Sample records for b-like proteins cbls

  1. Regulation of Microbe-Associated Molecular Pattern-Induced Hypersensitive Cell Death, Phytoalexin Production, and Defense Gene Expression by Calcineurin B-Like Protein-Interacting Protein Kinases, OsCIPK14/15, in Rice Cultured Cells1[W][OA

    PubMed Central

    Kurusu, Takamitsu; Hamada, Jumpei; Nokajima, Hiroshi; Kitagawa, Youichiro; Kiyoduka, Masahiro; Takahashi, Akira; Hanamata, Shigeru; Ohno, Ryoko; Hayashi, Teruyuki; Okada, Kazunori; Koga, Jinichiro; Hirochika, Hirohiko; Yamane, Hisakazu; Kuchitsu, Kazuyuki

    2010-01-01

    Although cytosolic free Ca2+ mobilization induced by microbe/pathogen-associated molecular patterns is postulated to play a pivotal role in innate immunity in plants, the molecular links between Ca2+ and downstream defense responses still remain largely unknown. Calcineurin B-like proteins (CBLs) act as Ca2+ sensors to activate specific protein kinases, CBL-interacting protein kinases (CIPKs). We here identified two CIPKs, OsCIPK14 and OsCIPK15, rapidly induced by microbe-associated molecular patterns, including chitooligosaccharides and xylanase (Trichoderma viride/ethylene-inducing xylanase [TvX/EIX]), in rice (Oryza sativa). Although they are located on different chromosomes, they have over 95% nucleotide sequence identity, including the surrounding genomic region, suggesting that they are duplicated genes. OsCIPK14/15 interacted with several OsCBLs through the FISL/NAF motif in yeast cells and showed the strongest interaction with OsCBL4. The recombinant OsCIPK14/15 proteins showed Mn2+-dependent protein kinase activity, which was enhanced both by deletion of their FISL/NAF motifs and by combination with OsCBL4. OsCIPK14/15-RNAi transgenic cell lines showed reduced sensitivity to TvX/EIX for the induction of a wide range of defense responses, including hypersensitive cell death, mitochondrial dysfunction, phytoalexin biosynthesis, and pathogenesis-related gene expression. On the other hand, TvX/EIX-induced cell death was enhanced in OsCIPK15-overexpressing lines. Our results suggest that OsCIPK14/15 play a crucial role in the microbe-associated molecular pattern-induced defense signaling pathway in rice cultured cells. PMID:20357140

  2. iCBLS: An interactive case-based learning system for medical education.

    PubMed

    Ali, Maqbool; Han, Soyeon Caren; Bilal, Hafiz Syed Muhammad; Lee, Sungyoung; Kang, Matthew Jee Yun; Kang, Byeong Ho; Razzaq, Muhammad Asif; Amin, Muhammad Bilal

    2018-01-01

    Medical students should be able to actively apply clinical reasoning skills to further their interpretative, diagnostic, and treatment skills in a non-obtrusive and scalable way. Case-Based Learning (CBL) approach has been receiving attention in medical education as it is a student-centered teaching methodology that exposes students to real-world scenarios that need to be solved using their reasoning skills and existing theoretical knowledge. In this paper, we propose an interactive CBL System, called iCBLS, which supports the development of collaborative clinical reasoning skills for medical students in an online environment. The iCBLS consists of three modules: (i) system administration (SA), (ii) clinical case creation (CCC) with an innovative semi-automatic approach, and (iii) case formulation (CF) through intervention of medical students' and teachers' knowledge. Two evaluations under the umbrella of the context/input/process/product (CIPP) model have been performed with a Glycemia study. The first focused on the system satisfaction, evaluated by 54 students. The latter aimed to evaluate the system effectiveness, simulated by 155 students. The results show a high success rate of 70% for students' interaction, 76.4% for group learning, 72.8% for solo learning, and 74.6% for improved clinical skills. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Genome-Wide Analyses of Calcium Sensors Reveal Their Involvement in Drought Stress Response and Storage Roots Deterioration after Harvest in Cassava.

    PubMed

    Hu, Wei; Yan, Yan; Tie, Weiwei; Ding, Zehong; Wu, Chunlai; Ding, Xupo; Wang, Wenquan; Xia, Zhiqiang; Guo, Jianchun; Peng, Ming

    2018-04-19

    Calcium (Ca 2+ ) plays a crucial role in plant development and responses to environmental stimuli. Currently, calmodulins (CaMs), calmodulin-like proteins (CMLs), and calcineurin B-like proteins (CBLs), such as Ca 2+ sensors, are not well understood in cassava ( Manihot esculenta Crantz), an important tropical crop. In the present study, 8 CaMs, 48 CMLs, and 9 CBLs were genome-wide identified in cassava, which were divided into two, four, and four groups, respectively, based on evolutionary relationship, protein motif, and gene structure analyses. Transcriptomic analysis revealed the expression diversity of cassava CaMs-CMLs-CBLs in distinct tissues and in response to drought stress in different genotypes. Generally, cassava CaMs-CMLs-CBLs showed different expression profiles between cultivated varieties (Arg7 and SC124) and wild ancestor (W14) after drought treatment. In addition, numerous CaMs-CMLs-CBLs were significantly upregulated at 6 h, 12 h, and 48 h after harvest, suggesting their possible role during storage roots (SR) deterioration. Further interaction network and co-expression analyses suggested that a CBL-mediated interaction network was widely involved in SR deterioration. Taken together, this study provides new insights into CaMs-CMLs-CBLs-mediated drought adaption and SR deterioration at the transcription level in cassava, and identifies some candidates for the genetic improvement of cassava.

  4. Calcineurin B-like 3 calcium sensor associates with and inhibits 5'-methylthioadenosine nucleosidase 2 in Arabidopsis.

    PubMed

    Ok, Sung Han; Cho, Joo Hyuk; Oh, Seung-Ick; Choi, Mi Na; Ma, Jae-Yeon; Shin, Jeong-Sheop; Kim, Kyung-Nam

    2015-09-01

    Calcineurin B-like (CBL) proteins constitute a unique family of calcium sensor relays in plants. It is well known that CBLs detect the calcium signals elicited by a variety of abiotic stresses and relay the information to a group of serine/threonine protein kinases called CBL-interacting protein kinases (CIPKs). In this study, we found that a few CBL members can also target another group of enzymes 5'-methylthioadenosine nucleosidases (MTANs), which are encoded by two genes in Arabidopsis, AtMTAN1 and AtMTAN2. In the yeast two-hybrid system, AtMTAN1 interacted with multiple CBL members such as CBL2, CBL3 and CBL6, whereas AtMTAN2 associated exclusively with CBL3. We further demonstrated that the CBL3-AtMTAN2 association occurs in a calcium-dependent manner, which results in a significant decrease in the enzyme activity of the AtMTAN2 protein. Taken together, these results clearly indicate that the CBL family can target at least two distinct groups of enzymes (CIPKs and MTANs), conferring an additional level of complexity on the CBL-mediated signaling networks. In addition, our finding also provides a novel molecular mechanism by which calcium signals are transduced to alter metabolite profiles in plants. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. The actin-like MreB proteins in Bacillus subtilis: a new turn.

    PubMed

    Chastanet, Arnaud; Carballido-Lopez, Rut

    2012-06-01

    A decade ago, two breakthrough descriptions were reported: 1) the first helix-like protein localization pattern of MreB and its paralog Mbl in Bacillus subtilis and 2) the crystal structure of Thermotoga maritima MreB1, which was remarkably similar to that of actin. These discoveries strongly stimulated the field of bacterial development, leading to the identification of many new cytoskeletal proteins (1) and the publication of many studies describing the helical patterns of protein, DNA and even lipid domains. However, today, new breakthroughs are shaking up what had become a dogma. Instead of helical structures, MreBs appear to form discrete patches that move circumferentially around the cell, questioning the idea of MreB cables forming an actin-like cytoskeleton. Furthermore, increasing evidence of biochemical properties that are unlike the properties of actin suggest that the molecular behavior of MreB proteins may be different. The aim of this review is to summarize the current knowledge of the so-called "actin-like" MreB cytoskeleton through a discussion of the model Gram-positive bacterium B. subtilis and the most recent findings in this rapidly evolving research field.

  6. Expression Patterns and Identified Protein-Protein Interactions Suggest That Cassava CBL-CIPK Signal Networks Function in Responses to Abiotic Stresses

    PubMed Central

    Mo, Chunyan; Wan, Shumin; Xia, Youquan; Ren, Ning; Zhou, Yang; Jiang, Xingyu

    2018-01-01

    Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL) protein and CBL-interacting protein kinase (CIPK) is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 CBL (MeCBL) and 26 CIPK (MeCIPK) genes were isolated from cassava by genome searching and cloning of cDNA sequences of Arabidopsis CBLs and CIPKs. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression levels of MeCBL and MeCIPK genes were different in different tissues throughout the life cycle. The expression patterns of 7 CBL and 26 CIPK genes in response to NaCl, PEG, heat and cold stresses were analyzed by quantitative real-time PCR (qRT-PCR), and it was found that the expression of each was induced by multiple stimuli. Furthermore, we found that many pairs of CBLs and CIPKs could interact with each other via investigating the interactions between 8 CBL and 25 CIPK proteins using a yeast two-hybrid system. Yeast cells co-transformed with cassava MeCIPK24, MeCBL10, and Na+/H+ antiporter MeSOS1 genes exhibited higher salt tolerance compared to those with one or two genes. These results suggest that the cassava CBL-CIPK signal network might play key roles in response to abiotic stresses. PMID:29552024

  7. Structure-function correlations of pulmonary surfactant protein SP-B and the saposin-like family of proteins.

    PubMed

    Olmeda, Bárbara; García-Álvarez, Begoña; Pérez-Gil, Jesús

    2013-03-01

    Pulmonary surfactant is a lipid-protein complex secreted by the respiratory epithelium of mammalian lungs, which plays an essential role in stabilising the alveolar surface and so reducing the work of breathing. The surfactant protein SP-B is part of this complex, and is strictly required for the assembly of pulmonary surfactant and its extracellular development to form stable surface-active films at the air-liquid alveolar interface, making the lack of SP-B incompatible with life. In spite of its physiological importance, a model for the structure and the mechanism of action of SP-B is still needed. The sequence of SP-B is homologous to that of the saposin-like family of proteins, which are membrane-interacting polypeptides with apparently diverging activities, from the co-lipase action of saposins to facilitate the degradation of sphingolipids in the lysosomes to the cytolytic actions of some antibiotic proteins, such as NK-lysin and granulysin or the amoebapore of Entamoeba histolytica. Numerous studies on the interactions of these proteins with membranes have still not explained how a similar sequence and a potentially related fold can sustain such apparently different activities. In the present review, we have summarised the most relevant features of the structure, lipid-protein and protein-protein interactions of SP-B and the saposin-like family of proteins, as a basis to propose an integrated model and a common mechanistic framework of the apparent functional versatility of the saposin fold.

  8. Bacillus subtilis actin-like protein MreB influences the positioning of the replication machinery and requires membrane proteins MreC/D and other actin-like proteins for proper localization.

    PubMed

    Defeu Soufo, Hervé Joël; Graumann, Peter L

    2005-03-03

    Bacterial actin-like proteins have been shown to perform essential functions in several aspects of cellular physiology. They affect cell growth, cell shape, chromosome segregation and polar localization of proteins, and localize as helical filaments underneath the cell membrane. Bacillus subtilis MreB and Mbl have been shown to perform dynamic motor like movements within cells, extending along helical tracks in a time scale of few seconds. In this work, we show that Bacillus subtilis MreB has a dual role, both in the formation of rod cell shape, and in chromosome segregation, however, its function in cell shape is distinct from that of MreC. Additionally, MreB is important for the localization of the replication machinery to the cell centre, which becomes aberrant soon after depletion of MreB. 3D image reconstructions suggest that frequently, MreB filaments consist of several discontinuous helical filaments with varying length. The localization of MreB was abnormal in cells with decondensed chromosomes, as well as during depletion of Mbl, MreBH and of the MreC/MreD proteins, which we show localize to the cell membrane. Thus, proper positioning of MreB filaments depends on and is affected by a variety of factors in the cell. Our data provide genetic and cytological links between MreB and the membrane, as well as with other actin like proteins, and further supports the connection of MreB with the chromosome. The functional dependence on MreB of the localization of the replication machinery suggests that the replisome is not anchored at the cell centre, but is positioned in a dynamic manner.

  9. Bacterial motility complexes require the actin-like protein, MreB and the Ras homologue, MglA.

    PubMed

    Mauriello, Emilia M F; Mouhamar, Fabrice; Nan, Beiyan; Ducret, Adrien; Dai, David; Zusman, David R; Mignot, Tâm

    2010-01-20

    Gliding motility in the bacterium Myxococcus xanthus uses two motility engines: S-motility powered by type-IV pili and A-motility powered by uncharacterized motor proteins and focal adhesion complexes. In this paper, we identified MreB, an actin-like protein, and MglA, a small GTPase of the Ras superfamily, as essential for both motility systems. A22, an inhibitor of MreB cytoskeleton assembly, reversibly inhibited S- and A-motility, causing rapid dispersal of S- and A-motility protein clusters, FrzS and AglZ. This suggests that the MreB cytoskeleton is involved in directing the positioning of these proteins. We also found that a DeltamglA motility mutant showed defective localization of AglZ and FrzS clusters. Interestingly, MglA-YFP localization mimicked both FrzS and AglZ patterns and was perturbed by A22 treatment, consistent with results indicating that both MglA and MreB bind to motility complexes. We propose that MglA and the MreB cytoskeleton act together in a pathway to localize motility proteins such as AglZ and FrzS to assemble the A-motility machineries. Interestingly, M. xanthus motility systems, like eukaryotic systems, use an actin-like protein and a small GTPase spatial regulator.

  10. Bacterial motility complexes require the actin-like protein, MreB and the Ras homologue, MglA

    PubMed Central

    Mauriello, Emilia M F; Mouhamar, Fabrice; Nan, Beiyan; Ducret, Adrien; Dai, David; Zusman, David R; Mignot, Tâm

    2010-01-01

    Gliding motility in the bacterium Myxococcus xanthus uses two motility engines: S-motility powered by type-IV pili and A-motility powered by uncharacterized motor proteins and focal adhesion complexes. In this paper, we identified MreB, an actin-like protein, and MglA, a small GTPase of the Ras superfamily, as essential for both motility systems. A22, an inhibitor of MreB cytoskeleton assembly, reversibly inhibited S- and A-motility, causing rapid dispersal of S- and A-motility protein clusters, FrzS and AglZ. This suggests that the MreB cytoskeleton is involved in directing the positioning of these proteins. We also found that a ΔmglA motility mutant showed defective localization of AglZ and FrzS clusters. Interestingly, MglA–YFP localization mimicked both FrzS and AglZ patterns and was perturbed by A22 treatment, consistent with results indicating that both MglA and MreB bind to motility complexes. We propose that MglA and the MreB cytoskeleton act together in a pathway to localize motility proteins such as AglZ and FrzS to assemble the A-motility machineries. Interestingly, M. xanthus motility systems, like eukaryotic systems, use an actin-like protein and a small GTPase spatial regulator. PMID:19959988

  11. Prokaryotic Ubiquitin-Like Protein Modification

    PubMed Central

    Maupin-Furlow, Julie A.

    2016-01-01

    Prokaryotes form ubiquitin (Ub)-like isopeptide bonds on the lysine residues of proteins by at least two distinct pathways that are reversible and regulated. In mycobacteria, the C-terminal Gln of Pup (prokaryotic ubiquitin-like protein) is deamidated and isopeptide linked to proteins by a mechanism distinct from ubiquitylation in enzymology yet analogous to ubiquitylation in targeting proteins for destruction by proteasomes. Ub-fold proteins of archaea (SAMPs, small archaeal modifier proteins) and Thermus (TtuB, tRNA-two-thiouridine B) that differ from Ub in amino acid sequence, yet share a common β-grasp fold, also form isopeptide bonds by a mechanism that appears streamlined compared with ubiquitylation. SAMPs and TtuB are found to be members of a small group of Ub-fold proteins that function not only in protein modification but also in sulfur-transfer pathways associated with tRNA thiolation and molybdopterin biosynthesis. These multifunctional Ub-fold proteins are thought to be some of the most ancient of Ub-like protein modifiers. PMID:24995873

  12. Overexpression of avenin-like b proteins in bread wheat (Triticum aestivum L.) improves dough mixing properties by their incorporation into glutenin polymers.

    PubMed

    Ma, Fengyun; Li, Miao; Li, Tingting; Liu, Wei; Liu, Yunyi; Li, Yin; Hu, Wei; Zheng, Qian; Wang, Yaqiong; Li, Kexiu; Chang, Junli; Chen, Mingjie; Yang, Guangxiao; Wang, Yuesheng; He, Guangyuan

    2013-01-01

    Avenin-like b proteins are a small family of wheat storage proteins, each containing 18 or 19 cysteine residues. The role of these proteins, with high numbers of cysteine residues, in determining the functional properties of wheat flour is unclear. In the present study, two transgenic lines of the bread wheat overexpressing avenin-like b gene were generated to investigate the effects of Avenin-like b proteins on dough mixing properties. Sodium dodecyl sulfate sedimentation (SDSS) test and Mixograph analysis of these lines demonstrated that overexpression of Avenin-like b proteins in both transgenic wheat lines significantly increased SDSS volume and improved dough elasticity, mixing tolerance and resistance to extension. These changes were associated with the increased proportion of polymeric proteins due to the incorporation of overexpressed Avenin-like b proteins into the glutenin polymers. The results of this study were critical to confirm the hypothesis that Avenin-like b proteins could be integrated into glutenin polymers by inter-chain disulphide bonds, which could help understand the mechanism behind strengthening wheat dough strength.

  13. Overexpression of Avenin-Like b Proteins in Bread Wheat (Triticum aestivum L.) Improves Dough Mixing Properties by Their Incorporation into Glutenin Polymers

    PubMed Central

    Ma, Fengyun; Li, Miao; Li, Tingting; Liu, Wei; Liu, Yunyi; Li, Yin; Hu, Wei; Zheng, Qian; Wang, Yaqiong; Li, Kexiu; Chang, Junli; Chen, Mingjie; Yang, Guangxiao; Wang, Yuesheng; He, Guangyuan

    2013-01-01

    Avenin-like b proteins are a small family of wheat storage proteins, each containing 18 or 19 cysteine residues. The role of these proteins, with high numbers of cysteine residues, in determining the functional properties of wheat flour is unclear. In the present study, two transgenic lines of the bread wheat overexpressing avenin-like b gene were generated to investigate the effects of Avenin-like b proteins on dough mixing properties. Sodium dodecyl sulfate sedimentation (SDSS) test and Mixograph analysis of these lines demonstrated that overexpression of Avenin-like b proteins in both transgenic wheat lines significantly increased SDSS volume and improved dough elasticity, mixing tolerance and resistance to extension. These changes were associated with the increased proportion of polymeric proteins due to the incorporation of overexpressed Avenin-like b proteins into the glutenin polymers. The results of this study were critical to confirm the hypothesis that Avenin-like b proteins could be integrated into glutenin polymers by inter-chain disulphide bonds, which could help understand the mechanism behind strengthening wheat dough strength. PMID:23843964

  14. SRC-like adaptor protein regulates B cell development and function.

    PubMed

    Dragone, Leonard L; Myers, Margaret D; White, Carmen; Sosinowski, Tomasz; Weiss, Arthur

    2006-01-01

    The avidity of BCRs and TCRs influences signal strength during processes of lymphocyte development. Avidity is determined by both the intrinsic affinity for Ag and surface levels of the Ag receptor. The Src-like adaptor protein (SLAP) is a regulator of TCR levels on thymocytes, and its deficiency alters thymocyte development. We hypothesized that SLAP, which is expressed in B cells, also is important in regulating BCR levels, signal strength, and B cell development. To test this hypothesis, we analyzed the B cell compartment in SLAP-deficient mice. We found increased splenic B cell numbers and decreased surface IgM levels on mature, splenic B cells deficient in SLAP. Immature bone marrow and splenic B cells from BCR-transgenic, SLAP-deficient mice were found to express higher surface levels of IgM. In contrast, mature splenic B cells from BCR-transgenic mice expressed decreased levels of surface BCR associated with decreased calcium flux and activation-induced markers, compared with controls. These data suggest that SLAP regulates BCR levels and signal strength during lymphocyte development.

  15. Dynamic localization and interaction with other Bacillus subtilis actin-like proteins are important for the function of MreB.

    PubMed

    Defeu Soufo, Hervé Joël; Graumann, Peter L

    2006-12-01

    Bacterial actin-like proteins play a key role in cell morphology and in chromosome segregation. Many bacteria, like Bacillus subtilis, contain three genes encoding actin-like proteins, called mreB, mbl and mreBH in B. subtilis. We show that MreB and Mbl colocalize extensively within live cells, and that all three B. subtilis actin paralogues interact with each other underneath the cell membrane. A mutation in the phosphate 2 motif of MreB had a dominant negative effect on cell morphology and on chromosome segregation. Expression of this mutant allele of MreB interfered with the dynamic localization of Mbl. These experiments show that the interaction between MreB and Mbl has physiological significance. An mreB deletion strain can grow under special media conditions, however, depletion of Mbl in this mutant background abolished growth, indicating that actin paralogues can partially complement each other. The membrane protein MreC was found to interact with Mbl, but not with MreB, revealing a clear distinction between the function of the two paralogues. The phosphate 2 mutant MreB protein allowed for filament formation of mutant or wild-type MreB, but abolished the dynamic reorganization of the filaments. The latter mutation led to a strong reduction, but not complete loss, of function of MreB, both in terms of chromosome segregation and of cell morphology. Our work shows that that the dynamic localization of MreB is essential for the proper activity of the actin-like protein and that the interactions between MreB paralogues have important physiological significance.

  16. The MreB-like protein Mbl of Streptomyces coelicolor A3(2) depends on MreB for proper localization and contributes to spore wall synthesis.

    PubMed

    Heichlinger, Andrea; Ammelburg, Moritz; Kleinschnitz, Eva-Maria; Latus, Annette; Maldener, Iris; Flärdh, Klas; Wohlleben, Wolfgang; Muth, Günther

    2011-04-01

    Most bacteria with a rod-shaped morphology contain an actin-like cytoskeleton consisting of MreB polymers, which form helical spirals underneath the cytoplasmic membrane to direct peptidoglycan synthesis for the elongation of the cell wall. In contrast, MreB of Streptomyces coelicolor is not required for vegetative growth but has a role in sporulation. Besides MreB, S. coelicolor encodes two further MreB-like proteins, Mbl and SCO6166, whose function is unknown. Whereas MreB and Mbl are highly similar, SCO6166 is shorter, lacking the subdomains IB and IIB of actin-like proteins. Here, we showed that MreB and Mbl are not functionally redundant but cooperate in spore wall synthesis. Expression analysis by semiquantitative reverse transcription-PCR revealed distinct expression patterns. mreB and mbl are induced predominantly during morphological differentiation. In contrast, sco6166 is strongly expressed during vegetative growth but switched off during sporulation. All genes could be deleted without affecting viability. Even a ΔmreB Δmbl double mutant was viable. Δsco6166 had a wild-type phenotype. ΔmreB, Δmbl, and ΔmreB Δmbl produced swollen, prematurely germinating spores that were sensitive to various kinds of stress, suggesting a defect in spore wall integrity. During aerial mycelium formation, an Mbl-mCherry fusion protein colocalized with an MreB-enhanced green fluorescent protein (MreB-eGFP) fusion protein at the sporulation septa. Whereas MreB-eGFP localized properly in the Δmbl mutant, Mbl-mCherry localization depended on the presence of a functional MreB protein. Our results revealed that MreB and Mbl cooperate in the synthesis of the thickened spore wall, while SCO6166 has a nonessential function during vegetative growth.

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

    PubMed

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

    2016-03-18

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

  18. Translation elongation factor EF-Tu modulates filament formation of actin-like MreB protein in vitro.

    PubMed

    Defeu Soufo, Hervé Joël; Reimold, Christian; Breddermann, Hannes; Mannherz, Hans G; Graumann, Peter L

    2015-04-24

    EF-Tu has been shown to interact with actin-like protein MreB and to affect its localization in Escherichia coli and in Bacillus subtilis cells. We have purified YFP-MreB in an active form, which forms filaments on glass slides in vitro and was active in dynamic light-scattering assays, polymerizing in milliseconds after addition of magnesium. Purified EF-Tu enhanced the amount of MreB filaments, as seen by sedimentation assays, the speed of filament formation and the length of MreB filaments in vitro. EF-Tu had the strongest impact on MreB filaments in a 1:1 ratio, and EF-Tu co-sedimented with MreB filaments, revealing a stoichiometric interaction between both proteins. This was supported by cross-linking assays where 1:1 species were well detectable. When expressed in E. coli cells, B. subtilis MreB formed filaments and induced the formation of co-localizing B. subtilis EF-Tu structures, indicating that MreB can direct the positioning of EF-Tu structures in a heterologous cell system. Fluorescence recovery after photobleaching analysis showed that MreB filaments have a higher turnover in B. subtilis cells than in E. coli cells, indicating different filament kinetics in homologous or heterologous cell systems. The data show that MreB can direct the localization of EF-Tu in vivo, which in turn positively affects the formation and dynamics of MreB filaments. Thus, EF-Tu is a modulator of the activity of a bacterial actin-like protein. Copyright © 2015. Published by Elsevier Ltd.

  19. The MreB-Like Protein Mbl of Streptomyces coelicolor A3(2) Depends on MreB for Proper Localization and Contributes to Spore Wall Synthesis▿ †

    PubMed Central

    Heichlinger, Andrea; Ammelburg, Moritz; Kleinschnitz, Eva-Maria; Latus, Annette; Maldener, Iris; Flärdh, Klas; Wohlleben, Wolfgang; Muth, Günther

    2011-01-01

    Most bacteria with a rod-shaped morphology contain an actin-like cytoskeleton consisting of MreB polymers, which form helical spirals underneath the cytoplasmic membrane to direct peptidoglycan synthesis for the elongation of the cell wall. In contrast, MreB of Streptomyces coelicolor is not required for vegetative growth but has a role in sporulation. Besides MreB, S. coelicolor encodes two further MreB-like proteins, Mbl and SCO6166, whose function is unknown. Whereas MreB and Mbl are highly similar, SCO6166 is shorter, lacking the subdomains IB and IIB of actin-like proteins. Here, we showed that MreB and Mbl are not functionally redundant but cooperate in spore wall synthesis. Expression analysis by semiquantitative reverse transcription-PCR revealed distinct expression patterns. mreB and mbl are induced predominantly during morphological differentiation. In contrast, sco6166 is strongly expressed during vegetative growth but switched off during sporulation. All genes could be deleted without affecting viability. Even a ΔmreB Δmbl double mutant was viable. Δsco6166 had a wild-type phenotype. ΔmreB, Δmbl, and ΔmreB Δmbl produced swollen, prematurely germinating spores that were sensitive to various kinds of stress, suggesting a defect in spore wall integrity. During aerial mycelium formation, an Mbl-mCherry fusion protein colocalized with an MreB-enhanced green fluorescent protein (MreB-eGFP) fusion protein at the sporulation septa. Whereas MreB-eGFP localized properly in the Δmbl mutant, Mbl-mCherry localization depended on the presence of a functional MreB protein. Our results revealed that MreB and Mbl cooperate in the synthesis of the thickened spore wall, while SCO6166 has a nonessential function during vegetative growth. PMID:21257777

  20. Brucella TIR-like protein TcpB/Btp1 specifically targets the host adaptor protein MAL/TIRAP to promote infection.

    PubMed

    Li, Wenna; Ke, Yuehua; Wang, Yufei; Yang, Mingjuan; Gao, Junguang; Zhan, Shaoxia; Xinying, Du; Huang, Liuyu; Li, Wenfeng; Chen, Zeliang; Li, Juan

    2016-08-26

    Brucella spp. are known to avoid host immune recognition and weaken the immune response to infection. Brucella like accomplish this by employing two clever strategies, called the stealth strategy and hijacking strategy. The TIR domain-containing protein (TcpB/Btp1) of Brucella melitensis is thought to be involved in inhibiting host NF-κB activation by binding to adaptors downstream of Toll-like receptors. However, of the five TIR domain-containing adaptors conserved in mammals, whether MyD88 or MAL, even other three adaptors, are specifically targeted by TcpB has not been identified. Here, we confirmed the effect of TcpB on B.melitensis virulence in mice and found that TcpB selectively targets MAL. By using siRNA against MAL, we found that TcpB from B.melitensis is involved in intracellular survival and that MAL affects intracellular replication of B.melitensis. Our results confirm that TcpB specifically targets MAL/TIRAP to disrupt downstream signaling pathways and promote intra-host survival of Brucella spp. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Novel cathepsin B and cathepsin B-like cysteine protease of Naegleria fowleri excretory-secretory proteins and their biochemical properties.

    PubMed

    Lee, Jinyoung; Kim, Jong-Hyun; Sohn, Hae-Jin; Yang, Hee-Jong; Na, Byoung-Kuk; Chwae, Yong-Joon; Park, Sun; Kim, Kyongmin; Shin, Ho-Joon

    2014-08-01

    Naegleria fowleri causes a lethal primary amoebic meningoencephalitis (PAM) in humans and experimental animals, which leads to death within 7-14 days. Cysteine proteases of parasites play key roles in nutrient uptake, excystment/encystment, host tissue invasion, and immune evasion. In this study, we cloned N. fowleri cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes from our cDNA library of N. fowleri. The full-length sequences of genes were 1,038 and 939 bp (encoded 345 and 313 amino acids), and molecular weights were 38.4 and 34 kDa, respectively. Also, nfcpb and nfcpb-L showed a 56 and 46 % identity to Naegleria gruberi cathepsin B and cathepsin B-like enzyme, respectively. Recombinant NfCPB (rNfCPB) and NfCPB-L (rNfCPB-L) proteins were expressed by the pEX5-NT/TOPO vector that was transformed into Escherichia coli BL21, and they showed 38.4 and 34 kDa bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using their respective antibodies. Proteolytic activity of refolded rNfCPB and rNfCPB-L was maximum at a pH of 4.5, and the most effective substrate was Z-LR-MCA. rNfCPB and rNfCPB-L showed proteolytic activity for several proteins such as IgA, IgG, IgM, collagen, fibronectin, hemoglobin, and albumin. These results suggested that NfCPB and NfCPB-L cysteine protease are important components of the N. fowleri ESP, and they may play important roles in host tissue invasion and immune evasion as pathogens that cause N. fowleri PAM.

  2. Production and characterization of monoclonal antibodies against cathepsin B and cathepsin B-Like proteins of Naegleria fowleri.

    PubMed

    Seong, Gi-Sang; Sohn, Hae-Jin; Kang, Heekyoung; Seo, Ga-Eun; Kim, Jong-Hyun; Shin, Ho-Joon

    2017-12-01

    Naegleria fowleri causes fatal primary amoebic meningoencephalitis (PAM) in humans and experimental animals. In previous studies, cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes of N. fowleri were cloned, and it was suggested that refolding rNfCPB and rNfCPB-L proteins could play important roles in host tissue invasion, immune response evasion and nutrient uptake. In this study, we produced anti-NfCPB and anti-NfCPB-L monoclonal antibodies (McAb) using a cell fusion technique, and observed their immunological characteristics. Seven hybridoma cells secreting rNfCPB McAbs and three hybridoma cells secreting rNfCPB-L McAbs were produced. Among these, 2C9 (monoclone for rNfCPB) and 1C8 (monoclone for rNfCPB-L) McAb showed high antibody titres and were finally selected for use. As determined by western blotting, 2C9 McAb bound to N. fowleri lysates, specifically the rNfCPB protein, which had bands of 28 kDa and 38.4 kDa. 1C8 McAb reacted with N. fowleri lysates, specifically the rNfCPB-L protein, which had bands of 24 kDa and 34 kDa. 2C9 and 1C8 monoclonal antibodies did not bind to lysates of other amoebae, such as N. gruberi, Acanthamoeba castellanii and A. polyphaga in western blot analyses. Immuno-cytochemistry analysis detected NfCPB and NfCPB-L proteins in the cytoplasm of N. fowleri trophozoites, particularly in the pseudopodia and food-cup. These results suggest that monoclonal antibodies produced against rNfCPB and rNfCPB-L proteins may be useful for further immunological study of PAM. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Comparative Effects of Parathion and Chlorpyrifos on Endocannabinoid and Endocannabinoid-Like Lipid Metabolites in Rat Striatum

    PubMed Central

    Liu, Jing; Parsons, Loren; Pope, Carey

    2015-01-01

    Parathion and chlorpyrifos are organophosphorus insecticides (OPs) that elicit acute toxicity by inhibiting acetylcholinesterase (AChE). The endocannabinoids (eCBs, N-arachidonoylethanolamine, AEA; 2-arachidonoylglycerol, 2AG) are endogenous neuromodulators that regulate presynaptic neurotransmitter release in neurons throughout the central and peripheral nervous systems. While substantial information is known about the eCBs, less is known about a number of endocannabinoid-like metabolites (eCBLs, e.g., N-palmitoylethanolamine, PEA; N-oleoylethanolamine, OEA). We report the comparative effects of parathion and chlorpyrifos on AChE and enzymes responsible for inactivation of the eCBs, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), and changes in the eCBs AEA and 2AG and eCBLs PEA and OEA, in rat striatum. Adult, male rats were treated with vehicle (peanut oil, 2 ml/kg, sc), parathion (27 mg/kg) or chlorpyrifos (280 mg/kg) 6-7 days after surgical implantation of microdialysis cannulae into the right striatum, followed by microdialysis two or four days later. Additional rats were similarly treated and sacrificed for evaluation of tissue levels of eCBs and eCBLs. Dialysates and tissue extracts were analyzed by LC-MS/MS. AChE and FAAH were extensively inhibited at both time-points (85-96%), while MAGL activity was significantly but lesser affected (37-62% inhibition) by parathion and chlorpyrifos. Signs of toxicity were noted only in parathion-treated rats. In general, chlorpyrifos increased eCB levels while parathion had no or lesser effects. Early changes in extracellular AEA, 2AG and PEA levels were significantly different between parathion and chlorpyrifos exposures. Differential changes in extracellular and/or tissue levels of eCBs and eCBLs could potentially influence a number of signaling pathways and contribute to selective neurological changes following acute OP intoxications. PMID:26215119

  4. Comparative effects of parathion and chlorpyrifos on endocannabinoid and endocannabinoid-like lipid metabolites in rat striatum.

    PubMed

    Liu, Jing; Parsons, Loren; Pope, Carey

    2015-09-01

    Parathion and chlorpyrifos are organophosphorus insecticides (OPs) that elicit acute toxicity by inhibiting acetylcholinesterase (AChE). The endocannabinoids (eCBs, N-arachidonoylethanolamine, AEA; 2-arachidonoylglycerol, 2AG) are endogenous neuromodulators that regulate presynaptic neurotransmitter release in neurons throughout the central and peripheral nervous systems. While substantial information is known about the eCBs, less is known about a number of endocannabinoid-like metabolites (eCBLs, e.g., N-palmitoylethanolamine, PEA; N-oleoylethanolamine, OEA). We report the comparative effects of parathion and chlorpyrifos on AChE and enzymes responsible for inactivation of the eCBs, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), and changes in the eCBs AEA and 2AG and eCBLs PEA and OEA, in rat striatum. Adult, male rats were treated with vehicle (peanut oil, 2 ml/kg, sc), parathion (27 mg/kg) or chlorpyrifos (280 mg/kg) 6-7 days after surgical implantation of microdialysis cannulae into the right striatum, followed by microdialysis two or four days later. Additional rats were similarly treated and sacrificed for evaluation of tissue levels of eCBs and eCBLs. Dialysates and tissue extracts were analyzed by LC-MS/MS. AChE and FAAH were extensively inhibited at both time-points (85-96%), while MAGL activity was significantly but lesser affected (37-62% inhibition) by parathion and chlorpyrifos. Signs of toxicity were noted only in parathion-treated rats. In general, chlorpyrifos increased eCB levels while parathion had no or lesser effects. Early changes in extracellular AEA, 2AG and PEA levels were significantly different between parathion and chlorpyrifos exposures. Differential changes in extracellular and/or tissue levels of eCBs and eCBLs could potentially influence a number of signaling pathways and contribute to selective neurological changes following acute OP intoxications. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Redox biology of Mycobacterium tuberculosis H37Rv: protein-protein interaction between GlgB and WhiB1 involves exchange of thiol-disulfide

    PubMed Central

    Garg, Saurabh; Alam, Md Suhail; Bajpai, Richa; Kishan, KV Radha; Agrawal, Pushpa

    2009-01-01

    Background Mycobacterium tuberculosis, an intracellular pathogen encounters redox stress throughout its life inside the host. In order to protect itself from the redox onslaughts of host immune system, M. tuberculosis appears to have developed accessory thioredoxin-like proteins which are represented by ORFs encoding WhiB-like proteins. We have earlier reported that WhiB1/Rv3219 is a thioredoxin like protein of M. tuberculosis and functions as a protein disulfide reductase. Generally thioredoxins have many substrate proteins. The current study aims to identify the substrate protein(s) of M. tuberculosis WhiB1. Results Using yeast two-hybrid screen, we identified alpha (1,4)-glucan branching enzyme (GlgB) of M. tuberculosis as a interaction partner of WhiB1. In vitro GST pull down assay confirmed the direct physical interaction between GlgB and WhiB1. Both mass spectrometry data of tryptic digests and in vitro labeling of cysteine residues with 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid showed that in GlgB, C95 and C658 are free but C193 and C617 form an intra-molecular disulfide bond. WhiB1 has a C37XXC40 motif thus a C40S mutation renders C37 to exist as a free thiol to form a hetero-disulfide bond with the cysteine residue of substrate protein. A disulfide mediated binary complex formation between GlgB and WhiB1C40S was shown by both in-solution protein-protein interaction and thioredoxin affinity chromatography. Finally, transfer of reducing equivalent from WhiB1 to GlgB disulfide was confirmed by 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid trapping by the reduced disulfide of GlgB. Two different thioredoxins, TrxB/Rv1471 and TrxC/Rv3914 of M. tuberculosis could not perform this reaction suggesting that the reduction of GlgB by WhiB1 is specific. Conclusion We conclude that M. tuberculosis GlgB has one intra-molecular disulfide bond which is formed between C193 and C617. WhiB1, a thioredoxin like protein interacts with GlgB and

  6. Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

    PubMed

    Morohashi, Kengo; Sahara, Hiroeki; Watashi, Koichi; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

    2011-04-29

    Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

  7. Cyclosporin A Associated Helicase-Like Protein Facilitates the Association of Hepatitis C Virus RNA Polymerase with Its Cellular Cyclophilin B

    PubMed Central

    Sahara, Hiroeki; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

    2011-01-01

    Background Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Principal Findings Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. Conclusions We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology. PMID:21559518

  8. Effect of protein solution components in the adsorption of Herbaspirillum seropedicae GlnB protein on mica.

    PubMed

    Ferreira, Cecília F G; Benelli, Elaine M; Klein, Jorge J; Schreiner, Wido; Camargo, Paulo C

    2009-10-15

    The adsorption of proteins and its buffer solution on mica surfaces was investigated by atomic force microscopy (AFM). Different salt concentration of the Herbaspirillum seropedicae GlnB protein (GlnB-Hs) solution deposited on mica was investigated. This protein is a globular, soluble homotrimer (36kDa), member of PII-like proteins family involved in signal transducing in prokaryote. Supramolecular structures were formed when this protein was deposited onto bare mica surface. The topographic AFM images of the GlnB-Hs films showed that at high salt concentration the supramolecular structures are spherical-like, instead of the typical doughnut-like shape for low salt concentration. AFM images of NaCl and Tris from the buffer solution showed structures with the same pattern as those observed for high salt protein solution, misleading the image interpretation. XPS experiments showed that GlnB protein film covers the mica surface without chemical reaction.

  9. Expression Patterns and Identified Protein-Protein Interactions Suggest That Cassava CBL-CIPK Signal Networks Function in Responses to Abiotic Stresses.

    PubMed

    Mo, Chunyan; Wan, Shumin; Xia, Youquan; Ren, Ning; Zhou, Yang; Jiang, Xingyu

    2018-01-01

    Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL) protein and CBL-interacting protein kinase (CIPK) is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 CBL ( MeCBL ) and 26 CIPK ( MeCIPK ) genes were isolated from cassava by genome searching and cloning of cDNA sequences of Arabidopsis CBL s and CIPK s. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression levels of MeCBL and MeCIPK genes were different in different tissues throughout the life cycle. The expression patterns of 7 CBL and 26 CIPK genes in response to NaCl, PEG, heat and cold stresses were analyzed by quantitative real-time PCR (qRT-PCR), and it was found that the expression of each was induced by multiple stimuli. Furthermore, we found that many pairs of CBLs and CIPKs could interact with each other via investigating the interactions between 8 CBL and 25 CIPK proteins using a yeast two-hybrid system. Yeast cells co-transformed with cassava MeCIPK24, MeCBL10 , and Na + /H + antiporter MeSOS1 genes exhibited higher salt tolerance compared to those with one or two genes. These results suggest that the cassava CBL-CIPK signal network might play key roles in response to abiotic stresses.

  10. Identification and comprehensive analyses of the CBL and CIPK gene families in wheat (Triticum aestivum L.).

    PubMed

    Sun, Tao; Wang, Yan; Wang, Meng; Li, Tingting; Zhou, Yi; Wang, Xiatian; Wei, Shuya; He, Guangyuan; Yang, Guangxiao

    2015-11-04

    Calcineurin B-like (CBL) proteins belong to a unique group of calcium sensors in plant that decode the Ca(2+) signature by interacting with CBL-interacting protein kinases (CIPKs). Although CBL-CIPK complexes have been shown to play important roles in the responses to various stresses in plants, little is known about their functions in wheat. A total of seven TaCBL and 20 TaCIPK genes were amplified from bread wheat, Triticum aestivum cv. Chinese Spring. Reverse-transcriptase-polymerase chain reaction (RT-PCR) and in silico expression analyses showed that TaCBL and TaCIPK genes were expressed at different levels in different tissues, or maintained at nearly constant expression levels during the whole life cycle of the wheat plant. Some TaCBL and TaCIPK genes showed up- or down-regulated expressions during seed germination. Preferential interactions between TaCBLs and TaCIPKs were observed in yeast two-hybrid and bimolecular fluorescence complementation experiments. Analyses of a deletion series of TaCIPK proteins with amino acid variations at the C-terminus provided new insights into the specificity of the interactions between TaCIPKs and TaCBLs, and indicated that the TaCBL-TaCIPK signaling pathway is very complex in wheat because of its hexaploid genome. The expressions of many TaCBLs and TaCIPKs were responsive to abiotic stresses (salt, cold, and simulated drought) and abscisic acid treatment. Transgenic Arabidopsis plants overexpressing TaCIPK24 exhibited improved salt tolerance through increased Na(+) efflux and an enhanced reactive oxygen species scavenging capacity. These results contribute to our understanding of the functions of CBL-CIPK complexes and provide the basis for selecting appropriate genes for in-depth functional studies of CBL-CIPK in wheat.

  11. Suppression of NF-κB signal pathway by NLRC3-like protein in stony coral Acropora aculeus under heat stress.

    PubMed

    Zhou, Zhi; Wu, Yibo; Zhang, Chengkai; Li, Can; Chen, Guangmei; Yu, Xiaopeng; Shi, Xiaowei; Xu, Yanlai; Wang, Lingui; Huang, Bo

    2017-08-01

    Heat stress is the most common factor for coral bleaching, which has increased both in frequency and severity due to global warming. In the present study, the stony coral Acropora aculeus was subjected to acute heat stress and entire transcriptomes were sequenced via the next generation sequencing platform. Four paired-end libraries were constructed and sequenced in two groups, including a control and a heat stress group. A total of 120,319,751 paired-end reads with lengths of 2 × 100 bp were assembled and 55,021 coral-derived genes were obtained. After read mapping and abundance estimation, 9110 differentially expressed genes were obtained in the comparison between the control and heat stress group, including 4465 significantly upregulated and 4645 significantly downregulated genes. Twenty-three GO terms in the Biological Process category were overrepresented for significantly upregulated genes, and divided into six groups according to their relationship. These three groups were related to the NF-κB signal pathway, and the remaining three groups were relevant for pathogen response, immunocyte activation and protein ubiquitination. Forty-three common genes were found in four GO terms, which were directly related to the NF-κB signal pathway. These included 2 NACHT, LRR, PYD domains-containing protein, 5 nucleotide-binding oligomerization domain-containing protein, 29 NLRC3-like protein, 4 NLRC5-like protein, and 3 uncharacterized protein. For significantly downregulated genes, 27 overrepresented GO terms were found in the Biological Process category, which were relevant to protein ubiquitination and ATP metabolism. Our results indicate that heat stress suppressed the immune response level via the NLRC3-like protein, the fine-tuning of protein turnover activity, and ATP metabolism. This might disrupt the balance of coral-zooxanthellae symbiosis and result in the bleaching of the coral A. aculeus. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

    PubMed

    Cai, Yingying; Liu, Yuting; Culhane, Kelly J; DeVree, Brian T; Yang, Yang; Sunahara, Roger K; Yan, Elsa C Y

    2017-01-01

    Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

  13. Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor

    PubMed Central

    Cai, Yingying; Liu, Yuting; Culhane, Kelly J.; DeVree, Brian T.; Yang, Yang; Sunahara, Roger K.; Yan, Elsa C. Y.

    2017-01-01

    Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms. PMID:28609478

  14. The C-terminal region of the non-structural protein 2B from Hepatitis A Virus demonstrates lipid-specific viroporin-like activity

    NASA Astrophysics Data System (ADS)

    Shukla, Ashutosh; Dey, Debajit; Banerjee, Kamalika; Nain, Anshu; Banerjee, Manidipa

    2015-10-01

    Viroporins are virally encoded, membrane-active proteins, which enhance viral replication and assist in egress of viruses from host cells. The 2B proteins in the picornaviridae family are known to have viroporin-like properties, and play critical roles during virus replication. The 2B protein of Hepatitis A Virus (2B), an unusual picornavirus, is somewhat dissimilar from its analogues in several respects. HAV 2B is approximately 2.5 times the length of other 2B proteins, and does not disrupt calcium homeostasis or glycoprotein trafficking. Additionally, its membrane penetrating properties are not yet clearly established. Here we show that the membrane interacting activity of HAV 2B is localized in its C-terminal region, which contains an alpha-helical hairpin motif. We show that this region is capable of forming small pores in membranes and demonstrates lipid specific activity, which partially rationalizes the intracellular localization of full-length 2B. Using a combination of biochemical assays and molecular dynamics simulation studies, we also show that HAV 2B demonstrates a marked propensity to dimerize in a crowded environment, and probably interacts with membranes in a multimeric form, a hallmark of other picornavirus viroporins. In sum, our study clearly establishes HAV 2B as a bona fide viroporin in the picornaviridae family.

  15. Marburg Virus VP24 Protein Relieves Suppression of the NF-κB Pathway Through Interaction With Kelch-like ECH-Associated Protein 1.

    PubMed

    Edwards, Megan R; Basler, Christopher F

    2015-10-01

    Marburg virus (MARV) is an emerging zoonotic pathogen that causes hemorrhagic fever. MARV VP24 (mVP24) protein interacts with the host cell protein Kelch-like-ECH-associated protein 1 (Keap1). Keap1 interacts with and promotes the degradation of IκB kinase β (IKKβ), a component of the IκB kinase (IKK) complex that regulates nuclear factor-κB (NF-κB) activity. We studied whether mVP24 could relieve Keap1 repression of the NF-κB pathway. Coimmunoprecipitation assays were used to examine the interaction between Keap1 and IKKβ in the presence of wild-type mVP24 and mutants of mVP24 defective for binding to Keap1. Western blotting was used to determine levels of IKKβ expression in the presence of Keap1 and mVP24. NF-κB promoter-luciferase assays were used to determine the effect of mVP24 on Keap1-induced repression of activity. Expression of wild-type mVP24 disrupted the interaction of IKKβ and Keap1, whereas weakly interacting and noninteracting mVP24 mutants did not disrupt the interaction between Keap1 and IKKβ. The interaction of mVP24 with Keap1 enhanced IKKβ levels in the presence of Keap1. The interaction of mVP24 with Keap1 also relieved Keap1 repression of NF-κB reporter activity. mVP24 can relieve Keap1 repression of the NF-κB pathway through its interaction with Keap1. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. The SsgA-like proteins in actinomycetes: small proteins up to a big task.

    PubMed

    Traag, Bjørn A; van Wezel, Gilles P

    2008-06-01

    Several unique protein families have been identified that play a role in the control of developmental cell division in streptomycetes. The SsgA-like proteins or SALPs, of which streptomycetes typically have at least five paralogues, control specific steps of sporulation-specific cell division in streptomycetes, affecting cell wall-related events such as septum localization and synthesis, thickening of the spore wall and autolytic spore separation. The expression level of SsgA, the best studied SALP, has a rather dramatic effect on septation and on hyphal morphology, which is not only of relevance for our understanding of (developmental) cell division but has also been successfully applied in industrial fermentation, to improve growth and production of filamentous actinomycetes. Recent observations suggest that SsgB most likely is the archetypal SALP, with only SsgB orthologues occurring in all morphologically complex actinomycetes. Here we review 10 years of research on the SsgA-like proteins in actinomycetes and discuss the most interesting regulatory, functional, phylogenetic and applied aspects of this relatively unknown protein family.

  17. Requirement of SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 for paired immunoglobulin-like receptor B (PIR-B)-mediated inhibitory signal.

    PubMed

    Maeda, A; Kurosaki, M; Ono, M; Takai, T; Kurosaki, T

    1998-04-20

    Paired immunoglobulin-like receptor B (PIR-B) (p91) molecule has been proposed to function as an inhibitory receptor in B cells and myeloid lineage cells. We demonstrate here that the cytoplasmic region of PIR-B is capable of inhibiting B cell activation. Mutational analysis of five cytoplasmic tyrosines indicate that tyrosine 771 in the motif VxYxxL plays the most crucial role in mediating the inhibitory signal. PIR-B-mediated inhibition was markedly reduced in the SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 double-deficient DT40 B cells, whereas this inhibition was unaffected in the inositol polyphosphate 5'-phosphatase SHIP-deficient cells. These data demonstrate that PIR-B can negatively regulate B cell receptor activation and that this PIR-B-mediated inhibition requires redundant functions of SHP-1 and SHP-2.

  18. Organometallic DNA-B12 Conjugates as Potential Oligonucleotide Vectors: Synthesis and Structural and Binding Studies with Human Cobalamin-Transport Proteins.

    PubMed

    Mutti, Elena; Hunger, Miriam; Fedosov, Sergey; Nexo, Ebba; Kräutler, Bernhard

    2017-11-16

    The synthesis and structural characterization of Co-(dN) 25 -Cbl (Cbl: cobalamin; dN: deoxynucleotide) and Co-(dN) 39 -Cbl, which are organometallic DNA-B 12 conjugates with single DNA strands consisting of 25 and 39 deoxynucleotides, respectively, and binding studies of these two DNA-Cbl conjugates to three homologous human Cbl transporting proteins, transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are reported. This investigation tests the suitability of such DNA-Cbls for the task of eventual in vivo oligonucleotide delivery. The binding of DNA-Cbl to TC, IF, and HC was investigated in competition with either a fluorescent Cbl derivative and Co-(dN) 25 -Cbl, or radiolabeled vitamin B 12 ( 57 Co-CNCbl) and Co-(dN) 25 -Cbl or Co-(dN) 39 -Cbl. Binding of the new DNA-Cbl conjugates was fast and tight with TC, but poorer with HC and IF, which extends a similar original finding with the simpler DNA-Cbl, Co-(dN) 18 -Cbl. The contrasting affinities of TC versus IF and HC for the DNA-Cbl conjugates are rationalized herein by a stepwise mechanism of Cbl binding. Critical contributions to overall affinity result from gradual conformational adaptations of the Cbl-binding proteins to the DNA-Cbl, which is first bound to the respective β domains. This transition is fast with TC, but slow with IF and HC, with which weaker binding results. The invariably tight interaction of the DNA-Cbl conjugates with TC makes the Cbl moiety a potential natural vector for the specific delivery of oligonucleotide loads from the blood into cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Antidepressant-like actions by silencing of neuronal ELAV-like RNA-binding proteins HuB and HuC in a model of depression in male mice.

    PubMed

    Sanna, Maria Domenica; Quattrone, Alessandro; Galeotti, Nicoletta

    2018-06-01

    Currently available antidepressant drugs often fail to achieve full remission and patients might evolve to treatment resistance, showing the need to achieve a better therapy of depressive disorders. Increasing evidence supports that post-transcriptional regulation of gene expression is important in neuronal development and survival and a relevant role is played by RNA binding proteins (RBP). To explore new therapeutic strategies, we investigated the role of the neuron-specific ELAV-like RBP (HuB, HuC, HuD) in a mouse model of depression. In this study, a 4-week unpredictable chronic mild stress (UCMS) protocol was applied to mice to induce a depressive-like phenotype. In the last 2 weeks of the UCMS regimen, silencing of HuB, HuC or HuD was performed by using specific antisense oligonucleotides (aODN). Treatment of UCMS-exposed mice with anti-HuB and anti-HuC aODN improved both anhedonia and behavioural despair, used as measures of depressive-like behaviour, without modifying the response of stressed mice to an anxiety-inducing environment. On the contrary, HuD silencing promoted an anxiolytic-like behaviour in UCMS-exposed mice without improving depressive-like behaviours. The antidepressant-like phenotype of anti-HuB and anti-HuC mice was not shown concurrently with the promotion of adult hippocampal neurogenesis in the dentate gyrus, and no increase in the BDNF and CREB content was detected. Conversely, in the CA3 hippocampal region, projection area of newly born neurons, HuB and HuC silencing increased the number of BrdU/NeuN positive cells. These results give the first indication of a role of nELAV in the modulation of emotional states in a mouse model of depression. Copyright © 2018 Elsevier Ltd. All rights reserved.

  20. TrkA and TrkC neurotrophin receptor-like proteins in the lizard gut.

    PubMed

    Lucini, C; de Girolamo, P; Lamanna, C; Botte, V; Vega, J A; Castaldo, L

    2001-03-01

    The tyrosine kinase proteins (Trk), encoded by the trk family of proto-oncogenes, mediate, in mammals, the action of neurotrophins, a family of growth factors acting on the development and maintenance of the nervous system. Neurotrophins and their specific receptors, TrkA, TrkB and TrkC, seem to be phylogenetically well preserved but, in reptiles, data regarding the occurrence of Trk-like proteins are very scarce, especially in non-nervous organs. Western blot analysis demonstrated that the lizard gut contains TrkA- and TrkC-like, but not TrkB-like, proteins. Consistently, TrkA- and TrkC-like immunoreactivity were both observed in neurons of the anterior intestine, whereas endocrine cells of the stomach and anterior intestine only displayed TrkA-like immunoreactivity. These results demonstrate for the first time the occurrence of Trk-like proteins in non-neuronal tissues of reptilians and provide further evidence for the evolutionary preservation of the molecular mass and cell distribution of Trk neurotrophin receptor-like proteins in the gut of vertebrates.

  1. A new TRAF-like protein from B. oleracea ssp. botrytis with lectin activity and its effect on macrophages.

    PubMed

    Duarte, Christiane E M; Abranches, Monise V; Silva, Patrick F; de Paula, Sérgio O; Cardoso, Silvia A; Oliveira, Leandro L

    2017-01-01

    Lectins are involved in a wide range of biological mechanisms, like immunomodulatory agent able to activate the innate immunity. In this study, we purified and characterized a new lectin from cauliflower (Brassica oleracea ssp. botrytis - BOL) by three sequential chromatographic steps and confirmed the purity by SDS-PAGE. Additionally, we evaluated the role of the lectin in innate immunity by a phagocytosis assay, production of H 2 O 2 and NO. BOL was characterized like a non-glycosylated protein that showed a molecular mass of ∼34kDa in SDS-PAGE. Its N-terminal sequence (ETRAFREERPSSKIVTIAG) did not reveal any similarity to the other lectins; nevertheless, it showed 100% homology to a putative TRAF-like protein from Brassica rapa and Brassica napus. This is a first report of the TRAF-protein with lectinic activity. The BOL retained its complete hemagglutination activity from 4°C up to 60°C, with stability being more apparent between pH 7.0 and 8.0. Moreover, the lectin was able to stimulate phagocytosis and induce the production of H 2 O 2 and NO. Therefore, BOL can be explored as an immunomodulatory agent by being able to activate the innate immunity and favor antigen removal. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 1 targets misfolded HLA-B27 dimers for endoplasmic reticulum-associated degradation.

    PubMed

    Guiliano, David B; Fussell, Helen; Lenart, Izabela; Tsao, Edward; Nesbeth, Darren; Fletcher, Adam J; Campbell, Elaine C; Yousaf, Nasim; Williams, Sarah; Santos, Susana; Cameron, Amy; Towers, Greg J; Kellam, Paul; Hebert, Daniel N; Gould, Keith G; Powis, Simon J; Antoniou, Antony N

    2014-11-01

    HLA-B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This study was undertaken to define the role of the UPR-induced ER-associated degradation (ERAD) pathway in the disposal of HLA-B27 dimeric conformers. HeLa cell lines expressing only 2 copies of a carboxy-terminally Sv5-tagged HLA-B27 were generated. The ER stress-induced protein ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1) was overexpressed by transfection, and dimer levels were monitored by immunoblotting. EDEM1, the UPR-associated transcription factor X-box binding protein 1 (XBP-1), the E3 ubiquitin ligase hydroxymethylglutaryl-coenzyme A reductase degradation 1 (HRD1), and the degradation-associated proteins derlin 1 and derlin 2 were inhibited using either short hairpin RNA or dominant-negative mutants. The UPR-associated ERAD of HLA-B27 was confirmed using ER stress-inducing pharamacologic agents in kinetic and pulse chase assays. We demonstrated that UPR-induced machinery can target HLA-B27 dimers and that dimer formation can be controlled by alterations to expression levels of components of the UPR-induced ERAD pathway. HLA-B27 dimers and misfolded major histocompatibility complex class I monomeric molecules bound to EDEM1 were detected, and overexpression of EDEM1 led to inhibition of HLA-B27 dimer formation. EDEM1 inhibition resulted in up-regulation of HLA-B27 dimers, while UPR-induced ERAD of dimers was prevented in the absence of EDEM1. HLA-B27 dimer formation was also enhanced in the absence of XBP-1, HRD1, and derlins 1 and 2. The present findings indicate that the UPR ERAD pathway can dispose of HLA-B27 dimers, thus presenting a potential novel therapeutic target for modulation of HLA-B27-associated inflammatory disease. Copyright © 2014 by the American College of Rheumatology.

  3. Molecular evolution of the actin-like MreB protein gene family in wall-less bacteria.

    PubMed

    Ku, Chuan; Lo, Wen-Sui; Kuo, Chih-Horng

    2014-04-18

    The mreB gene family encodes actin-like proteins that determine cell shape by directing cell wall synthesis and often exists in one to three copies in the genomes of non-spherical bacteria. Intriguingly, while most wall-less bacteria do not have this gene, five to seven mreB homologs are found in Spiroplasma and Haloplasma, which are both characterized by cell contractility. To investigate the molecular evolution of this gene family in wall-less bacteria, we sampled the available genome sequences from these two genera and other related lineages for comparative analysis. The gene phylogenies indicated that the mreB homologs in Haloplasma are more closely related to those in Firmicutes, whereas those in Spiroplasma form a separate clade. This finding suggests that the gene family expansions in these two lineages are the results of independent ancient duplications. Moreover, the Spiroplasma mreB homologs can be classified into five clades, of which the genomic positions are largely conserved. The inference of gene gains and losses suggests that there has been an overall trend to retain only one homolog from each of the five mreB clades in the evolutionary history of Spiroplasma. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  4. The SsgA-like proteins in actinomycetes: small proteins up to a big task

    PubMed Central

    Traag, Bjørn A.

    2008-01-01

    Several unique protein families have been identified that play a role in the control of developmental cell division in streptomycetes. The SsgA-like proteins or SALPs, of which streptomycetes typically have at least five paralogues, control specific steps of sporulation-specific cell division in streptomycetes, affecting cell wall-related events such as septum localization and synthesis, thickening of the spore wall and autolytic spore separation. The expression level of SsgA, the best studied SALP, has a rather dramatic effect on septation and on hyphal morphology, which is not only of relevance for our understanding of (developmental) cell division but has also been succesfully applied in industrial fermentation, to improve growth and production of filamentous actinomycetes. Recent observations suggest that SsgB most likely is the archetypal SALP, with only SsgB orthologues occurring in all morphologically complex actinomycetes. Here we review 10 years of research on the SsgA-like proteins in actinomycetes and discuss the most interesting regulatory, functional, phylogenetic and applied aspects of this relatively unknown protein family. Electronic supplementary material The online version of this article (doi:10.1007/s10482-008-9225-3) contains supplementary material, which is available to authorized users. PMID:18273689

  5. Effect of apolipoprotein B mRNA-editing catalytic polypeptide-like protein-3G in cervical cancer

    PubMed Central

    Xu, Yanhua; Leng, Junhong; Xue, Fang; Dong, Ruiqian

    2015-01-01

    Cervical cancer is one of the most common gynecologic cancers. The role of apolipoprotein B mRNA-editing catalytic polypeptide-like protein-3G (APCBEC-3G) in cervical cancer has yet to be elucidated. This study intends to explore the effect ofAPCBEC-3G on cervical cancer cell proliferation and invasion. In vitro, the cervical cancer cell line Hela was transfected by APCBEC-3G plasmid. The mRNA and protein expression levels of APCBEC-3G were detected by Real-time PCR and Western blot, respectively. Cervical cancer cell proliferation was determined by MTT. Transwell assay was applied to measure the effect of APCBEC-3G on cell invasion. APCBEC-3G mRNA and protein increased significantly after transfection (P<0.05) and cervical cancer cell proliferation and invasive ability were decreased significantly (P<0.05). APOBEC-3G serves as a suppressor of cervical cancer cell proliferation and invasion. Our research provides theoretical basis for further investigationAPOBEC-3G effect in cervical cancer occurrence and development. PMID:26722417

  6. Effect of apolipoprotein B mRNA-editing catalytic polypeptide-like protein-3G in cervical cancer.

    PubMed

    Xu, Yanhua; Leng, Junhong; Xue, Fang; Dong, Ruiqian

    2015-01-01

    Cervical cancer is one of the most common gynecologic cancers. The role of apolipoprotein B mRNA-editing catalytic polypeptide-like protein-3G (APCBEC-3G) in cervical cancer has yet to be elucidated. This study intends to explore the effect of APCBEC-3G on cervical cancer cell proliferation and invasion. In vitro, the cervical cancer cell line Hela was transfected by APCBEC-3G plasmid. The mRNA and protein expression levels of APCBEC-3G were detected by Real-time PCR and Western blot, respectively. Cervical cancer cell proliferation was determined by MTT. Transwell assay was applied to measure the effect of APCBEC-3G on cell invasion. APCBEC-3G mRNA and protein increased significantly after transfection (P<0.05) and cervical cancer cell proliferation and invasive ability were decreased significantly (P<0.05). APOBEC-3G serves as a suppressor of cervical cancer cell proliferation and invasion. Our research provides theoretical basis for further investigation APOBEC-3G effect in cervical cancer occurrence and development.

  7. A conserved region of leptospiral immunoglobulin-like A and B proteins as a DNA vaccine elicits a prophylactic immune response against leptospirosis.

    PubMed

    Forster, Karine M; Hartwig, Daiane D; Seixas, Fabiana K; Bacelo, Kátia L; Amaral, Marta; Hartleben, Cláudia P; Dellagostin, Odir A

    2013-05-01

    The leptospiral immunoglobulin-like (Lig) proteins LigA and LigB possess immunoglobulin-like domains with 90-amino-acid repeats and are adhesion molecules involved in pathogenicity. They are conserved in pathogenic Leptospira spp. and thus are of interest for use as serodiagnostic antigens and in recombinant vaccine formulations. The N-terminal amino acid sequences of the LigA and LigB proteins are identical, but the C-terminal sequences vary. In this study, we evaluated the protective potential of five truncated forms of LigA and LigB proteins from Leptospira interrogans serovar Canicola as DNA vaccines using the pTARGET mammalian expression vector. Hamsters immunized with the DNA vaccines were subjected to a heterologous challenge with L. interrogans serovar Copenhageni strain Spool via the intraperitoneal route. Immunization with a DNA vaccine encoding LigBrep resulted in the survival of 5/8 (62.5%) hamsters against lethal infection (P < 0.05). None of the control hamsters or animals immunized with the other vaccine preparations survived. The vaccine induced an IgG antibody response and, additionally, conferred sterilizing immunity in 80% of the surviving animals. Our results indicate that the LigBrep DNA vaccine is a promising candidate for inclusion in a protective leptospiral vaccine.

  8. A Conserved Region of Leptospiral Immunoglobulin-Like A and B Proteins as a DNA Vaccine Elicits a Prophylactic Immune Response against Leptospirosis

    PubMed Central

    Forster, Karine M.; Hartwig, Daiane D.; Seixas, Fabiana K.; Bacelo, Kátia L.; Amaral, Marta; Hartleben, Cláudia P.

    2013-01-01

    The leptospiral immunoglobulin-like (Lig) proteins LigA and LigB possess immunoglobulin-like domains with 90-amino-acid repeats and are adhesion molecules involved in pathogenicity. They are conserved in pathogenic Leptospira spp. and thus are of interest for use as serodiagnostic antigens and in recombinant vaccine formulations. The N-terminal amino acid sequences of the LigA and LigB proteins are identical, but the C-terminal sequences vary. In this study, we evaluated the protective potential of five truncated forms of LigA and LigB proteins from Leptospira interrogans serovar Canicola as DNA vaccines using the pTARGET mammalian expression vector. Hamsters immunized with the DNA vaccines were subjected to a heterologous challenge with L. interrogans serovar Copenhageni strain Spool via the intraperitoneal route. Immunization with a DNA vaccine encoding LigBrep resulted in the survival of 5/8 (62.5%) hamsters against lethal infection (P < 0.05). None of the control hamsters or animals immunized with the other vaccine preparations survived. The vaccine induced an IgG antibody response and, additionally, conferred sterilizing immunity in 80% of the surviving animals. Our results indicate that the LigBrep DNA vaccine is a promising candidate for inclusion in a protective leptospiral vaccine. PMID:23486420

  9. FLICE-like inhibitory protein (FLIP) protects against apoptosis and suppresses NF-kappaB activation induced by bacterial lipopolysaccharide.

    PubMed

    Bannerman, Douglas D; Eiting, Kristine T; Winn, Robert K; Harlan, John M

    2004-10-01

    Bacterial lipopolysaccharide (LPS) via its activation of Toll-like receptor-4 contributes to much of the vascular injury/dysfunction associated with gram-negative sepsis. Inhibition of de novo gene expression has been shown to sensitize endothelial cells (EC) to LPS-induced apoptosis, the onset of which correlates with decreased expression of FLICE-like inhibitory protein (FLIP). We now have data that conclusively establish a role for FLIP in protecting EC against LPS-induced apoptosis. Overexpression of FLIP protected against LPS-induced apoptosis, whereas down-regulation of FLIP using antisense oligonucleotides sensitized EC to direct LPS killing. Interestingly, FLIP overexpression suppressed NF-kappaB activation induced by LPS, but not by phorbol ester, suggesting a specific role for FLIP in mediating LPS activation. Conversely, mouse embryo fibroblasts (MEF) obtained from FLIP -/- mice showed enhanced LPS-induced NF-kappaB activation relative to those obtained from wild-type mice. Reconstitution of FLIP-/- MEF with full-length FLIP reversed the enhanced NF-kappaB activity elicited by LPS in the FLIP -/- cells. Changes in the expression of FLIP had no demonstrable effect on other known LPS/Tlr-4-activated signaling pathways including the p38, Akt, and Jnk pathways. Together, these data support a dual role for FLIP in mediating LPS-induced apoptosis and NF-kappaB activation.

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

    PubMed

    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.

  11. The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria.

    PubMed

    Muñoz-Espín, Daniel; Daniel, Richard; Kawai, Yoshikazu; Carballido-López, Rut; Castilla-Llorente, Virginia; Errington, Jeff; Meijer, Wilfried J J; Salas, Margarita

    2009-08-11

    Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage ϕ29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the ϕ29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the ϕ29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane.

  12. Molecular modeling of the human sperm associated antigen 11 B (SPAG11B) proteins.

    PubMed

    Narmadha, Ganapathy; Yenugu, Suresh

    2015-04-01

    Antimicrobial proteins and peptides are ubiquitous in nature with diverse structural and biological properties. Among them, the human beta-defensins are known to contribute to the innate immune response. Besides the defensins, a number of defensin-like proteins and peptides are expressed in many organ systems including the male reproductive system. Some of the protein isoforms encoded by the sperm associated antigen 11B (SPAG11) gene in humans are beta-defensin-like and exhibit structure dependent and salt tolerant antimicrobial activity, besides contributing to sperm maturation. Though some of the functional roles of these proteins are reported, the structural and molecular features that contribute to their antimicrobial activity is not yet reported. In this study, using in silico tools, we report the three dimensional structure of the human SPAG11B proteins and their C-terminal peptides. web-based hydropathy, amphipathicity, and topology (WHAT) analyses and grand average of hydropathy (GRAVY) indices show that these proteins and peptides are amphipathic and highly hydrophilic. Self-optimized prediction method with alignment (SOPMA) analyses and circular dichroism data suggest that the secondary structure of these proteins and peptides primarily contain beta-sheet and random coil structure and alpha-helix to a lesser extent. Ramachandran plots show that majority of the amino acids in these proteins and peptides fall in the permissible regions, thus indicating stable structures. The secondary structure of SPAG11B isoforms and their peptides were not perturbed with increasing NaCl concentration (0-300 mM) and at different pH (3, 7, and 10), thus reinforcing our previously reported observation that their antimicrobial activity is salt tolerant. To the best of our knowledge, for the first time, results of our study provide vital information on the structural features of SPAG11B protein isoforms and their contribution to antimicrobial activity.

  13. Leptospira Immunoglobulin-Like Protein B Interacts with the 20th Exon of Human Tropoelastin Contributing to Leptospiral Adhesion to Human Lung Cells

    PubMed Central

    Hsieh, Ching-Lin; Tseng, Andrew; He, Hongxuan; Kuo, Chih-Jung; Wang, Xuannian; Chang, Yung-Fu

    2017-01-01

    Leptospira immunoglobulin-like protein B (LigB), a surface adhesin, is capable of mediating the attachment of pathogenic leptospira to the host through interaction with various components of the extracellular matrix (ECM). Human tropoelastin (HTE), the building block of elastin, confers resilience and elasticity to lung, and other tissues. Previously identified Ig-like domains of LigB, including LigB4 and LigB12, bind to HTE, which is likely to promote Leptospira adhesion to lung tissue. However, the molecular mechanism that mediates the LigB-HTE interaction is unclear. In this study, the LigB-binding site on HTE was further pinpointed to a N-terminal region of the 20th exon of HTE (HTE20N). Alanine mutants of basic and aromatic residues on HTE20N significantly reduced binding to the LigB. Additionally, HTE-binding site was narrowed down to the first β-sheet of LigB12. On this binding surface, residues F1054, D1061, A1065, and D1066 were critical for the association with HTE. Most importantly, the recombinant HTE truncates could diminish the binding of LigB to human lung fibroblasts (WI-38) by 68%, and could block the association of LigA-expressing L. biflexa to lung cells by 61%. These findings should expand our understanding of leptospiral pathogenesis, particularly in pulmonary manifestations of leptospirosis. PMID:28536676

  14. Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes.

    PubMed

    Stone, Matthew B; Shelby, Sarah A; Núñez, Marcos F; Wisser, Kathleen; Veatch, Sarah L

    2017-02-01

    Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase-separated domains in model membranes. This concept remains controversial and lacks direct experimental support in intact cells. Here, we visualize ordered and disordered domains in mouse B lymphoma cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, and present a minimal, predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling and a plausible mechanism of BCR activation via receptor clustering that could be generalized to other signaling pathways. Overall, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that broadly impact signal transduction.

  15. The B1 Protein Guides the Biosynthesis of a Lasso Peptide

    NASA Astrophysics Data System (ADS)

    Zhu, Shaozhou; Fage, Christopher D.; Hegemann, Julian D.; Mielcarek, Andreas; Yan, Dushan; Linne, Uwe; Marahiel, Mohamed A.

    2016-10-01

    Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique lariat knot-like fold that endows them with extraordinary stability and biologically relevant activity. However, the biosynthetic mechanism of these fascinating molecules remains largely speculative. Generally, two enzymes (B for processing and C for cyclization) are required to assemble the unusual knot-like structure. Several subsets of lasso peptide gene clusters feature a “split” B protein on separate open reading frames (B1 and B2), suggesting distinct functions for the B protein in lasso peptide biosynthesis. Herein, we provide new insights into the role of the RiPP recognition element (RRE) PadeB1, characterizing its capacity to bind the paeninodin leader peptide and deliver its peptide substrate to PadeB2 for processing.

  16. A novel pair of immunoglobulin-like receptors expressed by B cells and myeloid cells

    PubMed Central

    Kubagawa, Hiromi; Burrows, Peter D.; Cooper, Max D.

    1997-01-01

    An Fcα receptor probe of human origin was used to identify novel members of the Ig gene superfamily in mice. Paired Ig-like receptors, named PIR-A and PIR-B, are predicted from sequence analysis of the cDNAs isolated from a mouse splenic library. Both type I transmembrane proteins possess similar ectodomains with six Ig-like loops, but have different transmembrane and cytoplasmic regions. The predicted PIR-A protein has a short cytoplasmic tail and a charged Arg residue in the transmembrane region that, by analogy with the FcαR relative, suggests the potential for association with an additional transmembrane protein to form a signal transducing unit. In contrast, the PIR-B protein has an uncharged transmembrane region and a long cytoplasmic tail containing four potential immunoreceptor tyrosine-based inhibitory motifs. These features are shared by the related killer inhibitory receptors. PIR-A proteins appear to be highly variable, in that predicted peptide sequences differ for seven randomly selected PIR-A clones, whereas PIR-B cDNA clones are invariant. Southern blot analysis with PIR-B and PIR-A-specific probes suggests only one PIR-B gene and multiple PIR-A genes. The PIR-A and PIR-B genes are expressed in B lymphocytes and myeloid lineage cells, wherein both are expressed simultaneously. The characteristics of the highly-conserved PIR-A and PIR-B genes and their coordinate cellular expression suggest a potential regulatory role in humoral, inflammatory, and allergic responses. PMID:9144225

  17. Drosophila Nora virus capsid proteins differ from those of other picorna-like viruses.

    PubMed

    Ekström, Jens-Ola; Habayeb, Mazen S; Srivastava, Vaibhav; Kieselbach, Thomas; Wingsle, Gunnar; Hultmark, Dan

    2011-09-01

    The recently discovered Nora virus from Drosophila melanogaster is a single-stranded RNA virus. Its published genomic sequence encodes a typical picorna-like cassette of replicative enzymes, but no capsid proteins similar to those in other picorna-like viruses. We have now done additional sequencing at the termini of the viral genome, extending it by 455 nucleotides at the 5' end, but no more coding sequence was found. The completeness of the final 12,333-nucleotide sequence was verified by the production of infectious virus from the cloned genome. To identify the capsid proteins, we purified Nora virus particles and analyzed their proteins by mass spectrometry. Our results show that the capsid is built from three major proteins, VP4A, B and C, encoded in the fourth open reading frame of the viral genome. The viral particles also contain traces of a protein from the third open reading frame, VP3. VP4A and B are not closely related to other picorna-like virus capsid proteins in sequence, but may form similar jelly roll folds. VP4C differs from the others and is predicted to have an essentially α-helical conformation. In a related virus, identified from EST database sequences from Nasonia parasitoid wasps, VP4C is encoded in a separate open reading frame, separated from VP4A and B by a frame-shift. This opens a possibility that VP4C is produced in non-equimolar quantities. Altogether, our results suggest that the Nora virus capsid has a different protein organization compared to the order Picornavirales. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form

    PubMed Central

    Zheng, Heping; Mandal, Arabinda; Shumilin, Igor A.; Chordia, Mahendra D.; Panneerdoss, Subbarayalu; Herr, John C.; Minor, Wladek

    2016-01-01

    Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75Å in diameter with a 25Å central pore comprised of six monomers per helix turn repeating every 33Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally-observed SLLP1/SAS1B interaction involved in fertilization. PMID:26198801

  19. A Calcium Sensor-Regulated Protein Kinase, CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE19, Is Required for Pollen Tube Growth and Polarity1[OPEN

    PubMed Central

    Zhou, Liming; Lan, Wenzhi; Chen, Binqing; Fang, Wei; Luan, Sheng

    2015-01-01

    Calcium plays an essential role in pollen tube tip growth. However, little is known concerning the molecular basis of the signaling pathways involved. Here, we identified Arabidopsis (Arabidopsis thaliana) CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE19 (CIPK19) as an important element to pollen tube growth through a functional survey for CIPK family members. The CIPK19 gene was specifically expressed in pollen grains and pollen tubes, and its overexpression induced severe loss of polarity in pollen tube growth. In the CIPK19 loss-of-function mutant, tube growth and polarity were significantly impaired, as demonstrated by both in vitro and in vivo pollen tube growth assays. Genetic analysis indicated that disruption of CIPK19 resulted in a male-specific transmission defect. Furthermore, loss of polarity induced by CIPK19 overexpression was associated with elevated cytosolic Ca2+ throughout the bulging tip, whereas LaCl3, a Ca2+ influx blocker, rescued CIPK19 overexpression-induced growth inhibition. Our results suggest that CIPK19 may be involved in maintaining Ca2+ homeostasis through its potential function in the modulation of Ca2+ influx. PMID:25713341

  20. Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

    PubMed

    Zheng, H; Mandal, A; Shumilin, I A; Chordia, M D; Panneerdoss, S; Herr, J C; Minor, W

    2015-07-01

    Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization. © 2015 American Society of Andrology and European Academy of Andrology.

  1. Src-like adaptor protein (SLAP) regulates B cell receptor levels in a c-Cbl-dependent manner.

    PubMed

    Dragone, Leonard L; Myers, Margaret D; White, Carmen; Gadwal, Shyam; Sosinowski, Tomasz; Gu, Hua; Weiss, Arthur

    2006-11-28

    Src-like adaptor protein (SLAP) and c-Cbl recently have been shown to cooperate in regulating T cell receptor (TCR) levels in developing T cells. SLAP also is expressed in developing B cells, and its deficiency leads to alterations in B cell receptor (BCR) levels and B cell development. Hence, we hypothesized that SLAP and c-Cbl may cooperate during B cell development to regulate BCR levels. In mice deficient in both SLAP and c-Cbl, we found that B cell development is altered, suggesting that they function through intersecting pathways. To study the mechanism by which SLAP and c-Cbl alter BCR levels, we coexpressed them in a mature mouse B cell line (Bal-17). First we determined that SLAP associates with proximal components of the BCR complex after stimulation and internalization. Coexpression of SLAP and c-Cbl in Bal-17 led to decreased surface and total BCR levels. This decrease in BCR levels depended on intact Src homology 2 (SH2) and C-terminal domains of SLAP. In addition, a mutation in the SH2 domain of SLAP blocked its colocalization with c-Cbl and the BCR complex, whereas deletion of the C terminus did not affect its localization. Last, coexpression of SLAP and c-Cbl altered BCR complex recycling. This alteration in BCR complex recycling depended on enzymatically active c-Cbl and Src family kinases, as well as the intact SH2 and C-terminal domains of SLAP. These data suggest that SLAP has a conserved function in B and T cells by adapting c-Cbl to the antigen-receptor complex and targeting it for degradation.

  2. Src-like adaptor protein (SLAP) regulates B cell receptor levels in a c-Cbl-dependent manner

    PubMed Central

    Dragone, Leonard L.; Myers, Margaret D.; White, Carmen; Gadwal, Shyam; Sosinowski, Tomasz; Gu, Hua; Weiss, Arthur

    2006-01-01

    Src-like adaptor protein (SLAP) and c-Cbl recently have been shown to cooperate in regulating T cell receptor (TCR) levels in developing T cells. SLAP also is expressed in developing B cells, and its deficiency leads to alterations in B cell receptor (BCR) levels and B cell development. Hence, we hypothesized that SLAP and c-Cbl may cooperate during B cell development to regulate BCR levels. In mice deficient in both SLAP and c-Cbl, we found that B cell development is altered, suggesting that they function through intersecting pathways. To study the mechanism by which SLAP and c-Cbl alter BCR levels, we coexpressed them in a mature mouse B cell line (Bal-17). First we determined that SLAP associates with proximal components of the BCR complex after stimulation and internalization. Coexpression of SLAP and c-Cbl in Bal-17 led to decreased surface and total BCR levels. This decrease in BCR levels depended on intact Src homology 2 (SH2) and C-terminal domains of SLAP. In addition, a mutation in the SH2 domain of SLAP blocked its colocalization with c-Cbl and the BCR complex, whereas deletion of the C terminus did not affect its localization. Last, coexpression of SLAP and c-Cbl altered BCR complex recycling. This alteration in BCR complex recycling depended on enzymatically active c-Cbl and Src family kinases, as well as the intact SH2 and C-terminal domains of SLAP. These data suggest that SLAP has a conserved function in B and T cells by adapting c-Cbl to the antigen-receptor complex and targeting it for degradation. PMID:17110436

  3. Identification of PDC-109-like protein(s) in buffalo seminal plasma.

    PubMed

    Harshan, Hiron M; Sankar, Surya; Singh, L P; Singh, Manish Kumar; Sudharani, S; Ansari, M R; Singh, S K; Majumdar, A C; Joshi, P

    2009-10-01

    The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma.

  4. Antigenic Determinants of Alpha-Like Proteins of Streptococcus agalactiae

    PubMed Central

    Maeland, Johan A.; Bevanger, Lars; Lyng, Randi Valsoe

    2004-01-01

    The majority of group B streptococcus (GBS) isolates express one or more of a family of surface-anchored proteins that vary by strain and that form ladder-like patterns on Western blotting due to large repeat units. These proteins, which are important as GBS serotype markers and as inducers of protective antibodies, include the alpha C (Cα) and R4 proteins and the recently described alpha-like protein 2 (Alp2), encoded by alp2, and Alp3, encoded by alp3. In this study, we examined antigenic determinants possessed by Alp2 and Alp3 by testing of antibodies raised in rabbits, mainly by using enzyme-linked immunosorbent assays (ELISA) and an ELISA absorption test. The results showed that Alp2 and Alp3 shared an antigenic determinant, which may be a unique immunological marker of the Alp variants of GBS proteins. Alp2, in addition, possessed an antigenic determinant which showed specificity for Alp2 and a third determinant which showed serological cross-reactivity with Cα. Alp3, in addition to the determinant common to Alp2 and Alp3, harbored an antigenic site which also was present in the R4 protein, whereas no Alp3-specific antigenic site was detected. These ELISA-based results were confirmed by Western blotting and a fluorescent-antibody test. The results are consistent with highly complex antigenic structures of the alpha-like proteins in a fashion which is in agreement with the recently described structural mosaicism of the alp2 and alp3 genes. The results are expected to influence GBS serotyping, immunoprotection studies, and GBS vaccine developments. PMID:15539502

  5. The structure of the BfrB-Bfd complex reveals protein-protein interactions enabling iron release from bacterioferritin

    PubMed Central

    Yao, Huili; Wang, Yan; Lovell, Scott; Kumar, Ritesh; Ruvinsky, Anatoly M.; Battaile, Kevin P.; Vakser, Ilya A.; Rivera, Mario

    2012-01-01

    Ferritin-like molecules are unique to cellular iron homeostasis because they can store iron at concentrations much higher than those dictated by the solubility of Fe3+. Very little is known about the protein interactions that deliver iron for storage, or promote the mobilization of stored iron from ferritin-like molecules. Here, we report the X-ray crystal structure of Pseudomonas aeruginosa bacterioferritin (Pa-BfrB) in complex with bacterioferritin-associated ferredoxin (Pa-Bfd) at 2.0 Å resolution. As the first example of a ferritin-like molecule in complex with a cognate partner, the structure provides unprecedented insight into the complementary interface that enables the [2Fe-2S] cluster of Pa-Bfd to promote heme-mediated electron transfer through the BfrB protein dielectric (~18 Å), a process that is necessary to reduce the core ferric mineral and facilitate mobilization of Fe2+. The Pa-BfrB-Bfd complex also revealed the first structure of a Bfd, thus providing a first view to what appears to be a versatile metal binding domain ubiquitous to the large Fer2_BFD family of proteins and enzymes with diverse functions. Residues at the Pa-BfrB-Bfd interface are highly conserved in Bfr and Bfd sequences from a number of pathogenic bacteria, suggesting that the specific recognition between Pa-BfrB and Pa-Bfd is of widespread significance to the understanding of bacterial iron homeostasis. PMID:22812654

  6. The family B1 GPCR: structural aspects and interaction with accessory proteins.

    PubMed

    Couvineau, Alain; Laburthe, Marc

    2012-01-01

    G protein coupled receptors (GPCRs) play a crucial role in physiology and pathophysiology in humans. Beside the large family A (rhodopsin-like receptors) and family C GPCR (metabotropic glutamate receptors), the small family B1 GPCR (secretin-like receptors) includes important receptors such as vasoactive intestinal peptide receptors (VPAC), pituitary adenylyl cyclase activating peptide receptor (PAC1R), secretin receptor (SECR), growth hormone releasing factor receptor (GRFR), glucagon receptor (GCGR), glucagon like-peptide 1 and 2 receptors (GLPR), gastric inhibitory peptide receptor (GIPR), parathyroid hormone receptors (PTHR), calcitonin receptors (CTR) and corticotropin-releasing factor receptors (CRFR). They represent very promising targets for the development of drugs having therapeutical impact on many diseases such as chronic inflammation, neurodegeneration, diabetes, stress and osteoporosis. Over the past decade, structure-function relationship studies have demonstrated that the N-terminal ectodomain (N-ted) of family B1 receptors plays a pivotal role in natural ligand recognition. Structural analysis of some family B1 GPCR N-teds revealed the existence of a Sushi domain fold consisting of two antiparallel β sheets stabilized by three disulfide bonds and a salt bridge. The family B1 GPCRs promote cellular responses through a signaling pathway including predominantly the Gsadenylyl cyclase-cAMP pathway activation. Family B1 GPCRs also interact with a few accessory proteins which play a role in cell signaling, receptor expression and/or pharmacological profiles of receptors. These accessory proteins may represent new targets for the design of new drugs. Here, we review the current knowledge regarding: i) the structure of family B1 GPCR binding domain for natural ligands and ii) the interaction of family B1 GPCRs with accessory proteins.

