result of DNA damage, induced by the genotoxic agent etoposide. These results suggest that PANDA stabilizes p53 protein in response to DNA damage, and provide new insight into the regulatory mechanisms of p53. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  5. Conserved amino acids within the N-terminus of the West Nile virus NS4A protein contribute to virus replication, protein stability and membrane proliferation

    International Nuclear Information System (INIS)

    Ambrose, R.L.; Mackenzie, J.M.

    2015-01-01

    The West Nile virus strain Kunjin virus (WNV KUN ) NS4A protein is a multifunctional protein involved in many aspects of the virus life-cycle and is a major component of the WNV KUN replication complex (RC). Previously we identified a conserved region in the C-terminus of NS4A regulating proteolytic processing and RC assembly, and now investigate key conserved residues in the N-terminus of NS4A and their contribution to WNV KUN replication. Mutation of P13 completely ablated replication, whereas, mutation of P48 and D49, near the first transmembrane helix, and G66 within the helix, showed variable defects in replication, virion secretion and membrane proliferation. Intriguingly, the P48 and G66 NS4A mutants resulted in specific proteasome depletion of NS4A that could in part be rescued with a proteasome inhibitor. Our results suggest that the N-terminus of NS4A contributes to correct folding and stability, essential for facilitating the essential roles of NS4A during replication. - Highlights: • Mutation of Proline13 of the WNV NS4A protein is lethal to replication. • 1st TMB helix of NS4A contributes to protein stability and membrane remodelling. • Unstable mutants of NS4A can be rescued with a proteasome inhibitor. • This study (and of others) contributes to a functional mapping of the NS4A protein

  6. Electrostatic contribution of surface charge residues to the stability of a thermophilic protein: benchmarking experimental and predicted pKa values.

    Directory of Open Access Journals (Sweden)

    Chi-Ho Chan

    Full Text Available Optimization of the surface charges is a promising strategy for increasing thermostability of proteins. Electrostatic contribution of ionizable groups to the protein stability can be estimated from the differences between the pKa values in the folded and unfolded states of a protein. Using this pKa-shift approach, we experimentally measured the electrostatic contribution of all aspartate and glutamate residues to the stability of a thermophilic ribosomal protein L30e from Thermococcus celer. The pKa values in the unfolded state were found to be similar to model compound pKas. The pKa values in both the folded and unfolded states obtained at 298 and 333 K were similar, suggesting that electrostatic contribution of ionizable groups to the protein stability were insensitive to temperature changes. The experimental pKa values for the L30e protein in the folded state were used as a benchmark to test the robustness of pKa prediction by various computational methods such as H++, MCCE, MEAD, pKD, PropKa, and UHBD. Although the predicted pKa values were affected by crystal contacts that may alter the side-chain conformation of surface charged residues, most computational methods performed well, with correlation coefficients between experimental and calculated pKa values ranging from 0.49 to 0.91 (p<0.01. The changes in protein stability derived from the experimental pKa-shift approach correlate well (r = 0.81 with those obtained from stability measurements of charge-to-alanine substituted variants of the L30e protein. Our results demonstrate that the knowledge of the pKa values in the folded state provides sufficient rationale for the redesign of protein surface charges leading to improved protein stability.

  7. Stability of Hyperthermophilic Proteins

    DEFF Research Database (Denmark)

    Stiefler-Jensen, Daniel

    stability by randomly generate mutants and lengthy screening processes to identify the best new mutants. However, with the increase in available genomic sequences of thermophilic or hyperthermophilic organisms a world of enzymes with intrinsic high stability are now available. As these organisms are adapted...... to life at high temperatures so are their enzymes, as a result the high stability is accompanied by low activity at moderate temperatures. Thus, much effort had been put into decoding the mechanisms behind the high stability of the thermophilic enzymes. The hope is to enable scientist to design enzymes...... in the high stability of hyperthermophilic enzymes. The thesis starts with an introduction to the field of protein and enzyme stability with special focus on the thermophilic and hyperthermophilic enzymes and proteins. After the introduction three original research manuscripts present the experimental data...

  8. Ion-ion interactions in the denatured state contribute to the stabilization of CutA1 proteins.

    Science.gov (United States)

    Yutani, Katsuhide; Matsuura, Yoshinori; Naitow, Hisashi; Joti, Yasumasa

    2018-05-16

    In order to elucidate features of the denatured state ensembles that exist in equilibrium with the native state under physiological conditions, we performed 1.4-μs molecular dynamics (MD) simulations at 400 K and 450 K using the monomer subunits of three CutA1 mutants from Escherichia coli: an SH-free mutant (Ec0SH) with denaturation temperature (T d ) = 85.6 °C, a hydrophobic mutant (Ec0VV) with T d  = 113.3 °C, and an ionic mutant (Ec0VV_6) with T d  = 136.8 °C. The occupancy of salt bridges by the six substituted charged residues in Ec0VV_6 was 140.1% at 300 K and 89.5% at 450 K, indicating that even in the denatured state, salt bridge occupancy was high, approximately 60% of that at 300 K. From these results, we can infer that proteins from hyperthermophiles with a high ratio of charged residues are stabilized by a decrease in conformational entropy due to ion-ion interactions in the denatured state. The mechanism must be comparable to the stabilization conferred by disulfide bonds within a protein. This suggests that introduction of charged residues, to promote formation of salt bridges in the denatured state, would be a simple way to rationally design stability-enhanced mutants.

  9. Protein stability: a crystallographer’s perspective

    International Nuclear Information System (INIS)

    Deller, Marc C.; Kong, Leopold; Rupp, Bernhard

    2016-01-01

    An understanding of protein stability is essential for optimizing the expression, purification and crystallization of proteins. In this review, discussion will focus on factors affecting protein stability on a somewhat practical level, particularly from the view of a protein crystallographer. Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed

  10. Protein stability: a crystallographer’s perspective

    Energy Technology Data Exchange (ETDEWEB)

    Deller, Marc C., E-mail: mdeller@stanford.edu [Stanford University, Shriram Center, 443 Via Ortega, Room 097, MC5082, Stanford, CA 94305-4125 (United States); Kong, Leopold [National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Building 8, Room 1A03, 8 Center Drive, Bethesda, MD 20814 (United States); Rupp, Bernhard [k.-k. Hofkristallamt, 91 Audrey Place, Vista, CA 92084 (United States); Medical University of Innsbruck, Schöpfstrasse 41, A-6020 Innsbruck (Austria)

    2016-01-26

    An understanding of protein stability is essential for optimizing the expression, purification and crystallization of proteins. In this review, discussion will focus on factors affecting protein stability on a somewhat practical level, particularly from the view of a protein crystallographer. Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed.

  11. Protein stability: a crystallographer’s perspective

    Science.gov (United States)

    Deller, Marc C.; Kong, Leopold; Rupp, Bernhard

    2016-01-01

    Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed. PMID:26841758

  12. Isomeric Detergent Comparison for Membrane Protein Stability

    DEFF Research Database (Denmark)

    Cho, Kyung Ho; Hariharan, Parameswaran; Mortensen, Jonas S.

    2016-01-01

    and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta...... and ortho isomers of the previously reported para-substituted xylene-linked maltoside amphiphiles (XMAs), along with alkyl chain-length variation. The isomeric XMAs were assessed with three membrane proteins, and the meta isomer with a C12 alkyl chain was most effective at maintaining solubility....../stability of the membrane proteins. We propose that interplay between the hydrophile–lipophile balance (HLB) and alkyl chain length is of central importance for high detergent efficacy. In addition, differences in inter-alkyl-chain distance between the isomers influence the ability of the detergents to stabilise membrane...

  13. Extracellular DNA Contributes to Dental Biofilm Stability

    DEFF Research Database (Denmark)

    Schlafer, Sebastian; Meyer, Rikke Louise; Dige, Irene

    2017-01-01

    dental biofilms. This study aimed to determine whether eDNA was part of the matrix in biofilms grown in situ in the absence of sucrose and whether treatment with DNase dispersed biofilms grown for 2.5, 5, 7.5, 16.5, or 24 h. Three hundred biofilms from 10 study participants were collected and treated...... the amount of biofilm in very early stages of growth (up to 7.5 h), but the treatment effect decreased with increasing biofilm age. This study proves the involvement of eDNA in dental biofilm formation and its importance for biofilm stability in the earliest stages. Further research is required to uncover...

  14. Untangling the biological contributions to soil stability in semiarid shrublands

    Science.gov (United States)

    Chaudhary, V. Bala; Bowker, Matthew A.; O'Dell, Thomas E.; Grace, James B.; Redman, Andrea E.; Rillig, Matthias C.; Johnson, Nancy C.

    2009-01-01

    Communities of plants, biological soil crusts (BSCs), and arbuscular mycorrhizal (AM) fungi are known to influence soil stability individually, but their relative contributions, interactions, and combined effects are not well understood, particularly in arid and semiarid ecosystems. In a landscape-scale field study we quantified plant, BSC, and AM fungal communities at 216 locations along a gradient of soil stability levels in southern Utah, USA. We used multivariate modeling to examine the relative influences of plants, BSCs, and AM fungi on surface and subsurface stability in a semiarid shrubland landscape. Models were found to be congruent with the data and explained 35% of the variation in surface stability and 54% of the variation in subsurface stability. The results support several tentative conclusions. While BSCs, plants, and AM fungi all contribute to surface stability, only plants and AM fungi contribute to subsurface stability. In both surface and subsurface models, the strongest contributions to soil stability are made by biological components of the system. Biological soil crust cover was found to have the strongest direct effect on surface soil stability (0.60; controlling for other factors). Surprisingly, AM fungi appeared to influence surface soil stability (0.37), even though they are not generally considered to exist in the top few millimeters of the soil. In the subsurface model, plant cover appeared to have the strongest direct influence on soil stability (0.42); in both models, results indicate that plant cover influences soil stability both directly (controlling for other factors) and indirectly through influences on other organisms. Soil organic matter was not found to have a direct contribution to surface or subsurface stability in this system. The relative influence of AM fungi on soil stability in these semiarid shrublands was similar to that reported for a mesic tallgrass prairie. Estimates of effects that BSCs, plants, and AM fungi have

  15. Structuring detergents for extracting and stabilizing functional membrane proteins.

    Directory of Open Access Journals (Sweden)

    Rima Matar-Merheb

    Full Text Available BACKGROUND: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. METHODOLOGY/PRINCIPAL FINDINGS: Anionic calix[4]arene based detergents (C4Cn, n=1-12 were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein, a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM. They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux much more efficiently than SDS (sodium dodecyl sulphate, FC12 (Foscholine 12 or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. CONCLUSION/SIGNIFICANCE: These compounds seem promising to extract in a functional state

  16. Mutation of exposed hydrophobic amino acids to arginine to increase protein stability

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    Czaplicki Jerzy

    2004-07-01

    Full Text Available Abstract Background One strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface. Results In order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion. Conclusion Altough the mutational effects were rather small, this strategy proved to be successful since half of the mutants showed an increased stability. This stability may originate from the suppression of unfavorable interactions of nonpolar residues with water or from addition of new hydrogen bonds with the solvent. Other mechanisms may also contribute to the increased stability observed with some mutants. For example, introduction of a charge at the surface of the protein may provide a new coulombic interaction on the protein surface.

  17. Contribution of simple saccharides to the stabilization of amyloid structure

    International Nuclear Information System (INIS)

    Fung, Justin; Darabie, Audrey A.; McLaurin, JoAnne

    2005-01-01

    The use of osmolytes or chaperones to stabilize proteins/peptides that misfold in neurodegenerative diseases is an attractive concept for drug development. We have investigated the role of a series of small carbohydrates for protection of the natively structured Alzheimer's amyloid-β peptides (Aβ). Using circular dichroism spectroscopy to follow the β-structural transitions and electron microscopy to examine tertiary structural characteristics, we demonstrate that the hydrogen bonding capacity of the carbohydrate determines the inhibition or promotion of fibrillogenesis. Three sugar molecules that vary only in their distribution of potential H-bonding partners promote various structural changes in Aβ. Two of these sugar molecules are excluded from Aβ during aggregation and promote mature fibre growth, while the other binds Aβ promoting nucleation and the accumulation of protofibrils. Our studies suggest that utilization of a combinatorial strategy to alter H-bonding capacity across a simple carbohydrate molecule may represent a novel drug design strategy

  18. Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding.

    Directory of Open Access Journals (Sweden)

    Chi-Ho Chan

    Full Text Available Most thermophilic proteins tend to have more salt bridges, and achieve higher thermostability by up-shifting and broadening their protein stability curves. While the stabilizing effect of salt-bridge has been extensively studied, experimental data on how salt-bridge influences protein stability curves are scarce. Here, we used double mutant cycles to determine the temperature-dependency of the pair-wise interaction energy and the contribution of salt-bridges to ΔC(p in a thermophilic ribosomal protein L30e. Our results showed that the pair-wise interaction energies for the salt-bridges E6/R92 and E62/K46 were stabilizing and insensitive to temperature changes from 298 to 348 K. On the other hand, the pair-wise interaction energies between the control long-range ion-pair of E90/R92 were negligible. The ΔC(p of all single and double mutants were determined by Gibbs-Helmholtz and Kirchhoff analyses. We showed that the two stabilizing salt-bridges contributed to a reduction of ΔC(p by 0.8-1.0 kJ mol⁻¹ K⁻¹. Taken together, our results suggest that the extra salt-bridges found in thermophilic proteins enhance the thermostability of proteins by reducing ΔC(p, leading to the up-shifting and broadening of the protein stability curves.

  19. Protein kinesis: The dynamics of protein trafficking and stability

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  20. Hydrophobic environment is a key factor for the stability of thermophilic proteins.

    Science.gov (United States)

    Gromiha, M Michael; Pathak, Manish C; Saraboji, Kadhirvel; Ortlund, Eric A; Gaucher, Eric A

    2013-04-01

    The stability of thermophilic proteins has been viewed from different perspectives and there is yet no unified principle to understand this stability. It would be valuable to reveal the most important interactions for designing thermostable proteins for such applications as industrial protein engineering. In this work, we have systematically analyzed the importance of various interactions by computing different parameters such as surrounding hydrophobicity, inter-residue interactions, ion-pairs and hydrogen bonds. The importance of each interaction has been determined by its predicted relative contribution in thermophiles versus the same contribution in mesophilic homologues based on a dataset of 373 protein families. We predict that hydrophobic environment is the major factor for the stability of thermophilic proteins and found that 80% of thermophilic proteins analyzed showed higher hydrophobicity than their mesophilic counterparts. Ion pairs, hydrogen bonds, and interaction energy are also important and favored in 68%, 50%, and 62% of thermophilic proteins, respectively. Interestingly, thermophilic proteins with decreased hydrophobic environments display a greater number of hydrogen bonds and/or ion pairs. The systematic elimination of mesophilic proteins based on surrounding hydrophobicity, interaction energy, and ion pairs/hydrogen bonds, led to correctly identifying 95% of the thermophilic proteins in our analyses. Our analysis was also applied to another, more refined set of 102 thermophilic-mesophilic pairs, which again identified hydrophobicity as a dominant property in 71% of the thermophilic proteins. Further, the notion of surrounding hydrophobicity, which characterizes the hydrophobic behavior of residues in a protein environment, has been applied to the three-dimensional structures of elongation factor-Tu proteins and we found that the thermophilic proteins are enriched with a hydrophobic environment. The results obtained in this work highlight the

  1. SUMOylation Confers Posttranslational Stability on NPM-ALK Oncogenic Protein

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    Deeksha Vishwamitra

    2015-09-01

    Full Text Available Nucleophosmin-anaplastic lymphoma kinase–expressing (NPM-ALK+ T-cell lymphoma is an aggressive form of cancer that commonly affects children and adolescents. The expression of NPM-ALK chimeric oncogene results from the chromosomal translocation t(2;5(p23;q35 that causes the fusion of the ALK and NPM genes. This translocation generates the NPM-ALK protein tyrosine kinase that forms the constitutively activated NPM-ALK/NPM-ALK homodimers. In addition, NPM-ALK is structurally associated with wild-type NPM to form NPM/NPM-ALK heterodimers, which can translocate to the nucleus. The mechanisms that sustain the stability of NPM-ALK are not fully understood. SUMOylation is a posttranslational modification that is characterized by the reversible conjugation of small ubiquitin-like modifiers (SUMOs with target proteins. SUMO competes with ubiquitin for substrate binding and therefore, SUMOylation is believed to protect target proteins from proteasomal degradation. Moreover, SUMOylation contributes to the subcellular distribution of target proteins. Herein, we found that the SUMOylation pathway is deregulated in NPM-ALK+ T-cell lymphoma cell lines and primary lymphoma tumors from patients. We also identified Lys24 and Lys32 within the NPM domain as the sites where NPM-ALK conjugates with SUMO-1 and SUMO-3. Importantly, antagonizing SUMOylation by the SENP1 protease decreased the accumulation of NPM-ALK and suppressed lymphoma cell viability, proliferation, and anchorage-independent colony formation. One possible mechanism for the SENP1-mediated decrease in NPM-ALK levels was the increase in NPM-ALK association with ubiquitin, which facilitates its degradation. Our findings propose a model in which aberrancies in SUMOylation contribute to the pathogenesis of NPM-ALK+ T-cell lymphoma. Unraveling such pathogenic mechanisms may lead to devising novel strategies to eliminate this aggressive neoplasm.

  2. Increasing protein stability by improving beta-turns.

    Science.gov (United States)

    Fu, Hailong; Grimsley, Gerald R; Razvi, Abbas; Scholtz, J Martin; Pace, C Nick

    2009-11-15

    Our goal was to gain a better understanding of how protein stability can be increased by improving beta-turns. We studied 22 beta-turns in nine proteins with 66-370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some beta-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein, Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase alpha-subunit, and Maltose binding protein. Of the 15 single proline mutations, 11 increased stability (Average = 0.8 +/- 0.3; Range = 0.3-1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. On the basis of this and our previous work, we conclude that proteins can generally be stabilized by replacing nonproline residues with proline residues at the i + 1 position of Type I and II beta-turns and at the i position in Type II beta-turns. Other turn positions can sometimes be used if the phi angle is near -60 degrees for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in beta-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in beta-turns that could be replaced by Gly to increase protein stability. Improving beta-turns by substituting Pro residues is a generally useful way of increasing protein stability. 2009 Wiley-Liss, Inc.

  3. Effects of protein phosphorylation on color stability of ground meat.

    Science.gov (United States)

    Li, Meng; Li, Xin; Xin, Jianzeng; Li, Zheng; Li, Guixia; Zhang, Yan; Du, Manting; Shen, Qingwu W; Zhang, Dequan

    2017-03-15

    The influence of protein phosphorylation on meat color stability was investigated in this study. Phosphatase and protein kinase inhibitors were added to minced ovine Longissimus thoracis et lumborum (LTL) muscle to manipulate the global phosphorylation of sarcoplasmic proteins. The data obtained show that the rate and extent of pH decline, along with lactate accumulation in postmortem muscle, were related to protein phosphorylation. Analysis of meat color and the relative content of myoglobin redox forms revealed that meat color stability was inversely related to the phosphorylation of sarcoplasmic proteins. Thus, this study suggests that protein phosphorylation may be involved in meat color development by regulating glycolysis and the redox stability of myoglobin. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. The contribution of coevolving residues to the stability of KDO8P synthase.

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    Sharon H Ackerman

    2011-03-01

    Full Text Available The evolutionary tree of 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P synthase (KDO8PS, a bacterial enzyme that catalyzes a key step in the biosynthesis of bacterial endotoxin, is evenly divided between metal and non-metal forms, both having similar structures, but diverging in various degrees in amino acid sequence. Mutagenesis, crystallographic and computational studies have established that only a few residues determine whether or not KDO8PS requires a metal for function. The remaining divergence in the amino acid sequence of KDO8PSs is apparently unrelated to the underlying catalytic mechanism.The multiple alignment of all known KDO8PS sequences reveals that several residue pairs coevolved, an indication of their possible linkage to a structural constraint. In this study we investigated by computational means the contribution of coevolving residues to the stability of KDO8PS. We found that about 1/4 of all strongly coevolving pairs probably originated from cycles of mutation (decreasing stability and suppression (restoring it, while the remaining pairs are best explained by a succession of neutral or nearly neutral covarions.Both sequence conservation and coevolution are involved in the preservation of the core structure of KDO8PS, but the contribution of coevolving residues is, in proportion, smaller. This is because small stability gains or losses associated with selection of certain residues in some regions of the stability landscape of KDO8PS are easily offset by a large number of possible changes in other regions. While this effect increases the tolerance of KDO8PS to deleterious mutations, it also decreases the probability that specific pairs of residues could have a strong contribution to the thermodynamic stability of the protein.

  5. Effect of pasteurization on the protein composition and oxidative stability of beer during storage.

    Science.gov (United States)

    Lund, Marianne N; Hoff, Signe; Berner, Torben S; Lametsch, René; Andersen, Mogens L

    2012-12-19

    The impacts of pasteurization of a lager beer on protein composition and the oxidative stability were studied during storage at 22 °C for 426 days in the dark. Pasteurization clearly improved the oxidative stability of beer determined by ESR spectroscopy, whereas it had a minor negative effect on the volatile profile by increasing volatile compounds that is generally associated with heat treatment and a loss of fruity ester aroma. A faster rate of radical formation in unpasteurized beer was consistent with a faster consumption of sulfite. Beer proteins in the unpasteurized beer were more degraded, most likely due to proteolytic enzyme activity of yeast remnants and more precipitation of proteins was also observed. The differences in soluble protein content and composition are suggested to result in differences in the contents of prooxidative metals as a consequence of the proteins ability to bind metals. This also contributes to the differences in oxidative stabilities of the beers.

  6. Applications of Protein Thermodynamic Database for Understanding Protein Mutant Stability and Designing Stable Mutants.

    Science.gov (United States)

    Gromiha, M Michael; Anoosha, P; Huang, Liang-Tsung

    2016-01-01

    Protein stability is the free energy difference between unfolded and folded states of a protein, which lies in the range of 5-25 kcal/mol. Experimentally, protein stability is measured with circular dichroism, differential scanning calorimetry, and fluorescence spectroscopy using thermal and denaturant denaturation methods. These experimental data have been accumulated in the form of a database, ProTherm, thermodynamic database for proteins and mutants. It also contains sequence and structure information of a protein, experimental methods and conditions, and literature information. Different features such as search, display, and sorting options and visualization tools have been incorporated in the database. ProTherm is a valuable resource for understanding/predicting the stability of proteins and it can be accessed at http://www.abren.net/protherm/ . ProTherm has been effectively used to examine the relationship among thermodynamics, structure, and function of proteins. We describe the recent progress on the development of methods for understanding/predicting protein stability, such as (1) general trends on mutational effects on stability, (2) relationship between the stability of protein mutants and amino acid properties, (3) applications of protein three-dimensional structures for predicting their stability upon point mutations, (4) prediction of protein stability upon single mutations from amino acid sequence, and (5) prediction methods for addressing double mutants. A list of online resources for predicting has also been provided.

  7. Nanostructured Mineral Coatings Stabilize Proteins for Therapeutic Delivery.

    Science.gov (United States)

    Yu, Xiaohua; Biedrzycki, Adam H; Khalil, Andrew S; Hess, Dalton; Umhoefer, Jennifer M; Markel, Mark D; Murphy, William L

    2017-09-01

    Proteins tend to lose their biological activity due to their fragile structural conformation during formulation, storage, and delivery. Thus, the inability to stabilize proteins in controlled-release systems represents a major obstacle in drug delivery. Here, a bone mineral inspired protein stabilization strategy is presented, which uses nanostructured mineral coatings on medical devices. Proteins bound within the nanostructured coatings demonstrate enhanced stability against extreme external stressors, including organic solvents, proteases, and ethylene oxide gas sterilization. The protein stabilization effect is attributed to the maintenance of protein conformational structure, which is closely related to the nanoscale feature sizes of the mineral coatings. Basic fibroblast growth factor (bFGF) released from a nanostructured mineral coating maintains its biological activity for weeks during release, while it maintains activity for less than 7 d during release from commonly used polymeric microspheres. Delivery of the growth factors bFGF and vascular endothelial growth factor using a mineral coated surgical suture significantly improves functional Achilles tendon healing in a rabbit model, resulting in increased vascularization, more mature collagen fiber organization, and a two fold improvement in mechanical properties. The findings of this study demonstrate that biomimetic interactions between proteins and nanostructured minerals provide a new, broadly applicable mechanism to stabilize proteins in the context of drug delivery and regenerative medicine. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Thermal precipitation fluorescence assay for protein stability screening.

    Science.gov (United States)

    Fan, Junping; Huang, Bo; Wang, Xianping; Zhang, Xuejun C

    2011-09-01

    A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Tandem Facial Amphiphiles for Membrane Protein Stabilization

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Gotfryd, Kamil; Pacyna, Jennifer

    2010-01-01

    We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles....

  10. Stabilization of Proteins and Noncovalent Protein Complexes during Electrospray Ionization by Amino Acid Additives.

    Science.gov (United States)

    Zhang, Hua; Lu, Haiyan; Chingin, Konstantin; Chen, Huanwen

    2015-07-21

    Ionization of proteins and noncovalent protein complexes with minimal disturbance to their native structure presents a great challenge for biological mass spectrometry (MS). In living organisms, the native structure of intracellular proteins is commonly stabilized by solute amino acids (AAs) accumulated in cells at very high concentrations. Inspired by nature, we hypothesized that AAs could also pose a stabilizing effect on the native structure of proteins and noncovalent protein complexes during ionization. To test this hypothesis, here we explored MS response for various protein complexes upon the addition of free AAs at mM concentrations into the electrospray ionization (ESI) solution. Thermal activation of ESI droplets in the MS inlet capillary was employed as a model destabilizing factor during ionization. Our results indicate that certain AAs, in particular proline (Pro), pose considerable positive effect on the stability of noncovalent protein complexes in ESI-MS without affecting the signal intensity of protein ions and original protein-ligand equilibrium, even when added at the 20 mM concentration. The data suggest that the degree of protein stabilization is primarily determined by the osmolytic and ampholytic characteristics of AA solutes. The highest stability and visibility of noncovalent protein complexes in ESI-MS are achieved using AA additives with neutral isoelectric point, moderate proton affinity, and unfavorable interaction with the native protein state. Overall, our results indicate that the simple addition of free amino acids into the working solution can notably improve the stability and accuracy of protein analysis by native ESI-MS.

  11. New insights into structural determinants of prion protein folding and stability.

    Science.gov (United States)

    Benetti, Federico; Legname, Giuseppe

    2015-01-01

    Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability.

  12. Physicochemical stability of lycopene-loaded emulsions stabilized by plant or dairy proteins

    NARCIS (Netherlands)

    Ho, Kacie; Schroen, C.G.P.H.; San Martín-González, M.F.; Berton-Carabin, C.C.

    2017-01-01

    Lycopene is a lipophilic bioactive compound that can be challenging to deliver in vivo. To mediate this, delivery strategies, such as protein-stabilized oil-in-water (O/W) emulsions, have been suggested to improve the physicochemical stability and bioavailability of lycopene. In this research, the

  13. Contributions to fuzzy polynomial techniques for stability analysis and control

    OpenAIRE

    Pitarch Pérez, José Luis

    2014-01-01

    The present thesis employs fuzzy-polynomial control techniques in order to improve the stability analysis and control of nonlinear systems. Initially, it reviews the more extended techniques in the field of Takagi-Sugeno fuzzy systems, such as the more relevant results about polynomial and fuzzy polynomial systems. The basic framework uses fuzzy polynomial models by Taylor series and sum-of-squares techniques (semidefinite programming) in order to obtain stability guarantees...

  14. Protein stability and enzyme activity at extreme biological temperatures

    International Nuclear Information System (INIS)

    Feller, Georges

    2010-01-01

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 0 C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins. (topical review)

  15. Protein engineering of subtilisins to improve stability in detergent formulations.

    Science.gov (United States)

    von der Osten, C; Branner, S; Hastrup, S; Hedegaard, L; Rasmussen, M D; Bisgård-Frantzen, H; Carlsen, S; Mikkelsen, J M

    1993-03-01

    Microbial proteases are used extensively in a large number of industrial processes and most importantly in detergent formulations facilitating the removal of proteinaceous stains. Site-directed mutagenesis has been employed in the construction of subtilisin variants with improved storage and oxidation stabilities. It is shown that in spite of significant structural homology between subtilisins subjected to protein engineering the effects of specific mutations can be quite different. Mutations that stabilize one subtilisin may destabilize another.

  16. Contribution of cation-π interactions to the stability of Sm/LSm oligomeric assemblies.

    Science.gov (United States)

    Mucić, Ivana D; Nikolić, Milan R; Stojanović, Srđan Đ

    2015-07-01

    In this work, we have analyzed the influence of cation-π interactions to the stability of Sm/LSm assemblies and their environmental preferences. The number of interactions formed by arginine is higher than lysine in the cationic group, while histidine is comparatively higher than phenylalanine and tyrosine in the π group. Arg-Tyr interactions are predominant among the various pairs analyzed. The furcation level of multiple cation-π interactions is much higher than that of single cation-π interactions in Sm/LSm interfaces. We have found hot spot residues forming cation-π interactions, and hot spot composition is similar for all aromatic residues. The Arg-Phe pair has the strongest interaction energy of -8.81 kcal mol(-1) among all the possible pairs of amino acids. The extent of burial of the residue side-chain correlates with the ΔΔG of binding for residues in the core and also for hot spot residues cation-π bonded across the interface. Secondary structure of the cation-π residues shows that Arg and Lys preferred to be in strand. Among the π residues, His prefers to be in helix, Phe prefers to be in turn, and Tyr prefers to be in strand. Stabilization centers for these proteins showed that all the five residues found in cation-π interactions are important in locating one or more of such centers. More than 50 % of the cation-π interacting residues are highly conserved. It is likely that the cation-π interactions contribute significantly to the overall stability of Sm/LSm proteins.

  17. Contributions of depth filter components to protein adsorption in bioprocessing.

    Science.gov (United States)

    Khanal, Ohnmar; Singh, Nripen; Traylor, Steven J; Xu, Xuankuo; Ghose, Sanchayita; Li, Zheng J; Lenhoff, Abraham M

    2018-04-16

    Depth filtration is widely used in downstream bioprocessing to remove particulate contaminants via depth straining and is therefore applied to harvest clarification and other processing steps. However, depth filtration also removes proteins via adsorption, which can contribute variously to impurity clearance and to reduction in product yield. The adsorption may occur on the different components of the depth filter, that is, filter aid, binder, and cellulose filter. We measured adsorption of several model proteins and therapeutic proteins onto filter aids, cellulose, and commercial depth filters at pH 5-8 and ionic strengths filter component in the adsorption of proteins with different net charges, using confocal microscopy. Our findings show that a complete depth filter's maximum adsorptive capacity for proteins can be estimated by its protein monolayer coverage values, which are of order mg/m 2 , depending on the protein size. Furthermore, the extent of adsorption of different proteins appears to depend on the nature of the resin binder and its extent of coating over the depth filter surface, particularly in masking the cation-exchanger-like capacity of the siliceous filter aids. In addition to guiding improved depth filter selection, the findings can be leveraged in inspiring a more intentional selection of components and design of depth filter construction for particular impurity removal targets. © 2018 Wiley Periodicals, Inc.

  18. Enhanced Stability of a Protein with Increasing Temperature

    DEFF Research Database (Denmark)

    Vinther, Joachim Møllesøe; Kristensen, Søren M; Led, Jens J

    2010-01-01

    The unusual stability of a structured but locally flexible protein, human growth hormone (hGH) at pH 2.7, was investigated using the temperature dependence of the nanosecond-picosecond dynamics of the backbone amide groups obtained from (15)N NMR relaxation data. It is found that the flexibility ...

  19. Salinity induced changes in cell membrane stability, protein and ...

    African Journals Online (AJOL)

    control), 4.7, 9.4 and 14.1 dS m-1 to determine the effect of salt on vegetative growth, relative water content, cell membrane stability, protein and RNA contents in sand culture experiment. Fresh and dry weights of plants, shoots and roots decreased ...

  20. Uric acid contributes greatly to hepatic antioxidant capacity besides protein.

    Science.gov (United States)

    Mikami, T; Sorimachi, M

    2017-12-20

    Uric acid is the end-product of purine nucleotide metabolism and an increase in uric acid concentration in the body results in hyperuricemia, ultimately leading to gout. However, uric acid is a potent antioxidant and interacts with reactive oxygen species (ROS) to be non-enzymatically converted to allantoin. Uric acid accounts for approximately 60 % of antioxidant capacity in the plasma; however, its contribution to tissue antioxidant capacity is unknown. In this study, the contribution of uric acid to tissue antioxidant capacity and its conversion to allantoin by scavenging ROS in tissue were examined. The results showed that a decrease in hepatic uric acid content via allopurinol administration significantly reduced hepatic total-radical trapping antioxidant parameter (TRAP) content in protein-free cytosol. Additionally, treating protein-free cytosol with uricase led to a further reduction of hepatic TRAP content. Allantoin was also detected in the solution containing protein-free cytosol that reacted with ROS. These findings suggest that in the absence of protein, uric acid contributes greatly to antioxidant capacity in the liver, where uric acid is converted to allantoin by scavenging ROS.

  1. Self-assembling peptides form nanodiscs that stabilize membrane proteins

    DEFF Research Database (Denmark)

    Midtgaard, Søren Roi; Pedersen, Martin Cramer; Kirkensgaard, Jacob Judas Kain

    2014-01-01

    -ray scattering (SAXS) and small-angle neutron scattering (SANS) supported by coarse-grained molecular dynamics simulations. The detailed structure of the discs was determined in unprecedented detail and it was found that they adopt a discoidal structure very similar to the ApoA1 based nanodiscs. We furthermore...... show that, like the ApoA1 and derived nanodiscs, these peptide discs can accommodate and stabilize a membrane protein. Finally, we exploit their dynamic properties and show that the 18A discs may be used for transferring membrane proteins and associated phospholipids directly and gently......New methods to handle membrane bound proteins, e.g. G-protein coupled receptors (GPCRs), are highly desirable. Recently, apoliprotein A1 (ApoA1) based lipoprotein particles have emerged as a new platform for studying membrane proteins, and it has been shown that they can self...

  2. StAR Protein Stability in Y1 and Kin-8 Mouse Adrenocortical Cells.

    Science.gov (United States)

    Clark, Barbara J; Hudson, Elizabeth A

    2015-03-04

    The steroidogenic acute regulatory protein (STAR) protein expression is required for cholesterol transport into mitochondria to initiate steroidogenesis in the adrenal and gonads. STAR is synthesized as a 37 kDa precursor protein which is targeted to the mitochondria and imported and processed to an intra-mitochondrial 30 kDa protein. Tropic hormone stimulation of the cAMP-dependent protein kinase A (PKA) signaling pathway is the major contributor to the transcriptional and post-transcriptional regulation of STAR synthesis. Many studies have focused on the mechanisms of cAMP-PKA mediated control of STAR synthesis while there are few reports on STAR degradation pathways. The objective of this study was to determine the effect of cAMP-PKA-dependent signaling on STAR protein stability. We have used the cAMP-PKA responsive Y1 mouse adrenocortical cells and the PKA-deficient Kin-8 cells to measure STAR phosphorylation and protein half-life. Western blot analysis and standard radiolabeled pulse-chase experiments were used to determine STAR phosphorylation status and protein half-life, respectively. Our data demonstrate that PKA-dependent STAR phosphorylation does not contribute to 30 kDa STAR protein stability in the mitochondria. We further show that inhibition of the 26S proteasome does not block precursor STAR phosphorylation or steroid production in Y1 cells. These data suggest STAR can maintain function and promote steroidogenesis under conditions of proteasome inhibition.

  3. Thermodynamic effects of proline introduction on protein stability.

    Science.gov (United States)

    Prajapati, Ravindra Singh; Das, Mili; Sreeramulu, Sridhar; Sirajuddin, Minhajuddin; Srinivasan, Sankaranarayanan; Krishnamurthy, Vaishnavi; Ranjani, Ranganathan; Ramakrishnan, C; Varadarajan, Raghavan

    2007-02-01

    The amino acid Pro is more rigid than other naturally occurring amino acids and, in proteins, lacks an amide hydrogen. To understand the structural and thermodynamic effects of Pro substitutions, it was introduced at 13 different positions in four different proteins, leucine-isoleucine-valine binding protein, maltose binding protein, ribose binding protein, and thioredoxin. Three of the maltose binding protein mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, fluorescence and CD spectroscopy were used to show the absence of structural change. Stabilities of wild type and mutant proteins were characterized by chemical denaturation at neutral pH and by differential scanning calorimetry as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, 6 of the 13 single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, 2 mutants showed no change, and 5 were destabilized. In five of the six cases, the stabilization was because of reduced entropy of unfolding. However, the magnitude of the reduction in entropy of unfolding was typically several fold larger than the theoretical estimate of -4 cal K(-1) mol(-1) derived from the relative areas in the Ramachandran map accessible to Pro and Ala residues, respectively. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were nonadditive. Copyright 2006 Wiley-Liss, Inc.

  4. Contribution of TyrB26 to the Function and Stability of Insulin

    Science.gov (United States)

    Pandyarajan, Vijay; Phillips, Nelson B.; Rege, Nischay; Lawrence, Michael C.; Whittaker, Jonathan; Weiss, Michael A.

    2016-01-01

    Crystallographic studies of insulin bound to receptor domains have defined the primary hormone-receptor interface. We investigated the role of TyrB26, a conserved aromatic residue at this interface. To probe the evolutionary basis for such conservation, we constructed 18 variants at B26. Surprisingly, non-aromatic polar or charged side chains (such as Glu, Ser, or ornithine (Orn)) conferred high activity, whereas the weakest-binding analogs contained Val, Ile, and Leu substitutions. Modeling of variant complexes suggested that the B26 side chains pack within a shallow depression at the solvent-exposed periphery of the interface. This interface would disfavor large aliphatic side chains. The analogs with highest activity exhibited reduced thermodynamic stability and heightened susceptibility to fibrillation. Perturbed self-assembly was also demonstrated in studies of the charged variants (Orn and Glu); indeed, the GluB26 analog exhibited aberrant aggregation in either the presence or absence of zinc ions. Thus, although TyrB26 is part of insulin's receptor-binding surface, our results suggest that its conservation has been enjoined by the aromatic ring's contributions to native stability and self-assembly. We envisage that such classical structural relationships reflect the implicit threat of toxic misfolding (rather than hormonal function at the receptor level) as a general evolutionary determinant of extant protein sequences. PMID:27129279

  5. On the effect of hydrostatic pressure on the conformational stability of globular proteins.

    Science.gov (United States)

    Graziano, Giuseppe

    2015-12-01

    The model developed for cold denaturation (Graziano, PCCP 2010, 12, 14245-14252) is extended to rationalize the dependence of protein conformational stability upon hydrostatic pressure, at room temperature. A pressure- volume work is associated with the process of cavity creation for the need to enlarge the liquid volume against hydrostatic pressure. This contribution destabilizes the native state that has a molecular volume slightly larger than the denatured state due to voids existing in the protein core. Therefore, there is a hydrostatic pressure value at which the pressure-volume contribution plus the conformational entropy loss of the polypeptide chain are able to overwhelm the stabilizing gain in translational entropy of water molecules, due to the decrease in water accessible surface area upon folding, causing denaturation. © 2015 Wiley Periodicals, Inc.

  6. Dissecting the critical factors for thermodynamic stability of modular proteins using molecular modeling approach.

    Directory of Open Access Journals (Sweden)

    Yuno Lee

    Full Text Available Repeat proteins have recently attracted much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural and biophysical features. In particular, repeat proteins show high stability against temperature and chaotic agents. Despite many studies, structural features for the stability of repeat proteins remain poorly understood. Here we present an interesting result from in silico analyses pursuing the factors which affect the stability of repeat proteins. Previously developed repebody structure based on variable lymphocytes receptors (VLRs which consists of leucine-rich repeat (LRR modules was used as initial structure for the present study. We constructed extra six repebody structures with varying numbers of repeat modules and those structures were used for molecular dynamics simulations. For the structures, the intramolecular interactions including backbone H-bonds, van der Waals energy, and hydrophobicity were investigated and then the radius of gyration, solvent-accessible surface area, ratio of secondary structure, and hydration free energy were also calculated to find out the relationship between the number of LRR modules and stability of the protein. Our results show that the intramolecular interactions lead to more compact structure and smaller surface area of the repebodies, which are critical for the stability of repeat proteins. The other features were also well compatible with the experimental results. Based on our observations, the repebody-5 was proposed as the best structure from the all repebodies in structure optimization process. The present study successfully demonstrated that our computer-based molecular modeling approach can significantly contribute to the experiment-based protein engineering challenge.

  7. The otolithic contribution to vertical ocular stability in the cat.

    Science.gov (United States)

    Pettorossi, V E; Draicchio, F; Ferraresi, A; Bruni, R

    1994-10-01

    enhancement of gain and reduction of phase lead at low frequency vestibular stimulation, resulting in similar vertical and horizontal gaze stability; 2) the equalization of the upward and downward responses of both vestibulo-ocular and optokinetic responses; 3) the reduction of the amplitude and frequency of vertical quick phases.

  8. USING BIOPOLYMERS TO STABILIZE THE PROTEIN OXYGEN FOAM

    Directory of Open Access Journals (Sweden)

    N. V. Nepovinnyh

    2013-01-01

    Full Text Available The cottage cheese whey as an oxygen cocktail foaming base and natural juices as a flavoring ingredient are analyzed. The lifetime of foam generated by the serum proteins is not long: foam falls off rapidly; because from the foam liquid is released (syneresis. The effects of plant polysaccharides on the stabilization of the protein foam oxygen cocktail is studied. It was shown that the use of plant polysaccharides (guar gum, high methoxyl citrus pectin, locust been gum prolong the life of the foam up to 20 times, compared with conventional blowing agents. It was found that oxygen foam properties depend on the molecular weight of guar gum.

  9. Direct measurements of protein-stabilized gold nanoparticle interactions.

    Science.gov (United States)

    Eichmann, Shannon L; Bevan, Michael A

    2010-09-21

    We report integrated video and total internal reflection microscopy measurements of protein stabilized 110 nm Au nanoparticles confined in 280 nm gaps in physiological media. Measured potential energy profiles display quantitative agreement with Brownian dynamic simulations that include hydrodynamic interactions and camera exposure time and noise effects. Our results demonstrate agreement between measured nonspecific van der Waals and adsorbed protein interactions with theoretical potentials. Confined, lateral nanoparticle diffusivity measurements also display excellent agreement with predictions. These findings provide a basis to interrogate specific biomacromolecular interactions in similar experimental configurations and to design future improved measurement methods.

  10. Mechanism of Stabilization of Labile Compounds by Silk Fibroin Proteins

    Science.gov (United States)

    2017-04-05

    saliva, or urine , and their collection and storage is critical to obtain reliable results. Without proper temperature regulation protein biomarkers in... samples for long-term ambient storage and subsequent on-demand recovery and laboratory analysis. Air dried silks provide a protective barrier that...silk in the stabilization of a range of different analytes, including entrapment, storage and recovery. Here, we successfully used silk fibroin as a

  11. Human sperm degradation of zona pellucida proteins contributes to fertilization.

    Science.gov (United States)

    Saldívar-Hernández, Analilia; González-González, María E; Sánchez-Tusié, Ana; Maldonado-Rosas, Israel; López, Pablo; Treviño, Claudia L; Larrea, Fernando; Chirinos, Mayel

    2015-09-02

    The mammalian oocyte extracellular matrix known as the zona pellucida (ZP) acts as a barrier to accomplish sperm fusion with the female gamete. Although penetration of the ZP is a limiting event to achieve fertilization, this is one of the least comprehended stages of gamete interaction. Even though previous studies suggest that proteases of sperm origin contribute to facilitate the passage of sperm through the ZP, in human this process is not yet fully understood. The aim of this study was to determine the ability of human sperm to degrade recombinant human ZP (rhZPs) proteins and to characterize the proteases involved in this process. Purified rhZP2, rhZP3 and rhZP4 proteins were incubated with capacitated sperm and the proteolytic activity was determined by Western blot analysis. To further characterize the proteases involved, parallel incubations were performed in the presence of the protease inhibitors o-phenanthroline, benzamidine and MG-132 meant to block the activity of metalloproteases, serine proteases and the proteasome, respectively. Additionally, protease inhibitors effect on sperm-ZP binding was evaluated by hemizona assay. The results showed that rhZPs were hydrolyzed in the presence of capacitated sperm. O-phenanthroline inhibited the degradation of rhZP3, MG-132 inhibited the degradation of rhZP4 and benzamidine inhibited the degradation of the three proteins under investigation. Moreover, hemizona assays demonstrated that sperm proteasome inhibition impairs sperm interaction with human native ZP. This study suggests that sperm proteasomes could participate in the degradation of ZP, particularly of the ZP4 protein. Besides, metalloproteases may be involved in specific degradation of ZP3 while serine proteases may contribute to unspecific degradation of the ZP. These findings suggest that localized degradation of ZP proteins by sperm is probably involved in ZP penetration and may be of help in understanding the mechanisms of fertilization in humans.

  12. Stability of halophilic proteins: from dipeptide attributes to discrimination classifier.

    Science.gov (United States)

    Zhang, Guangya; Huihua, Ge; Yi, Lin

    2013-02-01

    To investigate the molecular features responsible for protein halophilicity is of great significance for understanding the structure basis of protein halo-stability and would help to develop a practical strategy for designing halophilic proteins. In this work, we have systematically analyzed the dipeptide composition of the halophilic and non-halophilic protein sequences. We observed the halophilic proteins contained more DA, RA, AD, RR, AP, DD, PD, EA, VG and DV at the expense of LK, IL, II, IA, KK, IS, KA, GK, RK and AI. We identified some macromolecular signatures of halo-adaptation, and thought the dipeptide composition might contain more information than amino acid composition. Based on the dipeptide composition, we have developed a machine learning method for classifying halophilic and non-halophilic proteins for the first time. The accuracy of our method for the training dataset was 100.0%, and for the 10-fold cross-validation was 93.1%. We also discussed the influence of some specific dipeptides on prediction accuracy. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Effective stabilization of CLA by microencapsulation in pea protein.

    Science.gov (United States)

    Costa, A M M; Nunes, J C; Lima, B N B; Pedrosa, C; Calado, V; Torres, A G; Pierucci, A P T R

    2015-02-01

    CLA was microencapsulated by spray drying in ten varied wall systems (WS) consisting of pea protein isolate or pea protein concentrate (PPC) alone at varied core:WS ratios (1:2; 1:3 and 1:4), or blended with maltodextrin (M) and carboxymethylcellulose at a pea protein:carbohydrate ratio of 3:1. The physical-chemical properties of the CLA microparticles were characterised by core retention, microencapsulation efficiency (ME), particle size and moisture. CLA:M:PPC (1:1:3) showed the most promising results, thus we evaluated the effect of M addition in the WS on other physical-chemical characteristics and oxidative stability (CLA isomer profile, quantification of CLA and volatile compounds by SPME coupled with CG-MS) during two months of storage at room temperature, CLA:PPC (1:4) was selected for comparisons. CLA:M:PPC (1:1:3) microparticles demonstrated better morphology, solubility, dispersibility and higher glass-transition temperature values. M addition did not influence the oxidative stability of CLA, however its presence improved physical-chemical characteristics necessary for food applications. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer

    Science.gov (United States)

    Guinn, Emily J.; Pegram, Laurel M.; Capp, Michael W.; Pollock, Michelle N.; Record, M. Thomas

    2011-01-01

    To explain the large, opposite effects of urea and glycine betaine (GB) on stability of folded proteins and protein complexes, we quantify and interpret preferential interactions of urea with 45 model compounds displaying protein functional groups and compare with a previous analysis of GB. This information is needed to use urea as a probe of coupled folding in protein processes and to tune molecular dynamics force fields. Preferential interactions between urea and model compounds relative to their interactions with water are determined by osmometry or solubility and dissected using a unique coarse-grained analysis to obtain interaction potentials quantifying the interaction of urea with each significant type of protein surface (aliphatic, aromatic hydrocarbon (C); polar and charged N and O). Microscopic local-bulk partition coefficients Kp for the accumulation or exclusion of urea in the water of hydration of these surfaces relative to bulk water are obtained. Kp values reveal that urea accumulates moderately at amide O and weakly at aliphatic C, whereas GB is excluded from both. These results provide both thermodynamic and molecular explanations for the opposite effects of urea and glycine betaine on protein stability, as well as deductions about strengths of amide NH—amide O and amide NH—amide N hydrogen bonds relative to hydrogen bonds to water. Interestingly, urea, like GB, is moderately accumulated at aromatic C surface. Urea m-values for protein folding and other protein processes are quantitatively interpreted and predicted using these urea interaction potentials or Kp values. PMID:21930943

  15. New directions towards structure formation and stability of protein-rich foods from globular proteins

    NARCIS (Netherlands)

    Purwanti, N.; Goot, van der A.J.; Boom, R.M.; Vereijken, J.M.

    2010-01-01

    Concentrated protein-rich foods have strong potential to be developed in terms of health and well-being roles. Unfortunately, limitations in creating products with the rights texture and stability hinder the use of those products by consumers. Main reason is that the formation of micro- and

  16. Distribution, transition and thermodynamic stability of protein conformations in the denaturant-induced unfolding of proteins.

    Science.gov (United States)

    Bian, Liujiao; Ji, Xu

    2014-01-01

    Extensive and intensive studies on the unfolding of proteins require appropriate theoretical model and parameter to clearly illustrate the feature and characteristic of the unfolding system. Over the past several decades, four approaches have been proposed to describe the interaction between proteins and denaturants, but some ambiguity and deviations usually occur in the explanation of the experimental data. In this work, a theoretical model was presented to show the dependency of the residual activity ratio of the proteins on the molar denaturant concentration. Through the characteristic unfolding parameters ki and Δmi in this model, the distribution, transition and thermodynamic stability of protein conformations during the unfolding process can be quantitatively described. This model was tested with the two-state unfolding of bovine heart cytochrome c and the three-state unfolding of hen egg white lysozyme induced by both guanidine hydrochloride and urea, the four-state unfolding of bovine carbonic anhydrase b induced by guanidine hydrochloride and the unfolding of some other proteins induced by denaturants. The results illustrated that this model could be used accurately to reveal the distribution and transition of protein conformations in the presence of different concentrations of denaturants and to evaluate the unfolding tendency and thermodynamic stability of different conformations. In most denaturant-induced unfolding of proteins, the unfolding became increasingly hard in next transition step and the proteins became more unstable as they attained next successive stable conformation. This work presents a useful method for people to study the unfolding of proteins and may be used to describe the unfolding and refolding of other biopolymers induced by denaturants, inducers, etc.

  17. Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A. (UNL)

    2012-08-21

    DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1{alpha} and DJ-1{beta}) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1{beta}. The structure of D. melanogaster DJ-1{beta} is similar to that of human DJ-1, although two important residues in the human protein, Met26 and His126, are not conserved in DJ-1{beta}. His126 in human DJ-1 is substituted with a tyrosine in DJ-1{beta}, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO{sub 2{sup -}}) results in considerable thermal stabilization of both Drosophila DJ-1{beta} and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.

  18. Innovative aspects of protein stability in ionic liquid mixtures.

    Science.gov (United States)

    Kumar, Awanish; Venkatesu, Pannuru

    2018-06-01

    Mixtures of ionic liquids (ILs) have attracted our attention because of their extraordinary performances in extraction technologies and in absorbing large amount of CO 2 gas. It has been observed that when two or more ILs are mixed in different proportions, a new solvent is obtained which is much better than that of each component of ILs from which the mixture is obtained. Within a mixture of ILs, several unidentified interactions occur among several ions which give rise to unique solvent properties to the mixture. Herein, in this review, we have highlighted the utilization of the advantageous properties of the IL mixtures in protein stability studies. This approach is exceptional and opens new directions to the use of ILs in biotechnology.

  19. Characterisation of protein stability in rod-insert vaginal rings.

    Science.gov (United States)

    Pattani, Aditya; Lowry, Deborah; Curran, Rhonda M; McGrath, Stephanie; Kett, Vicky L; Andrews, Gavin P; Malcolm, R Karl

    2012-07-01

    A major goal in vaccine development is elimination of the 'cold chain', the transport and storage system for maintenance and distribution of the vaccine product. This is particularly pertinent to liquid formulation of vaccines. We have previously described the rod-insert vaginal ring (RiR) device, comprising an elastomeric body into which are inserted lyophilised, rod-shaped, solid drug dosage forms, and having potential for sustained mucosal delivery of biomacromolecules, such as HIV envelope protein-based vaccine candidates. Given the solid, lyophilised nature of these insert dosage forms, we hypothesised that antigen stability may be significantly increased compared with more conventional solubilised vaginal gel format. In this study, we prepared and tested vaginal ring devices fitted with lyophilised rod inserts containing the model antigen bovine serum albumin (BSA). Both the RiRs and the gels that were freeze-dried to prepare the inserts were evaluated for BSA stability using PAGE, turbidimetry, microbial load, MALDI-TOF and qualitative precipitate solubility measurements. When stored at 4 °C, but not when stored at 40 °C/75% RH, the RiR formulation offered protection against structural and conformational changes to BSA. The insert also retained matrix integrity and release characteristics. The results demonstrate that lypophilised gels can provide relative protection against degradation at lower temperatures compared to semi-solid gels. The major mechanism of degradation at 40 °C/75% RH was shown to be protein aggregation. Finally, in a preliminary study, we found that addition of trehalose to the formulation significantly reduces the rate of BSA degradation compared to the original formulation when stored at 40 °C/75% RH. Establishing the mechanism of degradation, and finding that degradation is decelerated in the presence of trehalose, will help inform further development of RiRs specifically and polymer based freeze-dried systems in general. Copyright

  20. The contribution of particle swarm optimization to three-dimensional slope stability analysis.

    Science.gov (United States)

    Kalatehjari, Roohollah; Rashid, Ahmad Safuan A; Ali, Nazri; Hajihassani, Mohsen

    2014-01-01

    Over the last few years, particle swarm optimization (PSO) has been extensively applied in various geotechnical engineering including slope stability analysis. However, this contribution was limited to two-dimensional (2D) slope stability analysis. This paper applied PSO in three-dimensional (3D) slope stability problem to determine the critical slip surface (CSS) of soil slopes. A detailed description of adopted PSO was presented to provide a good basis for more contribution of this technique to the field of 3D slope stability problems. A general rotating ellipsoid shape was introduced as the specific particle for 3D slope stability analysis. A detailed sensitivity analysis was designed and performed to find the optimum values of parameters of PSO. Example problems were used to evaluate the applicability of PSO in determining the CSS of 3D slopes. The first example presented a comparison between the results of PSO and PLAXI-3D finite element software and the second example compared the ability of PSO to determine the CSS of 3D slopes with other optimization methods from the literature. The results demonstrated the efficiency and effectiveness of PSO in determining the CSS of 3D soil slopes.

  1. The Contribution of Particle Swarm Optimization to Three-Dimensional Slope Stability Analysis

    Science.gov (United States)

    A Rashid, Ahmad Safuan; Ali, Nazri

    2014-01-01

    Over the last few years, particle swarm optimization (PSO) has been extensively applied in various geotechnical engineering including slope stability analysis. However, this contribution was limited to two-dimensional (2D) slope stability analysis. This paper applied PSO in three-dimensional (3D) slope stability problem to determine the critical slip surface (CSS) of soil slopes. A detailed description of adopted PSO was presented to provide a good basis for more contribution of this technique to the field of 3D slope stability problems. A general rotating ellipsoid shape was introduced as the specific particle for 3D slope stability analysis. A detailed sensitivity analysis was designed and performed to find the optimum values of parameters of PSO. Example problems were used to evaluate the applicability of PSO in determining the CSS of 3D slopes. The first example presented a comparison between the results of PSO and PLAXI-3D finite element software and the second example compared the ability of PSO to determine the CSS of 3D slopes with other optimization methods from the literature. The results demonstrated the efficiency and effectiveness of PSO in determining the CSS of 3D soil slopes. PMID:24991652

  2. The Contribution of Particle Swarm Optimization to Three-Dimensional Slope Stability Analysis

    Directory of Open Access Journals (Sweden)

    Roohollah Kalatehjari

    2014-01-01

    Full Text Available Over the last few years, particle swarm optimization (PSO has been extensively applied in various geotechnical engineering including slope stability analysis. However, this contribution was limited to two-dimensional (2D slope stability analysis. This paper applied PSO in three-dimensional (3D slope stability problem to determine the critical slip surface (CSS of soil slopes. A detailed description of adopted PSO was presented to provide a good basis for more contribution of this technique to the field of 3D slope stability problems. A general rotating ellipsoid shape was introduced as the specific particle for 3D slope stability analysis. A detailed sensitivity analysis was designed and performed to find the optimum values of parameters of PSO. Example problems were used to evaluate the applicability of PSO in determining the CSS of 3D slopes. The first example presented a comparison between the results of PSO and PLAXI-3D finite element software and the second example compared the ability of PSO to determine the CSS of 3D slopes with other optimization methods from the literature. The results demonstrated the efficiency and effectiveness of PSO in determining the CSS of 3D soil slopes.

  3. The Impact of O-Glycan Chemistry on the Stability of Intrinsically Disordered Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Beckham, Gregg T [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Prates, Erica T [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Crowley, Michael F [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Guan, Xiaoyang [University of Colorado; Li, Yaohao [University of Colorado; Wang, Xinfeng [University of Colorado; Chaffey, Patrick K. [University of Colorado; Skaf, Munir S. [University of Campinas; Tan, Zhongping [University of Colorado

    2018-03-02

    Protein glycosylation is a diverse post-translational modification that serves myriad biological functions. O-linked glycans in particular vary widely in extent and chemistry in eukaryotes, with secreted proteins from fungi and yeast commonly exhibiting O-mannosylation in intrinsically disordered regions of proteins, likely for proteolysis protection, among other functions. However, it is not well understood why mannose is often the preferred glycan, and more generally, if the neighboring protein sequence and glycan have coevolved to protect against proteolysis in glycosylated intrinsically disordered proteins (IDPs). Here, we synthesized variants of a model IDP, specifically a natively O-mannosylated linker from a fungal enzyme, with a-O-linked mannose, glucose, and galactose moieties, along with a non-glycosylated linker. Upon exposure to thermolysin, O-mannosylation, by far, provides the highest extent of proteolysis protection. To explain this observation, extensive molecular dynamics simulations were conducted, revealing that the axial configuration of the C2-hydroxyl group (2-OH) of a-mannose adjacent to the glycan-peptide bond strongly influences the conformational features of the linker. Specifically, a-mannose restricts the torsions of the IDP main chain more than other glycans whose equatorial 2-OH groups exhibit interactions that favor perpendicular glycan-protein backbone orientation. We suggest that IDP stiffening due to O-mannosylation impairs protease action, with contributions from protein-glycan interactions, protein flexibility, and protein stability. Our results further imply that resistance to proteolysis is an important driving force for evolutionary selection of a-mannose in eukaryotic IDPs, and more broadly, that glycan motifs for proteolysis protection likely coevolve with the protein sequence to which they attach.

  4. StaRProtein, A Web Server for Prediction of the Stability of Repeat Proteins

    Science.gov (United States)

    Xu, Yongtao; Zhou, Xu; Huang, Meilan

    2015-01-01

    Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins. PMID:25807112

  5. Analysis of protein stability and ligand interactions by thermal shift assay.

    Science.gov (United States)

    Huynh, Kathy; Partch, Carrie L

    2015-02-02

    Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye. Copyright © 2015 John Wiley & Sons, Inc.

  6. The Escherichia coli antiterminator protein BglG stabilizes the 5 ...

    Indian Academy of Sciences (India)

    Unknown

    Keywords. Antitermination; mRNA stability; RNA binding protein ... factor, Rho, and the pBR322 copy number protein, Rop, have been .... Transcription analysis using the oligo- ..... Retarded RNA turnover in Escherichia coli a means of main-.

  7. Stabilities and Dynamics of Protein Folding Nuclei by Molecular Dynamics Simulation

    Science.gov (United States)

    Song, Yong-Shun; Zhou, Xin; Zheng, Wei-Mou; Wang, Yan-Ting

    2017-07-01

    To understand how the stabilities of key nuclei fragments affect protein folding dynamics, we simulate by molecular dynamics (MD) simulation in aqueous solution four fragments cut out of a protein G, including one α-helix (seqB: KVFKQYAN), two β-turns (seqA: LNGKTLKG and seqC: YDDATKTF), and one β-strand (seqD: DGEWTYDD). The Markov State Model clustering method combined with the coarse-grained conformation letters method are employed to analyze the data sampled from 2-μs equilibrium MD simulation trajectories. We find that seqA and seqB have more stable structures than their native structures which become metastable when cut out of the protein structure. As expected, seqD alone is flexible and does not have a stable structure. Throughout our simulations, the native structure of seqC is stable but cannot be reached if starting from a structure other than the native one, implying a funnel-shape free energy landscape of seqC in aqueous solution. All the above results suggest that different nuclei have different formation dynamics during protein folding, which may have a major contribution to the hierarchy of protein folding dynamics. Supported by the National Basic Research Program of China under Grant No. 2013CB932804, the National Natural Science Foundation of China under Grant No. 11421063, and the CAS Biophysics Interdisciplinary Innovation Team Project

  8. Protein capped nanosilver free radical oxidation: role of biomolecule capping on nanoparticle colloidal stability and protein oxidation.

    Science.gov (United States)

    Ahumada, Manuel; Bohne, Cornelia; Oake, Jessy; Alarcon, Emilio I

    2018-05-03

    We studied the effect of human serum albumin protein capped spherical nanosilver on the nanoparticle stability upon peroxyl radical oxidation. The nanoparticle-protein composite is less prone to oxidation compared to the individual components. However, higher concentrations of hydrogen peroxide were formed in the nanoparticle-protein system.

  9. Contribution of gastroenteropancreatic appetite hormones to protein-induced satiety

    DEFF Research Database (Denmark)

    Sparre, Anita Belza; Ritz, Christian; Sørensen, Mejse Q

    2013-01-01

    BACKGROUND: Effects of protein intake on appetite-regulating hormones and their dynamics are unclear. OBJECTIVES: We investigated the satiating effects of meals with varying protein contents and whether there was an effect of dose on appetite-regulating hormones and appetite ratings.Design: Twenty...

  10. The contribution of polystyrene nanospheres towards the crystallization of proteins.

    Directory of Open Access Journals (Sweden)

    Johanna M Kallio

    Full Text Available BACKGROUND: Protein crystallization is a slow process of trial and error and limits the amount of solved protein structures. Search of a universal heterogeneous nucleant is an effort to facilitate crystallizability of proteins. METHODOLOGY: The effect of polystyrene nanospheres on protein crystallization were tested with three commercial proteins: lysozyme, xylanase, xylose isomerase, and with five research target proteins: hydrophobins HFBI and HFBII, laccase, sarcosine dimethylglycine N-methyltransferase (SDMT, and anti-testosterone Fab fragment 5F2. The use of nanospheres both in screening and as an additive for known crystallization conditions was studied. In screening, the addition of an aqueous solution of nanosphere to the crystallization drop had a significant positive effect on crystallization success in comparison to the control screen. As an additive in hydrophobin crystallization, the nanospheres altered the crystal packing, most likely due to the amphiphilic nature of hydrophobins. In the case of laccase, nanospheres could be used as an alternative for streak-seeding, which insofar had remained the only technique to produce high-diffracting crystals. With methyltransferase SDMT the nanospheres, used also as an additive, produced fewer, larger crystals in less time. Nanospheres, combined with the streak-seeding method, produced single 5F2 Fab crystals in shorter equilibration times. CONCLUSIONS: All in all, the use of nanospheres in protein crystallization proved to be beneficial, both when screening new crystallization conditions to promote nucleation and when used as an additive to produce better quality crystals, faster. The polystyrene nanospheres are easy to use, commercially available and close to being inert, as even with amphiphilic proteins only the crystal packing is altered and the nanospheres do not interfere with the structure and function of the protein.

  11. Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity

    Energy Technology Data Exchange (ETDEWEB)

    DuBois, Rebecca M.; Zaraket, Hassan; Reddivari, Muralidhar; Heath, Richard J.; White, Stephen W.; Russell, Charles J. (Tennessee-HSC); (SJCH)

    2012-12-10

    Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 {angstrom} resolution and two structures of HP HA at 2.95 and 3.10 {angstrom} resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.

  12. Squared ligament of the elbow: anatomy and contribution to forearm stability.

    Science.gov (United States)

    Otayek, Salma; Tayeb, Abd-el-Kader Ait; Assabah, Bouchra; Viard, Brice; Dayan, Romain; Lazure, Thierry; Soubeyrand, Marc

    2016-03-01

    The present study describes the macroscopic and microscopic features of the squared ligament of the elbow (SLE). In addition, the SLE biomechanical behavior and contribution to the forearm stability were also examined. Ten forearms from freshly frozen cadavers were used for this work. Each forearm was mounted in an experimental frame for quantification of longitudinal and transverse stability. Macroscopic features and biomechanical behavior were analyzed on dynamic videos obtained during forearm rotation. Then, the SLE was harvested from the 10 forearms for microscopic analysis on histological slices stained with hematoxylin-eosin-saffron. Two main SLE configurations were identified. One in which the SLE had three distinct bundles (anterior, middle, posterior) and another in which it was homogeneous. The anterior part of the SLE had a mean length of 11.2 mm (±2.4 mm) and a mean width of 1.2 mm (±0.2 mm) while the posterior part had a mean length of 9.9 mm (±2.2 mm) and a mean width of 1 mm (±0.2 mm). Microscopic examination showed that the SLE is composed of a thin layer of arranged collagen fibers. During forearm rotation, the SLE progressively tightens upon pronation and supination by wrapping around the radial neck. Tightening of the SLE during forearm rotation provides transverse and longitudinal stability to the forearm, mainly in maximal pronation and supination. The SLE is a true ligament and provides forearm stability when it is stretched in pronation and supination.

