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Sample records for coiled-coil interactions contribute

  1. The structure of the GemC1 coiled coil and its interaction with the Geminin family of coiled-coil proteins

    SciTech Connect

    Caillat, Christophe; Fish, Alexander; Pefani, Dafni-Eleftheria; Taraviras, Stavros; Lygerou, Zoi; Perrakis, Anastassis

    2015-10-31

    The GemC1 coiled-coil structure has subtle differences compared with its homologues Geminin and Idas. Co-expression experiments in cells and biophysical stability analysis of the Geminin-family coiled coils suggest that the GemC1 coiled coil alone is unstable. GemC1, together with Idas and Geminin, an important regulator of DNA-replication licensing and differentiation decisions, constitute a superfamily sharing a homologous central coiled-coil domain. To better understand this family of proteins, the crystal structure of a GemC1 coiled-coil domain variant engineered for better solubility was determined to 2.2 Å resolution. GemC1 shows a less typical coiled coil compared with the Geminin homodimer and the Geminin–Idas heterodimer structures. It is also shown that both in vitro and in cells GemC1 interacts with Geminin through its coiled-coil domain, forming a heterodimer that is more stable that the GemC1 homodimer. Comparative analysis of the thermal stability of all of the possible superfamily complexes, using circular dichroism to follow the unfolding of the entire helix of the coiled coil, or intrinsic tryptophan fluorescence of a unique conserved N-terminal tryptophan, shows that the unfolding of the coiled coil is likely to take place from the C-terminus towards the N-terminus. It is also shown that homodimers show a single-state unfolding, while heterodimers show a two-state unfolding, suggesting that the dimer first falls apart and the helices then unfold according to the stability of each protein. The findings argue that Geminin-family members form homodimers and heterodimers between them, and this ability is likely to be important for modulating their function in cycling and differentiating cells.

  2. IMPROVED COILED-COIL DESIGN ENHANCES INTERACTION WITH BCR-ABL AND INDUCES APOPTOSIS

    PubMed Central

    Dixon, Andrew S.; Miller, Geoffrey D.; Bruno, Benjamin J.; Constance, Jonathan E.; Woessner, David W.; Fidler, Trevor P.; Robertson, James C.; Cheatham, Thomas E.; Lim, Carol S.

    2012-01-01

    The oncoprotein Bcr-Abl drives aberrant downstream activity through trans-autophosphorylation of homo-oligomers in chronic myelogenous leukemia (CML).1,2 The formation of Bcr-Abl oligomers is achieved through the coiled-coil domain at the N-terminus of Bcr.3, 4 We have previously reported a modified version of this coiled-coil domain, CCmut2, which exhibits disruption of Bcr-Abl oligomeric complexes and results in decreased proliferation of CML cells and induction of apoptosis.5 A major contributing factor to these enhanced capabilities is the destabilization of the CCmut2 homo-dimers, increasing the availability to interact with and inhibit Bcr-Abl. Here, we included an additional mutation (K39E) that could in turn further destabilize the mutant homo-dimer. Incorporation of this modification into CCmut2 (C38A, S41R, L45D, E48R, Q60E) generated what we termed CCmut3, and resulted in further improvements in the binding properties with the wild-type coiled-coil domain representative of Bcr-Abl. A separate construct containing one revert mutation, CCmut4, did not demonstrate improved oligomeric properties and indicated the importance of the L45D mutation. CCmut3 demonstrated improved oligomerization via a two-hybrid assay as well as through colocalization studies, in addition to showing similar biologic activity as CCmut2. The improved binding between CCmut3 and the Bcr-Abl coiled-coil may be used to redirect Bcr-Abl to alternative subcellular locations with interesting therapeutic implications. PMID:22136227

  3. Accommodation of structural rearrangements in the huntingtin-interacting protein 1 coiled-coil domain

    SciTech Connect

    Wilbur, Jeremy D.; Hwang, Peter K.; Brodsky, Frances M.; Fletterick, Robert J.

    2010-03-01

    Variable packing interaction related to the conformational flexibility within the huntingtin-interacting protein 1 coiled coil domain. Huntingtin-interacting protein 1 (HIP1) is an important link between the actin cytoskeleton and clathrin-mediated endocytosis machinery. HIP1 has also been implicated in the pathogenesis of Huntington’s disease. The binding of HIP1 to actin is regulated through an interaction with clathrin light chain. Clathrin light chain binds to a flexible coiled-coil domain in HIP1 and induces a compact state that is refractory to actin binding. To understand the mechanism of this conformational regulation, a high-resolution crystal structure of a stable fragment from the HIP1 coiled-coil domain was determined. The flexibility of the HIP1 coiled-coil region was evident from its variation from a previously determined structure of a similar region. A hydrogen-bond network and changes in coiled-coil monomer interaction suggest that the HIP1 coiled-coil domain is uniquely suited to allow conformational flexibility.

  4. The structure of the GemC1 coiled coil and its interaction with the Geminin family of coiled-coil proteins

    PubMed Central

    Caillat, Christophe; Fish, Alexander; Pefani, Dafni-Eleftheria; Taraviras, Stavros; Lygerou, Zoi; Perrakis, Anastassis

    2015-01-01

    GemC1, together with Idas and Geminin, an important regulator of DNA-replication licensing and differentiation decisions, constitute a superfamily sharing a homologous central coiled-coil domain. To better understand this family of proteins, the crystal structure of a GemC1 coiled-coil domain variant engineered for better solubility was determined to 2.2 Å resolution. GemC1 shows a less typical coiled coil compared with the Geminin homodimer and the Geminin–Idas heterodimer structures. It is also shown that both in vitro and in cells GemC1 interacts with Geminin through its coiled-coil domain, forming a heterodimer that is more stable that the GemC1 homodimer. Comparative analysis of the thermal stability of all of the possible superfamily complexes, using circular dichroism to follow the unfolding of the entire helix of the coiled coil, or intrinsic tryptophan fluorescence of a unique conserved N-terminal tryptophan, shows that the unfolding of the coiled coil is likely to take place from the C-terminus towards the N-terminus. It is also shown that homodimers show a single-state unfolding, while heterodimers show a two-state unfolding, suggesting that the dimer first falls apart and the helices then unfold according to the stability of each protein. The findings argue that Geminin-family members form homodimers and heterodimers between them, and this ability is likely to be important for modulating their function in cycling and differentiating cells. PMID:26527144

  5. Data-Driven Prediction and Design of bZIP Coiled-Coil Interactions

    PubMed Central

    Potapov, Vladimir; Kaplan, Jenifer B.; Keating, Amy E.

    2015-01-01

    Selective dimerization of the basic-region leucine-zipper (bZIP) transcription factors presents a vivid example of how a high degree of interaction specificity can be achieved within a family of structurally similar proteins. The coiled-coil motif that mediates homo- or hetero-dimerization of the bZIP proteins has been intensively studied, and a variety of methods have been proposed to predict these interactions from sequence data. In this work, we used a large quantitative set of 4,549 bZIP coiled-coil interactions to develop a predictive model that exploits knowledge of structurally conserved residue-residue interactions in the coiled-coil motif. Our model, which expresses interaction energies as a sum of interpretable residue-pair and triplet terms, achieves a correlation with experimental binding free energies of R = 0.68 and significantly out-performs other scoring functions. To use our model in protein design applications, we devised a strategy in which synthetic peptides are built by assembling 7-residue native-protein heptad modules into new combinations. An integer linear program was used to find the optimal combination of heptads to bind selectively to a target human bZIP coiled coil, but not to target paralogs. Using this approach, we designed peptides to interact with the bZIP domains from human JUN, XBP1, ATF4 and ATF5. Testing more than 132 candidate protein complexes using a fluorescence resonance energy transfer assay confirmed the formation of tight and selective heterodimers between the designed peptides and their targets. This approach can be used to make inhibitors of native proteins, or to develop novel peptides for applications in synthetic biology or nanotechnology. PMID:25695764

  6. A coiled-coil interaction mediates cauliflower mosaic virus cell-to-cell movement

    PubMed Central

    Stavolone, Livia; Villani, Maria Elena; Leclerc, Denis; Hohn, Thomas

    2005-01-01

    The function of the virion-associated protein (VAP) of cauliflower mosaic virus (CaMV) has long been only poorly understood. VAP is associated with the virion but is dispensable for virus morphogenesis and replication. It mediates virus transmission by aphids through simultaneous interaction with both the aphid transmission factor and the virion. However, although insect transmission is not fundamental to CaMV survival, VAP is indispensable for spreading the virus infection within the host plant. We used a GST pull-down technique to demonstrate that VAP interacts with the viral movement protein through coiled-coil domains and surface plasmon resonance to measure the interaction kinetics. We mapped the movement protein coiled-coil to the C terminus of the protein and proved that it self-assembles as a trimer. Immunogold labeling/electron microscopy revealed that the VAP and viral movement protein colocalize on CaMV particles within plasmodesmata. These results highlight the multifunctional potential of the VAP protein conferred by its efficient coiled-coil interaction system and show a plant virus possessing a surface-exposed protein (VAP) mediating viral entry into host cells. PMID:15837934

  7. A coiled-coil interaction mediates cauliflower mosaic virus cell-to-cell movement

    NASA Astrophysics Data System (ADS)

    Stavolone, Livia; Villani, Maria Elena; Leclerc, Denis; Hohn, Thomas

    2005-04-01

    The function of the virion-associated protein (VAP) of cauliflower mosaic virus (CaMV) has long been only poorly understood. VAP is associated with the virion but is dispensable for virus morphogenesis and replication. It mediates virus transmission by aphids through simultaneous interaction with both the aphid transmission factor and the virion. However, although insect transmission is not fundamental to CaMV survival, VAP is indispensable for spreading the virus infection within the host plant. We used a GST pull-down technique to demonstrate that VAP interacts with the viral movement protein through coiled-coil domains and surface plasmon resonance to measure the interaction kinetics. We mapped the movement protein coiled-coil to the C terminus of the protein and proved that it self-assembles as a trimer. Immunogold labeling/electron microscopy revealed that the VAP and viral movement protein colocalize on CaMV particles within plasmodesmata. These results highlight the multifunctional potential of the VAP protein conferred by its efficient coiled-coil interaction system and show a plant virus possessing a surface-exposed protein (VAP) mediating viral entry into host cells. movement protein | virion-associated protein | Biacore

  8. Coiled coil interactions for the targeting of liposomes for nucleic acid delivery

    NASA Astrophysics Data System (ADS)

    Oude Blenke, Erik E.; van den Dikkenberg, Joep; van Kolck, Bartjan; Kros, Alexander; Mastrobattista, Enrico

    2016-04-01

    Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes encapsulating a splice correcting oligonucleotide or siRNA. These peptide-functionalized vesicles are highly stable in solution but start to cluster when vesicles modified with complementary peptides are mixed together, demonstrating that the peptides quickly coil and crosslink the vesicles. When one of the peptides was anchored to the cell membrane using a hydrophobic cholesterol anchor, vesicles functionalized with the complementary peptide could be docked to these cells, whereas non-functionalized cells did not show any vesicle tethering. Although the anchored peptides do not have a downstream signaling pathway, microscopy pictures revealed that after four hours, the majority of the docked vesicles were internalized by endocytosis. Finally, for the first time, it was shown that the coiled coil assembly at the interface between the vesicles and the cell membrane induces active uptake and leads to cytosolic delivery of the nucleic acid cargo. Both the siRNA and the splice correcting oligonucleotide were functionally delivered, resulting respectively in the silencing or recovery of luciferase expression in the appropriate cell lines. These results demonstrate that the docking to the cell by coiled coil interaction can induce active uptake and achieve the successful intracellular delivery of otherwise membrane impermeable nucleic acids in a highly specific manner.Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes

  9. Coiled coil interactions for the targeting of liposomes for nucleic acid delivery

    NASA Astrophysics Data System (ADS)

    Oude Blenke, Erik E.; van den Dikkenberg, Joep; van Kolck, Bartjan; Kros, Alexander; Mastrobattista, Enrico

    2016-04-01

    Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes encapsulating a splice correcting oligonucleotide or siRNA. These peptide-functionalized vesicles are highly stable in solution but start to cluster when vesicles modified with complementary peptides are mixed together, demonstrating that the peptides quickly coil and crosslink the vesicles. When one of the peptides was anchored to the cell membrane using a hydrophobic cholesterol anchor, vesicles functionalized with the complementary peptide could be docked to these cells, whereas non-functionalized cells did not show any vesicle tethering. Although the anchored peptides do not have a downstream signaling pathway, microscopy pictures revealed that after four hours, the majority of the docked vesicles were internalized by endocytosis. Finally, for the first time, it was shown that the coiled coil assembly at the interface between the vesicles and the cell membrane induces active uptake and leads to cytosolic delivery of the nucleic acid cargo. Both the siRNA and the splice correcting oligonucleotide were functionally delivered, resulting respectively in the silencing or recovery of luciferase expression in the appropriate cell lines. These results demonstrate that the docking to the cell by coiled coil interaction can induce active uptake and achieve the successful intracellular delivery of otherwise membrane impermeable nucleic acids in a highly specific manner.Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes

  10. The SH3 domain of UNC-89 (obscurin) interacts with paramyosin, a coiled-coil protein, in Caenorhabditis elegans muscle

    PubMed Central

    Qadota, Hiroshi; Mayans, Olga; Matsunaga, Yohei; McMurry, Jonathan L.; Wilson, Kristy J.; Kwon, Grace E.; Stanford, Rachel; Deehan, Kevin; Tinley, Tina L.; Ngwa, Verra M.; Benian, Guy M.

    2016-01-01

    UNC-89 is a giant polypeptide located at the sarcomeric M-line of Caenorhabditis elegans muscle. The human homologue is obscurin. To understand how UNC-89 is localized and functions, we have been identifying its binding partners. Screening a yeast two-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains and resides in thick filament cores. Minimally, this interaction requires UNC-89’s SH3 domain and residues 294–376 of paramyosin and has a KD of ∼1.1 μM. In unc-89 loss-of-function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mislocalized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89’s SH3 is α-helical and lacks prolines. Homology modeling of UNC-89’s SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a “skip residue,” which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity. PMID:27009202

  11. Interactions of HIV-1 Inhibitory Peptide T20 with the gp41 N-HR Coiled Coil*S⃞

    PubMed Central

    Champagne, Kelly; Shishido, Akira; Root, Michael J.

    2009-01-01

    Cellular entry of human immunodeficiency virus type 1 (HIV-1) involves fusion of viral and cellular membranes and is mediated by structural transitions in viral glycoprotein gp41. The antiviral C-peptide T20 targets the gp41 N-terminal heptad repeat region (N-HR), blocking gp41 conformational changes essential for the entry process. To probe the T20 structure-activity relationship, we engineered a molecular mimic of the entire gp41 N-HR coiled coil using the 5-Helix design strategy. T20 bound this artificial protein (denoted 5H-ex) with nanomolar affinity (KD = 30 nm), close to its IC50 concentration (∼3 nm) but much weaker than the affinity of a related inhibitory C-peptide C37 (KD = 0.0007 nm). T20/C37 competitive binding assays confirmed that T20 interacts with the hydrophobic groove on the surface of the N-HR coiled coil outside of a deep pocket region crucial for C37 binding. We used 5H-ex to investigate how the T20 N and C termini contributed to the inhibitor binding activity. Mutating three aromatic residues at the T20 C terminus (WNWF → ANAA) had no effect on affinity, suggesting that these amino acids do not participate in T20 binding to the gp41 N-HR. The results support recent evidence pointing to a different role for these residues in T20 inhibition (Peisajovich, S. G., Gallo, S. A., Blumenthal, R., and Shai, Y. (2003) J. Biol. Chem. 278, 21012–21017; Liu, S., Jing, W., Cheung, B., Lu, H., Sun, J., Yan, X., Niu, J., Farmar, J., Wu, S., and Jiang, S. (2007) J. Biol. Chem. 282, 9612–9620). By contrast, mutations near the T20 N terminus substantially influenced inhibitor binding strength. When Ile was substituted for Thr in the second T20 position, a 40-fold increase in binding affinity was measured (KD = 0.75 nm). The effect of this affinity enhancement on T20 inhibitory potency varied among different viral strains. The original T20 and the higher affinity T20 variant had similar potency against wild type HIV-1. However, the higher affinity T20

  12. Tropomyosin is an interaction partner of the Drosophila coiled coil protein yuri gagarin.

    PubMed

    Texada, Michael J; Simonette, Rebecca A; Deery, William J; Beckingham, Kathleen M

    2011-02-15

    The Drosophila gene yuri gagarin is a complex locus encoding three protein isoform classes that are ubiquitously expressed in the organism. Mutations to the gene affect processes as diverse as gravitactic behavior and spermatogenesis. The larger Yuri isoforms contain extensive coiled-coil regions. Our previous studies indicate that one of the large isoform classes (Yuri-65) is required for formation of specialized F-actin-containing structures generated during spermatogenesis, including the so-called actin "cones" that mediate spermatid individualization. We used the tandem affinity purification of a tagged version of Yuri-65 (the TAP-tagging technique) to identify proteins associated with Yuri-65 in the intact organism. Tropomyosin, primarily as the 284-residue isoform derived from the ubiquitously expressed Tropomyosin 1 gene was thus identified as a major Yuri interaction partner. Co-immunoprecipitation experiments confirmed this interaction. We have established that the stable F-actin cones of spermatogenesis contain Tropomyosin 1 (Tm1) and that in mutant yuri(F64), failure of F-actin cone formation is associated with failure of Tm1 to accumulate at the cone initiation sites. In investigating possible interactions of Tm1 and Yuri in other tissues, we discovered that Tm1 and Yuri frequently colocalize with the endoplasmic reticulum. Tropomyosin has been implicated in actin-mediated membrane trafficking activity in other systems. Our findings suggest that Yuri-Tm1 complexes participate in related functions. PMID:21126519

  13. TROPOMYOSIN IS AN INTERACTION PARTNER OF THE DROSOPHILA COILED COIL PROTEIN YURI GAGARIN

    PubMed Central

    Texada, Michael J.; Simonette, Rebecca A.; Deery, William J.; Beckingham, Kathleen M.

    2011-01-01

    The Drosophila gene yuri gagarin is a complex locus encoding three protein isoform classes that are ubiquitously expressed in the organism. Mutations to the gene affect processes as diverse as gravitactic behavior and spermatogenesis. The larger Yuri isoforms contain extensive coiled-coil regions. Our previous studies indicate that one of the large isoform classes (Yuri-65) is required for formation of specialized F-actin-containing structures generated during spermatogenesis, including the so-called actin “cones” that mediate spermatid individualization. We used tandem affinity purification of a tagged version of Yuri-65 (the TAP-tagging technique) to identify proteins associated with Yuri-65 in the intact organism. Tropomyosin, primarily as the 284-residue isoform derived from the ubiquitously expressed Tropomyosin 1 gene was thus identified as a major Yuri interaction partner. Co-immunoprecipitation experiments confirmed this interaction. We have established that the stable F-actin cones of spermatogenesis contain Tropomyosin 1 (Tm1) and that in mutant yuriF64, failure of F-actin cone formation is associated with failure of Tm1 to accumulate at the cone initiation sites. In investigating possible interactions of Tm1 and Yuri in other tissues, we discovered that Tm1 and Yuri frequently colocalize with the endoplasmic reticulum. Tropomyosin has been implicated in actin-mediated membrane trafficking activity in other systems. Our findings suggest that Yuri-Tm1 complexes participate in related functions. PMID:21126519

  14. Crystal Structure of Cytomegalovirus IE1 Protein Reveals Targeting of TRIM Family Member PML via Coiled-Coil Interactions

    PubMed Central

    Sevvana, Madhumati; Otto, Victoria; Schilling, Eva-Maria; Stump, Joachim D.; Müller, Regina; Reuter, Nina; Sticht, Heinrich; Muller, Yves A.; Stamminger, Thomas

    2014-01-01

    PML nuclear bodies (PML-NBs) are enigmatic structures of the cell nucleus that act as key mediators of intrinsic immunity against viral pathogens. PML itself is a member of the E3-ligase TRIM family of proteins that regulates a variety of innate immune signaling pathways. Consequently, viruses have evolved effector proteins to modify PML-NBs; however, little is known concerning structure-function relationships of viral antagonists. The herpesvirus human cytomegalovirus (HCMV) expresses the abundant immediate-early protein IE1 that colocalizes with PML-NBs and induces their dispersal, which correlates with the antagonization of NB-mediated intrinsic immunity. Here, we delineate the molecular basis for this antagonization by presenting the first crystal structure for the evolutionary conserved primate cytomegalovirus IE1 proteins. We show that IE1 consists of a globular core (IE1CORE) flanked by intrinsically disordered regions. The 2.3 Å crystal structure of IE1CORE displays an all α-helical, femur-shaped fold, which lacks overall fold similarity with known protein structures, but shares secondary structure features recently observed in the coiled-coil domain of TRIM proteins. Yeast two-hybrid and coimmunoprecipitation experiments demonstrate that IE1CORE binds efficiently to the TRIM family member PML, and is able to induce PML deSUMOylation. Intriguingly, this results in the release of NB-associated proteins into the nucleoplasm, but not of PML itself. Importantly, we show that PML deSUMOylation by IE1CORE is sufficient to antagonize PML-NB-instituted intrinsic immunity. Moreover, co-immunoprecipitation experiments demonstrate that IE1CORE binds via the coiled-coil domain to PML and also interacts with TRIM5α We propose that IE1CORE sequesters PML and possibly other TRIM family members via structural mimicry using an extended binding surface formed by the coiled-coil region. This mode of interaction might render the antagonizing activity less susceptible to

  15. Peptidyl Materials Formed Through Click Chemistry Enhanced Coiled-Coil Interactions

    NASA Astrophysics Data System (ADS)

    Koehler, Kenneth

    2014-03-01

    Biologically derived materials offer a level of sophistication synthetically fabricated materials have only attempted to mimic. This level of complexity may be found in materials such as peptides. Implementing new theory and modeling, peptides with the propensity to form coiled-coil (CC) bundles were designed and synthesized. Through the use of this de novo approach, modeling allowed prediction of the feasibility to include non-natural amino acids conducive to click chemistry into the peptide. Amino acids showcasing thiol or alkyne functionalities were considered owing to the ability of these moieties to participate in the thiol-ene and copper click reactions respectively. Once synthesized, the peptides decorated with these clickable motifs were placed in solution and allowed to self-assemble into CC's. CD spectroscopy and DLS experiments confirmed the formation and assembly of CC's. Click reactions were then incited to link the CC assemblies together and form a network with predictable dimensionality and pore size between CC bundles. To incite network formation, click reactions between CC side chain residues and suitably functionalized crosslinkers were implemented. The linking of coiled-coils and material formation were assessed using DLS and TEM.