  7. Nicotinamide Inhibits the Lysosomal Cathepsin b-like Protease and Kills African Trypanosomes*

    PubMed Central

    Unciti-Broceta, Juan D.; Maceira, José; Morales, Sonia; García-Pérez, Angélica; Muñóz-Torres, Manuel E.; Garcia-Salcedo, Jose A.

    2013-01-01

    Nicotinamide, a soluble compound of the vitamin B3 group, has antimicrobial activity against several microorganisms ranging from viruses to parasite protozoans. However, the mode of action of this antimicrobial activity is unknown. Here, we investigate the trypanocidal activity of nicotinamide on Trypanosoma brucei, the causative agent of African trypanosomiasis. Incubation of trypanosomes with nicotinamide causes deleterious defects in endocytic traffic, disruption of the lysosome, failure of cytokinesis, and, ultimately, cell death. At the same concentrations there was no effect on a cultured mammalian cell line. The effects on endocytosis and vesicle traffic were visible within 3 h and can be attributed to inhibition of lysosomal cathepsin b-like protease activity. The inhibitory effect of nicotinamide was confirmed by a direct activity assay of recombinant cathepsin b-like protein. Taken together, these data demonstrate that inhibition of the lysosomal protease cathepsin b-like blocks endocytosis, causing cell death. In addition, these results demonstrate for the first time the inhibitory effect of nicotinamide on a protease. PMID:23443665

  8. Uncoupling proteins (UCP) in unicellular eukaryotes: true UCPs or UCP1-like acting proteins?

    PubMed

    Luévano-Martínez, Luis Alberto

    2012-04-05

    Uncoupling proteins belong to the superfamily of mitochondrial anion carriers. They are apparently present throughout the Eukarya domain in which only some members have an established physiological function, i.e. UCP1 from brown adipose tissue is involved in non-shivering thermogenesis. However, the proteins responsible for the phenotype observed in unicellular organisms have not been characterized. In this report we analyzed functional evidence concerning unicellular UCPs and found that true UCPs are restricted to some taxonomical groups while proteins conferring a UCP1-like phenotype to fungi and most protists are the result of a promiscuous activity exerted by other mitochondrial anion carriers. We describe a possible evolutionary route followed by these proteins by which they acquire this promiscuous mechanism. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. Novel function of the endoplasmic reticulum degradation-enhancing α-mannosidase-like proteins in the human hepatitis B virus life cycle, mediated by the middle envelope protein.

    PubMed

    Lazar, Catalin; Uta, Mihaela; Petrescu, Stefana Maria; Branza-Nichita, Norica

    2017-02-01

    Cells replicating the human hepatitis B virus (HBV) express high levels of degradation-enhancing α-mannosidase-like proteins (EDEMs), a family of proteins involved in the endoplasmic reticulum associated degradation, one of the pathways activated during the unfolded protein response. Owing to their α-1,2 mannosidase activity, the EDEM1-3 proteins are able to process the N-linked glycans of misfolded or incompletely folded proteins, providing the recognition signal for their subsequent degradation. The HBV small (S), medium (M), and large (L) surface proteins bear an N-linked glycosylation site in the common S domain that is partially occupied in all proteins. The M protein contains an additional site in its preS2 domain, which is always functional. Here, we report that these oligosaccharides are processed by EDEMs, more efficiently by EDEM3, which induces degradation of L and S proteins, accompanied by a reduction of subviral particles production. In striking contrast, M not only is spared from degradation but its trafficking is also accelerated leading to an improved secretion. This unusual behavior of the M protein requires strictly the mannose trimming of the preS2 N-linked glycan. Furthermore, we show that HBV secretion is significantly inhibited under strong endoplasmic reticulum stress conditions when M expression is prevented by mutagenesis of the viral genome. These observations unfold unique properties of the M protein in the HBV life cycle during unfolded protein response and point to alternative mechanisms employed by EDEMs to alleviate this stress in case of necessity by promoting glycoprotein trafficking rather than degradation. © 2016 John Wiley & Sons Ltd.

  10. Advanced oxidation protein products induce inflammatory response in fibroblast-like synoviocytes through NADPH oxidase -dependent activation of NF-κB.

    PubMed

    Zheng, Shuai; Zhong, Zhao-Ming; Qin, Shuai; Chen, Guo-Xian; Wu, Qian; Zeng, Ji-Huan; Ye, Wen-Bin; Li, Wei; Yuan, Kai; Yao, Ling; Chen, Jian-Ting

    2013-01-01

    Advanced oxidation protein products (AOPPs), a marker of oxidative stress, are prevalent in many kinds of disorders. Rheumatoid arthritis (RA), mainly resulting from the dysfunction of fibroblast-like synoviocytes (FLSs), is related to oxidative stress. Although the increased levels of AOPPs in RA patients were reported, the effect of AOPPs on FLSs function still remains unclear. Therefore, our study aims to investigate whether AOPPs have an effect on the inflammatory response of FLSs in vitro. FLSs obtained from both knees of rats were treated with or without AOPPs-modified rat serum albumin (AOPPs-RSA) in vitro. The mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin(IL)-1β, matrix metalloproteinases(MMP)-3, MMP-13 and vascular endothelial growth factor (VEGF) were measured by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. Reactive oxygen species (ROS) generation was detected by fluorescent microscope and fluorescence microplate reader. Immunoprecipitation, Co-Immunoprecipitation and western blot were performed to examine the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nuclear factor kappa B (NF-κB). Exposure of FLSs to AOPPs upregulated the mRNA and protein expression of TNF-α, IL-1β, MMP-3, MMP-13 and VEGF in a concentration dependent manner. AOPPs treatment triggered ROS production in FLSs, which was significantly abolished by ROS scavenger N-acetyl-L-cysteine (NAC), superoxide dismutase (SOD), NADPH oxidase inhibitors diphenyleneiodonium (DPI) and apocynin. Challenged AOPPs induced phosphorylation of p47(phox), triggered an interaction between p47(phox), p22(phox) and gp91(phox), and significantly upregulated expression of NADPH oxidase subunits p47(phox), p22(phox) and gp91(phox). IκB degradation and nuclear translocation of NF-κB p65 induced by AOPPs were significantly blocked by SOD, NAC, DPI and apocynin. These data indicate that

  11. Lipid transfer proteins in the assembly of apoB-containing lipoproteins.

    PubMed

    Sirwi, Alaa; Hussain, M Mahmood

    2018-04-12

    A better understanding of intracellular lipoprotein assembly may help identify proteins with important roles in lipid disorders. ApoB-containing lipoproteins are macromolecular lipid and protein micelles that act as specialized transport vehicles for hydrophobic lipids. They are assembled predominantly in enterocytes and hepatocytes to transport dietary and endogenous fat, respectively, to different tissues. Assembly occurs in the endoplasmic reticulum and is dependent on lipid re-synthesis in the endoplasmic reticulum and on a chaperone, namely microsomal triglyceride transfer protein. Precursors for lipid synthesis are obtained from extracellular sources and from cytoplasmic lipid droplets. Microsomal triglyceride transfer protein is the major and essential lipid transfer protein that transfers phospholipids and triacylglycerols to nascent apoB for the assembly of lipoproteins. Assembly is aided by cell death-inducing DFF45-like effector B and by phospholipid transfer protein, which may facilitate additional deposition of triacylglycerols and phospholipids, respectively, to apoB. Here, we summarize the current understanding of the different steps in the assembly of apoB-containing lipoproteins and discuss the role of lipid transfer proteins in these steps to help identify new clinical targets for lipid-associated disorders, such as heart disease. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Crystal structure of cGMP-dependent protein kinase Iβ cyclic nucleotide-binding-B domain : Rp-cGMPS complex reveals an apo-like, inactive conformation

    DOE PAGES

    Campbell, James C.; VanSchouwen, Bryan; Lorenz, Robin; ...

    2016-12-23

    The R-diastereomer of phosphorothioate analogs of cGMP, Rp-cGMPS, is one of few known inhibitors of cGMP-dependent protein kinase I (PKG I); however, its mechanism of inhibition is currently not fully understood. We determined the crystal structure of the PKG Iβ cyclic nucleotide-binding domain (PKG Iβ CNB-B), considered a ‘gatekeeper’ for cGMP activation, bound to Rp-cGMPS at 1.3 Å. Our structural and NMR data show that PKG Iβ CNB-B bound to Rp-cGMPS displays an apo-like structure with its helical domain in an open conformation. Comparison with the cAMP-dependent protein kinase regulatory subunit (PKA RIα) showed that this conformation resembles the catalyticmore » subunit-bound inhibited state of PKA RIα more closely than the apo or Rp-cAMPS-bound conformations. Our results suggest that Rp-cGMPS inhibits PKG I by stabilizing the inactive conformation of CNB-B.« less

  13. Ubiquitin B in Cervical Cancer: Critical for the Maintenance of Cancer Stem-Like Cell Characters

    PubMed Central

    Wang, Yingying; Ji, Teng; Sun, Shujuan; Mo, Qingqing; Chen, Pingbo; Fang, Yong; Liu, Jia; Wang, Beibei; Zhou, Jianfeng; Ma, Ding; Wu, Peng

    2013-01-01

    Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate. Ubiquitin, which is a small, highly conserved protein expressed in all eukaryotic cells, can be covalently linked to certain target proteins to mark them for degradation by the ubiquitin-proteasome system. Previous studies highlight the essential role of Ubiquitin B (UbB) and UbB-dependent proteasomal protein degradation in histone deacetylase inhibitor (HDACi) -induced tumor selectivity. We hypothesized that UbB plays a critical role in the function of cervical cancer stem cells. We measured endogenous UbB levels in mammospheres in vitro by real-time PCR and Western blotting. The function of UbB in cancer stem-like cells was assessed after knockdown of UbB expression in prolonged Trichostatin A-selected HeLa cells (HeLa/TSA) by measuring in vitro cell proliferation, cell apoptosis, invasion, and chemotherapy resistance as well as by measuring in vivo growth in an orthotopic model of cervical cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human cervical cancer xenografts after UbB silencing. We found that HeLa/TSA were resistant to chemotherapy, highly expressed the UbB gene and the stem cell markers Sox2, Oct4 and Nanog. These cells also displayed induced differentiation abilities, including enhanced migration/invasion/malignancy capabilities in vitro and in vivo. Furthermore, an elevated expression of UbB was shown in the tumor samples of chemotherapy patients. Silencing of UbB inhibited tumorsphere formation, lowered the expression of stem cell markers and decreased cervical xenograft growth. Our results demonstrate that UbB was significantly increased in prolonged Trichostatin A-selected HeLa cells and it played a key role in the maintenance of cervical cancer stem-like cells. PMID:24367661

  14. Short toxin-like proteins abound in Cnidaria genomes.

    PubMed

    Tirosh, Yitshak; Linial, Itai; Askenazi, Manor; Linial, Michal

    2012-11-16

    Cnidaria is a rich phylum that includes thousands of marine species. In this study, we focused on Anthozoa and Hydrozoa that are represented by the Nematostella vectensis (Sea anemone) and Hydra magnipapillata genomes. We present a method for ranking the toxin-like candidates from complete proteomes of Cnidaria. Toxin-like functions were revealed using ClanTox, a statistical machine-learning predictor trained on ion channel inhibitors from venomous animals. Fundamental features that were emphasized in training ClanTox include cysteines and their spacing along the sequences. Among the 83,000 proteins derived from Cnidaria representatives, we found 170 candidates that fulfill the properties of toxin-like-proteins, the vast majority of which were previously unrecognized as toxins. An additional 394 short proteins exhibit characteristics of toxin-like proteins at a moderate degree of confidence. Remarkably, only 11% of the predicted toxin-like proteins were previously classified as toxins. Based on our prediction methodology and manual annotation, we inferred functions for over 400 of these proteins. Such functions include protease inhibitors, membrane pore formation, ion channel blockers and metal binding proteins. Many of the proteins belong to small families of paralogs. We conclude that the evolutionary expansion of toxin-like proteins in Cnidaria contributes to their fitness in the complex environment of the aquatic ecosystem.

  15. Bacillus licheniformis Contains Two More PerR-Like Proteins in Addition to PerR, Fur, and Zur Orthologues

    PubMed Central

    Ju, Shin-Yeong; Yang, Yoon-Mo; Ryu, Su-Hyun; Kwon, Yumi; Won, Young-Bin; Lee, Yeh-Eun; Youn, Hwan; Lee, Jin-Won

    2016-01-01

    The ferric uptake regulator (Fur) family proteins include sensors of Fe (Fur), Zn (Zur), and peroxide (PerR). Among Fur family proteins, Fur and Zur are ubiquitous in most prokaryotic organisms, whereas PerR exists mainly in Gram positive bacteria as a functional homologue of OxyR. Gram positive bacteria such as Bacillus subtilis, Listeria monocytogenes and Staphylococcus aureus encode three Fur family proteins: Fur, Zur, and PerR. In this study, we identified five Fur family proteins from B. licheniformis: two novel PerR-like proteins (BL00690 and BL00950) in addition to Fur (BL05249), Zur (BL03703), and PerR (BL00075) homologues. Our data indicate that all of the five B. licheniformis Fur homologues contain a structural Zn2+ site composed of four cysteine residues like many other Fur family proteins. Furthermore, we provide evidence that the PerR-like proteins (BL00690 and BL00950) as well as PerRBL (BL00075), but not FurBL (BL05249) and ZurBL (BL03703), can sense H2O2 by histidine oxidation with different sensitivity. We also show that PerR2 (BL00690) has a PerR-like repressor activity for PerR-regulated genes in vivo. Taken together, our results suggest that B. licheniformis contains three PerR subfamily proteins which can sense H2O2 by histidine oxidation not by cysteine oxidation, in addition to Fur and Zur. PMID:27176811

  16. Short Toxin-like Proteins Abound in Cnidaria Genomes

    PubMed Central

    Tirosh, Yitshak; Linial, Itai; Askenazi, Manor; Linial, Michal

    2012-01-01

    Cnidaria is a rich phylum that includes thousands of marine species. In this study, we focused on Anthozoa and Hydrozoa that are represented by the Nematostella vectensis (Sea anemone) and Hydra magnipapillata genomes. We present a method for ranking the toxin-like candidates from complete proteomes of Cnidaria. Toxin-like functions were revealed using ClanTox, a statistical machine-learning predictor trained on ion channel inhibitors from venomous animals. Fundamental features that were emphasized in training ClanTox include cysteines and their spacing along the sequences. Among the 83,000 proteins derived from Cnidaria representatives, we found 170 candidates that fulfill the properties of toxin-like-proteins, the vast majority of which were previously unrecognized as toxins. An additional 394 short proteins exhibit characteristics of toxin-like proteins at a moderate degree of confidence. Remarkably, only 11% of the predicted toxin-like proteins were previously classified as toxins. Based on our prediction methodology and manual annotation, we inferred functions for over 400 of these proteins. Such functions include protease inhibitors, membrane pore formation, ion channel blockers and metal binding proteins. Many of the proteins belong to small families of paralogs. We conclude that the evolutionary expansion of toxin-like proteins in Cnidaria contributes to their fitness in the complex environment of the aquatic ecosystem. PMID:23202321

  17. Prions and prion-like proteins.

    PubMed

    Fraser, Paul E

    2014-07-18

    Prions are self-replicating protein aggregates and are the primary causative factor in a number of neurological diseases in mammals. The prion protein (PrP) undergoes a conformational transformation leading to aggregation into an infectious cellular pathogen. Prion-like protein spreading and transmission of aggregates between cells have also been demonstrated for other proteins associated with Alzheimer disease and Parkinson disease. This protein-only phenomenon may therefore have broader implications in neurodegenerative disorders. The minireviews in this thematic series highlight the recent advances in prion biology and the roles these unique proteins play in disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. The VP7 Outer Capsid Protein of Rotavirus Induces Polyclonal B-Cell Activation

    PubMed Central

    Blutt, Sarah E.; Crawford, Sue E.; Warfield, Kelly L.; Lewis, Dorothy E.; Estes, Mary K.; Conner, Margaret E.

    2004-01-01

    The early response to a homologous rotavirus infection in mice includes a T-cell-independent increase in the number of activated B lymphocytes in the Peyer's patches. The mechanism of this activation has not been previously determined. Since rotavirus has a repetitively arranged triple-layered capsid and repetitively arranged antigens can induce activation of B cells, one or more of the capsid proteins could be responsible for the initial activation of B cells during infection. To address this question, we assessed the ability of rotavirus and virus-like particles to induce B-cell activation in vivo and in vitro. Using infectious rotavirus, inactivated rotavirus, noninfectious but replication-competent virus, and virus-like particles, we determined that neither infectivity nor RNA was necessary for B-cell activation but the presence of the rotavirus outer capsid protein, VP7, was sufficient for murine B-cell activation. Preincubation of the virus with neutralizing VP7 antibodies inhibited B-cell activation. Polymyxin B treatment and boiling of the virus preparation were performed, which ruled out possible lipopolysaccharide contamination as the source of activation and confirmed that the structural conformation of VP7 is important for B-cell activation. These findings indicate that the structure and conformation of the outer capsid protein, VP7, initiate intestinal B-cell activation during rotavirus infection. PMID:15194774

  19. Mitochondrial ADCK3 employs an atypical protein kinase-like fold to enable coenzyme Q biosynthesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Stefely, Jonathan A.; Reidenbach, Andrew G.; Ulbrich, Arne

    The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. In this paper, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flipsmore » this coenzyme selectivity and enables autophosphorylation but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Finally, our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways.« less

  20. Mitochondrial ADCK3 employs an atypical protein kinase-like fold to enable coenzyme Q biosynthesis

    DOE PAGES

    Stefely, Jonathan A.; Reidenbach, Andrew G.; Ulbrich, Arne; ...

    2014-12-11

    The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. In this paper, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flipsmore » this coenzyme selectivity and enables autophosphorylation but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Finally, our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways.« less

  1. Ring-like oligomers of Synaptotagmins and related C2 domain proteins

    PubMed Central

    Zanetti, Maria N; Bello, Oscar D; Wang, Jing; Coleman, Jeff; Cai, Yiying; Sindelar, Charles V; Rothman, James E; Krishnakumar, Shyam S

    2016-01-01

    We recently reported that the C2AB portion of Synaptotagmin 1 (Syt1) could self-assemble into Ca2+-sensitive ring-like oligomers on membranes, which could potentially regulate neurotransmitter release. Here we report that analogous ring-like oligomers assemble from the C2AB domains of other Syt isoforms (Syt2, Syt7, Syt9) as well as related C2 domain containing protein, Doc2B and extended Synaptotagmins (E-Syts). Evidently, circular oligomerization is a general and conserved structural aspect of many C2 domain proteins, including Synaptotagmins. Further, using electron microscopy combined with targeted mutations, we show that under physiologically relevant conditions, both the Syt1 ring assembly and its rapid disruption by Ca2+ involve the well-established functional surfaces on the C2B domain that are important for synaptic transmission. Our data suggests that ring formation may be triggered at an early step in synaptic vesicle docking and positions Syt1 to synchronize neurotransmitter release to Ca2+ influx. DOI: http://dx.doi.org/10.7554/eLife.17262.001 PMID:27434670

  2. SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family.

    PubMed

    Antonets, Denis V; Nepomnyashchikh, Tatyana S; Shchelkunov, Sergei N

    2010-10-27

    Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

  3. High yield bacterial expression, purification and characterisation of bioactive Human Tousled-like Kinase 1B involved in cancer.

    PubMed

    Bhoir, Siddhant; Shaik, Althaf; Thiruvenkatam, Vijay; Kirubakaran, Sivapriya

    2018-03-19

    Human Tousled-like kinases (TLKs) are highly conserved serine/threonine protein kinases responsible for cell proliferation, DNA repair, and genome surveillance. Their possible involvement in cancer via efficient DNA repair mechanisms have made them clinically relevant molecular targets for anticancer therapy. Innovative approaches in chemical biology have played a key role in validating the importance of kinases as molecular targets. However, the detailed understanding of the protein structure and the mechanisms of protein-drug interaction through biochemical and biophysical techniques demands a method for the production of an active protein of exceptional stability and purity on a large scale. We have designed a bacterial expression system to express and purify biologically active, wild-type Human Tousled-like Kinase 1B (hTLK1B) by co-expression with the protein phosphatase from bacteriophage λ. We have obtained remarkably high amounts of the soluble and homogeneously dephosphorylated form of biologically active hTLK1B with our unique, custom-built vector design strategy. The recombinant hTLK1B can be used for the structural studies and may further facilitate the development of new TLK inhibitors for anti-cancer therapy using a structure-based drug design approach.

  4. Genetic diversity of avenin-like b genes in Aegilops tauschii Coss.

    PubMed

    Cao, Dong; Wang, Hongxia; Zhang, Bo; Liu, Baolong; Liu, Dengcai; Chen, Wenjie; Zhang, Huaigang

    2018-02-01

    Avenin-like storage proteins influence the rheological properties and processing quality in common wheat, and the discovery of new alleles will benefit wheat quality improvement. In this study, 13 avenin-like b alleles (TaALPb7D-A-M) were discovered in 108 Aegilops tauschii Coss. accessions. Ten alleles were reported for the first time, while the remaining three alleles were the same as alleles in other species. A total of 15 nucleotide changes were detected in the 13 alleles, resulting in only 11 amino acid changes because of synonymous mutations. Alleles TaALPb7D-E, TaALPb7D-G, and TaALPb7D-J encoded the same protein. These polymorphic sites existed in the N-terminus, Repetitive region (Left), Repetitive region (Right) and C-terminus domains, with no polymorphisms in the signal peptide sequence nor in those encoding the 18 conserved cysteine residues. Phylogenetic analysis divided the TaALPb7Ds into four clades. The Ae. tauschii alleles were distributed in all four clades, while the alleles derived from common wheat, TaALPb7D-G and TaALPb7D-C, belonged to clade III and IV, respectively. Alleles TaALPb7D-G and TaALPb7D-C were the most widely distributed, being present in nine and six countries, respectively. Iran and Turkey exhibited the highest genetic diversity with respect to TaALPb7D alleles, accessions from these countries carrying seven and six alleles, respectively, which implied that these countries were the centers of origin of the avenin-like b gene. The new alleles discovered and the phylogenetic analysis of avenin-like b genes will provide breeding materials and a theoretical basis for wheat quality improvement.

  5. Insulin-like growth factor-binding protein-3 inhibits IGF-1-induced proliferation of human hepatocellular carcinoma cells by controlling bFGF and PDGF autocrine/paracrine loops

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ma, Yang; Han, Chen-chen; Li, Yifan

    Basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) produced by hepatocellular carcinoma (HCC) cells are responsible for the growth of HCC cells. Accumulating evidence shows that insulin-like growth factor-binding protein-3 (IGFBP-3) suppresses HCC cell proliferation in both IGF-dependent and independent manners. It's unknown, however, whether treatment with exogenous IGFBP-3 inhibits bFGF and PDGF production in HCC cells. The present study demonstrates that IGFBP-3 suppressed IGF-1-induced bFGF and PDGF expression while it does not affect their expression in the absence of IGF-1. To delineate the underlying mechanism, western-blot and RT-PCR assays confirmed that the transcription factor early growth responsemore » protein 1 (EGR1) is involved in IGFBP-3 regulation of bFGF and PDGF. IGFBP-3 inhibition of type 1 insulin-like growth factor receptor (IGF1R), ERK and AKT activation is IGF-1-dependent. Furthermore, transient transfection with constitutively activated AKT or MEK partially blocks the IGFBP-3 inhibition of EGR1, bFGF and PDGF expression. In conclusion, these findings suggest that IGFBP-3 suppresses transcription of EGR1 and its target genes bFGF and PDGF through inhibiting IGF-1-dependent ERK and AKT activation. It demonstrates the importance of IGFBP-3 in the regulation of HCC cell proliferation, suggesting that IGFBP-3 could be a target for the treatment of HCC. - Highlights: • IGFBP-3 plays an inhibition role in IGF1-induced HCC cell growth. • IGFBP-3 inhibits bFGF and PDGF production in the IGF-dependent manner. • EGR1 is involved in IGFBP-3 regulation of bFGF and PDGF in HCC cells. • IGFBP-3 suppresses EGR1 and its target genes bFGF and PDGF through inhibiting IGF-1-dependent ERK and AKT activation.« less

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

    PubMed Central

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

    2010-01-01

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

  7. Nucleophilic modification of human complement protein C3: correlation of conformational changes with acquisition of C3b-like functional properties.

    PubMed

    Isenman, D E; Kells, D I; Cooper, N R; Müller-Eberhard, H J; Pangburn, M K

    1981-07-21

    Inactivation of C3 by enzymatic cleavage, nucleophilic addition, or slow freezing and thawing resulted in the acquisition of similar end-state conformations as judged by near-UV circular dichroism. Although inactivation by the two nonenzymatic processes involves no peptide bond scission, the inactivated C3 resembled C3b in that it possessed a free sulfhydryl group not present in the native protein and an increased surface hydrophobicity as evidenced by enhanced binding of the fluorophore 8-anilino-1-naphthalensulfonate (ANS). The C3b-like functional properties of modified C3 [Pangburn, M. K., & Müller-Eberhard, H. J. (1980) J. Exp. Med. 152, 1102-1114] may thus be understood in terms of the similarity of its conformation to that of C3b. The rate of the conformational change following proteolytic cleavage was fast and appeared to be limited by the rate of the enzymatic reaction. In contrast, the rate of conformational change following addition of methylamine was slow and rate limited by the conformational rearrangement itself, not by the chemical modification. A kinetic analysis of the changes in circular dichroism and ANS fluorescence enhancement suggested that the nucleophilic addition was spectroscopically undetectable and was followed by a minimally biphasic, spectroscopically demonstrable conformational rearrangement. The appearance of C3b-like functional activity in nucleophile-modified C3 largely parallels the time course of the spectroscopically detectable conformational change but is distinctly slower than the rate at which hemolytic activity is lost. While fully transconformed methylamine-inactivated C3 can bind factor B and is susceptible to cleavage by C3b inactivator and its cofactor beta 1H, this cleavage occurs at a substantially slower rate than the equivalent process in C3b. The implications of these findings in terms of the mechanism through which the alterative pathway of complement is initiated are discussed.

  8. Salmonella enterica synthesizes 5,6-dimethylbenzimidazolyl-(DMB)-α-riboside. Why some Firmicutes do not require the canonical DMB activation system to synthesize adenosylcobalamin.

    PubMed

    Mattes, Theodoric A; Escalante-Semerena, Jorge C

    2017-01-01

    5,6-Dimethylbenzimidazolyl-(DMB)-α-ribotide [α-ribazole-5'-phosphate (α-RP)] is an intermediate in the biosynthesis of adenosylcobalamin (AdoCbl) in many prokaryotes. In such microbes, α-RP is synthesized by nicotinate mononucleotide (NaMN):DMB phosphoribosyltransferases (CobT in Salmonella enterica), in a reaction that is considered to be the canonical step for the activation of the base of the nucleotide present in adenosylcobamides. Some Firmicutes lack CobT-type enzymes but have a two-protein system comprised of a transporter (i.e., CblT) and a kinase (i.e., CblS) that can salvage exogenous α-ribazole (α-R) from the environment using CblT to take up α-R, followed by α-R phosphorylation by CblS. We report that Geobacillus kaustophilus CblT and CblS proteins restore α-RP synthesis in S. enterica lacking the CobT enzyme. We also show that a S. enterica cobT strain that synthesizes GkCblS ectopically makes only AdoCbl, even under growth conditions where the synthesis of pseudoCbl is favored. Our results indicate that S. enterica synthesizes α-R, a metabolite that had not been detected in this bacterium and that GkCblS has a strong preference for DMB-ribose over adenine-ribose as substrate. We propose that in some Firmicutes DMB is activated to α-RP via α-R using an as-yet-unknown route to convert DMB to α-R and CblS to convert α-R to α-RP. © 2016 John Wiley & Sons Ltd.

  9. SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family

    PubMed Central

    2010-01-01

    Background Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. Findings De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Conclusions Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein. PMID:20979600

  10. Insulin-like plant proteins as potential innovative drugs to treat diabetes-The Moringa oleifera case study.

    PubMed

    Paula, P C; Oliveira, J T A; Sousa, D O B; Alves, B G T; Carvalho, A F U; Franco, O L; Vasconcelos, I M

    2017-10-25

    Various plant species have long been used in traditional medicine worldwide to treat diabetes. Among the plant-based compounds with hypoglycemic properties, studies on insulin-like proteins isolated from leaves, fruits and seeds are rarely reported in the relevant literature. Our research group has been investigating the presence of insulin-like proteins in Moringa oleifera, a plant species native to India, and we have obtained a leaf protein isolate and semi-purified derived fractions, as well as a seed coat protein fraction (Mo-SC), with hypoglycemic activity in chemically induced diabetic mice that have increased tolerance to orally administered glucose. Equally importantly, Mo-SC possesses insulin-like antigenic epitopes. In this context, the present review aims to highlight that prospection of insulin-like proteins in plants is of the utmost importance both for finding new drugs for the treatment of diabetes and for shedding light on the mechanisms involved in diabetes. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter

    PubMed Central

    2012-01-01

    Background Bacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (PliaI) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. In the absence of a stimulus, this promoter is kept tightly inactive. Upon induction by cell wall antibiotics, it shows an over 100-fold increase in activity within 10 min. Results Based on these traits of PliaI, we developed a novel LiaRS-controlled gene expression system for B. subtilis (the “LIKE" system). Two expression vectors, the integrative pLIKE-int and the replicative pLIKE-rep, were constructed. To enhance the performance of the PliaI-derived system, site-directed mutagenesis was employed to optimize the ribosome binding site and alter its spacing to the initiation codon used for the translational fusion. The impact of these genetic modifications on protein production yield was measured using GFP as a model protein. Moreover, a number of tailored B. subtilis expression strains containing different markerless chromosomal deletions of the liaIH region were constructed to circumvent undesired protein production, enhance the positive autoregulation of the LiaRS system and thereby increase target gene expression strength from the PliaI promoter. Conclusions The LIKE protein expression system is a novel protein expression system, which offers a number of advantages over existing systems. Its major advantages are (i) a tightly switched-off promoter during exponential growth in the absence of a stimulus, (ii) a concentration-dependent activation of PliaI in the presence of suitable inducers, (iii) a very fast but transient response with a very high dynamic range of over 100-fold (up to 1,000-fold) induction, (iv) a choice from a range of well-defined, commercially available

  12. The Scaffold Protein TANK/I-TRAF Inhibits NF-κB Activation by Recruiting Polo-like Kinase 1

    PubMed Central

    Zhang, Wanqiao; Zhang, Ying; Yuan, Yanzhi; Guan, Wei; Jin, Chaozhi; Chen, Hui; Wang, Xiaohui

    2010-01-01

    TANK/I-TRAF is a TRAF-binding protein that negatively regulates NF-κB activation. The underlying mechanism of this activity remains unclear. Here we show that TANK directly interacts with PLK1, a conserved cell cycle–regulated kinase. PLK1 inhibits NF-κB transcriptional activation induced by TNF-α, IL-1β, or several activators, but not by nuclear transcription factor p65. PLK1 expression reduces the DNA-binding activity of NF-κB induced by TNF-α. Moreover, endogenous activation of PLK1 reduces the TNF-induced phosphorylation of endogenous IκBα. PLK1 is bound to NEMO (IKKγ) through TANK to form a ternary complex in vivo. We describe a new regulatory mechanism for PLK1: PLK1 negatively regulates TNF-induced IKK activation by inhibiting the ubiquitination of NEMO. These findings reveal that the scaffold protein TANK recruits PLK1 to negatively regulate NF-κB activation and provide direct evidence that PLK1 is required for the repression function of TANK. PMID:20484576

  13. Calcitonin and calcitonin receptor-like receptors: common themes with family B GPCRs?

    PubMed

    Barwell, James; Gingell, Joseph J; Watkins, Harriet A; Archbold, Julia K; Poyner, David R; Hay, Debbie L

    2012-05-01

    The calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR) are two of the 15 human family B (or Secretin-like) GPCRs. CTR and CLR are of considerable biological interest as their pharmacology is moulded by interactions with receptor activity-modifying proteins. They also have therapeutic relevance for many conditions, such as osteoporosis, diabetes, obesity, lymphatic insufficiency, migraine and cardiovascular disease. In light of recent advances in understanding ligand docking and receptor activation in both the family as a whole and in CLR and CTR specifically, this review reflects how applicable general family B GPCR themes are to these two idiosyncratic receptors. We review the main functional domains of the receptors; the N-terminal extracellular domain, the juxtamembrane domain and ligand interface, the transmembrane domain and the intracellular C-terminal domain. Structural and functional findings from the CLR and CTR along with other family B GPCRs are critically appraised to gain insight into how these domains may function. The ability for CTR and CLR to interact with receptor activity-modifying proteins adds another level of sophistication to these receptor systems but means careful consideration is needed when trying to apply generic GPCR principles. This review encapsulates current thinking in the realm of family B GPCR research by highlighting both conflicting and recurring themes and how such findings relate to two unusual but important receptors, CTR and CLR. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  14. BDNF/TrkB Pathway Mediates the Antidepressant-Like Role of H2S in CUMS-Exposed Rats by Inhibition of Hippocampal ER Stress.

    PubMed

    Wei, Le; Kan, Li-Yuan; Zeng, Hai-Ying; Tang, Yi-Yun; Huang, Hong-Lin; Xie, Ming; Zou, Wei; Wang, Chun-Yan; Zhang, Ping; Tang, Xiao-Qing

    2018-06-01

    Our previous works have shown that hydrogen sulfide (H 2 S) significantly attenuates chronic unpredictable mild stress (CUMS)-induced depressive-like behaviors and hippocampal endoplasmic reticulum (ER) stress. Brain-derived neurotrophic factor (BDNF) generates an antidepressant-like effect by its receptor tyrosine protein kinase B (TrkB). We have previously found that H 2 S upregulates the expressions of BDNF and p-TrkB in the hippocampus of CUMS-exposed rats. Therefore, the present work was to explore whether BDNF/TrkB pathway mediates the antidepressant-like role of H 2 S by blocking hippocampal ER stress. We found that treatment with K252a (an inhibitor of BDNF/TrkB pathway) significantly increased the immobility time in the forced swim test and tail suspension test and increased the latency to feed in the novelty-suppressed feeding test in the rats cotreated with sodium hydrosulfide (NaHS, a donor of H 2 S) and CUMS. Similarly, K252a reversed the protective effect of NaHS against CUMS-induced hippocampal ER stress, as evidenced by increases in the levels of ER stress-related proteins, glucose-regulated protein 78, CCAAT/enhancer binding protein homologous protein and cleaved caspase-12. Taken together, our results suggest that BDNF/TrkB pathway plays an important mediatory role in the antidepressant-like action of H 2 S in CUMS-exposed rats, which is by suppression of hippocampal ER stress. These data provide a novel mechanism underlying the protection of H 2 S against CUMS-induced depressive-like behaviors.

  15. Primary central nervous system diffuse large B-cell lymphoma shows an activated B-cell-like phenotype with co-expression of C-MYC, BCL-2, and BCL-6.

    PubMed

    Li, Xiaomei; Huang, Ying; Bi, Chengfeng; Yuan, Ji; He, Hong; Zhang, Hong; Yu, QiuBo; Fu, Kai; Li, Dan

    2017-06-01

    Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma, whose main prognostic factor is closely related to germinal center B-cell-like subtype (GCB- DLBCL) or activated B-cell-like type (non-GCB-DLBCL). The most common type of primary central nervous system lymphoma is diffuse large B-cell type with poor prognosis and the reason is unclear. This study aims to stratify primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL) according to the cell-of-origin (COO) and to investigate the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53, further to elucidate the reason why primary central nervous system diffuse large B-cell lymphoma possesses a poor clinical outcome as well. Nineteen cases of primary central nervous system DLBCL were stratified according to immunostaining algorithms of Hans, Choi and Meyer (Tally) and we investigated the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53. The Epstein-Barr virus and Borna disease virus infection were also detected. Among nineteen cases, most (15-17 cases) were assigned to the activated B-cell-like subtype, highly expression of C-MYC (15 cases, 78.9%), BCL-2 (10 cases, 52.6%), BCL-6 (15 cases, 78.9%). Unfortunately, two cases were positive for PD-L1 while PD-L2 was not expressed in any case. Two cases infected with BDV but no one infected with EBV. In conclusion, most primary central nervous system DLBCLs show an activated B-cell-like subtype characteristic and have multiple expressions of C-MYC, BCL-2, BCL-6 protein, these features might be significant factor to predict the outcome and guide treatment of PCNS-DLBCLs. Copyright © 2017 Elsevier GmbH. All rights reserved.

  16. Bacterial translation elongation factor EF-Tu interacts and colocalizes with actin-like MreB protein.

    PubMed

    Defeu Soufo, Hervé Joël; Reimold, Christian; Linne, Uwe; Knust, Tobias; Gescher, Johannes; Graumann, Peter L

    2010-02-16

    We show that translation initiation factor EF-Tu plays a second important role in cell shape maintenance in the bacterium Bacillus subtilis. EF-Tu localizes in a helical pattern underneath the cell membrane and colocalizes with MreB, an actin-like cytoskeletal element setting up rod cell shape. The localization of MreB and of EF-Tu is interdependent, but in contrast to the dynamic MreB filaments, EF-Tu structures are more static and may serve as tracks for MreB filaments. In agreement with this idea, EF-Tu and MreB interact in vivo and in vitro. Lowering of the EF-Tu levels had a minor effect on translation but a strong effect on cell shape and on the localization of MreB, and blocking of the function of EF-Tu in translation did not interfere with the localization of MreB, showing that, directly or indirectly, EF-Tu affects the cytoskeletal MreB structure and thus serves two important functions in a bacterium.

  17. Overproduction, purification, and ATPase activity of the Escherichia coli RuvB protein involved in DNA repair.

    PubMed Central

    Iwasaki, H; Shiba, T; Makino, K; Nakata, A; Shinagawa, H

    1989-01-01

    The ruvA and ruvB genes of Escherichia coli constitute an operon which belongs to the SOS regulon. Genetic evidence suggests that the products of the ruv operon are involved in DNA repair and recombination. To begin biochemical characterization of these proteins, we developed a plasmid system that overproduced RuvB protein to 20% of total cell protein. Starting from the overproducing system, we purified RuvB protein. The purified RuvB protein behaved like a monomer in gel filtration chromatography and had an apparent relative molecular mass of 38 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the value predicted from the DNA sequence. The amino acid sequence of the amino-terminal region of the purified protein was analyzed, and the sequence agreed with the one deduced from the DNA sequence. Since the deduced sequence of RuvB protein contained the consensus sequence for ATP-binding proteins, we examined the ATP-binding and ATPase activities of the purified RuvB protein. RuvB protein had a stronger affinity to ADP than to ATP and weak ATPase activity. The results suggest that the weak ATPase activity of RuvB protein is at least partly due to end product inhibition by ADP. Images PMID:2529252

  18. A Crayfish Insulin-like-binding Protein

    PubMed Central

    Rosen, Ohad; Weil, Simy; Manor, Rivka; Roth, Ziv; Khalaila, Isam; Sagi, Amir

    2013-01-01

    Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a “pulldown” methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23–38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development. PMID:23775079

  19. Olfactomedin-like 2 A and B (OLFML2A and OLFML2B) expression profile in primates (human and baboon).

    PubMed

    Pérez-Ibave, Diana Cristina; González-Alvarez, Rafael; de La Luz Martinez-Fierro, Margarita; Ruiz-Ayma, Gabriel; Luna-Muñoz, Maricela; Martínez-De-Villarreal, Laura Elia; De Lourdes Garza-Rodríguez, María; Reséndez-Pérez, Diana; Mohamed-Noriega, Jibran; Garza-Guajardo, Raquel; Bautista-De-Lucío, Víctor Manuel; Mohamed-Noriega, Karim; Barboza-Quintana, Oralia; Arámburo-De-La-Hoz, Carlos; Barrera-Saldaña, Hugo Alberto; Rodríguez-Sánchez, Irám Pablo

    2016-11-08

    The olfactomedin-like domain (OLFML) is present in at least four families of proteins, including OLFML2A and OLFML2B, which are expressed in adult rat retina cells. However, no expression of their orthologous has ever been reported in human and baboon. The aim of this study was to investigate the expression of OLFML2A and OLFML2B in ocular tissues of baboons (Papio hamadryas) and humans, as a key to elucidate OLFML function in eye physiology. OLFML2A and OLFML2B cDNA detection in ocular tissues of these species was performed by RT-PCR. The amplicons were cloned and sequenced, phylogenetically analyzed and their proteins products were confirmed by immunofluorescence assays. OLFML2A and OLFML2B transcripts were found in human cornea, lens and retina and in baboon cornea, lens, iris and retina. The baboon OLFML2A and OLFML2B ORF sequences have 96% similarity with their human's orthologous. OLFML2A and OLFML2B evolution fits the hypothesis of purifying selection. Phylogenetic analysis shows clear orthology in OLFML2A genes, while OLFML2B orthology is not clear. Expression of OLFML2A and OLFML2B in human and baboon ocular tissues, including their high similarity, make the baboon a powerful model to deduce the physiological and/or metabolic function of these proteins in the eye.

  20. Proteolysis of serum amyloid A and AA amyloid proteins by cysteine proteases: cathepsin B generates AA amyloid proteins and cathepsin L may prevent their formation

    PubMed Central

    Rocken, C; Menard, R; Buhling, F; Vockler, S; Raynes, J; Stix, B; Kruger, S; Roessner, A; Kahne, T

    2005-01-01

    Background: AA amyloidosis develops in patients with chronic inflammatory diseases. The AA amyloid proteins are proteolytic fragments obtained from serum amyloid A (SAA). Previous studies have provided evidence that endosomes or lysosomes might be involved in the processing of SAA, and contribute to the pathology of AA amyloidosis. Objective: To investigate the anatomical distribution of cathepsin (Cath) B and CathL in AA amyloidosis and their ability to process SAA and AA amyloid proteins. Methods and results: CathB and CathL were found immunohistochemically in every patient with AA amyloidosis and displayed a spatial relationship with amyloid in all the cases studied. Both degraded SAA and AA amyloid proteins in vitro. With the help of mass spectrometry 27 fragments were identified after incubation of SAA with CathB, nine of which resembled AA amyloid proteins, and seven fragments after incubation with CathL. CathL did not generate AA amyloid-like peptides. When native human AA amyloid proteins were used as a substrate 26 fragments were identified after incubation with CathB and 18 after incubation with CathL. Conclusion: The two most abundant and ubiquitously expressed lysosomal proteases can cleave SAA and AA amyloid proteins. CathB generates nine AA amyloid-like proteins by its carboxypeptidase activity, whereas CathL may prevent the formation of AA amyloid proteins by endoproteolytic activity within the N-terminal region of SAA. This is particularly interesting, because AA amyloidosis is a systemic disease affecting many organs and tissue types, almost all of which express CathB and CathL. PMID:15897303

  1. Follistatin-like protein 1 induction of matrix metalloproteinase 1, 3 and 13 gene expression in rheumatoid arthritis synoviocytes requires MAPK, JAK/STAT3 and NF-κB pathways.

    PubMed

    Ni, Su; Li, Chenkai; Xu, Nanwei; Liu, Xi; Wang, Wei; Chen, Wenyang; Wang, Yuji; van Wijnen, Andre J

    2018-06-22

    Elevated levels of follistatin-like protein 1 (FSTL1) have been found both in mouse models for human rheumatoid arthritis (RA) and collagen-induced arthritis (CIA). In this study, we elucidated the potential mechanisms by which FSTL1 contributes to the pathogenesis of RA. Fibroblast-like synoviocytes (FLSs) were established from synovial tissues of RA patients and stimulated with human recombinant FSTL1. Protein and mRNA expression levels of select matrix metalloproteinases (i.e., MMP1, MMP3, MMP13) in FLS were measured by, respectively, real-time RT-qPCR and ELISA. Activation of MAPK and other pathways that affect MMPs were evaluated by Western blotting. We also compared concentrations of MMPs in plasma in RA patients versus healthy controls (HC). Expression levels of MMP1, MMP3, and MMP13 were clearly stimulated by FSTL1 in vitro. FSTL1 activated the inflammation-related NF-κB signaling pathway, as well as all three mitogen-activated protein kinase (MAPK) pathways and the JAK/STAT3 pathway. Moreover, select chemical inhibitors that target p38 (SB203580), Erk1/2 (SP600125), JNK (SCH772984), STAT3 (AG490), and NF-κB (BAY 11-7082) significantly attenuated MMP expression. Inhibition of Toll-like receptor 4 by compound TAK-242 significantly abolished those effects of FSTL1. Importantly, elevated plasma concentrations of MMP3 were found to correlate with plasma FSTL1 levels in RA patients. These findings suggest that FSTL1 accelerates RA progression by activating MAPK, JAK/STAT3, and NF-κB pathways to enhance secretion of different MMPs and this enhancement is via TLR4. Targeting FSTL1 may provide a promising pharmacological drug therapy to ameliorate RA symptoms and perhaps reverse disease progression. © 2018 Wiley Periodicals, Inc.

  2. Derepression of microRNA-mediated protein translation inhibition by apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) and its family members.

    PubMed

    Huang, Jialing; Liang, Zhihui; Yang, Bin; Tian, Heng; Ma, Jin; Zhang, Hui

    2007-11-16

    The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) and its fellow cytidine deaminase family members are potent restrictive factors for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. A3G interacts with a vast spectrum of RNA-binding proteins and is located in processing bodies and stress granules. However, its cellular function remains to be further clarified. Using a luciferase reporter gene and green fluorescent protein reporter gene, we demonstrate that A3G and other APOBEC family members can counteract the inhibition of protein synthesis by various microRNAs (miRNAs) such as mir-10b, mir-16, mir-25, and let-7a. A3G could also enhance the expression level of miRNA-targeted mRNA. Further, A3G facilitated the association of microRNA-targeted mRNA with polysomes rather than with processing bodies. Intriguingly, experiments with a C288A/C291A A3G mutant indicated that this function of A3G is separable from its cytidine deaminase activity. Our findings suggest that the major cellular function of A3G, in addition to inhibiting the mobility of retrotransposons and replication of endogenous retroviruses, is most likely to prevent the decay of miRNA-targeted mRNA in processing bodies.

  3. Insights into the nature of DNA binding of AbrB-like transcription factors

    PubMed Central

    Sullivan, Daniel M.; Bobay, Benjamin G.; Kojetin, Douglas J.; Thompson, Richele J.; Rance, Mark; Strauch, Mark A.; Cavanagh, John

    2008-01-01

    Summary Understanding the DNA recognition and binding by the AbrB-like family of transcriptional regulators is of significant interest since these proteins enable bacteria to elicit the appropriate response to diverse environmental stimuli. Although these ‘transition-state regulator’ proteins have been well characterized at the genetic level, the general and specific mechanisms of DNA binding remain elusive. We present RDC-refined NMR solution structures and dynamic properties of the DNA-binding domains of three Bacillus subtilis transition-state regulators AbrB, Abh, and SpoVT. We combined previously investigated DNase I footprinting, DNA methylation, gel shift assays, mutagenic and NMR studies to generate a structural model of the complex between AbrBN55 and its cognate promoter, abrB8. These investigations have enabled us to generate the first model for the specific nature of the transition-state regulator-DNA interaction. PMID:19000822

  4. Proteomic analysis of rodent ribosomes revealed heterogeneity including ribosomal proteins L10-like, L22-like 1, and L39-like.

    PubMed

    Sugihara, Yoshihiko; Honda, Hiroki; Iida, Tomoharu; Morinaga, Takuma; Hino, Shingo; Okajima, Tetsuya; Matsuda, Tsukasa; Nadano, Daita

    2010-03-05

    Heterogeneity of ribosome structure, due to variations in ribosomal protein composition, has been shown to be of physiological significance in plants and yeast. Mammalian genomics have demonstrated numerous genes that are paralogous to genes encoding ribosomal proteins. Although the vast majority are considered to be pseudogenes, mRNA expression of a few paralogues, such as human ribosomal protein L39-like/L39-2, has been reported. In the present study, ribosomes from the liver, mammary gland, and testis of rodents were analyzed using a combination of two-dimensional gel electrophoresis under radical-free and highly reducing conditions, and mass spectrometry. This system allowed identification of 78 ribosomal proteins and Rack1 from a single gel. The degree of heterogeneity was far less than that reported for plant and yeast ribosomes, and was in accord with published biochemical and genetic data for mammalian ribosomes. Nevertheless, an uncharacterized paralogue of ribosomal protein L22, ribosomal protein L22-like 1, was identified as a minor ribosomal component. Ribosomal proteins L10-like and L39-like, paralogues of ribosomal proteins L10 and L39, respectively, were found in ribosomes only from the testis. Reverse transcription-polymerase chain reaction yielded supportive evidence for specific expression of L10-like and L39-like in the testis. Newly synthesized L39-like is likely to be transported to the nucleolus, where ribosome biosynthesis occurs, and then incorporated into translating ribosomes in the cytoplasm. Heterogeneity of mammalian testicular ribosomes is structurally non-negligible, and may offer valuable insights into the function of the customized ribosome.

  5. NopB, a type III secreted protein of Rhizobium sp. strain NGR234, is associated with pilus-like surface appendages.

    PubMed

    Saad, Maged M; Kobayashi, Hajime; Marie, Corinne; Brown, Ian R; Mansfield, John W; Broughton, William J; Deakin, William J

    2005-02-01

    Rhizobium sp. strain NGR234 possesses a functional type three secretion system (TTSS), through which a number of proteins, called nodulation outer proteins (Nops), are delivered to the outside of the cell. A major constraint to the identification of Nops is their low abundance in the supernatants of NGR234 strains grown in culture. To overcome this limitation, a more sensitive proteomics-based strategy was developed. Secreted proteins from wild-type NGR234 were separated by two-dimensional gel electrophoresis, and the gel was compared to similar gels containing the proteins from a TTSS mutant (NGROmegarhcN). To identify the proteins, spots unique to the NGR234 gels were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and the data were compared to the sequence of the symbiotic plasmid of NGR234. A nonpolar mutant of one of these proteins was generated called NopB. NopB is required for Nop secretion but inhibits the interaction with Pachyrhizus tuberosus and augments nodulation of Tephrosia vogelii. Flavonoids and a functional TTSS are required for the formation of some surface appendages on NGR234. In situ immunogold labeling and isolation of these pili showed that they contain NopB.

  6. NopB, a Type III Secreted Protein of Rhizobium sp. Strain NGR234, Is Associated with Pilus-Like Surface Appendages

    PubMed Central

    Saad, Maged M.; Kobayashi, Hajime; Marie, Corinne; Brown, Ian R.; Mansfield, John W.; Broughton, William J.; Deakin, William J.

    2005-01-01

    Rhizobium sp. strain NGR234 possesses a functional type three secretion system (TTSS), through which a number of proteins, called nodulation outer proteins (Nops), are delivered to the outside of the cell. A major constraint to the identification of Nops is their low abundance in the supernatants of NGR234 strains grown in culture. To overcome this limitation, a more sensitive proteomics-based strategy was developed. Secreted proteins from wild-type NGR234 were separated by two-dimensional gel electrophoresis, and the gel was compared to similar gels containing the proteins from a TTSS mutant (NGRΩrhcN). To identify the proteins, spots unique to the NGR234 gels were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and the data were compared to the sequence of the symbiotic plasmid of NGR234. A nonpolar mutant of one of these proteins was generated called NopB. NopB is required for Nop secretion but inhibits the interaction with Pachyrhizus tuberosus and augments nodulation of Tephrosia vogelii. Flavonoids and a functional TTSS are required for the formation of some surface appendages on NGR234. In situ immunogold labeling and isolation of these pili showed that they contain NopB. PMID:15659692

  7. Staphylococcus aureus FepA and FepB proteins drive heme iron utilization in Escherichia coli.

    PubMed

    Turlin, Evelyne; Débarbouillé, Michel; Augustyniak, Katarzyna; Gilles, Anne-Marie; Wandersman, Cécile

    2013-01-01

    EfeUOB-like tripartite systems are widespread in bacteria and in many cases they are encoded by genes organized into iron-regulated operons. They consist of: EfeU, a protein similar to the yeast iron permease Ftrp1; EfeO, an extracytoplasmic protein of unknown function and EfeB, also an extracytoplasmic protein with heme peroxidase activity, belonging to the DyP family. Many bacterial EfeUOB systems have been implicated in iron uptake, but a prefential iron source remains undetermined. Nevertheless, in the case of Escherichia coli, the EfeUOB system has been shown to recognize heme and to allow extracytoplasmic heme iron extraction via a deferrochelation reaction. Given the high level of sequence conservations between EfeUOB orthologs, we hypothesized that heme might be the physiological iron substrate for the other orthologous systems. To test this hypothesis, we undertook characterization of the Staphylococcus aureus FepABC system. Results presented here indicate: i) that the S. aureus FepB protein binds both heme and PPIX with high affinity, like EfeB, the E. coli ortholog; ii) that it has low peroxidase activity, comparable to that of EfeB; iii) that both FepA and FepB drive heme iron utilization, and both are required for this activity and iv) that the E. coli FepA ortholog (EfeO) cannot replace FepA in FepB-driven iron release from heme indicating protein specificity in these activities. Our results show that the function in heme iron extraction is conserved in the two orthologous systems.

  8. Purification and Characterization of a Novel Cold Shock Protein-Like Bacteriocin Synthesized by Bacillus thuringiensis

    PubMed Central

    Huang, Tianpei; Zhang, Xiaojuan; Pan, Jieru; Su, Xiaoyu; Jin, Xin; Guan, Xiong

    2016-01-01

    Bacillus thuringiensis (Bt), one of the most successful biopesticides, may expand its potential by producing bacteriocins (thuricins). The aim of this study was to investigate the antimicrobial potential of a novel Bt bacteriocin, thuricin BtCspB, produced by Bt BRC-ZYR2. The results showed that this bacteriocin has a high similarity with cold-shock protein B (CspB). BtCspB lost its activity after proteinase K treatment; however it was active at 60 °C for 30 min and was stable in the pH range 5–7. The partial loss of activity after the treatments of lipase II and catalase were likely due to the change in BtCspB structure and the partial degradation of BtCspB, respectively. The loss of activity at high temperatures and the activity variation at different pHs were not due to degradation or large conformational change. BtCspB did not inhibit four probiotics. It was only active against B. cereus strains 0938 and ATCC 10987 with MIC values of 3.125 μg/mL and 0.781 μg/mL, and MBC values of 12.5 μg/mL and 6.25 μg/mL, respectively. Taken together, these results provide new insights into a novel cold shock protein-like bacteriocin, BtCspB, which displayed promise for its use in food preservation and treatment of B. cereus-associated diseases. PMID:27762322

  9. Purification and Characterization of a Novel Cold Shock Protein-Like Bacteriocin Synthesized by Bacillus thuringiensis.

    PubMed

    Huang, Tianpei; Zhang, Xiaojuan; Pan, Jieru; Su, Xiaoyu; Jin, Xin; Guan, Xiong

    2016-10-20

    Bacillus thuringiensis (Bt), one of the most successful biopesticides, may expand its potential by producing bacteriocins (thuricins). The aim of this study was to investigate the antimicrobial potential of a novel Bt bacteriocin, thuricin BtCspB, produced by Bt BRC-ZYR2. The results showed that this bacteriocin has a high similarity with cold-shock protein B (CspB). BtCspB lost its activity after proteinase K treatment; however it was active at 60 °C for 30 min and was stable in the pH range 5-7. The partial loss of activity after the treatments of lipase II and catalase were likely due to the change in BtCspB structure and the partial degradation of BtCspB, respectively. The loss of activity at high temperatures and the activity variation at different pHs were not due to degradation or large conformational change. BtCspB did not inhibit four probiotics. It was only active against B. cereus strains 0938 and ATCC 10987 with MIC values of 3.125 μg/mL and 0.781 μg/mL, and MBC values of 12.5 μg/mL and 6.25 μg/mL, respectively. Taken together, these results provide new insights into a novel cold shock protein-like bacteriocin, BtCspB, which displayed promise for its use in food preservation and treatment of B. cereus-associated diseases.

  10. Functional diversification of B MADS-box homeotic regulators of flower development: Adaptive evolution in protein-protein interaction domains after major gene duplication events.

    PubMed

    Hernández-Hernández, Tania; Martínez-Castilla, León Patricio; Alvarez-Buylla, Elena R

    2007-02-01

    B-class MADS-box genes have been shown to be the key regulators of petal and stamen specification in several eudicot model species such as Arabidopsis thaliana, Antirrhinum majus, and Petunia hybrida. Orthologs of these genes have been found across angiosperms and gymnosperms, and it is thought that the basic regulatory function of B proteins is conserved in seed plant lineages. The evolution of B genes is characterized by numerous duplications that might represent key elements fostering the functional diversification of duplicates with a deep impact on their role in the evolution of the floral developmental program. To evaluate this, we performed a rigorous statistical analysis with B gene sequences. Using maximum likelihood and Bayesian methods, we estimated molecular substitution rates and determined the selective regimes operating at each residue of B proteins. We implemented tests that rely on phylogenetic hypotheses and codon substitution models to detect significant differences in substitution rates (DSRs) and sites under positive adaptive selection (PS) in specific lineages before and after duplication events. With these methods, we identified several protein residues fixed by PS shortly after the origin of PISTILLATA-like and APETALA3-like lineages in angiosperms and shortly after the origin of the euAP3-like lineage in core eudicots, the 2 main B gene duplications. The residues inferred to have been fixed by positive selection lie mostly within the K domain of the protein, which is key to promote heterodimerization. Additionally, we used a likelihood method that accommodates DSRs among lineages to estimate duplication dates for AP3-PI and euAP3-TM6, calibrating with data from the fossil record. The dates obtained are consistent with angiosperm origins and diversification of core eudicots. Our results strongly suggest that novel multimer formation with other MADS proteins could have been crucial for the functional divergence of B MADS-box genes. We thus

  11. The loss of Gnai2 and Gnai3 in B cells eliminates B lymphocyte compartments and leads to a hyper-IgM like syndrome.

    PubMed

    Hwang, Il-Young; Park, Chung; Luong, Thuyvi; Harrison, Kathleen A; Birnbaumer, Lutz; Kehrl, John H

    2013-01-01

    B lymphocytes are compartmentalized within lymphoid organs. The organization of these compartments depends upon signaling initiated by G-protein linked chemoattractant receptors. To address the importance of the G-proteins Gαi2 and Gαi3 in chemoattractant signaling we created mice lacking both proteins in their B lymphocytes. While bone marrow B cell development and egress is grossly intact; mucosal sites, splenic marginal zones, and lymph nodes essentially lack B cells. There is a partial block in splenic follicular B cell development and a 50-60% reduction in splenic B cells, yet normal numbers of splenic T cells. The absence of Gαi2 and Gαi3 in B cells profoundly disturbs the architecture of lymphoid organs with loss of B cell compartments in the spleen, thymus, lymph nodes, and gastrointestinal tract. This results in a severe disruption of B cell function and a hyper-IgM like syndrome. Beyond the pro-B cell stage, B cells are refractory to chemokine stimulation, and splenic B cells are poorly responsive to antigen receptor engagement. Gαi2 and Gαi3 are therefore critical for B cell chemoattractant receptor signaling and for normal B cell function. These mice provide a worst case scenario of the consequences of losing chemoattractant receptor signaling in B cells.

  12. Virulence of the Melioidosis Pathogen Burkholderia pseudomallei Requires the Oxidoreductase Membrane Protein DsbB

    PubMed Central

    2018-01-01

    ABSTRACT The naturally antibiotic-resistant bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a disease with stubbornly high mortality and a complex, protracted treatment regimen. The worldwide incidence of melioidosis is likely grossly underreported, though it is known to be highly endemic in northern Australia and Southeast Asia. Bacterial disulfide bond (DSB) proteins catalyze the oxidative folding and isomerization of disulfide bonds in substrate proteins. In the present study, we demonstrate that B. pseudomallei membrane protein disulfide bond protein B (BpsDsbB) forms a functional redox relay with the previously characterized virulence mediator B. pseudomallei disulfide bond protein A (BpsDsbA). Genomic analysis of diverse B. pseudomallei clinical isolates demonstrated that dsbB is a highly conserved core gene. Critically, we show that DsbB is required for virulence in B. pseudomallei. A panel of B. pseudomallei dsbB deletion strains (K96243, 576, MSHR2511, MSHR0305b, and MSHR5858) were phenotypically diverse according to the results of in vitro assays that assess hallmarks of virulence. Irrespective of their in vitro virulence phenotypes, two deletion strains were attenuated in a BALB/c mouse model of infection. A crystal structure of a DsbB-derived peptide complexed with BpsDsbA provides the first molecular characterization of their interaction. This work contributes to our broader understanding of DSB redox biology and will support the design of antimicrobial drugs active against this important family of bacterial virulence targets. PMID:29440370

  13. The human TRPV6 channel protein is associated with cyclophilin B in human placenta.

    PubMed

    Stumpf, Tobias; Zhang, Qi; Hirnet, Daniela; Lewandrowski, Urs; Sickmann, Albert; Wissenbach, Ulrich; Dörr, Janka; Lohr, Christian; Deitmer, Joachim W; Fecher-Trost, Claudia

    2008-06-27

    Transcellular calcium transport in the kidney, pancreas, small intestine, and placenta is partly mediated by transient receptor potential (TRP) channels. The highly selective TRPV6 calcium channel protein is most likely important for the calcium transfer in different specialized epithelial cells. In the human placenta the protein is expressed in trophoblast tissue, where it is implicated in the transepithelial calcium transfer from mother to the fetus. We enriched the TRPV6 channel protein endogenously expressed in placenta together with annexin A2 and cyclophilin B (CypB), which is a member of the huge immunophilin family. In the human placenta TRPV6 and CypB are mainly located intracellularly in the syncytiotrophoblast layer, but a small amount of the mature glycosylated TRPV6 channel protein and CypB is also expressed in microvilli apical membranes, the fetomaternal barrier. To understand the role of CypB on the TRPV6 channel function, we evaluated the effect of CypB co-expression on TRPV6-mediated calcium uptake into Xenopus laevis oocytes expressing TRPV6. A significant increase of TRPV6-mediated calcium uptake was observed after CypB/TRPV6 co-expression. This stimulatory effect of CypB was reversed by the immunosuppressive drug cyclosporin A, which inhibits the enzymatic activity of CypB. Cyclosporin A had no significant effect on TRPV6 and CypB protein expression levels in the oocytes. In summary, our results establish CypB as a new TRPV6 accessory protein with potential involvement in TRPV6 channel activation through its peptidyl-prolyl cis/trans isomerase activity.

  14. Heat shock proteins and toll-like receptors.

    PubMed

    Asea, Alexzander

    2008-01-01

    Researchers have only just begun to elucidate the relationship between heat shock proteins (HSP) and Toll-like receptors (TLR). HSP were originally described as an intracellular molecular chaperone of naïve, aberrantly folded, or mutated proteins and primarily implicated as a cytoprotective protein when cells are exposed to stressful stimuli. However, recent studies have ascribed novel functions to the Hsp70 protein depending on its localization: Surface-bound Hsp70 specifically activate natural killer (NK) cells, while Hsp70 released into the extracellular milieu specifically bind to Toll-like receptors (TLR) 2 and 4 on antigen-presenting cells (APC) and exerts immunoregulatory effects, including upregulation of adhesion molecules, co-stimulatory molecule expression, and cytokine and chemokine release-a process known as the chaperokine activity of Hsp70. This chapter discusses the most recent advances in the understanding of heat shock protein (HSP) and TLR interactions in general and highlights recent findings that demonstrate Hsp70 is a ligand for TLR and its biological significance.

  15. Recombinant human B7.2 IgV-like domain expressed in bacteria maintains its co-stimulatory activity in vitro.

    PubMed

    Yan, Xiaocai; Ma, Jun; Zheng, Jin; Lai, Baochang; Geng, Yiping; Wang, Yili; Si, Lüsheng

    2002-07-01

    To investigate which of the two immunoglobulin (Ig)-like domains, the immunoglobulin variable region homologous domain IgV (hB7.2 IgV) and the immunoglobulin constant region homologous domain IgC (hB7.2 IgC) on the human B7.2 molecule contains receptor binding sites, and to evaluate whether the B7.2 protein expressed in bacteria has biological activity in vitro. Three fragments of hB7.2 IgV,hB7.2 IgC and the complete extracellular region of human B7.2 containing both the IgV and IgC domains,hB7.2 Ig (V+C), were amplified by PCR and subcloned into pGEM-Teasy. Three recombinants,pGEX-4T-3-hB7.2 IgV,pGEX-4T-3-hB7.2 IgC and pGEX-4T-3-hB7.2 Ig (V+C), were generated by cloning the fragments into a prokaryote expression plasmid (pGEX-4T-3) and transformed into the host strain E. coli DH5alpha. The relevant target fusion proteins consisting of GST and hB7.2 IgV,hB7.2 IgC and hB7.2 Ig (V+C), were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7.2 fusion proteins by [(3)H]-TdR incorporation. Three recombinant fusion proteins of human B7.2, GST-hB7.2 IgV, GST-hB7.2 IgC and GST-hB7.2 Ig (V+C) were produced and detected in inclusion body form from engineered bacteria. With the first signal present,T lymphocytes proliferated when co-stimulated by bacterially-produced either GST-hB7.2 Ig (V+C) or GST-hB7.2 IgV fusion proteins, but not by GST-hB7.2 IgC. Functional human B7.2 fusion protein can be produced in bacteria. The IgV-like domain of human B7.2 is sufficient for B7.2 to interact with its counter-receptors and co-stimulate T lymphocytes.

  16. Conformational Rigidity and Protein Dynamics at Distinct Timescales Regulate PTP1B Activity and Allostery.

    PubMed

    Choy, Meng S; Li, Yang; Machado, Luciana E S F; Kunze, Micha B A; Connors, Christopher R; Wei, Xingyu; Lindorff-Larsen, Kresten; Page, Rebecca; Peti, Wolfgang

    2017-02-16

    Protein function originates from a cooperation of structural rigidity, dynamics at different timescales, and allostery. However, how these three pillars of protein function are integrated is still only poorly understood. Here we show how these pillars are connected in Protein Tyrosine Phosphatase 1B (PTP1B), a drug target for diabetes and cancer that catalyzes the dephosphorylation of numerous substrates in essential signaling pathways. By combining new experimental and computational data on WT-PTP1B and ≥10 PTP1B variants in multiple states, we discovered a fundamental and evolutionarily conserved CH/π switch that is critical for positioning the catalytically important WPD loop. Furthermore, our data show that PTP1B uses conformational and dynamic allostery to regulate its activity. This shows that both conformational rigidity and dynamics are essential for controlling protein activity. This connection between rigidity and dynamics at different timescales is likely a hallmark of all enzyme function. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Intermolecular cleavage by UmuD-like mutagenesis proteins

    PubMed Central

    McDonald, John P.; Frank, Ekaterina G.; Levine, Arthur S.; Woodgate, Roger

    1998-01-01

    The activity of a number of proteins is regulated by self-processing reactions. Elegant examples are the cleavage of the prokaryotic LexA and λCI transcriptional repressors and the UmuD-like mutagenesis proteins. Various studies support the hypothesis that LexA and λCI cleavage reactions are predominantly intramolecular in nature. The recently described crystal structure of the Escherichia coli UmuD′ protein (the posttranslational cleavage product of the UmuD protein) suggests, however, that the region of the protein corresponding to the cleavage site is at least 50 Å away from the catalytic active site. We considered the possibility, therefore, that the UmuD-like proteins might undergo self-processing that, in contrast to LexA and λCI, occurs via an intermolecular rather than intramolecular reaction. To test this hypothesis, we introduced into E. coli compatible plasmids with mutations at either the cleavage or the catalytic site of three UmuD-like proteins. Cleavage of these proteins only occurs in the presence of both plasmids, indicating that the reaction is indeed intermolecular in nature. Furthermore, this intermolecular reaction is completely dependent upon the multifunctional RecA protein and leads to the restoration of cellular mutagenesis in nonmutable E. coli strains. Intermolecular cleavage of a biotinylated UmuD active site mutant was also observed in vitro in the presence of the wild-type UmuD′ protein, indicating that in addition to the intact UmuD protein, the normal cleavage product (UmuD′) can also act as a classical enzyme. PMID:9465040

  18. Whole-Chain Tick Saliva Proteins Presented on Hepatitis B Virus Capsid-Like Particles Induce High-Titered Antibodies with Neutralizing Potential

    PubMed Central

    Kolb, Philipp; Wallich, Reinhard; Nassal, Michael

    2015-01-01

    Ticks are vectors for various, including pathogenic, microbes. Tick saliva contains multiple anti-host defense factors that enable ticks their bloodmeals yet also facilitate microbe transmission. Lyme disease-causing borreliae profit specifically from the broadly conserved tick histamine release factor (tHRF), and from cysteine-rich glycoproteins represented by Salp15 from Ixodes scapularis and Iric-1 from Ixodes ricinus ticks which they recruit to their outer surface protein C (OspC). Hence these tick proteins are attractive targets for anti-tick vaccines that simultaneously impair borrelia transmission. Main obstacles are the tick proteins´ immunosuppressive activities, and for Salp15 orthologs, the lack of efficient recombinant expression systems. Here, we exploited the immune-enhancing properties of hepatitis B virus core protein (HBc) derived capsid-like particles (CLPs) to generate, in E. coli, nanoparticulate vaccines presenting tHRF and, as surrogates for the barely soluble wild-type proteins, cysteine-free Salp15 and Iric-1 variants. The latter CLPs were exclusively accessible in the less sterically constrained SplitCore system. Mice immunized with tHRF CLPs mounted a strong anti-tHRF antibody response. CLPs presenting cysteine-free Salp15 and Iric-1 induced antibodies to wild-type, including glycosylated, Salp15 and Iric-1. The broadly distributed epitopes included the OspC interaction sites. In vitro, the anti-Salp15 antibodies interfered with OspC binding and enhanced human complement-mediated killing of Salp15 decorated borreliae. A mixture of all three CLPs induced high titered antibodies against all three targets, suggesting the feasibility of combination vaccines. These data warrant in vivo validation of the new candidate vaccines´ protective potential against tick infestation and Borrelia transmission. PMID:26352137

  19. Rye B chromosomes encode a functional Argonaute-like protein with in vitro slicer activities similar to its A chromosome paralog.

    PubMed

    Ma, Wei; Gabriel, Tobias Sebastian; Martis, Mihaela Maria; Gursinsky, Torsten; Schubert, Veit; Vrána, Jan; Doležel, Jaroslav; Grundlach, Heidrun; Altschmied, Lothar; Scholz, Uwe; Himmelbach, Axel; Behrens, Sven-Erik; Banaei-Moghaddam, Ali Mohammad; Houben, Andreas

    2017-01-01

    B chromosomes (Bs) are supernumerary, dispensable parts of the nuclear genome, which appear in many different species of eukaryote. So far, Bs have been considered to be genetically inert elements without any functional genes. Our comparative transcriptome analysis and the detection of active RNA polymerase II (RNAPII) in the proximity of B chromatin demonstrate that the Bs of rye (Secale cereale) contribute to the transcriptome. In total, 1954 and 1218 B-derived transcripts with an open reading frame were expressed in generative and vegetative tissues, respectively. In addition to B-derived transposable element transcripts, a high percentage of short transcripts without detectable similarity to known proteins and gene fragments from A chromosomes (As) were found, suggesting an ongoing gene erosion process. In vitro analysis of the A- and B-encoded AGO4B protein variants demonstrated that both possess RNA slicer activity. These data demonstrate unambiguously the presence of a functional AGO4B gene on Bs and that these Bs carry both functional protein coding genes and pseudogene copies. Thus, B-encoded genes may provide an additional level of gene control and complexity in combination with their related A-located genes. Hence, physiological effects, associated with the presence of Bs, may partly be explained by the activity of B-located (pseudo)genes. © 2016 IPK Gatersleben. New Phytologist © 2016 New Phytologist Trust.

  20. Evolutionary hierarchy of vertebrate-like heterotrimeric G protein families.

    PubMed

    Krishnan, Arunkumar; Mustafa, Arshi; Almén, Markus Sällman; Fredriksson, Robert; Williams, Michael J; Schiöth, Helgi B

    2015-10-01

    Heterotrimeric G proteins perform a crucial role as molecular switches controlling various cellular responses mediated by G protein-coupled receptor (GPCR) signaling pathway. Recent data have shown that the vertebrate-like G protein families are found across metazoans and their closest unicellular relatives. However, an overall evolutionary hierarchy of vertebrate-like G proteins, including gene family annotations and in particular mapping individual gene gain/loss events across diverse holozoan lineages is still incomplete. Here, with more expanded invertebrate taxon sampling, we have reconstructed phylogenetic trees for each of the G protein classes/families and provide a robust classification and hierarchy of vertebrate-like heterotrimeric G proteins. Our results further extend the evidence that the common ancestor (CA) of holozoans had at least five ancestral Gα genes corresponding to all major vertebrate Gα classes and contain a total of eight genes including two Gβ and one Gγ. Our results also indicate that the GNAI/O-like gene likely duplicated in the last CA of metazoans to give rise to GNAI- and GNAO-like genes, which are conserved across invertebrates. Moreover, homologs of GNB1-4 paralogon- and GNB5 family-like genes are found in most metazoans and that the unicellular holozoans encode two ancestral Gβ genes. Similarly, most bilaterian invertebrates encode two Gγ genes which include a representative of the GNG gene cluster and a putative homolog of GNG13. Interestingly, our results also revealed key evolutionary events such as the Drosophila melanogaster eye specific Gβ subunit that is found conserved in most arthropods and several previously unidentified species specific expansions within Gαi/o, Gαs, Gαq, Gα12/13 classes and the GNB1-4 paralogon. Also, we provide an overall proposed evolutionary scenario on the expansions of all G protein families in vertebrate tetraploidizations. Our robust classification/hierarchy is essential to further

  1. Leptospira Immunoglobulin-Like Proteins as a Serodiagnostic Marker for Acute Leptospirosis▿

    PubMed Central

    Croda, Julio; Ramos, João G. R.; Matsunaga, James; Queiroz, Adriano; Homma, Akira; Riley, Lee W.; Haake, David A.; Reis, Mitermayer G.; Ko, Albert I.

    2007-01-01

    There is an urgent need for improved diagnosis of leptospirosis, an emerging infectious disease which imparts a large disease burden in developing countries. We evaluated the use of Leptospira immunoglobulin (Ig)-like (Lig) proteins as a serodiagnostic marker for leptospirosis. Lig proteins have bacterial immunoglobulin-like (Big) tandem repeat domains, a moiety found in virulence factors in other pathogens. Sera from patients identified during urban outbreaks in Brazil reacted strongly with immunoblots of a recombinant fragment comprised of the second to sixth Big domains of LigB from L. interrogans serovar Copenhageni, the principal agent for transmission in this setting. Furthermore, the sera recognized an analogous LigB fragment derived from L. kirschneri serovar Grippotyphosa, a pathogenic serovar which is not endemic to the study area. The immunoblot assay detected anti-LigB IgM antibodies in sera from 92% (95% confidence interval, 85 to 96%) of patients during acute-phase leptospirosis. The assay had a sensitivity of 81% for sera from patients with less than 7 days of illness. Anti-LigB antibodies were found in sera from 57% of the patients who did not have detectable anti-whole-Leptospira responses as detected by IgM enzyme-linked immunosorbent assay and microagglutination test. The specificities of the assay were 93 to 100% and 90 to 97% among sera from healthy individuals and patients with diseases that have clinical presentations that overlap with those of leptospirosis, respectively. These findings indicate that the antibody response to this putative virulence determinant is a sensitive and specific marker for acute infection. The use of this marker may aid the prompt and timely diagnosis required to reduce the high mortality associated with severe forms of the disease. PMID:17360842

  2. Leptospira immunoglobulin-like proteins as a serodiagnostic marker for acute leptospirosis.

    PubMed

    Croda, Julio; Ramos, João G R; Matsunaga, James; Queiroz, Adriano; Homma, Akira; Riley, Lee W; Haake, David A; Reis, Mitermayer G; Ko, Albert I

    2007-05-01

    There is an urgent need for improved diagnosis of leptospirosis, an emerging infectious disease which imparts a large disease burden in developing countries. We evaluated the use of Leptospira immunoglobulin (Ig)-like (Lig) proteins as a serodiagnostic marker for leptospirosis. Lig proteins have bacterial immunoglobulin-like (Big) tandem repeat domains, a moiety found in virulence factors in other pathogens. Sera from patients identified during urban outbreaks in Brazil reacted strongly with immunoblots of a recombinant fragment comprised of the second to sixth Big domains of LigB from L. interrogans serovar Copenhageni, the principal agent for transmission in this setting. Furthermore, the sera recognized an analogous LigB fragment derived from L. kirschneri serovar Grippotyphosa, a pathogenic serovar which is not endemic to the study area. The immunoblot assay detected anti-LigB IgM antibodies in sera from 92% (95% confidence interval, 85 to 96%) of patients during acute-phase leptospirosis. The assay had a sensitivity of 81% for sera from patients with less than 7 days of illness. Anti-LigB antibodies were found in sera from 57% of the patients who did not have detectable anti-whole-Leptospira responses as detected by IgM enzyme-linked immunosorbent assay and microagglutination test. The specificities of the assay were 93 to 100% and 90 to 97% among sera from healthy individuals and patients with diseases that have clinical presentations that overlap with those of leptospirosis, respectively. These findings indicate that the antibody response to this putative virulence determinant is a sensitive and specific marker for acute infection. The use of this marker may aid the prompt and timely diagnosis required to reduce the high mortality associated with severe forms of the disease.

  3. Collagen-Like Proteins in Pathogenic E. coli Strains

    PubMed Central

    Ghosh, Neelanjana; McKillop, Thomas J.; Jowitt, Thomas A.; Howard, Marjorie; Davies, Heather; Holmes, David F.; Roberts, Ian S.; Bella, Jordi

    2012-01-01

    The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages. PMID:22701585

  4. Mechanism-based Proteomic Screening Identifies Targets of Thioredoxin-like Proteins*

    PubMed Central

    Nakao, Lia S.; Everley, Robert A.; Marino, Stefano M.; Lo, Sze M.; de Souza, Luiz E.; Gygi, Steven P.; Gladyshev, Vadim N.

    2015-01-01

    Thioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CXXC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nrx1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIF and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nrx1 could be detected, suggesting that their CXXC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CXXC motifs, providing important insights into thiol-based redox control of cellular processes. PMID:25561728

  5. A Coarse-Grained Protein Model in a Water-like Solvent

    NASA Astrophysics Data System (ADS)

    Sharma, Sumit; Kumar, Sanat K.; Buldyrev, Sergey V.; Debenedetti, Pablo G.; Rossky, Peter J.; Stanley, H. Eugene

    2013-05-01

    Simulations employing an explicit atom description of proteins in solvent can be computationally expensive. On the other hand, coarse-grained protein models in implicit solvent miss essential features of the hydrophobic effect, especially its temperature dependence, and have limited ability to capture the kinetics of protein folding. We propose a free space two-letter protein (``H-P'') model in a simple, but qualitatively accurate description for water, the Jagla model, which coarse-grains water into an isotropically interacting sphere. Using Monte Carlo simulations, we design protein-like sequences that can undergo a collapse, exposing the ``Jagla-philic'' monomers to the solvent, while maintaining a ``hydrophobic'' core. This protein-like model manifests heat and cold denaturation in a manner that is reminiscent of proteins. While this protein-like model lacks the details that would introduce secondary structure formation, we believe that these ideas represent a first step in developing a useful, but computationally expedient, means of modeling proteins.

  6. Does acute or habitual protein deprivation influence liking for monosodium glutamate?

    PubMed

    Masic, Una; Yeomans, Martin R

    2017-03-15

    The umami flavour generated by monosodium glutamate (MSG) has been proposed as the marker for the presence of protein in foods. As protein is the most closely regulated macronutrient in the diet, the present study addressed whether acute protein deprivation, habitual protein intake or a combination of the two influenced liking for the taste of MSG. 24 low-restraint male participants (mean age: 22; BMI: 23) consumed either their habitual breakfast (baseline), a low protein breakfast (breakfast meal with low protein milk and milkshake) or a high protein breakfast (breakfast meal with high protein milk and milkshake) on three different days, and then evaluated the acceptability of umami (MSG), salty (NaCl) or sweet (Acesulphame K) tastes at low or high concentrations in a soup context at lunchtime. Participants also completed a habitual protein intake questionnaire (39-item protein Food Frequency Questionnaire). Liking for all tastes was higher on the low than on the high protein day, and NaCl and Acesulphame K were liked less on both protein manipulation days when compared to the no added flavour control. Habitual protein intake was not related to liking for MSG stimuli alone but habitual high protein consumers rated a high concentration of MSG as more pleasant than any other taste when in protein deficit. Overall, these findings suggest that liking for high MSG concentrations may be moderated by nutritional need in high protein consumers. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  7. Molecular characterization and analysis of a novel protein disulfide isomerase-like protein of Eimeria tenella.