  13. Increasing the thermal stability of cellulase C using rules learned from thermophilic proteins: a pilot study.

    Science.gov (United States)

    Németh, Attila; Kamondi, Szilárd; Szilágyi, András; Magyar, Csaba; Kovári, Zoltán; Závodszky, Péter

    2002-05-02

    Some structural features underlying the increased thermostability of enzymes from thermophilic organisms relative to their homologues from mesophiles are known from earlier studies. We used cellulase C from Clostridium thermocellum to test whether thermostability can be increased by mutations designed using rules learned from thermophilic proteins. Cellulase C has a TIM barrel fold with an additional helical subdomain. We designed and produced a number of mutants with the aim to increase its thermostability. Five mutants were designed to create new electrostatic interactions. They all retained catalytic activity but exhibited decreased thermostability relative to the wild-type enzyme. Here, the stabilizing contributions are obviously smaller than the destabilization caused by the introduction of the new side chains. In another mutant, the small helical subdomain was deleted. This mutant lost activity but its melting point was only 3 degrees C lower than that of the wild-type enzyme, which suggests that the subdomain is an independent folding unit and is important for catalytic function. A double mutant was designed to introduce a new disulfide bridge into the enzyme. This mutant is active and has an increased stability (deltaT(m)=3 degrees C, delta(deltaG(u))=1.73 kcal/mol) relative to the wild-type enzyme. Reduction of the disulfide bridge results in destabilization and an altered thermal denaturation behavior. We conclude that rules learned from thermophilic proteins cannot be used in a straightforward way to increase the thermostability of a protein. Creating a crosslink such as a disulfide bond is a relatively sure-fire method but the stabilization may be smaller than calculated due to coupled destabilizing effects.

  14. Dissecting Protein Configurational Entropy into Conformational and Vibrational Contributions.

    Science.gov (United States)

    Chong, Song-Ho; Ham, Sihyun

    2015-10-01

    Quantifying how the rugged nature of the underlying free-energy landscape determines the entropic cost a protein must incur upon folding and ligand binding is a challenging problem. Here, we present a novel computational approach that dissects the protein configurational entropy on the basis of the classification of protein dynamics on the landscape into two separate components: short-term vibrational dynamics related to individual free-energy wells and long-term conformational dynamics associated with transitions between wells. We apply this method to separate the configurational entropy of the protein villin headpiece subdomain into its conformational and vibrational components. We find that the change in configurational entropy upon folding is dominated by the conformational entropy despite the fact that the magnitude of the vibrational entropy is the significantly larger component in each of the folded and unfolded states, which is in accord with the previous empirical estimations. The straightforward applicability of our method to unfolded proteins promises a wide range of applications, including those related to intrinsically disordered proteins.

  15. Ultra-High Pressure Homogenization improves oxidative stability and interfacial properties of soy protein isolate-stabilized emulsions.

    Science.gov (United States)

    Fernandez-Avila, C; Trujillo, A J

    2016-10-15

    Ultra-High Pressure Homogenization (100-300MPa) has great potential for technological, microbiological and nutritional aspects of fluid processing. Its effect on the oxidative stability and interfacial properties of oil-in-water emulsions prepared with 4% (w/v) of soy protein isolate and soybean oil (10 and 20%, v/v) were studied and compared to emulsions treated by conventional homogenization (15MPa). Emulsions were characterized by particle size, emulsifying activity index, surface protein concentration at the interface and by transmission electron microscopy. Primary and secondary lipid oxidation products were evaluated in emulsions upon storage. Emulsions with 20% oil treated at 100 and 200MPa exhibited the most oxidative stability due to higher amount of oil and protein surface load at the interface. This manuscript addresses the improvement in oxidative stability in emulsions treated by UHPH when compared to conventional emulsions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Regulation of PCNA-protein interactions for genome stability

    DEFF Research Database (Denmark)

    Mailand, Niels; Gibbs-Seymour, Ian; Bekker-Jensen, Simon

    2013-01-01

    Proliferating cell nuclear antigen (PCNA) has a central role in promoting faithful DNA replication, providing a molecular platform that facilitates the myriad protein-protein and protein-DNA interactions that occur at the replication fork. Numerous PCNA-associated proteins compete for binding...

  17. CD-loop Extension in Zika Virus Envelope Protein Key for Stability and Pathogenesis.

    Science.gov (United States)

    Gallichotte, Emily N; Dinnon, Kenneth H; Lim, Xin-Ni; Ng, Thiam-Seng; Lim, Elisa X Y; Menachery, Vineet D; Lok, Shee-Mei; Baric, Ralph S

    2017-12-05

    With severe disease manifestations including microcephaly, congenital malformation, and Guillain-Barré syndrome, Zika virus (ZIKV) remains a persistent global public health threat. Despite antigenic similarities with dengue viruses, structural studies have suggested the extended CD-loop and hydrogen-bonding interaction network within the ZIKV envelope protein contribute to stability differences between the viral families. This enhanced stability may lead to the augmented infection, disease manifestation, and persistence in body fluids seen following ZIKV infection. To examine the role of these motifs in infection, we generated a series of ZIKV recombinant viruses that disrupted the hydrogen-bonding network (350A, 351A, and 350A/351A) or the CD-loop extension (Δ346). Our results demonstrate a key role for the ZIKV extended CD-loop in cell-type-dependent replication, virion stability, and in vivo pathogenesis. Importantly, the Δ346 mutant maintains similar antigenicity to wild-type virus, opening the possibility for its use as a live-attenuated vaccine platform for ZIKV and other clinically relevant flaviviruses. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  18. Efficient protein-repelling thin films regulated by chain mobility of low-Tg polymers with increased stability via crosslinking

    Science.gov (United States)

    Zhang, Jinghui; Huang, Zhiwei; Liu, Dan

    2017-12-01

    Polymer thin films are generally employed as coatings on implants to prevent protein adsorption. Polymer chain mobility and surface softness have been found to contribute to the protein resistance, but also bring film instability in a liquid protein medium. We investigated the protein resistance ability of three low-Tg polymers, including hydrophobic polymers polyisoprene (PI), poly(n-butyl methacrylate) (PnBMA) and hydrophilic polyethylene oxide (PEO), by overcoming the instability issue with crosslinking. We found that the Tgs of PI and PEO can be increased to around 0 °C after crosslinking. The remained strong chain mobility of both films can still resist protein adsorption regardless the hydrophobicity, yet greatly increases the film stability under an aqueous circumstance. The PnBMA film increased its Tg to around room temperature after crosslinking, which deteriorated the protein-resistance ability having the surface covered by BSA molecules. Our results support that the chain mobility of a polymer film plays an important role in resisting protein adsorption due to the increased entropy associated with more mobile polymer chains. By tune the degree of crosslinking, the stability of polymer in aqueous environment can be increased while the protein resistant ability can be remained. Our results provide a new strategy to design polymer materials for effective antifouling.

  19. Comparison of the colloidal stability, bioaccessibility and antioxidant activity of corn protein hydrolysate and sodium caseinate stabilized curcumin nanoparticles.

    Science.gov (United States)

    Wang, Yong-Hui; Yuan, Yang; Yang, Xiao-Quan; Wang, Jin-Mei; Guo, Jian; Lin, Yuan

    2016-07-01

    The aims of this work were to construct corn protein hydrolysate (CPH)-based curcumin nanoparticles (Cur NPs) and to compare the colloidal stability, bioaccessibility and antioxidant activity of the Cur NPs stabilized CPH and sodium caseinate (NaCas) respectively. The results indicated that Cur solubility could be considerably improved after the Cur NPs fabrication. The spectroscopy results demonstrated that the solubilization of Cur should be attributed to its complexation with CPH or NaCas. The Cur NPs exhibited good colloidal stability after 1 week's storage but showed smaller (40 nm) size in CPH than in NaCas (100 nm). After lyophilization, the Cur NPs powders showed good rehydration properties and chemical stability, and compared with NaCas, the size of Cur NPs stabilized by CPH was still smaller. Additionally, the Cur NPs exhibited higher chemical stability against the temperature compared with free Cur, and the CPH could protect Cur from degradation more efficiently. Comparing with NaCas, the Cur NPs stabilized by CPH exhibited better bioaccessibility and antioxidant activity. This study demonstrated that CPH may be better than NaCas in Cur NPs fabrication and it opens up the possibility of using hydrophobic protein hydrolysate to construct the NPs delivery system.

  20. The cartilage protein melanoma inhibitory activity contributes to inflammatory arthritis

    NARCIS (Netherlands)

    Yeremenko, Nataliya; Härle, Peter; Cantaert, Tineke; van Tok, Melissa; van Duivenvoorde, Leonie M.; Bosserhoff, Anja; Baeten, Dominique

    2014-01-01

    Melanoma inhibitory activity (MIA) is a small chondrocyte-specific protein with unknown function. MIA knockout mice (MIA(-/-)) have a normal phenotype with minor microarchitectural alterations of cartilage. Our previous study demonstrated that immunodominant epitopes of MIA are actively presented in

  1. Trade-off between positive and negative design of protein stability: from lattice models to real proteins.

    Directory of Open Access Journals (Sweden)

    Orly Noivirt-Brik

    2009-12-01

    Full Text Available Two different strategies for stabilizing proteins are (i positive design in which the native state is stabilized and (ii negative design in which competing non-native conformations are destabilized. Here, the circumstances under which one strategy might be favored over the other are explored in the case of lattice models of proteins and then generalized and discussed with regard to real proteins. The balance between positive and negative design of proteins is found to be determined by their average "contact-frequency", a property that corresponds to the fraction of states in the conformational ensemble of the sequence in which a pair of residues is in contact. Lattice model proteins with a high average contact-frequency are found to use negative design more than model proteins with a low average contact-frequency. A mathematical derivation of this result indicates that it is general and likely to hold also for real proteins. Comparison of the results of correlated mutation analysis for real proteins with typical contact-frequencies to those of proteins likely to have high contact-frequencies (such as disordered proteins and proteins that are dependent on chaperonins for their folding indicates that the latter tend to have stronger interactions between residues that are not in contact in their native conformation. Hence, our work indicates that negative design is employed when insufficient stabilization is achieved via positive design owing to high contact-frequencies.

  2. Recent advances in the applications of ionic liquids in protein stability and activity: a review.

    Science.gov (United States)

    Patel, Rajan; Kumari, Meena; Khan, Abbul Bashar

    2014-04-01

    Room temperatures ionic liquids are considered as miraculous solvents for biological system. Due to their inimitable properties and large variety of applications, they have been widely used in enzyme catalysis and protein stability and separation. The related information present in the current review is helpful to the researchers working in the field of biotechnology and biochemistry to design or choose an ionic liquid that can serve as a noble and selective solvent for any particular enzymatic reaction, protein preservation and other protein based applications. We have extensively analyzed the methods used for studying the protein-IL interaction which is useful in providing information about structural and conformational dynamics of protein. This can be helpful to develop and understanding about the effect of ionic liquids on stability and activity of proteins. In addition, the affect of physico-chemical properties of ionic liquids, viz. hydrogen bond capacity and hydrophobicity on protein stability are discussed.

  3. A rapid, ensemble and free energy based method for engineering protein stabilities.

    Science.gov (United States)

    Naganathan, Athi N

    2013-05-02

    Engineering the conformational stabilities of proteins through mutations has immense potential in biotechnological applications. It is, however, an inherently challenging problem given the weak noncovalent nature of the stabilizing interactions. In this regard, we present here a robust and fast strategy to engineer protein stabilities through mutations involving charged residues using a structure-based statistical mechanical model that accounts for the ensemble nature of folding. We validate the method by predicting the absolute changes in stability for 138 experimental mutations from 16 different proteins and enzymes with a correlation of 0.65 and importantly with a success rate of 81%. Multiple point mutants are predicted with a higher success rate (90%) that is validated further by comparing meosphile-thermophile protein pairs. In parallel, we devise a methodology to rapidly engineer mutations in silico which we benchmark against experimental mutations of ubiquitin (correlation of 0.95) and check for its feasibility on a larger therapeutic protein DNase I. We expect the method to be of importance as a first and rapid step to screen for protein mutants with specific stability in the biotechnology industry, in the construction of stability maps at the residue level (i.e., hot spots), and as a robust tool to probe for mutations that enhance the stability of protein-based drugs.

  4. Tight junction proteins contribute to barrier properties in human pleura.

    Science.gov (United States)

    Markov, Alexander G; Voronkova, Maria A; Volgin, George N; Yablonsky, Piotr K; Fromm, Michael; Amasheh, Salah

    2011-03-15

    The permeability of pleural mesothelium helps to control the volume and composition of the liquid lubricating pleural surfaces. Information on pleural barrier function in health and disease, however, is scarce. Tissue specimens of human pleura were mounted in Ussing chambers for measurement of transmesothelial resistance. Expression of tight junction (TJ) proteins was studied by Western blots and immune fluorescence confocal microscopy. Both visceral and parietal pleura showed barrier properties represented by transmesothelial resistance. Occludin, claudin-1, -3, -5, and -7, were detected in visceral pleura. In parietal pleura, the same TJ proteins were detected, except claudin-7. In tissues from patients with pleural inflammation these tightening claudins were decreased and in visceral pleura claudin-2, a paracellular channel former, became apparent. We report that barrier function in human pleura coincides with expression of claudins known to be key determinants of epithelial barrier properties. In inflamed tissue, claudin expression indicates a reduced barrier function. Copyright © 2010 Elsevier B.V. All rights reserved.

  5. Protein modification by acrolein: Formation and stability of cysteine adducts

    OpenAIRE

    Cai, Jian; Bhatnagar, Aruni; Pierce, William M.

    2009-01-01

    The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to iden...

  6. Variable Domain N-Linked Glycans Acquired During Antigen-Specific Immune Responses Can Contribute to Immunoglobulin G Antibody Stability

    Directory of Open Access Journals (Sweden)

    Fleur S. van de Bovenkamp

    2018-04-01

    Full Text Available Immunoglobulin G (IgG can contain N-linked glycans in the variable domains, the so-called Fab glycans, in addition to the Fc glycans in the CH2 domains. These Fab glycans are acquired following introduction of N-glycosylation sites during somatic hypermutation and contribute to antibody diversification. We investigated whether Fab glycans may—in addition to affecting antigen binding—contribute to antibody stability. By analyzing thermal unfolding profiles of antibodies with or without Fab glycans, we demonstrate that introduction of Fab glycans can improve antibody stability. Strikingly, removal of Fab glycans naturally acquired during antigen-specific immune responses can deteriorate antibody stability, suggesting in vivo selection of stable, glycosylated antibodies. Collectively, our data show that variable domain N-linked glycans acquired during somatic hypermutation can contribute to IgG antibody stability. These findings indicate that introducing Fab glycans may represent a mechanism to improve therapeutic/diagnostic antibody stability.

  7. The stability and formation of native proteins from unfolded monomers is increased through interactions with unrelated proteins.

    Directory of Open Access Journals (Sweden)

    Claudia Rodríguez-Almazán

    Full Text Available The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins.

  8. Correlation between mechanical behavior of protein films at the air/water interface and intrinsic stability of protein molecules

    NARCIS (Netherlands)

    Martin, A.H.; Cohen Stuart, M.A.; Bos, M.A.; Vliet, T. van

    2005-01-01

    The relation between mechanical film properties of various adsorbed protein layers at the air/water interface and intrinsic stability of the corresponding proteins is discussed. Mechanical film properties were determined by surface deformation in shear and dilation. In shear, fracture stress, σf,

  9. Tumor protein D52 expression is post-transcriptionally regulated by T-cell intercellular antigen (TIA) 1 and TIA-related protein via mRNA stability.

    Science.gov (United States)

    Motohashi, Hiromi; Mukudai, Yoshiki; Ito, Chihiro; Kato, Kosuke; Shimane, Toshikazu; Kondo, Seiji; Shirota, Tatsuo

    2017-05-04

    Although tumor protein D52 (TPD52) family proteins were first identified nearly 20 years ago, their molecular regulatory mechanisms remain unclear. Therefore, we investigated the post-transcriptional regulation of TPD52 family genes. An RNA immunoprecipitation (RIP) assay showed the potential binding ability of TPD52 family mRNAs to several RNA-binding proteins, and an RNA degradation assay revealed that TPD52 is subject to more prominent post-transcriptional regulation than are TPD53 and TPD54. We subsequently focused on the 3'-untranslated region (3'-UTR) of TPD52 as a cis -acting element in post-transcriptional gene regulation. Several deletion mutants of the 3'-UTR of TPD52 mRNA were constructed and ligated to the 3'-end of a reporter green fluorescence protein gene. An RNA degradation assay revealed that a minimal cis -acting region, located in the 78-280 region of the 5'-proximal region of the 3'-UTR, stabilized the reporter mRNA. Biotin pull-down and RIP assays revealed specific binding of the region to T-cell intracellular antigen 1 (TIA-1) and TIA-1-related protein (TIAR). Knockdown of TIA-1/TIAR decreased not only the expression, but also the stability of TPD52 mRNA; it also decreased the expression and stability of the reporter gene ligated to the 3'-end of the 78-280 fragment. Stimulation of transforming growth factor-β and epidermal growth factor decreased the binding ability of these factors, resulting in decreased mRNA stability. These results indicate that the 78-280 fragment and TIA-1/TIAR concordantly contribute to mRNA stability as a cis -acting element and trans -acting factor(s), respectively. Thus, we here report the specific interactions between these elements in the post-transcriptional regulation of the TPD52 gene. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  10. Contributing Factors for Protein Calorie Malnutrition in Distsrict Mardan

    International Nuclear Information System (INIS)

    Khan, D.M.; Khan, S.A.

    2017-01-01

    Background: Protein calorie malnutrition (PCM) is a common health problem in developing countries resulting in high mortality in children under five years of age. It is also known as protein energy malnutrition. Objectives: To calculate the incidence and risk factors for Protein Calorie Malnutrition in children attending hospitals of district Mardan, Khyber Pakhtunkhwa. Study design, settings and duration: Retrospective hospital case record analysis of admitted children diagnosed as suffers of PCM and were admitted in hospitals from 2011-15 was done. Subjects and Methods: Children < 5 years fulfilling the inclusion criteria and reporting at four major hospitals of district Mardan from 2011-15 were included in the study. Data of children fulfilling the definition of PCM were further analyzed using SPSS software. Chi-square test and logistic regression model was used to determine the significance of the risk factors with the PCM disease. Results: Out of 448 children, 58.5 percent (n=262) had PCM and 41.5 percent (n=186) did not have PCM. The significant risk factors in the logistic model fitted for male children included economic status, number of living children, environmental sanitation, immunization, skin changes. Risk factors for PCM in female children were economic status, weight, height, number of living children, environmental sanitation, immunization, hair changes, time to time monitoring of the child and clean water availability. In the logistic model for both genders; the risk factors that showed significant association with PCM were economic status, weight, height, number of living children, environmental sanitation, immunization, hair changes, time to time monitoring of the child health, clean water availability and hypothermia. Conclusion: Almost 58 percent children admitted in different hospitals of district Mardan had PCM and the significant risk factors were economic status, weight, height, number of living children, environmental sanitation

  11. The Phosphocarrier Protein HPr Contributes to Meningococcal Survival during Infection.

    Directory of Open Access Journals (Sweden)

    Ana Antunes

    Full Text Available Neisseria meningitidis is an exclusively human pathogen frequently carried asymptomatically in the nasopharynx but it can also provoke invasive infections such as meningitis and septicemia. N. meningitidis uses a limited range of carbon sources during infection, such as glucose, that is usually transported into bacteria via the phosphoenolpyruvate (PEP:sugar phosphotransferase system (PTS, in which the phosphocarrier protein HPr (encoded by the ptsH gene plays a central role. Although N. meningitidis possesses an incomplete PTS, HPr was found to be required for its virulence. We explored the role of HPr using bioluminescent wild-type and ΔptsH strains in experimental infection in transgenic mice expressing the human transferrin. The wild-type MC58 strain was recovered at higher levels from the peritoneal cavity and particularly from blood compared to the ΔptsH strain. The ΔptsH strain provoked lower levels of septicemia in mice and was more susceptible to complement-mediated killing than the wild-type strain. We tested whether meningococcal structures impacted complement resistance and observed that only the capsule level was decreased in the ΔptsH mutant. We therefore compared the transcriptomic profiles of wild-type and ΔptsH strains and identified 49 differentially expressed genes. The HPr regulon contains mainly hypothetical proteins (43% and several membrane-associated proteins that could play a role during host interaction. Some other genes of the HPr regulon are involved in stress response. Indeed, the ΔptsH strain showed increased susceptibility to environmental stress conditions. Our data suggest that HPr plays a pleiotropic role in host-bacteria interactions most likely through the innate immune response that may be responsible for the enhanced clearance of the ΔptsH strain from blood.

  12. Membrane Protein Stability Analyses by Means of Protein Energy Profiles in Case of Nephrogenic Diabetes Insipidus

    Directory of Open Access Journals (Sweden)

    Florian Heinke

    2012-01-01

    Full Text Available Diabetes insipidus (DI is a rare endocrine, inheritable disorder with low incidences in an estimated one per 25,000–30,000 live births. This disease is characterized by polyuria and compensatory polydypsia. The diverse underlying causes of DI can be central defects, in which no functional arginine vasopressin (AVP is released from the pituitary or can be a result of defects in the kidney (nephrogenic DI, NDI. NDI is a disorder in which patients are unable to concentrate their urine despite the presence of AVP. This antidiuretic hormone regulates the process of water reabsorption from the prourine that is formed in the kidney. It binds to its type-2 receptor (V2R in the kidney induces a cAMP-driven cascade, which leads to the insertion of aquaporin-2 water channels into the apical membrane. Mutations in the genes of V2R and aquaporin-2 often lead to NDI. We investigated a structure model of V2R in its bound and unbound state regarding protein stability using a novel protein energy profile approach. Furthermore, these techniques were applied to the wild-type and selected mutations of aquaporin-2. We show that our results correspond well to experimental water ux analysis, which confirms the applicability of our theoretical approach to equivalent problems.

  13. Interdependence of laforin and malin proteins for their stability and ...

    Indian Academy of Sciences (India)

    https://www.ias.ac.in/article/fulltext/jbsc/040/05/0863-0871. Keywords. Epilepsy; locus heterogeneity; post-translational modification; protein-protein interaction. Abstract. Lafora disease (LD), an autosomal recessive and fatal form of neurodegenerative disorder, is characterized by the presence of polyglucosan inclusions in ...

  14. Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state.

    Science.gov (United States)

    Nicolas, A; Egmond, M; Verrips, C T; de Vlieg, J; Longhi, S; Cambillau, C; Martinez, C

    1996-01-16

    Cutinase from the fungus Fusarium solani pisi is a lipolytic enzyme able to hydrolyze both aggregated and soluble substrates. It therefore provides a powerful tool for probing the mechanisms underlying lipid hydrolysis. Lipolytic enzymes have a catalytic machinery similar to those present in serine proteinases. It is characterized by the triad Ser, His, and Asp (Glu) residues, by an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups, and possibly by other determinants. It has been suggested on the basis of a covalently bond inhibitor that the cutinase oxyanion hole may consist not only of two main chain amide groups but also of the Ser42 O gamma side chain. Among the esterases and the serine and the cysteine proteases, only Streptomyces scabies esterase, subtilisin, and papain, respectively, have a side chain residue which is involved in the oxyanion hole formation. The position of the cutinase Ser42 side chain is structurally conserved in Rhizomucor miehei lipase with Ser82 O gamma, in Rhizopus delemar lipase with Thr83 O gamma 1, and in Candida antartica B lipase with Thr40 O gamma 1. To evaluate the increase in the tetrahedral intermediate stability provided by Ser42 O gamma, we mutated Ser42 into Ala. Furthermore, since the proper orientation of Ser42 O gamma is directed by Asn84, we mutated Asn84 into Ala, Leu, Asp, and Trp, respectively, to investigate the contribution of this indirect interaction to the stabilization of the oxyanion hole. The S42A mutation resulted in a drastic decrease in the activity (450-fold) without significantly perturbing the three-dimensional structure. The N84A and N84L mutations had milder kinetic effects and did not disrupt the structure of the active site, whereas the N84W and N84D mutations abolished the enzymatic activity due to drastic steric and electrostatic effects, respectively.

  15. Interplay between black carbon and minerals contributes to long term carbon stabilization and mineral transformation

    Science.gov (United States)

    Liang, B.; Weng, Y. T.; Wang, C. C.; Chiang, C. C.; Liu, C. C.; Lehmann, J.

    2017-12-01

    Black carbon receives increasing global wide research attention due to its role in carbon sequestration, soil fertility enhancement and remediation application. Generally considered chemically stable in bulk, the reactive surface of BC can interplays with minerals and form strong chemical bondage, which renders physical protection of BC and contributes to its long term stabilization. Using historical BC-rich Amazonian Dark Earth (ADE), we probe the in-situ organo-mineral association and transformation of BC and minerals over a millennium scale using various synchrotron-based spectroscopic (XANES, FTIR) and microscopic (TXM) methods. Higher content of SRO minerals was found in BC-rich ADE compare to adjacent tropical soils. The iron signature found in BC-rich ADE was mainly ferrihydrite/lepidocrocite, a more reactive form of Fe compared to goethite, which was dominant in adjacent soil. Abundant nano minerals particles were observed in-situ associated with BC surface, in clusters and layers. The organo-mineral interaction lowers BC bioavailability and enhances its long-term stabilization in environment, while at the same time, transforms associated minerals into more reactive forms under rapid redox/weathering environment. The results suggest that mineral physical protection for BC sequestration may be more important than previous understanding. The scale up application of BC/biochar into agricultural systems and natural environments have long lasting impact on the in-situ transformation of associated minerals.

  16. Relative contributions of plantar fascia and ligaments on the arch static stability: a finite element study.

    Science.gov (United States)

    Tao, Kai; Ji, Wen-Ting; Wang, Dong-Mei; Wang, Cheng-Tao; Wang, Xu

    2010-10-01

    The plantar fascia (PF) and major ligaments play important roles in keeping the static foot arch structure. Their functions and relative contributions to the arch stability have not been well studied. A three-dimensional finite element foot model was created based on the reconstruction of magnetic resonance images. During balanced standing, four cases after individual releases of the PF, spring ligament (SL), and long and short plantar ligaments (LPL and SPL) were simulated, to compare their biomechanical consequences with the normal predictions under the intact structure. Although the predictions showed the arch did not collapse obviously after each structure sectioning, the internal mechanical behaviors changed considerably. The PF release resulted in the maximal increases of approximately 91%, 65% and 47% in the tensions of the LPF, SPL and SL, produced the largest changes in all bone rotations, and brought an obvious shift of high stress from the medial metatarsals to the lateral metatarsals. The SL release mainly enhanced bone rotation angles and weakened the joint stability of the arch structure. The LPL and the SPL performed the roles of mutual compensation as either one was released. The influence of the LPL on the load distribution among metatarsals was greater than for the SPL and the SL.

  17. Clinical application for the preservation of phospho-proteins through in-situ tissue stabilization

    Directory of Open Access Journals (Sweden)

    Ding Wei

    2010-11-01

    Full Text Available Abstract Background Protein biomarkers will play a pivotal role in the future of personalized medicine for both diagnosis and treatment decision-making. While the results of several pre-clinical and small-scale clinical studies have demonstrated the value of protein biomarkers, there have been significant challenges to translating these findings into routine clinical care. Challenges to the use of protein biomarkers include inter-sample variability introduced by differences in post-collection handling and ex vivo degradation of proteins and protein modifications. Results In this report, we re-create laboratory and clinical scenarios for sample collection and test the utility of a new tissue stabilization technique in preserving proteins and protein modifications. In the laboratory setting, tissue stabilization with the Denator Stabilizor T1 resulted in a significantly higher yield of phospho-protein when compared to standard snap freeze preservation. Furthermore, in a clinical scenario, tissue stabilization at collection resulted in a higher yield of total phospho-protein, total phospho-tyrosine, pErkT202/Y204 and pAktS473 when compared to standard methods. Tissue stabilization did not have a significant effect on other post-translational modifications such as acetylation and glycosylation, which are more stable ex-vivo. Tissue stabilization did decrease total RNA quantity and quality. Conclusion Stabilization at the time of collection offers the potential to better preserve tissue protein and protein modification levels, as well as reduce the variability related to tissue processing delays that are often associated with clinical samples.

  18. Mutation of exposed hydrophobic amino acids to arginine to increase protein stability

    OpenAIRE

    Strub, Caroline; Alies, Carole; Lougarre, Andrée; Ladurantie, Caroline; Czaplicki, Jerzy; Fournier, Didier

    2004-01-01

    Abstract Background One strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface. Results In order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion. Conclusion Altough the mutational effects were rather small, this strategy proved to be successful...

  19. Can Stabilization and Inhibition of Aquaporins Contribute to Future Development of Biomimetic Membranes?

    Science.gov (United States)

    To, Janet; Torres, Jaume

    2015-08-10

    In recent years, the use of biomimetic membranes that incorporate membrane proteins, i.e., biomimetic-hybrid membranes, has increased almost exponentially. Key membrane proteins in these systems have been aquaporins, which selectively permeabilize cellular membranes to water. Aquaporins may be incorporated into synthetic lipid bilayers or to more stable structures made of block copolymers or solid-state nanopores. However, translocation of aquaporins to these alien environments has adverse consequences in terms of performance and stability. Aquaporins incorporated in biomimetic membranes for use in water purification and desalination should also withstand the harsh environment that may prevail in these conditions, such as high pressure, and presence of salt or other chemicals. In this respect, modified aquaporins that can be adapted to these new environments should be developed. Another challenge is that biomimetic membranes that incorporate high densities of aquaporin should be defect-free, and this can only be efficiently ascertained with the availability of completely inactive mutants that behave otherwise like the wild type aquaporin, or with effective non-toxic water channel inhibitors that are so far inexistent. In this review, we describe approaches that can potentially be used to overcome these challenges.

  20. Can Stabilization and Inhibition of Aquaporins Contribute to Future Development of Biomimetic Membranes?

    Directory of Open Access Journals (Sweden)

    Janet To

    2015-08-01

    Full Text Available In recent years, the use of biomimetic membranes that incorporate membrane proteins, i.e., biomimetic-hybrid membranes, has increased almost exponentially. Key membrane proteins in these systems have been aquaporins, which selectively permeabilize cellular membranes to water. Aquaporins may be incorporated into synthetic lipid bilayers or to more stable structures made of block copolymers or solid-state nanopores. However, translocation of aquaporins to these alien environments has adverse consequences in terms of performance and stability. Aquaporins incorporated in biomimetic membranes for use in water purification and desalination should also withstand the harsh environment that may prevail in these conditions, such as high pressure, and presence of salt or other chemicals. In this respect, modified aquaporins that can be adapted to these new environments should be developed. Another challenge is that biomimetic membranes that incorporate high densities of aquaporin should be defect-free, and this can only be efficiently ascertained with the availability of completely inactive mutants that behave otherwise like the wild type aquaporin, or with effective non-toxic water channel inhibitors that are so far inexistent. In this review, we describe approaches that can potentially be used to overcome these challenges.

  1. Stabilization of structure in near-infrared fluorescent proteins by binding of biliverdin chromophore

    Science.gov (United States)

    Stepanenko, Olesya V.; Stepanenko, Olga V.; Bublikov, G. S.; Kuznetsova, I. M.; Verkhusha, V. V.; Turoverov, K. K.

    2017-07-01

    Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes and their mutants with different location of Cys residues, which able to bind a biliverdin chromophore, or without these Cys residues were studied using intrinsic tryptophan fluorescence, NIR fluorescence and circular dichroism. It was shown that a covalent binding of the biliverdin chromophore to a Cys residue via thioether group substantially stabilizes the spatial structure of NIR FPs. The stability of the protein structure and the chromophore association strength strongly depends on the location of Cys residues and decreases in the following order: a protein with Cys residues in both domains, a protein with Cys in PAS domains, and a protein with Cys in GAF domains. NIR FPs without Cys residues capable to covalently attach biliverdin have the lowest stability, comparable to NIR FP apoforms.

  2. Water-in-Oil Microemulsions for Protein Delivery: Loading Optimization and Stability.

    Science.gov (United States)

    Perinelli, Diego R; Cespi, Marco; Pucciarelli, Stefania; Vincenzetti, Silvia; Casettari, Luca; Lam, Jenny K W; Logrippo, Serena; Canala, Elisa; Soliman, Mahmoud E; Bonacucina, Giulia; Palmieri, Giovanni F

    2017-01-01

    Microemulsions are attractive delivery systems for therapeutic proteins and peptides due to their ability to enhance bioavailability. Although different proteins and peptides have been successfully delivered through such ternary systems, no information can be found about protein loading and the formulation stability when such microemulsions are prepared with pharmaceuticallyapproved oils and surfactants. The aim of this work was to optimise a ternary system consisting of water/ ethyl oleate/Span® 80-Tween® 80 and to determine its protein loading capacity and stability, using bovine serum albumin (BSA) as a model of biomolecule. The optimization was carried out using a Central Composite Design and all the prepared formulations were characterised through dynamic light scattering, rheology, optical and polarized microscopy. Subsequently, the maximum loading capacity was determined and the stability of the final microemulsion with the highest content of protein was followed over six months. To investigate the structural features of the protein, BSA was recovered from the microemulsion and analysed through fluorescence spectroscopy. After incorporation of the protein in the microemulsion, a decrease of its aqueous solubility was observed. However, the formulation remained stable over six months and the native-like state of the recovered protein was demonstrated by fluorescence spectroscopy Conclusion: This study demonstrated the feasibility of preparing microemulsions with the highest content of protein and their long-term stability. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. Exceptional heat stability of high protein content dispersions containing whey protein particles

    NARCIS (Netherlands)

    Saglam, D.; Venema, P.; Vries, de R.J.; Linden, van der E.

    2014-01-01

    Due to aggregation and/or gelation during thermal treatment, the amount of whey proteins that can be used in the formulation of high protein foods e.g. protein drinks, is limited. The aim of this study was to replace whey proteins with whey protein particles to increase the total protein content and

  4. Interdependence of laforin and malin proteins for their stability and ...

    Indian Academy of Sciences (India)

    eisic

    2015-11-28

    Nov 28, 2015 ... Since the clinical symptoms of LD patients representing the two genetic groups are very similar and since malin is ... proteins were also implicated cellular quality control ... Discovery Series software of Bio-Rad Laboratories.

  5. Astatine-211 labelled proteins and their stability in vivo

    International Nuclear Information System (INIS)

    Yi Changhou; Jin Jannan; Zhang Shuyuan; Wang Ketai; Zhang Dayuan; Zhou Maolun

    1989-01-01

    211 At or 131 I labelled proteins, e.g. 211 At-IgG or 211 At-BSA (bovine serum albumin) were prepared by 211 At reaction with the diazo-compound of para-aminobenzoic acid, which is then conjugated with IgG or BSA via an acylation reaction. The 211 At-carbon bond was found metabolically stable under in vivo conditions. For the labelling of proteins with 211 At or 131 I, other methods of direct oxidation are also described. The results show that for the labelling of proteins with 211 At, high rate of incorporation can be obtained with hydrogen peroxide as oxidant, but the labelling of proteins with 131 I is more favourable with the strong oxidant Chloramine-T. (author) 12 refs.; 6 figs

  6. ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus.

    Science.gov (United States)

    Frankel, Matthew B; Wojcik, Brandon M; DeDent, Andrea C; Missiakas, Dominique M; Schneewind, Olaf

    2010-10-01

    The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. © 2010 Blackwell Publishing Ltd.

  7. Improved Model for Predicting the Free Energy Contribution of Dinucleotide Bulges to RNA Duplex Stability.

    Science.gov (United States)

    Tomcho, Jeremy C; Tillman, Magdalena R; Znosko, Brent M

    2015-09-01

    Predicting the secondary structure of RNA is an intermediate in predicting RNA three-dimensional structure. Commonly, determining RNA secondary structure from sequence uses free energy minimization and nearest neighbor parameters. Current algorithms utilize a sequence-independent model to predict free energy contributions of dinucleotide bulges. To determine if a sequence-dependent model would be more accurate, short RNA duplexes containing dinucleotide bulges with different sequences and nearest neighbor combinations were optically melted to derive thermodynamic parameters. These data suggested energy contributions of dinucleotide bulges were sequence-dependent, and a sequence-dependent model was derived. This model assigns free energy penalties based on the identity of nucleotides in the bulge (3.06 kcal/mol for two purines, 2.93 kcal/mol for two pyrimidines, 2.71 kcal/mol for 5'-purine-pyrimidine-3', and 2.41 kcal/mol for 5'-pyrimidine-purine-3'). The predictive model also includes a 0.45 kcal/mol penalty for an A-U pair adjacent to the bulge and a -0.28 kcal/mol bonus for a G-U pair adjacent to the bulge. The new sequence-dependent model results in predicted values within, on average, 0.17 kcal/mol of experimental values, a significant improvement over the sequence-independent model. This model and new experimental values can be incorporated into algorithms that predict RNA stability and secondary structure from sequence.

  8. Contribution of Electrostatics in the Fibril Stability of a Model Ionic-Complementary Peptide.

    Science.gov (United States)

    Owczarz, Marta; Casalini, Tommaso; Motta, Anna C; Morbidelli, Massimo; Arosio, Paolo

    2015-12-14

    In this work we quantified the role of electrostatic interactions in the self-assembly of a model amphiphilic peptide (RADA 16-I) into fibrillar structures by a combination of size exclusion chromatography and molecular simulations. For the peptide under investigation, it is found that a net charge of +0.75 represents the ideal condition to promote the formation of regular amyloid fibrils. Lower net charges favor the formation of amorphous precipitates, while larger net charges destabilize the fibrillar aggregates and promote a reversible dissociation of monomers from the ends of the fibrils. By quantifying the dependence of the equilibrium constant of this reversible reaction on the pH value and the peptide net charge, we show that electrostatic interactions contribute largely to the free energy of fibril formation. The addition of both salt and a charged destabilizer (guanidinium hydrochloride) at moderate concentration (0.3-1 M) shifts the monomer-fibril equilibrium toward the fibrillar state. Whereas the first effect can be explained by charge screening of electrostatic repulsion only, the promotion of fibril formation in the presence of guanidinium hydrochloride is also attributed to modifications of the peptide conformation. The results of this work indicate that the global peptide net charge is a key property that correlates well with the fibril stability, although the peptide conformation and the surface charge distribution also contribute to the aggregation propensity.

  9. Pickering emulsions stabilized by whey protein nanoparticles prepared by thermal cross-linking

    NARCIS (Netherlands)

    Wu, Jiande; Shi, Mengxuan; Li, Wei; Zhao, Luhai; Wang, Ze; Yan, Xinzhong; Norde, Willem; Li, Yuan

    2015-01-01

    A Pickering (o/w) emulsion was formed and stabilized by whey protein isolate nanoparticles (WPI NPs). Those WPI NPs were prepared by thermal cross-linking of denatured WPI proteins within w/o emulsion droplets at 80. °C for 15. min. During heating of w/o emulsions containing 10% (w/v) WPI

  10. Adsorption of plasma proteins : adsorption behaviour on apolar surfaces and effect on colloid stability

    NARCIS (Netherlands)

    van der Scheer, Albert

    1978-01-01

    In this thesis the adsorption of some plasma proteins (human albumin (HSA) and fibrinogen (HFb)) on non polar surfaces is studied, together with the influence of these proteins on the stability of polystyrene latices. The aim of these investigations is a better understanding of the processes

  11. 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

  12. 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

  13. Protein Charge and Mass Contribute to the Spatio-temporal Dynamics of Protein-Protein Interactions in a Minimal Proteome

    Science.gov (United States)

    Xu, Yu; Wang, Hong; Nussinov, Ruth; Ma, Buyong

    2013-01-01

    We constructed and simulated a ‘minimal proteome’ model using Langevin dynamics. It contains 206 essential protein types which were compiled from the literature. For comparison, we generated six proteomes with randomized concentrations. We found that the net charges and molecular weights of the proteins in the minimal genome are not random. The net charge of a protein decreases linearly with molecular weight, with small proteins being mostly positively charged and large proteins negatively charged. The protein copy numbers in the minimal genome have the tendency to maximize the number of protein-protein interactions in the network. Negatively charged proteins which tend to have larger sizes can provide large collision cross-section allowing them to interact with other proteins; on the other hand, the smaller positively charged proteins could have higher diffusion speed and are more likely to collide with other proteins. Proteomes with random charge/mass populations form less stable clusters than those with experimental protein copy numbers. Our study suggests that ‘proper’ populations of negatively and positively charged proteins are important for maintaining a protein-protein interaction network in a proteome. It is interesting to note that the minimal genome model based on the charge and mass of E. Coli may have a larger protein-protein interaction network than that based on the lower organism M. pneumoniae. PMID:23420643

  14. Characterization of milk proteins-lutein complexes and the impact on lutein chemical stability.

    Science.gov (United States)

    Yi, Jiang; Fan, Yuting; Yokoyama, Wallace; Zhang, Yuzhu; Zhao, Liqing

    2016-06-01

    In this study, the interaction of WPI (whey protein isolate) and SC (sodium caseinate) with hydrophobic lutein was investigated through UV-vis spectroscopy and circular dichroism (CD) as well as fluorescence. The effects on lutein's chemical stability were also examined. The decrease of turbidity of lutein suggested that lutein's aqueous solubility was improved after binding with milk proteins. CD analysis indicated lutein had little impact on the secondary structures of both proteins. Different preparation methods have significant impacts on the binding constant. Fluorescence results indicated that WPI and SC interact with lutein by hydrophobic contacts. Milk proteins have protective effects on lutein against oxidation and decomposition, and SC showed better capability in protecting lutein from oxidation than WPI during 16 days storage. The lutein's chemical stability was increased with increasing of proteins concentration. The results indicated that milk proteins may act as effective carriers for lipophilic nutraceuticals. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Contribution of Eucalyptus Harvest Residues and Nitrogen Fertilization to Carbon Stabilization in Ultisols of Southern Bahia

    Directory of Open Access Journals (Sweden)

    Fernanda Cristina Caparelli Oliveira

    2018-02-01

    Full Text Available ABSTRACT: Eucalyptus forests in southern Bahia (BA are planted in soils with a sandy surface layer and humid tropical climate, conditions that lead to soil carbon (C decomposition. Recent studies have shown that nitrogen (N may be important for soil C stabilization. The aim of this study was to evaluate the contribution of Eucalyptus harvest residues and nitrogen fertilization to C stabilization in Ultisols of southern BA. The experiment was conducted in Eucalyptus clonal plantations cultivated in two regions of Eunápolis, BA, Brazil, with different clay content: southern region (140 g kg-1 of clay and western region (310 g kg-1 of clay. Five treatments were evaluated: one control (CTR, without Eucalyptus harvest residues and N fertilization, and four treatments with harvest residues combined with four rates of N fertilization: 0, 25, 50, and 100 kg ha-1. Soil samples were collected from the 0.00-0.10, 0.10-0.20, 0.20-0.40, and 0.40-0.60 m layers at the beginning and the end of the experiment (36 months. The amount of C and N and the C and N isotopic ratio (δ13C and δ15N of particulate organic matter (POM and mineral-associated organic matter (MAOM were determined. In the southern region after 36 months, the C-MAOM stocks in the 0.00-0.10 m layer of the CTR decreased by 33 %. The addition of harvest residue followed by 100 kg ha-1 N increased C-POM and N-POM stocks (0.00-0.10 m compared to the CTR, and the final N-POM stocks and residue-C recovery in the surface soil layer were positively correlated with the increase in N fertilization rates. In the western region, residue maintenance resulted in increased C-MAOM stocks (0.00-0.10 m compared to the CTR, but an increase in N availability reduced this increment. The increase in N fertilization rates did not alter C stocks, but reduced N stocks of POM and MAOM in the upper soil layer. At the end of the experiment, N fertilizer recovery (0.00-0.60 m was similar among the regions evaluated. In

  16. Poly(zwitterionic)protein conjugates offer increased stability without sacrificing binding affinity or bioactivity

    Science.gov (United States)

    Keefe, Andrew J.; Jiang, Shaoyi

    2012-01-01

    Treatment with therapeutic proteins is an attractive approach to targeting a number of challenging diseases. Unfortunately, the native proteins themselves are often unstable in physiological conditions, reducing bioavailability and therefore increasing the dose that is required. Conjugation with poly(ethylene glycol) (PEG) is often used to increase stability, but this has a detrimental effect on bioactivity. Here, we introduce conjugation with zwitterionic polymers such as poly(carboxybetaine). We show that poly(carboxybetaine) conjugation improves stability in a manner similar to PEGylation, but that the new conjugates retain or even improve the binding affinity as a result of enhanced protein-substrate hydrophobic interactions. This chemistry opens a new avenue for the development of protein therapeutics by avoiding the need to compromise between stability and affinity.

  17. A New Class of Amphiphiles Bearing Rigid Hydrophobic Groups for Solubilization and Stabilization of Membrane Proteins

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Rasmussen, Søren G F; Rana, Rohini R

    2012-01-01

    Non-traditional amphiphiles: Conferring aqueous solubility on membrane proteins generally requires the use of a detergent or other amphiphilic agent. A new class of amphiphiles was synthesized, based on steroidal lipophilic groups, and evaluated with several membrane proteins. The results show th...... that the new amphiphile, "glyco-diosgenin" (GDN; see figure), confers enhanced stability to a variety of membrane proteins in solution relative to popular conventional detergents, such as dodecylmaltoside (DDM)....

  18. Protein stability in pulmonary drug delivery via nebulization.

    Science.gov (United States)

    Hertel, Sebastian P; Winter, Gerhard; Friess, Wolfgang

    2015-10-01

    Protein inhalation is a delivery route which offers high potential for direct local lung application of proteins. Liquid formulations are usually available in early stages of biopharmaceutical development and nebulizers are the device of choice for atomization avoiding additional process steps like drying and enabling fast progression to clinical trials. While some proteins were proven to remain stable throughout aerosolization e.g. DNase, many biopharmaceuticals are more susceptible towards the stresses encountered during nebulization. The main reason for protein instability is unfolding and aggregation at the air-liquid interface, a problem which is of particular challenge in the case of ultrasound and jet nebulizers due to recirculation of much of the generated droplets. Surfactants are an important formulation component to protect the sensitive biomolecules. A second important challenge is warming of ultrasound and vibrating mesh devices, which can be overcome by overfilling, precooled solutions or cooling of the reservoir. Ultimately, formulation development has to go hand in hand with device evaluation. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. The contribution of fine roots to peatland stability under changing environmental conditions

    Science.gov (United States)

    Malhotra, A.; Brice, D. J.; Childs, J.; Phillips, J.; Hanson, P. J.; Iversen, C. M.

    2017-12-01

    Fine-root production and traits are closely linked with ecosystem nutrient and water fluxes, and may regulate these fluxes in response to environmental change. Plant strategies can shift to favoring below- over aboveground biomass allocation when nutrients or moisture are limited. Fine-roots traits such as root tissue density (RTD) or specific root length (SRL) can also adapt to the environment, for example, by maximizing the area of soil exploited by decreasing RTD and increasing SRL during dry conditions. Fine-root trait plasticity could contribute to the stability of peatland carbon function in response to environmental change. However, the extent and mechanisms of peatland fine-root plasticity are unknown. We investigated fine-root growth and traits and their link to environmental factors and aboveground dynamics at SPRUCE (Spruce and Peatland Responses Under Changing Environments), a warming and elevated CO2 (eCO2) experiment in an ombrotrophic peatland. In the first growing season of whole ecosystem warming, fine-root production increased with warming and drying. Above- versus belowground allocation strategies varied by plant functional type (PFT). In shrubs, contrary to our expectation, aboveground- to fine-root production allocation ratio increased with dryer conditions, perhaps as a response to a concurrent increase in nutrients. Trait response hypotheses were largely supported, with RTD decreasing and SRL increasing with warming; however, response varied among PFTs. Once eCO2 was turned on in the second growing season, preliminary results suggest interactive effects of warming and eCO2 on total fine-root production: production decreased or increased with warming in ambient or elevated CO2 plots, respectively. Both trait and production responses to warming and eCO2 varied by microtopography and depth. Our results highlight plasticity of fine-root traits and biomass allocation strategies; the extent and mechanism of which varies by PFT. We will summarize

  20. Urea Recycling Contributes to Nitrogen Retention in Calves Fed Milk Replacer and Low-Protein Solid Feed

    NARCIS (Netherlands)

    Berends, H.; Borne, van den J.J.G.C.; Røjen, B.A.; Baal, van J.; Gerrits, W.J.J.

    2014-01-01

    Urea recycling, with urea originating from catabolism of amino acids and hepatic detoxification of ammonia, is particularly relevant for ruminant animals, in which microbial protein contributes substantially to the metabolizable protein supply. However, the quantitative contribution of urea

  1. Caffeine stabilizes Cdc25 independently of Rad3 in S chizosaccharomyces pombe contributing to checkpoint override

    Science.gov (United States)

    Alao, John P; Sjölander, Johanna J; Baar, Juliane; Özbaki-Yagan, Nejla; Kakoschky, Bianca; Sunnerhagen, Per

    2014-01-01

    Cdc25 is required for Cdc2 dephosphorylation and is thus essential for cell cycle progression. Checkpoint activation requires dual inhibition of Cdc25 and Cdc2 in a Rad3-dependent manner. Caffeine is believed to override activation of the replication and DNA damage checkpoints by inhibiting Rad3-related proteins in both S chizosaccharomyces pombe and mammalian cells. In this study, we have investigated the impact of caffeine on Cdc25 stability, cell cycle progression and checkpoint override. Caffeine induced Cdc25 accumulation in S . pombe independently of Rad3. Caffeine delayed cell cycle progression under normal conditions but advanced mitosis in cells treated with replication inhibitors and DNA-damaging agents. In the absence of Cdc25, caffeine inhibited cell cycle progression even in the presence of hydroxyurea or phleomycin. Caffeine induces Cdc25 accumulation in S . pombe by suppressing its degradation independently of Rad3. The induction of Cdc25 accumulation was not associated with accelerated progression through mitosis, but rather with delayed progression through cytokinesis. Caffeine-induced Cdc25 accumulation appears to underlie its ability to override cell cycle checkpoints. The impact of Cdc25 accumulation on cell cycle progression is attenuated by Srk1 and Mad2. Together our findings suggest that caffeine overrides checkpoint enforcement by inducing the inappropriate nuclear localization of Cdc25. PMID:24666325

  2. Physicochemical properties and storage stability of soybean protein nanoemulsions prepared by ultra-high pressure homogenization.

    Science.gov (United States)

    Xu, Jing; Mukherjee, Dipaloke; Chang, Sam K C

    2018-02-01

    This study investigated the effects of the ultrahigh pressure homogenization (pressure, protein concentration, oil phase fraction, pH, temperature, and ionic strength) and storage on the properties of nanoemulsions (100-500nm range), which were stabilized by laboratory-prepared soybean protein isolate (SPI), β-conglycinin (7S) and glycinin (11S). The nanoemulsions made with SPI, 7S and 11S proteins exhibited considerable stability over various ionic strengths (0-500mM NaCl), pH (7), thermal treatments (30-60°C) and storage (0-45days). The far-UV spectra of SPI, 7S, 11S dispersions, and SPI-, 7S-, 11S protein-stabilized nanoemulsions were analyzed for the protein structural changes following lipid removal. The ultra-high pressure homogenization changed the secondary structure of SPI, 7S, 11S proteins in the nanoemulsions, and enhanced their stability. This study demonstrated that SPI, 7S, and 11S proteins can be used as effective emulsifiers in nanoemulsions prepared by ultra-high pressure homogenization. Copyright © 2017. Published by Elsevier Ltd.

  3. Benzalkonium Chloride Provides Remarkable Stability to Liquid Protein Lures for Trapping Anastrepha obliqua (Diptera: Tephritidae).

    Science.gov (United States)

    Lasa, R; Williams, T

    2017-12-05

    Hydrolyzed protein lures are widely used to monitor fruit fly pests but are rapidly degraded by microbial activity and must be replaced frequently. To improve the stability of lures, the quaternary ammonium biocide, benzalkonium chloride (BC), was evaluated in mixtures with two hydrolyzed proteins commonly used to monitor Anastrepha spp. The mean number of Anastrepha obliqua adults captured during six consecutive weeks using Captor + borax with the addition of 240 mg BC/liter, not renewed during the test, was similar to Captor + borax that was replaced at weekly intervals and was more effective than Captor + borax without BC. Numbers of A. obliqua flies captured in 30% CeraTrap diluted in water containing 240 mg BC/liter were similar to those caught in traps baited with Captor + borax or 30% CeraTrap without BC in the first 9 d of evaluation but was significantly more effective than both lures after 56 d. After >2 mo of use, 30% CeraTrap containing 240 mg BC/liter remained as effective as newly prepared 30% CeraTrap. The addition of BC to lures reduced surface tension of liquid lures by ~40-50%. However, when BC was increased to 720 mg BC/liter, only a small additional reduction in surface tension was observed and higher concentrations of BC did not increase capture rates. These findings could contribute to reduced costs for trapping networks and the development of long-lasting formulations of liquid protein lures for bait stations and mass-trapping targeted at major tephritid pests. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Proteins contribute insignificantly to the intrinsic buffering capacity of yeast cytoplasm

    International Nuclear Information System (INIS)

    Poznanski, Jaroslaw; Szczesny, Pawel; Ruszczyńska, Katarzyna; Zielenkiewicz, Piotr; Paczek, Leszek

    2013-01-01

    Highlights: ► We predicted buffering capacity of yeast proteome from protein abundance data. ► We measured total buffering capacity of yeast cytoplasm. ► We showed that proteins contribute insignificantly to buffering capacity. -- Abstract: Intracellular pH is maintained by a combination of the passive buffering of cytoplasmic dissociable compounds and several active systems. Over the years, a large portion of and possibly most of the cell’s intrinsic (i.e., passive non-bicarbonate) buffering effect was attributed to proteins, both in higher organisms and in yeast. This attribution was not surprising, given that the concentration of proteins with multiple protonable/deprotonable groups in the cell exceeds the concentration of free protons by a few orders of magnitude. Using data from both high-throughput experiments and in vitro laboratory experiments, we tested this concept. We assessed the buffering capacity of the yeast proteome using protein abundance data and compared it to our own titration of yeast cytoplasm. We showed that the protein contribution is less than 1% of the total intracellular buffering capacity. As confirmed with NMR measurements, inorganic phosphates play a crucial role in the process. These findings also shed a new light on the role of proteomes in maintaining intracellular pH. The contribution of proteins to the intrinsic buffering capacity is negligible, and proteins might act only as a recipient of signals for changes in pH.

  5. TOPICAL REVIEW: Protein stability and enzyme activity at extreme biological temperatures

    Science.gov (United States)

    Feller, Georges

    2010-08-01

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.

  6. Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

    Science.gov (United States)

    Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

    2003-01-01

    Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

  7. Identification of herpesvirus proteins that contribute to G1/S arrest.

    Science.gov (United States)

    Paladino, Patrick; Marcon, Edyta; Greenblatt, Jack; Frappier, Lori

    2014-04-01

    Lytic infection by herpesviruses induces cell cycle arrest at the G1/S transition. This appears to be a function of multiple herpesvirus proteins, but only a minority of herpesvirus proteins have been examined for cell cycle effects. To gain a more comprehensive understanding of the viral proteins that contribute to G1/S arrest, we screened a library of over 200 proteins from herpes simplex virus type 1, human cytomegalovirus, and Epstein-Barr virus (EBV) for effects on the G1/S interface, using HeLa fluorescent, ubiquitination-based cell cycle indicator (Fucci) cells in which G1/S can be detected colorimetrically. Proteins from each virus were identified that induce accumulation of G1/S cells, predominantly tegument, early, and capsid proteins. The identification of several capsid proteins in this screen suggests that incoming viral capsids may function to modulate cellular processes. The cell cycle effects of selected EBV proteins were further verified and examined for effects on p53 and p21 as regulators of the G1/S transition. Two EBV replication proteins (BORF2 and BMRF1) were found to induce p53 but not p21, while a previously uncharacterized tegument protein (BGLF2) was found to induce p21 protein levels in a p53-independent manner. Proteomic analyses of BGLF2-interacting proteins identified interactions with the NIMA-related protein kinase (NEK9) and GEM-interacting protein (GMIP). Silencing of either NEK9 or GMIP induced p21 without affecting p53 and abrogated the ability of BGLF2 to further induce p21. Collectively, these results suggest multiple viral proteins contribute to G1/S arrest, including BGLF2, which induces p21 levels likely by interfering with the functions of NEK9 and GMIP. Most people are infected with multiple herpesviruses, whose proteins alter the infected cells in several ways. During lytic infection, the viral proteins block cell proliferation just before the cellular DNA replicates. We used a novel screening method to identify proteins

  8. Synergistic and antagonistic effects of plant and dairy protein blends on the physicochemical stability of lycopene-loaded emulsions

    NARCIS (Netherlands)

    Ho, Kacie K.H.Y.; Schroën, Karin; San Martín-González, M.F.; Berton-Carabin, Claire C.

    2018-01-01

    Whey-plant protein-based emulsions had high physicochemical stability. Whey and plant protein blend-based interfaces were viscoelastic while casein-based interfaces were relatively viscous. Whey-plant and plant-plant protein blends behaved synergistically leading to enhanced emulsion stability.

  9. The Effect of Protein PEGylation on Physical Stability in Liquid Formulation

    DEFF Research Database (Denmark)

    Holm, Louise Stenstrup; Mcumber, Aaron; Rasmussen, Jakob Ewald

    2014-01-01

    The presence of micron aggregates in protein formulations has recently attracted increased interest from regulatory authorities, industry, and academia because of the potential undesired side effects of their presence. In this study, we characterized the micron aggregate formation of hen egg...... approximately half as many particles as Lyz, despite its lower apparent thermodynamic stability and more loose protein fold. Further characterization showed that the PEGylation led to a change from attractive to repulsive protein-protein interactions, which may partly explain the reduced particle formation...

  10. Insights into the role of hydration in protein structure and stability obtained through hydrostatic pressure studies

    Directory of Open Access Journals (Sweden)

    C.A. Royer

    2005-08-01

    Full Text Available A thorough understanding of protein structure and stability requires that we elucidate the molecular basis for the effects of both temperature and pressure on protein conformational transitions. While temperature effects are relatively well understood and the change in heat capacity upon unfolding has been reasonably well parameterized, the state of understanding of pressure effects is much less advanced. Ultimately, a quantitative parameterization of the volume changes (at the basis of pressure effects accompanying protein conformational transitions will be required. The present report introduces a qualitative hypothesis based on available model compound data for the molecular basis of volume change upon protein unfolding and its dependence on temperature.

  11. Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases

    OpenAIRE

    Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

    2012-01-01

    Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only...

  12. Principles and equations for measuring and interpreting protein stability: From monomer to tetramer.

    Science.gov (United States)

    Bedouelle, Hugues

    2016-02-01

    The ability to measure the thermodynamic stability of proteins with precision is important for both academic and applied research. Such measurements rely on mathematical models of the protein denaturation profile, i.e. the relation between a global protein signal, corresponding to the folding states in equilibrium, and the variable value of a denaturing agent, either heat or a chemical molecule, e.g. urea or guanidinium hydrochloride. In turn, such models rely on a handful of physical laws: the laws of mass action and conservation, the law that relates the protein signal and concentration, and the one that relates stability and denaturant value. So far, equations have been derived mainly for the denaturation profiles of homomeric proteins. Here, we review the underlying basic physical laws and show in detail how to derive model equations for the unfolding equilibria of homomeric or heteromeric proteins up to trimers and potentially tetramers, with or without folding intermediates, and give full demonstrations. We show that such equations cannot be derived for pentamers or higher oligomers except in special degenerate cases. We expand the method to signals that do not correspond to extensive protein properties. We review and expand methods for uncovering hidden intermediates of unfolding. Finally, we review methods for comparing and interpreting the thermodynamic parameters that derive from stability measurements for cognate wild-type and mutant proteins. This work should provide a robust theoretical basis for measuring the stability of complex proteins. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  13. Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases.

    Science.gov (United States)

    Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

    2012-08-01

    Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior. Copyright © 2012 The Protein Society.

  14. High-quality Thermodynamic Data on the Stability Changes of Proteins Upon Single-site Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Pucci, Fabrizio, E-mail: fapucci@ulb.ac.be; Bourgeas, Raphaël, E-mail: rbourgeas@ulb.ac.be; Rooman, Marianne, E-mail: mrooman@ulb.ac.be [Department of BioModeling, BioInformatics and BioProcesses, Université Libre de Bruxelles, CP 165/61, Roosevelt Avenue 50, 1050 Brussels, Belgium and Interuniversity Institute of Bioinformatics in Brussels, CP 263, Triumph Bld, 1050 Brussels (Belgium)

    2016-06-15

    We have set up and manually curated a dataset containing experimental information on the impact of amino acid substitutions in a protein on its thermal stability. It consists of a repository of experimentally measured melting temperatures (T{sub m}) and their changes upon point mutations (ΔT{sub m}) for proteins having a well-resolved x-ray structure. This high-quality dataset is designed for being used for the training or benchmarking of in silico thermal stability prediction methods. It also reports other experimentally measured thermodynamic quantities when available, i.e., the folding enthalpy (ΔH) and heat capacity (ΔC{sub P}) of the wild type proteins and their changes upon mutations (ΔΔH and ΔΔC{sub P}), as well as the change in folding free energy (ΔΔG) at a reference temperature. These data are analyzed in view of improving our insights into the correlation between thermal and thermodynamic stabilities, the asymmetry between the number of stabilizing and destabilizing mutations, and the difference in stabilization potential of thermostable versus mesostable proteins.

  15. Contribution of the maculo-ocular reflex to gaze stability in the rabbit.

    Science.gov (United States)

    Pettorossi, V E; Errico, P; Santarelli, R M

    1991-01-01

    The contribution of the maculo-ocular reflex to gaze stability was studied in 10 pigmented rabbits by rolling the animals at various angles of sagittal inclination of the rotation and/or longitudinal animal axes. At low frequencies (0.005-0.01 Hz) of sinusoidal stimulation the vestibulo-ocular reflex (VOR) was due to macular activation, while at intermediate and high frequencies it was mainly due to ampullar activation. The following results were obtained: 1) maculo-ocular reflex gain decreased as a function of the cosine of the angle between the rotation axis and the earth's horizontal plane. No change in gain was observed when longitudinal animal axis alone was inclined. 2) At 0 degrees of rotation axis and with the animal's longitudinal axis inclination also set at 0 degrees, the maculo-ocular reflex was oriented about 20 degrees forward and upward with respect to the earth's vertical axis. This orientation remained constant with sagittal inclinations of the rotation and/or longitudinal animal axes ranging from approximately 5 degrees upward to 30 degrees downward. When the longitudinal animal axis was inclined beyond these limits, the eye trajectory tended to follow the axis inclination. In the upside down position, the maculo-ocular reflex was anticompensatory, oblique and fixed with respect to orbital coordinates. 3) Ampullo-ocular reflex gain did not change with inclinations of the rotation and/or longitudinal animal axes. The ocular responses were consistently oriented to the stimulus plane. At intermediate frequencies the eye movement trajectory was elliptic because of directional differences between the ampullo- and maculo-ocular reflexes.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. How the industry can contribute to grid stability. Possibilities and impediments; Netzstabilisierende Beitraege der Industrie. Moeglichkeiten und Hemmnisse

    Energy Technology Data Exchange (ETDEWEB)

    Loske, Annette; Bier, Christoph [Verband der Industriellen Energie- und Kraftwirtschaft (VIK) e.V., Essen (Germany)

    2011-10-15

    The increase in volatile power infeed from renewable resources and load flow changes caused by restructuring measures in the power plant fleet pose major challenges in maintaining grid stability. Industrial final consumers can contribute to the solution of this problem by load shifting. However this presupposes suitable incentives.

  17. Sample Stability and Protein Composition of Saliva: Implications for Its Use as a Diagnostic Fluid

    Directory of Open Access Journals (Sweden)

    Han Roelofsen

    2008-01-01

    Full Text Available Saliva is an easy accessible plasma ultra-filtrate. Therefore, saliva can be an attractive alternative to blood for measurement of diagnostic protein markers. Our aim was to determine stability and protein composition of saliva. Protein stability at room temperature was examined by incubating fresh whole saliva with and without inhibitors of proteases and bacterial metabolism followed by Surface Enhanced Laser Desorption/Ionization (SELDI analyses. Protein composition was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE fractionation of saliva proteins followed by digestion of excised bands and identification by liquid chromatography tandem mass spectrometry (LC-MS/MS. Results show that rapid protein degradation occurs within 30 minutes after sample collection. Degradation starts already during collection. Protease inhibitors partly prevented degradation while inhibition of bacterial metabolism did not affect degradation. Three stable degradation products of 2937 Da, 3370 Da and 4132 Da were discovered which can be used as markers to monitor sample quality. Saliva proteome analyses revealed 218 proteins of which 84 can also be found in blood plasma. Based on a comparison with seven other proteomics studies on whole saliva we identified 83 new saliva proteins. We conclude that saliva is a promising diagnostic fl uid when precautions are taken towards protein breakdown.

  18. Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin.

    Science.gov (United States)

    Oulhen, Nathalie; Wessel, Gary M

    2016-10-01

    Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3'UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Whole-protein alanine-scanning mutagenesis of allostery: A large percentage of a protein can contribute to mechanism.

    Science.gov (United States)

    Tang, Qingling; Fenton, Aron W

    2017-09-01

    Many studies of allosteric mechanisms use limited numbers of mutations to test whether residues play "key" roles. However, if a large percentage of the protein contributes to allosteric function, mutating any residue would have a high probability of modifying allostery. Thus, a predicted mechanism that is dependent on only a few residues could erroneously appear to be supported. We used whole-protein alanine-scanning mutagenesis to determine which amino acid sidechains of human liver pyruvate kinase (hL-PYK; approved symbol PKLR) contribute to regulation by fructose-1,6-bisphosphate (Fru-1,6-BP; activator) and alanine (inhibitor). Each nonalanine/nonglycine residue of hL-PYK was mutated to alanine to generate 431 mutant proteins. Allosteric functions in active proteins were quantified by following substrate affinity over a concentration range of effectors. Results show that different residues contribute to the two allosteric functions. Only a small fraction of mutated residues perturbed inhibition by alanine. In contrast, a large percentage of mutated residues influenced activation by Fru-1,6-BP; inhibition by alanine is not simply the reverse of activation by Fru-1,6-BP. Moreover, the results show that Fru-1,6-BP activation would be extremely difficult to elucidate using a limited number of mutations. Additionally, this large mutational data set will be useful to train and test computational algorithms aiming to predict allosteric mechanisms. © 2017 Wiley Periodicals, Inc.