  16. SAS-6 coiled-coil structure and interaction with SAS-5 suggest a regulatory mechanism in C. elegans centriole assembly

    PubMed Central

    Qiao, Renping; Cabral, Gabriela; Lettman, Molly M; Dammermann, Alexander; Dong, Gang

    2012-01-01

    The centriole is a conserved microtubule-based organelle essential for both centrosome formation and cilium biogenesis. Five conserved proteins for centriole duplication have been identified. Two of them, SAS-5 and SAS-6, physically interact with each other and are codependent for their targeting to procentrioles. However, it remains unclear how these two proteins interact at the molecular level. Here, we demonstrate that the short SAS-5 C-terminal domain (residues 390–404) specifically binds to a narrow central region (residues 275–288) of the SAS-6 coiled coil. This was supported by the crystal structure of the SAS-6 coiled-coil domain (CCD), which, together with mutagenesis studies, indicated that the association is mediated by synergistic hydrophobic and electrostatic interactions. The crystal structure also shows a periodic charge pattern along the SAS-6 CCD, which gives rise to an anti-parallel tetramer. Overall, our findings establish the molecular basis of the specific interaction between SAS-5 and SAS-6, and suggest that both proteins individually adopt an oligomeric conformation that is disrupted upon the formation of the hetero-complex to facilitate the correct assembly of the nine-fold symmetric centriole. PMID:23064147

  17. Molecular basis of coiled-coil formation.

    PubMed

    Steinmetz, Michel O; Jelesarov, Ilian; Matousek, William M; Honnappa, Srinivas; Jahnke, Wolfgang; Missimer, John H; Frank, Sabine; Alexandrescu, Andrei T; Kammerer, Richard A

    2007-04-24

    Coiled coils have attracted considerable interest as design templates in a wide range of applications. Successful coiled-coil design strategies therefore require a detailed understanding of coiled-coil folding. One common feature shared by coiled coils is the presence of a short autonomous helical folding unit, termed "trigger sequence," that is indispensable for folding. Detailed knowledge of trigger sequences at the molecular level is thus key to a general understanding of coiled-coil formation. Using a multidisciplinary approach, we identify and characterize here the molecular determinants that specify the helical conformation of the monomeric early folding intermediate of the GCN4 coiled coil. We demonstrate that a network of hydrogen-bonding and electrostatic interactions stabilize the trigger-sequence helix. This network is rearranged in the final dimeric coiled-coil structure, and its destabilization significantly slows down GCN4 leucine zipper folding. Our findings provide a general explanation for the molecular mechanism of coiled-coil formation. PMID:17438295

  18. The Conserved RIC-3 Coiled-Coil Domain Mediates Receptor-specific Interactions with Nicotinic Acetylcholine Receptors

    PubMed Central

    Biala, Yoav; Liewald, Jana F.; Ben-Ami, Hagit Cohen; Gottschalk, Alexander

    2009-01-01

    RIC-3 belongs to a conserved family of proteins influencing nicotinic acetylcholine receptor (nAChR) maturation. RIC-3 proteins are integral membrane proteins residing in the endoplasmic reticulum (ER), and containing a C-terminal coiled-coil domain (CC-I). Conservation of CC-I in all RIC-3 family members indicates its importance; however, previous studies could not show its function. To examine the role of CC-I, we studied effects of its deletion on Caenorhabditis elegans nAChRs in vivo. Presence of CC-I promoted maturation of particular nAChRs expressed in body-wall muscle, whereas it was not required for other nAChR subtypes expressed in neurons or pharyngeal muscles. This effect is receptor-specific, because it could be reproduced after heterologous expression. Consistently, coimmunoprecipitation analysis showed that CC-I enhances the interaction of RIC-3 with a nAChR that requires CC-I in vivo; thus CC-I appears to enhance affinity of RIC-3 to specific nAChRs. However, we found that this function of CC-I is redundant with functions of sequences downstream to CC-I, potentially a second coiled-coil. Alternative splicing in both vertebrates and invertebrates generates RIC-3 transcripts that lack the entire C-terminus, or only CC-I. Thus, our results suggest that RIC-3 alternative splicing enables subtype specific regulation of nAChR maturation. PMID:19116311

  19. CCBuilder: an interactive web-based tool for building, designing and assessing coiled-coil protein assemblies

    PubMed Central

    Wood, Christopher W.; Bruning, Marc; Ibarra, Amaurys Á.; Bartlett, Gail J.; Thomson, Andrew R.; Sessions, Richard B.; Brady, R Leo; Woolfson, Derek N.

    2014-01-01

    Motivation: The ability to accurately model protein structures at the atomistic level underpins efforts to understand protein folding, to engineer natural proteins predictably and to design proteins de novo. Homology-based methods are well established and produce impressive results. However, these are limited to structures presented by and resolved for natural proteins. Addressing this problem more widely and deriving truly ab initio models requires mathematical descriptions for protein folds; the means to decorate these with natural, engineered or de novo sequences; and methods to score the resulting models. Results: We present CCBuilder, a web-based application that tackles the problem for a defined but large class of protein structure, the α-helical coiled coils. CCBuilder generates coiled-coil backbones, builds side chains onto these frameworks and provides a range of metrics to measure the quality of the models. Its straightforward graphical user interface provides broad functionality that allows users to build and assess models, in which helix geometry, coiled-coil architecture and topology and protein sequence can be varied rapidly. We demonstrate the utility of CCBuilder by assembling models for 653 coiled-coil structures from the PDB, which cover >96% of the known coiled-coil types, and by generating models for rarer and de novo coiled-coil structures. Availability and implementation: CCBuilder is freely available, without registration, at http://coiledcoils.chm.bris.ac.uk/app/cc_builder/ Contact: D.N.Woolfson@bristol.ac.uk or Chris.Wood@bristol.ac.uk PMID:25064570

  20. A Coiled-coil Clamp Controls Both Conformation and Clustering of Stromal Interaction Molecule 1 (STIM1)*

    PubMed Central

    Fahrner, Marc; Muik, Martin; Schindl, Rainer; Butorac, Carmen; Stathopulos, Peter; Zheng, Le; Jardin, Isaac; Ikura, Mitsuhiko; Romanin, Christoph

    2014-01-01

    Store-operated Ca2+ entry, essential for the adaptive immunity, is initiated by the endoplasmic reticulum (ER) Ca2+ sensor STIM1. Ca2+ entry occurs through the plasma membrane resident Ca2+ channel Orai1 that directly interacts with the C-terminal STIM1 domain, named SOAR/CAD. Depletion of the ER Ca2+ store controls this STIM1/Orai1 interaction via transition to an extended STIM1 C-terminal conformation, exposure of the SOAR/CAD domain, and STIM1/Orai1 co-clustering. Here we developed a novel approach termed FRET-derived Interaction in a Restricted Environment (FIRE) in an attempt to dissect the interplay of coiled-coil (CC) interactions in controlling STIM1 quiescent as well as active conformation and cluster formation. We present evidence of a sequential activation mechanism in the STIM1 cytosolic domains where the interaction between CC1 and CC3 segment regulates both SOAR/CAD exposure and CC3-mediated higher-order oligomerization as well as cluster formation. These dual levels of STIM1 auto-inhibition provide efficient control over the coupling to and activation of Orai1 channels. PMID:25342749

  1. An intramolecular interaction between the FHA domain and a coiled coil negatively regulates the kinesin motor KIF1A

    PubMed Central

    Lee, Jae-Ran; Shin, Hyewon; Choi, Jeonghoon; Ko, Jaewon; Kim, Seho; Lee, Hyun Woo; Kim, Karam; Rho, Seong-Hwan; Lee, Jun Hyuck; Song, Hye-Eun; Eom, Soo Hyun; Kim, Eunjoon

    2004-01-01

    Motor proteins not actively involved in transporting cargoes should remain inactive at sites of cargo loading to save energy and remain available for loading. KIF1A/Unc104 is a monomeric kinesin known to dimerize into a processive motor at high protein concentrations. However, the molecular mechanisms underlying monomer stabilization and monomer-to-dimer transition are not well understood. Here, we report an intramolecular interaction in KIF1A between the forkhead-associated (FHA) domain and a coiled-coil domain (CC2) immediately following the FHA domain. Disrupting this interaction by point mutations in the FHA or CC2 domains leads to a dramatic accumulation of KIF1A in the periphery of living cultured neurons and an enhancement of the microtubule (MT) binding and self-multimerization of KIF1A. In addition, point mutations causing rigidity in the predicted flexible hinge disrupt the intramolecular FHA–CC2 interaction and increase MT binding and peripheral accumulation of KIF1A. These results suggest that the intramolecular FHA–CC2 interaction negatively regulates KIF1A activity by inhibiting MT binding and dimerization of KIF1A, and point to a novel role of the FHA domain in the regulation of kinesin motors. PMID:15014437

  2. Study on the interaction between methyl jasmonate and the coiled-coil domain of rice blast resistance protein Pi36 by spectroscopic methods

    NASA Astrophysics Data System (ADS)

    Liu, Xin Q.; Zhang, Dan; Zhang, Xiang M.; Wang, Chun T.; Liu, Xue Q.; Tan, Yan P.; Wu, Yun H.

    2012-03-01

    Interaction between the coiled-coil domain of rice blast resistance protein Pi36 and methyl-jasmonate (MeJA) was studied by fluorescence and UV-vis spectroscopic techniques. The quenching mechanism of fluorescence of MeJA by this domain was discussed to be a static quenching procedure. Fluorescence quenching was explored to measure the number of binding sites n and apparent binding constants K. The thermodynamics parameters ΔH, ΔG, ΔS were also calculated. The results indicate the binding reaction was not entropy-driven but enthalpy-driven, and hydrophobic binding played major role in the interaction. The binding sites of MeJA with the coiled-coil structural domain of rice blast resistance protein Pi36 were found to approach the microenvironment of both Tyr and Trp by the synchronous fluorescence spectrometry. The distance r between donor (the coiled-coil domain of rice blast resistance protein Pi36) and acceptor (MeJA) was obtained according to Förster theory of non-radioactive energy transfer.

  3. Disruption of Bcr-Abl Coiled Coil Oligomerization by Design*

    PubMed Central

    Dixon, Andrew S.; Pendley, Scott S.; Bruno, Benjamin J.; Woessner, David W.; Shimpi, Adrian A.; Cheatham, Thomas E.; Lim, Carol S.

    2011-01-01

    Oligomerization is an important regulatory mechanism for many proteins, including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity, suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges, resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations, which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl, reduced cell proliferation, and increased caspase-3/7 activity and DNA segmentation. Importantly, the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention. PMID:21659527

  4. SUMO modification of TBK1 at the adaptor-binding C-terminal coiled-coil domain contributes to its antiviral activity.

    PubMed

    Saul, Vera V; Niedenthal, Rainer; Pich, Andreas; Weber, Friedemann; Schmitz, M Lienhard

    2015-01-01

    The non-canonical IKK kinase TBK1 serves as an important signal transmitter of the antiviral interferon response, but is also involved in the regulation of further processes such as autophagy. The activity of TBK1 is regulated by posttranslational modifications comprising phosphorylation and ubiquitination. This study identifies SUMOylation as a novel posttranslational TBK1 modification. TBK1 kinase activity is required to allow the attachment of SUMO1 or SUMO2/3 proteins. Since TBK1 does not bind to the E2 enzyme Ubc9, this modification most likely proceeds via trans-SUMOylation. Mass spectrometry allowed identifying K694 as the SUMO acceptor site, a residue located in the C-terminal coiled-coil domain which is exclusively responsible for the association with the adaptor proteins NAP1, Sintbad and TANK. SUMO modification at K694 contributes to the antiviral function of TBK1 and accordingly the viral protein Gam1 antagonizes this posttranslational modification. PMID:25409927

  5. The basic amino acids in the coiled-coil domain of CIN85 regulate its interaction with c-Cbl and phosphatidic acid during epidermal growth factor receptor (EGFR) endocytosis

    PubMed Central

    2014-01-01

    Background During EGFR internalization CIN85 bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of CIN85 with c-Cbl, endophilins and phosphatidic acid. These protein-protein and protein-lipid interactions are mediated or regulated by the positively charged C-terminal coiled-coil domain of CIN85. However, the details of CIN85-lipid interaction remain unknown. The present study suggested a possible electric interaction between the negative charge of phosphatidic acid and the positive charge of basic amino acids in coiled-coil domain. Results Mutations of the basic amino acids in the coiled-coil domain, especially K645, K646, R648 and R650, into neutral amino acid alanine completely blocked the interaction of CIN85 with c-Cbl or phosphatidic acid. However, they did not affect CIN85-endophilin interaction. In addition, CIN85 was found to associate with the internalized EGFR endosomes. It interacted with several ESCRT (Endosomal Sorting Complex Required for Transport) component proteins for ESCRT assembly on endosomal membrane. Mutations in the coiled-coil domain (deletion of the coiled-coil domain or point mutations of the basic amino acids) dissociated CIN85 from endosomes. These mutants bound the ESCRT components in cytoplasm to prevent them from assembly on endosomal membrane and inhibited EGFR sorting for degradation. Conclusions As an adaptor protein, CIN85 interacts with variety of partners through several domains. The positive charges of basic amino acids in the coiled-coil domain are not only involved in the interaction with phosphatidic acid, but also regulate the interaction of CIN85 with c-Cbl. CIN85 also interacts with ESCRT components for protein sorting in endosomes. These CIN85-protein and CIN85-lipid interactions enable CIN85 to link EGFR-Cbl endocytic complex with fusible membrane during EGFR endocytosis and subsequently to facilitate ESCRT formation on endosomal membrane for EGFR sorting and degradation. PMID

  6. α/β coiled coils

    PubMed Central

    Hartmann, Marcus D; Mendler, Claudia T; Bassler, Jens; Karamichali, Ioanna; Ridderbusch, Oswin; Lupas, Andrei N; Hernandez Alvarez, Birte

    2016-01-01

    Coiled coils are the best-understood protein fold, as their backbone structure can uniquely be described by parametric equations. This level of understanding has allowed their manipulation in unprecedented detail. They do not seem a likely source of surprises, yet we describe here the unexpected formation of a new type of fiber by the simple insertion of two or six residues into the underlying heptad repeat of a parallel, trimeric coiled coil. These insertions strain the supercoil to the breaking point, causing the local formation of short β-strands, which move the path of the chain by 120° around the trimer axis. The result is an α/β coiled coil, which retains only one backbone hydrogen bond per repeat unit from the parent coiled coil. Our results show that a substantially novel backbone structure is possible within the allowed regions of the Ramachandran space with only minor mutations to a known fold. DOI: http://dx.doi.org/10.7554/eLife.11861.001 PMID:26771248

  7. Coiled-coil networking shapes cell molecular machinery

    PubMed Central

    Wang, Yongqiang; Zhang, Xinlei; Zhang, Hong; Lu, Yi; Huang, Haolong; Dong, Xiaoxi; Chen, Jinan; Dong, Jiuhong; Yang, Xiao; Hang, Haiying; Jiang, Taijiao

    2012-01-01

    The highly abundant α-helical coiled-coil motif not only mediates crucial protein–protein interactions in the cell but is also an attractive scaffold in synthetic biology and material science and a potential target for disease intervention. Therefore a systematic understanding of the coiled-coil interactions (CCIs) at the organismal level would help unravel the full spectrum of the biological function of this interaction motif and facilitate its application in therapeutics. We report the first identified genome-wide CCI network in Saccharomyces cerevisiae, which consists of 3495 pair-wise interactions among 598 predicted coiled-coil regions. Computational analysis revealed that the CCI network is specifically and functionally organized and extensively involved in the organization of cell machinery. We further show that CCIs play a critical role in the assembly of the kinetochore, and disruption of the CCI network leads to defects in kinetochore assembly and cell division. The CCI network identified in this study is a valuable resource for systematic characterization of coiled coils in the shaping and regulation of a host of cellular machineries and provides a basis for the utilization of coiled coils as domain-based probes for network perturbation and pharmacological applications. PMID:22875988

  8. The Chloroplastic Protein THF1 Interacts with the Coiled-Coil Domain of the Disease Resistance Protein N' and Regulates Light-Dependent Cell Death.

    PubMed

    Hamel, Louis-Philippe; Sekine, Ken-Taro; Wallon, Thérèse; Sugiwaka, Yuji; Kobayashi, Kappei; Moffett, Peter

    2016-05-01

    One branch of plant immunity is mediated through nucleotide-binding/Leu-rich repeat (NB-LRR) family proteins that recognize specific effectors encoded by pathogens. Members of the I2-like family constitute a well-conserved subgroup of NB-LRRs from Solanaceae possessing a coiled-coil (CC) domain at their N termini. We show here that the CC domains of several I2-like proteins are able to induce a hypersensitive response (HR), a form of programmed cell death associated with disease resistance. Using yeast two-hybrid screens, we identified the chloroplastic protein Thylakoid Formation1 (THF1) as an interacting partner for several I2-like CC domains. Co-immunoprecipitations and bimolecular fluorescence complementation assays confirmed that THF1 and I2-like CC domains interact in planta and that these interactions take place in the cytosol. Several HR-inducing I2-like CC domains have a negative effect on the accumulation of THF1, suggesting that the latter is destabilized by active CC domains. To confirm this model, we investigated N', which recognizes the coat protein of most Tobamoviruses, as a prototypical member of the I2-like family. Transient expression and gene silencing data indicated that THF1 functions as a negative regulator of cell death and that activation of full-length N' results in the destabilization of THF1. Consistent with the known function of THF1 in maintaining chloroplast homeostasis, we show that the HR induced by N' is light-dependent. Together, our results define, to our knowledge, novel molecular mechanisms linking light and chloroplasts to the induction of cell death by a subgroup of NB-LRR proteins. PMID:26951433

  9. The Chloroplastic Protein THF1 Interacts with the Coiled-Coil Domain of the Disease Resistance Protein N′ and Regulates Light-Dependent Cell Death1[OPEN

    PubMed Central

    Sekine, Ken-Taro; Wallon, Thérèse; Sugiwaka, Yuji; Kobayashi, Kappei

    2016-01-01

    One branch of plant immunity is mediated through nucleotide-binding/Leu-rich repeat (NB-LRR) family proteins that recognize specific effectors encoded by pathogens. Members of the I2-like family constitute a well-conserved subgroup of NB-LRRs from Solanaceae possessing a coiled-coil (CC) domain at their N termini. We show here that the CC domains of several I2-like proteins are able to induce a hypersensitive response (HR), a form of programmed cell death associated with disease resistance. Using yeast two-hybrid screens, we identified the chloroplastic protein Thylakoid Formation1 (THF1) as an interacting partner for several I2-like CC domains. Co-immunoprecipitations and bimolecular fluorescence complementation assays confirmed that THF1 and I2-like CC domains interact in planta and that these interactions take place in the cytosol. Several HR-inducing I2-like CC domains have a negative effect on the accumulation of THF1, suggesting that the latter is destabilized by active CC domains. To confirm this model, we investigated N′, which recognizes the coat protein of most Tobamoviruses, as a prototypical member of the I2-like family. Transient expression and gene silencing data indicated that THF1 functions as a negative regulator of cell death and that activation of full-length N′ results in the destabilization of THF1. Consistent with the known function of THF1 in maintaining chloroplast homeostasis, we show that the HR induced by N′ is light-dependent. Together, our results define, to our knowledge, novel molecular mechanisms linking light and chloroplasts to the induction of cell death by a subgroup of NB-LRR proteins. PMID:26951433

  10. Transforming acidic coiled-coil 3 and Aurora-A interact in human thyrocytes and their expression is deregulated in thyroid cancer tissues

    PubMed Central

    Ulisse, Salvatore; Baldini, Enke; Toller, Matteo; Delcros, Jean-Guy; Guého, Aurélie; Curcio, Francesco; De Antoni, Enrico; Giacomelli, Laura; Ambesi-Impiombato, Francesco S; Bocchini, Sarah; D'Armiento, Massimino; Arlot-Bonnemains, Yannick

    2007-01-01

    Aurora-A kinase has recently been shown to be deregulated in thyroid cancer cells and tissues. Among the Aurora-A substrates identified, transforming acidic coiled-coil (TACC3), a member of the TACC family, plays an important role in cell cycle progression and alterations of its expression occur in different cancer tissues. In this study, we demonstrated the expression of the TACC3 gene in normal human thyroid cells (HTU5), and its modulation at both mRNA and protein levels during cell cycle. Its expression was found, with respect to HTU5 cells, unchanged in cells derived from a benign thyroid follicular tumor (HTU42), and significantly reduced in cell lines derived from follicular (FTC-133), papillary (B-CPAP), and anaplastic thyroid carcinomas (CAL-62 and 8305C). Moreover, in 16 differentiated thyroid cancer tissues, TACC3 mRNA levels were found, with respect to normal matched tissues, reduced by twofold in 56% of cases and increased by twofold in 44% of cases. In the same tissues, a correlation between the expression of the TACC3 and Aurora-A mRNAs was observed. TACC3 and Aurora-A interact in vivo in thyroid cells and both proteins localized onto the mitotic structure of thyroid cells. Finally, TACC3 localization on spindle microtubule was no more observed following the inhibition of Aurora kinase activity by VX-680. We propose that Aurora-A and TACC3 interaction is important to control the mitotic spindle organization required for proper chromosome segregation. PMID:17914111

  11. A Parallel Coiled-Coil Tetramer with Offset Helices

    SciTech Connect

    Liu,J.; Deng, Y.; Zheng, Q.; Cheng, C.; Kallenbach, N.; Lu, M.

    2006-01-01

    Specific helix-helix interactions are fundamental in assembling the native state of proteins and in protein-protein interfaces. Coiled coils afford a unique model system for elucidating principles of molecular recognition between {alpha} helices. The coiled-coil fold is specified by a characteristic seven amino acid repeat containing hydrophobic residues at the first (a) and fourth (d) positions. Nonpolar side chains spaced three and four residues apart are referred to as the 3-4 hydrophobic repeat. The presence of apolar amino acids at the e or g positions (corresponding to a 3-3-1 hydrophobic repeat) can provide new possibilities for close-packing of {alpha}-helices that includes examples such as the lac repressor tetramerization domain. Here we demonstrate that an unprecedented coiled-coil interface results from replacement of three charged residues at the e positions in the dimeric GCN4 leucine zipper by nonpolar valine side chains. Equilibrium circular dichroism and analytical ultracentrifugation studies indicate that the valine-containing mutant forms a discrete {alpha}-helical tetramer with a significantly higher stability than the parent leucine-zipper molecule. The 1.35 {angstrom} resolution crystal structure of the tetramer reveals a parallel four-stranded coiled coil with a three-residue interhelical offset. The local packing geometry of the three hydrophobic positions in the tetramer conformation is completely different from that seen in classical tetrameric structures yet bears resemblance to that in three-stranded coiled coils. These studies demonstrate that distinct van der Waals interactions beyond the a and d side chains can generate a diverse set of helix-helix interfaces and three-dimensional supercoil structures.

  12. Contributed Review: Absolute spectral radiance calibration of fiber-optic shock-temperature pyrometers using a coiled-coil irradiance standard lamp

    NASA Astrophysics Data System (ADS)

    Fat'yanov, O. V.; Asimow, P. D.

    2015-10-01

    We describe an accurate and precise calibration procedure for multichannel optical pyrometers such as the 6-channel, 3-ns temporal resolution instrument used in the Caltech experimental geophysics laboratory. We begin with a review of calibration sources for shock temperatures in the 3000-30 000 K range. High-power, coiled tungsten halogen standards of spectral irradiance appear to be the only practical alternative to NIST-traceable tungsten ribbon lamps, which are no longer available with large enough calibrated area. However, non-uniform radiance complicates the use of such coiled lamps for reliable and reproducible calibration of pyrometers that employ imaging or relay optics. Careful analysis of documented methods of shock pyrometer calibration to coiled irradiance standard lamps shows that only one technique, not directly applicable in our case, is free of major radiometric errors. We provide a detailed description of the modified Caltech pyrometer instrument and a procedure for its absolute spectral radiance calibration, accurate to ±5%. We employ a designated central area of a 0.7× demagnified image of a coiled-coil tungsten halogen lamp filament, cross-calibrated against a NIST-traceable tungsten ribbon lamp. We give the results of the cross-calibration along with descriptions of the optical arrangement, data acquisition, and processing. We describe a procedure to characterize the difference between the static and dynamic response of amplified photodetectors, allowing time-dependent photodiode correction factors for spectral radiance histories from shock experiments. We validate correct operation of the modified Caltech pyrometer with actual shock temperature experiments on single-crystal NaCl and MgO and obtain very good agreement with the literature data for these substances. We conclude with a summary of the most essential requirements for error-free calibration of a fiber-optic shock-temperature pyrometer using a high-power coiled tungsten halogen

  13. Contributed Review: Absolute spectral radiance calibration of fiber-optic shock-temperature pyrometers using a coiled-coil irradiance standard lamp.