    PubMed

    Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

    2014-01-01

    Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC). BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells. These results

  8. Molecular Characterization and Analysis of a Novel Protein Disulfide Isomerase-Like Protein of Eimeria tenella

    PubMed Central

    Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

    2014-01-01

    Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC). BLAST analysis showed that the EtPDIL protein was 55–59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells. These results

  9. The 2.5 Å Structure of the Enterococcus Conjugation Protein TraM resembles VirB8 Type IV Secretion Proteins*

    PubMed Central

    Goessweiner-Mohr, Nikolaus; Grumet, Lukas; Arends, Karsten; Pavkov-Keller, Tea; Gruber, Christian C.; Gruber, Karl; Birner-Gruenberger, Ruth; Kropec-Huebner, Andrea; Huebner, Johannes; Grohmann, Elisabeth; Keller, Walter

    2013-01-01

    Conjugative plasmid transfer is the most important means of spreading antibiotic resistance and virulence genes among bacteria and therefore presents a serious threat to human health. The process requires direct cell-cell contact made possible by a multiprotein complex that spans cellular membranes and serves as a channel for macromolecular secretion. Thus far, well studied conjugative type IV secretion systems (T4SS) are of Gram-negative (G−) origin. Although many medically relevant pathogens (e.g., enterococci, staphylococci, and streptococci) are Gram-positive (G+), their conjugation systems have received little attention. This study provides structural information for the transfer protein TraM of the G+ broad host range Enterococcus conjugative plasmid pIP501. Immunolocalization demonstrated that the protein localizes to the cell wall. We then used opsonophagocytosis as a novel tool to verify that TraM was exposed on the cell surface. In these assays, antibodies generated to TraM recruited macrophages and enabled killing of pIP501 harboring Enteroccocus faecalis cells. The crystal structure of the C-terminal, surface-exposed domain of TraM was determined to 2.5 Å resolution. The structure, molecular dynamics, and cross-linking studies indicated that a TraM trimer acts as the biological unit. Despite the absence of sequence-based similarity, TraM unexpectedly displayed a fold similar to the T4SS VirB8 proteins from Agrobacterium tumefaciens and Brucella suis (G−) and to the transfer protein TcpC from Clostridium perfringens plasmid pCW3 (G+). Based on the alignments of secondary structure elements of VirB8-like proteins from mobile genetic elements and chromosomally encoded T4SS from G+ and G− bacteria, we propose a new classification scheme of VirB8-like proteins. PMID:23188825

  10. The hepatitis B virus large surface protein (LHBs) is a transcriptional activator.

    PubMed

    Hildt, E; Saher, G; Bruss, V; Hofschneider, P H

    1996-11-01

    It has been shown that a C-terminally truncated form of the middle-sized hepatitis B virus (HBV) surface protein (MHBst) functions as a transcriptional activator. This function is dependent on the cytosolic orientation of the N-terminal PreS2 domain of MHBst, but in the case of wild-type MHBs, the PreS2 domain is contranslationally translocated into the ER lumen. Recent reports demonstrated that the PreS2 domain of the large HBV surface protein (LHBs) initially remains on the cytosolic side of the ER membrane after translation. Therefore, the question arose as to whether the LHBs protein exhibits the same transcriptional activator function as MHBst. We show that LHBs, like MHBst, is indeed able to activate a variety of promoter elements. There is evidence for a PKC-dependent activation of AP-1 and NF-kappa B by LHBs. Downstream of the PKC the functionality of c-Raf-1 kinase is a prerequisite for LHBs-dependent activation of AP-1 and NF-kappa B since inhibition of c-Raf-1 kinase abolishes LHBs-dependent transcriptional activation of AP-1 and NF-kappa B.

  11. The low density lipoprotein receptor-related protein 1B retains beta-amyloid precursor protein at the cell surface and reduces amyloid-beta peptide production.

    PubMed

    Cam, Judy A; Zerbinatti, Celina V; Knisely, Jane M; Hecimovic, Silva; Li, Yonghe; Bu, Guojun

    2004-07-09

    The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a newly identified member of the LDL receptor family that shares high homology with the LDL receptor-related protein (LRP). LRP1B was originally described as a putative tumor suppressor in lung cancer cells; however, its expression profile in several regions of adult human brain suggests it may have additional functions in the central nervous system. Since LRP1B has overlapping ligand binding properties with LRP, we investigated whether LRP1B, like LRP, could interact with the beta-amyloid precursor protein (APP) and modulate its processing to amyloid-beta peptides (Abetas). Using an LRP1B minireceptor (mLRP1B4) generated to study the trafficking of LRP1B, we found that mLRP1B4 and APP form an immunoprecipitable complex. Furthermore mLRP1B4 bound and facilitated the degradation of a soluble isoform of APP containing a Kunitz proteinase inhibitor domain but not soluble APP lacking a Kunitz proteinase inhibitor domain. A functional consequence of mLRP1B4 expression was a significant accumulation of APP at the cell surface, which is likely related to the slow endocytosis rate of LRP1B. More importantly, mLRP1B4-expressing cells that accumulated cell surface APP produced less Abeta and secreted more soluble APP. These findings reveal that LRP1B is a novel binding partner of APP that functions to decrease APP processing to Abeta. Consequently LRP1B expression could function to protect against the pathogenesis of Alzheimer's disease.

  12. Human Tamm-Horsfall protein, a renal specific protein, serves as a cofactor in complement 3b degradation

    PubMed Central

    2017-01-01

    Tamm-Horsfall protein (THP) is an abundant urinary protein of renal origin. We hypothesize that THP can act as an inhibitor of complement since THP binds complement 1q (C1q) of the classical complement pathway, inhibits activation of this pathway, and is important in decreasing renal ischemia-reperfusion injury (a complement-mediated condition). In this study, we began to investigate whether THP interacted with the alternate complement pathway via complement factor H (CFH). THP was shown to bind CFH using ligand blots and in an ELISA (KD of 1 × 10−6 M). Next, the ability of THP to alter CFH’s normal action as it functioned as a cofactor in complement factor I (CFI)–mediated complement 3b (C3b) degradation was investigated. Unexpectedly, control experiments in these in vitro assays suggested that THP, without added CFH, could act as a cofactor in CFI-mediated C3b degradation. This cofactor activity was present equally in THP isolated from 10 different individuals. While an ELISA demonstrated small amounts of CFH contaminating THP samples, these CFH amounts were insufficient to explain the degree of cofactor activity present in THP. An ELISA demonstrated that THP directly bound C3b (KD ~ 5 × 10−8 m), a prerequisite for a protein acting as a C3b degradation cofactor. The cofactor activity of THP likely resides in the protein portion of THP since partially deglycosylated THP still retained cofactor activity. In conclusion, THP appears to participate directly in complement inactivation by its ability to act as a cofactor for C3b degradation, thus adding support to the hypothesis that THP might act as an endogenous urinary tract inhibitor of complement. PMID:28742158

  13. Expression and Purification of Neurotrophin-Elastin-Like Peptide Fusion Proteins for Neural Regeneration.

    PubMed

    Johnson, Tamina; Koria, Piyush

    2016-04-01

    Neural injuries such as spinal cord injuries, traumatic brain injuries, or nerve transection injuries pose a major health problem. Neurotrophins such as nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) have been shown to improve the outcome of neural injuries in several pre-clinical models, but their use in clinics is limited by the lack of a robust delivery system that enhances their bioavailability and half-life. We describe two fusion proteins comprising NGF or BDNF fused with elastin-like peptides (ELPs). The aim of this study was to investigate the biological activity of neurotrophin-ELP (N-ELP) fusion proteins via in vitro culture models. NGF and BDNF were cloned in front of an elastin-like polypeptide sequence V40C2. These proteins were expressed in bacteria as inclusion bodies. These fusion proteins underwent solubilization via 8 M urea and purification via inverse transition cycling (ITC). We measured the particle size and the effect of temperature on precipitated particles using dynamic light scattering (DLS). We used western blot analysis to confirm the specificity of NGF-ELP to tropomyosin receptor kinase A (TrkA) antibody and to confirm the specificity of BDNF-ELP to TrkB antibody. PC12 cells were used to perform a neurite outgrowth assay to determine the biological activity of NGF-ELP. Bioactivity of BDNF-ELP was ascertained via transfecting human epithelial kidney (HEK 293-T) cells to express the TrkB receptor. The proteins were successfully purified to high homogeneity by exploiting the phase transition property of ELPs and urea, which solubilize inclusion bodies. Using PC12 neurite outgrowth assay, we further demonstrated that the biological activity of NGF was retained in the fusion. Similarly, BDNF-ELP phosphorylated the TrkB receptor, suggesting the biological activity of BDNF was also retained in the fusion. We further show that owing to the phase transition property of ELPs in the fusion, these proteins self-assembled into

  14. IMPDHII Protein Inhibits Toll-like Receptor 2-mediated Activation of NF-κB*

    PubMed Central

    Toubiana, Julie; Rossi, Anne-Lise; Grimaldi, David; Belaidouni, Nadia; Chafey, Philippe; Clary, Guilhem; Courtine, Emilie; Pene, Frederic; Mira, Jean-Paul; Claessens, Yann-Erick; Chiche, Jean-Daniel

    2011-01-01

    Toll-like receptor 2 (TLR2) plays an essential role in innate immunity by the recognition of a large variety of pathogen-associated molecular patterns. It induces its recruitment to lipid rafts induces the formation of a membranous activation cluster necessary to enhance, amplify, and control downstream signaling. However, the exact composition of the TLR2-mediated molecular complex is unknown. We performed a proteomic analysis in lipopeptide-stimulated THP1 and found IMPDHII protein rapidly recruited to lipid raft. Whereas IMPDHII is essential for lymphocyte proliferation, its biologic function within innate immune signal pathways has not been established yet. We report here that IMPDHII plays an important role in the negative regulation of TLR2 signaling by modulating PI3K activity. Indeed, IMPDHII increases the phosphatase activity of SHP1, which participates to the inactivation of PI3K. PMID:21460227

  15. A Plasmodesmal Glycosyltransferase-Like Protein

    PubMed Central

    Zalepa-King, Lisa; Citovsky, Vitaly

    2013-01-01

    Plasmodesmata (Pd) are plant intercellular connections that represent cytoplasmic conduits for a wide spectrum of cellular transport cargoes, from ions to house-keeping proteins to transcription factors and RNA silencing signals; furthermore, Pd are also utilized by most plant viruses for their spread between host cells. Despite this central role of Pd in the plant life cycle, their structural and functional composition remains poorly characterized. In this study, we used a known Pd-associated calreticulin protein AtCRT1 as bait to isolate other Pd associated proteins in Arabidopsis thaliana. These experiments identified a beta-1,6-N-acetylglucosaminyl transferase-like enzyme (AtGnTL). Subcellular localization studies using confocal microscopy observed AtGnTL at Pd within living plant cells and demonstrated colocalization with a Pd callose-binding protein (AtPDCB1). That AtGnTL is resident in Pd was consistent with its localization within the plant cell wall following plasmolysis. Initial characterization of an Arabidopsis T-DNA insertional mutant in the AtGnTL gene revealed defects in seed germination and delayed plant growth. PMID:23469135

  16. The Tp0684 (MglB-2) Lipoprotein of Treponema pallidum: A Glucose-Binding Protein with Divergent Topology.

    PubMed

    Brautigam, Chad A; Deka, Ranjit K; Liu, Wei Z; Norgard, Michael V

    2016-01-01

    Treponema pallidum, the bacterium that causes syphilis, is an obligate human parasite. As such, it must acquire energy, in the form of carbon sources, from the host. There is ample evidence that the principal source of energy for this spirochete is D-glucose acquired from its environment, likely via an ABC transporter. Further, there is genetic evidence of a D-glucose chemotaxis system in T. pallidum. Both of these processes may be dependent on a single lipidated chemoreceptor: Tp0684, also called TpMglB-2 for its sequence homology to MglB of Escherichia coli. To broaden our understanding of this potentially vital protein, we determined a 2.05-Å X-ray crystal structure of a soluble form of the recombinant protein. Like its namesake, TpMglB-2 adopts a bilobed fold that is similar to that of the ligand-binding proteins (LBPs) of other ABC transporters. However, the protein has an unusual, circularly permuted topology. This feature prompted a series of biophysical studies that examined whether the protein's topological distinctiveness affected its putative chemoreceptor functions. Differential scanning fluorimetry and isothermal titration calorimetry were used to confirm that the protein bound D-glucose in a cleft between its two lobes. Additionally, analytical ultracentrifugation was employed to reveal that D-glucose binding is accompanied by a significant conformational change. TpMglB-2 thus appears to be fully functional in vitro, and given the probable central importance of the protein to T. pallidum's physiology, our results have implications for the viability and pathogenicity of this obligate human pathogen.

  17. [Structure and evolution of the eukaryotic FANCJ-like proteins].

    PubMed

    Wuhe, Jike; Zefeng, Wu; Sanhong, Fan; Xuguang, Xi

    2015-02-01

    The FANCJ-like protein family is a class of ATP-dependent helicases that can catalytically unwind duplex DNA along the 5'-3' direction. It is involved in the processes of DNA damage repair, homologous recombination and G-quadruplex DNA unwinding, and plays a critical role in maintaining genome integrity. In this study, we systemically analyzed FNACJ-like proteins from 47 eukaryotic species and discussed their sequences diversity, origin and evolution, motif organization patterns and spatial structure differences. Four members of FNACJ-like proteins, including XPD, CHL1, RTEL1 and FANCJ, were found in eukaryotes, but some of them were seriously deficient in most fungi and some insects. For example, the Zygomycota fungi lost RTEL1, Basidiomycota and Ascomycota fungi lost RTEL1 and FANCJ, and Diptera insect lost FANCJ. FANCJ-like proteins contain canonical motor domains HD1 and HD2, and the HD1 domain further integrates with three unique domains Fe-S, Arch and Extra-D. Fe-S and Arch domains are relatively conservative in all members of the family, but the Extra-D domain is lost in XPD and differs from one another in rest members. There are 7, 10 and 2 specific motifs found from the three unique domains respectively, while 5 and 12 specific motifs are found from HD1 and HD2 domains except the conserved motifs reported previously. By analyzing the arrangement pattern of these specific motifs, we found that RTEL1 and FANCJ are more closer and share two specific motifs Vb2 and Vc in HD2 domain, which are likely related with their G-quadruplex DNA unwinding activity. The evidence of evolution showed that FACNJ-like proteins were originated from a helicase, which has a HD1 domain inserted by extra Fe-S domain and Arch domain. By three continuous gene duplication events and followed specialization, eukaryotes finally possessed the current four members of FANCJ-like proteins.

  18. Assembly of Lipopolysaccharide in Escherichia coli Requires the Essential LapB Heat Shock Protein*

    PubMed Central

    Klein, Gracjana; Kobylak, Natalia; Lindner, Buko; Stupak, Anna; Raina, Satish

    2014-01-01

    Here, we describe two new heat shock proteins involved in the assembly of LPS in Escherichia coli, LapA and LapB (lipopolysaccharide assembly protein A and B). lapB mutants were identified based on an increased envelope stress response. Envelope stress-responsive pathways control key steps in LPS biogenesis and respond to defects in the LPS assembly. Accordingly, the LPS content in ΔlapB or Δ(lapA lapB) mutants was elevated, with an enrichment of LPS derivatives with truncations in the core region, some of which were pentaacylated and exhibited carbon chain polymorphism. Further, the levels of LpxC, the enzyme that catalyzes the first committed step of lipid A synthesis, were highly elevated in the Δ(lapA lapB) mutant. Δ(lapA lapB) mutant accumulated extragenic suppressors that mapped either to lpxC, waaC, and gmhA, or to the waaQ operon (LPS biosynthesis) and lpp (Braun's lipoprotein). Increased synthesis of either FabZ (3-R-hydroxymyristoyl acyl carrier protein dehydratase), slrA (novel RpoE-regulated non-coding sRNA), lipoprotein YceK, toxin HicA, or MurA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase) suppressed some of the Δ(lapA lapB) defects. LapB contains six tetratricopeptide repeats and, at the C-terminal end, a rubredoxin-like domain that was found to be essential for its activity. In pull-down experiments, LapA and LapB co-purified with LPS, Lpt proteins, FtsH (protease), DnaK, and DnaJ (chaperones). A specific interaction was also observed between WaaC and LapB. Our data suggest that LapB coordinates assembly of proteins involved in LPS synthesis at the plasma membrane and regulates turnover of LpxC, thereby ensuring balanced biosynthesis of LPS and phospholipids consistent with its essentiality. PMID:24722986

  19. Tight regulation between cell survival and programmed cell death in GBM stem-like cells by EGFR/GSK3b/PP2A signaling.

    PubMed

    Gürsel, Demirkan B; Banu, Matei A; Berry, Nicholas; Marongiu, Roberta; Burkhardt, Jan-Karl; Kobylarz, Keith; Kaplitt, Michael G; Rafii, Shahin; Boockvar, John A

    2015-01-01

    Malignant gliomas represent one of the most aggressive forms of cancer, displaying high mortality rates and limited treatment options. Specific subpopulations of cells residing in the tumor niche with stem-like characteristics have been postulated to initiate and maintain neoplasticity while resisting conventional therapies. The study presented here aims to define the role of glycogen synthase kinase 3 beta (GSK3b) in patient-derived glioblastoma (GBM) stem-like cell (GSC) proliferation, apoptosis and invasion. To evaluate the potential role of GSK3b in GBM, protein profiles from 68 GBM patients and 20 normal brain samples were analyzed for EGFR-mediated PI3kinase/Akt and GSK3b signaling molecules including protein phosphatase 2A (PP2A). To better understand the function of GSK3b in GBM, GSCs were isolated from GBM patient samples. Blocking GSK3b phosphorylation at Serine 9 attenuated cell proliferation while concomitantly stimulating apoptosis through activation of Caspase-3 in patient-derived GSCs. Increasing GSK3b protein content resulted in the inhibition of cell proliferation, colony formation and stimulated programmed cell death. Depleting GSK3b in GSCs down regulated PP2A. Furthermore, knocking down PP2A or blocking its activity by okadaic acid inactivated GSK3b by increasing GSK3b phosphorylation at Serine 9. Our data suggests that GSK3b may function as a regulator of apoptosis and tumorigenesis in GSCs. Therapeutic approaches targeting GSK3b in glioblastoma stem-like cells may be a useful addition to our current therapeutic armamentarium.

  20. A cryptochrome-like protein is involved in the regulation of photosynthesis genes in Rhodobacter sphaeroides.

    PubMed

    Hendrischk, Anne-Kathrin; Frühwirth, Sebastian Walter; Moldt, Julia; Pokorny, Richard; Metz, Sebastian; Kaiser, Gebhard; Jäger, Andreas; Batschauer, Alfred; Klug, Gabriele

    2009-11-01

    Blue light receptors belonging to the cryptochrome/photolyase family are found in all kingdoms of life. The functions of photolyases in repair of UV-damaged DNA as well as of cryptochromes in the light-dependent regulation of photomorphogenetic processes and in the circadian clock in plants and animals are well analysed. In prokaryotes, the only role of members of this protein family that could be demonstrated is DNA repair. Recently, we identified a gene for a cryptochrome-like protein (CryB) in the alpha-proteobacterium Rhodobacter sphaeroides. The protein lacks the typical C-terminal extension of cryptochromes, and is not related to the Cry DASH family. Here we demonstrate that CryB binds flavin adenine dinucleotide that can be photoreduced by blue light. CryB binds single-stranded DNA with very high affinity (K(d) approximately 10(-8) M) but double-stranded DNA and single-stranded RNA with far lower affinity (K(d) approximately 10(-6) M). Despite of that, no in vitro repair activity for pyrimidine dimers in single-stranded DNA could be detected. However, we show that CryB clearly affects the expression of genes for pigment-binding proteins and consequently the amount of photosynthetic complexes in R. sphaeroides. Thus, for the first time a role of a bacterial cryptochrome in gene regulation together with a biological function is demonstrated.

  1. Glutamine-rich toxic proteins GrtA, GrtB and GrtC together with the antisense RNA AsgR constitute a toxin-antitoxin-like system in Corynebacterium glutamicum.

    PubMed

    Maeda, Tomoya; Tanaka, Yuya; Inui, Masayuki

    2018-06-01

    The Corynebacterium glutamicum R grtA (cgR_2936), grtB (cgR_2934) and grtC (cgR_2933) genes were identified as paralogs encoding glutamine-rich toxic proteins. We also identified a new antisense small RNA AsgR (antisense sRNA for grtA) that overlaps the 3' end of the grtA gene. Single over-expressions of grtA, grtB and grtC resulted in complete inhibition of Escherichia coli cell growth. This growth was rescued by co-expression of AsgR. Similar effects were observed in C. glutamicum, although the toxicities of these proteins were moderate. Inhibition of AsgR transcription resulted in increased levels and prolonged half-lives of grtA, grtB and grtC mRNAs. We also found that the expression levels of grtA, grtB and grtC were increased in an RNase III deletion mutant. Primer extension analysis revealed the RNase III cleavage site to be in the 3' untranslated region (3'-UTR) of the grtA mRNA. The expression levels of grtA, grtB and grtC were increased after exposure to several stresses, including heat shock, treatment with penicillin G, lysozyme or H 2 O 2 . The deletions of grtABC and asgR genes resulted in decreased survival rate under several stresses. These results indicate that GrtABC and AsgR constitute a type I toxin-antitoxin-like system in C. glutamicum. © 2018 John Wiley & Sons Ltd.

  2. Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell-mediated cytotoxicity.

    PubMed

    Tuli, Amit; Thiery, Jerome; James, Ashley M; Michelet, Xavier; Sharma, Mahak; Garg, Salil; Sanborn, Keri B; Orange, Jordan S; Lieberman, Judy; Brenner, Michael B

    2013-12-01

    Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell-mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity.

  3. Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell–mediated cytotoxicity

    PubMed Central

    Tuli, Amit; Thiery, Jerome; James, Ashley M.; Michelet, Xavier; Sharma, Mahak; Garg, Salil; Sanborn, Keri B.; Orange, Jordan S.; Lieberman, Judy; Brenner, Michael B.

    2013-01-01

    Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell–mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity. PMID:24088571

  4. GW domains of the Listeria monocytogenes invasion protein InlB are SH3-like and mediate binding to host ligands

    PubMed Central

    Marino, Michael; Banerjee, Manidipa; Jonquières, Renaud; Cossart, Pascale; Ghosh, Partho

    2002-01-01

    InlB, a surface-localized protein of Listeria monocytogenes, induces phagocytosis in non-phagocytic mammalian cells by activating Met, a receptor tyrosine kinase. InlB also binds glycosaminoglycans and the protein gC1q-R, two additional host ligands implicated in invasion. We present the structure of InlB, revealing a highly elongated molecule with leucine-rich repeats that bind Met at one end, and GW domains that dissociably bind the bacterial surface at the other. Surprisingly, the GW domains are seen to resemble SH3 domains. Despite this, GW domains are unlikely to act as functional mimics of SH3 domains since their potential proline-binding sites are blocked or destroyed. However, we do show that the GW domains, in addition to binding glycosaminoglycans, bind gC1q-R specifically, and that this binding requires release of InlB from the bacterial surface. Dissociable attachment to the bacterial surface via the GW domains may be responsible for restricting Met activation to a small, localized area of the host cell and for coupling InlB-induced host membrane dynamics with bacterial proximity during invasion. PMID:12411480

  5. Tandem Fusion of Hepatitis B Core Antigen Allows Assembly of Virus-Like Particles in Bacteria and Plants with Enhanced Capacity to Accommodate Foreign Proteins

    PubMed Central

    Peyret, Hadrien; Gehin, Annick; Thuenemann, Eva C.; Blond, Donatienne; El Turabi, Aadil; Beales, Lucy; Clarke, Dean; Gilbert, Robert J. C.; Fry, Elizabeth E.; Stuart, David I.; Holmes, Kris; Stonehouse, Nicola J.; Whelan, Mike; Rosenberg, William; Lomonossoff, George P.; Rowlands, David J.

    2015-01-01

    The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody. PMID:25830365

  6. Mössbauer spectroscopy of the chloroplast-targeted DnaJ-like proteins CDJ3 and CDJ4

    NASA Astrophysics Data System (ADS)

    Auerbach, H.; Kalienkova, V.; Schroda, M.; Schünemann, V.

    2017-11-01

    The heat shock protein 70 (HSP70) in the chloroplast of Chlamydomonas reinhardtii, termed HSP70B, interacts with chloroplast-targeted DnaJ-like proteins (CDJs). In this work we focus on two CDJ co-chaperones (CDJ3 and CDJ4) of HSP70B which contain a redox-active Fe-S cluster (Dorn et al. Biochem. J. 427, 205 [2010]). We have performed Mössbauer spectroscopy on 57Fe enriched CDJ3 an) CDJ4. Our results indicate that both proteins have unusual [4Fe4S] 2+ clusters showing structural inhomogeneity of the two [Fe 2.5+-Fe 2.5+] pairs. The spectra have been analyzed by means of two components with δ-values characteristic for Fe 2.5+ centers, but the differences in Δ E Q indicate variations in their tetrahedral coordination spheres.

  7. Structure of Haze Forming Proteins in White Wines: Vitis vinifera Thaumatin-Like Proteins

    PubMed Central

    Marangon, Matteo; Van Sluyter, Steven C.; Waters, Elizabeth J.; Menz, Robert I.

    2014-01-01

    Grape thaumatin-like proteins (TLPs) play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. Different isoforms of TLPs have different hazing potential and aggregation behavior. Here we present the elucidation of the molecular structures of three grape TLPs that display different hazing potential. The three TLPs have very similar structures despite belonging to two different classes (F2/4JRU is a thaumatin-like protein while I/4L5H and H2/4MBT are VVTL1), and having different unfolding temperatures (56 vs. 62°C), with protein F2/4JRU being heat unstable and forming haze, while I/4L5H does not. These differences in properties are attributable to the conformation of a single loop and the amino acid composition of its flanking regions. PMID:25463627

  8. Structure of haze forming proteins in white wines: Vitis vinifera thaumatin-like proteins.

    PubMed

    Marangon, Matteo; Van Sluyter, Steven C; Waters, Elizabeth J; Menz, Robert I

    2014-01-01

    Grape thaumatin-like proteins (TLPs) play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. Different isoforms of TLPs have different hazing potential and aggregation behavior. Here we present the elucidation of the molecular structures of three grape TLPs that display different hazing potential. The three TLPs have very similar structures despite belonging to two different classes (F2/4JRU is a thaumatin-like protein while I/4L5H and H2/4MBT are VVTL1), and having different unfolding temperatures (56 vs. 62°C), with protein F2/4JRU being heat unstable and forming haze, while I/4L5H does not. These differences in properties are attributable to the conformation of a single loop and the amino acid composition of its flanking regions.

  9. Somatomedin-1 binding protein-3: insulin-like growth factor-1 binding protein-3, insulin-like growth factor-1 carrier protein.

    PubMed

    2003-01-01

    Somatomedin-1 binding protein-3 [insulin-like growth factor-1 binding protein-3, SomatoKine] is a recombinant complex of insulin-like growth factor-1 (rhIGF-1) and binding protein-3 (IGFBP-3), which is the major circulating somatomedin (insulin-like growth factor) binding protein; binding protein-3 regulates the delivery of somatomedin-1 to target tissues. Somatomedin-1 binding protein-3 has potential as replacement therapy for somatomedin-1 which may become depleted in indications such as major surgery, organ damage/failure and traumatic injury, resulting in catabolism. It also has potential for the treatment of osteoporosis; diseases associated with protein wasting including chronic renal failure, cachexia and severe trauma; and to attenuate cardiac dysfunction in a variety of disease states, including after severe burn trauma. Combined therapy with somatomedin-1 and somatomedin-1 binding protein-3 would prolong the duration of action of somatomedin-1 and would reduce or eliminate some of the undesirable effects associated with somatomedin-1 monotherapy. Somatomedin-1 is usually linked to binding protein-3 in the normal state of the body, and particular proteases clip them apart in response to stresses and release somatomedin-1 as needed. Therefore, somatomedin-1 binding protein-3 is a self-dosing system and SomatoKine would augment the natural supply of these linked compounds. Somatomedin-1 binding protein-3 was developed by Celtrix using its proprietary recombinant protein production technology. Subsequently, Celtrix was acquired by Insmed Pharmaceuticals on June 1 2000. Insmed and Avecia, UK, have signed an agreement for the manufacturing of SomatoKine and its components, IGF-1 and binding protein-3. CGMP clinical production of SomatoKine and its components will be done in Avecia's Advanced Biologics Centre, Billingham, UK, which manufactures recombinant-based medicines and vaccines with a capacity of up to 1000 litres. In 2003, manufacturing of SomatoKine is

  10. Functional characterization of Arabidopsis thaliana transthyretin-like protein.

    PubMed

    Pessoa, João; Sárkány, Zsuzsa; Ferreira-da-Silva, Frederico; Martins, Sónia; Almeida, Maria R; Li, Jianming; Damas, Ana M

    2010-02-18

    Arabidopsis thaliana transthyretin-like (TTL) protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. Moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase (N-terminal domain) and 5-hydroxyisourate (5-HIU) hydrolase (C-terminal domain). TTL is a member of the transthyretin-related protein family (TRP), which comprises a number of proteins with sequence homology to transthyretin (TTR) and the characteristic C-terminal sequence motif Tyr-Arg-Gly-Ser. TRPs are single domain proteins that form tetrameric structures with 5-HIU hydrolase activity. Experimental evidence is fundamental for knowing if TTL is a tetrameric protein, formed by the association of the 5-HIU hydrolase domains and, in this case, if the structural arrangement allows for OHCU decarboxylase activity. This work reports about the biochemical and functional characterization of TTL. The TTL gene was cloned and the protein expressed and purified for biochemical and functional characterization. The results show that TTL is composed of four subunits, with a moderately elongated shape. We also found evidence for 5-HIU hydrolase and OHCU decarboxylase activities in vitro, in the full-length protein. The Arabidopsis thaliana transthyretin-like (TTL) protein is a tetrameric bifunctional enzyme, since it has 5-HIU hydrolase and OHCU decarboxylase activities, which were simultaneously observed in vitro.

  11. Convulxin, a C-type lectin-like protein, inhibits HCASMCs functions via WAD-motif/integrin-αv interaction and NF-κB-independent gene suppression of GRO and IL-8.

    PubMed

    Shih, Chun-Ho; Chiang, Tin-Bin; Wang, Wen-Jeng

    2017-03-15

    Convulxin (CVX), a C-type lectin-like protein (CLPs), is a potent platelet aggregation inducer. To evaluate its potential applications in angiogenic diseases, the multimeric CVX were further explored on its mode of actions toward human coronary artery smooth muscle cells (HCASMCs). The N-terminus of β-chain of CVX (CVX-β) contains a putative disintegrin-like domain with a conserved motif upon the sequence comparison with other CLPs. Importantly, native CVX had no cytotoxic activity as examined by electrophoretic pattern. A Trp-Ala-Asp (WAD)-containing octapeptide, MTWADAEK, was thereafter synthesized and analyzed in functional assays. In the case of specific integrin antagonists as positive controls, the anti-angiogenic effects of CVX on HCASMCs were investigated by series of functional analyses. CVX showed to exhibit multiple inhibitory activities toward HCASMCs proliferation, adhesion and invasion with a dose- and integrin αvβ3-dependent fashion. However, the WAD-octapeptide exerting a minor potency could also work as an active peptidomimetic. In addition, flow cytometric analysis demonstrated both the intact CVX and synthetic peptide can specifically interact with integrin-αv on HCASMCs and CVX was shown to have a down-regulatory effect on the gene expression of CXC-chemokines, such as growth-related oncogene and interleukin-8. According to nuclear factor-κB (NF-κB) p65 translocation assay and Western blotting analysis, the NF-κB activation was not involved in the signaling events of CVX-induced gene expression. In conclusion, CVX may act as a disintegrin-like protein via the interactions of WAD-motif in CVX-β with integrin-αv on HCASMCs and it also is a gene suppressor with the ability to diminish the expression of two CXC-chemokines in a NF-κB-independent manner. Indeed, more extensive investigations are needed and might create a new avenue for the development of a novel angiostatic agent. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Big domains are novel Ca²+-binding modules: evidences from big domains of Leptospira immunoglobulin-like (Lig) proteins.

    PubMed

    Raman, Rajeev; Rajanikanth, V; Palaniappan, Raghavan U M; Lin, Yi-Pin; He, Hongxuan; McDonough, Sean P; Sharma, Yogendra; Chang, Yung-Fu

    2010-12-29

    Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca²+. Leptospiral immunoglobulin-like (Lig) proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big) domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca²+ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold. We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9(th) (Lig A9) and 10(th) repeats (Lig A10); and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon). All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca²+ with dissociation constants of 2-4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm), probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold. We demonstrate that the Lig are Ca²+-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca²+. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca²+-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca²+ binding.

  13. Phylogenetic and functional analyses of a plant protein related to human B-cell receptor-associated proteins.

    PubMed

    Atabekova, Anastasia K; Pankratenko, Anna V; Makarova, Svetlana S; Lazareva, Ekaterina A; Owens, Robert A; Solovyev, Andrey G; Morozov, Sergey Y

    2017-01-01

    Human B-cell receptor-associated protein BAP31 (HsBAP31) is the endoplasmic reticulum-resident protein involved in protein sorting and transport as well as pro-apoptotic signaling. Plant orthologs of HsBAP31 termed 'plant BAP-like proteins' (PBL proteins) have thus far remained unstudied. Recently, the PBL protein from Nicotiana tabacum (NtPBL) was identified as an interactor of Nt-4/1, a plant protein known to interact with plant virus movement proteins and affect the long-distance transport of potato spindle tuber viroid (PSTVd) via the phloem. Here, we have compared the sequences of PBL proteins and studied the biochemical properties of NtPBL. Analysis of a number of fully sequenced plant genomes revealed that PBL-encoding genes represent a small multigene family with up to six members per genome. Two conserved motifs were identified in the C-terminal region of PBL proteins. The NtPBL C-terminal hydrophilic region (NtPBL-C) was expressed in bacterial cells, purified, and used for analysis of its RNA binding properties in vitro. In gel shift experiments, NtPBL-C was found to bind several tested RNAs, showing the most efficient binding to microRNA precursors (pre-miRNA) and less efficient interaction with PSTVd. Mutational analysis suggested that NtPBL-C has a composite RNA-binding site, with two conserved lysine residues in the most C-terminal protein region being involved in binding of pre-miRNA but not PSTVd RNA. Virus-mediated transient expression of NtPBL-C in plants resulted in stunting and leaf malformation, developmental abnormalities similar to those described previously for blockage of miRNA biogenesis/function. We hypothesize that the NtPBL protein represents a previously undiscovered component of the miRNA pathway. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  14. Loss of calreticulin function decreases NFκB activity by stabilizing IκB protein.

    PubMed

    Massaeli, Hamid; Jalali, Shahrzad; Viswanathan, Divya; Mesaeli, Nasrin

    2014-11-01

    Transcription factor NFκB is activated by several processes including inflammation, endoplasmic-reticulum (ER) stress, increase in Akt signaling and enhanced proteasomal degradation. Calreticulin (CRT) is an ER Ca(2+)-binding chaperone that regulates many cellular processes. Gene-targeted deletion of CRT has been shown to induce ER stress that is accompanied with a significant increase in the proteasome activity. Loss of CRT function increases the resistance of CRT-deficient (crt-/-) cells to UV- and drug-induced apoptosis. Based on these reports we hypothesized that loss of CRT will activate NFκB signaling thus contributing to enhanced resistance to apoptosis. In contrast to our hypothesis, we observed a significant decrease in the basal transcriptional activity of NFκB in CRT-deficient cells. Treatment with lipopolysaccharide failed to increase the transcriptional activity of NFκB in the crt-/- cells to the same level as in the wt cells. Our data illustrate that the mechanism of decreased NFκB activity in CRT-deficient cells is mediated by a significant increase in IκB protein expression. Furthermore, we showed a significant increase in protein phosphatase 2A activity inhibition which resulted in decreased IκBα protein level in CRT-deficient cells. Based on our data we concluded that loss of CRT increases the stability of IκB protein thus reducing NFκB activity. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Glucagon Like Peptide-1 (GLP-1) Modulates OVA-Induced Airway Inflammation and Mucus Secretion Involving a Protein Kinase A (PKA)-Dependent Nuclear Factor-κB (NF-κB) Signaling Pathway in Mice.

    PubMed

    Zhu, Tao; Wu, Xiao-Ling; Zhang, Wei; Xiao, Min

    2015-08-26

    Asthma is a common chronic pulmonary inflammatory disease, featured with mucus hyper-secretion in the airway. Recent studies found that glucagon like peptide-1 (GLP-1) analogs, including liraglutide and exenatide, possessed a potent anti-inflammatory property through a protein kinase A (PKA)-dependent signaling pathway. Therefore, the aim of current study was to investigate the value of GLP-1 analog therapy liraglutide in airway inflammation and mucus secretion in a murine model of ovalbumin (OVA)-induced asthma, and its underlying molecular mechanism. In our study, BALB/c mice were sensitized and challenged by OVA to induce chronic asthma. Pathological alterations, the number of cells and the content of inflammatory mediators in bronchoalveolar lavage fluid (BALF), and mucus secretion were observed and measured. In addition, the mRNA and protein expression of E-selectin and MUC5AC were analyzed by qPCR and Western blotting. Then, the phosphorylation of PKA and nuclear factor-κB (NF-κB) p65 were also measured by Western blotting. Further, NF-κB p65 DNA binding activity was detected by ELISA. OVA-induced airway inflammation, airway mucus hyper-secretion, the up-regulation of E-selectin and MUC5AC were remarkably inhibited by GLP-1 in mice (all p < 0.01). Then, we also found that OVA-reduced phosphorylation of PKA, and OVA-enhanced NF-κB p65 activation and NF-κB p65 DNA binding activity were markedly improved by GLP-1 (all p < 0.01). Furthermore, our data also figured out that these effects of GLP-1 were largely abrogated by the PKA inhibitor H-89 (all p < 0.01). Taken together, our results suggest that OVA-induced asthma were potently ameliorated by GLP-1 possibly through a PKA-dependent inactivation of NF-κB in mice, indicating that GLP-1 analogs may be considered an effective and safe drug for the potential treatment of asthma in the future.

  16. Molecular Characterization, Structural Modeling, and Evaluation of Antimicrobial Activity of Basrai Thaumatin-Like Protein against Fungal Infection.

    PubMed

    Yasmin, Nusrat; Saleem, Mahjabeen; Naz, Mamoona; Gul, Roquyya; Rehman, Hafiz Muzzammel

    2017-01-01

    A thaumatin-like protein gene from Basrai banana was cloned and expressed in Escherichia coli . Amplified gene product was cloned into pTZ57R/T vector and subcloned into expression vector pET22b(+) and resulting pET22b-basrai TLP construct was introduced into E. coli BL21. Maximum protein expression was obtained at 0.7 mM IPTG concentration after 6 hours at 37°C. Western blot analysis showed the presence of approximately 20 kDa protein in induced cells. Basrai antifungal TLP was tried as pharmacological agent against fungal disease. Independently Basrai antifungal protein and amphotericin B exhibited their antifungal activity against A. fumigatus ; however combined effect of both agents maximized activity against the pathogen. Docking studies were performed to evaluate the antimicrobial potential of TLP against A. fumigatus by probing binding pattern of antifungal protein with plasma membrane ergosterol of targeted fungal strain. Ice crystallization primarily damages frozen food items; however addition of antifreeze proteins limits the growth of ice crystal in frozen foods. The potential of Basrai TLP protein, as an antifreezing agent, in controlling the ice crystal formation in frozen yogurt was also studied. The scope of this study ranges from cost effective production of pharmaceutics to antifreezing and food preserving agent as well as other real life applications.

  17. Molecular Characterization, Structural Modeling, and Evaluation of Antimicrobial Activity of Basrai Thaumatin-Like Protein against Fungal Infection

    PubMed Central

    Yasmin, Nusrat; Naz, Mamoona; Gul, Roquyya; Rehman, Hafiz Muzzammel

    2017-01-01

    A thaumatin-like protein gene from Basrai banana was cloned and expressed in Escherichia coli. Amplified gene product was cloned into pTZ57R/T vector and subcloned into expression vector pET22b(+) and resulting pET22b-basrai TLP construct was introduced into E. coli BL21. Maximum protein expression was obtained at 0.7 mM IPTG concentration after 6 hours at 37°C. Western blot analysis showed the presence of approximately 20 kDa protein in induced cells. Basrai antifungal TLP was tried as pharmacological agent against fungal disease. Independently Basrai antifungal protein and amphotericin B exhibited their antifungal activity against A. fumigatus; however combined effect of both agents maximized activity against the pathogen. Docking studies were performed to evaluate the antimicrobial potential of TLP against A. fumigatus by probing binding pattern of antifungal protein with plasma membrane ergosterol of targeted fungal strain. Ice crystallization primarily damages frozen food items; however addition of antifreeze proteins limits the growth of ice crystal in frozen foods. The potential of Basrai TLP protein, as an antifreezing agent, in controlling the ice crystal formation in frozen yogurt was also studied. The scope of this study ranges from cost effective production of pharmaceutics to antifreezing and food preserving agent as well as other real life applications. PMID:28875151

  18. Active G protein-coupled receptors (GPCR), matrix metalloproteinases 2/9 (MMP2/9), heparin-binding epidermal growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) are necessary for trenbolone acetate-induced alterations in protein turnover rate of fused bovine satellite cell cultures.

    PubMed

    Thornton, K J; Kamanga-Sollo, E; White, M E; Dayton, W R

    2016-06-01

    Trenbolone acetate (TBA), a testosterone analog, increases protein synthesis and decreases protein degradation in fused bovine satellite cell (BSC) cultures. However, the mechanism through which TBA alters these processes remains unknown. Recent studies indicate that androgens improve rate and extent of muscle growth through a nongenomic mechanism involving G protein-coupled receptors (GPCR), matrix metalloproteinases (MMP), heparin-binding epidermal growth factor (hbEGF), the epidermal growth factor receptor (EGFR), erbB2, and the insulin-like growth factor-1 receptor (IGF-1R). We hypothesized that TBA activates GPCR, resulting in activation of MMP2/9 that releases hbEGF, which activates the EGFR and/or erbB2. To determine whether the proposed nongenomic pathway is involved in TBA-mediated alterations in protein turnover, fused BSC cultures were treated with TBA in the presence or absence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R, and resultant protein synthesis and degradation rates were analyzed. Assays were replicated at least 9 times for each inhibitor experiment utilizing BSC cultures obtained from at least 3 different steers that had no previous exposure to steroid compounds. As expected, fused BSC cultures treated with 10 n TBA exhibited increased ( < 0.05) protein synthesis rates and decreased ( < 0.05) protein degradation rates when compared to control cultures. Treatment of fused BSC cultures with 10 n TBA in the presence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R suppressed ( < 0.05) TBA-mediated increases in protein synthesis rate. Alternatively, inhibition of GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R in the presence of 10 n TBA each had no ( > 0.05) effect on TBA-mediated decreases in protein degradation. However, inhibition of both EGFR and erbB2 in the presence of 10 n TBA resulted in decreased ( < 0.05) ability of TBA to decrease protein degradation rate. Additionally, fused BSC cultures treated with 10 n

  19. DNA-binding by Haemophilus influenzae and Escherichia coli YbaB, members of a widely-distributed bacterial protein family.

    PubMed

    Cooley, Anne E; Riley, Sean P; Kral, Keith; Miller, M Clarke; DeMoll, Edward; Fried, Michael G; Stevenson, Brian

    2009-07-13

    Genes orthologous to the ybaB loci of Escherichia coli and Haemophilus influenzae are widely distributed among eubacteria. Several years ago, the three-dimensional structures of the YbaB orthologs of both E. coli and H. influenzae were determined, revealing a novel "tweezer"-like structure. However, a function for YbaB had remained elusive, with an early study of the H. influenzae ortholog failing to detect DNA-binding activity. Our group recently determined that the Borrelia burgdorferi YbaB ortholog, EbfC, is a DNA-binding protein. To reconcile those results, we assessed the abilities of both the H. influenzae and E. coli YbaB proteins to bind DNA to which B. burgdorferi EbfC can bind. Both the H. influenzae and the E. coli YbaB proteins bound to tested DNAs. DNA-binding was not well competed with poly-dI-dC, indicating some sequence preferences for those two proteins. Analyses of binding characteristics determined that both YbaB orthologs bind as homodimers. Different DNA sequence preferences were observed between H. influenzae YbaB, E. coli YbaB and B. burgdorferi EbfC, consistent with amino acid differences in the putative DNA-binding domains of these proteins. Three distinct members of the YbaB/EbfC bacterial protein family have now been demonstrated to bind DNA. Members of this protein family are encoded by a broad range of bacteria, including many pathogenic species, and results of our studies suggest that all such proteins have DNA-binding activities. The functions of YbaB/EbfC family members in each bacterial species are as-yet unknown, but given the ubiquity of these DNA-binding proteins among Eubacteria, further investigations are warranted.

  20. Influence of heterologous MreB proteins on cell morphology of Bacillus subtilis.

    PubMed

    Schirner, Kathrin; Errington, Jeff

    2009-11-01

    The prokaryotic cytoskeletal protein MreB is thought to govern cell shape by positioning the cell wall synthetic apparatus at growth sites in the cell. In rod-shaped bacteria it forms helical filaments that run around the periphery of the rod during elongation. Gram-positive bacteria often contain more than one mreB gene. Bacillus subtilis has three mreB-like genes, mreB, mbl and mreBH, the first two of which have been shown to be essential under normal growth conditions. Expression of an mreB homologue from the closely related organism Bacillus licheniformis did not have any effect on cell growth or morphology. In contrast, expression of mreB from the phylogenetically more distant bacterium Clostridium perfringens produced shape defects and ultimately cell death, due to disruption of the endogenous MreB cytoskeleton. However, expression of either mreB(B. licheniformis) (mreB(Bl)) or mreB(C. perfringens) (mreB(Cp)) was sufficient to confer a rod shape to B. subtilis deleted for the three mreB isologues, supporting the idea that the three proteins have largely redundant functions in cell morphogenesis. Expression of mreBCD(Bl) could fully compensate for the loss of mreBCD in B. subtilis and led to the formation of rod-shaped cells. In contrast, expression of mreBCD(Cp) was not sufficient to confer a rod shape to B. subtilis Delta mreBCD, indicating that a complex of these three cell shape determinants is not enough for cell morphogenesis of B. subtilis.

  1. The effect of in silico targeting Pseudomonas aeruginosa patatin-like protein D, for immunogenic administration.

    PubMed

    Chirani, Alireza Salimi; Majidzadeh, Robabeh; Pouriran, Ramin; Heidary, Mohsen; Nasiri, Mohammad Javad; Gholami, Mehrdad; Goudarzi, Mehdi; Omrani, Vahid Fallah

    2018-02-05

    The vaccine candidates that have been introduced for immunization against Pseudomonas aeruginosa (P. aeruginosa) strains are quite diverse. In fact, there has been no proper antigen to act as an effective immunogenic substance against this ubiquitous pathogen in the market as yet. The complications caused by this bacterium due to the rapid development of multiple drug resistant strains have led to clinical problems worldwide. P. aeruginosa encodes many specific virulence elements that could be used as appropriate vaccine candidates. Type Vd secretion system, also known as patatin-like protein D, is a novel P. aeruginosa auto-transporter system. It is known that cellular or humoral immune responses could be elevated by chimeric proteins carrying epitopes. It has been recognized that in silico tools are essential for the evaluation of new chimeric antigens. In this study, we have considered the patatin-like protein D (PlpD) molecule from P. aeruginosa and predicted some immunogenic properties of this strong cytotoxic phospholipase A2 with the use of in-depth computational and immunoinformatics assessment methods The novelty of our in silico study is the modeling and assessment of both humoral and cellular immune potential against the PlpD molecule. The molecule was considered by multiple sequence alignment and homology valuation. The extremely conserved regions in the PlpD were predicted. The allergenic and physicochemical property predictions on the PlpD state that the molecule is a non-allergic and stable molecule. High-resolution secondary and tertiary conformations were created. Indeed, the B-cell and T-cell epitope mapping on the chimeric target protein confirmed that the engineered protein contained a tremendous number of both B-cell and T-cell corresponding epitopes. This investigation magnificently attained the chimeric molecule as being a potent lipolytic enzyme composed of numerous B-cell and T-cell restricted epitopes and could induce both humoral and

  2. Autoimmune limbic encephalitis with anti-contactin-associated protein-like 2 antibody secondary to pembrolizumab therapy.

    PubMed

    Brown, Michael P; Hissaria, Pravin; Hsieh, Amy Hc; Kneebone, Christopher; Vallat, Wilson

    2017-04-15

    Immune checkpoint inhibitors such as Pembrolizumab are used to restore antitumour immune response. It is important to be vigilant of immune mediated adverse events related to such therapy. We report a case of autoimmune limbic encephalitis with Contactin-Associated Protein-like 2 (CASPR2) antibody secondary to Pembrolizumab therapy for metastatic melanoma. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Strong Keratin-like Nanofibers Made of Globular Protein

    NASA Astrophysics Data System (ADS)

    Dror, Yael; Makarov, Vadim; Admon, Arie; Zussman, Eyal

    2008-03-01

    Protein fibers as elementary structural and functional elements in nature inspire the engineering of protein-based products for versatile bio-medical applications. We have recently used the electrospinning process to fabricate strong sub-micron fibers made solely of serum albumin (SA). This raises the challenges of turning a globular non-viscous protein solution into a polymer--like spinnable solution and producing keratin-like fibers enriched in inter S-S bridges. A stable spinning process was achieved by using SA solution in a rich trifluoroethanol-water mixture with β-mercaptoethanol. The breakage of the intra disulfide bridges, as identified by mass spectrometry, together with the denaturing alcohol, enabled a pronounced expansion of the protein. This in turn, affects the rheological properties of the solution. X-ray diffraction pattern of the fibers revealed equatorial orientation, indicating the alignment of structures along the fiber axis. The mechanical properties reached remarkable average values (Young's modulus of 1.6GPa, and max stress of 36MPa) as compared to other fibrous protein nanofibers. These significant results are attributed to both the alignment and inter disulfide bonds (cross linking) that were formed by spontaneous post-spinning oxidation.

  4. Gβ-Like CpcB Plays a Crucial Role for Growth and Development of Aspergillus nidulans and Aspergillus fumigatus

    PubMed Central

    Kong, Qing; Wang, Long; Liu, Zengran; Kwon, Nak-Jung; Kim, Sun Chang; Yu, Jae-Hyuk

    2013-01-01

    Growth, development, virulence and secondary metabolism in fungi are governed by heterotrimeric G proteins (G proteins). A Gβ-like protein called Gib2 has been shown to function as an atypical Gβ in Gpa1-cAMP signaling in Cryptococcus neoformans. We found that the previously reported CpcB (cross pathway control B) protein is the ortholog of Gib2 in Aspergillus nidulans and Aspergillus fumigatus. In this report, we further characterize the roles of CpcB in governing growth, development and toxigenesis in the two aspergilli. The deletion of cpcB results in severely impaired cellular growth, delayed spore germination, and defective asexual sporulation (conidiation) in both aspergilli. Moreover, CpcB is necessary for proper expression of the key developmental activator brlA during initiation and progression of conidiation in A. nidulans and A. fumigatus. Somewhat in accordance with the previous study, the absence of cpcB results in the formation of fewer, but not micro-, cleistothecia in A. nidulans in the presence of wild type veA, an essential activator of sexual development. However, the cpcB deletion mutant cleistothecia contain no ascospores, validating that CpcB is required for progression and completion of sexual fruiting including ascosporogenesis. Furthermore, unlike the canonical GβSfaD, CpcB is not needed for the biosynthesis of the mycotoxin sterigmatocystin (ST) as the cpcB null mutant produced reduced amount of ST with unaltered STC gene expression. However, in A. fumigatus, the deletion of cpcB results in the blockage of gliotoxin (GT) production. Further genetic analyses in A. nidulans indicate that CpcB may play a central role in vegetative growth, which might be independent of FadA- and GanB-mediated signaling. A speculative model summarizing the roles of CpcB in conjunction with SfaD in A. nidulans is presented. PMID:23936193

  5. Gβ-like CpcB plays a crucial role for growth and development of Aspergillus nidulans and Aspergillus fumigatus.

    PubMed

    Kong, Qing; Wang, Long; Liu, Zengran; Kwon, Nak-Jung; Kim, Sun Chang; Yu, Jae-Hyuk

    2013-01-01

    Growth, development, virulence and secondary metabolism in fungi are governed by heterotrimeric G proteins (G proteins). A Gβ-like protein called Gib2 has been shown to function as an atypical Gβ in Gpa1-cAMP signaling in Cryptococcus neoformans. We found that the previously reported CpcB (cross pathway control B) protein is the ortholog of Gib2 in Aspergillus nidulans and Aspergillus fumigatus. In this report, we further characterize the roles of CpcB in governing growth, development and toxigenesis in the two aspergilli. The deletion of cpcB results in severely impaired cellular growth, delayed spore germination, and defective asexual sporulation (conidiation) in both aspergilli. Moreover, CpcB is necessary for proper expression of the key developmental activator brlA during initiation and progression of conidiation in A. nidulans and A. fumigatus. Somewhat in accordance with the previous study, the absence of cpcB results in the formation of fewer, but not micro-, cleistothecia in A. nidulans in the presence of wild type veA, an essential activator of sexual development. However, the cpcB deletion mutant cleistothecia contain no ascospores, validating that CpcB is required for progression and completion of sexual fruiting including ascosporogenesis. Furthermore, unlike the canonical GβSfaD, CpcB is not needed for the biosynthesis of the mycotoxin sterigmatocystin (ST) as the cpcB null mutant produced reduced amount of ST with unaltered STC gene expression. However, in A. fumigatus, the deletion of cpcB results in the blockage of gliotoxin (GT) production. Further genetic analyses in A. nidulans indicate that CpcB may play a central role in vegetative growth, which might be independent of FadA- and GanB-mediated signaling. A speculative model summarizing the roles of CpcB in conjunction with SfaD in A. nidulans is presented.

  6. Mu-Like Prophage in Serogroup B Neisseria meningitidis Coding for Surface-Exposed Antigens

    PubMed Central

    Masignani, Vega; Giuliani, Marzia Monica; Tettelin, Hervé; Comanducci, Maurizio; Rappuoli, Rino; Scarlato, Vincenzo

    2001-01-01

    Sequence analysis of the genome of Neisseria meningititdis serogroup B revealed the presence of an ∼35-kb region inserted within a putative gene coding for an ABC-type transporter. The region contains 46 open reading frames, 29 of which are colinear and homologous to the genes of Escherichia coli Mu phage. Two prophages with similar organizations were also found in serogroup A meningococcus, and one was found in Haemophilus influenzae. Early and late phage functions are well preserved in this family of Mu-like prophages. Several regions of atypical nucleotide content were identified. These likely represent genes acquired by horizontal transfer. Three of the acquired genes are shown to code for surface-associated antigens, and the encoded proteins are able to induce bactericidal antibodies. PMID:11254622

  7. Can natural proteins designed with 'inverted' peptide sequences adopt native-like protein folds?

    PubMed

    Sridhar, Settu; Guruprasad, Kunchur

    2014-01-01

    We have carried out a systematic computational analysis on a representative dataset of proteins of known three-dimensional structure, in order to evaluate whether it would possible to 'swap' certain short peptide sequences in naturally occurring proteins with their corresponding 'inverted' peptides and generate 'artificial' proteins that are predicted to retain native-like protein fold. The analysis of 3,967 representative proteins from the Protein Data Bank revealed 102,677 unique identical inverted peptide sequence pairs that vary in sequence length between 5-12 and 18 amino acid residues. Our analysis illustrates with examples that such 'artificial' proteins may be generated by identifying peptides with 'similar structural environment' and by using comparative protein modeling and validation studies. Our analysis suggests that natural proteins may be tolerant to accommodating such peptides.

  8. Osthole protects against inflammation in a rat model of chronic kidney failure via suppression of nuclear factor-κB, transforming growth factor-β1 and activation of phosphoinositide 3-kinase/protein kinase B/nuclear factor (erythroid-derived 2)-like 2 signaling.

    PubMed

    Huang, Tao; Dong, Zhen

    2017-10-01

    Multiple pharmacological applications of osthole have been previously recognized, including antioxidant, anti-inflammatory, anti‑platelet and estrogenic effects, and resistance to pain. The present study investigated the protective effects of osthole against inflammation in a rat model of chronic kidney failure (CRF) and the underlying mechanisms. Osthole treatment with significantly reversed CRF‑induced changes in serum creatinine, calcium, phosphorus and blood urea nitrogen levels in CRF rats. Male Sprague‑Dawley rats (age, 8 weeks) received 200 mg/kg 2% adenine suspension to induce CRF in the model group. In the osthole‑treated group, rats received 200 mg/kg 2% adenine suspension + osthole (40 mg/kg, intravenously). The results revealed that treatment with osthole significantly inhibited CRF‑induced tumor necrosis factor‑α, interleukin (IL)‑8 and IL‑6 expression, and suppressed nuclear factor‑κB (NF‑κB) protein expression in CRF rats. Osthole treatment significantly attenuated the protein expression of transforming growth factor‑β1 (TGF‑β1), reduced monocyte chemoattractant protein‑1 activity and increased the phosphoinositide 3‑kinase (PI3K)/protein kinase B (Akt) ratio in CRF rats. These results suggested that osthole protects against inflammation in a rat model of CRF via suppression of NF‑κB and TGF‑β1, and activation of PI3K/Akt/nuclear factor (erythroid‑derived 2)‑like 2 signaling. Therefore, osthole may represent a potential therapeutic agent for the treatment of CRF.

  9. Eight RGS and RGS-like Proteins Orchestrate Growth, Differentiation, and Pathogenicity of Magnaporthe oryzae

    PubMed Central

    Zhang, Haifeng; Tang, Wei; Liu, Kaiyue; Huang, Qian; Zhang, Xin; Yan, Xia; Chen, Yue; Wang, Jiansheng; Qi, Zhongqiang; Wang, Zhengyi; Zheng, Xiaobo; Wang, Ping; Zhang, Zhengguang

    2011-01-01

    A previous study identified MoRgs1 as an RGS protein that negative regulates G-protein signaling to control developmental processes such as conidiation and appressorium formation in Magnaporthe oryzae. Here, we characterized additional seven RGS and RGS-like proteins (MoRgs2 through MoRgs8). We found that MoRgs1 and MoRgs4 positively regulate surface hydrophobicity, conidiation, and mating. Indifference to MoRgs1, MoRgs4 has a role in regulating laccase and peroxidase activities. MoRgs1, MoRgs2, MoRgs3, MoRgs4, MoRgs6, and MoRgs7 are important for germ tube growth and appressorium formation. Interestingly, MoRgs7 and MoRgs8 exhibit a unique domain structure in which the RGS domain is linked to a seven-transmembrane motif, a hallmark of G-protein coupled receptors (GPCRs). We have also shown that MoRgs1 regulates mating through negative regulation of Gα MoMagB and is involved in the maintenance of cell wall integrity. While all proteins appear to be involved in the control of intracellular cAMP levels, only MoRgs1, MoRgs3, MoRgs4, and MoRgs7 are required for full virulence. Taking together, in addition to MoRgs1 functions as a prominent RGS protein in M. oryzae, MoRgs4 and other RGS and RGS-like proteins are also involved in a complex process governing asexual/sexual development, appressorium formation, and pathogenicity. PMID:22241981

  10. A20-binding inhibitor of NF-κB (ABIN1) controls Toll-like receptor-mediated CCAAT/enhancer-binding protein β activation and protects from inflammatory disease.

    PubMed

    Zhou, Jingran; Wu, Ruiqiong; High, Anthony A; Slaughter, Clive A; Finkelstein, David; Rehg, Jerold E; Redecke, Vanessa; Häcker, Hans

    2011-11-01

    Toll-like receptors (TLRs) are expressed on innate immune cells and trigger inflammation upon detection of pathogens and host tissue injury. TLR-mediated proinflammatory-signaling pathways are counteracted by partially characterized anti-inflammatory mechanisms that prevent exaggerated inflammation and host tissue damage as manifested in inflammatory diseases. We biochemically identified a component of TLR-signaling pathways, A20-binding inhibitor of NF-κB (ABIN1), which recently has been linked by genome-wide association studies to the inflammatory diseases systemic lupus erythematosus and psoriasis. We generated ABIN1-deficient mice to study the function of ABIN1 in vivo and during TLR activation. Here we show that ABIN1-deficient mice develop a progressive, lupus-like inflammatory disease characterized by expansion of myeloid cells, leukocyte infiltrations in different parenchymatous organs, activated T and B lymphocytes, elevated serum Ig levels, and the appearance of autoreactive antibodies. Kidneys develop glomerulonephritis and proteinuria, reflecting tissue injury. Surprisingly, ABIN1-deficient macrophages exhibit normal regulation of major proinflammatory signaling pathways and mediators but show selective deregulation of the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) and its target genes, such as colony-stimulating factor 3 (Csf3), nitric oxide synthase, inducible (Nos2), and S100 calcium-binding protein A8 (S100a8). Their gene products, which are intimately linked to innate immune cell expansion (granulocyte colony-stimulating factor), cytotoxicity (inducible nitric oxide synthase), and host factor-derived inflammation (S100A8), may explain, at least in part, the inflammatory phenotype observed. Together, our data reveal ABIN1 as an essential anti-inflammatory component of TLR-signaling pathways that controls C/EBPβ activity.

  11. Filament formation of the Escherichia coli actin-related protein, MreB, in fission yeast.

    PubMed

    Srinivasan, Ramanujam; Mishra, Mithilesh; Murata-Hori, Maki; Balasubramanian, Mohan K

    2007-02-06

    Proteins structurally related to eukaryotic actins have recently been identified in several prokaryotic organisms. These actin-like proteins (MreB and ParM) and the deviant Walker A ATPase (SopA) play a key role in DNA segregation and assemble into polymers in vitro and in vivo. MreB also plays a role in cellular morphogenesis. Whereas the dynamic properties of eukaryotic actins have been extensively characterized, those of bacterial actins are only beginning to emerge. We have established the fission yeast Schizosaccharomyces pombe as a cellular model for the functional analysis of the Escherichia coli actin-related protein MreB. We show that MreB organizes into linear bundles that grow in a symmetrically bidirectional manner at 0.46 +/- 0.03 microm/min, with new monomers and/or oligomers being added along the entire length of the bundle. Organization of linear arrays was dependent on the ATPase activity of MreB, and their alignment along the cellular long axis was achieved by sliding along the cortex of the cylindrical part of the cell. The cell ends appeared to provide a physical barrier for bundle elongation. These experiments provide new insights into the mechanism of assembly and organization of the bacterial actin cytoskeleton.

  12. Chicken TREM-B1, an Inhibitory Ig-Like Receptor Expressed on Chicken Thrombocytes

    PubMed Central

    Turowski, Vanessa; Sperling, Beatrice; Hanczaruk, Matthias A.; Göbel, Thomas W.; Viertlboeck, Birgit C.

    2016-01-01

    Triggering receptors expressed on myeloid cells (TREM) form a multigene family of immunoregulatory Ig-like receptors and play important roles in the regulation of innate and adaptive immunity. In chickens, three members of the TREM family have been identified on chromosome 26. One of them is TREM-B1 which possesses two V-set Ig-domains, an uncharged transmembrane region and a long cytoplasmic tail with one ITSM and two ITIMs indicating an inhibitory function. We generated specific monoclonal antibodies by immunizing a Balb/c mouse with a TREM-B1-FLAG transfected BWZ.36 cell line and tested the hybridoma supernatants on TREM-B1-FLAG transfected 2D8 cells. We obtained two different antibodies specific for TREM-B1, mab 7E8 (mouse IgG1) and mab 1E9 (mouse IgG2a) which were used for cell surface staining. Single and double staining of different tissues, including whole blood preparations, revealed expression on thrombocytes. Next we investigated the biochemical properties of TREM-B1 by using the specific mab 1E9 for immunoprecipitation of either lysates of surface biotinylated peripheral blood cells or stably transfected 2D8 cells. Staining with streptavidin coupled horse radish peroxidase revealed a glycosylated monomeric protein of about 50 kDa. Furthermore we used the stably transfected 2D8 cell line for analyzing the cytoplasmic tyrosine based signaling motifs. After pervanadate treatment, we detected phosphorylation of the tyrosine residues and subsequent recruitment of the tyrosine specific protein phosphatase SHP-2, indicating an inhibitory potential for TREM-B1. We also showed the inhibitory effect of TREM-B1 in chicken thrombocytes using a CD107 degranulation assay. Crosslinking of TREM-B1 on activated primary thrombocytes resulted in decreased CD107 surface expression of about 50–70%. PMID:26967520

  13. Protein A-like activity and streptococcal cross-reactions.

    PubMed Central

    Kingston, D

    1981-01-01

    Recognition of the protein A-like activity of some strains of group A streptococci has thrown doubt on much previous work suggesting antigenic cross-reactions between these streptococci and mammalian tissues. The strains used in our previous studies have now been examined by the mixed reverse passive antiglobulin reaction (MRPAH) for the 'non-specific' absorption of purified Fc portion of human IgG. They were found to have only traces of activity. The strain of Staphylococcus aureus used to control 'non-specific' absorption by bacterial cell walls was strongly positive. Protein A-like material as detected in this way was not therefore responsible for our earlier results. PMID:7039880

  14. Immunolocalization of integrin-like proteins in Arabidopsis and Chara

    NASA Technical Reports Server (NTRS)

    Katembe, W. J.; Swatzell, L. J.; Makaroff, C. A.; Kiss, J. Z.

    1997-01-01

    Integrins are a large family of integral plasma membrane proteins that link the extracellular matrix to the cytoskeleton in animal cells. As a first step in determining if integrin-like proteins are involved in gravitropic signal transduction pathways, we have used a polyclonal antibody against the chicken beta1 integrin subunit in western blot analyses and immunofluorescence microscopy to gain information on the size and location of these proteins in plants. Several different polypeptides are recognized by the anti-integrin antibody in roots and shoots of Arabidopsis and in the internodal cells and rhizoids of Chara. These cross-reactive polypeptides are associated with cellular membranes, a feature which is consistent with the known location of integrins in animal systems. In immunofluorescence studies of Arabidopsis roots, a strong signal was obtained from labeling integrin-like proteins in root cap cells, and there was little or no immunolabel in other regions of the root tip. While the antibody stained throughout Chara rhizoids, the highest density of immunolabel was at the tip. Thus, in both Arabidopsis roots and Chara rhizoids, the sites of gravity perception/transduction appear to be enriched in integrin-like molecules.

  15. HnRNP-like proteins as post-transcriptional regulators.

    PubMed

    Yeap, Wan-Chin; Namasivayam, Parameswari; Ho, Chai-Ling

    2014-10-01

    Plant cells contain a diverse repertoire of RNA-binding proteins (RBPs) that coordinate a network of post-transcriptional regulation. RBPs govern diverse developmental processes by modulating the gene expression of specific transcripts. Recent gene annotation and RNA sequencing clearly showed that heterogeneous nuclear ribonucleoprotein (hnRNP)-like proteins which form a family of RBPs, are also expressed in higher plants and serve specific plant functions. In addition to their involvement in post-transcriptional regulation from mRNA capping to translation, they are also involved in telomere regulation, gene silencing and regulation in chloroplast. Here, we review the involvement of plant hnRNP-like proteins in post-transcription regulation of RNA processes and their functional roles in control of plant developmental processes especially plant-specific functions including flowering, chloroplastic-specific mRNA regulation, long-distance phloem transportation and plant responses to environmental stresses. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. Protein patterns of black fungi under simulated Mars-like conditions

    NASA Astrophysics Data System (ADS)

    Zakharova, Kristina; Marzban, Gorji; de Vera, Jean-Pierre; Lorek, Andreas; Sterflinger, Katja

    2014-05-01

    Two species of microcolonial fungi - Cryomyces antarcticus and Knufia perforans - and a species of black yeasts-Exophiala jeanselmei - were exposed to thermo-physical Mars-like conditions in the simulation chamber of the German Aerospace Center. In this study the alterations at the protein expression level from various fungi species under Mars-like conditions were analyzed for the first time using 2D gel electrophoresis. Despite of the expectations, the fungi did not express any additional proteins under Mars simulation that could be interpreted as stress induced HSPs. However, up-regulation of some proteins and significant decreasing of protein number were detected within the first 24 hours of the treatment. After 4 and 7 days of the experiment protein spot number was increased again and the protein patterns resemble the protein patterns of biomass from normal conditions. It indicates the recovery of the metabolic activity under Martian environmental conditions after one week of exposure.

  17. Protein patterns of black fungi under simulated Mars-like conditions

    PubMed Central

    Zakharova, Kristina; Marzban, Gorji; de Vera, Jean-Pierre; Lorek, Andreas; Sterflinger, Katja

    2014-01-01

    Two species of microcolonial fungi – Cryomyces antarcticus and Knufia perforans - and a species of black yeasts–Exophiala jeanselmei - were exposed to thermo-physical Mars-like conditions in the simulation chamber of the German Aerospace Center. In this study the alterations at the protein expression level from various fungi species under Mars-like conditions were analyzed for the first time using 2D gel electrophoresis. Despite of the expectations, the fungi did not express any additional proteins under Mars simulation that could be interpreted as stress induced HSPs. However, up-regulation of some proteins and significant decreasing of protein number were detected within the first 24 hours of the treatment. After 4 and 7 days of the experiment protein spot number was increased again and the protein patterns resemble the protein patterns of biomass from normal conditions. It indicates the recovery of the metabolic activity under Martian environmental conditions after one week of exposure. PMID:24870977

  18. Protein patterns of black fungi under simulated Mars-like conditions.

    PubMed

    Zakharova, Kristina; Marzban, Gorji; de Vera, Jean-Pierre; Lorek, Andreas; Sterflinger, Katja

    2014-05-29

    Two species of microcolonial fungi - Cryomyces antarcticus and Knufia perforans - and a species of black yeasts-Exophiala jeanselmei - were exposed to thermo-physical Mars-like conditions in the simulation chamber of the German Aerospace Center. In this study the alterations at the protein expression level from various fungi species under Mars-like conditions were analyzed for the first time using 2D gel electrophoresis. Despite of the expectations, the fungi did not express any additional proteins under Mars simulation that could be interpreted as stress induced HSPs. However, up-regulation of some proteins and significant decreasing of protein number were detected within the first 24 hours of the treatment. After 4 and 7 days of the experiment protein spot number was increased again and the protein patterns resemble the protein patterns of biomass from normal conditions. It indicates the recovery of the metabolic activity under Martian environmental conditions after one week of exposure.

  19. The Drosophila muscle LIM protein, Mlp84B, cooperates with D-titin to maintain muscle structural integrity.

    PubMed

    Clark, Kathleen A; Bland, Jennifer M; Beckerle, Mary C

    2007-06-15

    Muscle LIM protein (MLP) is a cytoskeletal LIM-only protein expressed in striated muscle. Mutations in human MLP are associated with cardiomyopathy; however, the molecular mechanism by which MLP functions is not established. A Drosophila MLP homolog, mlp84B, displays many of the same features as the vertebrate protein, illustrating the utility of the fly for the study of MLP function. Animals lacking Mlp84B develop into larvae with a morphologically intact musculature, but the mutants arrest during pupation with impaired muscle function. Mlp84B displays muscle-specific expression and is a component of the Z-disc and nucleus. Preventing nuclear retention of Mlp84B does not affect its function, indicating that Mlp84B site of action is likely to be at the Z-disc. Within the Z-disc, Mlp84B is colocalized with the N-terminus of D-titin, a protein crucial for sarcomere organization and stretch mechanics. The mlp84B mutants phenotypically resemble weak D-titin mutants. Furthermore, reducing D-titin activity in the mlp84B background leads to pronounced enhancement of the mlp84B muscle defects and loss of muscle structural integrity. The genetic interactions between mlp84B and D-titin reveal a role for Mlp84B in maintaining muscle structural integrity that was not obvious from analysis of the mlp84B mutants themselves, and suggest Mlp84B and D-titin cooperate to stabilize muscle sarcomeres.

  20. Big Domains Are Novel Ca2+-Binding Modules: Evidences from Big Domains of Leptospira Immunoglobulin-Like (Lig) Proteins

    PubMed Central

    Palaniappan, Raghavan U. M.; Lin, Yi-Pin; He, Hongxuan; McDonough, Sean P.; Sharma, Yogendra; Chang, Yung-Fu

    2010-01-01

    Background Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca2+. Leptospiral immunoglobulin-like (Lig) proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big) domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca2+ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold. Principal Findings We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9th (Lig A9) and 10th repeats (Lig A10); and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon). All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca2+ with dissociation constants of 2–4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm), probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold. Conclusions We demonstrate that the Lig are Ca2+-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca2+. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca2+-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca2+ binding. PMID:21206924

  1. A parapoxviral virion protein targets the retinoblastoma protein to inhibit NF-κB signaling

    PubMed Central

    Nagendraprabhu, Ponnuraj; Khatiwada, Sushil; Chaulagain, Sabal

    2017-01-01

    Poxviruses have evolved multiple strategies to subvert signaling by Nuclear Factor κB (NF-κB), a crucial regulator of host innate immune responses. Here, we describe an orf virus (ORFV) virion-associated protein, ORFV119, which inhibits NF-κB signaling very early in infection (≤ 30 min post infection). ORFV119 NF-κB inhibitory activity was found unimpaired upon translation inhibition, suggesting that virion ORFV119 alone is responsible for early interference in signaling. A C-terminal LxCxE motif in ORFV119 enabled the protein to interact with the retinoblastoma protein (pRb) a multifunctional protein best known for its tumor suppressor activity. Notably, experiments using a recombinant virus containing an ORFV119 mutation which abrogates its interaction with pRb together with experiments performed in cells lacking or with reduced pRb levels indicate that ORFV119 mediated inhibition of NF-κB signaling is largely pRb dependent. ORFV119 was shown to inhibit IKK complex activation early in infection. Consistent with IKK inhibition, ORFV119 also interacted with TNF receptor associated factor 2 (TRAF2), an adaptor protein recruited to signaling complexes upstream of IKK in infected cells. ORFV119-TRAF2 interaction was enhanced in the presence of pRb, suggesting that ORFV119-pRb complex is required for efficient interaction with TRAF2. Additionally, transient expression of ORFV119 in uninfected cells was sufficient to inhibit TNFα-induced IKK activation and NF-κB signaling, indicating that no other viral proteins are required for the effect. Infection of sheep with ORFV lacking the ORFV119 gene led to attenuated disease phenotype, indicating that ORFV119 contributes to virulence in the natural host. ORFV119 represents the first poxviral protein to interfere with NF-κB signaling through interaction with pRb. PMID:29244863

  2. Identification of the likely translational start of Mycobacterium tuberculosis GyrB.

    PubMed

    Karkare, Shantanu; Brown, Amanda C; Parish, Tanya; Maxwell, Anthony

    2013-07-15

    Bacterial DNA gyrase is a validated target for antibacterial chemotherapy. It consists of two subunits, GyrA and GyrB, which form an A₂B₂ complex in the active enzyme. Sequence alignment of Mycobacterium tuberculosis GyrB with other bacterial GyrBs predicts the presence of 40 potential additional amino acids at the GyrB N-terminus. There are discrepancies between the M. tuberculosis GyrB sequences retrieved from different databases, including sequences annotated with or without the additional 40 amino acids. This has resulted in differences in the GyrB sequence numbering that has led to the reporting of previously known fluoroquinolone-resistance mutations as novel mutations. We have expressed M. tuberculosis GyrB with and without the extra 40 amino acids in Escherichia coli and shown that both can be produced as soluble, active proteins. Supercoiling and other assays of the two proteins show no differences, suggesting that the additional 40 amino acids have no effect on the enzyme in vitro. RT-PCR analysis of M. tuberculosis mRNA shows that transcripts that could yield both the longer and shorter protein are present. However, promoter analysis showed that only the promoter elements leading to the shorter GyrB (lacking the additional 40 amino acids) had significant activity. We conclude that the most probable translational start codon for M. tuberculosis GyrB is GTG (Val) which results in translation of a protein of 674 amino acids (74 kDa).

  3. Intrinsic disorder in the partitioning protein KorB persists after co-operative complex formation with operator DNA and KorA.

    PubMed

    Hyde, Eva I; Callow, Philip; Rajasekar, Karthik V; Timmins, Peter; Patel, Trushar R; Siligardi, Giuliano; Hussain, Rohanah; White, Scott A; Thomas, Christopher M; Scott, David J

    2017-08-30

    The ParB protein, KorB, from the RK2 plasmid is required for DNA partitioning and transcriptional repression. It acts co-operatively with other proteins, including the repressor KorA. Like many multifunctional proteins, KorB contains regions of intrinsically disordered structure, existing in a large ensemble of interconverting conformations. Using NMR spectroscopy, circular dichroism and small-angle neutron scattering, we studied KorB selectively within its binary complexes with KorA and DNA, and within the ternary KorA/KorB/DNA complex. The bound KorB protein remains disordered with a mobile C-terminal domain and no changes in the secondary structure, but increases in the radius of gyration on complex formation. Comparison of wild-type KorB with an N-terminal deletion mutant allows a model of the ensemble average distances between the domains when bound to DNA. We propose that the positive co-operativity between KorB, KorA and DNA results from conformational restriction of KorB on binding each partner, while maintaining disorder. © 2017 The Author(s).

  4. Hepatitis B virus X protein modulates peroxisome proliferator-activated receptor gamma through protein-protein interaction.

    PubMed

    Choi, Youn-Hee; Kim, Ha-il; Seong, Je Kyung; Yu, Dae-Yeul; Cho, Hyeseong; Lee, Mi-Ock; Lee, Jae Myun; Ahn, Yong-ho; Kim, Se Jong; Park, Jeon Han

    2004-01-16

    Ligand activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been reported to induce growth inhibition and apoptosis in various cancers including hepatocellular carcinoma (HCC). However, the effect of hepatitis B virus X protein (HBx) on PPARgamma activation has not been characterized in hepatitis B virus (HBV)-associated HCC. Herein, we demonstrated that HBx counteracted growth inhibition caused by PPARgamma ligand in HBx-associated HCC cells. We found that HBx bound to DNA binding domain of PPARgamma and HBx/PPARgamma interaction blocked nuclear localization and binding to recognition site of PPARgamma. HBx significantly suppressed a PPARgamma-mediated transactivation. These results suggest that HBx modulates PPARgamma function through protein-protein interaction.

  5. LeCTR2, a CTR1-like protein kinase from tomato, plays a role in ethylene signalling, development and defence

    PubMed Central

    Lin, Zhefeng; Alexander, Lucy; Hackett, Rachel; Grierson, Don

    2008-01-01

    Arabidopsis AtCTR1 is a Raf-like protein kinase that interacts with ETR1 and ERS and negatively regulates ethylene responses. In tomato, several CTR1-like proteins could perform this role. We have characterized LeCTR2, which has similarity to AtCTR1 and also to EDR1, a CTR1-like Arabidopsis protein involved in defence and stress responses. Protein–protein interactions between LeCTR2 and six tomato ethylene receptors indicated that LeCTR2 interacts preferentially with the subfamily I ETR1-type ethylene receptors LeETR1 and LeETR2, but not the NR receptor or the subfamily II receptors LeETR4, LeETR5 and LeETR6. The C-terminus of LeCTR2 possesses serine/threonine kinase activity and is capable of auto-phosphorylation and phosphorylation of myelin basic protein in vitro. Overexpression of the LeCTR2 N-terminus in tomato resulted in altered growth habit, including reduced stature, loss of apical dominance, highly branched inflorescences and fruit trusses, indeterminate shoots in place of determinate flowers, and prolific adventitious shoot development from the rachis or rachillae of the leaves. Expression of the ethylene-responsive genes E4 and chitinase B was upregulated in transgenic plants, but ethylene production and the level of mRNA for the ethylene biosynthetic gene ACO1 was unaffected. The leaves and fruit of transgenic plants also displayed enhanced susceptibility to infection by the fungal pathogen Botrytis cinerea, which was associated with much stronger induction of pathogenesis-related genes such as PR1b1 and chitinase B compared with the wild-type. The results suggest that LeCTR2 plays a role in ethylene signalling, development and defence, probably through its interactions with the ETR1-type ethylene receptors of subfamily I. PMID:18346193

  6. A germin-like protein with superoxide dismutase activity in pea nodules with high protein sequence identity to a putative rhicadhesin receptor.

    PubMed

    Gucciardo, Sébastian; Wisniewski, Jean-Pierre; Brewin, Nicholas J; Bornemann, Stephen

    2007-01-01

    The cDNAs encoding three germin-like proteins (PsGER1, PsGER2a, and PsGER2b) were isolated from Pisum sativum. The coding sequence of PsGER1 transiently expressed in tobacco leaves gave a protein with superoxide dismutase activity but no detectable oxalate oxidase activity according to in-gel activity stains. The transient expression of wheat germin gf-2.8 oxalate oxidase showed oxalate oxidase but no superoxide dismutase activity under the same conditions. The superoxide dismutase activity of PsGER1 was resistant to high temperature, denaturation by detergent, and high concentrations of hydrogen peroxide. In salt-stressed pea roots, a heat-resistant superoxide dismutase activity was observed with an electrophoretic mobility similar to that of the PsGER1 protein, but this activity was below the detection limit in non-stressed or H(2)O(2)-stressed pea roots. Oxalate oxidase activity was not detected in either pea roots or nodules. Following in situ hybridization in developing pea nodules, PsGER1 transcript was detected in expanding cells just proximal to the meristematic zone and also in the epidermis, but to a lesser extent. PsGER1 is the first known germin-like protein with superoxide dismutase activity to be associated with nodules. It shared protein sequence identity with the N-terminal sequence of a putative plant receptor for rhicadhesin, a bacterial attachment protein. However, its primary location in nodules suggests functional roles other than as a rhicadhesin receptor required for the first stage of bacterial attachment to root hairs.

  7. Antigenicity analysis of human parvovirus B19-VP1u protein in the induction of anti-phospholipid syndrome.

    PubMed

    Lin, Chun-Yu; Chiu, Chun-Ching; Cheng, Ju; Lin, Chia-Yun; Shi, Ya-Fang; Tsai, Chun-Chou; Tzang, Bor-Show; Hsu, Tsai-Ching

    2018-01-01

    Mounting evidence suggests a connection between human parvovirus B19 (B19) and autoimmune diseases, and especially an association between the B19-VP1 unique region (VP1u) and anti-phospholipid syndrome (APS). However, little is known about the antigenicity of B19-VP1u in the induction of APS-like syndrome. To elucidate the antigenicity of B19-VP1u in the induction of APS, N-terminal truncated B19-VP1u (tVP1u) proteins were prepared to immunize Balb/c mice to generate antibodies against B19-tVP1u proteins. The secreted phospholipase A2 (sPLA2) activities and binding specificity of mice anti-B19-tVP1u antibodies with cardiolipin (CL) and beta-2-glycoprotein I (β2GPI) were evaluated by performing immunoblot, ELISA and absorption experiments. A mice model of passively induced APS was adopted. Although sPLA2 activities were identified in all B19-tVP1u proteins, only amino acid residues 61-227 B19-tVP1u exhibited a higher sPLA2 activity. Autoantibodies against CL and β2GPI exhibited binding activities with all B19-tVP1u proteins. IgG that was purified from mice that had been immunized with amino acid residues 21-227 to 121-227 B19-tVP1u proteins exhibited significantly higher binding activity with CL. IgG that was purified from mice that had been immunized with amino acid residues 21-227, 31-227, 82-227 and 91-227 B19-tVP1u proteins exhibited significantly higher binding activity with β2GPI. Accordingly, significantly higher binding inhibition of CL was detected in the presence of amino acid residues 61-227 and 101-227 B19-tVP1u. Significantly higher binding inhibition of β2GPI was detected in the presence of amino acid residues 21-227, 31-227, 82-227 and 91-227 B19-tVP1u. The mice that received amino acid residues 31-227 or 61-227 anti-tB19-VP1u IgG revealed significant thrombocytopenia and those that received amino acid residues 21-227, 31-227, 61-227, 71-227, 82-227, 91-227, 101-227 or 114-227 anti-tB19-VP1u IgG exhibited significantly prolonged aPTT. These

  8. Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways.

    PubMed

    Yankee, Thomas M; Solow, Sasha A; Draves, Kevin D; Clark, Edward A

    2003-01-01

    Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.

  9. A Novel Kinesin-Like Protein with a Calmodulin-Binding Domain

    NASA Technical Reports Server (NTRS)

    Wang, W.; Takezawa, D.; Narasimhulu, S. B.; Reddy, A. S. N.; Poovaiah, B. W.

    1996-01-01

    Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with S-35-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca(2+)-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCKI is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca(2+)/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.

  10. Expression and localization of a novel phosducin-like protein from amphioxus Branchiostoma belcheri

    NASA Astrophysics Data System (ADS)

    Saren, Gaowa; Zhao, Yonggang

    2009-05-01

    A full length amphioxus cDNA, encoding a novel phosducin-like protein ( Amphi-PhLP), was identified for the first time from the gut cDNA library of Branchiostoma belcheri. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histochemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-N1/ Amphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri.

  11. Cutaneous water loss and covalently bound lipids of the stratum corneum in nestling house sparrows (Passer domesticus L.) from desert and mesic habitats.