  20. Stabilizing Protein Effects on the Pressure Sensitivity of Fluorescent Gold Nanoclusters

    Science.gov (United States)

    2016-01-13

    affected by the environment of the stabilizing protein, allowing these hybrid systems to act as sensors in many applications.2,9,14–19 This has led...Biosens Bioelectron. 2012;32:297–299. 8. Joseph D, Geckeler KE. Synthesis of highly fluorescent gold nanoclusters using egg white proteins. Colloids Surf...Chang HW, Chien YC, Hsiao JK, Cheng JT, Chou PT. Insulin -directed synthesis of fluorescent gold nanoclusters: preservation of insulin bioactivity and

  1. Stability and structure of the membrane protein transporter Ffh is modulated by substrates and lipids

    DEFF Research Database (Denmark)

    Reinau, Marika Ejby; Otzen, Daniel

    2009-01-01

    the apoprotein. Escherichia coli lipid and DOPG (and to a smaller extent DOPC) increase Ffh's α-helical content, possibly related to Ffh's role in guiding membrane proteins to the membrane. Binding is largely mediated by electrostatic interactions but does not protect Ffh against trypsinolysis. We conclude...... that Ffh is a structurally flexible and dynamic protein whose stability is significantly modulated by the environment. © 2009 Elsevier Inc. All rights reserved....

  2. Contribution of Human Oral Cells to Astringency by Binding Salivary Protein/Tannin Complexes.

    Science.gov (United States)

    Soares, Susana; Ferrer-Galego, Raúl; Brandão, Elsa; Silva, Mafalda; Mateus, Nuno; Freitas, Victor de

    2016-10-10

    The most widely accepted mechanism to explain astringency is the interaction and precipitation of salivary proteins by food tannins, in particular proline-rich proteins. However, other mechanisms have been arising to explain astringency, such as binding of tannins to oral cells. In this work, an experimental method was adapted to study the possible contribution of both salivary proteins and oral cells to astringency induced by grape seed procyanidin fractions. Overall, in the absence of salivary proteins, the extent of procyanidin complexation with oral cells increased with increasing procyanidin degree of polymerization (mDP). Procyanidin fractions rich in monomers were the ones with the lowest ability to bind to oral cells. In the presence of salivary proteins and for procyanidins with mDP 2 the highest concentrations (1.5 and 2.0 mM) resulted in an increased binding of procyanidins to oral cells. This was even more evident for fractions III and IV at 1.0 mM and upper concentrations. Regarding the salivary proteins affected, it was possible to observe a decrease of P-B peptide and aPRP proteins for fractions II and III. This decrease is greater as the procyanidins' mDP increases. In fact, for fraction IV an almost total depletion of all salivary proteins was observed. This decrease is due to the formation of insoluble salivary protein/procyanidin complexes. Altogether, these data suggest that some procyanidins are able to bind to oral cells and that the salivary proteins interact with procyanidins forming salivary protein/procyanidin complexes that are also able to link to oral cells. The procyanidins that remain unbound to oral cells are able to bind to salivary proteins forming a large network of salivary protein/procyanidin complexes. Overall, the results presented herein provide one more step to understand food oral astringency onset.

  3. Stabilization and delivery approaches for protein and peptide pharmaceuticals: an extensive review of patents.

    Science.gov (United States)

    Swain, Suryakanta; Mondal, Debanik; Beg, Sarwar; Patra, Chinam Niranjan; Dinda, Subas Chandra; Sruti, Jammula; Rao, Muddana Eswara Bhanoji

    2013-04-01

    Proteins and peptides are the building blocks of human body and act as the arsenal to combat against the invading pathogenic organisms for treatment and management of diseases. Majority of such biomacromolecules are synthesized by the human body itself. However, entry of disease causing pathogens causes misleading in the synthesis of desired proteins for antibody formation. In such alarming situations, the delivery of requisite protein and peptide from external source helps in augmenting the body's immunity. The major drawbacks underlying poor biopharmaceutical performance of high molecular weight protein and peptide drugs are due to poor oral absorption, formulation stability, degradation in the gastric milieu, susceptible to presystemic metabolism. Numerous literature recounts the application of myriad drug delivery strategies for the effective delivery of protein and peptides viz. parentral, oral, transdermal, nasal, pulmonary, rectal, buccal and ocular drug delivery systems. There are many reviews on various delivery strategies for protein and peptide pharmaceuticals, but the present review article provides a bird's eye view on various novel drug delivery systems used for enhanced delivery of protein and peptide pharmaceuticals in the light of patent literature. Apart from this, the present manuscript endeavor provides idea on possible causes and major degradation pathways responsible for poor stability of protein and peptide drugs along with recent market instances on them utilizing novel drug delivery systems.

  4. In vivo stability and inertness of various direct labelled and chelate-tagged protein

    International Nuclear Information System (INIS)

    Janoki, A.; Korosi, L.; Klivenyi, G.; Spett, B.

    1987-01-01

    There were looking for methods giving precise information about composition and activity distribution of protein components, both in the initial samples and serum samples after intravenous administration. It was tested the applicability of electroimmunoassay, polyacrilamide gel electrophoresis and high performance liquid chromatography for the assessment of in vivo stability and labelled proteins. The model compound was human serum albumin (HSA) labelled with 99m Tc and 125 I, respectively. Bifunctional chelate labelling was done with desferrioxamine, in this case protein was labelled with 67 Ga. Biodistribution of the labelled compounds and their elimination from the blood were studied in rabbits. Experience with various labelling proteins, especially with Tc-Sn-HSA system indicate that in vivo stability of this compounds are generally low. Following intravenous injection of proteins labelled with metal isotopes, due to dilution and to the presence of considerable amount of compatitive protein in the serum, part of the label is being detached from the carrier protein. Distribution of the detached metal is different from the original distribution of the protein. This problem arises also with radiopharmaceuticals based on monoclonal antibodies. (M.E.L.) [es

  5. Nanoporous microbead supported bilayers: stability, physical characterization, and incorporation of functional transmembrane proteins.

    Energy Technology Data Exchange (ETDEWEB)

    Davis, Ryan W. (University of New Mexico, Albuquerque, NM); Brozik, James A. (University of New Mexico, Albuquerque, NM); Brozik, Susan Marie; Cox, Jason M. (University of New Mexico, Albuquerque, NM); Lopez, Gabriel P. (University of New Mexico, Albuquerque, NM); Barrick, Todd A. (University of New Mexico, Albuquerque, NM); Flores, Adrean (University of New Mexico, Albuquerque, NM)

    2007-03-01

    The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform.

  6. A Comparison of Protein Stability in Prefillable Syringes Made of Glass and Plastic.

    Science.gov (United States)

    Waxman, Lloyd; Vilivalam, Vinod D

    2017-01-01

    The development of protein therapeutics requires stabilization of these labile molecules during shipment and storage. Biologics, particularly monoclonal antibodies, are frequently packaged at high concentration in prefillable syringes traditionally made of glass. However, some biologics are unstable in glass due to sensitivity to silicone oil, tungsten, glue, or metal ions. Syringes made from the plastic cyclic olefin polymer, Daikyo Crystal Zenith® (CZ), with a Flurotec-laminated piston, have none of these issues. This study compared the stability of several proteins including biotherapeutics when stored up to 14 months at 5 °C and 25 °C in prefillable siliconized syringes made of glass or silicone oil-free CZ syringes, and when subjected to mild agitation by end-over-end rotation at room temperature. At each time point, proteins were analyzed by several techniques including turbidity, size exclusion high-performance liquid chromatography, reversed phase high-performance liquid chromatography, ion-exchange chromatography, electrophoresis, and light scattering to monitor changes in aggregation and degradation. The results show that proteins have comparable stability when stored in glass syringes or in syringes made of CZ sterilized by E-beam or autoclave. In addition, proteins stressed by agitation were generally more stable and aggregated less in syringes made of CZ than in ones made of glass. LAY ABSTRACT: Biotherapeutic protein drugs such as monoclonal antibodies are frequently packaged at high concentration in prefillable syringes, which allows the drug to be directly administered by the patient or caregiver. Protein drugs, or biologics, can be unstable, and may aggregate, particularly when shaken. These aggregates can be immunogenic, stimulating the body's immune system to produce antibodies that can reduce the drug's efficacy. Although prefillable syringes are traditionally made of glass, some biologics are unstable in glass syringes due to the presence of

  7. Long-term manure amendments reduced soil aggregate stability via redistribution of the glomalin-related soil protein in macroaggregates

    Science.gov (United States)

    Xie, Hongtu; Li, Jianwei; Zhang, Bin; Wang, Lianfeng; Wang, Jingkuan; He, Hongbo; Zhang, Xudong

    2015-01-01

    Glomalin-related soil protein (GRSP) contributes to the formation and maintenance of soil aggregates, it is however remains unclear whether long-term intensive manure amendments alter soil aggregates stability and whether GRSP regulates these changes. Based on a three-decade long fertilization experiment in northeast China, this study examined the impact of long-term manure input on soil organic carbon (SOC), total and easily extractable GRSP (GRSPt and GRSPe) and their respective allocations in four soil aggregates (>2000 μm; 2000–250 μm; 250–53 μm; and soil and SOC in each aggregate generally increased with increasing manure input, GRSPt and GRSPe in each aggregate showed varying changes with manure input. Both GRSP in macroaggregates (2000–250 μm) were significantly higher under low manure input, a pattern consistent with changes in soil aggregate stability. Constituting 38~49% of soil mass, macroaggregates likely contributed to the nonlinear changes of aggregate stability under manure amendments. The regulatory process of GRSP allocations in soil aggregates has important implications for manure management under intensive agriculture. PMID:26423355

  8. Protein content in gallstone patients can be a contributing factor in cholelithiasis

    International Nuclear Information System (INIS)

    Channa, N.A.; Memon, A.H.; Awan, A.Y.; Noorani, L.

    2017-01-01

    To find out the protein contents as contributing factor in the cholelithiasis patients. Methodology: The study was conducted during January 2016 to December 2016 at Institute of Biochemistry, University of Sindh, Jamshoro, Pakistan. A total number of 109 cholelithiasis patients admitted at Liaquat University Hospital, Jamshoro, Wali Bhai Rajputana Hospital, Hyderabad and Memon Charitable Hospital, Hyderabad, Pakistan and 294 age and gender matched control subjects were selected for the study. The serum samples of gallstone patients and control subjects were analyzed for the total protein contents by biuret kit method, gallstones recovered from the patients were also analyzed for the protein contents by Bradford method. Results: Cholelithiasis patients showed that gallstone protein contents increased with increasing age. We found increased amount of protein contents in mixed composition of gallstones as compared to pure cholesterol gallstones. Serum protein contents were increased in patients with age group 31-45 years, followed by age group 16-30 years. Female cholelithiasis patients were found with increased serum protein contents (but within normal limits) as compared to male cholelithiasis patients. Increased protein contents were also observed in serum as well as in gallstone samples recovered from multiparous female patients in comparison to triparous female patients. Conclusion: Protein may play a role in the pathogenesis of cholelithiasis. (author)

  9. Kinetic Stability of Proteins in Beans and Peas: Implications for Protein Digestibility, Seed Germination, and Plant Adaptation.

    Science.gov (United States)

    Xia, Ke; Pittelli, Sandy; Church, Jennifer; Colón, Wilfredo

    2016-10-12

    Kinetically stable proteins (KSPs) are resistant to the denaturing detergent sodium dodecyl sulfate (SDS). Such resilience makes KSPs resistant to proteolytic degradation and may have arisen in nature as a mechanism for organismal adaptation and survival against harsh conditions. Legumes are well-known for possessing degradation-resistant proteins that often diminish their nutritional value. Here we applied diagonal two-dimensional (D2D) SDS-polyacrylamide gel electrophoresis (PAGE), a method that allows for the proteomics-level identification of KSPs, to a group of 12 legumes (mostly beans and peas) of agricultural and nutritional importance. Our proteomics results show beans that are more difficult to digest, such as soybean, lima beans, and various common beans, have high contents of KSPs. In contrast, mung bean, red lentil, and various peas that are highly digestible contain low amounts of KSPs. Identified proteins with high kinetic stability are associated with warm-season beans, which germinate at higher temperatures. In contrast, peas and red lentil, which are cool-season legumes, contain low levels of KSPs. Thus, our results show protein kinetic stability is an important factor in the digestibility of legume proteins and may relate to nutrition efficiency, timing of seed germination, and legume resistance to biotic stressors. Furthermore, we show D2D SDS-PAGE is a powerful method that could be applied for determining the abundance and identity of KSPs in engineered and wild legumes and for advancing basic research and associated applications.

  10. Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization

    Energy Technology Data Exchange (ETDEWEB)

    Rutz, Natalja; Heilbronn, Regine; Weger, Stefan, E-mail: stefan.weger@charite.de

    2015-08-28

    Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 induced polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. - Highlights: • KCTD5 nuclear translocation depends upon M phase and protein oligomerization. • Identification of MCM7, ZNF711 and FAM193 as KCTD5 interaction partners. • Formation of trimeric complexes of KCTD5/cullin3 with MCM7, ZNF711 and FAM193B. • KCTD5 is not involved in polyubiquitylation of MCM7 replication factor. • The KCTD5/cullin3 complex stabilizes ZNF711 transcription factor.

  11. Rapid directed evolution of stabilized proteins with cellular high-throughput encapsulation solubilization and screening (CHESS).

    Science.gov (United States)

    Yong, K J; Scott, D J

    2015-03-01

    Directed evolution is a powerful method for engineering proteins towards user-defined goals and has been used to generate novel proteins for industrial processes, biological research and drug discovery. Typical directed evolution techniques include cellular display, phage display, ribosome display and water-in-oil compartmentalization, all of which physically link individual members of diverse gene libraries to their translated proteins. This allows the screening or selection for a desired protein function and subsequent isolation of the encoding gene from diverse populations. For biotechnological and industrial applications there is a need to engineer proteins that are functional under conditions that are not compatible with these techniques, such as high temperatures and harsh detergents. Cellular High-throughput Encapsulation Solubilization and Screening (CHESS), is a directed evolution method originally developed to engineer detergent-stable G proteins-coupled receptors (GPCRs) for structural biology. With CHESS, library-transformed bacterial cells are encapsulated in detergent-resistant polymers to form capsules, which serve to contain mutant genes and their encoded proteins upon detergent mediated solubilization of cell membranes. Populations of capsules can be screened like single cells to enable rapid isolation of genes encoding detergent-stable protein mutants. To demonstrate the general applicability of CHESS to other proteins, we have characterized the stability and permeability of CHESS microcapsules and employed CHESS to generate thermostable, sodium dodecyl sulfate (SDS) resistant green fluorescent protein (GFP) mutants, the first soluble proteins to be engineered using CHESS. © 2014 Wiley Periodicals, Inc.

  12. Ultra-High Pressure Homogenization enhances physicochemical properties of soy protein isolate-stabilized emulsions.

    Science.gov (United States)

    Fernández-Ávila, C; Escriu, R; Trujillo, A J

    2015-09-01

    The effect of Ultra-High Pressure Homogenization (UHPH, 100-300MPa) on the physicochemical properties of oil-in-water emulsions prepared with 4.0% (w/v) of soy protein isolate (SPI) and soybean oil (10 and 20%, v/v) was studied and compared to emulsions treated by conventional homogenization (CH, 15MPa). CH emulsions were prepared with non-heated and heated (95°C for 15min) SPI dispersions. Emulsions were characterized by particle size determination with laser diffraction, rheological properties using a rotational rheometer by applying measurements of flow curve and by transmission electron microscopy. The variation on particle size and creaming was assessed by Turbiscan® analysis, and visual observation of the emulsions was also carried out. UHPH emulsions showed much smaller d 3.2 values and greater physical stability than CH emulsions. The thermal treatment of SPI prior CH process did not improve physical stability properties. In addition, emulsions containing 20% of oil exhibited greater physical stability compared to emulsions containing 10% of oil. Particularly, UHPH emulsions treated at 100 and 200MPa with 20% of oil were the most stable due to low particle size values (d 3.2 and Span), greater viscosity and partial protein denaturation. These results address the physical stability improvement of protein isolate-stabilized emulsions by using the emerging UHPH technology. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. CAN THE STABILITY OF PROTEIN MUTANTS BE PREDICTED BY FREE-ENERGY CALCULATIONS

    NARCIS (Netherlands)

    YUNYU, S; MARK, AE; WANG, CX; HUANG, FH; BERENDSEN, HJC; VANGUNSTEREN, WF

    The use of free energy simulation techniques in the study of protein stability is critically evaluated. Results from two simulations of the thermostability mutation Asn218 to Ser218 in Subtilisin are presented. It is shown that components of the free energy change can be highly sensitive to the

  14. Carotenoid-protein complexes and their stability towards oxygen and radiation

    International Nuclear Information System (INIS)

    Ramakrishnan, T.V.; Francis, F.J.

    1980-01-01

    Carotenoid-protein complexes isolated from fresh mangoes were found to be more stable to oxygen and radiation when dissolved in water as compared with β-carotene in petroleum ether. Part of the pigment could be released from the complex by gamma irradiation. Observations on the stability of the carotenoid (98% β-carotene) in the complex indicated that the pigment is either associated with the lipid prosthetic group of the protein or loosely attached to the protein by weak hydrophobic bonds. (author)

  15. Stabilizing additives added during cell lysis aid in the solubilization of recombinant proteins.

    Directory of Open Access Journals (Sweden)

    David J Leibly

    Full Text Available Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl(2, proline, xylitol, NDSB 201, CTAB and K(2PO(4 solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40% were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher.

  16. A Breast Tissue Protein Expression Profile Contributing to Early Parity-Induced Protection Against Breast Cancer

    Directory of Open Access Journals (Sweden)

    Christina Marie Gutierrez

    2015-11-01

    Full Text Available Background/Aims: Early parity reduces breast cancer risk, whereas, late parity and nulliparity increase breast cancer risk. Despite substantial efforts to understand the protective effects of early parity, the precise molecular circuitry responsible for these changes is not yet fully defined. Methods: Here, we have conducted the first study assessing protein expression profiles in normal breast tissue of healthy early parous, late parous, and nulliparous women. Breast tissue biopsies were obtained from 132 healthy parous and nulliparous volunteers. These samples were subjected to global protein expression profiling and immunohistochemistry. GeneSpring and MetaCore bioinformatics analysis software were used to identify protein expression profiles associated with early parity (low risk versus late/nulliparity (high risk. Results: Early parity reduces expression of key proteins involved in mitogenic signaling pathways in breast tissue through down regulation of EGFR1/3, ESR1, AKT1, ATF, Fos, and SRC. Early parity is also characterized by greater genomic stability and reduced tissue inflammation based on differential expression of aurora kinases, p53, RAD52, BRCA1, MAPKAPK-2, ATF-1, ICAM1, and NF-kappaB compared to late and nulli parity. Conclusions: Early parity reduces basal cell proliferation in breast tissue, which translates to enhanced genomic stability, reduced cellular stress/inflammation, and thus reduced breast cancer risk.

  17. Influence of Pea Protein Aggregates on the Structure and Stability of Pea Protein/Soybean Polysaccharide Complex Emulsions

    Directory of Open Access Journals (Sweden)

    Baoru Yin

    2015-03-01

    Full Text Available The applications of plant proteins in the food and beverage industry have been hampered by their precipitation in acidic solution. In this study, pea protein isolate (PPI with poor dispersibility in acidic solution was used to form complexes with soybean soluble polysaccharide (SSPS, and the effects of PPI aggregates on the structure and stability of PPI/SSPS complex emulsions were investigated. Under acidic conditions, high pressure homogenization disrupts the PPI aggregates and the electrostatic attraction between PPI and SSPS facilitates the formation of dispersible PPI/SSPS complexes. The PPI/SSPS complex emulsions prepared from the PPI containing aggregates prove to possess similar droplet structure and similar stability compared with the PPI/SSPS emulsions produced from the PPI in which the aggregates have been previously removed by centrifugation. The oil droplets are protected by PPI/SSPS complex interfacial films and SSPS surfaces. The emulsions show long-term stability against pH and NaCl concentration changes. This study demonstrates that PPI aggregates can also be used to produce stable complex emulsions, which may promote the applications of plant proteins in the food and beverage industry.

  18. Influence of pea protein aggregates on the structure and stability of pea protein/soybean polysaccharide complex emulsions.

    Science.gov (United States)

    Yin, Baoru; Zhang, Rujing; Yao, Ping

    2015-03-20

    The applications of plant proteins in the food and beverage industry have been hampered by their precipitation in acidic solution. In this study, pea protein isolate (PPI) with poor dispersibility in acidic solution was used to form complexes with soybean soluble polysaccharide (SSPS), and the effects of PPI aggregates on the structure and stability of PPI/SSPS complex emulsions were investigated. Under acidic conditions, high pressure homogenization disrupts the PPI aggregates and the electrostatic attraction between PPI and SSPS facilitates the formation of dispersible PPI/SSPS complexes. The PPI/SSPS complex emulsions prepared from the PPI containing aggregates prove to possess similar droplet structure and similar stability compared with the PPI/SSPS emulsions produced from the PPI in which the aggregates have been previously removed by centrifugation. The oil droplets are protected by PPI/SSPS complex interfacial films and SSPS surfaces. The emulsions show long-term stability against pH and NaCl concentration changes. This study demonstrates that PPI aggregates can also be used to produce stable complex emulsions, which may promote the applications of plant proteins in the food and beverage industry.

  19. 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.

  20. Cysteine residue is not essential for CPM protein thermal-stability assay.

    Science.gov (United States)

    Wang, Zhaoshuai; Ye, Cui; Zhang, Xinyi; Wei, Yinan

    2015-05-01

    A popular thermal-stability assay developed especially for the study of membrane proteins uses a thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). The fluorescence emission of CPM surges when it forms a covalent bond with the side chain of a free Cys, which becomes more readily accessible upon protein thermal denaturation. Interestingly, the melting temperatures of membrane proteins determined using the CPM assay in literature are closely clustered in the temperature range 45-55 °C. A thorough understanding of the mechanism behind the observed signal change is critical for the accurate interpretation of the protein unfolding. Here we used two α-helical membrane proteins, AqpZ and AcrB, as model systems to investigate the nature of the fluorescence surge in the CPM assay. We found that the transition temperatures measured using circular-dichroism (CD) spectroscopy and the CPM assay were significantly different. To eliminate potential artifact that might arise from the presence of detergent, we monitored the unfolding of two soluble proteins. We found that, contrary to current understanding, the presence of a sulfhydryl group was not a prerequisite for the CPM thermal-stability assay. The observed fluorescence increase is probably caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding.

  1. Increased helix and protein stability through the introduction of a new tertiary hydrogen bond.

    Science.gov (United States)

    Peterson, R W; Nicholson, E M; Thapar, R; Klevit, R E; Scholtz, J M

    1999-03-12

    In an effort to quantify the importance of hydrogen bonding and alpha-helix formation to protein stability, a capping box motif was introduced into the small phosphocarrier protein HPr. Previous studies had confirmed that Ser46, at the N-cap position of the short helix-B in HPr, serves as an N-cap in solution. Thus, only a single-site mutation was required to produce a canonical S-X-X-E capping box: Lys49 at the N3 position was substituted with a glutamic acid residue. Thermal and chemical denaturation studies on the resulting K49E HPr show that the designed variant is approximately 2 kcal mol-1 more stable than the wild-type protein. However, NMR studies indicate that the side-chain of Glu49 does not participate in the expected capping H-bond interaction, but instead forms a new tertiary H-bond that links helix-B to the four-stranded beta-sheet of HPr. Here, we demonstrate that a strategy in which new non-native H-bonds are introduced can generate proteins with increased stability. We discuss why the original capping box design failed, and compare the energetic consequences of the new tertiary side-chain to main-chain H-bond with a local (helix-capping) side-chain to main-chain H-bond on the protein's global stability. Copyright 1999 Academic Press.

  2. Stability of spray-dried beetroot extract using oligosaccharides and whey proteins.

    Science.gov (United States)

    Carmo, Eloá Lourenço do; Teodoro, Rhana Amanda Ribeiro; Félix, Pedro Henrique Campelo; Fernandes, Regiane Victória de Barros; Oliveira, Érica Resende de; Veiga, Taís Regina Lima Abreu; Borges, Soraia Vilela; Botrel, Diego Alvarenga

    2018-05-30

    The properties and stability of spray-dried beetroot extract using maltodextrin (MD), inulin (IN), and whey protein isolate (WPI) as carrier agents were evaluated. The values of moisture, betalains content, and retention were 3.33-4.24%, 348.79-385.47 mg/100 g (dry-basis), and 88.45-95.69%, respectively. Higher values of antioxidant activity were observed for the treatments using WPI. The treatment with inulin alone presented higher hygroscopicity in the moisture adsorption isotherms at 25 °C and lower thermal stability when evaluating the thermogravimetric curves. When stored at 60 °C, the use of WPI alone conferred lower stability to the beetroot extract powder. In general, the simultaneous use of IN and WPI as carrier agents resulted in good stability of the beetroot extract powder, representing an opportunity for innovation in food products. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins.

    Science.gov (United States)

    Chae, Pil Seok; Rasmussen, Søren G F; Rana, Rohini R; Gotfryd, Kamil; Chandra, Richa; Goren, Michael A; Kruse, Andrew C; Nurva, Shailika; Loland, Claus J; Pierre, Yves; Drew, David; Popot, Jean-Luc; Picot, Daniel; Fox, Brian G; Guan, Lan; Gether, Ulrik; Byrne, Bernadette; Kobilka, Brian; Gellman, Samuel H

    2010-12-01

    The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family show favorable behavior relative to conventional detergents, as manifested in multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.

  4. Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Overton, K. Wesley; Park, Dan M.; Yung, Mimi C.; Dohnalkova, Alice; Smit, John; Jiao, Yongqin

    2016-09-23

    ABSTRACT

    Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions, including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport the S-layer protein from the cytoplasm to the cell exterior.Caulobacter crescentusis unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFaand RsaFb, which together with other components form a type I protein translocation pathway for S-layer export. These proteins have homology toEscherichia coliTolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaFaand RsaFbare not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFaand RsaFbare required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFaand RsaFbled to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and transcriptome sequencing (RNA-seq), we show that loss of both RsaFaand RsaFbled to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein misfolding and degradation pathways. These findings provide new insight into the requirement for RsaFaand RsaFbin cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels in

  5. Quantifying the contribution of the root system of alpine vegetation in the soil aggregate stability of moraine

    Directory of Open Access Journals (Sweden)

    Csilla Hudek

    2017-03-01

    Full Text Available One fifth of the world's population is living in mountains or in their surrounding areas. This anthropogenic pressure continues to grow with the increasing number of settlements, especially in areas connected to touristic activities, such as the Italian Alps. The process of soil formation on high mountains is particularly slow and these soils are particularly vulnerable to soil degradation. In alpine regions, extreme meteorological events are increasingly frequent due to climate change, speeding up the process of soil degradation and increasing the number of severe erosion processes, shallow landslides and debris flows. Vegetation cover plays a crucial role in the stabilization of mountain soils thereby reducing the risk of natural hazards effecting downslope areas. Soil aggregate stability is one of the main soil properties that can be linked to soil loss processes. Soils developed on moraines in recently deglaciated areas typically have low levels of soil aggregation, and a limited or discontinuous vegetation cover making them more susceptible to degradation. However, soil structure can be influenced by the root system of the vegetation. Roots are actively involved in the formation of water-stable soil aggregation, increasing the stability of the soil and its nutrient content. In the present study, we aim to quantify the effect of the root system of alpine vegetation on the soil aggregate stability of the forefield of the Lys glacier, in the Aosta Valley (NW-Italy. This proglacial area provides the opportunity to study how the root system of ten pioneer alpine species from different successional stages can contribute to soil development and soil stabilization. To quantify the aggregate stability of root permeated soils, a modified wet sieving method was employed. The root length per soil volume of the different species was also determined and later correlated with the aggregate stability results. The results showed that soil aggregate

  6. Molecular Dynamics Driven Design of pH-Stabilized Mutants of MNEI, a Sweet Protein.

    Directory of Open Access Journals (Sweden)

    Serena Leone

    Full Text Available MNEI is a single chain derivative of monellin, a plant protein that can interact with the human sweet taste receptor, being therefore perceived as sweet. This unusual physiological activity makes MNEI a potential template for the design of new sugar replacers for the food and beverage industry. Unfortunately, applications of MNEI have been so far limited by its intrinsic sensitivity to some pH and temperature conditions, which could occur in industrial processes. Changes in physical parameters can, in fact, lead to irreversible protein denaturation, as well as aggregation and precipitation. It has been previously shown that the correlation between pH and stability in MNEI derives from the presence of a single glutamic residue in a hydrophobic pocket of the protein. We have used molecular dynamics to study the consequences, at the atomic level, of the protonation state of such residue and have identified the network of intramolecular interactions responsible for MNEI stability at acidic pH. Based on this information, we have designed a pH-independent, stabilized mutant of MNEI and confirmed its increased stability by both molecular modeling and experimental techniques.

  7. Molecular Dynamics Driven Design of pH-Stabilized Mutants of MNEI, a Sweet Protein.

    Science.gov (United States)

    Leone, Serena; Picone, Delia

    2016-01-01

    MNEI is a single chain derivative of monellin, a plant protein that can interact with the human sweet taste receptor, being therefore perceived as sweet. This unusual physiological activity makes MNEI a potential template for the design of new sugar replacers for the food and beverage industry. Unfortunately, applications of MNEI have been so far limited by its intrinsic sensitivity to some pH and temperature conditions, which could occur in industrial processes. Changes in physical parameters can, in fact, lead to irreversible protein denaturation, as well as aggregation and precipitation. It has been previously shown that the correlation between pH and stability in MNEI derives from the presence of a single glutamic residue in a hydrophobic pocket of the protein. We have used molecular dynamics to study the consequences, at the atomic level, of the protonation state of such residue and have identified the network of intramolecular interactions responsible for MNEI stability at acidic pH. Based on this information, we have designed a pH-independent, stabilized mutant of MNEI and confirmed its increased stability by both molecular modeling and experimental techniques.

  8. Effect of hydrogen peroxide on improving the heat stability of whey protein isolate solutions.

    Science.gov (United States)

    Sutariya, Suresh; Patel, Hasmukh

    2017-05-15

    Whey protein isolate (WPI) solutions (12.8%w/w protein) were treated with varying concentrations of H 2 O 2 in the range of 0-0.144 H 2 O 2 to protein ratios (HTPR) by the addition of the required quantity of H 2 O 2 and deionized water. The samples were analyzed for heat stability, rheological properties, denaturation level of β-lactoglobulin (β-LG) and α-lactalbumin (α-LA). The samples treated with H 2 O 2 concentration >0.072 (HTPR) showed significant improvement in the heat stability, and decreased whey protein denaturation and aggregation. The WPI solution treated with H 2 O 2 (>0.072 HTPR) remained in the liquid state after heat treatment at 120°C, whereas the control samples formed gel upon heat treatment. Detailed analysis of these samples suggested that the improvement in the heat stability of H 2 O 2 treated WPI solution was attributed to the significant reduction in the sulfhydryl-disulfide interchange reaction during denaturation of β-LG and α-LA. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Weak Links: Stabilizers of Complex Systems from Proteins to Social Networks

    Science.gov (United States)

    Csermely, Peter

    Why do women stabilize our societies? Why can we enjoy and understand Shakespeare? Why are fruitflies uniform? Why do omnivorous eating habits aid our survival? Why is Mona Lisa's smile beautiful? -- Is there any answer to these questions? This book shows that the statement: "weak links stabilize complex systems" holds the answers to all of the surprising questions above. The author (recipientof several distinguished science communication prizes) uses weak (low affinity, low probability) interactions as a thread to introduce a vast varietyof networks from proteins to ecosystems.

  10. Protein adsorption at the electrified air-water interface: implications on foam stability.

    Science.gov (United States)

    Engelhardt, Kathrin; Rumpel, Armin; Walter, Johannes; Dombrowski, Jannika; Kulozik, Ulrich; Braunschweig, Björn; Peukert, Wolfgang

    2012-05-22

    The surface chemistry of ions, water molecules, and proteins as well as their ability to form stable networks in foams can influence and control macroscopic properties such as taste and texture of dairy products considerably. Despite the significant relevance of protein adsorption at liquid interfaces, a molecular level understanding on the arrangement of proteins at interfaces and their interactions has been elusive. Therefore, we have addressed the adsorption of the model protein bovine serum albumin (BSA) at the air-water interface with vibrational sum-frequency generation (SFG) and ellipsometry. SFG provides specific information on the composition and average orientation of molecules at interfaces, while complementary information on the thickness of the adsorbed layer can be obtained with ellipsometry. Adsorption of charged BSA proteins at the water surface leads to an electrified interface, pH dependent charging, and electric field-induced polar ordering of interfacial H(2)O and BSA. Varying the bulk pH of protein solutions changes the intensities of the protein related vibrational bands substantially, while dramatic changes in vibrational bands of interfacial H(2)O are simultaneously observed. These observations have allowed us to determine the isoelectric point of BSA directly at the electrolyte-air interface for the first time. BSA covered air-water interfaces with a pH near the isoelectric point form an amorphous network of possibly agglomerated BSA proteins. Finally, we provide a direct correlation of the molecular structure of BSA interfaces with foam stability and new information on the link between microscopic properties of BSA at water surfaces and macroscopic properties such as the stability of protein foams.

  11. IGF-IR promotes prostate cancer growth by stabilizing α5β1 integrin protein levels.

    Directory of Open Access Journals (Sweden)

    Aejaz Sayeed

    Full Text Available Dynamic crosstalk between growth factor receptors, cell adhesion molecules and extracellular matrix is essential for cancer cell migration and invasion. Integrins are transmembrane receptors that bind extracellular matrix proteins and enable cell adhesion and cytoskeletal organization. They also mediate signal transduction to regulate cell proliferation and survival. The type 1 insulin-like growth factor receptor (IGF-IR mediates tumor cell growth, adhesion and inhibition of apoptosis in several types of cancer. We have previously demonstrated that β1 integrins regulate anchorage-independent growth of prostate cancer (PrCa cells by regulating IGF-IR expression and androgen receptor-mediated transcriptional functions. Furthermore, we have recently reported that IGF-IR regulates the expression of β1 integrins in PrCa cells. We have dissected the mechanism through which IGF-IR regulates β1 integrin expression in PrCa. Here we report that IGF-IR is crucial for PrCa cell growth and that β1 integrins contribute to the regulation of proliferation by IGF-IR. We demonstrate that β1 integrin regulation by IGF-IR does not occur at the mRNA level. Exogenous expression of a CD4 - β1 integrin cytoplasmic domain chimera does not interfere with such regulation and fails to stabilize β1 integrin expression in the absence of IGF-IR. This appears to be due to the lack of interaction between the β1 cytoplasmic domain and IGF-IR. We demonstrate that IGF-IR stabilizes the β1 subunit by protecting it from proteasomal degradation. The α5 subunit, one of the binding partners of β1, is also downregulated along with β1 upon IGF-IR knockdown while no change is observed in the expression of the α2, α3, α4, α6 and α7 subunits. Our results reveal a crucial mechanistic role for the α5β1 integrin, downstream of IGF-IR, in regulating cancer growth.

  12. Activation of the Unfolded Protein Response Contributes toward the Antitumor Activity of Vorinostat

    Directory of Open Access Journals (Sweden)

    Soumen Kahali

    2010-01-01

    Full Text Available Histone deacetylase (HDAC inhibitors represent an emerging class of anticancer agents progressing through clinical trials. Although their primary target is thought to involve acetylation of core histones, several nonhistone substrates have been identified, including heat shock protein (HSP 90, which may contribute towards their antitumor activity. Glucose-regulated protein 78 (GRP78 is a member of the HSP family of molecular chaperones and plays a central role in regulating the unfolded protein response (UPR. Emerging data suggest that GRP78 is critical in cellular adaptation and survival associated with oncogenesis and may serve as a cancer-specific therapeutic target. On the basis of shared homology with HSP family proteins, we sought to determine whether GRP78 could serve as a molecular target of the HDAC inhibitor vorinostat. Vorinostat treatment led to GRP78 acetylation, dissociation, and subsequent activation of its client protein double-stranded RNA-activated protein-like endoplasmic reticulum kinase (PERK. Investigations in a panel of cancer cell lines identified that UPR activation after vorinostat exposure is specific to certain lines. Mass spectrometry performed on immunoprecipitated GRP78 identified lysine-585 as a specific vorinostat-induced acetylation site of GRP78. Downstream activation of the UPR was confirmed, including eukaryotic initiating factor 2α phosphorylation and increase in ATF4 and C/EBP homologous protein expression. To determine the biologic relevance of UPR activation after vorinostat, RNA interference of PERK was performed, demonstrating significantly decreased sensitivity to vorinostat-induced cytotoxicity. Collectively, these findings indicate that GRP78 is a biologic target of vorinostat, and activation of the UPR through PERK phosphorylation contributes toward its antitumor activity.

  13. Effects of lysine residues on structural characteristics and stability of tau proteins

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Myeongsang; Baek, Inchul; Choi, Hyunsung; Kim, Jae In; Na, Sungsoo, E-mail: nass@korea.ac.kr

    2015-10-23

    Pathological amyloid proteins have been implicated in neuro-degenerative diseases, specifically Alzheimer's, Parkinson's, Lewy-body diseases and prion related diseases. In prion related diseases, functional tau proteins can be transformed into pathological agents by environmental factors, including oxidative stress, inflammation, Aβ-mediated toxicity and covalent modification. These pathological agents are stable under physiological conditions and are not easily degraded. This un-degradable characteristic of tau proteins enables their utilization as functional materials to capturing the carbon dioxides. For the proper utilization of amyloid proteins as functional materials efficiently, a basic study regarding their structural characteristic is necessary. Here, we investigated the basic tau protein structure of wild-type (WT) and tau proteins with lysine residues mutation at glutamic residue (Q2K) on tau protein at atomistic scale. We also reported the size effect of both the WT and Q2K structures, which allowed us to identify the stability of those amyloid structures. - Highlights: • Lysine mutation effect alters the structure conformation and characteristic of tau. • Over the 15 layers both WT and Q2K models, both tau proteins undergo fractions. • Lysine mutation causes the increment of non-bonded energy and solvent accessible surface area. • Structural instability of Q2K model was proved by the number of hydrogen bonds analysis.

  14. Effects of lysine residues on structural characteristics and stability of tau proteins

    International Nuclear Information System (INIS)

    Lee, Myeongsang; Baek, Inchul; Choi, Hyunsung; Kim, Jae In; Na, Sungsoo

    2015-01-01

    Pathological amyloid proteins have been implicated in neuro-degenerative diseases, specifically Alzheimer's, Parkinson's, Lewy-body diseases and prion related diseases. In prion related diseases, functional tau proteins can be transformed into pathological agents by environmental factors, including oxidative stress, inflammation, Aβ-mediated toxicity and covalent modification. These pathological agents are stable under physiological conditions and are not easily degraded. This un-degradable characteristic of tau proteins enables their utilization as functional materials to capturing the carbon dioxides. For the proper utilization of amyloid proteins as functional materials efficiently, a basic study regarding their structural characteristic is necessary. Here, we investigated the basic tau protein structure of wild-type (WT) and tau proteins with lysine residues mutation at glutamic residue (Q2K) on tau protein at atomistic scale. We also reported the size effect of both the WT and Q2K structures, which allowed us to identify the stability of those amyloid structures. - Highlights: • Lysine mutation effect alters the structure conformation and characteristic of tau. • Over the 15 layers both WT and Q2K models, both tau proteins undergo fractions. • Lysine mutation causes the increment of non-bonded energy and solvent accessible surface area. • Structural instability of Q2K model was proved by the number of hydrogen bonds analysis.

  15. New aspects of protein stability and turnover in the regulation of genome integrity

    DEFF Research Database (Denmark)

    Gallina, Irene

    of DNA repair is the control of protein abundance, both at a global cellular level, and locally at the site of damage. This is achieved through transcriptional regulation of protein synthesis and through the control of protein stability and turnover. In this study, we investigate the role of Rad56...... sensitivity when mutant. Prior to the work presented here,all these loci have been mapped to a specific gene except RAD56. We map the rad56-1 mutation to the NAT3 gene, which encodes the catalytic subunit of the NatB N-terminal acetyltransferase in yeast. Deletion of RAD56 causes sensitivity to X-rays, methyl......-scale studies investigating factors involved in DNA metabolism, but no specific function has been assigned to Cmr1. Taking advantage of a series of high-throughput screens we characterize Cmr1 as a chromatinassociated protein, involved in the regulation of fork progression in the presence of replication stress...

  16. Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability

    DEFF Research Database (Denmark)

    Lauritsen, Ida; Martinez, Virginia; Ronda, Carlotta

    2018-01-01

    In molecular biology and cell factory engineering, tools that enable control of protein production and stability are highly important. Here, we describe protocols for tagging genes in Escherichia coli allowing for inducible degradation and transcriptional control of any soluble protein of interest....... The underlying molecular biology is based on the two cross-kingdom tools CRISPRi and the N-end rule for protein degradation. Genome editing is performed with the CRMAGE technology and randomization of the translational initiation region minimizes the polar effects of tag insertion. The approach has previously...... been applied for targeting proteins originating from essential operon-located genes and has potential to serve as a universal synthetic biology tool....

  17. Stability of some Cactaceae proteins based on fluorescence, circular dichroism, and differential scanning calorimetry measurements.

    Science.gov (United States)

    Gorinstein, S; Zemser, M; Vargas-Albores, F; Ochoa, J L; Paredes-Lopez, O; Scheler, C; Aksu, S; Salnikow, J

    1999-02-01

    Characterization of three cactus proteins (native and denatured) from Machaerocereus gummosus (Pitahaya agria), Lophocereu schottii (Garambullo), and Cholla opuntia (Cholla), was based on electrophoretic, fluorescence, CD (circular dichroism), DSC (differential scanning calorimetry), and FT-IR (Fourier transform infrared) measurements. The obtained results of intrinsic fluorescence, DSC, and CD were dissimilar for the three species of cactus, providing evidence of differences in secondary and tertiary structures. Cactus proteins may be situated in the following order corresponding to their relative stability: Machaerocereus gummosus (Pitahaya agria) > Cholla opuntia (Cholla) > Lophocereu schottii (Garambullo). Thermodynamic properties of proteins and their changes upon denaturation (temperature of denaturation, enthalphy, and the number of ruptured hydrogen bonds) were correlated with the secondary structure of proteins and disappearance of alpha-helix.

  18. Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation.

    Science.gov (United States)

    Schlimpert, Susan; Wasserstrom, Sebastian; Chandra, Govind; Bibb, Maureen J; Findlay, Kim C; Flärdh, Klas; Buttner, Mark J

    2017-07-25

    During sporulation, the filamentous bacteria Streptomyces undergo a massive cell division event in which the synthesis of ladders of sporulation septa convert multigenomic hyphae into chains of unigenomic spores. This process requires cytokinetic Z-rings formed by the bacterial tubulin homolog FtsZ, and the stabilization of the newly formed Z-rings is crucial for completion of septum synthesis. Here we show that two dynamin-like proteins, DynA and DynB, play critical roles in this process. Dynamins are a family of large, multidomain GTPases involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. Using a cell biological approach, we show that the two Streptomyces dynamins specifically localize to sporulation septa in an FtsZ-dependent manner. Moreover, dynamin mutants have a cell division defect due to the decreased stability of sporulation-specific Z-rings, as demonstrated by kymographs derived from time-lapse images of FtsZ ladder formation. This defect causes the premature disassembly of individual Z-rings, leading to the frequent abortion of septum synthesis, which in turn results in the production of long spore-like compartments with multiple chromosomes. Two-hybrid analysis revealed that the dynamins are part of the cell division machinery and that they mediate their effects on Z-ring stability during developmentally controlled cell division via a network of protein-protein interactions involving DynA, DynB, FtsZ, SepF, SepF2, and the FtsZ-positioning protein SsgB.

  19. Structure and stability insights into tumour suppressor p53 evolutionary related proteins.

    Directory of Open Access Journals (Sweden)

    Bruno Pagano

    Full Text Available The p53 family of genes and their protein products, namely, p53, p63 and p73, have over one billion years of evolutionary history. Advances in computational biology and genomics are enabling studies of the complexities of the molecular evolution of p53 protein family to decipher the underpinnings of key biological conditions spanning from cancer through to various metabolic and developmental disorders and facilitate the design of personalised medicines. However, a complete understanding of the inherent nature of the thermodynamic and structural stability of the p53 protein family is still lacking. This is due, to a degree, to the lack of comprehensive structural information for a large number of homologous proteins and to an incomplete knowledge of the intrinsic factors responsible for their stability and how these might influence function. Here we investigate the thermal stability, secondary structure and folding properties of the DNA-binding domains (DBDs of a range of proteins from the p53 family using biophysical methods. While the N- and the C-terminal domains of the p53 family show sequence diversity and are normally targets for post-translational modifications and alternative splicing, the central DBD is highly conserved. Together with data obtained from Molecular Dynamics simulations in solution and with structure based homology modelling, our results provide further insights into the molecular properties of evolutionary related p53 proteins. We identify some marked structural differences within the p53 family, which could account for the divergence in biological functions as well as the subtleties manifested in the oligomerization properties of this family.

  20. Protein Glycosylation in Archaea: A Post-Translational Modification to Enhance Extremophilic Protein Stability

    Science.gov (United States)

    2010-01-15

    Analysis of the chemical composition of the Asn-linked polysaccharides decorating many archaeal proteins has revealed the use of a wider variety of sugar...reminiscent of the eukaryal glycan-charged lipid, linked to a variety of monosaccharides , including glucose, mannose, and N-acetylglucosamine (GlcNAc

  1. Folding 19 proteins to their native state and stability of large proteins from a coarse-grained model.

    Science.gov (United States)

    Kapoor, Abhijeet; Travesset, Alex

    2014-03-01

    We develop an intermediate resolution model, where the backbone is modeled with atomic resolution but the side chain with a single bead, by extending our previous model (Proteins (2013) DOI: 10.1002/prot.24269) to properly include proline, preproline residues and backbone rigidity. Starting from random configurations, the model properly folds 19 proteins (including a mutant 2A3D sequence) into native states containing β sheet, α helix, and mixed α/β. As a further test, the stability of H-RAS (a 169 residue protein, critical in many signaling pathways) is investigated: The protein is stable, with excellent agreement with experimental B-factors. Despite that proteins containing only α helices fold to their native state at lower backbone rigidity, and other limitations, which we discuss thoroughly, the model provides a reliable description of the dynamics as compared with all atom simulations, but does not constrain secondary structures as it is typically the case in more coarse-grained models. Further implications are described. Copyright © 2013 Wiley Periodicals, Inc.

  2. The role of PEG conformation in mixed layers: from protein corona substrate to steric stabilization avoiding protein adsorption

    Directory of Open Access Journals (Sweden)

    Joan Comenge

    2015-03-01

    Full Text Available Although nanoparticles (NPs have been traditionally modified with a single ligand layer, mixture of ligands might help to combine different functionalities and to further engineer the NP surface. A detailed study of the competition between an alkanethiol (11-mercaptoundecanoic acid and SH-PEG for the surface of AuNPs and the resultant behaviors of this model nanoconjugate is presented here. As a result, the physicochemical properties of these conjugates can be progressively tuned by controlling the composition and especially the conformation of the mixed monolayer. This has implications in the physiological stability. The controlled changes on the SH-PEG conformation rather than its concentration induce a change in the stabilization mechanism from electrostatic repulsion to steric hindrance, which changes the biological fate of NPs. Importantly, the adsorption of proteins on the conjugates can be tailored by tuning the composition and conformation of the mixed layer.

  3. Carboxymethylated chitosan-stabilized copper nanoparticles: a promise to contribute a potent antifungal and antibacterial agent

    Energy Technology Data Exchange (ETDEWEB)

    Tantubay, Sangeeta, E-mail: sang.chem2@gmail.com [Indian Institute of Technology Kharagpur, Department of Chemistry (India); Mukhopadhyay, Sourav K. [Indian Institute of Technology Kharagpur, Department of Biotechnology (India); Kalita, Himani; Konar, Suraj [Indian Institute of Technology Kharagpur, Department of Chemistry (India); Dey, Satyahari [Indian Institute of Technology Kharagpur, Department of Biotechnology (India); Pathak, Amita, E-mail: ami@chem.iitkgp.ernet.in; Pramanik, Panchanan, E-mail: ppramanik1946@yahoo.in, E-mail: pramanik1946@gmail.com [Indian Institute of Technology Kharagpur, Department of Chemistry (India)

    2015-06-15

    Carboxymethylated chitosan (CMC)-stabilized copper nanoparticles (Cu-NPs) have been synthesized via chemical reduction of copper(II)–CMC complex in aqueous medium by hydrazine under microwave irradiation in ambient atmosphere. Structural morphology, phase, and chemical compositions of CMC-stabilized Cu-NPs (CMC–Cu-NPs) have been analyzed through high-resolution transmission electron microscopy, field emission scanning electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. Antifungal and antibacterial activities of CMC–Cu-NPs have been evaluated against Candida tropicalis and Escherichia coli through agar well diffusion method, broth microdilution assay, live–dead assay, and microscopic observation. Antimicrobial activity of spherical CMC–Cu-NPs (∼4–15 nm of diameters) has been observed to be significant for both C. tropicalis and E. coli. The cytotoxicity study indicates that CMC–Cu-NPs have no significant toxic effect against normal cell line, L929.

  4. Security Analysis and the Contribution of UPFC for Improving Voltage Stability

    Directory of Open Access Journals (Sweden)

    Asma Meddeb

    2018-02-01

    Full Text Available The occurrence of many failures in the power system can lead to power instability and affects the system parameters to go beyond its operating limits. It may lead to obstructing the secure operations and reliability of power systems. Ensuring power system security needs proper actions to be taken for the undesirable contingency. Thus, security analysis is important tasks in modern energy management systems. This paper proposes an approach based on the Newton Raphson power flow method for power system security analysis. Firstly, the contingencies will be specified to assess their impact on the transient stability. Secondly, the selected contingencies will be classified in the order of severity. In addition, the integration of the Unified Power Flow Controller (UPFC to enhance the transient stability of the power system is considered. The proposed method is implemented on the IEEE-14 bus system. We performed this case study using the well-known software EUROSTAG.

  5. Changes in predicted protein disorder tendency may contribute to disease risk

    Directory of Open Access Journals (Sweden)

    Hu Yang

    2011-12-01

    Full Text Available Abstract Background Recent studies suggest that many proteins or regions of proteins lack 3D structure. Defined as intrinsically disordered proteins, these proteins/peptides are functionally important. Recent advances in next generation sequencing technologies enable genome-wide identification of novel nucleotide variations in a specific population or cohort. Results Using the exonic single nucleotide variations (SNVs identified in the 1,000 Genomes Project and distributed by the Genetic Analysis Workshop 17, we systematically analysed the genetic and predicted disorder potential features of the non-synonymous variations. The result of experiments suggests that a significant change in the tendency of a protein region to be structured or disordered caused by SNVs may lead to malfunction of such a protein and contribute to disease risk. Conclusions After validation with functional SNVs on the traits distributed by GAW17, we conclude that it is valuable to consider structure/disorder tendencies while prioritizing and predicting mechanistic effects arising from novel genetic variations.

  6. EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells

    Directory of Open Access Journals (Sweden)

    Kim Suk

    2009-12-01

    Full Text Available Abstract Background The uptake of abortion-inducing pathogens by trophoblast giant (TG cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70 contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this. Methods Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice. Results The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70. Conclusions Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.

  7. Thermodynamic Stabilization of the Folded Domain of Prion Protein Inhibits Prion Infection in Vivo

    Directory of Open Access Journals (Sweden)

    Qingzhong Kong

    2013-07-01

    Full Text Available Prion diseases, or transmissible spongiform encephalopathies (TSEs, are associated with the conformational conversion of the cellular prion protein, PrPC, into a protease-resistant form, PrPSc. Here, we show that mutation-induced thermodynamic stabilization of the folded, α-helical domain of PrPC has a dramatic inhibitory effect on the conformational conversion of prion protein in vitro, as well as on the propagation of TSE disease in vivo. Transgenic mice expressing a human prion protein variant with increased thermodynamic stability were found to be much more resistant to infection with the TSE agent than those expressing wild-type human prion protein, in both the primary passage and three subsequent subpassages. These findings not only provide a line of evidence in support of the protein-only model of TSEs but also yield insight into the molecular nature of the PrPC→PrPSc conformational transition, and they suggest an approach to the treatment of prion diseases.

  8. How plants connect pollination and herbivory networks and their contribution to community stability.

    Science.gov (United States)

    Sauve, Alix M C; Thébault, Elisa; Pocock, Michael J O; Fontaine, Colin

    2016-04-01

    Pollination and herbivory networks have mainly been studied separately, highlighting their distinct structural characteristics and the related processes and dynamics. However, most plants interact with both pollinators and herbivores, and there is evidence that both types of interaction affect each other. Here we investigated the way plants connect these mutualistic and antagonistic networks together, and the consequences for community stability. Using an empirical data set, we show that the way plants connect pollination and herbivory networks is not random and promotes community stability. Analyses of the structure of binary and quantitative networks show different results: the plants' generalism with regard to pollinators is positively correlated to their generalism with regard to herbivores when considering binary interactions, but not when considering quantitative interactions. We also show that plants that share the same pollinators do not share the same herbivores. However, the way plants connect pollination and herbivory networks promotes stability for both binary and quantitative networks. Our results highlight the relevance of considering the diversity of interaction types in ecological communities, and stress the need to better quantify the costs and benefits of interactions, as well as to develop new metrics characterizing the way different interaction types are combined within ecological networks.

  9. Co-encapsulation of lyoprotectants improves the stability of protein-loaded PLGA nanoparticles upon lyophilization

    DEFF Research Database (Denmark)

    Fonte, Pedro; Araújo, Francisca; Seabra, Vítor

    2015-01-01

    The purpose of this work was to evaluate the influence of the co-encapsulation of lyoprotectants with insulin into PLGA nanoparticles, on the stability of the protein and nanoparticles upon lyophilization. Different lyoprotectants were used, namely trehalose, glucose, sucrose, fructose and sorbitol...... formulations with externally added lyoprotectants, except trehalose, showed crystallinity. FTIR assessment showed that co-encapsulating lyoprotectants better preserved insulin structure upon lyophilization with a spectral area overlap of 82-87%, compared to only 72% in lyoprotectant absence. These results were...... confirmed by circular dichroism spectroscopy. Surprisingly, the simultaneous co-encapsulation and addition of lyoprotectants was detrimental to protein stabilization. The insulin in vitro release studies demonstrated that formulations with co-encapsulated trehalose, glucose, sucrose, fructose and sorbitol...

  10. Homogenization Pressure and Temperature Affect Protein Partitioning and Oxidative Stability of Emulsions

    DEFF Research Database (Denmark)

    Horn, Anna Frisenfeldt; Barouh, Nathalie; Nielsen, Nina Skall

    2013-01-01

    The oxidative stability of 10 % fish oil-in-water emulsions was investigated for emulsions prepared under different homogenization conditions. Homogenization was conducted at two different pressures (5 or 22.5 MPa), and at two different temperatures (22 and 72 °C). Milk proteins were used...... prior to homogenization did not have any clear effect on lipid oxidation in either of the two types of emulsions....

  11. Impact of Power Ultrasound on Antihypertensive Activity, Functional Properties, and Thermal Stability of Rapeseed Protein Hydrolysates

    Directory of Open Access Journals (Sweden)

    Asif Wali

    2017-01-01

    Full Text Available The effects of power ultrasound pretreatments on the degree of hydrolysis (DH, angiotensin-I-converting enzyme (ACE inhibitory activity, amino acid composition, surface hydrophobicity, protein solubility, and thermal stability of ACE inhibition of rapeseed protein hydrolysates were evaluated. Ultrasonic pretreatments before enzymolysis in terms of power and exposure time increased the DH and ACE inhibitory activities over the control (without sonication. In this study, maximum DH 22.07% and ACE inhibitory activity 72.13% were achieved at 600 W and 12 min pretreatment. Compared to the hydrolysates obtained without sonication, the amino acid profile of ultrasound pretreated hydrolysates showed significant changes particularly in the proline content and hydrophobic amino acids with an increased rate of 2.47% and 6.31%, respectively. Ultrasound pretreatment (600 watts, 12 min improved functional properties of protein hydrolysates over control by enhancing surface hydrophobicity and solubility index with an increased rate of 130.76% and 34.22%. Moreover, the stability test showed that the ACE inhibitory activity remains stable against heat treatments. However, extensive heat, prolonged heating time, and alkaline conditions were not in the favor of stability test, while under mild heat and acidic conditions their ACE inhibitory activities were not significantly different from unheated samples.

  12. Disulfide bond effects on protein stability: designed variants of Cucurbita maxima trypsin inhibitor-V.

    Science.gov (United States)

    Zavodszky, M; Chen, C W; Huang, J K; Zolkiewski, M; Wen, L; Krishnamoorthi, R

    2001-01-01

    Attempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross-links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross-links, protein flexibility in the vicinity of a cross-link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor-V (CMTI-V; 7 kD) by differential scanning calorimetry. CMTI-V offers the advantage of a large, flexible, and solvent-exposed loop not involved in extensive intra-molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column chromatography, a measure of protein hydrophobicity, and melting temperature (T(m)), an indicator of native state stabilization, for CMTI-V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide-removed C3S/C48S (Delta Delta G(d)(50 degrees C) = -4 kcal/mole; Delta T(m) = -22 degrees C), the native state is destabilized more than the denatured state; this also applies to the less-stable CMTI-V* (Delta Delta G(d)(50 degrees C) = -3 kcal/mole; Delta T(m) = -11 degrees C), in which the disulfide-containing loop is opened by specific hydrolysis of the Lys(44)-Asp(45) peptide bond; (2) In the less stable, disulfide-inserted E38C/W54C (Delta Delta G(d)(50 degrees C) = -1 kcal/mole; Delta T(m) = +2 degrees C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide-engineered V42C/R52C (Delta Delta G(d)(50 degrees C) = +1 kcal/mole; Delta T(m) = +17 degrees C), the native state is more stabilized than the denatured state. These results show that a cross-link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen

  13. Immobilization-stabilization of proteins on nanofibrillated cellulose derivatives and their bioactive film formation.

    Science.gov (United States)

    Arola, Suvi; Tammelin, Tekla; Setälä, Harri; Tullila, Antti; Linder, Markus B

    2012-03-12

    In a number of different applications for enzymes and specific binding proteins a key technology is the immobilization of these proteins to different types of supports. In this work we describe a concept for protein immobilization that is based on nanofibrillated cellulose (NFC). NFC is a form of cellulose where fibers have been disintegrated into fibrils that are only a few nanometers in diameter and have a very large aspect ratio. Proteins were conjugated through three different strategies using amine, epoxy, and carboxylic acid functionalized NFC. The conjugation chemistries were chosen according to the reactive groups on the NFC derivatives; epoxy amination, heterobifunctional modification of amino groups, and EDC/s-NHS activation of carboxylic acid groups. The conjugation reactions were performed in solution and immobilization was performed by spin coating the protein-NCF conjugates. The structure of NFC was shown to be advantageous for both protein performance and stability. The use of NFC allows all covalent chemistry to be performed in solution, while the immobilization is achieved by a simple spin coating or spreading of the protein-NFC conjugates on a support. This allows more scalable methods and better control of conditions compared to the traditional methods that depend on surface reactions.

  14. Rapid Determination of Protein Solubility and Stability Conditions for NMR Studies Using Incomplete Factorial Design

    International Nuclear Information System (INIS)

    Ducat, Thierry; Declerck, Nathalie; Gostan, Thierry; Kochoyan, Michel; Demene, Helene

    2006-01-01

    Sample preparation constitutes a crucial and limiting step in structural studies of proteins by NMR. The determination of the solubility and stability (SAS) conditions of biomolecules at millimolar concentrations stays today empirical and hence time- and material-consuming. Only few studies have been recently done in this field and they have highlighted the interest of using crystallogenesis tools to optimise sample conditions. In this study, we have adapted a method based on incomplete factorial design and making use of crystallisation plates to quantify the influence of physico-chemical parameters such as buffer pH and salts on protein SAS. A description of the experimental set up and an evaluation of the method are given by case studies on two functional domains from the bacterial regulatory protein LicT as well as two other proteins. Using this method, we could rapidly determine optimised conditions for extracting soluble proteins from bacterial cells and for preparing purified protein samples sufficiently concentrated and stable for NMR characterisation. The drastic reduction in the time and number of experiments required for searching protein SAS conditions makes this method particularly well-adapted for a systematic investigation on a large range of physico-chemical parameters

  15. A novel mechanism for antiglycative action of limonene through stabilization of protein conformation.

    Science.gov (United States)

    Joglekar, Madhav M; Panaskar, Shrimant N; Chougale, Ashok D; Kulkarni, Mahesh J; Arvindekar, Akalpita U

    2013-10-01

    Inhibition of protein glycation is known to ameliorate secondary complications in diabetes. In the present study antiglycative properties of limonene, a natural product, were evaluated using BSA as a model protein. AMG (aminoguanidine) was used as a positive control. Measurement of total AGEs (Advanced Glycation End-products) and specific AGEs revealed that limonene could inhibit protein glycation to the extent of 56.3% and 75.1% respectively at 50 μM concentration as against 54.4% and 82.2% by AMG at 1 mM. Congo red binding and CD (Circular Dichroism) analysis revealed inhibition of α-helix to β-sheet transition wherein 18.5% β-sheet structures were observed in glycated BSA (bovine serum albumin) as against 4.9% with limonene. Glycation of protein in the presence of urea was enhanced by 18%, while in the presence of limonene it was reduced by 23% revealing the stabilizing effect of limonene. Electrophoretic mobility was similar to the normal control and a zeta potential value of -12.1 mV as against -15.1 mV in diabetic control was observed. Inhibition of glycation in limonene treated samples was confirmed through LC-MS analysis wherein AGEs such as pentosidine, CML (N(ε)-(carboxymethyl)lysine), CEL (N(ε)-(carboxyethyl)lysine), MOLD (methylglyoxal-lysine dimer) and imidazolone observed in glycated samples were absent in limonene treated samples. PatchDock studies revealed that limonene could bind to the major glycation sites IB, IIA and IIB sub domains and AMG to the IIIA sub domain. Thus limonene is a potent protein glycation inhibitor that prevents protein glycation through a novel mechanism of stabilization of protein structure through hydrophobic interactions.

  16. Axin and GSK3- control Smad3 protein stability and modulate TGF- signaling.

    Science.gov (United States)

    Guo, Xing; Ramirez, Alejandro; Waddell, David S; Li, Zhizhong; Liu, Xuedong; Wang, Xiao-Fan

    2008-01-01

    The broad range of biological responses elicited by transforming growth factor-beta (TGF-beta) in various types of tissues and cells is mainly determined by the expression level and activity of the effector proteins Smad2 and Smad3. It is not fully understood how the baseline properties of Smad3 are regulated, although this molecule is in complex with many other proteins at the steady state. Here we show that nonactivated Smad3, but not Smad2, undergoes proteasome-dependent degradation due to the concerted action of the scaffolding protein Axin and its associated kinase, glycogen synthase kinase 3-beta (GSK3-beta). Smad3 physically interacts with Axin and GSK3-beta only in the absence of TGF-beta. Reduction in the expression or activity of Axin/GSK3-beta leads to increased Smad3 stability and transcriptional activity without affecting TGF-beta receptors or Smad2, whereas overexpression of these proteins promotes Smad3 basal degradation and desensitizes cells to TGF-beta. Mechanistically, Axin facilitates GSK3-beta-mediated phosphorylation of Smad3 at Thr66, which triggers Smad3 ubiquitination and degradation. Thr66 mutants of Smad3 show altered protein stability and hence transcriptional activity. These results indicate that the steady-state stability of Smad3 is an important determinant of cellular sensitivity to TGF-beta, and suggest a new function of the Axin/GSK3-beta complex in modulating critical TGF-beta/Smad3-regulated processes during development and tumor progression.

  17. The breaking of a delayed ring neural network contributes to stability: The rule and exceptions.

    Science.gov (United States)

    Khokhlova, T N; Kipnis, M M

    2013-12-01

    We prove that in our mathematical model the breaking of a delayed ring neural network extends the stability region in the parameters space, if the number of the neurons is sufficiently large. If the number of neurons is small, then a "paradoxical" region exists in the parameters space, wherein the ring neural configuration is stable, while the linear one is unstable. We study the conditions under which the paradoxical region is nonempty. We discuss how our mathematical modeling reflects neurosurgical operations with the severing of particular connections in the brain. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Stability of buffer-free freeze-dried formulations: A feasibility study of a monoclonal antibody at high protein concentrations.

    Science.gov (United States)

    Garidel, Patrick; Pevestorf, Benjamin; Bahrenburg, Sven

    2015-11-01

    We studied the stability of freeze-dried therapeutic protein formulations over a range of initial concentrations (from 40 to 160 mg/mL) and employed a variety of formulation strategies (including buffer-free freeze dried formulations, or BF-FDF). Highly concentrated, buffer-free liquid formulations of therapeutic monoclonal antibodies (mAbs) have been shown to be a viable alternative to conventionally buffered preparations. We considered whether it is feasible to use the buffer-free strategy in freeze-dried formulations, as an answer to some of the known drawbacks of conventional buffers. We therefore conducted an accelerated stability study (24 weeks at 40 °C) to assess the feasibility of stabilizing freeze-dried formulations without "classical" buffer components. Factors monitored included pH stability, protein integrity, and protein aggregation. Because the protein solutions are inherently self-buffering, and the system's buffer capacity scales with protein concentration, we included highly concentrated buffer-free freeze-dried formulations in the study. The tested formulations ranged from "fully formulated" (containing both conventional buffer and disaccharide stabilizers) to "buffer-free" (including formulations with only disaccharide lyoprotectant stabilizers) to "excipient-free" (with neither added buffers nor stabilizers). We evaluated the impacts of varying concentrations, buffering schemes, pHs, and lyoprotectant additives. At the end of 24 weeks, no change in pH was observed in any of the buffer-free formulations. Unbuffered formulations were found to have shorter reconstitution times and lower opalescence than buffered formulations. Protein stability was assessed by visual inspection, sub-visible particle analysis, protein monomer content, charge variants analysis, and hydrophobic interaction chromatography. All of these measures found the stability of buffer-free formulations that included a disaccharide stabilizer comparable to buffer

  19. Alkaline transition of pseudoazurin Met16X mutant proteins: protein stability influenced by the substitution of Met16 in the second sphere coordination.