    PubMed

    Fat'yanov, O V; Asimow, P D

    2015-10-01

    We describe an accurate and precise calibration procedure for multichannel optical pyrometers such as the 6-channel, 3-ns temporal resolution instrument used in the Caltech experimental geophysics laboratory. We begin with a review of calibration sources for shock temperatures in the 3000-30,000 K range. High-power, coiled tungsten halogen standards of spectral irradiance appear to be the only practical alternative to NIST-traceable tungsten ribbon lamps, which are no longer available with large enough calibrated area. However, non-uniform radiance complicates the use of such coiled lamps for reliable and reproducible calibration of pyrometers that employ imaging or relay optics. Careful analysis of documented methods of shock pyrometer calibration to coiled irradiance standard lamps shows that only one technique, not directly applicable in our case, is free of major radiometric errors. We provide a detailed description of the modified Caltech pyrometer instrument and a procedure for its absolute spectral radiance calibration, accurate to ±5%. We employ a designated central area of a 0.7× demagnified image of a coiled-coil tungsten halogen lamp filament, cross-calibrated against a NIST-traceable tungsten ribbon lamp. We give the results of the cross-calibration along with descriptions of the optical arrangement, data acquisition, and processing. We describe a procedure to characterize the difference between the static and dynamic response of amplified photodetectors, allowing time-dependent photodiode correction factors for spectral radiance histories from shock experiments. We validate correct operation of the modified Caltech pyrometer with actual shock temperature experiments on single-crystal NaCl and MgO and obtain very good agreement with the literature data for these substances. We conclude with a summary of the most essential requirements for error-free calibration of a fiber-optic shock-temperature pyrometer using a high-power coiled tungsten halogen

  14. Contributed Review: Absolute spectral radiance calibration of fiber-optic shock-temperature pyrometers using a coiled-coil irradiance standard lamp

    SciTech Connect

    Fat’yanov, O. V. Asimow, P. D.

    2015-10-15

    We describe an accurate and precise calibration procedure for multichannel optical pyrometers such as the 6-channel, 3-ns temporal resolution instrument used in the Caltech experimental geophysics laboratory. We begin with a review of calibration sources for shock temperatures in the 3000-30 000 K range. High-power, coiled tungsten halogen standards of spectral irradiance appear to be the only practical alternative to NIST-traceable tungsten ribbon lamps, which are no longer available with large enough calibrated area. However, non-uniform radiance complicates the use of such coiled lamps for reliable and reproducible calibration of pyrometers that employ imaging or relay optics. Careful analysis of documented methods of shock pyrometer calibration to coiled irradiance standard lamps shows that only one technique, not directly applicable in our case, is free of major radiometric errors. We provide a detailed description of the modified Caltech pyrometer instrument and a procedure for its absolute spectral radiance calibration, accurate to ±5%. We employ a designated central area of a 0.7× demagnified image of a coiled-coil tungsten halogen lamp filament, cross-calibrated against a NIST-traceable tungsten ribbon lamp. We give the results of the cross-calibration along with descriptions of the optical arrangement, data acquisition, and processing. We describe a procedure to characterize the difference between the static and dynamic response of amplified photodetectors, allowing time-dependent photodiode correction factors for spectral radiance histories from shock experiments. We validate correct operation of the modified Caltech pyrometer with actual shock temperature experiments on single-crystal NaCl and MgO and obtain very good agreement with the literature data for these substances. We conclude with a summary of the most essential requirements for error-free calibration of a fiber-optic shock-temperature pyrometer using a high-power coiled tungsten halogen

  15. Transport Vesicle Tethering at the Trans Golgi Network: Coiled Coil Proteins in Action

    PubMed Central

    Cheung, Pak-yan P.; Pfeffer, Suzanne R.

    2016-01-01

    The Golgi complex is decorated with so-called Golgin proteins that share a common feature: a large proportion of their amino acid sequences are predicted to form coiled-coil structures. The possible presence of extensive coiled coils implies that these proteins are highly elongated molecules that can extend a significant distance from the Golgi surface. This property would help them to capture or trap inbound transport vesicles and to tether Golgi mini-stacks together. This review will summarize our current understanding of coiled coil tethers that are needed for the receipt of transport vesicles at the trans Golgi network (TGN). How do long tethering proteins actually catch vesicles? Golgi-associated, coiled coil tethers contain numerous binding sites for small GTPases, SNARE proteins, and vesicle coat proteins. How are these interactions coordinated and are any or all of them important for the tethering process? Progress toward understanding these questions and remaining, unresolved mysteries will be discussed. PMID:27014693

  16. Immune responses to coiled coil supramolecular biomaterials

    PubMed Central

    Rudra, Jai S.; Tripathi, Pulak; Hildeman, David A.; Jung, Jangwook P.; Collier, Joel H.

    2010-01-01

    Self-assembly has been increasingly utilized in recent years to create peptide-based biomaterials for 3D cell culture, tissue engineering, and regenerative medicine, but the molecular determinants of these materials' immunogenicity have remained largely unexplored. In this study, a set of molecules that self-assembled through coiled coil oligomerization was designed and synthesized, and immune responses against them were investigated in mice. Experimental groups spanned a range of oligomerization behaviors and included a peptide from the coiled coil region of mouse fibrin that did not form supramolecular structures, an engineered version of this peptide that formed coiled coil bundles, and a peptide-PEG-peptide triblock bioconjugate that formed coiled coil multimers and supramolecular aggregates. In mice, the native peptide and engineered peptide did not produce any detectable antibody response, and none of the materials elicited detectable peptide-specific T cell responses, as evidenced by the absence of IL-2 and interferon-gamma in cultures of peptide-challenged splenocytes or draining lymph node cells. However, specific antibody responses were elevated in mice injected with the multimerizing peptide-PEG-peptide. Minimal changes in secondary structure were observed between the engineered peptide and the triblock peptide-PEG-peptide, making it possible that the triblock's multimerization was responsible for this antibody response. PMID:20708258

  17. A Non-perturbing Probe of Coiled Coil Formation Based on Electron Transfer Mediated Fluorescence Quenching.

    PubMed

    Watson, Matthew D; Peran, Ivan; Raleigh, Daniel P

    2016-07-01

    Coiled coils are abundant in nature, occurring in ∼3% of proteins across sequenced genomes, and are found in proteins ranging from transcription factors to structural proteins. The motif continues to be an important model system for understanding protein-protein interactions and is finding increased use in bioinspired materials and synthetic biology. Knowledge of the thermodynamics of self-assembly, particularly the dissociation constant KD, is essential for the application of designed coiled coils and for understanding the in vivo specificity of natural coiled coils. Standard methods for measuring KD typically rely on concentration dependent circular dichroism (CD). Fluorescence methods are an attractive alternative; however Trp is rarely found in an interior position of a coiled coil, and appending unnatural fluorophores can perturb the system. We demonstrate a simple, non-perturbing method to monitor coiled coil formation using p-cyanophenylalanine (FCN) and selenomethionine (MSe), the Se analogue of Met. FCN fluorescence can be selectively excited and is effectively quenched by electron transfer with MSe. Both FCN and MSe represent minimally perturbing substitutions in coiled coils. MSe quenching of FCN fluorescence is shown to offer a non-perturbing method for following coiled coil formation and for accurately determining dissociation constants. The method is validated using a designed heterodimeric coiled coil. The KD deduced by fluorescence monitored titration is in excellent agreement with the value deduced from concentration dependent CD measurements to within the uncertainty of the measurement. However, the fluorescence approach requires less protein, is less time-consuming, can be applied to lower concentrations and could be applied to high throughput screens. PMID:27258904

  18. Kinking the coiled coil--negatively charged residues at the coiled-coil interface.

    PubMed

    Straussman, Ravid; Ben-Ya'acov, Ami; Woolfson, Derek N; Ravid, Shoshana

    2007-03-01

    The coiled coil is one of the most common protein-structure motifs. It is believed to be adopted by 3-5% of all amino acids in proteins. It comprises two or more alpha-helical chains wrapped around one another. The sequences of most coiled coils are characterized by a seven-residue (heptad) repeat, denoted (abcdefg)(n). Residues at the a and d positions define the helical interface (core) and are usually hydrophobic, though about 20% are polar or charged. We show that parallel coiled-coils have a unique pattern of their negatively charged residues at the core positions: aspartic acid is excluded from these positions while glutamic acid is not. In contrast the antiparallel structures are more permissive in their amino acid usage. We show further, and for the first time, that incorporation of Asp but not Glu into the a positions of a parallel coiled coil creates a flexible hinge and that the maximal hinge angle is being directly related to the number of incorporated mutations. These new computational and experimental observations will be of use in improving protein-structure predictions, and as rules to guide rational design of novel coiled-coil motifs and coiled coil-based materials. PMID:17207815

  19. A Synthetic Coiled-Coil Interactome Provides Heterospecific Modules for Molecular Engineering

    SciTech Connect

    Reinke, Aaron W.; Grant, Robert A.; Keating, Amy E.

    2010-06-21

    The versatile coiled-coil protein motif is widely used to induce and control macromolecular interactions in biology and materials science. Yet the types of interaction patterns that can be constructed using known coiled coils are limited. Here we greatly expand the coiled-coil toolkit by measuring the complete pairwise interactions of 48 synthetic coiled coils and 7 human bZIP coiled coils using peptide microarrays. The resulting 55-member protein 'interactome' includes 27 pairs of interacting peptides that preferentially heteroassociate. The 27 pairs can be used in combinations to assemble sets of 3 to 6 proteins that compose networks of varying topologies. Of special interest are heterospecific peptide pairs that participate in mutually orthogonal interactions. Such pairs provide the opportunity to dimerize two separate molecular systems without undesired crosstalk. Solution and structural characterization of two such sets of orthogonal heterodimers provide details of their interaction geometries. The orthogonal pair, along with the many other network motifs discovered in our screen, provide new capabilities for synthetic biology and other applications.

  20. High-resolution structures of a heterochiral coiled coil

    PubMed Central

    Mortenson, David E.; Steinkruger, Jay D.; Kreitler, Dale F.; Perroni, Dominic V.; Sorenson, Gregory P.; Huang, Lijun; Mittal, Ritesh; Yun, Hyun Gi; Travis, Benjamin R.; Mahanthappa, Mahesh K.; Forest, Katrina T.; Gellman, Samuel H.

    2015-01-01

    Interactions between polypeptide chains containing amino acid residues with opposite absolute configurations have long been a source of interest and speculation, but there is very little structural information for such heterochiral associations. The need to address this lacuna has grown in recent years because of increasing interest in the use of peptides generated from d amino acids (d peptides) as specific ligands for natural proteins, e.g., to inhibit deleterious protein–protein interactions. Coiled–coil interactions, between or among α-helices, represent the most common tertiary and quaternary packing motif in proteins. Heterochiral coiled–coil interactions were predicted over 50 years ago by Crick, and limited experimental data obtained in solution suggest that such interactions can indeed occur. To address the dearth of atomic-level structural characterization of heterochiral helix pairings, we report two independent crystal structures that elucidate coiled-coil packing between l- and d-peptide helices. Both structures resulted from racemic crystallization of a peptide corresponding to the transmembrane segment of the influenza M2 protein. Networks of canonical knobs-into-holes side-chain packing interactions are observed at each helical interface. However, the underlying patterns for these heterochiral coiled coils seem to deviate from the heptad sequence repeat that is characteristic of most homochiral analogs, with an apparent preference for a hendecad repeat pattern. PMID:26460035

  1. Tropomyosin lysine reactivities and relationship to coiled-coil structure.

    PubMed

    Hitchcock-DeGregori, S E; Lewis, S F; Chou, T M

    1985-06-18

    We have carried out a detailed analysis of tropomyosin structure using lysines as specific probes for the protein surface in regions of the molecule that have not been investigated by other methods. We have measured the relative reactivities of lysines in rabbit skeletal muscle alpha, alpha-tropomyosin with acetic anhydride using a competitive labeling procedure. We have identified 37 of 39 lysines and find that they range 20-fold in reactivity. The observed reactivities are related to the coiled-coil model of the tropomyosin molecule [Crick, F.H.C. (1953) Acta Crystallogr. 6, 689-697; McLachlan, A.D., Stewart, M., & Smillie, L.B. (1975) J. Mol. Biol. 98, 281-291] and other available chemical and physical information about the structure. In most cases, the observed lysine reactivities can be explained by allowable interactions with neighboring amino acid side chains on the same or facing alpha-helix. However, we found no correlation between reactivity and helical position of a given lysine. For example, lysines in the outer helical positions included lysines of low as well as high reactivity, indicating that they vary widely in their accessibility to solvent and that the coiled coil is heterogeneous along its length. Furthermore, the middle of the molecule (residues 126-182) that is susceptible to proteolysis and known to be the least stable region of the protein also contains some of the least and most reactive lysines. We have discussed the implications of our results on our understanding the structures of tropomyosin and other coiled-coil proteins as well as globular proteins containing helical regions. PMID:3927977

  2. Crystal Structure of the Central Coiled-Coil Domain from Human Liprin-[beta]2

    SciTech Connect

    Stafford, Ryan L.; Tang, Ming-Yun; Sawaya, Michael R.; Phillips, Martin L.; Bowie, James U.

    2012-02-07

    Liprins are a conserved family of scaffolding proteins important for the proper regulation and development of neuronal synapses. Humans have four liprin-{alpha}s and two liprin-{beta}s which all contain long coiled-coil domains followed by three tandem SAM domains. Complex interactions between the coiled-coil and SAM domains are thought to create liprin scaffolds, but the structural and biochemical properties of these domains remain largely uncharacterized. In this study we find that the human liprin-{beta}2 coiled-coil forms an extended dimer. Several protease-resistant subdomains within the liprin-{beta}1 and liprin-{beta}2 coiled-coils were also identified. A 2.0 {angstrom} crystal structure of the central, protease-resistant core of the liprin-{beta}2 coiled-coil reveals a parallel helix orientation. These studies represent an initial step toward determining the overall architecture of liprin scaffolds and understanding the molecular basis for their synaptic functions.

  3. Finding the Golgi: Golgin Coiled-Coil Proteins Show the Way.

    PubMed

    Gillingham, Alison K; Munro, Sean

    2016-06-01

    The Golgi apparatus lies at the centre of the secretory pathway. It consists of a series of flattened compartments typically organised into a stack that, in mammals, is connected to additional stacks to form a Golgi ribbon. The Golgi is responsible for the maturation and modification of proteins and lipids, and receives and exports vesicles to and from multiple destinations within the cell. This complex trafficking network requires that only the correct vesicles fuse with the correct destination membrane. Recently, a group of coiled-coil proteins called golgins were shown to not only capture incoming vesicles but to also provide specificity to the tethering step. This raises many interesting questions about how they interact with other components of membrane traffic, some of which may also contribute to specificity. PMID:26972448

  4. Subunit b-Dimer of the Escherichia coli ATP Synthase Can Form Left-Handed Coiled-Coils

    PubMed Central

    Wise, John G.; Vogel, Pia D.

    2008-01-01

    One remaining challenge to our understanding of the ATP synthase concerns the dimeric coiled-coil stator subunit b of bacterial synthases. The subunit b-dimer has been implicated in important protein interactions that appear necessary for energy conservation and that may be instrumental in energy conservation during rotary catalysis by the synthase. Understanding the stator structure and its interactions with the rest of the enzyme is crucial to the understanding of the overall catalytic mechanism. Controversy exists on whether subunit b adopts a classic left-handed or a presumed right-handed dimeric coiled-coil and whether or not staggered pairing between nonhomologous residues in the homodimer is required for intersubunit packing. In this study we generated molecular models of the Escherichia coli subunit b-dimer that were based on the well-established heptad-repeat packing exhibited by left-handed, dimeric coiled-coils by employing simulated annealing protocols with structural restraints collected from known structures. In addition, we attempted to create hypothetical right-handed coiled-coil models and left- and right-handed models with staggered packing in the coiled-coil domains. Our analyses suggest that the available structural and biochemical evidence for subunit b can be accommodated by classic left-handed, dimeric coiled-coil quaternary structures. PMID:18326648

  5. Antiparallel Four-Stranded Coiled Coil Specified by a 3-3-1 Hyrdrophobic Heptad Repeat

    SciTech Connect

    Deng,Y.; Liu, J.; Zheng, Q.; Eliezer, D.; Kallenbach, N.; Lu, M.

    2006-01-01

    Coiled-coil sequences in proteins commonly share a seven-amino acid repeat with nonpolar side chains at the first (a) and fourth (d) positions. We investigate here the role of a 3-3-1 hydrophobic repeat containing nonpolar amino acids at the a, d, and g positions in determining the structures of coiled coils using mutants of the GCN4 leucine zipper dimerization domain. When three charged residues at the g positions in the parental sequence are replaced by nonpolar alanine or valine side chains, stable four-helix structures result. The X-ray crystal structures of the tetramers reveal antiparallel, four-stranded coiled coils in which the a, d, and g side chains interlock in a combination of knobs-into-knobs and knobs-into-holes packing. Interfacial interactions in a coiled coil can therefore be prescribed by hydrophobic-polar patterns beyond the canonical 3-4 heptad repeat. The results suggest that the conserved, charged residues at the g positions in the GCN4 leucine zipper can impart a negative design element to disfavor thermodynamically more stable, antiparallel tetramers.

  6. Forced Unfolding of the Coiled-Coils of Fibrinogen by Single-Molecule AFM

    NASA Astrophysics Data System (ADS)

    Brown, Andre; Litvinov, Rustem; Discher, Dennis; Weisel, John

    2007-03-01

    A blood clot needs to have the right degree of stiffness and plasticity for hemostasis, but the origin of these mechanical properties is unknown. Here we report the first measurements using single molecule atomic force microscopy (AFM) to study the forced unfolding of fibrinogen to begin addressing this problem. To generate longer reproducible curves than are possible using monomer, factor XIIIa cross-linked, single chain fibrinogen oligomers were used. When extended under force, these oligomers showed sawtooth shaped force-extension patterns characteristic of unfolding proteins with a peak-to-peak separation of approximately 26 nm, consistent with the independent unfolding of the coiled-coils. These results were then reproduced using a Monte Carlo simulation with parameters in the same range as those previously used for unfolding globular domains. In particular, we found that the refolding time was negligible on experimental time and force scales in contrast to previous work on simpler coiled-coils. We suggest that this difference may be due to fibrinogen's structurally and topologically more complex coiled-coils and that an interaction between the alpha C and central domains may be involved. These results suggest a new functional property of fibrinogen and that the coiled-coil is more than a passive structural element of this molecule.

  7. Essential role of coiled-coils for aggregation and activity of Q/N-rich prions and polyQ proteins

    PubMed Central

    Fiumara, Ferdinando; Fioriti, Luana

    2012-01-01

    SUMMARY The functional switch of glutamine/asparagine (Q/N)-rich prions and the neurotoxicity of polyQ-expanded proteins involve complex aggregation-prone structural transitions, commonly presumed to be forming β-sheets. By analyzing sequences of interaction partners of these proteins, we discovered a recurrent presence of coiled-coil domains both in the partners and in segments that flank or overlap Q/N-rich and polyQ domains. Since coiled-coils can mediate protein interactions and multimerization, we studied their possible involvement in Q/N-rich and polyQ aggregations. Using circular dichroism and chemical cross-linking, we found that Q/N-rich and polyQ peptides form α-helical coiled-coils in vitro and assemble into multimers. Using structure-guided mutagenesis, we found that coiled-coil domains modulate in vivo properties of two Q/N-rich prions and polyQ-expanded huntingtin. Mutations that disrupt coiled-coils impair aggregation and activity, whereas mutations that enhance coiled-coil propensity promote aggregation. These findings support a coiled-coil model for the functional switch of Q/N-rich prions and for the pathogenesis of polyQ-expansion diseases. PMID:21183075

  8. Protein destabilization by electrostatic repulsions in the two-stranded alpha-helical coiled-coil/leucine zipper.

    PubMed Central

    Kohn, W. D.; Kay, C. M.; Hodges, R. S.

    1995-01-01

    The destabilizing effect of electrostatic repulsions on protein stability has been studied by using synthetic two-stranded alpha-helical coiled-coils as a model system. The native coiled-coil consists of two identical 35-residue polypeptide chains with a heptad repeat QgVaGbAcLdQeKf and a Cys residue at position 2 to allow formation of an interchain disulfide bridge. This peptide, designed to contain no intrahelical or interhelical electrostatic interactions, forms a stable coiled-coil structure at 20 degrees C in benign medium (50 mM KCl, 25 mM PO4, pH 7) with a [urea]1/2 value of 6.1 M. Four mutant coiled-coils were designed to contain one or two Glu substitutions for Gln per polypeptide chain. The resulting coiled-coils contained potential i to i' + 5 Glu-Glu interchain repulsions (denoted as peptide E2(15,20)), i to i' + 2 Glu-Glu interchain repulsions (denoted E2(20,22)), or no interchain ionic interactions (denoted E2(13,22) and E1(20)). The stabilities of the coiled-coils were determined by measuring the ellipticities at 222 nm as a function of urea or guanidine hydrochloride concentration at 20 degrees C in the presence and absence of an interchain disulfide bridge. At pH 7, in the presence of urea, the stabilities of E2(13,22) and E2(20,22) were identical suggesting that the potential i to i' + 2 interchain Glu-Glu repulsion in the E2(20,22) coiled-coil does not occur. In contrast, the mutant E2(15,20) is substantially less stable than E2(13,22) or E2(15,20) by 0.9 kcal/mol due to the presence of two i to i' + 5 interchain Glu-Glu repulsions, which destabilize the coiled-coil by 0.45 kcal/mol each. At pH 3 the coiled-coils were found to increase in stability as the number of Glu substitutions were increased. This, combined with reversed-phase HPLC results at pH 7 and pH 2, supports the conclusion that the protonated Glu side chains present at low pH are significantly more hydrophobic than Gln side chains which are in turn more hydrophobic than the ionized

  9. X-ray crystal structure of a TRPM assembly domain reveals an antiparallel four-stranded coiled-coil

    PubMed Central

    Fujiwara, Yuichiro; Minor, Daniel L.