    PubMed

    Clement, Michelle E; Muñoz-Garcia, Agustí; Williams, Joseph B

    2012-04-01

    Lipids of the stratum corneum (SC), the outer layer of the epidermis of birds and mammals, provide a barrier to water vapor diffusion through the skin. The SC of birds consists of flat dead cells, called corneocytes, and two lipid compartments: an intercellular matrix and a monolayer of covalently bound lipids (CBLs) attached to the outer surface of the corneocytes. We previously found two classes of sphingolipids, ceramides and cerebrosides, covalently bound to corneocytes in the SC of house sparrows (Passer domesticus L.); these lipids were associated with cutaneous water loss (CWL). In this study, we collected adult and nestling house sparrows from Ohio and nestlings from Saudi Arabia, acclimated them to either high or low humidity, and measured their rates of CWL. We also measured CWL for natural populations of nestlings from Ohio and Saudi Arabia, beginning when chicks were 2 days old until they fledged. We then evaluated the composition of the CBLs of the SC of sparrows using thin layer chromatography. We found that adult house sparrows had a greater diversity of CBLs in their SC than previously described. During ontogeny, nestling sparrows increased the amount of CBLs and developed their CBLs differently, depending on their habitat. Acclimating nestlings to different humidity regimes did not alter the ontogeny of the CBLs, suggesting that these lipids represent a fundamental component of SC organization that does not respond to short-term environmental change.

  12. Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells.

    PubMed

    Morel, Etienne; Ghezzal, Sara; Lucchi, Géraldine; Truntzer, Caroline; Pais de Barros, Jean-Paul; Simon-Plas, Françoise; Demignot, Sylvie; Mineo, Chieko; Shaul, Philip W; Leturque, Armelle; Rousset, Monique; Carrière, Véronique

    2018-02-01

    Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Functional characterization of chitin-binding lectin from Solanum integrifolium containing anti-fungal and insecticidal activities.

    PubMed

    Chen, Chang-Shan; Chen, Chun-Yi; Ravinath, Divya Malathy; Bungahot, Agustina; Cheng, Chi-Ping; You, Ren-In

    2018-01-03

    Along with the rapid development of glycomic tools, the study of lectin-carbohydrate interactions has expanded, opening the way for applications in the fields of analytic, diagnostic, and drug delivery. Chitin-binding lectins (CBLs) play roles in immune defense against chitin-containing pathogens. CBLs from species of the Solanaceae family, such as tomato, potato and jimsonweed, display different binding specificities to sugar chains containing poly-N-acetyllactosamine. In this report, CBLs from Solanum integrifolium were isolated by ion exchange chromatography. The fractions showed hemagglutination activity (HA). The recombinant CBL in the 293F cell culture supernatant was able to inhibit the growth of Rhizoctonia solani and Colletotrichum gloeosporioide. Furthermore, the carbohydrate-binding property of CBLs was confirmed with the inhibition of HA. Binding of CBL to Spodoptera frugiperda (sf21) insect cells can partly be inhibited by N-Acetylglucosamine (GlcNAc), which is related to decrease mitochondrial membrane potential of sf21 cells. The results showed that CBL exhibited antifungal properties and inhibited insect cell growth, which is directly correlated to the lectin-carbohydrate interaction. Further identification and characterization of CBLs will help to broaden their scope of application in plant defense and in biomedical applications.

  14. Plant Hsp90 Proteins Interact with B-Cells and Stimulate Their Proliferation

    PubMed Central

    Corigliano, Mariana G.; Maglioco, Andrea; Laguía Becher, Melina; Goldman, Alejandra; Martín, Valentina; Angel, Sergio O.; Clemente, Marina

    2011-01-01

    Background The molecular chaperone heat shock protein 90 (Hsp90) plays an important role in folding stabilization and activation of client proteins. Besides, Hsp90 of mammals and mammalian pathogens displays immunostimulatory properties. Here, we investigated the role of plant-derived Hsp90s as B-cell mitogens by measuring their proliferative responses in vitro. Methodology Plant cytosolic Hsp90 isoforms from Arabidopsis thaliana (AtHsp81.2) and Nicotiana benthamiana (NbHsp90.3) were expressed in E. coli. Over-expression of recombinant plant Hsp90s (rpHsp90s) was confirmed by SDS-PAGE and western blot using and anti-AtHsp81.2 polyclonal anti-body. Both recombinant proteins were purified by Ni-NTA affinity chromatography and their identity confirmed by MALDI-TOF-TOF. Recombinant AtHsp81.2 and NbHsp90.3 proteins induced prominent proliferative responses in spleen cells form BALB/c mice. Polymyxin-B, a potent inhibitor of lipopolysaccharide (LPS), did not eliminate the rpHsp90-induced proliferation. In addition, in vitro incubation of spleen cells with rpHsp90 led to the expansion of CD19-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. This effect was confirmed by immunofluorescence analysis, where a direct binding of rpHsp90 to B- but not to T-cells was observed in cells from BALB/c and C3H/HeN mice. Finally, we examined the involvement of Toll Like Receptor 4 (TLR4) molecules in the rpHsp90s induction of B-cell proliferation. Spleen cells from C3H/HeJ mice, which carry a point mutation in the cytoplasmic region of TLR4, responded poorly to prAtHsp90. However, the interaction between rpHsp90 and B-cells from C3H/HeJ mice was not altered, suggesting that the mutation on TLR4 would be affecting the signal cascade but not the rpHsp90-TLR4 receptor interaction. Conclusions Our results show for the first time that spleen cell proliferation can be stimulated by a non-pathogen-derived Hsp90. Furthermore, our data provide a new example of

  15. Cross-Reactivity of Antibodies against Leptospiral Recurrent Uveitis-Associated Proteins A and B (LruA and LruB) with Eye Proteins

    PubMed Central

    Verma, Ashutosh; Kumar, Pawan; Babb, Kelly; Timoney, John F.; Stevenson, Brian

    2010-01-01

    Infection by Leptospira interrogans has been causally associated with human and equine uveitis. Studies in our laboratories have demonstrated that leptospiral lipoprotein LruA and LruB are expressed in the eyes of uveitic horses, and that antibodies directed against LruA and LruB react with equine lenticular and retinal extracts, respectively. These reactivities were investigated further by performing immunofluorescent assays on lenticular and retinal tissue sections. Incubation of lens tissue sections with LruA-antiserum and retinal sections with LruB-antiserum resulted in positive fluorescence. By employing two-dimensional gel analyses followed by immunoblotting and mass spectrometry, lens proteins cross-reacting with LruA antiserum were identified to be α-crystallin B and vimentin. Similarly, mass spectrometric analyses identified β-crystallin B2 as the retinal protein cross-reacting with LruB-antiserum. Purified recombinant human α-crystallin B and vimentin were recognized by LruA-directed antiserum, but not by control pre-immune serum. Recombinant β-crystallin B2 was likewise recognized by LruB-directed antiserum, but not by pre-immune serum. Moreover, uveitic eye fluids contained significantly higher levels of antiibodies that recognized α-crystallin B, β-crystallin B2 and vimentin than did normal eye fluids. Our results indicate that LruA and LruB share immuno-relevant epitopes with eye proteins, suggesting that cross-reactive antibody interactions with eye antigens may contribute to immunopathogenesis of Leptospira-associated recurrent uveitis. PMID:20689825

  16. Cross-reactivity of antibodies against leptospiral recurrent uveitis-associated proteins A and B (LruA and LruB) with eye proteins.

    PubMed

    Verma, Ashutosh; Kumar, Pawan; Babb, Kelly; Timoney, John F; Stevenson, Brian

    2010-08-03

    Infection by Leptospira interrogans has been causally associated with human and equine uveitis. Studies in our laboratories have demonstrated that leptospiral lipoprotein LruA and LruB are expressed in the eyes of uveitic horses, and that antibodies directed against LruA and LruB react with equine lenticular and retinal extracts, respectively. These reactivities were investigated further by performing immunofluorescent assays on lenticular and retinal tissue sections. Incubation of lens tissue sections with LruA-antiserum and retinal sections with LruB-antiserum resulted in positive fluorescence. By employing two-dimensional gel analyses followed by immunoblotting and mass spectrometry, lens proteins cross-reacting with LruA antiserum were identified to be alpha-crystallin B and vimentin. Similarly, mass spectrometric analyses identified beta-crystallin B2 as the retinal protein cross-reacting with LruB-antiserum. Purified recombinant human alpha-crystallin B and vimentin were recognized by LruA-directed antiserum, but not by control pre-immune serum. Recombinant beta-crystallin B2 was likewise recognized by LruB-directed antiserum, but not by pre-immune serum. Moreover, uveitic eye fluids contained significantly higher levels of antiibodies that recognized alpha-crystallin B, beta-crystallin B2 and vimentin than did normal eye fluids. Our results indicate that LruA and LruB share immuno-relevant epitopes with eye proteins, suggesting that cross-reactive antibody interactions with eye antigens may contribute to immunopathogenesis of Leptospira-associated recurrent uveitis.

  17. Structural Basis for the Acyltransferase Activity of Lecithin:Retinol Acyltransferase-like Proteins*

    PubMed Central

    Golczak, Marcin; Kiser, Philip D.; Sears, Avery E.; Lodowski, David T.; Blaner, William S.; Palczewski, Krzysztof

    2012-01-01

    Lecithin:retinol acyltransferase-like proteins, also referred to as HRAS-like tumor suppressors, comprise a vertebrate subfamily of papain-like or NlpC/P60 thiol proteases that function as phospholipid-metabolizing enzymes. HRAS-like tumor suppressor 3, a representative member of this group, plays a key role in regulating triglyceride accumulation and energy expenditure in adipocytes and therefore constitutes a novel pharmacological target for treatment of metabolic disorders causing obesity. Here, we delineate a catalytic mechanism common to lecithin:retinol acyltransferase-like proteins and provide evidence for their alternative robust lipid-dependent acyltransferase enzymatic activity. We also determined high resolution crystal structures of HRAS-like tumor suppressor 2 and 3 to gain insight into their active site architecture. Based on this structural analysis, two conformational states of the catalytic Cys-113 were identified that differ in reactivity and thus could define the catalytic properties of these two proteins. Finally, these structures provide a model for the topology of these enzymes and allow identification of the protein-lipid bilayer interface. This study contributes to the enzymatic and structural understanding of HRAS-like tumor suppressor enzymes. PMID:22605381

  18. An integrated genomic analysis of Tudor domain-containing proteins identifies PHD finger protein 20-like 1 (PHF20L1) as a candidate oncogene in breast cancer.

    PubMed

    Jiang, Yuanyuan; Liu, Lanxin; Shan, Wenqi; Yang, Zeng-Quan

    2016-02-01

    Tudor domain-containing proteins (TDRDs), which recognize and bind to methyl-lysine/arginine residues on histones and non-histone proteins, play critical roles in regulating chromatin architecture, transcription, genomic stability, and RNA metabolism. Dysregulation of several TDRDs have been observed in various types of cancer. However, neither the genomic landscape nor clinical significance of TDRDs in breast cancer has been explored comprehensively. Here, we performed an integrated genomic and transcriptomic analysis of 41 TDRD genes in breast cancer (TCGA and METABRIC datasets) and identified associations among recurrent copy number alterations, gene expressions, clinicopathological features, and survival of patients. Among seven TDRDs that had the highest frequency (>10%) of gene amplification, the plant homeodomain finger protein 20-like 1 (PHF20L1) was the most commonly amplified (17.62%) TDRD gene in TCGA breast cancers. Different subtypes of breast cancer had different patterns of copy number and expression for each TDRD. Notably, amplification and overexpression of PHF20L1 were more prevalent in aggressive basal-like and Luminal B subtypes and were significantly associated with shorter survival of breast cancer patients. Furthermore, knockdown of PHF20L1 inhibited cell proliferation in PHF20L1-amplified breast cancer cell lines. PHF20L1 protein contains N-terminal Tudor and C-terminal plant homeodomain domains. Detailed characterization of PHF20L1 in breast cancer revealed that the Tudor domain likely plays a critical role in promoting cancer. Mechanistically, PHF20L1 might participate in regulating DNA methylation by stabilizing DNA methyltransferase 1 (DNMT1) protein in breast cancer. Thus, our results demonstrated the oncogenic potential of PHF20L1 and its association with poor prognostic parameters in breast cancer. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  19. Binding Affects the Tertiary and Quaternary Structures of the Shigella Translocator Protein IpaB and its Chaperone IpgC†

    PubMed Central

    Adam, Philip R.; Patil, Mrinalini K.; Dickenson, Nicholas E.; Choudhari, Shyamal; Barta, Michael; Geisbrecht, Brian V.; Picking, Wendy L.; Picking, William D.

    2012-01-01

    Shigella flexneri uses its type III secretion system (T3SS) to promote invasion of human intestinal epithelial cells as the first step in causing shigellosis, a life threatening form of dysentery. The Shigella type III secretion apparatus (T3SA) consists of a basal body that spans the bacterial envelope and an exposed needle that injects effector proteins into target cells. The nascent Shigella T3SA needle is topped with a pentamer of the needle tip protein invasion plasmid antigen D (IpaD). Bile salts trigger recruitment of the first hydrophobic translocator protein, IpaB, to the tip complex where it senses contact with a host membrane. In the bacterial cytoplasm, IpaB exists in a complex with its chaperone IpgC. Several structures of IpgC have been solved and we recently reported the 2.1-Å crystal structure of the N-terminal domain (IpaB74.224) of IpaB. Like IpgC, the IpaB N-terminal domain exists as a homodimer in solution. We now report that when the two are mixed, these homodimers dissociate and form heterodimers having a nanomolar dissociation constant. This is consistent with the equivalent complexes co-purified after being co-expressed in E. coli. Fluorescence data presented here also indicate that the N-terminal domain of IpaB possesses two regions that appear to contribute additively to chaperone binding. It is also likely that the IpaB N terminus adopts an alternative conformation as a result of chaperone binding. The importance of these findings within the functional context of these proteins is discussed. PMID:22497344

  20. Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1.

    PubMed

    Ishikawa-Sasaki, Kumiko; Nagashima, Shigeo; Taniguchi, Koki; Sasaki, Jun

    2018-04-15

    Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs. IMPORTANCE Positive-strand RNA viruses utilize host pathways to modulate the lipid composition of

  1. Circulating CD21low B cells in common variable immunodeficiency resemble tissue homing, innate-like B cells

    PubMed Central

    Rakhmanov, Mirzokhid; Keller, Baerbel; Gutenberger, Sylvia; Foerster, Christian; Hoenig, Manfred; Driessen, Gertjan; van der Burg, Mirjam; van Dongen, Jacques J.; Wiech, Elisabeth; Visentini, Marcella; Quinti, Isabella; Prasse, Antje; Voelxen, Nadine; Salzer, Ulrich; Goldacker, Sigune; Fisch, Paul; Eibel, Hermann; Schwarz, Klaus; Peter, Hans-Hartmut; Warnatz, Klaus

    2009-01-01

    The homeostasis of circulating B cell subsets in the peripheral blood of healthy adults is well regulated, but in disease it can be severely disturbed. Thus, a subgroup of patients with common variable immunodeficiency (CVID) presents with an extraordinary expansion of an unusual B cell population characterized by the low expression of CD21. CD21low B cells are polyclonal, unmutated IgM+IgD+ B cells but carry a highly distinct gene expression profile which differs from conventional naïve B cells. Interestingly, while clearly not representing a memory population, they do share several features with the recently defined memory-like tissue, Fc receptor-like 4 positive B cell population in the tonsils of healthy donors. CD21low B cells show signs of previous activation and proliferation in vivo, while exhibiting defective calcium signaling and poor proliferation in response to B cell receptor stimulation. CD21low B cells express decreased amounts of homeostatic but increased levels of inflammatory chemokine receptors. This might explain their preferential homing to peripheral tissues like the bronchoalveolar space of CVID or the synovium of rheumatoid arthritis patients. Therefore, as a result of the close resemblance to the gene expression profile, phenotype, function and preferential tissue homing of murine B1 B cells, we suggest that CD21low B cells represent a human innate-like B cell population. PMID:19666505

  2. Lactoferrin binding protein B – a bi-functional bacterial receptor protein

    PubMed Central

    Ostan, Nicholas K. H.; Yu, Rong-Hua; Ng, Dixon; Lai, Christine Chieh-Lin; Sarpe, Vladimir; Schriemer, David C.

    2017-01-01

    Lactoferrin binding protein B (LbpB) is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf) receptor complex in Neisseria meningitidis and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB), there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation. PMID:28257520

  3. In vivo reconstitution of a homodimeric cytochrome b559 like structure: The role of the N-terminus α-subunit from Synechocystis sp. PCC 6803.

    PubMed

    Luján, María A; Martínez, Jesús I; Alonso, Pablo J; Torrado, Alejandro; Roncel, Mercedes; Ortega, José M; Sancho, Javier; Picorel, Rafael

    2015-11-01

    The cytochrome b559 is a heme-bridged heterodimeric protein with two subunits, α and β. Both subunits from Synechocystis sp. PCC 6803 have previously been cloned and overexpressed in Escherichia coli and in vivo reconstitution experiments have been carried out. The formation of homodimers in the bacterial membrane with endogenous heme was only observed in the case of the β-subunit (β/β) but not with the full length α-subunit. In the present work, reconstitution of a homodimer (α/α) cytochrome b559 like structure was possible using a chimeric N-terminus α-subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic α-helix that lays on the surface of the membrane. Overexpression and in vivo reconstitution in the bacteria was clearly demonstrated by the brownish color of the culture pellet and the use of a commercial monoclonal antibody against the fusion protein carrier, the maltoside binding protein, and polyclonal antibodies against a synthetic peptide of the α-subunit from Thermosynechococcus elongatus. Moreover, a simple partial purification after membrane solubilization with Triton X-100 confirmed that the overexpressed protein complex corresponded with the maltoside binding protein-chimeric α-subunit cytochrome b559 like structure. The features of the new structure were determined by UV-Vis, electron paramagnetic resonance and redox potentiometric techniques. Ribbon representations of all possible structures are also shown to better understand the mechanism of the cytochrome b559 maturation in the bacterial cytoplasmic membrane. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Protein-like fully reversible tetramerisation and super-association of an aminocellulose

    NASA Astrophysics Data System (ADS)

    Nikolajski, Melanie; Adams, Gary G.; Gillis, Richard B.; Besong, David Tabot; Rowe, Arthur J.; Heinze, Thomas; Harding, Stephen E.

    2014-01-01

    Unusual protein-like, partially reversible associative behaviour has recently been observed in solutions of the water soluble carbohydrates known as 6-deoxy-6-(ω-aminoalkyl)aminocelluloses, which produce controllable self-assembling films for enzyme immobilisation and other biotechnological applications. Now, for the first time, we have found a fully reversible self-association (tetramerisation) within this family of polysaccharides. Remarkably these carbohydrate tetramers are then seen to associate further in a regular way into supra-molecular complexes. Fully reversible oligomerisation has been hitherto completely unknown for carbohydrates and instead resembles in some respects the assembly of polypeptides and proteins like haemoglobin and its sickle cell mutation. Our traditional perceptions as to what might be considered ``protein-like'' and what might be considered as ``carbohydrate-like'' behaviour may need to be rendered more flexible, at least as far as interaction phenomena are concerned.

  5. Bacterially produced human B7-1 protein encompassing its complete extracellular domain maintains its costimulatory activity in vitro.

    PubMed

    Shen, W; Wang, Y; Geng, Y; Si, L

    2000-08-01

    To investigate which of the two immunoglobulin (Ig)-like domains, immunoglobulin variable region homologous domain IgV (hB7-1 IgV), or immunoglobulin constant region homologous domain IgC (hB7-1 IgC) on human B7-1 molecule contain the receptor binding sites, and to evaluate if the B7-1 molecule expressed in bacteria has biological activity. PCR was used to amplify three fragments of hB7-1 IgV, hB7-1 IgC and complete extracellular region of human B7-1 containing both the IgV and IgC domains (hB7-1 IgV + IgC). Three recombinants, pQE9-hB7-1 IgV, pQE9-hB7-1 IgC and pQE9-Hb7-1 (IgV + IgC) were generated by cloning the PCR products into a prokaryote expression plasmid (pQE-9) and were introduced into the host stain M15. The relevant target hexahistidine-tagged proteins were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7-1 proteins and [3H]-TdR incorporation. Three recombinant proteins of human B7-1, hB7-1 IgV, hB7-1 IgC and hB7-1 (IgV + IgC) were produced and detected in both soluble and inclusive body forms from engineered bacterial cells. With the presence of anti-CD3 antibody, T lymphocytes proliferated when co-stimulated by bacterially produced hB7-1 (IgV + IgC), but not by either hB7-1 IgV or hB7-1 IgC. Functional glycoprotein human B7-1 could be produced in bacterial cells. Both extracellular immunoglobulin-like domains are necessary for B7-1 to react with its counter receptors.

  6. The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions.

    PubMed

    Treuner-Lange, Anke; Macia, Eric; Guzzo, Mathilde; Hot, Edina; Faure, Laura M; Jakobczak, Beata; Espinosa, Leon; Alcor, Damien; Ducret, Adrien; Keilberg, Daniela; Castaing, Jean Philippe; Lacas Gervais, Sandra; Franco, Michel; Søgaard-Andersen, Lotte; Mignot, Tâm

    2015-07-20

    In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate-bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA-MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein-cytoskeleton interactions are a universally conserved feature. © 2015 Treuner-Lange et al.

  7. Coarse-grained model for colloidal protein interactions, B(22), and protein cluster formation.

    PubMed

    Blanco, Marco A; Sahin, Erinc; Robinson, Anne S; Roberts, Christopher J

    2013-12-19

    Reversible protein cluster formation is an important initial step in the processes of native and non-native protein aggregation, but involves relatively long time and length scales for detailed atomistic simulations and extensive mapping of free energy landscapes. A coarse-grained (CG) model is presented to semiquantitatively characterize the thermodynamics and key configurations involved in the landscape for protein oligomerization, as well as experimental measures of interactions such as the osmotic second virial coefficient (B22). Based on earlier work (Grüenberger et al., J. Phys. Chem. B 2013, 117, 763), this CG model treats proteins as rigid bodies composed of one bead per amino acid, with each amino acid having specific parameters for its size, hydrophobicity, and charge. The net interactions are a combination of steric repulsions, short-range attractions, and screened long-range charge-charge interactions. Model parametrization was done by fitting simulation results against experimental value of B22 as a function of solution ionic strength for α-chymotrypsinogen A and γD-Crystallin (gD-Crys). The CG model is applied to characterize the pairwise interactions and dimerization of gD-Crys and the dependence on temperature, protein concentration, and ionic strength. The results illustrate that at experimentally relevant conditions where stable dimers do not form, the entropic contributions are predominant in the free-energy of protein cluster formation and colloidal protein interactions, arguing against interpretations that treat B22 primarily from energetic considerations alone. Additionally, the results suggest that electrostatic interactions help to modulate the population of the different stable configurations for protein nearest-neighbor pairs, while short-range attractions determine the relative orientations of proteins within these configurations. Finally, simulation results are combined with Principal Component Analysis to identify those amino

  8. How large B-factors can be in protein crystal structures.

    PubMed

    Carugo, Oliviero

    2018-02-23

    Protein crystal structures are potentially over-interpreted since they are routinely refined without any restraint on the upper limit of atomic B-factors. Consequently, some of their atoms, undetected in the electron density maps, are allowed to reach extremely large B-factors, even above 100 square Angstroms, and their final positions are purely speculative and not based on any experimental evidence. A strategy to define B-factors upper limits is described here, based on the analysis of protein crystal structures deposited in the Protein Data Bank prior 2008, when the tendency to allow B-factor to arbitrary inflate was limited. This B-factor upper limit (B_max) is determined by extrapolating the relationship between crystal structure average B-factor and percentage of crystal volume occupied by solvent (pcVol) to pcVol =100%, when, ab absurdo, the crystal contains only liquid solvent, the structure of which is, by definition, undetectable in electron density maps. It is thus possible to highlight structures with average B-factors larger than B_max, which should be considered with caution by the users of the information deposited in the Protein Data Bank, in order to avoid scientifically deleterious over-interpretations.

  9. Genome-Wide Protein Interaction Screens Reveal Functional Networks Involving Sm-Like Proteins

    PubMed Central

    Fromont-Racine, Micheline; Mayes, Andrew E.; Brunet-Simon, Adeline; Rain, Jean-Christophe; Colley, Alan; Dix, Ian; Decourty, Laurence; Joly, Nicolas; Ricard, Florence; Beggs, Jean D.

    2000-01-01

    A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein–protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale. PMID:10900456

  10. Compare the Difference of B-cell Epitopes of EgAgB1 and EgAgB3 Proteins Selected through Bioinformatic Analysis

    NASA Astrophysics Data System (ADS)

    An, Mengting; Zhang, Fengbo; Zhu, Yuejie; Zhao, Xiao; Ding, Jianbing

    2018-01-01

    Cystic echinococcosis, as a zoonosis, seriously endangers humans and animals, so early diagnosis of this disease is particularly important. Therefore, this study is to predict B-cell epitopes of EgAgB1 and EgAgB3 proteins by bioinformatics software. B-cell epitopes of EgAgB1 and EgAgB3 proteins are predicted using DNAStar and IEDB software. The results suggest that there are two potential B-cell epitopes in EgAgB1, which located in the 8-15 and 31-37 amino acid residue segments. And two potential B-cell epitopes in EgAgB2, located in the 20∼27 and 47∼53 amino acid residue segments. This study predicted the B-cell epitopes of EgAgB1 and EgAgB3 proteins, which laid the foundation for the early diagnosis of Cystic echinococcosis.

  11. Prokaryotic histone-like protein interacting with RNA polymerase.

    PubMed Central

    Lathe, R; Buc, H; Lecocq, J P; Bautz, E K

    1980-01-01

    firA mutation of Escherichia coli can render RNA synthesis thermosensitive and confer abnormal sensitivity to rifampicin, an antibiotic that specifically inhibits the activity of RNA polymerase. We previously described the cloning of a chromosomal HindIII fragment containing the firA gene, and we now present strong evidence that the product of this gene is a 17,000-dalton polypeptide which, by various criteria, closely resembles the eukaryotic histones. This protein forms the largest of a unique set of three abundant histone-like proteins (HLP) found in E. coli and is hence referred to as HLPI. We discuss possible routes by which these proteins might affect transcription. Images PMID:6447875

  12. Paired immunoglobulin-like receptor B knockout does not enhance axonal regeneration or locomotor recovery after spinal cord injury.

    PubMed

    Nakamura, Yuka; Fujita, Yuki; Ueno, Masaki; Takai, Toshiyuki; Yamashita, Toshihide

    2011-01-21

    Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced in NgR-knock-out mice after spinal cord injury. Therefore, we assessed CST regeneration in PIR-B-knock-out mice. We found that hindlimb motor function, as assessed using the Basso mouse scale, footprint test, inclined plane test, and beam walking test, did not differ between the PIR-B-knock-out and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sprouting in the CST of the PIR-B-knock-out mice. Systemic administration of NEP1-40, a NgR antagonist, to PIR-B knock-out mice did not enhance the regenerative response. These results indicate that PIR-B knock-out is not sufficient to induce extensive axonal regeneration after spinal cord injury.

  13. Modulation of the nuclear factor-kappa B (NF-κB) signalling pathway by glutamine in peritoneal macrophages of a murine model of protein malnutrition.

    PubMed

    da Silva Lima, Fabiana; Rogero, Marcelo Macedo; Ramos, Mayara Caldas; Borelli, Primavera; Fock, Ricardo Ambrósio

    2013-06-01

    Protein malnutrition affects resistance to infection by impairing the inflammatory response, modifying the function of effector cells, such as macrophages. Recent studies have revealed that glutamine-a non-essential amino acid, which could become conditionally essential in some situations like trauma, infection, post-surgery and sepsis-is able to modulate the synthesis of cytokines. The aim of this study was to evaluate the effect of glutamine on the expression of proteins involved in the nuclear factor-kappa B (NF-κB) signalling pathway of peritoneal macrophages from malnourished mice. Two-month-old male Balb/c mice were submitted to protein-energy malnutrition (n = 10) with a low-protein diet containing 2 % protein, whereas control mice (n = 10) were fed a 12 % protein-containing diet. The haemogram and analysis of plasma glutamine and corticosterone were evaluated. Peritoneal macrophages were pre-treated in vitro with glutamine (0, 0.6, 2 and 10 mmol/L) for 24 h and then stimulated with 1.25 μg LPS for 30 min, and the synthesis of TNF-α and IL-1α and the expression of proteins related to the NF-κB pathway were evaluated. Malnourished animals had anaemia, leucopoenia, lower plasma glutamine and increased corticosterone levels. TNF-α production of macrophages stimulated with LPS was significantly lower in cells from malnourished animals when cultivated in supraphysiological (2 and 10 mmol/L) concentrations of glutamine. Further, glutamine has a dose-dependent effect on the activation of macrophages, in both groups, when stimulated with LPS, inducing a decrease in TNF-α and IL-1α production and negatively modulating the NF-κB signalling pathway. These data lead us to infer that the protein malnutrition state interferes with the activation of macrophages and that higher glutamine concentrations, in vitro, have the capacity to act negatively in the NF-κB signalling pathway.

  14. Environmental Pollutant Ozone Causes Damage to Lung Surfactant Protein B (SP-B)

    PubMed Central

    2015-01-01

    Lung surfactant protein B (SP-B) is an essential protein found in the surfactant fluid at the air–water interface of the lung. Exposure to the air pollutant ozone could potentially damage SP-B and lead to respiratory distress. We have studied two peptides, one consisting of the N-terminus of SP-B [SP-B(1–25)] and the other a construct of the N- and C-termini of SP-B [SP-B(1–25,63–78)], called SMB. Exposure to dilute levels of ozone (∼2 ppm) of monolayers of each peptide at the air–water interface leads to a rapid reaction, which is evident from an increase in the surface tension. Fluorescence experiments revealed that this increase in surface tension is accompanied by a loss of fluorescence from the tryptophan residue at the interface. Neutron and X-ray reflectivity experiments show that, in contrast to suggestions in the literature, the peptides are not solubilized upon oxidation but rather remain at the interface with little change in their hydration. Analysis of the product material reveals that no cleavage of the peptides occurs, but a more hydrophobic product is slowly formed together with an increased level of oligomerization. We attributed this to partial unfolding of the peptides. Experiments conducted in the presence of phospholipids reveal that the presence of the lipids does not prevent oxidation of the peptides. Our results strongly suggest that exposure to low levels of ozone gas will damage SP-B, leading to a change in its structure. The implication is that the oxidized protein will be impaired in its ability to interact at the air–water interface with negatively charged phosphoglycerol lipids, thus compromising what is thought to be its main biological function. PMID:26270023

  15. Ontogeny of a novel pituitary protein (7B2) in the human fetal intestine.

    PubMed

    Suzuki, H; Christofides, N D; Adrian, T E; Chretien, M; Seidah, N G; Polak, J M; Bloom, S R

    1985-11-28

    The developmental profile of the concentration of a novel pituitary protein (7B2) was studied immunochemically in the human gastrointestinal tract from 12 weeks of gestation to 4 months after birth and was compared to the distribution in the adult. 7B2-like immunoreactivity (IR-7B2) was detected in all segments studied, but no gross changes were seen through fetal life. At term higher concentrations of IR-7B2 were found in the duodenum and the antrum, which is similar to the distribution of adult man. Gel permeation chromatography revealed that the main peak of 7B2 immunoreactivity in the fetal intestinal extract eluted with a Kav of 0.3. Similar elution profiles were also observed in extracts of human adult intestine.

  16. Functional Fluorescent Protein Insertions in Herpes Simplex Virus gB Report on gB Conformation before and after Execution of Membrane Fusion

    PubMed Central

    Gallagher, John R.; Atanasiu, Doina; Saw, Wan Ting; Paradisgarten, Matthew J.; Whitbeck, J. Charles; Eisenberg, Roselyn J.; Cohen, Gary H.

    2014-01-01

    Entry of herpes simplex virus (HSV) into a target cell requires complex interactions and conformational changes by viral glycoproteins gD, gH/gL, and gB. During viral entry, gB transitions from a prefusion to a postfusion conformation, driving fusion of the viral envelope with the host cell membrane. While the structure of postfusion gB is known, the prefusion conformation of gB remains elusive. As the prefusion conformation of gB is a critical target for neutralizing antibodies, we set out to describe its structure by making genetic insertions of fluorescent proteins (FP) throughout the gB ectodomain. We created gB constructs with FP insertions in each of the three globular domains of gB. Among 21 FP insertion constructs, we found 8 that allowed gB to remain membrane fusion competent. Due to the size of an FP, regions in gB that tolerate FP insertion must be solvent exposed. Two FP insertion mutants were cell-surface expressed but non-functional, while FP insertions located in the crown were not surface expressed. This is the first report of placing a fluorescent protein insertion within a structural domain of a functional viral fusion protein, and our results are consistent with a model of prefusion HSV gB constructed from the prefusion VSV G crystal structure. Additionally, we found that functional FP insertions from two different structural domains could be combined to create a functional form of gB labeled with both CFP and YFP. FRET was measured with this construct, and we found that when co-expressed with gH/gL, the FRET signal from gB was significantly different from the construct containing CFP alone, as well as gB found in syncytia, indicating that this construct and others of similar design are likely to be powerful tools to monitor the conformation of gB in any model system accessible to light microscopy. PMID:25233449

  17. Alfalfa Mob1-like proteins are involved in cell proliferation and are localized in the cell division plane during cytokinesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Citterio, Sandra; Piatti, Simonetta; Albertini, Emidio

    2006-04-15

    Mps-one-binder (Mob) proteins play a crucial role in yeast cytokinesis. After cloning two Mob1-like genes, MsMob1-A and MsMob1-B from alfalfa (Medicago sativa L.) we show that, although they are constitutively expressed in roots, stems, leaves, flowers and pods, their transcripts and proteins are mostly produced in actively proliferating tissues. A polyclonal antibody specifically raised against MsMob1 proteins was used for immunolocalization studies in synchronized root tip cells. The subcellular localization of MsMob1-like proteins is demonstrated to be cell cycle-regulated. Cytoplasmic localization is faint and diffused during G{sub 1} and S. It becomes concentrated in punctuate and fibrillar structures in G{submore » 2} as well as M phase. At the stage of cytokinesis, the protein is found at the emerging cell plate marking the progressive formation of the septum. Mob1 proteins partially co-localize with microtubules structures functionally related to the spindles and important for cytokinesis in eukaryotic cells. The MsMob1 expression cannot rescue the lethality of the yeast mob1 mutant, suggesting that interaction of Mob1 proteins with their effectors may be species-specific. Localization of Mob1 proteins in the inner layer of the root cap indicates an additional function for this class of proteins in plants, which is likely related to the onset of programmed cell death.« less

  18. The chitinase-like protein YKL-40 increases mucin5AC production in human bronchial epithelial cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Chunyi; Li, Qi; Zhou, Xiangdong, E-mail: zxd999@263.net

    2013-11-01

    Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases. However, the regulatory mechanisms that mediate excessive mucin production remain elusive. Recently, the level of YKL-40, a chitinase-like protein, has been found to be significantly increased in chronic inflammatory airway diseases and has been shown to be associated with the severity of these diseases. In this study, we sought to explore the effect of YKL-40 on mucin5AC (MUC5AC) production in chronic inflammatory airway diseases and the potential signaling pathways involved in this process. We found that elevated YKL-40 levels increased the mRNA and protein expression of MUC5ACmore » in a dose- and time-dependent manner, in association with the phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB), reflecting their activation. These responses were significantly suppressed by the knockdown of protease-activating receptor 2 (PAR2) with specific small interfering RNA or the inhibitors of ERK and NF-κB. YKL-40-induced MUC5AC overproduction was also effectively attenuated by the inhibitor of focal adhesion kinase (FAK). Taken together, these results imply that YKL-40 can stimulate excessive MUC5AC production through PAR2- and FAK-mediated mechanisms. - Highlights: • MUC5AC is the major secreted mucin in chronic inflammatory airway diseases. • YKL-40 is a prototype of the chitinase-like protein in mammals. • YKL-40 is an active player in chronic inflammatory airway diseases. • YKL-40 can increase MUC5AC production via PAR2-mediated pathway. • FAK is another candidate to mediate YKL-40-induced MUC5AC overexpression.« less

  19. Fragmentation of the CRISPR-Cas Type I-B signature protein Cas8b.

    PubMed

    Richter, Hagen; Rompf, Judith; Wiegel, Julia; Rau, Kristina; Randau, Lennart

    2017-11-01

    CRISPR arrays are transcribed into long precursor RNA species, which are further processed into mature CRISPR RNAs (crRNAs). Cas proteins utilize these crRNAs, which contain spacer sequences that can be derived from mobile genetic elements, to mediate immunity during a reoccurring virus infection. Type I CRISPR-Cas systems are defined by the presence of different Cascade interference complexes containing large and small subunits that play major roles during target DNA selection. Here, we produce the protein and crRNA components of the Type I-B CRISPR-Cas complex of Clostridium thermocellum and Methanococcus maripaludis. The C. thermocellum Cascade complexes were reconstituted and analyzed via size-exclusion chromatography. Activity of the heterologous M. maripaludis CRISPR-Cas system was followed using phage lambda plaques assays. The reconstituted Type-I-B Cascade complex contains Cas7, Cas5, Cas6b and the large subunit Cas8b. Cas6b can be omitted from the reconstitution protocol. The large subunit Cas8b was found to be represented by two tightly associated protein fragments and a small C-terminal Cas8b segment was identified in recombinant complexes and C. thermocellum cell lysate. Production of Cas8b generates a small C-terminal fragment, which is suggested to fulfill the role of the missing small subunit. A heterologous, synthetic M. maripaludis Type I-B system is active in E. coli against phage lambda, highlighting a potential for genome editing using endogenous Type-I-B CRISPR-Cas machineries. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Molecular identification and characterisation of catalase and catalase-like protein genes in urease-positive thermophilic Campylobacter (UPTC).

    PubMed

    Nakajima, T; Kuribayashi, T; Moore, J E; Millar, B C; Yamamoto, S; Matsuda, Motoo

    2016-01-01

    Thermophilic Campylobacter are important bacterial pathogens of foodborne diseases worldwide. These organisms' physiology requires a microaerophilic atmosphere. To date, little is known about the protective catalase mechanism in urease-positive thermophilic campylobacters (UPTC); hence, it was the aim of this study to identify and characterise catalase and catalase-like protein genes in these organisms. Catalase (katA) and catalase (Kat)-like protein genes from the Japanese UPTC CF89-12 strain were molecularly analysed and compared with C. lari RM2100 and other C. lari and thermophilic Campylobacter reference isolates. A possible open reading frame of 1,422 base pairs, predicted to encode a peptide of 474 amino acid residues, with calculated molecular weight of 52.7 kilo Daltons for katA, was identified within UPTC CF89-12. A probable ribosome binding site, two putative promoters and a putative ρ-independent transcription terminator were also identified within katA. A similar katA cluster also existed in the C. lari RM2100 strain, except that this strain carries no DcuB genes. However, the Kat-like protein gene or any other homologue(s) were never identified in the C. lari RM2100 strain, or in C. jejuni and C. upsaliensis. This study demonstrates the presence of catalase/catalase-like protein genes in UPTC organisms. These findings are significant in that they suggest that UPTC organisms have the protective genetic capability of helping protect the organisms from toxic oxygen stress, which may help them to survive in physiologically harsh environments, both within human and animal hosts, as well as in the natural environment.

  1. A comprehensive proteomics and genomics analysis reveals novel transmembrane proteins in human platelets and mouse megakaryocytes including G6b-B, a novel ITIM protein

    PubMed Central

    Senis, Yotis A.; Tomlinson, Michael G.; García, Ángel; Dumon, Stephanie; Heath, Victoria L.; Herbert, John; Cobbold, Stephen P.; Spalton, Jennifer C.; Ayman, Sinem; Antrobus, Robin; Zitzmann, Nicole; Bicknell, Roy; Frampton, Jon; Authi, Kalwant; Martin, Ashley; Wakelam, Michael J.O.; Watson, Stephen P.

    2007-01-01

    Summary The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we have identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomic and genomic approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography; biotin/NeutrAvidin affinity chromatography; and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68 and 22 surface membrane, intracellular membrane and membrane proteins of unknown sub-cellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomic studies, we analysed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing importance of mutant mouse models in establishing protein function in platelets. This approach identified all of the major classes of platelet transmembrane receptors, including multi-transmembrane proteins. Strikingly, 17 of the 25 most megakaryocyte-specific genes (relative to 30 other SAGE libraries) were transmembrane proteins, illustrating the unique nature of the megakaryocyte/platelet surface. The list of novel plasma membrane proteins identified using proteomics includes the immunoglobulin superfamily member G6b, which undergoes extensive alternate splicing. Specific antibodies were used to demonstrate expression of the G6b-B isoform, which contains an immunoreceptor tyrosine-based inhibition motif. G6b-B undergoes tyrosine phosphorylation and association with the SH2-containing phosphatase, SHP-1, in stimulated

  2. Toll-Like Receptor Stimulation Induces Nondefensin Protein Expression and Reverses Antibiotic-Induced Gut Defense Impairment

    PubMed Central

    Wu, Ying-Ying; Hsu, Ching-Mei; Chen, Pei-Hsuan; Fung, Chang-Phone

    2014-01-01

    Prior antibiotic exposure is associated with increased mortality in Gram-negative bacteria-induced sepsis. However, how antibiotic-mediated changes of commensal bacteria promote the spread of enteric pathogenic bacteria in patients remains unclear. In this study, the effects of systemic antibiotic treatment with or without Toll-like receptor (TLR) stimulation on bacterium-killing activity, antibacterial protein expression in the intestinal mucosa, and bacterial translocation were examined in mice receiving antibiotics with or without oral supplementation of dead Escherichia coli or Staphylococcus aureus. We developed a systemic ampicillin, vancomycin, and metronidazole treatment protocol to simulate the clinical use of antibiotics. Antibiotic treatment decreased the total number of bacteria, including aerobic bacteria belonging to the family Enterobacteriaceae and the genus Enterococcus as well as organisms of the anaerobic genera Lactococcus and Bifidobacterium in the intestinal mucosa and lumen. Antibiotic treatment significantly decreased the bacterium-killing activity of the intestinal mucosa and the expression of non-defensin-family proteins, such as RegIIIβ, RegIIIγ, C-reactive protein-ductin, and RELMβ, but not the defensin-family proteins, and increased Klebsiella pneumoniae translocation. TLR stimulation after antibiotic treatment increased NF-κB DNA binding activity, nondefensin protein expression, and bacterium-killing activity in the intestinal mucosa and decreased K. pneumoniae translocation. Moreover, germfree mice showed a significant decrease in nondefensin proteins as well as intestinal defense against pathogen translocation. Since TLR stimulation induced NF-κB DNA binding activity, TLR4 expression, and mucosal bacterium-killing activity in germfree mice, we conclude that the commensal microflora is critical in maintaining intestinal nondefensin protein expression and the intestinal barrier. In turn, we suggest that TLR stimulation induces

  3. Toll-like receptor stimulation induces nondefensin protein expression and reverses antibiotic-induced gut defense impairment.

    PubMed

    Wu, Ying-Ying; Hsu, Ching-Mei; Chen, Pei-Hsuan; Fung, Chang-Phone; Chen, Lee-Wei

    2014-05-01

    Prior antibiotic exposure is associated with increased mortality in Gram-negative bacteria-induced sepsis. However, how antibiotic-mediated changes of commensal bacteria promote the spread of enteric pathogenic bacteria in patients remains unclear. In this study, the effects of systemic antibiotic treatment with or without Toll-like receptor (TLR) stimulation on bacterium-killing activity, antibacterial protein expression in the intestinal mucosa, and bacterial translocation were examined in mice receiving antibiotics with or without oral supplementation of dead Escherichia coli or Staphylococcus aureus. We developed a systemic ampicillin, vancomycin, and metronidazole treatment protocol to simulate the clinical use of antibiotics. Antibiotic treatment decreased the total number of bacteria, including aerobic bacteria belonging to the family Enterobacteriaceae and the genus Enterococcus as well as organisms of the anaerobic genera Lactococcus and Bifidobacterium in the intestinal mucosa and lumen. Antibiotic treatment significantly decreased the bacterium-killing activity of the intestinal mucosa and the expression of non-defensin-family proteins, such as RegIIIβ, RegIIIγ, C-reactive protein-ductin, and RELMβ, but not the defensin-family proteins, and increased Klebsiella pneumoniae translocation. TLR stimulation after antibiotic treatment increased NF-κB DNA binding activity, nondefensin protein expression, and bacterium-killing activity in the intestinal mucosa and decreased K. pneumoniae translocation. Moreover, germfree mice showed a significant decrease in nondefensin proteins as well as intestinal defense against pathogen translocation. Since TLR stimulation induced NF-κB DNA binding activity, TLR4 expression, and mucosal bacterium-killing activity in germfree mice, we conclude that the commensal microflora is critical in maintaining intestinal nondefensin protein expression and the intestinal barrier. In turn, we suggest that TLR stimulation induces

  4. Single prolonged stress enhances hippocampal glucocorticoid receptor and phosphorylated protein kinase B levels

    PubMed Central

    Eagle, Andrew L.; Knox, Dayan; Roberts, Megan M.; Mulo, Kostika; Liberzon, Israel; Galloway, Matthew P.; Perrine, Shane A.

    2012-01-01

    Animal models of posttraumatic stress disorder (PTSD) can explore neurobiological mechanisms by which trauma enhances fear and anxiety reactivity. Single prolonged stress (SPS) shows good validity in producing PTSD-like behavior. While SPS-induced behaviors have been linked to enhanced glucocorticoid receptor (GR) expression, the molecular ramifications of enhanced GR expression have yet to be identified. Phosphorylated protein kinase B (pAkt) is critical for stress-mediated enhancement in general anxiety and memory, and may be regulated by GRs. However, it is currently unknown if pAkt levels are modulated by SPS, as well as if the specificity of GR and pAkt related changes contribute to anxiety-like behavior after SPS. The current study set out to examine the effects of SPS on GR and pAkt protein levels in the amygdala and hippocampus and to examine the specificity of these changes to unconditioned anxiety-like behavior. Levels of GR and pAkt were increased in the hippocampus, but not amygdala. Furthermore, SPS had no effect on unconditioned anxiety-like behavior suggesting that generalized anxiety is not consistently observed following SPS. The results suggest that SPS-enhanced GR expression is associated with phosphorylation of Akt, and also suggest that these changes are not related to an anxiogenic phenotype. PMID:23201176

  5. Neuron-Like Networks Between Ribosomal Proteins Within the Ribosome

    NASA Astrophysics Data System (ADS)

    Poirot, Olivier; Timsit, Youri

    2016-05-01

    From brain to the World Wide Web, information-processing networks share common scale invariant properties. Here, we reveal the existence of neural-like networks at a molecular scale within the ribosome. We show that with their extensions, ribosomal proteins form complex assortative interaction networks through which they communicate through tiny interfaces. The analysis of the crystal structures of 50S eubacterial particles reveals that most of these interfaces involve key phylogenetically conserved residues. The systematic observation of interactions between basic and aromatic amino acids at the interfaces and along the extension provides new structural insights that may contribute to decipher the molecular mechanisms of signal transmission within or between the ribosomal proteins. Similar to neurons interacting through “molecular synapses”, ribosomal proteins form a network that suggest an analogy with a simple molecular brain in which the “sensory-proteins” innervate the functional ribosomal sites, while the “inter-proteins” interconnect them into circuits suitable to process the information flow that circulates during protein synthesis. It is likely that these circuits have evolved to coordinate both the complex macromolecular motions and the binding of the multiple factors during translation. This opens new perspectives on nanoscale information transfer and processing.

  6. A Caleosin-Like Protein with Peroxygenase Activity Mediates Aspergillus flavus Development, Aflatoxin Accumulation, and Seed Infection.

    PubMed

    Hanano, Abdulsamie; Almousally, Ibrahem; Shaban, Mouhnad; Blee, Elizabeth

    2015-09-01

    Caleosins are a small family of calcium-binding proteins endowed with peroxygenase activity in plants. Caleosin-like genes are present in fungi; however, their functions have not been reported yet. In this work, we identify a plant caleosin-like protein in Aspergillus flavus that is highly expressed during the early stages of spore germination. A recombinant purified 32-kDa caleosin-like protein supported peroxygenase activities, including co-oxidation reactions and reduction of polyunsaturated fatty acid hydroperoxides. Deletion of the caleosin gene prevented fungal development. Alternatively, silencing of the gene led to the increased accumulation of endogenous polyunsaturated fatty acid hydroperoxides and antioxidant activities but to a reduction of fungal growth and conidium formation. Two key genes of the aflatoxin biosynthesis pathway, aflR and aflD, were downregulated in the strains in which A. flavus PXG (AfPXG) was silenced, leading to reduced aflatoxin B1 production in vitro. Application of caleosin/peroxygenase-derived oxylipins restored the wild-type phenotype in the strains in which AfPXG was silenced. PXG-deficient A. flavus strains were severely compromised in their capacity to infect maize seeds and to produce aflatoxin. Our results uncover a new branch of the fungal oxylipin pathway and may lead to the development of novel targets for controlling fungal disease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. The Ferredoxin-Like Proteins HydN and YsaA Enhance Redox Dye-Linked Activity of the Formate Dehydrogenase H Component of the Formate Hydrogenlyase Complex.

    PubMed

    Pinske, Constanze

    2018-01-01

    Formate dehydrogenase H (FDH-H) and [NiFe]-hydrogenase 3 (Hyd-3) form the catalytic components of the hydrogen-producing formate hydrogenlyase (FHL) complex, which disproportionates formate to H 2 and CO 2 during mixed acid fermentation in enterobacteria. FHL comprises minimally seven proteins and little is understood about how this complex is assembled. Early studies identified a ferredoxin-like protein, HydN, as being involved in FDH-H assembly into the FHL complex. In order to understand how FDH-H and its small subunit HycB, which is also a ferredoxin-like protein, attach to the FHL complex, the possible roles of HydN and its paralogue, YsaA, in FHL complex stability and assembly were investigated. Deletion of the hycB gene reduced redox dye-mediated FDH-H activity to approximately 10%, abolished FHL-dependent H 2 -production, and reduced Hyd-3 activity. These data are consistent with HycB being an essential electron transfer component of the FHL complex. The FDH-H activity of the hydN and the ysaA deletion strains was reduced to 59 and 57% of the parental, while the double deletion reduced activity of FDH-H to 28% and the triple deletion with hycB to 1%. Remarkably, and in contrast to the hycB deletion, the absence of HydN and YsaA was without significant effect on FHL-dependent H 2 -production or total Hyd-3 activity; FDH-H protein levels were also unaltered. This is the first description of a phenotype for the E. coli ysaA deletion strain and identifies it as a novel factor required for optimal redox dye-linked FDH-H activity. A ysaA deletion strain could be complemented for FDH-H activity by hydN and ysaA , but the hydN deletion strain could not be complemented. Introduction of these plasmids did not affect H 2 production. Bacterial two-hybrid interactions showed that YsaA, HydN, and HycB interact with each other and with the FDH-H protein. Further novel anaerobic cross-interactions of 10 ferredoxin-like proteins in E. coli were also discovered and described

  8. NFκB is a central regulator of protein quality control in response to protein aggregation stresses via autophagy modulation

    PubMed Central

    Nivon, Mathieu; Fort, Loïc; Muller, Pascale; Richet, Emma; Simon, Stéphanie; Guey, Baptiste; Fournier, Maëlenn; Arrigo, André-Patrick; Hetz, Claudio; Atkin, Julie D.; Kretz-Remy, Carole

    2016-01-01

    During cell life, proteins often misfold, depending on particular mutations or environmental changes, which may lead to protein aggregates that are toxic for the cell. Such protein aggregates are the root cause of numerous diseases called “protein conformational diseases,” such as myofibrillar myopathy and familial amyotrophic lateral sclerosis. To fight against aggregates, cells are equipped with protein quality control mechanisms. Here we report that NFκB transcription factor is activated by misincorporation of amino acid analogues into proteins, inhibition of proteasomal activity, expression of the R120G mutated form of HspB5 (associated with myofibrillar myopathy), or expression of the G985R and G93A mutated forms of superoxide dismutase 1 (linked to familial amyotrophic lateral sclerosis). This noncanonical stimulation of NFκB triggers the up-regulation of BAG3 and HspB8 expression, two activators of selective autophagy, which relocalize to protein aggregates. Then NFκB-dependent autophagy allows the clearance of protein aggregates. Thus NFκB appears as a central and major regulator of protein aggregate clearance by modulating autophagic activity. In this context, the pharmacological stimulation of this quality control pathway might represent a valuable strategy for therapies against protein conformational diseases. PMID:27075172

  9. miRNA-135b Contributes to Triple Negative Breast Cancer Molecular Heterogeneity: Different Expression Profile in Basal-like Versus non-Basal-like Phenotypes.

    PubMed

    Uva, Paolo; Cossu-Rocca, Paolo; Loi, Federica; Pira, Giovanna; Murgia, Luciano; Orrù, Sandra; Floris, Matteo; Muroni, Maria Rosaria; Sanges, Francesca; Carru, Ciriaco; Angius, Andrea; De Miglio, Maria Rosaria

    2018-01-01

    The clinical and genetic heterogeneity of Triple Negative Breast Cancer (TNBC) and the lack of unambiguous molecular targets contribute to the inadequacy of current therapeutic options for these variants. MicroRNAs (miRNA) are a class of small highly conserved regulatory endogenous non-coding RNA, which can alter the expression of genes encoding proteins and may play a role in the dysregulation of cellular pathways. Our goal was to improve the knowledge of the molecular pathogenesis of TNBC subgroups analyzing the miRNA expression profile, and to identify new prognostic and predictive biomarkers. We conducted a human miRNome analysis by TaqMan Low Density Array comparing different TNBC subtypes, defined by immunohistochemical basal markers EGFR and CK5/6. RT-qPCR confirmed differential expression of microRNAs. To inspect the function of the selected targets we perform Gene Ontology and KEGG enrichment analysis. We identified a single miRNA signature given by miR-135b expression level, which was strictly related to TNBC with basal-like phenotype. miR-135b target analysis revealed a role in the TGF-beta, WNT and ERBB pathways. A significant positive correlation was identified between neoplastic proliferative index and miR-135b expression. These findings confirm the oncogenic roles of miR-135b in the pathogenesis of TNBC expressing basal markers. A potential negative prognostic role of miR-135b overexpression might be related to the positive correlation with high proliferative index. Our study implies potential clinical applications: miR-135b could be a potential therapeutic target in basal-like TNBCs.

  10. miRNA-135b Contributes to Triple Negative Breast Cancer Molecular Heterogeneity: Different Expression Profile in Basal-like Versus non-Basal-like Phenotypes

    PubMed Central

    Uva, Paolo; Cossu-Rocca, Paolo; Loi, Federica; Pira, Giovanna; Murgia, Luciano; Orrù, Sandra; Floris, Matteo; Muroni, Maria Rosaria; Sanges, Francesca; Carru, Ciriaco; Angius, Andrea; De Miglio, Maria Rosaria

    2018-01-01

    The clinical and genetic heterogeneity of Triple Negative Breast Cancer (TNBC) and the lack of unambiguous molecular targets contribute to the inadequacy of current therapeutic options for these variants. MicroRNAs (miRNA) are a class of small highly conserved regulatory endogenous non-coding RNA, which can alter the expression of genes encoding proteins and may play a role in the dysregulation of cellular pathways. Our goal was to improve the knowledge of the molecular pathogenesis of TNBC subgroups analyzing the miRNA expression profile, and to identify new prognostic and predictive biomarkers. We conducted a human miRNome analysis by TaqMan Low Density Array comparing different TNBC subtypes, defined by immunohistochemical basal markers EGFR and CK5/6. RT-qPCR confirmed differential expression of microRNAs. To inspect the function of the selected targets we perform Gene Ontology and KEGG enrichment analysis. We identified a single miRNA signature given by miR-135b expression level, which was strictly related to TNBC with basal-like phenotype. miR-135b target analysis revealed a role in the TGF-beta, WNT and ERBB pathways. A significant positive correlation was identified between neoplastic proliferative index and miR-135b expression. These findings confirm the oncogenic roles of miR-135b in the pathogenesis of TNBC expressing basal markers. A potential negative prognostic role of miR-135b overexpression might be related to the positive correlation with high proliferative index. Our study implies potential clinical applications: miR-135b could be a potential therapeutic target in basal-like TNBCs. PMID:29725243

  11. Host-derived, pore-forming toxin–like protein and trefoil factor complex protects the host against microbial infection

    PubMed Central

    Xiang, Yang; Yan, Chao; Guo, Xiaolong; Zhou, Kaifeng; Li, Sheng’an; Gao, Qian; Wang, Xuan; Zhao, Feng; Liu, Jie; Lee, Wen-Hui; Zhang, Yun

    2014-01-01

    Aerolysins are virulence factors belonging to the bacterial β-pore–forming toxin superfamily. Surprisingly, numerous aerolysin-like proteins exist in vertebrates, but their biological functions are unknown. βγ-CAT, a complex of an aerolysin-like protein subunit (two βγ-crystallin domains followed by an aerolysin pore-forming domain) and two trefoil factor subunits, has been identified in frogs (Bombina maxima) skin secretions. Here, we report the rich expression of this protein, in the frog blood and immune-related tissues, and the induction of its presence in peritoneal lavage by bacterial challenge. This phenomena raises the possibility of its involvement in antimicrobial infection. When βγ-CAT was administrated in a peritoneal infection model, it greatly accelerated bacterial clearance and increased the survival rate of both frogs and mice. Meanwhile, accelerated Interleukin-1β release and enhanced local leukocyte recruitments were determined, which may partially explain the robust and effective antimicrobial responses observed. The release of interleukin-1β was potently triggered by βγ-CAT from the frog peritoneal cells and murine macrophages in vitro. βγ-CAT was rapidly endocytosed and translocated to lysosomes, where it formed high molecular mass SDS-stable oligomers (>170 kDa). Lysosomal destabilization and cathepsin B release were detected, which may explain the activation of caspase-1 inflammasome and subsequent interleukin-1β maturation and release. To our knowledge, these results provide the first functional evidence of the ability of a host-derived aerolysin-like protein to counter microbial infection by eliciting rapid and effective host innate immune responses. The findings will also largely help to elucidate the possible involvement and action mechanisms of aerolysin-like proteins and/or trefoil factors widely existing in vertebrates in the host defense against pathogens. PMID:24733922

  12. Virus-like particle expression and assembly in plants: hepatitis B and Norwalk viruses.

    PubMed

    Huang, Zhong; Elkin, Galina; Maloney, Bryan J; Beuhner, Norene; Arntzen, Charles J; Thanavala, Yasmin; Mason, Hugh S

    2005-03-07

    Expression of vaccine antigens in plants and delivery via ingestion of transgenic plant material has shown promise in numerous pre-clinical animal studies and in a few clinical trials. A number of different viral antigens have been tested, and among the most promising are those that can assemble virus-like particles (VLP), which mimic the form of authentic virions and display neutralizing antibody epitopes. We have extensively studied plant expression, VLP assembly, and immunogenicity of hepatitis B surface antigen (HBsAg) and Norwalk virus capsid protein (NVCP). The HBsAg small protein (S protein) was found by TEM to assemble tubular membrane complexes derived from endoplasmic reticulum in suspension cultured cells of tobacco and soybean, and in potato leaf and tuber tissues. The potato material was immunogenic in mice upon delivery by ingestion. Here we describe the plant expression and immunogenicity of HBsAg middle protein (M protein or pre-S2 + S) which contains additional 55 amino acid pre-S2 region at N-terminus of the S protein. Plant-derived recombinant M protein provoked stronger serum antibody responses against HBsAg than did S protein when injected systemically in mice. We discuss implications for use of fusion proteins for enhanced immunogenicity and mucosal targeting of HBsAg, as well as delivery of heterologous fused antigens. NVCP expressed in plants assembled 38 nm virion-size icosahedral (T = 3) VLP, similar to those produced in insect cells. The VLP stimulated serum IgG and IgA responses in mice and humans when they were delivered by ingestion of fresh potato tuber. Here we show that freeze-drying of transgenic NVCP tomato fruit yielded stable preparations that stimulated excellent IgG and IgA responses against NVCP when fed to mice. However, the predominant VLP form in tomato fruit was the small 23 nm particle also observed in insect cell-derived NVCP.

  13. Discovery and study of novel protein tyrosine phosphatase 1B inhibitors

    NASA Astrophysics Data System (ADS)

    Zhang, Qian; Chen, Xi; Feng, Changgen

    2017-10-01

    Protein tyrosine phosphatase 1B (PTP1B) is considered to be a target for therapy of type II diabetes and obesity. So it is of great significance to take advantage of a computer aided drug design protocol involving the structured-based virtual screening with docking simulations for fast searching small molecule PTP1B inhibitors. Based on optimized complex structure of PTP1B bound with specific inhibitor of IX1, structured-based virtual screening against a library of natural products containing 35308 molecules, which was constructed based on Traditional Chinese Medicine database@ Taiwan (TCM database@ Taiwan), was conducted to determine the occurrence of PTP1B inhibitors using the Lubbock module and CDOCKER module from Discovery Studio 3.1 software package. The results were further filtered by predictive ADME simulation and predictive toxic simulation. As a result, 2 good drug-like molecules, namely para-benzoquinone compound 1 and Clavepictine analogue 2 were identified ultimately with the dock score of original inhibitor (IX1) and the receptor as a threshold. Binding model analyses revealed that these two candidate compounds have good interactions with PTP1B. The PTP1B inhibitory activity of compound 2 hasn't been reported before. The optimized compound 2 has higher scores and deserves further study.

  14. Impedimetric aptasensor for nuclear factor kappa B with peroxidase-like mimic coupled DNA nanoladders as enhancer.

    PubMed

    Peng, Kanfu; Zhao, Hongwen; Xie, Pan; Hu, Shuang; Yuan, Yali; Yuan, Ruo; Wu, Xiongfei

    2016-07-15

    In this work, we developed a sensitive and universal aptasensor for nuclear factor kappa B (NF-κB) detection based on peroxidase-like mimic coupled DNA nanoladders for signal amplification. The dsDNA formed by capture DNA S1 and NF-κB binding aptamer (NBA) was firstly assembled on electrode surface. The presence of target NF-κB then led to the leave of NBA from electrode surface and thus provided the binding sites for immobilizing initiator to trigger in situ formation of DNA nanoladders on electrode surface. Since the peroxidase-like mimic manganese (III) meso-tetrakis (4-Nmethylpyridyl)-porphyrin (MnTMPyP) interacts with DNA nanoladders via groove binding, the insoluble benzo-4-chlorohexadienone (4-CD) precipitation derived from the oxidation of 4-chloro-1-naphthol (4-CN) could be formed on electrode surface in the presence of H2O2, resulting in a significantly amplified EIS signal output for quantitative target analysis. As a result, the developed aptasensor showed a low detection limit of 7pM and a wide linear range of 0.01-20nM. Featured with high sensitivity and label-free capability, the proposed sensing scheme can thus offer new opportunities for achieving sensitive, selective and stable detection of different types of target proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. A family of GFP-like proteins with different spectral properties in lancelet Branchiostoma floridae

    PubMed Central

    Baumann, Diana; Cook, Malcolm; Ma, Limei; Mushegian, Arcady; Sanders, Erik; Schwartz, Joel; Yu, C Ron

    2008-01-01

    Background Members of the green fluorescent protein (GFP) family share sequence similarity and the 11-stranded β-barrel fold. Fluorescence or bright coloration, observed in many members of this family, is enabled by the intrinsic properties of the polypeptide chain itself, without the requirement for cofactors. Amino acid sequence of fluorescent proteins can be altered by genetic engineering to produce variants with different spectral properties, suitable for direct visualization of molecular and cellular processes. Naturally occurring GFP-like proteins include fluorescent proteins from cnidarians of the Hydrozoa and Anthozoa classes, and from copepods of the Pontellidae family, as well as non-fluorescent proteins from Anthozoa. Recently, an mRNA encoding a fluorescent GFP-like protein AmphiGFP, related to GFP from Pontellidae, has been isolated from the lancelet Branchiostoma floridae, a cephalochordate (Deheyn et al., Biol Bull, 2007 213:95). Results We report that the nearly-completely sequenced genome of Branchiostoma floridae encodes at least 12 GFP-like proteins. The evidence for expression of six of these genes can be found in the EST databases. Phylogenetic analysis suggests that a gene encoding a GFP-like protein was present in the common ancestor of Cnidaria and Bilateria. We synthesized and expressed two of the lancelet GFP-like proteins in mammalian cells and in bacteria. One protein, which we called LanFP1, exhibits bright green fluorescence in both systems. The other protein, LanFP2, is identical to AmphiGFP in amino acid sequence and is moderately fluorescent. Live imaging of the adult animals revealed bright green fluorescence at the anterior end and in the basal region of the oral cirri, as well as weaker green signals throughout the body of the animal. In addition, red fluorescence was observed in oral cirri, extending to the tips. Conclusion GFP-like proteins may have been present in the primitive Metazoa. Their evolutionary history includes

  16. Autism-like behavior caused by deletion of vaccinia-related kinase 3 is improved by TrkB stimulation

    PubMed Central

    Kang, Myung-Su; Lee, Dohyun; Lee, Seung-Hyun

    2017-01-01

    Vaccinia-related kinases (VRKs) are multifaceted serine/threonine kinases that play essential roles in various aspects of cell signaling, cell cycle progression, apoptosis, and neuronal development and differentiation. However, the neuronal function of VRK3 is still unknown despite its etiological potential in human autism spectrum disorder (ASD). Here, we report that VRK3-deficient mice exhibit typical symptoms of autism-like behavior, including hyperactivity, stereotyped behaviors, reduced social interaction, and impaired context-dependent spatial memory. A significant decrease in dendritic spine number and arborization were identified in the hippocampus CA1 of VRK3-deficient mice. These mice also exhibited a reduced rectification of AMPA receptor–mediated current and changes in expression of synaptic and signaling proteins, including tyrosine receptor kinase B (TrkB), Arc, and CaMKIIα. Notably, TrkB stimulation with 7,8-dihydroxyflavone reversed the altered synaptic structure and function and successfully restored autism-like behavior in VRK3-deficient mice. These results reveal that VRK3 plays a critical role in neurodevelopmental disorders and suggest a potential therapeutic strategy for ASD. PMID:28899869

  17. Autism-like behavior caused by deletion of vaccinia-related kinase 3 is improved by TrkB stimulation.

    PubMed

    Kang, Myung-Su; Choi, Tae-Yong; Ryu, Hye Guk; Lee, Dohyun; Lee, Seung-Hyun; Choi, Se-Young; Kim, Kyong-Tai

    2017-10-02

    Vaccinia-related kinases (VRKs) are multifaceted serine/threonine kinases that play essential roles in various aspects of cell signaling, cell cycle progression, apoptosis, and neuronal development and differentiation. However, the neuronal function of VRK3 is still unknown despite its etiological potential in human autism spectrum disorder (ASD). Here, we report that VRK3 -deficient mice exhibit typical symptoms of autism-like behavior, including hyperactivity, stereotyped behaviors, reduced social interaction, and impaired context-dependent spatial memory. A significant decrease in dendritic spine number and arborization were identified in the hippocampus CA1 of VRK3 -deficient mice. These mice also exhibited a reduced rectification of AMPA receptor-mediated current and changes in expression of synaptic and signaling proteins, including tyrosine receptor kinase B (TrkB), Arc, and CaMKIIα. Notably, TrkB stimulation with 7,8-dihydroxyflavone reversed the altered synaptic structure and function and successfully restored autism-like behavior in VRK3 -deficient mice. These results reveal that VRK3 plays a critical role in neurodevelopmental disorders and suggest a potential therapeutic strategy for ASD. © 2017 Kang et al.

  18. Unfolding study of a trimeric membrane protein AcrB.

    PubMed

    Ye, Cui; Wang, Zhaoshuai; Lu, Wei; Wei, Yinan

    2014-07-01

    The folding of a multi-domain trimeric α-helical membrane protein, Escherichia coli inner membrane protein AcrB, was investigated. AcrB contains both a transmembrane domain and a large periplasmic domain. Protein unfolding in sodium dodecyl sulfate (SDS) and urea was monitored using the intrinsic fluorescence and circular dichroism spectroscopy. The SDS denaturation curve displayed a sigmoidal profile, which could be fitted with a two-state unfolding model. To investigate the unfolding of separate domains, a triple mutant was created, in which all three Trp residues in the transmembrane domain were replaced with Phe. The SDS unfolding profile of the mutant was comparable to that of the wild type AcrB, suggesting that the observed signal change was largely originated from the unfolding of the soluble domain. Strengthening of trimer association through the introduction of an inter-subunit disulfide bond had little effect on the unfolding profile, suggesting that trimer dissociation was not the rate-limiting step in unfolding monitored by fluorescence emission. Under our experimental condition, AcrB unfolding was not reversible. Furthermore, we experimented with the refolding of a monomeric mutant, AcrBΔloop , from the SDS unfolded state. The CD spectrum of the refolded AcrBΔloop superimposed well onto the spectra of the original folded protein, while the fluorescence spectrum was not fully recovered. In summary, our results suggested that the unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re-association of the trimer might be the limiting factor to obtain folded wild-type AcrB. © 2014 The Protein Society.

  19. A PR-1-like Protein of Fusarium oxysporum Functions in Virulence on Mammalian Hosts*

    PubMed Central

    Prados-Rosales, Rafael C.; Roldán-Rodríguez, Raquel; Serena, Carolina; López-Berges, Manuel S.; Guarro, Josep; Martínez-del-Pozo, Álvaro; Di Pietro, Antonio

    2012-01-01

    The pathogenesis-related PR-1-like protein family comprises secreted proteins from the animal, plant, and fungal kingdoms whose biological function remains poorly understood. Here we have characterized a PR-1-like protein, Fpr1, from Fusarium oxysporum, an ubiquitous fungal pathogen that causes vascular wilt disease on a wide range of plant species and can produce life-threatening infections in immunocompromised humans. Fpr1 is secreted and proteolytically processed by the fungus. The fpr1 gene is required for virulence in a disseminated immunodepressed mouse model, and its function depends on the integrity of the proposed active site of PR-1-like proteins. Fpr1 belongs to a gene family that has expanded in plant pathogenic Sordariomycetes. These results suggest that secreted PR-1-like proteins play important roles in fungal pathogenicity. PMID:22553200

  20. Coarse-Grained Model for Colloidal Protein Interactions, B22, and Protein Cluster Formation

    PubMed Central

    Blanco, Marco A.; Sahin, Eric; Robinson, Anne S.; Roberts, Christopher J.

    2014-01-01

    Reversible protein cluster formation is an important initial step in the processes of native and non-native protein aggregation, but involves relatively long time and length scales for detailed atomistic simulations and extensive mapping of free energy landscapes. A coarse-grained (CG) model is presented to semi-quantitatively characterize the thermodynamics and key configurations involved in the landscape for protein oligomerization, as well as experimental measures of interactions such as the osmotic second virial coefficient (B22). Based on earlier work, this CG model treats proteins as rigid bodies composed of one bead per amino acid, with each amino acid having specific parameters for its size, hydrophobicity, and charge. The net interactions are a combination of steric repulsions, short-range attractions, and screened long-range charge-charge interactions. Model parametrization was done by fitting simulation results against experimental values of the B22 as a function of solution ionic strength for α-chymotrypsinogen A and γD-crystallin (gD-Crys). The CG model is applied to characterize the pairwise interactions and dimerization of gD-Crys and the dependance on temperature, protein concentration, and ionic strength. The results illustrate that at experimentally relevant conditions where stable dimers do not form, the entropic contributions are predominant in the free-energy of protein cluster formation and colloidal protein interactions, arguing against interpretations that treat B22 primarily from energetic considerations alone. Additionally, the results suggest that electrostatic interactions help to modulate the population of the different stable configurations for protein nearest-neighbor pairs, while short-range attractions determine the relative orientations of proteins within these configurations. Finally, simulation results are combined with Principal Component Analysis to identify those amino-acids / surface patches that form inter-protein contacts

  1. Probing the Interaction of Brain Fatty Acid Binding Protein (B-FABP) with Model Membranes

    PubMed Central

    Dyszy, Fábio; Pinto, Andressa P. A.; Araújo, Ana P. U.; Costa-Filho, Antonio J.

    2013-01-01

    Brain fatty acid-binding protein (B-FABP) interacts with biological membranes and delivers polyunsaturated fatty acids (FAs) via a collisional mechanism. The binding of FAs in the protein and the interaction with membranes involve a motif called “portal region”, formed by two small α-helices, A1 and A2, connected by a loop. We used a combination of site-directed mutagenesis and electron spin resonance to probe the changes in the protein and in the membrane model induced by their interaction. Spin labeled B-FABP mutants and lipidic spin probes incorporated into a membrane model confirmed that B-FABP interacts with micelles through the portal region and led to structural changes in the protein as well in the micelles. These changes were greater in the presence of LPG when compared to the LPC models. ESR spectra of B-FABP labeled mutants showed the presence of two groups of residues that responded to the presence of micelles in opposite ways. In the presence of lysophospholipids, group I of residues, whose side chains point outwards from the contact region between the helices, had their mobility decreased in an environment of lower polarity when compared to the same residues in solution. The second group, composed by residues with side chains situated at the interface between the α-helices, experienced an increase in mobility in the presence of the model membranes. These modifications in the ESR spectra of B-FABP mutants are compatible with a less ordered structure of the portal region inner residues (group II) that is likely to facilitate the delivery of FAs to target membranes. On the other hand, residues in group I and micelle components have their mobilities decreased probably as a result of the formation of a collisional complex. Our results bring new insights for the understanding of the gating and delivery mechanisms of FABPs. PMID:23555925

  2. The role of chitin, chitinases, and chitinase-like proteins in pediatric lung diseases.

    PubMed

    Mack, Ines; Hector, Andreas; Ballbach, Marlene; Kohlhäufl, Julius; Fuchs, Katharina J; Weber, Alexander; Mall, Marcus A; Hartl, Dominik

    2015-12-01

    Chitin, after cellulose, the second most abundant biopolymer on earth, is a key component of insects, fungi, and house-dust mites. Lower life forms are endowed with chitinases to defend themselves against chitin-bearing pathogens. Unexpectedly, humans were also found to express chitinases as well as chitinase-like proteins that modulate immune responses. Particularly, increased levels of the chitinase-like protein YKL-40 have been associated with severe asthma, cystic fibrosis, and other inflammatory disease conditions. Here, we summarize and discuss the potential role of chitin, chitinases, and chitinase-like proteins in pediatric lung diseases.

  3. Protein v. carbohydrate intake differentially affects liking- and wanting-related brain signalling.

    PubMed

    Born, Jurriaan M; Martens, Mieke J I; Lemmens, Sofie G T; Goebel, Rainer; Westerterp-Plantenga, Margriet S

    2013-01-28

    Extreme macronutrient intakes possibly lead to different brain signalling. The aim of the present study was to determine the effects of ingesting high-protein v. high-carbohydrate food on liking and wanting task-related brain signalling (TRS) and subsequent macronutrient intake. A total of thirty female subjects (21.6 (SD 2.2) years, BMI 25.0 (SD 3.7) kg/m²) completed four functional MRI scans: two fasted and two satiated on two different days. During the scans, subjects rated all food items for liking and wanting, thereby choosing the subsequent meal. The results show that high-protein (PROT) v. high-carbohydrate (CARB) conditions were generated using protein or carbohydrate drinks at the first meal. Energy intake and hunger were recorded. PROT (protein: 53.7 (SD 2.1) percentage of energy (En%); carbohydrate: 6.4 (SD 1.3) En%) and CARB conditions (protein: 11.8 (SD 0.6) En%; carbohydrate: 70.0 (SD 2.4) En%) were achieved during the first meal, while the second meals were not different between the conditions. Hunger, energy intake, and behavioural liking and wanting ratings were decreased after the first meal (P< 0.001). Comparing the first with the second meal, the macronutrient content changed: carbohydrate -26.9 En% in the CARB condition, protein -37.8 En% in the PROT condition. After the first meal in the CARB condition, wanting TRS was increased in the hypothalamus. After the first meal in the PROT condition, liking TRS was decreased in the putamen (P< 0.05). The change in energy intake from the first to the second meal was inversely related to the change in liking TRS in the striatum and hypothalamus in the CARB condition and positively related in the PROT condition (P< 0.05). In conclusion, wanting and liking TRS were affected differentially with a change in carbohydrate or protein intake, underscoring subsequent energy intake and shift in macronutrient composition.

  4. Bombyx mori nucleopolyhedrovirus nucleic acid binding proteins BRO-B and BRO-E associate with host T-cell intracellular antigen 1 homologue BmTRN-1 to influence protein synthesis during infection.

    PubMed

    Kotani, Eiji; Muto, Sayaka; Ijiri, Hiroshi; Mori, Hajime

    2015-07-01

    Previous reports have indicated that the Bombyx mori nucleopolyhedrovirus (BmNPV) nucleic acid binding proteins BRO-B and BRO-E are expressed during the early stage of infection and that the BRO family likely supports the regulation of mRNA; however, no study has directly examined the function of BRO family proteins in virus-permissive cells. Here, we show that BRO-B and BRO-E associate with cellular T-cell intracellular antigen 1 homologue (BmTRN-1), a translational regulator, and other cellular translation-related proteins in silkworm cells during viral infection. We created BM-N cells that expressed BRO-B/E to study molecular interactions between BmTRN-1 and BRO-B/E and how they influenced protein synthesis. Fluorescent microscopy revealed that BmTRN-1 was localized in cytoplasmic foci during BmNPV infection. Immunofluorescence studies confirmed that BmTRN-1 and BRO-B/E were colocalized in the amorphous conspicuous cytoplasmic foci. Reporter gene studies revealed that co-expression of BRO-B/E synergistically led to a significant decrease in protein synthesis from a designed transcript carrying the 5'untranslated region of a cellular mRNA with no significant change of transcript abundance. Additionally, RNA interference-mediated knockdown of BmTRN-1 resulted in a marked inhibition of the ability of BRO-B/E to regulate the transcript. These results suggested that the association of BmTRN-1 with BRO-B/E is responsible for the inhibitory regulation of certain mRNAs at the post-transcriptional level and add an additional mechanism for how baculoviruses control protein synthesis during infection.

  5. Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Swatzell, L. J.; Edelmann, R. E.; Makaroff, C. A.; Kiss, J. Z.

    1999-01-01

    Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

  6. Identification of a polymorphic collagen-like protein in the crustacean bacteria Pasteuria ramosa.

    PubMed

    Mouton, Laurence; Traunecker, Emmanuel; McElroy, Kerensa; Du Pasquier, Louis; Ebert, Dieter

    2009-12-01

    Pasteuria ramosa is a spore-forming bacterium that infects Daphnia species. Previous results demonstrated a high specificity of host clone/parasite genotype interactions. Surface proteins of bacteria often play an important role in attachment to host cells prior to infection. We analyzed surface proteins of P. ramosa spores by two-dimensional gel electrophoresis. For the first time, we prove that two isolates selected for their differences in infectivity reveal few but clear-cut differences in protein patterns. Using internal sequencing and LC/MS/MS, we identified a collagen-like protein named Pcl1a (Pasteuria collagen-like protein 1a). This protein, reconstructed with the help of Pasteuria genome sequences, contains three domains: a 75-amino-acid amino-terminal domain with a potential transmembrane helix domain, a central collagen-like region (CLR) containing Gly-Xaa-Yaa (GXY) repeats, and a 7-amino-acid carboxy-terminal domain. The CLR region is polymorphic among the two isolates with amino-acid substitutions and a variable number of GXY triplets. Collagen-like proteins are rare in prokaryotes, although they have been described in several pathogenic bacteria, including Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis, closely related to Pasteuria species, in which they could be involved in the adherence of bacteria to host cells.

  7. Purification and Functional Characterization of a Protein: Bombyx mori Human Growth Hormone Like Protein in Silkworm Pupa

    PubMed Central

    Lv, Zhengbing; Nie, Zuoming; Chen, Jian; Chen, Hao; Yu, Wei; Gai, Qijing; Zhang, Yaozhou

    2014-01-01

    Human growth hormone (hGH) is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the β-trefoil superfamily, with β domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people’s interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs. PMID:25469649

  8. Purification and functional characterization of a protein: Bombyx mori human growth hormone like protein in silkworm pupa.

    PubMed

    Chen, Jianqing; Shu, Tejun; Lv, Zhengbing; Nie, Zuoming; Chen, Jian; Chen, Hao; Yu, Wei; Gai, Qijing; Zhang, Yaozhou

    2014-01-01

    Human growth hormone (hGH) is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the β-trefoil superfamily, with β domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people's interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs.

  9. Overlooked Short Toxin-Like Proteins: A Shortcut to Drug Design

    PubMed Central

    Linial, Michal

    2017-01-01

    Short stable peptides have huge potential for novel therapies and biosimilars. Cysteine-rich short proteins are characterized by multiple disulfide bridges in a compact structure. Many of these metazoan proteins are processed, folded, and secreted as soluble stable folds. These properties are shared by both marine and terrestrial animal toxins. These stable short proteins are promising sources for new drug development. We developed ClanTox (classifier of animal toxins) to identify toxin-like proteins (TOLIPs) using machine learning models trained on a large-scale proteomic database. Insects proteomes provide a rich source for protein innovations. Therefore, we seek overlooked toxin-like proteins from insects (coined iTOLIPs). Out of 4180 short (<75 amino acids) secreted proteins, 379 were predicted as iTOLIPs with high confidence, with as many as 30% of the genes marked as uncharacterized. Based on bioinformatics, structure modeling, and data-mining methods, we found that the most significant group of predicted iTOLIPs carry antimicrobial activity. Among the top predicted sequences were 120 termicin genes from termites with antifungal properties. Structural variations of insect antimicrobial peptides illustrate the similarity to a short version of the defensin fold with antifungal specificity. We also identified 9 proteins that strongly resemble ion channel inhibitors from scorpion and conus toxins. Furthermore, we assigned functional fold to numerous uncharacterized iTOLIPs. We conclude that a systematic approach for finding iTOLIPs provides a rich source of peptides for drug design and innovative therapeutic discoveries. PMID:29109389

  10. Association of Takayasu arteritis with HLA-B 67:01 and two amino acids in HLA-B protein.

    PubMed

    Terao, Chikashi; Yoshifuji, Hajime; Ohmura, Koichiro; Murakami, Kosaku; Kawabata, Daisuke; Yurugi, Kimiko; Tazaki, Junichi; Kinoshita, Hideyuki; Kimura, Akinori; Akizuki, Masashi; Kawaguchi, Yasushi; Yamanaka, Hisashi; Miura, Yasuo; Maekawa, Taira; Saji, Hiroo; Mimori, Tsuneyo; Matsuda, Fumihiko

    2013-10-01

    Takayasu arteritis (TAK) is a rare autoimmune arteritis that affects large arteries. Although the association between TAK and HLA-B 52:01 is established, the other susceptibility HLA-B alleles are not fully known. We performed genetic association studies to determine independent HLA-B susceptibility alleles other than HLA-B 52:01 and to identify important amino acids of HLA-B protein in TAK susceptibility. One hundred patients with TAK and 1000 unrelated healthy controls were genotyped for HLA-B alleles in the first set, followed by a replication set containing 73 patients with TAK and 1000 controls to compare the frequencies of HLA-B alleles. Step-up logistic regression analysis was performed to identify susceptibility amino acids of HLA-B protein. Strong associations of susceptibility to TAK with HLA-B 52:01 and HLA-B 67:01 were observed (P = 1.0 × 10(-16) and 9.5 × 10(-6), respectively). An independent susceptibility effect of HLA-B 67:01 from HLA-B 52:01 was also detected (P = 1.8 × 10(-7)). Amino acid residues of histidine at position 171 and phenylalanine at position 67, both of which are located in antigen binding grooves of the HLA-B protein, were associated with TAK susceptibility (P ≤ 3.8 × 10(-5)) with a significant difference from other amino acid variations (ΔAIC ≥ 9.65). HLA-B 67:01 is associated with TAK independently from HLA-B 52:01. Two amino acids in HLA-B protein are strongly associated with TAK susceptibility.

  11. Effects of hepatitis C virus core protein and nonstructural protein 4B on the Wnt/β-catenin pathway.

    PubMed

    Jiang, Xiao-Hua; Xie, Yu-Tao; Cai, Ya-Ping; Ren, Jing; Ma, Tao

    2017-05-25

    Hepatitis C virus (HCV) core protein and nonstructural protein 4B (NS4B) are potentially oncogenic. Aberrant activation of the Wnt/β-catenin signaling pathway is closely associated with hepatocarcinogenesis. We investigated the effects of HCV type 1b core protein and NS4B on Wnt/β-catenin signaling in various liver cells, and explored the molecular mechanism underlying HCV-related hepatocarcinogenesis. Compared with the empty vector control, HCV core protein and NS4B demonstrated the following characteristics in the Huh7 cells: significantly enhanced β-catenin/Tcf-dependent transcriptional activity (F = 40.87, P < 0.01); increased nuclear translocation of β-catenin (F = 165.26, P < 0.01); upregulated nuclear β-catenin, cytoplasmic β-catenin, Wnt1, c-myc, and cyclin D1 protein expression (P < 0.01); and promoted proliferation of Huh7 cells (P < 0.01 or P < 0.05). Neither protein enhanced β-catenin/Tcf-dependent transcriptional activity in the LO2 cells (F = 0.65, P > 0.05), but they did significantly enhance Wnt3a-induced β-catenin/Tcf-dependent transcriptional activity (F = 64.25, P < 0.01), and promoted the nuclear translocation of β-catenin (F = 66.54, P < 0.01) and the Wnt3a-induced proliferation of LO2 cells (P < 0.01 or P < 0.05). Moreover, activation of the Wnt/β-catenin signaling pathway was greater with the core protein than with NS4B (P < 0.01 or P < 0.05). HCV core protein and NS4B directly activate the Wnt/β-catenin signaling pathway in Huh7 cells and LO2 cells induced by Wnt3a. These data suggest that HCV core protein and NS4B contribute to HCV-associated hepatocellular carcinogenesis.