    Science.gov (United States)

    Abdelhamid, Rehab F; Obara, Yuji; Kohzuma, Takamitsu

    2008-01-01

    Several blue copper proteins are known to change the active site structure at alkaline pH (alkaline transition). Spectroscopic studies of Met16Phe, Met16Tyr, Met16Trp, and Met16Val pseudoazurin variants were performed to investigate the second sphere role through alkaline transition. The visible electronic absorption and resonance Raman spectra of Met16Phe, Met16Tyr, and Met16Trp variants showed the increasing of axial component at pH approximately 11 like wild-type PAz. The visible electronic absorption and far-UV CD spectra of Met16Val demonstrated that the destabilization of the protein structure was triggered at pH>11. Resonance Raman (RR) spectra of PAz showed that the intensity-weighted averaged Cu-S(Cys) stretching frequency was shifted to higher frequency region at pH approximately 11. The higher frequency shift of Cu-S(Cys) bond is implied the stronger Cu-S(Cys) bond at alkaline transition pH approximately 11. The visible electronic absorption and far-UV CD spectra of Met16X PAz revealed that the Met16Val variant is denatured at pH>11, but Met16Phe, Met16Tyr, and Met16Trp mutant proteins are not denatured even at pH>11. These observations suggest that Met16 is important to maintain the protein structure through the possible weak interaction between methionine -SCH3 part and coordinated histidine imidazole moiety. The introduction of pi-pi interaction in the second coordination sphere may be contributed to the enhancement of protein structure stability.

  20. Analysis of ABCB phosphoglycoproteins (PGPs) and their contribution to monocot biomass, structural stability, and productivity

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, Angus Stuart [Purdue University

    2014-09-23

    Efforts to manipulate production of plant secondary cell walls to improve the quality of biofuel feedstocks are currently limited by an inability to regulate the transport of small molecule components out of the cell. Plant ABCB p-glycoproteins are a small family of plasma membrane organic molecule transporters that have become primary targets for this effort, as they can potentially be harnessed to control the export of aromatic compounds and organic acids. However, unlike promiscuous mammalian ABCBs that function in multidrug resistance, all plant ABCB proteins characterized to date exhibit relatively narrow substrate specificity. Although ABCBs exhibit a highly conserved architecture, efforts to modify ABCB activity have been hampered by a lack of structural information largely because an eukaryotic ABCB protein crystal structure has yet to be obtained. Structure/ function analyses have been further impeded by the lack of a common heterologous expression system that can be used to characterize recombinant ABCB proteins, as many cannot be functionally expressed in S. cereviseae or other systems where proteins with analogous function can be readily knocked out. Using experimentally-determined plant ABCB substrate affinities and the crystal structure of the bacterial Sav1866 “half” ABC transporter, we have developed sequence/structure models for ABCBs that provide a testable context for mutational analysis of plant ABCB transporters. We have also developed a flexible heterologous expression system in Schizosaccharomyces pombe in which all endogenous ABC transporters have been knocked out. The effectiveness of this system for transport studies has been demonstrated by the successful functional expression all of the known PIN, AUX/LAX and ABCB auxin transporters. Our central hypothesis is that the domains of the ABCB proteins that we have identified as substrate docking sites and regulators of transport directionality can be altered or swapped to alter the

  1. Golgi Outpost Synthesis Impaired by Toxic Polyglutamine Proteins Contributes to Dendritic Pathology in Neurons

    Directory of Open Access Journals (Sweden)

    Chang Geon Chung

    2017-07-01

    Full Text Available Dendrite aberration is a common feature of neurodegenerative diseases caused by protein toxicity, but the underlying mechanisms remain largely elusive. Here, we show that nuclear polyglutamine (polyQ toxicity resulted in defective terminal dendrite elongation accompanied by a loss of Golgi outposts (GOPs and a decreased supply of plasma membrane (PM in Drosophila class IV dendritic arborization (da (C4 da neurons. mRNA sequencing revealed that genes downregulated by polyQ proteins included many secretory pathway-related genes, including COPII genes regulating GOP synthesis. Transcription factor enrichment analysis identified CREB3L1/CrebA, which regulates COPII gene expression. CrebA overexpression in C4 da neurons restores the dysregulation of COPII genes, GOP synthesis, and PM supply. Chromatin immunoprecipitation (ChIP-PCR revealed that CrebA expression is regulated by CREB-binding protein (CBP, which is sequestered by polyQ proteins. Furthermore, co-overexpression of CrebA and Rac1 synergistically restores the polyQ-induced dendrite pathology. Collectively, our results suggest that GOPs impaired by polyQ proteins contribute to dendrite pathology through the CBP-CrebA-COPII pathway.

  2. Computational study of elements of stability of a four-helix bundle protein biosurfactant

    Science.gov (United States)

    Schaller, Andrea; Connors, Natalie K.; Dwyer, Mirjana Dimitrijev; Oelmeier, Stefan A.; Hubbuch, Jürgen; Middelberg, Anton P. J.

    2015-01-01

    Biosurfactants are surface-active molecules produced principally by microorganisms. They are a sustainable alternative to chemically-synthesized surfactants, having the advantages of being non-toxic, highly functional, eco-friendly and biodegradable. However they are currently only used in a few industrial products due to costs associated with production and purification, which exceed those for commodity chemical surfactants. DAMP4, a member of a four-helix bundle biosurfactant protein family, can be produced in soluble form and at high yield in Escherichia coli, and can be recovered using a facile thermal phase-separation approach. As such, it encompasses an interesting synergy of biomolecular and chemical engineering with prospects for low-cost production even for industrial sectors. DAMP4 is highly functional, and due to its extraordinary thermal stability it can be purified in a simple two-step process, in which the combination of high temperature and salt leads to denaturation of all contaminants, whereas DAMP4 stays stable in solution and can be recovered by filtration. This study aimed to characterize and understand the fundamental drivers of DAMP4 stability to guide further process and surfactant design studies. The complementary use of experiments and molecular dynamics simulation revealed a broad pH and temperature tolerance for DAMP4, with a melting point of 122.4 °C, suggesting the hydrophobic core as the major contributor to thermal stability. Simulation of systematically created in silico variants of DAMP4 showed an influence of number and location of hydrophilic mutations in the hydrophobic core on stability, demonstrating a tolerance of up to three mutations before a strong loss in stability occurred. The results suggest a consideration of a balance of stability, functionality and kinetics for new designs according to their application, aiming for maximal functionality but at adequate stability to allow for cost-efficient production using thermal

  3. Influence of degree correlations on network structure and stability in protein-protein interaction networks

    Directory of Open Access Journals (Sweden)

    Zimmer Ralf

    2007-08-01

    Full Text Available Abstract Background The existence of negative correlations between degrees of interacting proteins is being discussed since such negative degree correlations were found for the large-scale yeast protein-protein interaction (PPI network of Ito et al. More recent studies observed no such negative correlations for high-confidence interaction sets. In this article, we analyzed a range of experimentally derived interaction networks to understand the role and prevalence of degree correlations in PPI networks. We investigated how degree correlations influence the structure of networks and their tolerance against perturbations such as the targeted deletion of hubs. Results For each PPI network, we simulated uncorrelated, positively and negatively correlated reference networks. Here, a simple model was developed which can create different types of degree correlations in a network without changing the degree distribution. Differences in static properties associated with degree correlations were compared by analyzing the network characteristics of the original PPI and reference networks. Dynamics were compared by simulating the effect of a selective deletion of hubs in all networks. Conclusion Considerable differences between the network types were found for the number of components in the original networks. Negatively correlated networks are fragmented into significantly less components than observed for positively correlated networks. On the other hand, the selective deletion of hubs showed an increased structural tolerance to these deletions for the positively correlated networks. This results in a lower rate of interaction loss in these networks compared to the negatively correlated networks and a decreased disintegration rate. Interestingly, real PPI networks are most similar to the randomly correlated references with respect to all properties analyzed. Thus, although structural properties of networks can be modified considerably by degree

  4. EX4 stabilizes and activates Nrf2 via PKCδ, contributing to the prevention of oxidative stress-induced pancreatic beta cell damage

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Mi-Hwi; Kim, Eung-Hwi [College of Pharmacy, Gachon Institute of Pharmaceutical Science, Gachon University, Yeonsu-ku, Incheon (Korea, Republic of); Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Yeonsu-ku, Incheon (Korea, Republic of); Jung, Hye Seung [Department of Internal Medicine, Seoul National University College of Medicine, Seoul (Korea, Republic of); Yang, Dongki [Department of Physiology, Gachon University College of Medicine, Incheon (Korea, Republic of); Park, Eun-Young, E-mail: parkey@mokpo.ac.kr [College of Pharmacy, Natural Medicine Research Institute, Mokpo National University, Muan-gun, Jeonnam (Korea, Republic of); Jun, Hee-Sook, E-mail: hsjun@gachon.ac.kr [College of Pharmacy, Gachon Institute of Pharmaceutical Science, Gachon University, Yeonsu-ku, Incheon (Korea, Republic of); Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Yeonsu-ku, Incheon (Korea, Republic of); Gachon Medical Research Institute, Gil Hospital, Incheon (Korea, Republic of)

    2017-01-15

    Oxidative stress in pancreatic beta cells can inhibit insulin secretion and promote apoptotic cell death. Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, can suppress beta cell apoptosis, improve beta cell function and protect against oxidative damage. In this study, we investigated the molecular mechanisms for antioxidative effects of EX4 in pancreatic beta cells. INS-1 cells, a rat insulinoma cell line, were pretreated with EX4 and exposed to palmitate or H{sub 2}O{sub 2}. Reactive oxygen species (ROS) production, and glutathione and insulin secretion were measured. The mRNA and protein expression levels of antioxidant genes were examined. The level of nuclear factor erythroid 2-related factor 2 (Nrf2), its binding to antioxidant response element (ARE), and its ubiquination in the presence of EX4 were determined. The Nrf2 signaling pathway was determined using rottlerin (protein kinase [PK]Cδ inhibitor), H89 (PKA inhibitor) and LY294002 (phosphatidylinositide 3-kinase [PI3K] inhibitor). EX4 treatment decreased ROS production, recovered cellular glutathione levels and insulin secretion in the presence of oxidative stress in INS-1 cells. The expression levels of glutamate-cysteine ligase catalytic subunit and heme oxygenase-1 were increased by EX4 treatment. EX4 promoted Nrf2 translocation, ARE binding activity and enhanced stabilization of Nrf2 by inhibition of ubiquitination. Knockdown of Nrf2 abolished the effect of EX4 on increased insulin secretion. Inhibition of PKCδ attenuated Nrf2 translocation and antioxidative gene expression by EX4 treatment. We suggest that EX4 activates and stabilizes Nrf2 through PKCδ activation, contributing to the increase of antioxidant gene expression and consequently improving beta cell function in the presence of oxidative stress. - Highlights: • EX4 protects against oxidative stress-induced pancreatic beta cell dysfunction. • EX4 increases antioxidant gene expression. • Antioxidative effect of EX4 is

  5. EX4 stabilizes and activates Nrf2 via PKCδ, contributing to the prevention of oxidative stress-induced pancreatic beta cell damage

    International Nuclear Information System (INIS)

    Kim, Mi-Hwi; Kim, Eung-Hwi; Jung, Hye Seung; Yang, Dongki; Park, Eun-Young; Jun, Hee-Sook

    2017-01-01

    Oxidative stress in pancreatic beta cells can inhibit insulin secretion and promote apoptotic cell death. Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, can suppress beta cell apoptosis, improve beta cell function and protect against oxidative damage. In this study, we investigated the molecular mechanisms for antioxidative effects of EX4 in pancreatic beta cells. INS-1 cells, a rat insulinoma cell line, were pretreated with EX4 and exposed to palmitate or H 2 O 2 . Reactive oxygen species (ROS) production, and glutathione and insulin secretion were measured. The mRNA and protein expression levels of antioxidant genes were examined. The level of nuclear factor erythroid 2-related factor 2 (Nrf2), its binding to antioxidant response element (ARE), and its ubiquination in the presence of EX4 were determined. The Nrf2 signaling pathway was determined using rottlerin (protein kinase [PK]Cδ inhibitor), H89 (PKA inhibitor) and LY294002 (phosphatidylinositide 3-kinase [PI3K] inhibitor). EX4 treatment decreased ROS production, recovered cellular glutathione levels and insulin secretion in the presence of oxidative stress in INS-1 cells. The expression levels of glutamate-cysteine ligase catalytic subunit and heme oxygenase-1 were increased by EX4 treatment. EX4 promoted Nrf2 translocation, ARE binding activity and enhanced stabilization of Nrf2 by inhibition of ubiquitination. Knockdown of Nrf2 abolished the effect of EX4 on increased insulin secretion. Inhibition of PKCδ attenuated Nrf2 translocation and antioxidative gene expression by EX4 treatment. We suggest that EX4 activates and stabilizes Nrf2 through PKCδ activation, contributing to the increase of antioxidant gene expression and consequently improving beta cell function in the presence of oxidative stress. - Highlights: • EX4 protects against oxidative stress-induced pancreatic beta cell dysfunction. • EX4 increases antioxidant gene expression. • Antioxidative effect of EX4 is mediated by

  6. A specific role for the ZipA protein in cell division: stabilization of the FtsZ protein.

    Science.gov (United States)

    Pazos, Manuel; Natale, Paolo; Vicente, Miguel

    2013-02-01

    In Escherichia coli, the cell division protein FtsZ is anchored to the cytoplasmic membrane by the action of the bitopic membrane protein ZipA and the cytoplasmic protein FtsA. Although the presence of both ZipA and FtsA is strictly indispensable for cell division, an FtsA gain-of-function mutant FtsA* (R286W) can bypass the ZipA requirement for cell division. This observation casts doubts on the role of ZipA and its need for cell division. Maxicells are nucleoid-free bacterial cells used as a whole cell in vitro system to probe protein-protein interactions without the need of protein purification. We show that ZipA protects FtsZ from the ClpXP-directed degradation observed in E. coli maxicells and that ZipA-stabilized FtsZ forms membrane-attached spiral-like structures in the bacterial cytoplasm. The overproduction of the FtsZ-binding ZipA domain is sufficient to protect FtsZ from degradation, whereas other C-terminal ZipA partial deletions lacking it are not. Individual overproduction of the proto-ring component FtsA or its gain-of-function mutant FtsA* does not result in FtsZ protection. Overproduction of FtsA or FtsA* together with ZipA does not interfere with the FtsZ protection. Moreover, neither FtsA nor FtsA* protects FtsZ when overproduced together with ZipA mutants lacking the FZB domain. We propose that ZipA protects FtsZ from degradation by ClpP by making the FtsZ site of interaction unavailable to the ClpX moiety of the ClpXP protease. This role cannot be replaced by either FtsA or FtsA*, suggesting a unique function for ZipA in proto-ring stability.

  7. Controlled formation of emulsion gels stabilized by salted myofibrillar protein under malondialdehyde (MDA)-induced oxidative stress.

    Science.gov (United States)

    Zhou, Feibai; Sun, Weizheng; Zhao, Mouming

    2015-04-15

    This study presented the cold-set gelation of emulsions stabilized by salted myofibrillar protein (MP) under oxidative stress originated from malondialdehyde (MDA). Gel properties were compared over a range of MDA/NaCl concentrations including gel viscoelastic properties, strength, water-holding capacity (WHC), amount of protein entrapped, and microstructure. The oxidative stability of emulsion gels as indicated by lipid hydroperoxide was further determined and compared. Results indicated that emulsion stabilized by MP at swollen state under certain ionic strengths (0.2-0.6 M) was the premise of gel formation under MDA. In the presence of intermediate MDA concentrations (2.5-10 mM), the emulsion gels showed an improved elasticity, strength, WHC, and oxidative stability. This improvement should be mainly attributed to the enhanced protein-protein cross-linkings via MDA, which were homogeneously formed among absorbed and/or unabsorbed proteins, entrapping a greater amount and fractions of protein within network. Therefore, the oil droplets were better adherent to the gel matrix. Nevertheless, addition of high MDA concentrations (25-50 mM) led to the formation of excessive covalent bonds, which might break protein-protein bonds and trigger the desorption of protein from the interface. This ultimately caused "oil leak" phenomena as well as the collapse of gel structure and, thus, overall decreased gel properties and oxidative stability.

  8. Critical lysine residues of Klf4 required for protein stabilization and degradation

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Key-Hwan; Kim, So-Ra; Ramakrishna, Suresh; Baek, Kwang-Hyun, E-mail: baek@cha.ac.kr

    2014-01-24

    Highlights: • Klf4 undergoes the 26S proteasomal degradation by ubiquitination on its multiple lysine residues. • Essential Klf4 ubiquitination sites are accumulated between 190–263 amino acids. • A mutation of lysine at 232 on Klf4 elongates protein turnover. • Klf4 mutants dramatically suppress p53 expression both under normal and UV irradiated conditions. - Abstract: The transcription factor, Krüppel-like factor 4 (Klf4) plays a crucial role in generating induced pluripotent stem cells (iPSCs). As the ubiquitination and degradation of the Klf4 protein have been suggested to play an important role in its function, the identification of specific lysine sites that are responsible for protein degradation is of prime interest to improve protein stability and function. However, the molecular mechanism regulating proteasomal degradation of the Klf4 is poorly understood. In this study, both the analysis of Klf4 ubiquitination sites using several Klf4 deletion fragments and bioinformatics predictions showed that the lysine sites which are signaling for Klf4 protein degradation lie in its N-terminal domain (aa 1–296). The results also showed that Lys32, 52, 232, and 252 of Klf4 are responsible for the proteolysis of the Klf4 protein. These results suggest that Klf4 undergoes proteasomal degradation and that these lysine residues are critical for Klf4 ubiquitination.

  9. Structure-function relationships governing activity and stability of a DNA alkylation damage repair thermostable protein.

    Science.gov (United States)

    Perugino, Giuseppe; Miggiano, Riccardo; Serpe, Mario; Vettone, Antonella; Valenti, Anna; Lahiri, Samarpita; Rossi, Franca; Rossi, Mosè; Rizzi, Menico; Ciaramella, Maria

    2015-10-15

    Alkylated DNA-protein alkyltransferases repair alkylated DNA bases, which are among the most common DNA lesions, and are evolutionary conserved, from prokaryotes to higher eukaryotes. The human ortholog, hAGT, is involved in resistance to alkylating chemotherapy drugs. We report here on the alkylated DNA-protein alkyltransferase, SsOGT, from an archaeal species living at high temperature, a condition that enhances the harmful effect of DNA alkylation. The exceptionally high stability of SsOGT gave us the unique opportunity to perform structural and biochemical analysis of a protein of this class in its post-reaction form. This analysis, along with those performed on SsOGT in its ligand-free and DNA-bound forms, provides insights in the structure-function relationships of the protein before, during and after DNA repair, suggesting a molecular basis for DNA recognition, catalytic activity and protein post-reaction fate, and giving hints on the mechanism of alkylation-induced inactivation of this class of proteins. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Stereochemical criteria for prediction of the effects of proline mutations on protein stability.

    Directory of Open Access Journals (Sweden)

    Kanika Bajaj

    2007-12-01

    Full Text Available When incorporated into a polypeptide chain, proline (Pro differs from all other naturally occurring amino acid residues in two important respects. The phi dihedral angle of Pro is constrained to values close to -65 degrees and Pro lacks an amide hydrogen. Consequently, mutations which result in introduction of Pro can significantly affect protein stability. In the present work, we describe a procedure to accurately predict the effect of Pro introduction on protein thermodynamic stability. Seventy-seven of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to Pro, and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or nonperturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties including main chain dihedral angle phi and main chain amide H-bonds (hydrogen bonds were determined from 3D models of the mutant proteins built using MODELLER. We assessed the performance of the decision tree on a large dataset of 163 single-site Pro mutations of T4 lysozyme, 74 nsSNPs, and 52 other Pro substitutions from the literature. The overall accuracy of this algorithm was found to be 81% in the case of CcdB, 77% in the case of lysozyme, 76% in the case of nsSNPs, and 71% in the case of other Pro substitution data. The accuracy of Pro scanning mutagenesis for secondary structure assignment was also assessed and found to be at best 69%. Our prediction procedure will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design.

  11. Stereochemical criteria for prediction of the effects of proline mutations on protein stability.

    Science.gov (United States)

    Bajaj, Kanika; Madhusudhan, M S; Adkar, Bharat V; Chakrabarti, Purbani; Ramakrishnan, C; Sali, Andrej; Varadarajan, Raghavan

    2007-12-01

    When incorporated into a polypeptide chain, proline (Pro) differs from all other naturally occurring amino acid residues in two important respects. The phi dihedral angle of Pro is constrained to values close to -65 degrees and Pro lacks an amide hydrogen. Consequently, mutations which result in introduction of Pro can significantly affect protein stability. In the present work, we describe a procedure to accurately predict the effect of Pro introduction on protein thermodynamic stability. Seventy-seven of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to Pro, and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or nonperturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties including main chain dihedral angle phi and main chain amide H-bonds (hydrogen bonds) were determined from 3D models of the mutant proteins built using MODELLER. We assessed the performance of the decision tree on a large dataset of 163 single-site Pro mutations of T4 lysozyme, 74 nsSNPs, and 52 other Pro substitutions from the literature. The overall accuracy of this algorithm was found to be 81% in the case of CcdB, 77% in the case of lysozyme, 76% in the case of nsSNPs, and 71% in the case of other Pro substitution data. The accuracy of Pro scanning mutagenesis for secondary structure assignment was also assessed and found to be at best 69%. Our prediction procedure will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design.

  12. 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.

  13. The contribution of riparian vegetation to the stability of agricultural channels banks in the Lombardy plane

    Science.gov (United States)

    Spelta, E.; Chiaradia, E. A.; Bischetti, G. B.

    2009-04-01

    Vegetation is well-known to affect the stability of slopes and banks in several ways, influencing both the water content and the mechanical properties of the soil. From the mechanical point of view, in particular, vegetation acts by means of the root system, which reinforces the soil. Three reinforcement mechanisms are generally recognised to prevent mass movements: the first is due to soil-roots interaction, which allows the mobilisation of the root tensile strength and increases the compound matrix (soil-fibre) strength, the second is due to great size roots intersecting the shear surface, which act as individual anchors, the third is due to the whole root system which exerts buttressing and arching actions. All these effects can be quantified via modelling if appropriate parameters are provided. Due to the scarcity of data, however, only the fibre reinforcement mechanism is generally considered and it is quantified in terms of additional root cohesion, which can be easily incorporated into stability models. Root cohesion values can be estimated by means of direct shear tests (in situ or in laboratory), by means of back analysis of collapsed slopes and by means of modelling. Direct shear tests and back analysis, however, due to site-specific development of root systems (which leads to a dramatic space variability of root density and size), provide results that are valid only for the specific (or highly similar) conditions that occur in the location where the investigations are carried out. Reinforcement modelling, on the contrary, represents a more general way of estimating root cohesion along the soil profile. The scheme commonly adopted in modelling is the Wu (1976) and Waldron (1977) approach (W&W model), which estimates root cohesion values basing on root tensile strength and root density (in terms of Root Area Ratio). Despite its simplicity and some questions about the hypotheses involved in, it still represents the benchmark. To overcome some of such

  14. Trimethylamine N-oxide stabilizes proteins via a distinct mechanism compared with betaine and glycine

    Science.gov (United States)

    Liao, Yi-Ting; Manson, Anthony C.; DeLyser, Michael R.; Noid, William G.; Cremer, Paul S.

    2017-01-01

    We report experimental and computational studies investigating the effects of three osmolytes, trimethylamine N-oxide (TMAO), betaine, and glycine, on the hydrophobic collapse of an elastin-like polypeptide (ELP). All three osmolytes stabilize collapsed conformations of the ELP and reduce the lower critical solution temperature (LSCT) linearly with osmolyte concentration. As expected from conventional preferential solvation arguments, betaine and glycine both increase the surface tension at the air–water interface. TMAO, however, reduces the surface tension. Atomically detailed molecular dynamics (MD) simulations suggest that TMAO also slightly accumulates at the polymer–water interface, whereas glycine and betaine are strongly depleted. To investigate alternative mechanisms for osmolyte effects, we performed FTIR experiments that characterized the impact of each cosolvent on the bulk water structure. These experiments showed that TMAO red-shifts the OH stretch of the IR spectrum via a mechanism that was very sensitive to the protonation state of the NO moiety. Glycine also caused a red shift in the OH stretch region, whereas betaine minimally impacted this region. Thus, the effects of osmolytes on the OH spectrum appear uncorrelated with their effects upon hydrophobic collapse. Similarly, MD simulations suggested that TMAO disrupts the water structure to the least extent, whereas glycine exerts the greatest influence on the water structure. These results suggest that TMAO stabilizes collapsed conformations via a mechanism that is distinct from glycine and betaine. In particular, we propose that TMAO stabilizes proteins by acting as a surfactant for the heterogeneous surfaces of folded proteins. PMID:28228526

  15. Stability and in vitro digestibility of emulsions containing lecithin and whey proteins.

    Science.gov (United States)

    Mantovani, Raphaela Araujo; Cavallieri, Ângelo Luiz Fazani; Netto, Flavia Maria; Cunha, Rosiane Lopes

    2013-09-01

    The effect of pH and high-pressure homogenization on the properties of oil-in-water (O/W) emulsions stabilized by lecithin and/or whey proteins (WPI) was evaluated. For this purpose, emulsions were characterized by visual analysis, droplet size distribution, zeta potential, electrophoresis, rheological measurements and their response to in vitro digestion. Lecithin emulsions were stable even after 7 days of storage and WPI emulsions were unstable only at pH values close to the isoelectric point (pI) of proteins. Systems containing the mixture of lecithin and WPI showed high kinetic instability at pH 3, which was attributed to the electrostatic interaction between the emulsifiers oppositely charged at this pH value. At pH 5.5 and 7, the mixture led to reduction of the droplet size with enhanced emulsion stability compared to the systems with WPI or lecithin. The stability of WPI emulsions after the addition of lecithin, especially at pH 5.5, was associated with the increase of droplet surface charge density. The in vitro digestion evaluation showed that WPI emulsion was more stable against gastrointestinal conditions.

  16. Protein Stabilization and Enzyme Activation in Ionic Liquids: Specific Ion Effects

    Science.gov (United States)

    Zhao, Hua

    2015-01-01

    There are still debates on whether the hydration of ions perturbs the water structure, and what is the degree of such disturbance; therefore, the origin of Hofmeister effect on protein stabilization continues being questioned. For this reason, it is suggested to use the ‘specific ion effect’ instead of other misleading terms such as Hofmeister effect, Hofmeister series, lyotropic effect, and lyotropic series. In this review, we firstly discuss the controversial aspect of inorganic ion effects on water structures, and several possible contributors to the specific ion effect of protein stability. Due to recent overwhelming attraction of ionic liquids (ILs) as benign solvents in many enzymatic reactions, we further evaluate the structural properties and molecular-level interactions in neat ILs and their aqueous solutions. Next, we systematically compare the specific ion effects of ILs on enzyme stability and activity, and conclude that (a) the specificity of many enzymatic systems in diluted aqueous IL solutions is roughly in line with the traditional Hofmeister series albeit some exceptions; (b) however, the specificity follows a different track in concentrated or neat ILs because other factors (such as hydrogen-bond basicity, nucelophilicity, and hydrophobicity, etc) are playing leading roles. In addition, we demonstrate some examples of biocatalytic reactions in IL systems that are guided by the empirical specificity rule. PMID:26949281

  17. The actin family protein ARP6 contributes to the structure and the function of the nucleolus

    International Nuclear Information System (INIS)

    Kitamura, Hiroshi; Matsumori, Haruka; Kalendova, Alzbeta; Hozak, Pavel; Goldberg, Ilya G.; Nakao, Mitsuyoshi; Saitoh, Noriko; Harata, Masahiko

    2015-01-01

    The actin family members, consisting of actin and actin-related proteins (ARPs), are essential components of chromatin remodeling complexes. ARP6, one of the nuclear ARPs, is part of the Snf-2-related CREB-binding protein activator protein (SRCAP) chromatin remodeling complex, which promotes the deposition of the histone variant H2A.Z into the chromatin. In this study, we showed that ARP6 influences the structure and the function of the nucleolus. ARP6 is localized in the central region of the nucleolus, and its knockdown induced a morphological change in the nucleolus. We also found that in the presence of high concentrations of glucose ARP6 contributed to the maintenance of active ribosomal DNA (rDNA) transcription by placing H2A.Z into the chromatin. In contrast, under starvation, ARP6 was required for cell survival through the repression of rDNA transcription independently of H2A.Z. These findings reveal novel pleiotropic roles for the actin family in nuclear organization and metabolic homeostasis. - Highlights: • ARP6, an actin related protein, is important for nucleolar function and structure. • A population of ARP6 is localized in the center of nucleolus. • Depletion of ARP6 resulted in aberrant shape of the nucleolus. • ARP6 maintains the active rDNA transcription under high glucose. • ARP6 is required for the repression of rDNA transcription under starvation

  18. Endogenous contributions to egg protein formation in lesser scaup Aythya affinis

    Science.gov (United States)

    Cutting, Kyle A.; Hobson, Keith A.; Rotella, Jay J.; Warren, Jeffrey M.; Wainwright-de la Cruz, Susan E.; Takekawa, John Y.

    2011-01-01

    Lesser scaup Aythya affinis populations have declined throughout the North American continent for the last three decades. It has been hypothesized that the loss and degradation of staging habitats has resulted in reduced female body condition on the breeding grounds and a concomitant decline in productivity. We explored the importance of body (endogenous) reserves obtained prior to arrival on the breeding ground in egg protein formation in southwestern Montana during 2006–2008 using stable-carbon (δ13C) and nitrogen (δ15N) isotope analyses of scaup egg components, female tissue, and local prey items. From arrival on the breeding grounds through the egg-laying period, δ15N values of scaup red blood cells decreased while δ13C values became less variable; a pattern consistent with endogenous tissues equilibrating with local (freshwater) dietary sources. In 2006 and 2008, isotopic values for egg albumen and yolk protein indicated that most (>90%) protein used to produce these components was obtained on the breeding grounds. However, in 2007, a year with an exceptionally warm and dry spring, endogenous reserves contributed on average 41% of yolk and 29% of albumen. Results from this study suggest that female scaup can meet the protein needs of egg production largely from local dietary food sources. This highlights the importance of providing high-quality breeding habitats for scaup. Whether this pattern holds in areas with similar breeding season lengths but longer migration routes, such as those found in the western boreal forest, should be investigated.

  19. The actin family protein ARP6 contributes to the structure and the function of the nucleolus

    Energy Technology Data Exchange (ETDEWEB)

    Kitamura, Hiroshi [Laboratory of Molecular Biology, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi 1-1, Aoka-ku, Sendai 981-8555 (Japan); Matsumori, Haruka [Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811 (Japan); Kalendova, Alzbeta; Hozak, Pavel [Department of Biology of the Cell Nucleus, Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20 Prague (Czech Republic); Goldberg, Ilya G. [Image Informatics and Computational Biology Unit, Laboratory of Genetics, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Suite 100, Baltimore, MD 21224 (United States); Nakao, Mitsuyoshi [Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811 (Japan); Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo 102-0076 (Japan); Saitoh, Noriko [Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811 (Japan); Harata, Masahiko, E-mail: mharata@biochem.tohoku.ac.jp [Laboratory of Molecular Biology, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi 1-1, Aoka-ku, Sendai 981-8555 (Japan)

    2015-08-21

    The actin family members, consisting of actin and actin-related proteins (ARPs), are essential components of chromatin remodeling complexes. ARP6, one of the nuclear ARPs, is part of the Snf-2-related CREB-binding protein activator protein (SRCAP) chromatin remodeling complex, which promotes the deposition of the histone variant H2A.Z into the chromatin. In this study, we showed that ARP6 influences the structure and the function of the nucleolus. ARP6 is localized in the central region of the nucleolus, and its knockdown induced a morphological change in the nucleolus. We also found that in the presence of high concentrations of glucose ARP6 contributed to the maintenance of active ribosomal DNA (rDNA) transcription by placing H2A.Z into the chromatin. In contrast, under starvation, ARP6 was required for cell survival through the repression of rDNA transcription independently of H2A.Z. These findings reveal novel pleiotropic roles for the actin family in nuclear organization and metabolic homeostasis. - Highlights: • ARP6, an actin related protein, is important for nucleolar function and structure. • A population of ARP6 is localized in the center of nucleolus. • Depletion of ARP6 resulted in aberrant shape of the nucleolus. • ARP6 maintains the active rDNA transcription under high glucose. • ARP6 is required for the repression of rDNA transcription under starvation.

  20. Effects of monohydric alcohols and polyols on the thermal stability of a protein

    Energy Technology Data Exchange (ETDEWEB)

    Murakami, Shota [Graduate School of Energy Science, Kyoto University, Uji, Kyoto 611-0011 (Japan); Kinoshita, Masahiro, E-mail: kinoshit@iae.kyoto-u.ac.jp [Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2016-03-28

    The thermal stability of a protein is lowered by the addition of a monohydric alcohol, and this effect becomes larger as the size of hydrophobic group in an alcohol molecule increases. By contrast, it is enhanced by the addition of a polyol possessing two or more hydroxyl groups per molecule, and this effect becomes larger as the number of hydroxyl groups increases. Here, we show that all of these experimental observations can be reproduced even in a quantitative sense by rigid-body models focused on the entropic effect originating from the translational displacement of solvent molecules. The solvent is either pure water or water-cosolvent solution. Three monohydric alcohols and five polyols are considered as cosolvents. In the rigid-body models, a protein is a fused hard spheres accounting for the polyatomic structure in the atomic detail, and the solvent is formed by hard spheres or a binary mixture of hard spheres with different diameters. The effective diameter of cosolvent molecules and the packing fractions of water and cosolvent, which are crucially important parameters, are carefully estimated using the experimental data of properties such as the density of solid crystal of cosolvent, parameters in the pertinent cosolvent-cosolvent interaction potential, and density of water-cosolvent solution. We employ the morphometric approach combined with the integral equation theory, which is best suited to the physical interpretation of the calculation result. It is argued that the degree of solvent crowding in the bulk is the key factor. When it is made more serious by the cosolvent addition, the solvent-entropy gain upon protein folding is magnified, leading to the enhanced thermal stability. When it is made less serious, the opposite is true. The mechanism of the effects of monohydric alcohols and polyols is physically the same as that of sugars. However, when the rigid-body models are employed for the effect of urea, its addition is predicted to enhance the

  1. Centromere Protein (CENP)-W Interacts with Heterogeneous Nuclear Ribonucleoprotein (hnRNP) U and May Contribute to Kinetochore-Microtubule Attachment in Mitotic Cells

    Science.gov (United States)

    Chun, Younghwa; Kim, Raehyung; Lee, Soojin

    2016-01-01

    Background Recent studies have shown that heterogeneous nuclear ribonucleoprotein U (hnRNP U), a component of the hnRNP complex, contributes to stabilize the kinetochore-microtubule interaction during mitosis. CENP-W was identified as an inner centromere component that plays crucial roles in the formation of a functional kinetochore complex. Results We report that hnRNP U interacts with CENP-W, and the interaction between hnRNP U and CENP-W mutually increased each other’s protein stability by inhibiting the proteasome-mediated degradation. Further, their co-localization was observed chiefly in the nuclear matrix region and at the microtubule-kinetochore interface during interphase and mitosis, respectively. Both microtubule-stabilizing and microtubule-destabilizing agents significantly decreased the protein stability of CENP-W. Furthermore, loss of microtubules and defects in microtubule organization were observed in CENP-W-depleted cells. Conclusion Our data imply that CENP-W plays an important role in the attachment and interaction between microtubules and kinetochore during mitosis. PMID:26881882

  2. Further contributions to the understanding of nitrogen removal in waste stabilization ponds.

    Science.gov (United States)

    Bastos, R K X; Rios, E N; Sánchez, I A

    2018-06-01

    A set of experiments were conducted in Brazil in a pilot-scale waste stabilization pond (WSP) system (a four-maturation-pond series) treating an upflow anaerobic sludge blanket (UASB) reactor effluent. Over a year and a half the pond series was monitored under two flow rate conditions, hence also different hydraulic retention times and surface loading rates. On-site and laboratory trials were carried out to assess: (i) ammonia losses by volatilization using acrylic capture chambers placed at the surface of the ponds; (ii) organic nitrogen sedimentation rates using metal buckets placed at the bottom of the ponds for collecting settled particulate matter; (iii) nitrogen removal by algal uptake based on the nitrogen content of the suspended particulate matter in samples from the ponds' water column. In addition, nitrification and denitrification rates were measured in laboratory-based experiments using pond water and sediment samples. The pond system achieved high nitrogen removal (69% total nitrogen and 92% ammonia removal). The average total nitrogen removal rates varied from 10,098 to 3,849 g N/ha·d in the first and the last ponds, respectively, with the following fractions associated with the various removal pathways: (i) 23.5-45.6% sedimentation of organic nitrogen; (ii) 13.1-27.8% algal uptake; (iii) 1.2-3.1% ammonia volatilization; and (iv) 0.15-0.34% nitrification-denitrification.

  3. Stability of foot-and-mouth disease virus, its genome and proteins at 37 grad C

    International Nuclear Information System (INIS)

    Razdan, R.; Sen, A.K.; Rao, B.V.; Suryanarayana, V.V.S.

    1996-01-01

    Infectivity titers of foot-and-mouth disease virus (FMDV) types Asia 1 and 0 were reduced by 4 and 2 log units, respectively, after incubation at 37 grad C for 12 hours. The stability of the FMDV RNA genome at 37 grad C was studied using 32 P-labelled virus. The RNA of FMDV type 0 was found to be more stable than that of type Asia 1. Oligo(dT)-cellulose chromatography showed that 21 % and 31 % of the labelled RNA were bound to the column in the case of types Asia 1 and 0, respectively. Possible correlation between the poly(A) tail length, accessibility of the genome to nucleases and thermo-stability of the infective virus is discussed. A possible correlation between the thermo-stability of the genome and general distribution of a particular virus type seems to exist. A stable genome associated with poor virus immunogenicity may be responsible for the prevalence of FMDV type 0 in the nature. The isoelectric focussing of structural proteins isolated from the virus samples incubated at 37 grad C revealed charge differences in the major immuno-gen between the two FMDV types. A rapid proteolytic degradation of the viral immuno-gen and stability of the genome may be responsible for frequent outbreaks of FMDV, at least, in the endemic countries. (author)

  4. An Improved Methodology for Multidimensional High-Throughput Preformulation Characterization of Protein Conformational Stability

    Science.gov (United States)

    Maddux, Nathaniel R.; Rosen, Ilan T.; Hu, Lei; Olsen, Christopher M.; Volkin, David B.; Middaugh, C. Russell

    2013-01-01

    The Empirical Phase Diagram (EPD) technique is a vector-based multidimensional analysis method for summarizing large data sets from a variety of biophysical techniques. It can be used to provide comprehensive preformulation characterization of a macromolecule’s higher-order structural integrity and conformational stability. In its most common mode, it represents a type of stimulus-response diagram using environmental variables such as temperature, pH, and ionic strength as the stimulus, with alterations in macromolecular structure being the response. Until now EPD analysis has not been available in a high throughput mode because of the large number of experimental techniques and environmental stressor/stabilizer variables typically employed. A new instrument has been developed that combines circular dichroism, UV-absorbance, fluorescence spectroscopy and light scattering in a single unit with a 6-position temperature controlled cuvette turret. Using this multifunctional instrument and a new software system we have generated EPDs for four model proteins. Results confirm the reproducibility of the apparent phase boundaries and protein behavior within the boundaries. This new approach permits two EPDs to be generated per day using only 0.5 mg of protein per EPD. Thus, the new methodology generates reproducible EPDs in high-throughput mode, and represents the next step in making such determinations more routine. PMID:22447621

  5. A single mutation in the envelope protein modulates flavivirus antigenicity, stability, and pathogenesis.

    Directory of Open Access Journals (Sweden)

    Leslie Goo

    2017-02-01

    Full Text Available The structural flexibility or 'breathing' of the envelope (E protein of flaviviruses allows virions to sample an ensemble of conformations at equilibrium. The molecular basis and functional consequences of virus conformational dynamics are poorly understood. Here, we identified a single mutation at residue 198 (T198F of the West Nile virus (WNV E protein domain I-II hinge that regulates virus breathing. The T198F mutation resulted in a ~70-fold increase in sensitivity to neutralization by a monoclonal antibody targeting a cryptic epitope in the fusion loop. Increased exposure of this otherwise poorly accessible fusion loop epitope was accompanied by reduced virus stability in solution at physiological temperatures. Introduction of a mutation at the analogous residue of dengue virus (DENV, but not Zika virus (ZIKV, E protein also increased accessibility of the cryptic fusion loop epitope and decreased virus stability in solution, suggesting that this residue modulates the structural ensembles sampled by distinct flaviviruses at equilibrium in a context dependent manner. Although the T198F mutation did not substantially impair WNV growth kinetics in vitro, studies in mice revealed attenuation of WNV T198F infection. Overall, our study provides insight into the molecular basis and the in vitro and in vivo consequences of flavivirus breathing.

  6. Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

    Directory of Open Access Journals (Sweden)

    Thijs Roebroek

    2017-09-01

    Full Text Available Reversibly switchable fluorescent proteins (RSFPs enable advanced fluorescence imaging, though the performance of this imaging crucially depends on the properties of the labels. We report on the use of an existing small binding peptide, named Enhancer, to modulate the spectroscopic properties of the recently developed rsGreen series of RSFPs. Fusion constructs of Enhancer with rsGreen1 and rsGreenF revealed an increased molecular brightness and pH stability, although expression in living E. coli or HeLa cells resulted in a decrease of the overall emission. Surprisingly, Enhancer binding also increased off-switching speed and resistance to switching fatigue. Further investigation suggested that the RSFPs can interconvert between fast- and slow-switching emissive states, with the overall protein population gradually converting to the slow-switching state through irradiation. The Enhancer modulates the spectroscopic properties of both states, but also preferentially stabilizes the fast-switching state, supporting the increased fatigue resistance. This work demonstrates how the photo-physical properties of RSFPs can be influenced by their binding to other small proteins, which opens up new horizons for applications that may require such modulation. Furthermore, we provide new insights into the photoswitching kinetics that should be of general consideration when developing new RSFPs with improved or different photochromic properties.

  7. Structural stability of human protein tyrosine phosphatase ρ catalytic domain: effect of point mutations.

    Directory of Open Access Journals (Sweden)

    Alessandra Pasquo

    Full Text Available Protein tyrosine phosphatase ρ (PTPρ belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ~4.0 M urea.

  8. Can a pairwise contact potential stabilize native protein folds against decoys obtained by threading?

    Science.gov (United States)

    Vendruscolo, M; Najmanovich, R; Domany, E

    2000-02-01

    We present a method to derive contact energy parameters from large sets of proteins. The basic requirement on which our method is based is that for each protein in the database the native contact map has lower energy than all its decoy conformations that are obtained by threading. Only when this condition is satisfied one can use the proposed energy function for fold identification. Such a set of parameters can be found (by perceptron learning) if Mp, the number of proteins in the database, is not too large. Other aspects that influence the existence of such a solution are the exact definition of contact and the value of the critical distance Rc, below which two residues are considered to be in contact. Another important novel feature of our approach is its ability to determine whether an energy function of some suitable proposed form can or cannot be parameterized in a way that satisfies our basic requirement. As a demonstration of this, we determine the region in the (Rc, Mp) plane in which the problem is solvable, i.e., we can find a set of contact parameters that stabilize simultaneously all the native conformations. We show that for large enough databases the contact approximation to the energy cannot stabilize all the native folds even against the decoys obtained by gapless threading.

  9. Stability of milk fat globule membrane proteins toward human enzymatic gastrointestinal digestion.

    Science.gov (United States)

    Le, T T; Van de Wiele, T; Do, T N H; Debyser, G; Struijs, K; Devreese, B; Dewettinck, K; Van Camp, J

    2012-05-01

    The milk fat globule membrane (MFGM) fraction refers to the thin film of polar lipids and membrane proteins that surrounds fat globules in milk. It is its unique biochemical composition that renders MFGM with some beneficial biological activities, such as anti-adhesive effects toward pathogens. However, a prerequisite for the putative bioactivity of MFGM is its stability during gastrointestinal digestion. We, therefore, subjected MFGM material, isolated from raw milk, to an in vitro enzymatic gastrointestinal digestion. Sodium dodecyl sulfate PAGE, in combination with 2 staining methods, Coomassie Blue and periodic acid Schiff staining, was used to evaluate polypeptide patterns of the digest, whereas mass spectrometry was used to confirm the presence of specific MFGM proteins. Generally, it was observed that glycoproteins showed higher resistance to endogenous proteases compared with non-glycosylated proteins. Mucin 1 displayed the highest resistance to digestion and a considerable part of this protein was still detected at its original molecular weight after gastric and small intestine digestion. Cluster of differentiation 36 was also quite resistant to pepsin. A significant part of periodic acid Schiff 6/7 survived the gastric digestion, provided that the lipid moiety was not removed from the MFGM material. Overall, MFGM glycoproteins are generally more resistant to gastrointestinal digestion than serum milk proteins and the presence of lipids, besides glycosylation, may protect MFGM glycoproteins from gastrointestinal digestion. This gastrointestinal stability makes MFGM glycoproteins amenable to further studies in which their putative health-promoting effects can be explored. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  10. The disordered C-terminal domain of human DNA glycosylase NEIL1 contributes to its stability via intramolecular interactions.

    Science.gov (United States)

    Hegde, Muralidhar L; Tsutakawa, Susan E; Hegde, Pavana M; Holthauzen, Luis Marcelo F; Li, Jing; Oezguen, Numan; Hilser, Vincent J; Tainer, John A; Mitra, Sankar

    2013-07-10

    NEIL1 [Nei (endonuclease VIII)-like protein 1], one of the five mammalian DNA glycosylases that excise oxidized DNA base lesions in the human genome to initiate base excision repair, contains an intrinsically disordered C-terminal domain (CTD; ~100 residues), not conserved in its Escherichia coli prototype Nei. Although dispensable for NEIL1's lesion excision and AP lyase activities, this segment is required for efficient in vivo enzymatic activity and may provide an interaction interface for many of NEIL1's interactions with other base excision repair proteins. Here, we show that the CTD interacts with the folded domain in native NEIL1 containing 389 residues. The CTD is poised for local folding in an ordered structure that is induced in the purified fragment by osmolytes. Furthermore, deletion of the disordered tail lacking both Tyr and Trp residues causes a red shift in NEIL1's intrinsic Trp-specific fluorescence, indicating a more solvent-exposed environment for the Trp residues in the truncated protein, which also exhibits reduced stability compared to the native enzyme. These observations are consistent with stabilization of the native NEIL1 structure via intramolecular, mostly electrostatic, interactions that were disrupted by mutating a positively charged (Lys-rich) cluster of residues (amino acids 355-360) near the C-terminus. Small-angle X-ray scattering (SAXS) analysis confirms the flexibility and dynamic nature of NEIL1's CTD, a feature that may be critical to providing specificity for NEIL1's multiple, functional interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Conformational co-dependence between Plasmodium berghei LCCL proteins promotes complex formation and stability.

    Science.gov (United States)

    Saeed, Sadia; Tremp, Annie Z; Dessens, Johannes T

    2012-10-01

    Malaria parasites express a conserved family of LCCL-lectin adhesive-like domain proteins (LAPs) that have essential functions in sporozoite transmission. In Plasmodium falciparum all six family members are expressed in gametocytes and form a multi-protein complex. Intriguingly, knockout of P. falciparum LCCL proteins adversely affects expression of other family members at protein, but not at mRNA level, a phenomenon termed co-dependent expression. Here, we investigate this in Plasmodium berghei by crossing a PbLAP1 null mutant parasite with a parasite line expressing GFP-tagged PbLAP3 that displays strong fluorescence in gametocytes. Selected and validated double mutants show normal synthesis and subcellular localization of PbLAP3::GFP. However, GFP-based fluorescence is dramatically reduced without PbLAP1 present, indicating that PbLAP1 and PbLAP3 interact. Moreover, absence of PbLAP1 markedly reduces the half-life of PbLAP3, consistent with a scenario of misfolding. These findings unveil a potential mechanism of conformational interdependence that facilitates assembly and stability of the functional LCCL protein complex. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Stress proteins in lymphocytes: Membrane stabilization does not affect the heat shock response

    International Nuclear Information System (INIS)

    Hughes, C.S.; Repasky, E.A.; Subjeck, J.R.

    1987-01-01

    Temperatures which have been used to induce heat shock proteins (hsps) have been at the upper physiologic limit or well above this limit. In addition, little attention has been given to the effects of physiologic heat exposures on hsp induction in lymphocytes. The author examined temperatures between 39 0 C and 41 0 C on protein synthesis in the following lymphoid cell lines and cells: BDK, EL-4, JM, DO.11, and in dispersed lymph nodes and thymic tissues. In these studies, 39.5 0 appears to be the threshold for hsp induction (as distinguished by gel electrophoresis). At this temperature the induction of the major hsps at 70 and 89 kDa are observed. Hsp 89 appears to be the most strongly induced in all cells examined. In JM cells, a human cell line, heat shock also induces hsp 68, the non-constitutive hsp at this size. These temperatures do not depress normal levels of protein synthesis. When stearic acid or cholesterol was added to lymphocyte cultures prior to heating (which stabilize membranes), hsp induction appears to occur in a manner indistinguishable from cells heated in normal media. This suggests that membrane fluidity (as influenced by these agents) does not affect or depress the heat shock response in these cells. Finally, the authors observed that 2-deoxyglucose and other inducers of glucose regulated proteins in fibroblasts also induce the major glucose regulated proteins in lymphocytes

  13. Characterization of polyacrylamide-stabilized Pf1 phage liquid crystals for protein NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Trempe, Jean-Francois; Morin, Frederick G.; Xia Zhicheng; Marchessault, Robert H.; Gehring, Kalle [McGill University, Department of Biochemistry and Department of Chemistry (Canada)], E-mail: kalle@bri.nrc.ca

    2002-01-15

    A new polymer-stabilized nematic liquid crystal has been characterized for the measurement of biomolecular residual dipolar couplings. Filamentous Pf1 phage were embedded in a polyacrylamide matrix that fixes the orientation of the particles. The alignment was characterized by the quadrupolar splitting of the {sup 2}H NMR water signal and by the measurement of {sup 1}H-{sup 15}N residual dipolar couplings (RDC) in the archeal translation elongation factor 1{beta}. Protein dissolved in the polymer-stabilized medium orients quantitatively as in media without polyacrylamide. We show that the quadrupolar splitting and RDCs are zero in media in which the Pf1 phage particles are aligned at the magic angle. This allows measurement of J and dipolar couplings in a single sample.

  14. Characterization of polyacrylamide-stabilized Pf1 phage liquid crystals for protein NMR spectroscopy

    International Nuclear Information System (INIS)

    Trempe, Jean-Francois; Morin, Frederick G.; Xia Zhicheng; Marchessault, Robert H.; Gehring, Kalle

    2002-01-01

    A new polymer-stabilized nematic liquid crystal has been characterized for the measurement of biomolecular residual dipolar couplings. Filamentous Pf1 phage were embedded in a polyacrylamide matrix that fixes the orientation of the particles. The alignment was characterized by the quadrupolar splitting of the 2 H NMR water signal and by the measurement of 1 H- 15 N residual dipolar couplings (RDC) in the archeal translation elongation factor 1β. Protein dissolved in the polymer-stabilized medium orients quantitatively as in media without polyacrylamide. We show that the quadrupolar splitting and RDCs are zero in media in which the Pf1 phage particles are aligned at the magic angle. This allows measurement of J and dipolar couplings in a single sample

  15. TRF2 Protein Interacts with Core Histones to Stabilize Chromosome Ends*

    Science.gov (United States)

    Izumi, Takashi; Shimizu, Shigeomi

    2016-01-01

    Mammalian chromosome ends are protected by a specialized nucleoprotein complex called telomeres. Both shelterin, a telomere-specific multi-protein complex, and higher order telomeric chromatin structures combine to stabilize the chromosome ends. Here, we showed that TRF2, a component of shelterin, binds to core histones to protect chromosome ends from inappropriate DNA damage response and loss of telomeric DNA. The N-terminal Gly/Arg-rich domain (GAR domain) of TRF2 directly binds to the globular domain of core histones. The conserved arginine residues in the GAR domain of TRF2 are required for this interaction. A TRF2 mutant with these arginine residues substituted by alanine lost the ability to protect telomeres and induced rapid telomere shortening caused by the cleavage of a loop structure of the telomeric chromatin. These findings showed a previously unnoticed interaction between the shelterin complex and nucleosomal histones to stabilize the chromosome ends. PMID:27514743

  16. Detergent Isolation Stabilizes and Activates the Shigella Type III Secretion System Translocator Protein IpaC.

    Science.gov (United States)

    Bernard, Abram R; Duarte, Shari M; Kumar, Prashant; Dickenson, Nicholas E

    2016-07-01

    Shigella rely on a type III secretion system as the primary virulence factor for invasion and colonization of human hosts. Although there are an estimated 90 million Shigella infections, annually responsible for more than 100,000 deaths worldwide, challenges isolating and stabilizing many type III secretion system proteins have prevented a full understanding of the Shigella invasion mechanism and additionally slowed progress toward a much needed Shigella vaccine. Here, we show that the non-denaturing zwitterionic detergent N, N-dimethyldodecylamine N-oxide (LDAO) and non-ionic detergent n-octyl-oligo-oxyethylene efficiently isolated the hydrophobic Shigella translocator protein IpaC from the co-purified IpaC/IpgC chaperone-bound complex. Both detergents resulted in monomeric IpaC that exhibits strong membrane binding and lysis characteristics while the chaperone-bound complex does not, suggesting that the stabilizing detergents provide a means of following IpaC "activation" in vitro. Additionally, biophysical characterization found that LDAO provides significant thermal and temporal stability to IpaC, protecting it for several days at room temperature and brief exposure to temperatures reaching 90°C. In summary, this work identified and characterized conditions that provide stable, membrane active IpaC, providing insight into key interactions with membranes and laying a strong foundation for future vaccine formulation studies taking advantage of the native immunogenicity of IpaC and the stability provided by LDAO. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  17. Dissolution, agglomerate morphology, and stability limits of protein-coated silver nanoparticles.

    Science.gov (United States)

    Martin, Matthew N; Allen, Andrew J; MacCuspie, Robert I; Hackley, Vincent A

    2014-09-30

    Little is understood regarding the impact that molecular coatings have on nanoparticle dissolution kinetics and agglomerate formation in a dilute nanoparticle dispersion. Dissolution and agglomeration processes compete in removing isolated nanoparticles from the dispersion, making quantitative time-dependent measurements of the mechanisms of nanoparticle loss particularly challenging. In this article, we present in situ ultra-small-angle X-ray scattering (USAXS) results, simultaneously quantifying dissolution, agglomeration, and stability limits of silver nanoparticles (AgNPs) coated with bovine serum albumin (BSA) protein. When the BSA corona is disrupted, we find that the loss of silver from the nanoparticle core is well matched by a second-order kinetic rate reaction, arising from the oxidative dissolution of silver. Dissolution and agglomeration are quantified, and morphological transitions throughout the process are qualified. By probing the BSA-AgNP suspension around its stability limits, we provide insight into the destabilization mechanism by which individual particles rapidly dissolve as a whole rather than undergo slow dissolution from the aqueous interface inward, once the BSA layer is breached. Because USAXS rapidly measures over the entire nanometer to micrometer size range during the dissolution process, many insights are also gained into the stabilization of NPs by protein and its ability to protect the labile metal core from the solution environment by prohibiting the diffusion of reactive species. This approach can be extended to a wide variety of coating molecules and reactive metal nanoparticle systems to carefully survey their stability limits, revealing the likely mechanisms of coating breakdown and ensuing reactions.

  18. Heat shock protein 90β stabilizes focal adhesion kinase and enhances cell migration and invasion in breast cancer cells

    International Nuclear Information System (INIS)

    Xiong, Xiangyang; Wang, Yao; Liu, Chengmei; Lu, Quqin; Liu, Tao; Chen, Guoan; Rao, Hai; Luo, Shiwen

    2014-01-01

    Focal adhesion kinase (FAK) acts as a regulator of cellular signaling and may promote cell spreading, motility, invasion and survival in malignancy. Elevated expression and activity of FAK frequently correlate with tumor cell metastasis and poor prognosis in breast cancer. However, the mechanisms by which the turnover of FAK is regulated remain elusive. Here we report that heat shock protein 90β (HSP90β) interacts with FAK and the middle domain (amino acids 233–620) of HSP90β is mainly responsible for this interaction. Furthermore, we found that HSP90β regulates FAK stability since HSP90β inhibitor 17-AAG triggers FAK ubiquitylation and subsequent proteasome-dependent degradation. Moreover, disrupted FAK-HSP90β interaction induced by 17-AAG contributes to attenuation of tumor cell growth, migration, and invasion. Together, our results reveal how HSP90β regulates FAK stability and identifies a potential therapeutic strategy to breast cancer. - Highlights: • HSP90β protects FAK from degradation by the ubiquitin-proteasome pathway. • Inhibition of HSP90β or FAK attenuates tumorigenesis of breast cancer cells. • Genetic repression of HSP90β or FAK inhibits tumor cell migration and proliferation. • Inhibition of HSP90β or FAK interferes cell invasion and cytoskeleton

  19. Heat shock protein 90β stabilizes focal adhesion kinase and enhances cell migration and invasion in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Xiangyang [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanchang University, Nanchang, Jiangxi 330006 (China); State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047 (China); Wang, Yao [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Liu, Chengmei [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047 (China); Lu, Quqin [Department of Biostatistics and Epidemiology, School of Public Health, Nanchang University, Nanchang, Jiangxi 330006 (China); Liu, Tao [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Chen, Guoan [Department of Hematology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006 (China); Rao, Hai [Department of Molecular Medicine, University of Texas Health Science Center, San Antonio, TX 78229 (United States); Luo, Shiwen, E-mail: shiwenluo@ncu.edu.cn [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China)

    2014-08-01

    Focal adhesion kinase (FAK) acts as a regulator of cellular signaling and may promote cell spreading, motility, invasion and survival in malignancy. Elevated expression and activity of FAK frequently correlate with tumor cell metastasis and poor prognosis in breast cancer. However, the mechanisms by which the turnover of FAK is regulated remain elusive. Here we report that heat shock protein 90β (HSP90β) interacts with FAK and the middle domain (amino acids 233–620) of HSP90β is mainly responsible for this interaction. Furthermore, we found that HSP90β regulates FAK stability since HSP90β inhibitor 17-AAG triggers FAK ubiquitylation and subsequent proteasome-dependent degradation. Moreover, disrupted FAK-HSP90β interaction induced by 17-AAG contributes to attenuation of tumor cell growth, migration, and invasion. Together, our results reveal how HSP90β regulates FAK stability and identifies a potential therapeutic strategy to breast cancer. - Highlights: • HSP90β protects FAK from degradation by the ubiquitin-proteasome pathway. • Inhibition of HSP90β or FAK attenuates tumorigenesis of breast cancer cells. • Genetic repression of HSP90β or FAK inhibits tumor cell migration and proliferation. • Inhibition of HSP90β or FAK interferes cell invasion and cytoskeleton.

  20. Structure and stability of complexes of agmatine with some functional receptor residues of proteins

    Science.gov (United States)

    Remko, Milan; Broer, Ria; Remková, Anna; Van Duijnen, Piet Th.

    2017-04-01

    The paper reports the results of a theoretical study of the conformational behavior and basicity of biogenic amine agmatine. The complexes modelling of agmatine - protein interaction are also under scrutiny of our investigation using the Becke3LYP and B97D levels of the density functional theory. The relative stabilities (Gibbs energies) of individual complexes are by both DFT methods described equally. Hydration has a dramatic effect on the hydrogen bonded complexes studied. The pairing acidic carboxylate group with different agmatine species resulted in charged hydrogen bond complexes containing negatively charged acetate species acting as proton acceptors.

  1. Protein thermal stability enhancement by designing salt bridges: a combined computational and experimental study.

    Directory of Open Access Journals (Sweden)

    Chi-Wen Lee

    Full Text Available Protein thermal stability is an important factor considered in medical and industrial applications. Many structural characteristics related to protein thermal stability have been elucidated, and increasing salt bridges is considered as one of the most efficient strategies to increase protein thermal stability. However, the accurate simulation of salt bridges remains difficult. In this study, a novel method for salt-bridge design was proposed based on the statistical analysis of 10,556 surface salt bridges on 6,493 X-ray protein structures. These salt bridges were first categorized based on pairing residues, secondary structure locations, and Cα-Cα distances. Pairing preferences generalized from statistical analysis were used to construct a salt-bridge pair index and utilized in a weighted electrostatic attraction model to find the effective pairings for designing salt bridges. The model was also coupled with B-factor, weighted contact number, relative solvent accessibility, and conservation prescreening to determine the residues appropriate for the thermal adaptive design of salt bridges. According to our method, eight putative salt-bridges were designed on a mesophilic β-glucosidase and 24 variants were constructed to verify the predictions. Six putative salt-bridges leaded to the increase of the enzyme thermal stability. A significant increase in melting temperature of 8.8, 4.8, 3.7, 1.3, 1.2, and 0.7°C of the putative salt-bridges N437K-D49, E96R-D28, E96K-D28, S440K-E70, T231K-D388, and Q277E-D282 was detected, respectively. Reversing the polarity of T231K-D388 to T231D-D388K resulted in a further increase in melting temperatures by 3.6°C, which may be caused by the transformation of an intra-subunit electrostatic interaction into an inter-subunit one depending on the local environment. The combination of the thermostable variants (N437K, E96R, T231D and D388K generated a melting temperature increase of 15.7°C. Thus, this study

  2. Improved glucose-neopentyl glycol (GNG) amphiphiles for membrane protein solubilization and stabilization.

    Science.gov (United States)

    Cho, Kyung Ho; Bae, Hyoung Eun; Das, Manabendra; Gellman, Samuel H; Chae, Pil Seok

    2014-02-01

    Membrane proteins are inherently amphipathic and undergo dynamic conformational changes for proper function within native membranes. Maintaining the functional structures of these biomacromolecules in aqueous media is necessary for structural studies but difficult to achieve with currently available tools, thus necessitating the development of novel agents with favorable properties. This study introduces several new glucose-neopentyl glycol (GNG) amphiphiles and reveals some agents that display favorable behaviors for the solubilization and stabilization of a large, multi-subunit membrane protein assembly. Furthermore, a detergent structure-property relationship that could serve as a useful guideline for the design of novel amphiphiles is discussed. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Data on the role of accessible surface area on osmolytes-induced protein stabilization

    Directory of Open Access Journals (Sweden)

    Safikur Rahman

    2017-02-01

    Full Text Available This paper describes data related to the research article “Testing the dependence of stabilizing effect of osmolytes on the fractional increase in the accessible surface area on thermal and chemical denaturations of proteins” [1]. Heat- and guanidinium chloride (GdmCl-induced denaturation of three disulfide free proteins (bovine cytochrome c (b-cyt-c, myoglobin (Mb and barstar in the presence of different concentrations of methylamines (sarcosine, glycine-betaine (GB and trimethylamine-N-oxide (TMAO was monitored by [ϴ]222, the mean residue ellipticity at 222 nm at pH 7.0. Methylamines belong to a class of osmolytes known to protect proteins from deleterious effect of urea. This paper includes comprehensive thermodynamic data obtained from the heat- and GdmCl-induced denaturations of barstar, b-cyt-c and Mb.

  4. The actin family protein ARP6 contributes to the structure and the function of the nucleolus.

    Science.gov (United States)

    Kitamura, Hiroshi; Matsumori, Haruka; Kalendova, Alzbeta; Hozak, Pavel; Goldberg, Ilya G; Nakao, Mitsuyoshi; Saitoh, Noriko; Harata, Masahiko

    2015-08-21

    The actin family members, consisting of actin and actin-related proteins (ARPs), are essential components of chromatin remodeling complexes. ARP6, one of the nuclear ARPs, is part of the Snf-2-related CREB-binding protein activator protein (SRCAP) chromatin remodeling complex, which promotes the deposition of the histone variant H2A.Z into the chromatin. In this study, we showed that ARP6 influences the structure and the function of the nucleolus. ARP6 is localized in the central region of the nucleolus, and its knockdown induced a morphological change in the nucleolus. We also found that in the presence of high concentrations of glucose ARP6 contributed to the maintenance of active ribosomal DNA (rDNA) transcription by placing H2A.Z into the chromatin. In contrast, under starvation, ARP6 was required for cell survival through the repression of rDNA transcription independently of H2A.Z. These findings reveal novel pleiotropic roles for the actin family in nuclear organization and metabolic homeostasis. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Effect of homogenization and pasteurization on the structure and stability of whey protein in milk.