    2008-01-01

    Transient receptor potential (TRP) channels comprise a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Previous studies have shown that members of the TRPM subclass possess a self-assembling tetrameric C-terminal cytoplasmic coiled-coil domain that underlies channel assembly and trafficking. Here, we present the high-resolution crystal structure of the coiled-coil domain of the channel enzyme TRPM7. The crystal structure, together with biochemical experiments, reveals an unexpected four-stranded antiparallel coiled-coil architecture that bears unique features relative to other antiparallel coiled-coils. Structural analysis indicates that a limited set of interactions encode assembly specificity determinants and uncovers a previously unnoticed segregation of TRPM assembly domains into two families that correspond with the phylogenetic divisions seen for the complete subunits. Together, the data provide a framework for understanding the mechanism of the TRPM channel assembly and highlight the diversity of forms found in the coiled-coil fold. PMID:18782578

  10. X-ray crystal structure of a TRPM assembly domain reveals an antiparallel four-stranded coiled-coil.

    PubMed

    Fujiwara, Yuichiro; Minor, Daniel L

    2008-11-21

    Transient receptor potential (TRP) channels comprise a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Earlier studies showed that members of the TRPM subclass possess a self-assembling tetrameric C-terminal cytoplasmic coiled-coil domain that underlies channel assembly and trafficking. Here, we present the high-resolution crystal structure of the coiled-coil domain of the channel enzyme TRPM7. The crystal structure, together with biochemical experiments, reveals an unexpected four-stranded antiparallel coiled-coil architecture that bears unique features relative to other antiparallel coiled-coils. Structural analysis indicates that a limited set of interactions encode assembly specificity determinants and uncovers a previously unnoticed segregation of TRPM assembly domains into two families that correspond with the phylogenetic divisions seen for the complete subunits. Together, the data provide a framework for understanding the mechanism of TRPM channel assembly and highlight the diversity of forms found in the coiled-coil fold. PMID:18782578

  11. X-Ray Crystal Structure of a TRPM Assembly Domain Reveals An Antiparallel Four-Stranded Coiled-Coil

    SciTech Connect

    Fujiwara, Y.; Minor, D.L.; Jr.

    2009-05-18

    Transient receptor potential (TRP) channels comprise a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Earlier studies showed that members of the TRPM subclass possess a self-assembling tetrameric C-terminal cytoplasmic coiled-coil domain that underlies channel assembly and trafficking. Here, we present the high-resolution crystal structure of the coiled-coil domain of the channel enzyme TRPM7. The crystal structure, together with biochemical experiments, reveals an unexpected four-stranded antiparallel coiled-coil architecture that bears unique features relative to other antiparallel coiled-coils. Structural analysis indicates that a limited set of interactions encode assembly specificity determinants and uncovers a previously unnoticed segregation of TRPM assembly domains into two families that correspond with the phylogenetic divisions seen for the complete subunits. Together, the data provide a framework for understanding the mechanism of TRPM channel assembly and highlight the diversity of forms found in the coiled-coil fold.

  12. GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

    SciTech Connect

    Singh, Pratibha; Savithri, H.S.

    2015-08-15

    Plant viruses exploit the host machinery for targeting the viral genome–movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of this domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER–GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER–GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130–138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm

  13. Structural mapping of the coiled-coil domain of a bacterial condensin and comparative analyses across all domains of life suggest conserved features of SMC proteins.

    PubMed

    Waldman, Vincent M; Stanage, Tyler H; Mims, Alexandra; Norden, Ian S; Oakley, Martha G

    2015-06-01

    The structural maintenance of chromosomes (SMC) proteins form the cores of multisubunit complexes that are required for the segregation and global organization of chromosomes in all domains of life. These proteins share a common domain structure in which N- and C- terminal regions pack against one another to form a globular ATPase domain. This "head" domain is connected to a central, globular, "hinge" or dimerization domain by a long, antiparallel coiled coil. To date, most efforts for structural characterization of SMC proteins have focused on the globular domains. Recently, however, we developed a method to map interstrand interactions in the 50-nm coiled-coil domain of MukB, the divergent SMC protein found in γ-proteobacteria. Here, we apply that technique to map the structure of the Bacillus subtilis SMC (BsSMC) coiled-coil domain. We find that, in contrast to the relatively complicated coiled-coil domain of MukB, the BsSMC domain is nearly continuous, with only two detectable coiled-coil interruptions. Near the middle of the domain is a break in coiled-coil structure in which there are three more residues on the C-terminal strand than on the N-terminal strand. Close to the head domain, there is a second break with a significantly longer insertion on the same strand. These results provide an experience base that allows an informed interpretation of the output of coiled-coil prediction algorithms for this family of proteins. A comparison of such predictions suggests that these coiled-coil deviations are highly conserved across SMC types in a wide variety of organisms, including humans. PMID:25664627

  14. Coiled coil rich proteins (Ccrp) influence molecular pathogenicity of Helicobacter pylori.

    PubMed

    Schätzle, Sarah; Specht, Mara; Waidner, Barbara

    2015-01-01

    Pathogenicity of the human pathogen Helicobacter pylori relies on its capacity to adapt to a hostile environment and to escape the host response. Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its contribution to virulence. In this study we have explored the influence of coiled coil rich proteins (Ccrp) cytoskeletal elements on pathogenicity factors of H. pylori. Deletion of any of the ccrp resulted in a strongly decreased activity of the main pathogenicity factor urease. We further investigated their role using in vitro co-culture experiments with the human gastric adenocarcinoma cell line AGS modeling H. pylori - host cell interactions. Intriguingly, host cell showed only a weak "scattering/hummingbird" phenotype, in which host cells are transformed from a uniform polygonal shape into a severely elongated state characterized by the formation of needle-like projections, after co-incubation with any ccrp deletion mutant. Furthermore, co-incubation with the ccrp59 mutant resulted in reduced type IV secretion system associated activities, e.g. IL-8 production and CagA translocation/phosphorylation. Thus, in addition to their role in maintaining the helical cell shape of H. pylori Ccrp proteins influence many cellular processes and are thereby crucial for the virulence of this human pathogen. PMID:25822999

  15. Growth factor identity is encoded by discrete coiled coil rotamers in the EGFR juxtamembrane region

    PubMed Central

    Doerner, Amy; Scheck, Rebecca; Schepartz, Alanna

    2015-01-01

    Summary Binding of the growth factor TGF-α to the EGFR extracellular domain is encoded through the formation of a unique anti-parallel coiled coil within the juxtamembrane segment. This new coiled coil is an ‘inside-out’ version of the coiled coil formed in the presence of EGF. A third, intermediary coiled coil interface is formed in the juxtamembrane segment when EGFR is stimulated with betacellulin. The seven growth factors that activate EGFR in mammalian systems (EGF, TGF-α, epigen, epiregulin, betacellulin, heparin-binding EGF, and amphiregulin) fall into distinct categories in which the structure of the coiled coil induced within the juxtamembrane segment correlates with cell state. The observation that coiled coil state tracks with the downstream signaling profiles for each ligand provides evidence for growth factor functional selectivity by EGFR. Encoding growth factor identity in alternative coiled coil rotamers provides a simple and elegant method for communicating chemical information across the plasma membrane. PMID:26091170

  16. Coiled-coil intermediate filament stutter instability and molecular unfolding.

    PubMed

    Arslan, Melis; Qin, Zhao; Buehler, Markus J

    2011-05-01

    Intermediate filaments (IFs) are the key components of cytoskeleton in eukaryotic cells and are critical for cell mechanics. The building block of IFs is a coiled-coil alpha-helical dimer, consisting of several domains that include linkers and other structural discontinuities. One of the discontinuities in the dimer's coiled-coil region is the so-called 'stutter' region. The stutter is a region where a variation of the amino acid sequence pattern from other parts of the alpha-helical domains of the protein is found. It was suggested in earlier works that due to this sequence variation, the perfect coiled-coil arrangement ceases to exist. Here, we show using explicit water molecular dynamics and well-tempered metadynamics that for the coil2 domain of vimentin IFs the stutter is more stable in a non-alpha-helical, unfolded state. This causes a local structural disturbance in the alpha helix, which has a global effect on the nanomechanics of the structure. Our analysis suggests that the stutter features an enhanced tendency to unfolding even under the absence of external forces, implying a much greater structural instability than previously assumed. As a result it features a smaller local bending stiffness than other segments and presents a seed for the initiation of molecular bending and unfolding at large deformation. PMID:21516532

  17. Effect of chain length on the formation and stability of synthetic alpha-helical coiled coils.

    PubMed

    Su, J Y; Hodges, R S; Kay, C M

    1994-12-27

    A series of polypeptides containing 9, 12, 16, 19, 23, 26, 30, 33, and 35 amino acid residues was designed to investigate the effects of peptide chain length on the formation and stability of two-stranded alpha-helical dimers or coiled coils. These peptides were synthesized by the solid-phase method, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and characterized by RP-HPLC, amino acid composition analysis, and mass spectrometry. The amphipathic alpha-helical peptides were designed to dimerize by interchain hydrophobic interactions at positions a and d and interchain salt bridges between lysine and glutamic acid residues at positions e and g of the repeating heptad sequence of Glu-Ile-Glu-Ala-Leu-Lys-Ala (g-a-b-c-d-e-f). The ability of these peptides to form alpha-helical structures in the presence and absence of a helix-inducing reagent (trifluoroethanol) was monitored by circular dichroism spectroscopy. The helicity of the peptides increased with increasing chain length in a cooperative manner. A minimum of three heptads corresponding to six helical turns was required for a peptide to adopt the two-stranded alpha-helical coiled coil conformation in aqueous medium. The increased stability of the peptides as a result of an increase in hydrophobic interactions (chain length) was demonstrated by the shift in the transitions of the guanidine hydrochloride (Gdn.HCl) denaturation and thermal unfolding profiles. The concentrations of denaturant (Gdn.HCl) required to achieve 50% denaturation are 3.2, 4.9, 6.9, and 7.5 M for peptides 23r, 26r, 30r, and 33r, respectively, in aqueous medium. However, the effect of a chain length increase on coiled-coil stability was not additive. The melting temperature, Tm, at which 50% of the helicity is lost, increased by 34 degrees C in changing the peptide chain length from 23 to 26; however, that shift was only 14 degrees C when the chain length was increased from 30 to 33 residues. These results are

  18. Designed coiled coils promote folding of a recombinant bacterial collagen.

    PubMed

    Yoshizumi, Ayumi; Fletcher, Jordan M; Yu, Zhuoxin; Persikov, Anton V; Bartlett, Gail J; Boyle, Aimee L; Vincent, Thomas L; Woolfson, Derek N; Brodsky, Barbara

    2011-05-20

    Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the Streptococcus pyogenes collagen-like protein Scl2, a V domain predicted to be largely α-helical, occurs N-terminal to the collagen triple helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, three-stranded, α-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ∼37 °C and CC at >90 °C. Heating the hybrid proteins to 50 °C unfolds CL, leaving CC intact, and upon cooling, the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ∼37 °C thermal stability for CL but shows less triple helix at low temperature and less denaturation at 50 °C. Most strikingly however, in CC-CL-CC, the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end, whereas initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this because, where present, there is generally only one coiled-coil domain close to the triple helix, and it is nearly always N-terminal to the collagen repeat. PMID:21454493

  19. Transmembrane and coiled-coil domain family 1 is a novel protein of the endoplasmic reticulum.

    PubMed

    Zhang, Chao; Kho, Yik-Shing; Wang, Zhe; Chiang, Yan Ting; Ng, Gary K H; Shaw, Pang-Chui; Wang, Yuzhuo; Qi, Robert Z

    2014-01-01

    The endoplasmic reticulum (ER) is a continuous membrane network in eukaryotic cells comprising the nuclear envelope, the rough ER, and the smooth ER. The ER has multiple critical functions and a characteristic structure. In this study, we identified a new protein of the ER, TMCC1 (transmembrane and coiled-coil domain family 1). The TMCC family consists of at least 3 putative proteins (TMCC1-3) that are conserved from nematode to human. We show that TMCC1 is an ER protein that is expressed in diverse human cell lines. TMCC1 contains 2 adjacent transmembrane domains near the C-terminus, in addition to coiled-coil domains. TMCC1 was targeted to the rough ER through the transmembrane domains, whereas the N-terminal region and C-terminal tail of TMCC1 were found to reside in the cytoplasm. Moreover, the cytosolic region of TMCC1 formed homo- or hetero-dimers or oligomers with other TMCC proteins and interacted with ribosomal proteins. Notably, overexpression of TMCC1 or its transmembrane domains caused defects in ER morphology. Our results suggest roles of TMCC1 in ER organization. PMID:24454821

  20. Selective amine labeling of cell surface proteins guided by coiled-coil assembly.

    PubMed

    Yano, Yoshiaki; Furukawa, Nami; Ono, Satoshi; Takeda, Yuki; Matsuzaki, Katsumi

    2016-11-01

    Covalent labeling of target proteins in living cells is useful for both fluorescence live-cell imaging and the subsequent biochemical analyses of the proteins. Here, we report an efficient method for the amine labeling of membrane proteins on the cell surface, guided by a noncovalent coiled-coil interaction. A carboxyl sulfosuccinimidyl ester introduced at the C-terminus of the coiled-coil probe reacted with target proteins under mild labeling conditions ([probe] = 150 nM, pH 7.4, 25°C) for 20 min. Various fluorescent moieties with different hydrophobicities are available for covalent labeling with high signal/background labeling ratios. Using this method, oligomeric states of glycophorin A (GpA) were compared in mammalian CHO-K1 cells and sodium dodecyl sulfate (SDS) micelles. In the cell membranes, no significant self-association of GpA was detected, whereas SDS-PAGE suggested partial dimerization of the proteins. Membrane cholesterol was found to be an important factor that suppressed the dimerization of GpA. Thus, the covalent functionality enables direct comparison of the oligomeric state of membrane proteins under various conditions. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 484-490, 2016. PMID:26285787

  1. Dimerization of the DYT6 dystonia protein, THAP1, requires residues within the coiled-coil domain.

    PubMed

    Sengel, Cem; Gavarini, Sophie; Sharma, Nutan; Ozelius, Laurie J; Bragg, D Cristopher

    2011-09-01

    Thanatos-associated [THAP] domain-containing apoptosis-associated protein 1 (THAP1) is a DNA-binding protein that has been recently associated with DYT6 dystonia, a hereditary movement disorder involving sustained, involuntary muscle contractions. A large number of dystonia-related mutations have been identified in THAP1 in diverse patient populations worldwide. Previous reports have suggested that THAP1 oligomerizes with itself via a C-terminal coiled-coil domain, raising the possibility that DYT6 mutations in this region might affect this interaction. In this study, we examined the ability of wild-type THAP1 to bind itself and the effects on this interaction of the following disease mutations: C54Y, F81L, ΔF132, T142A, I149T, Q154fs180X, and A166T. The results confirmed that wild-type THAP1 associated with itself and most of the DYT6 mutants tested, except for the Q154fs180X variant, which loses most of the coiled-coil domain because of a frameshift at position 154. However, deletion of C-terminal residues after position 166 produced a truncated variant of THAP1 that was able to bind the wild-type protein. The interaction of THAP1 with itself therefore required residues within a 13-amino acid region (aa 154-166) of the coiled-coil domain. Further inspection of this sequence revealed elements highly consistent with previous descriptions of leucine zippers, which serve as dimerization domains in other transcription factor families. Based on this similarity, a structural model was generated to predict how hydrophobic residues in this region may mediate dimerization. These observations offer additional insight into the role of the coiled-coil domain in THAP1, which may facilitate future analyses of DYT6 mutations in this region. PMID:21752024

  2. Dimerization of the DYT6 dystonia protein, THAP1, requires residues within the coiled-coil domain

    PubMed Central

    Sengel, Cem; Gavarini, Sophie; Sharma, Nutan; Ozelius, Laurie J.; Bragg, D. Cristopher

    2011-01-01

    THAP1 is a DNA binding protein that has been recently associated with DYT6 dystonia, a hereditary movement disorder involving sustained, involuntary muscle contractions. A large number of dystonia-related mutations have been identified in THAP1 in diverse patient populations worldwide. Previous reports have suggested that THAP1 oligomerizes with itself via a C-terminal coiled-coil domain, raising the possibility that DYT6 mutations in this region might affect this interaction. In this study we examined the ability of wild-type THAP1 to bind itself and the effects on this interaction of the following disease mutations: C54Y, F81L, ΔF132, T142A, I149T, Q154fs180X, and A166T. The results confirmed that wild-type THAP1 associated with itself and most of the DYT6 mutants tested, except for the Q154fs180X variant, which loses most of the coiled-coil domain due to a frameshift at position 154. However, deletion of C-terminal residues after position 166 produced a truncated variant of THAP1 that was able to bind the wild-type protein. The interaction of THAP1 with itself therefore required residues within a 13-amino acid region (aa 154–166) of the coiled-coil domain. Further inspection of this sequence revealed elements highly consistent with previous descriptions of leucine zippers, which serve as dimerization domains in other transcription factor families. Based on this similarity, a structural model was generated to predict how hydrophobic residues in this region may mediate dimerization. These observations offer additional insight into the role of the coiled-coil domain in THAP1, which may facilitate future analyses of DYT6 mutations in this region. PMID:21752024

  3. The structure of human SFPQ reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation

    PubMed Central

    Lee, Mihwa; Sadowska, Agata; Bekere, Indra; Ho, Diwei; Gully, Benjamin S.; Lu, Yanling; Iyer, K. Swaminathan; Trewhella, Jill; Fox, Archa H.; Bond, Charles S.

    2015-01-01

    SFPQ, (a.k.a. PSF), is a human tumor suppressor protein that regulates many important functions in the cell nucleus including coordination of long non-coding RNA molecules into nuclear bodies. Here we describe the first crystal structures of Splicing Factor Proline and Glutamine Rich (SFPQ), revealing structural similarity to the related PSPC1/NONO heterodimer and a strikingly extended structure (over 265 Å long) formed by an unusual anti-parallel coiled-coil that results in an infinite linear polymer of SFPQ dimers within the crystals. Small-angle X-ray scattering and transmission electron microscopy experiments show that polymerization is reversible in solution and can be templated by DNA. We demonstrate that the ability to polymerize is essential for the cellular functions of SFPQ: disruptive mutation of the coiled-coil interaction motif results in SFPQ mislocalization, reduced formation of nuclear bodies, abrogated molecular interactions and deficient transcriptional regulation. The coiled-coil interaction motif thus provides a molecular explanation for the functional aggregation of SFPQ that directs its role in regulating many aspects of cellular nucleic acid metabolism. PMID:25765647

  4. Tailoring Supramolecular Peptide-Poly(ethylene glycol) Hydrogels by Coiled Coil Self-Assembly and Self-Sorting.

    PubMed

    Dånmark, Staffan; Aronsson, Christopher; Aili, Daniel

    2016-06-13

    Physical hydrogels are extensively used in a wide range of biomedical applications. However, different applications require hydrogels with different mechanical and structural properties. Tailoring these properties demands exquisite control over the supramolecular interactions involved. Here we show that it is possible to control the mechanical properties of hydrogels using de novo designed coiled coil peptides with different affinities for dimerization. Four different nonorthogonal peptides, designed to fold into four different coiled coil heterodimers with dissociation constants spanning from μM to pM, were conjugated to star-shaped 4-arm poly(ethylene glycol) (PEG). The different PEG-coiled coil conjugates self-assemble as a result of peptide heterodimerization. Different combinations of PEG-peptide conjugates assemble into PEG-peptide networks and hydrogels with distinctly different thermal stabilities, supramolecular, and rheological properties, reflecting the peptide dimer affinities. We also demonstrate that it is possible to rationally modulate the self-assembly process by means of thermodynamic self-sorting by sequential additions of nonpegylated peptides. The specific interactions involved in peptide dimerization thus provides means for programmable and reversible self-assembly of hydrogels with precise control over rheological properties, which can significantly facilitate optimization of their overall performance and adaption to different processing requirements and applications. PMID:27219681

  5. Structural basis for cargo binding and autoinhibition of Bicaudal-D1 by a parallel coiled-coil with homotypic registry

    SciTech Connect

    Terawaki, Shin-ichi; Yoshikane, Asuka; Higuchi, Yoshiki; Wakamatsu, Kaori

    2015-05-01

    Bicaudal-D1 (BICD1) is an α-helical coiled-coil protein mediating the attachment of specific cargo to cytoplasmic dynein. It plays an essential role in minus end-directed intracellular transport along microtubules. The third C-terminal coiled-coil region of BICD1 (BICD1 CC3) has an important role in cargo sorting, including intracellular vesicles associating with the small GTPase Rab6 and the nuclear pore complex Ran binding protein 2 (RanBP2), and inhibiting the association with cytoplasmic dynein by binding to the first N-terminal coiled-coil region (CC1). The crystal structure of BICD1 CC3 revealed a parallel homodimeric coiled-coil with asymmetry and complementary knobs-into-holes interactions, differing from Drosophila BicD CC3. Furthermore, our binding study indicated that BICD1 CC3 possesses a binding surface for two distinct cargos, Rab6 and RanBP2, and that the CC1-binding site overlaps with the Rab6-binding site. These findings suggest a molecular basis for cargo recognition and autoinhibition of BICD proteins during dynein-dependent intracellular retrograde transport. - Highlights: • BICD1 CC3 is a parallel homodimeric coiled-coil with axial asymmetry. • The coiled-coil packing of BICD1 CC3 is adapted to the equivalent heptad position. • BICD1 CC3 has distinct binding sites for two classes of cargo, Rab6 and RanBP2. • The CC1-binding site of BICD1 CC3 overlaps with the Rab6-binding site.

  6. A Switch from Parallel to Antiparallel Strand Orientation in a Coiled-Coil X-Ray Structure via Two Core Hydrophobic Mutations

    PubMed Central

    Malashkevich, Vladimir N.; Higgins, Chelsea D.; Almo, Steven C.; Lai, Jonathan R.

    2016-01-01

    The coiled-coil is one of the most ubiquitous and well studied protein structural motifs. Significant effort has been devoted to dissecting subtle variations of the typical heptad repeat sequence pattern that can designate larger topological features such as relative α-helical orientation and oligomer size. Here we report the X-ray structure of a model coiled-coil peptide, HA2-Del-L2seM, which forms an unanticipated core antiparallel dimer with potential sites for discrete higher-order multimerization (trimer or tetramer). In the X-ray structure, a third, partially-ordered α-helix is weakly associated with the antiparallel dimer and analytical ultracentrifugation experiments indicate the peptide forms a well-defined tetramer in solution. The HA2-Del-L2seM sequence is closely related to a parent model peptide, HA2-Del, which we previously reported adopts a parallel trimer; HA2-Del-L2seM differs by only hydrophobic leucine to selenomethione mutations and thus this subtle difference is sufficient to switch both relative α-helical topology and number of α-helices participating in the coiled-coil. Comparison of the X-ray structures of HA2-Del-L2seM (reported here) with the HA2-Del parent (reported previously) reveals novel interactions involving the selenomethionine residues that promote antiparallel coiled-coil configuration and preclude parallel trimer formation. These novel atomic insights are instructive for understanding subtle features that can affect coiled-coil topology and provide additional information for design of antiparallel coiled-coils. PMID:25753192

  7. Structure of Leishmania donovani coronin coiled coil domain reveals an antiparallel 4 helix bundle with inherent asymmetry.