  12. Fixed points of contractive mappings in b-metric-like spaces.

    PubMed

    Hussain, Nawab; Roshan, Jamal Rezaei; Parvaneh, Vahid; Kadelburg, Zoran

    2014-01-01

    We discuss topological structure of b-metric-like spaces and demonstrate a fundamental lemma for the convergence of sequences. As an application we prove certain fixed point results in the setup of such spaces for different types of contractive mappings. Finally, some periodic point results in b-metric-like spaces are obtained. Two examples are presented in order to verify the effectiveness and applicability of our main results.

  13. Fixed Points of Contractive Mappings in b-Metric-Like Spaces

    PubMed Central

    Hussain, Nawab; Roshan, Jamal Rezaei

    2014-01-01

    We discuss topological structure of b-metric-like spaces and demonstrate a fundamental lemma for the convergence of sequences. As an application we prove certain fixed point results in the setup of such spaces for different types of contractive mappings. Finally, some periodic point results in b-metric-like spaces are obtained. Two examples are presented in order to verify the effectiveness and applicability of our main results. PMID:25143980

  14. The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions

    PubMed Central

    Treuner-Lange, Anke; Macia, Eric; Guzzo, Mathilde; Hot, Edina; Faure, Laura M.; Jakobczak, Beata; Espinosa, Leon; Alcor, Damien; Ducret, Adrien; Keilberg, Daniela; Castaing, Jean Philippe; Lacas Gervais, Sandra; Franco, Michel

    2015-01-01

    In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate–bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA–MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein–cytoskeleton interactions are a universally conserved feature. PMID:26169353

  15. Purification of a novel, nucleoplasmin-like protein from somatic nuclei.

    PubMed Central

    Cotten, M; Chalkley, R

    1987-01-01

    We have purified a nucleoplasmin-like protein from the nuclei of somatic Xenopus laevis cells. This protein possesses a number of the distinctive features of nucleoplasmin isolated from oocytes or unfertilized eggs. The protein is recognized by both monoclonal and polyclonal antisera raised against egg nucleoplasmin. The protein has an oligomeric structure, which must be heated in SDS to completely dissociate, is acidic, phosphorylated and efficiently promotes the in vitro formation of chromatin. We have partially characterized this novel protein and because of its resemblance to nucleoplasmin isolated from oocytes or unfertilized eggs we have named this protein nucleoplasmin S. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:3443097

  16. The Wilms tumor protein WT1 stimulates transcription of the gene encoding insulin-like growth factor binding protein 5 (IGFBP5).

    PubMed

    Müller, Miriam; Persson, Anja Bondke; Krueger, Katharina; Kirschner, Karin M; Scholz, Holger

    2017-07-01

    Insulin-like growth factor (IGF) binding proteins (IGFBPs) constitute a family of six secreted proteins that regulate the signaling of insulin-like growth factors (IGFs). IGFBP5 is the most conserved family member in vertebrates and the major IGF binding protein in bone. IGFBP5 is required for normal development of the musculoskeletal system, and various types of cancer frequently express high levels of IGFP5. Here we identify the gene encoding IGFBP5 as a novel downstream target of the Wilms tumor protein WT1. IGFBP5 and WT1 are expressed in an overlapping pattern in the condensing metanephric mesenchyme of embryonic murine kidneys. Down-regulation of WT1 by transfection with antisense vivo-morpholino significantly decreased Igfbp5 transcripts in murine embryonic kidney explants. Likewise, silencing of Wt1 in a mouse mesonephros-derived cell line reduced Igfbp5 mRNA levels by approximately 80%. Conversely, induction of the WT1(-KTS) isoform, whose role as transcriptional regulator has been firmly established, significantly increased IGFBP5 mRNA and protein levels in osteosarcoma cells. IGFBP5 expression was not significantly changed by WT1(+KTS) protein, which exhibits lower DNA binding affinity than the WT1(-KTS) isoform and has a presumed role in post-transcriptional gene regulation. Luciferase reporter constructs harboring 0.8 and 1.6 kilobases of the murine Igfbp5 promoter, respectively, were stimulated approximately 5-fold by co-transfection of WT1(-KTS). The WT1(+KTS) variant had no significant effect on IGFBP5 promoter activity. Binding of WT1(-KTS), but not of WT1(+KTS) protein, to the IGFBP5 promoter in human osteosarcoma cells was proven by chromatin immunoprecipitation (ChIP) and confirmed by electrophoretic mobility shift assay. These findings demonstrate that WT1 activates transcription of the IGFBP5 gene with possible implications for kidney development and bone (patho)physiology. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Interaction of Charged Patchy Protein Models with Like-Charged Polyelectrolyte Brushes.

    PubMed

    Yigit, Cemil; Kanduč, Matej; Ballauff, Matthias; Dzubiella, Joachim

    2017-01-10

    We study the adsorption of charged patchy particle models (CPPMs) on a thin film of a like-charged and dense polyelectrolyte (PE) brush (of 50 monomers per chain) by means of implicit-solvent, explicit-salt Langevin dynamics computer simulations. Our previously introduced set of CPPMs embraces well-defined one- and two-patched spherical globules, each of the same net charge and (nanometer) size, with mono- and multipole moments comparable to those of small globular proteins. We focus on electrostatic effects on the adsorption far away from the isoelectric point of typical proteins, i.e., where charge regulation plays no role. Despite the same net charge of the brush and globule, we observe large binding affinities up to tens of the thermal energy, k B T, which are enhanced by decreasing salt concentration and increasing charge of the patch(es). Our analysis of the distance-resolved potentials of mean force together with a phenomenological description of all leading interaction contributions shows that the attraction is strongest at the brush surface, driven by multipolar, Born (self-energy), and counterion-release contributions, dominating locally over the monopolar and steric repulsions.

  18. Reduced functionality of PSE-like chicken breast meat batter resulting from alterations in protein conformation.

    PubMed

    Li, K; Zhao, Y Y; Kang, Z L; Wang, P; Han, M Y; Xu, X L; Zhou, G H

    2015-01-01

    The objectives of this study were to evaluate protein thermal stability, water-protein interaction, microstructure, and protein conformation between PSE-like and normal chicken breast meat batters. Sixty pale, soft, and exudative (PSE)-like (L*>53, pH24 h<5.7) and 60 normal (46protein and 2% salt, and they were analyzed for the protein changes and the microstructure using differential scanning calorimetry, low-field (LF)-NMR, SEM, and Raman spectroscopy. PSE-like meat batter had lower gel strength, water-holding capacity, and salt-soluble protein extraction (P<0.05). Heated PSE-like meat batter formed an aggregated gel matrix, while normal meat batter produced a compact gel network with fine, cross-linked strands by many protein filaments. LF-NMR revealed an increase in the water mobility in heated PSE-like meat batter with an increasing amount of loosely bound water (P<0.05). No significant changes were observed in the electrophoretic patterns of salt-soluble protein extracts by SDS-PAGE. However, differential scanning calorimetry showed that PSE-like meat had greater myosin and sarcoplasmic proteins/collagen denaturation (P<0.05). In PSE-like meat, actin denaturation was particular evident after salt addition (P<0.05) using differential scanning calorimetry. Moreover, Raman spectroscopy indicated that PSE-like meat batter had less unfolded α-helix and β-sheet structure formation, reduced exposure of hydrophobic and tyrosine residues (P<0.05), and changes in the microenvironment of aliphatic residues and tryptophan, which affected salt-soluble protein extraction, gel properties, and water-holding capacity. In conclusion, the inferior functional properties of PSE-like meat were attributed to not only myosin denaturation, but also actin denaturation after salt addition and different

  19. Immunogenicity of virus-like particles containing modified goose parvovirus VP2 protein.

    PubMed

    Chen, Zongyan; Li, Chuanfeng; Zhu, Yingqi; Wang, Binbin; Meng, Chunchun; Liu, Guangqing

    2012-10-01

    The major capsid protein VP2 of goose parvovirus (GPV) expressed using a baculovirus expression system (BES) assembles into virus-like particles (VLPs). To optimize VP2 gene expression in Sf9 cells, we converted wild-type VP2 (VP2) codons into codons that are more common in insect genes. This change greatly increased VP2 protein production in Sf9 cells. The protein generated from the codon-optimized VP2 (optVP2) was detected by immunoblotting and an indirect immunofluorescence assay (IFA). Transmission electron microscopy analysis revealed the formation of VLPs. These findings indicate that optVP2 yielded stable and high-quality VLPs. Immunogenicity assays revealed that the VLPs are highly immunogenic, elicit a high level of neutralizing antibodies and provide protection against lethal challenge. The antibody levels appeared to be directly related to the number of GP-Ag-positive hepatocytes. The variation trends for GP-Ag-positive hepatocytes were similar in the vaccine groups. In comparison with the control group, the optVP2 VLPs groups exhibited obviously better responses. These data indicate that the VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Thus, GPV optVP2 appears to be a good candidate for the vaccination of goslings. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Li2 B12 and Li3 B12 : Prediction of the Smallest Tubular and Cage-like Boron Structures.

    PubMed

    Dong, Xue; Jalife, Said; Vásquez-Espinal, Alejandro; Ravell, Estefanía; Pan, Sudip; Cabellos, José Luis; Liang, Wei-Yan; Cui, Zhong-Hua; Merino, Gabriel

    2018-04-16

    An intriguing structural transition from the quasi-planar form of B 12 cluster upon the interaction with lithium atoms is reported. High-level computations show that the lowest energy structures of LiB 12 , Li 2 B 12 , and Li 3 B 12 have quasi-planar (C s ), tubular (D 6d ), and cage-like (C s ) geometries, respectively. The energetic cost of distorting the B 12 quasi-planar fragment is overcompensated by an enhanced electrostatic interaction between the Li cations and the tubular or cage-like B 12 fragments, which is the main reason of such drastic structural changes, resulting in the smallest tubular (Li 2 B 12 ) and cage-like (Li 3 B 12 ) boron structures reported to date. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. An anti-interferon activity shared by paramyxovirus C proteins: inhibition of Toll-like receptor 7/9-dependent alpha interferon induction.

    PubMed

    Yamaguchi, Mayu; Kitagawa, Yoshinori; Zhou, Min; Itoh, Masae; Gotoh, Bin

    2014-01-03

    Paramyxovirus C protein targets the host interferon (IFN) system for virus immune evasion. To identify its unknown anti-IFN activity, we examined the effect of Sendai virus C protein on activation of the IFN-α promoter via various signaling pathways. This study uncovers a novel ability of C protein to block Toll-like receptor (TLR) 7- and TLR9-dependent IFN-α induction, which is specific to plasmacytoid dendritic cells. C protein interacts with a serine/threonine kinase IKKα and inhibits phosphorylation of IRF7. This anti-IFN activity of C protein is shared across genera of the Paramyxovirinae, and thus appears to play an important role in paramyxovirus immune evasion. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  2. Whole-body protein turnover in malnourished patients with child class B and C cirrhosis on diets low to high in protein energy.

    PubMed

    Dichi, J B; Dichi, I; Maio, R; Correa, C R; Angeleli, A Y; Bicudo, M H; Rezende, T A; Burini, R C

    2001-03-01

    The purpose of this study was to determine the rate of whole-body protein turnover in moderately and severely alcoholic, malnourished, cirrhotic patients fed with different amounts of protein or energy. Six male patients (Child classes B and C) and four age- and sex-matched healthy control subjects were studied for 18 d in fasting and feeding states; a single oral dose of [(15)N]glycine was used as a tracer and urinary ammonia was the end product. The kinetic study showed that patients had higher protein catabolism while fasting (patients: 3.14 +/- 1.2 g of lean body mass/9 h; controls: 1.8 +/- 0.3 g of lean body mass/9 h; P < 0.02). Although not statistically significant, protein catabolism (grams of lean body mass/9 h) was lower with the hyperproteic/hyperenergetic diet when compared with fasting. Nitrogen retention was consistent with the lower protein-catabolism rate; a statistically significant increase in nitrogen balance was observed when patients were fed with the hyperproteic/hyperenergetic diet compared with fasting (4.3 +/- 3.2 g of nitrogen/d and -2.2 +/- 1.9 g of nitrogen/d, respectively; P < 0.01). These data indicate that Child class B and C cirrhotic patients are hypercatabolic and that long-term nutritional intervention with a hyperproteic/hyperenergetic diet is likely needed to improve their clinical and nutritional status.

  3. PrkC-mediated phosphorylation of overexpressed YvcK protein regulates PBP1 protein localization in Bacillus subtilis mreB mutant cells.

    PubMed

    Foulquier, Elodie; Pompeo, Frédérique; Freton, Céline; Cordier, Baptiste; Grangeasse, Christophe; Galinier, Anne

    2014-08-22

    The YvcK protein has been shown to be necessary for growth under gluconeogenic conditions in Bacillus subtilis. Amazingly, its overproduction rescues growth and morphology defects of the actin-like protein MreB deletion mutant by restoration of PBP1 localization. In this work, we observed that YvcK was phosphorylated at Thr-304 by the protein kinase PrkC and that phosphorylated YvcK was dephosphorylated by the cognate phosphatase PrpC. We show that neither substitution of this threonine with a constitutively phosphorylated mimicking glutamic acid residue or a phosphorylation-dead mimicking alanine residue nor deletion of prkC or prpC altered the ability of B. subtilis to grow under gluconeogenic conditions. However, we observed that a prpC mutant and a yvcK mutant were more sensitive to bacitracin compared with the WT strain. In addition, the bacitracin sensitivity of strains in which YvcK Thr-304 was replaced with either an alanine or a glutamic acid residue was also affected. We also analyzed rescue of the mreB mutant strain by overproduction of YvcK in which the phosphorylation site was substituted. We show that YvcK T304A overproduction did not rescue the mreB mutant aberrant morphology due to PBP1 mislocalization. The same observation was made in an mreB prkC double mutant overproducing YvcK. Altogether, these data show that YvcK may have two distinct functions: 1) in carbon source utilization independent of its phosphorylation level and 2) in cell wall biosynthesis and morphogenesis through its phosphorylation state. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Constitutive and UV-B modulated transcription of Nod-like receptors and their functional partners in human corneal epithelial cells.

    PubMed

    Benko, Szilvia; Tozser, Jozsef; Miklossy, Gabriella; Varga, Aliz; Kadas, Janos; Csutak, Adrienne; Berta, Andras; Rajnavolgyi, Eva

    2008-08-29

    To determine the transcription pattern of Nod-like receptors (NLRs) and inflammasome components (apoptosis-associated speck-like protein containing a CARD [ASC], CARD inhibitor of NFkB-activating ligands [Cardinal], and caspase-1) in human corneal epithelial cells obtained from healthy individuals undergoing photorefractive keratectomy and in immortalized human corneal epithelial cells (HCE-T). Human corneal epithelial cells were taken from the eyes of healthy individuals by epithelial ablation for photorefractive keratectomy (PRK). The SV-40 immortalized human corneal epithelial cell line (HCE-T) was cultured. mRNA obtained from the cells was reverse transcribed and subjected to quantitative real-time polymerase chain reaction (PCR) measurements. Protein obtained from HCE-T cells was studied using the western blot technique. HCE-T cells were irradiated by UV-B light or treated with ultrapure peptidoglycan, and the effects were studied at the mRNA and protein level while the supernatant of the cells was tested for the presence of various cytokines by using enzyme-linked immunosorbent assay (ELISA) methods. mRNA levels of the studied proteins in the primary cells of the donors were similar in most cases. The transcription of Nod1, Nod2, NLRX1, Nalp1, and Cardinal was similar in the two cell types. While the expression of Nalp3 and Nalp10 was higher in HCE-T cells, ASC and caspase-1 showed higher transcription levels in the primary cells. NLRC5 and Nalp7 were hardly detectable in the studied cells. Functionality of the Nod1/Nod2 system was demonstrated by increased phosphorylation of IkB upon Nod1/Nod2 agonist ultrapure peptidoglycan treatment in HCE-T cells. While UV-B irradiation exerted a downregulation of both Nalp and Nod mRNAs as well as those of inflammasome components in HCE-T cells, longer incubation of the cells after exposure resulted in recovery or upregulation only of the Nalp sensors. At the protein level, we detected a short isoform of Nalp1 and its

  5. A Light Harvesting Complex-Like Protein in Maintenance of Photosynthetic Components in Chlamydomonas1[OPEN

    PubMed Central

    Zhao, Lei; Cheng, Dongmei; Huang, Xiahe; Chen, Mei; Xing, Jiale; Gao, Liyan; Li, Lingyu; Wang, Yale; Peng, Lianwei; Wang, Yingchun

    2017-01-01

    Using a genetic approach, we have identified and characterized a novel protein, named Msf1 (Maintenance factor for photosystem I), that is required for the maintenance of specific components of the photosynthetic apparatus in the green alga Chlamydomonas reinhardtii. Msf1 belongs to the superfamily of light-harvesting complex proteins with three transmembrane domains and consensus chlorophyll-binding sites. Loss of Msf1 leads to reduced accumulation of photosystem I and chlorophyll-binding proteins/complexes. Msf1is a component of a thylakoid complex containing key enzymes of the tetrapyrrole biosynthetic pathway, thus revealing a possible link between Msf1 and chlorophyll biosynthesis. Protein interaction assays and greening experiments demonstrate that Msf1 interacts with Copper target homolog1 (CHL27B) and accumulates concomitantly with chlorophyll in Chlamydomonas, implying that chlorophyll stabilizes Msf1. Contrary to other light-harvesting complex-like genes, the expression of Msf1 is not stimulated by high-light stress, but its protein level increases significantly under heat shock, iron and copper limitation, as well as in stationary cells. Based on these results, we propose that Msf1 is required for the maintenance of photosystem I and specific protein-chlorophyll complexes especially under certain stress conditions. PMID:28637830

  6. Brachyspira hyodysenteriae and B. pilosicoli Proteins Recognized by Sera of Challenged Pigs.

    PubMed

    Casas, Vanessa; Rodríguez-Asiain, Arantza; Pinto-Llorente, Roberto; Vadillo, Santiago; Carrascal, Montserrat; Abian, Joaquin

    2017-01-01

    The spirochetes Brachyspira hyodysenteriae and B. pilosicoli are pig intestinal pathogens that are the causative agents of swine dysentery (SD) and porcine intestinal spirochaetosis (PIS), respectively. Although some inactivated bacterin and recombinant vaccines have been explored as prophylactic treatments against these species, no effective vaccine is yet available. Immunoproteomics approaches hold the potential for the identification of new, suitable candidates for subunit vaccines against SD and PIS. These strategies take into account the gene products actually expressed and present in the cells, and thus susceptible of being targets of immune recognition. In this context, we have analyzed the immunogenic pattern of two B. pilosicoli porcine isolates (the Spanish farm isolate OLA9 and the commercial P43/6/78 strain) and one B. hyodysenteriae isolate (the Spanish farm V1). The proteins from the Brachyspira lysates were fractionated by preparative isoelectric focusing, and the fractions were analyzed by Western blot with hyperimmune sera from challenged pigs. Of the 28 challenge-specific immunoreactive bands detected, 21 were identified as single proteins by MS, while the other 7 were shown to contain several major proteins. None of these proteins were detected in the control immunoreactive bands. The proteins identified included 11 from B. hyodysenteriae and 28 from the two B. pilosicoli strains. Eight proteins were common to the B. pilosicoli strains (i.e., elongation factor G, aspartyl-tRNA synthase, biotin lipoyl, TmpB outer membrane protein, flagellar protein FlaA, enolase, PEPCK, and VspD), and enolase and PEPCK were common to both species . Many of the identified proteins were flagellar proteins or predicted to be located on the cell surface and some of them had been previously described as antigenic or as bacterial virulence factors. Here we report on the identification and semiquantitative data of these immunoreactive proteins which constitute a unique

  7. Identification of Substituted Pyrimido[5,4-b]indoles as Selective Toll-Like Receptor 4 Ligands

    PubMed Central

    2013-01-01

    A cell-based high-throughput screen to identify small molecular weight stimulators of the innate immune system revealed substituted pyrimido[5,4-b]indoles as potent NFκB activators. The most potent hit compound selectively stimulated Toll-like receptor 4 (TLR4) in human and mouse cells. Synthetic modifications of the pyrimido[5,4-b]indole scaffold at the carboxamide, N-3, and N-5 positions revealed differential TLR4 dependent production of NFκB and type I interferon associated cytokines, IL-6 and interferon γ-induced protein 10 (IP-10) respectively. Specifically, a subset of compounds bearing phenyl and substituted phenyl carboxamides induced lower IL-6 release while maintaining higher IP-10 production, skewing toward the type I interferon pathway. Substitution at N-5 with short alkyl substituents reduced the cytotoxicity of the leading hit compound. Computational studies supported that active compounds appeared to bind primarily to MD-2 in the TLR4/MD-2 complex. These small molecules, which stimulate innate immune cells with minimal toxicity, could potentially be used as adjuvants or immune modulators. PMID:23656327

  8. Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx.

    PubMed

    Jiang, Tianyu; Liu, Minhao; Wu, Jianping; Shi, Yigong

    2016-02-23

    HBx is a hepatitis B virus protein that is required for viral infectivity and replication. Anti-apoptotic Bcl-2 family members are thought to be among the important host targets of HBx. However, the structure and function of HBx are poorly understood and the molecular mechanism of HBx-induced carcinogenesis remains unknown. In this study, we report biochemical and structural characterization of HBx. The recombinant HBx protein contains metal ions, in particular iron and zinc. A BH3-like motif in HBx (residues 110-135) binds Bcl-2 with a dissociation constant of ∼193 μM, which is drastically lower than that for a canonical BH3 motif from Bim or Bad. Structural analysis reveals that, similar to other BH3 motifs, the BH3-like motif of HBx adopts an amphipathic α-helix and binds the conserved BH3-binding groove on Bcl-2. Unlike the helical Bim or Bad BH3 motif, the C-terminal portion of the bound HBx BH3-like motif has an extended conformation and makes considerably fewer interactions with Bcl-2. These observations suggest that HBx may modulate Bcl-2 function in a way that is different from that of the classical BH3-only proteins.

  9. Ubiquitin-like and ubiquitin-associated domain proteins: significance in proteasomal degradation

    PubMed Central

    Lau, Alan F.

    2009-01-01

    The ubiquitin–proteasome pathway of protein degradation is one of the major mechanisms that are involved in the maintenance of the proper levels of cellular proteins. The regulation of proteasomal degradation thus ensures proper cell functions. The family of proteins containing ubiquitin-like (UbL) and ubiquitin-associated (UBA) domains has been implicated in proteasomal degradation. UbL–UBA domain containing proteins associate with substrates destined for degradation as well as with subunits of the proteasome, thus regulating the proper turnover of proteins. PMID:19468686

  10. Bay11-7082 attenuates neuropathic pain via inhibition of nuclear factor-kappa B and nucleotide-binding domain-like receptor protein 3 inflammasome activation in dorsal root ganglions in a rat model of lumbar disc herniation

    PubMed Central

    Zhang, Ailiang; Wang, Kun; Ding, Lianghua; Bao, Xinnan; Wang, Xuan; Qiu, Xubin; Liu, Jinbo

    2017-01-01

    Lumbar disc herniation (LDH) is an important cause of radiculopathy, but the underlying mechanisms are incompletely understood. Many studies suggested that local inflammation, rather than mechanical compression, results in radiculopathy induced by LDH. On the molecular and cellular level, nuclear factor-kappa B (NF-κB) and nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome have been implicated in the regulation of neuroinflammation formation and progression. In this study, the autologous nucleus pulposus (NP) was implanted in the left L5 dorsal root ganglion (DRG) to mimic LDH in rats. We investigated the expression of NF-κB and the components of NLRP3 inflammasome in the DRG neurons in rats. Western blotting and immunofluorescence for the related molecules, including NLRP3, apoptosis-associated speck-like protein containing caspase-1 activator domain (ASC), caspase-1, interleukin (IL)-1β, IL-18, IκBα, p-IκBα, p65, p-p65, and calcitonin gene-related peptide (CGRP) were examined. In the NP-treated group, the activations of NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65 in DRG neurons in rats were elevated at 1 day after surgery, and the peak occurred at 7 days. Treatment with Bay11-7082, an inhibitor of the actions of IKK-β, was able to inhibit expression and activation of the molecules (NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65) and relieve the pain in rats. Our study shows that NF-κB and NLRP3 inflammasome are involved in the maintenance of NP-induced pain, and that Bay11-7082 could alleviate mechanical allodynia and thermal hyperalgesia by inhibiting NF-κB and NLRP3 inflammasome activation. PMID:28243141

  11. Leptospira Immunoglobulin-Like Protein B (LigB) Binds to Both the C-Terminal 23 Amino Acids of Fibrinogen αC Domain and Factor XIII: Insight into the Mechanism of LigB-Mediated Blockage of Fibrinogen α Chain Cross-Linking.

    PubMed

    Hsieh, Ching-Lin; Chang, Eric; Tseng, Andrew; Ptak, Christopher; Wu, Li-Chen; Su, Chun-Li; McDonough, Sean P; Lin, Yi-Pin; Chang, Yung-Fu

    2016-09-01

    The coagulation system provides a primitive but effective defense against hemorrhage. Soluble fibrinogen (Fg) monomers, composed of α, β and γ chains, are recruited to provide structural support for the formation of a hemostatic plug. Fg binds to platelets and is processed into a cross-linked fibrin polymer by the enzymatic clotting factors, thrombin and Factor XIII (FXIII). The newly formed fibrin-platelet clot can act as barrier to protect against pathogens from entering the bloodstream. Further, injuries caused by bacterial infections can be confined to the initial wound site. Many pathogenic bacteria have Fg-binding adhesins that can circumvent the coagulation pathway and allow the bacteria to sidestep containment. Fg expression is upregulated during lung infection providing an attachment surface for bacteria with the ability to produce Fg-binding adhesins. Fg binding by leptospira might play a crucial factor in Leptospira-associated pulmonary hemorrhage, the main factor contributing to lethality in severe cases of leptospirosis. The 12th domain of Leptospira immunoglobulin-like protein B (LigB12), a leptospiral adhesin, interacts with the C-terminus of FgαC (FgαCC). In this study, the binding site for LigB12 was mapped to the final 23 amino acids at the C-terminal end of FgαCC (FgαCC8). The association of FgαCC8 with LigB12 (ELISA, KD = 0.76 μM; SPR, KD = 0.96 μM) was reduced by mutations of both charged residues (R608, R611 and H614 from FgαCC8; D1061 from LigB12) and hydrophobic residues (I613 from FgαCC8; F1054 and A1065 from LigB12). Additionally, LigB12 bound strongly to FXIII and also inhibited fibrin formation, suggesting that LigB can disrupt coagulation by suppressing FXIII activity. Here, the detailed binding mechanism of a leptospiral adhesin to a host hemostatic factor is characterized for the first time and should provide better insight into the pathogenesis of leptospirosis.

  12. Leptospira Immunoglobulin-Like Protein B (LigB) Binds to Both the C-Terminal 23 Amino Acids of Fibrinogen αC Domain and Factor XIII: Insight into the Mechanism of LigB-Mediated Blockage of Fibrinogen α Chain Cross-Linking

    PubMed Central

    Hsieh, Ching-Lin; Chang, Eric; Tseng, Andrew; Ptak, Christopher; Wu, Li-Chen; Su, Chun-Li; McDonough, Sean P.; Lin, Yi-Pin; Chang, Yung-Fu

    2016-01-01

    The coagulation system provides a primitive but effective defense against hemorrhage. Soluble fibrinogen (Fg) monomers, composed of α, β and γ chains, are recruited to provide structural support for the formation of a hemostatic plug. Fg binds to platelets and is processed into a cross-linked fibrin polymer by the enzymatic clotting factors, thrombin and Factor XIII (FXIII). The newly formed fibrin-platelet clot can act as barrier to protect against pathogens from entering the bloodstream. Further, injuries caused by bacterial infections can be confined to the initial wound site. Many pathogenic bacteria have Fg-binding adhesins that can circumvent the coagulation pathway and allow the bacteria to sidestep containment. Fg expression is upregulated during lung infection providing an attachment surface for bacteria with the ability to produce Fg-binding adhesins. Fg binding by leptospira might play a crucial factor in Leptospira-associated pulmonary hemorrhage, the main factor contributing to lethality in severe cases of leptospirosis. The 12th domain of Leptospira immunoglobulin-like protein B (LigB12), a leptospiral adhesin, interacts with the C-terminus of FgαC (FgαCC). In this study, the binding site for LigB12 was mapped to the final 23 amino acids at the C-terminal end of FgαCC (FgαCC8). The association of FgαCC8 with LigB12 (ELISA, KD = 0.76 μM; SPR, KD = 0.96 μM) was reduced by mutations of both charged residues (R608, R611 and H614 from FgαCC8; D1061 from LigB12) and hydrophobic residues (I613 from FgαCC8; F1054 and A1065 from LigB12). Additionally, LigB12 bound strongly to FXIII and also inhibited fibrin formation, suggesting that LigB can disrupt coagulation by suppressing FXIII activity. Here, the detailed binding mechanism of a leptospiral adhesin to a host hemostatic factor is characterized for the first time and should provide better insight into the pathogenesis of leptospirosis. PMID:27622634

  13. Mutations in STX1B, encoding a presynaptic protein, cause fever-associated epilepsy syndromes.

    PubMed

    Schubert, Julian; Siekierska, Aleksandra; Langlois, Mélanie; May, Patrick; Huneau, Clément; Becker, Felicitas; Muhle, Hiltrud; Suls, Arvid; Lemke, Johannes R; de Kovel, Carolien G F; Thiele, Holger; Konrad, Kathryn; Kawalia, Amit; Toliat, Mohammad R; Sander, Thomas; Rüschendorf, Franz; Caliebe, Almuth; Nagel, Inga; Kohl, Bernard; Kecskés, Angela; Jacmin, Maxime; Hardies, Katia; Weckhuysen, Sarah; Riesch, Erik; Dorn, Thomas; Brilstra, Eva H; Baulac, Stephanie; Møller, Rikke S; Hjalgrim, Helle; Koeleman, Bobby P C; Jurkat-Rott, Karin; Lehman-Horn, Frank; Roach, Jared C; Glusman, Gustavo; Hood, Leroy; Galas, David J; Martin, Benoit; de Witte, Peter A M; Biskup, Saskia; De Jonghe, Peter; Helbig, Ingo; Balling, Rudi; Nürnberg, Peter; Crawford, Alexander D; Esguerra, Camila V; Weber, Yvonne G; Lerche, Holger

    2014-12-01

    Febrile seizures affect 2-4% of all children and have a strong genetic component. Recurrent mutations in three main genes (SCN1A, SCN1B and GABRG2) have been identified that cause febrile seizures with or without epilepsy. Here we report the identification of mutations in STX1B, encoding syntaxin-1B, that are associated with both febrile seizures and epilepsy. Whole-exome sequencing in independent large pedigrees identified cosegregating STX1B mutations predicted to cause an early truncation or an in-frame insertion or deletion. Three additional nonsense or missense mutations and a de novo microdeletion encompassing STX1B were then identified in 449 familial or sporadic cases. Video and local field potential analyses of zebrafish larvae with antisense knockdown of stx1b showed seizure-like behavior and epileptiform discharges that were highly sensitive to increased temperature. Wild-type human syntaxin-1B but not a mutated protein rescued the effects of stx1b knockdown in zebrafish. Our results thus implicate STX1B and the presynaptic release machinery in fever-associated epilepsy syndromes.

  14. Functional characterisation of a TLR accessory protein, UNC93B1, in Atlantic salmon (Salmo salar).

    PubMed

    Lee, P T; Zou, J; Holland, J W; Martin, S A M; Scott, C J W; Kanellos, T; Secombes, C J

    2015-05-01

    Toll-like receptors (TLRs) are indispensable components of the innate immune system, which recognise conserved pathogen associated molecular patterns (PAMPs) and induce a series of defensive immune responses to protect the host. Biosynthesis, localisation and activation of TLRs are dependent on TLR accessory proteins. In this study, we identified the accessory protein, UNC93B1, from Atlantic salmon (Salmo salar) whole-genome shotgun (WGS) contigs aided by the conserved gene synteny of genes flanking UNC93B1 in fish, birds and mammals. Phylogenetic analysis showed that salmon UNC93B1 grouped with other vertebrate UNC93B1 molecules, and had highest amino acid identity and similarity to zebrafish UNC93B1. The salmon UNC93B1 gene organisation was also similar in structure to mammalian UNC93B1. Our gene expression studies revealed that salmon UNC93B1 was more highly expressed in spleen, liver and gill tissues but was expressed at a lower level in head kidney tissue in post-smolts relative to parr. Moreover, salmon UNC93B1 mRNA transcripts were up-regulated in vivo in spleen tissue from polyI:C treated salmon and in vitro in polyI:C or IFNγ stimulated Salmon Head Kidney-1 (SHK-1) cells. Initial studies into the functional role of salmon UNC93B1 in fish TLR signalling found that both wild type salmon UNC93B1 and a molecule with a site-directed mutation (H424R) co-immunoprecipitated with salmon TLR19, TLR20a and TLR20d. Overall, these data illustrate the potential importance of UNC93B1 as an accessory protein in fish TLR signalling. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity.

    PubMed

    Shagin, Dmitry A; Barsova, Ekaterina V; Yanushevich, Yurii G; Fradkov, Arkady F; Lukyanov, Konstantin A; Labas, Yulii A; Semenova, Tatiana N; Ugalde, Juan A; Meyers, Ann; Nunez, Jose M; Widder, Edith A; Lukyanov, Sergey A; Matz, Mikhail V

    2004-05-01

    Homologs of the green fluorescent protein (GFP), including the recently described GFP-like domains of certain extracellular matrix proteins in Bilaterian organisms, are remarkably similar at the protein structure level, yet they often perform totally unrelated functions, thereby warranting recognition as a superfamily. Here we describe diverse GFP-like proteins from previously undersampled and completely new sources, including hydromedusae and planktonic Copepoda. In hydromedusae, yellow and nonfluorescent purple proteins were found in addition to greens. Notably, the new yellow protein seems to follow exactly the same structural solution to achieving the yellow color of fluorescence as YFP, an engineered yellow-emitting mutant variant of GFP. The addition of these new sequences made it possible to resolve deep-level phylogenetic relationships within the superfamily. Fluorescence (most likely green) must have already existed in the common ancestor of Cnidaria and Bilateria, and therefore GFP-like proteins may be responsible for fluorescence and/or coloration in virtually any animal. At least 15 color diversification events can be inferred following the maximum parsimony principle in Cnidaria. Origination of red fluorescence and nonfluorescent purple-blue colors on several independent occasions provides a remarkable example of convergent evolution of complex features at the molecular level.

  16. Crystal structure of the protein At3g01520, a eukaryotic universal stress protein-like protein from Arabidopsis thaliana in complex with AMP.

    PubMed

    Kim, Do Jin; Bitto, Eduard; Bingman, Craig A; Kim, Hyun-Jung; Han, Byung Woo; Phillips, George N

    2015-07-01

    Members of the universal stress protein (USP) family are conserved in a phylogenetically diverse range of prokaryotes, fungi, protists, and plants and confer abilities to respond to a wide range of environmental stresses. Arabidopsis thaliana contains 44 USP domain-containing proteins, and USP domain is found either in a small protein with unknown physiological function or in an N-terminal portion of a multi-domain protein, usually a protein kinase. Here, we report the first crystal structure of a eukaryotic USP-like protein encoded from the gene At3g01520. The crystal structure of the protein At3g01520 was determined by the single-wavelength anomalous dispersion method and refined to an R factor of 21.8% (Rfree = 26.1%) at 2.5 Å resolution. The crystal structure includes three At3g01520 protein dimers with one AMP molecule bound to each protomer, comprising a Rossmann-like α/β overall fold. The bound AMP and conservation of residues in the ATP-binding loop suggest that the protein At3g01520 also belongs to the ATP-binding USP subfamily members. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

  17. On the mineral core of ferritin-like proteins: structural and magnetic characterization

    NASA Astrophysics Data System (ADS)

    García-Prieto, A.; Alonso, J.; Muñoz, D.; Marcano, L.; Abad Díaz de Cerio, A.; Fernández de Luis, R.; Orue, I.; Mathon, O.; Muela, A.; Fdez-Gubieda, M. L.

    2015-12-01

    It is generally accepted that the mineral core synthesized by ferritin-like proteins consists of a ferric oxy-hydroxide mineral similar to ferrihydrite in the case of horse spleen ferritin (HoSF) and an oxy-hydroxide-phosphate phase in plant and prokaryotic ferritins. The structure reflects a dynamic process of deposition and dissolution, influenced by different biological, chemical and physical variables. In this work we shed light on this matter by combining a structural (High Resolution Transmission Electron Microscopy (HRTEM) and Fe K-edge X-ray Absorption Spectroscopy (XAS)) and a magnetic study of the mineral core biomineralized by horse spleen ferritin (HoSF) and three prokaryotic ferritin-like proteins: bacterial ferritin (FtnA) and bacterioferritin (Bfr) from Escherichia coli and archaeal ferritin (PfFtn) from Pyrococcus furiosus. The prokaryotic ferritin-like proteins have been studied under native conditions and inside the cells for the sake of preserving their natural attributes. They share with HoSF a nanocrystalline structure rather than an amorphous one as has been frequently reported. However, the presence of phosphorus changes drastically the short-range order and magnetic response of the prokaryotic cores with respect to HoSF. The superparamagnetism observed in HoSF is absent in the prokaryotic proteins, which show a pure atomic-like paramagnetic behaviour attributed to phosphorus breaking the Fe-Fe exchange interaction.It is generally accepted that the mineral core synthesized by ferritin-like proteins consists of a ferric oxy-hydroxide mineral similar to ferrihydrite in the case of horse spleen ferritin (HoSF) and an oxy-hydroxide-phosphate phase in plant and prokaryotic ferritins. The structure reflects a dynamic process of deposition and dissolution, influenced by different biological, chemical and physical variables. In this work we shed light on this matter by combining a structural (High Resolution Transmission Electron Microscopy (HRTEM

  18. A nitrogen response pathway regulates virulence functions in Fusarium oxysporum via the protein kinase TOR and the bZIP protein MeaB.

    PubMed

    López-Berges, Manuel S; Rispail, Nicolas; Prados-Rosales, Rafael C; Di Pietro, Antonio

    2010-07-01

    During infection, fungal pathogens activate virulence mechanisms, such as host adhesion, penetration and invasive growth. In the vascular wilt fungus Fusarium oxysporum, the mitogen-activated protein kinase Fmk1 is required for plant infection and controls processes such as cellophane penetration, vegetative hyphal fusion, or root adhesion. Here, we show that these virulence-related functions are repressed by the preferred nitrogen source ammonium and restored by treatment with l-methionine sulfoximine or rapamycin, two specific inhibitors of Gln synthetase and the protein kinase TOR, respectively. Deletion of the bZIP protein MeaB also resulted in nitrogen source-independent activation of virulence mechanisms. Activation of these functions did not require the global nitrogen regulator AreA, suggesting that MeaB-mediated repression of virulence functions does not act through inhibition of AreA. Tomato plants (Solanum lycopersicum) supplied with ammonium rather than nitrate showed a significant reduction in vascular wilt symptoms when infected with the wild type but not with the DeltameaB strain. Nitrogen source also affected invasive growth in the rice blast fungus Magnaporthe oryzae and the wheat head blight pathogen Fusarium graminearum. We propose that a conserved nitrogen-responsive pathway might operate via TOR and MeaB to control virulence in plant pathogenic fungi.

  19. Plasmonic photocatalyst-like fluorescent proteins for generating reactive oxygen species

    NASA Astrophysics Data System (ADS)

    Leem, Jung Woo; Kim, Seong-Ryul; Choi, Kwang-Ho; Kim, Young L.

    2018-03-01

    The recent advances in photocatalysis have opened a variety of new possibilities for energy and biomedical applications. In particular, plasmonic photocatalysis using hybridization of semiconductor materials and metal nanoparticles has recently facilitated the rapid progress in enhancing photocatalytic efficiency under visible or solar light. One critical underlying aspect of photocatalysis is that it generates and releases reactive oxygen species (ROS) as intermediate or final products upon light excitation or activation. Although plasmonic photocatalysis overcomes the limitation of UV irradiation, synthesized metal/semiconductor nanomaterial photocatalysts often bring up biohazardous and environmental issues. In this respect, this review article is centered in identifying natural photosensitizing organic materials that can generate similar types of ROS as those of plasmonic photocatalysis. In particular, we propose the idea of plasmonic photocatalyst-like fluorescent proteins for ROS generation under visible light irradiation. We recapitulate fluorescent proteins that have Type I and Type II photosensitization properties in a comparable manner to plasmonic photocatalysis. Plasmonic photocatalysis and protein photosensitization have not yet been compared systemically in terms of ROS photogeneration under visible light, although the phototoxicity and cytotoxicity of some fluorescent proteins are well recognized. A comprehensive understanding of plasmonic photocatalyst-like fluorescent proteins and their potential advantages will lead us to explore new environmental, biomedical, and defense applications.

  20. Regulated binding of PTP1B-like phosphatase to N-cadherin: control of cadherin-mediated adhesion by dephosphorylation of beta-catenin

    PubMed Central

    1996-01-01

    Cadherins are a family of cell-cell adhesion molecules which play a central role in controlling morphogenetic movements during development. Cadherin function is regulated by its association with the actin containing cytoskeleton, an association mediated by a complex of cytoplasmic proteins, the catenins: alpha, beta, and gamma. Phosphorylated tyrosine residues on beta-catenin are correlated with loss of cadherin function. Consistent with this, we find that only nontyrosine phosphorylated beta-catenin is associated with N-cadherin in E10 chick retina tissue. Moreover, we demonstrate that a PTP1B-like tyrosine phosphatase associates with N-cadherin and may function as a regulatory switch controlling cadherin function by dephosphorylating beta-catenin, thereby maintaining cells in an adhesion-competent state. The PTP1B-like phosphatase is itself tyrosine phosphorylated. Moreover, both direct binding experiments performed with phosphorylated and dephosphorylated molecules, and treatment of cells with tyrosine kinase inhibitors indicate that the interaction of the PTP1B-like phosphatase with N-cadherin depends on its tyrosine phosphorylation. Concomitant with the tyrosine kinase inhibitor-induced loss of the PTP1B-like phosphatase from its association with N-cadherin, phosphorylated tyrosine residues are retained on beta-catenin, the association of N- cadherin with the actin containing cytoskeleton is lost and N-cadherin- mediated cell adhesion is prevented. Tyrosine phosphatase inhibitors also result in the accumulation of phosphorylated tyrosine residues on beta-catenin, loss of the association of N-cadherin with the actin- containing cytoskeleton, and prevent N-cadherin mediated adhesion, presumably by directly blocking the function of the PTP1B-like phosphatase. We previously showed that the binding of two ligands to the cell surface N-acetylgalactosaminylphosphotransferase (GalNAcPTase), the monoclonal antibody 1B11 and a proteoglycan with a 250-kD core protein

  1. ACBD3 functions as a scaffold to organize the Golgi stacking proteins and a Rab33b-GAP.

    PubMed

    Yue, Xihua; Bao, Mengjing; Christiano, Romain; Li, Siyang; Mei, Jia; Zhu, Lianhui; Mao, Feifei; Yue, Qiang; Zhang, Panpan; Jing, Shuaiyang; Rothman, James E; Qian, Yi; Lee, Intaek

    2017-09-01

    Golgin45 plays important roles in Golgi stack assembly and is known to bind both the Golgi stacking protein GRASP55 and Rab2 in the medial-Golgi cisternae. In this study, we sought to further characterize the cisternal adhesion complex using a proteomics approach. We report here that Acyl-CoA binding domain containing 3 (ACBD3) is likely to be a novel binding partner of Golgin45. ACBD3 interacts with Golgin45 via its GOLD domain, while its co-expression significantly increases Golgin45 targeting to the Golgi. Furthermore, ACBD3 recruits TBC1D22, a Rab33b GTPase activating protein (GAP), to a large multi-protein complex containing Golgin45 and GRASP55. These results suggest that ACBD3 may provide a scaffolding to organize the Golgi stacking proteins and a Rab33b-GAP at the medial-Golgi. © 2017 Federation of European Biochemical Societies.

  2. PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice.

    PubMed

    Zinker, Bradley A; Rondinone, Cristina M; Trevillyan, James M; Gum, Rebecca J; Clampit, Jill E; Waring, Jeffrey F; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E; Reilly, Regina M; Koterski, Sandra; Opgenorth, Terry J; Ulrich, Roger G; Crosby, Seth; Butler, Madeline; Murray, Susan F; McKay, Robert A; Bhanot, Sanjay; Monia, Brett P; Jirousek, Michael R

    2002-08-20

    The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA(1C). Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50alpha, were increased and PI3-kinase p85alpha expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes.

  3. Synthesis, purification and crystallographic studies of the C-terminal sterol carrier protein type 2 (SCP-2) domain of human hydroxysteroid dehydrogenase-like protein 2.

    PubMed

    Cheng, Zhong; Li, Yao; Sui, Chun; Sun, Xiaobo; Xie, Yong

    2015-07-01

    Human hydroxysteroid dehydrogenase-like protein 2 (HSDL2) is a member of the short-chain dehydrogenase/reductase (SDR) subfamily of oxidoreductases and contains an N-terminal catalytic domain and a C-termianl sterol carrier protein type 2 (SCP-2) domain. In this study, the C-terminal SCP-2 domain of human HSDL2, including residues Lys318-Arg416, was produced in Escherichia coli, purified and crystallized. X-ray diffraction data were collected to 2.10 Å resolution. The crystal belonged to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a = b = 70.4, c = 60.6 Å, α = β = 90, γ = 120°. Two protein molecules are present in the asymmetric unit, resulting in a Matthews coefficient of 2.16 Å(3) Da(-1) and an approximate solvent content of 43%.

  4. Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1

    PubMed Central

    Sun, Fei; Huang, Li

    2013-01-01

    Strand displacement by a DNA polymerase serves a key role in Okazaki fragment maturation, which involves displacement of the RNA primer of the preexisting Okazaki fragment into a flap structure, and subsequent flap removal and fragment ligation. We investigated the role of Sulfolobus chromatin proteins Sso7d and Cren7 in strand displacement by DNA polymerase B1 (PolB1) from the hyperthermophilic archaeon Sulfolobus solfataricus. PolB1 showed a robust strand displacement activity and was capable of synthesizing thousands of nucleotides on a DNA-primed 72-nt single-stranded circular DNA template. This activity was inhibited by both Sso7d and Cren7, which limited the flap length to 3–4 nt at saturating concentrations. However, neither protein inhibited RNA displacement on an RNA-primed single-stranded DNA minicircle by PolB1. Strand displacement remained sensitive to modulation by the chromatin proteins when PolB1 was in association with proliferating cell nuclear antigen. Inhibition of DNA instead of RNA strand displacement by the chromatin proteins is consistent with the finding that double-stranded DNA was more efficiently bound and stabilized than an RNA:DNA duplex by these proteins. Our results suggest that Sulfolobus chromatin proteins modulate strand displacement by PolB1, permitting efficient removal of the RNA primer while inhibiting excessive displacement of the newly synthesized DNA strand during Okazaki fragment maturation. PMID:23821667

  5. Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions.

    PubMed

    Gillette, William K; Esposito, Dominic; Abreu Blanco, Maria; Alexander, Patrick; Bindu, Lakshman; Bittner, Cammi; Chertov, Oleg; Frank, Peter H; Grose, Carissa; Jones, Jane E; Meng, Zhaojing; Perkins, Shelley; Van, Que; Ghirlando, Rodolfo; Fivash, Matthew; Nissley, Dwight V; McCormick, Frank; Holderfield, Matthew; Stephen, Andrew G

    2015-11-02

    Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer's disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5-10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ.

  6. Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions

    PubMed Central

    Gillette, William K.; Esposito, Dominic; Abreu Blanco, Maria; Alexander, Patrick; Bindu, Lakshman; Bittner, Cammi; Chertov, Oleg; Frank, Peter H.; Grose, Carissa; Jones, Jane E.; Meng, Zhaojing; Perkins, Shelley; Van, Que; Ghirlando, Rodolfo; Fivash, Matthew; Nissley, Dwight V.; McCormick, Frank; Holderfield, Matthew; Stephen, Andrew G.

    2015-01-01

    Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer’s disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5–10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ. PMID:26522388

  7. Plasmin cleaves fibrinogen and the human complement proteins C3b and C5 in the presence of Leptospira interrogans proteins: A new role of LigA and LigB in invasion and complement immune evasion.

    PubMed

    Castiblanco-Valencia, Mónica Marcela; Fraga, Tatiana Rodrigues; Pagotto, Ana Helena; Serrano, Solange Maria de Toledo; Abreu, Patricia Antonia Estima; Barbosa, Angela Silva; Isaac, Lourdes

    2016-05-01

    Plasminogen is a single-chain glycoprotein found in human plasma as the inactive precursor of plasmin. When converted to proteolytically active plasmin, plasmin(ogen) regulates both complement and coagulation cascades, thus representing an important target for pathogenic microorganisms. Leptospira interrogans binds plasminogen, which is converted to active plasmin. Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules that interact with extracellular matrix components and complement regulators, including proteins of the FH family and C4BP. In this work, we demonstrate that these multifunctional molecules also bind plasminogen through both N- and C-terminal domains. These interactions are dependent on lysine residues and are affected by ionic strength. Competition assays suggest that plasminogen does not share binding sites with C4BP or FH on Lig proteins at physiological molar ratios. Plasminogen bound to Lig proteins is converted to proteolytic active plasmin in the presence of urokinase-type plasminogen activator (uPA). Lig-bound plasmin is able to cleave the physiological substrates fibrinogen and the complement proteins C3b and C5. Taken together, our data point to a new role of LigA and LigB in leptospiral invasion and complement immune evasion. Plasmin(ogen) acquisition by these versatile proteins may contribute to Leptospira infection, favoring bacterial survival and dissemination inside the host. Copyright © 2016. Published by Elsevier GmbH.

  8. Intragenome Diversity of Gene Families Encoding Toxin-like Proteins in Venomous Animals.

    PubMed

    Rodríguez de la Vega, Ricardo C; Giraud, Tatiana

    2016-11-01

    The evolution of venoms is the story of how toxins arise and of the processes that generate and maintain their diversity. For animal venoms these processes include recruitment for expression in the venom gland, neofunctionalization, paralogous expansions, and functional divergence. The systematic study of these processes requires the reliable identification of the venom components involved in antagonistic interactions. High-throughput sequencing has the potential of uncovering the entire set of toxins in a given organism, yet the existence of non-venom toxin paralogs and the misleading effects of partial census of the molecular diversity of toxins make necessary to collect complementary evidence to distinguish true toxins from their non-venom paralogs. Here, we analyzed the whole genomes of two scorpions, one spider and one snake, aiming at the identification of the full repertoires of genes encoding toxin-like proteins. We classified the entire set of protein-coding genes into paralogous groups and monotypic genes, identified genes encoding toxin-like proteins based on known toxin families, and quantified their expression in both venom-glands and pooled tissues. Our results confirm that genes encoding toxin-like proteins are part of multigene families, and that these families arise by recruitment events from non-toxin genes followed by limited expansions of the toxin-like protein coding genes. We also show that failing to account for sequence similarity with non-toxin proteins has a considerable misleading effect that can be greatly reduced by comparative transcriptomics. Our study overall contributes to the understanding of the evolutionary dynamics of proteins involved in antagonistic interactions. © The Author 2016. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

  9. Engineering a pharmacologically superior form of granulocyte-colony-stimulating factor by fusion with gelatin-like-protein polymer.

    PubMed

    Huang, Yan-Shan; Wen, Xiao-Fang; Wu, Yi-Liang; Wang, Ye-Fei; Fan, Min; Yang, Zhi-Yu; Liu, Wei; Zhou, Lin-Fu

    2010-03-01

    The plasma half-life of therapeutic proteins is a critical factor in many clinical applications. Therefore, new strategies to prolong plasma half-life of long-acting peptides and protein drugs are in high demand. Here, we designed an artificial gelatin-like protein (GLK) and fused this hydrophilic GLK polymer to granulocyte-colony-stimulating factor (G-CSF) to generate a chimeric GLK/G-CSF fusion protein. The genetically engineered recombinant GLK/G-CSF (rGLK/G-CSF) fusion protein was purified from Pichia pastoris. In vitro studies demonstrated that rGLK/G-CSF possessed an enlarged hydrodynamic radius, improved thermal stability and retained full bioactivity compared to unfused G-CSF. Following a single subcutaneous administration to rats, the rGLK/G-CSF fusion protein displayed a slower plasma clearance rate and stimulated greater and longer lasting increases in circulating white blood cells than G-CSF. Our findings indicate that fusion with this artificial, hydrophilic, GLK polymer provides many advantages in the construction of a potent hematopoietic factor with extended plasma half-life. This approach could be easily applied to other therapeutic proteins and have important clinical applications. (c) 2009 Elsevier B.V. All rights reserved.

  10. Inhibition of interferon-inducible MxA protein expression by hepatitis B virus capsid protein.

    PubMed

    Rosmorduc, O; Sirma, H; Soussan, P; Gordien, E; Lebon, P; Horisberger, M; Bréchot, C; Kremsdorf, D

    1999-05-01

    Chronic hepatitis B treatment has been significantly improved by interferon (IFN) treatment. However, some studies have suggested that hepatitis B virus (HBV) might have a direct effect on the resistance to IFN. Defective particles, generated by spliced HBV RNA and associated with chronic hepatitis B, have been previously characterized; expression of these particles leads to cytoplasmic accumulation of the capsid protein. The aim of this study was to investigate the role of these defective genomes in IFN resistance. The global antiviral activity of IFN was studied by virus yield reduction assays, the expression of three IFN-induced antiviral proteins was analysed by Western blotting and confocal microscopy, and the regulation of MxA gene expression was studied by Northern blotting and the luciferase assay, in Huh7 cells transfected with a complete or the defective HBV genome. Results showed that the expression of the defective genome reduces the antiviral activity of IFN and that this modulation involves a selective inhibition of MxA protein induction by the HBV capsid protein. Our results also show the trans-suppressive effect of the HBV capsid on the MxA promoter, which might participate in this phenomenon. In conclusion, this study shows a direct interplay between the IFN-sensitive pathway and the capsid protein and might implicate this defective HBV genome in virus persistence.

  11. Anti-viral role of toll like receptor 4 in hepatitis B virus infection: An in vitro study.

    PubMed

    Das, Dipanwita; Sarkar, Neelakshi; Sengupta, Isha; Pal, Ananya; Saha, Debraj; Bandopadhyay, Manikankana; Das, Chandrima; Narayan, Jimmy; Singh, Shivram Prasad; Chakravarty, Runu

    2016-12-21

    Toll like receptors plays a significant anti-viral role in different infections. The aim of this study was to look into the role of toll like receptor 4 (TLR4) in hepatitis B virus (HBV) infection. Real time PCR was used to analyze the transcription of TLR4 signaling molecules, cell cycle regulators and HBV DNA viral load after triggering the HepG2.2.15 cells with TLR4 specific ligand. Nuclear factor (NF)-κB translocation on TLR4 activation was analyzed using microscopic techniques. Protein and cell cycle analysis was done using Western Blot and FACS respectively. The present study shows that TLR4 activation represses HBV infection. As a result of HBV suppression, there are several changes in host factors which include partial release in G1/S cell cycle arrest and changes in host epigenetic marks. Finally, it was observed that anti-viral action of TLR4 takes place through the NF-κB pathway. The study shows that TLR4 activation in HBV infection brings about changes in hepatocyte microenvironment and can be used for developing a promising therapeutic target in future.

  12. Inhibition of protein glycation by procyanidin-B2 enriched fraction of cinnamon: delay of diabetic cataract in rats.

    PubMed

    Muthenna, Puppala; Raghu, Ganugula; Akileshwari, Chandrasekhar; Sinha, Sukesh Narayana; Suryanarayana, Palla; Reddy, Geereddy Bhanuprakash

    2013-11-01

    Accumulation of advanced glycation endproducts (AGE) from nonenzymatic glycation of proteins has been implicated in several diabetic complications including diabetic cataract. Previously, we have reported that extracts of dietary agents such as cinnamon have the potential to inhibit AGE formation. In this study, we have shown procyanidin-B2 as the active component of cinnamon that is involved in AGE inhibition using bioassay-guided fractionation of eye lens proteins under in vitro conditions. The data indicate that procyanidin-B2 enriched fraction scavenges dicarbonyls. Further, procyanidin-B2 fraction of cinnamon inhibited the formation of glycosylated hemoglobin in human blood under ex vivo conditions. We have also demonstrated the physiological significance of procyanidin-B2 fraction in terms of delay of diabetic cataract through inhibition of AGE in diabetic rats. These findings establish the antiglycating potential of procyanidin-B2 fraction of cinnamon which suggests a scope for controlling AGE-mediated diabetic complications by food sources that are rich in proanthocyanidins like procyanidin-B2. © 2013 International Union of Biochemistry and Molecular Biology.

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

    DOE PAGES

    Fu, Xian; Liu, Rui; Sanchez, Iona; ...

    2016-05-17

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

  14. Diversities and similarities in pH dependency among bacterial NhaB-like Na+/H+ antiporters.

    PubMed

    Kiriyama, Wakako; Honma, Kei; Hiratsuka, Tomoaki; Takahashi, Itsuka; Nomizu, Takahiro; Takashima, Yuta; Ohtsuka, Masataka; Takahashi, Daiki; Moriyama, Kazuya; Mori, Sayoko; Nishiyama, Shiho; Fukuhara, Masahiro; Nakamura, Tatsunosuke; Shigematsu, Toru; Yamaguchi, Toshio

    2013-10-01

    NhaB-like antiporters were the second described class of Na(+)/H(+) antiporters, identified in bacteria more than 20 years ago. While nhaB-like gene sequences have been found in a number of bacterial genomes, only a few of the NhaB-like antiporters have been functionally characterized to date. Although earlier studies have identified a few pH-sensitive and -insensitive NhaB-like antiporters, the mechanisms that determine their pH responses still remain elusive. In this study, we sought to investigate the diversities and similarities among bacterial NhaB-like antiporters, with particular emphasis on their pH responsiveness. Our phylogenetic analysis of NhaB-like antiporters, combined with pH profile analyses of activities for representative members of several phylogenetic groups, demonstrated that NhaB-like antiporters could be classified into three distinct types according to the degree of their pH dependencies. Interestingly, pH-insensitive NhaB-like antiporters were only found in a limited proportion of enterobacterial species, which constitute a subcluster that appears to have diverged relatively recently among enterobacterial NhaB-like antiporters. Furthermore, kinetic property analyses of NhaB-like antiporters at different pH values revealed that the degree of pH sensitivity of antiport activities was strongly correlated with the magnitude of pH-dependent change in apparent Km values, suggesting that the dramatic pH sensitivities observed for several NhaB-like antiporters might be mainly due to the significant increases of apparent Km at lower pH. These results strongly suggested the possibility that the loss of pH sensitivity of NhaB-like antiporters had occurred relatively recently, probably via accumulation of the mutations that impair pH-dependent change of Km in the course of molecular evolution.

  15. Quantification of chitinase and thaumatin-like proteins in grape juices and wines.

    PubMed

    Le Bourse, D; Conreux, A; Villaume, S; Lameiras, P; Nuzillard, J-M; Jeandet, P

    2011-09-01

    Chitinases and thaumatin-like proteins are important grape proteins as they have a great influence on wine quality. The quantification of these proteins in grape juices and wines, along with their purification, is therefore crucial to study their intrinsic characteristics and the exact role they play in wines. The main isoforms of these two proteins from Chardonnay grape juice were thus purified by liquid chromatography. Two fast protein liquid chromatography (FLPC) steps allowed the fractionation and purification of the juice proteins, using cation exchange and hydrophobic interaction media. A further high-performance liquid chromatography (HPLC) step was used to achieve higher purity levels. Fraction assessment was achieved by mass spectrometry. Fraction purity was determined by HPLC to detect the presence of protein contaminants, and by nuclear magnetic resonance (NMR) spectroscopy to detect the presence of organic contaminants. Once pure fractions of lyophilized chitinase and thaumatin-like protein were obtained, ultra-HPLC (UHPLC) and enzyme-linked immunosorbent assay (ELISA) calibration curves were constructed. The quantification of these proteins in different grape juice and wine samples was thus achieved for the first time with both techniques through comparison with the purified protein calibration curve. UHPLC and ELISA showed very consistent results (less than 16% deviation for both proteins) and either could be considered to provide an accurate and reliable quantification of proteins in the oenology field.

  16. Structure of the substrate-binding b′ domain of the Protein disulfide isomerase-like protein of the testis

    PubMed Central

    Bastos-Aristizabal, Sara; Kozlov, Guennadi; Gehring, Kalle

    2014-01-01

    Protein Disulfide Isomerase-Like protein of the Testis (PDILT) is a testis-specific member of the PDI family. PDILT displays similar domain architecture to PDIA1, the founding member of this protein family, but lacks catalytic cysteines needed for oxidoreduction reactions. This suggests special importance of chaperone activity of PDILT, but how it recognizes misfolded protein substrates is unknown. Here, we report the high-resolution crystal structure of the b′ domain of human PDILT. The structure reveals a conserved hydrophobic pocket, which is likely a principal substrate-binding site in PDILT. In the crystal, this pocket is occupied by side chains of tyrosine and tryptophan residues from another PDILT molecule, suggesting a preference for binding exposed aromatic residues in protein substrates. The lack of interaction of the b′ domain with the P-domains of calreticulin-3 and calmegin hints at a novel way of interaction between testis-specific lectin chaperones and PDILT. Further studies of this recently discovered PDI member would help to understand the important role that PDILT plays in the differentiation and maturation of spermatozoids. PMID:24662985

  17. Cellular and functional specificity among ferritin-like proteins in the multicellular cyanobacterium Nostoc punctiforme.

    PubMed

    Ekman, Martin; Sandh, Gustaf; Nenninger, Anja; Oliveira, Paulo; Stensjö, Karin

    2014-03-01

    Ferritin-like proteins constitute a remarkably heterogeneous protein family, including ferritins, bacterioferritins and Dps proteins. The genome of the filamentous heterocyst-forming cyanobacterium Nostoc punctiforme encodes five ferritin-like proteins. In the present paper, we report a multidimensional characterization of these proteins. Our phylogenetic and bioinformatics analyses suggest both structural and physiological differences among the ferritin-like proteins. The expression of these five genes responded differently to hydrogen peroxide treatment, with a significantly higher rise in transcript level for Npun_F3730 as compared with the other four genes. A specific role for Npun_F3730 in the cells tolerance against hydrogen peroxide was also supported by the inactivation of Npun_F3730, Npun_R5701 and Npun_R6212; among these, only the ΔNpun_F3730 strain showed an increased sensitivity to hydrogen peroxide compared with wild type. Analysis of promoter-GFP reporter fusions of the ferritin-like genes indicated that Npun_F3730 and Npun_R5701 were expressed in all cell types of a diazotrophic culture, while Npun_F6212 was expressed specifically in heterocysts. Our study provides the first comprehensive analysis combining functional differentiation and cellular specificity within this important group of proteins in a multicellular cyanobacterium. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

  18. Nuclear actions of insulin-like growth factor binding protein-3.

    PubMed

    Baxter, Robert C

    2015-09-10

    In addition to its actions outside the cell, cellular uptake and nuclear import of insulin-like growth factor binding protein-3 (IGFBP-3) has been recognized for almost two decades, but knowledge of its nuclear actions has been slow to emerge. IGFBP-3 has a functional nuclear localization signal and interacts with the nuclear transport protein importin-β. Within the nucleus IGFBP-3 appears to have a role in transcriptional regulation. It can bind to the nuclear receptor, retinoid X receptor-α and several of its dimerization partners, including retinoic acid receptor, vitamin D receptor (VDR), and peroxisome proliferator-activated receptor-γ (PPARγ). These interactions modulate the functions of these receptors, for example inhibiting VDR-dependent transcription in osteoblasts and PPARγ-dependent transcription in adipocytes. Nuclear IGFBP-3 can be detected by immunohistochemistry in cancer and other tissues, and its presence in the nucleus has been shown in many cell culture studies to be necessary for its pro-apoptotic effect, which may also involve interaction with the nuclear receptor Nur77, and export from the nucleus. IGFBP-3 is p53-inducible and in response to DNA damage, forms a complex with the epidermal growth factor receptor (EGFR), translocating to the nucleus to interact with DNA-dependent protein kinase. Inhibition of EGFR kinase activity or downregulation of IGFBP-3 can inhibit DNA double strand-break repair by nonhomologous end joining. IGFBP-3 thus has the ability to influence many cell functions through its interactions with intranuclear pathways, but the importance of these interactions in vivo, and their potential to be targeted for therapeutic benefit, require further investigation. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35★

    PubMed Central

    Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; Pan, Gaofeng; Fu, Zhiping

    2012-01-01

    PC12 cell injury was induced using 20 μM amyloid β-protein 25–35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25–35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25–35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein. PMID:25745458

  20. Heat Shock Protein B1-Deficient Mice Display Impaired Wound Healing

    PubMed Central

    McNamee, Kay; Przybycien, Paulina M.; Lu, Xin; Williams, Richard O.; Bou-Gharios, George; Saklatvala, Jeremy; Dean, Jonathan L. E.

    2013-01-01

    There is large literature describing in vitro experiments on heat shock protein (hsp)B1 but understanding of its function in vivo is limited to studies in mice overexpressing human hspB1 protein. Experiments in cells have shown that hspB1 has chaperone activity, a cytoprotective role, regulates inflammatory gene expression, and drives cell proliferation. To investigate the function of the protein in vivo we generated hspB1-deficient mice. HspB1-deficient fibroblasts display increased expression of the pro-inflammatory cytokine, interleukin-6, compared to wild-type cells, but reduced proliferation. HspB1-deficient fibroblasts exhibit reduced entry into S phase and increased expression of cyclin-dependent kinase inhibitors p27kip1 and p21waf1. The expression of hspB1 protein and mRNA is also controlled by the cell cycle. To investigate the physiological function of hspB1 in regulating inflammation and cell proliferation we used an excisional cutaneous wound healing model. There was a significant impairment in the rate of healing of wounds in hspB1-deficient mice, characterised by reduced re-epithelialisation and collagen deposition but also increased inflammation. HspB1 deficiency augments neutrophil infiltration in wounds, driven by increased chemokine (C-X-C motif) ligand 1 expression. This appears to be a general mechanism as similar results were obtained in the air-pouch and peritonitis models of acute inflammation. PMID:24143227

  1. Molecular and Biochemical Characterization of a Cathepsin B-Like Protease Family Unique to Trypanosoma congolense▿ †

    PubMed Central

    Mendoza-Palomares, Carlos; Biteau, Nicolas; Giroud, Christiane; Coustou, Virginie; Coetzer, Theresa; Authié, Edith; Boulangé, Alain; Baltz, Théo

    2008-01-01

    Cysteine proteases have been shown to be essential virulence factors and drug targets in trypanosomatids and an attractive antidisease vaccine candidate for Trypanosoma congolense. Here, we describe an important amplification of genes encoding cathepsin B-like proteases unique to T. congolense. More than 13 different genes were identified, whereas only one or two highly homologous genes have been identified in other trypanosomatids. These proteases grouped into three evolutionary clusters: TcoCBc1 to TcoCBc5 and TcoCBc6, which possess the classical catalytic triad (Cys, His, and Asn), and TcoCBs7 to TcoCBs13, which contains an unusual catalytic site (Ser, Xaa, and Asn). Expression profiles showed that members of the TcoCBc1 to TcoCBc5 and the TcoCBs7 to TcoCBs13 groups are expressed mainly in bloodstream forms and localize in the lysosomal compartment. The expression of recombinant representatives of each group (TcoCB1, TcoCB6, and TcoCB12) as proenzymes showed that TcoCBc1 and TcoCBc6 are able to autocatalyze their maturation 21 and 31 residues, respectively, upstream of the predicted start of the catalytic domain. Both displayed a carboxydipeptidase function, while only TcoCBc1 behaved as an endopeptidase. TcoCBc1 exhibited biochemical differences regarding inhibitor sensitivity compared to that of other cathepsin B-like proteases. Recombinant pro-TcoCBs12 did not automature in vitro, and the pepsin-matured enzyme was inactive in tests with cathepsin B fluorogenic substrates. In vivo inhibition studies using CA074Me (a cell-permeable cathepsin B-specific inhibitor) demonstrated that TcoCB are involved in lysosomal protein degradation essential for survival in bloodstream form. Furthermore, TcoCBc1 elicited an important immune response in experimentally infected cattle. We propose this family of proteins as a potential therapeutic target and as a plausible antigen for T. congolense diagnosis. PMID:18281598

  2. PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice

    PubMed Central

    Zinker, Bradley A.; Rondinone, Cristina M.; Trevillyan, James M.; Gum, Rebecca J.; Clampit, Jill E.; Waring, Jeffrey F.; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E.; Reilly, Regina M.; Koterski, Sandra; Opgenorth, Terry J.; Ulrich, Roger G.; Crosby, Seth; Butler, Madeline; Murray, Susan F.; McKay, Robert A.; Bhanot, Sanjay; Monia, Brett P.; Jirousek, Michael R.

    2002-01-01

    The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50α, were increased and PI3-kinase p85α expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes. PMID:12169659

  3. Functional Redundancy of the B9 Proteins and Nephrocystins in Caenorhabditis elegans Ciliogenesis

    PubMed Central

    Williams, Corey L.; Winkelbauer, Marlene E.; Schafer, Jenny C.; Michaud, Edward J.

    2008-01-01

    Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and Joubert syndrome (JBTS) are a group of heterogeneous cystic kidney disorders with partially overlapping loci. Many of the proteins associated with these diseases interact and localize to cilia and/or basal bodies. One of these proteins is MKS1, which is disrupted in some MKS patients and contains a B9 motif of unknown function that is found in two other mammalian proteins, B9D2 and B9D1. Caenorhabditis elegans also has three B9 proteins: XBX-7 (MKS1), TZA-1 (B9D2), and TZA-2 (B9D1). Herein, we report that the C. elegans B9 proteins form a complex that localizes to the base of cilia. Mutations in the B9 genes do not overtly affect cilia formation unless they are in combination with a mutation in nph-1 or nph-4, the homologues of human genes (NPHP1 and NPHP4, respectively) that are mutated in some NPHP patients. Our data indicate that the B9 proteins function redundantly with the nephrocystins to regulate the formation and/or maintenance of cilia and dendrites in the amphid and phasmid ciliated sensory neurons. Together, these data suggest that the human homologues of the novel B9 genes B9D2 and B9D1 will be strong candidate loci for pathologies in human MKS, NPHP, and JBTS. PMID:18337471

  4. The Murine Factor H-Related Protein FHR-B Promotes Complement Activation.

    PubMed

    Cserhalmi, Marcell; Csincsi, Ádám I; Mezei, Zoltán; Kopp, Anne; Hebecker, Mario; Uzonyi, Barbara; Józsi, Mihály

    2017-01-01

    Factor H-related (FHR) proteins consist of varying number of complement control protein domains that display various degrees of sequence identity to respective domains of the alternative pathway complement inhibitor factor H (FH). While such FHR proteins are described in several species, only human FHRs were functionally investigated. Their biological role is still poorly understood and in part controversial. Recent studies on some of the human FHRs strongly suggest a role for FHRs in enhancing complement activation via competing with FH for binding to certain ligands and surfaces. The aim of the current study was the functional characterization of a murine FHR, FHR-B. To this end, FHR-B was expressed in recombinant form. Recombinant FHR-B bound to human C3b and was able to compete with human FH for C3b binding. FHR-B supported the assembly of functionally active C3bBb alternative pathway C3 convertase via its interaction with C3b. This activity was confirmed by demonstrating C3 activation in murine serum. In addition, FHR-B bound to murine pentraxin 3 (PTX3), and this interaction resulted in murine C3 fragment deposition due to enhanced complement activation in mouse serum. FHR-B also induced C3 deposition on C-reactive protein, the extracellular matrix (ECM) extract Matrigel, and endothelial cell-derived ECM when exposed to mouse serum. Moreover, mouse C3 deposition was strongly enhanced on necrotic Jurkat T cells and the mouse B cell line A20 by FHR-B. FHR-B also induced lysis of sheep erythrocytes when incubated in mouse serum with FHR-B added in excess. Altogether, these data demonstrate that, similar to human FHR-1 and FHR-5, mouse FHR-B modulates complement activity by promoting complement activation via interaction with C3b and via competition with murine FH.

  5. Crystallization and preliminary X-ray diffraction studies of NP24-I, an isoform of a thaumatin-like protein from ripe tomato fruits

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ghosh, Raka; Chakrabarti, Chandana, E-mail: chandana.chakrabarti@saha.ac.in

    2005-08-01

    A thaumatin-like antifungal protein, NP24-I, has been isolated from ripe tomato fruits. It was crystallized by the vapour-diffusion method and data were collected to 2.45 Å. The structure was solved by molecular replacement. NP24 is a 24 kDa (207-amino-acid) antifungal thaumatin-like protein (TLP) found in tomato fruits. An isoform of the protein, NP24-I, is reported to play a possible role in ripening of the fruit in addition to its antifungal properties. The protein has been isolated and purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the tetragonal space group P4{sub 3}, with unit-cell parameters a =more » b = 61.01, c = 62.90 Å and one molecule per asymmetric unit. X-ray diffraction data were processed to a resolution of 2.45 Å and the structure was solved by molecular replacement.« less

  6. Campylobacter jejuni transducer like proteins: Chemotaxis and beyond.

    PubMed

    Chandrashekhar, Kshipra; Kassem, Issmat I; Rajashekara, Gireesh

    2017-07-04

    Chemotaxis, a process that mediates directional motility toward or away from chemical stimuli (chemoeffectors/ligands that can be attractants or repellents) in the environment, plays an important role in the adaptation of Campylobacter jejuni to disparate niches. The chemotaxis system consists of core signal transduction proteins and methyl-accepting-domain-containing Transducer like proteins (Tlps). Ligands binding to Tlps relay a signal to chemotaxis proteins in the cytoplasm which initiate a signal transduction cascade, culminating into a directional flagellar movement. Tlps facilitate substrate-specific chemotaxis in C. jejuni, which plays an important role in the pathogen's adaptation, pathobiology and colonization of the chicken gastrointestinal tract. However, the role of Tlps in C. jejuni's host tissue specific colonization, physiology and virulence remains not completely understood. Based on recent studies, it can be predicted that Tlps might be important targets for developing strategies to control C. jejuni via vaccines and antimicrobials.

  7. Classical autophagy proteins LC3B and ATG4B facilitate melanosome movement on cytoskeletal tracks.

    PubMed

    Ramkumar, Amrita; Murthy, Divya; Raja, Desingu Ayyappa; Singh, Archana; Krishnan, Anusha; Khanna, Sangeeta; Vats, Archana; Thukral, Lipi; Sharma, Pushkar; Sivasubbu, Sridhar; Rani, Rajni; Natarajan, Vivek T; Gokhale, Rajesh S

    2017-08-03

    Macroautophagy/autophagy is a dynamic and inducible catabolic process that responds to a variety of hormonal and environmental cues. Recent studies highlight the interplay of this central pathway in a variety of pathophysiological diseases. Although defective autophagy is implicated in melanocyte proliferation and pigmentary disorders, the mechanistic relationship between the 2 pathways has not been elucidated. In this study, we show that autophagic proteins LC3B and ATG4B mediate melanosome trafficking on cytoskeletal tracks. While studying melanogenesis, we observed spatial segregation of LC3B-labeled melanosomes with preferential absence at the dendritic ends of melanocytes. This LC3B labeling of melanosomes did not impact the steady-state levels of these organelles but instead facilitated their intracellular positioning. Melanosomes primarily traverse on microtubule and actin cytoskeletal tracks and our studies reveal that LC3B enables the assembly of microtubule translocon complex. At the microtubule-actin crossover junction, ATG4B detaches LC3B from melanosomal membranes by enzymatic delipidation. Further, by live-imaging we show that melanosomes transferred to keratinocytes lack melanocyte-specific LC3B. Our study thus elucidates a new role for autophagy proteins in directing melanosome movement and reveal the unconventional use of these proteins in cellular trafficking pathways. Such crosstalk between the central cellular function and housekeeping pathway may be a crucial mechanism to balance melanocyte bioenergetics and homeostasis.

  8. Toll-like receptor 4 promotes proliferation and apoptosis resistance in human papillomavirus-related cervical cancer cells through the Toll-like receptor 4/nuclear factor-κB pathway.

    PubMed

    Jiang, Ninghong; Xie, Feng; Guo, Qisang; Li, Ming-Qing; Xiao, Jingjing; Sui, Long

    2017-06-01

    Toll-like receptor 4 is overexpressed in various tumors, including cervical carcinoma. However, the role of Toll-like receptor 4 in cervical cancer remains controversial, and the underlying mechanisms are largely elusive. Therefore, Toll-like receptor 4 in cervical cancer and related mechanisms were investigated in this study. Quantitative reverse transcription polymerase chain reaction and western blot analyses were used to detect messenger RNA and protein levels in HeLa, Caski, and C33A cells with different treatments. Proliferation was quantified using Cell Counting Kit-8. Cell cycle distribution and apoptosis were assessed by flow cytometry. Higher levels of Toll-like receptor 4 expression were found in human papillomavirus-positive cells compared to human papillomavirus-negative cells. Proliferation of HeLa and Caski cells was promoted in lipopolysaccharide-stimulated groups but suppressed in short hairpin RNA-transfected groups. Apoptosis rates were lower in lipopolysaccharide-stimulated groups relative to short hairpin RNA-transfected groups. In addition, G2-phase distribution was enhanced when Toll-like receptor 4 was downregulated. Moreover, the pNF-κBp65 level was positively correlated with the Toll-like receptor 4 level in HeLa and Caski cells, though when an nuclear factor-κB inhibitor was applied to lipopolysaccharide-stimulated groups, the patterns of proliferation and apoptosis were opposite to those of the lipopolysaccharide-stimulated groups without inhibitor treatment. In conclusion, these data suggest that Toll-like receptor 4 promotes proliferation and apoptosis resistance in human papillomavirus-related cervical cancer cells at least in part through the Toll-like receptor 4/nuclear factor-κB pathway, which may be correlated with the occurrence and development of cervical carcinoma.

  9. ProClaT, a new bioinformatics tool for in silico protein reclassification: case study of DraB, a protein coded from the draTGB operon in Azospirillum brasilense.

    PubMed

    Rubel, Elisa Terumi; Raittz, Roberto Tadeu; Coimbra, Nilson Antonio da Rocha; Gehlen, Michelly Alves Coutinho; Pedrosa, Fábio de Oliveira

    2016-12-15

    Azopirillum brasilense is a plant-growth promoting nitrogen-fixing bacteria that is used as bio-fertilizer in agriculture. Since nitrogen fixation has a high-energy demand, the reduction of N 2 to NH 4 + by nitrogenase occurs only under limiting conditions of NH 4 + and O 2 . Moreover, the synthesis and activity of nitrogenase is highly regulated to prevent energy waste. In A. brasilense nitrogenase activity is regulated by the products of draG and draT. The product of the draB gene, located downstream in the draTGB operon, may be involved in the regulation of nitrogenase activity by an, as yet, unknown mechanism. A deep in silico analysis of the product of draB was undertaken aiming at suggesting its possible function and involvement with DraT and DraG in the regulation of nitrogenase activity in A. brasilense. In this work, we present a new artificial intelligence strategy for protein classification, named ProClaT. The features used by the pattern recognition model were derived from the primary structure of the DraB homologous proteins, calculated by a ProClaT internal algorithm. ProClaT was applied to this case study and the results revealed that the A. brasilense draB gene codes for a protein highly similar to the nitrogenase associated NifO protein of Azotobacter vinelandii. This tool allowed the reclassification of DraB/NifO homologous proteins, hypothetical, conserved hypothetical and those annotated as putative arsenate reductase, ArsC, as NifO-like. An analysis of co-occurrence of draB, draT, draG and of other nif genes was performed, suggesting the involvement of draB (nifO) in nitrogen fixation, however, without the definition of a specific function.

  10. Functional dissection of the Hox protein Abdominal-B in Drosophila cell culture

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhai, Zongzhao; CellNetworks - Cluster of Excellence, Centre for Organismal Studies; Graduate School of Chinese Academy of Sciences, Beijing 100039

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer ct340 CRM was identified to be the posterior spiracle enhancer of gene cut. Black-Right-Pointing-Pointer ct340 is under the direct transcriptional control of Hox protein Abd-B. Black-Right-Pointing-Pointer An efficient cloning system was developed to assay protein-DNA interaction. Black-Right-Pointing-Pointer New features of Abd-B dependent target gene regulation were detected. -- Abstract: Hox transcription factors regulate the morphogenesis along the anterior-posterior (A/P) body axis through the interaction with small cis-regulatory modules (CRMs) of their target gene, however so far very few Hox CRMs are known and have been analyzed in detail. In this study we have identified a new Hox CRM,more » ct340, which guides the expression of the cell type specification gene cut (ct) in the posterior spiracle under the direct control of the Hox protein Abdominal-B (Abd-B). Using the ct340 enhancer activity as readout, an efficient cloning system to generate VP16 activation domain fusion protein was developed to unambiguously test protein-DNA interaction in Drosophila cell culture. By functionally dissecting the Abd-B protein, new features of Abd-B dependent target gene regulation were detected. Due to its easy adaptability, this system can be generally used to map functional domains within sequence-specific transcriptional factors in Drosophila cell culture, and thus provide preliminary knowledge of the protein functional domain structure for further in vivo analysis.« less

  11. HIV-1 Vif can directly inhibit apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G-mediated cytidine deamination by using a single amino acid interaction and without protein degradation.

    PubMed

    Santa-Marta, Mariana; da Silva, Frederico Aires; Fonseca, Ana Margarida; Goncalves, Joao

    2005-03-11

    The human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G), also known as CEM-15, is a host-cell factor involved in innate resistance to retroviral infection. HIV-1 viral infectivity factor (Vif) protein was shown to protect the virus from APOBEC3G-mediated viral cDNA hypermutation. The mechanism proposed for protection of the virus by HIV-1 Vif is mediated by APOBEC3G degradation through ubiquitination and the proteasomal pathway. Here we show that in Escherichia coli the APOBEC3G-induced cytidine deamination is inhibited by expression of Vif without depletion of deaminase. Moreover, inhibition of deaminase-mediated bacterial hypermutation is dependent on a single amino acid substitution D128K that renders APOBEC3G resistant to Vif inhibition. This single amino acid was elegantly proven by other authors to determine species-specific sensitivity. Our results show that in bacteria this single amino acid substitution controls Vif-dependent blocking of APOBEC3G that is dependent on a strong protein interaction. The C-terminal region of Vif is responsible for this strong protein-protein interaction. In conclusion, our experiments suggest a complement to the model of Vif-induced degradation of APOBEC3G by bringing to relevance that deaminase inhibition can also result from a direct interaction with Vif protein.