    Science.gov (United States)

    Qi, Phoebe X; Ren, Daxi; Xiao, Yingping; Tomasula, Peggy M

    2015-05-01

    The effect of homogenization alone or in combination with high-temperature, short-time (HTST) pasteurization or UHT processing on the whey fraction of milk was investigated using highly sensitive spectroscopic techniques. In pilot plant trials, 1-L quantities of whole milk were homogenized in a 2-stage homogenizer at 35°C (6.9 MPa/10.3 MPa) and, along with skim milk, were subjected to HTST pasteurization (72°C for 15 s) or UHT processing (135°C for 2 s). Other whole milk samples were processed using homogenization followed by either HTST pasteurization or UHT processing. The processed skim and whole milk samples were centrifuged further to remove fat and then acidified to pH 4.6 to isolate the corresponding whey fractions, and centrifuged again. The whey fractions were then purified using dialysis and investigated using the circular dichroism, Fourier transform infrared, and Trp intrinsic fluorescence spectroscopic techniques. Results demonstrated that homogenization combined with UHT processing of milk caused not only changes in protein composition but also significant secondary structural loss, particularly in the amounts of apparent antiparallel β-sheet and α-helix, as well as diminished tertiary structural contact. In both cases of homogenization alone and followed by HTST treatments, neither caused appreciable chemical changes, nor remarkable secondary structural reduction. But disruption was evident in the tertiary structural environment of the whey proteins due to homogenization of whole milk as shown by both the near-UV circular dichroism and Trp intrinsic fluorescence. In-depth structural stability analyses revealed that even though processing of milk imposed little impairment on the secondary structural stability, the tertiary structural stability of whey protein was altered significantly. The following order was derived based on these studies: raw whole>HTST, homogenized, homogenized and pasteurized>skimmed and pasteurized, and skimmed UHT

  6. Stabilization

    Directory of Open Access Journals (Sweden)

    Muhammad H. Al-Malack

    2016-07-01

    Full Text Available Fuel oil flyash (FFA produced in power and water desalination plants firing crude oils in the Kingdom of Saudi Arabia is being disposed in landfills, which increases the burden on the environment, therefore, FFA utilization must be encouraged. In the current research, the effect of adding FFA on the engineering properties of two indigenous soils, namely sand and marl, was investigated. FFA was added at concentrations of 5%, 10% and 15% to both soils with and without the addition of Portland cement. Mixtures of the stabilized soils were thoroughly evaluated using compaction, California Bearing Ratio (CBR, unconfined compressive strength (USC and durability tests. Results of these tests indicated that stabilized sand mixtures could not attain the ACI strength requirements. However, marl was found to satisfy the ACI strength requirement when only 5% of FFA was added together with 5% of cement. When the FFA was increased to 10% and 15%, the mixture’s strength was found to decrease to values below the ACI requirements. Results of the Toxicity Characteristics Leaching Procedure (TCLP, which was performed on samples that passed the ACI requirements, indicated that FFA must be cautiously used in soil stabilization.

  7. The nuclear protein Artemis promotes AMPK activation by stabilizing the LKB1–AMPK complex

    International Nuclear Information System (INIS)

    Nakagawa, Koji; Uehata, Yasuko; Natsuizaka, Mitsuteru; Kohara, Toshihisa; Darmanin, Stephanie; Asaka, Masahiro; Takeda, Hiroshi; Kobayashi, Masanobu

    2012-01-01

    Highlights: ► The nuclear protein Artemis physically interacts with AMPKα2. ► Artemis co-localizes with AMPKα2 in the nucleus. ► Artemis promotes phosphorylation and activation of AMPK. ► The interaction between AMPKα2 and LKB1 is stabilized by Artemis. -- Abstract: AMP-activated protein kinase (AMPK) is a hetero-trimeric Ser/Thr kinase composed of a catalytic α subunit and regulatory β and γ subunits; it functions as an energy sensor that controls cellular energy homeostasis. In response to an increased cellular AMP/ATP ratio, AMPK is activated by phosphorylation at Thr172 in the α-subunit by upstream AMPK kinases (AMPKKs), including tumor suppressor liver kinase B1 (LKB1). To elucidate more precise molecular mechanisms of AMPK activation, we performed yeast two-hybrid screening and isolated the complementary DNA (cDNA) encoding the nuclear protein Artemis/DNA cross-link repair 1C (DCLRE1C) as an AMPKα2-binding protein. Artemis was found to co-immunoprecipitate with AMPKα2, and the co-localization of Artemis with AMPKα2 in the nucleus was confirmed by immunofluorescence staining in U2OS cells. Moreover, over-expression of Artemis enhanced the phosphorylation of AMPKα2 and the AMPK substrate acetyl-CoA carboxylase (ACC). Conversely, RNAi-mediated knockdown of Artemis reduced AMPK and ACC phosphorylation. In addition, Artemis markedly increased the physical association between AMPKα2 and LKB1. Taken together, these results suggest that Artemis functions as a positive regulator of AMPK signaling by stabilizing the LKB1–AMPK complex.

  8. The nuclear protein Artemis promotes AMPK activation by stabilizing the LKB1-AMPK complex

    Energy Technology Data Exchange (ETDEWEB)

    Nakagawa, Koji, E-mail: k_nakagawa@pharm.hokudai.ac.jp [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan); Uehata, Yasuko; Natsuizaka, Mitsuteru; Kohara, Toshihisa; Darmanin, Stephanie [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Asaka, Masahiro [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Takeda, Hiroshi [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan); Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Kobayashi, Masanobu [Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); School of Nursing and Social Services, Health Sciences University of Hokkaido, Ishikari-Toubetsu, Hokkaido 061-0293 (Japan)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer The nuclear protein Artemis physically interacts with AMPK{alpha}2. Black-Right-Pointing-Pointer Artemis co-localizes with AMPK{alpha}2 in the nucleus. Black-Right-Pointing-Pointer Artemis promotes phosphorylation and activation of AMPK. Black-Right-Pointing-Pointer The interaction between AMPK{alpha}2 and LKB1 is stabilized by Artemis. -- Abstract: AMP-activated protein kinase (AMPK) is a hetero-trimeric Ser/Thr kinase composed of a catalytic {alpha} subunit and regulatory {beta} and {gamma} subunits; it functions as an energy sensor that controls cellular energy homeostasis. In response to an increased cellular AMP/ATP ratio, AMPK is activated by phosphorylation at Thr172 in the {alpha}-subunit by upstream AMPK kinases (AMPKKs), including tumor suppressor liver kinase B1 (LKB1). To elucidate more precise molecular mechanisms of AMPK activation, we performed yeast two-hybrid screening and isolated the complementary DNA (cDNA) encoding the nuclear protein Artemis/DNA cross-link repair 1C (DCLRE1C) as an AMPK{alpha}2-binding protein. Artemis was found to co-immunoprecipitate with AMPK{alpha}2, and the co-localization of Artemis with AMPK{alpha}2 in the nucleus was confirmed by immunofluorescence staining in U2OS cells. Moreover, over-expression of Artemis enhanced the phosphorylation of AMPK{alpha}2 and the AMPK substrate acetyl-CoA carboxylase (ACC). Conversely, RNAi-mediated knockdown of Artemis reduced AMPK and ACC phosphorylation. In addition, Artemis markedly increased the physical association between AMPK{alpha}2 and LKB1. Taken together, these results suggest that Artemis functions as a positive regulator of AMPK signaling by stabilizing the LKB1-AMPK complex.

  9. Root reinforcement and its contribution to slope stability in the Western Ghats of Kerala, India

    Science.gov (United States)

    Lukose Kuriakose, Sekhar; van Beek, L. P. H.

    2010-05-01

    The Western Ghats of Kerala, India is prone to shallow landslides and consequent debris flows. An earlier study (Kuriakose et al., DOI:10.1002/esp.1794) with limited data had already demonstrated the possible effects of vegetation on slope hydrology and stability. Spatially distributed root cohesion is one of the most important data necessary to assess the effects of anthropogenic disturbances on the probability of shallow landslide initiation, results of which are reported in sessions GM6.1 and HS13.13/NH3.16. Thus it is necessary to the know the upper limits of reinforcement that the roots are able to provide and its spatial and vertical distribution in such an anthropogenically intervened terrain. Root tensile strength and root pull out tests were conducted on nine species of plants that are commonly found in the region. They are 1) Rubber (Hevea Brasiliensis), 2) Coconut Palm (Cocos nucifera), 3) Jackfruit trees (Artocarpus heterophyllus), 4) Teak (Tectona grandis), 5) Mango trees (Mangifera indica), 6) Lemon grass (Cymbopogon citratus), 7) Gambooge (Garcinia gummi-gutta), 8) Coffee (Coffea Arabica) and 9) Tea (Camellia sinensis). About 1500 samples were collected of which only 380 could be tested (in the laboratory) due to breakage of roots during the tests. In the successful tests roots failed in tension. Roots having diameters between 2 mm and 12 mm were tested. Each sample tested had a length of 15 cm. Root pull out tests were conducted in the field. Root tensile strength vs root diameter, root pull out strength vs diameter, root diameter vs root depth and root count vs root depth relationships were derived. Root cohesion was computed for nine most dominant plants in the region using the perpendicular root model of Wu et al. (1979) modified by Schimidt et al. (2001). A soil depth map was derived using regression kriging as suggested by Kuriakose et al., (doi:10.1016/j.catena.2009.05.005) and used along with the land use map of 2008 to distribute the

  10. Polycystin-1 Is a Cardiomyocyte Mechanosensor That Governs L-Type Ca2+ Channel Protein Stability.

    Science.gov (United States)

    Pedrozo, Zully; Criollo, Alfredo; Battiprolu, Pavan K; Morales, Cyndi R; Contreras-Ferrat, Ariel; Fernández, Carolina; Jiang, Nan; Luo, Xiang; Caplan, Michael J; Somlo, Stefan; Rothermel, Beverly A; Gillette, Thomas G; Lavandero, Sergio; Hill, Joseph A

    2015-06-16

    L-type calcium channel activity is critical to afterload-induced hypertrophic growth of the heart. However, the mechanisms governing mechanical stress-induced activation of L-type calcium channel activity are obscure. Polycystin-1 (PC-1) is a G protein-coupled receptor-like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. We subjected neonatal rat ventricular myocytes to mechanical stretch by exposing them to hypo-osmotic medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on L-type calcium channel activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Overexpression of a C-terminal fragment of PC-1 was sufficient to trigger neonatal rat ventricular myocyte hypertrophy. Exposing neonatal rat ventricular myocytes to hypo-osmotic medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown-dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 knockout) and subjected them to mechanical stress in vivo (transverse aortic constriction). At baseline, PC-1 knockout mice manifested decreased cardiac function relative to littermate controls, and α1C L-type calcium channel protein levels were significantly lower in PC-1 knockout hearts. Whereas control mice manifested robust transverse aortic constriction-induced increases in cardiac mass, PC-1 knockout mice showed no significant growth. Likewise, transverse aortic constriction-elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals PC-1 is a cardiomyocyte mechanosensor that is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein. © 2015 American Heart Association, Inc.

  11. Protein samples for NMR: expression and analysis without purification, and stabilization by covalent cyclization

    International Nuclear Information System (INIS)

    Otting, G.; Ozawa, K.; Prosselkov, P.; Williams, N.K.; Dixon, N.E.; Liepinsh, E.

    2002-01-01

    Full text: A modified cell-free in vitro expression system was established for the expression of milligram quantities of protein per mL reaction medium. Expression levels of the E coli cytoplasmic peptidyl-prolyl cis-trans isomerase, PpiB, in 0 6 mL reaction medium were sufficient for the direct recording of clean 15N-HSQC spectra without chromatographic purification or sample concentration steps, using a 600 MHz NMR spectrometer with cryoprobe. Besides providing a route to high-throughput sample preparation, in vitro expression systems are known to be highly economic in their utilization of selectively labelled ammo acids. Using dual-selective labelling with 15N- and 13C-labelled amino acids, the 15N-HSQC cross peaks of strategically selected ammo acids can readily be identified and monitored for their response to the presence of ligand molecules, again without sample purification. 2) The N-terminal domain of E coli DnaB is a protein of ca 110 residues with a structured core composed of 6 helices. Additional segments of 10 residues each at the N- and C-termini are highly mobile. Both ends are close in space and can be linked together in a covalent peptide bond using intern technology. The core structures of linear (lin-DnaB-N) and cyclized (cz-DnaB-N) protein are conserved, as evidenced by superimposable NOESY spectra and chemical shifts. The linker segment in cz-DnaB-N is mobile as shown by 1H-15N NOEs. Yet, the cyclic protein melts about 10 degrees higher than the linear version. A stabilization free energy of ca 2 kcal/mol is in agreement with predictions based on the reduced entropy in the unfolded state. Amide proton exchange rates are much slower in the cyclic protein and reveal cooperative exchange through total, global unfolding at a rate of once every 100 minutes in the linear protein

  12. The Role of Stress Proteins in Cell Stabilization: A Perspective from an Extremophile

    Science.gov (United States)

    Trent, Jonathan

    2001-01-01

    The existence of organisms that live at near boiling temperatures is living proof that all of the complex biochemical machinery of life can be adapted to function under these harsh conditions. The purpose of our research is to elucidate the role of a group of proteins known as heat shock proteins or HSP60s in this adaptation to high temperatures. HSP60s are found in all organisms and they are among the most highly conserved proteins known. We are investigating HSP60s in an organism growing at 80 C and pH 2.0 (Sulfolobus shibatae). This organism produces three closely-related HSP60 proteins, referred to as HSP60 alpha, beta, and gamma. Our DOE-funded research during the last two years has focused on clarifying the role of FiSP60 alpha and beta. These are among the two most abundant proteins in S. shibatae grown at high temperatures and significantly increase in abundance when the cells are exposed to near-lethal temperatures. We have demonstrated that these proteins protect the cells from lethal temperatures by stabilizing their membranes. During this last year we have been studying gamma, which was discovered by genome sequence analysis but nothing was known about its function. We have determined that gamma is only expressed at low temperatures. that it interacts with alpha and beta, and that it influences their ability to form higher-order structures critical to their function. We propose that gamma modulates HSP60 function at low temperatures.

  13. Conserved residues and their role in the structure, function, and stability of acyl-coenzyme A binding protein

    DEFF Research Database (Denmark)

    Kragelund, B B; Poulsen, K; Andersen, K V

    1999-01-01

    In the family of acyl-coenzyme A binding proteins, a subset of 26 sequence sites are identical in all eukaryotes and conserved throughout evolution of the eukaryotic kingdoms. In the context of the bovine protein, the importance of these 26 sequence positions for structure, function, stability...

  14. Molecular mechanisms underlying the pilsicainide-induced stabilization of hERG proteins in transfected mammalian cells

    Directory of Open Access Journals (Sweden)

    Takeshi Onohara, MD

    2017-06-01

    Conclusions: Pilsicainide penetrates the plasma membrane, stabilizes WT-hERG proteins by acting as a chemical chaperone, and enhances WT-hERG channel currents. This mechanism could also be applicable to modulations of certain mutant-hERG proteins.

  15. Cellular uptake of beta-carotene from protein stabilized solid lipid nano-particles prepared by homogenization-evaporation method

    Science.gov (United States)

    Using a homogenization-evaporation method, beta-carotene (BC) loaded nano-particles were prepared with different ratios of food-grade sodium caseinate (SC), whey protein isolate (WPI), or soy protein isolate (SPI) to BC and evaluated for their physiochemical stability, in vitro cytotoxicity, and cel...

  16. The Assembly-Activating Protein Promotes Stability and Interactions between AAV’s Viral Proteins to Nucleate Capsid Assembly

    Directory of Open Access Journals (Sweden)

    Anna C. Maurer

    2018-05-01

    Full Text Available Summary: The adeno-associated virus (AAV vector is a preferred delivery platform for in vivo gene therapy. Natural and engineered variations of the AAV capsid affect a plurality of phenotypes relevant to gene therapy, including vector production and host tropism. Fundamental to these aspects is the mechanism of AAV capsid assembly. Here, the role of the viral co-factor assembly-activating protein (AAP was evaluated in 12 naturally occurring AAVs and 9 putative ancestral capsid intermediates. The results demonstrate increased capsid protein stability and VP-VP interactions in the presence of AAP. The capsid’s dependence on AAP can be partly overcome by strengthening interactions between monomers within the assembly, as illustrated by the transfer of a minimal motif defined by a phenotype-to-phylogeny mapping method. These findings suggest that the emergence of AAP within the Dependovirus genus relaxes structural constraints on AAV assembly in favor of increasing the degrees of freedom for the capsid to evolve. : Maurer et al. describe a phenotype-to-phylogeny mapping strategy correlating phenotypic variation in AAVs to a reconstructed phylogeny, revealing capsid structure-function relationships relevant to that phenotype. Dependence on the viral co-factor AAP for capsid assembly is examined, and capsid functional motifs, in addition to mechanistic roles of AAP, are elucidated. Keywords: AAV, AAP, adeno-associated virus, capsid assembly, manufacturing, capsid, vector engineering, structure-function, gene therapy

  17. The Surface-Exposed Protein SntA Contributes to Complement Evasion in Zoonotic Streptococcus suis.

    Science.gov (United States)

    Deng, Simin; Xu, Tong; Fang, Qiong; Yu, Lei; Zhu, Jiaqi; Chen, Long; Liu, Jiahui; Zhou, Rui

    2018-01-01

    Streptococcus suis is an emerging zoonotic pathogen causing streptococcal toxic shock like syndrome (STSLS), meningitis, septicemia, and even sudden death in human and pigs. Serious septicemia indicates this bacterium can evade the host complement surveillance. In our previous study, a functionally unknown protein SntA of S. suis has been identified as a heme-binding protein, and contributes to virulence in pigs. SntA can interact with the host antioxidant protein AOP2 and consequently inhibit its antioxidant activity. In the present study, SntA is identified as a cell wall anchored protein that functions as an important player in S. suis complement evasion. The C3 deposition and membrane attack complex (MAC) formation on the surface of sntA -deleted mutant strain Δ sntA are demonstrated to be significantly higher than the parental strain SC-19 and the complementary strain CΔ sntA . The abilities of anti-phagocytosis, survival in blood, and in vivo colonization of Δ sntA are obviously reduced. SntA can interact with C1q and inhibit hemolytic activity via the classical pathway. Complement activation assays reveal that SntA can also directly activate classical and lectin pathways, resulting in complement consumption. These two complement evasion strategies may be crucial for the pathogenesis of this zoonotic pathogen. Concerning that SntA is a bifunctional 2',3'-cyclic nucleotide 2'-phosphodiesterase/3'-nucleotidase in many species of Gram-positive bacteria, these complement evasion strategies may have common biological significance.

  18. Reactivity of polymeric proanthocyanidins toward salivary proteins and their contribution to young red wine astringency.

    Science.gov (United States)

    Sun, Baoshan; de Sá, Marta; Leandro, Conceição; Caldeira, Ilda; Duarte, Filomena L; Spranger, Isabel

    2013-01-30

    Recent studies have indicated the presence of significant amount of highly polymerized and soluble proanthocyanidins in red wine and such compounds interacted readily with proteins, suggesting that they might be particularly astringent. Thus, the objective of this work was to verify the astringency of polymeric proanthocyanidins and their contribution to red wine astringency. The precipitation reactions of the purified oligomeric procyanidins (degree of polymerization ranging from 2 to 12-15) and polymeric procyanidins (degree of polymerization ranging from 12-15 to 32-34) with human salivary proteins were studied; salivary proteins composition changes before and after the reaction was verified by SDS-PAGE and procyanidins composition changes by spectrometric, direct HPLC and thiolysis-HPLC methods. The astringency intensity of these two procyanidin fractions was evaluated by a sensory analysis panel. For verifying the correlation between polymeric proanthocyanidins and young red wine astringency, the levels of total oligomeric and total polymeric proanthocyanidins and other phenolic composition in various young red wines were quantified and the astringency intensities of these wines were evaluated by a sensory panel. The results showed that polymeric proanthocyanidins had much higher reactivity toward human salivary proteins and higher astringency intensity than the oligomeric ones. Furthermore, young red wine astringency intensities were highly correlated to levels of polymeric proanthocyanidins, particularly at low concentration range (correlation coefficient r = 0.9840) but not significant correlated to total polyphenols (r = 0.2343) or other individual phenolic compounds (generally r wine astringency and the levels of polymeric polyphenols in red wines may be used as an indicator for its astringency.

  19. Contribution of glucan-binding protein A to firm and stable biofilm formation by Streptococcus mutans.

    Science.gov (United States)

    Matsumi, Y; Fujita, K; Takashima, Y; Yanagida, K; Morikawa, Y; Matsumoto-Nakano, M

    2015-06-01

    Glucan-binding proteins (Gbps) of Streptococcus mutans, a major pathogen of dental caries, mediate the binding of glucans synthesized from sucrose by the action of glucosyltransferases (GTFs) encoded by gtfB, gtfC, and gtfD. Several stress proteins, including DnaK and GroEL encoded by dnaK and groEL, are related to environmental stress tolerance. The contribution of Gbp expression to biofilm formation was analyzed by focusing on the expression levels of genes encoding GTFs and stress proteins. Biofilm-forming assays were performed using GbpA-, GbpB-, and GbpC-deficient mutant strains and the parental strain MT8148. The expression levels of gtfB, gtfC, gtfD, dnaK, and groEL were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Furthermore, the structure of biofilms formed by these Gbp-deficient mutant strains was observed using confocal laser scanning microscopy (CLSM). Biofilm-forming assay findings demonstrated that the amount formed by the GbpA-deficient mutant strain (AD1) was nearly the same as that by the parental strain, while the GbpB- and GbpC-deficient mutant strains produced lower amounts than MT8148. Furthermore, RT-qPCR assay results showed that the expressions of gtfB, dnaK, and groEL in AD1 were elevated compared with MT8148. CLSM also revealed that the structure of biofilm formed by AD1 was prominently different compared with that formed by the parental strain. These results suggest that a defect in GbpA influences the expression of genes controlling biofilm formation, indicating its importance as a protein for firm and stable biofilm formation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Biophysical evaluation of aminoclay as an effective protectant for protein stabilization during freeze-drying and storage

    Directory of Open Access Journals (Sweden)

    Song JG

    2016-12-01

    Full Text Available Jae Geun Song, Sang Hoon Lee, Hyo-Kyung Han College of Pharmacy, Dongguk University, Goyang, South Korea Abstract: This study aimed to evaluate aminoclay (3-aminopropyl-functionalized magnesium phyllosilicate as an effective protectant for the stabilization of protein formulation in freeze-drying. Bovine serum albumin (BSA, as a model protein, was freeze-dried with aminoclay at various concentrations, and the effects of aminoclay on the structural stability of proteins were compared with those of the conventional stabilizers. The structural characteristics of the protein were determined by size exclusion chromatography (SEC, circular dichroism (CD, and Fourier transform infrared (FTIR spectroscopy. Furthermore, physicochemical and morphological characteristics were examined by X-ray powder diffraction (XRPD, differential scanning calorimetry (DSC, and scanning electron microscopy (SEM. XRPD and DSC patterns indicated that the glass transition temperature (Tg of the amorphous formulation of aminoclay mixed with proteins was gradually elevated as the concentration of aminoclay increased. FTIR and CD spectral analysis suggested that the protein structure was well maintained with aminoclay during the freeze-drying process and 3 months of storage at 4°C and 40°C. Furthermore, aminoclay conferred the greatest protection against aggregation and retained the monomer content of BSA even at a high temperature. The morphological characteristics of lyophilized proteins were also well conserved during the storage with aminoclay. These results suggested that aminoclay may be useful as an alternative stabilizer for maintaining the structural stability of protein formulations. Keywords: aminoclay, cryoprotectant, lyoprotectant, freeze-drying, protein, stability

  1. Structural Stability of Light-harvesting Protein LH2 Adsorbed on Mesoporous Silica Supports.

    Science.gov (United States)

    Shibuya, Yuuta; Itoh, Tetsuji; Matsuura, Shun-ichi; Yamaguchi, Akira

    2015-01-01

    In the present study, we examined the reversible thermal deformation of the membrane protein light-harvesting complex LH2 adsorbed on mesoporous silica (MPS) supports. The LH2 complex from Thermochromatium tepidum cells was conjugated to MPS supports with a series of pore diameter (2.4 to 10.6 nm), and absorption spectra of the resulting LH2/MPS conjugates were observed over a temperature range of 273 - 313 K in order to examine the structure of the LH2 adsorbed on the MPS support. The experimental results confirmed that a slight ellipsoidal deformation of LH2 was induced by adsorption on the MPS supports. On the other hand, the structural stability of LH2 was not perturbed by the adsorption. Since the pore diameter of MPS support did not influence the structural stability of LH2, it could be considered that the spatial confinement of LH2 in size-matches pore did not improve the structural stability of LH2.

  2. Slow Histidine H/D Exchange Protocol for Thermodynamic Analysis of Protein Folding and Stability using Mass Spectrometry

    OpenAIRE

    Tran, Duc T.; Banerjee, Sambuddha; Alayash, Abdu I.; Crumbliss, Alvin L.; Fitzgerald, Michael C.

    2012-01-01

    Described here is a mass spectrometry based protocol to study the thermodynamic stability of proteins and protein-ligand complexes using the slow H/D exchange reaction of the imidazole C2 proton in histidine side chains. The protocol, which involves evaluating the denaturant dependence of this slow H/D exchange reaction in proteins, allows the global and/or subglobal unfolding/refolding properties of proteins and protein-ligand complexes to be probed. The protocol is developed using several m...

  3. Temporal stability of naturally acquired immunity to Merozoite Surface Protein-1 in Kenyan Adults

    Directory of Open Access Journals (Sweden)

    Crabb Brendan S

    2009-07-01

    Full Text Available Abstract Background Naturally acquired immunity to blood-stage Plasmodium falciparum infection develops with age and after repeated infections. In order to identify immune surrogates that can inform vaccine trials conducted in malaria endemic populations and to better understand the basis of naturally acquired immunity it is important to appreciate the temporal stability of cellular and humoral immune responses to malaria antigens. Methods Blood samples from 16 adults living in a malaria holoendemic region of western Kenya were obtained at six time points over the course of 9 months. T cell immunity to the 42 kDa C-terminal fragment of Merozoite Surface Protein-1 (MSP-142 was determined by IFN-γ ELISPOT. Antibodies to the 42 kDa and 19 kDa C-terminal fragments of MSP-1 were determined by serology and by functional assays that measure MSP-119 invasion inhibition antibodies (IIA to the E-TSR (3D7 allele and growth inhibitory activity (GIA. The haplotype of MSP-119 alleles circulating in the population was determined by PCR. The kappa test of agreement was used to determine stability of immunity over the specified time intervals of 3 weeks, 6 weeks, 6 months, and 9 months. Results MSP-1 IgG antibodies determined by serology were most consistent over time, followed by MSP-1 specific T cell IFN-γ responses and GIA. MSP-119 IIA showed the least stability over time. However, the level of MSP-119 specific IIA correlated with relatively higher rainfall and higher prevalence of P. falciparum infection with the MSP-119 E-TSR haplotype. Conclusion Variation in the stability of cellular and humoral immune responses to P. falciparum blood stage antigens needs to be considered when interpreting the significance of these measurements as immune endpoints in residents of malaria endemic regions.

  4. E11/Podoplanin Protein Stabilization Through Inhibition of the Proteasome Promotes Osteocyte Differentiation in Murine in Vitro Models.

    Science.gov (United States)

    Staines, Katherine A; Prideaux, Matt; Allen, Steve; Buttle, David J; Pitsillides, Andrew A; Farquharson, Colin

    2016-06-01

    The transmembrane glycoprotein E11 is considered critical in early osteoblast-osteocyte transitions (osteocytogenesis), however its function and regulatory mechanisms are still unknown. Using the late osteoblast MLO-A5 cell line we reveal increased E11 protein/mRNA expression (P < 0.001) concomitant with extensive osteocyte dendrite formation and matrix mineralization (P < 0.001). Transfection with E11 significantly increased mRNA levels (P < 0.001), but immunoblotting failed to detect any correlative increases in E11 protein levels, suggestive of post-translational degradation. We found that exogenous treatment of MLO-A5 and osteocytic IDG-SW3 cells with 10 μM ALLN (calpain and proteasome inhibitor) stabilized E11 protein levels and induced a profound increase in osteocytic dendrite formation (P < 0.001). Treatment with other calpain inhibitors failed to promote similar osteocytogenic changes, suggesting that these effects of ALLN rely upon its proteasome inhibitor actions. Accordingly we found that proteasome-selective inhibitors (MG132/lactacystin/ Bortezomib/Withaferin-A) produced similar dose-dependent increases in E11 protein levels in MLO-A5 and primary osteoblast cells. This proteasomal targeting was confirmed by immunoprecipitation of ubiquitinylated proteins, which included E11, and by increased levels of ubiquitinylated E11 protein upon addition of the proteasome inhibitors MG132/Bortezomib. Activation of RhoA, the small GTPase, was found to be increased concomitant with the peak in E11 levels and its downstream signaling was also observed to promote MLO-A5 cell dendrite formation. Our data indicate that a mechanism reliant upon blockade of proteasome-mediated E11 destabilization contributes to osteocytogenesis and that this may involve downstream targeting of RhoA. This work adds to our mechanistic understanding of the factors regulating bone homeostasis, which may lead to future therapeutic approaches. © 2015 The Authors. Journal of

  5. Preliminary protein corona formation stabilizes gold nanoparticles and improves deposition efficiency

    Science.gov (United States)

    Luby, Alexandra O.; Breitner, Emily K.; Comfort, Kristen K.

    2016-08-01

    Due to their advantageous characteristics, gold nanoparticles (AuNPs) are being increasingly utilized in a vast array of biomedical applications. However, the efficacy of these procedures are highly dependent upon strong interactions between AuNPs and the surrounding environment. While the field of nanotechnology has grown exponentially, there is still much to be discovered with regards to the complex interactions between NPs and biological systems. One area of particular interest is the generation of a protein corona, which instantaneously forms when NPs encounter a protein-rich environment. Currently, the corona is viewed as an obstacle and has been identified as the cause for loss of application efficiency in physiological systems. To date, however, no study has explored if the protein corona could be designed and advantageously utilized to improve both NP behavior and application efficacy. Therefore, we sought to identify if the formation of a preliminary protein corona could modify both AuNP characteristics and association with the HaCaT cell model. In this study, a corona comprised solely of epidermal growth factor (EGF) was successfully formed around 10-nm AuNPs. These EGF-AuNPs demonstrated augmented particle stability, a modified corona composition, and increased deposition over stock AuNPs, while remaining biocompatible. Analysis of AuNP dosimetry was repeated under dynamic conditions, with lateral flow significantly disrupting deposition and the nano-cellular interface. Taken together, this study demonstrated the plausibility and potential of utilizing the protein corona as a means to influence NP behavior; however, fluid dynamics remains a major challenge to progressing NP dosimetry.

  6. Protein Comparability Assessments and Potential Applicability of High Throughput Biophysical Methods and Data Visualization Tools to Compare Physical Stability Profiles

    Directory of Open Access Journals (Sweden)

    Mohammad A. Alsenaidy

    2014-03-01

    Full Text Available In this review, some of the challenges and opportunities encountered during protein comparability assessments are summarized with an emphasis on developing new analytical approaches to better monitor higher-order protein structures. Several case studies are presented using high throughput biophysical methods to collect protein physical stability data as function of temperature, agitation, ionic strength and/or solution pH. These large data sets were then used to construct empirical phase diagrams (EPDs, radar charts, and comparative signature diagrams (CSDs for data visualization and structural comparisons between the different proteins. Protein samples with different sizes, post-translational modifications, and inherent stability are presented: acidic fibroblast growth factor (FGF-1 mutants, different glycoforms of an IgG1 mAb prepared by deglycosylation, as well as comparisons of different formulations of an IgG1 mAb and granulocyte colony stimulating factor (GCSF. Using this approach, differences in structural integrity and conformational stability profiles were detected under stress conditions that could not be resolved by using the same techniques under ambient conditions (i.e., no stress. Thus, an evaluation of conformational stability differences may serve as an effective surrogate to monitor differences in higher-order structure between protein samples. These case studies are discussed in the context of potential utility in protein comparability studies.

  7. Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles.

    Science.gov (United States)

    Alsenaidy, Mohammad A; Jain, Nishant K; Kim, Jae H; Middaugh, C Russell; Volkin, David B

    2014-01-01

    In this review, some of the challenges and opportunities encountered during protein comparability assessments are summarized with an emphasis on developing new analytical approaches to better monitor higher-order protein structures. Several case studies are presented using high throughput biophysical methods to collect protein physical stability data as function of temperature, agitation, ionic strength and/or solution pH. These large data sets were then used to construct empirical phase diagrams (EPDs), radar charts, and comparative signature diagrams (CSDs) for data visualization and structural comparisons between the different proteins. Protein samples with different sizes, post-translational modifications, and inherent stability are presented: acidic fibroblast growth factor (FGF-1) mutants, different glycoforms of an IgG1 mAb prepared by deglycosylation, as well as comparisons of different formulations of an IgG1 mAb and granulocyte colony stimulating factor (GCSF). Using this approach, differences in structural integrity and conformational stability profiles were detected under stress conditions that could not be resolved by using the same techniques under ambient conditions (i.e., no stress). Thus, an evaluation of conformational stability differences may serve as an effective surrogate to monitor differences in higher-order structure between protein samples. These case studies are discussed in the context of potential utility in protein comparability studies.

  8. Influence of pH value on microstructure of oil-in-water emulsions stabilized by chickpea protein flour.

    Science.gov (United States)

    Felix, Manuel; Isurralde, Nadia; Romero, Alberto; Guerrero, Antonio

    2018-01-01

    Food industry is highly interested in the development of healthier formulations of oil-in-water emulsions, stabilized by plant proteins instead of egg or milk proteins. These emulsions would avoid allergic issues or animal fat. Among other plant proteins, legumes are a cost-competitive product. This work evaluates the influence of pH value (2.5, 5.0 and 7.5) on emulsions stabilized by chickpea-based emulsions at two different protein concentration (2.0 and 4.0 wt%). Microstructure of chickpea-based emulsions is assessed by means of backscattering, droplet size distributions and small amplitude oscillatory shear measurements. Visual appearances as well as confocal laser scanning microscopy images are obtained to provide useful information on the emulsions structure. Interestingly, results indicate that the pH value and protein concentration have a strong influence on emulsion microstructure and stability. Thus, the system which contains protein surfaces positively charged shows the highest viscoelastic properties, a good droplet size distribution profile and non-apparent destabilization phenomena. Interestingly, results also reveal the importance of rheological measurements in the prediction of protein interactions and emulsion stability since this technique is able to predict destabilization mechanisms sooner than other techniques such as backscattering or droplet size distribution measurements.

  9. Application of a high-throughput relative chemical stability assay to screen therapeutic protein formulations by assessment of conformational stability and correlation to aggregation propensity.

    Science.gov (United States)

    Rizzo, Joseph M; Shi, Shuai; Li, Yunsong; Semple, Andrew; Esposito, Jessica J; Yu, Shenjiang; Richardson, Daisy; Antochshuk, Valentyn; Shameem, Mohammed

    2015-05-01

    In this study, an automated high-throughput relative chemical stability (RCS) assay was developed in which various therapeutic proteins were assessed to determine stability based on the resistance to denaturation post introduction to a chaotrope titration. Detection mechanisms of both intrinsic fluorescence and near UV circular dichroism (near-UV CD) are demonstrated. Assay robustness was investigated by comparing multiple independent assays and achieving r(2) values >0.95 for curve overlays. The complete reversibility of the assay was demonstrated by intrinsic fluorescence, near-UV CD, and biologic potency. To highlight the method utility, we compared the RCS assay with differential scanning calorimetry and dynamic scanning fluorimetry methodologies. Utilizing C1/2 values obtained from the RCS assay, formulation rank-ordering of 12 different mAb formulations was performed. The prediction of long-term stability on protein aggregation is obtained by demonstrating a good correlation with an r(2) of 0.83 between RCS and empirical aggregation propensity data. RCS promises to be an extremely useful tool to aid in candidate formulation development efforts based on the complete reversibility of the method to allow for multiple assessments without protein loss and the strong correlation between the C1/2 data obtained and accelerated stability under stressed conditions. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  10. Influence of yogurt fermentation and refrigerated storage on the stability of protein toxin contaminants.

    Science.gov (United States)

    Jackson, Lauren S; Triplett, Odbert A; Tolleson, William H

    2015-06-01

    Dairy products sold in a ready-to-eat form present the risk that adulterants persisting through manufacturing, storage, and distribution would reach consumers. Pathogenic microbes, including shigatoxigenic strains of Escherichia coli and the toxins they produce, are common food safety hazards associated with dairy products. Ricin and abrin are plant-derived ribosome-inactivating protein toxins related to the shiga-like toxins produced by E. coli. Limited information exists on the effects of manufacturing processes on the stabilities of these heat-resistant ribosome-inactivating proteins in the presence of foods. The goal of this study was to determine how typical yogurt manufacturing and storage processes influence ribosome-inactivating protein toxins. Ricin and abrin were added to skim or whole milk and batch pasteurized. Complete inactivation of both toxins was observed after 30 minutes at 85 °C. If the toxins were added after pasteurization, the levels of ricin and abrin in yogurt and their cytotoxic activities did not change significantly during fermentation or refrigerated storage for 4 weeks. The activities of ricin and abrin were inhibited by skim milk, nonfat yogurt, whole milk, and whole milk yogurt. The results showed minimal effects of the toxins on yogurt pH and %titratable acidity but inhibitory effects of yogurt on toxin activity. Published by Elsevier Ltd.

  11. An Outlook on Biothermodynamics: Needs, Problems, and New Developments. I. Stability and Hydration of Proteins

    Science.gov (United States)

    Keller, Jürgen U.

    2008-12-01

    The application of concepts, principles, and methods of thermodynamics of equilibria and processes to bioengineering systems has led to a new and growing field: engineering biothermodynamics. This article, which is meant as the first in a series, gives an outline of basic aspects, changes, and actual examples in this field. After a few introductory remarks, the basic concepts and laws of thermodynamics extended to systems with internal variables, which serve as models for biofluids and other biosystems, are given. The method of thermodynamics is then applied to the problem of thermal stability of aqueous protein solutions, especially to that of myoglobin solutions. After this, the phenomenon of hydration of proteins by adsorption and intrusion of water molecules is considered. Several other phenomena like the adsorption of proteins on solid surfaces or cell membranes and their temperature and pressure-related behavior represented by an equation of state, or the thermodynamics of bacterial solutions including chemical reactions like wine fermentation, etc., will be presented in Parts II and III of this article.

  12. Storage stability of cauliflower soup powder: The effect of lipid oxidation and protein degradation reactions.

    Science.gov (United States)

    Raitio, Riikka; Orlien, Vibeke; Skibsted, Leif H

    2011-09-15

    Soups based on cauliflower soup powders, prepared by dry mixing of ingredients and rapeseed oil, showed a decrease in quality, as evaluated by a sensory panel, during the storage of the soup powder in the dark for up to 12weeks under mildly accelerated conditions of 40°C and 75% relative humidity. Antioxidant, shown to be effective in protecting the rapeseed bulk oil, used for the powder preparation, had no effect on storage stability of the soup powder. The freshly prepared soup powder had a relatively high concentration of free radicals, as measured by electron spin resonance spectroscopy, which decreased during storage, and most remarkably during the first two weeks of storage, with only marginal increase in lipid hydroperoxides as primary lipid oxidation products, and without any increase in secondary lipid oxidation products. Analyses of volatiles by SPME-GC-MS revealed a significant increase in concentrations of 2-methyl- and 3-methyl butanals, related to Maillard reactions, together with an increase in 2-acetylpyrrole concentration. The soup powders became more brown during storage, as indicated by a decreasing Hunter L-value, in accord with non-enzymatic browning reactions. A significant increase in the concentrations of dimethyl disulfide in soup powder headspace indicated free radical-initiated protein oxidation. Protein degradation, including Maillard reactions and protein oxidation, is concluded to be more important than lipid oxidation in determining the shelf-life of dry cauliflower soup powder. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Screening of mutations affecting protein stability and dynamics of FGFR1—A simulation analysis

    Directory of Open Access Journals (Sweden)

    C. George Priya Doss

    2012-12-01

    Full Text Available Single amino acid substitutions in Fibroblast Growth Factor Receptor 1 (FGFR1 destabilize protein and have been implicated in several genetic disorders like various forms of cancer, Kallamann syndrome, Pfeiffer syndrome, Jackson Weiss syndrome, etc. In order to gain functional insight into mutation caused by amino acid substitution to protein function and expression, special emphasis was laid on molecular dynamics simulation techniques in combination with in silico tools such as SIFT, PolyPhen 2.0, I-Mutant 3.0 and SNAP. It has been estimated that 68% nsSNPs were predicted to be deleterious by I-Mutant, slightly higher than SIFT (37%, PolyPhen 2.0 (61% and SNAP (58%. From the observed results, P722S mutation was found to be most deleterious by comparing results of all in silico tools. By molecular dynamics approach, we have shown that P722S mutation leads to increase in flexibility, and deviated more from the native structure which was supported by the decrease in the number of hydrogen bonds. In addition, biophysical analysis revealed a clear insight of stability loss due to P722S mutation in FGFR1 protein. Majority of mutations predicted by these in silico tools were in good concordance with the experimental results.

  14. Development of a Synthetic Switch to Control Protein Stability in Eukaryotic Cells with Light.

    Science.gov (United States)

    Taxis, Christof

    2017-01-01

    In eukaryotic cells, virtually all regulatory processes are influenced by proteolysis. Thus, synthetic control of protein stability is a powerful approach to influence cellular behavior. To achieve this, selected target proteins are modified with a conditional degradation sequence (degron) that responds to a distinct signal. For development of a synthetic degron, an appropriate sensor domain is fused with a degron such that activity of the degron is under control of the sensor. This chapter describes the development of a light-activated, synthetic degron in the model organism Saccharomyces cerevisiae. This photosensitive degron module is composed of the light-oxygen-voltage (LOV) 2 photoreceptor domain of Arabidopsis thaliana phototropin 1 and a degron derived from murine ornithine decarboxylase (ODC). Excitation of the photoreceptor with blue light induces a conformational change that leads to exposure and activation of the degron. Subsequently, the protein is targeted for degradation by the proteasome. Here, the strategy for degron module development and optimization is described in detail together with experimental aspects, which were pivotal for successful implementation of light-controlled proteolysis. The engineering of the photosensitive degron (psd) module may well serve as a blueprint for future development of sophisticated synthetic switches.

  15. Possible linkage of SP6 transcriptional activity with amelogenesis by protein stabilization.

    Science.gov (United States)

    Utami, Trianna W; Miyoshi, Keiko; Hagita, Hiroko; Yanuaryska, Ryna Dwi; Horiguchi, Taigo; Noma, Takafumi

    2011-01-01

    Ameloblasts produce enamel matrix proteins such as amelogenin, ameloblastin, and amelotin during tooth development. The molecular mechanisms of ameloblast differentiation (amelogenesis) are currently not well understood. SP6 is a transcription factor of the Sp/KLF family that was recently found to regulate cell proliferation in a cell-type-specific manner. Sp6-deficient mice demonstrate characteristic tooth anomalies such as delayed eruption of the incisors and supernumerary teeth with disorganized amelogenesis. However, it remains unclear how Sp6 controls amelogenesis. In this study, we used SP6 high producer cells to identify SP6 target genes. Based on the observations that long-term culture of SP6 high producer cells reduced SP6 protein expression but not Sp6 mRNA expression, we found that SP6 is short lived and specifically degraded through a proteasome pathway. We established an in vitro inducible SP6 expression system coupled with siRNA knockdown and found a possible linkage between SP6 and amelogenesis through the regulation of amelotin and Rock1 gene expression by microarray analysis. Our findings suggest that the regulation of SP6 protein stability is one of the crucial steps in amelogenesis.

  16. Possible Linkage of SP6 Transcriptional Activity with Amelogenesis by Protein Stabilization

    Directory of Open Access Journals (Sweden)

    Trianna W. Utami

    2011-01-01

    Full Text Available Ameloblasts produce enamel matrix proteins such as amelogenin, ameloblastin, and amelotin during tooth development. The molecular mechanisms of ameloblast differentiation (amelogenesis are currently not well understood. SP6 is a transcription factor of the Sp/KLF family that was recently found to regulate cell proliferation in a cell-type-specific manner. Sp6-deficient mice demonstrate characteristic tooth anomalies such as delayed eruption of the incisors and supernumerary teeth with disorganized amelogenesis. However, it remains unclear how Sp6 controls amelogenesis. In this study, we used SP6 high producer cells to identify SP6 target genes. Based on the observations that long-term culture of SP6 high producer cells reduced SP6 protein expression but not Sp6 mRNA expression, we found that SP6 is short lived and specifically degraded through a proteasome pathway. We established an in vitro inducible SP6 expression system coupled with siRNA knockdown and found a possible linkage between SP6 and amelogenesis through the regulation of amelotin and Rock1 gene expression by microarray analysis. Our findings suggest that the regulation of SP6 protein stability is one of the crucial steps in amelogenesis.

  17. Antioxidant activity and emulsion-stabilizing effect of pectic enzyme treated pectin in soy protein isolate-stabilized oil/water emulsion.

    Science.gov (United States)

    Huang, Ping-Hsiu; Lu, Hao-Te; Wang, Yuh-Tai; Wu, Ming-Chang

    2011-09-14

    The antioxidant activity of pectic enzyme treated pectin (PET-pectin) prepared from citrus pectin by enzymatic hydrolysis and its potential use as a stabilizer and an antioxidant for soy protein isolate (SPI)-stabilized oil in water (O/W) emulsion were investigated. Trolox equivalent antioxidant capacity (TEAC) was found to be positively associated with molecular weight (M(w)) of PET-pectin and negatively associated with degree of esterification (DE) of PET-pectin. PET-pectin (1 kDa and 11.6% DE) prepared from citrus pectin after 24 h of hydrolysis by commercial pectic enzyme produced by Aspergillus niger expressed higher α,α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity, TEAC, and reducing power than untreated citrus pectin (353 kDa and 60% DE). The addition of PET-pectin could increase both emulsifying activity (EA) and emulsion stability (ES) of SPI-stabilized O/W emulsion. When the SPI-stabilized lipid droplet was coated with the mixture of PET-pectin and pectin, the EA and ES of the emulsion were improved more than they were when the lipid droplet was coated with either pectin or PET-pectin alone. The amount of secondary oxidation products (thiobarbituric acid reactive substances) produced in the emulsion prepared with the mixture of SPI and PET-pectin was less than the amount produced in the emulsion prepared with either SPI or SPI/pectin. These results suggest that PET-pectin has an emulsion-stabilizing effect and lipid oxidation inhibition ability on SPI-stabilized emulsion. Therefore, PET-pectin can be used as a stabilizer as well as an antioxidant in plant origin in SPI-stabilized O/W emulsion and thus prolong the shelf life of food emulsion.

  18. Utilizing whey protein isolate and polysaccharide complexes to stabilize aerated dairy gels.

    Science.gov (United States)

    O'Chiu, Emily; Vardhanabhuti, Bongkosh

    2017-05-01

    Heated soluble complexes of whey protein isolate (WPI) with polysaccharides may be used to modify the properties of aerated dairy gels, which could be formulated into novel-textured high-protein desserts. The objective of this study was to determine the effect of polysaccharide charge density and concentration within a WPI-polysaccharide complex on the physical properties of aerated gels. Three polysaccharides having different degrees of charge density were chosen: low-methoxyl pectin, high-methoxyl type D pectin, and guar gum. Heated complexes were prepared by heating the mixed dispersions (8% protein, 0 to 1% polysaccharide) at pH 7. To form aerated gels, 2% glucono-δ-lactone was added to the dispersions of skim milk powder and heated complex and foam was generated by whipping with a handheld frother. The foam set into a gel as the glucono-δ-lactone acidified to a final pH of 4.5. The aerated gels were evaluated for overrun, drainage, gel strength, and viscoelastic properties. Without heated complexes, stable aerated gels could not be formed. Overrun of aerated gel decreased (up to 73%) as polysaccharide concentration increased from 0.105 to 0.315% due to increased viscosity, which limited air incorporation. A negative relationship was found between percent drainage and dispersion viscosity. However, plotting of drainage against dispersion viscosity separated by polysaccharide type revealed that drainage decreased most in samples with high-charge-density, low-methoxyl pectin followed by those with low-charge-density, high-methoxyl type D pectin. Aerated gels with guar gum (no charge) did not show improvement to stability. Rheological results showed no significant difference in gelation time among samples; therefore, stronger interactions between WPI and high-charge-density polysaccharide were likely responsible for increased stability. Stable dairy aerated gels can be created from WPI-polysaccharide complexes. High-charge-density polysaccharides, at

  19. 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.

  20. Accurate prediction of stability changes in protein mutants by combining machine learning with structure based computational mutagenesis.

    Science.gov (United States)

    Masso, Majid; Vaisman, Iosif I

    2008-09-15

    Accurate predictive models for the impact of single amino acid substitutions on protein stability provide insight into protein structure and function. Such models are also valuable for the design and engineering of new proteins. Previously described methods have utilized properties of protein sequence or structure to predict the free energy change of mutants due to thermal (DeltaDeltaG) and denaturant (DeltaDeltaG(H2O)) denaturations, as well as mutant thermal stability (DeltaT(m)), through the application of either computational energy-based approaches or machine learning techniques. However, accuracy associated with applying these methods separately is frequently far from optimal. We detail a computational mutagenesis technique based on a four-body, knowledge-based, statistical contact potential. For any mutation due to a single amino acid replacement in a protein, the method provides an empirical normalized measure of the ensuing environmental perturbation occurring at every residue position. A feature vector is generated for the mutant by considering perturbations at the mutated position and it's ordered six nearest neighbors in the 3-dimensional (3D) protein structure. These predictors of stability change are evaluated by applying machine learning tools to large training sets of mutants derived from diverse proteins that have been experimentally studied and described. Predictive models based on our combined approach are either comparable to, or in many cases significantly outperform, previously published results. A web server with supporting documentation is available at http://proteins.gmu.edu/automute.

  1. Spray dried microparticles of chia oil using emulsion stabilized by whey protein concentrate and pectin by electrostatic deposition.

    Science.gov (United States)

    Noello, C; Carvalho, A G S; Silva, V M; Hubinger, M D

    2016-11-01

    Chia seed oil has a high content of α-linolenic acid (60%) and linoleic acid (20%). Use of this oil in different products is limited due to its liquid state, and the presence of insaturation is a trigger for oxidation. In this context, to facilitate the incorporation of chia oil in food products and increase its protection against oxidation, the aim of this work was to produce chia oil microparticles by spray drying using emulsions stabilized by whey protein concentrate (ζ-potential +13.4 at pH3.8) and pectin (ζ-potential -40.4 at pH3.8) through the electrostatic layer-by-layer deposition technique and emulsions prepared with only whey protein concentrate. Emulsions stabilized by whey protein concentrate and stabilized by whey protein concentrate-pectin were prepared using maltodextrin (10 DE) and modified starch (Hi-Cap® 100). They were characterized in relation to stability, droplet size, ζ-Potential and optical microscopy. The microparticles were characterized in relation to moisture content, water activity, particle size, microstructure and oxidative stability by the Rancimat method. Emulsions stabilized by whey protein concentrate-pectin with added maltodextrin 10 DE and emulsions stabilized by whey protein concentrate with added modified starch (Hi-Cap® 100) were stable after 24h. Emulsions stabilized by whey protein concentrate and by whey protein concentrate-pectin showed droplets with mean diameter ranging from 0.80 to 1.31μm, respectively and ζ-potential varying from -6.9 to -27.43mV, respectively. After spray drying, the microparticles showed an mean diameter ranging from 7.00 to 9.00μm. All samples presented high encapsulation efficiency values, above 99%. Microparticles produced with modified starch showed a smoother spherical surface than particles with maltodextrin 10 DE, which presented a wrinkled surface. All microparticles exhibited higher oxidative stability than chia oil in pure form. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Influence of Tableting on the Conformation and Thermal Stability of Trypsin as a Model Protein

    DEFF Research Database (Denmark)

    Klukkert, Marten; Van De Weert, Marco; Fanø, Mathias

    2015-01-01

    was performed to determine the Tm as well as the folding reversibility after thermal denaturation of the reconstituted samples. It was found that compacted samples showed reduced activity accompanied by an altered secondary structure. Conformational changes that occur in the solid state were partially...... reversible upon tablet reconstitution. Aqueous-state IR spectroscopy combined with partial least squares was shown to be a powerful tool to follow irreversible structural changes and evaluate sample bioactivity. Besides its conformation, the thermal stability of trypsin was altered as a result of the applied...... compaction pressure, indicated by a reduced folding reversibility. In conclusion, this study reveals that tableting can have a negative impact on the biological quality of protein APIs....

  3. Entropic formulation for the protein folding process: Hydrophobic stability correlates with folding rates

    Science.gov (United States)

    Dal Molin, J. P.; Caliri, A.

    2018-01-01

    possible connection between the hydrophobic component of protein stability and the native structural topology. We simulated those same 200 targets again with the Mq A, only. However, this time we evaluated the relative frequency {ϕq } in which each target visits its corresponding native structure along an appropriate simulation time. Due to the presence of the hydrophobic effect in our approach we obtained a strong correlation between the stability and the folding rate (R = 0 . 85). So, as faster a sequence found its target, as larger is the hydrophobic component of its stability. The strong correlation fulfills our last goal. This final finding suggests that the hydrophobic effect could not be a general stabilizing factor for proteins.

  4. Interfacial composition and stability of emulsions made with mixtures of commercial sodium caseinate and whey protein concentrate.

    Science.gov (United States)

    Ye, Aiqian

    2008-10-15

    The interfacial composition and the stability of oil-in-water emulsion droplets (30% soya oil, pH 7.0) made with mixtures of sodium caseinate and whey protein concentrate (WPC) (1:1 by protein weight) at various total protein concentrations were examined. The average volume-surface diameter (d32) and the total surface protein concentration of emulsion droplets were similar to those of emulsions made with both sodium caseinate alone and WPC alone. Whey proteins were adsorbed in preference to caseins at low protein concentrations (caseins were adsorbed in preference to whey proteins at high protein concentrations. The creaming stability of the emulsions decreased markedly as the total protein concentration of the system was increased above 2% (sodium caseinate >1%). This was attributed to depletion flocculation caused by the sodium caseinate in these emulsions. Whey proteins did not retard this instability in the emulsions made with mixtures of sodium caseinate and WPC. Copyright © 2008 Elsevier Ltd. All rights reserved.

  5. Silver nanoparticles in complex biological media: assessment of colloidal stability and protein corona formation

    Energy Technology Data Exchange (ETDEWEB)

    Argentiere, Simona, E-mail: simona.argentiere@fondazionefilarete.com; Cella, Claudia, E-mail: claudia.cella@unimi.it [Fondazione Filarete (Italy); Cesaria, Maura, E-mail: maura.cesaria@le.infn.it [Università del Salento, Dipartimento di Matematica e Fisica “Ennio De Giorgi” (Italy); Milani, Paolo, E-mail: paolo.milani@mi.infn.it; Lenardi, Cristina, E-mail: cristina.lenardi@mi.infn.it [Università degli Studi di Milano, CIMAINA and Dipartimento di Fisica (Italy)

    2016-08-15

    Engineered silver nanoparticles (AgNPs) are among the most used nanomaterials in consumer products, therefore concerns are raised about their potential for adverse effects in humans and environment. Although an increasing number of studies in vitro and in vivo are being reported on the toxicity of AgNPs, most of them suffer from incomplete characterization of AgNPs in the tested biological media. As a consequence, the comparison of toxicological data is troublesome and the toxicity evaluation still remains an open critical issue. The development of a reliable protocol to evaluate interactions of AgNPs with surrounding proteins as well as to assess their colloidal stability is therefore required. In this regard, it is of importance not only to use multiple, easy-to-access and simple techniques but also to understand limitations of each characterization methods. In this work, the morphological and structural behaviour of AgNPs has been studied in two relevant biological media, namely 10 % FBS and MP. Three different techniques (Dynamic Light Scattering, Transmission Electron Microscopy, UV–Vis spectroscopy) were tested for their suitability in detecting AgNPs of three different sizes (10, 40 and 100 nm) coated with either citrate or polyvinylpyrrolidone. Results showed that UV–Vis spectroscopy is the most versatile and informative technique to gain information about interaction between AgNPs and surrounding proteins and to determine their colloidal stability in the tested biological media. These findings are expected to provide useful insights in characterizing AgNPs before performing any further in vitro/in vivo experiment.

  6. Cables1 controls p21/Cip1 protein stability by antagonizing proteasome subunit alpha type 3.

    Science.gov (United States)

    Shi, Z; Li, Z; Li, Z J; Cheng, K; Du, Y; Fu, H; Khuri, F R

    2015-05-07

    The cyclin-dependent kinase (CDK) inhibitor 1A, p21/Cip1, is a vital cell cycle regulator, dysregulation of which has been associated with a large number of human malignancies. One critical mechanism that controls p21 function is through its degradation, which allows the activation of its associated cell cycle-promoting kinases, CDK2 and CDK4. Thus delineating how p21 is stabilized and degraded will enhance our understanding of cell growth control and offer a basis for potential therapeutic interventions. Here we report a novel regulatory mechanism that controls the dynamic status of p21 through its interaction with Cdk5 and Abl enzyme substrate 1 (Cables1). Cables1 has a proposed role as a tumor suppressor. We found that upregulation of Cables1 protein was correlated with increased half-life of p21 protein, which was attributed to Cables1/p21 complex formation and supported by their co-localization in the nucleus. Mechanistically, Cables1 interferes with the proteasome (Prosome, Macropain) subunit alpha type 3 (PSMA3) binding to p21 and protects p21 from PSMA3-mediated proteasomal degradation. Moreover, silencing of p21 partially reverses the ability of Cables1 to induce cell death and inhibit cell proliferation. In further support of a potential pathophysiological role of Cables1, the expression level of Cables1 is tightly associated with p21 in both cancer cell lines and human lung cancer patient tumor samples. Together, these results suggest Cables1 as a novel p21 regulator through maintaining p21 stability and support the model that the tumor-suppressive function of Cables1 occurs at least in part through enhancing the tumor-suppressive activity of p21.

  7. Albumen foam stability and s-ovalbumin contents in eggs coated with whey protein concentrate

    Directory of Open Access Journals (Sweden)

    ACC Alleoni

    2004-06-01

    Full Text Available Food products such as breads, cakes, crackers, meringues, ice creams and several bakery items depend on air incorporation to maintain their texture and structure during or after processing. Proteins are utilized in the food industry since they improve texture attributes through their ability to encapsulate and retain air. The objectives of this work were to quantify s-ovalbumin contents in albumen and to determine alterations in egg white foam stability in fresh eggs, and in eggs coated and non-coated with a whey protein-based concentrate film (WPC, stored at 25°C for 28 days. The volume of drained liquid was higher in non-coated eggs than in coated eggs stored at 25°C at all storage periods. The difference on the third day of storage was in the order of 59% between coated and non-coated eggs, while on the twenty-eighth day it was 202%. During the storage period, an increase in pH and drainage volume was observed for non-coated eggs. After three days, the non-coated eggs showed a s-ovalbumin content 33% higher than coated eggs; this increase jumped to 205% at 28 days of storage. There was a positive correlation between s-ovalbumin content and the volume of drained liquid for coated and non-coated eggs; in other words, when the s-ovalbumin content increased, there was an increase in the volume of drained liquid and a decrease in foam stability. WPC coating maintain egg quality, since it is an effective barrier against the loss of CO2, avoiding changes in the pH of egg white.

  8. Physicochemical and functional properties, microstructure, and storage stability of whey protein/polyvinylpyrrolidone based glue sticks

    Directory of Open Access Journals (Sweden)

    Guorong Wang

    2012-11-01

    Full Text Available A glue stick is comprised of solidified adhesive mounted in a lipstick-like push-up tube. Whey is a byproduct of cheese making. Direct disposal of whey can cause environmental pollution. The objective of this study was to use whey protein isolate (WPI as a natural polymer along with polyvinylpyrrolidone (PVP to develop safe glue sticks. Pre-dissolved WPI solution, PVP, sucrose, 1,2-propanediol (PG, sodium stearate, defoamer, and preservative were mixed and dissolved in water at 90 oC and then molded in push-up tubes. Chemical composition, functional properties (bonding strength, glue setting time, gel hardness, extension/retraction, and spreading properties, microstructure, and storage stability of the prototypes were evaluated in comparison with a commercial control. Results showed that all WPI/PVP prototypes had desirable bonding strength and exhibited faster setting than PVP prototypes and control. WPI could reduce gel hardness and form less compact and rougher structures than that of PVP, but there was no difference in bonding strength. PVP and sucrose could increase the hygroscopicity of glue sticks, thus increasing storage stability. Finally, the optimized prototype GS3 (major components: WPI 8.0%, PVP 12.0%, 1,2-propanediol 10.0%, sucrose 10.0%, and stearic sodium 7.0% had a comparable functionality to the commercial control. Results indicated that whey protein could be used as an adhesive polymer for glue stick formulations, which could be used to bond fiber or cellulose derived substrates such as paper.

  9. Thermal stability of chemically denatured green fluorescent protein (GFP) A preliminary study

    Energy Technology Data Exchange (ETDEWEB)

    Nagy, Attila; Malnasi-Csizmadia, Andras; Somogyi, Bela; Lorinczy, Denes

    2004-02-09

    Green fluorescent protein (GFP) is a light emitter in the bioluminescence reaction of the jellyfish Aequorea victoria. The protein consist of 238 amino acids and produces green fluorescent light ({lambda}{sub max}=508 nm), when irradiated with near ultraviolet light. The fluorescence is due to the presence of chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser{sup 65}-Tyr{sup 66}-Gly{sup 67}-, which buried into {beta}-barrel. GFP is extremely compact and heat stable molecule. In this work, we present data for the effect of chemical denaturing agent on the thermal stability of GFP. When denaturing agent is applied, global thermal stability and the melting point of the molecule is decreases, that can be monitored with differential scanning calorimetry. The results indicate, that in 1-6 M range of GuHCl the melting temperature is decreasing continuously from 83 to 38 deg. C. Interesting finding, that the calculated calorimetric enthalpy decreases with GuHCl concentration up to 3 M (5.6-0.2 kJ mol{sup -1}), but at 4 M it jumps to 8.4 and at greater concentration it is falling down to 1.1 kJ mol{sup -1}. First phenomena, i.e. the decrease of melting point with increasing GuHCl concentration can be easily explained by the effect of the extended chemical denaturation, when less and less amount of heat required to diminish the remaining hydrogen bonds in {beta}-barrel. The surprising increase of calorimetric enthalpy at 4 M concentration of GuHCl could be the consequence of a dimerization or a formation of stable complex between GFP and denaturing agent as well as a precipitation at an extreme GuHCl concentration. We are planning further experiments to elucidate fluorescent consequence of these processes.

  10. Protein-silver nanoparticle interactions to colloidal stability in acidic environments.

    Science.gov (United States)

    Tai, Jui-Ting; Lai, Chao-Shun; Ho, Hsin-Chia; Yeh, Yu-Shan; Wang, Hsiao-Fang; Ho, Rong-Ming; Tsai, De-Hao

    2014-11-04

    We report a kinetic study of Ag nanoparticles (AgNPs) under acidic environments (i.e., pH 2.3 to pH ≈7) and systematically investigate the impact of protein interactions [i.e., bovine serum albumin (BSA) as representative] to the colloidal stability of AgNPs. Electrospray-differential mobility analysis (ES-DMA) was used to characterize the particle size distributions and the number concentrations of AgNPs. Transmission electron microscopy was employed orthogonally to provide visualization of AgNPs. For unconjugated AgNPs, the extent of aggregation, or the average particle size, was shown to be increased significantly with an increase of acidity, where a partial coalescence was found between the primary particles of unconjugated AgNP clusters. Aggregation rate constant, kD, was also shown to be proportional to acidity, following a correlation of log(kD) = -1.627(pH)-9.3715. Using ES-DMA, we observe BSA had a strong binding affinity (equilibrium binding constant, ≈ 1.1 × 10(6) L/mol) to the surface of AgNPs, with an estimated maximum molecular surface density of ≈0.012 nm(-2). BSA-functionalized AgNPs exhibited highly-improved colloidal stability compared to the unconjugated AgNPs under acidic environments, where both the acid-induced interfacial dissolution and the particle aggregation became negligible. Results confirm a complex mechanism of colloidal stability of AgNPs: the aggregation process was shown to be dominant, and the formation of BSA corona on AgNPs suppressed both particle aggregation and interfacial dissolution of AgNP samples under acidic environments.

  11. Polypeptide N-Acetylgalactosaminyltransferase 13 Contributes to Neurogenesis via Stabilizing the Mucin-type O-Glycoprotein Podoplanin.

    Science.gov (United States)

    Xu, Yingjiao; Pang, Wenjie; Lu, Jishun; Shan, Aidong; Zhang, Yan

    2016-11-04

    Mucin-type O-glycosylation is initiated by an evolutionarily conserved family of polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). Previously, it was reported that ppGalNAc-T13 is restrictively expressed at a high level in the brain. Here we provide evidence for the critical role of ppGalNAc-T13 in neural differentiation. In detail, we show that the expression of ppGalNAc-T13 was dramatically up-regulated during early neurogenesis in mouse embryonic brains. Similar changes were also observed in cell models of neuronal differentiation by using either primary mouse cortical neural precursor cells or murine embryonal carcinoma P19 cells. Knockout of ppGalNAc-T13 in P19 cells suppressed not only neural induction but also neuronal differentiation. These effects are at least partly mediated by the mucin-type O-glycoprotein podoplanin (PDPN), as knockdown of PDPN led to a similar inhibition of neuronal differentiation and PDPN was significantly reduced at the posttranscriptional level after ppGalNAc-T13 knockout. Further data demonstrate that PDPN acts as a substrate of ppGalNAc-T13 and that the ppGalNAc-T13-mediated O-glycosylation on PDPN is important for its stability. Taken together, this study suggests that ppGalNAc-T13 contributes to neuronal differentiation through glycosylating and stabilizing PDPN, which provides insights into the regulatory roles of O-glycosylation in mammalian neural development. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. ATM Mediates pRB Function To Control DNMT1 Protein Stability and DNA Methylation

    Science.gov (United States)

    Suzuki, Misa; Hayashi, Naoyuki; Kobayashi, Masahiko; Sasaki, Nobunari; Nishiuchi, Takumi; Doki, Yuichiro; Okamoto, Takahiro; Kohno, Susumu; Muranaka, Hayato; Kitajima, Shunsuke; Yamamoto, Ken-ichi

    2013-01-01

    The retinoblastoma tumor suppressor gene (RB) product has been implicated in epigenetic control of gene expression owing to its ability to physically bind to many chromatin modifiers. However, the biological and clinical significance of this activity was not well elucidated. To address this, we performed genetic and epigenetic analyses in an Rb-deficient mouse thyroid C cell tumor model. Here we report that the genetic interaction of Rb and ATM regulates DNMT1 protein stability and hence controls the DNA methylation status in the promoters of at least the Ink4a, Shc2, FoxO6, and Noggin genes. Furthermore, we demonstrate that inactivation of pRB promotes Tip60 (acetyltransferase)-dependent ATM activation; allows activated ATM to physically bind to DNMT1, forming a complex with Tip60 and UHRF1 (E3 ligase); and consequently accelerates DNMT1 ubiquitination driven by Tip60-dependent acetylation. Our results indicate that inactivation of the pRB pathway in coordination with aberration in the DNA damage response deregulates DNMT1 stability, leading to an abnormal DNA methylation pattern and malignant progression. PMID:23754744

  13. TRF2 Protein Interacts with Core Histones to Stabilize Chromosome Ends.

    Science.gov (United States)

    Konishi, Akimitsu; Izumi, Takashi; Shimizu, Shigeomi

    2016-09-23

    Mammalian chromosome ends are protected by a specialized nucleoprotein complex called telomeres. Both shelterin, a telomere-specific multi-protein complex, and higher order telomeric chromatin structures combine to stabilize the chromosome ends. Here, we showed that TRF2, a component of shelterin, binds to core histones to protect chromosome ends from inappropriate DNA damage response and loss of telomeric DNA. The N-terminal Gly/Arg-rich domain (GAR domain) of TRF2 directly binds to the globular domain of core histones. The conserved arginine residues in the GAR domain of TRF2 are required for this interaction. A TRF2 mutant with these arginine residues substituted by alanine lost the ability to protect telomeres and induced rapid telomere shortening caused by the cleavage of a loop structure of the telomeric chromatin. These findings showed a previously unnoticed interaction between the shelterin complex and nucleosomal histones to stabilize the chromosome ends. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Ageing and smoking contribute to plasma surfactant proteins and protease imbalance with correlations to airway obstruction

    Directory of Open Access Journals (Sweden)

    Ishikawa Nobuhisa

    2011-04-01

    Full Text Available Abstract Background A significant number of young people start smoking at an age of 13-15, which means that serious smoking-evoked changes may have been occurred by their twenties. Surfactant proteins (SP and matrix metalloproteinases (MMPs and their tissue inhibitors (TIMPs have been linked to cigarette smoke induced lung remodelling and chronic obstructive pulmonary disease (COPD. However, the level of these proteins has not been examined during ageing or in young individuals with short smoking histories. Methods Plasma levels of SP-A, SP-D, MMP-9, and TIMP-1 were measured by EIA/ELISA from young (18-23 years non-smoking controls (YNS (n = 36, smokers (YS (n = 51, middle aged/elderly (37-77 years non-smoking controls (ONS (n = 40, smokers (OS (n = 64 (FEV1/FVC >0.7 in all subjects and patients with COPD (n = 44, 35-79 years. Results Plasma levels of SP-A increased with age and in the older group in relation to smoking and COPD. Plasma SP-D and MMP-9 levels did not change with age but were elevated in OS and COPD as compared to ONS. The TIMP-1 level declined with age but increased in chronic smokers when compared to ONS. The clearest correlations could be detected between plasma SP-A vs. age, pack years and FEV1/FVC. The receiver operating characteristic (ROC curve analysis revealed SP-A to be the best marker for discriminating between patients with COPD and the controls (area under ROC curve of 0.842; 95% confidence interval, 0.785-0.899; p Conclusions Age has a significant contribution to potential markers related to smoking and COPD; SP-A seems to be the best factor in differentiating COPD from the controls.