    PubMed

    Nayak, Ashok Ranjan; Karade, Sharanbasappa Shrimant; Srivastava, Vijay Kumar; Rana, Ajay Kumar; Gupta, C M; Sahasrabuddhe, Amogh A; Pratap, J Venkatesh

    2016-07-01

    Coiled coils are ubiquitous structural motifs that serve as a platform for protein-protein interactions and play a central role in myriad physiological processes. Though the formation of a coiled coil requires only the presence of suitably spaced hydrophobic residues, sequence specificities have also been associated with specific oligomeric states. RhXXhE is one such sequence motif, associated with parallel trimers, found in coronins and other proteins. Coronin, present in all eukaryotes, is an actin-associated protein involved in regulating actin turnover. Most eukaryotic coronins possess the RhXXhE trimerization motif. However, a unique feature of parasitic kinetoplastid coronin is that the positions of R and E are swapped within their coiled coil domain, but were still expected to form trimers. To understand the role of swapped motif in oligomeric specificity, we determined the X-ray crystal structure of Leishmania donovani coronin coiled coil domain (LdCoroCC) at 2.2Å, which surprisingly, reveals an anti-parallel tetramer assembly. Small angle X-ray scattering studies and chemical crosslinking confirm the tetramer in solution and is consistent with the oligomerization observed in the full length protein. Structural analyses reveal that LdCoroCC possesses an inherent asymmetry, in that one of the helices of the bundle is axially shifted with respect to the other three. The analysis also identifies steric reasons that cause this asymmetry. The bundle adapts an extended a-d-e core packing, the e residue being polar (with an exception) which results in a thermostable bundle with polar and apolar interfaces, unlike the existing a-d-e core antiparallel homotetramers with apolar core. Functional implications of the anti-parallel association in kinetoplastids are discussed. PMID:26940672

  8. L1 retrotransposition requires rapid ORF1p oligomerization, a novel coiled coil-dependent property conserved despite extensive remodeling

    PubMed Central

    Naufer, M. Nabuan; Callahan, Kathryn E.; Cook, Pamela R.; Perez-Gonzalez, Cesar E.; Williams, Mark C.; Furano, Anthony V.

    2016-01-01

    Detailed mechanistic understanding of L1 retrotransposition is sparse, particularly with respect to ORF1p, a coiled coil-mediated homotrimeric nucleic acid chaperone that can form tightly packed oligomers on nucleic acids. Although the coiled coil motif is highly conserved, it is uniquely susceptible to evolutionary change. Here we studied three ORF1 proteins: a modern human one (111p), its resuscitated primate ancestor (555p) and a mosaic modern protein (151p) wherein 9 of the 30 coiled coil substitutions retain their ancestral state. While 111p and 555p equally supported retrotransposition, 151p was inactive. Nonetheless, they were fully active in bulk assays of nucleic acid interactions including chaperone activity. However, single molecule assays showed that 151p trimers form stably bound oligomers on ssDNA at <1/10th the rate of the active proteins, revealing that oligomerization rate is a novel critical parameter of ORF1p activity in retrotransposition conserved for at least the last 25 Myr of primate evolution. PMID:26673717

  9. N-Terminal Coiled-Coil Structure of ATPase Subunits of 26S Proteasome Is Crucial for Proteasome Function

    PubMed Central

    Inobe, Tomonao; Genmei, Reiko

    2015-01-01

    The proteasome is an essential proteolytic machine in eukaryotic cells, where it removes damaged proteins and regulates many cellular activities by degrading ubiquitinated proteins. Its heterohexameric AAA+ ATPase Rpt subunits play a central role in proteasome activity by the engagement of substrate unfolding and translocation for degradation; however, its detailed mechanism remains poorly understood. In contrast to AAA+ ATPase domains, their N-terminal regions of Rpt subunits substantially differ from each other. Here, to investigate the requirements and roles of the N-terminal regions of six Rpt subunits derived from Saccharomyces cerevisiae, we performed systematic mutational analysis using conditional knockdown yeast strains for each Rpt subunit and bacterial heterologous expression system of the base subcomplex. We showed that the formation of the coiled-coil structure was the most important for the N-terminal region of Rpt subunits. The primary role of coiled-coil structure would be the maintenance of the ring structure with the defined order. However, the coiled-coil region would be also be involved in substrate recognition and an interaction between lid and base subcomplexes. PMID:26208326

  10. Hierarchical Cascades of Instability Govern the Mechanics of Coiled Coils: Helix Unfolding Precedes Coil Unzipping

    PubMed Central

    Hamed, Elham; Keten, Sinan

    2014-01-01

    Coiled coils are a fundamental emergent motif in proteins found in structural biomaterials, consisting of α-helical secondary structures wrapped in a supercoil. A fundamental question regarding the thermal and mechanical stability of coiled coils in extreme environments is the sequence of events leading to the disassembly of individual oligomers from the universal coiled-coil motifs. To shed light on this phenomenon, here we report atomistic simulations of a trimeric coiled coil in an explicit water solvent and investigate the mechanisms underlying helix unfolding and coil unzipping in the assembly. We employ advanced sampling techniques involving steered molecular dynamics and metadynamics simulations to obtain the free-energy landscapes of single-strand unfolding and unzipping in a three-stranded assembly. Our comparative analysis of the free-energy landscapes of instability pathways shows that coil unzipping is a sequential process involving multiple intermediates. At each intermediate state, one heptad repeat of the coiled coil first unfolds and then unzips due to the loss of contacts with the hydrophobic core. This observation suggests that helix unfolding facilitates the initiation of coiled-coil disassembly, which is confirmed by our 2D metadynamics simulations showing that unzipping of one strand requires less energy in the unfolded state compared with the folded state. Our results explain recent experimental findings and lay the groundwork for studying the hierarchical molecular mechanisms that underpin the thermomechanical stability/instability of coiled coils and similar protein assemblies. PMID:25028889

  11. De Novo Design of Ln(III) Coiled Coils for Imaging Applications

    PubMed Central

    2014-01-01

    A new peptide sequence (MB1) has been designed which, in the presence of a trivalent lanthanide ion, has been programmed to self-assemble to form a three stranded metallo-coiled coil, Ln(III)(MB1)3. The binding site has been incorporated into the hydrophobic core using natural amino acids, restricting water access to the lanthanide. The resulting terbium coiled coil displays luminescent properties consistent with a lack of first coordination sphere water molecules. Despite this the gadolinium coiled coil, the first to be reported, displays promising magnetic resonance contrast capabilities. PMID:24405157

  12. Molecular basis of the STIL coiled coil oligomerization explains its requirement for de-novo formation of centrosomes in mammalian cells

    PubMed Central

    David, Ahuvit; Amartely, Hadar; Rabinowicz, Noa; Shamir, Mai; Friedler, Assaf; Izraeli, Shai

    2016-01-01

    The STIL protein is essential for centriole replication and for the non-templated, de novo centriole biogenesis that is required for mammalian embryogenesis. Here we performed quantitative biophysical and structural analysis of the central short coiled coil domain (CCD) of STIL that is critical for its function. Using biophysical, biochemical and cell biology approaches, we identified the specific residues in the CCD that mediate the oligomerization, centrosomal localization and protein interactions of STIL. We characterized the structural properties of the coiled coil peptide using circular dichroism spectroscopy and size exclusion chromatography. We identified two regions in this domain, containing eight hydrophobic residues, which mediate the coiled coil oligomerization. Mutations in these residues destabilized the coiled coil thermodynamically but in most cases did not affect its secondary structure. Reconstituting mouse embryonic fibroblasts lacking endogenous Stil, we show that STIL oligomerization mediated by these residues is not only important for the centrosomal functions of STIL during the canonical duplication process but also for de-novo formation of centrosomes. PMID:27075531

  13. The Golgin Family of Coiled-Coil Tethering Proteins

    PubMed Central

    Witkos, Tomasz M.; Lowe, Martin

    2016-01-01

    The golgins are a family of predominantly coiled-coil proteins that are localized to the Golgi apparatus. Golgins are present in all eukaryotes, suggesting an evolutionary conserved function. Golgins are anchored to the Golgi membrane by their carboxy terminus and are predicted to adopt an extended conformation that projects into the surrounding cytoplasm. This arrangement is ideal for the capture or tethering of nearby membranes or cytoskeletal elements. Golgin-mediated tethering is thought to be important for vesicular traffic at the Golgi apparatus, the maintenance of Golgi architecture, as well as the positioning of the Golgi apparatus within cells. In addition to acting as tethers, some golgins can also sequester various factors at the Golgi membrane, allowing for the spatiotemporal regulation of downstream cellular functions. Although it is now established that golgins are membrane and cytoskeleton tethers, the mechanisms underlying tethering remain poorly defined. Moreover, the importance of golgin-mediated tethering in a physiological context remains to be fully explored. This review will describe our current understanding of golgin function, highlighting recent progress that has been made, and goes on to discuss outstanding questions and potential avenues for future research with regard to this family of conserved Golgi-associated proteins. PMID:26793708

  14. pH sensitive coiled coils: a strategy for enhanced liposomal drug delivery.

    PubMed

    Reja, Rahi M; Khan, Mohsina; Singh, Sumeet K; Misra, Rajkumar; Shiras, Anjali; Gopi, Hosahudya N

    2016-03-01

    Stimuli responsive controlled release from liposome based vesicles is a promising strategy for the site specific delivery of drugs. Herein, we report the design of pH sensitive coiled coils and their incorporation into the liposome as triggers for the controlled release of encapsulated drugs. The designed coiled coil peptides with the incorporation of environment sensitive fluorescent amino acids were found to be stable at physiological pH and unstructured while changing the pH of the environment to either acidic or basic. This pH dependent conformational switch of the coiled-coil polypeptides was exploited as triggers for the enhanced release of the encapsulated drug molecules from liposomes. The SEM, DLS and TEM analysis revealed the uniform morphology of the peptide liposome hybrid vesicles. Further, the drug encapsulated liposome internalization experiments with cancer cells revealed the enhanced release and accumulation of drugs in the acidic lysosomal compartments in comparison with liposomes without coiled coils. PMID:26876788

  15. Coiled-coil protein composition of 22 proteomes – differences and common themes in subcellular infrastructure and traffic control

    PubMed Central

    Rose, Annkatrin; Schraegle, Shannon J; Stahlberg, Eric A; Meier, Iris

    2005-01-01

    Background Long alpha-helical coiled-coil proteins are involved in diverse organizational and regulatory processes in eukaryotic cells. They provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems and tissues, motors, levers, rotating arms, and possibly springs. Mutations in long coiled-coil proteins have been implemented in a growing number of human diseases. Using the coiled-coil prediction program MultiCoil, we have previously identified all long coiled-coil proteins from the model plant Arabidopsis thaliana and have established a searchable Arabidopsis coiled-coil protein database. Results Here, we have identified all proteins with long coiled-coil domains from 21 additional fully sequenced genomes. Because regions predicted to form coiled-coils interfere with sequence homology determination, we have developed a sequence comparison and clustering strategy based on masking predicted coiled-coil domains. Comparing and grouping all long coiled-coil proteins from 22 genomes, the kingdom-specificity of coiled-coil protein families was determined. At the same time, a number of proteins with unknown function could be grouped with already characterized proteins from other organisms. Conclusion MultiCoil predicts proteins with extended coiled-coil domains (more than 250 amino acids) to be largely absent from bacterial genomes, but present in archaea and eukaryotes. The structural maintenance of chromosomes proteins and their relatives are the only long coiled-coil protein family clearly conserved throughout all kingdoms, indicating their ancient nature. Motor proteins, membrane tethering and vesicle transport proteins are the dominant eukaryote-specific long coiled-coil proteins, suggesting that coiled-coil proteins have gained functions in the increasingly complex processes of subcellular infrastructure maintenance and trafficking control of the eukaryotic cell. PMID:16288662

  16. Critical evaluation of in silico methods for prediction of coiled-coil domains in proteins.

    PubMed

    Li, Chen; Ching Han Chang, Catherine; Nagel, Jeremy; Porebski, Benjamin T; Hayashida, Morihiro; Akutsu, Tatsuya; Song, Jiangning; Buckle, Ashley M

    2016-03-01

    Coiled-coils refer to a bundle of helices coiled together like strands of a rope. It has been estimated that nearly 3% of protein-encoding regions of genes harbour coiled-coil domains (CCDs). Experimental studies have confirmed that CCDs play a fundamental role in subcellular infrastructure and controlling trafficking of eukaryotic cells. Given the importance of coiled-coils, multiple bioinformatics tools have been developed to facilitate the systematic and high-throughput prediction of CCDs in proteins. In this article, we review and compare 12 sequence-based bioinformatics approaches and tools for coiled-coil prediction. These approaches can be categorized into two classes: coiled-coil detection and coiled-coil oligomeric state prediction. We evaluated and compared these methods in terms of their input/output, algorithm, prediction performance, validation methods and software utility. All the independent testing data sets are available at http://lightning.med.monash.edu/coiledcoil/. In addition, we conducted a case study of nine human polyglutamine (PolyQ) disease-related proteins and predicted CCDs and oligomeric states using various predictors. Prediction results for CCDs were highly variable among different predictors. Only two peptides from two proteins were confirmed to be CCDs by majority voting. Both domains were predicted to form dimeric coiled-coils using oligomeric state prediction. We anticipate that this comprehensive analysis will be an insightful resource for structural biologists with limited prior experience in bioinformatics tools, and for bioinformaticians who are interested in designing novel approaches for coiled-coil and its oligomeric state prediction. PMID:26177815

  17. Crystal Structure of a Super Leucine Zipper an Extended Two-Stranded Super Long Coiled Coil

    SciTech Connect

    J Diao

    2011-12-31

    Coiled coil is a ubiquitous structural motif in proteins, with two to seven alpha helices coiled together like the strands of a rope, and coiled coil folding and assembly is not completely understood. A GCN4 leucine zipper mutant with four mutations of K3A, D7A, Y17W, and H18N has been designed, and the crystal structure has been determined at 1.6 {angstrom} resolution. The peptide monomer shows a helix trunk with short curved N- and C-termini. In the crystal, two monomers cross in 35{sup o} and form an X-shaped dimer, and each X-shaped dimer is welded into the next one through sticky hydrophobic ends, thus forming an extended two-stranded, parallel, super long coiled coil rather than a discrete, two-helix coiled coil of the wild-type GCN4 leucine zipper. Leucine residues appear at every seventh position in the super long coiled coil, suggesting that it is an extended super leucine zipper. Compared to the wild-type leucine zipper, the N-terminus of the mutant has a dramatic conformational change and the C-terminus has one more residue Glu 32 determined. The mutant X-shaped dimer has a large crossing angle of 35{sup o} instead of 18{sup o} in the wild-type dimer. The results show a novel assembly mode and oligomeric state of coiled coil, and demonstrate that mutations may affect folding and assembly of the overall coiled coil. Analysis of the formation mechanism of the super long coiled coil may help understand and design self-assembling protein fibers.

  18. Structural Characteristics of the Redox-sensing Coiled Coil in the Voltage-gated H+ Channel*

    PubMed Central

    Fujiwara, Yuichiro; Takeshita, Kohei; Nakagawa, Atsushi; Okamura, Yasushi

    2013-01-01

    Oxidation is an important biochemical defense mechanism, but it also elicits toxicity; therefore, oxidation must be under strict control. In phagocytotic events in neutrophils, the voltage-gated H+ (Hv) channel is a key regulator of the production of reactive oxygen species against invading bacteria. The cytoplasmic domain of the Hv channel forms a dimeric coiled coil underpinning a dimerized functional unit. Importantly, in the alignment of the coiled-coil core, a conserved cysteine residue forms a potential intersubunit disulfide bond. In this study, we solved the crystal structures of the coiled-coil domain in reduced, oxidized, and mutated (Cys → Ser) states. The crystal structures indicate that a pair of Cys residues forms an intersubunit disulfide bond dependent on the redox conditions. CD spectroscopy revealed that the disulfide bond increases the thermal stability of the coiled-coil protein. We also reveal that two thiol modifier molecules are able to bind to Cys in a redox-dependent manner without disruption of the dimeric coiled-coil assembly. Thus, the biochemical properties of the cytoplasmic coiled-coil domain in the Hv channel depend on the redox condition, which may play a role in redox sensing in the phagosome. PMID:23667254

  19. Natural templates for coiled-coil biomaterials from praying mantis egg cases.

    PubMed

    Walker, Andrew A; Weisman, Sarah; Kameda, Tsunenori; Sutherland, Tara D

    2012-12-10

    Whereas there is growing interest in producing biomaterials containing coiled-coils, relatively few studies have made use of naturally occurring fibrous proteins. In this study, we have characterized fibrous proteins used by mother praying mantises to produce an extensive covering for their eggs called an ootheca and demonstrate the production of artificial ootheca using recombinantly produced proteins. Examination of natural oothecae by infrared spectroscopy and solid-state nuclear magnetic resonance revealed the material to consist of proteins organized predominately as coiled-coils. Two structural proteins, Mantis Fibroin 1 and Mantis Fibroin 2, were identified in ootheca from each of three species. Between species, the primary sequences of both proteins had diverged considerably, but other features were tightly conserved, including low molecular weight, high abundance of Ala, Glu, Lys, and Ser, and a triblock-like architecture with extensive central coiled-coil domain. Mantis fibroin hydrophobic cores had an unusual composition containing high levels of alanine and aromatic residues. Recombinantly produced mantis fibroins folded into coiled-coils in solution and could be fabricated into solid materials with high coiled-coil content. The structural features of mantis fibroins and their straightforward recombinant production make them promising templates for the production of coiled-coil biomimetics materials. PMID:23137042

  20. AAFreqCoil: a new classifier to distinguish parallel dimeric and trimeric coiled coils.

    PubMed

    Wang, Xiaofeng; Zhou, Yuan; Yan, Renxiang

    2015-07-01

    Coiled coils are characteristic rope-like protein structures, constituted by one or more heptad repeats. Native coiled-coil structures play important roles in various biological processes, while the designed ones are widely employed in medicine and industry. To date, two major oligomeric states (i.e. dimeric and trimeric states) of a coiled-coil structure have been observed, plausibly exerting different biological functions. Therefore, exploration of the relationship between heptad repeat sequences and coiled coil structures is highly important. In this paper, we develop a new method named AAFreqCoil to classify parallel dimeric and trimeric coiled coils. Our method demonstrated its competitive performance when benchmarked based on 10-fold cross validation and jackknife cross validation. Meanwhile, the rules that can explicitly explain the prediction results of the test coiled coil can be extracted from the AAFreqCoil model for a better explanation of user predictions. A web server and stand-alone program implementing the AAFreqCoil algorithm are freely available at . PMID:25918905

  1. Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3

    SciTech Connect

    Amano, Yosuke; Ishikawa, Rie; Sakatani, Toshio; Ichinose, Junji; Sunohara, Mitsuhiro; Watanabe, Kousuke; Kage, Hidenori; Nakajima, Jun; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2015-02-13

    Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation. - Highlights: • TPM3-ALK fusion protein dimerizes through the coiled-coil structure of TPM3. • Longer coiled-coil structure of TPM3 leads to higher TPM3-ALK dimer formation. • Presence of TPM3-ALK dimer leads to ALK, STAT3, and ERK1/2 phosphorylation. • Presence of TPM3-ALK leads to loss of contact inhibition. • BN-PAGE is a simple technique for visualizing oncogenic dimerization.

  2. Structural domains of vault proteins: a role for the coiled coil domain in vault assembly.

    PubMed

    van Zon, Arend; Mossink, Marieke H; Schoester, Martijn; Scheffer, George L; Scheper, Rik J; Sonneveld, Pieter; Wiemer, Erik A C

    2002-03-01

    Vaults consist of multiple copies of three proteins (MVP, VPARP, and TEP1) and several untranslated RNAs. The function of vaults is unknown but the typical and evolutionary conserved structure indicates a role in intracellular transport. Although all vault components have been identified and characterized, not much is known about vault protein assembly. In this study we identified and analyzed structural domains involved in vault assembly with emphasis on protein-protein interactions. Using a yeast two-hybrid system, we demonstrate within MVP an intramolecular binding site and show that MVP molecules interact with each other via their coiled coil domain. We show that purified MVP is able to bind calcium, most likely at calcium-binding EF-hands. No interactions could be detected between TEP1 and other vault proteins. However, the N-terminal half of MVP binds to a specific domain in the C-terminus of VPARP. Furthermore, VPARP contains amino acid stretches mediating intramolecular binding. PMID:11855821

  3. Crystal Structure of a Coiled-Coil Domain from Human ROCK I

    PubMed Central

    Tu, Daqi; Li, Yiqun; Song, Hyun Kyu; Toms, Angela V.; Gould, Christopher J.; Ficarro, Scott B.; Marto, Jarrod A.; Goode, Bruce L.; Eck, Michael J.

    2011-01-01

    The small GTPase Rho and one of its targets, Rho-associated kinase (ROCK), participate in a variety of actin-based cellular processes including smooth muscle contraction, cell migration, and stress fiber formation. The ROCK protein consists of an N-terminal kinase domain, a central coiled-coil domain containing a Rho binding site, and a C-terminal pleckstrin homology domain. Here we present the crystal structure of a large section of the central coiled-coil domain of human ROCK I (amino acids 535–700). The structure forms a parallel α-helical coiled-coil dimer that is structurally similar to tropomyosin, an actin filament binding protein. There is an unusual discontinuity in the coiled-coil; three charged residues (E613, R617 and D620) are positioned at what is normally the hydrophobic core of coiled-coil packing. We speculate that this conserved irregularity could function as a hinge that allows ROCK to adopt its autoinhibited conformation. PMID:21445309

  4. Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif

    PubMed Central

    Hirtreiter, Angela; Damsma, Gerke E.; Cheung, Alan C. M.; Klose, Daniel; Grohmann, Dina; Vojnic, Erika; Martin, Andrew C. R.; Cramer, Patrick; Werner, Finn

    2010-01-01

    Spt5 is the only known RNA polymerase-associated factor that is conserved in all three domains of life. We have solved the structure of the Methanococcus jannaschii Spt4/5 complex by X-ray crystallography, and characterized its function and interaction with the archaeal RNAP in a wholly recombinant in vitro transcription system. Archaeal Spt4 and Spt5 form a stable complex that associates with RNAP independently of the DNA–RNA scaffold of the elongation complex. The association of Spt4/5 with RNAP results in a stimulation of transcription processivity, both in the absence and the presence of the non-template strand. A domain deletion analysis reveals the molecular anatomy of Spt4/5—the Spt5 Nus-G N-terminal (NGN) domain is the effector domain of the complex that both mediates the interaction with RNAP and is essential for its elongation activity. Using a mutagenesis approach, we have identified a hydrophobic pocket on the Spt5 NGN domain as binding site for RNAP, and reciprocally the RNAP clamp coiled-coil motif as binding site for Spt4/5. PMID:20197319

  5. Accessing Three-Dimensional Crystals with Incorporated Guests through Metal-Directed Coiled-Coil Peptide Assembly.

    PubMed

    Nepal, Manish; Sheedlo, Michael J; Das, Chittaranjan; Chmielewski, Jean

    2016-08-31

    Obtaining three-dimensional (3D) protein and peptide crystals on demand requires a precisely orchestrated hierarchical assembly of biopolymer building blocks. In this work, we disclose a metal-ion-mediated strategy to assemble trimeric coiled-coil peptides in a head-to-tail fashion into linear strands with interstrand interactions. This design led to hexagonal 3D peptide crystal formation within 30 min in the presence of divalent metal ions. The crystal morphology could be controlled by varying the metal ion/peptide ratio, resulting in hexagonal discs to rods. Diffraction studies elucidated the head-to-tail arrangement of the coiled-coil linear strands and their hexagonal, antiparallel packing within the crystal. Unsatisfied ligands at the hexagonal ends of the crystals were harnessed as a powerful means to direct His-tagged fluorophores to distinct locations within the crystals. Overall, the designed hierarchical assembly provides a facile means to obtain 3D peptide crystals and incorporate His-tag-based cargoes and may have potential use in drug delivery and sensor design. PMID:27500907

  6. X-ray Crystallographic Structure and Solution Behavior of an Antiparallel Coiled-Coil Hexamer Formed by de Novo Peptides.