  12. Comparative genome analysis reveals a conserved family of actin-like proteins in apicomplexan parasites

    PubMed Central

    Gordon, Jennifer L; Sibley, L David

    2005-01-01

    Background The phylum Apicomplexa is an early-branching eukaryotic lineage that contains a number of important human and animal pathogens. Their complex life cycles and unique cytoskeletal features distinguish them from other model eukaryotes. Apicomplexans rely on actin-based motility for cell invasion, yet the regulation of this system remains largely unknown. Consequently, we focused our efforts on identifying actin-related proteins in the recently completed genomes of Toxoplasma gondii, Plasmodium spp., Cryptosporidium spp., and Theileria spp. Results Comparative genomic and phylogenetic studies of apicomplexan genomes reveals that most contain only a single conventional actin and yet they each have 8–10 additional actin-related proteins. Among these are a highly conserved Arp1 protein (likely part of a conserved dynactin complex), and Arp4 and Arp6 homologues (subunits of the chromatin-remodeling machinery). In contrast, apicomplexans lack canonical Arp2 or Arp3 proteins, suggesting they lost the Arp2/3 actin polymerization complex on their evolutionary path towards intracellular parasitism. Seven of these actin-like proteins (ALPs) are novel to apicomplexans. They show no phylogenetic associations to the known Arp groups and likely serve functions specific to this important group of intracellular parasites. Conclusion The large diversity of actin-like proteins in apicomplexans suggests that the actin protein family has diverged to fulfill various roles in the unique biology of intracellular parasites. Conserved Arps likely participate in vesicular transport and gene expression, while apicomplexan-specific ALPs may control unique biological traits such as actin-based gliding motility. PMID:16343347

  13. Enteric glial-derived S100B protein stimulates nitric oxide production in celiac disease.

    PubMed

    Esposito, Giuseppe; Cirillo, Carla; Sarnelli, Giovanni; De Filippis, Daniele; D'Armiento, Francesco Paolo; Rocco, Alba; Nardone, Gerardo; Petruzzelli, Raffaella; Grosso, Michela; Izzo, Paola; Iuvone, Teresa; Cuomo, Rosario

    2007-09-01

    Enteric glia participates to the homeostasis of the gastrointestinal tract. In the central nervous system, increased expression of astroglial-derived S100B protein has been associated with the onset and maintaining of inflammation. The role of enteric glial-derived S100B protein in gastrointestinal inflammation has never been investigated in humans. In this study, we evaluated the expression of S100B and its relationship with nitric oxide production in celiac disease. Duodenal biopsy specimens from untreated and on gluten-free diet patients with celiac disease and controls were respectively processed for S100B and inducible nitric oxide synthase (iNOS) protein expression and nitrite production. To evaluate the direct involvement of S100B in the inflammation, control biopsy specimens were exposed to exogenous S100B, and iNOS protein expression and nitrite production were measured. We also tested gliadin induction of S100B-dependent inflammation in cultured biopsy specimens deriving from on gluten-free diet patients in the absence or presence of the specific S100B antibody. S100B messenger RNA and protein expression, iNOS protein expression, and nitrite production were significantly increased in untreated patients but not in on gluten-free diet patients vs controls. Addition of S100B to control biopsy specimens resulted in a significant increase of iNOS protein expression and nitrite production. In celiac disease patients but not in controls biopsy specimens, gliadin challenge significantly increased S100B messenger RNA and protein expression, iNOS protein expression, and nitrite production, but these effects were completely inhibited by S100B antibody. Enteric glial-derived S100B is increased in the duodenum of patients with celiac disease and plays a role in nitric oxide production.

  14. TsAg5, a Taenia solium cysticercus protein with a marginal trypsin-like activity in the diagnosis of human neurocysticercosis.

    PubMed

    Rueda, Analiz; Sifuentes, Cecilia; Gilman, Robert H; Gutiérrez, Andrés H; Piña, Ruby; Chile, Nancy; Carrasco, Sebastián; Larson, Sandra; Mayta, Holger; Verástegui, Manuela; Rodriguez, Silvia; Gutiérrez-Correa, Marcel; García, Héctor H; Sheen, Patricia; Zimic, Mirko

    2011-12-01

    Neurocysticercosis is an endemic parasitic disease caused by Taenia solium larva. Although the mechanism of infection is not completely understood, it is likely driven by proteolytic activity that degrades the intestinal wall to facilitate oncosphere penetration and further infection. We analyzed the publicly available T. solium EST/DNA library and identified two contigs comprising a full-length cDNA fragment very similar to Echinococcus granulosus Ag5 protein. The T. solium cDNA sequence included a proteolytic trypsin-like-domain in the C-terminal region, and a thrombospondin type-1 adherence-domain in the N-terminal region. Both the trypsin-like and adherence domains were expressed independently as recombinant proteins in bacterial systems. TsAg5 showed marginal trypsin-like activity and high sequence similarity to Ag5. The purified antigens were tested in a Western immunoblot assay to diagnose human neurocysticercosis. The sensitivity of the trypsin-like-domain was 96.36% in patients infected with extraparenchymal cysts, 75.44% in patients infected with multiple cysts, and 39.62% in patients with a single cyst. Specificity was 76.70%. The thrombospondin type-1 adherence-domain was not specific for neurocysticercosis. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. Naloxone inhibits nod-like receptor protein 3 inflammasome.

    PubMed

    Lin, Han-Yu; Chang, Ya-Ying; Kao, Ming-Chang; Huang, Chun-Jen

    2017-11-01

    Naloxone, an opioid receptor antagonist, possesses potent anti-inflammation effects. We previously confirmed the effects of naloxone on inhibiting upregulation of inflammatory cytokine interleukin-1β (IL-1β). Production of mature form IL-1β is mediated by the nod-like receptor protein 3 (NLRP3) inflammasome, a multiprotein complex composed of NLRP3, and the adaptor protein apoptosis-associated speck-like protein contains a caspase recruitment domain (ASC). We elucidated whether naloxone could inhibit the activation of NLRP3 inflammasome. To induce IL-1β production and NLRP3 inflammasome activation, the human monocytic leukemia cell line THP-1 cells were first primed with lipopolysaccharide (LPS, 1 μg/mL) and then activated with adenosine triphosphate (ATP, 1 mM). For NLRP3 transcription, THP-1 cells were only treated with LPS priming. Enzyme-link immunosorbent assay data revealed that the concentration of IL-1β in THP-1 cells treated with LPS plus ATP was significantly higher than that in THP-1 cells treated with LPS plus ATP plus naloxone (0.1 μM) (P < 0.001). Real-time quantitative reverse transcription and polymerase chain reaction data also revealed that NLRP3 mRNA concentration in THP-1 cells treated with LPS was significantly higher than that in THP-1 cells treated with LPS plus naloxone (P = 0.001). ASC speck formation, that is, ASC assembles into a large protein complex, is an indicator for NLRP3 inflammasome activation. Our data revealed that the percentage of cells containing ASC specks in THP-1 cells treated with LPS plus ATP was also significantly higher than that in THP-1 cells treated with LPS plus ATP plus naloxone (P < 0.001). Naloxone inhibits NLRP3 inflammasome activation. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Regulation of ciliary retrograde protein trafficking by the Joubert syndrome proteins ARL13B and INPP5E.

    PubMed

    Nozaki, Shohei; Katoh, Yohei; Terada, Masaya; Michisaka, Saki; Funabashi, Teruki; Takahashi, Senye; Kontani, Kenji; Nakayama, Kazuhisa

    2017-02-01

    ARL13B (a small GTPase) and INPP5E (a phosphoinositide 5-phosphatase) are ciliary proteins encoded by causative genes of Joubert syndrome. We here showed, by taking advantage of a visible immunoprecipitation assay, that ARL13B interacts with the IFT46 -: IFT56 (IFT56 is also known as TTC26) dimer of the intraflagellar transport (IFT)-B complex, which mediates anterograde ciliary protein trafficking. However, the ciliary localization of ARL13B was found to be independent of its interaction with IFT-B, but dependent on the ciliary-targeting sequence RVEP in its C-terminal region. ARL13B-knockout cells had shorter cilia than control cells and exhibited aberrant localization of ciliary proteins, including INPP5E. In particular, in ARL13B-knockout cells, the IFT-A and IFT-B complexes accumulated at ciliary tips, and GPR161 (a negative regulator of Hedgehog signaling) could not exit cilia in response to stimulation with Smoothened agonist. This abnormal phenotype was rescued by the exogenous expression of wild-type ARL13B, as well as by its mutant defective in the interaction with IFT-B, but not by its mutants defective in INPP5E binding or in ciliary localization. Thus, ARL13B regulates IFT-A-mediated retrograde protein trafficking within cilia through its interaction with INPP5E. © 2017. Published by The Company of Biologists Ltd.

  17. Comparison of potential protection conferred by three immunization strategies (protein/protein, DNA/DNA, and DNA/protein) against Brucella infection using Omp2b in BALB/c Mice.

    PubMed

    Golshani, Maryam; Rafati, Sima; Nejati-Moheimani, Mehdi; Ghasemian, Melina; Bouzari, Saeid

    2016-12-25

    In the present study, immunogenicity and protective efficacy of the Brucella outer membrane protein 2b (Omp2b) was evaluated in BALB/c mice using Protein/Protein, DNA/DNA and DNA/Protein vaccine strategies. Immunization of mice with three vaccine regimens elicited a strong specific IgG response (higher IgG2a titers over IgG1 titers) and provided Th1-oriented immune response. Vaccination of BALB/c mice with the DNA/Pro regimen induced higher levels of IFN-γ/IL-2 and conferred more protection levels against B. melitenisis and B. abortus challenge than did the protein or DNA alone. In conclusion, Omp2b is able to stimulate specific immune responses and to confer cross protection against B. melitensis and B. abortus infection. Therefore, it could be introduced as a new potential candidate for the development of a subunit vaccine against Brucella infection. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Trastuzumab-Resistant Luminal B Breast Cancer Cells Show Basal-Like Cell Growth Features Through NF-κB-Activation

    PubMed Central

    Kanzaki, Hirotaka; Mukhopadhya, Nishit K.; Cui, Xiaojiang; Ramanujan, V. Krishnan

    2016-01-01

    A major clinical problem in the treatment of breast cancer is mortality due to metastasis. Understanding the molecular mechanisms associated with metastasis should aid in designing new therapeutic approaches for breast cancer. Trastuzumab is the main therapeutic option for HER2+ breast cancer patients; however, the molecular basis for trastuzumab resistance (TZR) and subsequent metastasis is not known. Earlier, we found expression of basal-like molecular markers in TZR tissues from patients with invasive breast cancer.(1) The basal-like phenotype is a particularly aggressive form of breast cancer. This observation suggests that TZR might contribute to an aggressive phenotype. To understand if resistance to TZR can lead to basal-like phenotype, we generated a trastuzumab-resistant human breast cancer cell line (BT-474-R) that maintained human epidermal growth factor receptor 2 (HER2) overexpression and HER2 mediated signaling. Analysis showed that nuclear factor-kappa B (NF-κB) was constitutively activated in the BT-474-R cells, a feature similar to the basal-like tumor phenotype. Pharmacologic inhibition of NF-κB improved sensitivity of BT-474-R cells to trastuzumab. Interestingly, activation of HER2 independent NF-κB is not shown in luminal B breast cancer cells. Our study suggests that by activating the NF-κB pathway, luminal B cells may acquire a HER2+ basal-like phenotype in which NF-κB is constitutively activated; this notion is consistent with the recently proposed “progression through grade” or “evolution of resistance” hypothesis. Furthermore, we identified IKK-α/IKK-β and nuclear accumulation of RelA/p65 as the major determinants in the resistant cells. Thus our study additionally suggests that the nuclear accumulation of p65 may be a useful marker for identifying metastasis-initiating tumor cells and targeting RelA/p65 may limit metastasis of breast and other cancers associated with NF-κB activation. PMID:26871511

  19. Four Proteins Encoded in the gspB-secY2A2 Operon of Streptococcus gordonii Mediate the Intracellular Glycosylation of the Platelet-Binding Protein GspB

    PubMed Central

    Takamatsu, Daisuke; Bensing, Barbara A.; Sullam, Paul M.

    2004-01-01

    Platelet binding by Streptococcus gordonii strain M99 is mediated predominantly by the cell surface glycoprotein GspB. This adhesin consists of a putative N-terminal signal peptide, two serine-rich regions (SRR1 and SRR2), a basic region between SRR1 and SRR2, and a C-terminal cell wall anchoring domain. The glycosylation of GspB is mediated at least in part by Gly and Nss, which are encoded in the secY2A2 locus immediately downstream of gspB. This region also encodes two proteins (Gtf and Orf4) that are required for the expression of GspB but whose functions have not been delineated. In this study, we further characterized the roles of Gly, Nss, Gtf, and Orf4 by investigating the expression and glycosylation of a series of glutathione S-transferase-GspB fusion proteins in M99 and in gly, nss, gtf, and orf4 mutants. Compared with fusion proteins expressed in the wild-type background, fusion proteins expressed in the mutant strain backgrounds showed altered electrophoretic mobility. In addition, the fusion proteins formed insoluble aggregates in protoplasts of the gtf and orf4 mutants. Glycan detection and lectin blot analysis revealed that SRR1 and SRR2 were glycosylated but that the basic region was unmodified. When the fusion protein was expressed in Escherichia coli, glycosylation of this protein was observed only in the presence of both gtf and orf4. These results demonstrate that Gly, Nss, Gtf, and Orf4 are all involved in the intracellular glycosylation of SRRs. Moreover, Gtf and Orf4 are essential for glycosylation, which in turn is important for the solubility of GspB. PMID:15489421

  20. Identification of Novel STAT6-Regulated Proteins in Mouse B Cells by Comparative Transcriptome and Proteome Analysis.

    PubMed

    Mokada-Gopal, Lavanya; Boeser, Alexander; Lehmann, Christian H K; Drepper, Friedel; Dudziak, Diana; Warscheid, Bettina; Voehringer, David

    2017-05-01

    The transcription factor STAT6 plays a key role in mediating signaling downstream of the receptors for IL-4 and IL-13. In B cells, STAT6 is required for class switch recombination to IgE and for germinal center formation during type 2 immune responses directed against allergens or helminths. In this study, we compared the transcriptomes and proteomes of primary mouse B cells from wild-type and STAT6-deficient mice cultured for 4 d in the presence or absence of IL-4. Microarray analysis revealed that 214 mRNAs were upregulated and 149 were downregulated >3-fold by IL-4 in a STAT6-dependent manner. Across all samples, ∼5000 proteins were identified by label-free quantitative liquid chromatography/mass spectrometry. A total of 149 proteins was found to be differentially expressed >3-fold between IL-4-stimulated wild-type and STAT6 -/- B cells (75 upregulated and 74 downregulated). Comparative analysis of the proteome and transcriptome revealed that expression of these proteins was mainly regulated at the transcriptional level, which argues against a major role for posttranscriptional mechanisms that modulate the STAT6-dependent proteome. Nine proteins were selected for confirmation by flow cytometry or Western blot. We show that CD30, CD79b, SLP-76, DEC205, IL-5Rα, STAT5, and Thy1 are induced by IL-4 in a STAT6-dependent manner. In contrast, Syk and Fc receptor-like 1 were downregulated. This dataset provides a framework for further functional analysis of newly identified IL-4-regulated proteins in B cells that may contribute to germinal center formation and IgE switching in type 2 immunity. Copyright © 2017 by The American Association of Immunologists, Inc.

  1. MCP-1-Induced Protein Promotes Endothelial-Like and Angiogenic Properties in Human Bone Marrow Monocytic Cells

    PubMed Central

    Wang, Kangkai; Zhelyabovska, Olga; Saad, Yasser; Kolattukudy, Pappachan E.

    2013-01-01

    Monocytic cells enhance neovascularization by releasing proangiogenic mediators and/or by transdifferentiating into endothelial-like cells. However, the mechanisms that govern this transdifferentiation process are largely unknown. Recently, monocyte chemotactic protein-1 (MCP-1)-induced protein (MCPIP) has been identified as a novel CCCH-type zinc-finger protein expressed primarily in monocytic cells. Here, we analyzed whether MCPIP might exert angiogenic effects by promoting differentiation of monocytic cells into endothelial cell (EC)-like phenotype. The expression of MCPIP increased during MCP-1-induced transdifferentiation in human bone marrow mononuclear cells (BMNCs). Knockdown of MCPIP with small interfering RNA (siRNA) abolished MCP-1-induced expression of EC markers Flk-1 and Tie-2 in human BMNCs. BMNCs transfected with MCPIP expression vector displayed EC-like morphology accompanied by downregulation of monocytic markers CD14 and CD11b, upregulation of EC markers Flk-1 and Tie-2, induction of cadherin (cdh)-12 and -19, activation of endoplasmic reticulum (ER) stress, and autophagy. Knockdown of cdh-12 or cdh-19 markedly inhibited MCPIP-induced enhancement of cell attachment and EC-marker expression. Inhibition of ER stress by tauroursodeoxycholate abolished MCPIP-induced expression of EC markers. Inhibition of autophagy by knockdown of Beclin-1 with siRNA or by an autophagy inhibitor 3′-methyladenine inhibited MCPIP-induced expression of EC markers. Expression of MCPIP in BMNCs enhanced uptake of acetylated low-density lipoprotein (acLDL), formation of EC-colony, incorporation of cells into capillary-like structure on Matrigel, and exhibited increased neovascularization in the ischemic hindlimb in mice. These results demonstrate that MCPIP may be an important regulator of inflammatory angiogenesis and provide novel mechanistic insights into the link between MCP-1 and cardiovascular diseases. PMID:24008336

  2. Optochemical Control of Protein Localization and Activity within Cell-like Compartments.

    PubMed

    Caldwell, Reese M; Bermudez, Jessica G; Thai, David; Aonbangkhen, Chanat; Schuster, Benjamin S; Courtney, Taylor; Deiters, Alexander; Hammer, Daniel A; Chenoweth, David M; Good, Matthew C

    2018-05-08

    We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.

  3. A rare polyglycine type II-like helix motif in naturally occurring proteins.

    PubMed

    Warkentin, Eberhard; Weidenweber, Sina; Schühle, Karola; Demmer, Ulrike; Heider, Johann; Ermler, Ulrich

    2017-11-01

    Common structural elements in proteins such as α-helices or β-sheets are characterized by uniformly repeating, energetically favorable main chain conformations which additionally exhibit a completely saturated hydrogen-bonding network of the main chain NH and CO groups. Although polyproline or polyglycine type II helices (PP II or PG II ) are frequently found in proteins, they are not considered as equivalent secondary structure elements because they do not form a similar self-contained hydrogen-bonding network of the main chain atoms. In this context our finding of an unusual motif of glycine-rich PG II -like helices in the structure of the acetophenone carboxylase core complex is of relevance. These PG II -like helices form hexagonal bundles which appear to fulfill the criterion of a (largely) saturated hydrogen-bonding network of the main-chain groups and therefore may be regarded in this sense as a new secondary structure element. It consists of a central PG II -like helix surrounded by six nearly parallel PG II -like helices in a hexagonal array, plus an additional PG II -like helix extending the array outwards. Very related structural elements have previously been found in synthetic polyglycine fibers. In both cases, all main chain NH and CO groups of the central PG II -helix are saturated by either intra- or intermolecular hydrogen-bonds, resulting in a self-contained hydrogen-bonding network. Similar, but incomplete PG II -helix patterns were also previously identified in a GTP-binding protein and an antifreeze protein. © 2017 Wiley Periodicals, Inc.

  4. In nucleoli, the steady state of nucleolar proteins is leptomycin B-sensitive.

    PubMed

    Muro, Eleonora; Hoang, Thang Q; Jobart-Malfait, Aude; Hernandez-Verdun, Danièle

    2008-05-01

    The nucleolus is a dynamic structure. It has been demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in continuous exchanges with the nucleoplasm using GFP (green fluorescent protein)-tagged proteins. However, how the exchanges within one nucleolus and between nucleoli within the nuclear volume occurred is still poorly understood. The movement of PAGFP (photoactivatable GFP)-tagged proteins that become visible after photoactivation can be followed. In the present study, we establish the protocol allowing quantification of the traffic of PAGFP-tagged nucleolar proteins in nuclei containing two nucleoli. The traffic in the activated area, at the periphery of the activated area and to the neighbouring nucleolus is measured. Protein B23 is rapidly replaced in the activated area, and at the periphery of the activated area the steady state suggests intranucleolar recycling of B23; this recycling is LMB (leptomycin B)-sensitive. The pool of activated B23 is equally distributed in the volume of the two nucleoli within 2 min. The three-dimensional distribution of the proteins Nop52 and fibrillarin is less rapid than that of B23 but is also LMB-sensitive. In contrast, traffic of fibrillarin from the nucleoli to the CB (Cajal body) was not modified by LMB. We propose that the steady state of nucleolar proteins in nucleoli depends on the affinity of the proteins for their partners and on intranucleolar recycling. This steady state can be impaired by LMB but not the uptake in the neighbouring nucleolus or the CB.

  5. Ribosome hijacking: a role for small protein B during trans-translation

    PubMed Central

    Nonin-Lecomte, Sylvie; Germain-Amiot, Noella; Gillet, Reynald; Hallier, Marc; Ponchon, Luc; Dardel, Frédéric; Felden, Brice

    2009-01-01

    Tight recognition of codon–anticodon pairings by the ribosome ensures the accuracy and fidelity of protein synthesis. In eubacteria, translational surveillance and ribosome rescue are performed by the ‘tmRNA–SmpB' system (transfer messenger RNA–small protein B). Remarkably, entry and accommodation of aminoacylated-tmRNA into stalled ribosomes occur without a codon–anticodon interaction but in the presence of SmpB. Here, we show that within a stalled ribosome, SmpB interacts with the three universally conserved bases G530, A1492 and A1493 that form the 30S subunit decoding centre, in which canonical codon–anticodon pairing occurs. The footprints at positions A1492 and A1493 of a small decoding centre, as well as on a set of conserved SmpB amino acids, were identified by nuclear magnetic resonance. Mutants at these residues display the same growth defects as for ΔsmpB strains. The SmpB protein has functional and structural similarities with initiation factor 1, and is proposed to be a functional mimic of the pairing between a codon and an anticodon. PMID:19132006

  6. Ribosome hijacking: a role for small protein B during trans-translation.

    PubMed

    Nonin-Lecomte, Sylvie; Germain-Amiot, Noella; Gillet, Reynald; Hallier, Marc; Ponchon, Luc; Dardel, Frédéric; Felden, Brice

    2009-02-01

    Tight recognition of codon-anticodon pairings by the ribosome ensures the accuracy and fidelity of protein synthesis. In eubacteria, translational surveillance and ribosome rescue are performed by the 'tmRNA-SmpB' system (transfer messenger RNA-small protein B). Remarkably, entry and accommodation of aminoacylated-tmRNA into stalled ribosomes occur without a codon-anticodon interaction but in the presence of SmpB. Here, we show that within a stalled ribosome, SmpB interacts with the three universally conserved bases G530, A1492 and A1493 that form the 30S subunit decoding centre, in which canonical codon-anticodon pairing occurs. The footprints at positions A1492 and A1493 of a small decoding centre, as well as on a set of conserved SmpB amino acids, were identified by nuclear magnetic resonance. Mutants at these residues display the same growth defects as for DeltasmpB strains. The SmpB protein has functional and structural similarities with initiation factor 1, and is proposed to be a functional mimic of the pairing between a codon and an anticodon.

  7. Mechanistic Insight into the Nitrosylation of the [4Fe−4S] Cluster of WhiB-like Proteins

    PubMed Central

    2010-01-01

    The reactivity of protein bound iron−sulfur clusters with nitric oxide (NO) is well documented, but little is known about the actual mechanism of cluster nitrosylation. Here, we report studies of members of the Wbl family of [4Fe−4S] containing proteins, which play key roles in regulating developmental processes in actinomycetes, including Streptomyces and Mycobacteria, and have been shown to be NO responsive. Streptomyces coelicolor WhiD and Mycobacterium tuberculosis WhiB1 react extremely rapidly with NO in a multiphasic reaction involving, remarkably, 8 NO molecules per [4Fe−4S] cluster. The reaction is 104-fold faster than that observed with O2 and is by far the most rapid iron−sulfur cluster nitrosylation reaction reported to date. An overall stoichiometry of [Fe4S4(Cys)4]2− + 8NO → 2[FeI2(NO)4(Cys)2]0 + S2− + 3S0 has been established by determination of the sulfur products and their oxidation states. Kinetic analysis leads to a four-step mechanism that accounts for the observed NO dependence. DFT calculations suggest the possibility that the nitrosylation product is a novel cluster [FeI4(NO)8(Cys)4]0 derived by dimerization of a pair of Roussin’s red ester (RRE) complexes. PMID:21182249

  8. Free energy landscape of protein-like chains with discontinuous potentials

    NASA Astrophysics Data System (ADS)

    Movahed, Hanif Bayat; van Zon, Ramses; Schofield, Jeremy

    2012-06-01

    In this article the configurational space of two simple protein models consisting of polymers composed of a periodic sequence of four different kinds of monomers is studied as a function of temperature. In the protein models, hydrogen bond interactions, electrostatic repulsion, and covalent bond vibrations are modeled by discontinuous step, shoulder, and square-well potentials, respectively. The protein-like chains exhibit a secondary alpha helix structure in their folded states at low temperatures, and allow a natural definition of a configuration by considering which beads are bonded. Free energies and entropies of configurations are computed using the parallel tempering method in combination with hybrid Monte Carlo sampling of the canonical ensemble of the discontinuous potential system. The probability of observing the most common configuration is used to analyze the nature of the free energy landscape, and it is found that the model with the least number of possible bonds exhibits a funnel-like free energy landscape at low enough temperature for chains with fewer than 30 beads. For longer proteins, the free landscape consists of several minima, where the configuration with the lowest free energy changes significantly by lowering the temperature and the probability of observing the most common configuration never approaches one due to the degeneracy of the lowest accessible potential energy.

  9. Fullerene-like organization of HIV gag-protein shell in virus-like particles produced by recombinant baculovirus.

    PubMed

    Nermut, M V; Hockley, D J; Jowett, J B; Jones, I M; Garreau, M; Thomas, D

    1994-01-01

    Virus-like particles produced by a recombinant baculovirus containing the HIV gag gene were examined by negative staining after delipidization. This technique demonstrated that the gag-protein shell consisted of radially arranged short rods which formed a network of ring-like structures. Similar structures were observed at the plasma membrane of infected cells which had been opened by wet-cleaving. Occasionally five or six subunits were observed forming a ring. These findings suggest that the gag-encoded precursor (pr55) is a rod-like molecule about 34 A in diameter and 85 A in length. A protein cylinder of such dimensions would have a molecular weight of 56K. The center-to-center distance of two neighboring rings formed by the rods was 66 +/- 8 A (N = 200) by direct measurements and 65 A as obtained from averaged images. This morphology and these dimensions indicate that the virus-like particles contain the gag precursor in the form of a near-spherical "fullerene-like" icosahedral shell. Our data indicate that the triangulation number of the rings equals 63. However, since one rod of pr55 is shared by two rings, the number of copies of the precursor will be 1890 as opposed to 2522 if the molecules were closely packed. The particle diameter of 102 nm deduced from the proposed model was close to the diameter obtained from thin sections of low-temperature-embedded specimens (103-108 nm).

  10. Overexpression of a SNARE protein AtBS14b alters BR response in Arabidopsis.

    PubMed

    Zhu, Zhong Xin; Ye, Hong Bo; Xuan, Yuan Hu; Yao, Da Nian

    2014-12-01

    N-ethyl-maleimide sensitive factor adaptor protein receptor (SNAREs) domain-containing proteins were known as key players in vesicle-associated membrane fusion. Genetic screening has revealed the function of SNAREs in different aspects of plant biology, but the role of many SNAREs are still unknown. In this study, we have characterized the role of Arabidopsis Qc-SNARE protein AtBS14b in brassinosteroids (BRs) signaling pathway. AtBS14b overexpression (AtBS14b ox) plants exhibited short hypocotyl and petioles lengths as well as insensitivity to exogenously supplied BR, while AtBS14b mutants did not show any visible BR-dependent morphological differences. BR biosynthesis enzyme BR6OX2 expression was slightly lower in AtBS14b ox than in wild type plants. Further BR-mediated repression of BR6OX2, CPD and DWF4 was inhibited in AtBS14b ox plants. AtBS14b-mCherry fusion protein localized in vesicular compartments surrounding plasma membrane in N. benthamiana leaves. In addition, isolation of AtBS14b-interacting BR signaling protein, which localized in plasma membrane, showed that AtBS14b directly interacted with membrane steroid binding protein 1 (MSBP1), but did not interact with BAK1 or BRI1. These data suggested that Qc-SNARE protein AtBS14b is the first SNARE protein identified that interacts with MSBP1, and the overexpression of AtBS14b modulates BR response in Arabidopsis.

  11. Sel1-like repeat proteins in signal transduction.

    PubMed

    Mittl, Peer R E; Schneider-Brachert, Wulf

    2007-01-01

    Solenoid proteins, which are distinguished from general globular proteins by their modular architectures, are frequently involved in signal transduction pathways. Proteins from the tetratricopeptide repeat (TPR) and Sel1-like repeat (SLR) families share similar alpha-helical conformations but different consensus sequence lengths and superhelical topologies. Both families are characterized by low sequence similarity levels, rendering the identification of functional homologous difficult. Therefore current knowledge of the molecular and cellular functions of the SLR proteins Sel1, Hrd3, Chs4, Nif1, PodJ, ExoR, AlgK, HcpA, Hsp12, EnhC, LpnE, MotX, and MerG has been reviewed. Although SLR proteins possess different cellular functions they all seem to serve as adaptor proteins for the assembly of macromolecular complexes. Sel1, Hrd3, Hsp12 and LpnE are activated under cellular stress. The eukaryotic Sel1 and Hrd3 proteins are involved in the ER-associated protein degradation, whereas the bacterial LpnE, EnhC, HcpA, ExoR, and AlgK proteins mediate the interactions between bacterial and eukaryotic host cells. LpnE and EnhC are responsible for the entry of L. pneumophila into epithelial cells and macrophages. ExoR from the symbiotic microorganism S. melioti and AlgK from the pathogen P. aeruginosa regulate exopolysaccaride synthesis. Nif1 and Chs4 from yeast are responsible for the regulation of mitosis and septum formation during cell division, respectively, and PodJ guides the cellular differentiation during the cell cycle of the bacterium C. crescentus. Taken together the SLR motif establishes a link between signal transduction pathways from eukaryotes and bacteria. The SLR motif is so far absent from archaea. Therefore the SLR could have developed in the last common ancestor between eukaryotes and bacteria.

  12. RNA buffers the phase separation behavior of prion-like RNA binding proteins.

    PubMed

    Maharana, Shovamayee; Wang, Jie; Papadopoulos, Dimitrios K; Richter, Doris; Pozniakovsky, Andrey; Poser, Ina; Bickle, Marc; Rizk, Sandra; Guillén-Boixet, Jordina; Franzmann, Titus M; Jahnel, Marcus; Marrone, Lara; Chang, Young-Tae; Sterneckert, Jared; Tomancak, Pavel; Hyman, Anthony A; Alberti, Simon

    2018-05-25

    Prion-like RNA binding proteins (RBPs) such as TDP43 and FUS are largely soluble in the nucleus but form solid pathological aggregates when mislocalized to the cytoplasm. What keeps these proteins soluble in the nucleus and promotes aggregation in the cytoplasm is still unknown. We report here that RNA critically regulates the phase behavior of prion-like RBPs. Low RNA/protein ratios promote phase separation into liquid droplets, whereas high ratios prevent droplet formation in vitro. Reduction of nuclear RNA levels or genetic ablation of RNA binding causes excessive phase separation and the formation of cytotoxic solid-like assemblies in cells. We propose that the nucleus is a buffered system in which high RNA concentrations keep RBPs soluble. Changes in RNA levels or RNA binding abilities of RBPs cause aberrant phase transitions. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  13. RNA Binding Protein RBM38 Regulates Expression of the 11-Kilodalton Protein of Parvovirus B19, Which Facilitates Viral DNA Replication.

    PubMed

    Ganaie, Safder S; Chen, Aaron Yun; Huang, Chun; Xu, Peng; Kleiboeker, Steve; Du, Aifang; Qiu, Jianming

    2018-04-15

    Human parvovirus B19 (B19V) expresses a single precursor mRNA (pre-mRNA), which undergoes alternative splicing and alternative polyadenylation to generate 12 viral mRNA transcripts that encode two structural proteins (VP1 and VP2) and three nonstructural proteins (NS1, 7.5-kDa protein, and 11-kDa protein). Splicing at the second 5' donor site (D2 site) of the B19V pre-mRNA is essential for the expression of VP2 and the 11-kDa protein. We previously identified that cis -acting intronic splicing enhancer 2 (ISE2) that lies immediately after the D2 site facilitates the recognition of the D2 donor for its efficient splicing. In this study, we report that ISE2 is critical for the expression of the 11-kDa viral nonstructural protein. We found that ISE2 harbors a consensus RNA binding motif protein 38 (RBM38) binding sequence, 5'-UGUGUG-3'. RBM38 is expressed during the middle stage of erythropoiesis. We first confirmed that RBM38 binds specifically with the ISE2 element in vitro The knockdown of RBM38 significantly decreases the level of spliced mRNA at D2 that encodes the 11-kDa protein but not that of the D2-spliced mRNA that encodes VP2. Importantly, we found that the 11-kDa protein enhances viral DNA replication and virion release. Accordingly, the knockdown of RBM38 decreases virus replication via downregulating 11-kDa protein expression. Taken together, these results suggest that the 11-kDa protein facilitates B19V DNA replication and that RBM38 is an essential host factor for B19V pre-mRNA splicing and for the expression of the 11-kDa protein. IMPORTANCE B19V is a human pathogen that can cause fifth disease, arthropathy, anemia in immunocompromised patients and sickle cell disease patients, myocarditis, and hydrops fetalis in pregnant women. Human erythroid progenitor cells (EPCs) are most susceptible to B19V infection and fully support viral DNA replication. The exclusive tropism of B19V for erythroid-lineage cells is dependent not only on the expression of viral

  14. Campylobacter jejuni transducer like proteins: Chemotaxis and beyond

    PubMed Central

    Chandrashekhar, Kshipra; Kassem, Issmat I.; Rajashekara, Gireesh

    2017-01-01

    ABSTRACT Chemotaxis, a process that mediates directional motility toward or away from chemical stimuli (chemoeffectors/ligands that can be attractants or repellents) in the environment, plays an important role in the adaptation of Campylobacter jejuni to disparate niches. The chemotaxis system consists of core signal transduction proteins and methyl-accepting-domain-containing Transducer like proteins (Tlps). Ligands binding to Tlps relay a signal to chemotaxis proteins in the cytoplasm which initiate a signal transduction cascade, culminating into a directional flagellar movement. Tlps facilitate substrate-specific chemotaxis in C. jejuni, which plays an important role in the pathogen's adaptation, pathobiology and colonization of the chicken gastrointestinal tract. However, the role of Tlps in C. jejuni's host tissue specific colonization, physiology and virulence remains not completely understood. Based on recent studies, it can be predicted that Tlps might be important targets for developing strategies to control C. jejuni via vaccines and antimicrobials. PMID:28080213

  15. Development of the Cellular Immune System of Drosophila Requires the Membrane Attack Complex/Perforin-Like Protein Torso-Like.

    PubMed

    Forbes-Beadle, Lauren; Crossman, Tova; Johnson, Travis K; Burke, Richard; Warr, Coral G; Whisstock, James C

    2016-10-01

    Pore-forming members of the membrane attack complex/perforin-like (MACPF) protein superfamily perform well-characterized roles as mammalian immune effectors. For example, complement component 9 and perforin function to directly form pores in the membrane of Gram-negative pathogens or virally infected/transformed cells, respectively. In contrast, the only known MACPF protein in Drosophila melanogaster, Torso-like, plays crucial roles during development in embryo patterning and larval growth. Here, we report that in addition to these functions, Torso-like plays an important role in Drosophila immunity. However, in contrast to a hypothesized effector function in, for example, elimination of Gram-negative pathogens, we find that torso-like null mutants instead show increased susceptibility to certain Gram-positive pathogens such as Staphylococcus aureus and Enterococcus faecalis We further show that this deficit is due to a severely reduced number of circulating immune cells and, as a consequence, an impaired ability to phagocytose bacterial particles. Together these data suggest that Torso-like plays an important role in controlling the development of the Drosophila cellular immune system. Copyright © 2016 by the Genetics Society of America.

  16. The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis.

    PubMed

    Silva, Everton F; Medeiros, Marco A; McBride, Alan J A; Matsunaga, Jim; Esteves, Gabriela S; Ramos, João G R; Santos, Cleiton S; Croda, Júlio; Homma, Akira; Dellagostin, Odir A; Haake, David A; Reis, Mitermayer G; Ko, Albert I

    2007-08-14

    Subunit vaccines are a potential intervention strategy against leptospirosis, which is a major public health problem in developing countries and a veterinary disease in livestock and companion animals worldwide. Leptospiral immunoglobulin-like (Lig) proteins are a family of surface-exposed determinants that have Ig-like repeat domains found in virulence factors such as intimin and invasin. We expressed fragments of the repeat domain regions of LigA and LigB from Leptospira interrogans serovar Copenhageni. Immunization of Golden Syrian hamsters with Lig fragments in Freund's adjuvant induced robust antibody responses against recombinant protein and native protein, as detected by ELISA and immunoblot, respectively. A single fragment, LigANI, which corresponds to the six carboxy-terminal Ig-like repeat domains of the LigA molecule, conferred immunoprotection against mortality (67-100%, P<0.05) in hamsters which received a lethal inoculum of L. interrogans serovar Copenhageni. However, immunization with this fragment did not confer sterilizing immunity. These findings indicate that the carboxy-terminal portion of LigA is an immunoprotective domain and may serve as a vaccine candidate for human and veterinary leptospirosis.

  17. Survey for the presence of a vitronectin-like protein in micro- and macroalgae and cyanobacteria.

    PubMed

    Field, Lacey M; Fagerberg, Wayne R; Böttger, S Anne

    2018-04-01

    Vitronectin (Vn) is a glycoprotein that serves a wide variety of roles in multicellular organisms. It was first identified in multicellular animals but has also been isolated from land plants and some algae, where it appears to serve as an extracellular adhesive molecule. In order to further elucidate presence and localization of a Vn-like protein and its potential role in algae, we surveyed different morphological regions of 24 species of macro- and microalgae and three species of cyanobacteria for the presence of a Vn-like protein. Vn-like proteins were not detected in any of the species of cyanobacteria, microalgae or Rhodophyta investigated. They were detected in several species of the Phaeophyceae and Chlorophyta where their localization was limited to the holdfast and rhizoids of these organisms, respectively. Detection of a Vn-like protein (between 0.0125 and 0.097 μg · μL -1 protein extract) was therefore limited to locations associated with substrate attachment. © 2017 Phycological Society of America.

  18. Natural products possessing protein tyrosine phosphatase 1B (PTP1B) inhibitory activity found in the last decades

    PubMed Central

    Jiang, Cheng-shi; Liang, Lin-fu; Guo, Yue-wei

    2012-01-01

    This article provides an overview of approximately 300 secondary metabolites with inhibitory activity against protein tyrosine phosphatase 1B (PTP1B), which were isolated from various natural sources or derived from synthetic process in the last decades. The structure-activity relationship and the selectivity of some compounds against other protein phosphatases were also discussed. Potential pharmaceutical applications of several PTP1B inhibitors were presented. PMID:22941286

  19. Camellianols A-G, Barrigenol-like Triterpenoids with PTP1B Inhibitory Effects from the Endangered Ornamental Plant Camellia crapnelliana.

    PubMed

    Xiong, Juan; Wan, Jiang; Ding, Jie; Wang, Pei-Pei; Ma, Guang-Lei; Li, Jia; Hu, Jin-Feng

    2017-11-22

    Seven new naturally occurring barrigenol-like compounds, camellianols A-G (1-7), and 10 known triterpenoids were isolated from the twigs and leaves of the cultivated endangered ornamental plant Camellia crapnelliana. According to the ECD octant rule for saturated cyclohexanones, the absolute configurations of camellianols D (4) and E (5) were defined. The backbones of the remaining new isolates are assumed to have the same absolute configuration as compounds 4, 5, and harpullone (12). Compounds 2, 3, 9, 10, 13, and 16 exhibited inhibitory effects on the protein tyrosine phosphatase 1B (PTP1B) enzyme, with IC 50 values less than 10 μM.

  20. DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats

    PubMed Central

    de Lange, Orlando; Wolf, Christina; Thiel, Philipp; Krüger, Jens; Kleusch, Christian; Kohlbacher, Oliver; Lahaye, Thomas

    2015-01-01

    Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats. PMID:26481363

  1. A new look on protein-polyphenol complexation during honey storage: is this a random or organized event with the help of dirigent-like proteins?

    PubMed

    Brudzynski, Katrina; Sjaarda, Calvin; Maldonado-Alvarez, Liset

    2013-01-01

    Honey storage initiates melanoidin formation that involves a cascade of seemingly unguided redox reactions between amino acids/proteins, reducing sugars and polyphenols. In the process, high molecular weight protein-polyphenol complexes are formed, but the mechanism involved remains unknown. The objective of this study was twofold: to determine quantitative and qualitative changes in proteins in honeys stored for prolonged times and in different temperatures and to relate these changes to the formation of protein-polyphenol complexes. Six -month storage decreased the protein content by 46.7% in all tested honeys (t-test, p<0.002) with the rapid reduction occurring during the first three month. The changes in protein levels coincided with alterations in molecular size and net charge of proteins on SDS -PAGE. Electro-blotted proteins reacted with a quinone-specific nitro blue tetrazolium (NBT) on nitrocellulose membranes indicating that quinones derived from oxidized polyphenols formed covalent bonds with proteins. Protein-polyphenol complexes isolated by size-exclusion chromatography differed in size and stoichiometry and fall into two categories: (a) high molecular weight complexes (230-180 kDa) enriched in proteins but possessing a limited reducing activity toward the NBT and (b) lower molecular size complexes (110-85 kDa) enriched in polyphenols but strongly reducing the dye. The variable stoichiometry suggest that the large, "protein-type" complexes were formed by protein cross-linking, while in the smaller, "polyphenol-type" complexes polyphenols were first polymerized prior to protein binding. Quinones preferentially bound a 31 kDa protein which, by the electrospray quadrupole time of flight mass spectrometry (ESI-Qtof-MS) analysis, showed homology to dirigent-like proteins known for assisting in radical coupling and polymerization of phenolic compounds. These findings provide a new look on protein-polyphenol interaction in honey where the reaction of quinones

  2. Solubilization of protein aggregates by the acid stress chaperones HdeA and HdeB.

    PubMed

    Malki, Abderrahim; Le, Hai-Tuong; Milles, Sigrid; Kern, Renée; Caldas, Teresa; Abdallah, Jad; Richarme, Gilbert

    2008-05-16

    The acid stress chaperones HdeA and HdeB of Escherichia coli prevent the aggregation of periplasmic proteins at acidic pH. We show in this report that they also form mixed aggregates with proteins that have failed to be solubilized at acidic pH and allow their subsequent solubilization at neutral pH. HdeA, HdeB, and HdeA and HdeB together display an increasing efficiency for the solubilization of protein aggregates at pH 3. They are less efficient for the solubilization of aggregates at pH 2, whereas HdeB is the most efficient. Increasing amounts of periplasmic proteins draw increasing amounts of chaperone into pellets, suggesting that chaperones co-aggregate with their substrate proteins. We observed a decrease in the size of protein aggregates in the presence of HdeA and HdeB, from very high molecular mass aggregates to 100-5000-kDa species. Moreover, a marked decrease in the exposed hydrophobicity of aggregated proteins in the presence of HdeA and HdeB was revealed by 1,1'-bis(4-anilino)naphtalene-5,5'-disulfonic acid binding experiments. In vivo, during the recovery at neutral pH of acid stressed bacterial cells, HdeA and HdeB allow the solubilization and renaturation of protein aggregates, including those formed by the maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ. Thus, HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH, by decreasing the size of protein aggregates and the exposed hydrophobicity of aggregated proteins.

  3. Purification of a PHA-like chitin-binding protein from Acacia farnesiana seeds: a time-dependent oligomerization protein.

    PubMed

    Santi-Gadelha, T; Rocha, B A M; Oliveira, C C; Aragão, K S; Marinho, E S; Gadelha, C A A; Toyama, M H; Pinto, V P T; Nagano, C S; Delatorre, P; Martins, J L; Galvani, F R; Sampaio, A H; Debray, H; Cavada, B S

    2008-07-01

    A lectin-like protein from the seeds of Acacia farnesiana was isolated from the albumin fraction, characterized, and sequenced by tandem mass spectrometry. The albumin fraction was extracted with 0.5 M NaCl, and the lectin-like protein of A. farnesiana (AFAL) was purified by ion-exchange chromatography (Mono-Q) followed by chromatofocusing. AFAL agglutinated rabbit erythrocytes and did not agglutinate human ABO erythrocytes either native or treated with proteolytic enzymes. In sodium dodecyl sulfate gel electrophoresis under reducing and nonreducing conditions, AFAL separated into two bands with a subunit molecular mass of 35 and 50 kDa. The homogeneity of purified protein was confirmed by chromatofocusing with a pI = 4.0 +/- 0.5. Molecular exclusion chromatography confirmed time-dependent oligomerization in AFAL, in accordance with mass spectrometry analysis, which confers an alteration in AFAL affinity for chitin. The protein sequence was obtained by a liquid chromatography quadrupole time-of-flight experiment and showed that AFAL has 68% and 63% sequence similarity with lectins of Phaseolus vulgaris and Dolichos biflorus, respectively.

  4. Mechanism of DNA binding enhancement by hepatitis B virus protein pX.

    PubMed

    Palmer, C R; Gegnas, L D; Schepartz, A

    1997-12-09

    At least three hundred million people worldwide are infected with the hepatitis B virus (HBV), and epidemiological studies show a clear correlation between chronic HBV infection and the development of hepatocellular carcinoma. HBV encodes a protein, pX, which abducts the cellular transcriptional machinery in several ways including direct interactions with bZIP transcription factors. These interactions increase the DNA affinities of target bZIP proteins in a DNA sequence-dependent manner. Here we use a series of bZIP peptide models to explore the mechanism by which pX interacts with bZIP proteins. Our results suggest that pX increases bZIP.DNA stability by increasing the stability of the bZIP dimer as well as the affinity of the dimer for DNA. Additional experiments provide evidence for a mechanism in which pX recognizes the composite structure of the peptide.DNA complex, not simply the primary peptide sequence. These experiments provide a framework for understanding how pX alters the patterns of transcription within the nucleus. The similarities between the mechanism proposed for pX and the mechanism previously proposed for the human T-cell leukemia virus protein Tax are discussed.

  5. New procyanidin B3-human salivary protein complexes by mass spectrometry. Effect of salivary protein profile, tannin concentration, and time stability.

    PubMed

    Perez-Gregorio, Maria Rosa; Mateus, Nuno; De Freitas, Victor

    2014-10-15

    Several factors could influence the tannin-protein interaction such as the human salivary protein profile, the tannin tested, and the tannin/protein ratio. The goal of this study aims to study the effect of different salivas (A, B, and C) and different tannin concentrations (0.5 and 1 mg/mL) on the interaction process as well as the complex's stability over time. This study is focused on the identification of new procyanidin B3-human salivary protein complexes. Thus, 48 major B3-human salivary protein aggregates were identified regardless of the saliva and tannin concentration tested. A higher number of aggregates was found at lower tannin concentration. Moreover, the number of protein moieties involved in the aggregation process was higher when the tannin concentration was also higher. The selectivity of the different groups of proteins to bind tannin was also confirmed. It was also verified that the B3-human salivary protein complexes formed evolved over time.

  6. Activation of the pacidamycin PacL adenylation domain by MbtH-like proteins.

    PubMed

    Zhang, Wenjun; Heemstra, John R; Walsh, Christopher T; Imker, Heidi J

    2010-11-23

    Nonribosomal peptide synthetase (NRPS) assembly lines are major avenues for the biosynthesis of a vast array of peptidyl natural products. Several hundred bacterial NRPS gene clusters contain a small (∼70-residue) protein belonging to the MbtH family for which no function has been defined. Here we show that two strictly conserved Trp residues in MbtH-like proteins contribute to stimulation of amino acid adenylation in some NRPS modules. We also demonstrate that adenylation can be stimulated not only by cognate MbtH-like proteins but also by homologues from disparate natural product pathways.

  7. S100B protein in benzodiazepine overdose.

    PubMed

    Ambrozic, J; Bunc, M; Osredkar, J; Brvar, M

    2008-02-01

    Severe benzodiazepine overdose can result in coma and respiratory depression that might cause brain hypoxia, necrosis and delayed post-anoxic leucoencephalopathy with permanent neurological sequelae. The aim of this study was to assess the possible role of S100B, a structural protein of astroglial cells, as a biochemical marker of brain injury in acute benzodiazepine overdose. Serum S100B determination was performed in 38 consecutive patients admitted to the emergency department (ED) in Ljubljana with benzodiazepine overdose. The level of consciousness and respiratory insufficiency on the scene were assessed by responsiveness to a verbal stimulus and pulse oximetry. Blood samples were taken immediately after arrival at the ED and S100B concentrations were measured with a commercial immunoluminometric assay. 20 healthy sex- and age-matched volunteers formed a control group. There were significant differences in S100B levels between the control group and the patients with benzodiazepine overdose according to their responsiveness to a verbal stimulus. Post hoc test results showed that S100B levels in patients with benzodiazepine overdose who were unresponsive to a verbal stimulus were significantly higher than those in patients responsive to a verbal stimulus (median 0.31 vs 0.11 microg/l; p = 0.001). Both groups of patients with benzodiazepine overdose had significantly higher S100B levels than the control group (median 0.07 microg/; both p = 0.001). Arterial oxygen saturation of patients with benzodiazepine overdose unresponsive to a verbal stimulus was significantly lower than in patients responsive to a verbal stimulus (median 83% vs 94%; p = 0.001). There was no significant difference in the systolic blood pressure of patients with benzodiazepine overdose responsive or unresponsive to a verbal stimulus. Raised levels of S100B protein are associated with depressed levels of consciousness and respiratory insufficiency in patients with benzodiazepine overdose.

  8. Purification, partial characterization, crystallization and preliminary X-ray diffraction of a novel cardiotoxin-like basic protein from Naja naja atra (South Anhui) venom

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rong, Hui; Li, Yan; Lou, Xiao-hua

    2007-02-01

    A novel cardiotoxin-like basic protein from Naja naja atra was crystallized and diffraction data were collected to 2.35 Å resolution. A novel cardiotoxin-like basic protein was isolated from the venom of the Chinese cobra (Naja naja atra) from the south of Anhui in China. The protein inhibits the expression of vascular endothelial growth factor and basic fibroblast growth factor in human lung cancer cell line H1299 and induces the haemolysis of rabbit erythrocytes under low-lecithin conditions. After a two-step chromatographic purification, the resultant 7 kDa protein was crystallized by the hanging-drop vapour-diffusion method at room temperature. A complete data setmore » was collected to 2.35 Å resolution using an in-house X-ray diffraction system. The crystal belongs to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 43.2, c = 147.9 Å. There are two molecules in the crystallographic asymmetric unit.« less

  9. Conformational Stability of the NH2-Terminal Propeptide of the Precursor of Pulmonary Surfactant Protein SP-B

    PubMed Central

    Bañares-Hidalgo, Ángeles; Estrada, Pilar

    2016-01-01

    Assembly of pulmonary surfactant lipid-protein complexes depends on conformational changes coupled with proteolytic maturation of proSP-B, the precursor of pulmonary surfactant protein B (SP-B), along the surfactant biogenesis pathway in pneumocytes. Conformational destabilization of the N-terminal propeptide of proSP-B (SP-BN) triggers exposure of the mature SP-B domain for insertion into surfactant lipids. We have studied the conformational stability during GdmCl- or urea-promoted unfolding of SP-BN with trp fluorescence and circular dichroism spectroscopies. Binding of the intermediate states to bis-ANS suggests their molten globule-like character. ΔG0H2O was ~ 12.7 kJ·mol-1 either with urea or GdmCl. None of the thermal transitions of SP-BN detected by CD correspond to protein unfolding. Differential scanning calorimetry of SP-BN evidenced two endothermic peaks involved in oligomer dissociation as confirmed with 2 M urea. Ionic strength was relevant since at 150 mM NaCl, the process originating the endotherm at the highest temperature was irreversible (Tm2 = 108.5°C) with an activation energy of 703.8 kJ·mol-1. At 500 mM NaCl the process became reversible (Tm2 = 114.4°C) and data were fitted to the Non-two States model with two subpeaks. No free thiols in the propeptide could be titrated by DTNB with or without 5.7 M GdmCl, indicating disulfide bonds establishment. PMID:27380171

  10. Purification and characterization of Escherichia coli MreB protein.

    PubMed

    Nurse, Pearl; Marians, Kenneth J

    2013-02-01

    The actin homolog MreB is required in rod-shaped bacteria for maintenance of cell shape and is intimately connected to the holoenzyme that synthesizes the peptidoglycan layer. The protein has been reported variously to exist in helical loops under the cell surface, to rotate, and to move in patches in both directions around the cell surface. Studies of the Escherichia coli protein in vitro have been hampered by its tendency to aggregate. Here we report the purification and characterization of native E. coli MreB. The protein requires ATP hydrolysis for polymerization, forms bundles with a left-hand twist that can be as long as 4 μm, forms sheets in the presence of calcium, and has a critical concentration for polymerization of 1.5 μM.

  11. Purification and Characterization of Escherichia coli MreB Protein*

    PubMed Central

    Nurse, Pearl; Marians, Kenneth J.

    2013-01-01

    The actin homolog MreB is required in rod-shaped bacteria for maintenance of cell shape and is intimately connected to the holoenzyme that synthesizes the peptidoglycan layer. The protein has been reported variously to exist in helical loops under the cell surface, to rotate, and to move in patches in both directions around the cell surface. Studies of the Escherichia coli protein in vitro have been hampered by its tendency to aggregate. Here we report the purification and characterization of native E. coli MreB. The protein requires ATP hydrolysis for polymerization, forms bundles with a left-hand twist that can be as long as 4 μm, forms sheets in the presence of calcium, and has a critical concentration for polymerization of 1.5 μm. PMID:23235161

  12. A Nitrogen Response Pathway Regulates Virulence Functions in Fusarium oxysporum via the Protein Kinase TOR and the bZIP Protein MeaB[C][W

    PubMed Central

    López-Berges, Manuel S.; Rispail, Nicolas; Prados-Rosales, Rafael C.; Di Pietro, Antonio

    2010-01-01

    During infection, fungal pathogens activate virulence mechanisms, such as host adhesion, penetration and invasive growth. In the vascular wilt fungus Fusarium oxysporum, the mitogen-activated protein kinase Fmk1 is required for plant infection and controls processes such as cellophane penetration, vegetative hyphal fusion, or root adhesion. Here, we show that these virulence-related functions are repressed by the preferred nitrogen source ammonium and restored by treatment with l-methionine sulfoximine or rapamycin, two specific inhibitors of Gln synthetase and the protein kinase TOR, respectively. Deletion of the bZIP protein MeaB also resulted in nitrogen source–independent activation of virulence mechanisms. Activation of these functions did not require the global nitrogen regulator AreA, suggesting that MeaB-mediated repression of virulence functions does not act through inhibition of AreA. Tomato plants (Solanum lycopersicum) supplied with ammonium rather than nitrate showed a significant reduction in vascular wilt symptoms when infected with the wild type but not with the ΔmeaB strain. Nitrogen source also affected invasive growth in the rice blast fungus Magnaporthe oryzae and the wheat head blight pathogen Fusarium graminearum. We propose that a conserved nitrogen-responsive pathway might operate via TOR and MeaB to control virulence in plant pathogenic fungi. PMID:20639450

  13. Identification, inheritance, and linkage of B-G-like and MHC class I genes in cranes

    USGS Publications Warehouse

    Jarvi, S.I.; Goto, R.M.; Gee, G.F.; Briles, W.E.; Miller, M.M.

    1999-01-01

    We identified B-G-like genes in the whooping and Florida sandhill cranes and linked them to the major histocompatibility complex (MHC). We evaluated the inheritance of B-G-like genes in families of whooping and Florida sandhill cranes using restriction fragment patterns (RFPs). Two B-G-like genes, designated wcbgl and wcbg2, were located within 8 kb of one another. The fully sequenced wcbg2 gene encodes a B-G IgV-like domain, an additional Ig-like domain, a transmembrane domain, and a single heptad domain typical of '-helical coiled coils. Patterns of restriction fragments in DNA from the whooping crane and from a number of other species indicate that the B-G-like gene families of cranes are large with diverse sequences. Segregation of RFPs in families of Florida sandhill cranes provide evidence for genetic polymorphism in the B-G-like genes. The restriction fragments generally segregated in concert with MHC haplotypes assigned by serological typing and by single stranded conformational polymorphism (SSCP) assays based in the second exon of the crane MHC class I genes. This study supports the concept of a long-term association of polymorphic B-G-like genes with the MHC. It also establishes SSCP as a means for evaluating MHC genetic variability in cranes.

  14. Identification, inheritance, and linkage of B-G-like and MHC class I genes in cranes.

    PubMed

    Jarvi, S I; Goto, R M; Gee, G F; Briles, W E; Miller, M M

    1999-01-01

    We identified B-G-like genes in the whooping and Florida sandhill cranes and linked them to the major histocompatibility complex (MHC). We evaluated the inheritance of B-G-like genes in families of whooping and Florida sandhill cranes using restriction fragment patterns (RFPs). Two B-G-like genes, designated wcbg1 and wcbg2, were located within 8 kb of one another. The fully sequenced wcbg2 gene encodes a B-G IgV-like domain, an additional Ig-like domain, a transmembrane domain, and a single heptad domain typical of alpha-helical coiled coils. Patterns of restriction fragments in DNA from the whooping crane and from a number of other species indicate that the B-G-like gene families of cranes are large with diverse sequences. Segregation of RFPs in families of Florida sandhill cranes provide evidence for genetic polymorphism in the B-G-like genes. The restriction fragments generally segregated in concert with MHC haplotypes assigned by serological typing and by single stranded conformational polymorphism (SSCP) assays based in the second exon of the crane MHC class I genes. This study supports the concept of a long-term association of polymorphic B-G-like genes with the MHC. It also establishes SSCP as a means for evaluating MHC genetic variability in cranes.

  15. Isolation and Identification of Putative Protein Substrates of the AAA+ Molecular Chaperone ClpB from the Pathogenic Spirochaete Leptospira interrogans.

    PubMed

    Krajewska, Joanna; Arent, Zbigniew; Zolkiewski, Michal; Kędzierska-Mieszkowska, Sabina

    2018-04-18

    Bacterial ClpB is an ATP-dependent Hsp100 chaperone that reactivates aggregated proteins in cooperation with the DnaK chaperone system and promotes survival of bacteria under stress conditions. A large number of publications also indicate that ClpB supports the virulence of bacteria, including a pathogenic spirochaete Leptospira interrogans responsible for leptospirosis in both animals and humans. However, the exact role of ClpB in bacterial pathogenicity remains poorly characterized. It can be assumed that ClpB, due to its role as the molecular chaperone, mediates refolding of essential bacterial proteins, including the known virulence factors, which may become prone to aggregation under infection-induced stresses. In this study, we identified putative substrates of ClpB from L. interrogans (ClpB Li ). For this purpose, we used a proteomic approach combining the ClpB-Trap affinity pull-down assays, Liquid chromatography-tandem mass spectrometry (LC-MS-MS/MS), and bioinformatics analyses. Most of the identified proteins were enzymes predominantly associated with major metabolic pathways like the tricarboxylic acid (TCA) cycle, glycolysis–gluconeogenesis and amino acid and fatty acid metabolism. Based on our proteomic study, we suggest that ClpB can support the virulence of L. interrogans by protecting the conformational integrity and catalytic activity of multiple metabolic enzymes, thus maintaining energy homeostasis in pathogen cells.

  16. RED CELL STROMA PROTEIN RICH IN VITAMIN B12 DURING ACTIVE REGENERATION

    PubMed Central

    Whipple, G. H.; Robscheit-Robbins, F. S.; Bale, W. F.

    1955-01-01

    During active blood regeneration in anemia in dogs an increase occurs in the stroma protein of the red cells. When vitamin B12 with radioactive cobalt is given at the start of this blood regeneration one finds concentration of labeled B 12 in the stroma protein but not in the hemoglobin. After the acute phase of red cell regeneration is ended the concentration of B12 in stroma protein falls rapidly to very low levels within 2 weeks. Subsequent episodes of red blood cell regeneration seems not to cause remobilization of radioactive cobalt into red cells from other body stores. It appears that the vitamin B12 is a factor of importance in the first steps of stroma protein formation in the first few days of the life of the red cell in the dog. This response in dogs and the response in pernicious anemia to vitamin B12 may have some points in common. Distribution of the B12-radioactive cobalt in the organs and tissues at autopsy has been recorded. Some very suggestive localizations were noted and some variation 1 week and 7 weeks after B12 injections. Radioactive cobalt escapes in the urine during the weeks following B12 injections. PMID:13271685

  17. Enteral B-hydroxy-B-methylbutyrate supplementation increases protein synthesis in skeletal muscle of neonatal pigs

    USDA-ARS?s Scientific Manuscript database

    Many low-birth weight infants are at risk for poor growth due to an inability to achieve adequate protein intake. Administration of the amino acid leucine stimulates protein synthesis in skeletal muscle of neonates. To determine the effects of enteral supplementation of the leucine metabolite B-hydr...

  18. Identification and biochemical characterization of an acid sphingomyelinase-like protein from the bacterial plant pathogen Ralstonia solanacearum that hydrolyzes ATP to AMP but not sphingomyelin to ceramide.

    PubMed

    Airola, Michael V; Tumolo, Jessica M; Snider, Justin; Hannun, Yusuf A

    2014-01-01

    Acid sphingomyelinase (aSMase) is a human enzyme that catalyzes the hydrolysis of sphingomyelin to generate the bioactive lipid ceramide and phosphocholine. ASMase deficiency is the underlying cause of the genetic diseases Niemann-Pick Type A and B and has been implicated in the onset and progression of a number of other human diseases including cancer, depression, liver, and cardiovascular disease. ASMase is the founding member of the aSMase protein superfamily, which is a subset of the metallophosphatase (MPP) superfamily. To date, MPPs that share sequence homology with aSMase, termed aSMase-like proteins, have been annotated and presumed to function as aSMases. However, none of these aSMase-like proteins have been biochemically characterized to verify this. Here we identify RsASML, previously annotated as RSp1609: acid sphingomyelinase-like phosphodiesterase, as the first bacterial aSMase-like protein from the deadly plant pathogen Ralstonia solanacearum based on sequence homology with the catalytic and C-terminal domains of human aSMase. A biochemical characterization of RsASML does not support a role in sphingomyelin hydrolysis but rather finds RsASML capable of acting as an ATP diphosphohydrolase, catalyzing the hydrolysis of ATP and ADP to AMP. In addition, RsASML displays a neutral, not acidic, pH optimum and prefers Ni2+ or Mn2+, not Zn2+, for catalysis. This alters the expectation that all aSMase-like proteins function as acid SMases and expands the substrate possibilities of this protein superfamily to include nucleotides. Overall, we conclude that sequence homology with human aSMase is not sufficient to predict substrate specificity, pH optimum for catalysis, or metal dependence. This may have implications to the biochemically uncharacterized human aSMase paralogs, aSMase-like 3a (aSML3a) and aSML3b, which have been implicated in cancer and kidney disease, respectively, and assumed to function as aSMases.

  19. EPI64B Acts as a GTPase-activating Protein for Rab27B in Pancreatic Acinar Cells*

    PubMed Central

    Hou, Yanan; Chen, Xuequn; Tolmachova, Tatyana; Ernst, Stephen A.; Williams, John A.

    2013-01-01

    The small GTPase Rab27B localizes to the zymogen granule membranes and plays an important role in regulating protein secretion by pancreatic acinar cells, as does Rab3D. A common guanine nucleotide exchange factor (GEF) for Rab3 and Rab27 has been reported; however, the GTPase-activating protein (GAP) specific for Rab27B has not been identified. In this study, the expression in mouse pancreatic acini of two candidate Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins, EPI64 (TBC1D10A) and EPI64B (TBC1D10B), was first demonstrated. Their GAP activity on digestive enzyme secretion was examined by adenovirus-mediated overexpression of EPI64 and EPI64B in isolated pancreatic acini. EPI64B almost completely abolished the GTP-bound form of Rab27B, without affecting GTP-Rab3D. Overexpression of EPI64B also enhanced amylase release. This enhanced release was independent of Rab27A, but dependent on Rab27B, as shown using acini from genetically modified mice. EPI64 had a mild effect on both GTP-Rab27B and amylase release. Co-overexpression of EPI64B with Rab27B can reverse the inhibitory effect of Rab27B on amylase release. Mutations that block the GAP activity decreased the inhibitory effect of EPI64B on the GTP-bound state of Rab27B and abolished the enhancing effect of EPI64B on the amylase release. These data suggest that EPI64B can serve as a potential physiological GAP for Rab27B and thereby participate in the regulation of exocytosis in pancreatic acinar cells. PMID:23671284

  20. The promiscuous protein binding ability of erythrosine B studied by metachromasy (metachromasia).

    PubMed

    Ganesan, Lakshmi; Buchwald, Peter

    2013-04-01

    The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein-protein interactions (PPIs) with a remarkably consistent median inhibitory concentration (IC50 ) in the 5- to 30-μM range. Because ErB exhibits metachromasy, that is, color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd ) and stoichiometry (nb ) using spectrophotometric methods. Binding was reversible, and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 μM for BSA and CD40L, respectively) were in good agreement with that expected from the PPI inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5-6 and 8-9 for BSA and CD40L, respectively), indicating the possibility of nonspecific binding of the flat and rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. Copyright © 2013 John Wiley & Sons, Ltd.

  1. The Promiscuous Protein Binding Ability of Erythrosine B Studied by Metachromasy (Metachromasia)

    PubMed Central

    Ganesan, Lakshmi; Buchwald, Peter

    2013-01-01

    The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein–protein interactions (PPI) with a remarkably consistent median inhibitory concentration (IC50) in the 5–30 µM range. Because ErB exhibits metachromasy, i.e., color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd) and stoichiometry (nb) using spectrophotometric methods. Binding was reversible and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 µM for BSA and CD40L, respectively) were in good agreement with that expected from the protein–protein interaction (PPI) inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5–6 and 8–9 for BSA and CD40L, respectively) indicating the possibility of nonspecific binding of the flat an rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. PMID:23456742

  2. The 8th and 9th tandem spectrin-like repeats of utrophin cooperatively form a functional unit to interact with polarity-regulating kinase PAR-1b.

    PubMed

    Yamashita, Kazunari; Suzuki, Atsushi; Satoh, Yoshinori; Ide, Mariko; Amano, Yoshiko; Masuda-Hirata, Maki; Hayashi, Yukiko K; Hamada, Keisuke; Ogata, Kazuhiro; Ohno, Shigeo

    2010-01-01

    Utrophin is a widely expressed paralogue of dystrophin, the protein responsible for Duchenne muscular dystrophy. Utrophin is a large spectrin-like protein whose C-terminal domain mediates anchorage to a laminin receptor, dystroglycan (DG). The rod domain, composed of 22 spectrin-like repeats, connects the N-terminal actin-binding domain and the C-terminal DG binding domain, and thus mediates molecular linkage between intracellular F-actin and extracellular basement membrane. Previously, we demonstrated that a cell polarity-regulating kinase, PAR-1b, interacts with the utrophin-DG complex, and positively regulates the interaction between utrophin and DG. In this study, we demonstrate that the 8th and 9th spectrin-like repeats (R8 and R9) of utrophin cooperatively form a PAR-1b-interacting domain, and that Ser1258 within R9 is specifically phosphorylated by PAR-1b. Substitution of Ser1258 to alanine reduces the interaction between utrophin and DG, suggesting that the Ser1258 phosphorylation contributes to the stabilization of the utrophin-DG complex. Interestingly, PAR-1b also binds and phosphorylates R8-9 of dystrophin, and colocalizes with dystrophin at the skeletal muscle membrane. These results reveal a novel function of the rod domain of utrophin beyond that of a passive structural linker connecting the N- and C-terminal domain. Copyright 2009 Elsevier Inc. All rights reserved.

  3. Regulatory Interactions of Csr Components: the RNA Binding Protein CsrA Activates csrB Transcription in Escherichia coli

    PubMed Central

    Gudapaty, Seshagirirao; Suzuki, Kazushi; Wang, Xin; Babitzke, Paul; Romeo, Tony

    2001-01-01

    The global regulator CsrA (carbon storage regulator) of Escherichia coli is a small RNA binding protein that represses various metabolic pathways and processes that are induced in the stationary phase of growth, while it activates certain exponential phase functions. Both repression and activation by CsrA involve posttranscriptional mechanisms, in which CsrA binding to mRNA leads to decreased or increased transcript stability, respectively. CsrA also binds to a small untranslated RNA, CsrB, forming a ribonucleoprotein complex, which antagonizes CsrA activity. We have further examined the regulatory interactions of CsrA and CsrB RNA. The 5′ end of the CsrB transcript was mapped, and a csrB::cam null mutant was constructed. CsrA protein and CsrB RNA levels were estimated throughout the growth curves of wild-type and isogenic csrA, csrB, rpoS, or csrA rpoS mutant strains. CsrA levels exhibited modest or negligible effects of these mutations. The intracellular concentration of CsrA exceeded the total CsrA-binding capacity of intracellular CsrB RNA. In contrast, CsrB levels were drastically decreased (∼10-fold) in the csrA mutants. CsrB transcript stability was unaffected by csrA. The expression of a csrB-lacZ transcriptional fusion containing the region from −242 to +4 bp of the csrB gene was decreased ∼20-fold by a csrA::kanR mutation in vivo but was unaffected by CsrA protein in vitro. These results reveal a significant, though most likely indirect, role for CsrA in regulating csrB transcription. Furthermore, our findings suggest that CsrA mediates an intriguing form of autoregulation, whereby its activity, but not its levels, is modulated through effects on an RNA antagonist, CsrB. PMID:11567002

  4. Characterization of Three Novel Linear Neutralizing B-Cell Epitopes in the Capsid Protein of Swine Hepatitis E Virus.

    PubMed

    Chen, Yiyang; Liu, Baoyuan; Sun, Yani; Li, Huixia; Du, Taofeng; Nan, Yuchen; Hiscox, Julian A; Zhou, En-Min; Zhao, Qin

    2018-07-01

    Hepatitis E virus (HEV) causes liver disease in humans and is thought to be a zoonotic infection, with domestic animals, including swine and rabbits, being a reservoir. One of the proteins encoded by the virus is the capsid protein. This is likely the major immune-dominant protein and a target for vaccination. Four monoclonal antibodies (MAbs), three novel, 1E4, 2C7, and 2G9, and one previously characterized, 1B5, were evaluated for binding to the capsid protein from genotype 4 swine HEV. The results indicated that 625 DFCP 628 , 458 PSRPF 462 , and 407 EPTV 410 peptides on the capsid protein comprised minimal amino acid sequence motifs recognized by 1E4, 2C7, and 2G9, respectively. The data suggested that 2C7 and 2G9 epitopes were partially exposed on the surface of the capsid protein. Truncated genotype 4 swine HEV capsid protein (sp239, amino acids 368 to 606) can exist in multimeric forms. Preincubation of swine HEV with 2C7, 2G9, or 1B5 before addition to HepG2 cells partially blocked sp239 cell binding and inhibited swine HEV infection. The study indicated that 2C7, 2G9, and 1B5 partially blocked swine HEV infection of rabbits better than 1E4 or normal mouse IgG. The cross-reactivity of antibodies suggested that capsid epitopes recognized by 2C7 and 2G9 are common to HEV strains infecting most host species. Collectively, MAbs 2C7, 2G9, and 1B5 were shown to recognize three novel linear neutralizing B-cell epitopes of genotype 4 HEV capsid protein. These results enhance understanding of HEV capsid protein structure to guide vaccine and antiviral design. IMPORTANCE Genotype 3 and 4 HEVs are zoonotic viruses. Here, genotype 4 HEV was studied due to its prevalence in human populations and pig herds in China. To improve HEV disease diagnosis and prevention, a better understanding of the antigenic structure and neutralizing epitopes of HEV capsid protein are needed. In this study, the locations of three novel linear B-cell recognition epitopes within genotype 4

  5. Identification of a nuclear localization signal in the retinitis pigmentosa-mutated RP26 protein, ceramide kinase-like protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Inagaki, Yuichi; Mitsutake, Susumu; Igarashi, Yasuyuki

    2006-05-12

    Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. A mutation in a new ceramide kinase (CERK) homologous gene, named CERK-like protein (CERKL), was found to cause autosomal recessive retinitis pigmentosa (RP26). Here, we show a point mutation of one of two putative nuclear localization signal (NLS) sequences inhibited the nuclear localization of the protein. Furthermore, the tetra-GFP-tagged NLS, which cannot passively enter the nucleus, was observed not only in the nucleus but also in the nucleolus. Our results provide First evidence of the active nuclear import of CERKL and suggest that the identified NLSmore » might be responsible for nucleolar retention of the protein. As recent studies have shown other RP-related proteins are localized in the nucleus or the nucleolus, our identification of NLS in CERKL suggests that CERKL likely plays important roles for retinal functions in the nucleus and the nucleolus.« less

  6. Distinct activities of Bartonella henselae type IV secretion effector proteins modulate capillary-like sprout formation.

    PubMed

    Scheidegger, F; Ellner, Y; Guye, P; Rhomberg, T A; Weber, H; Augustin, H G; Dehio, C

    2009-07-01

    The zoonotic pathogen Bartonella henselae (Bh) can lead to vasoproliferative tumour lesions in the skin and inner organs known as bacillary angiomatosis and bacillary peliosis. The knowledge on the molecular and cellular mechanisms involved in this pathogen-triggered angiogenic process is confined by the lack of a suitable animal model and a physiologically relevant cell culture model of angiogenesis. Here we employed a three-dimensional in vitro angiogenesis assay of collagen gel-embedded endothelial cell (EC) spheroids to study the angiogenic properties of Bh. Spheroids generated from Bh-infected ECs displayed a high capacity to form sprouts, which represent capillary-like projections into the collagen gel. The VirB/VirD4 type IV secretion system and a subset of its translocated Bartonella effector proteins (Beps) were found to profoundly modulate this Bh-induced sprouting activity. BepA, known to protect ECs from apoptosis, strongly promoted sprout formation. In contrast, BepG, triggering cytoskeletal rearrangements, potently inhibited sprouting. Hence, the here established in vitro model of Bartonella- induced angiogenesis revealed distinct and opposing activities of type IV secretion system effector proteins, which together with a VirB/VirD4-independent effect may control the angiogenic activity of Bh during chronic infection of the vasculature.

  7. Inferring the effects of compositional boundary layers on crystal nucleation, growth textures, and mineral chemistry in natural volcanic tephras through submicron-resolution imaging

    NASA Astrophysics Data System (ADS)

    Zellmer, Georg; Sakamoto, Naoya; Hwang, Shyh-Lung; Matsuda, Nozomi; Iizuka, Yoshiyuki; Moebis, Anja; Yurimoto, Hisayoshi

    2016-09-01

    Crystal nucleation and growth are first order processes captured in volcanic rocks and record important information about the rates of magmatic processes and chemical evolution of magmas during their ascent and eruption. We have studied glass-rich andesitic tephras from the Central Plateau of the Southern Taupo Volcanic Zone by electron- and ion-microbeam imaging techniques to investigate down to sub-micrometre scale the potential effects of compositional boundary layers (CBLs) of melt around crystals on the nucleation and growth of mineral phases and the chemistry of crystal growth zones. We find that CBLs may influence the types of mineral phases nucleating and growing, and growth textures such as the development of swallowtails. The chemistry of the CBLs also has the capacity to trigger intermittent overgrowths of nanometre-scale bands of different phases in rapidly growing crystals, resulting in what we refer to as cryptic phase zoning. The existence of cryptic phase zoning has implications for the interpretation of microprobe compositional data, and the resulting inferences made on the conditions of magmatic evolution. Identification of cryptic phase zoning may in future lead to more accurate thermobarometric estimates and thus geospeedometric constraints. In future, a more quantitative characterization of CBL formation and its effects on crystal nucleation and growth may contribute to a better understanding of melt rheology and magma ascent processes at the onset of explosive volcanic eruptions, and will likely be of benefit to hazard mitigation efforts.

  8. Characterization of the Bacteroides fragilis bfr Gene Product Identifies a Bacterial DPS-Like Protein and Suggests Evolutionary Links in the Ferritin Superfamily

    PubMed Central

    Gauss, George H.; Reott, Michael A.; Rocha, Edson R.; Young, Mark J.; Douglas, Trevor

    2012-01-01

    A factor contributing to the pathogenicity of Bacteroides fragilis, the most common anaerobic species isolated from clinical infections, is the bacterium's extreme aerotolerance, which allows survival in oxygenated tissues prior to anaerobic abscess formation. We investigated the role of the bacterioferritin-related (bfr) gene in the B. fragilis oxidative stress response. The bfr mRNA levels are increased in stationary phase or in response to O2 or iron. In addition, bfr null mutants exhibit reduced aerotolerance, and the bfr gene product protects DNA from hydroxyl radical cleavage in vitro. Crystallographic studies revealed a protein with a dodecameric structure and greater similarity to an archaeal DNA protection in starved cells (DPS)-like protein than to the 24-subunit bacterioferritins. Similarity to the DPS-like (DPSL) protein extends to the subunit and includes a pair of conserved cysteine residues juxtaposed to a buried dimetal binding site within the four-helix bundle. Compared to archaeal DPSLs, however, this bacterial DPSL protein contains several unique features, including a significantly different conformation in the C-terminal tail that alters the number and location of pores leading to the central cavity and a conserved metal binding site on the interior surface of the dodecamer. Combined, these characteristics confirm this new class of miniferritin in the bacterial domain, delineate the similarities and differences between bacterial DPSL proteins and their archaeal homologs, allow corrected annotations for B. fragilis bfr and other dpsl genes within the bacterial domain, and suggest an evolutionary link within the ferritin superfamily that connects dodecameric DPS to the (bacterio)ferritin 24-mer. PMID:22020642

  9. Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes

    PubMed Central

    Gedvilaite, Alma; Kucinskaite-Kodze, Indre; Lasickiene, Rita; Timinskas, Albertas; Vaitiekaite, Ausra; Ziogiene, Danguole; Zvirbliene, Aurelija

    2015-01-01

    Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes. PMID:26230706

  10. Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

    NASA Technical Reports Server (NTRS)

    Li, H.; Roux, S. J.

    1992-01-01

    A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

  11. Members of the Meloidogyne avirulence protein family contain multiple plant ligand-like motifs.

    PubMed

    Rutter, William B; Hewezi, Tarek; Maier, Tom R; Mitchum, Melissa G; Davis, Eric L; Hussey, Richard S; Baum, Thomas J

    2014-08-01

    Sedentary plant-parasitic nematodes engage in complex interactions with their host plants by secreting effector proteins. Some effectors of both root-knot nematodes (Meloidogyne spp.) and cyst nematodes (Heterodera and Globodera spp.) mimic plant ligand proteins. Most prominently, cyst nematodes secrete effectors that mimic plant CLAVATA3/ESR-related (CLE) ligand proteins. However, only cyst nematodes have been shown to secrete such effectors and to utilize CLE ligand mimicry in their interactions with host plants. Here, we document the presence of ligand-like motifs in bona fide root-knot nematode effectors that are most similar to CLE peptides from plants and cyst nematodes. We have identified multiple tandem CLE-like motifs conserved within the previously identified Meloidogyne avirulence protein (MAP) family that are secreted from root-knot nematodes and have been shown to function in planta. By searching all 12 MAP family members from multiple Meloidogyne spp., we identified 43 repetitive CLE-like motifs composing 14 unique variants. At least one CLE-like motif was conserved in each MAP family member. Furthermore, we documented the presence of other conserved sequences that resemble the variable domains described in Heterodera and Globodera CLE effectors. These findings document that root-knot nematodes appear to use CLE ligand mimicry and point toward a common host node targeted by two evolutionarily diverse groups of nematodes. As a consequence, it is likely that CLE signaling pathways are important in other phytonematode pathosystems as well.

  12. The multifunctional LigB adhesin binds homeostatic proteins with potential roles in cutaneous infection by pathogenic Leptospira interrogans.

    PubMed

    Choy, Henry A; Kelley, Melissa M; Croda, Julio; Matsunaga, James; Babbitt, Jane T; Ko, Albert I; Picardeau, Mathieu; Haake, David A

    2011-02-09

    Leptospirosis is a potentially fatal zoonotic disease in humans and animals caused by pathogenic spirochetes, such as Leptospira interrogans. The mode of transmission is commonly limited to the exposure of mucous membrane or damaged skin to water contaminated by leptospires shed in the urine of carriers, such as rats. Infection occurs during seasonal flooding of impoverished tropical urban habitats with large rat populations, but also during recreational activity in open water, suggesting it is very efficient. LigA and LigB are surface localized proteins in pathogenic Leptospira strains with properties that could facilitate the infection of damaged skin. Their expression is rapidly induced by the increase in osmolarity encountered by leptospires upon transition from water to host. In addition, the immunoglobulin-like repeats of the Lig proteins bind proteins that mediate attachment to host tissue, such as fibronectin, fibrinogen, collagens, laminin, and elastin, some of which are important in cutaneous wound healing and repair. Hemostasis is critical in a fresh injury, where fibrinogen from damaged vasculature mediates coagulation. We show that fibrinogen binding by recombinant LigB inhibits fibrin formation, which could aid leptospiral entry into the circulation, dissemination, and further infection by impairing healing. LigB also binds fibroblast fibronectin and type III collagen, two proteins prevalent in wound repair, thus potentially enhancing leptospiral adhesion to skin openings. LigA or LigB expression by transformation of a nonpathogenic saprophyte, L. biflexa, enhances bacterial adhesion to fibrinogen. Our results suggest that by binding homeostatic proteins found in cutaneous wounds, LigB could facilitate leptospirosis transmission. Both fibronectin and fibrinogen binding have been mapped to an overlapping domain in LigB comprising repeats 9-11, with repeat 11 possibly enhancing binding by a conformational effect. Leptospirosis patient antibodies react

  13. The Multifunctional LigB Adhesin Binds Homeostatic Proteins with Potential Roles in Cutaneous Infection by Pathogenic Leptospira interrogans

    PubMed Central

    Choy, Henry A.; Kelley, Melissa M.; Croda, Julio; Matsunaga, James; Babbitt, Jane T.; Ko, Albert I.; Picardeau, Mathieu; Haake, David A.

    2011-01-01

    Leptospirosis is a potentially fatal zoonotic disease in humans and animals caused by pathogenic spirochetes, such as Leptospira interrogans. The mode of transmission is commonly limited to the exposure of mucous membrane or damaged skin to water contaminated by leptospires shed in the urine of carriers, such as rats. Infection occurs during seasonal flooding of impoverished tropical urban habitats with large rat populations, but also during recreational activity in open water, suggesting it is very efficient. LigA and LigB are surface localized proteins in pathogenic Leptospira strains with properties that could facilitate the infection of damaged skin. Their expression is rapidly induced by the increase in osmolarity encountered by leptospires upon transition from water to host. In addition, the immunoglobulin-like repeats of the Lig proteins bind proteins that mediate attachment to host tissue, such as fibronectin, fibrinogen, collagens, laminin, and elastin, some of which are important in cutaneous wound healing and repair. Hemostasis is critical in a fresh injury, where fibrinogen from damaged vasculature mediates coagulation. We show that fibrinogen binding by recombinant LigB inhibits fibrin formation, which could aid leptospiral entry into the circulation, dissemination, and further infection by impairing healing. LigB also binds fibroblast fibronectin and type III collagen, two proteins prevalent in wound repair, thus potentially enhancing leptospiral adhesion to skin openings. LigA or LigB expression by transformation of a nonpathogenic saprophyte, L. biflexa, enhances bacterial adhesion to fibrinogen. Our results suggest that by binding homeostatic proteins found in cutaneous wounds, LigB could facilitate leptospirosis transmission. Both fibronectin and fibrinogen binding have been mapped to an overlapping domain in LigB comprising repeats 9–11, with repeat 11 possibly enhancing binding by a conformational effect. Leptospirosis patient antibodies react

  14. DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats.

    PubMed

    de Lange, Orlando; Wolf, Christina; Thiel, Philipp; Krüger, Jens; Kleusch, Christian; Kohlbacher, Oliver; Lahaye, Thomas

    2015-11-16

    Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Immunological Properties of Hepatitis B Core Antigen Fusion Proteins

    NASA Astrophysics Data System (ADS)

    Francis, Michael J.; Hastings, Gillian Z.; Brown, Alan L.; Grace, Ken G.; Rowlands, David J.; Brown, Fred; Clarke, Berwyn E.