  15. Human Sterol Regulatory Element-Binding Protein 1a Contributes Significantly to Hepatic Lipogenic Gene Expression

    Directory of Open Access Journals (Sweden)

    Andreas Bitter

    2015-01-01

    Full Text Available Background/Aims: Sterol regulatory element-binding protein (SREBP 1, the master regulator of lipogenesis, was shown to be associated with non-alcoholic fatty liver disease, which is attributed to its major isoform SREBP1c. Based on studies in mice, the minor isoform SREBP1a is regarded as negligible for hepatic lipogenesis. This study aims to elucidate the expression and functional role of SREBP1a in human liver. Methods: mRNA expression of both isoforms was quantified in cohorts of human livers and primary human hepatocytes. Hepatocytes were treated with PF-429242 to inhibit the proteolytic activation of SREBP precursor protein. SREBP1a-specifc and pan-SREBP1 knock-down were performed by transfection of respective siRNAs. Lipogenic SREBP-target gene expression was analyzed by real-time RT-PCR. Results: In human liver, SREBP1a accounts for up to half of the total SREBP1 pool. Treatment with PF-429242 indicated SREBP-dependent auto-regulation of SREBP1a, which however was much weaker than of SREBP1c. SREBP1a-specifc knock-down also reduced significantly the expression of SREBP1c and of SREBP-target genes. Regarding most SREBP-target genes, simultaneous knock-down of both isoforms resulted in effects of only similar extent as SREBP1a-specific knock-down. Conclusion: We here showed that SREBP1a is significantly contributing to the human hepatic SREBP1 pool and has a share in human hepatic lipogenic gene expression.

  16. Selection for Protein Kinetic Stability Connects Denaturation Temperatures to Organismal Temperatures and Provides Clues to Archaean Life.

    Directory of Open Access Journals (Sweden)

    M Luisa Romero-Romero

    Full Text Available The relationship between the denaturation temperatures of proteins (Tm values and the living temperatures of their host organisms (environmental temperatures: TENV values is poorly understood. Since different proteins in the same organism may show widely different Tm's, no simple universal relationship between Tm and TENV should hold, other than Tm≥TENV. Yet, when analyzing a set of homologous proteins from different hosts, Tm's are oftentimes found to correlate with TENV's but this correlation is shifted upward on the Tm axis. Supporting this trend, we recently reported Tm's for resurrected Precambrian thioredoxins that mirror a proposed environmental cooling over long geological time, while remaining a shocking ~50°C above the proposed ancestral ocean temperatures. Here, we show that natural selection for protein kinetic stability (denaturation rate can produce a Tm↔TENV correlation with a large upward shift in Tm. A model for protein stability evolution suggests a link between the Tm shift and the in vivo lifetime of a protein and, more specifically, allows us to estimate ancestral environmental temperatures from experimental denaturation rates for resurrected Precambrian thioredoxins. The TENV values thus obtained match the proposed ancestral ocean cooling, support comparatively high Archaean temperatures, and are consistent with a recent proposal for the environmental temperature (above 75°C that hosted the last universal common ancestor. More generally, this work provides a framework for understanding how features of protein stability reflect the environmental temperatures of the host organisms.

  17. Loss of ABHD15 Impairs the Anti-lipolytic Action of Insulin by Altering PDE3B Stability and Contributes to Insulin Resistance.

    Science.gov (United States)

    Xia, Wenmin; Pessentheiner, Ariane R; Hofer, Dina C; Amor, Melina; Schreiber, Renate; Schoiswohl, Gabriele; Eichmann, Thomas O; Walenta, Evelyn; Itariu, Bianca; Prager, Gerhard; Hackl, Hubert; Stulnig, Thomas; Kratky, Dagmar; Rülicke, Thomas; Bogner-Strauss, Juliane G

    2018-05-15

    Elevated circulating fatty acids (FAs) contribute to obesity-associated metabolic complications, but the mechanisms by which insulin suppresses lipolysis are poorly understood. We show that α/β-hydrolase domain-containing 15 (ABHD15) is required for the anti-lipolytic action of insulin in white adipose tissue (WAT). Neither insulin nor glucose treatments can suppress FA mobilization in global and conditional Abhd15-knockout (KO) mice. Accordingly, insulin signaling is impaired in Abhd15-KO adipocytes, as indicated by reduced AKT phosphorylation, glucose uptake, and de novo lipogenesis. In vitro data reveal that ABHD15 associates with and stabilizes phosphodiesterase 3B (PDE3B). Accordingly, PDE3B expression is decreased in the WAT of Abhd15-KO mice, mechanistically explaining increased protein kinase A (PKA) activity, hormone-sensitive lipase (HSL) phosphorylation, and undiminished FA release upon insulin signaling. Ultimately, Abhd15-KO mice develop insulin resistance. Notably, ABHD15 expression is decreased in humans with obesity and diabetes compared to humans with obesity and normal glucose tolerance, identifying ABHD15 as a potential therapeutic target to mitigate insulin resistance. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Effect of chitosan on the heat stability of whey protein solution as a function of pH.

    Science.gov (United States)

    Zhao, Zhengtao; Xiao, Qian

    2017-03-01

    Chitosan was reported to interact with proteins through electrostatic interactions. Their interaction was influenced by pH, which was not fully characterized. Further research on the interactions between protein and chitosan at different pH and their influence on the thermal denaturation of proteins is necessary. In this research, the effect of chitosan on the heat stability of whey protein solution at pH 4.0-6.0 was studied. At pH 4.0, a small amount chitosan was able to prevent the heat-induced denaturation and aggregation of whey protein molecules. At higher pH values (5.5 and 6.0), whey proteins complexed with chitosan through electrostatic attraction. The formation of chitosan-whey protein complexes at pH 5.5 improved the heat stability of dispersions and no precipitation could be detected up to 20 days. The dispersion with a medium amount of chitosan (chitosan:whey protein 1:5) produced the most stable particles, which had an average radius of 135 ± 14 nm and a zeta potential value of 36 ± 1 mV. In contrast, at pH 6.0 only the dispersion with a high amount of chitosan (chitosan:whey protein 1:2) showed good shelf stability up to 20 days. It was possible to produce heat-stable whey protein beverages by regulating the interaction between chitosan and whey protein molecules. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  19. A robust method to screen detergents for membrane protein stabilization, revisited.

    Science.gov (United States)

    Champeil, Philippe; Orlowski, Stéphane; Babin, Simon; Lund, Sten; le Maire, Marc; Møller, Jesper; Lenoir, Guillaume; Montigny, Cédric

    2016-10-15

    This report is a follow up of our previous paper (Lund, Orlowski, de Foresta, Champeil, le Maire and Møller (1989), J Biol Chem 264:4907-4915) showing that solubilization in detergent of a membrane protein may interfere with its long-term stability, and proposing a protocol to reveal the kinetics of such irreversible inactivation. We here clarify the fact that when various detergents are tested for their effects, special attention has of course to be paid to their critical micelle concentration. We also investigate the effects of a few more detergents, some of which have been recently advertised in the literature, and emphasize the role of lipids together with detergents. Among these detergents, lauryl maltose neopentyl glycol (LMNG) exerts a remarkable ability, even higher than that of β-dodecylmaltoside (DDM), to protect our test enzyme, the paradigmatic P-type ATPase SERCA1a from sarcoplasmic reticulum. Performing such experiments for one's favourite protein probably remains useful in pre-screening assays testing various detergents. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants.

    Science.gov (United States)

    Zheng, Xuan; Dong, Shuangshuang; Zheng, Jie; Li, Duanhua; Li, Feng; Luo, Zhongli

    2014-01-01

    G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Physicochemical Properties of Whey-Protein-Stabilized Astaxanthin Nanodispersion and Its Transport via a Caco-2 Monolayer.

    Science.gov (United States)

    Shen, Xue; Zhao, Changhui; Lu, Jing; Guo, Mingruo

    2018-02-14

    Astaxanthin nanodispersion was prepared using whey protein isolate (WPI) and polymerized whey protein (PWP) through an emulsification-evaporation technique. The physicochemical properties of the astaxanthin nanodispersion were evaluated, and the transport of astaxanthin was assessed using a Caco-2 cell monolayer model. The astaxanthin nanodispersions stabilized by WPI and PWP (2.5%, w/w) had a small particle size (121 ± 4.9 and 80.4 ± 5.9 nm, respectively), negative ζ potential (-19.3 ± 1.5 and -35.0 ± 2.2 mV, respectively), and high encapsulation efficiency (92.1 ± 2.9 and 93.5 ± 2.4%, respectively). Differential scanning calorimetry curves indicated that amorphous astaxanthin existed in both astaxanthin nanodispersions. Whey-protein-stabilized astaxanthin nanodispersion showed resistance to pepsin digestion but readily released astaxanthin after trypsin digestion. The nanodispersions showed no cytotoxicity to Caco-2 cells at a protein concentration below 10 mg/mL. WPI- and PWP-stabilized nanodispersions improved the apparent permeability coefficient (P app ) of Caco-2 cells to astaxanthin by 10.3- and 16.1-fold, respectively. The results indicated that whey-protein-stabilized nanodispersion is a good vehicle to deliver lipophilic bioactive compounds, such as astaxanthin, and to improve their bioavailability.

  2. The impact of particle preparation methods and polymorphic stability of lipid excipients on protein distribution in microparticles

    DEFF Research Database (Denmark)

    Liu, Jingying; Christophersen, Philip C; Yang, Mingshi

    2017-01-01

    OBJECTIVE: The present study aimed at elucidating the influence of polymorphic stability of lipid excipients on the physicochemical characters of different solid lipid microparticles (SLM), with the focus on the alteration of protein distribution in SLM. METHODS: Labeled lysozyme was incorporated...... provides updated knowledge for rational development of lipid-based formulations for oral delivery of peptide or protein drugs.......OBJECTIVE: The present study aimed at elucidating the influence of polymorphic stability of lipid excipients on the physicochemical characters of different solid lipid microparticles (SLM), with the focus on the alteration of protein distribution in SLM. METHODS: Labeled lysozyme was incorporated...... into SLM prepared with different excipients, i.e. trimyristin (TG14), glyceryl distearate (GDS), and glyceryl monostearate (GMS), by water-oil-water (w/o/w) or solid-oil-water (s/o/w) method. The distribution of lysozyme in SLM and the release of the protein from SLM were evaluated by confocal laser...

  3. Missense mutation Lys18Asn in dystrophin that triggers X-linked dilated cardiomyopathy decreases protein stability, increases protein unfolding, and perturbs protein structure, but does not affect protein function.

    Directory of Open Access Journals (Sweden)

    Surinder M Singh

    Full Text Available Genetic mutations in a vital muscle protein dystrophin trigger X-linked dilated cardiomyopathy (XLDCM. However, disease mechanisms at the fundamental protein level are not understood. Such molecular knowledge is essential for developing therapies for XLDCM. Our main objective is to understand the effect of disease-causing mutations on the structure and function of dystrophin. This study is on a missense mutation K18N. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD. We created and expressed the wild-type (WT N-ABD and its K18N mutant, and purified to homogeneity. Reversible folding experiments demonstrated that both mutant and WT did not aggregate upon refolding. Mutation did not affect the protein's overall secondary structure, as indicated by no changes in circular dichroism of the protein. However, the mutant is thermodynamically less stable than the WT (denaturant melts, and unfolds faster than the WT (stopped-flow kinetics. Despite having global secondary structure similar to that of the WT, mutant showed significant local structural changes at many amino acids when compared with the WT (heteronuclear NMR experiments. These structural changes indicate that the effect of mutation is propagated over long distances in the protein structure. Contrary to these structural and stability changes, the mutant had no significant effect on the actin-binding function as evident from co-sedimentation and depolymerization assays. These results summarize that the K18N mutation decreases thermodynamic stability, accelerates unfolding, perturbs protein structure, but does not affect the function. Therefore, K18N is a stability defect rather than a functional defect. Decrease in stability and increase in unfolding decrease the net population of dystrophin molecules available for function, which might trigger XLDCM. Consistently, XLDCM patients have decreased levels of dystrophin in cardiac muscle.

  4. Increased bone morphogenetic protein signaling contributes to age-related declines in neurogenesis and cognition.

    Science.gov (United States)

    Meyers, Emily A; Gobeske, Kevin T; Bond, Allison M; Jarrett, Jennifer C; Peng, Chian-Yu; Kessler, John A

    2016-02-01

    Aging is associated with decreased neurogenesis in the hippocampus and diminished hippocampus-dependent cognitive functions. Expression of bone morphogenetic protein 4 (BMP4) increases with age by more than 10-fold in the mouse dentate gyrus while levels of the BMP inhibitor, noggin, decrease. This results in a profound 30-fold increase in phosphorylated-SMAD1/5/8, the effector of canonical BMP signaling. Just as observed in mice, a profound increase in expression of BMP4 is observed in the dentate gyrus of humans with no known cognitive abnormalities. Inhibition of BMP signaling either by overexpression of noggin or transgenic manipulation not only increases neurogenesis in aging mice, but remarkably, is associated with a rescue of cognitive deficits to levels comparable to young mice. Additive benefits are observed when combining inhibition of BMP signaling and environmental enrichment. These findings indicate that increased BMP signaling contributes significantly to impairments in neurogenesis and to cognitive decline associated with aging, and identify this pathway as a potential druggable target for reversing age-related changes in cognition. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Contribution of Kunitz protease inhibitor and transmembrane domains to amyloid precursor protein homodimerization.

    Science.gov (United States)

    Ben Khalifa, N; Tyteca, D; Courtoy, P J; Renauld, J C; Constantinescu, S N; Octave, J N; Kienlen-Campard, P

    2012-01-01

    The two major isoforms of the human amyloid precursor protein (APP) are APP695 and APP751. They differ by the insertion of a Kunitz-type protease inhibitor (KPI) sequence in the extracellular domain of APP751. APP-KPI isoforms are increased in Alzheimer's disease brains, and they could be associated with disease progression. Recent studies have shown that APP processing to Aβ is regulated by homodimerization, which involves both extracellular and juxtamembrane/transmembrane (JM/TM) regions. Our aim is to understand the mechanisms controlling APP dimerization and the contribution of the ectodomain and JM/TM regions to this process. We used bimolecular fluorescence complementation approaches coupled to fluorescence-activated cell sorting analysis to measure the dimerization level of different APP isoforms and APP C-terminal fragments (C99) mutated in their JM/TM region. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain of APP or C99 did not significantly affect fluorescence complementation. These findings indicate that the KPI domain plays a major role in APP dimerization. They set the basis for further investigation of the relation between dimerization, metabolism and function of APP. Copyright © 2012 S. Karger AG, Basel.

  6. TMV-Cg Coat Protein stabilizes DELLA proteins and in turn negatively modulates salicylic acid-mediated defense pathway during Arabidopsis thaliana viral infection.

    Science.gov (United States)

    Rodriguez, Maria Cecilia; Conti, Gabriela; Zavallo, Diego; Manacorda, Carlos Augusto; Asurmendi, Sebastian

    2014-08-03

    Plant viral infections disturb defense regulatory networks during tissue invasion. Emerging evidence demonstrates that a significant proportion of these alterations are mediated by hormone imbalances. Although the DELLA proteins have been reported to be central players in hormone cross-talk, their role in the modulation of hormone signaling during virus infections remains unknown. This work revealed that TMV-Cg coat protein (CgCP) suppresses the salicylic acid (SA) signaling pathway without altering defense hormone SA or jasmonic acid (JA) levels in Arabidopsis thaliana. Furthermore, it was observed that the expression of CgCP reduces plant growth and delays the timing of floral transition. Quantitative RT-qPCR analysis of DELLA target genes showed that CgCP alters relative expression of several target genes, indicating that the DELLA proteins mediate transcriptional changes produced by CgCP expression. Analyses by fluorescence confocal microscopy showed that CgCP stabilizes DELLA proteins accumulation in the presence of gibberellic acid (GA) and that the DELLA proteins are also stabilized during TMV-Cg virus infections. Moreover, DELLA proteins negatively modulated defense transcript profiles during TMV-Cg infection. As a result, TMV-Cg accumulation was significantly reduced in the quadruple-DELLA mutant Arabidopsis plants compared to wild type plants. Taken together, these results demonstrate that CgCP negatively regulates the salicylic acid-mediated defense pathway by stabilizing the DELLA proteins during Arabidopsis thaliana viral infection, suggesting that CgCP alters the stability of DELLAs as a mechanism of negative modulation of antiviral defense responses.

  7. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

    International Nuclear Information System (INIS)

    Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

    1988-01-01

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn 2+ , while Fe 2+ and Mn 2+ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca 2+ , phorbol ester, or antigen

  8. Urea recycling contributes to nitrogen retention in calves fed milk replacer and low-protein solid feed

    DEFF Research Database (Denmark)

    Berends, Harma; van den Borne, Joost J G C; Røjen, Betina A.

    2014-01-01

    Urea recycling, with urea originating from catabolism of amino acids and hepatic detoxification of ammonia, is particularly relevant for ruminant animals, in which microbial protein contributes substantially to the metabolizable protein supply. However, the quantitative contribution of urea...... recycling to protein anabolism in calves during the transition from preruminants (milk-fed calves) to ruminants [solid feed (SF)-fed calves] is unknown. The aim of this study was to quantify urea recycling in milk-fed calves when provided with low-protein SF. Forty-eight calves [164 ± 1.6 kg body weight (BW......)] were assigned to 1 of 4 SF levels [0, 9, 18, and 27 g of dry matter (DM) SF · kg BW2-0.75 . d-1] provided in addition to an identical amount of milk replacer. Urea recycling was quantified after a 24-h intravenous infusion of [15N2]urea by analyzing urea isotopomers in 68-h fecal and urinary...

  9. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

    1988-05-15

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn/sup 2 +/, while Fe/sup 2 +/ and Mn/sup 2 +/ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca/sup 2 +/, phorbol ester, or antigen.

  10. Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants.

    Science.gov (United States)

    Jutras, Philippe V; D'Aoust, Marc-André; Couture, Manon M-J; Vézina, Louis-Philippe; Goulet, Marie-Claire; Michaud, Dominique; Sainsbury, Frank

    2015-09-01

    Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host-cell engineering approaches are being developed to ameliorate host-cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid-susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co-expression. The co-expression of a heterologous ion channel to stabilize acid-labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. The type III protein secretion system contributes to Xanthomonas citri subsp. citri biofilm formation

    KAUST Repository

    Zimaro, Tamara; Thomas, Ludivine; Marondedze, Claudius; Sgro, Germá n G; Garofalo, Cecilia G; Ficarra, Florencia A; Gehring, Christoph A; Ottado, Jorgelina; Gottig, Natalia

    2014-01-01

    Background: Several bacterial plant pathogens colonize their hosts through the secretion of effector proteins by a Type III protein secretion system (T3SS). The role of T3SS in bacterial pathogenesis is well established but whether this system

  12. Preparation and in vivo evaluation of a novel stabilized linker for 211At labeling of protein

    International Nuclear Information System (INIS)

    Talanov, Vladimir S.; Garmestani, Kayhan; Regino, Celeste A.S.; Milenic, Diane E.; Plascjak, Paul S.; Waldmann, Thomas A.; Brechbiel, Martin W.

    2006-01-01

    Significant improvement of in vivo stability of 211 At-labeled radioimmunoconjugates achieved upon employment of a recently reported new linker, succinimidyl N-2-(4-[ 211 At]astatophenethyl)succinamate (SAPS), prompted additional studies of its chemistry. The 211 At radiolabeling of succinimidyl N-2-(4-tributylstannylphenethyl)succinamate (1) was noted to decline after storage at -15 o C for greater than 6 months. Compound 1 was found to degrade via a ring closure reaction with the formation of N-2-(4-tributylstannylphenethyl)succinimide (3), and a modified procedure for the preparation of 1 was developed. The N-methyl structural analog of 1, succinimidyl N-2-(4-tributylstannylphenethyl)-N-methyl succinamate (SPEMS), was synthesized to investigate the possibility of improving the stability of reagent-protein linkage chemistry. Radiolabeling of SPEMS with 211 At generates succinimidyl N-2-(4-[ 211 At]astatophenethyl)-N-methyl succinamate (Methyl-SAPS), with yields being consistent for greater than 1 year. Radiolabelings of 1 and SPEMS with 125 I generated succinimidyl N-2-(4-[ 125 I]iodophenethyl)succinamate (SIPS) and succinimidyl N-2-(4-[ 125 I]iodophenethyl)-N-methyl succinamate (Methyl-SIPS), respectively, and showed no decline in yields. Methyl-SAPS, SAPS, Methyl-SIPS and SIPS were conjugated to Herceptin for a comparative assessment in LS-174T xenograft-bearing mice. The conjugates of Herceptin with Methyl-SAPS or Methyl-SIPS demonstrated immunoreactivity equivalent to if not superior to the SAPS and SIPS paired analogs. The in vivo studies also revealed that the N-methyl modification resulted in a superior statinated product

  13. Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins

    International Nuclear Information System (INIS)

    Roosild, Tarmo P.; Castronovo, Samantha; Choe, Senyon

    2006-01-01

    The X-ray crystallographic analysis of anti-FLAG M2 Fab is reported and the implications of the structure on FLAG epitope binding are described as a first step in the development of a tool for the structural and biophysical study of membrane proteins. The inherent difficulties of stabilizing detergent-solubilized integral membrane proteins for biophysical or structural analysis demand the development of new methodologies to improve success rates. One proven strategy is the use of antibody fragments to increase the ‘soluble’ portion of any membrane protein, but this approach is limited by the difficulties and expense associated with producing monoclonal antibodies to an appropriate exposed epitope on the target protein. Here, the stabilization of a detergent-solubilized K + channel protein, KvPae, by engineering a FLAG-binding epitope into a known loop region of the protein and creating a complex with Fab fragments from commercially available anti-FLAG M2 monoclonal antibodies is reported. Although well diffracting crystals of the complex have not yet been obtained, during the course of crystallization trials the structure of the anti-FLAG M2 Fab domain was solved to 1.86 Å resolution. This structure, which should aid future structure-determination efforts using this approach by facilitating molecular-replacement phasing, reveals that the binding pocket appears to be specific only for the first four amino acids of the traditional FLAG epitope, namely DYKD. Thus, the use of antibody fragments for improving the stability of target proteins can be rapidly applied to the study of membrane-protein structure by placing the short DKYD motif within a predicted peripheral loop of that protein and utilizing commercially available anti-FLAG M2 antibody fragments

  14. Effect of the Freezing Step in the Stability and Bioactivity of Protein-Loaded PLGA Nanoparticles Upon Lyophilization

    DEFF Research Database (Denmark)

    Fonte, Pedro; Andrade, Fernanda; Azevedo, Cláudia

    2016-01-01

    , sucrose and sorbitol as cryoprotectants was evaluated. METHODS: Cryoprotectants were co-encapsulated with insulin into PLGA nanoparticles and lyophilized using an optimized cycle with freezing at -80°C, in liquid nitrogen, or ramped cooling at -40°C. Upon lyophilization, the stability of protein structure...

  15. Stabilization of sulfide cations: mechanisms relevant to oxidation of peptides and proteins containing methionine

    International Nuclear Information System (INIS)

    Bobrowski, K.; Hug, G.L.; Pogocki, D.; Horner, G.; Marciniak, B.; Schoneich, C.

    2006-01-01

    Sulfide radical cations (R 2 S +. ) have recently attracted considerable attention. In particular they are implicated in assorted biological electron transfers where they are likely intermediates in biological redox-processes. There is unambigous theoretical and experimental evidence that R 2 S +. can be stabilized through intramolecular complexation with nucleophiles that are present in neighboring groups. Reactions of this type are of special interest to biology when stabilization of sulfide radical cations derived from methionine, Met(>S +. ) occurs in peptides and proteins. The methionine (Met) residues in these biopolymers are susceptible to attack by Reactive Oxygen Species (ROS) during oxidative stress and biological aging. Moreover, the pathogenesis of some neurodegenerative diseases (Alzheimer's, Jacob-Creutzfeld's, and Parkinson's) seems to be strongly linked to the presence in brain tissue of β-amyloid peptide (βAP), human prion protein (hPrP), and an aggregated form of α-synuclein, respectively. These macro- molecules contain methionine(s) with βAP having a Met 35 residue in its C-terminal α-helical domain, hPrP having three out of nine Met-residues (namely Met 205 , Met 206 , and Met 213 ) located within its α-helical segments, and α-synuclein having four Met-residues. The effective neighboring-group interactions would likely involve nucleophilic functionalities in the side chain of amino acids residues. However, very often heteroatoms in peptide bonds are the only nucleophiles present in the vicinity of Met(>S +. ). In this regard, it was recently shown that such interactions play an important role in N-acetylmethione amide and in oligopeptides of the form N-Ac-Gly-Met-Gly and N-Ac-Gly-(Gly) 2 -Met-(Gly) 3 . Intramolecularly bonded sulfide radical cations, Met(>S +. ), were directly observed in these systems with the bonding partner being either the carbonyl oxygen or the amide nitrogen of a peptide bond. Cyclic dipeptides are suitable model

  16. Conserved CPEs in the p53 3' untranslated region influence mRNA stability and protein synthesis

    DEFF Research Database (Denmark)

    Rosenstierne, Maiken W; Vinther, Jeppe; Mittler, Gerhard

    2008-01-01

    CaT skin and MCF-7 breast cancer cell lines were established. Quantitative PCR and an enzymatic assay were used to quantify the reporter mRNA and protein levels, respectively. Proteins binding to the CPEs were identified by RNA-immunoprecipitation (IP) and quantitative mass spectroscopy. RESULTS: The wild...... irradiation. Several proteins (including GAPDH, heterogeneous nuclear ribonucleoprotein (hnRNP) D and A/B) were identified from the MCF-7 cytoplasmic extracts that bound specifically to the CPEs. CONCLUSION: Two conserved CPEs in the p53 3'UTR regulate stability and translation of a reporter mRNA in non...

  17. Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax

    DEFF Research Database (Denmark)

    Friis, Dennis Steven; Johnsen, Johannes Lørup; Kristiansen, Erlend

    2014-01-01

    The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect......, the RmAFP1 has only one disulfide bridge. The melting temperature, Tm, of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show...

  18. DUB3 Deubiquitylating Enzymes Regulate Hippo Pathway Activity by Regulating the Stability of ITCH, LATS and AMOT Proteins

    DEFF Research Database (Denmark)

    Nguyen, Thanh Hung; Kugler, Jan-Michael; Cohen, Stephen Michael

    2017-01-01

    /TAZ, is regulated by ubiquitin mediated protein turnover and several ubiquitin ligase complexes have been implicated in human cancer. However, little is known about the deubiquitylating enzymes that counteract these ubiquitin ligases in regulation of the Hippo pathway. Here we identify the DUB3 family...... deubiquitylating enzymes as regulators of Hippo pathway activity. We provide evidence that DUB3 proteins regulate YAP/TAZ activity by controlling the stability of the E3 ligase ITCH, the LATS kinases and the AMOT family proteins. As a novel Hippo pathway regulator, DUB3 has the potential to act a tumor suppressor...

  19. Variation in Orthologous Shell-Forming Proteins Contribute to Molluscan Shell Diversity.

    Science.gov (United States)

    Jackson, Daniel J; Reim, Laurin; Randow, Clemens; Cerveau, Nicolas; Degnan, Bernard M; Fleck, Claudia

    2017-11-01

    Despite the evolutionary success and ancient heritage of the molluscan shell, little is known about the molecular details of its formation, evolutionary origins, or the interactions between the material properties of the shell and its organic constituents. In contrast to this dearth of information, a growing collection of molluscan shell-forming proteomes and transcriptomes suggest they are comprised of both deeply conserved, and lineage specific elements. Analyses of these sequence data sets have suggested that mechanisms such as exon shuffling, gene co-option, and gene family expansion facilitated the rapid evolution of shell-forming proteomes and supported the diversification of this phylum specific structure. In order to further investigate and test these ideas we have examined the molecular features and spatial expression patterns of two shell-forming genes (Lustrin and ML1A2) and coupled these observations with materials properties measurements of shells from a group of closely related gastropods (abalone). We find that the prominent "GS" domain of Lustrin, a domain believed to confer elastomeric properties to the shell, varies significantly in length between the species we investigated. Furthermore, the spatial expression patterns of Lustrin and ML1A2 also vary significantly between species, suggesting that both protein architecture, and the regulation of spatial gene expression patterns, are important drivers of molluscan shell evolution. Variation in these molecular features might relate to certain materials properties of the shells of these species. These insights reveal an important and underappreciated source of variation within shell-forming proteomes that must contribute to the diversity of molluscan shell phenotypes. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  20. Physicochemical stability and in vitro bioaccessibility of ß-carotene nanoemulsions stabilized with whey protein-dextran conjugates

    Science.gov (United States)

    In this study, ß-carotene (BC)-loaded nanoemulsions encapsulated with native whey protein isolate (WPI) and WPI-dextran (DT, 5 kDa, 20 kDa, and 70 kDa) conjugates were prepared and the effects of glycosylation with various molecular weight DTs on the physicochemical property, lipolysis, and BC bioac...

  1. ZMS regulation of M2 muscarinic receptor mRNA stability requires protein factor

    International Nuclear Information System (INIS)

    Zhang Yongfang; Xia Zongqin; Hu Ya'er

    2010-01-01

    Aim The aim of this work is to study the elevation mechanism of ZMS on muscarinic M2 receptor mRNA expression. Methods Actinomycin D was added to cultured CHOm2 cells to stop the de novo synthesis of M2 receptor mRNA and samples were taken at various times to determine the time course of mRNA of M2 receptor with real-time quantitative RT-PCR. Half-life of M2 receptor mRNA and the effect of ZMS on the half-life was obtained from the slope of the exponential curves. Cycloheximide was added at 4 h prior to and 24 h after the addition of ZMS to examine the effect of de novo protein synthesis on the action of ZMS. Results The half-life of m2 mRNA was prolonged by ZMS treatment without cycloheximide (4.75±0.54 h and 2.13 h±0.23 h for ZMS and vehicle treated groups, respectively, P<0.05). When cycloheximide was added to the culture medium 4h prior to the addition of ZMS, the effect of ZMS in prolonging the half-life of m2 mRNA disappeared (3.06 h±0.23 h and 3.00 h±l.20 h for cells with and without ZMS, respectively). However, when the ZMS was added to the medium 24h prior to the addition of cycloheximide, the action of ZMS was not abolished by cycloheximide (half-life was 5.43 h±1.13 h and 2.46 h±0.09 h for cells with and without ZMS, respectively). Conclusion These data suggest that de novo protein synthesis was required for the increase in M2 mRNA stability induced by ZMS. (authors)

  2. Cross-linking proteins by laccase: Effects on the droplet size and rheology of emulsions stabilized by sodium caseinate.

    Science.gov (United States)

    Sato, A C K; Perrechil, F A; Costa, A A S; Santana, R C; Cunha, R L

    2015-09-01

    The aim of this work was to evaluate the influence of laccase and ferulic acid on the characteristics of oil-in-water emulsions stabilized by sodium caseinate at different pH (3, 5 and 7). Emulsions were prepared by high pressure homogenization of soybean oil with sodium caseinate solution containing varied concentrations of laccase (0, 1 and 5mg/mL) and ferulic acid (5 and 10mM). Laccase treatment and pH exerted a strong influence on the properties with a consequent effect on stability, structure and rheology of emulsions stabilized by Na-caseinate. At pH7, O/W emulsions were kinetically stable due to the negative protein charge which enabled electrostatic repulsion between oil droplets resulting in an emulsion with small droplet size, low viscosity, pseudoplasticity and viscoelastic properties. The laccase treatment led to emulsions showing shear-thinning behavior as a result of a more structured system. O/W emulsions at pH5 and 3 showed phase separation due to the proximity to protein pI, but the laccase treatment improved their stability of emulsions especially at pH3. At pH3, the addition of ferulic acid and laccase produced emulsions with larger droplet size but with narrower droplet size distribution, increased viscosity, pseudoplasticity and viscoelastic properties (gel-like behavior). Comparing laccase treatments, the combined addition of laccase and ferulic acid generally produced emulsions with lower stability (pH5), larger droplet size (pH3, 5 and 7) and higher pseudoplasticity (pH5 and 7) than emulsion with only ferulic acid. The results suggested that the cross-linking of proteins by laccase and ferulic acid improved protein emulsifying properties by changing functional mechanisms of the protein on emulsion structure and rheology, showing that sodium caseinate can be successfully used in acid products when treated with laccase. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. The F box protein Fbx6 regulates Chk1 stability and cellular sensitivity to replication stress.

    Science.gov (United States)

    Zhang, You-Wei; Brognard, John; Coughlin, Chris; You, Zhongsheng; Dolled-Filhart, Marisa; Aslanian, Aaron; Manning, Gerard; Abraham, Robert T; Hunter, Tony

    2009-08-28

    ATR and Chk1 are two key protein kinases in the replication checkpoint. Activation of ATR-Chk1 has been extensively investigated, but checkpoint termination and replication fork restart are less well understood. Here, we report that DNA damage not only activates Chk1, but also exposes a degron-like region at the carboxyl terminus of Chk1 to an Fbx6-containing SCF (Skp1-Cul1-F box) E3 ligase, which mediates the ubiquitination and degradation of Chk1 and, in turn, terminates the checkpoint. The protein levels of Chk1 and Fbx6 showed an inverse correlation in both cultured cancer cells and in human breast tumor tissues. Further, we show that low levels of Fbx6 and consequent impairment of replication stress-induced Chk1 degradation are associated with cancer cell resistance to the chemotherapeutic agent, camptothecin. We propose that Fbx6-dependent Chk1 degradation contributes to S phase checkpoint termination and that a defect in this mechanism might increase tumor cell resistance to certain anticancer drugs.

  4. Formulating food protein-stabilized indomethacin nanosuspensions into pellets by fluid-bed coating technology: physical characterization, redispersibility, and dissolution.

    Science.gov (United States)

    He, Wei; Lu, Yi; Qi, Jianping; Chen, Lingyun; Yin, Lifang; Wu, Wei

    2013-01-01

    Drug nanosuspensions are very promising for enhancing the dissolution and bioavailability of drugs that are poorly soluble in water. However, the poor stability of nanosuspensions, reflected in particle growth, aggregation/agglomeration, and change in crystallinity state greatly limits their applications. Solidification of nanosuspensions is an ideal strategy for addressing this problem. Hence, the present work aimed to convert drug nanosuspensions into pellets using fluid-bed coating technology. Indomethacin nanosuspensions were prepared by the precipitation-ultrasonication method using food proteins (soybean protein isolate, whey protein isolate, β-lactoglobulin) as stabilizers. Dried nanosuspensions were prepared by coating the nanosuspensions onto pellets. The redispersibility, drug dissolution, solid-state forms, and morphology of the dried nanosuspensions were evaluated. The mean particle size for the nanosuspensions stabilized using soybean protein isolate, whey protein isolate, and β-lactoglobulin was 588 nm, 320 nm, and 243 nm, respectively. The nanosuspensions could be successfully layered onto pellets with high coating efficiency. Both the dried nanosuspensions and nanosuspensions in their original amorphous state and not influenced by the fluid-bed coating drying process could be redispersed in water, maintaining their original particle size and size distribution. Both the dried nanosuspensions and the original drug nanosuspensions showed similar dissolution profiles, which were both much faster than that of the raw crystals. Fluid-bed coating technology has potential for use in the solidification of drug nanosuspensions.

  5. Identification of cytoskeleton-associated proteins essential for lysosomal stability and survival of human cancer cells

    DEFF Research Database (Denmark)

    Groth-Pedersen, Line; Aits, Sonja; Corcelle-Termeau, Elisabeth

    2012-01-01

    Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library...... in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 si......), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, i.e. photo-oxidation, siramesine, etoposide...

  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. Effects of debt mutualization in a monetary union with endogenous risk premia : Can Eurobonds contribute to debt stabilization?

    NARCIS (Netherlands)

    van Aarle, B.; Engwerda, Jacob; Weeren, A.J.T.M.

    2017-01-01

    This paper analyses debt stabilization in a monetary union that features endogenous risk premia. In particular, debt stabilization in two diametrically opposed regimes is compared. In the first regime, the “national fiscal discipline regime”, financial markets impose sovereign risk premia based on

  8. Osmolyte Effects on Monoclonal Antibody Stability and Concentration-Dependent Protein Interactions with Water and Common Osmolytes.

    Science.gov (United States)

    Barnett, Gregory V; Razinkov, Vladimir I; Kerwin, Bruce A; Blake, Steven; Qi, Wei; Curtis, Robin A; Roberts, Christopher J

    2016-04-07

    Preferential interactions of proteins with water and osmolytes play a major role in controlling the thermodynamics of protein solutions. While changes in protein stability and shifts in phase behavior are often reported with the addition of osmolytes, the underlying protein interactions with water and/or osmolytes are typically inferred rather than measured directly. In this work, Kirkwood-Buff integrals for protein-water interactions (G12) and protein-osmolyte interactions (G23) were determined as a function of osmolyte concentration from density measurements of antistreptavidin immunoglobulin gamma-1 (AS-IgG1) in ternary aqueous solutions for a set of common neutral osmolytes: sucrose, trehalose, sorbitol, and poly(ethylene glycol) (PEG). For sucrose and PEG solutions, both protein-water and protein-osmolyte interactions depend strongly on osmolyte concentrations (c3). Strikingly, both osmolytes change from being preferentially excluded to preferentially accumulated with increasing c3. In contrast, sorbitol and trehalose solutions do not show large enough preferential interactions to be detected by densimetry. G12 and G23 values are used to estimate the transfer free energy for native AS-IgG1 (Δμ2N) and compared with existing models. AS-IgG1 unfolding via calorimetry shows a linear increase in midpoint temperatures as a function of trehalose, sucrose, and sorbitol concentrations, but the opposite behavior for PEG. Together, the results highlight limitations of existing models and common assumptions regarding the mechanisms of protein stabilization by osmolytes. Finally, PEG preferential interactions destabilize the Fab regions of AS-IgG1 more so than the CH2 or CH3 domains, illustrating preferential interactions can be specific to different protein domains.

  9. Muscle protein turnover in the elderly and its potential contribution to the development of sarcopenia.

    Science.gov (United States)

    Murton, Andrew J

    2015-11-01

    The underlying aetiology of sarcopenia appears multifaceted and not yet fully defined, but ultimately involves the gradual loss of muscle protein content over time. The present evidence suggests that the loss of lean tissue in the elderly is exacerbated by low dietary protein intake. Moreover, acute stable-isotope-based methodologies have demonstrated that the muscle anabolic response to a given amount of protein may decline with age, a phenomenon that has been termed anabolic resistance. Although the mechanism responsible for the inability of muscle to mount a satisfactory anabolic response to protein provision with increasing age is presently unknown, it does not appear due to impaired digestion or absorption of dietary protein. Rather, the issue could reside with any combination of: a diminished delivery of amino acids to peripheral tissues, impaired uptake of amino acids into muscle cells, or an inability of amino acids to elicit intracellular events pivotal for anabolism to occur. Despite the presence of anabolic resistance to dietary protein, present evidence suggests that protein supplementation may be able to overcome these issues, particularly when combined with resistance exercise programmes. As such, protein supplementation may prove to be an effective approach to delay the loss of muscle mass with age and has led to calls for the recommended daily intake of protein to be increased for the elderly population.

  10. Effect of the geometry of confining media on the stability and folding rate of α -helix proteins

    Science.gov (United States)

    Wang, Congyue; Piroozan, Nariman; Javidpour, Leili; Sahimi, Muhammad

    2018-05-01

    Protein folding in confined media has attracted wide attention over the past 15 years due to its importance to both in vivo and in vitro applications. It is generally believed that protein stability increases by decreasing the size of the confining medium, if the medium's walls are repulsive, and that the maximum folding temperature in confinement is in a pore whose size D0 is only slightly larger than the smallest dimension of a protein's folded state. Until recently, the stability of proteins in pores with a size very close to that of the folded state has not received the attention it deserves. In a previous paper [L. Javidpour and M. Sahimi, J. Chem. Phys. 135, 125101 (2011)], we showed that, contrary to the current theoretical predictions, the maximum folding temperature occurs in larger pores for smaller α-helices. Moreover, in very tight pores, the free energy surface becomes rough, giving rise to a new barrier for protein folding close to the unfolded state. In contrast to unbounded domains, in small nanopores proteins with an α-helical native state that contain the β structures are entropically stabilized implying that folding rates decrease notably and that the free energy surface becomes rougher. In view of the potential significance of such results to interpretation of many sets of experimental data that could not be explained by the current theories, particularly the reported anomalously low rates of folding and the importance of entropic effects on proteins' misfolded states in highly confined environments, we address the following question in the present paper: To what extent the geometry of a confined medium affects the stability and folding rates of proteins? Using millisecond-long molecular dynamics simulations, we study the problem in three types of confining media, namely, cylindrical and slit pores and spherical cavities. Most importantly, we find that the prediction of the previous theories that the dependence of the maximum folding

  11. A single cysteine post-translational oxidation suffices to compromise globular proteins kinetic stability and promote amyloid formation

    Directory of Open Access Journals (Sweden)

    Patrizia Marinelli

    2018-04-01

    Full Text Available Oxidatively modified forms of proteins accumulate during aging. Oxidized protein conformers might act as intermediates in the formation of amyloids in age-related disorders. However, it is not known whether this amyloidogenic conversion requires an extensive protein oxidative damage or it can be promoted just by a discrete, localized post-translational modification of certain residues. Here, we demonstrate that the irreversible oxidation of a single free Cys suffices to severely perturb the folding energy landscape of a stable globular protein, compromise its kinetic stability, and lead to the formation of amyloids under physiological conditions. Experiments and simulations converge to indicate that this specific oxidation-promoted protein aggregation requires only local unfolding. Indeed, a large scale analysis indicates that many cellular proteins are at risk of undergoing this kind of deleterious transition; explaining how oxidative stress can impact cell proteostasis and subsequently lead to the onset of pathological states. Keywords: Protein oxidation, Protein misfolding, Protein aggregation, Oxidative stress, Post-translational modification

  12. Stabilization of Nrf2 protein by D3T provides protection against ethanol-induced apoptosis in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Jian Dong

    2011-02-01

    Full Text Available Previous studies have demonstrated that maternal ethanol exposure induces a moderate increase in Nrf2 protein expression in mouse embryos. Pretreatment with the Nrf2 inducer, 3H-1, 2-dithiole-3-thione (D3T, significantly increases the Nrf2 protein levels and prevents apoptosis in ethanol-exposed embryos. The present study, using PC12 cells, was designed to determine whether increased Nrf2 stability is a mechanism by which D3T enhances Nrf2 activation and subsequent antioxidant protection. Ethanol and D3T treatment resulted in a significant accumulation of Nrf2 protein in PC 12 cells. CHX chase analysis has shown that ethanol treatment delayed the degradation of Nrf2 protein in PC12 cells. A significantly greater decrease in Nrf2 protein degradation was observed in the cells treated with D3T alone or with both ethanol and D3T. In addition, D3T treatment significantly reduced ethanol-induced apoptosis. These results demonstrate that the stabilization of Nrf2 protein by D3T confers protection against ethanol-induced apoptosis.

  13. Prospects in the use of aptamers for characterizing the structure and stability of bioactive proteins and peptides in food.

    Science.gov (United States)

    Agyei, Dominic; Acquah, Caleb; Tan, Kei Xian; Hii, Hieng Kok; Rajendran, Subin R C K; Udenigwe, Chibuike C; Danquah, Michael K

    2018-01-01

    Food-derived bioactive proteins and peptides have gained acceptance among researchers, food manufacturers and consumers as health-enhancing functional food components that also serve as natural alternatives for disease prevention and/or management. Bioactivity in food proteins and peptides is determined by their conformations and binding characteristics, which in turn depend on their primary and secondary structures. To maintain their bioactivities, the molecular integrity of bioactive peptides must remain intact, and this warrants the study of peptide form and structure, ideally with robust, highly specific and sensitive techniques. Short single-stranded nucleic acids (i.e. aptamers) are known to have high affinity for cognate targets such as proteins and peptides. Aptamers can be produced cost-effectively and chemically derivatized to increase their stability and shelf life. Their improved binding characteristics and minimal modification of the target molecular signature suggests their suitability for real-time detection of conformational changes in both proteins and peptides. This review discusses the developmental progress of systematic evolution of ligands by exponential enrichment (SELEX), an iterative technology for generating cost-effective aptamers with low dissociation constants (K d ) for monitoring the form and structure of bioactive proteins and peptides. The review also presents case studies of this technique in monitoring the structural stability of bioactive peptide formulations to encourage applications in functional foods. The challenges and potential of aptamers in this research field are also discussed. Graphical abstract Advancing bioactive proteins and peptide functionality via aptameric ligands.

  14. Contribution to the study of proteins and peptides structure by hydrogen isotopic exchange

    International Nuclear Information System (INIS)

    Nabedryk-Viala, Eliane.

    1978-01-01

    Development of hydrogen exchange measurement methods to study the structure and the molecular interaction of globular protein molecules in aqueous solution (ribonuclease A, cytochrome c, coupling factors of chloroplasts), in peptide hormones in trifluoroethanol solution (angiotensin II, corticotropin) and in proteins of membranes (rhodopsin) [fr

  15. G-protein-coupled inward rectifier potassium current contributes to ventricular repolarization

    DEFF Research Database (Denmark)

    Liang, Bo; Nissen, Jakob D; Laursen, Morten

    2014-01-01

    The purpose of this study was to investigate the functional role of G-protein-coupled inward rectifier potassium (GIRK) channels in the cardiac ventricle.......The purpose of this study was to investigate the functional role of G-protein-coupled inward rectifier potassium (GIRK) channels in the cardiac ventricle....

  16. Analysis of contributions of herpes simplex virus type 1 UL43 protein ...

    African Journals Online (AJOL)

    Purpose: To investigate whether UL43 protein, which is highly conserved in alpha- and gamma herpes viruses, and a non-glycosylated transmembrane protein, is involved in virus entry and virus-induced cell fusion. Methods: Mutagenesis was accomplished by a markerless two-step Red recombination mutagenesis system ...

  17. Clusters of basic amino acids contribute to RNA binding and nucleolar localization of ribosomal protein L22.

    Directory of Open Access Journals (Sweden)

    Jennifer L Houmani

    Full Text Available The ribosomal protein L22 is a component of the 60S eukaryotic ribosomal subunit. As an RNA-binding protein, it has been shown to interact with both cellular and viral RNAs including 28S rRNA and the Epstein-Barr virus encoded RNA, EBER-1. L22 is localized to the cell nucleus where it accumulates in nucleoli. Although previous studies demonstrated that a specific amino acid sequence is required for nucleolar localization, the RNA-binding domain has not been identified. Here, we investigated the hypothesis that the nucleolar accumulation of L22 is linked to its ability to bind RNA. To address this hypothesis, mutated L22 proteins were generated to assess the contribution of specific amino acids to RNA binding and protein localization. Using RNA-protein binding assays, we demonstrate that basic amino acids 80-93 are required for high affinity binding of 28S rRNA and EBER-1 by L22. Fluorescence localization studies using GFP-tagged mutated L22 proteins further reveal that basic amino acids 80-93 are critical for nucleolar accumulation and for incorporation into ribosomes. Our data support the growing consensus that the nucleolar accumulation of ribosomal proteins may not be mediated by a defined localization signal, but rather by specific interaction with established nucleolar components such as rRNA.

  18. Spinal atypical protein kinase C activity is necessary to stabilize inactivity-induced phrenic motor facilitation

    Science.gov (United States)

    Strey, K.A.; Nichols, N.L.; Baertsch, N.A.; Broytman, O.; Baker-Herman, T.L.

    2012-01-01

    The neural network controlling breathing must establish rhythmic motor output at a level adequate to sustain life. Reduced respiratory neural activity elicits a novel form of plasticity in circuits driving the diaphragm known as inactivity-induced phrenic motor facilitation (iPMF), a rebound increase in phrenic inspiratory output observed once respiratory neural drive is restored. The mechanisms underlying iPMF are unknown. Here, we demonstrate in anesthetized rats that spinal mechanisms give rise to iPMF, and that iPMF consists of at least two mechanistically distinct phases: 1) an early, labile phase that requires atypical PKC (PKCζ and/or PKCΙ/λ) activity to transition to a 2) late, stable phase. Early (but not late) iPMF is associated with increased interactions between PKCζ/Ι and the scaffolding protein ZIP/p62 in spinal regions associated with the phrenic motor pool. Although PKCζ/Ι activity is necessary for iPMF, spinal aPKC activity is not necessary for phrenic long-term facilitation (pLTF) following acute intermittent hypoxia, an activity-independent form of spinal respiratory plasticity. Thus, while iPMF and pLTF both manifest as prolonged increases in phrenic burst amplitude, they arise from distinct spinal cellular pathways. Our data are consistent with the hypotheses that: 1) local mechanisms sense and respond to reduced respiratory-related activity in the phrenic motor pool, and 2) inactivity-induced increases in phrenic inspiratory output require local PKCζ/Ι activity to stabilize into a long-lasting iPMF. Although the physiological role of iPMF is unknown, we suspect that iPMF represents a compensatory mechanism, assuring adequate motor output in a physiological system where prolonged inactivity ends life. PMID:23152633

  19. Interfacial properties of whey protein and whey protein hydrolysates and their influence on O/W emulsion stability

    NARCIS (Netherlands)

    Schroder, A.J.; Berton-Carabin, C.C.; Venema, P.; Cornacchia, L.

    2017-01-01

    Protein hydrolysates are commonly used in high-tolerance or hypoallergenic formulae. The relation between the physicochemical properties of hydrolysed proteins (i.e., size, molecular weight distribution, charge, hydrophobicity), and their emulsifying properties is not fully understood. In this work,

  20. Mechanical properties of protein adsorption layers at the air/water and oil/water interface: a comparison in light of the thermodynamical stability of proteins.

    Science.gov (United States)

    Mitropoulos, Varvara; Mütze, Annekathrin; Fischer, Peter

    2014-04-01

    Over the last decades numerous studies on the interfacial rheological response of protein adsorption layers have been published. The comparison of these studies and the retrieval of a common parameter to compare protein interfacial activity are hampered by the fact that different boundary conditions (e.g. physico-chemical, instrumental, interfacial) were used. In the present work we review previous studies and attempt a unifying approach for the comparison between bulk protein properties and their adsorption films. Among many common food grade proteins we chose bovine serum albumin, β-lactoglobulin and lysozyme for their difference in thermodynamic stability and studied their adsorption at the air/water and limonene/water interface. In order to achieve this we have i) systematically analyzed protein adsorption kinetics in terms of surface pressure rise using a drop profile analysis tensiometer and ii) we addressed the interfacial layer properties under shear stress using an interfacial shear rheometer under the same experimental conditions. We could show that thermodynamically less stable proteins adsorb generally faster and yield films with higher shear rheological properties at air/water interface. The same proteins showed an analog behavior when adsorbing at the limonene/water interface but at slower rates. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Small molecule PGC-1α1 protein stabilizers induce adipocyte Ucp1 expression and uncoupled mitochondrial respiration

    Directory of Open Access Journals (Sweden)

    A.T. Pettersson-Klein

    2018-03-01

    Full Text Available Objective: The peroxisome proliferator-activated receptor-γ coactivator-1α1 (PGC-1α1 regulates genes involved in energy metabolism. Increasing adipose tissue energy expenditure through PGC-1α1 activation is potentially beneficial for systemic metabolism. Pharmacological PGC-1α1 activators could be valuable tools in the fight against obesity and metabolic disease. Finding such compounds has been challenging partly because PGC-1α1 is a transcriptional coactivator with no known ligand-binding properties. While, PGC-1α1 activation is regulated by several mechanisms, protein stabilization is a crucial limiting step due to its short half-life under unstimulated conditions. Methods: We designed a cell-based high-throughput screening system to identify PGC-1α1 protein stabilizers. Positive hits were tested for their ability to induce endogenous PGC-1α1 protein accumulation and activate target gene expression in brown adipocytes. Select compounds were analyzed for their effects on global gene expression and cellular respiration in adipocytes. Results: Among 7,040 compounds screened, we highlight four small molecules with high activity as measured by: PGC-1α1 protein accumulation, target gene expression, and uncoupled mitochondrial respiration in brown adipocytes. Conclusions: We identify compounds that induce PGC-1α1 protein accumulation and show that this increases uncoupled respiration in brown adipocytes. This screening platform establishes the foundation for a new class of therapeutics with potential use in obesity and associated disorders. Keywords: Small molecule screening, PGC-1a, PGC-1alpha, PGC-1alpha1, Protein stabilization, UCP1, Mitochondrial respiration, Brown adipose tissue

  2. Low levels of the AhR in chronic obstructive pulmonary disease (COPD)-derived lung cells increases COX-2 protein by altering mRNA stability.

    Science.gov (United States)

    Zago, Michela; Sheridan, Jared A; Traboulsi, Hussein; Hecht, Emelia; Zhang, Yelu; Guerrina, Necola; Matthews, Jason; Nair, Parameswaran; Eidelman, David H; Hamid, Qutayba; Baglole, Carolyn J

    2017-01-01

    Heightened inflammation, including expression of COX-2, is associated with chronic obstructive pulmonary disease (COPD) pathogenesis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is reduced in COPD-derived lung fibroblasts. The AhR also suppresses COX-2 in response to cigarette smoke, the main risk factor for COPD, by destabilizing the Cox-2 transcript by mechanisms that may involve the regulation of microRNA (miRNA). Whether reduced AhR expression is responsible for heightened COX-2 in COPD is not known. Here, we investigated the expression of COX-2 as well as the expression of miR-146a, a miRNA known to regulate COX-2 levels, in primary lung fibroblasts derived from non-smokers (Normal) and smokers (At Risk) with and without COPD. To confirm the involvement of the AhR, AhR knock-down via siRNA in Normal lung fibroblasts and MLE-12 cells was employed as were A549-AhRko cells. Basal expression of COX-2 protein was higher in COPD lung fibroblasts compared to Normal or Smoker fibroblasts but there was no difference in Cox-2 mRNA. Knockdown of AhR in lung structural cells increased COX-2 protein by stabilizing the Cox-2 transcript. There was less induction of miR-146a in COPD-derived lung fibroblasts but this was not due to the AhR. Instead, we found that RelB, an NF-κB protein, was required for transcriptional induction of both Cox-2 and miR-146a. Therefore, we conclude that the AhR controls COX-2 protein via mRNA stability by a mechanism independent of miR-146a. Low levels of the AhR may therefore contribute to the heightened inflammation common in COPD patients.

  3. Calculation of the relative chemical stabilities of proteins as a function of temperature and redox chemistry in a hot spring.

    Directory of Open Access Journals (Sweden)

    Jeffrey M Dick

    Full Text Available Uncovering the chemical and physical links between natural environments and microbial communities is becoming increasingly amenable owing to geochemical observations and metagenomic sequencing. At the hot spring known as Bison Pool in Yellowstone National Park, the cooling of the water in the outflow channel is associated with an increase in oxidation potential estimated from multiple field-based measurements. Representative groups of proteins whose sequences were derived from metagenomic data also exhibit an increase in average oxidation state of carbon in the protein molecules with distance from the hot-spring source. The energetic requirements of reactions to form selected proteins used in the model were computed using amino-acid group additivity for the standard molal thermodynamic properties of the proteins, and the relative chemical stabilities of the proteins were investigated by varying temperature, pH and oxidation state, expressed as activity of dissolved hydrogen. The relative stabilities of the proteins were found to track the locations of the sampling sites when the calculations included a function for hydrogen activity that increases with temperature and is higher, or more reducing, than values consistent with measurements of dissolved oxygen, sulfide and oxidation-reduction potential in the field. These findings imply that spatial patterns in the amino acid compositions of proteins can be linked, through energetics of overall chemical reactions representing the formation of the proteins, to the environmental conditions at this hot spring, even if microbial cells maintain considerably different internal conditions. Further applications of the thermodynamic calculations are possible for other natural microbial ecosystems.

  4. A Novel Acidic Matrix Protein, PfN44, Stabilizes Magnesium Calcite to Inhibit the Crystallization of Aragonite*

    Science.gov (United States)

    Pan, Cong; Fang, Dong; Xu, Guangrui; Liang, Jian; Zhang, Guiyou; Wang, Hongzhong; Xie, Liping; Zhang, Rongqing

    2014-01-01

    Magnesium is widely used to control calcium carbonate deposition in the shell of pearl oysters. Matrix proteins in the shell are responsible for nucleation and growth of calcium carbonate crystals. However, there is no direct evidence supporting a connection between matrix proteins and magnesium. Here, we identified a novel acidic matrix protein named PfN44 that affected aragonite formation in the shell of the pearl oyster Pinctada fucata. Using immunogold labeling assays, we found PfN44 in both the nacreous and prismatic layers. In shell repair, PfN44 was repressed, whereas other matrix proteins were up-regulated. Disturbing the function of PfN44 by RNAi led to the deposition of porous nacreous tablets with overgrowth of crystals in the nacreous layer. By in vitro circular dichroism spectra and fluorescence quenching, we found that PfN44 bound to both calcium and magnesium with a stronger affinity for magnesium. During in vitro calcium carbonate crystallization and calcification of amorphous calcium carbonate, PfN44 regulated the magnesium content of crystalline carbonate polymorphs and stabilized magnesium calcite to inhibit aragonite deposition. Taken together, our results suggested that by stabilizing magnesium calcite to inhibit aragonite deposition, PfN44 participated in P. fucata shell formation. These observations extend our understanding of the connections between matrix proteins and magnesium. PMID:24302723

  5. Developmentally regulated GTP-binding protein 2 is required for stabilization of Rac1-positive membrane tubules.

    Science.gov (United States)

    Mani, Muralidharan; Lee, Unn Hwa; Yoon, Nal Ae; Yoon, Eun Hye; Lee, Byung Ju; Cho, Wha Ja; Park, Jeong Woo

    2017-11-04

    Previously we have reported that developmentally regulated GTP-binding protein 2 (DRG2) localizes on Rab5 endosomes and plays an important role in transferrin (Tfn) recycling. We here identified DRG2 as a key regulator of membrane tubule stability. At 30 min after Tfn treatment, DRG2 localized to membrane tubules which were enriched with phosphatidylinositol 4-monophosphate [PI(4)P] and did not contain Rab5. DRG2 interacted with Rac1 more strongly with GTP-bound Rac1 and tubular localization of DRG2 depended on Rac1 activity. DRG2 depletion led to destabilization of membrane tubules, while ectopic expression of DRG2 rescued the stability of the membrane tubules in DRG2-depleted cells. Our results reveal a novel mechanism for regulation of membrane tubule stability mediated by DRG2. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. The Drosophila Microtubule-Associated Protein Mars Stabilizes Mitotic Spindles by Crosslinking Microtubules through Its N-Terminal Region

    Science.gov (United States)

    Zhang, Gang; Beati, Hamze; Nilsson, Jakob; Wodarz, Andreas

    2013-01-01

    Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs) are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs) have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs. PMID:23593258

  7. The Drosophila microtubule-associated protein mars stabilizes mitotic spindles by crosslinking microtubules through its N-terminal region.

    Directory of Open Access Journals (Sweden)

    Gang Zhang

    Full Text Available Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs.

  8. Stabilization of alanine substituted p53 protein at Ser15, Thr18, and Ser20 in response to ionizing radiation

    International Nuclear Information System (INIS)

    Yamauchi, Motohiro; Suzuki, Keiji; Kodama, Seiji; Watanabe, Masami

    2004-01-01

    Phosphorylation of p53 at Ser15, Thr18, and Ser20 has been thought to be important for p53 stabilization in response to ionizing radiation. In the present study, we examined the X-ray-induced stabilization of Ala-substituted p53 protein at Ser15, Thr18, and Ser20, whose gene expression was controlled under an ecdyson-inducible promoter. We found that all single-, double-, or triple-Ala-substituted p53 at Ser15, Yhr18, and Ser20 were accumulated in the nucleus similarly to wild-type p53 after X-irradiation. These results indicate that the phosphorylation of p53 at Ser15, Thr18, and Ser20 is not necessarily needed for p53 stabilization in response to ionizing radiation

  9. The Drosophila microtubule-associated protein mars stabilizes mitotic spindles by crosslinking microtubules through its N-terminal region.

    Science.gov (United States)

    Zhang, Gang; Beati, Hamze; Nilsson, Jakob; Wodarz, Andreas

    2013-01-01

    Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs) are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs) have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs.

  10. The Contribution of Tissue Level Organization to Genomic Stability Following Low Dose/Low Dose Rate Gamma and Proton Irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Cheryl G. Burrell, Ph.D.

    2012-05-14

    The formation of functional tissue units is necessary in maintaining homeostasis within living systems, with individual cells contributing to these functional units through their three-dimensional organization with integrin and adhesion proteins to form a complex extra-cellular matrix (ECM). This is of particular importance in those tissues susceptible to radiation-induced tumor formation, such as epithelial glands. The assembly of epithelial cells of the thyroid is critical to their normal receipt of, and response to, incoming signals. Traditional tissue culture and live animals present significant challenges to radiation exposure and continuous sampling, however, the production of bioreactor-engineered tissues aims to bridge this gap by improve capabilities in continuous sampling from the same functional tissue, thereby increasing the ability to extrapolate changes induced by radiation to animals and humans in vivo. Our study proposes that the level of tissue organization will affect the induction and persistence of low dose radiation-induced genomic instability. Rat thyroid cells, grown in vitro as 3D tissue analogs in bioreactors and as 2D flask grown cultures were exposed to acute low dose (1, 5, 10 and 200 cGy) gamma rays. To assess immediate (6 hours) and delayed (up to 30 days) responses post-irradiation, various biological endpoints were studied including cytogenetic analyses, apoptosis analysis and cell viability/cytotoxicity analyses. Data assessing caspase 3/7 activity levels show that, this activity varies with time post radiation and that, overall, 3D cultures display more genomic instability (as shown by the lower levels of apoptosis over time) when compared to the 2D cultures. Variation in cell viability levels were only observed at the intermediate and late time points post radiation. Extensive analysis of chromosomal aberrations will give further insight on the whether the level of tissue organization influences genomic instability patterns after

  11. Yeast hnRNP-related proteins contribute to the maintenance of telomeres

    Energy Technology Data Exchange (ETDEWEB)

    Lee-Soety, Julia Y., E-mail: jlee04@sju.edu [Department of Biology, Saint Joseph' s University, PA 19131 (United States); Jones, Jennifer; MacGibeny, Margaret A.; Remaly, Erin C.; Daniels, Lynsey; Ito, Andrea; Jean, Jessica; Radecki, Hannah; Spencer, Shannon [Department of Biology, Saint Joseph' s University, PA 19131 (United States)

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Yeast hnRNP-related proteins are able to prevent faster senescence in telomerase-null cells. Black-Right-Pointing-Pointer The conserved RRMs in Npl3 are important for telomere maintenance. Black-Right-Pointing-Pointer Human hnRNP A1 is unable to complement the lack of NPL3 in yeast. Black-Right-Pointing-Pointer Npl3 and Cbc2 may work as telomere capping proteins. -- Abstract: Telomeres protect the ends of linear chromosomes, which if eroded to a critical length can become uncapped and lead to replicative senescence. Telomerase maintains telomere length in some cells, but inappropriate expression facilitates the immortality of cancer cells. Recently, proteins involved in RNA processing and ribosome assembly, such as hnRNP (heterogeneous nuclear ribonucleoprotein) A1, have been found to participate in telomere maintenance in mammals. The Saccharomyces cerevisiae protein Npl3 shares significant amino acid sequence similarities with hnRNP A1. We found that deleting NPL3 accelerated the senescence of telomerase null cells. The highly conserved RNA recognition motifs (RRM) in Npl3 appear to be important for preventing faster senescence. Npl3 preferentially binds telomere sequences in vitro, suggesting that Npl3 may affect telomeres directly. Despite similarities between the two proteins, human hnRNP A1 is unable to complement the lack of Npl3 to rescue accelerated senescence in tlc1 npl3 cells. Deletion of CBC2, which encodes another hnRNP-related protein that associates with Npl3, also accelerates senescence. Potential mechanisms by which hnRNP-related proteins maintain telomeres are discussed.