    PubMed

    Spencer, Ryan K; Hochbaum, Allon I

    2016-06-14

    The self-assembly of peptides and proteins into higher-ordered structures is encoded in the amino acid sequence of each peptide or protein. Understanding the relationship among the amino acid sequence, the assembly dynamics, and the structure of well-defined peptide oligomers expands the synthetic toolbox for these structures. Here, we present the X-ray crystallographic structure and solution behavior of de novo peptides that form antiparallel coiled-coil hexamers (ACC-Hex) by an interaction motif neither found in nature nor predicted by existing peptide design software. The 1.70 Å X-ray crystallographic structure of peptide 1a shows six α-helices associating in an antiparallel arrangement around a central axis comprising hydrophobic and aromatic residues. Size-exclusion chromatography studies suggest that peptides 1 form stable oligomers in solution, and circular dichroism experiments show that peptides 1 are stable to relatively high temperatures. Small-angle X-ray scattering studies of the solution behavior of peptide 1a indicate an equilibrium of dimers, hexamers, and larger aggregates in solution. The structures presented here represent a new motif of biomolecular self-assembly not previously observed for de novo peptides and suggest supramolecular design principles for material scaffolds based on coiled-coil motifs containing aromatic residues. PMID:27192036

  7. An engineered coiled-coil polypeptide assembled onto quantum dots for targeted cell imaging

    NASA Astrophysics Data System (ADS)

    Yao, Ming-Hao; Yang, Jie; Song, Ji-Tao; Zhang, Lin; Fang, Bi-Yun; Zhao, Dong-Hui; Xia, Rui-Xue; Jin, Rui-Mei; Zhao, Yuan-Di; Liu, Bo

    2015-12-01

    Quantum dot (QD)-polypeptide probes have been developed through the specific metal-affinity interaction between polypeptides appended with N-terminal polyhistidine sequences and CdSe/ZnS core-shell QDs. The size and charge of a QD-polypeptide can be tuned by using different coiled-coil polypeptides. Compared to glutathione-capped QDs (QD-GSH), QD-polypeptide probes showed an approximately two- to three-fold luminescence increase, and the luminescence increase was not obviously related to the charge of the polypeptide. QD-polypeptide probes with different charge have a great effect on nonspecific cellular uptake. QD-polypeptide probes with negative charge exhibited lower nonspecific cellular uptake in comparison to the QD-GSH, while positively charged QD-polypeptide probes presented higher cellular uptake than the QD-GSH. A targeted QD-ARGD probe can obviously increase targeted cellular uptake in α v β 3 overexpressing HeLa cells compared to QD-A. In addition, QD-polypeptide probes showed lower in vitro cytotoxicity compared to the original QDs. These results demonstrate that these QD-polypeptide probes with high specific cellular uptake, high fluorescence intensity and low background noise are expected to have great potential applications in targeted cell imaging.

  8. pH sensitive coiled coils: a strategy for enhanced liposomal drug delivery

    NASA Astrophysics Data System (ADS)

    Reja, Rahi M.; Khan, Mohsina; Singh, Sumeet K.; Misra, Rajkumar; Shiras, Anjali; Gopi, Hosahudya N.

    2016-02-01

    Stimuli responsive controlled release from liposome based vesicles is a promising strategy for the site specific delivery of drugs. Herein, we report the design of pH sensitive coiled coils and their incorporation into the liposome as triggers for the controlled release of encapsulated drugs. The designed coiled coil peptides with the incorporation of environment sensitive fluorescent amino acids were found to be stable at physiological pH and unstructured while changing the pH of the environment to either acidic or basic. This pH dependent conformational switch of the coiled-coil polypeptides was exploited as triggers for the enhanced release of the encapsulated drug molecules from liposomes. The SEM, DLS and TEM analysis revealed the uniform morphology of the peptide liposome hybrid vesicles. Further, the drug encapsulated liposome internalization experiments with cancer cells revealed the enhanced release and accumulation of drugs in the acidic lysosomal compartments in comparison with liposomes without coiled coils.Stimuli responsive controlled release from liposome based vesicles is a promising strategy for the site specific delivery of drugs. Herein, we report the design of pH sensitive coiled coils and their incorporation into the liposome as triggers for the controlled release of encapsulated drugs. The designed coiled coil peptides with the incorporation of environment sensitive fluorescent amino acids were found to be stable at physiological pH and unstructured while changing the pH of the environment to either acidic or basic. This pH dependent conformational switch of the coiled-coil polypeptides was exploited as triggers for the enhanced release of the encapsulated drug molecules from liposomes. The SEM, DLS and TEM analysis revealed the uniform morphology of the peptide liposome hybrid vesicles. Further, the drug encapsulated liposome internalization experiments with cancer cells revealed the enhanced release and accumulation of drugs in the acidic

  9. Self-Assembling Peptide-Polymer Hydrogels Designed From the Coiled Coil Region of Fibrin

    PubMed Central

    Jing, Peng; Rudra, Jai S.; Herr, Andrew B.; Collier, Joel H.

    2010-01-01

    Biomaterials constructed from self-assembling peptides, peptide derivatives, and peptide-polymer conjugates are receiving increasing attention as defined matrices for tissue engineering, controlled therapeutic release, and in vitro cell expansion, but many are constructed from peptide structures not typically found in the human extracellular matrix. Here we report a self-assembling biomaterial constructed from a designed peptide inspired by the coiled coil domain of human fibrin, the major protein constituent of blood clots and the provisional scaffold of wound healing. Targeted substitutions were made in the residues forming the interface between coiled coil strands for a 37-amino acid peptide from human fibrinogen to stabilize the coiled coil peptide bundle, while the solvent-exposed residues were left unchanged to provide a surface similar to that of the native protein. This peptide, which self-assembled into coiled coil dimers and tetramers, was then used to produce triblock peptide-PEG-peptide bioconjugates that self-assembled into viscoelastic hydrogel biomaterials. PMID:18712921

  10. Self-sorting heterodimeric coiled coil peptides with defined and tuneable self-assembly properties

    PubMed Central

    Aronsson, Christopher; Dånmark, Staffan; Zhou, Feng; Öberg, Per; Enander, Karin; Su, Haibin; Aili, Daniel

    2015-01-01

    Coiled coils with defined assembly properties and dissociation constants are highly attractive components in synthetic biology and for fabrication of peptide-based hybrid nanomaterials and nanostructures. Complex assemblies based on multiple different peptides typically require orthogonal peptides obtained by negative design. Negative design does not necessarily exclude formation of undesired species and may eventually compromise the stability of the desired coiled coils. This work describe a set of four promiscuous 28-residue de novo designed peptides that heterodimerize and fold into parallel coiled coils. The peptides are non-orthogonal and can form four different heterodimers albeit with large differences in affinities. The peptides display dissociation constants for dimerization spanning from the micromolar to the picomolar range. The significant differences in affinities for dimerization make the peptides prone to thermodynamic social self-sorting as shown by thermal unfolding and fluorescence experiments, and confirmed by simulations. The peptides self-sort with high fidelity to form the two coiled coils with the highest and lowest affinities for heterodimerization. The possibility to exploit self-sorting of mutually complementary peptides could hence be a viable approach to guide the assembly of higher order architectures and a powerful strategy for fabrication of dynamic and tuneable nanostructured materials. PMID:26370878

  11. The tripartite motif coiled-coil is an elongated antiparallel hairpin dimer

    PubMed Central

    Sanchez, Jacint G.; Okreglicka, Katarzyna; Chandrasekaran, Viswanathan; Welker, Jordan M.; Sundquist, Wesley I.; Pornillos, Owen

    2014-01-01

    Tripartite motif (TRIM) proteins make up a large family of coiled-coil-containing RING E3 ligases that function in many cellular processes, particularly innate antiviral response pathways. Both dimerization and higher-order assembly are important elements of TRIM protein function, but the atomic details of TRIM tertiary and quaternary structure have not been fully understood. Here, we present crystallographic and biochemical analyses of the TRIM coiled-coil and show that TRIM proteins dimerize by forming interdigitating antiparallel helical hairpins that position the N-terminal catalytic RING domains at opposite ends of the dimer and the C-terminal substrate-binding domains at the center. The dimer core comprises an antiparallel coiled-coil with a distinctive, symmetric pattern of flanking heptad and central hendecad repeats that appear to be conserved across the entire TRIM family. Our studies reveal how the coiled-coil organizes TRIM25 to polyubiquitylate the RIG-I/viral RNA recognition complex and how dimers of the TRIM5α protein are arranged within hexagonal arrays that recognize the HIV-1 capsid lattice and restrict retroviral replication. PMID:24550273

  12. A coiled-coil domain acts as a molecular ruler in LPS chain length regulation

    PubMed Central

    Tuukkanen, Anne; Danciu, Iulia; Svergun, Dmitri I.; Hussain, Rohanah; Liu, Huanting; Whitfield, Chris; Naismith, James H.

    2014-01-01

    Long-chain bacterial polysaccharides play important roles in pathogenicity. In Escherichia coli O9a, a model for ABC transporter dependent polysaccharide assembly, a large extracellular carbohydrate with a narrow distribution of size is polymerized from monosaccharides by a complex of two proteins, WbdA (polymerase) and WbdD (terminating protein). Such careful control of polymerization is recurring theme in biology. Combining crystallography and small angle X-ray scattering, we show that the C-terminal domain of WbdD contains an extended coiled-coil that physically separates WbdA from the catalytic domain of WbdD. The effects of insertions and deletions within the coiled-coil region were analyzed in vivo, revealing that polymer size is controlled by varying the length of the coiled-coil domain. Thus, the coiled-coil domain of WbdD functions as a molecular ruler that, along with WbdA:WbdD stoichiometry, controls the chain length of a model bacterial polysaccharide. PMID:25504321

  13. Coiled-Coil Irregularities and Instabilities in Group A Streptococcus M1 Are Required for Virulence

    SciTech Connect

    McNamara, Case; Zinkernagel, Annelies S.; Macheboeuf, Pauline; Cunningham, Madeleine W.; Nizet, Victor; Ghosh, Partho

    2008-07-21

    Antigenically variable M proteins are major virulence factors and immunogens of the human pathogen group A Streptococcus (GAS). Here, we report the -3 angstrom resolution structure of a GAS M1 fragment containing the regions responsible for eliciting type-specific, protective immunity and for binding fibrinogen, which promotes M1 proinflammatory and antiphagocytic functions. The structure revealed substantial irregularities and instabilities throughout the coiled coil of the M1 fragment. Similar structural irregularities occur in myosin and tropomyosin, explaining the patterns of cross-reactivity seen in autoimmune sequelae of GAS infection. Sequence idealization of a large segment of the M1 coiled coil enhanced stability but diminished fibrinogen binding, proinflammatory effects, and antibody cross-reactivity, whereas it left protective immunogenicity undiminished. Idealized M proteins appear to have promise as vaccine immunogens.

  14. Short peptide tag for covalent protein labeling based on coiled coils.

    PubMed

    Wang, Jianpeng; Yu, Yongsheng; Xia, Jiang

    2014-01-15

    To label proteins covalently, one faces a trade-off between labeling a protein specifically and using a small tag. Often one must compromise one parameter for the other or use additional components, such as an enzyme, to satisfy both requirements. Here, we report a new reaction that covalently labels proteins by using engineered coiled-coil peptides. Harnessing the concept of "proximity-induced reactivity", the 21-amino-acid three-heptad peptides CCE/CCK were modified with a nucleophilic cysteine and an α-chloroacetyl group at selected positions. When pairs of coiled coils associated, an irreversible covalent bond spontaneously formed between the peptides. The specificity of the cross-linking reaction was characterized, the probes were improved by making them bivalent, and the system was used to label a protein in vitro and receptors on the surface of mammalian cells. PMID:24341800

  15. Application of Coiled Coil Peptides in Liposomal Anticancer Drug Delivery Using a Zebrafish Xenograft Model.

    PubMed

    Yang, Jian; Shimada, Yasuhito; Olsthoorn, René C L; Snaar-Jagalska, B Ewa; Spaink, Herman P; Kros, Alexander

    2016-08-23

    The complementary coiled coil forming peptides E4 [(EIAALEK)4] and K4 [(KIAALKE)4] are known to trigger liposomal membrane fusion when tethered to lipid vesicles in the form of lipopeptides. In this study, we examined whether these coiled coil forming peptides can be used for drug delivery applications. First, we prepared E4 peptide modified liposomes containing the far-red fluorescent dye TO-PRO-3 iodide (E4-Lipo-TP3) and confirmed that E4-liposomes could deliver TP3 into HeLa cells expressing K4 peptide on the membrane (HeLa-K) under cell culture conditions in a selective manner. Next, we prepared doxorubicin-containing E4-liposomes (E4-Lipo-DOX) and confirmed that E4-liposomes could also deliver DOX into HeLa-K cells. Moreover, E4-Lipo-DOX showed enhanced cytotoxicity toward HeLa-K cells compared to free doxorubicin. To prove the suitability of E4/K4 coiled coil formation for in vivo drug delivery, we injected E4-Lipo-TP3 or E4-Lipo-DOX into zebrafish xenografts of HeLa-K. As a result, E4-liposomes delivered TP3 to the implanted HeLa-K cells, and E4-Lipo-DOX could suppress cancer proliferation in the xenograft when compared to nontargeted conditions (i.e., zebrafish xenograft with free DOX injection). These data demonstrate that coiled coil formation enables drug selectivity and efficacy in vivo. It is envisaged that these findings are a step forward toward biorthogonal targeting systems as a tool for clinical drug delivery. PMID:27504667

  16. Dynamics of the coiled-coil unfolding transition of myosin rod probed by dissipation force spectrum.

    PubMed

    Taniguchi, Yukinori; Khatri, Bhavin S; Brockwell, David J; Paci, Emanuele; Kawakami, Masaru

    2010-07-01

    The motor protein myosin II plays a crucial role in muscle contraction. The mechanical properties of its coiled-coil region, the myosin rod, are important for effective force transduction during muscle function. Previous studies have investigated the static elastic response of the myosin rod. However, analogous to the study of macroscopic complex fluids, how myosin will respond to physiological time-dependent loads can only be understood from its viscoelastic response. Here, we apply atomic force microscopy using a magnetically driven oscillating cantilever to measure the dissipative properties of single myosin rods that provide unique dynamical information about the coiled-coil structure as a function of force. We find that the friction constant of the single myosin rod has a highly nontrivial variation with force; in particular, the single-molecule friction constant is reduced dramatically and increases again as it passes through the coiled-uncoiled transition. This is a direct indication of a large free-energy barrier to uncoiling, which may be related to a fine-tuned dynamic mechanosignaling response to large and unexpected physiological loads. Further, from the critical force at which the minimum in friction occurs we determine the asymmetry of the bistable landscape that controls uncoiling of the coiled coil. This work highlights the sensitivity of the dissipative signal in force unfolding to dynamic molecular structure that is hidden to the elastic signal. PMID:20655854

  17. Leveraging intrinsic chain anisotropy to align coil-coil block copolymers with magnetic fields

    NASA Astrophysics Data System (ADS)

    Rokhlenko, Yekaterina; Zhang, Kai; Gopinadhan, Manesh; Larson, Steve; Majewski, Pawel; Yager, Kevin; Gopalan, Padma; O'Hern, Corey; Osuji, Chinedum

    Magnetic field alignment of block copolymers (BCPs) has typically relied on the presence of liquid crystalline or crystalline assemblies to provide sufficient magnetic anisotropy to drive alignment. Recent experiments however show that alignment is also possible in simple coil-coil BCPs. In particular, alignment of lamellae was observed in poly(styrene-b-4-vinylpyridine) (PS-P4VP) on cooling across the order-disorder transition at field strengths as low as 1 T, with alignment improving markedly with increasing field strength and decreasing cooling rate. Here we discuss the intrinsic chain anisotropy which drives the observed alignment, and its display as a net microdomain anisotropy due to chain tethering at the block interface. We use in-situ X-ray scattering to study the phase behavior and temperature-, time-, and field- dependent dynamics of magnetic alignment in coil-coil BCPs, highlighting the important roles of chain anisotropy and grain size in alignment. For the right combination of field strength and grain size, we can leverage intrinsic chain anisotropy to magnetically direct self-assembly in other coil-coil systems, including cylinder-forming poly(styrene-b-dimethylsiloxane). Field alignment of PS-P4VP with PEO and other blends provides a route to form functional materials such as nanoporous films and ion conducting polymers.

  18. Cloning and expression analysis of mouse Cclp1, a new gene encoding a coiled-coil-like protein.

    PubMed

    Noben-Trauth, K; Naggert, J K; Nishina, P M

    1997-05-30

    Here we describe the nucleotide sequence and expression pattern of a novel gene termed Coiled-coil-like protein 1 (Cclp1). A 2646bp open reading frame encodes a 882 amino acid protein with a predicted coiled-coil domain at the amino terminus. Cclp1 is expressed in a variety of adult tissues and during different stages of embryogenesis. The broad expression pattern suggests a general cellular function of CCLP1. PMID:9199242

  19. Domain organization, folding and stability of bacteriophage T4 fibritin, a segmented coiled-coil protein.

    PubMed

    Boudko, Sergei P; Londer, Yuri Y; Letarov, Andrei V; Sernova, Natalia V; Engel, Juergen; Mesyanzhinov, Vadim V

    2002-02-01

    Fibritin is a segmented coiled-coil homotrimer of the 486-residue product of phage T4 gene wac. This protein attaches to a phage particle by the N-terminal region and forms fibrous whiskers of 530 A, which perform a chaperone function during virus assembly. The short C-terminal region has a beta-annulus-like structure. We engineered a set of fibritin deletion mutants sequentially truncated from the N-termini, and the mutants were studied by differential scanning calorimetry (DSC) and CD measurements. The analysis of DSC curves indicates that full-length fibritin exhibits three thermal-heat-absorption peaks centred at 321 K (Delta H=1390 kJ x mol trimer(-1)), at 336 K (Delta H=7600 kJ x mol trimer(-1)), and at 345 K (Delta H=515 kJ x mol trimer(-1)). These transitions were assigned to the N-terminal, segmented coiled-coil, and C-terminal functional domains, respectively. The coiled-coil region, containing 13 segments, melts co-operatively as a single domain with a mean enthalpy Delta Hres=21 kJ x mol residue(-1). The ratio of Delta HVH/Delta Hcal for the coiled-coil part of the 120-, 182-, 258- and 281-residue per monomer mutants, truncated from the N-termini, and for full-length fibritin are 0.91, 0.88, 0.42, 0.39, and 0.13, respectively. This gives an indication of the decrease of the 'all-or-none' character of the transition with increasing protein size. The deletion of the 12-residue-long loop in the 120-residue fibritin increases the thermal stability of the coiled-coil region. According to CD data, full-length fibritin and all the mutants truncated from the N-termini refold properly after heat denaturation. In contrast, fibritin XN, which is deleted for the C-terminal domain, forms aggregates inside the cell. The XN protein can be partially refolded by dilution from urea and does not refold after heat denaturation. These results confirm that the C-terminal domain is essential for correct fibritin assembly both in vivo and in vitro and acts as a foldon. PMID

  20. STIM1/Orai1 coiled-coil interplay in the regulation of store-operated calcium entry

    PubMed Central

    Stathopulos, Peter B.; Schindl, Rainer; Fahrner, Marc; Zheng, Le; Gasmi-Seabrook, Geneviève M.; Muik, Martin; Romanin, Christoph; Ikura, Mitsuhiko

    2013-01-01

    Orai1 calcium channels in the plasma membrane are activated by stromal interaction molecule-1 (STIM1), an endoplasmic reticulum calcium sensor, to mediate store-operated calcium entry (SOCE). The cytosolic region of STIM1 contains a long putative coiled-coil (CC)1 segment and shorter CC2 and CC3 domains. Here we present solution nuclear magnetic resonance structures of a trypsin-resistant CC1–CC2 fragment in the apo and Orai1-bound states. Each CC1–CC2 subunit forms a U-shaped structure that homodimerizes through antiparallel interactions between equivalent α-helices. The CC2:CC2′ helix pair clamps two identical acidic Orai1 C-terminal helices at opposite ends of a hydrophobic/basic STIM–Orai association pocket. STIM1 mutants disrupting CC1:CC1′ interactions attenuate, while variants promoting CC1 stability spontaneously activate Orai1 currents. CC2 mutations cause remarkable variability in Orai1 activation because of a dual function in binding Orai1 and autoinhibiting STIM1 oligomerization via interactions with CC3. We conclude that SOCE is activated through dynamic interplay between STIM1 and Orai1 helices. PMID:24351972

  1. Type I macrophage scavenger receptor contains α-helical and collagen-like coiled coils

    NASA Astrophysics Data System (ADS)

    Kodama, Tatsuhiko; Freeman, Mason; Rohrer, Lucia; Zabrecky, James; Matsudaira, Paul; Krieger, Monty

    1990-02-01

    The macrophage scavenger receptor is a trimeric membrane glycoprotein with unusual ligand-binding properties which has been implicated in the development of atherosclerosis. The trimeric structure of the bovine type I scavenger receptor, deduced by complementary DNA cloning, contains three extracellular C-terminal cysteine-rich domains connected to the transmembrane domain by a long fibrous stalk. This stalk structure, composed of an a-helical coiled coil and a collagen-like triple helix, has not previously been observed in an integral membrane protein.

  2. Structural characterization of the C-terminal coiled-coil domains of wild-type and kidney disease-associated mutants of apolipoprotein L1.