    1990-04-01

    The immunogenicity of a 19 amino acid peptide from foot-and-mouth disease virus has previously been shown to approach that of the inactivated virus from which it was derived after multimeric particulate presentation as an N-terminal fusion with hepatitis B core antigen. In this report we demonstrate that rhinovirus peptide-hepatitis B core antigen fusion proteins are 10-fold more immunogenic than peptide coupled to keyhole limpet hemocyanin and 100-fold more immunogenic than uncoupled peptide with an added helper T-cell epitope. The fusion proteins can be readily administered without adjuvant or with adjuvants acceptable for human and veterinary application and can elicit a response after nasal or oral dosing. The fusion proteins can also act as T-cell-independent antigens. These properties provide further support for their suitability as presentation systems for "foreign" epitopes in the development of vaccines.

  16. Analysis of Native-Like Proteins and Protein Complexes Using Cation to Anion Proton Transfer Reactions (CAPTR)

    NASA Astrophysics Data System (ADS)

    Laszlo, Kenneth J.; Bush, Matthew F.

    2015-12-01

    Mass spectra of native-like protein complexes often exhibit narrow charge-state distributions, broad peaks, and contributions from multiple, coexisting species. These factors can make it challenging to interpret those spectra, particularly for mixtures with significant heterogeneity. Here we demonstrate the use of ion/ion proton transfer reactions to reduce the charge states of m/ z-selected, native-like ions of proteins and protein complexes, a technique that we refer to as cation to anion proton transfer reactions (CAPTR). We then demonstrate that CAPTR can increase the accuracy of charge state assignments and the resolution of interfering species in native mass spectrometry. The CAPTR product ion spectra for pyruvate kinase exhibit ~30 peaks and enable unambiguous determination of the charge state of each peak, whereas the corresponding precursor spectra exhibit ~6 peaks and the assigned charge states have an uncertainty of ±3%. 15+ bovine serum albumin and 21+ yeast enolase dimer both appear near m/ z 4450 and are completely unresolved in a mixture. After a single CAPTR event, the resulting product ions are baseline resolved. The separation of the product ions increases dramatically after each subsequent CAPTR event; 12 events resulted in a 3000-fold improvement in separation relative to the precursor ions. Finally, we introduce a framework for interpreting and predicting the figures of merit for CAPTR experiments. More generally, these results suggest that CAPTR strongly complements other mass spectrometry tools for analyzing proteins and protein complexes, particularly those in mixtures.

  17. Molecular and functional characterization of single-box high-mobility group B (HMGB) chromosomal protein from Aedes aegypti.

    PubMed

    de Abreu da Silva, Isabel Caetano; Vicentino, Amanda Roberta Revoredo; Dos Santos, Renata Coutinho; da Fonseca, Rodrigo Nunes; de Mendonça Amarante, Anderson; Carneiro, Vitor Coutinho; de Amorim Pinto, Marcia; Aguilera, Estefania Anahi; Mohana-Borges, Ronaldo; Bisch, Paulo Mascarello; da Silva-Neto, Mario Alberto Cardoso; Fantappié, Marcelo Rosado

    2018-05-30

    High-mobility group B (HMGB) proteins have highly conserved, unique DNA-binding domains, HMG boxes, that can bind non-B-type DNA structures, such as bent, kinked and unwound structures, with high affinity. HMGB proteins also promote DNA bending, looping and unwinding. In this study, we determined the role of the Aedes aegypti single HMG-box domain protein AaHMGB; characterized its structure, spatiotemporal expression levels, subcellular localization, and nucleic acid binding activities; and compared these properties with those of its double-HMG-box counterpart protein, AaHMGB1. Via qRT-PCR, we showed that AaHMGB is expressed at much higher levels than AaHMGB1 throughout mosquito development. In situ hybridization results suggested a role for AaHMGB and AaHMGB1 during embryogenesis. Immunolocalization in the midgut revealed that AaHMGB is exclusively nuclear. Circular dichroism and fluorescence spectroscopy analyses showed that AaHMGB exhibits common features of α-helical structures and is more stably folded than AaHMGB1, likely due to the presence of one or two HMG boxes. Using several DNA substrates or single-stranded RNAs as probes, we observed significant differences between AaHMGB and AaHMGB1 in terms of their binding patterns, activity and/or specificity. Importantly, we showed that the phosphorylation of AaHMGB plays a critical role in its DNA-binding activity. Our study provides additional insight into the roles of single- versus double-HMG-box-containing proteins in nucleic acid interactions for better understanding of mosquito development, physiology and homeostasis. Copyright © 2017. Published by Elsevier B.V.

  18. Mitotic MELK-eIF4B signaling controls protein synthesis and tumor cell survival

    PubMed Central

    Wang, Yubao; Begley, Michael; Li, Qing; Huang, Hai-Tsang; Lako, Ana; Eck, Michael J.; Gray, Nathanael S.; Mitchison, Timothy J.; Cantley, Lewis C.; Zhao, Jean J.

    2016-01-01

    The protein kinase maternal and embryonic leucine zipper kinase (MELK) is critical for mitotic progression of cancer cells; however, its mechanisms of action remain largely unknown. By combined approaches of immunoprecipitation/mass spectrometry and peptide library profiling, we identified the eukaryotic translation initiation factor 4B (eIF4B) as a MELK-interacting protein during mitosis and a bona fide substrate of MELK. MELK phosphorylates eIF4B at Ser406, a modification found to be most robust in the mitotic phase of the cell cycle. We further show that the MELK–eIF4B signaling axis regulates protein synthesis during mitosis. Specifically, synthesis of myeloid cell leukemia 1 (MCL1), an antiapoptotic protein known to play a role in cancer cell survival during cell division, depends on the function of MELK-elF4B. Inactivation of MELK or eIF4B results in reduced protein synthesis of MCL1, which, in turn, induces apoptotic cell death of cancer cells. Our study thus defines a MELK–eIF4B signaling axis that regulates protein synthesis during mitosis, and consequently influences cancer cell survival. PMID:27528663

  19. Poly-Small Ubiquitin-like Modifier (PolySUMO)-binding Proteins Identified through a String Search*

    PubMed Central

    Sun, Huaiyu; Hunter, Tony

    2012-01-01

    Polysumoylation is a crucial cellular response to stresses against genomic integrity or proteostasis. Like the small ubiquitin-like modifier (SUMO)-targeted ubiquitin ligase RNF4, proteins with clustered SUMO-interacting motifs (SIMs) can be important signal transducers downstream of polysumoylation. To identify novel polySUMO-binding proteins, we conducted a computational string search with a custom Python script. We found clustered SIMs in another RING domain protein Arkadia/RNF111. Detailed biochemical analysis of the Arkadia SIMs revealed that dominant SIMs in a SIM cluster often contain a pentameric VIDLT ((V/I/L/F/Y)(V/I)DLT) core sequence that is also found in the SIMs in PIAS family E3s and is likely the best-fitted structure for SUMO recognition. This idea led to the identification of additional novel SIM clusters in FLASH/CASP8AP2, C5orf25, and SOBP/JXC1. We suggest that the clustered SIMs in these proteins form distinct SUMO binding domains to recognize diverse forms of protein sumoylation. PMID:23086935

  20. Identification of Proteins in Promastigote and Amastigote-like Leishmania Using an Immunoproteomic Approach

    PubMed Central

    Coelho, Vinicio T. S.; Oliveira, Jamil S.; Valadares, Diogo G.; Chávez-Fumagalli, Miguel A.; Duarte, Mariana C.; Lage, Paula S.; Soto, Manuel; Santoro, Marcelo M.; Tavares, Carlos A. P.; Fernandes, Ana Paula; Coelho, Eduardo A. F.

    2012-01-01

    Background The present study aims to identify antigens in protein extracts of promastigote and amastigote-like Leishmania (Leishmania) chagasi syn. L. (L.) infantum recognized by antibodies present in the sera of dogs with asymptomatic and symptomatic visceral leishmaniasis (VL). Methodology/Principal Findings Proteins recognized by sera samples were separated by two-dimensional electrophoresis (2DE) and identified by mass spectrometry. A total of 550 spots were observed in the 2DE gels, and approximately 104 proteins were identified. Several stage-specific proteins could be identified by either or both classes of sera, including, as expected, previously known proteins identified as diagnosis, virulence factors, drug targets, or vaccine candidates. Three, seven, and five hypothetical proteins could be identified in promastigote antigenic extracts; while two, eleven, and three hypothetical proteins could be identified in amastigote-like antigenic extracts by asymptomatic and symptomatic sera, as well as a combination of both, respectively. Conclusions/Significance The present study represents a significant contribution not only in identifying stage-specific L. infantum molecules, but also in revealing the expression of a large number of hypothetical proteins. Moreover, when combined, the identified proteins constitute a significant source of information for the improvement of diagnostic tools and/or vaccine development to VL. PMID:22272364

  1. Molecular weights and subunit structure of LamB proteins.

    PubMed

    Nakae, T; Ishii, J N

    1982-01-01

    Phage lambda-receptor proteins of Escherichia coli, LamB proteins, form oligomeric aggregates to build transmembrane diffusion pores selective for maltose and maltodextrins. The molecular weights (MW) of functional oligomers as well as dissociated monomers were determined by sedimentation equilibrium analysis in homogeneous non-ionic surfactant and deuterium oxide and in 6 M guanidine-HCl, respectively. The MW of oligomers and monomers appeared as 135 600 and 45 900, respectively. Thus, functional Lamb proteins consisted of three identical subunits.

  2. Identifying intrinsically disordered protein regions likely to undergo binding-induced helical transitions.

    PubMed

    Glover, Karen; Mei, Yang; Sinha, Sangita C

    2016-10-01

    Many proteins contain intrinsically disordered regions (IDRs) lacking stable secondary and ordered tertiary structure. IDRs are often implicated in macromolecular interactions, and may undergo structural transitions upon binding to interaction partners. However, as binding partners of many protein IDRs are unknown, these structural transitions are difficult to verify and often are poorly understood. In this study we describe a method to identify IDRs that are likely to undergo helical transitions upon binding. This method combines bioinformatics analyses followed by circular dichroism spectroscopy to monitor 2,2,2-trifluoroethanol (TFE)-induced changes in secondary structure content of these IDRs. Our results demonstrate that there is no significant change in the helicity of IDRs that are not predicted to fold upon binding. IDRs that are predicted to fold fall into two groups: one group does not become helical in the presence of TFE and includes examples of IDRs that form β-strands upon binding, while the other group becomes more helical and includes examples that are known to fold into helices upon binding. Therefore, we propose that bioinformatics analyses combined with experimental evaluation using TFE may provide a general method to identify IDRs that undergo binding-induced disorder-to-helix transitions. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Proteins that interact with calgranulin B in the human colon cancer cell line HCT-116.

    PubMed

    Myung, Jae Kyung; Yeo, Seung-Gu; Kim, Kyung Hee; Baek, Kwang-Soo; Shin, Daye; Kim, Jong Heon; Cho, Jae Youl; Yoo, Byong Chul

    2017-01-24

    Calgranulin B is released from immune cells and can be internalized into colon cancer cells to prevent proliferation. The present study aimed to identify proteins that interact with calgranulin B to suppress the proliferation of colon cancer cells, and to obtain information on the underlying anti-tumor mechanism(s) of calgranulin B. Calgranulin B expression was induced in colon cancer cell line HCT-116 by infection with calgranulin B-FLAG expressing lentivirus, and it led to a significant suppression of cell proliferation. Proteins that interacted with calgranulin B were obtained by immunoprecipitation using whole homogenate of lentivirus-infected HCT-116 cells which expressing calgranulin B-FLAG, and identified using liquid chromatography-mass spectrometry/mass spectrometry analysis. A total of 454 proteins were identified that potentially interact with calgranulin B, and most identified proteins were associated with RNA processing, post-transcriptional modifications and the EIF2 signaling pathway. Direct interaction of calgranulin B with flotillin-1, dynein intermediate chain 1, and CD59 glycoprotein has been confirmed, and the molecules N-myc proto-oncogene protein, rapamycin-insensitive companion of mTOR, and myc proto-oncogene protein were shown to regulate calgranulin B-interacting proteins. Our results provide new insight and useful information to explain the possible mechanism(s) underlying the role of calgranulin B as an anti-tumor effector in colon cancer cells.

  4. Preparation, characterization, and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates

    PubMed Central

    1980-01-01

    A method is presented for covalently bonding Haemophilus influenzae type b capsular polysaccharide (HIB Ps) to several proteins. The method is efficient and relies upon the use of adipic dihydrazide as a spacer between the capsular polysaccharide and the carrier protein. In contrast to the poor immunogenicity of the purified HIB Ps in mice and rabbits, the HIB Ps-protein conjugates induced serum anti-type b antibodies having bactericidal activity at levels shown to be protective in humans when low doses were injected subcutaneously in a saline solution. The antibody response in mice was related to the dose of the conjugates, increased with the number of injections, and could be primed by the previous injection of the carrier protein. The HIB Ps- protein conjugates were immunogenic in three different mouse strains. The importance of the carrier molecule for the enhanced immunogenicity of the HIB Ps-protein conjugates was shown by the failure of HIB Ps hybrids prepared with either the homologous polysaccharide or pneumococcus type 3 polysaccharide to induce antibodie in mice. Rabbits injected with the HIB Ps-protein conjugates emulsified in Freund's adjuvant produced high levels of serum anti-type b antibodies which induced a bactericidal effect upon H. influenzae type b organisms. It is proposed that the HIB Ps component of the polysaccharide protein conjugates has been converted to a thymic-dependent immunogen. This method may be used to prepare protein-polysaccharide conjugates with HIB Ps and other polysaccharides to be considered for human use. PMID:6967514

  5. Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain

    PubMed Central

    de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

    2014-01-01

    The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution. PMID:24792163

  6. B61 is a ligand for the ECK receptor protein-tyrosine kinase.

    PubMed

    Bartley, T D; Hunt, R W; Welcher, A A; Boyle, W J; Parker, V P; Lindberg, R A; Lu, H S; Colombero, A M; Elliott, R L; Guthrie, B A

    1994-04-07

    A protein ligand for the ECK receptor protein-tyrosine kinase has been isolated by using the extracellular domain (ECK-X) of the receptor as an affinity reagent. Initially, concentrated cell culture supernatants were screened for receptor binding activity using immobilized ECK-X in a surface plasmon resonance detection system. Subsequently, supernatants from selected cell lines were fractionated directly by receptor affinity chromatography, resulting in the single-step purification of B61, a protein previously identified as the product of an early response gene induced by tumour necrosis factor-alpha. We report here that recombinant B61 induces autophosphorylation of ECK in intact cells, consistent with B61 being an authentic ligand for ECK. ECK is a member of a large orphan receptor protein-tyrosine kinase family headed by EPH, and we suggest that ligands for other members of this family will be related to B61, and can be isolated in the same way.

  7. Fibroblast growth factor regulates insulin-like growth factor-binding protein production by vascular smooth muscle cells.

    PubMed

    Ververis, J; Ku, L; Delafontaine, P

    1994-02-01

    Insulin-like growth factor I is an important mitogen for vascular smooth muscle cells, and its effects are regulated by several binding proteins. Western ligand blotting of conditioned medium from rat aortic smooth muscle cells detected a 24 kDa binding protein and a 28 kDa glycosylated variant of this protein, consistent with insulin-like growth factor binding protein-4 by size. Low amounts of a glycosylated 38 to 42 kDa doublet (consistent with binding protein-3) and a 31 kDa non-glycosylated protein also were present. Basic fibroblast growth factor markedly increased secretion of the 24 kDa binding protein and its 28 kDa glycosylated variant. This effect was dose- and time-dependent and was inhibited by co-incubation with cycloheximide. Crosslinking of [125I]-insulin-like growth factor I to cell monolayers revealed no surface-associated binding proteins, either basally or after agonist treatment. Induction of binding protein production by fibroblast growth factor at sites of vascular injury may be important in vascular proliferative responses in vivo.

  8. Role of the disaggregase ClpB in processing of proteins aggregated as inclusion bodies.

    PubMed

    Zblewska, Kamila; Krajewska, Joanna; Zolkiewski, Michal; Kędzierska-Mieszkowska, Sabina

    2014-08-01

    Overproduction of heterologous proteins in bacterial systems often results in the formation of insoluble inclusion bodies (IBs), which is a major impediment in biochemical research and biotechnology. In principle, the activity of molecular chaperones could be employed to gain control over the IB formation and to improve the recombinant protein yields, but the potential of each of the major bacterial chaperones (DnaK/J, GroEL/ES, and ClpB) to process IBs has not been fully established yet. We investigated the formation of inclusion bodies (IBs) of two aggregation-prone proteins, VP1LAC and VP1GFP, overproduced in Escherichiacoli in the presence and absence of the chaperone ClpB. We found that both ClpB isoforms, ClpB95 and ClpB80 accumulated in E. coli cells during the production of IBs. The amount of IB proteins increased in the absence of ClpB. ClpB supported the resolubilization and reactivation of the aggregated VP1LAC and VP1GFP in E. coli cells. The IB disaggregation was optimal in the presence of both ClpB95 and ClpB80. Our results indicate an essential role of ClpB in controlling protein aggregation and inclusion body formation in bacteria. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. A period (per)-like protein exhibits daily rhythmicity in the suprachiasmatic nuclei of the rat.

    PubMed

    Rosewell, K L; Siwicki, K K; Wise, P M

    1994-10-03

    The period (per) gene of Drosophila melanogaster is considered an important biological clock gene, since it regulates multiple behavioral rhythms. Per mRNA and protein exhibit circadian rhythms in the fruitfly brain and these rhythms appear to influence each other through a feedback loop. More recently, using the same antibody as was used in the Drosophila studies, PER-like proteins were detected in the suprachiasmatic nuclei (SCN) of male rats. This region of the brain is considered to be a major neural circadian pacemaker in mammals. The purpose of this study was to confirm that PER-like proteins are detectable in the SCN of female rats and to determine whether PER-like proteins exhibit a circadian rhythm. Female rats were killed at several times of day under both light/dark and constant conditions. Using the same anti-PER antibody in Western blots with Enhanced Chemiluminescence (Western-ECL) detection, the levels of the PER-like proteins were quantified in the SCN and cerebral cortex. The antibody identified a doublet band of approximately 170-160 kDa and a single band at 115 kDa. Of the three PER-like proteins only the largest exhibited a daily rhythm in the SCN, which peaked in the middle of the dark and attained its nadir around lights off; levels during the light were intermediate with a tendency towards a second drop around lights on. This rhythm did not persist under constant dim red light.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane▿

    PubMed Central

    Kaserer, Wallace A.; Jiang, Xiaoxu; Xiao, Qiaobin; Scott, Daniel C.; Bauler, Matthew; Copeland, Daniel; Newton, Salete M. C.; Klebba, Phillip E.

    2008-01-01

    We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB+ bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of

  11. Asperentin B, a New Inhibitor of the Protein Tyrosine Phosphatase 1B.

    PubMed

    Wiese, Jutta; Aldemir, Hülya; Schmaljohann, Rolf; Gulder, Tobias A M; Imhoff, Johannes F

    2017-06-21

    In the frame of studies on secondary metabolites produced by fungi from deep-sea environments we have investigated inhibitors of enzymes playing key roles in signaling cascades of biochemical pathways relevant for the treatment of diseases. Here we report on a new inhibitor of the human protein tyrosine phosphatase 1B (PTP1B), a target in the signaling pathway of insulin. A new asperentin analog is produced by an Aspergillus sydowii strain isolated from the sediment of the deep Mediterranean Sea. Asperentin B ( 1 ) contains an additional phenolic hydroxy function at C-6 and exhibits an IC 50 value against PTP1B of 2 μM in vitro, which is six times stronger than the positive control, suramin. Interestingly, asperentin ( 2 ) did not show any inhibition of this enzymatic activity. Asperentin B ( 1 ) is discussed as possible therapeutic agents for type 2 diabetes and sleeping sickness.

  12. Asperentin B, a New Inhibitor of the Protein Tyrosine Phosphatase 1B

    PubMed Central

    Wiese, Jutta; Aldemir, Hülya; Schmaljohann, Rolf; Gulder, Tobias A. M.; Imhoff, Johannes F.

    2017-01-01

    In the frame of studies on secondary metabolites produced by fungi from deep-sea environments we have investigated inhibitors of enzymes playing key roles in signaling cascades of biochemical pathways relevant for the treatment of diseases. Here we report on a new inhibitor of the human protein tyrosine phosphatase 1B (PTP1B), a target in the signaling pathway of insulin. A new asperentin analog is produced by an Aspergillus sydowii strain isolated from the sediment of the deep Mediterranean Sea. Asperentin B (1) contains an additional phenolic hydroxy function at C-6 and exhibits an IC50 value against PTP1B of 2 μM in vitro, which is six times stronger than the positive control, suramin. Interestingly, asperentin (2) did not show any inhibition of this enzymatic activity. Asperentin B (1) is discussed as possible therapeutic agents for type 2 diabetes and sleeping sickness. PMID:28635658

  13. Isolation of 16,000-dalton parathyroid hormone-like proteins from two animal tumors causing humoral hypercalcemia of malignancy.

    PubMed

    Weir, E C; Burtis, W J; Morris, C A; Brady, T G; Insogna, K L

    1988-12-01

    A 16K PTH-like protein with a unique primary structure has recently been isolated from several human tumors associated with the syndrome of humoral hypercalcemia of malignancy. Certain spontaneous and transplantable animal tumors also cause this syndrome. The responsible mediator in these animal tumors is not known. We report the isolation of 16K proteins from the rat H500 Leydig cell tumor and the canine apocrine cell adenocarcinoma of the anal sac. Both proteins are potent activators of PTH receptor-coupled adenylate cyclase in bone cells. Both proteins demonstrate similarities in amino acid composition to one another and to the human PTH-like protein. Limited amino-terminal sequence information from the canine protein demonstrates homology with the human PTH-like protein. Antibodies raised to a synthetic human PTH-(1-36)-like peptide cross-react with both the rat and canine proteins in an immunoradiometric assay. These data demonstrate that by physical and immunological criteria PTH-like peptides are present in these animal tumors that appear to be closely related to the human PTH-like peptide. These data further suggest that this protein is not unique to humans, but has an evolutionary origin which extends back at least 65-80 million yr.

  14. Development of an FHbp-CTB holotoxin-like chimera and the elicitation of bactericidal antibodies against serogroup B Neisseria meningitidis.

    PubMed

    Price, Gregory A; Bash, Margaret C

    2018-01-29

    The Neisseria meningitidis factor H binding protein (FHbp) is an important virulence factor and vaccine antigen contained in both USA licensed serogroup B meningococcal vaccines. Recent studies in human factor H (hFH) transgenic mice suggest that hFH-FHbp interactions lower FHbp-elicited immunogenicity. To provide tools with which to characterize and potentially improve FHbp immunogenicity, we developed an FHbp-cholera holotoxin-like chimera vaccine expression system in Escherichia coli that utilizes cholera toxin B (CTB) as both a scaffold and adjuvant for FHbp. We developed FHbp-CTB chimeras using a wild-type (WT) FHbp and a low hFH-binding FHbp mutant R41S. Both chimeras bound to G M1 ganglioside and were recognized by the FHbp-specific monoclonal antibody JAR4. The R41S mutant had greatly reduced hFH binding compared to the WT FHbp-CTB chimera. WT and R41S FHbp-CTB chimeric antigens were compared to equimolar amounts of FHbp admixed with CTB or FHbp alone in mouse immunogenicity studies. The chimeras were significantly more immunogenic than FHbp alone or mixed with CTB, and elicited bactericidal antibodies against a panel of MenB isolates. This study demonstrates a unique and simple method for studying FHbp immunogenicity. The chimeric approach may facilitate studies of other protein-based antigens targeting pathogenic Neisseria and lay groundwork for the development of new protein based vaccines against meningococcal and gonococcal disease. Published by Elsevier Ltd.

  15. Protein tyrosine phosphatase-1B (PTP1B) helps regulate EGF-induced stimulation of S-phase entry in human corneal endothelial cells

    PubMed Central

    Ishino, Yutaka; Zhu, Cheng; Harris, Deshea L.

    2008-01-01

    Purpose Human corneal endothelial cells (HCEC), particularly from older donors, only proliferate weakly in response to EGF. The protein tyrosine phosphatase, PTP1B, is known to negatively regulate EGF-induced signaling in several cell types by dephosphorylating the epidermal growth factor receptor (EGFR). The current studies were conducted to determine whether PTP1B plays a role in regulating cell cycle entry in HCEC in response to EGF stimulation. Methods Donor corneas were obtained from the National Disease Research Interchange and accepted for study based on established exclusion criteria. PTP1B was localized in the endothelium of ex vivo corneas and in cultured cells by immunocytochemistry. Western blot analysis verified PTP1B protein expression in HCEC and then compared the relative expression of EGFR and PTP1B in HCEC from young (<3 years old) and older donors (>60 years old). The effect of inhibiting the activity of PTP1B on S-phase entry was tested by comparing time-dependent BrdU incorporation in subconfluent HCEC incubated in the presence or absence of the PTP1B inhibitor, CinnGEL 2Me, before EGF stimulation. Results PTP1B was localized in a punctate pattern mainly within the cytoplasm of HCEC in ex vivo corneas and cultured cells. Western blots revealed the presence of three PTP1B-positive bands in HCEC and the control. Further western blot analysis showed no significant age-related difference in expression of EGFR (p=0.444>0.05); however, PTP1B expression was significantly higher in HCEC from older donors (p=0.024<0.05). Pre-incubation of HCEC with the PTP1B inhibitor significantly increased (p=0.019<0.05) the number of BrdU positive cells by 48 h after EGF stimulation. Conclusions Both immunolocalization and western blot studies confirmed that PTP1B is expressed in HCEC. Staining patterns strongly suggest that at least a subset of PTP1B is localized to the cytoplasm and most likely to the endoplasmic reticulum, the known site of EGFR/PTP1B interaction

  16. Crystallization and preliminary X-ray crystallographic analysis of the variable domain of Scl2.3, a streptococcal collagen-like protein from invasive M3-type Streptococcus pyogenes.

    PubMed

    Squeglia, Flavia; Bachert, Beth; Romano, Maria; Lukomski, Slawomir; Berisio, Rita

    2013-09-01

    Streptococcal collagen-like proteins (Scls) are widely expressed by the well recognized human pathogen Streptococcus pyogenes. These surface proteins contain a signature central collagen-like region and an amino-terminal globular domain, termed the variable domain, which is protruded away from the cell surface by the collagen-like domain. Despite their recognized importance in bacterial pathogenicity, no structural information is presently available on proteins of the Scl class. The variable domain of Scl2 from invasive M3-type S. pyogenes has successfully been crystallized using vapour-diffusion methods. The crystals diffracted to 1.5 Å resolution and belonged to space group H32, with unit-cell parameters a = 44.23, b = 44.23, c = 227.83 Å. The crystal structure was solved by single-wavelength anomalous dispersion using anomalous signal from a europium chloride derivative.|

  17. Interacting protein partners of Arabidopsis RNA binding protein AtRBP45b

    USDA-ARS?s Scientific Manuscript database

    RNA binding proteins (RBPs) are important players in post-transcriptional gene regulation and shown to play an important role in normal development and in response to environmental perturbations. Arabidopsis RBP, AtRBP45b with triple RNA recognition motifs (RRMs) have are closely related to the yeas...

  18. The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis

    PubMed Central

    Silva, Éverton F.; Medeiros, Marco A.; McBride, Alan J. A.; Matsunaga, Jim; Esteves, Gabriela S.; Ramos, João G. R.; Santos, Cleiton S.; Croda, Júlio; Homma, Akira; Dellagostin, Odir A.; Haake, David A.; Reis, Mitermayer G.; Ko, Albert I.

    2007-01-01

    Subunit vaccines are a potential intervention strategy against leptospirosis, which is a major public health problem in developing countries and a veterinary disease in livestock and companion animals worldwide. Leptospiral immunoglobulin-like (Lig) proteins are a family of surface-exposed determinants that have Ig-like repeat domains found in virulence factors such as intimin and invasin. We expressed fragments of the repeat domain regions of LigA and LigB from Leptospira interrogans serovar Copenhageni. Immunization of Golden Syrian hamsters with Lig fragments in Freund’s adjuvant induced robust antibody responses against recombinant protein and native protein, as detected by ELISA and immunoblot, respectively. A single fragment, LigANI, which corresponds to the six carboxy-terminal Ig-like repeat domains of the LigA molecule, conferred immunoprotection against mortality (67-100%, P <0.05) in hamsters which received a lethal inoculum of L. interrogans serovar Copenhageni. However, immunization with this fragment did not confer sterilizing immunity. These findings indicate that the carboxy-terminal portion of LigA is an immunoprotective domain and may serve as a vaccine candidate for human and veterinary leptospirosis. PMID:17629368

  19. Akt1/protein kinase B enhances transcriptional reprogramming of fibroblasts to functional cardiomyocytes

    PubMed Central

    Zhou, Huanyu; Dickson, Matthew E.; Kim, Min Soo; Bassel-Duby, Rhonda; Olson, Eric N.

    2015-01-01

    Conversion of fibroblasts to functional cardiomyocytes represents a potential approach for restoring cardiac function after myocardial injury, but the technique thus far has been slow and inefficient. To improve the efficiency of reprogramming fibroblasts to cardiac-like myocytes (iCMs) by cardiac transcription factors [Gata4, Hand2, Mef2c, and Tbx5 (GHMT)], we screened 192 protein kinases and discovered that Akt/protein kinase B dramatically accelerates and amplifies this process in three different types of fibroblasts (mouse embryo, adult cardiac, and tail tip). Approximately 50% of reprogrammed mouse embryo fibroblasts displayed spontaneous beating after 3 wk of induction by Akt plus GHMT. Furthermore, addition of Akt1 to GHMT evoked a more mature cardiac phenotype for iCMs, as seen by enhanced polynucleation, cellular hypertrophy, gene expression, and metabolic reprogramming. Insulin-like growth factor 1 (IGF1) and phosphoinositol 3-kinase (PI3K) acted upstream of Akt whereas the mitochondrial target of rapamycin complex 1 (mTORC1) and forkhead box o3 (Foxo3a) acted downstream of Akt to influence fibroblast-to-cardiomyocyte reprogramming. These findings provide insights into the molecular basis of cardiac reprogramming and represent an important step toward further application of this technique. PMID:26354121

  20. Trichohyalin-like 1 protein, a member of fused S100 proteins, is expressed in normal and pathologic human skin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yamakoshi, Takako; Makino, Teruhiko, E-mail: tmakino@med.u-toyama.ac.jp; Ur Rehman, Mati

    2013-03-01

    Highlights: ► Trichohyalin-like 1 protein is a member of the fused-type S100 protein gene family. ► Specific antibodies against the C-terminus of the TCHHL1 protein were generated. ► TCHHL1 proteins were expressed in the basal layer of the normal epidermis. ► TCHHL1 proteins were strongly expressed in tumor nests of BCC and SCC. ► The expression of TCHHL1 proteins increased in epidermis of psoriasis vulgaris. - Abstract: Trichohyalin-like 1 (TCHHL1) protein is a novel member of the fused-type S100 protein gene family. The deduced amino acid sequence of TCHHL1 contains an EF-hand domain in the N-terminus, one trans-membrane domain andmore » a nuclear localization signal. We generated specific antibodies against the C-terminus of the TCHHL1 protein and examined the expression of TCHHL1 proteins in normal and pathological human skin. An immunohistochemical study showed that TCHHL1 proteins were expressed in the basal layer of the normal epidermis. In addition, signals of TCHHL1 proteins were observed around the nuclei of cultured growing keratinocytes. Accordingly, TCHHL1 mRNA has been detected in normal skin and cultured growing keratinocytes. Furthermore, TCHHL1 proteins were strongly expressed in the peripheral areas of tumor nests in basal cell carcinomas and squamous cell carcinomas. A dramatic increase in the number of Ki67 positive cells was observed in TCHHL1-expressing areas. The expression of TCHHL1 proteins also increased in non-cancerous hyperproliferative epidermal tissues such as those of psoriasis vulgaris and lichen planus. These findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocytes.« less

  1. The Env-like open reading frame of the baculovirus-integrated retrotransposon TED encodes a retrovirus-like envelope protein.

    PubMed

    Ozers, M S; Friesen, P D

    1996-12-15

    TED is a 7.5-kbp member of the gypsy family of retrotransposons that was first identified by its integration within the baculovirus DNA genome. This lepidopteran (moth) transposon contains three retrovirus-like genes, including functional gag and pol that yield reverse transcriptase-containing virus-like particles. To identify and characterize the product(s) of the third env-like open reading frame, TED ORF3 was expressed in homologous lepidopteran cells by using a baculovirus vector, vENV. Immunoblots and immunoprecipitations with antiserum raised against a bacterial ORF3-fusion protein detected two ORF3-encoded proteins, p68env and gp75env. On the basis of selective incorporation of [3H]mannose and inhibition of modification by tunicamycin which blocks N-linked glycosylation, gp75env is a glycoprotein derived from core precursor p68env. As predicted by the presence of a transmembrane domain near the carboxyl terminus, both p68env and gp75env were associated with heavy membranes of vENV-infected cells. Thus, TED ORF3 encodes a membrane glycoprotein with properties characteristic of retroviral env proteins. These data are consistent with the hypothesis that TED is an invertebrate retrovirus. Moreover, TED integration within the baculovirus genome provides an example of retroelement-mediated acquisition of host genes that may contribute to virus evolution.

  2. Drug-like density: a method of quantifying the "bindability" of a protein target based on a very large set of pockets and drug-like ligands from the Protein Data Bank.

    PubMed

    Sheridan, Robert P; Maiorov, Vladimir N; Holloway, M Katharine; Cornell, Wendy D; Gao, Ying-Duo

    2010-11-22

    One approach to estimating the "chemical tractability" of a candidate protein target where we know the atomic resolution structure is to examine the physical properties of potential binding sites. A number of other workers have addressed this issue. We characterize ~290,000 "pockets" from ~42,000 protein crystal structures in terms of a three parameter "pocket space": volume, buriedness, and hydrophobicity. A metric DLID (drug-like density) measures how likely a pocket is to bind a drug-like molecule. This is calculated from the count of other pockets in its local neighborhood in pocket space that contain drug-like cocrystallized ligands and the count of total pockets in the neighborhood. Surprisingly, despite being defined locally, a global trend in DLID can be predicted by a simple linear regression on log(volume), buriedness, and hydrophobicity. Two levels of simplification are necessary to relate the DLID of individual pockets to "targets": taking the best DLID per Protein Data Bank (PDB) entry (because any given crystal structure can have many pockets), and taking the median DLID over all PDB entries for the same target (because different crystal structures of the same protein can vary because of artifacts and real conformational changes). We can show that median DLIDs for targets that are detectably homologous in sequence are reasonably similar and that median DLIDs correlate with the "druggability" estimate of Cheng et al. (Nature Biotechnology 2007, 25, 71-75).

  3. Inhibition of protein tyrosine phosphatase 1B as a potential treatment of diabetes and obesity.

    PubMed

    Pei, Zhonghua; Liu, Gang; Lubben, Thomas H; Szczepankiewicz, Bruce G

    2004-01-01

    Diabetes is a prevalent disease which effects over 150 million people worldwide and there is a great medical need for new therapeutic agents to treat it. Inhibition of protein tyrosine phosphatase 1B (PTP1B) has emerged as a highly validated, attractive target for treatment of not only diabetes but also obesity. Discovery of small-molecule inhibitors has been pursued extensively in both academia and industry and a number of very potent and selective inhibitors have been identified. With X-ray crystallography, the binding interactions of several classes of inhibitors have been elucidated. This has resulted in significant progress in understanding important interactions between inhibitors and specific residues of PTP1B, which could help the design of future inhibitors. However, since the active site of PTP1B that most of these inhibitors bind to is highly hydrophilic, it remains a challenge to identify inhibitors with both excellent in vitro potency and drug-like physiochemical properties which would lead to good in vivo activities.

  4. Nuclear-cytoplasmic partitioning of cucumber mosaic virus protein 2b determines the balance between its roles as a virulence determinant and an RNA-silencing suppressor.

    PubMed

    Du, Zhiyou; Chen, Aizhong; Chen, Wenhu; Liao, Qiansheng; Zhang, Hengmu; Bao, Yiming; Roossinck, Marilyn J; Carr, John P

    2014-05-01

    The Cucumber Mosaic Virus (CMV) 2b protein is an RNA-silencing suppressor that plays roles in CMV accumulation and virulence. The 2b proteins of subgroup IA CMV strains partition between the nucleus and cytoplasm, but the biological significance of this is uncertain. We fused an additional nuclear localization signal (NLS) to the 2b protein of subgroup IA strain Fny-CMV to create 2b-NLS and tested its effects on subcellular distribution, silencing, and virulence. The additional NLS enhanced 2b protein nuclear and nucleolar accumulation, but nuclear and nucleolar enrichment correlated with markedly diminished silencing suppressor activity in patch assays and abolished 2b protein-mediated disruption of microRNA activity in transgenic Arabidopsis. Nucleus/nucleolus-localized 2b protein possesses at least some ability to inhibit antiviral silencing, but this was not sufficient to prevent recovery from disease in younger, developing leaves in Arabidopsis. However, enhanced nuclear and nucleolar accumulation of 2b increased virulence and accelerated symptom appearance in older leaves. Experiments with Arabidopsis lines carrying mutant Dicer-like alleles demonstrated that compromised suppressor activity explained the diminished ability of 2b-NLS to enhance virus accumulation. Remarkably, the increased virulence that 2b-NLS engendered was unrelated to effects on microRNA- or short interfering RNA-regulated host functions. Thus, although nucleus- and nucleolus-localized 2b protein is less efficient at silencing suppression than cytoplasm-localized 2b, it enhances CMV virulence. We propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus. In this work, the main finding is that nucleus/nucleolus-localized 2b protein is strongly associated with CMV virulence, which is independent of its effect on

  5. Localization of the Kinesin-like Protein Xklp2 to Spindle Poles Requires a Leucine Zipper, a Microtubule-associated Protein, and Dynein

    PubMed Central

    Wittmann, Torsten; Boleti, Haralabia; Antony, Claude; Karsenti, Eric; Vernos, Isabelle

    1998-01-01

    Xklp2 is a plus end–directed Xenopus kinesin-like protein localized at spindle poles and required for centrosome separation during spindle assembly in Xenopus egg extracts. A glutathione-S-transferase fusion protein containing the COOH-terminal domain of Xklp2 (GST-Xklp2-Tail) was previously found to localize to spindle poles (Boleti, H., E. Karsenti, and I. Vernos. 1996. Cell. 84:49–59). Now, we have examined the mechanism of localization of GST-Xklp2-Tail. Immunofluorescence and electron microscopy showed that Xklp2 and GST-Xklp2-Tail localize specifically to the minus ends of spindle pole and aster microtubules in mitotic, but not in interphase, Xenopus egg extracts. We found that dimerization and a COOH-terminal leucine zipper are required for this localization: a single point mutation in the leucine zipper prevented targeting. The mechanism of localization is complex and two additional factors in mitotic egg extracts are required for the targeting of GST-Xklp2-Tail to microtubule minus ends: (a) a novel 100-kD microtubule-associated protein that we named TPX2 (Targeting protein for Xklp2) that mediates the binding of GST-Xklp2-Tail to microtubules and (b) the dynein–dynactin complex that is required for the accumulation of GST-Xklp2-Tail at microtubule minus ends. We propose two molecular mechanisms that could account for the localization of Xklp2 to microtubule minus ends. PMID:9813089

  6. Nasal Colonisation by Staphylococcus aureus Depends upon Clumping Factor B Binding to the Squamous Epithelial Cell Envelope Protein Loricrin

    PubMed Central

    Mulcahy, Michelle E.; Geoghegan, Joan A.; Monk, Ian R.; O'Keeffe, Kate M.; Walsh, Evelyn J.

    2012-01-01

    Staphylococcus aureus asymptomatically colonises the anterior nares, but the host and bacterial factors that facilitate colonisation remain incompletely understood. The S. aureus surface protein ClfB has been shown to mediate adherence to squamous epithelial cells in vitro and to promote nasal colonisation in both mice and humans. Here, we demonstrate that the squamous epithelial cell envelope protein loricrin represents the major target ligand for ClfB during S. aureus nasal colonisation. In vitro adherence assays indicated that bacteria expressing ClfB bound loricrin most likely by the “dock, lock and latch” mechanism. Using surface plasmon resonance we showed that ClfB bound cytokeratin 10 (K10), a structural protein of squamous epithelial cells, and loricrin with similar affinities that were in the low µM range. Loricrin is composed of three separate regions comprising GS-rich omega loops. Each loop was expressed separately and found to bind ClfB, However region 2 bound with highest affinity. To investigate if the specific interaction between ClfB and loricrin was sufficient to facilitate S. aureus nasal colonisation, we compared the ability of ClfB+ S. aureus to colonise the nares of wild-type and loricrin-deficient (Lor−/−) mice. In the absence of loricrin, S. aureus nasal colonisation was significantly impaired. Furthermore a ClfB− mutant colonised wild-type mice less efficiently than the parental ClfB+ strain whereas a similar lower level of colonisation was observed with both the parental strain and the ClfB− mutant in the Lor−/− mice. The ability of ClfB to support nasal colonisation by binding loricrin in vivo was confirmed by the ability of Lactococcus lactis expressing ClfB to be retained in the nares of WT mice but not in the Lor−/− mice. By combining in vitro biochemical analysis with animal model studies we have identified the squamous epithelial cell envelope protein loricrin as the target ligand for ClfB during nasal

  7. Zona pellucida-binding protein 2 (ZPBP2) and several proteins containing BX7B motifs in human sperm may have hyaluronic acid binding or recognition properties.

    PubMed

    Torabi, F; Bogle, O A; Estanyol, J M; Oliva, R; Miller, D

    2017-12-01

    Are there novel hyaladherins in human sperm? Zona pellucida-binding protein 2 (ZPBP2), containing a Link-like hyaluronic acid (HA)-binding domain, and several other proteins containing BX7B motifs, such as ADAM32 and Midkine, may be novel hyaladherins with HA-binding properties. HA-binding proteins (hyaladherins), which can bind HA surrounding the cumulus-oophorus complex, are distinct from hyases such as PH 20 (SPAM1) and are expressed by mature spermatozoa. Although HABP1 and CD44 are reasonably well characterized hyaladherins and the former has been implicated in sperm-oocyte interactions, the overall significance of sperm hyaladherins for male fertility is still poorly understood. This was a laboratory-based investigation into human sperm hyaladherins undertaken as part of a three year PhD programme sponsored by the EU Marie Curie Training network, Reprotrain. Protein homogenates of sperm obtained from young men of unknown fertility (N = 4) were partitioned into HA-binding and non-binding fractions by a protein affinity 'panning' method; their subsequent characterization was by liquid chromatography-tandem mass spectrometry (LC-MS-MS) and partitioning behaviour was confirmed by western blotting. Sequences of proteins from both fractions were submitted to PDBsum to look for orthologous entries (PDB codes) and all returned codes were queried against the matching protein using SAS (Sequences Annotated by Structure) looking for structural similarities between them. A systematic search for other common features of hyaladherins was also undertaken. The presence of BX7B sequence motifs found in several well-described hyaladherins including RHAMM was used to assess efficacy of potential hyaladherin partitioning by the HA substrate. The data showed that 50% (14/28) and 34.5% (28/81) of proteins in the bound and unbound fractions, respectively, contained these motifs (one-tailed Z-score = 1.45; P = 0.074), indicating weak discrimination by the substrate. Querying PDBsum

  8. Identification and application of self-binding zipper-like sequences in SARS-CoV spike protein.

    PubMed

    Zhang, Si Min; Liao, Ying; Neo, Tuan Ling; Lu, Yanning; Liu, Ding Xiang; Vahlne, Anders; Tam, James P

    2018-05-22

    Self-binding peptides containing zipper-like sequences, such as the Leu/Ile zipper sequence within the coiled coil regions of proteins and the cross-β spine steric zippers within the amyloid-like fibrils, could bind to the protein-of-origin through homophilic sequence-specific zipper motifs. These self-binding sequences represent opportunities for the development of biochemical tools and/or therapeutics. Here, we report on the identification of a putative self-binding β-zipper-forming peptide within the severe acute respiratory syndrome-associated coronavirus spike (S) protein and its application in viral detection. Peptide array scanning of overlapping peptides covering the entire length of S protein identified 34 putative self-binding peptides of six clusters, five of which contained octapeptide core consensus sequences. The Cluster I consensus octapeptide sequence GINITNFR was predicted by the Eisenberg's 3D profile method to have high amyloid-like fibrillation potential through steric β-zipper formation. Peptide C6 containing the Cluster I consensus sequence was shown to oligomerize and form amyloid-like fibrils. Taking advantage of this, C6 was further applied to detect the S protein expression in vitro by fluorescence staining. Meanwhile, the coiled-coil-forming Leu/Ile heptad repeat sequences within the S protein were under-represented during peptide array scanning, in agreement with that long peptide lengths were required to attain high helix-mediated interaction avidity. The data suggest that short β-zipper-like self-binding peptides within the S protein could be identified through combining the peptide scanning and predictive methods, and could be exploited as biochemical detection reagents for viral infection. Copyright © 2018. Published by Elsevier Ltd.

  9. MreB-Dependent Organization of the E. coli Cytoplasmic Membrane Controls Membrane Protein Diffusion.

    PubMed

    Oswald, Felix; Varadarajan, Aravindan; Lill, Holger; Peterman, Erwin J G; Bollen, Yves J M

    2016-03-08

    The functional organization of prokaryotic cell membranes, which is essential for many cellular processes, has been challenging to analyze due to the small size and nonflat geometry of bacterial cells. Here, we use single-molecule fluorescence microscopy and three-dimensional quantitative analyses in live Escherichia coli to demonstrate that its cytoplasmic membrane contains microdomains with distinct physical properties. We show that the stability of these microdomains depends on the integrity of the MreB cytoskeletal network underneath the membrane. We explore how the interplay between cytoskeleton and membrane affects trans-membrane protein (TMP) diffusion and reveal that the mobility of the TMPs tested is subdiffusive, most likely caused by confinement of TMP mobility by the submembranous MreB network. Our findings demonstrate that the dynamic architecture of prokaryotic cell membranes is controlled by the MreB cytoskeleton and regulates the mobility of TMPs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  10. The immunoglobulin-like domains 1 and 2 of the protein tyrosine phosphatase LAR adopt an unusual horseshoe-like conformation

    PubMed Central

    Biersmith, Bridget H.; Hammel, Michal; Geisbrecht, Erika R.; Bouyain, Samuel

    2011-01-01

    Neurogenesis depends on exquisitely regulated interactions between macromolecules on the cell surface and in the extracellular matrix. In particular, interactions between proteoglycans and members of the type IIa subgroup of receptor protein tyrosine phosphatases underlie critical developmental processes such as the formation of synapses at the neuromuscular junction and the migration of axons to their appropriate targets. We report here the crystal structures of the first and second immunoglobulin-like domains of the Drosophila type IIa receptor Dlar and its mouse homologue LAR. These two domains adopt an unusual antiparallel arrangement that has not been previously observed in tandem repeats of immunoglobulin-like domains and that is presumably conserved in all type IIa receptor protein tyrosine phosphatases. PMID:21402080

  11. SNAP dendrimers: multivalent protein display on dendrimer-like DNA for directed evolution.

    PubMed

    Kaltenbach, Miriam; Stein, Viktor; Hollfelder, Florian

    2011-09-19

    Display systems connect a protein with the DNA encoding it. Such systems (e.g., phage or ribosome display) have found widespread application in the directed evolution of protein binders and constitute a key element of the biotechnological toolkit. In this proof-of-concept study we describe the construction of a system that allows the display of multiple copies of a protein of interest in order to take advantage of avidity effects during affinity panning. To this end, dendrimer-like DNA is used as a scaffold with docking points that can join the coding DNA with multiple protein copies. Each DNA construct is compartmentalised in water-in-oil emulsion droplets. The corresponding protein is expressed, in vitro, inside the droplets as a SNAP-tag fusion. The covalent bond between DNA and the SNAP-tag is created by reaction with dendrimer-bound benzylguanine (BG). The ability to form dendrimer-like DNA straightforwardly from oligonucleotides bearing BG allowed the comparison of a series of templates differing in size, valency and position of BG. In model selections the most efficient constructs show recoveries of up to 0.86 % and up to 400-fold enrichments. The comparison of mono- and multivalent constructs suggests that the avidity effect enhances enrichment by up to fivefold and recovery by up to 25-fold. Our data establish a multivalent format for SNAP-display based on dendrimer-like DNA as the first in vitro display system with defined tailor-made valencies and explore a new application for DNA nanostructures. These data suggest that multivalent SNAP dendrimers have the potential to facilitate the selection of protein binders especially during early rounds of directed evolution, allowing a larger diversity of candidate binders to be recovered. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Phosphodiesterase inhibitors suppress Lactobacillus casei cell-wall-induced NF-κB and MAPK activations and cell proliferation through protein kinase A--or exchange protein activated by cAMP-dependent signal pathway.

    PubMed

    Saito, Takekatsu; Sugimoto, Naotoshi; Ohta, Kunio; Shimizu, Tohru; Ohtani, Kaori; Nakayama, Yuko; Nakamura, Taichi; Hitomi, Yashiaki; Nakamura, Hiroyuki; Koizumi, Shoichi; Yachie, Akihiro

    2012-01-01

    Specific strains of Lactobacillus have been found to be beneficial in treating some types of diarrhea and vaginosis. However, a high mortality rate results from underlying immunosuppressive conditions in patients with Lactobacillus casei bacteremia. Cyclic AMP (cAMP) is a small second messenger molecule that mediates signal transduction. The onset and progression of inflammatory responses are sensitive to changes in steady-state cAMP levels. L. casei cell wall extract (LCWE) develops arteritis in mice through Toll-like receptor-2 signaling. The purpose of this study was to investigate whether intracellular cAMP affects LCWE-induced pathological signaling. LCWE was shown to induce phosphorylation of the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways and cell proliferation in mice fibroblast cells. Theophylline and phosphodiesterase inhibitor increased intracellular cAMP and inhibited LCWE-induced cell proliferation as well as phosphorylation of NF-κB and MAPK. Protein kinase A inhibitor H89 prevented cAMP-induced MAPK inhibition, but not cAMP-induced NF-κB inhibition. An exchange protein activated by cAMP (Epac) agonist inhibited NF-κB activation but not MAPK activation. These results indicate that an increase in intracellular cAMP prevents LCWE induction of pathological signaling pathways dependent on PKA and Epac signaling.

  13. [Expression of angiopoietin-like proteins for animal breeding: a review].

    PubMed

    Fu, Weiwei; Ma, Yun; Chen, Ningbo; Li, He; Bai, Yueyu

    2015-11-01

    Angiopoietin-like proteins are a family of proteins that are closely related to lipid, glucose and energy metabolism, as well as angiogenesis. To date, eight Angptls have been discovered, namely Angptl1 to Angptl8 that play key roles in metabolic regulation and marker assisted selection. In this review, we summarized current progress on the structure, signaling pathways, upstream regulatory genes and metabolic network of Angptl1-8. Finally, in combination with our work, the status and problems of animal breeding as well as the future prospects for Angptls were discussed.

  14. The RING finger/B-box factor TAM-1 and a retinoblastoma-like protein LIN-35 modulate context-dependent gene silencing in Caenorhabditis elegans.

    PubMed

    Hsieh, J; Liu, J; Kostas, S A; Chang, C; Sternberg, P W; Fire, A

    1999-11-15

    Context-dependent gene silencing is used by many organisms to stably modulate gene activity for large chromosomal regions. We have used tandem array transgenes as a model substrate in a screen for Caenorhabditis elegans mutants that affect context-dependent gene silencing in somatic tissues. This screen yielded multiple alleles of a previously uncharacterized gene, designated tam-1 (for tandem-array-modifier). Loss-of-function mutations in tam-1 led to a dramatic reduction in the activity of numerous highly repeated transgenes. These effects were apparently context dependent, as nonrepetitive transgenes retained activity in a tam-1 mutant background. In addition to the dramatic alterations in transgene activity, tam-1 mutants showed modest alterations in expression of a subset of endogenous cellular genes. These effects include genetic interactions that place tam-1 into a group called the class B synMuv genes (for a Synthetic Multivulva phenotype); this family plays a negative role in the regulation of RAS pathway activity in C. elegans. Loss-of-function mutants in other members of the class-B synMuv family, including lin-35, which encodes a protein similar to the tumor suppressor Rb, exhibit a hypersilencing in somatic transgenes similar to that of tam-1 mutants. Molecular analysis reveals that tam-1 encodes a broadly expressed nuclear protein with RING finger and B-box motifs.

  15. A New Look on Protein-Polyphenol Complexation during Honey Storage: Is This a Random or Organized Event with the Help of Dirigent-Like Proteins?

    PubMed Central

    Brudzynski, Katrina; Sjaarda, Calvin; Maldonado-Alvarez, Liset

    2013-01-01

    Honey storage initiates melanoidin formation that involves a cascade of seemingly unguided redox reactions between amino acids/proteins, reducing sugars and polyphenols. In the process, high molecular weight protein-polyphenol complexes are formed, but the mechanism involved remains unknown. The objective of this study was twofold: to determine quantitative and qualitative changes in proteins in honeys stored for prolonged times and in different temperatures and to relate these changes to the formation of protein-polyphenol complexes. Six -month storage decreased the protein content by 46.7% in all tested honeys (t-test, p<0.002) with the rapid reduction occurring during the first three month. The changes in protein levels coincided with alterations in molecular size and net charge of proteins on SDS –PAGE. Electro-blotted proteins reacted with a quinone-specific nitro blue tetrazolium (NBT) on nitrocellulose membranes indicating that quinones derived from oxidized polyphenols formed covalent bonds with proteins. Protein-polyphenol complexes isolated by size-exclusion chromatography differed in size and stoichiometry and fall into two categories: (a) high molecular weight complexes (230–180 kDa) enriched in proteins but possessing a limited reducing activity toward the NBT and (b) lower molecular size complexes (110–85 kDa) enriched in polyphenols but strongly reducing the dye. The variable stoichiometry suggest that the large, “protein-type” complexes were formed by protein cross-linking, while in the smaller, “polyphenol-type” complexes polyphenols were first polymerized prior to protein binding. Quinones preferentially bound a 31 kDa protein which, by the electrospray quadrupole time of flight mass spectrometry (ESI-Qtof-MS) analysis, showed homology to dirigent-like proteins known for assisting in radical coupling and polymerization of phenolic compounds. These findings provide a new look on protein-polyphenol interaction in honey where the

  16. Tobacco LSU-like protein couples sulphur-deficiency response with ethylene signalling pathway.

    PubMed

    Moniuszko, Grzegorz; Skoneczny, Marek; Zientara-Rytter, Katarzyna; Wawrzyńska, Anna; Głów, Dawid; Cristescu, Simona M; Harren, Frans J M; Sirko, Agnieszka

    2013-11-01

    Most genes from the plant-specific family encoding Response to Low Sulphur (LSU)-like proteins are strongly induced in sulphur (S)-deficient conditions. The exact role of these proteins remains unclear; however, some data suggest their importance for plants' adjustment to nutrient deficiency and other environmental stresses. This work established that the regulation of ethylene signalling is a part of plants' response to S deficiency and showed the interaction between UP9C, a tobacco LSU family member, and one of the tobacco isoforms of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO2A). Increase in ethylene level induced by S deficiency does not take place in tobacco plants with UP9C expressed in an antisense orientation. Based on transcriptomics data, this work also demonstrated that the majority of tobacco's response to S deficiency is misregulated in plants expressing UP9C-antisense. A link between response to S deficiency, ethylene sensing, and LSU-like proteins was emphasized by changes in expression of the genes encoding ethylene receptors and F-box proteins specific for the ethylene pathway.

  17. Expression of Surface Protein LapB by a Wide Spectrum of Listeria monocytogenes Serotypes as Demonstrated with Anti-LapB Monoclonal Antibodies

    PubMed Central

    Boivin, Teela; Elmgren, Cathie; Brooks, Brian W.; Huang, Hongsheng; Pagotto, Franco

    2016-01-01

    ABSTRACT Protein antigens expressed on the surface of all strains of Listeria monocytogenes and absent from nonpathogenic Listeria spp. are presumably useful targets for pathogen identification, detection, and isolation using specific antibodies (Abs). To seek such surface proteins expressed in various strains of L. monocytogenes for diagnostic applications, we focused on a set of surface proteins known to be involved or putatively involved in L. monocytogenes virulence and identified Listeria adhesion protein B (LapB) as a candidate based on the bioinformatics analysis of whole-genome sequences showing that the gene coding for LapB was present in L. monocytogenes strains and absent from strains of other Listeria spp. Immunofluorescence microscopy (IFM), performed with rabbit polyclonal antibodies against the recombinant LapB protein (rLapB) of L. monocytogenes serotype 4b strain L10521, confirmed expression of LapB on the surface. A panel of 48 mouse monoclonal antibodies (MAbs) to rLaB was generated, and 7 of them bound strongly to the surface of L. monocytogenes cells as demonstrated using IFM. Further characterization of these 7 anti-LapB MAbs, using an enzyme-linked immunosorbent assay (ELISA), revealed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) reacted strongly with 46 (86.8%) of 53 strains representing 10 of the 12 serotypes tested (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4ab, 4b, 4d, and 4e). These results indicate that LapB, together with companion anti-LapB MAbs, can be targeted as a biomarker for the detection and isolation of various L. monocytogenes strains from contaminated foods. IMPORTANCE Strains of L. monocytogenes are traditionally grouped into serotypes. Identification of a surface protein expressed in all or the majority of at least 12 serotypes would aid in the development of surface-binding monoclonal antibodies (MAbs) for detection and isolation of L. monocytogenes from foods. Bioinformatics analysis revealed that the gene

  18. Expression of Surface Protein LapB by a Wide Spectrum of Listeria monocytogenes Serotypes as Demonstrated with Anti-LapB Monoclonal Antibodies.

    PubMed

    Boivin, Teela; Elmgren, Cathie; Brooks, Brian W; Huang, Hongsheng; Pagotto, Franco; Lin, Min

    2016-11-15

    Protein antigens expressed on the surface of all strains of Listeria monocytogenes and absent from nonpathogenic Listeria spp. are presumably useful targets for pathogen identification, detection, and isolation using specific antibodies (Abs). To seek such surface proteins expressed in various strains of L. monocytogenes for diagnostic applications, we focused on a set of surface proteins known to be involved or putatively involved in L. monocytogenes virulence and identified Listeria adhesion protein B (LapB) as a candidate based on the bioinformatics analysis of whole-genome sequences showing that the gene coding for LapB was present in L. monocytogenes strains and absent from strains of other Listeria spp. Immunofluorescence microscopy (IFM), performed with rabbit polyclonal antibodies against the recombinant LapB protein (rLapB) of L. monocytogenes serotype 4b strain L10521, confirmed expression of LapB on the surface. A panel of 48 mouse monoclonal antibodies (MAbs) to rLaB was generated, and 7 of them bound strongly to the surface of L. monocytogenes cells as demonstrated using IFM. Further characterization of these 7 anti-LapB MAbs, using an enzyme-linked immunosorbent assay (ELISA), revealed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) reacted strongly with 46 (86.8%) of 53 strains representing 10 of the 12 serotypes tested (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4ab, 4b, 4d, and 4e). These results indicate that LapB, together with companion anti-LapB MAbs, can be targeted as a biomarker for the detection and isolation of various L. monocytogenes strains from contaminated foods. Strains of L. monocytogenes are traditionally grouped into serotypes. Identification of a surface protein expressed in all or the majority of at least 12 serotypes would aid in the development of surface-binding monoclonal antibodies (MAbs) for detection and isolation of L. monocytogenes from foods. Bioinformatics analysis revealed that the gene coding for Listeria

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

    PubMed

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

    2017-10-13

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

  20. Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain.

    PubMed

    de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

    2014-06-01

    The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Myelin protein zero gene mutated in Charcot-Marie-Tooth type 1B patients

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Su, Ying; Li, Lanying; Lepercq, J.

    1993-11-15

    The autosomal dominant of Charcot-Marie-Tooth disease (CMT), whose gene is type 1B (CMT1B), has slow nerve conduction with demyelinated Schwann cells. In this study the abundant peripheral myelin protein zero (MPZ) gene, MPZ, was mapped 130 kb centromeric to the Fc receptor immunoglobulin gene cluster in band 1q22, and a major MPZ point mutation was found to cosegregate with CMT1B in one large CMT1B family. The MPZ point mutation in 18 of 18 related CMT1B pedigree 1 patients converts a positively charged lysine in codon 96 to a negatively charged glutamate. The same MPZ locus cosegregates with the CMT1B diseasemore » gene in a second CMT1B family [total multipoint logarithm of odds (lod) = 11.4 at [theta] = 0.00] with a splice junction mutation. Both mutations occur in MPZ protein regions otherwise conserved identically in human, rat, and cow since these species diverged 100 million years ago. MPZ protein, expressed exclusively in myelinated peripheral nerve Schwann cells, constitutes >50% of myelin protein. These mutations are anticipated to disrupt homophilic MPZ binding and result in CMT1B peripheral nerve demyelination.« less

  2. The RtcB RNA ligase is an essential component of the metazoan unfolded protein response

    PubMed Central

    Kosmaczewski, Sara Guckian; Edwards, Tyson James; Han, Sung Min; Eckwahl, Matthew J; Meyer, Benjamin Isaiah; Peach, Sally; Hesselberth, Jay R; Wolin, Sandra L; Hammarlund, Marc

    2014-01-01

    RNA ligation can regulate RNA function by altering RNA sequence, structure and coding potential. For example, the function of XBP1 in mediating the unfolded protein response requires RNA ligation, as does the maturation of some tRNAs. Here, we describe a novel in vivo model in Caenorhabditis elegans for the conserved RNA ligase RtcB and show that RtcB ligates the xbp-1 mRNA during the IRE-1 branch of the unfolded protein response. Without RtcB, protein stress results in the accumulation of unligated xbp-1 mRNA fragments, defects in the unfolded protein response, and decreased lifespan. RtcB also ligates endogenous pre-tRNA halves, and RtcB mutants have defects in growth and lifespan that can be bypassed by expression of pre-spliced tRNAs. In addition, animals that lack RtcB have defects that are independent of tRNA maturation and the unfolded protein response. Thus, RNA ligation by RtcB is required for the function of multiple endogenous target RNAs including both xbp-1 and tRNAs. RtcB is uniquely capable of performing these ligation functions, and RNA ligation by RtcB mediates multiple essential processes in vivo. Subject Categories Protein Biosynthesis & Quality Control; RNA Biology PMID:25366321

  3. Direct observation of transcription activator-like effector (TALE) protein dynamics

    NASA Astrophysics Data System (ADS)

    Cuculis, Luke; Abil, Zhanar; Zhao, Huimin; Schroeder, Charles M.

    2014-03-01

    In this work, we describe a single molecule assay to probe the site-search dynamics of transcription activator-like effector (TALE) proteins along DNA. In modern genetics, the ability to selectively edit the human genome is an unprecedented development, driven by recent advances in targeted nuclease proteins. Specific gene editing can be accomplished using TALE proteins, which are programmable DNA-binding proteins that can be fused to a nuclease domain. In this way, TALENs are a leading technology that has shown great success in the genomic editing of pluripotent stem cells. A major hurdle facing clinical implementation, however, is the potential for deleterious off-target binding events. For these reasons, a molecular-level understanding of TALE binding and target sequence search on DNA is essential. To this end, we developed a single-molecule fluorescence imaging assay that provides a first-of-its-kind view of the 1-D diffusion of TALE proteins along stretched DNA. Taken together with co-crystal structures of DNA-bound TALEs, our results suggest a rotationally-coupled, major groove tracking model for diffusion. We further report diffusion constants for TALE proteins as a function of salt concentration, consistent with previously described models of 1-D protein diffusion.

  4. Amino Acid Distribution Rules Predict Protein Fold: Protein Grammar for Beta-Strand Sandwich-Like Structures

    PubMed Central

    Kister, Alexander

    2015-01-01

    We present an alternative approach to protein 3D folding prediction based on determination of rules that specify distribution of “favorable” residues, that are mainly responsible for a given fold formation, and “unfavorable” residues, that are incompatible with that fold, in polypeptide sequences. The process of determining favorable and unfavorable residues is iterative. The starting assumptions are based on the general principles of protein structure formation as well as structural features peculiar to a protein fold under investigation. The initial assumptions are tested one-by-one for a set of all known proteins with a given structure. The assumption is accepted as a “rule of amino acid distribution” for the protein fold if it holds true for all, or near all, structures. If the assumption is not accepted as a rule, it can be modified to better fit the data and then tested again in the next step of the iterative search algorithm, or rejected. We determined the set of amino acid distribution rules for a large group of beta sandwich-like proteins characterized by a specific arrangement of strands in two beta sheets. It was shown that this set of rules is highly sensitive (~90%) and very specific (~99%) for identifying sequences of proteins with specified beta sandwich fold structure. The advantage of the proposed approach is that it does not require that query proteins have a high degree of homology to proteins with known structure. So long as the query protein satisfies residue distribution rules, it can be confidently assigned to its respective protein fold. Another advantage of our approach is that it allows for a better understanding of which residues play an essential role in protein fold formation. It may, therefore, facilitate rational protein engineering design. PMID:25625198

  5. Mapping the Complement Factor H-Related Protein 1 (CFHR1):C3b/C3d Interactions

    PubMed Central

    Laskowski, Jennifer; Thurman, Joshua M.; Hageman, Gregory S.; Holers, V. Michael

    2016-01-01

    Complement factor H-related protein 1 (CFHR1) is a complement regulator which has been reported to regulate complement by blocking C5 convertase activity and interfering with C5b surface association. CFHR1 also competes with complement factor H (CFH) for binding to C3b, and may act as an antagonist of CFH-directed regulation on cell surfaces. We have employed site-directed mutagenesis in conjunction with ELISA-based and functional assays to isolate the binding interaction that CFHR1 undertakes with complement components C3b and C3d to a single shared interface. The C3b/C3d:CFHR1 interface is identical to that which occurs between the two C-terminal domains (SCR19-20) of CFH and C3b. Moreover, we have been able to corroborate that dimerization of CFHR1 is necessary for this molecule to bind effectively to C3b and C3d, or compete with CFH. Finally, we have established that CFHR1 competes with complement factor H-like protein 1 (CFHL-1) for binding to C3b. CFHL-1 is a CFH gene splice variant, which is almost identical to the N-terminal 7 domains of CFH (SCR1-7). CFHR1, therefore, not only competes with the C-terminus of CFH for binding to C3b, but also sterically blocks the interaction that the N-terminus of CFH undertakes with C3b, and which is required for CFH-regulation. PMID:27814381

  6. DjhnRNPA2/B1-like gene is required for planarian regeneration and tissue homeostasis.

    PubMed

    Dong, Zimei; Yang, Tong; Yang, Yibo; Dou, He; Chen, Guangwen

    2017-10-30

    The hnRNPs play important roles in physiological processes in eukaryotic organisms by regulation of pre-mRNA after transcription, including pre-mRNA splicing, mRNA stability, DNA replication and repair and telomere maintenance and so on. However, it remains unclear about the specific functions of these genes. In this study, the full-length cDNA sequence of hnRNPA2/B1-like was first cloned from Dugesia japonica, and its roles were investigated by WISH and RNAi. The results showed that: (1) DjhnRNPA2/B1-like was highly conserved during animal evolution; (2) DjhnRNPA2/B1-like mRNA was mainly distributed each side of the body in intact worms and regenerative blastemas, and its expression levels were up-regulated on days 0 and 5 after amputation; (3) the intact and regenerating worms gradually lysed or lost regeneration capacity after DjhnRNPA2/B1-like RNAi; and (4) DjhnRNPA2/B1-like expression is induced by temperature and heavy metal ion stress. The data suggests that DjhnRNPA2/B1-like is a multiple functional gene, it plays important roles in regeneration and homeostatic maintenance and it is also involved in stress responses in planarians. Our work provides basic data for the study of regenerative mechanism and stress responses in freshwater planarians. Copyright © 2017. Published by Elsevier B.V.

  7. Prion-like domains in RNA binding proteins are essential for building subnuclear paraspeckles

    PubMed Central

    Hennig, Sven; Kong, Geraldine; Mannen, Taro; Sadowska, Agata; Kobelke, Simon; Blythe, Amanda; Knott, Gavin J.; Iyer, K. Swaminathan; Ho, Diwei; Newcombe, Estella A.; Hosoki, Kana; Goshima, Naoki; Kawaguchi, Tetsuya; Hatters, Danny; Trinkle-Mulcahy, Laura; Hirose, Tetsuro; Bond, Charles S.

    2015-01-01

    Prion-like domains (PLDs) are low complexity sequences found in RNA binding proteins associated with the neurodegenerative disorder amyotrophic lateral sclerosis. Recently, PLDs have been implicated in mediating gene regulation via liquid-phase transitions that drive ribonucleoprotein granule assembly. In this paper, we report many PLDs in proteins associated with paraspeckles, subnuclear bodies that form around long noncoding RNA. We mapped the interactome network of paraspeckle proteins, finding enrichment of PLDs. We show that one protein, RBM14, connects key paraspeckle subcomplexes via interactions mediated by its PLD. We further show that the RBM14 PLD, as well as the PLD of another essential paraspeckle protein, FUS, is required to rescue paraspeckle formation in cells in which their endogenous counterpart has been knocked down. Similar to FUS, the RBM14 PLD also forms hydrogels with amyloid-like properties. These results suggest a role for PLD-mediated liquid-phase transitions in paraspeckle formation, highlighting this nuclear body as an excellent model system for understanding the perturbation of such processes in neurodegeneration. PMID:26283796

  8. Salmonella typhimurium PtsJ is a novel MocR-like transcriptional repressor involved in regulating the vitamin B6 salvage pathway.

    PubMed

    Tramonti, Angela; Milano, Teresa; Nardella, Caterina; di Salvo, Martino L; Pascarella, Stefano; Contestabile, Roberto

    2017-02-01

    The vitamin B 6 salvage pathway, involving pyridoxine 5'-phosphate oxidase (PNPOx) and pyridoxal kinase (PLK), recycles B 6 vitamers from nutrients and protein turnover to produce pyridoxal 5'-phosphate (PLP), the catalytically active form of the vitamin. Regulation of this pathway, widespread in living organisms including humans and many bacteria, is very important to vitamin B 6 homeostasis but poorly understood. Although some information is available on the enzymatic regulation of PNPOx and PLK, little is known on their regulation at the transcriptional level. In the present work, we identified a new MocR-like regulator, PtsJ from Salmonella typhimurium, which controls the expression of the pdxK gene encoding one of the two PLKs expressed in this organism (PLK1). Analysis of pdxK expression in a ptsJ knockout strain demonstrated that PtsJ acts as a transcriptional repressor. This is the first case of a MocR-like regulator acting as repressor of its target gene. Expression and purification of PtsJ allowed a detailed characterisation of its effector and DNA-binding properties. PLP is the only B 6 vitamer acting as effector molecule for PtsJ. A DNA-binding region composed of four repeated nucleotide sequences is responsible for binding of PtsJ to its target promoter. Analysis of binding stoichiometry revealed that protein subunits/DNA molar ratio varies from 4 : 1 to 2 : 1, depending on the presence or absence of PLP. Structural characteristics of DNA transcriptional factor-binding sites suggest that PtsJ binds DNA according to a different model with respect to other characterised members of the MocR subgroup. © 2016 Federation of European Biochemical Societies.

  9. Monothiol CGFS Glutaredoxins and BolA-like Proteins: [2Fe-2S] Binding Partners in Iron Homeostasis

    PubMed Central

    Li, Haoran; Outten, Caryn E.

    2012-01-01

    Monothiol glutaredoxins (Grxs) with a signature CGFS active site and BolA-like proteins have recently emerged as novel players in iron homeostasis. Elegant genetic and biochemical studies examining the functional and physical interactions of CGFS Grxs in the fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe have unveiled their essential roles in intracellular iron signaling, iron trafficking, and the maturation of Fe-S cluster proteins. Biophysical and biochemical analyses of the [2Fe-2S]-bridging interaction between CGFS Grxs and a BolA-like protein in S. cerevisiae provided the first molecular-level understanding of the iron regulation mechanism in this model eukaryote, and established the ubiquitous CGFS Grxs and BolA-like proteins as novel Fe-S cluster-binding regulatory partners. Parallel studies focused on E. coli and human homologues for CGFS Grxs and BolA-like proteins have supported the studies in yeast and provided additional clues to their involvement in cellular iron metabolism. Herein we review recent progress in uncovering the cellular and molecular mechanisms by which CGFS Grxs and BolA-like proteins help regulate iron metabolism in both eukaryotic and prokaryotic organisms. PMID:22583368

  10. Secretome protein signature of human pancreatic cancer stem-like cells.

    PubMed

    Brandi, Jessica; Dalla Pozza, Elisa; Dando, Ilaria; Biondani, Giulia; Robotti, Elisa; Jenkins, Rosalind; Elliott, Victoria; Park, Kevin; Marengo, Emilio; Costello, Eithne; Scarpa, Aldo; Palmieri, Marta; Cecconi, Daniela

    2016-03-16

    Emerging research has demonstrated that pancreatic ductal adenocarcinoma (PDAC) contains a sub-population of cancer stem cells (CSCs) characterized by self-renewal, anchorage-independent-growth, long-term proliferation and chemoresistance. The secretome analysis of pancreatic CSCs has not yet been performed, although it may provide insight into tumour/microenvironment interactions and intracellular processes, as well as to identify potential biomarkers. To characterize the secreted proteins of pancreatic CSCs, we performed an iTRAQ-based proteomic analysis to compare the secretomes of Panc1 cancer stem-like cells (Panc1 CSCs) and parental cell line. A total of 72 proteins were found up-/down-regulated in the conditioned medium of Panc1 CSCs. The pathway analysis revealed modulation of vital physiological pathways including glycolysis, gluconeogenesis and pentose phosphate. Through ELISA immunoassays we analysed the presence of the three proteins most highly secreted by Panc1 CSCs (ceruloplasmin, galectin-3, and MARCKS) in sera of PDAC patient. ROC curve analysis suggests ceruloplasmin as promising marker for patients negative for CA19-9. Overall, our study provides a systemic secretome analysis of pancreatic CSCs revealing a number of secreted proteins which participate in pathological conditions including cancer differentiation, invasion and metastasis. They may serve as a valuable pool of proteins from which biomarkers and therapeutic targets can be identified. The secretome of CSCs is a rich reservoir of biomarkers of cancer progression and molecular therapeutic targets, and thus is a topic of great interest for cancer research. The secretome analysis of pancreatic CSCs has not yet been performed. Recently, our group has demonstrated that Panc-1 CSCs isolated from parental cell line by using the CSC selective medium, represent a model of great importance to deepen the understanding of the biology of pancreatic adenocarcinoma. To our knowledge, this is the first

  11. Exaptation of Bornavirus-Like Nucleoprotein Elements in Afrotherians

    PubMed Central

    Kobayashi, Yuki; Horie, Masayuki; Nakano, Ayumi; Murata, Koichi; Itou, Takuya; Suzuki, Yoshiyuki

    2016-01-01

    Endogenous bornavirus-like nucleoprotein elements (EBLNs), the nucleotide sequence elements derived from the nucleoprotein gene of ancient bornavirus-like viruses, have been identified in many animal genomes. Here we show evidence that EBLNs encode functional proteins in their host. Some afrotherian EBLNs were observed to have been maintained for more than 83.3 million years under negative selection. Splice variants were expressed from the genomic loci of EBLNs in elephant, and some were translated into proteins. The EBLN proteins appeared to be localized to the rough endoplasmic reticulum in African elephant cells, in contrast to the nuclear localization of bornavirus N. These observations suggest that afrotherian EBLNs have acquired a novel function in their host. Interestingly, genomic sequences of the first exon and its flanking regions in these EBLN loci were homologous to those of transmembrane protein 106B (TMEM106B). The upstream region of the first exon in the EBLN loci exhibited a promoter activity, suggesting that the ability of these EBLNs to be transcribed in the host cell was gained through capturing a partial duplicate of TMEM106B. In conclusion, our results strongly support for exaptation of EBLNs to encode host proteins in afrotherians. PMID:27518265

  12. CIKS, a connection to IκB kinase and stress-activated protein kinase

    PubMed Central

    Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich

    2000-01-01

    Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033

  13. CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

    PubMed

    Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

    2000-09-12

    Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

  14. HTLV-I Tax protein binds to MEKK1 to stimulate IkappaB kinase activity and NF-kappaB activation.

    PubMed

    Yin, M J; Christerson, L B; Yamamoto, Y; Kwak, Y T; Xu, S; Mercurio, F; Barbosa, M; Cobb, M H; Gaynor, R B

    1998-05-29

    NF-kappaB, a key regulator of the cellular inflammatory and immune response, is activated by the HTLV-I transforming and transactivating protein Tax. We show that Tax binds to the amino terminus of the protein kinase MEKK1, a component of an IkappaB kinase complex, and stimulates MEKK1 kinase activity. Tax expression increases the activity of IkappaB kinase beta (IKKbeta) to enhance phosphorylation of serine residues in IkappaB alpha that lead to its degradation. Dominant negative mutants of both IKKbeta and MEKK1 prevent Tax activation of the NF-kappaB pathway. Furthermore, recombinant MEKK1 stimulates IKKbeta phosphorylation of IkappaB alpha. Thus, Tax-mediated increases in NF-kappaB nuclear translocation result from direct interactions of Tax and MEKK1 leading to enhanced IKKbeta phosphorylation of IkappaB alpha.

  15. Characterization of B61, the ligand for the Eck receptor protein-tyrosine kinase.

    PubMed

    Shao, H; Pandey, A; O'Shea, K S; Seldin, M; Dixit, V M

    1995-03-10

    B61 was originally described as a novel secreted tumor necrosis factor-alpha-inducible gene product in endothelial cells (Holzman, L. B., Marks, R. M., and Dixit, V. M. (1990) Mol. Cell. Biol. 10, 5830-5838). It was recently discovered that soluble recombinant B61 could serve as a ligand for the Eck receptor protein-tyrosine kinase, a member of the Eph/Eck subfamily of receptor protein-tyrosine kinases (Bartley, T.D., Hunt, R. W., Welcher, A. A., Boyle, W. J., Parker, V. P., Lindberg, R. A., Lu, H. S., Colombero, A. M., Elliott, R. L., Guthrie, R. A., Holst, P. L., Skrine, J. D., Toso, R. J., Zhang, M., Fernandez, E., Trail, G., Yarnum, B., Yarden, Y., Hunter, T., and Fox, G. M. (1994) Nature 368, 558-560). We now show that B61 can also exist as a cell surface glycosylphosphatidyl-inositol-linked protein that is capable of activating the Eck receptor protein-tyrosine kinase, the first such report of a receptor protein-tyrosine kinase ligand that is glycosylphosphatidylinositol-linked. In addition, the expression patterns of B61 and Eck during mouse ontogeny were determined by in situ hybridization. Both were found to be highly expressed in the developing lung and gut, while Eck was preferentially expressed in the thymus. Finally, the gene for B61 was localized to a specific position on mouse chromosome 3 by interspecific back-cross analysis.

  16. CBX7 regulates stem cell-like properties of gastric cancer cells via p16 and AKT-NF-κB-miR-21 pathways.

    PubMed

    Ni, Su-Jie; Zhao, Li-Qin; Wang, Xiao-Feng; Wu, Zhen-Hua; Hua, Rui-Xi; Wan, Chun-Hua; Zhang, Jie-Yun; Zhang, Xiao-Wei; Huang, Ming-Zhu; Gan, Lu; Sun, Hua-Lin; Dimri, Goberdhan P; Guo, Wei-Jian

    2018-02-08

    Chromobox protein homolog 7 (CBX7), a member of the polycomb group (PcG) family of proteins, is involved in the regulation of cell proliferation and cancer progression. PcG family members, such as BMI, Mel-18, and EZH2, are integral constituents of the polycomb repressive complexes (PRCs) and have been known to regulate cancer stem cell (CSC) phenotype. However, the role of other PRCs' constituents such as CBX7 in the regulation of CSC phenotype remains largely elusive. This study was to investigate the role of CBX7 in regulating stem cell-like properties of gastric cancer and the underlying mechanisms. Firstly, the role of CBX7 in regulating stem cell-like properties of gastric cancer was investigated using sphere formation, Western blot, and xenograft tumor assays. Next, RNA interference and ectopic CBX7 expression were employed to determine the impact of CBX7 on the expression of CSC marker proteins and CSC characteristics. The expression of CBX7, its downstream targets, and stem cell markers were analyzed in gastric stem cell spheres, common cancer cells, and gastric cancer tissues. Finally, the pathways by which CBX7 regulates stem cell-like properties of gastric cancer were explored. We found that CBX7, a constituent of the polycomb repressive complex 1 (PRC1), plays an important role in maintaining stem cell-like characteristics of gastric cancer cells via the activation of AKT pathway and the downregulation of p16. Spearman rank correlation analysis showed positive correlations among the expression of CBX7 and phospho-AKT (pAKT), stem cell markers OCT-4, and CD133 in gastric cancer tissues. In addition, CBX7 was found to upregulate microRNA-21 (miR-21) via the activation of AKT-NF-κB pathway, and miR-21 contributes to CBX7-mediated CSC characteristics. CBX7 positively regulates stem cell-like characteristics of gastric cancer cells by inhibiting p16 and activating AKT-NF-κB-miR-21 pathway.

  17. Drosophila Shaking-B protein forms gap junctions in paired Xenopus oocytes.

    PubMed

    Phelan, P; Stebbings, L A; Baines, R A; Bacon, J P; Davies, J A; Ford, C

    1998-01-08

    In most multicellular organisms direct cell-cell communication is mediated by the intercellular channels of gap junctions. These channels allow the exchange of ions and molecules that are believed to be essential for cell signalling during development and in some differentiated tissues. Proteins called connexins, which are products of a multigene family, are the structural components of vertebrate gap junctions. Surprisingly, molecular homologues of the connexins have not been described in any invertebrate. A separate gene family, which includes the Drosophila genes shaking-B and l(1)ogre, and the Caenorhabditis elegans genes unc-7 and eat-5, encodes transmembrane proteins with a predicted structure similar to that of the connexins. shaking-B and eat-5 are required for the formation of functional gap junctions. To test directly whether Shaking-B is a channel protein, we expressed it in paired Xenopus oocytes. Here we show that Shaking-B localizes to the membrane, and that its presence induces the formation of functional intercellular channels. To our knowledge, this is the first structural component of an invertebrate gap junction to be characterized.

  18. Disruption of protein-tyrosine phosphatase 1B expression in the pancreas affects β-cell function.

    PubMed

    Liu, Siming; Xi, Yannan; Bettaieb, Ahmed; Matsuo, Kosuke; Matsuo, Izumi; Kulkarni, Rohit N; Haj, Fawaz G

    2014-09-01

    Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and energy balance. However, the role of PTP1B in pancreatic endocrine function remains largely unknown. To investigate the metabolic role of pancreatic PTP1B, we generated mice with pancreas PTP1B deletion (panc-PTP1B KO). Mice were fed regular chow or a high-fat diet, and metabolic parameters, insulin secretion and glucose tolerance were determined. On regular chow, panc-PTP1B KO and control mice exhibited comparable glucose tolerance whereas aged panc-PTP1B KO exhibited mild glucose intolerance. Furthermore, high-fat feeding promoted earlier impairment of glucose tolerance and attenuated glucose-stimulated insulin secretion in panc-PTP1B KO mice. The secretory defect in glucose-stimulated insulin secretion was recapitulated in primary islets ex vivo, suggesting that the effects were likely cell-autonomous. At the molecular level, PTP1B deficiency in vivo enhanced basal and glucose-stimulated tyrosyl phosphorylation of EphA5 in islets. Consistently, PTP1B overexpression in the glucose-responsive MIN6 β-cell line attenuated EphA5 tyrosyl phosphorylation, and substrate trapping identified EphA5 as a PTP1B substrate. In summary, these studies identify a novel role for PTP1B in pancreatic endocrine function.

  19. Cross-Specificities between cII-like Proteins and pRE-like Promoters of Lambdoid Bacteriophages

    PubMed Central

    Wulff, Daniel L.; Mahoney, Michael E.