  12. Surface rheological properties of liquid-liquid interfaces stabilized by protein fibrillar aggregates and protein-polysaccharide complexes

    NARCIS (Netherlands)

    Humblet-Hua, K.N.P.; Linden, van der E.; Sagis, L.M.C.

    2013-01-01

    In this study we have investigated the surface rheological properties of oil-water interfaces stabilized by fibrils from lysozyme (long and semi-flexible and short and rigid ones), fibrils from ovalbumin (short and semi-flexible), lysozyme-pectin complexes, or ovalbumin-pectin complexes. We have

  13. Reassessing Domain Architecture Evolution of Metazoan Proteins: The Contribution of Different Evolutionary Mechanisms

    Directory of Open Access Journals (Sweden)

    Laszlo Patthy

    2011-08-01

    Full Text Available In the accompanying papers we have shown that sequence errors of public databases and confusion of paralogs and epaktologs (proteins that are related only through the independent acquisition of the same domain types significantly distort the picture that emerges from comparison of the domain architecture (DA of multidomain Metazoan proteins since they introduce a strong bias in favor of terminal over internal DA change. The issue of whether terminal or internal DA changes occur with greater probability has very important implications for the DA evolution of multidomain proteins since gene fusion can add domains only at terminal positions, whereas domain-shuffling is capable of inserting domains both at internal and terminal positions. As a corollary, overestimation of terminal DA changes may be misinterpreted as evidence for a dominant role of gene fusion in DA evolution. In this manuscript we show that in several recent studies of DA evolution of Metazoa the authors used databases that are significantly contaminated with incomplete, abnormal and mispredicted sequences (e.g., UniProtKB/TrEMBL, EnsEMBL and/or the authors failed to separate paralogs and epaktologs, explaining why these studies concluded that the major mechanism for gains of new domains in metazoan proteins is gene fusion. In contrast with the latter conclusion, our studies on high quality orthologous and paralogous Swiss-Prot sequences confirm that shuffling of mobile domains had a major role in the evolution of multidomain proteins of Metazoa and especially those formed in early vertebrates.

  14. Multiple DNA binding proteins contribute to timing of chromosome replication in E. coli

    DEFF Research Database (Denmark)

    Riber, Leise; Frimodt-Møller, Jakob; Charbon, Godefroid

    2016-01-01

    Chromosome replication in Escherichia coli is initiated from a single origin, oriC. Initiation involves a number of DNA binding proteins, but only DnaA is essential and specific for the initiation process. DnaA is an AAA+ protein that binds both ATP and ADP with similar high affinities. Dna...... replication is initiated, or the time window in which all origins present in a single cell are initiated, i.e. initiation synchrony, or both. Overall, these DNA binding proteins modulate the initiation frequency from oriC by: (i) binding directly to oriC to affect DnaA binding, (ii) altering the DNA topology...... in or around oriC, (iii) altering the nucleotide bound status of DnaA by interacting with non-coding chromosomal sequences, distant from oriC, that are important for DnaA activity. Thus, although DnaA is the key protein for initiation of replication, other DNA-binding proteins act not only on ori...

  15. Proteolytic stability in colloidal systems : interaction of proteins with the solid-water interface

    NARCIS (Netherlands)

    Maste, M.C.L.

    1996-01-01


    Proteolytic enzymes in liquid detergents suffer from lack of stability in the sense that activity diminishes with time. Although the phenomenon could be attributed to several factors, the influence of colloidal surfaces on the enzymatic stability was investigated. Besides the types of

  16. Contribution to the study of the stability of water-cooled reactors; Contribution a l'etude de la stabilite des reacteurs refroidis par de l'eau

    Energy Technology Data Exchange (ETDEWEB)

    Coudert, C [Commissariat a l' Energie Atomique, Cadarache (France). Centre d' Etudes Nucleaires

    1969-06-01

    This work is devoted to the study of the stability of reactors cooled by water subjected only to natural convection. It is made up of two parts, a theoretical study and experimental work, each of these parts being devoted to a consideration of linear and non-linear conditions: - calculation of the transfer function of the reactor using neutronic and hydrodynamic linear equations with the determination of the instability threshold; - demonstration of the existence of the limiting oscillation cycle in the case of a linear feedback using MALKIN'S method; - measurement and interpretation of the reactor's transfer functions and of the hydrodynamic transfer functions; and - analysis of the noise due to boiling. (author) [French] Dans ce travail on etudie la stabilite des piles refroidies par de l'eau circulant en convection naturelle. Cette etude se divise en deux parties: un travail theorique et un travail experimental, chacune de ces parties comportant une etude lineaire et une etude non-lineaire: - calcul de la fonction de transfert du reacteur a partir des equations lineaires de la neutronique et de l'hydrodynamique avec determination du seuil d'instabilite; - demonstration de l'existence du cycle limite des oscillations dans le cas d'une retroaction lineaire en utilisant la methode de MALKIN; - mesure et interpretation de la fonction de transfert du reacteur et des fonctions de transfert hydrodynamiques; et - analyse du bruit d'ebullition. (auteur)

  17. Engineering nutritious proteins: improvement of stability in the designer protein MB-1 via introduction of disulfide bridges.

    Science.gov (United States)

    Doucet, Alain; Williams, Martin; Gagnon, Mylene C; Sasseville, Maxime; Beauregard, Marc

    2002-01-02

    Protein design is currently used for the creation of new proteins with desirable traits. In this laboratory the focus has been on the synthesis of proteins with high essential amino acid content having potential applications in animal nutrition. One of the limitations faced in this endeavor is achieving stable proteins despite a highly biased amino acid content. Reported here are the synthesis and characterization of two disulfide-bridged mutants derived from the MB-1 designer protein. Both mutants outperformed their parent protein MB-1 with their bridge formed, as shown by circular dichroism, size exclusion chromatography, thermal denaturation, and proteolytic degradation experiments. When the disulfide bridges were cleaved, the mutants' behavior changed: the mutants significantly unfolded, suggesting that the introduction of Cys residues was deleterious to MB-1-folding. In an attempt to compensate for the mutations used, a Tyr62-Trp mutation was performed, leading to an increase in bulk and hydrophobicity in the core. The Trp-containing disulfide-bridged mutants did not behave as well as the original MB-1Trp, suggesting that position 62 might not be adequate for a compensatory mutation.

  18. Truncation of the C-terminal region of Toscana Virus NSs protein is critical for interferon-β antagonism and protein stability.

    Science.gov (United States)

    Gori Savellini, Gianni; Gandolfo, Claudia; Cusi, Maria Grazia

    2015-12-01

    Toscana Virus (TOSV) is a Phlebovirus responsible for central nervous system (CNS) injury in humans. The TOSV non-structural protein (NSs), which interacting with RIG-I leads to its degradation, was analysed in the C terminus fragment in order to identify its functional domains. To this aim, two C-terminal truncated NSs proteins, Δ1C-NSs (aa 1-284) and Δ2C-NSs (aa 1-287) were tested. Only Δ1C-NSs did not present any inhibitory effect on RIG-I and it showed a greater stability than the whole NSs protein. Moreover, the deletion of the TLQ aa sequence interposed between the two ΔC constructs caused a greater accumulation of the protein with a weak inhibitory effect on RIG-I, indicating some involvement of these amino acids in the NSs activity. Nevertheless, all the truncated proteins were still able to interact with RIG-I, suggesting that the domains responsible for RIG-I signaling and RIG-I interaction are mapped on different regions of the protein. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Modulating weak interactions for molecular recognition: a dynamic combinatorial analysis for assessing the contribution of electrostatics to the stability of CH-π bonds in water.

    Science.gov (United States)

    Jiménez-Moreno, Ester; Gómez, Ana M; Bastida, Agatha; Corzana, Francisco; Jiménez-Oses, Gonzalo; Jiménez-Barbero, Jesús; Asensio, Juan Luis

    2015-03-27

    Electrostatic and charge-transfer contributions to CH-π complexes can be modulated by attaching electron-withdrawing substituents to the carbon atom. While clearly stabilizing in the gas phase, the outcome of this chemical modification in water is more difficult to predict. Herein we provide a definitive and quantitative answer to this question employing a simple strategy based on dynamic combinatorial chemistry. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes.

    Science.gov (United States)

    Csermely, P; Szamel, M; Resch, K; Somogyi, J

    1988-05-15

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested (Parker, P.J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M.D., and Ullrich, A. (1986) Science 233, 853-859). In the present report, we demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes, and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn2+, while Fe2+ and Mn2+ are only partially counteractive. Our results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca2+, phorbol ester, or antigen.

  1. Contribution to the study of protein deficiency: Use of radioisotope techniques

    International Nuclear Information System (INIS)

    Dubois, J.; Colard, J.; Vis, H.L.

    1970-01-01

    Research methods based on the use of radioisotopes have already been used for some time to study the physiopathogenesis of malnutritional and denutritional conditions in childhood. Two very specific aspects of malnutrition are studied primarily by means of these techniques: plasma protein metabolism proper and hydroelectrolytic disorders, which are an integral part of the physiopathogenetic picture of the disease. The authors have attempted, in so far as is possible, to define the clinical condition of children from along Lake Kivu, in the east of Kivu Province, who are suffering from protein and calorie deficiency. (author) [fr

  2. Effect of different fining agents and additives in white wine protein stability

    OpenAIRE

    Ribeiro, Tânia Isabel Monteiro; Cosme, Fernanda; Filipe-Ribeiro, L.; Fernandes, Conceição; Mendes-Faia, Arlete

    2012-01-01

    Proteins in white wine could become insoluble and precipitate causing the appearance of a haze in bottled wine. Protein instability may be due to intrinsically or extrinsically factor such as protein molecular weight, isoelectric point, ionic strength, alcohol degree and wine pH or storage temperature. These modifications may occur during aging, storage or when diverse wines are blended. The type and concentration of proteins in the wine depends on grape variety, maturation deg...

  3. Contribution of Adsorbed Protein Films to Nanoscopic Vibrations Exhibited by Bacteria Adhering through Ligand-Receptor Bonds.

    Science.gov (United States)

    Song, Lei; Sjollema, Jelmer; Norde, Willem; Busscher, Henk J; van der Mei, Henny C

    2015-09-29

    Bacteria adhering to surfaces exhibit nanoscopic vibrations that depend on the viscoelasticity of the bond. The quantification of the nanoscopic vibrations of bacteria adhering to surfaces provides new opportunities to better understand the properties of the bond through which bacteria adhere and the mechanisms by which they resist detachment. Often, however, bacteria do not adhere to bare surfaces but to adsorbed protein films, on which adhesion involves highly specific ligand-receptor binding next to nonspecific DLVO interaction forces. Here we determine the contribution of adsorbed salivary protein and fibronectin films to vibrations exhibited by adhering streptococci and staphylococci, respectively. The streptococcal strain used has the ability to adhere to adsorbed salivary proteins films through antigen I/II ligand-receptor binding, while the staphylococcal strain used adheres to adsorbed fibronectin films through a proteinaceous ligand-receptor bond. In the absence of ligand-receptor binding, electrostatic interactions had a large impact on vibration amplitudes of adhering bacteria on glass. On an adsorbed salivary protein film, vibration amplitudes of adhering streptococci depended on the film softness as determined by QCM-D and were reduced after film fixation using glutaraldehyde. On a relatively stiff fibronectin film, cross-linking the film in glutaraldehyde hardly reduced its softness, and accordingly fibronectin film softness did not contribute to vibration amplitudes of adhering staphylococci. However, fixation of the staphylococcus-fibronectin bond further decreased vibration amplitudes, while fixation of the streptococcus bond hardly impacted vibration amplitudes. Summarizing, this study shows that both the softness of adsorbed protein films and the properties of the bond between an adhering bacterium and an adsorbed protein film play an important role in bacterial vibration amplitudes. These nanoscopic vibrations reflect the viscoelasticity of the

  4. Contributions of Racial and Sociobehavioral Homophily to Friendship Stability and Quality among Same-Race and Cross-Race Friends

    Science.gov (United States)

    McDonald, Kristina L.; Dashiell-Aje, Ebony; Menzer, Melissa M.; Rubin, Kenneth H.; Oh, Wonjung; Bowker, Julie C.

    2013-01-01

    The current study examined how racial and sociobehavioral similarities were associated with friendship stability and friendship quality. Cross-race friends were not significantly similar to each other in peer-nominated shyness/withdrawal, victimization, exclusion, and popularity/sociability. Relative to same-race friends, cross-race friends were…

  5. How the EU can contribute to security and stability in South Caucasus? / Harri Kämäräinen

    Index Scriptorium Estoniae

    Kämäräinen, Harri

    2003-01-01

    Ettekanne 22.-23. septembrini 2003 Vilniuses toimunud seminaril "South Caucasus: making the best use of external assistance for stability building and for co-operation with NATO" käsitleb Euroopa Liidu eesmärke ja programme Lõuna-Kaukaasias, mille täitmist koordineerib Euroopa Liidu eriesindaja, suursaadik Heikki Talvitie

  6. The type III protein secretion system contributes to Xanthomonas citri subsp. citri biofilm formation

    KAUST Repository

    Zimaro, Tamara

    2014-04-18

    Background: Several bacterial plant pathogens colonize their hosts through the secretion of effector proteins by a Type III protein secretion system (T3SS). The role of T3SS in bacterial pathogenesis is well established but whether this system is involved in multicellular processes, such as bacterial biofilm formation has not been elucidated. Here, the phytopathogen Xanthomonas citri subsp. citri (X. citri) was used as a model to gain further insights about the role of the T3SS in biofilm formation. Results: The capacity of biofilm formation of different X. citri T3SS mutants was compared to the wild type strain and it was observed that this secretion system was necessary for this process. Moreover, the T3SS mutants adhered proficiently to leaf surfaces but were impaired in leaf-associated growth. A proteomic study of biofilm cells showed that the lack of the T3SS causes changes in the expression of proteins involved in metabolic processes, energy generation, exopolysaccharide (EPS) production and bacterial motility as well as outer membrane proteins. Furthermore, EPS production and bacterial motility were also altered in the T3SS mutants. Conclusions: Our results indicate a novel role for T3SS in X. citri in the modulation of biofilm formation. Since this process increases X. citri virulence, this study reveals new functions of T3SS in pathogenesis. 2014 Zimaro et al.; licensee BioMed Central Ltd.

  7. Contribution of residual proteins to the thermomechanical performance of cellulosic nanofibrils isolated from green macroalgae

    Science.gov (United States)

    Jiaqi Guo; Khan Mohammad Ahsan Uddin; Karl Mihhels; Wenwen Fang; Päivi Laaksonen; J. Y. Zhu; Orlando J. Rojas

    2017-01-01

    Cellulosic nanofibrils (CNFs) were isolated from one of the most widespread freshwater macroalgae, Aegagropila linnaei. The algae were first carboxylated with a recyclable dicarboxylic acid, which facilitated deconstruction into CNFs via microfluidization while preserving the protein component. For comparison, cellulosic fibrils were also isolated by chemical treatment...

  8. Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

    Science.gov (United States)

    Tong, X; Kono, T; Evans-Molina, C

    2015-06-18

    The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b

  9. Some factors contributing to protein-energy malnutrition in the middle belt of Nigeria.

    Science.gov (United States)

    Ighogboja, S I

    1992-10-01

    A number of risk factors leading to malnutrition were investigated among 400 mothers of malnourished children in the middle belt of Nigeria. Poverty, family instability, poor environmental sanitation, faulty weaning practices, illiteracy, ignorance, large family size and preventable infections are the main factors responsible for malnutrition. The strategies for intervention are in the area of health education emphasizing the importance of breastfeeding, family stability, responsible parenthood and small family sizes through culturally acceptable family planning methods. There is need to improve weaning methods through nutrition education, growth monitoring and food demonstration with community participation. Political will is needed to improve literacy status, farming methods and general living conditions.

  10. Stability of Proteins in Carbohydrates and Other Additives during Freezing: The Human Growth Hormone as a Case Study.

    Science.gov (United States)

    Arsiccio, Andrea; Pisano, Roberto

    2017-09-21

    Molecular dynamics is here used to elucidate the mechanism of protein stabilization by carbohydrates and other additives during freezing. More specifically, we used molecular dynamics simulations to obtain a quantitative estimation of the capability of various cryoprotectants to preserve a model protein, the human growth hormone, against freezing stresses. Three mechanisms were investigated, preferential exclusion, water replacement, and vitrification. Model simulations were finally validated upon experimental data in terms of the ability of excipients to prevent protein aggregation. Overall, we found that the preferential exclusion and vitrification mechanisms are important during the whole freezing process, while water replacement becomes dominant only toward the end of the cryoconcentration phase. The disaccharides were found to be the most efficient excipients, in regard to both preferential exclusion and water replacement. Moreover, sugars were in general more efficient than other excipients, such as glycine or sorbitol.

  11. Ubiquitin-specific protease 11 (USP11) functions as a tumor suppressor through deubiquitinating and stabilizing VGLL4 protein

    Science.gov (United States)

    Zhang, Encheng; Shen, Bing; Mu, Xingyu; Qin, Yan; Zhang, Fang; Liu, Yong; Xiao, Jiantao; Zhang, Pingzhao; Wang, Chenji; Tan, Mingyue; Fan, Yu

    2016-01-01

    VGLL4 is a transcriptional repressor that interacts with transcription factors TEADs and inhibits YAP-induced overgrowth and tumorigenesis. VGLL4 protein was dramatically reduced in various types of human cancers. But how VGLL4 protein is post-transcriptional regulated is poorly understood. In this study, we identify deubiquitinating enzyme USP11 as a novel VGLL4 interactor. We reveal that the USP domain of USP11 and the N-terminal region of VGLL4 are required for mutual binding. USP11 controls VGLL4 protein stability by promoting its deubiquitination. Furthermore, our results show that knockdown of USP11 promotes cell growth, migration, and invasion in a YAP-dependent manner. Together, our results suggest that USP11 may exert its tumor suppressor role by modulating VGLL4/YAP-TEADs regulatory loop. PMID:28042509

  12. Antigenic stability of pecan [Carya illinoinensis (Wangenh.) K. Koch] proteins: effects of thermal treatments and in vitro digestion.

    Science.gov (United States)

    Venkatachalam, Mahesh; Teuber, Suzanne S; Peterson, W Rich; Roux, Kenneth H; Sathe, Shridhar K

    2006-02-22

    Rabbit polyclonal antibody-based inhibition ELISA as well as immunoblotting analyses of proteins extracted from variously processed pecans (cv. Desirable) indicate that pecan proteins are antigenically stable. Pecan antigens were more sensitive to moist heat than dry heat processing treatments. SDS-PAGE and immunoblotting analysis of the native and heat-denatured proteins that were previously subjected to in vitro simulated gastric fluid digestions indicate that stable antigenic peptides were produced. Both enzyme-to-substrate ratio and digestion time were influential in determining the stability of pecan polypeptides. The stable antigenic polypeptides may serve as useful markers in developing assays suitable for the detection of trace amounts of pecans in foods.

  13. Characterization of Whey Protein Oil-In-Water Emulsions with Different Oil Concentrations Stabilized by Ultra-High Pressure Homogenization

    Directory of Open Access Journals (Sweden)

    Essam Hebishy

    2017-02-01

    Full Text Available In this study, the effect of ultra-high-pressure homogenization (UHPH: 100 or 200 MPa at 25 °C, in comparison to colloid mill (CM: 5000 rpm at 20 °C and conventional homogenization (CH: 15 MPa at 60 °C, on the stability of oil-in-water emulsions with different oil concentrations (10, 30 or 50 g/100 g emulsified by whey protein isolate (4 g/100 g was investigated. Emulsions were characterized for their microstructure, rheological properties, surface protein concentration (SPC, stability to creaming and oxidative stability under light (2000 lux/m2. UHPH produced emulsions containing lipid droplets in the sub-micron range (100–200 nm and with low protein concentrations on droplet surfaces. Droplet size (d3.2, µm was increased in CH and UHPH emulsions by increasing the oil concentration. CM emulsions exhibited Newtonian flow behaviour at all oil concentrations studied; however, the rheological behaviour of CH and UHPH emulsions varied from Newtonian flow (n ≈ 1 to shear-thinning (n ˂ 1 and thixotropic behaviour in emulsions containing 50% oil. This was confirmed by the non-significant differences in the d4.3 (µm value between the top and bottom of emulsions in tubes left at room temperature for nine days and also by a low migration velocity measured with a Turbiscan LAB instrument. UHPH emulsions showed significantly lower oxidation rates during 10 days storage in comparison to CM and CH emulsions as confirmed by hydroperoxides and thiobarbituric acid-reactive substances (TBARS. UHPH emulsions treated at 100 MPa were less oxidized than those treated at 200 MPa. The results from this study suggest that UHPH treatment generates emulsions that have a higher stability to creaming and lipid oxidation compared to colloid mill and conventional treatments.

  14. Urea recycling contributes to nitrogen retention in calves fed milk replacer and low-protein solid feed.

    Science.gov (United States)

    Berends, Harma; van den Borne, Joost J G C; Røjen, Betina A; van Baal, Jürgen; Gerrits, Walter J J

    2014-07-01

    Urea recycling, with urea originating from catabolism of amino acids and hepatic detoxification of ammonia, is particularly relevant for ruminant animals, in which microbial protein contributes substantially to the metabolizable protein supply. However, the quantitative contribution of urea recycling to protein anabolism in calves during the transition from preruminants (milk-fed calves) to ruminants [solid feed (SF)-fed calves] is unknown. The aim of this study was to quantify urea recycling in milk-fed calves when provided with low-protein SF. Forty-eight calves [164 ± 1.6 kg body weight (BW)] were assigned to 1 of 4 SF levels [0, 9, 18, and 27 g of dry matter (DM) SF · kg BW(-0.75) · d⁻¹] provided in addition to an identical amount of milk replacer. Urea recycling was quantified after a 24-h intravenous infusion of [¹⁵N₂]urea by analyzing urea isotopomers in 68-h fecal and urinary collections. Real-time qPCR was used to measure gene expression levels of bovine urea transporter B (bUTB) and aquaglyceroporin-3 and aquaglyceroporin-7 in rumen wall tissues. For every incremental gram of DM SF intake (g DM · kg(0.75)), nitrogen intake increased by 0.70 g, and nitrogen retention increased by 0.55 g (P intake, but aquaglyceroporin-7 expression did not. We conclude that in addition to the increase in digested nitrogen, urea recycling contributes to the observed increase in nitrogen retention with increasing SF intake in milk-fed calves. Furthermore, ruminal bUTB and aquaglyceroporin-3 expression are upregulated with SF intake, which might be associated with urea recycling. © 2014 American Society for Nutrition.

  15. Changing folding and binding stability in a viral coat protein: a comparison between substitutions accessible through mutation and those fixed by natural selection.

    Science.gov (United States)

    Miller, Craig R; Lee, Kuo Hao; Wichman, Holly A; Ytreberg, F Marty

    2014-01-01

    Previous studies have shown that most random amino acid substitutions destabilize protein folding (i.e. increase the folding free energy). No analogous studies have been carried out for protein-protein binding. Here we use a structure-based model of the major coat protein in a simple virus, bacteriophage φX174, to estimate the free energy of folding of a single coat protein and binding of five coat proteins within a pentameric unit. We confirm and extend previous work in finding that most accessible substitutions destabilize both protein folding and protein-protein binding. We compare the pool of accessible substitutions with those observed among the φX174-like wild phage and in experimental evolution with φX174. We find that observed substitutions have smaller effects on stability than expected by chance. An analysis of adaptations at high temperatures suggests that selection favors either substitutions with no effect on stability or those that simultaneously stabilize protein folding and slightly destabilize protein binding. We speculate that these mutations might involve adjusting the rate of capsid assembly. At normal laboratory temperature there is little evidence of directional selection. Finally, we show that cumulative changes in stability are highly variable; sometimes they are well beyond the bounds of single substitution changes and sometimes they are not. The variation leads us to conclude that phenotype selection acts on more than just stability. Instances of larger cumulative stability change (never via a single substitution despite their availability) lead us to conclude that selection views stability at a local, not a global, level.

  16. Activity dependent protein degradation is critical for the formation and stability of fear memory in the amygdala.

    Directory of Open Access Journals (Sweden)

    Timothy J Jarome

    Full Text Available Protein degradation through the ubiquitin-proteasome system [UPS] plays a critical role in some forms of synaptic plasticity. However, its role in memory formation in the amygdala, a site critical for the formation of fear memories, currently remains unknown. Here we provide the first evidence that protein degradation through the UPS is critically engaged at amygdala synapses during memory formation and retrieval. Fear conditioning results in NMDA-dependent increases in degradation-specific polyubiquitination in the amygdala, targeting proteins involved in translational control and synaptic structure and blocking the degradation of these proteins significantly impairs long-term memory. Furthermore, retrieval of fear memory results in a second wave of NMDA-dependent polyubiquitination that targets proteins involved in translational silencing and synaptic structure and is critical for memory updating following recall. These results indicate that UPS-mediated protein degradation is a major regulator of synaptic plasticity necessary for the formation and stability of long-term memories at amygdala synapses.

  17. LncRNA NEAT1 promotes autophagy in MPTP-induced Parkinson's disease through stabilizing PINK1 protein.

    Science.gov (United States)

    Yan, Wang; Chen, Zhao-Ying; Chen, Jia-Qi; Chen, Hui-Min

    2018-02-19

    Long non-coding RNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) was found to be closely related to the pathological changes in brain and nervous system. However, the role of NEAT1 and its potential mechanism in Parkinson's disease (PD) largely remain uncharacterized. In this study, PD mouse model was established by intraperitoneal injection of MPTP. The numbers of TH + neurons, NEAT1 expression and the level of PINK1, LC3-II, LC3-I protein were assessed in PD mice. SH-SY5Y cells were treated with MPP + as PD cell model. RNA pull-down assay was used to identify the interaction between NEAT1 and PINK1 in vitro. The endogenous expression of NEAT1 was modified by lentiviral vector carrying interference sequence for NEAT1 in vivo. The numbers of TH + neurons significantly decreased in PD mice compared with the control. The expressions of NEAT1, PINK1 protein and LC3-II/LC3-I level were increased by MPTP in vitro and in vivo. Moreover, NEAT1 positively regulated the protein level of PINK1 through inhibition of PINK1 protein degradation. And NEAT1 mediated the effects of MPP + on SH-SY5Y cells through stabilization of PINK1 protein. The results of in vivo experiments revealed that NEAT1 knockdown could effectively suppress MPTP-induced autophagy in vivo that alleviated dopaminergic neuronal injury. LncRNA NEAT1 promoted the MPTP-induced autophagy in PD through stabilization of PINK1 protein. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Characterization, stoichiometry, and stability of salivary protein-tannin complexes by ESI-MS and ESI-MS/MS.

    Science.gov (United States)

    Canon, Francis; Paté, Franck; Meudec, Emmanuelle; Marlin, Thérèse; Cheynier, Véronique; Giuliani, Alexandre; Sarni-Manchado, Pascale

    2009-12-01

    Numerous protein-polyphenol interactions occur in biological and food domains particularly involving proline-rich proteins, which are representative of the intrinsically unstructured protein group (IUP). Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), which also gives access to ligand binding stoichiometry. Surprisingly, the study of interactions between polyphenolic molecules and proteins is still an area where ESI-MS has poorly benefited, whereas it has been extensively applied to the detection of noncovalent complexes. Electrospray ionization mass spectrometry has been applied to the detection and the characterization of the complexes formed between tannins and a human salivary proline-rich protein (PRP), namely IB5. The study of the complex stability was achieved by low-energy collision-induced dissociation (CID) measurements, which are commonly implemented using triple quadrupole, hybrid quadrupole time-of-flight, or ion trap instruments. Complexes composed of IB5 bound to a model polyphenol EgCG have been detected by ESI-MS and further analyzed by MS/MS. Mild ESI interface conditions allowed us to observe intact noncovalent PRP-tannin complexes with stoichiometries ranging from 1:1 to 1:5. Thus, ESI-MS shows its efficiency for (1) the study of PRP-tannin interactions, (2) the determination of stoichiometry, and (3) the study of complex stability. We were able to establish unambiguously both their stoichiometries and their overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Our results prove that IB5.EgCG complexes are maintained intact in the gas phase.

  19. Contributions of herpes simplex virus type 1 envelope proteins to entry by endocytosis

    Science.gov (United States)

    Herpes simplex virus (HSV) proteins specifically required for endocytic entry but not direct penetration have not been identified. HSVs deleted of gE, gG, gI, gJ, gM, UL45, or Us9 entered cells via either pH-dependent or pH-independent endocytosis and were inactivated by mildly acidic pH. Thus, the ...

  20. The Contribution of Serine 194 Phosphorylation to Steroidogenic Acute Regulatory Protein Function

    OpenAIRE

    Sasaki, Goro; Zubair, Mohamad; Ishii, Tomohiro; Mitsui, Toshikatsu; Hasegawa, Tomonobu; Auchus, Richard J.

    2014-01-01

    The steroidogenic acute regulatory protein (StAR) facilitates the delivery of cholesterol to the inner mitochondrial membrane, where the cholesterol side-chain cleavage enzyme catalyzes the initial step of steroid hormone biosynthesis. StAR was initially identified in adrenocortical cells as a phosphoprotein, the expression and phosphorylation of which were stimulated by corticotropin. A number of in vitro studies have implicated cAMP-dependent phosphorylation at serine 194 (S194, S195 in hum...

  1. Selection for a Zinc-Finger Protein Contributes to Seed Oil Increase during Soybean Domestication.

    Science.gov (United States)

    Li, Qing-Tian; Lu, Xiang; Song, Qing-Xin; Chen, Hao-Wei; Wei, Wei; Tao, Jian-Jun; Bian, Xiao-Hua; Shen, Ming; Ma, Biao; Zhang, Wan-Ke; Bi, Ying-Dong; Li, Wei; Lai, Yong-Cai; Lam, Sin-Man; Shui, Guang-Hou; Chen, Shou-Yi; Zhang, Jin-Song

    2017-04-01

    Seed oil is a momentous agronomical trait of soybean ( Glycine max ) targeted by domestication in breeding. Although multiple oil-related genes have been uncovered, knowledge of the regulatory mechanism of seed oil biosynthesis is currently limited. We demonstrate that the seed-preferred gene GmZF351 , encoding a tandem CCCH zinc finger protein, is selected during domestication. Further analysis shows that GmZF351 facilitates oil accumulation by directly activating WRINKLED1 , BIOTIN CARBOXYL CARRIER PROTEIN2 , 3-KETOACYL-ACYL CARRIER PROTEIN SYNTHASE III , DIACYLGLYCEROL O-ACYLTRANSFERASE1 , and OLEOSIN2 in transgenic Arabidopsis ( Arabidopsis thaliana ) seeds. Overexpression of GmZF351 in transgenic soybean also activates lipid biosynthesis genes, thereby accelerating seed oil accumulation. The ZF351 haplotype from the cultivated soybean group and the wild soybean ( Glycine soja ) subgroup III correlates well with high gene expression level, seed oil contents and promoter activity, suggesting that selection of GmZF351 expression leads to increased seed oil content in cultivated soybean. Our study provides novel insights into the regulatory mechanism for seed oil accumulation, and the manipulation of GmZF351 may have great potential in the improvement of oil production in soybean and other related crops. © 2017 American Society of Plant Biologists. All Rights Reserved.

  2. The transient nature of Bunyamwera orthobunyavirus NSs protein expression: effects of increased stability of NSs protein on virus replication.

    Science.gov (United States)

    van Knippenberg, Ingeborg; Fragkoudis, Rennos; Elliott, Richard M

    2013-01-01

    The NSs proteins of bunyaviruses are the viral interferon antagonists, counteracting the host's antiviral response to infection. During high-multiplicity infection of cultured mammalian cells with Bunyamwera orthobunyavirus (BUNV), NSs is rapidly degraded after reaching peak levels of expression at 12hpi. Through the use of inhibitors this was shown to be the result of proteasomal degradation. A recombinant virus (rBUN4KR), in which all four lysine residues in NSs were replaced by arginine residues, expresses an NSs protein (NSs4KR) that is resistant to degradation, confirming that degradation is lysine-dependent. However, despite repeated attempts, no direct ubiquitylation of NSs in infected cells could be demonstrated. This suggests that degradation of NSs, although lysine-dependent, may be achieved through an indirect mechanism. Infection of cultured mammalian cells or mice indicated no disadvantage for the virus in having a non-degradable NSs protein: in fact rBUN4KR had a slight growth advantage over wtBUNV in interferon-competent cells, presumably due to the increased and prolonged presence of NSs. In cultured mosquito cells there was no difference in growth between wild-type BUNV and rBUN4KR, but surprisingly NSs4KR was not stabilised compared to the wild-type NSs protein.

  3. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins

    OpenAIRE

    Chae, Pil Seok; Rasmussen, Søren G. F.; Rana, Rohini; Gotfryd, Kamil; Chandra, Richa; Goren, Michael A.; Kruse, Andrew C.; Nurva, Shailika; Loland, Claus J.; Pierre, Yves; Drew, David; Popot, Jean-Luc; Picot, Daniel; Fox, Brian G.; Guan, Lan

    2010-01-01

    The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces displayed by native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each of which is built around a central quaternary carbon atom derived from...

  4. Respective contribution of CML8 and CML9, two arabidopsis calmodulin-like proteins, to plant stress responses.

    Science.gov (United States)

    Zhu, Xiaoyang; Perez, Manon; Aldon, Didier; Galaud, Jean-Philippe

    2017-05-04

    In their natural environment, plants have to continuously face constraints such as biotic and abiotic stresses. To achieve their life cycle, plants have to perceive and interpret the nature, but also the strength of environmental stimuli to activate appropriate physiological responses. Nowadays, it is well established that signaling pathways are crucial steps in the implementation of rapid and efficient plant responses such as genetic reprogramming. It is also reported that rapid raises in calcium (Ca 2+ ) levels within plant cells participate in these early signaling steps and are essential to coordinate adaptive responses. However, to be informative, calcium increases need to be decoded and relayed by calcium-binding proteins also referred as calcium sensors to carry-out the appropriate responses. In a recent study, we showed that CML8, an Arabidopsis calcium sensor belonging to the calmodulin-like (CML) protein family, promotes plant immunity against the phytopathogenic bacteria Pseudomonas syringae pv tomato (strain DC3000). Interestingly, other CML proteins such as CML9 were also reported to contribute to plant immunity using the same pathosystem. In this addendum, we propose to discuss about the specific contribution of these 2 CMLs in stress responses.

  5. Combining structural modeling with ensemble machine learning to accurately predict protein fold stability and binding affinity effects upon mutation.

    Directory of Open Access Journals (Sweden)

    Niklas Berliner

    Full Text Available Advances in sequencing have led to a rapid accumulation of mutations, some of which are associated with diseases. However, to draw mechanistic conclusions, a biochemical understanding of these mutations is necessary. For coding mutations, accurate prediction of significant changes in either the stability of proteins or their affinity to their binding partners is required. Traditional methods have used semi-empirical force fields, while newer methods employ machine learning of sequence and structural features. Here, we show how combining both of these approaches leads to a marked boost in accuracy. We introduce ELASPIC, a novel ensemble machine learning approach that is able to predict stability effects upon mutation in both, domain cores and domain-domain interfaces. We combine semi-empirical energy terms, sequence conservation, and a wide variety of molecular details with a Stochastic Gradient Boosting of Decision Trees (SGB-DT algorithm. The accuracy of our predictions surpasses existing methods by a considerable margin, achieving correlation coefficients of 0.77 for stability, and 0.75 for affinity predictions. Notably, we integrated homology modeling to enable proteome-wide prediction and show that accurate prediction on modeled structures is possible. Lastly, ELASPIC showed significant differences between various types of disease-associated mutations, as well as between disease and common neutral mutations. Unlike pure sequence-based prediction methods that try to predict phenotypic effects of mutations, our predictions unravel the molecular details governing the protein instability, and help us better understand the molecular causes of diseases.

  6. Dissecting the Photoprotective Mechanism Encoded by the flv4-2 Operon: a Distinct Contribution of Sll0218 in Photosystem II Stabilization.

    Science.gov (United States)

    Bersanini, Luca; Allahverdiyeva, Yagut; Battchikova, Natalia; Heinz, Steffen; Lespinasse, Maija; Ruohisto, Essi; Mustila, Henna; Nickelsen, Jörg; Vass, Imre; Aro, Eva-Mari

    2017-03-01

    In Synechocystis sp. PCC 6803, the flv4-2 operon encodes the flavodiiron proteins Flv2 and Flv4 together with a small protein, Sll0218, providing photoprotection for Photosystem II (PSII). Here, the distinct roles of Flv2/Flv4 and Sll0218 were addressed, using a number of flv4-2 operon mutants. In the ∆sll0218 mutant, the presence of Flv2/Flv4 rescued PSII functionality as compared with ∆sll0218-flv2, where neither Sll0218 nor the Flv2/Flv4 heterodimer are expressed. Nevertheless, both the ∆sll0218 and ∆sll0218-flv2 mutants demonstrated deficiency in accumulation of PSII proteins suggesting a role for Sll0218 in PSII stabilization, which was further supported by photoinhibition experiments. Moreover, the accumulation of PSII assembly intermediates occurred in Sll0218-lacking mutants. The YFP-tagged Sll0218 protein localized in a few spots per cell at the external side of the thylakoid membrane, and biochemical membrane fractionation revealed clear enrichment of Sll0218 in the PratA-defined membranes, where the early biogenesis steps of PSII occur. Further, the characteristic antenna uncoupling feature of the ∆flv4-2 operon mutants is shown to be related to PSII destabilization in the absence of Sll0218. It is concluded that the Flv2/Flv4 heterodimer supports PSII functionality, while the Sll0218 protein assists PSII assembly and stabilization, including optimization of light harvesting. © 2016 The Authors. Plant, Cell & Enviroment Published by John Wiley & Sons Ltd.

  7. Sedimentation equilibrium of a small oligomer-forming membrane protein: effect of histidine protonation on pentameric stability.

    Science.gov (United States)

    Surya, Wahyu; Torres, Jaume

    2015-04-02

    Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single α-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.

  8. The actin family protein ARP6 contributes to the structure and the function of the nucleolus

    Czech Academy of Sciences Publication Activity Database

    Kitamura, H.; Matsumori, H.; Kalendová, Alžběta; Hozák, Pavel; Goldberg, I.G.; Nakao, M.; Saitoh, N.; Harata, M.

    2015-01-01

    Roč. 464, č. 2 (2015), s. 554-560 ISSN 0006-291X R&D Projects: GA MŠk EE2.3.30.0050; GA MŠk(CZ) ED1.1.00/02.0109 Grant - others:Human Frontier in Science programe(FR) RGP0017/2013 Institutional support: RVO:68378050 Keywords : Actin-related protein * ARP6 * Histone H2A.Z * Nucleolus * Wndchrm Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.371, year: 2015

  9. The transient nature of bunyamwera orthobunyavirus NSs protein expression : effects of increased stability of NSs protein on virus replication

    OpenAIRE

    van Knippenberg, Ingeborg; Fragkoudis, Rennos; Elliott, Richard M.

    2013-01-01

    The NSs proteins of bunyaviruses are the viral interferon antagonists, counteracting the host's antiviral response to infection. During high-multiplicity infection of cultured mammalian cells with Bunyamwera orthobunyavirus (BUNV), NSs is rapidly degraded after reaching peak levels of expression at 12hpi. Through the use of inhibitors this was shown to be the result of proteasomal degradation. A recombinant virus (rBUN4KR), in which all four lysine residues in NSs were replaced by arginine re...

  10. Coevolving residues of (beta/alpha)(8)-barrel proteins play roles in stabilizing active site architecture and coordinating protein dynamics.

    Science.gov (United States)

    Shen, Hongbo; Xu, Feng; Hu, Hairong; Wang, Feifei; Wu, Qi; Huang, Qiang; Wang, Honghai

    2008-12-01

    Indole-3-glycerol phosphate synthase (IGPS) is a representative of (beta/alpha)(8)-barrel proteins-the most common enzyme fold in nature. To better understand how the constituent amino-acids work together to define the structure and to facilitate the function, we investigated the evolutionary and dynamical coupling of IGPS residues by combining statistical coupling analysis (SCA) and molecular dynamics (MD) simulations. The coevolving residues identified by the SCA were found to form a network which encloses the active site completely. The MD simulations showed that these coevolving residues are involved in the correlated and anti-correlated motions. The correlated residues are within van der Waals contact and appear to maintain the active site architecture; the anti-correlated residues are mainly distributed on opposite sides of the catalytic cavity and coordinate the motions likely required for the substrate entry and product release. Our findings might have broad implications for proteins with the highly conserved (betaalpha)(8)-barrel in assessing the roles of amino-acids that are moderately conserved and not directly involved in the active site of the (beta/alpha)(8)-barrel. The results of this study could also provide useful information for further exploring the specific residue motions for the catalysis and protein design based on the (beta/alpha)(8)-barrel scaffold.

  11. Glucose-Neopentyl Glycol (GNG) Amphiphiles for Membrane Protein Solubilization, Stabilization and Crystallization

    Science.gov (United States)

    Rana, Rohini R.; Gotfryd, Kamil; Rasmussen, Søren G. F.; Kruse, Andrew C.; Cho, Kyung Ho; Capaldi, Stefano; Carlsson, Emil; Kobilka, Brian; Loland, Claus J.; Gether, Ulrik; Banerjee, Surajit

    2012-01-01

    The development of a new class of surfactants for membrane protein manipulation, “GNG amphiphiles”, is reported. These amphiphiles display promising behavior for membrane proteins, as demonstrated recently by the high resolution structure of a sodium-pumping pyrophosphatase reported by Kellosalo et al. PMID:23165475

  12. Glucose-Neopentyl Glycol (GNG) Amphiphiles for Membrane Protein Solubilization, Stabilization and Crystallization

    OpenAIRE

    Chae, Pil Seok; Rana, Rohini R.; Gotfryd, Kamil; Rasmussen, Søren G. F.; Kruse, Andrew C.; Cho, Kyung Ho; Capaldi, Stefano; Carlsson, Emil; Kobilka, Brian; Loland, Claus J.; Gether, Ulrik; Banerjee, Surajit; Byrne, Bernadette; Lee, John K.; Gellman, Samuel H.

    2013-01-01

    The development of a new class of surfactants for membrane protein manipulation, “GNG amphiphiles”, is reported. These amphiphiles display promising behavior for membrane proteins, as demonstrated recently by the high resolution structure of a sodium-pumping pyrophosphatase reported by Kellosalo et al.

  13. 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

  14. Light assisted drying (LAD) for protein stabilization: optical characterization of samples

    Science.gov (United States)

    Young, Madison A.; McKinnon, Madison E.; Elliott, Gloria D.; Trammell, Susan R.

    2018-02-01

    Light-Assisted Drying (LAD) is a novel biopreservation technique which allows proteins to be immobilized in a dry, amorphous solid at room temperature. Indicator proteins are used in a variety of diagnostic assays ranging from highthroughput 96-well plates to new microfluidic devices. A challenge in the development of protein-based assays is preserving the structure of the protein during production and storage of the assay, as the structure of the protein is responsible for its functional activity. Freeze-drying or freezing are currently the standard for the preservation of proteins, but these methods are expensive and can be challenging in some environments due to a lack of available infrastructure. An inexpensive, simple processing method that enables supra-zero temperature storage of proteins used in assays is needed. Light-assisted drying offers a relatively inexpensive method for drying samples. Proteins suspended in a trehalose solution are dehydrated using near-infrared laser light. The laser radiation speeds drying and as water is removed the sugar forms a protective matrix. The goal of this study is optically characterize samples processed with LAD. We use polarized light imaging (PLI) to look at crystallization kinetics of samples and determine optimal humidity. PLI shows a 62.5% chance of crystallization during LAD processing and negligible crystallization during low RH storage.

  15. CNA web server: rigidity theory-based thermal unfolding simulations of proteins for linking structure, (thermo-)stability, and function.

    Science.gov (United States)

    Krüger, Dennis M; Rathi, Prakash Chandra; Pfleger, Christopher; Gohlke, Holger

    2013-07-01

    The Constraint Network Analysis (CNA) web server provides a user-friendly interface to the CNA approach developed in our laboratory for linking results from rigidity analyses to biologically relevant characteristics of a biomolecular structure. The CNA web server provides a refined modeling of thermal unfolding simulations that considers the temperature dependence of hydrophobic tethers and computes a set of global and local indices for quantifying biomacromolecular stability. From the global indices, phase transition points are identified where the structure switches from a rigid to a floppy state; these phase transition points can be related to a protein's (thermo-)stability. Structural weak spots (unfolding nuclei) are automatically identified, too; this knowledge can be exploited in data-driven protein engineering. The local indices are useful in linking flexibility and function and to understand the impact of ligand binding on protein flexibility. The CNA web server robustly handles small-molecule ligands in general. To overcome issues of sensitivity with respect to the input structure, the CNA web server allows performing two ensemble-based variants of thermal unfolding simulations. The web server output is provided as raw data, plots and/or Jmol representations. The CNA web server, accessible at http://cpclab.uni-duesseldorf.de/cna or http://www.cnanalysis.de, is free and open to all users with no login requirement.

  16. Characterization of the influence of 1-butyl-3-methylimidazolium chloride on the structure and thermal stability of green fluorescent protein

    International Nuclear Information System (INIS)

    Heller, William T.; O'Neill, Hugh Michael; Zhang, Qiu; Baker, Gary A.

    2010-01-01

    Ionic liquids (ILs) are finding a vast array of applications as novel solvents for a wide variety of processes that include enzymatic chemistry, particularly as more biocompatible ILs are designed and discovered. While it is assumed that a native or near-native structure is required for enzymatic activity, there is some evidence that ILs alter protein structure and oligomerization states in a manner than can negatively impact function. The IL 1-butyl-3-methylimidazolium chloride, (bmim)Cl, is a well-studied, water-miscible member of the popular 1-alkyl-3-methylimidazolium IL family. To improve our understanding of the impact of water-miscible ILs on proteins, we have characterized the structure and oligomerization state of green fluorescent protein (GFP) in aqueous solutions containing 25 and 50 vol % (bmim)Cl using a combination of optical spectroscopy and small-angle neutron scattering (SANS). Measurements were also performed as a function of temperature to provide insight into the effect of the IL on the thermal stability of GFP. While GFP exists as a dimer in water, the presence of 25 vol % (bmim)Cl causes GFP to transition to a monomeric state. The SANS data indicate that GFP is a great deal less compact in 50 vol % (bmim)Cl than in neat water, indicative of unfolding from the native structure. The oligomerization state of the protein in IL-containing aqueous solution changes from a dimer to a monomer in response to the IL, but does not change as a function of temperature in the IL-containing solution. The SANS and spectroscopic results also demonstrate that the addition of (bmim)Cl to the solution decreases the thermal stability of GFP, allowing the protein to unfold at lower temperatures than in aqueous solution.

  17. Contribution of cellular retinol-binding protein type 1 to retinol metabolism during mouse development.

    Science.gov (United States)

    Matt, Nicolas; Schmidt, Carsten K; Dupé, Valérie; Dennefeld, Christine; Nau, Heinz; Chambon, Pierre; Mark, Manuel; Ghyselinck, Norbert B

    2005-05-01

    Within cells, retinol (ROL) is bound to cytoplasmic proteins (cellular retinol-binding proteins [CRBPs]), whose proposed function is to protect it from unspecific enzymes through channeling to retinoid-metabolizing pathways. We show that, during development, ROL and retinyl ester levels are decreased in CRBP type 1 (CRBP1) -deficient embryos and fetuses by 50% and 80%, respectively. The steady state level of retinoic acid (RA) is also decreased but to a lesser extent. However, CRBP1-null fetuses do not exhibit the abnormalities characteristic of a vitamin A-deficiency syndrome. Neither CRBP1 deficiency alters the expression patterns of RA-responding genes during development, nor does CRBP1 availability modify the expression of an RA-dependent gene in primary embryonic fibroblasts treated with ROL. Therefore, CRBP1 is required in prenatal life to maintain normal amounts of ROL and to ensure its efficient storage but seems of secondary importance for RA synthesis, at least under conditions of maternal vitamin A sufficiency. Copyright 2005 Wiley-Liss, Inc.

  18. Contribution of a natural polymorphism, protein kinase G, modulates electroconvulsive seizure recovery in D. melanogaster.

    Science.gov (United States)

    Kelly, Stephanie P; Risley, Monica G; Miranda, Leonor E; Dawson-Scully, Ken

    2018-05-24

    Drosophila melanogaster is a well-characterized model for neurological disorders and is widely used for investigating causes of altered neuronal excitability leading to seizure-like behavior. One method used to analyze behavioral output of neuronal perturbance is recording the time to locomotor recovery from an electroconvulsive shock. Based on this behavior, we sought to quantify seizure susceptibility in larval D. melanogaster with differences in the enzymatic activity levels of a major protein, cGMP-dependent protein kinase (PKG). PKG, encoded by foraging , has two natural allelic variants and has previously been implicated in several important physiological characteristics including: foraging patterns, learning and memory, and environmental stress tolerance. The well-established NO/cGMP/PKG signaling pathway found in the fly, which potentially targets downstream K + channel(s), which ultimately impacts membrane excitability; leading to our hypothesis: altering PKG enzymatic activity modulates time to recovery from an electroconvulsive seizure. Our results show that by both genetically and pharmacologically increasing PKG enzymatic activity, we can decrease the locomotor recovery time from an electroconvulsive seizure in larval D. melanogaster . © 2018. Published by The Company of Biologists Ltd.

  19. Physical Stability of Octenyl Succinate-Modified Polysaccharides and Whey Proteins for Potential Use as Bioactive Carriers in Food Systems.

    Science.gov (United States)

    Puerta-Gomez, Alex F; Castell-Perez, M Elena

    2015-06-01

    The high cost and potential toxicity of biodegradable polymers like poly(lactic-co-glycolic)acid (PLGA) has increased the interest in natural and modified biopolymers as bioactive carriers. This study characterized the physical stability (water sorption and state transition behavior) of selected starch and proteins: octenyl succinate-modified depolymerized waxy corn starch (DWxCn), waxy rice starch (DWxRc), phytoglycogen, whey protein concentrate (80%, WPC), whey protein isolate (WPI), and α-lactalbumin (α-L) to determine their potential as carriers of bioactive compounds under different environmental conditions. After enzyme modification and particle size characterization, glass transition temperature and moisture isotherms were used to characterize the systems. DWxCn and DWxRc had increased water sorption compared to native starch. The level of octenyl succinate anhydrate (OSA) modification (3% and 7%) did not reduce the water sorption of the DWxCn and phytoglycogen samples. The Guggenheim-Andersen-de Boer model indicated that native waxy corn had significantly (P whey proteins had higher glass transition temperature (Tg) values. On the other hand, depolymerized waxy starches at 7%-OSA modification had a "melted" appearance when exposed to environments with high relative humidity (above 70%) after 10 days at 23 °C. The use of depolymerized and OSA-modified polysaccharides blended with proteins created more stable blends of biopolymers. Hence, this biopolymer would be suitable for materials exposed to high humidity environments in food applications. © 2015 Institute of Food Technologists®

  20. The amino terminal end determines the stability and assembling capacity of eukaryotic ribosomal stalk proteins P1 and P2.

    Science.gov (United States)

    Camargo, Hendricka; Nusspaumer, Gretel; Abia, David; Briceño, Verónica; Remacha, Miguel; Ballesta, Juan P G

    2011-05-01

    The eukaryotic ribosomal proteins P1 and P2 bind to protein P0 through their N-terminal domain to form the essential ribosomal stalk. A mutational analysis points to amino acids at positions 2 and 3 as determinants for the drastic difference of Saccharomyces cerevisiae P1 and P2 half-life, and suggest different degradation mechanisms for each protein type. Moreover, the capacity to form P1/P2 heterodimers is drastically affected by mutations in the P2β four initial amino acids, while these mutations have no effect on P1β. Binding of P2β and, to a lesser extent, P1β to the ribosome is also seriously affected showing the high relevance of the amino acids in the first turn of the NTD α-helix 1 for the stalk assembly. The negative effect of some mutations on ribosome binding can be reversed by the presence of the second P1/P2 couple in the ribosome, indicating a stabilizing structural influence between the two heterodimers. Unexpectedly, some mutations totally abolish heterodimer formation but allow significant ribosome binding and, therefore, a previous P1 and P2 association seems not to be an absolute requirement for stalk assembly. Homology modeling of the protein complexes suggests that the mutated residues can affect the overall protein conformation. © The Author(s) 2011. Published by Oxford University Press.

  1. [Blood plasma protein adsorption capacity of perfluorocarbon emulsion stabilized by proxanol 268 (in vitro and in vivo studies)].

    Science.gov (United States)

    Sklifas, A N; Zhalimov, V K; Temnov, A A; Kukushkin, N I

    2012-01-01

    The adsorption abilities of the perfluorocarbon emulsion stabilized by Proxanol 268 were investigated in vitro and in vivo. In vitro, the saturation point for the blood plasma proteins was nearly reached after five minutes of incubation of the emulsion with human/rabbit blood plasma and was stable for all incubation periods studied. The decrease in volume ratio (emulsion/plasma) was accompanied by the increase in the adsorptive capacity of the emulsion with maximal values at 1/10 (3.2 and 1.5 mg of proteins per 1 ml of the emulsion, for human and rabbit blood plasma, respectively) that was unchanged at lower ratios. In vivo, in rabbits, intravenously injected with the emulsion, the proteins with molecular masses of 12, 25, 32, 44, 55, 70, and 200 kDa were adsorbed by the emulsion (as in vitro) if it was used 6 hours or less before testing. More delayed testing (6 h) revealed elimination of proteins with molecular masses of 25 and 44 kDa and an additional pool of adsorpted new ones of 27, 50, and 150 kDa. Specific adsorptive capacity of the emulsion enhanced gradually after emulsion injection and reached its maximum (3.5-5 mg of proteins per 1 ml of the emulsion) after 24 hours.

  2. Stability of cardamom (Elettaria cardamomum) essential oil in microcapsules made of whey protein isolate, guar gum, and carrageenan.

    Science.gov (United States)

    Mehyar, Ghadeer F; Al-Ismail, Khalid M; Al-Isamil, Khalid M; Al-Ghizzawi, Hana'a M; Holley, Richard A

    2014-10-01

    The effects of microencapsulating cardamom essential oil (CEO) in whey protein isolate (WPI) alone and combined with guar gum (GG) and carrageen (CG) on microencapsulation efficiency, oil chemical stability, and microcapsule structure were investigated. Freeze-dried microcapsules were prepared from emulsions containing (w/w): 15% and 30% WPI; 0.1% GG, and 0.2% CG as wall materials with CEO (at 10% of polymer concentration) as core material, and physical properties and chemical stability were compared. Bulk density of microcapsules was highest in WPI without GG or CG and in 30% WPI + GG microcapsules, and was more affected by moisture content (r = -0.6) than by mean particle diameter (d43 ; r = -0.2) and span (r = 0.1). Microcapsules containing only WPI had the highest entrapped oil (7.5%) and microencapsulation efficiency (98.5%). The concentrations of 1,8-cineole and d-limonene were used as indicators for microcapsule chemical stability since they were the main components of CEO. Microcapsules retained higher (P ≤ 0.05) concentrations of both components than non-microencapsulated CEO during 16 wk storage at 20 ºC, but higher loss of both components was noted at 35 ºC. Microencapsulated d-limonene was reduced faster than 1,8-cineole regardless of temperature. The 30% WPI and 30% WPI + GG microcapsules retained CEO best throughout storage at both storage temperatures. Scanning electron micrographs revealed that WPI microcapsules had smooth surfaces, were relatively homogenous and regular in shape, whereas GG and CG addition increased visual surface porosity and reduced shape regularity. It was concluded that the best formulation for encapsulating CEO was 30% WPI. Encapsulating cardamom essential oil in whey protein isolate alone or combined with guar gum produced dried powders that effectively retained and chemically stabilized CEO, and therefore enhanced its handling and storability. © 2014 Institute of Food Technologists®

  3. Delayed expression of cell cycle proteins contributes to astroglial scar formation and chronic inflammation after rat spinal cord contusion

    Directory of Open Access Journals (Sweden)

    Wu Junfang

    2012-07-01

    Full Text Available Abstract Background Traumatic spinal cord injury (SCI induces secondary tissue damage that is associated with astrogliosis and inflammation. We previously reported that acute upregulation of a cluster of cell-cycle-related genes contributes to post-mitotic cell death and secondary damage after SCI. However, it remains unclear whether cell cycle activation continues more chronically and contributes to more delayed glial change. Here we examined expression of cell cycle-related proteins up to 4 months following SCI, as well as the effects of the selective cyclin-dependent kinase (CDKs inhibitor CR8, on astrogliosis and microglial activation in a rat SCI contusion model. Methods Adult male rats were subjected to moderate spinal cord contusion injury at T8 using a well-characterized weight-drop model. Tissue from the lesion epicenter was obtained 4 weeks or 4 months post-injury, and processed for protein expression and lesion volume. Functional recovery was assessed over the 4 months after injury. Results Immunoblot analysis demonstrated a marked continued upregulation of cell cycle-related proteins − including cyclin D1 and E, CDK4, E2F5 and PCNA − for 4 months post-injury that were highly expressed by GFAP+ astrocytes and microglia, and co-localized with inflammatory-related proteins. CR8 administrated systemically 3 h post-injury and continued for 7 days limited the sustained elevation of cell cycle proteins and immunoreactivity of GFAP, Iba-1 and p22PHOX − a key component of NADPH oxidase − up to 4 months after SCI. CR8 treatment significantly reduced lesion volume, which typically progressed in untreated animals between 1 and 4 months after trauma. Functional recovery was also significantly improved by CR8 treatment after SCI from week 2 through week 16. Conclusions These data demonstrate that cell cycle-related proteins are chronically upregulated after SCI and may contribute to astroglial scar

  4. The role of nonlinear torsional contributions on the stability of flexural-torsional oscillations of open-cross section beams

    Science.gov (United States)

    Di Egidio, Angelo; Contento, Alessandro; Vestroni, Fabrizio

    2015-12-01

    An open-cross section thin-walled beam model, already developed by the authors, has been conveniently simplified while maintaining the capacity of accounting for the significant nonlinear warping effects. For a technical range of geometrical and mechanical characteristics of the beam, the response is characterized by the torsional curvature prevailing over the flexural ones. A Galerkin discretization is performed by using a suitable expansion of displacements based on shape functions. The attention is focused on the dynamic response of the beam to a harmonic force, applied at the free end of the cantilever beam. The excitation is directed along the symmetry axis of the beam section. The stability of the one-component oscillations has been investigated using the analytical model, showing the importance of the internal resonances due to the nonlinear warping coupling terms. Comparison with the results provided by a computational finite element model has been performed. The good agreement among the results of the analytical and the computational models confirms the effectiveness of the simplified model of a nonlinear open-cross section thin-walled beam and overall the important role of the warping and of the torsional elongation in the study of the one-component dynamic oscillations and their stability.

  5. Protein Kinase-C Beta Contributes to Impaired Endothelial Insulin Signaling in Humans with Diabetes Mellitus

    Science.gov (United States)

    Tabit, Corey E; Shenouda, Sherene M; Holbrook, Monica; Fetterman, Jessica L; Kiani, Soroosh; Frame, Alissa A; Kluge, Matthew A; Held, Aaron; Dohadwala, Mustali; Gokce, Noyan; Farb, Melissa; Rosenzweig, James; Ruderman, Neil; Vita, Joseph A; Hamburg, Naomi M

    2013-01-01

    Background Abnormal endothelial function promotes atherosclerotic vascular disease in diabetes. Experimental studies indicate that disruption of endothelial insulin signaling through the activity of protein kinase C-β (PKCβ) and nuclear factor κB (NFκB) reduces nitric oxide availability. We sought to establish whether similar mechanisms operate in the endothelium in human diabetes mellitus. Methods and Results We measured protein expression and insulin response in freshly isolated endothelial cells from patients with Type 2 diabetes mellitus (n=40) and non-diabetic controls (n=36). Unexpectedly, we observed 1.7-fold higher basal endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 in patients with diabetes (P=0.007) without a difference in total eNOS expression. Insulin stimulation increased eNOS phosphorylation in non-diabetic subjects but not in diabetic patients (P=0.003) consistent with endothelial insulin resistance. Nitrotyrosine levels were higher in diabetic patients indicating endothelial oxidative stress. PKCβ expression was higher in diabetic patients and was associated with lower flow-mediated dilation (r=−0.541, P=0.02) Inhibition of PKCβ with LY379196 reduced basal eNOS phosphorylation and improved insulin-mediated eNOS activation in patients with diabetes. Endothelial NFκB activation was higher in diabetes and was reduced with PKCβ inhibition. Conclusions We provide evidence for the presence of altered eNOS activation, reduced insulin action and inflammatory activation in the endothelium of patients with diabetes. Our findings implicate PKCβ activity in endothelial insulin resistance. PMID:23204109

  6. BAG3-mediated Mcl-1 stabilization contributes to drug resistance via interaction with USP9X in ovarian cancer.

    Science.gov (United States)

    Habata, Shutaro; Iwasaki, Masahiro; Sugio, Asuka; Suzuki, Miwa; Tamate, Masato; Satohisa, Seiro; Tanaka, Ryoichi; Saito, Tsuyoshi

    2016-07-01

    Paclitaxel in combination with carboplatin improves survival among patients with susceptible ovarian cancers, but no strategy has been established against resistant ovarian cancers. BAG3 (Bcl-2-associated athanogene 3) is one of six BAG family proteins, which are involved in such cellular processes as proliferation, migration and apoptosis. In addition, expression of BAG3 with Mcl-1, a Bcl-2 family protein, reportedly associates with resistance to chemotherapy. Our aim in this study was to evaluate the functional role of BAG3 and Mcl-1 in ovarian cancer chemoresistance and explore possible new targets for treatment. We found that combined expression of BAG3 and Mcl-1 was significantly associated with a poor prognosis in ovarian cancer patients. In vitro, BAG3 knockdown in ES2 clear ovarian cancer cells significantly increased the efficacy of paclitaxel in combination with the Mcl-1 antagonist MIM1, with or without the Bcl-2 family antagonist ABT737. Moreover, BAG3 was found to positively regulate Mcl-1 levels by binding to and inhibiting USP9X. Our data show that BAG3 and Mcl-1 are key mediators of resistance to chemotherapy in ovarian cancer. In BAG3 knockdown ES2 clear ovarian cancer cells, combination with ABT737 and MIM1 enhanced the efficacy of paclitaxel. These results suggest that inhibiting BAG3 in addition to anti-apoptotic Bcl-2 family proteins may be a useful therapeutic strategy for the treatment of chemoresistant ovarian cancers.

  7. Pre-analytical factors influencing the stability of cerebrospinal fluid proteins

    DEFF Research Database (Denmark)

    Simonsen, Anja H; Bahl, Justyna M C; Danborg, Pia B

    2013-01-01

    Cerebrospinal fluid (CSF) is a potential source for new biomarkers due to its proximity to the brain. This study aimed to clarify the stability of the CSF proteome when undergoing pre-analytical factors. We investigated the effects of repeated freeze/thaw cycles, protease inhibitors and delayed s...

  8. ERRα protein is stabilized by LSD1 in a demethylation-independent manner.

    Directory of Open Access Journals (Sweden)

    Julie Carnesecchi

    Full Text Available The LSD1 histone demethylase is highly expressed in breast tumors where it constitutes a factor of poor prognosis and promotes traits of cancer aggressiveness such as cell invasiveness. Recent work has shown that the Estrogen-Related Receptor α (ERRα induces LSD1 to demethylate the Lys 9 of histone H3. This results in the transcriptional activation of a number of common target genes, several of which being involved in cellular invasion. High expression of ERRα protein is also a factor of poor prognosis in breast tumors. Here we show that, independently of its demethylase activities, LSD1 protects ERRα from ubiquitination, resulting in overexpression of the latter protein. Our data also suggests that the elevation of LSD1 mRNA and protein in breast cancer (as compared to normal tissue may be a key event to increase ERRα protein, independently of its corresponding mRNA.

  9. ERRα protein is stabilized by LSD1 in a demethylation-independent manner.

    Science.gov (United States)

    Carnesecchi, Julie; Cerutti, Catherine; Vanacker, Jean-Marc; Forcet, Christelle

    2017-01-01

    The LSD1 histone demethylase is highly expressed in breast tumors where it constitutes a factor of poor prognosis and promotes traits of cancer aggressiveness such as cell invasiveness. Recent work has shown that the Estrogen-Related Receptor α (ERRα) induces LSD1 to demethylate the Lys 9 of histone H3. This results in the transcriptional activation of a number of common target genes, several of which being involved in cellular invasion. High expression of ERRα protein is also a factor of poor prognosis in breast tumors. Here we show that, independently of its demethylase activities, LSD1 protects ERRα from ubiquitination, resulting in overexpression of the latter protein. Our data also suggests that the elevation of LSD1 mRNA and protein in breast cancer (as compared to normal tissue) may be a key event to increase ERRα protein, independently of its corresponding mRNA.

  10. The challenges in and importance of analysing protein structure and physical stability in complex formulations

    DEFF Research Database (Denmark)

    Jorgensen, L.; Jensen, Minna Grønning; Roest, N.

    2013-01-01

    In this review several analytical challenges that may be encountered during protein formulation development of complex formulations are discussed through recent examples. These examples show how selected advanced biophysical methods can greatly increase our understanding of the system under...