    PubMed

    Sharma, Alok K; Friedman, David J; Pollak, Martin R; Alper, Seth L

    2016-05-01

    Trypanosomes that cause sleeping sickness endocytose apolipoprotein L1 (APOL1)-containing trypanolytic factors from human serum, leading to trypanolytic death through generation of APOL1-associated lytic pores in trypanosomal membranes. The trypanosome Trypanosoma brucei rhodesiense counteracts trypanolysis by expressing the surface protein serum response-associated (SRA), which can bind APOL1 common variant G0 to block its trypanolytic activity. However, two missense variants in the C terminal predicted coiled-coil (CC) domains of human APOL1 G1 (S342G/I384M) and G2 (ΔN388Y389) decrease or abrogate APOL1 binding to T. brucei rhodesiense SRA, thus preserving APOL1 trypanolytic activity. These evolutionarily selected APOL1 missense variants, found at a high frequency in some populations of African descent, also confer elevated risk of kidney disease. Understanding the SRA-APOL1 interaction and the role of APOL1 G1 and G2 variants in kidney disease demands structural characterization of the APOL1 CC domain. Using CD, heteronuclear NMR, and molecular dynamics (MD) simulation on structural homology models, we report here unique and dynamic solution conformations of nephropathy variants G1 and G2 as compared with the common variant G0. Conformational plasticity in G1 and G2 CC domains led to interhelical α1-α2 approximation coupled with secondary structural changes and delimited motional properties absent in the G0 CC domain. The G1 substitutions conferred local structural changes principally along helix α1, whereas the G2 deletion altered the structure of both helix α2 and helix α1. These dynamic features of APOL1 CC variants likely reflect their intrinsic structural properties, and should help interpret future APOL1 structural studies and define the contribution of APOL1 risk variants to kidney disease. PMID:26945671

  3. Protein-based hydrogels self-assembled from genetically engineered triblock polypeptides containing coiled-coil domains

    NASA Astrophysics Data System (ADS)

    Xu, Chunyu

    Protein-based biomaterials have great potential in biomedical applications due to their similar composition with biological organisms. Environment-sensitive hydrogels based on proteins can undergo sol-gel transition due to the conformational change of the proteins in response to external stimuli. The physical properties of these hydrogels can be tailored by modification of the protein structures. Two major hypotheses were made in this dissertation. One was that coiled-coil folding motifs could be a good candidate for physical crosslinking in protein-based hydrogels, and the other was that the conformational change of coiled-coils in response to external stimuli could mediate the sol-gel transition of the protein-based hydrogels. The first part established synthesis strategies of the coiled-coil containing proteins using a genetic engineering technique. An important observation was made that the fusion sequence on the proteins could influence the thermal stability of the proteins. In the second part of the research, the self-assembly of hydrogels from a series of triblock polypeptides containing coiled-coils was evaluated. It was found that the hydrogels had a porous interconnected network microstructure. The hydrogels responded to temperature and pH, which correlated to the temperature- and pH-triggered structural transition of the coiled-coil domains. In addition, the formation of hydrogels was reversible in the present or absence of guanidine hydrochloride (GdnHCl). The last part of the research attempted to explore the relationship between the structure of the protein polymers and the physical property of the hydrogels, and to investigate the parameters influencing the hydrogel formation and physical properties. Triblock and diblock polypeptides were designed to contain different lengths of coiled-coil domains. Tyrosine residues were incorporated at selected solvent-exposed positions in order to increase the hydrophobicity of the coiled-coil domains. The

  4. The coiled-coil domain of the Nop56/58 core protein is dispensable for sRNP assembly but is critical for archaeal box C/D sRNP-guided nucleotide methylation

    PubMed Central

    Zhang, Xinxin; Champion, Erica A.; Tran, Elizabeth J.; Brown, Bernard A.; Baserga, Susan J.; Maxwell, E. Stuart

    2006-01-01

    Archaeal box C/D sRNAs guide the methylation of specific nucleotides in archaeal ribosomal and tRNAs. Three Methanocaldococcus jannaschii sRNP core proteins (ribosomal protein L7, Nop56/58, and fibrillarin) bind the box C/D sRNAs to assemble the sRNP complex, and these core proteins are essential for nucleotide methylation. A distinguishing feature of the Nop56/58 core protein is the coiled-coil domain, established by α-helices 4 and 5, that facilitates Nop56/58 self-dimerization in vitro. The function of this coiled-coil domain has been assessed for box C/D sRNP assembly, sRNP structure, and sRNP-guided nucleotide methylation by mutating or deleting this protein domain. Protein pull-down experiments demonstrated that Nop56/58 self-dimerization and Nop56/58 dimerization with the core protein fibrillarin are mutually exclusive protein:protein interactions. Disruption of Nop56/58 homodimerization by alteration of specific amino acids or deletion of the entire coiled-coil domain had no obvious effect upon core protein binding and sRNP assembly. Site-directed mutation of the Nop56/58 homodimerization domain also had no apparent effect upon either box C/D RNP- or C′/D′ RNP-guided nucleotide modification. However, deletion of this domain disrupted guided methylation from both RNP complexes. Nuclease probing of the sRNP assembled with Nop56/58 proteins mutated in the coiled-coil domain indicated that while functional complexes were assembled, box C/D and C′/D′ RNPs were altered in structure. Collectively, these experiments revealed that the self-dimerization of the Nop56/58 coiled-coil domain is not required for assembly of a functional sRNP, but the coiled-coil domain is important for the establishment of wild-type box C/D and C′/D′ RNP structure essential for nucleotide methylation. PMID:16601205

  5. Arabidopsis COP1 SUPPRESSOR 2 Represses COP1 E3 Ubiquitin Ligase Activity through Their Coiled-Coil Domains Association

    PubMed Central

    Jiang, Yan; Ling, Junjie; Hettiarachchi, Chamari; Tellgren-Roth, Christian; Wei, Ning; Deng, Xing Wang

    2015-01-01

    CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) functions as an E3 ubiquitin ligase and mediates a variety of developmental processes in Arabidopsis by targeting a number of key regulators for ubiquitination and degradation. Here, we identify a novel COP1 interacting protein, COP1 SUPPRESSOR 2 (CSU2). Loss of function mutations in CSU2 suppress the constitutive photomorphogenic phenotype of cop1-6 in darkness. CSU2 directly interacts with COP1 via their coiled-coil domains and is recruited by COP1 into nuclear speckles in living plant cells. Furthermore, CSU2 inhibits COP1 E3 ubiquitin ligase activity in vitro, and represses COP1 mediated turnover of HY5 in cell-free extracts. We propose that in csu2 cop1-6 mutants, the lack of CSU2’s repression of COP1 allows the low level of COP1 to exhibit higher activity that is sufficient to prevent accumulation of HY5 in the dark, thus restoring the etiolated phenotype. In addition, CSU2 is required for primary root development under normal light growth condition. PMID:26714275

  6. Principles Governing the Self-Assembly of Coiled-Coil Protein Nanoparticles.

    PubMed

    Indelicato, Giuliana; Wahome, Newton; Ringler, Philippe; Müller, Shirley A; Nieh, Mu-Ping; Burkhard, Peter; Twarock, Reidun

    2016-02-01

    Self-assembly refers to the spontaneous organization of individual building blocks into higher order structures. It occurs in biological systems such as spherical viruses, which utilize icosahedral symmetry as a guiding principle for the assembly of coat proteins into a capsid shell. In this study, we characterize the self-assembling protein nanoparticle (SAPN) system, which was inspired by such viruses. To facilitate self-assembly, monomeric building blocks have been designed to contain two oligomerization domains. An N-terminal pentameric coiled-coil domain is linked to a C-terminal coiled-coil trimer by two glycine residues. By combining monomers with inherent propensity to form five- and threefold symmetries in higher order agglomerates, the supposition is that nanoparticles will form that exhibit local and global symmetry axes of order 3 and 5. This article explores the principles that govern the assembly of such a system. Specifically, we show that the system predominantly forms according to a spherical core-shell morphology using a combination of scanning transmission electron microscopy and small angle neutron scattering. We introduce a mathematical toolkit to provide a specific description of the possible SAPN morphologies, and we apply it to characterize all particles with maximal symmetry. In particular, we present schematics that define the relative positions of all individual chains in the symmetric SAPN particles, and provide a guide of how this approach can be generalized to nonspherical morphologies, hence providing unprecedented insights into their geometries that can be exploited in future applications. PMID:26840729

  7. An Autoinhibited Coiled-Coil Design Strategy for Split-Protein Protease Sensors

    PubMed Central

    Shekhawat, Sujan S.; Porter, Jason R.; Sriprasad, Akshay; Ghosh, Indraneel

    2009-01-01

    Proteases are widely studied as they are integral players in cell cycle control and apoptosis. We report a new approach for the design of a family of genetically encoded turn-on protease biosensors. In our design, an auto-inhibited coiled-coil switch is turned on upon proteolytic cleavage, which results in the complementation of split-protein reporters. Utilizing this new auto-inhibition design paradigm, we present the rational construction and optimization of three generations of protease biosensors, with the final design providing a 1000 fold increase in bioluminescent signal upon addition of the TEV protease. We demonstrate the generality of the approach utilizing two different split-protein reporters, firefly luciferase and beta-lactamase, while also testing our design in the context of a therapeutically relevant protease, caspase-3. Finally, we present a dual-protease sensor geometry that allows for the use of these turn-on sensors as potential AND logic gates. Thus these studies potentially provide a new method for the design and implementation of genetically encoded turn-on protease sensors while also providing a general auto-inhibited coiled-coil strategy for controlling the activity of fragmented proteins. PMID:19803505

  8. Directed surface attachment of nanomaterials via coiled-coil-driven self-assembly

    NASA Astrophysics Data System (ADS)

    White, Simon J.; Johnson, Steven; Szymonik, Michal; Wardingley, Richard A.; Pye, Douglas; Davies, A. Giles; Wälti, Christoph; Stockley, Peter G.

    2012-12-01

    Numerous nanoscale devices and materials have been fabricated in recent years using a variety of biological scaffolds. However, the interfacing of these devices and materials into existing circuits and ordered arrays has proved problematic. Here, we describe a simple solution to this problem using self-assembly of the peptide coiled-coil heterodimer ACID:BASE to immobilize M13 bacteriophage particles to specific locations on a patterned gold surface. Surface plasmon resonance demonstrated that free ACID peptides will assemble onto a surface derivatized with BASE. We then displayed the ACID peptide on the pIX coat protein of M13 and showed that these phage particles permit formation of the coiled-coil resulting in specific surface attachment. The ACID:immobilized BASE affinities appear to be similar for free peptide and phage-displayed ACID. Finally, we fabricated two gold electrodes, separated by a 200 nm gap, coated one of them with BASE and showed that this allows localization of the M13:ACID onto the functionalized electrode.

  9. Directed surface attachment of nanomaterials via coiled-coil-driven self-assembly.

    PubMed

    White, Simon J; Johnson, Steven; Szymonik, Michal; Wardingley, Richard A; Pye, Douglas; Davies, A Giles; Wälti, Christoph; Stockley, Peter G

    2012-12-14

    Numerous nanoscale devices and materials have been fabricated in recent years using a variety of biological scaffolds. However, the interfacing of these devices and materials into existing circuits and ordered arrays has proved problematic. Here, we describe a simple solution to this problem using self-assembly of the peptide coiled-coil heterodimer ACID:BASE to immobilize M13 bacteriophage particles to specific locations on a patterned gold surface. Surface plasmon resonance demonstrated that free ACID peptides will assemble onto a surface derivatized with BASE. We then displayed the ACID peptide on the pIX coat protein of M13 and showed that these phage particles permit formation of the coiled-coil resulting in specific surface attachment. The ACID:immobilized BASE affinities appear to be similar for free peptide and phage-displayed ACID. Finally, we fabricated two gold electrodes, separated by a 200 nm gap, coated one of them with BASE and showed that this allows localization of the M13:ACID onto the functionalized electrode. PMID:23154792

  10. Evidence of α-helical coiled coils and β-sheets in hornet silk.

    PubMed

    Kameda, Tsunenori; Nemoto, Takashi; Ogawa, Tetsuya; Tosaka, Masatoshi; Kurata, Hiroki; Schaper, Andreas K

    2014-03-01

    α-Helical coiled coil and β-sheet complexes are essential structural building elements of silk proteins produced by different species of the Hymenoptera. Beside X-ray scattering at wide and small angles we applied cryo-electron diffraction and microscopy to demonstrate the presence and the details of such structures in silk of the giant hornet Vespa mandarinia japonica. Our studies on the assembly of the fibrous silk proteins and their internal organization in relation to the primary chain structure suggest a 172 Å pitch supercoil consisting of four intertwined alanine-rich α-helical strands. The axial periodicity may adopt even multiples of the pitch value. Coiled coil motifs form the largest portion of the hornet silk structure and are aligned nearly parallel to the cocoon fiber axis in the same way as the membrane-like parts of the cocoon are molecularly orientated in the spinning direction. Supercoils were found to be associated with β-crystals, predominantly localized in the l-serine-rich chain sequences terminating each of the four predominant silk proteins. Such β-sheet blocks are considered resulting from transformation of random coil molecular sequences due to the action of elongational forces during the spinning process. PMID:24345346

  11. The Orientations of Large Aspect-Ratio Coiled-Coil Proteins Attached to Gold Nanostructures.

    PubMed

    Chang, Jae-Byum; Kim, Yong Ho; Thompson, Evan; No, Young Hyun; Kim, Nam Hyeong; Arrieta, Jose; Manfrinato, Vitor R; Keating, Amy E; Berggren, Karl K

    2016-03-01

    Methods for patterning biomolecules on a substrate at the single molecule level have been studied as a route to sensors with single-molecular sensitivity or as a way to probe biological phenomena at the single-molecule level. However, the arrangement and orientation of single biomolecules on substrates has been less investigated. Here, the arrangement and orientation of two rod-like coiled-coil proteins, cortexillin and tropomyosin, around patterned gold nanostructures is examined. The high aspect ratio of the coiled coils makes it possible to study their orientations and to pursue a strategy of protein orientation via two-point attachment. The proteins are anchored to the surfaces using thiol groups, and the number of cysteine residues in tropomyosin is varied to test how this variation affects the structure and arrangement of the surface-attached proteins. Molecular dynamics studies are used to interpret the observed positional distributions. Based on initial studies of protein attachment to gold post structures, two 31-nm-long tropomyosin molecules are aligned between the two sidewalls of a trench with a width of 68 nm. Because the approach presented in this study uses one of twenty natural amino acids, this method provides a convenient way to pattern biomolecules on substrates using standard chemistry. PMID:26799936

  12. Structure of a Designed, Right-Handed Coiled-Coil Tetramer Containing All Biological Amino Acids

    SciTech Connect

    Sales, M.; Plecs, J.J.; Holton, J.M.; Alber, T.

    2009-06-04

    The previous design of an unprecedented family of two-, three-, and four-helical, right-handed coiled coils utilized nonbiological amino acids to efficiently pack spaces in the oligomer cores. Here we show that a stable, right-handed parallel tetrameric coiled coil, called RH4B, can be designed entirely using biological amino acids. The X-ray crystal structure of RH4B was determined to 1.1 {angstrom} resolution using a designed metal binding site to coordinate a single Yb{sup 2+} ion per 33-amino acid polypeptide chain. The resulting experimental phases were particularly accurate, and the experimental electron density map provided an especially clear, unbiased view of the molecule. The RH4B structure closely matched the design, with equivalent core rotamers and an overall root-mean-square deviation for the N-terminal repeat of the tetramer of 0.24 {angstrom}. The clarity and resolution of the electron density map, however, revealed alternate rotamers and structural differences between the three sequence repeats in the molecule. These results suggest that the RH4B structure populates an unanticipated variety of structures.

  13. Improvement of probe peptides for coiled-coil labeling by introducing phosphoserines.

    PubMed

    Ono, Satoshi; Yano, Yoshiaki; Matsuzaki, Katsumi

    2012-01-01

    We have developed a method of rapidly labeling membrane proteins in living cells using a high-affinity heterodimeric coiled-coil construct containing an E3 tag (EIAALEK)(3) genetically fused to the target protein and a K4 probe (KIAALKE)(4) labeled with a fluorophore such as tetramethylrhodamine (TMR) at its N-terminus (TMR-K4). However, coiled-coil labeling cannot be applied to highly negatively charged cell lines such as HEK293, because of the nonspecific adsorption of the positively charged K4 probes to cell membranes. To reduce the net positive charge, we synthesized new probes that include phosphoserine residues (pSer) between the K4 sequence and TMR fluorophore (TMR-(pSer)(n)-K4, [n = 1-3]). The affinity of the pSer-introduced probes was comparable to that of the TMR-K4 probe. However, the TMR-(pSer)(2)-K4 and TMR-(pSer)(3)-K4 probes tended to aggregate during labeling. In contrast, TMR-pSer-K4, which was as soluble as TMR-K4, achieved higher signal/background ratios (30-100) for four host cell lines (HEK293, HeLa, SH-SY5Y, and PC12) than did TMR-K4 (~10 for HEK293 cells), demonstrating that the improved probe can be used for various types of cells. PMID:22782565

  14. Immunogenicity of coiled-coil based drug-free macromolecular therapeutics

    PubMed Central

    Kverka, Miloslav; Hartley, Jonathan M.; Chu, Te-Wei; Yang, Jiyuan; Heidchen, Regina; Kopeček, Jindřich

    2014-01-01

    A two-component CD20 (non-internalizing) receptor crosslinking system based on the biorecognition of complementary coiled-coil forming peptides was evaluated. Exposure of B cells to Fab’-peptide1 conjugate decorates the cell surface with peptide1; further exposure of the decorated cells to P-(peptide2)x (P is the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer backbone) results in the formation of coiled-coil heterodimers at the cell surface with concomitant induction of apoptosis. The aim of this study was to determine the potential immunogenicity of this therapeutic system that does not contain low molecular weight drugs. Enantiomeric peptides (L- and D-CCE and L- and D-CCK), HPMA copolymer-peptide conjugates, and Fab’ fragment-peptide conjugates were synthesized and the immunological properties of peptide conjugates evaluated in vitro on RAW264.7 macrophages and in vivo on immunocompetent BALB/c mice. HPMA copolymer did not induce immune response in vitro and in vivo. Administration of P-peptide conjugates with strong adjuvant resulted in antibody response directed to the peptide. Fab’ was responsible for macrophage activation of Fab’-peptide conjugates and a major factor in the antibody induction following i.v. administration of Fab’ conjugates. There was no substantial difference in the ability of conjugates of D-peptides and conjugates of L-peptides to induce Ab response. PMID:24767787

  15. Identification of a region in the coiled-coil domain of Smc3 that is essential for cohesin activity.

    PubMed

    Orgil, Ola; Mor, Hadar; Matityahu, Avi; Onn, Itay

    2016-07-27

    The cohesin complex plays an important role in sister chromatin cohesion. Cohesin's core is composed of two structural maintenance of chromosome (SMC) proteins, called Smc1 and Smc3. SMC proteins are built from a globular hinge domain, a rod-shaped domain composed of long anti-parallel coiled-coil (CC), and a second globular adenosine triphosphatase domain called the head. The functions of both head and hinge domains have been studied extensively, yet the function of the CC region remains elusive. We identified a mutation in the CC of smc3 (L217P) that disrupts the function of the protein. Cells carrying the smc3-L217P allele have a strong cohesion defect and complexes containing smc3-L217P are not loaded onto the chromosomes. However, the mutation does not affect inter-protein interactions in either the core complex or with the Scc2 loader. We show by molecular dynamics and biochemistry that wild-type Smc3 can adopt distinct conformations, and that adenosine triphosphate (ATP) induces the conformational change. The L217P mutation restricts the ability of the mutated protein to switch between the conformations. We suggest that the function of the CC is to transfer ATP binding/hydrolysis signals between the head and the hinge domains. The results provide a new insight into the mechanism of cohesin activity. PMID:27307603

  16. ELKS controls the pool of readily releasable vesicles at excitatory synapses through its N-terminal coiled-coil domains

    PubMed Central

    Held, Richard G; Liu, Changliang; Kaeser, Pascal S

    2016-01-01

    In a presynaptic nerve terminal, synaptic strength is determined by the pool of readily releasable vesicles (RRP) and the probability of release (P) of each RRP vesicle. These parameters are controlled at the active zone and vary across synapses, but how such synapse specific control is achieved is not understood. ELKS proteins are enriched at vertebrate active zones and enhance P at inhibitory hippocampal synapses, but ELKS functions at excitatory synapses are not known. Studying conditional knockout mice for ELKS, we find that ELKS enhances the RRP at excitatory synapses without affecting P. Surprisingly, ELKS C-terminal sequences, which interact with RIM, are dispensable for RRP enhancement. Instead, the N-terminal ELKS coiled-coil domains that bind to Liprin-α and Bassoon are necessary to control RRP. Thus, ELKS removal has differential, synapse-specific effects on RRP and P, and our findings establish important roles for ELKS N-terminal domains in synaptic vesicle priming. DOI: http://dx.doi.org/10.7554/eLife.14862.001 PMID:27253063

  17. Coiled-Coils at the Edge of Configurational Heterogeneity. Structural Analyses of Parallel and Antiparallel Homotetrameric Coiled-Coils Reveal Configurational Sensitivity to a Single Solvent-Exposed Amino Acid Substitution.†§

    PubMed Central

    Yadav, Maneesh K.; Leman, Luke J.; Price, Daniel J.; Brooks, Charles L.; Stout, C. David; Ghadiri, M. Reza

    2007-01-01

    A detailed understanding of the mechanisms by which particular amino acid sequences can give rise to more than one folded structure, such as for proteins that undergo large conformational changes or misfolding, is a long-standing objective of protein chemistry. Here we describe the crystal structures of a single coiled-coil peptide in distinct parallel and antiparallel tetrameric configurations and further describe the parallel or antiparallel crystal structures of several related peptide sequences; the antiparallel tetrameric assemblies represents the first crystal structures of GCN4-derived peptides exhibiting such a configuration. Intriguingly, substitution of a single solvent-exposed residue enabled the parallel coiled-coil tetramer GCN4-pLI to populate the antiparallel configuration, suggesting that the two configurations are close enough in energy for subtle sequence changes to have important structural consequences. We present a structural analysis of the small changes to helix register and side chain conformations that accommodate the two configurations, and have supplemented these results using solution studies and a molecular dynamics energetic analysis using a replica exchange methodology. Considering the previous examples of structural nonspecificity in coiled-coil peptides, the findings reported here not only emphasize the predisposition of the coiled-coil motif to adopt multiple configurations, but also call attention to the associated risk that observed crytstal structures may not represent the only (or even the major) species present in solution. PMID:16584182

  18. Gene Delivery from Supercharged Coiled-coil Protein and Cationic Lipid Hybrid Complex

    PubMed Central

    More, Haresh T.; Frezzo, Joseph A.; Dai, Jisen; Yamano, Seiichi; Montclare, Jin K.

    2014-01-01

    A lipoproteoplex comprised of an engineered supercharged coiled-coil protein (CSP) bearing multiple arginines and the cationic lipid formulation FuGENE HD (FG) was developed for effective condensation and delivery of nucleic acids. The CSP was able to maintain helical structure and self-assembly properties while exhibiting binding to plasmid DNA. The ternary CSP•DNA(8:1)•FG lipoproteoplex complex demonstrated enhanced transfection of β-galactosidase DNA into MC3T3-E1 mouse preosteoblasts. The lipoproteoplexes showed significant increases in transfection efficiency when compared to conventional FG and an mTat•FG lipopolyplex with a 6- and 2.5-fold increase in transfection, respectively. The CSP•DNA(8:1)•FG lipoproteoplex assembled into spherical particles with a net positive surface charge, enabling efficient gene delivery. These results support the application of lipoproteoplexes with protein engineered CSP for non-viral gene delivery. PMID:24875765

  19. Designed Coiled-Coil Peptides Inhibit the Type Three Secretion System of Enteropathogenic Escherichia coli

    PubMed Central

    Larzábal, Mariano; Mercado, Elsa C.; Vilte, Daniel A.; Salazar-González, Hector; Cataldi, Angel; Navarro-Garcia, Fernando

    2010-01-01

    Background Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are two categories of E. coli strains associated with human disease. A major virulence factor of both pathotypes is the expression of a type three secretion system (TTSS), responsible for their ability to adhere to gut mucosa causing a characteristic attaching and effacing lesion (A/E). The TTSS translocates effector proteins directly into the host cell that subvert mammalian cell biochemistry. Methods/Principal Findings We examined synthetic peptides designed to inhibit the TTSS. CoilA and CoilB peptides, both representing coiled-coil regions of the translocator protein EspA, and CoilD peptide, corresponding to a coiled–coil region of the needle protein EscF, were effective in inhibiting the TTSS dependent hemolysis of red blood cells by the EPEC E2348/69 strain. CoilA and CoilB peptides also reduced the formation of actin pedestals by the same strain in HEp-2 cells and impaired the TTSS-mediated protein translocation into the epithelial cell. Interestingly, CoilA and CoilB were able to block EspA assembly, destabilizing the TTSS and thereby Tir translocation. This blockage of EspA polymerization by CoilA or CoilB peptides, also inhibited the correct delivery of EspB and EspD as detected by immunoblotting. Interestingly, electron microscopy of bacteria incubated with the CoilA peptide showed a reduction of the length of EspA filaments. Conclusions Our data indicate that coiled-coil peptides can prevent the assembly and thus the functionality of the TTSS apparatus and suggest that these peptides could provide an attractive tool to block EPEC and EHEC pathogenesis. PMID:20140230

  20. pH-Dependent Assembly and Segregation of the Coiled-Coil Segments of Yeast Putative Cargo Receptors Emp46p and Emp47p

    PubMed Central

    Noda, Masanori; Kajino, Megumi; Kim, Akemi; Kurimoto, Eiji; Sato, Ken; Nakano, Akihiko; Kobayashi, Yuji; Yagi, Hirokazu; Uchiyama, Susumu; Kato, Koichi

    2015-01-01

    Emp46p and Emp47p are yeast putative cargo receptors that recycle between the endoplasmic reticulum and the Golgi apparatus. These receptors can form complexes in a pH-dependent manner, but their molecular mechanisms remain unclear. Here, we successfully reproduced their interactions in vitro solely with their coiled-coil segments, which form stable heterotetramers in the neutral condition but segregate at lower pH. Mutational data identified a key glutamate residue of Emp46p that serves as the pH-sensing switch of their oligomer formation. Our findings elucidate the mechanisms of the dynamic cargo receptor interactions in the secretory pathway and the design framework of the environment-responsive molecular assembly and disassembly systems. PMID:26447473

  1. Truncated and Helix-Constrained Peptides with High Affinity and Specificity for the cFos Coiled-Coil of AP-1

    PubMed Central

    Rao, Tara; Ruiz-Gómez, Gloria; Hill, Timothy A.; Hoang, Huy N.; Fairlie, David P.; Mason, Jody M.