    1987-01-01

    We have investigated the activation of transcription from the pRE promoters of phages λ, 21 and P22 by the λ and 21 cII proteins and the P22 c1 (cII-like) protein, using an in vivo system in which cII protein from a derepressed prophage activates transcription from a pRE DNA fragment on a multicopy plasmid. We find that each protein is highly specific for its own cognate pRE promoter, although measureable cross-reactions are observed. The primary recognition sequence for cII protein on λ pRE is a pair of TTGC repeat sequences in the sequence 5'-TTGCN 6TTGC-3' at the -35 region of the promoter. This same sequence is found in 21 pRE, while P22 pRE has the sequence 5'-TTGCN6TTGT-3', which is the same as that of λctr1, a pRE+ variant of λ. λctr1 pRE is half as active as λ + pRE when assayed with either the λ cII or the P22 c1 proteins. Therefore, the single base change in the P22 repeat sequence cannot explain why the P22 c1 protein is much more active with P22 pRE than λ p RE. The dya5 mutation, a G→A change at position -43 of pRE, makes pRE a stronger promoter when assayed with either the λ or 21 cII proteins or the P22 c1 protein. We conclude that efficient activation of a cII-dependent promoter by a cII protein requires sequence information in addition to the TTGC repeat sequences. We do not know the characteristics of the proteins which are responsible for the specificity of each protein for its own cognate promoter. However, λdya8, which has a Glu27→Lys alteration in the λ cII protein and a cII+ phenotype, results in a mutant cII protein that is much more highly specific than wild-type cII protein for its own cognate λ p RE promoter. This is especially remarkable because the dya8 amino acid alteration makes the helix-2 region (the region of the protein predicted to make contact with the phosphodiester backbone of the DNA) of λ cII protein conform exactly with the helix-2 region of the P22 c1 protein in both charge and charge distribution. PMID

  20. Expression of membrane-associated proteins within single emulsion cell facsimiles.

    PubMed

    Chanasakulniyom, Mayuree; Martino, Chiara; Paterson, David; Horsfall, Louise; Rosser, Susan; Cooper, Jonathan M

    2012-07-07

    MreB is a structural membrane-associated protein which is one of the key components of the bacterial cytoskeleton. Although it plays an important role in shape maintenance of rod-like bacteria, the understanding of its mechanism of action is still not fully understood. This study shows how segmented flow and microdroplet technology can be used as a new tool for biological in vitro investigation of this protein. In this paper, we demonstrate cell-free expression in a single emulsion system to express red fluorescence protein (RFP) and MreB linked RFP (MreB-RFP). We follow the aggregation and localisation of the fusion protein MreB-RFP in this artificial cell-like environment. The expression of MreB-RFP in single emulsion droplets leads to the formation of micrometer-scale protein patches distributed at the water/oil interface.

  1. NETWORKED 3B: a novel protein in the actin cytoskeleton-endoplasmic reticulum interaction.

    PubMed

    Wang, Pengwei; Hussey, Patrick J

    2017-03-01

    In plants movement of the endoplasmic reticulum (ER) is dependent on the actin cytoskeleton. However little is known about proteins that link the ER membrane and the actin cytoskeleton. Here we identified a novel protein, NETWORKED 3B (NET3B), which is associated with the ER and actin cytoskeleton in vivo. NET3B belongs to a superfamily of plant specific actin binding proteins, the NETWORKED family. NET3B associates with the actin cytoskeleton in vivo through an N-terminal NET actin binding (NAB) domain, which has been well-characterized in other members of the NET family. A three amino acid insertion, Val-Glu-Asp, in the NAB domain of NET3B appears to lower its ability to localize to the actin cytoskeleton compared with NET1A, the founding member of the NET family. The C-terminal domain of NET3B links the protein to the ER. Overexpression of NET3B enhanced the association between the ER and the actin cytoskeleton, and the extent of this association was dependent on the amount of NET3B available. Another effect of NET3B overexpression was a reduction in ER membrane diffusion. In conclusion, our results revealed that NET3B modulates ER and actin cytoskeleton interactions in higher plants. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  2. Lassa virus Z protein is a matrix protein and sufficient for the release of virus-like particles [corrected].

    PubMed

    Strecker, Thomas; Eichler, Robert; Meulen, Jan ter; Weissenhorn, Winfried; Dieter Klenk, Hans; Garten, Wolfgang; Lenz, Oliver

    2003-10-01

    Lassa virus is an enveloped virus with glycoprotein spikes on its surface. It contains an RNA ambisense genome that encodes the glycoprotein precursor GP-C, the nucleoprotein NP, the polymerase L, and the Z protein. Here we demonstrate that the Lassa virus Z protein (i). is abundant in viral particles, (ii). is strongly membrane associated, (iii). is sufficient in the absence of all other viral proteins to release enveloped particles, and (iv). contains two late domains, PTAP and PPXY, necessary for the release of virus-like particles. Our data provide evidence that Z is the Lassa virus matrix protein that is the driving force for virus particle release.

  3. p53 Protein interacts specifically with the meiosis-specific mammalian RecA-like protein DMC1 in meiosis.

    PubMed

    Habu, Toshiyuki; Wakabayashi, Nobunao; Yoshida, Kayo; Yomogida, Kenntaro; Nishimune, Yoshitake; Morita, Takashi

    2004-06-01

    The tumor suppressor protein p53 is specifically expressed during meiosis in spermatocytes. Subsets of p53 knockout mice exhibit testicular giant cell degenerative syndrome, which suggests p53 may be associated with meiotic cell cycle and/or DNA metabolism. Here, we show that p53 binds to the mouse meiosis-specific RecA-like protein Mus musculus DMC1 (MmDMC1). The C-terminal domain (amino acid 234-340) of MmDMC1 binds to DNA-binding domain of p53 protein. p53 might be involved in homologous recombination and/or checkpoint function by directly binding to DMC1 protein to repress genomic instability in meiotic germ cells.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dahms, Sven O., E-mail: sdahms@fli-leibniz.de; Mayer, Magnus C.; Miltenyi Biotec GmbH, Robert-Koch-Strasse 1, 17166 Teterow

    2015-03-01

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

  5. A Mutation Directs the Structural Switch of DNA Binding Proteins under Starvation to a Ferritin-like Protein Cage.

    PubMed

    Williams, Sunanda Margrett; Chandran, Anu Vijayakumari; Prakash, Sunita; Vijayan, Mamannamana; Chatterji, Dipankar

    2017-09-05

    Proteins of the ferritin family are ubiquitous in living organisms. With their spherical cage-like structures they are the iron storehouses in cells. Subfamilies of ferritins include 24-meric ferritins and bacterioferritins (maxiferritins), and 12-meric Dps (miniferritins). Dps safeguards DNA by direct binding, affording physical protection and safeguards from free radical-mediated damage by sequestering iron in its core. The maxiferritins can oxidize and store iron but cannot bind DNA. Here we show that a mutation at a critical interface in Dps alters its assembly from the canonical 12-mer to a ferritin-like 24-mer under crystallization. This structural switch was attributed to the conformational alteration of a highly conserved helical loop and rearrangement of the C-terminus. Our results demonstrate a novel concept of mutational switch between related protein subfamilies and corroborate the popular model for evolution by which subtle substitutions in an amino acid sequence lead to diversification among proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Control of B-cell responses by Toll-like receptors

    NASA Astrophysics Data System (ADS)

    Pasare, Chandrashekhar; Medzhitov, Ruslan

    2005-11-01

    Toll-like receptors (TLRs) detect microbial infection and have an essential role in the induction of immune responses. TLRs can directly induce innate host defence responses, but the mechanisms of TLR-mediated control of adaptive immunity are not fully understood. Although TLR-induced dendritic cell maturation is required for activation of T-helper (TH) cells, the role of TLRs in B-cell activation and antibody production in vivo is not yet known. Here we show that activation and differentiation of TH cells is not sufficient for the induction of T-dependent B-cell responses. We find that, in addition to CD4+ T-cell help, generation of T-dependent antigen-specific antibody responses requires activation of TLRs in B cells.

  7. Astrocyte Resilience to Oxidative Stress Induced by Insulin-like Growth Factor I (IGF-I) Involves Preserved AKT (Protein Kinase B) Activity*

    PubMed Central

    Dávila, David; Fernández, Silvia; Torres-Alemán, Ignacio

    2016-01-01

    Disruption of insulin-like growth factor I (IGF-I) signaling is a key step in the development of cancer or neurodegeneration. For example, interference of the prosurvival IGF-I/AKT/FOXO3 pathway by redox activation of the stress kinases p38 and JNK is instrumental in neuronal death by oxidative stress. However, in astrocytes, IGF-I retains its protective action against oxidative stress. The molecular mechanisms underlying this cell-specific protection remain obscure but may be relevant to unveil new ways to combat IGF-I/insulin resistance. Here, we describe that, in astrocytes exposed to oxidative stress by hydrogen peroxide (H2O2), p38 activation did not inhibit AKT (protein kinase B) activation by IGF-I, which is in contrast to our previous observations in neurons. Rather, stimulation of AKT by IGF-I was significantly higher and more sustained in astrocytes than in neurons either under normal or oxidative conditions. This may be explained by phosphorylation of the phosphatase PTEN at the plasma membrane in response to IGF-I, inducing its cytosolic translocation and preserving in this way AKT activity. Stimulation of AKT by IGF-I, mimicked also by a constitutively active AKT mutant, reduced oxidative stress levels and cell death in H2O2-exposed astrocytes, boosting their neuroprotective action in co-cultured neurons. These results indicate that armoring of AKT activation by IGF-I is crucial to preserve its cytoprotective effect in astrocytes and may form part of the brain defense mechanism against oxidative stress injury. PMID:26631726

  8. Ethanol specifically decreases peroxisome proliferator activated receptor beta in B12 oligodendrocyte-like cells.

    PubMed

    Leisewitz, Andrea V; Jung, Juan E; Perez-Alzola, Patricia; Fuenzalida, Karen M; Roth, Alejandro; Inestrosa, Nibaldo C; Bronfman, Miguel

    2003-04-01

    Peroxisome proliferator activated receptors (PPARs) are nuclear receptors that control important genes involved in lipid metabolism. Their role in nerve cells is uncertain, although anomalous myelination of the corpus callosum has been described in the PPARbeta-null mouse, and abnormalities of this tissue have been documented in fetal alcohol syndrome in humans. We report here that ethanol treatment of B12 oligodendrocyte-like cells induces a concentration- and time-dependent decrease in the mRNA and protein levels of PPARbeta, with no effect on PPARalpha or PPARgamma. The effect on PPARbeta is seen as an increase in mRNA degradation, as assessed by run-off assays, due to a significant decrease in PPARbeta mRNA half-life, with no observed changes in intracellular localization. Our results suggest a possible link between PPARbeta function and ethanol-induced abnormal myelination in oligodendrocytes.

  9. A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

    USDA-ARS?s Scientific Manuscript database

    Chitin-binding proteins (CBPs) existed in various species and involved in different biology processes. In the present study, we cloned a full length cDNA of chitin-binding protein-like (PpCBP-like) from Pteromalus puparum, a pupal endoparasitoid of Pieris rapae. PpCBP-like encoded a 96 putative amin...

  10. Multiple antibacterial histone H2B proteins are expressed in tissues of American oyster.

    PubMed

    Seo, Jung-Kil; Stephenson, Jeana; Noga, Edward J

    2011-03-01

    We have previously identified a histone H2B isomer (cvH2B-1) from tissue extracts of the bivalve mollusk, the American oyster (Crassostrea virginica). In this paper, we isolate an additional three antibacterial proteins from acidified gill extract by preparative acid-urea-polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography. Extraction of these proteins from tissue was best accomplished by briefly boiling the tissues in a weak acetic acid solution. Addition of protease inhibitors while boiling resulted in somewhat lower yields, with one protein being totally absent with this method. Via mass spectrometry, the masses of one of these purified proteins was 13607.0Da (peak 2), which is consistent with the molecular weight of histone H2B. In addition, via western-blotting using anti-calf histone H2B antibody, all three proteins were positive and were thus named cvH2B-2, cvH2B-3 and cvH2B-4. The antibacterial activity of cvH2B-2 was similar to that of cvH2B-1, with activity against a Gram-positive bacterium (Lactococcus lactis subsp. lactis; minimum effective concentration [MEC] 52-57μg/mL) but inactive against Staphylococcus aureus (MEC>250μg/mL). However, both proteins had relatively potent activity against the Gram-negative oyster pathogen Vibrio parahemolyticus (MEC 11.5-14μg/mL) as well as the human pathogen Vibrio vulnificus (MEC 21.3-25.3μg/mL). cvH2B-3 and cvH2B-4 also had similarly strong activity against Vibrio vulnificus. These data provide further evidence for the antimicrobial function of histone H2B isomers in modulating bacterial populations in oyster tissues. The combined estimated concentrations of these histone H2B isomers were far above the inhibitory concentrations for the tested vibrios, including human pathogens. Our results indicate that the highly conserved histone proteins might be important components not only of immune defenses in oysters but have the potential to influence the abundance of a

  11. Protein arginine methyltransferase 7 has a novel homodimer-like structure formed by tandem repeats.

    PubMed

    Hasegawa, Morio; Toma-Fukai, Sachiko; Kim, Jun-Dal; Fukamizu, Akiyoshi; Shimizu, Toshiyuki

    2014-05-21

    Protein arginine methyltransferase 7 (PRMT7) is a member of a family of enzymes that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to nitrogen atoms on arginine residues. Here, we describe the crystal structure of Caenorhabditis elegans PRMT7 in complex with its reaction product S-adenosyl-L-homocysteine. The structural data indicated that PRMT7 harbors two tandem repeated PRMT core domains that form a novel homodimer-like structure. S-adenosyl-L-homocysteine bound to the N-terminal catalytic site only; the C-terminal catalytic site is occupied by a loop that inhibits cofactor binding. Mutagenesis demonstrated that only the N-terminal catalytic site of PRMT7 is responsible for cofactor binding. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  12. Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillus oryzae

    PubMed Central

    Tanaka, Mizuki; Hiramoto, Tetsuya; Tada, Hinako; Shintani, Takahiro

    2017-01-01

    ABSTRACT Aspergillus oryzae produces copious amount of amylolytic enzymes, and MalP, a major maltose permease, is required for the expression of amylase-encoding genes. The expression of these genes is strongly repressed by carbon catabolite repression (CCR) in the presence of glucose. MalP is transported from the plasma membrane to the vacuole by endocytosis, which requires the homolog of E6-AP carboxyl terminus ubiquitin ligase HulA, an ortholog of yeast Rsp5. In yeast, arrestin-like proteins mediate endocytosis as adaptors of Rsp5 and transporters. In the present study, we examined the involvement of CreD, an arrestin-like protein, in glucose-induced MalP endocytosis and CCR of amylase-encoding genes. Deletion of creD inhibited the glucose-induced endocytosis of MalP, and CreD showed physical interaction with HulA. Phosphorylation of CreD was detected by Western blotting, and two serine residues were determined as the putative phosphorylation sites. However, the phosphorylation state of the serine residues did not regulate MalP endocytosis and its interaction with HulA. Although α-amylase production was significantly repressed by creD deletion, both phosphorylation and dephosphorylation mimics of CreD had a negligible effect on α-amylase activity. Interestingly, dephosphorylation of CreD was required for CCR relief of amylase genes that was triggered by disruption of the deubiquitinating enzyme-encoding gene creB. The α-amylase activity of the creB mutant was 1.6-fold higher than that of the wild type, and the dephosphorylation mimic of CreD further improved the α-amylase activity by 2.6-fold. These results indicate that a combination of the dephosphorylation mutation of CreD and creB disruption increased the production of amylolytic enzymes in A. oryzae. IMPORTANCE In eukaryotes, glucose induces carbon catabolite repression (CCR) and proteolytic degradation of plasma membrane transporters via endocytosis. Glucose-induced endocytosis of transporters is

  13. Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillus oryzae.

    PubMed

    Tanaka, Mizuki; Hiramoto, Tetsuya; Tada, Hinako; Shintani, Takahiro; Gomi, Katsuya

    2017-07-01

    Aspergillus oryzae produces copious amount of amylolytic enzymes, and MalP, a major maltose permease, is required for the expression of amylase-encoding genes. The expression of these genes is strongly repressed by carbon catabolite repression (CCR) in the presence of glucose. MalP is transported from the plasma membrane to the vacuole by endocytosis, which requires the homolog of E6-AP carboxyl terminus ubiquitin ligase HulA, an ortholog of yeast Rsp5. In yeast, arrestin-like proteins mediate endocytosis as adaptors of Rsp5 and transporters. In the present study, we examined the involvement of CreD, an arrestin-like protein, in glucose-induced MalP endocytosis and CCR of amylase-encoding genes. Deletion of creD inhibited the glucose-induced endocytosis of MalP, and CreD showed physical interaction with HulA. Phosphorylation of CreD was detected by Western blotting, and two serine residues were determined as the putative phosphorylation sites. However, the phosphorylation state of the serine residues did not regulate MalP endocytosis and its interaction with HulA. Although α-amylase production was significantly repressed by creD deletion, both phosphorylation and dephosphorylation mimics of CreD had a negligible effect on α-amylase activity. Interestingly, dephosphorylation of CreD was required for CCR relief of amylase genes that was triggered by disruption of the deubiquitinating enzyme-encoding gene creB The α-amylase activity of the creB mutant was 1.6-fold higher than that of the wild type, and the dephosphorylation mimic of CreD further improved the α-amylase activity by 2.6-fold. These results indicate that a combination of the dephosphorylation mutation of CreD and creB disruption increased the production of amylolytic enzymes in A. oryzae IMPORTANCE In eukaryotes, glucose induces carbon catabolite repression (CCR) and proteolytic degradation of plasma membrane transporters via endocytosis. Glucose-induced endocytosis of transporters is mediated by

  14. ArfB links protein lipidation and endocytosis to polarized growth of Aspergillus nidulans

    PubMed Central

    Lee, Soo Chan

    2008-01-01

    Aspergillus nidulans undergoes polarized hyphal growth during the majority of its life cycle. Regulatory mechanisms for hyphal polarity have been intensively investigated in a variety of filamentous fungi. Two important cellular processes, which have received recent attention, include protein myristoylation and endocytosis. It is clear that protein myristoylation is essential for polarity establishment because germinating A. nidulans conidia lost polarity in the presence of cerulenin, a lipid metabolism inhibitor and in an N-myristoyl transferase mutant background. Only 41 predicted proteins encoded by A. nidulans posses an N-myristoylation motif, one of which is ADP ribosylation factor B (ArfB). Disruption of ArfB leads to failure of polarity establishment and maintenance during early morphogenesis and in a delay in endocytosis. Therefore, ArfB connects N-myristoylation and endocytosis to polarized growth. Exocytotic vesicle trafficking through the Spitzenkörper may also require Arf proteins in their role in vesicle formation. Taken together, ArfB is one of the important key components for the fungal hyphal growth. PMID:19704790

  15. Unpredictable chronic mild stress induces anxiety and depression-like behaviors and inactivates AMP-activated protein kinase in mice.

    PubMed

    Zhu, Shenghua; Wang, Junhui; Zhang, Yanbo; Li, Victor; Kong, Jiming; He, Jue; Li, Xin-Min

    2014-08-12

    The unpredictable chronic mild stress (UCMS) model was developed based upon the stress-diathesis hypothesis of depression. Most effects of UCMS can be reversed by antidepressants, demonstrating a strong predictive validity of this model for depression. However, the mechanisms underlying the effects induced by UCMS remain incompletely understood. Increasing evidence has shown that AMP-activated protein kinase (AMPK) regulates intracellular energy metabolism and is especially important for neurons because neurons are known to have small energy reserves. Abnormalities in the AMPK pathway disturb normal brain functions and synaptic integrity. In the present study, we first investigated the effects of UCMS on a battery of different tests measuring anxiety and depression-like behaviors in female C57BL/6N mice after 4 weeks of UCMS exposure. Stressed mice showed suppressed body weight gain, heightened anxiety, and increased immobility in the forced swim and tail suspension tests. These results are representative of some of the core symptoms of depression. Simultaneously, we observed decrease of synaptic proteins in the cortex of mice subjected to UCMS, which is associated with decreased levels of phosphorylated AMP-activated protein kinase α (AMPKα) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase). Our findings suggest that AMPKα inactivation might be a mechanism by which UCMS causes anxiety/depression-like behaviors in mice. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Ectromelia virus encodes a family of Ankyrin/F-box proteins that regulate NFκB

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Burles, Kristin, E-mail: burles@ualberta.ca; Buuren, Nicholas van; Barry, Michele

    2014-11-15

    A notable feature of poxviruses is their ability to inhibit the antiviral response, including the nuclear factor kappa B (NFκB) pathway. NFκB is a transcription factor that is sequestered in the cytoplasm until cell stimulation, and relies on the SCF (Skp1, culllin-1, F-box) ubiquitin ligase to target its inhibitor, IκBα, for degradation. IκBα is recruited to the SCF by the F-box domain-containing protein βTrCP. Here, we show that ectromelia virus, the causative agent of mousepox, encodes four F-box-containing proteins, EVM002, EVM005, EVM154, and EVM165, all of which contain Ankyrin (Ank) domains. The Ank/F-box proteins inhibit NFκB nuclear translocation, and thismore » inhibition is dependent on the F-box domain. We also demonstrate that EVM002, EVM005, EVM154, and EVM165 prevent IκBα degradation, suggesting that they target the SCF. This study identifies a new mechanism by which ectromelia virus inhibits NFκB. - Highlights: • Ectromelia virus encodes four Ank/F-box proteins, EVM002, EVM005, EVM154 and EVM165. • The Ank/F-box proteins inhibit NFκB nuclear translocation, dependent on the F-box. • The Ank/F-box proteins prevent IκBα degradation, suggesting they target the SCF. • Deletion of a single Ank/F-box gene from ECTV does not prevent viral NFκB inhibition. • This study identifies a new mechanism by which ectromelia virus inhibits NFκB.« less

  17. Molecular evolution of miraculin-like proteins in soybean Kunitz super-family.

    PubMed

    Selvakumar, Purushotham; Gahloth, Deepankar; Tomar, Prabhat Pratap Singh; Sharma, Nidhi; Sharma, Ashwani Kumar

    2011-12-01

    Miraculin-like proteins (MLPs) belong to soybean Kunitz super-family and have been characterized from many plant families like Rutaceae, Solanaceae, Rubiaceae, etc. Many of them possess trypsin inhibitory activity and are involved in plant defense. MLPs exhibit significant sequence identity (~30-95%) to native miraculin protein, also belonging to Kunitz super-family compared with a typical Kunitz family member (~30%). The sequence and structure-function comparison of MLPs with that of a classical Kunitz inhibitor have demonstrated that MLPs have evolved to form a distinct group within Kunitz super-family. Sequence analysis of new genes along with available MLP sequences in the literature revealed three major groups for these proteins. A significant feature of Rutaceae MLP type 2 sequences is the presence of phosphorylation motif. Subtle changes are seen in putative reactive loop residues among different MLPs suggesting altered specificities to specific proteases. In phylogenetic analysis, Rutaceae MLP type 1 and type 2 proteins clustered together on separate branches, whereas native miraculin along with other MLPs formed distinct clusters. Site-specific positive Darwinian selection was observed at many sites in both the groups of Rutaceae MLP sequences with most of the residues undergoing positive selection located in loop regions. The results demonstrate the sequence and thereby the structure-function divergence of MLPs as a distinct group within soybean Kunitz super-family due to biotic and abiotic stresses of local environment.

  18. Mechanism for accurate, protein-assisted DNA annealing by Deinococcus radiodurans DdrB

    PubMed Central

    Sugiman-Marangos, Seiji N.; Weiss, Yoni M.; Junop, Murray S.

    2016-01-01

    Accurate pairing of DNA strands is essential for repair of DNA double-strand breaks (DSBs). How cells achieve accurate annealing when large regions of single-strand DNA are unpaired has remained unclear despite many efforts focused on understanding proteins, which mediate this process. Here we report the crystal structure of a single-strand annealing protein [DdrB (DNA damage response B)] in complex with a partially annealed DNA intermediate to 2.2 Å. This structure and supporting biochemical data reveal a mechanism for accurate annealing involving DdrB-mediated proofreading of strand complementarity. DdrB promotes high-fidelity annealing by constraining specific bases from unauthorized association and only releases annealed duplex when bound strands are fully complementary. To our knowledge, this mechanism provides the first understanding for how cells achieve accurate, protein-assisted strand annealing under biological conditions that would otherwise favor misannealing. PMID:27044084

  19. An Inducible DamID System for Profiling Interactions of Nuclear Lamina Protein Component Lamin B1 with Chromosomes in Mouse Cells.

    PubMed

    Kozhevnikova, E N; Leshchenko, A E; Pindyurin, A V

    2018-05-01

    At the level of DNA organization into chromatin, there are mechanisms that define gene expression profiles in specialized cell types. Genes within chromatin regions that are located at the nuclear periphery are generally expressed at lower levels; however, the nature of this phenomenon remains unclear. These parts of chromatin interact with nuclear lamina proteins like Lamin B1 and, therefore, can be identified in a given cell type by chromatin profiling of these proteins. In this study, we created and tested a Dam Identification (DamID) system induced by Cre recombinase using Lamin B1 and mouse embryonic fibroblasts. This inducible system will help to generate genome-wide profiles of chromatin proteins in given cell types and tissues with no need to dissect tissues from organs or separate cells from tissues, which is achieved by using specific regulatory DNA elements and due to the high sensitivity of the method.

  20. A novel method for preparation of HAMLET-like protein complexes.

    PubMed

    Permyakov, Sergei E; Knyazeva, Ekaterina L; Leonteva, Marina V; Fadeev, Roman S; Chekanov, Aleksei V; Zhadan, Andrei P; Håkansson, Anders P; Akatov, Vladimir S; Permyakov, Eugene A

    2011-09-01

    Some natural proteins induce tumor-selective apoptosis. α-Lactalbumin (α-LA), a milk calcium-binding protein, is converted into an antitumor form, called HAMLET/BAMLET, via partial unfolding and association with oleic acid (OA). Besides triggering multiple cell death mechanisms in tumor cells, HAMLET exhibits bactericidal activity against Streptococcus pneumoniae. The existing methods for preparation of active complexes of α-LA with OA employ neutral pH solutions, which greatly limit water solubility of OA. Therefore these methods suffer from low scalability and/or heterogeneity of the resulting α-LA - OA samples. In this study we present a novel method for preparation of α-LA - OA complexes using alkaline conditions that favor aqueous solubility of OA. The unbound OA is removed by precipitation under acidic conditions. The resulting sample, bLA-OA-45, bears 11 OA molecules and exhibits physico-chemical properties similar to those of BAMLET. Cytotoxic activities of bLA-OA-45 against human epidermoid larynx carcinoma and S. pneumoniae D39 cells are close to those of HAMLET. Treatment of S. pneumoniae with bLA-OA-45 or HAMLET induces depolarization and rupture of the membrane. The cells are markedly rescued from death upon pretreatment with an inhibitor of Ca(2+) transport. Hence, the activation mechanisms of S. pneumoniae death are analogous for these two complexes. The developed express method for preparation of active α-LA - OA complex is high-throughput and suited for development of other protein complexes with low-molecular-weight amphiphilic substances possessing valuable cytotoxic properties. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  1. OCD-like behavior is caused by dysfunction of thalamo-amygdala circuits and upregulated TrkB/ERK-MAPK signaling as a result of SPRED2 deficiency

    PubMed Central

    Ullrich, M; Weber, M; Post, A M; Popp, S; Grein, J; Zechner, M; Guerrero González, H; Kreis, A; Schmitt, A G; Üçeyler, N; Lesch, K-P; Schuh, K

    2018-01-01

    Obsessive-compulsive disorder (OCD) is a common neuropsychiatric disease affecting about 2% of the general population. It is characterized by persistent intrusive thoughts and repetitive ritualized behaviors. While gene variations, malfunction of cortico-striato-thalamo-cortical (CSTC) circuits, and dysregulated synaptic transmission have been implicated in the pathogenesis of OCD, the underlying mechanisms remain largely unknown. Here we show that OCD-like behavior in mice is caused by deficiency of SPRED2, a protein expressed in various brain regions and a potent inhibitor of Ras/ERK-MAPK signaling. Excessive self-grooming, reflecting OCD-like behavior in rodents, resulted in facial skin lesions in SPRED2 knockout (KO) mice. This was alleviated by treatment with the selective serotonin reuptake inhibitor fluoxetine. In addition to the previously suggested involvement of cortico-striatal circuits, electrophysiological measurements revealed altered transmission at thalamo-amygdala synapses and morphological differences in lateral amygdala neurons of SPRED2 KO mice. Changes in synaptic function were accompanied by dysregulated expression of various pre- and postsynaptic proteins in the amygdala. This was a result of altered gene transcription and triggered upstream by upregulated tropomyosin receptor kinase B (TrkB)/ERK-MAPK signaling in the amygdala of SPRED2 KO mice. Pathway overactivation was mediated by increased activity of TrkB, Ras, and ERK as a specific result of SPRED2 deficiency and not elicited by elevated brain-derived neurotrophic factor levels. Using the MEK inhibitor selumetinib, we suppressed TrkB/ERK-MAPK pathway activity in vivo and reduced OCD-like grooming in SPRED2 KO mice. Altogether, this study identifies SPRED2 as a promising new regulator, TrkB/ERK-MAPK signaling as a novel mediating mechanism, and thalamo-amygdala synapses as critical circuitry involved in the pathogenesis of OCD. PMID:28070119

  2. Virus-Like Particles Derived from HIV-1 for Delivery of Nuclear Proteins: Improvement of Production and Activity by Protein Engineering.

    PubMed

    Robert, Marc-André; Lytvyn, Viktoria; Deforet, Francis; Gilbert, Rénald; Gaillet, Bruno

    2017-01-01

    Virus-like particles (VLPs) derived from retroviruses and lentiviruses can be used to deliver recombinant proteins without the fear of causing insertional mutagenesis to the host cell genome. In this study we evaluate the potential of an inducible lentiviral vector packaging cell line for VLP production. The Gag gene from HIV-1 was fused to a gene encoding a selected protein and it was transfected into the packaging cells. Three proteins served as model: the green fluorescent protein and two transcription factors-the cumate transactivator (cTA) of the inducible CR5 promoter and the human Krüppel-like factor 4 (KLF4). The sizes of the VLPs were 120-150 nm in diameter and they were resistant to freeze/thaw cycles. Protein delivery by the VLPs reached up to 100% efficacy in human cells and was well tolerated. Gag-cTA triggered up to 1100-fold gene activation of the reporter gene in comparison to the negative control. Protein engineering was required to detect Gag-KLF4 activity. Thus, insertion of the VP16 transactivation domain increased the activity of the VLPs by eightfold. An additional 2.4-fold enhancement was obtained by inserting nuclear export signal. In conclusion, our platform produced VLPs capable of efficient protein transfer, and it was shown that protein engineering can be used to improve the activity of the delivered proteins as well as VLP production.

  3. The C-Terminal Sequence of RhoB Directs Protein Degradation through an Endo-Lysosomal Pathway

    PubMed Central

    Ramos, Irene; Herrera, Mónica; Stamatakis, Konstantinos

    2009-01-01

    Background Protein degradation is essential for cell homeostasis. Targeting of proteins for degradation is often achieved by specific protein sequences or posttranslational modifications such as ubiquitination. Methodology/Principal Findings By using biochemical and genetic tools we have monitored the localization and degradation of endogenous and chimeric proteins in live primary cells by confocal microscopy and ultra-structural analysis. Here we identify an eight amino acid sequence from the C-terminus of the short-lived GTPase RhoB that directs the rapid degradation of both RhoB and chimeric proteins bearing this sequence through a lysosomal pathway. Elucidation of the RhoB degradation pathway unveils a mechanism dependent on protein isoprenylation and palmitoylation that involves sorting of the protein into multivesicular bodies, mediated by the ESCRT machinery. Moreover, RhoB sorting is regulated by late endosome specific lipid dynamics and is altered in human genetic lipid traffic disease. Conclusions/Significance Our findings characterize a short-lived cytosolic protein that is degraded through a lysosomal pathway. In addition, we define a novel motif for protein sorting and rapid degradation, which allows controlling protein levels by means of clinically used drugs. PMID:19956591

  4. Hantaan virus nucleocapsid protein binds to importin alpha proteins and inhibits tumor necrosis factor alpha-induced activation of nuclear factor kappa B.

    PubMed

    Taylor, Shannon L; Frias-Staheli, Natalia; García-Sastre, Adolfo; Schmaljohn, Connie S

    2009-02-01

    Hantaviruses such as Hantaan virus (HTNV) and Andes virus cause two human diseases, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome, respectively. For both, disease pathogenesis is thought to be immunologically mediated and there have been numerous reports of patients with elevated levels of proinflammatory and inflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), in their sera. Multiple viruses have developed evasion strategies to circumvent the host cell inflammatory process, with one of the most prevalent being the disruption of nuclear factor kappa B (NF-kappaB) activation. We hypothesized that hantaviruses might also moderate host inflammation by interfering with this pathway. We report here that the nucleocapsid (N) protein of HTNV was able to inhibit TNF-alpha-induced activation of NF-kappaB, as measured by a reporter assay, and the activation of endogenous p65, an NF-kappaB subunit. Surprisingly, there was no defect in the degradation of the inhibitor of NF-kappaB (IkappaB) protein, nor was there any alteration in the level of p65 expression in HTNV N-expressing cells. However, immunofluorescence antibody staining demonstrated that cells expressing HTNV N protein and a green fluorescent protein-p65 fusion had limited p65 nuclear translocation. Furthermore, we were able to detect an interaction between HTNV N protein and importin alpha, a nuclear import molecule responsible for shuttling NF-kappaB to the nucleus. Collectively, our data suggest that HTNV N protein can sequester NF-kappaB in the cytoplasm, thus inhibiting NF-kappaB activity. These findings, which were obtained using cells transfected with cDNA representing the HTNV N gene, were confirmed using HTNV-infected cells.

  5. Comparison of Palivizumab-Like Antibody Binding to Different Conformations of the RSV F Protein in RSV-Infected Adult Hematopoietic Cell Transplant Recipients.

    PubMed

    Ye, Xunyan; Iwuchukwu, Obinna P; Avadhanula, Vasanthi; Aideyan, Letisha O; McBride, Trevor J; Ferlic-Stark, Laura L; Patel, Kirtida D; Piedra, Felipe-Andres; Shah, Dimpy P; Chemaly, Roy F; Piedra, Pedro A

    2018-03-28

    Most respiratory syncytial virus (RSV) vaccine candidates include fusion (F) protein in different conformations. Antigenic site II found in the different F conformations is the target of palivizumab, the only US Food and Drug Administration approved monoclonal antibody (mAb). Serum palivizumab-like antibody (PLA) is a potential serologic correlate of immunity. Our objective was to determine if different conformations of F protein in a palivizumab competitive antibody (PCA) assay affect the PLA concentrations. Four PCA assays were standardized using mAbs. Each contained prefusion, postfusion, or intermediate F forms. PLA concentrations were measured in acute and convalescent sera from 22 RSV/A and 18 RSV/B-infected adult hematopoietic cell transplant (HCT) recipients. PLA concentrations were calculated using a 4-parameter logistic regression model and analyzed for statistical significance. PCA assays revealed significantly greater PLA concentrations in convalescent sera; comparable increases in PLA concentration in RSV/A and RSV/B-infected HCT recipients; and significantly reduced PLA concentrations in HCT recipients who shed RSV ≥14 days. A significant positive correlation was observed between PCA assays and RSV neutralizing antibody titers. F protein conformation does not appear to have a measurable impact on PCA assays for measuring PLA induced by RSV/A or RSV/B infection.

  6. Conjugate-like immunogens produced as protein capsular matrix vaccines.

    PubMed

    Thanawastien, Ann; Cartee, Robert T; Griffin, Thomas J; Killeen, Kevin P; Mekalanos, John J

    2015-03-10

    Capsular polysaccharides are the primary antigenic components involved in protective immunity against encapsulated bacterial pathogens. Although immunization of adolescents and adults with polysaccharide antigens has reduced pathogen disease burden, pure polysaccharide vaccines have proved ineffective at conferring protective immunity to infants and the elderly, age cohorts that are deficient in their adaptive immune responses to such antigens. However, T-cell-independent polysaccharide antigens can be converted into more potent immunogens by chemically coupling to a "carrier protein" antigen. Such "conjugate vaccines" efficiently induce antibody avidity maturation, isotype switching, and immunological memory in immunized neonates. These immune responses have been attributed to T-cell recognition of peptides derived from the coupled carrier protein. The covalent attachment of polysaccharide antigens to the carrier protein is thought to be imperative to the immunological properties of conjugate vaccines. Here we provide evidence that covalent attachment to carrier proteins is not required for conversion of T-independent antigens into T-dependent immunogens. Simple entrapment of polysaccharides or a d-amino acid polymer antigen in a cross-linked protein matrix was shown to be sufficient to produce potent immunogens that possess the key characteristics of conventional conjugate vaccines. The versatility and ease of manufacture of these antigen preparations, termed protein capsular matrix vaccines (PCMVs), will likely provide improvements in the manufacture of vaccines designed to protect against encapsulated microorganisms. This in turn could improve the availability of such vaccines to the developing world, which has shown only a limited capacity to afford the cost of conventional conjugate vaccines.

  7. The RtcB RNA ligase is an essential component of the metazoan unfolded protein response.

    PubMed

    Kosmaczewski, Sara Guckian; Edwards, Tyson James; Han, Sung Min; Eckwahl, Matthew J; Meyer, Benjamin Isaiah; Peach, Sally; Hesselberth, Jay R; Wolin, Sandra L; Hammarlund, Marc

    2014-12-01

    RNA ligation can regulate RNA function by altering RNA sequence, structure and coding potential. For example, the function of XBP1 in mediating the unfolded protein response requires RNA ligation, as does the maturation of some tRNAs. Here, we describe a novel in vivo model in Caenorhabditis elegans for the conserved RNA ligase RtcB and show that RtcB ligates the xbp-1 mRNA during the IRE-1 branch of the unfolded protein response. Without RtcB, protein stress results in the accumulation of unligated xbp-1 mRNA fragments, defects in the unfolded protein response, and decreased lifespan. RtcB also ligates endogenous pre-tRNA halves, and RtcB mutants have defects in growth and lifespan that can be bypassed by expression of pre-spliced tRNAs. In addition, animals that lack RtcB have defects that are independent of tRNA maturation and the unfolded protein response. Thus, RNA ligation by RtcB is required for the function of multiple endogenous target RNAs including both xbp-1 and tRNAs. RtcB is uniquely capable of performing these ligation functions, and RNA ligation by RtcB mediates multiple essential processes in vivo. © 2014 The Authors.

  8. Structure and Calcium Binding Properties of a Neuronal Calcium-Myristoyl Switch Protein, Visinin-Like Protein 3.

    PubMed

    Li, Congmin; Lim, Sunghyuk; Braunewell, Karl H; Ames, James B

    2016-01-01

    Visinin-like protein 3 (VILIP-3) belongs to a family of Ca2+-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca2+ binding, characterize Ca2+-induced conformational changes, and determine the NMR structure of myristoylated VILIP-3. Three Ca2+ bind cooperatively to VILIP-3 at EF2, EF3 and EF4 (KD = 0.52 μM and Hill slope of 1.8). NMR assignments, mutagenesis and structural analysis indicate that the covalently attached myristoyl group is solvent exposed in Ca2+-bound VILIP-3, whereas Ca2+-free VILIP-3 contains a sequestered myristoyl group that interacts with protein residues (E26, Y64, V68), which are distinct from myristate contacts seen in other Ca2+-myristoyl switch proteins. The myristoyl group in VILIP-3 forms an unusual L-shaped structure that places the C14 methyl group inside a shallow protein groove, in contrast to the much deeper myristoyl binding pockets observed for recoverin, NCS-1 and GCAP1. Thus, the myristoylated VILIP-3 protein structure determined in this study is quite different from those of other known myristoyl switch proteins (recoverin, NCS-1, and GCAP1). We propose that myristoylation serves to fine tune the three-dimensional structures of neuronal calcium sensor proteins as a means of generating functional diversity.

  9. Taste sensitivity for monosodium glutamate and an increased liking of dietary protein.

    PubMed

    Luscombe-Marsh, Natalie D; Smeets, Astrid J P G; Westerterp-Plantenga, Margriet S

    2008-04-01

    The aim of the present study was to determine individuals' taste threshold for monosodium glutamate (MSG) alone and in combination with inosine 5'-monophosphate (IMP-5) and to examine if this threshold was related to an increase in sensory properties (including pleasantness of taste) and/or to one's preference for dietary protein over carbohydrate and fat. Using the triangle tasting method, the taste threshold was determined for thirty-six women and twenty-four men. Thresholds varied from zero to infinite as determined using a clear soup with added MSG in the concentration range of 0.1 to 0.8 % (w/w) MSG. Subjects rated fourteen sensory properties of the soup and also their 'liking', 'eating frequency' and 'preference' of twenty-two common high-protein, high-carbohydrate and high-fat food items. The taste threshold (and therefore sensitivity) of MSG was lowered from 0.33 (sem 0.24) to 0.26 (sem 0.22) % MSG when 0.25 % (w/w) IMP-5 was added. None of the sensory properties assessed was associated with the taste threshold of MSG +/- 0.25 % IMP-5 in the overall study population. However, the taste descriptor 'meatiness' was associated with the threshold data for individuals who could taste concentrations of Liking' and 'preference' scores for protein were found to be related to the threshold of MSG +/- 0.25 % IMP-5. From this study population we conclude that the taste threshold of MSG in combination with IMP-5 does appear to predict one's 'liking' of as well as 'preference' for high-protein foods.

  10. Parvovirus B19 induced lupus-like syndrome with nephritis.

    PubMed

    Georges, Elodie; Rihova, Zuzana; Cmejla, Radek; Decleire, Pierre-Yves; Langen, Corinne

    2016-12-01

    We report a case of a 65-year-old man who developed an acute illness with fever, arthralgia and nephritic syndrome. Antinuclear antibodies were slightly positive and complement levels were low. Renal biopsy showed exudative diffuse proliferative endocapillary glomerulonephritis with diffuse immunoglobulin (IgG, IgA, IgM) and complement deposition (C3d, C4d, C1q) on immunofluorescence. The patient was first treated with corticosteroids and mycophenolate mofetil for suspected lupus with WHO class IV glomerulonephritis. The diagnosis was questioned and a diagnosis of parvovirus B19-associated nephritis was made based on elevation of serum IgM antibodies for parvovirus B19 and detection of parvovirus B19 DNA on renal biopsy. The immunosuppressive treatment was stopped and progressive spontaneous regression of clinical and laboratory abnormalities was observed. We conclude that human parvovirus B19 infection should be considered as a cause of lupus-like symptomatology and acute glomerulonephritis.

  11. Phosducin-like protein: an ethanol-responsive potential modulator of guanine nucleotide-binding protein function.

    PubMed

    Miles, M F; Barhite, S; Sganga, M; Elliott, M

    1993-11-15

    Acute and chronic exposure to ethanol produces specific changes in several signal transduction cascades. Such alterations in signaling are thought to be a crucial aspect of the central nervous system's adaptive response, which occurs with chronic exposure to ethanol. We have recently identified and isolated several genes whose expression is specifically induced by ethanol in neural cell cultures. The product of one of these genes has extensive sequence homology to phosducin, a phosphoprotein expressed in retina and pineal gland that modulates trimeric guanine nucleotide-binding protein (G protein) function by binding to G-protein beta gamma subunits. We identified from a rat brain cDNA library an isolate encoding the phosducin-like protein (PhLP), which has 41% identity and 65% amino acid homology to phosducin. PhLP cDNA is expressed in all tissues screened by RNA blot-hybridization analysis and shows marked evolutionary conservation on Southern hybridization. We have identified four forms of PhLP cDNA varying only in their 5' ends, probably due to alternative splicing. This 5'-end variation generates two predicted forms of PhLP protein that differ by 79 aa at the NH2 terminus. Treatment of NG108-15 cells for 24 hr with concentrations of ethanol seen in actively drinking alcoholics (25-100 mM) causes up to a 3-fold increase in PhLP mRNA levels. Induction of PhLP by ethanol could account for at least some of the widespread alterations in signal transduction and G-protein function that are known to occur with chronic exposure to ethanol.

  12. Immunolocalization of an annexin-like protein in corn

    NASA Astrophysics Data System (ADS)

    Clark, G. B.; Dauwalder, M.; Roux, S. J.

    1994-08-01

    Although calcium has been proposed to be an important regulatory element in plant gravitropic growth, as yet no specific function of Ca2+ in growth regulation has been discovered. Our recent studies on a Ca2+-binding protein in pea seedlings called p35 indicate that it is a member of the annexin family of proteins and may play a key role in growth regulation through its function in delivering polysaccharides needed for wall construction. We previously reported the isolation of p35 from pea plumules and the production of polyclonal antibodies to it. Immunolocalization analyses of p35 in pea tissues revealed high levels of staining in secretory cell types such as developing vascular cells and outer root cap cells. To test how general was the occurrence and distribution of this annexin-like protein in plant cells we initiated an analysis of annexins in the monocot corn using immunological techniques. Our results indicate the immunochemical properties and localization of corn annexins are very similar to those reported for pea. They are consistent with the postulate that annexins may play a general role in the regulation of the secretion of wall polysaccharides needed for growth, and thus could be an important target of calcium action during gravitropic growth.

  13. Immunolocalization of an annexin-like protein in corn

    NASA Technical Reports Server (NTRS)

    Clark, G. B.; Dauwalder, M.; Roux, S. J.

    1994-01-01

    Although calcium has been proposed to be an important regulatory element in plant gravitropic growth, as yet no specific function of Ca2(+) in growth regulation has been discovered. Our recent studies on a Ca2(+)-binding protein in pea seedlings called p35 indicate that it is a member of the annexin family of proteins and may play a key role in growth regulation through its function in delivering polysaccharides needed for wall construction. We previously reported the isolation of p35 from pea plumules and the production of polyclonal antibodies to it. Immunolocalization analyses of p35 in pea tissues revealed high levels of staining in secretory cell types such as developing vascular cells and outer root cap cells. To test how general was the occurrence and distribution of this annexin-like protein in plant cells we initiated an analysis of annexins in the monocot corn using immunological techniques. Our results indicate the immunochemical properties and localization of corn annexins are very similar to those reported for pea. They are consistent with the postulate that annexins may play a general role in the regulation of the secretion of wall polysaccharides needed for growth, and thus could be an important target of calcium action during gravitropic growth.

  14. Topology of transmembrane channel-like gene 1 protein.

    PubMed

    Labay, Valentina; Weichert, Rachel M; Makishima, Tomoko; Griffith, Andrew J

    2010-10-05

    Mutations of transmembrane channel-like gene 1 (TMC1) cause hearing loss in humans and mice. TMC1 is the founding member of a family of genes encoding proteins of unknown function that are predicted to contain multiple transmembrane domains. The goal of our study was to define the topology of mouse TMC1 expressed heterologously in tissue culture cells. TMC1 was retained in the endoplasmic reticulum (ER) membrane of five tissue culture cell lines that we tested. We used anti-TMC1 and anti-HA antibodies to probe the topologic orientation of three native epitopes and seven HA epitope tags along full-length TMC1 after selective or complete permeabilization of transfected cells with digitonin or Triton X-100, respectively. TMC1 was present within the ER as an integral membrane protein containing six transmembrane domains and cytosolic N- and C-termini. There is a large cytoplasmic loop, between the fourth and fifth transmembrane domains, with two highly conserved hydrophobic regions that might associate with or penetrate, but do not span, the plasma membrane. Our study is the first to demonstrate that TMC1 is a transmembrane protein. The topologic organization revealed by this study shares some features with that of the shaker-TRP superfamily of ion channels.

  15. Detection of the Assembly and Disassembly of PCV2b Virus-Like Particles Using Fluorescence Spectroscopy Analysis.

    PubMed

    Fang, Mingli; Diao, Wenzhen; Dong, Boqi; Wei, Hongfei; Liu, Jialin; Hua, Li; Zhang, Miaomin; Guo, Sheng; Xiao, Yue; Yu, Yongli; Wang, Liying; Wan, Min

    2015-01-01

    Monitoring the assembly and disassembly of virus-like particles (VLPs) is important in developing effective VLP-based vaccines. We tried to establish a simple and rapid method to evaluate the status of VLP assembly using fluorescence spectroscopic analysis (FSA) while developing a VLP-based vaccine against porcine circovirus type 2b (PCV2b). We synthesized the gene coding for PCV2b capsid protein (CP). The CP was expressed in Escherichia coli in a soluble form, dialyzed into three different buffers, and assembled into VLPs. The immunogenicity of the VLPs was evaluated by an enzyme-linked immunosorbent assay using the sera of mice immunized with inactivated PCV2b. The VLP assembly was detected using transmission electron microscopy and FSA. The assembled VLPs showed a distinct FSA curve with a peak at 320 nm. We found that the assembly status was related to the immunogenicity, fluorescence intensity, and morphology of the VLP. The FSA assay was able to monitor the various denatured statuses of PCV2b VLPs treated with β-mercaptoethanol or β-mercaptoethanol plus urea. We have demonstrated that FSA can be used to detect the assembly of PCV2b VLPs produced in E. coli. This provides a simple solution for monitoring VLP assembly during the production of VLP-based vaccines. © 2016 S. Karger AG, Basel.

  16. The PBII gene of the human salivary proline-rich protein P-B produces another protein, Q504X8, with an opiorphin homolog, QRGPR.

    PubMed

    Saitoh, Eiichi; Sega, Takuya; Imai, Akane; Isemura, Satoko; Kato, Tetsuo; Ochiai, Akihito; Taniguchi, Masayuki

    2018-04-01

    The NCBI gene database and human-transcriptome database for alternative splicing were used to determine the expression of mRNAs for P-B (SMR3B) and variant form of P-B. The translational product from the former mRNA was identified as the protein named P-B, whereas that from the latter has not yet been elucidated. In the present study, we investigated the expression of P-B and its variant form at the protein level. To identify the variant protein of P-B, (1) cationic proteins with a higher isoelectric point in human pooled whole saliva were purified by a two dimensional liquid chromatography; (2) the peptide fragments generated from the in-solution of all proteins digested with trypsin separated and analyzed by MALDI-TOF-MS; and (3) the presence or absence of P-B in individual saliva was examined by 15% SDS-PAGE. The peptide sequences (I 37 PPPYSCTPNMNNCSR 52 , C 53 HHHHKRHHYPCNYCFCYPK 72 , R 59 HHYPCNYCFCYPK 72 and H 60 HYPCNYCFCYPK 72 ) present in the variant protein of P-B were identified. The peptide sequence (G 6 PYPPGPLAPPQPFGPGFVPPPPPPPYGPGR 36 ) in P-B (or the variant) and sequence (I 37 PPPPPAPYGPGIFPPPPPQP 57 ) in P-B were identified. The sum of the sequences identified indicated a 91.23% sequence identity for P-B and 79.76% for the variant. There were cases in which P-B existed in individual saliva, but there were cases in which it did not exist in individual saliva. The variant protein is produced by excising a non-canonical intron (CC-AC pair) from the 3'-noncoding sequence of the PBII gene. Both P-B and the variant are subject to proteolysis in the oral cavity. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. African swine fever virus IAP-like protein induces the activation of nuclear factor kappa B.

    PubMed

    Rodríguez, Clara I; Nogal, María L; Carrascosa, Angel L; Salas, María L; Fresno, Manuel; Revilla, Yolanda

    2002-04-01

    African swine fever virus (ASFV) encodes a homologue of the inhibitor of apoptosis (IAP) that promotes cell survival by controlling the activity of caspase-3. Here we show that ASFV IAP is also able to activate the transcription factor NF-kappaB. Thus, transient transfection of the viral IAP increases the activity of an NF-kappaB reporter gene in a dose-responsive manner in Jurkat cells. Similarly, stably transfected cells expressing ASFV IAP have elevated basal levels of c-rel, an NF-kappaB-dependent gene. NF-kappaB complexes in the nucleus were increased in A224L-expressing cells compared with control cells upon stimulation with phorbol myristate acetate (PMA) plus ionomycin. This resulted in greater NF-kappaB-dependent promoter activity in ASFV IAP-expressing than in control cells, both in basal conditions and after PMA plus ionophore stimulation. The elevated NF-kappaB activity seems to be the consequence of higher IkappaB kinase (IKK) basal activity in these cells. The NF-kappaB-inducing activity of ASFV IAP was abrogated by an IKK-2 dominant negative mutant and enhanced by expression of tumor necrosis factor receptor-associated factor 2.

  18. African Swine Fever Virus IAP-Like Protein Induces the Activation of Nuclear Factor Kappa B

    PubMed Central

    Rodríguez, Clara I.; Nogal, María L.; Carrascosa, Angel L.; Salas, María L.; Fresno, Manuel; Revilla, Yolanda

    2002-01-01

    African swine fever virus (ASFV) encodes a homologue of the inhibitor of apoptosis (IAP) that promotes cell survival by controlling the activity of caspase-3. Here we show that ASFV IAP is also able to activate the transcription factor NF-κB. Thus, transient transfection of the viral IAP increases the activity of an NF-κB reporter gene in a dose-responsive manner in Jurkat cells. Similarly, stably transfected cells expressing ASFV IAP have elevated basal levels of c-rel, an NF-κB-dependent gene. NF-κB complexes in the nucleus were increased in A224L-expressing cells compared with control cells upon stimulation with phorbol myristate acetate (PMA) plus ionomycin. This resulted in greater NF-κB-dependent promoter activity in ASFV IAP-expressing than in control cells, both in basal conditions and after PMA plus ionophore stimulation. The elevated NF-κB activity seems to be the consequence of higher IκB kinase (IKK) basal activity in these cells. The NF-κB-inducing activity of ASFV IAP was abrogated by an IKK-2 dominant negative mutant and enhanced by expression of tumor necrosis factor receptor-associated factor 2. PMID:11907233

  19. The nanoscale spatial organization of B-cell receptors on immunoglobulin M- and G-expressing human B-cells.

    PubMed

    Lee, Jinmin; Sengupta, Prabuddha; Brzostowski, Joseph; Lippincott-Schwartz, Jennifer; Pierce, Susan K

    2017-02-15

    B-cell activation is initiated by the binding of antigen to the B-cell receptor (BCR). Here we used dSTORM superresolution imaging to characterize the nanoscale spatial organization of immunoglobulin M (IgM) and IgG BCRs on the surfaces of resting and antigen--activated human peripheral blood B-cells. We provide insights into both the fundamental process of antigen-driven BCR clustering and differences in the spatial organization of IgM and IgG BCRs that may contribute to the characteristic differences in the responses of naive and memory B-cells to antigen. We provide evidence that although both IgM and IgG BCRs reside in highly heterogeneous protein islands that vary in size and number of BCR single-molecule localizations, both resting and activated B-cells intrinsically maintain a high -frequency of single isolated BCR localizations, which likely represent BCR monomers. IgG BCRs are more clustered than IgM BCRs on resting cells and form larger protein islands after antigen activation. Small, dense BCR clusters likely formed via protein-protein interactions are present on the surface of resting cells, and antigen activation induces these to come together to form less dense, larger islands, a process likely governed, at least in part, by protein-lipid interactions. © 2017 Lee, Sengupta, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  20. Chlorophyll a/b-binding proteins, pigment conversions, and early light-induced proteins in a chlorophyll b-less barley mutant.

    PubMed Central

    Król, M; Spangfort, M D; Huner, N P; Oquist, G; Gustafsson, P; Jansson, S

    1995-01-01

    Monospecific polyclonal antibodies have been raised against synthetic peptides derived from the primary sequences from different plant light-harvesting Chl a/b-binding (LHC) proteins. Together with other monospecific antibodies, these were used to quantify the levels of the 10 different LHC proteins in wild-type and chlorina f2 barley (Hordeum vulgare L.), grown under normal and intermittent light (ImL). Chlorina f2, grown under normal light, lacked Lhcb1 (type I LHC II) and Lhcb6 (CP24) and had reduced amounts of Lhcb2, Lhcb3 (types II and III LHC II), and Lhcb4 (CP 29). Chlorina f2 grown under ImL lacked all LHC proteins, whereas wild-type ImL plants contained Lhcb5 (CP 26) and a small amount of Lhcb2. The chlorina f2 ImL thylakoids were organized in large parallel arrays, but wild-type ImL thylakoids had appressed regions, indicating a possible role for Lhcb5 in grana stacking. Chlorina f2 grown under ImL contained considerable amounts of violaxanthin (2-3/reaction center), representing a pool of phototransformable xanthophyll cycle pigments not associated with LHC proteins. Chlorina f2 and the plants grown under ImL also contained early light-induced proteins (ELIPs) as monitored by western blotting. The levels of both ELIPs and xanthophyll cycle pigments increased during a 1 h of high light treatment, without accumulation of LHC proteins. These data are consistent with the hypothesis that ELIPs are pigment-binding proteins, and we suggest that ELIPs bind photoconvertible xanthophylls and replace "normal" LHC proteins under conditions of light stress. PMID:7748263

  1. 2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1.

    PubMed

    Dhar, Jayeeta; Cuevas, Rolando A; Goswami, Ramansu; Zhu, Jianzhong; Sarkar, Saumendra N; Barik, Sailen

    2015-10-01

    2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. The PPE18 protein of Mycobacterium tuberculosis inhibits NF-κB/rel-mediated proinflammatory cytokine production by upregulating and phosphorylating suppressor of cytokine signaling 3 protein.

    PubMed

    Nair, Shiny; Pandey, Akhilesh Datt; Mukhopadhyay, Sangita

    2011-05-01

    Mycobacterium tuberculosis bacteria are known to suppress proinflammatory cytokines like IL-12 and TNF-α for a biased Th2 response that favors a successful infection and its subsequent intracellular survival. However, the signaling pathways targeted by the bacilli to inhibit production of these cytokines are not fully understood. In this study, we demonstrate that the PPE18 protein of M. tuberculosis inhibits LPS-induced IL-12 and TNF-α production by blocking nuclear translocation of p50, p65 NF-κB, and c-rel transcription factors. We found that PPE18 upregulates the expression as well as tyrosine phosphorylation of suppressor of cytokine signaling 3 (SOCS3), and the phosphorylated SOCS3 physically interacts with IκBα-NF-κB/rel complex, inhibiting phosphorylation of IκBα at the serine 32/36 residues by IκB kinase-β, and thereby prevents nuclear translocation of the NF-κB/rel subunits in LPS-activated macrophages. Specific knockdown of SOCS3 by small interfering RNA enhanced IκBα phosphorylation, leading to increased nuclear levels of NF-κB/rel transcription factors vis-a-vis IL-12 p40 and TNF-α production in macrophages cotreated with PPE18 and LPS. The PPE18 protein did not affect the IκB kinase-β activity. Our study describes a novel mechanism by which phosphorylated SOCS3 inhibits NF-κB activation by masking the phosphorylation site of IκBα. Also, this study highlights the possible mechanisms by which the M. tuberculosis suppresses production of proinflammatory cytokines using PPE18.

  3. Like-charged protein-polyelectrolyte complexation driven by charge patches

    NASA Astrophysics Data System (ADS)

    Yigit, Cemil; Heyda, Jan; Ballauff, Matthias; Dzubiella, Joachim

    2015-08-01

    We study the pair complexation of a single, highly charged polyelectrolyte (PE) chain (of 25 or 50 monomers) with like-charged patchy protein models (CPPMs) by means of implicit-solvent, explicit-salt Langevin dynamics computer simulations. Our previously introduced set of CPPMs embraces well-defined zero-, one-, and two-patched spherical globules each of the same net charge and (nanometer) size with mono- and multipole moments comparable to those of globular proteins with similar size. We observe large binding affinities between the CPPM and the like-charged PE in the tens of the thermal energy, kBT, that are favored by decreasing salt concentration and increasing charge of the patch(es). Our systematic analysis shows a clear correlation between the distance-resolved potentials of mean force, the number of ions released from the PE, and CPPM orientation effects. In particular, we find a novel two-site binding behavior for PEs in the case of two-patched CPPMs, where intermediate metastable complex structures are formed. In order to describe the salt-dependence of the binding affinity for mainly dipolar (one-patched) CPPMs, we introduce a combined counterion-release/Debye-Hückel model that quantitatively captures the essential physics of electrostatic complexation in our systems.

  4. Bacterial-like PPP protein phosphatases: novel sequence alterations in pathogenic eukaryotes and peculiar features of bacterial sequence similarity.

    PubMed

    Kerk, David; Uhrig, R Glen; Moorhead, Greg B

    2013-01-01

    Reversible phosphorylation is a widespread modification affecting the great majority of eukaryotic cellular proteins, and whose effects influence nearly every cellular function. Protein phosphatases are increasingly recognized as exquisitely regulated contributors to these changes. The PPP (phosphoprotein phosphatase) family comprises enzymes, which catalyze dephosphorylation at serine and threonine residues. Nearly a decade ago, "bacterial-like" enzymes were recognized with similarity to proteins from various bacterial sources: SLPs (Shewanella-like phosphatases), RLPHs (Rhizobiales-like phosphatases), and ALPHs (ApaH-like phosphatases). A recent article from our laboratory appearing in Plant Physiology characterizes their extensive organismal distribution, abundance in plant species, predicted subcellular localization, motif organization, and sequence evolution. One salient observation is the distinct evolutionary trajectory followed by SLP genes and proteins in photosynthetic eukaryotes vs. animal and plant pathogens derived from photosynthetic ancestors. We present here a closer look at sequence data that emphasizes the distinctiveness of pathogen SLP proteins and that suggests that they might represent novel drug targets. A second observation in our original report was the high degree of similarity between the bacterial-like PPPs of eukaryotes and closely related proteins of the "eukaryotic-like" phyla Myxococcales and Planctomycetes. We here reflect on the possible implications of these observations and their importance for future research.

  5. A Unique Set of the Burkholderia Collagen-Like Proteins Provides Insight into Pathogenesis, Genome Evolution and Niche Adaptation, and Infection Detection.

    PubMed

    Bachert, Beth A; Choi, Soo J; Snyder, Anna K; Rio, Rita V M; Durney, Brandon C; Holland, Lisa A; Amemiya, Kei; Welkos, Susan L; Bozue, Joel A; Cote, Christopher K; Berisio, Rita; Lukomski, Slawomir

    2015-01-01

    Burkholderia pseudomallei and Burkholderia mallei, classified as category B priority pathogens, are significant human and animal pathogens that are highly infectious and broad-spectrum antibiotic resistant. Currently, the pathogenicity mechanisms utilized by Burkholderia are not fully understood, and correct diagnosis of B. pseudomallei and B. mallei infection remains a challenge due to limited detection methods. Here, we provide a comprehensive analysis of a set of 13 novel Burkholderia collagen-like proteins (Bucl) that were identified among B. pseudomallei and B. mallei select agents. We infer that several Bucl proteins participate in pathogenesis based on their noncollagenous domains that are associated with the components of a type III secretion apparatus and membrane transport systems. Homology modeling of the outer membrane efflux domain of Bucl8 points to a role in multi-drug resistance. We determined that bucl genes are widespread in B. pseudomallei and B. mallei; Fischer's exact test and Cramer's V2 values indicate that the majority of bucl genes are highly associated with these pathogenic species versus nonpathogenic B. thailandensis. We designed a bucl-based quantitative PCR assay which was able to detect B. pseudomallei infection in a mouse with a detection limit of 50 CFU. Finally, chromosomal mapping and phylogenetic analysis of bucl loci revealed considerable genomic plasticity and adaptation of Burkholderia spp. to host and environmental niches. In this study, we identified a large set of phylogenetically unrelated bucl genes commonly found in Burkholderia select agents, encoding predicted pathogenicity factors, detection targets, and vaccine candidates.

  6. A Unique Set of the Burkholderia Collagen-Like Proteins Provides Insight into Pathogenesis, Genome Evolution and Niche Adaptation, and Infection Detection

    PubMed Central

    Bachert, Beth A.; Choi, Soo J.; Snyder, Anna K.; Rio, Rita V. M.; Durney, Brandon C.; Holland, Lisa A.; Amemiya, Kei; Welkos, Susan L.; Bozue, Joel A.; Cote, Christopher K.; Berisio, Rita; Lukomski, Slawomir

    2015-01-01

    Burkholderia pseudomallei and Burkholderia mallei, classified as category B priority pathogens, are significant human and animal pathogens that are highly infectious and broad-spectrum antibiotic resistant. Currently, the pathogenicity mechanisms utilized by Burkholderia are not fully understood, and correct diagnosis of B. pseudomallei and B. mallei infection remains a challenge due to limited detection methods. Here, we provide a comprehensive analysis of a set of 13 novel Burkholderia collagen-like proteins (Bucl) that were identified among B. pseudomallei and B. mallei select agents. We infer that several Bucl proteins participate in pathogenesis based on their noncollagenous domains that are associated with the components of a type III secretion apparatus and membrane transport systems. Homology modeling of the outer membrane efflux domain of Bucl8 points to a role in multi-drug resistance. We determined that bucl genes are widespread in B. pseudomallei and B. mallei; Fischer’s exact test and Cramer’s V2 values indicate that the majority of bucl genes are highly associated with these pathogenic species versus nonpathogenic B. thailandensis. We designed a bucl-based quantitative PCR assay which was able to detect B. pseudomallei infection in a mouse with a detection limit of 50 CFU. Finally, chromosomal mapping and phylogenetic analysis of bucl loci revealed considerable genomic plasticity and adaptation of Burkholderia spp. to host and environmental niches. In this study, we identified a large set of phylogenetically unrelated bucl genes commonly found in Burkholderia select agents, encoding predicted pathogenicity factors, detection targets, and vaccine candidates. PMID:26356298

  7. B23/nucleophosmin interacts with bovine immunodeficiency virus Rev protein and facilitates viral replication.

    PubMed

    Passos-Castilho, Ana Maria; Marchand, Claude; Archambault, Denis

    2018-02-01

    The bovine immunodeficiency virus (BIV) Rev shuttling protein contains nuclear/nucleolar localization signals and nuclear import/export mechanisms that are novel among lentivirus Rev proteins. Several viral proteins localize to the nucleolus, which may play a role in processes that are essential to the outcome of viral replication. Although BIV Rev localizes to the nucleoli of transfected/infected cells and colocalizes with one of its major proteins, nucleophosmin (NPM1, also known as B23), the role of the nucleolus and B23 in BIV replication remains to be determined. Here, we demonstrate for the first time that BIV Rev interacts with nucleolar phosphoprotein B23 in cells. Using small interfering RNA (siRNA) technology, we show that depletion of B23 expression inhibits virus production by BIV-infected cells, indicating that B23 plays an important role in BIV replication. The interaction between Rev and B23 may represent a potential new target for the development of antiviral drugs against lentiviruses. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Interactions of RadB, a DNA repair protein in archaea, with DNA and ATP.

    PubMed

    Guy, Colin P; Haldenby, Sam; Brindley, Amanda; Walsh, David A; Briggs, Geoffrey S; Warren, Martin J; Allers, Thorsten; Bolt, Edward L

    2006-04-21

    The RecA family of recombinases (RecA, Rad51, RadA and UvsX) catalyse strand-exchange between homologous DNA molecules by utilising conserved DNA-binding modules and a common core ATPase domain. RadB was identified in archaea as a Rad51-like protein on the basis of conserved ATPase sequences. However, RadB does not catalyse strand exchange and does not turn over ATP efficiently. RadB does bind DNA, and here we report a triplet of residues (Lys-His-Arg) that is highly conserved at the RadB C terminus, and is crucial for DNA binding. This is consistent with the motif forming a "basic patch" of highly conserved residues identified in an atomic structure of RadB from Thermococcus kodakaraensis. As the triplet motif is conserved at the C terminus of XRCC2 also, a mammalian Rad51-paralogue, we present a phylogenetic analysis that clarifies the relationship between RadB, Rad51-paralogues and recombinases. We investigate interactions between RadB and ATP using genetics and biochemistry; ATP binding by RadB is needed to promote survival of Haloferax volcanii after UV irradiation, and ATP, but not other NTPs, induces pronounced conformational change in RadB. This is the first genetic analysis of radB, and establishes its importance for maintaining genome stability in archaea. ATP-induced conformational change in RadB may explain previous reports that RadB controls Holliday junction resolution by Hjc, depending on the presence or the absence of ATP.

  9. Apoplastic Venom Allergen-like Proteins of Cyst Nematodes Modulate the Activation of Basal Plant Innate Immunity by Cell Surface Receptors

    PubMed Central

    Lozano-Torres, Jose L.; Wilbers, Ruud H. P.; Warmerdam, Sonja; Finkers-Tomczak, Anna; Diaz-Granados, Amalia; van Schaik, Casper C.; Helder, Johannes; Bakker, Jaap; Goverse, Aska; Schots, Arjen; Smant, Geert

    2014-01-01

    Despite causing considerable damage to host tissue during the onset of parasitism, nematodes establish remarkably persistent infections in both animals and plants. It is thought that an elaborate repertoire of effector proteins in nematode secretions suppresses damage-triggered immune responses of the host. However, the nature and mode of action of most immunomodulatory compounds in nematode secretions are not well understood. Here, we show that venom allergen-like proteins of plant-parasitic nematodes selectively suppress host immunity mediated by surface-localized immune receptors. Venom allergen-like proteins are uniquely conserved in secretions of all animal- and plant-parasitic nematodes studied to date, but their role during the onset of parasitism has thus far remained elusive. Knocking-down the expression of the venom allergen-like protein Gr-VAP1 severely hampered the infectivity of the potato cyst nematode Globodera rostochiensis. By contrast, heterologous expression of Gr-VAP1 and two other venom allergen-like proteins from the beet cyst nematode Heterodera schachtii in plants resulted in the loss of basal immunity to multiple unrelated pathogens. The modulation of basal immunity by ectopic venom allergen-like proteins in Arabidopsis thaliana involved extracellular protease-based host defenses and non-photochemical quenching in chloroplasts. Non-photochemical quenching regulates the initiation of the defense-related programmed cell death, the onset of which was commonly suppressed by venom allergen-like proteins from G. rostochiensis, H. schachtii, and the root-knot nematode Meloidogyne incognita. Surprisingly, these venom allergen-like proteins only affected the programmed cell death mediated by surface-localized immune receptors. Furthermore, the delivery of venom allergen-like proteins into host tissue coincides with the enzymatic breakdown of plant cell walls by migratory nematodes. We, therefore, conclude that parasitic nematodes most likely utilize

  10. Apoplastic venom allergen-like proteins of cyst nematodes modulate the activation of basal plant innate immunity by cell surface receptors.

    PubMed

    Lozano-Torres, Jose L; Wilbers, Ruud H P; Warmerdam, Sonja; Finkers-Tomczak, Anna; Diaz-Granados, Amalia; van Schaik, Casper C; Helder, Johannes; Bakker, Jaap; Goverse, Aska; Schots, Arjen; Smant, Geert

    2014-12-01

    Despite causing considerable damage to host tissue during the onset of parasitism, nematodes establish remarkably persistent infections in both animals and plants. It is thought that an elaborate repertoire of effector proteins in nematode secretions suppresses damage-triggered immune responses of the host. However, the nature and mode of action of most immunomodulatory compounds in nematode secretions are not well understood. Here, we show that venom allergen-like proteins of plant-parasitic nematodes selectively suppress host immunity mediated by surface-localized immune receptors. Venom allergen-like proteins are uniquely conserved in secretions of all animal- and plant-parasitic nematodes studied to date, but their role during the onset of parasitism has thus far remained elusive. Knocking-down the expression of the venom allergen-like protein Gr-VAP1 severely hampered the infectivity of the potato cyst nematode Globodera rostochiensis. By contrast, heterologous expression of Gr-VAP1 and two other venom allergen-like proteins from the beet cyst nematode Heterodera schachtii in plants resulted in the loss of basal immunity to multiple unrelated pathogens. The modulation of basal immunity by ectopic venom allergen-like proteins in Arabidopsis thaliana involved extracellular protease-based host defenses and non-photochemical quenching in chloroplasts. Non-photochemical quenching regulates the initiation of the defense-related programmed cell death, the onset of which was commonly suppressed by venom allergen-like proteins from G. rostochiensis, H. schachtii, and the root-knot nematode Meloidogyne incognita. Surprisingly, these venom allergen-like proteins only affected the programmed cell death mediated by surface-localized immune receptors. Furthermore, the delivery of venom allergen-like proteins into host tissue coincides with the enzymatic breakdown of plant cell walls by migratory nematodes. We, therefore, conclude that parasitic nematodes most likely utilize

  11. Morris Water Maze Training in Mice Elevates Hippocampal Levels of Transcription Factors Nuclear Factor (Erythroid-derived 2)-like 2 and Nuclear Factor Kappa B p65

    PubMed Central

    Snow, Wanda M.; Pahlavan, Payam S.; Djordjevic, Jelena; McAllister, Danielle; Platt, Eric E.; Alashmali, Shoug; Bernstein, Michael J.; Suh, Miyoung; Albensi, Benedict C.

    2015-01-01

    Research has identified several transcription factors that regulate activity-dependent plasticity and memory, with cAMP-response element binding protein (CREB) being the most well-studied. In neurons, CREB activation is influenced by the transcription factor nuclear factor kappa B (NF-κB), considered central to immunity but more recently implicated in memory. The transcription factor early growth response-2 (Egr-2), an NF-κB gene target, is also associated with learning and memory. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an antioxidant transcription factor linked to NF-κB in pathological conditions, has not been studied in normal memory. Given that numerous transcription factors implicated in activity-dependent plasticity demonstrate connections to NF-κB, this study simultaneously evaluated protein levels of NF-κB, CREB, Egr-2, Nrf2, and actin in hippocampi from young (1 month-old) weanling CD1 mice after training in the Morris water maze, a hippocampal-dependent spatial memory task. After a 6-day acquisition period, time to locate the hidden platform decreased in the Morris water maze. Mice spent more time in the target vs. non-target quadrants of the maze, suggestive of recall of the platform location. Western blot data revealed a decrease in NF-κB p50 protein after training relative to controls, whereas NF-κB p65, Nrf2 and actin increased. Nrf2 levels were correlated with platform crosses in nearly all tested animals. These data demonstrate that training in a spatial memory task results in alterations in and associations with particular transcription factors in the hippocampus, including upregulation of NF-κB p65 and Nrf2. Training-induced increases in actin protein levels caution against its use as a loading control in immunoblot studies examining activity-dependent plasticity, learning, and memory. PMID:26635523

  12. A novel signal transduction protein: Combination of solute binding and tandem PAS-like sensor domains in one polypeptide chain: Periplasmic Ligand Binding Protein Dret_0059

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wu, R.; Wilton, R.; Cuff, M. E.

    We report the structural and biochemical characterization of a novel periplasmic ligand-binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from the Salt Lake Retba in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N-terminal and a tandem PAS-like sensor domain at the C-terminal region. SBP domains are found ubiquitously and their best known function is in solute transport across membranes. PAS-like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes butmore » have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS-like domain of the tandem PAS-like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS-like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non-redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport.« less

  13. A CheR/CheB fusion protein is involved in cyst cell development and chemotaxis in Azospirillum brasilense Sp7.

    PubMed

    Wu, Lixian; Cui, Yanhua; Hong, Yuanyuan; Chen, Sanfeng

    2011-12-20

    We here report the sequence and functional analysis of cstB of Azospirillum brasilense Sp7. The predicted cstB contains C-terminal two PAS domains and N-terminal part which has similarity with CheB-CheR fusion protein. cstB mutants had reduced swarming ability compared to that of A. brasilense wild-type strain, implying that cstB was involved in chemotaxis in A. brasilense. A microscopic analysis revealed that cstB mutants developed mature cyst cells more quickly than wild type, indicating that cstB is involved in cyst formation. cstB mutants were affected in colony morphology and the production of exopolysaccharides (EPS) which are essential for A. brasilense cells to differentiate into cyst-like forms. These observations suggested that cstB was a multi-effector involved in cyst development and chemotaxis in A. brasilense. Copyright © 2010 Elsevier GmbH. All rights reserved.

  14. Detection of a New cfr-Like Gene, cfr(B), in Enterococcus faecium Isolates Recovered from Human Specimens in the United States as Part of the SENTRY Antimicrobial Surveillance Program.

    PubMed

    Deshpande, Lalitagauri M; Ashcraft, Deborah S; Kahn, Heather P; Pankey, George; Jones, Ronald N; Farrell, David J; Mendes, Rodrigo E

    2015-10-01

    Two linezolid-resistant Enterococcus faecium isolates (MICs, 8 μg/ml) from unique patients of a medical center in New Orleans were included in this study. Isolates were initially investigated for the presence of mutations in the V domain of 23S rRNA genes and L3, L4, and L22 ribosomal proteins, as well as cfr. Isolates were subjected to pulsed-field gel electrophoresis (just one band difference), and one representative strain was submitted to whole-genome sequencing. Gene location was also determined by hybridization, and cfr genes were cloned and expressed in a Staphylococcus aureus background. The two isolates had one out of six 23S rRNA alleles mutated (G2576T), had wild-type L3, L4, and L22 sequences, and were positive for a cfr-like gene. The sequence of the protein encoded by the cfr-like gene was most similar (99.7%) to that found in Peptoclostridium difficile, which shared only 74.9% amino acid identity with the proteins encoded by genes previously identified in staphylococci and non-faecium enterococci and was, therefore, denominated Cfr(B). When expressed in S. aureus, the protein conferred a resistance profile similar to that of Cfr. Two copies of cfr(B) were chromosomally located and embedded in a Tn6218 similar to the cfr-carrying transposon described in P. difficile. This study reports the first detection of cfr genes in E. faecium clinical isolates in the United States and characterization of a new cfr variant, cfr(B). cfr(B) has been observed in mobile genetic elements in E. faecium and P. difficile, suggesting potential for dissemination. However, further analysis is necessary to access the resistance levels conferred by cfr(B) when expressed in enterococci. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. Ionic strength-dependent conformations of a ubiquitin-like small archaeal modifier protein (SAMP1) from Haloferax volcanii

    PubMed Central

    Ye, Kaiqin; Liao, Shanhui; Zhang, Wen; Fan, Kai; Zhang, Xuecheng; Zhang, Jiahai; Xu, Chao; Tu, Xiaoming

    2013-01-01

    Eukaryotic ubiquitin and ubiquitin-like systems play crucial roles in various cellular biological processes. In this work, we determined the solution structure of SAMP1 from Haloferax volcanii by NMR spectroscopy. Under low ionic conditions, SAMP1 presented two distinct conformations, one folded β-grasp and the other disordered. Interestingly, SAMP1 underwent a conformational conversion from disorder to order with ion concentration increasing, indicating that the ordered conformation is the functional form of SAMP1 under the physiological condition of H. volcanii. Furthermore, SAMP1 could interact with proteasome-activating nucleotidase B, supposing a potential role of SAMP1 in the protein degradation pathway mediated by proteasome. PMID:23818097

  16. Cloning and expression of Bartonella henselae sucB gene encoding an immunogenic dihydrolipoamide succinyltransferase homologous protein.

    PubMed

    Kabeya, Hidenori; Maruyama, Soichi; Hirano, Kouji; Mikami, Takeshi

    2003-01-01

    Immunoscreening of a ZAP genomic library of Bartonella henselae strain Houston-1 expressed in Escherichia coli resulted in the isolation of a clone containing 3.5 kb BamHI genomic DNA fragment. This 3.5 kb DNA fragment was found to contain a sequence of a gene encoding a protein with significant homology to the dihydrolipoamide succinyltransferase of Brucella melitensis (sucB). Subsequent cloning and DNA sequence analysis revealed that the deduced amino acid sequence from the cloned gene showed 66.5% identity to SucB protein of B. melitensis, and 43.4 and 47.2% identities to those of Coxiella burnetii and E. coli, respectively. The gene was expressed as a His-Nus A-tagged fusion protein. The recombinant SucB protein (rSucB) was shown to be an immunoreactive protein of about 115 kDa by Western blot analysis with sera from B. henselae-immunized mice. Therefore the rSucB may be a candidate antigen for a specific serological diagnosis of B. henselae infection.

  17. MET-activating Residues in the B-repeat of the Listeria monocytogenes Invasion Protein InlB*

    PubMed Central

    Bleymüller, Willem M.; Lämmermann, Nina; Ebbes, Maria; Maynard, Daniel; Geerds, Christina; Niemann, Hartmut H.

    2016-01-01

    The facultative intracellular pathogen Listeria monocytogenes causes listeriosis, a rare but life-threatening disease. Host cell entry begins with activation of the human receptor tyrosine kinase MET through the bacterial invasion protein InlB, which contains an internalin domain, a B-repeat, and three GW domains. The internalin domain is known to bind MET, but no interaction partner is known for the B-repeat. Adding the B-repeat to the internalin domain potentiates MET activation and is required to stimulate Madin-Darby canine kidney (MDCK) cell scatter. Therefore, it has been hypothesized that the B-repeat may bind a co-receptor on host cells. To test this hypothesis, we mutated residues that might be important for binding an interaction partner. We identified two adjacent residues in strand β2 of the β-grasp fold whose mutation abrogated induction of MDCK cell scatter. Biophysical analysis indicated that these mutations do not alter protein structure. We then tested these mutants in human HT-29 cells that, in contrast to the MDCK cells, were responsive to the internalin domain alone. These assays revealed a dominant negative effect, reducing the activity of a construct of the internalin domain and mutated B-repeat below that of the individual internalin domain. Phosphorylation assays of MET and its downstream targets AKT and ERK confirmed the dominant negative effect. Attempts to identify a host cell receptor for the B-repeat were not successful. We conclude that there is limited support for a co-receptor hypothesis and instead suggest that the B-repeat contributes to MET activation through low affinity homodimerization. PMID:27789707

  18. Novel protein interactions with an actin homolog (MreB) of Helicobacter pylori determined by bacterial two-hybrid system.

    PubMed

    Zepeda Gurrola, Reyna Cristina; Fu, Yajuan; Rodríguez Luna, Isabel Cristina; Benítez Cardoza, Claudia Guadalupe; López López, María de Jesús; López Vidal, Yolanda; Gutíerrez, Germán Rubén Aguilar; Rodríguez Pérez, Mario A; Guo, Xianwu

    2017-08-01

    The bacterium Helicobacter pylori infects more than 50% of the world population and causes several gastroduodenal diseases, including gastric cancer. Nevertheless, we still need to explore some protein interactions that may be involved in pathogenesis. MreB, an actin homolog, showed some special characteristics in previous studies, indicating that it could have different functions. Protein functions could be realized via protein-protein interactions. In the present study, the MreB protein from H. pylori 26695 fused with two tags 10×His and GST in tandem was overexpressed and purified from Escherchia coli. The purified recombinant protein was used to perform a pull-down assay with H. pylori 26695 cell lysate. The pulled-down proteins were identified by mass spectrometry (MALDI-TOF), in which the known important proteins related to morphogenesis were absent but several proteins related to pathogenesis process were observed. The bacterial two-hybrid system was further used to evaluate the protein interactions and showed that new interactions of MreB respectively with VacA, UreB, HydB, HylB and AddA were confirmed but the interaction MreB-MreC was not validated. These results indicated that the protein MreB in H. pylori has a distinct interactome, does not participate in cell morphogenesis via MreB-MreC but could be related to pathogenesis. Copyright © 2017 Elsevier GmbH. All rights reserved.

  19. Proteomic analysis identifies insulin-like growth factor-binding protein-related protein-1 as a podocyte product.

    PubMed

    Matsumoto, Takayuki; Hess, Sonja; Kajiyama, Hiroshi; Sakairi, Toru; Saleem, Moin A; Mathieson, Peter W; Nojima, Yoshihisa; Kopp, Jeffrey B

    2010-10-01

    The podocyte secretory proteome may influence the phenotype of adjacent podocytes, endothelial cells, parietal epithelial cells, and tubular epithelial cells but has not been systematically characterized. We have initiated studies to characterize this proteome, with the goal of further understanding the podocyte cell biology. We cultured differentiated conditionally immortalized human podocytes and subjected the proteins in conditioned medium to mass spectrometry. At a false discovery rate of <3%, we identified 111 candidates from conditioned medium, including 44 proteins that have signal peptides or are described as secreted proteins in the UniProt database. As validation, we confirmed that one of these proteins, insulin-like growth factor-binding protein-related protein-1 (IGFBP-rP1), was expressed in mRNA and protein of cultured podocytes. In addition, transforming growth factor-β1 stimulation increased IGFBP-rP1 in conditioned medium. We analyzed IGFBP-rP1 glomerular expression in a mouse model of human immunodeficiency virus-associated nephropathy. IGFBP-rP1 was absent from podocytes of normal mice and was expressed in podocytes and pseudocrescents of transgenic mice, where it was coexpressed with desmin, a podocyte injury marker. We conclude that IGFBP-rP1 may be a product of injured podocytes. Further analysis of the podocyte secretory proteome may identify biomarkers of podocyte injury.

  20. Use of Vitelline Protein B as a Microencapsulating Additive

    NASA Technical Reports Server (NTRS)

    Ficht, Allison R. (Inventor); Carson, Ken (Inventor); Waite, John Herbert (Inventor); Sheffield, Cynthia (Inventor)

    2017-01-01

    The present invention includes compositions and methods for the use of an encapsulation additive having between about 0.1 to about 30 percent isolated and purified vitelline protein B to provide for mixed and extended release formulations.