  11. Electrochemical sensing of tumor suppressor protein p53-deoxyribonucleic acid complex stability at an electrified interface

    Czech Academy of Sciences Publication Activity Database

    Paleček, Emil; Černocká, Hana; Ostatná, Veronika; Navrátilová, Lucie; Brázdová, Marie

    2014-01-01

    Roč. 828, MAY2014 (2014), s. 1-8 ISSN 0003-2670 R&D Projects: GA ČR(CZ) GAP301/11/2055; GA ČR(CZ) GA13-00956S; GA ČR(CZ) GA13-36108S Institutional support: RVO:68081707 Keywords : Deoxyribonucleic acid-protein binding * Tumor suppressor protein p53 * Electrochemical sensing Subject RIV: BO - Biophysics Impact factor: 4.513, year: 2014

  12. Impact of egg white protein on the quality and stability of corn oil-in-water emulsion

    International Nuclear Information System (INIS)

    Iqbal, S.; Batool, J.; Ajaz, M.

    2017-01-01

    The effect of egg albumin has been examined on the texture and stability of O/W emulsion. The corn oil was used as dispersed phase while the aqueous phase as continuous phase of the emulsion. The aqueous phase was designed with the protein contents (0.5- 4 wt. %) at pH 7. The different oil phase (10-40 wt. %) were homogenized in aqueous phase (90-60 wt. %). It was observed that the viscosity and turbidity of the emulsion were increased with the increase of protein concentration and oil phase contents. Flow profile showed that shear stress was increased with increase of shear rate but it decreased at higher shear rate (100 s-1) in heated emulsion. On the other hand the emulsion viscosity was decreased with the increase of shear rate showing non- Newtonian behavior. This work may be useful in the formulation and physicochemical properties of food products i.e. sauces, mayonnaise etc. (author)

  13. Molecular features contributing to virus-independent intracellular localization and dynamic behavior of the herpesvirus transport protein US9.

    Directory of Open Access Journals (Sweden)

    Manuela Pedrazzi

    Full Text Available Reaching the right destination is of vital importance for molecules, proteins, organelles, and cargoes. Thus, intracellular traffic is continuously controlled and regulated by several proteins taking part in the process. Viruses exploit this machinery, and viral proteins regulating intracellular transport have been identified as they represent valuable tools to understand and possibly direct molecules targeting and delivery. Deciphering the molecular features of viral proteins contributing to (or determining this dynamic phenotype can eventually lead to a virus-independent approach to control cellular transport and delivery. From this virus-independent perspective we looked at US9, a virion component of Herpes Simplex Virus involved in anterograde transport of the virus inside neurons of the infected host. As the natural cargo of US9-related vesicles is the virus (or its parts, defining its autonomous, virus-independent role in vesicles transport represents a prerequisite to make US9 a valuable molecular tool to study and possibly direct cellular transport. To assess the extent of this autonomous role in vesicles transport, we analyzed US9 behavior in the absence of viral infection. Based on our studies, Us9 behavior appears similar in different cell types; however, as expected, the data we obtained in neurons best represent the virus-independent properties of US9. In these primary cells, transfected US9 mostly recapitulates the behavior of US9 expressed from the viral genome. Additionally, ablation of two major phosphorylation sites (i.e. Y32Y33 and S34ES36 have no effect on protein incorporation on vesicles and on its localization on both proximal and distal regions of the cells. These results support the idea that, while US9 post-translational modification may be important to regulate cargo loading and, consequently, virion export and delivery, no additional viral functions are required for US9 role in intracellular transport.

  14. The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes

    Science.gov (United States)

    Hari, Yvonne; Dugovič, Branislav; Istrate, Alena; Fignolé, Annabel; Leumann, Christian J.; Schürch, Stefan

    2016-07-01

    Tricyclo-DNA (tcDNA) is a sugar-modified analogue of DNA currently tested for the treatment of Duchenne muscular dystrophy in an antisense approach. Tandem mass spectrometry plays a key role in modern medical diagnostics and has become a widespread technique for the structure elucidation and quantification of antisense oligonucleotides. Herein, mechanistic aspects of the fragmentation of tcDNA are discussed, which lay the basis for reliable sequencing and quantification of the antisense oligonucleotide. Excellent selectivity of tcDNA for complementary RNA is demonstrated in direct competition experiments. Moreover, the kinetic stability and fragmentation pattern of matched and mismatched tcDNA heteroduplexes were investigated and compared with non-modified DNA and RNA duplexes. Although the separation of the constituting strands is the entropy-favored fragmentation pathway of all nucleic acid duplexes, it was found to be only a minor pathway of tcDNA duplexes. The modified hybrid duplexes preferentially undergo neutral base loss and backbone cleavage. This difference is due to the low activation entropy for the strand dissociation of modified duplexes that arises from the conformational constraint of the tc-sugar-moiety. The low activation entropy results in a relatively high free activation enthalpy for the dissociation comparable to the free activation enthalpy of the alternative reaction pathway, the release of a nucleobase. The gas-phase behavior of tcDNA duplexes illustrates the impact of the activation entropy on the fragmentation kinetics and suggests that tandem mass spectrometric experiments are not suited to determine the relative stability of different types of nucleic acid duplexes.

  15. Both noncoding and protein-coding RNAs contribute to gene expression evolution in the primate brain.

    Science.gov (United States)

    Babbitt, Courtney C; Fedrigo, Olivier; Pfefferle, Adam D; Boyle, Alan P; Horvath, Julie E; Furey, Terrence S; Wray, Gregory A

    2010-01-18

    Despite striking differences in cognition and behavior between humans and our closest primate relatives, several studies have found little evidence for adaptive change in protein-coding regions of genes expressed primarily in the brain. Instead, changes in gene expression may underlie many cognitive and behavioral differences. Here, we used digital gene expression: tag profiling (here called Tag-Seq, also called DGE:tag profiling) to assess changes in global transcript abundance in the frontal cortex of the brains of 3 humans, 3 chimpanzees, and 3 rhesus macaques. A substantial fraction of transcripts we identified as differentially transcribed among species were not assayed in previous studies based on microarrays. Differentially expressed tags within coding regions are enriched for gene functions involved in synaptic transmission, transport, oxidative phosphorylation, and lipid metabolism. Importantly, because Tag-Seq technology provides strand-specific information about all polyadenlyated transcripts, we were able to assay expression in noncoding intragenic regions, including both sense and antisense noncoding transcripts (relative to nearby genes). We find that many noncoding transcripts are conserved in both location and expression level between species, suggesting a possible functional role. Lastly, we examined the overlap between differential gene expression and signatures of positive selection within putative promoter regions, a sign that these differences represent adaptations during human evolution. Comparative approaches may provide important insights into genes responsible for differences in cognitive functions between humans and nonhuman primates, as well as highlighting new candidate genes for studies investigating neurological disorders.

  16. Heat shock protein-derived T-cell epitopes contribute to autoimmune inflammation in pediatric Crohn's disease.

    Directory of Open Access Journals (Sweden)

    Gisella L Puga Yung

    Full Text Available Pediatric Crohn's disease is a chronic auto inflammatory bowel disorder affecting children under the age of 17 years. A putative etiopathogenesis of Crohn's disease (CD is associated with disregulation of immune response to antigens commonly present in the gut microenvironment. Heat shock proteins (HSP have been identified as ubiquitous antigens with the ability to modulate inflammatory responses associated with several autoimmune diseases. The present study tested the contribution of immune responses to HSP in the amplification of autoimmune inflammation in chronically inflamed mucosa of pediatric CD patients. Colonic biopsies obtained from normal and CD mucosa were stimulated with pairs of Pan HLA-DR binder HSP60-derived peptides (human/bacterial homologues. The modulation of RNA and protein levels of induced proinflammatory cytokines were measured. We identified two epitopes capable of sustaining proinflammatory responses, specifically TNF< and IFN induction, in the inflamed intestinal mucosa in CD patients. The responses correlated positively with clinical and histological measurements of disease activity, thus suggesting a contribution of immune responses to HSP in pediatric CD site-specific mucosal inflammation.

  17. Stability of Antiradical Activity of Protein Extracts and Hydrolysates from Dry-Cured Pork Loins with Probiotic Strains of LAB

    Directory of Open Access Journals (Sweden)

    Paulina Kęska

    2018-04-01

    Full Text Available The application of starter cultures to improve quality and safety has become a very common practice in the meat industry. Probiotic strains of lactic acid bacteria (LAB can also bring health benefits by releasing bioactive peptides. The aim of this work was to evaluate the stability of antiradical activity of protein extracts from LAB-inoculated dry-cured pork loins during long-term aging and evaluate their hydrolysates after simulated gastrointestinal digestion. Analyses of hydrolysates by using liquid chromatography-tandem mass spectrometry (LC-MS/MS were strengthened with in silico analysis. The highest antiradical activity of the protein extracts was observed after 180 days of aging. The influence of the strain used (LOCK, BAUER, or BB12 on the inactivation ability of ABTS radicals varied during long-term aging. The IC50 values indicated the higher antiradical properties of salt-soluble (SSF compared to water-soluble fraction (WSF of proteins. The peptides generated by in vitro digestion have MW between 700 and 4232 Da and their length ranged from 5 to 47 amino acids in a sequence where Leu, Pro, Lys, Glu, and His had the largest share. This study demonstrates that the degradation of pork muscle proteins during gastrointestinal digestion may give rise to a wide variety of peptides with antiradical properties.

  18. Protein cross-linking by chlorinated polyamines and transglutamylation stabilizes neutrophil extracellular traps.

    Science.gov (United States)

    Csomós, Krisztián; Kristóf, Endre; Jakob, Bernadett; Csomós, István; Kovács, György; Rotem, Omri; Hodrea, Judit; Bagoly, Zsuzsa; Muszbek, Laszlo; Balajthy, Zoltán; Csősz, Éva; Fésüs, László

    2016-08-11

    Neutrophil extracellular trap (NET) ejected from activated dying neutrophils is a highly ordered structure of DNA and selected proteins capable to eliminate pathogenic microorganisms. Biochemical determinants of the non-randomly formed stable NETs have not been revealed so far. Studying the formation of human NETs we have observed that polyamines were incorporated into the NET. Inhibition of myeloperoxidase, which is essential for NET formation and can generate reactive chlorinated polyamines through hypochlorous acid, decreased polyamine incorporation. Addition of exogenous primary amines that similarly to polyamines inhibit reactions catalyzed by the protein cross-linker transglutaminases (TGases) has similar effect. Proteomic analysis of the highly reproducible pattern of NET components revealed cross-linking of NET proteins through chlorinated polyamines and ɛ(γ-glutamyl)lysine as well as bis-γ-glutamyl polyamine bonds catalyzed by the TGases detected in neutrophils. Competitive inhibition of protein cross-linking by monoamines disturbed the cross-linking pattern of NET proteins, which resulted in the loss of the ordered structure of the NET and significantly reduced capacity to trap bacteria. Our findings provide explanation of how NETs are formed in a reproducible and ordered manner to efficiently neutralize microorganisms at the first defense line of the innate immune system.

  19. Effects of solute-solute interactions on protein stability studied using various counterions and dendrimers.

    Directory of Open Access Journals (Sweden)

    Curtiss P Schneider

    Full Text Available Much work has been performed on understanding the effects of additives on protein thermodynamics and degradation kinetics, in particular addressing the Hofmeister series and other broad empirical phenomena. Little attention, however, has been paid to the effect of additive-additive interactions on proteins. Our group and others have recently shown that such interactions can actually govern protein events, such as aggregation. Here we use dendrimers, which have the advantage that both size and surface chemical groups can be changed and therein studied independently. Dendrimers are a relatively new and broad class of materials which have been demonstrated useful in biological and therapeutic applications, such as drug delivery, perturbing amyloid formation, etc. Guanidinium modified dendrimers pose an interesting case given that guanidinium can form multiple attractive hydrogen bonds with either a protein surface or other components in solution, such as hydrogen bond accepting counterions. Here we present a study which shows that the behavior of such macromolecule species (modified PAMAM dendrimers is governed by intra-solvent interactions. Attractive guanidinium-anion interactions seem to cause clustering in solution, which inhibits cooperative binding to the protein surface but at the same time, significantly suppresses nonnative aggregation.

  20. Characterization of the stability and bio-functionality of tethered proteins on bioengineered scaffolds: implications for stem cell biology and tissue repair.

    Science.gov (United States)

    Wang, Ting-Yi; Bruggeman, Kiara A F; Sheean, Rebecca K; Turner, Bradley J; Nisbet, David R; Parish, Clare L

    2014-05-23

    Various engineering applications have been utilized to deliver molecules and compounds in both innate and biological settings. In the context of biological applications, the timely delivery of molecules can be critical for cellular and organ function. As such, previous studies have demonstrated the superiority of long-term protein delivery, by way of protein tethering onto bioengineered scaffolds, compared with conventional delivery of soluble protein in vitro and in vivo. Despite such benefits little knowledge exists regarding the stability, release kinetics, longevity, activation of intracellular pathway, and functionality of these proteins over time. By way of example, here we examined the stability, degradation and functionality of a protein, glial-derived neurotrophic factor (GDNF), which is known to influence neuronal survival, differentiation, and neurite morphogenesis. Enzyme-linked immunosorbent assays (ELISA) revealed that GDNF, covalently tethered onto polycaprolactone (PCL) electrospun nanofibrous scaffolds, remained present on the scaffold surface for 120 days, with no evidence of protein leaching or degradation. The tethered GDNF protein remained functional and capable of activating downstream signaling cascades, as revealed by its capacity to phosphorylate intracellular Erk in a neural cell line. Furthermore, immobilization of GDNF protein promoted cell survival and differentiation in culture at both 3 and 7 days, further validating prolonged functionality of the protein, well beyond the minutes to hours timeframe observed for soluble proteins under the same culture conditions. This study provides important evidence of the stability and functionality kinetics of tethered molecules. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. UV-Visible intensity ratio (aggregates/single particles) as a measure to obtain stability of gold nanoparticles conjugated with protein A

    Energy Technology Data Exchange (ETDEWEB)

    Rios-Corripio, M. A. [Instituto Politecnico Nacional, CIBA-Tlaxcala (Mexico); Garcia-Perez, B. E. [Instituto Politecnico Nacional, Departamento de Inmunologia, ENCB (Mexico); Jaramillo-Flores, M. E. [Instituto Politecnico Nacional, Departamento de Ingenieria Bioquimica, ENCB (Mexico); Gayou, V. L.; Rojas-Lopez, M., E-mail: marlonrl@yahoo.com.mx [Instituto Politecnico Nacional, CIBA-Tlaxcala (Mexico)

    2013-05-15

    We have analyzed the titration process of gold nanoparticles with several amounts of protein A (0.3, 0.5, 1, 3, 6, and 9 {mu}g/ml) in the presence of NaCl, which induces aggregation if the surface of particles is not fully covered with protein A. The colloidal solutions with different particle size (16, 18, 20, 33 nm) were synthesized by citrate reduction to be conjugated with protein A. UV-Visible spectroscopy was used to measure the absorption of the surface plasmon resonance of gold nanoparticles as a function of the concentration of protein A. Such dependence shows an aggregation region (0 < x<6 {mu}g/ml), where the amount of protein A was insufficient to cover the surface of particles, obtaining aggregation caused by NaCl. The next part is the stability region (x {>=} 6 {mu}g/ml), where the amount of protein used covers the surface of particles and protects it from the aggregation. In addition to that the ratio between the intensities of both: the aggregates and of the gold nanoparticle bands was plotted as a function of the concentration of protein A. It was determined that 6 {mu}g/ml is a sufficient value of protein A to stabilize the gold nanoparticle-protein A system. This method provides a simple way to stabilize gold nanoparticles obtained by citrate reduction, with protein A.

  2. Stability, protein binding and clearance studies of [99mTc]DTPA. Evaluation of a commercially available dry-kit

    DEFF Research Database (Denmark)

    Rehling, M

    1988-01-01

    [99mTc]DTPA has achieved widespread use for the measurement of glomerular filtration rate (GFR) with the single injection plasma clearance technique and for gamma-camera renography. However, the quality of the commercial preparations varies. The purpose of the present investigation was to study...... the quality of a commercial [99mTc]DTPA preparation (C.I.S., France) with reference to stability, protein binding and accuracy of the determined plasma clearance values as a measure of GFR. The stability of the preparations was studied by thin-layer chromatography, the in vitro protein binding by Sephadex.......7% and 1.1%, respectively. The plasma clearance of [99mTc]DTPA was on an average 3.7% higher than that of [51Cr]EDTA in 27 patients. It is concluded that the [99mTc]DTPA preparation is reliable for the measurement of GFR. The preparation is stable for at least six hours at room temperature...

  3. Evaluation of the Potency, Neutralizing Antibody Response, and Stability of a Recombinant Fusion Protein Vaccine for Streptococcus pyogenes.

    Science.gov (United States)

    Burlet, E; HogenEsch, H; Dunham, A; Morefield, G

    2017-05-01

    Streptococcus pyogenes or group A streptococcus (GAS) is a Gram-positive bacterium that can cause a wide range of diseases, including pharyngitis, impetigo, scarlet fever, necrotizing fasciitis, rheumatic fever, and streptococcal toxic shock syndrome. Despite the increasing burden on global health caused by GAS, there is currently no licensed vaccine available. In this study, we evaluated immunogenicity, induction of neutralizing antibodies, and stability of a new recombinant fusion protein vaccine that targets infections from GAS. The recombinant fusion protein (SpeAB) combines inactive mutant forms of streptococcal pyrogenic exotoxin A (SpeA) and streptococcal pyrogenic exotoxin B (SpeB). The SpeAB vaccine evaluated in this study was adsorbed to an aluminum adjuvant and demonstrated robust immunogenicity, eliciting production of specific neutralizing antibodies against SpeA and SpeB, two major virulence factors of S. pyogenes. Stability studies suggest that the vaccine will retain immunogenicity for at least 2 years when stored at refrigerated temperatures. This novel vaccine shows great potential to provide protection against GAS infections and to reduce the burden of GAS disease globally.

  4. Extensive Lysine Methylation in Hyperthermophilic Crenarchaea: Potential Implications for Protein Stability and Recombinant Enzymes

    Directory of Open Access Journals (Sweden)

    Catherine H. Botting

    2010-01-01

    Full Text Available In eukarya and bacteria, lysine methylation is relatively rare and is catalysed by sequence-specific lysine methyltransferases that typically have only a single-protein target. Using RNA polymerase purified from the thermophilic crenarchaeum Sulfolobus solfataricus, we identified 21 methyllysines distributed across 9 subunits of the enzyme. The modified lysines were predominantly in α-helices and showed no conserved sequence context. A limited survey of the Thermoproteus tenax proteome revealed widespread modification with 52 methyllysines in 30 different proteins. These observations suggest the presence of an unusual lysine methyltransferase with relaxed specificity in the crenarchaea. Since lysine methylation is known to enhance protein thermostability, this may be an adaptation to a thermophilic lifestyle. The implications of this modification for studies and applications of recombinant crenarchaeal enzymes are discussed.

  5. Contributions to the stability analysis of self-similar supersonic heat waves related to inertial confinement fusion

    International Nuclear Information System (INIS)

    Dastugue, Laurent

    2013-01-01

    Exact self-similar solutions of gas dynamics equations with nonlinear heat conduction for semi-infinite slabs of perfect gases are used for studying the stability of flows in inertial confinement fusion. Both the similarity solutions and their linear perturbations are computed with a multi domain Chebyshev pseudo-spectral method, allowing us to account for, without any other approximation, compressibility and unsteadiness. Following previous results (Clarisse et al., 2008; Lombard, 2008) representative of the early ablation of a target by a nonuniform laser flux (electronic conduction, subsonic heat front downstream of a quasi-perfect shock front), we explore here other configurations. For this early ablation phase, but for a nonuniform incident X-radiation (radiative conduction), we study a compressible and a weakly compressible flow. In both cases, we recover the behaviours obtained for compressible flows with electronic heat conduction with a maximal instability for a zero wavenumber. Besides, the spectral method is extended to compute similarity solutions taking into account the supersonic heat wave ahead of the shock front. Based on an analysis of the reduced equations singularities (infinitely stiff front), this method allows us to describe the supersonic heat wave regime proper to the initial irradiation of the target and to recover the ablative solutions which were obtained under a negligible fore-running heat wave approximation. (author) [fr

  6. Protein secondary structure and stability determined by combining exoproteolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Villanueva, Josep; Villegas, Virtudes; Querol, Enrique; Avilés, Francesc X; Serrano, Luis

    2002-09-01

    In the post-genomic era, several projects focused on the massive experimental resolution of the three-dimensional structures of all the proteins of different organisms have been initiated. Simultaneously, significant progress has been made in the ab initio prediction of protein three-dimensional structure. One of the keys to the success of such a prediction is the use of local information (i.e. secondary structure). Here we describe a new limited proteolysis methodology, based on the use of unspecific exoproteases coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), to map quickly secondary structure elements of a protein from both ends, the N- and C-termini. We show that the proteolytic patterns (mass spectra series) obtained can be interpreted in the light of the conformation and local stability of the analyzed proteins, a direct correlation being observed between the predicted and the experimentally derived protein secondary structure. Further, this methodology can be easily applied to check rapidly the folding state of a protein and characterize mutational effects on protein conformation and stability. Moreover, given global stability information, this methodology allows one to locate the protein regions of increased or decreased conformational stability. All of this can be done with a small fraction of the amount of protein required by most of the other methods for conformational analysis. Thus limited exoproteolysis, together with MALDI-TOF MS, can be a useful tool to achieve quickly the elucidation of protein structure and stability. Copyright 2002 John Wiley & Sons, Ltd.

  7. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Rasmussen, Søren G F; Rana, Rohini R

    2010-01-01

    proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile...

  8. Differential effects of divalent cations on elk prion protein fibril formation and stability

    Science.gov (United States)

    Misfolding of the normally folded prion protein of mammals (PrPC) into infectious fibrils causes a variety of different diseases, from scrapie in sheep to bovine spongiform encephalopathy in cattle to chronic wasting disease (CWD) in deer and elk. The misfolded form of PrPC, termed PrPSc, or in this...

  9. Nucleophosmin is required for DNA integrity and p19Arf protein stability

    DEFF Research Database (Denmark)

    Colombo, Emanuela; Bonetti, Paola; Lazzerini Denchi, Eros

    2005-01-01

    , such as mutated Ras or overexpressed Myc. In the absence of NPM, Arf protein is excluded from nucleoli and is markedly less stable. Our data demonstrate that NPM regulates DNA integrity and, through Arf, inhibits cell proliferation and are consistent with a putative tumor-suppressive function of NPM....

  10. Probing Bio-Nano Interactions between Blood Proteins and Monolayer-Stabilized Graphene Sheets

    DEFF Research Database (Denmark)

    Gan, Shiyu; Zhong, Lijie; Han, Dongxue

    2015-01-01

    Meeting proteins is regarded as the starting event for nanostructures to enter biological systems. Understanding their interactions is thus essential for a newly emerging field, nanomedicine. Chemically converted graphene (CCG) is a wonderful two-dimesional (2D) material for nanomedecine, but its...

  11. Dietary citrus pulp improves protein stability in lamb meat stored under aerobic conditions

    DEFF Research Database (Denmark)

    Gravador, Rufielyn Sungcaya; Jongberg, Sisse; Andersen, Mogens Larsen

    2014-01-01

    The antioxidant effects of dried citrus pulp on proteins in lamb meat, when used as a replacement of concentrate in the feed, was studied using meat from 26 male Comisana lambs. The lambs of age 90. days had been grouped randomly to receive one of the three dietary treatments: (1) commercial...

  12. Contributions of F-BAR and SH2 domains of Fes protein tyrosine kinase for coupling to the FcepsilonRI pathway in mast cells.

    Science.gov (United States)

    McPherson, Victor A; Everingham, Stephanie; Karisch, Robert; Smith, Julie A; Udell, Christian M; Zheng, Jimin; Jia, Zongchao; Craig, Andrew W B

    2009-01-01

    This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcepsilonRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcepsilonRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcepsilonRI beta chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcepsilonRI signaling and potential regulation the actin reorganization in mast cells.

  13. Cables1 controls p21/Cip1 protein stability by antagonizing proteasome subunit alpha type 3

    OpenAIRE

    Shi, Zhi; Li, Zenggang; Li, Zijian; Cheng, Kejun; Du, Yuhong; Fu, Haian; Khuri, Fadlo R.

    2014-01-01

    The cyclin-dependent kinase inhibitor 1A (CDKN1A), p21/Cip1, is a vital cell cycle regulator, dysregulation of which has been associated with a large number of human malignancies. One critical mechanism that controls p21 function is through its degradation, which allows the activation of its associated cell cycle promoting kinases, CDK2 and CDK4. Thus, delineating how p21 is stabilized and degraded will enhance our understanding of cell growth control and offer a basis for potential therapeut...

  14. Replication protein A, the laxative that keeps DNA regular: The importance of RPA phosphorylation in maintaining genome stability.

    Science.gov (United States)

    Byrne, Brendan M; Oakley, Gregory G

    2018-04-20

    The eukaryotic ssDNA-binding protein, Replication protein A (RPA), was first discovered almost three decades ago. Since then, much progress has been made to elucidate the critical roles for RPA in DNA metabolic pathways that help promote genomic stability. The canonical RPA heterotrimer (RPA1-3) is an essential coordinator of DNA metabolism that interacts with ssDNA and numerous protein partners to coordinate its roles in DNA replication, repair, recombination and telomere maintenance. An alternative form of RPA, termed aRPA, is formed by a complex of RPA4 with RPA1 and RPA3. aRPA is expressed differentially in cells compared to canonical RPA and has been shown to inhibit canonical RPA function while allowing for regular maintenance of cell viability. Interestingly, while aRPA is defective in DNA replication and cell cycle progression, it was shown to play a supporting role in nucleotide excision repair and recombination. The binding domains of canonical RPA interact with a growing number of partners involved in numerous genome maintenance processes. The protein interactions of the RPA-ssDNA complex are not only governed by competition between the binding proteins but also by post-translation modifications such as phosphorylation. Phosphorylation of RPA2 is an important post-translational modification of the RPA complex, and is essential for directing context-specific functions of the RPA complex in the DNA damage response. Due to the importance of RPA in cellular metabolism, it was identified as an appealing target for chemotherapeutic drug development that could be used in future cancer treatment regimens. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Hemoglobin bioconjugates with surface-protected gold nanoparticles in aqueous media: The stability depends on solution pH and protein properties.

    Science.gov (United States)

    Del Caño, Rafael; Mateus, Lucia; Sánchez-Obrero, Guadalupe; Sevilla, José Manuel; Madueño, Rafael; Blázquez, Manuel; Pineda, Teresa

    2017-11-01

    The identification of the factors that dictate the formation and physicochemical properties of protein-nanomaterial bioconjugates are important to understand their behavior in biological systems. The present work deals with the formation and characterization of bioconjugates made of the protein hemoglobin (Hb) and gold nanoparticles (AuNP) capped with three different molecular layers (citrate anions (c), 6-mercaptopurine (MP) and ω-mercaptoundecanoic acid (MUA)). The main focus is on the behavior of the bioconjugates in aqueous buffered solutions in a wide pH range. The stability of the bioconjugates have been studied by UV-visible spectroscopy by following the changes in the localized surface resonance plasmon band (LSRP), Dynamic light scattering (DLS) and zeta-potential pH titrations. It has been found that they are stable in neutral and alkaline solutions and, at pH lower than the protein isoelectric point, aggregation takes place. Although the surface chemical properties of the AuNPs confer different properties in respect to colloidal stability, once the bioconjugates are formed their properties are dictated by the Hb protein corona. The protein secondary structure, as analyzed by Attenuated total reflectance infrared (ATR-IR) spectroscopy, seems to be maintained under the conditions of colloidal stability but some small changes in protein conformation take place when the bioconjugates aggregate. These findings highlight the importance to keep the protein structure upon interaction with nanomaterials to drive the stability of the bioconjugates. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Ion pairs in non-redundant protein structures

    Indian Academy of Sciences (India)

    Ion pairs contribute to several functions including the activity of catalytic triads, fusion of viral membranes, stability in thermophilic proteins and solvent–protein interactions. Furthermore, they have the ability to affect the stability of protein structures and are also a part of the forces that act to hold monomers together.

  17. The influence of selection for protein stability on dN/dS estimations

    DEFF Research Database (Denmark)

    Dasmeh, Pouria; Serohijos, Adrian W. R.; Kepp, Kasper Planeta

    2014-01-01

    Understanding the relative contributions of various evolutionary processes-purifying selection, neutral drift, and adaptation-is fundamental to evolutionary biology. A common metric to distinguish these processes is the ratio of nonsynonymous to synonymous substitutions (i.e., dN/dS) interpreted ...

  18. Thermostable trypsin conjugates immobilized to biogenic magnetite show a high operational stability and remarkable reusability for protein digestion

    Science.gov (United States)

    Pečová, M.; Šebela, M.; Marková, Z.; Poláková, K.; Čuda, J.; Šafářová, K.; Zbořil, R.

    2013-03-01

    In this work, magnetosomes produced by microorganisms were chosen as a suitable magnetic carrier for covalent immobilization of thermostable trypsin conjugates with an expected applicability for efficient and rapid digestion of proteins at elevated temperatures. First, a biogenic magnetite was isolated from Magnetospirillum gryphiswaldense and its free surface was coated with the natural polysaccharide chitosan containing free amino and hydroxy groups. Prior to covalent immobilization, bovine trypsin was modified by conjugating with α-, β- and γ-cyclodextrin. Modified trypsin was bound to the magnetic carriers via amino groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling reagents. The magnetic biomaterial was characterized by magnetometric analysis and electron microscopy. With regard to their biochemical properties, the immobilized trypsin conjugates showed an increased resistance to elevated temperatures, eliminated autolysis, had an unchanged pH optimum and a significant storage stability and reusability. Considering these parameters, the presented enzymatic system exhibits properties that are superior to those of trypsin forms obtained by other frequently used approaches. The proteolytic performance was demonstrated during in-solution digestion of model proteins (horseradish peroxidase, bovine serum albumin and hen egg white lysozyme) followed by mass spectrometry. It is shown that both magnetic immobilization and chemical modification enhance the characteristics of trypsin making it a promising tool for protein digestion.

  19. Using the fluorescence red edge effect to assess the long-term stability of lyophilized protein formulations.

    Science.gov (United States)

    Qian, Ken K; Grobelny, Pawel J; Tyagi, Madhusudan; Cicerone, Marcus T

    2015-04-06

    Nanosecond relaxation processes in sugar matrices are causally linked through diffusional processes to protein stability in lyophilized formulations. Long-term protein degradation rates track mean-squared displacement (⟨u(2)⟩) of hydrogen atoms in sugar glasses, a parameter describing dynamics on a time scale of picoseconds to nanoseconds. However, measurements of ⟨u(2)⟩ are usually performed by neutron scattering, which is not conducive to rapid formulation screening in early development. Here, we present a benchtop technique to derive a ⟨u(2)⟩ surrogate based on the fluorescence red edge effect. Glycerol, lyophilized trehalose, and lyophilized sucrose were used as model systems. Samples containing 10(-6) mole fraction of rhodamine 6G, a fluorophore, were excited at either 532 nm (main peak) or 566 nm (red edge), and the ⟨u(2)⟩ surrogate was determined based the corresponding Stokes shifts. Results showed reasonable agreement between ⟨u(2)⟩ from neutron scattering and the surrogate from fluorescence, although deviations were observed at very low temperatures. We discuss the sources of the deviations and suggest technique improvements to ameliorate these. We expect that this method will be a valuable tool to evaluate lyophilized sugar matrices with respect to their ability to protect proteins from diffusion-limited degradation processes during long-term storage. Additionally, the method may have broader applications in amorphous pharmaceutical solids.

  20. Theoretical Insights into the Biophysics of Protein Bi-stability and Evolutionary Switches.

    Directory of Open Access Journals (Sweden)

    Tobias Sikosek

    2016-06-01

    Full Text Available Deciphering the effects of nonsynonymous mutations on protein structure is central to many areas of biomedical research and is of fundamental importance to the study of molecular evolution. Much of the investigation of protein evolution has focused on mutations that leave a protein's folded structure essentially unchanged. However, to evolve novel folds of proteins, mutations that lead to large conformational modifications have to be involved. Unraveling the basic biophysics of such mutations is a challenge to theory, especially when only one or two amino acid substitutions cause a large-scale conformational switch. Among the few such mutational switches identified experimentally, the one between the GA all-α and GB α+β folds is extensively characterized; but all-atom simulations using fully transferrable potentials have not been able to account for this striking switching behavior. Here we introduce an explicit-chain model that combines structure-based native biases for multiple alternative structures with a general physical atomic force field, and apply this construct to twelve mutants spanning the sequence variation between GA and GB. In agreement with experiment, we observe conformational switching from GA to GB upon a single L45Y substitution in the GA98 mutant. In line with the latent evolutionary potential concept, our model shows a gradual sequence-dependent change in fold preference in the mutants before this switch. Our analysis also indicates that a sharp GA/GB switch may arise from the orientation dependence of aromatic π-interactions. These findings provide physical insights toward rationalizing, predicting and designing evolutionary conformational switches.

  1. Differential stability of TATA box binding proteins from archaea with different optimal growth temperatures

    Science.gov (United States)

    Kopitz, Annette; Soppa, Jörg; Krejtschi, Carsten; Hauser, Karin

    2009-09-01

    The TATA box binding protein (TBP) is involved in promoter recognition, the first step of transcription initiation. TBP is universally conserved and essential in archaea and eukaryotes. In archaea, TBPs have to be stable and to function in species that cover an extremely wide range of optimal growth temperatures (OGTs), from below 0 °C to more than 100 °C. Thus, the archaeal TBP family is ideally suited to study the evolutionary adaptation of proteins to an extremely wide range of temperatures. We characterized the thermostability of one mesophilic and one thermophilic TBP by infrared spectroscopy. Transition temperatures ( Tms) of thermal unfolding have been determined using TBPs from Methanosarcina mazei (OGT 37 °C) and from Methanothermobacter thermautotrophicus (OGT 65 °C). Furthermore, the influence of protein and salt concentration on thermostability has been characterized. Together with previous studies, our results reveal that the Tms of archaeal TBPs are closely correlated with the OGTs of the respective species. Noteworthy, this is also true for the TBP from M. mazei representing the first characterized TBP from a mesophilic archaeon. In contrast, the only characterized eukaryotic TBP of the mesophilic plant Arabidopsis thaliana has a Tm more than 40 °C above the OGT.

  2. The hetero-hexameric nature of a chloroplast AAA+ FtsH protease contributes to its thermodynamic stability.

    Directory of Open Access Journals (Sweden)

    Ofer Moldavski

    Full Text Available FtsH is an evolutionary conserved membrane-bound metalloprotease complex. While in most prokaryotes FtsH is encoded by a single gene, multiple FtsH genes are found in eukaryotes. Genetic and biochemical data suggest that the Arabidopsis chloroplast FtsH is a hetero-hexamer. This raises the question why photosynthetic organisms require a heteromeric complex, whereas in most bacteria a homomeric one is sufficient. To gain structural information of the possible complexes, the Arabidopsis FtsH2 (type B and FtsH5 (type A were modeled. An in silico study with mixed models of FtsH2/5 suggests that heteromeric hexamer structure with ratio of 4:2 is more likely to exists. Specifically, calculation of the buried surface area at the interfaces between neighboring subunits revealed that a hetero-complex should be thermodynamically more stable than a homo-hexamer, due to the presence of additional hydrophobic and hydrophilic interactions. To biochemically assess this model, we generated Arabidopsis transgenic plants, expressing epitope-tagged FtsH2 and immuno-purified the protein. Mass-spectrometry analysis showed that FtsH2 is associated with FtsH1, FtsH5 and FtsH8. Interestingly, we found that 'type B' subunits (FtsH2 and FtsH8 were 2-3 fold more abundant than 'type A' (FtsH1 and FtsH5. The biochemical data corroborate the in silico model and suggest that the thylakoid FtsH hexamer is composed of two 'type A' and four 'type B' subunits.

  3. Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation

    Science.gov (United States)

    Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

    2016-11-01

    Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD+-dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some “loose-binding” substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

  4. Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis.

    Science.gov (United States)

    Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Ramaiah, M Janaki; Chowdhury, Debabani Roy; Bhadra, Utpal; Pal-Bhadra, Manika

    2013-01-24

    In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof¹/+; mnkp⁶/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry out the interaction of MOF

  5. Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis

    Directory of Open Access Journals (Sweden)

    Pushpavalli Sreerangam NCVL

    2013-01-01

    Full Text Available Abstract Background In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. Results Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof1/+; mnkp6/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. Conclusion mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using

  6. SIRT1 promotes N-Myc oncogenesis through a positive feedback loop involving the effects of MKP3 and ERK on N-Myc protein stability.

    Directory of Open Access Journals (Sweden)

    Glenn M Marshall

    2011-06-01

    Full Text Available The N-Myc oncoprotein is a critical factor in neuroblastoma tumorigenesis which requires additional mechanisms converting a low-level to a high-level N-Myc expression. N-Myc protein is stabilized when phosphorylated at Serine 62 by phosphorylated ERK protein. Here we describe a novel positive feedback loop whereby N-Myc directly induced the transcription of the class III histone deacetylase SIRT1, which in turn increased N-Myc protein stability. SIRT1 binds to Myc Box I domain of N-Myc protein to form a novel transcriptional repressor complex at gene promoter of mitogen-activated protein kinase phosphatase 3 (MKP3, leading to transcriptional repression of MKP3, ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62, and N-Myc protein stabilization. Importantly, SIRT1 was up-regulated, MKP3 down-regulated, in pre-cancerous cells, and preventative treatment with the SIRT1 inhibitor Cambinol reduced tumorigenesis in TH-MYCN transgenic mice. Our data demonstrate the important roles of SIRT1 in N-Myc oncogenesis and SIRT1 inhibitors in the prevention and therapy of N-Myc-induced neuroblastoma.

  7. Protein Kinase B/Akt Binds and Phosphorylates PED/PEA-15, Stabilizing Its Antiapoptotic Action

    OpenAIRE

    Trencia, Alessandra; Perfetti, Anna; Cassese, Angela; Vigliotta, Giovanni; Miele, Claudia; Oriente, Francesco; Santopietro, Stefania; Giacco, Ferdinando; Condorelli, Gerolama; Formisano, Pietro; Beguinot, Francesco

    2003-01-01

    The antiapoptotic protein PED/PEA-15 features an Akt phosphorylation motif upstream from Ser116. In vitro, recombinant PED/PEA-15 was phosphorylated by Akt with a stoichiometry close to 1. Based on Western blotting with specific phospho-Ser116 PED/PEA-15 antibodies, Akt phosphorylation of PED/PEA-15 occurred mainly at Ser116. In addition, a mutant of PED/PEA-15 featuring the substitution of Ser116→Gly (PEDS116→G) showed 10-fold-decreased phosphorylation by Akt. In intact 293 cells, Akt also i...

  8. Identifying stabilizing key residues in proteins using interresidue interaction energy matrix

    Czech Academy of Sciences Publication Activity Database

    Biedermannová, Lada; Hobza, Pavel; Vondrášek, Jiří

    2008-01-01

    Roč. 72, č. 1 (2008), s. 402-413 ISSN 0887-3585 R&D Projects: GA ČR GA203/05/0009; GA ČR GA203/06/1727; GA ČR(CZ) GD203/05/H001; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506 Keywords : protein stabilisation * an-initio calculation * interaction energy Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.419, year: 2008

  9. Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

    Science.gov (United States)

    Protein tyrosine phosphorylation mediates signal transduction of cellular processes, with protein tyrosine kinases (PTKs) regulating virtually all signaling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into ce...

  10. SIRT6 stabilizes DNA-dependent protein kinase at chromatin for DNA double-strand break repair

    DEFF Research Database (Denmark)

    McCord, Ronald A; Michishita, Eriko; Hong, Tao

    2009-01-01

    -PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor......-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA......, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA...

  11. Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells.

    Science.gov (United States)

    Grosse, Stefanie; Penaud-Budloo, Magalie; Herrmann, Anne-Kathrin; Börner, Kathleen; Fakhiri, Julia; Laketa, Vibor; Krämer, Chiara; Wiedtke, Ellen; Gunkel, Manuel; Ménard, Lucie; Ayuso, Eduard; Grimm, Dirk

    2017-10-15

    -like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans -complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production. Copyright © 2017 American Society for Microbiology.

  12. Carbohydrate particles as protein carriers and scaffolds: physico-chemical characterization and collagen stability

    International Nuclear Information System (INIS)

    Peres, Ivone; Rocha, Sandra; Loureiro, Joana A.; Carmo Pereira, Maria do; Ivanova, Galya; Coelho, Manuel

    2012-01-01

    The preservation of protein properties after entrapping into polymeric matrices and the effects of drying the emulsions still remains uncertain and controversial. Carbohydrate particles were designed and prepared by homogenization of gum arabic and maltodextrin mixture, with collagen hydrolysate (CH) followed by spray-drying. The encapsulation of CH in the carbohydrate matrix was achieved with an efficiency of 85 ± 2 %. The morphology and the size of the particles, before (40–400 nm) and after spray-drying (<20 μm), were characterized by scanning electron microscopy and dynamic light scattering. Measurements of the nuclear relaxation times and application of diffusion ordered spectroscopy, obtained through pulsed field gradient NMR experiments, have been performed to determine the structure of the CH–polysaccharide conjugates and to clarify the mechanism of CH immobilization in the polysaccharide matrix. In vitro release profiles in ultrapure water and in cellular medium reveal that the diffusion rate of CH from the polymeric matrix to the dialysis solution decreases in average 30–50 % over time, compared to free CH molecules. In cellular medium at 37 °C, the complete release of CH from the particles is achieved only after 24 h, demonstrating a significant decrease in the CH mass transfer process when compared with free CH. The findings of this study outline the ability of gum arabic/maltodextrin matrices to entrap and preserve CH original properties after the spray-drying process and support the potential of the polymeric scaffold for protein delivery and tissue engineering.

  13. A Network of Multi-Tasking Proteins at the DNA Replication Fork Preserves Genome Stability.

    Directory of Open Access Journals (Sweden)

    2005-12-01

    Full Text Available To elucidate the network that maintains high fidelity genome replication, we have introduced two conditional mutant alleles of DNA2, an essential DNA replication gene, into each of the approximately 4,700 viable yeast deletion mutants and determined the fitness of the double mutants. Fifty-six DNA2-interacting genes were identified. Clustering analysis of genomic synthetic lethality profiles of each of 43 of the DNA2-interacting genes defines a network (consisting of 322 genes and 876 interactions whose topology provides clues as to how replication proteins coordinate regulation and repair to protect genome integrity. The results also shed new light on the functions of the query gene DNA2, which, despite many years of study, remain controversial, especially its proposed role in Okazaki fragment processing and the nature of its in vivo substrates. Because of the multifunctional nature of virtually all proteins at the replication fork, the meaning of any single genetic interaction is inherently ambiguous. The multiplexing nature of the current studies, however, combined with follow-up supporting experiments, reveals most if not all of the unique pathways requiring Dna2p. These include not only Okazaki fragment processing and DNA repair but also chromatin dynamics.

  14. Carbohydrate particles as protein carriers and scaffolds: physico-chemical characterization and collagen stability

    Energy Technology Data Exchange (ETDEWEB)

    Peres, Ivone; Rocha, Sandra; Loureiro, Joana A.; Carmo Pereira, Maria do [University of Porto, LEPAE, Chemical Engineering Department, Faculty of Engineering (Portugal); Ivanova, Galya [Universidade do Porto, REQUIMTE, Departamento de Quimica, Faculdade de Ciencias (Portugal); Coelho, Manuel, E-mail: mcoelho@fe.up.pt [University of Porto, LEPAE, Chemical Engineering Department, Faculty of Engineering (Portugal)

    2012-09-15

    The preservation of protein properties after entrapping into polymeric matrices and the effects of drying the emulsions still remains uncertain and controversial. Carbohydrate particles were designed and prepared by homogenization of gum arabic and maltodextrin mixture, with collagen hydrolysate (CH) followed by spray-drying. The encapsulation of CH in the carbohydrate matrix was achieved with an efficiency of 85 {+-} 2 %. The morphology and the size of the particles, before (40-400 nm) and after spray-drying (<20 {mu}m), were characterized by scanning electron microscopy and dynamic light scattering. Measurements of the nuclear relaxation times and application of diffusion ordered spectroscopy, obtained through pulsed field gradient NMR experiments, have been performed to determine the structure of the CH-polysaccharide conjugates and to clarify the mechanism of CH immobilization in the polysaccharide matrix. In vitro release profiles in ultrapure water and in cellular medium reveal that the diffusion rate of CH from the polymeric matrix to the dialysis solution decreases in average 30-50 % over time, compared to free CH molecules. In cellular medium at 37 Degree-Sign C, the complete release of CH from the particles is achieved only after 24 h, demonstrating a significant decrease in the CH mass transfer process when compared with free CH. The findings of this study outline the ability of gum arabic/maltodextrin matrices to entrap and preserve CH original properties after the spray-drying process and support the potential of the polymeric scaffold for protein delivery and tissue engineering.

  15. Comparison of the thermal stabilization of proteins by oligosaccharides and monosaccharide mixtures: Measurement and analysis in the context of excluded volume theory.

    Science.gov (United States)

    Beg, Ilyas; Minton, Allen P; Islam, Asimul; Hassan, Md Imtaiyaz; Ahmad, Faizan

    2018-06-01

    The thermal stability of apo α-lactalbumin (α-LA) and lysozyme was measured in the presence of mixtures of glucose, fructose, and galactose. Mixtures of these monosaccharides in the appropriate stoichiometric ratio were found to have a greater stabilizing effect on each of the two proteins than equal weight/volume concentrations of di- tri- and tetrasaccharides with identical subunit composition (sucrose, trehalose, raffinose, and stachyose). The excluded volume model for the effect of a single saccharide on the stability of a protein previously proposed by Beg et al. [Biochemistry 54 (2015) 3594] was extended to treat the case of saccharide mixtures. The extended model predicts quantitatively the stabilizing effect of all monosaccharide mixtures on α-LA and lysozyme reported here, as well as previously published results obtained for ribonuclease A [Biophys. Chem. 138 (2008) 120] to within experimental uncertainty. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Heat shock protein 90 positively regulates Chikungunya virus replication by stabilizing viral non-structural protein nsP2 during infection.

    Directory of Open Access Journals (Sweden)

    Indrani Das

    Full Text Available BACKGROUND: The high morbidity and socio-economic loss associated with the recent massive global outbreak of Chikungunya virus (CHIKV emphasize the need to understand the biology of the virus for developing effective antiviral therapies. METHODS AND FINDINGS: In this study, an attempt was made to understand the molecular mechanism involved in Heat shock protein 90 (Hsp90 mediated regulation of CHIKV infection in mammalian cells using CHIKV prototype strain (S 27 and Indian outbreak strain of 2006 (DRDE-06. Our results showed that Hsp90 is required at a very early stage of viral replication and Hsp90 inhibitor Geldanamycin (GA can abrogate new virus particle formation more effectively in the case of S 27 than that of DRDE-06. Further analysis revealed that CHIKV nsP2 protein level is specifically reduced by GA treatment as well as HSP90-siRNA transfection; however, viral RNA remains unaltered. Immunoprecipitation analysis showed that nsP2 interacts with Hsp90 during infection; however this interaction is reduced in the presence of GA. In addition, our analysis on Hsp90 associated PI3K/Akt/mTOR signaling pathway demonstrated that CHIKV infection stabilizes Raf1 and activates Hsp90 client protein Akt, which in turn phosphorylates mTOR. Subsequently, this phosphorylation leads to the activation of two important downstream effectors, S6K and 4EBP1, which may facilitate translation of viral as well as cellular mRNAs. Hence, the data suggests that CHIKV infection is regulated by Hsp90 associated Akt phosphorylation and DRDE-06 is more efficient than S 27 in enhancing the activation of host signaling molecules for its efficient replication and virus production. CONCLUSION: Hsp90 positively regulates Chikungunya virus replication by stabilizing CHIKV-nsP2 through its interaction during infection. The study highlights the possible molecular mechanism of GA mediated inhibition of CHIKV replication and differential effect of this drug on S 27 and DRDE-06

  17. HNRNPLL stabilizes mRNAs for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells.

    Science.gov (United States)

    Sakuma, Keiichiro; Sasaki, Eiichi; Kimura, Kenya; Komori, Koji; Shimizu, Yasuhiro; Yatabe, Yasushi; Aoki, Masahiro

    2018-06-05

    HNRNPLL (heterogeneous nuclear ribonucleoprotein L-like), an RNA-binding protein that regulates alternative splicing of pre-mRNAs, has been shown to regulate differentiation of lymphocytes, as well as metastasis of colorectal cancer cells. Here we show that HNRNPLL promotes cell cycle progression and hence proliferation of colorectal cancer cells. Functional annotation analysis of those genes whose expression levels were changed by three-fold or more in RNA sequencing analysis between SW480 cells overexpressing HNRNPLL and those knocked down for HNRNPLL revealed enrichment of DNA replication-related genes by HNRNPLL overexpression. Among 13 genes detected in the DNA replication pathway, PCNA, RFC3, and FEN1 showed reproducible upregulation by HNRNPLL overexpression both at mRNA and protein levels in SW480 and HT29 cells. Importantly, knockdown of any of these genes alone suppressed the proliferation promoting effect induced by HNRNPLL overexpression. RNA-immunoprecipitation assay presented a binding of FLAG-tagged HNRNPLL to mRNA of these genes, and HNRNPLL overexpression significantly suppressed the downregulation of these genes during 12 hours of actinomycin D treatment, suggesting a role of HNRNPLL in mRNA stability. Finally, analysis of a public RNA sequencing dataset of clinical samples suggested a link between overexpression of HNRNPLL and that of PCNA, RFC3, and FEN1. This link was further supported by immunohistochemistry of colorectal cancer clinical samples, whereas expression of CDKN1A, which is known to inhibit the cooperative function of PCNA, RFC3, and FEN1, was negatively associated with HNRNPLL expression. These results indicate that HNRNPLL stabilizes mRNAs encoding regulators of DNA replication and promotes colorectal cancer cell proliferation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  18. The Effect of Membrane Environment on Surfactant Protein C Stability Studied by Constant-pH Molecular Dynamics.

    Science.gov (United States)

    Carvalheda, Catarina A; Campos, Sara R R; Baptista, António M

    2015-10-26

    Pulmonary surfactant protein C (SP-C) is a small peptide with two covalently linked fatty acyl chains that plays a crucial role in the formation and stabilization of the pulmonary surfactant reservoirs during the compression and expansion steps of the respiratory cycle. Although its function is known to be tightly related to its highly hydrophobic character and key interactions maintained with specific lipid components, much is left to understand about its molecular mechanism of action. Also, although it adopts a mainly helical structure while associated with the membrane, factors as pH variation and deacylation have been shown to affect its stability and function. In this work, the conformational behavior of both the acylated and deacylated SP-C isoforms was studied in a DPPC bilayer under different pH conditions using constant-pH molecular dynamics simulations. Our findings show that both protein isoforms are remarkably stable over the studied pH range, even though the acylated isoform exhibits a labile helix-turn-helix motif rarely observed in the other isoform. We estimate similar tilt angles for the two isoforms over the studied pH range, with a generally higher degree of internalization of the basic N-terminal residues in the deacylated case, and observe and discuss some protonation-conformation coupling effects. Both isoforms establish contacts with the surrounding lipid molecules (preferentially with the sn-2 ester bonds) and have a local effect on the conformational behavior of the surrounding lipid molecules, the latter being more pronounced for acylated SP-C.

  19. Electron beam irradiation induces abnormal development and the stabilization of p53 protein of American serpentine leafminer, Liriomyza trifolii (Burgess)

    Energy Technology Data Exchange (ETDEWEB)

    Koo, Hyun-Na; Yun, Seung-Hwan; Yoon, Changmann [Department of Plant Medicine, College of Agriculture, Life and Environment Sciences, Chungbuk National University, Cheongju 361-763 (Korea, Republic of); Kim, Gil-Hah, E-mail: khkim@chungbuk.ac.kr [Department of Plant Medicine, College of Agriculture, Life and Environment Sciences, Chungbuk National University, Cheongju 361-763 (Korea, Republic of)

    2012-01-15

    The American serpentine leafminer fly, Liriomyza trifolii (Burgess), is one of the most destructive polyphagous pests worldwide. In this study, we determined electron beam doses for inhibition of normal development of the leaf miner and investigated the effect of electron beam irradiation on DNA damage and p53 stability. Eggs (0-24 h old), larvae (2nd instar), puparia (0-24 h old after pupariation) and adults (24 h after emergence) were irradiated with increasing doses of electron beam irradiation (six levels between 30 and 200 Gy). At 150 Gy, the number of adults that developed from irradiated eggs, larvae and puparia was lower than in the untreated control. Fecundity and egg hatchability decreased depending on the doses applied. Reciprocal crosses between irradiated and unirradiated flies demonstrated that males were more radiotolerant than females. Adult longevity was not affected in all stages. The levels of DNA damage in L. trifolii adults were evaluated using the alkaline comet assay. Our results indicate that electron beam irradiation increased levels of DNA damage in a dose-dependent manner. Moreover, low doses of electron beam irradiation led to the rapid appearance of p53 protein within 6 h; however, it decreased after exposure to high doses (150 Gy and 200 Gy). These results suggest that electron beam irradiation induced not only abnormal development and reproduction but also p53 stability caused by DNA damage in L. trifolii. We conclude that a minimum dose of 150 Gy should be sufficient for female sterilization of L. trifolii. - Highlights: > Electron beam irradiation inhibited normal development of the leaf miner. > Electron beam irradiation inhibited normal reproduction of the leaf miner. > Electron beam irradiation increased levels of DNA damage. > Electron beam irradiation induced p53 stability.

  20. Effect of phospholipid, detergent and protein-protein interaction on stability and phosphoenzyme isomerization of soluble sarcoplasmic reticulum Ca-ATPase.

    Science.gov (United States)

    Vilsen, B; Andersen, J P

    1987-12-30

    The purpose of the present study was to elucidate the separate roles of lipid, detergent and protein-protein interaction for stability and catalytic properties of sarcoplasmic reticulum Ca-ATPase solubilized in the non-ionic detergent octa(ethylene glycol) monododecyl ether (C12E8). The use of large-zone high-performance liquid chromatography permitted us to define the self-association state of Ca-ATPase peptide at various detergent, phospholipid and protein concentrations, and also during enzymatic turnover with ATP. Conditions were established for monomerization of Ca-ATPase in the presence of a high concentration of phospholipid relative to detergent. The lipid-saturated monomeric preparation was relatively resistant to inactivation in the absence of Ca2+, whereas delipidated enzyme in monomeric or in oligomeric form was prone to inactivation. Kinetics of phosphoenzyme turnover were examined in the presence and absence of Mg2+. Dephosphorylation rates were sensitive to Mg2+, irrespective of whether the peptide was present in soluble monomeric form or was membrane-bound. C12E8-solubilized monomer without added phospholipid was, however, characterized by a fast initial phase of dephosphorylation in the absence of Mg2+. This was not observed with monomer saturated with phospholipid or with monomer solubilized in myristoylglycerophosphocholine or deoxycholate. The mechanism underlying this difference was shown to be a C12E8-induced acceleration of conversion of ADP-sensitive phosphoenzyme (E1P) to ADP-insensitive phosphoenzyme (E2P). The phosphoenzyme isomerization rate was also found to be enhanced by low-affinity binding of ATP. This was demonstrated both in membrane-bound and in soluble monomeric Ca-ATPase. Our results indicate that a single peptide chain constitutes the target for modulation of phosphoenzyme turnover by Mg2+ and ATP, and that detergent effects, distinct from those arising from disruption of protein-protein contacts, are the major determinants of

  1. The stress protein BAG3 stabilizes Mcl-1 protein and promotes survival of cancer cells and resistance to antagonist ABT-737.

    Science.gov (United States)

    Boiani, Mariana; Daniel, Cristina; Liu, Xueyuan; Hogarty, Michael D; Marnett, Lawrence J

    2013-03-08

    Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery.

  2. The Stress Protein BAG3 Stabilizes Mcl-1 Protein and Promotes Survival of Cancer Cells and Resistance to Antagonist ABT-737*

    Science.gov (United States)

    Boiani, Mariana; Daniel, Cristina; Liu, Xueyuan; Hogarty, Michael D.; Marnett, Lawrence J.

    2013-01-01

    Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery. PMID:23341456

  3. Climate, soil texture, and soil types affect the contributions of fine-fraction-stabilized carbon to total soil organic carbon in different land uses across China.

    Science.gov (United States)

    Cai, Andong; Feng, Wenting; Zhang, Wenju; Xu, Minggang

    2016-05-01

    Mineral-associated organic carbon (MOC), that is stabilized by fine soil particles (i.e., silt plus clay, organic carbon (SOC) persistence and sequestration, due to its large contribution to total SOC (TSOC) and long turnover time. Our objectives were to investigate how climate, soil type, soil texture, and agricultural managements affect MOC contributions to TSOC in China. We created a dataset from 103 published papers, including 1106 data points pairing MOC and TSOC across three major land use types: cropland, grassland, and forest. Overall, the MOC/TSOC ratio ranged from 0.27 to 0.80 and varied significantly among soil groups in cropland, grassland, and forest. Croplands and forest exhibited significantly higher median MOC/TSOC ratios than in grassland. Moreover, forest and grassland soils in temperate regions had higher MOC/TSOC ratios than in subtropical regions. Furthermore, the MOC/TSOC ratio was much higher in ultisol, compared with the other soil types. Both the MOC content and MOC/TSOC ratio were positively correlated with the amount of fine fraction (silt plus clay) in soil, highlighting the importance of soil texture in stabilizing organic carbon across various climate zones. In cropland, different fertilization practices and land uses (e.g., upland, paddy, and upland-paddy rotation) significantly altered MOC/TSOC ratios, but not in cropping systems (e.g., mono- and double-cropping) characterized by climatic differences. This study demonstrates that the MOC/TSOC ratio is mainly driven by soil texture, soil types, and related climate and land uses, and thus the variations in MOC/TSOC ratios should be taken into account when quantitatively estimating soil C sequestration potential of silt plus clay particles on a large scale. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Binding stability of peptides on major histocompatibility complex class I proteins: role of entropy and dynamics

    Science.gov (United States)

    Gul, Ahmet; Erman, Burak

    2018-03-01

    Prediction of peptide binding on specific human leukocyte antigens (HLA) has long been studied with successful results. We herein describe the effects of entropy and dynamics by investigating the binding stabilities of 10 nanopeptides on various HLA Class I alleles using a theoretical model based on molecular dynamics simulations. The fluctuational entropies of the peptides are estimated over a temperature range of 310-460 K. The estimated entropies correlate well with experimental binding affinities of the peptides: peptides that have higher binding affinities have lower entropies compared to non-binders, which have significantly larger entropies. The computation of the entropies is based on a simple model that requires short molecular dynamics trajectories and allows for approximate but rapid determination. The paper draws attention to the long neglected dynamic aspects of peptide binding, and provides a fast computation scheme that allows for rapid scanning of large numbers of peptides on selected HLA antigens, which may be useful in defining the right peptides for personal immunotherapy.

  5. The stability of human, bovine and avian tuberculin purified protein derivative (PPD).

    Science.gov (United States)

    Maes, Mailis; Giménez, José Francisco; D'Alessandro, Adriana; De Waard, Jacobus H

    2011-11-15

    Guidelines recommend storing tuberculin purified protein derivative (PPD) refrigerated. However, especially in developing countries, maintaining the product refrigerated under field conditions can be difficult, limiting its use. Here we determine the effect of prolonged exposure to high temperatures on the potency of human, bovine and avian tuberculin PPD. Human, bovine and avian tuberculin PPD were stored for several weeks exposed to temperatures ranging from 37º to 100ºC. The potency was evaluated in vivo, in sensitized or naturally infected animals. Most test situations didn't affect the biological activity of the tuberculin PPDs and only very long and extreme incubations (several days at 100 °C) compromised the potency. Tuberculin PPD is very stable and can be stored or transported for long periods without refrigeration. 

  6. Purification of barley dimeric α-amylase inhibitor-1 (BDAI-1) and avenin-like protein-a (ALP) from beer and their impact on beer foam stability.

    Science.gov (United States)

    Iimure, Takashi; Kihara, Makoto; Sato, Kazuhiro; Ogushi, Kensuke

    2015-04-01

    Foam stability is a key factor of beer quality for consumers and brewers. Recent beer proteome analyses have suggested that barley dimeric α-amylase inhibitor-1 (BDAI-1) and avenin-like protein-a (ALP) derived from barley are important for beer foam stability. In this study, BDAI-1 and ALP were purified from a Japanese commercial beer sample using salt precipitation and column chromatography. The purification level was verified using two-dimensional gel electrophoresis, mass spectrometry, and database searches. Purified BDAI-1 and ALP were added to a beer sample to compare the foam stability to that of a control beer sample. As a result, beer foam stability was significantly improved by BDAI-1 but not by ALP, thereby suggesting that BDAI-1 affects beer foam stability whereas ALP does not. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Diblock-copolymer-mediated self-assembly of protein-stabilized iron oxide nanoparticle clusters for magnetic resonance imaging.

    Science.gov (United States)

    Tähkä, Sari; Laiho, Ari; Kostiainen, Mauri A

    2014-03-03

    Superparamagnetic iron oxide nanoparticles (SPIONs) can be used as efficient transverse relaxivity (T2 ) contrast agents in magnetic resonance imaging (MRI). Organizing small (Doxide) diblock copolymer (P2QVP-b-PEO) to mediate the self-assembly of protein-cage-encapsulated iron oxide (γ-Fe2 O3 ) nanoparticles (magnetoferritin) into stable PEO-coated clusters. This approach relies on electrostatic interactions between the cationic N-methyl-2-vinylpyridinium iodide block and magnetoferritin protein cage surface (pI≈4.5) to form a dense core, whereas the neutral ethylene oxide block provides a stabilizing biocompatible shell. Formation of the complexes was studied in aqueous solvent medium with dynamic light scattering (DLS) and cryogenic transmission electron microcopy (cryo-TEM). DLS results indicated that the hydrodynamic diameter (Dh ) of the clusters is approximately 200 nm, and cryo-TEM showed that the clusters have an anisotropic stringlike morphology. MRI studies showed that in the clusters the longitudinal relaxivity (r1 ) is decreased and the transverse relaxivity (r2 ) is increased relative to free magnetoferritin (MF), thus indicating that clusters can provide considerable contrast enhancement. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Protein kinase C ϵ stabilizes β-catenin and regulates its subcellular localization in podocytes.

    Science.gov (United States)

    Duong, Michelle; Yu, Xuejiao; Teng, Beina; Schroder, Patricia; Haller, Hermann; Eschenburg, Susanne; Schiffer, Mario

    2017-07-21

    Kidney disease has been linked to dysregulated signaling via PKC in kidney cells such as podocytes. PKCα is a conventional isoform of PKC and a well-known binding partner of β-catenin, which promotes its degradation. β-Catenin is the main effector of the canonical Wnt pathway and is critical in cell adhesion. However, whether other PKC isoforms interact with β-catenin has not been studied systematically. Here we demonstrate that PKCϵ-deficient mice, which develop proteinuria and glomerulosclerosis, display lower β-catenin expression compared with PKC wild-type mice, consistent with an altered phenotype of podocytes in culture. Remarkably, β-catenin showed a reversed subcellular localization pattern: Although β-catenin exhibited a perinuclear pattern in undifferentiated wild-type cells, it predominantly localized to the nucleus in PKCϵ knockout cells. Phorbol 12-myristate 13-acetate stimulation of both cell types revealed that PKCϵ positively regulates β-catenin expression and stabilization in a glycogen synthase kinase 3β-independent manner. Further, β-catenin overexpression in PKCϵ-deficient podocytes could restore the wild-type phenotype, similar to rescue with a PKCϵ construct. This effect was mediated by up-regulation of P-cadherin and the β-catenin downstream target fascin1. Zebrafish studies indicated three PKCϵ-specific phosphorylation sites in β-catenin that are required for full β-catenin function. Co-immunoprecipitation and pulldown assays confirmed PKCϵ and β-catenin as binding partners and revealed that ablation of the three PKCϵ phosphorylation sites weakens their interaction. In summary, we identified a novel pathway for regulation of β-catenin levels and define PKCϵ as an important β-catenin interaction partner and signaling opponent of other PKC isoforms in podocytes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. TM9/Phg1 and SadA proteins control surface expression and stability of SibA adhesion molecules in Dictyostelium.

    Science.gov (United States)

    Froquet, Romain; le Coadic, Marion; Perrin, Jackie; Cherix, Nathalie; Cornillon, Sophie; Cosson, Pierre

    2012-02-01

    TM9 proteins form a family of conserved proteins with nine transmembrane domains essential for cellular adhesion in many biological systems, but their exact role in this process remains unknown. In this study, we found that genetic inactivation of the TM9 protein Phg1A dramatically decreases the surface levels of the SibA adhesion molecule in Dictyostelium amoebae. This is due to a decrease in sibA mRNA levels, in SibA protein stability, and in SibA targeting to the cell surface. A similar phenotype was observed in cells devoid of SadA, a protein that does not belong to the TM9 family but also exhibits nine transmembrane domains and is essential for cellular adhesion. A contact site A (csA)-SibA chimeric protein comprising only the transmembrane and cytosolic domains of SibA and the extracellular domain of the Dictyostelium surface protein csA also showed reduced stability and relocalization to endocytic compartments in phg1A knockout cells. These results indicate that TM9 proteins participate in cell adhesion by controlling the levels of adhesion proteins present at the cell surface.

  10. Characterization and stability of transthyretin isoforms in cerebrospinal fluid examined by immunoprecipitation and high-resolution mass spectrometry of intact protein

    DEFF Research Database (Denmark)

    Poulsen, Keld; Bahl, Justyna M C; Tanassi, Julia T

    2012-01-01

    Post-translational modifications (PTMs) contribute significantly to the complexity of proteins. PTMs may vary in certain patterns according to diseases and microenviroments making them potential markers for pathological processes. Human transthyretin (TTR) is a transporter of thyroxine and retino...

  11. Study of the effect of presence or absence of protozoa on rumen fermentation and microbial protein contribution to the chyme.

    Science.gov (United States)

    Belanche, A; Abecia, L; Holtrop, G; Guada, J A; Castrillo, C; de la Fuente, G; Balcells, J

    2011-12-01

    The aim of this study was to investigate the effect of presence or absence of protozoa on rumen fermentation and efficiency of microbial protein synthesis under different diets. Of 20 twin paired lambs, 1 lamb of each pair was isolated from the ewe within 24 h after birth and reared in a protozoa-free environment (n = 10), whereas their respective twin-