    2013-01-01

    Protein-based therapeutics feature large interacting surfaces. Protein folding endows structural stability to localised surface epitopes, imparting high affinity and target specificity upon interactions with binding partners. However, short synthetic peptides with sequences corresponding to such protein epitopes are unstructured in water and promiscuously bind to proteins with low affinity and specificity. Here we combine structural stability and target specificity of proteins, with low cost and rapid synthesis of small molecules, towards meeting the significant challenge of binding coiled coil proteins in transcriptional regulation. By iteratively truncating a Jun-based peptide from 37 to 22 residues, strategically incorporating i→i+4 helix-inducing constraints, and positioning unnatural amino acids, we have produced short, water-stable, α-helical peptides that bind cFos. A three-dimensional NMR-derived structure for one peptide (24) confirmed a highly stable α-helix which was resistant to proteolytic degradation in serum. These short structured peptides are entropically pre-organized for binding with high affinity and specificity to cFos, a key component of the oncogenic transcriptional regulator Activator Protein-1 (AP-1). They competitively antagonized the cJun–cFos coiled-coil interaction. Truncating a Jun-based peptide from 37 to 22 residues decreased the binding enthalpy for cJun by ∼9 kcal/mol, but this was compensated by increased conformational entropy (TΔS ≤7.5 kcal/mol). This study demonstrates that rational design of short peptides constrained by α-helical cyclic pentapeptide modules is able to retain parental high helicity, as well as high affinity and specificity for cFos. These are important steps towards small antagonists of the cJun-cFos interaction that mediates gene transcription in cancer and inflammatory diseases. PMID:23544065

  2. Structural Basis of HIV-1 Neutralization by Affinity Matured Fabs Directed against the Internal Trimeric Coiled-Coil of gp41

    SciTech Connect

    Gustchina, Elena; Li, Mi; Louis, John M.; Anderson, D.Eric; Lloyd, John; Frisch, Christian; Bewley, Carole A.; Gustchina, Alla; Wlodawer, Alexander; Clore, G.Marius

    2010-12-03

    The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naive human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Furthermore, modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.

  3. Missense mutation in DISC1 C-terminal coiled-coil has GSK3β signaling and sex-dependent behavioral effects in mice

    PubMed Central

    Dachtler, James; Elliott, Christina; Rodgers, R. John; Baillie, George S.; Clapcote, Steven J.

    2016-01-01

    Disrupted-in-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and affective disorders. The full-length DISC1 protein consists of an N-terminal ‘head’ domain and a C-terminal tail domain that contains several predicted coiled-coils, structural motifs involved in protein-protein interactions. To probe the in vivo effects of missense mutation of DISC1’s C-terminal tail, we tested mice carrying mutation D453G within a predicted α-helical coiled-coil region. We report that, relative to wild-type littermates, female DISC1D453G mice exhibited novelty-induced hyperlocomotion, an anxiogenic profile in the elevated plus-maze and open field tests, and reduced social exploration of unfamiliar mice. Male DISC1D453G mice displayed a deficit in passive avoidance, while neither males nor females exhibited any impairment in startle reactivity or prepulse inhibition. Whole brain homogenates showed normal levels of DISC1 protein, but decreased binding of DISC1 to GSK3β, decreased phospho-inhibition of GSK3β at serine 9, and decreased levels of β-catenin in DISC1D453G mice of either sex. Interrupted GSK3β signaling may thus be part of the mechanism underlying the behavioral phenotype associated with D453G, in common with the previously described N-terminal domain mutations Q31L and L100P in mice, and the schizophrenia risk-conferring variant R264Q in humans. PMID:26728762

  4. Missense mutation in DISC1 C-terminal coiled-coil has GSK3β signaling and sex-dependent behavioral effects in mice.

    PubMed

    Dachtler, James; Elliott, Christina; Rodgers, R John; Baillie, George S; Clapcote, Steven J

    2016-01-01

    Disrupted-in-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and affective disorders. The full-length DISC1 protein consists of an N-terminal 'head' domain and a C-terminal tail domain that contains several predicted coiled-coils, structural motifs involved in protein-protein interactions. To probe the in vivo effects of missense mutation of DISC1's C-terminal tail, we tested mice carrying mutation D453G within a predicted α-helical coiled-coil region. We report that, relative to wild-type littermates, female DISC1(D453G) mice exhibited novelty-induced hyperlocomotion, an anxiogenic profile in the elevated plus-maze and open field tests, and reduced social exploration of unfamiliar mice. Male DISC1(D453G) mice displayed a deficit in passive avoidance, while neither males nor females exhibited any impairment in startle reactivity or prepulse inhibition. Whole brain homogenates showed normal levels of DISC1 protein, but decreased binding of DISC1 to GSK3β, decreased phospho-inhibition of GSK3β at serine 9, and decreased levels of β-catenin in DISC1(D453G) mice of either sex. Interrupted GSK3β signaling may thus be part of the mechanism underlying the behavioral phenotype associated with D453G, in common with the previously described N-terminal domain mutations Q31L and L100P in mice, and the schizophrenia risk-conferring variant R264Q in humans. PMID:26728762

  5. Rapid Identification of Malaria Vaccine Candidates Based on α-Helical Coiled Coil Protein Motif

    PubMed Central

    Villard, Viviane; Agak, George W.; Frank, Géraldine; Jafarshad, Ali; Servis, Catherine; Nébié, Issa; Sirima, Sodiomon B.; Felger, Ingrid; Arevalo-Herrera, Myriam; Herrera, Socrates; Heitz, Frederic; Bäcker, Volker; Druilhe, Pierre; Kajava, Andrey V.; Corradin, Giampietro

    2007-01-01

    To identify malaria antigens for vaccine development, we selected α-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally “native” epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high α-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens. PMID:17653272

  6. Ndm, a coiled-coil domain protein that suppresses macropinocytosis and has effects on cell migration.

    PubMed

    Kelsey, Jessica S; Fastman, Nathan M; Noratel, Elizabeth F; Blumberg, Daphne D

    2012-09-01

    The ampA gene has a role in cell migration in Dictyostelium discoideum. Cells overexpressing AmpA show an increase in cell migration, forming large plaques on bacterial lawns. A second-site suppressor of this ampA-overexpressing phenotype identified a previously uncharacterized gene, ndm, which is described here. The Ndm protein is predicted to contain a coiled-coil BAR-like domain-a domain involved in endocytosis and membrane bending. ndm-knockout and Ndm-monomeric red fluorescent protein-expressing cell lines were used to establish a role for ndm in suppressing endocytosis. An increase in the rate of endocytosis and in the number of endosomes was detected in ndm(-) cells. During migration ndm(-) cells formed numerous endocytic cups instead of the broad lamellipodia structure characteristic of moving cells. A second lamellipodia-based function-cell spreading-was also defective in the ndm(-) cells. The increase in endocytosis and the defect in lamellipodia formation were associated with reduced chemotaxis in ndm(-) cells. Immunofluorescence results and glutathione S-transferase pull-down assays revealed an association of Ndm with coronin and F-actin. The results establish ndm as a gene important in regulating the balance between formation of endocytic cups and lamellipodia structures. PMID:22809629

  7. The coiled-coil domain of EHD2 mediates inhibition of LeEix2 endocytosis and signaling.

    PubMed

    Bar, Maya; Sharfman, Miya; Schuster, Silvia; Avni, Adi

    2009-01-01

    Endocytosis has been suggested to be crucial for the induction of plant immunity in several cases. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and LeEix2. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD2 that are required for its inhibitory activity on endocytosis remained unknown. In this work we demonstrate that the coiled-coil domain of EHD2 is crucial for the ability of EHD2 to inhibit endocytosis in plants, as mutant EHD2 forms lacking the coiled-coil lost the ability to inhibit endocytosis and signaling of LeEix2. The coiled-coil was also required for binding of EHD2 to the LeEix2 receptor. It is therefore probable that binding of EHD2 to the LeEix2 receptor is required for inhibition of LeEix2 internalization. We also show herein that the P-loop of EHD2 is important for EHD2 to function properly. The EH domain of AtEHD2 does not appear to be involved in inhibition of endocytosis. Moreover, AtEHD2 influences actin organization and may exert its inhibitory effect on endocytosis through actin re-distribution. The coiled-coil domain of EHD2 functions in inhibition of endocytosis, while the EH domain does not appear to be involved in inhibition of endocytosis. PMID:19936242

  8. The Coiled-Coil Domain of EHD2 Mediates Inhibition of LeEix2 Endocytosis and Signaling

    PubMed Central

    Bar, Maya; Sharfman, Miya; Schuster, Silvia; Avni, Adi

    2009-01-01

    Endocytosis has been suggested to be crucial for the induction of plant immunity in several cases. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and LeEix2. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD2 that are required for its inhibitory activity on endocytosis remained unknown. In this work we demonstrate that the coiled-coil domain of EHD2 is crucial for the ability of EHD2 to inhibit endocytosis in plants, as mutant EHD2 forms lacking the coiled-coil lost the ability to inhibit endocytosis and signaling of LeEix2. The coiled-coil was also required for binding of EHD2 to the LeEix2 receptor. It is therefore probable that binding of EHD2 to the LeEix2 receptor is required for inhibition of LeEix2 internalization. We also show herein that the P-loop of EHD2 is important for EHD2 to function properly. The EH domain of AtEHD2 does not appear to be involved in inhibition of endocytosis. Moreover, AtEHD2 influences actin organization and may exert its inhibitory effect on endocytosis through actin re-distribution. The coiled-coil domain of EHD2 functions in inhibition of endocytosis, while the EH domain does not appear to be involved in inhibition of endocytosis. PMID:19936242

  9. A thermodynamic model for the helix-coil transition coupled to dimerization of short coiled-coil peptides.

    PubMed Central

    Qian, H

    1994-01-01

    A simple thermodynamic formalism is presented to model the conformational transition between a random-coil monomeric peptide and a coiled-coil helical dimer. The coiled-coil helical dimer is the structure of a class of proteins also called leucine zipper, which has been studied intensively in recent years. Our model, which is appropriate particularly for short peptides, is an alternative to the theory developed by Skolnick and Holtzer. Using the present formalism, we discuss the multi-equilibriatory nature of this transition and provide an explanation for the apparent two-state behavior of coiled-coil formation when the helix-coil transition is coupled to dimerization. It is found that such coupling between multi-equilibria and a true two-state transition can simplify the data analysis, but care must be taken in using the overall association constant to determine helix propensities (w) of single residues. Successful use of the two-state model does not imply that the helix-coil transition is all-or-none. The all-or-none assumption can provide good numerical estimates when w is around unity (0.35 < or = w < or = 1.35), but when w is small (w < 0.01), similar estimations can lead to large errors. The theory of the helix-coil transition in denaturation experiments is also discussed. PMID:7919005

  10. Balance between Coiled-Coil Stability and Dynamics Regulates Activity of BvgS Sensor Kinase in Bordetella

    PubMed Central

    Lesne, E.; Krammer, E.-M.; Dupre, E.; Locht, C.; Lensink, M. F.

    2016-01-01

    ABSTRACT The two-component system BvgAS controls the expression of the virulence regulon of Bordetella pertussis. BvgS is a prototype of bacterial sensor kinases with extracytoplasmic Venus flytrap perception domains. Following its transmembrane segment, BvgS harbors a cytoplasmic Per-Arnt-Sim (PAS) domain and then a predicted 2-helix coiled coil that precede the dimerization-histidine-phosphotransfer domain of the kinase. BvgS homologs have a similar domain organization, or they harbor only a predicted coiled coil between the transmembrane and the dimerization-histidine-phosphotransfer domains. Here, we show that the 2-helix coiled coil of BvgS regulates the enzymatic activity in a mechanical manner. Its marginally stable hydrophobic interface enables a switch between a state of great rotational dynamics in the kinase mode and a more rigid conformation in the phosphatase mode in response to signal perception by the periplasmic domains. We further show that the activity of BvgS is controlled in the same manner if its PAS domain is replaced with the natural α-helical sequences of PAS-less homologs. Clamshell motions of the Venus flytrap domains trigger the shift of the coiled coil’s dynamics. Thus, we have uncovered a general mechanism of regulation for the BvgS family of Venus flytrap-containing two-component sensor kinases. PMID:26933056

  11. High-resolution spot-scan electron microscopy of microcrystals of an alpha-helical coiled-coil protein.

    PubMed

    Bullough, P A; Tulloch, P A

    1990-09-01

    We describe the electron microscopy of a crystalline assembly of an alpha-helical coiled-coil protein extracted from the ootheca of the praying mantis. Electron diffraction patterns of unstained crystals show crystal lattice sampling of the coiled-coil molecular transform to a resolution beyond 1.5 A. Using a "spot-scan" method of electron imaging, micrographs of unstained crystals have been obtained that visibly diffract laser light from crystal spacings as small as 4.3 A. A projection map was calculated to 4 A using electron diffraction amplitudes and phases from computer-processed images. The projection map clearly shows modulations in density arising from the 5.1 A alpha-helical repeat, the first time this type of modulation has been revealed by electron microscopy. The crystals have p2 plane group symmetry with a = 92.4 A, b = 150.7 A, y = 92.4 degrees. Examination of tilted specimens shows that c is approximately 18 A, indicating that the unit cell is only one molecule thick. A preliminary interpretation shows tightly packed molecules some 400 A long lying with their long axes in the plane of the projection. The molecules have a coiled-coil configuration for most of their length. The possible modes of packing of the molecules in three dimensions are discussed. PMID:2398496

  12. Mechanical Transition from α-Helical Coiled-Coils to β-Sheets in Fibrin(ogen)

    PubMed Central

    Zhmurov, Artem; Kononova, Olga; Litvinov, Rustem I.; Dima, Ruxandra I.; Barsegov, Valeri; Weisel, John W.

    2012-01-01

    We characterized the α-to-β transition in α-helical coiled-coil connectors of human fibrin(ogen) molecule using biomolecular simulations of their forced elongation, and theoretical modeling. The force (F) - extension (X) profiles show three distinct regimes: (1) the elastic regime, in which the coiled-coils act as entropic springs (F < 100–125 pN; X < 7–8 nm); (2) the constant-force plastic regime, characterized by a force-plateau (F≈150 pN; X≈10–35 nm); and (3) the non-linear regime (F >175–200 pN; X > 40–50 nm). In the plastic regime, the three-stranded α-helices undergo a non-cooperative phase transition to form parallel three-stranded β-sheets. The critical extension of α-helices is 0.25 nm, and the energy difference between the α-helices and β-sheets is 4.9 kcal/mol per helical pitch. The soft α-to-β phase transition in coiled-coils might be a universal mechanism underlying mechanical properties of filamentous α-helical proteins. PMID:22953986

  13. Structural Correlation of the Neck Coil with the Coiled-coil (CC1)-Forkhead-associated (FHA) Tandem for Active Kinesin-3 KIF13A.

    PubMed

    Ren, Jinqi; Huo, Lin; Wang, Wenjuan; Zhang, Yong; Li, Wei; Lou, Jizhong; Xu, Tao; Feng, Wei

    2016-02-12

    Processive kinesin motors often contain a coiled-coil neck that controls the directionality and processivity. However, the neck coil (NC) of kinesin-3 is too short to form a stable coiled-coil dimer. Here, we found that the coiled-coil (CC1)-forkhead-associated (FHA) tandem (that is connected to NC by Pro-390) of kinesin-3 KIF13A assembles as an extended dimer. With the removal of Pro-390, the NC-CC1 tandem of KIF13A unexpectedly forms a continuous coiled-coil dimer that can be well aligned into the CC1-FHA dimer. The reverse introduction of Pro-390 breaks the NC-CC1 coiled-coil dimer but provides the intrinsic flexibility to couple NC with the CC1-FHA tandem. Mutations of either NC, CC1, or the FHA domain all significantly impaired the motor activity. Thus, the three elements within the NC-CC1-FHA tandem of KIF13A are structurally interrelated to form a stable dimer for activating the motor. This work also provides the first direct structural evidence to support the formation of a coiled-coil neck by the short characteristic neck domain of kinesin-3. PMID:26680000

  14. Linear free-energy analysis of mercury(II) and cadmium(II) binding to three-stranded coiled coils.

    PubMed

    Ghosh, Debdip; Lee, Kyung-Hoon; Demeler, Borries; Pecoraro, Vincent L

    2005-08-01

    Investigators have studied how proteins enforce nonstandard geometries on metal centers to assess the question of how protein structures can define the coordination geometry and binding affinity of an active-site metal cofactor. We have shown that cysteine-substituted versions of the TRI peptide series [AcG-(LKALEEK)(4)G-NH(2)] bind Hg(II) and Cd(II) in geometries that are different from what is normally found with thiol ligands in aqueous solution. A fundamental question has been whether this structural perturbation is due to protein influence or a change in the metal geometry preference. To address this question, we have completed linear free-energy analyses that correlate the association of three-stranded coiled coils in the absence of a metal with the binding affinity of the peptides to the heavy metals, Hg(II) and Cd(II). In this paper, six new members of this family have been synthesized, replacing core leucine residues with smaller and less hydrophobic residues, consequently leading to varying degrees of self-association affinities. At the same time, studies with some smaller and longer sequenced peptides have also been examined. All of these peptides are seen to sequester Hg(II) and Cd(II) in an uncommon trigonal environment. For both metals, the binding is strong with micromolar dissociation constants. For binding of Hg(II) to the peptides, the dissociation constants range from 2.4 x 10(-)(5) M for Baby L12C to 2.5 x 10(-)(9) M for Grand L9C for binding of the third thiolate to a linear Hg(II)(pep)(2) species. The binding of Hg(II) to the peptide Grand L9C is similar in energetics for metal binding in the metalloregulatory protein, mercury responsive (merR), displaying approximately 50% trigonal Hg(II) formation at nanomolar metal concentrations. Approximately, 11 kcal/mol of the Hg(II)(Grand L9C)(3)(-) stability is due to peptide interactions, whereas only 1-4 kcal/mol stabilization results from Hg(II)(RS)(2) binding the third thiolate ligand. This

  15. Association of polyalanine and polyglutamine coiled coils mediates expansion disease-related protein aggregation and dysfunction

    PubMed Central

    Pelassa, Ilaria; Corà, Davide; Cesano, Federico; Monje, Francisco J.; Montarolo, Pier Giorgio; Fiumara, Ferdinando

    2014-01-01

    The expansion of homopolymeric glutamine (polyQ) or alanine (polyA) repeats in certain proteins owing to genetic mutations induces protein aggregation and toxicity, causing at least 18 human diseases. PolyQ and polyA repeats can also associate in the same proteins, but the general extent of their association in proteomes is unknown. Furthermore, the structural mechanisms by which their expansion causes disease are not well understood, and these repeats are generally thought to misfold upon expansion into aggregation-prone β-sheet structures like amyloids. However, recent evidence indicates a critical role for coiled-coil (CC) structures in triggering aggregation and toxicity of polyQ-expanded proteins, raising the possibility that polyA repeats may as well form these structures, by themselves or in association with polyQ. We found through bioinformatics screenings that polyA, polyQ and polyQA repeats have a phylogenetically graded association in human and non-human proteomes and associate/overlap with CC domains. Circular dichroism and cross-linking experiments revealed that polyA repeats can form—alone or with polyQ and polyQA—CC structures that increase in stability with polyA length, forming higher-order multimers and polymers in vitro. Using structure-guided mutagenesis, we studied the relevance of polyA CCs to the in vivo aggregation and toxicity of RUNX2—a polyQ/polyA protein associated with cleidocranial dysplasia upon polyA expansion—and found that the stability of its polyQ/polyA CC controls its aggregation, localization and toxicity. These findings indicate that, like polyQ, polyA repeats form CC structures that can trigger protein aggregation and toxicity upon expansion in human genetic diseases. PMID:24497578

  16. Association of polyalanine and polyglutamine coiled coils mediates expansion disease-related protein aggregation and dysfunction.

    PubMed

    Pelassa, Ilaria; Corà, Davide; Cesano, Federico; Monje, Francisco J; Montarolo, Pier Giorgio; Fiumara, Ferdinando

    2014-07-01

    The expansion of homopolymeric glutamine (polyQ) or alanine (polyA) repeats in certain proteins owing to genetic mutations induces protein aggregation and toxicity, causing at least 18 human diseases. PolyQ and polyA repeats can also associate in the same proteins, but the general extent of their association in proteomes is unknown. Furthermore, the structural mechanisms by which their expansion causes disease are not well understood, and these repeats are generally thought to misfold upon expansion into aggregation-prone β-sheet structures like amyloids. However, recent evidence indicates a critical role for coiled-coil (CC) structures in triggering aggregation and toxicity of polyQ-expanded proteins, raising the possibility that polyA repeats may as well form these structures, by themselves or in association with polyQ. We found through bioinformatics screenings that polyA, polyQ and polyQA repeats have a phylogenetically graded association in human and non-human proteomes and associate/overlap with CC domains. Circular dichroism and cross-linking experiments revealed that polyA repeats can form--alone or with polyQ and polyQA--CC structures that increase in stability with polyA length, forming higher-order multimers and polymers in vitro. Using structure-guided mutagenesis, we studied the relevance of polyA CCs to the in vivo aggregation and toxicity of RUNX2--a polyQ/polyA protein associated with cleidocranial dysplasia upon polyA expansion--and found that the stability of its polyQ/polyA CC controls its aggregation, localization and toxicity. These findings indicate that, like polyQ, polyA repeats form CC structures that can trigger protein aggregation and toxicity upon expansion in human genetic diseases. PMID:24497578

  17. Novel Anti-Nicotine Vaccine Using a Trimeric Coiled-Coil Hapten Carrier

    PubMed Central

    Miller, Keith D.; Roque, Richard; Clegg, Christopher H.

    2014-01-01

    Tobacco addiction represents one of the largest public health problems in the world and is the leading cause of cancer and heart disease, resulting in millions of deaths a year. Vaccines for smoking cessation have shown considerable promise in preclinical models, although functional antibody responses induced in humans are only modestly effective in preventing nicotine entry into the brain. The challenge in generating serum antibodies with a large nicotine binding capacity is made difficult by the fact that this drug is non-immunogenic and must be conjugated as a hapten to a protein carrier. To circumvent the limitations of traditional carriers like keyhole limpet hemocyanin (KLH), we have synthesized a short trimeric coiled-coil peptide (TCC) that creates a series of B and T cell epitopes with uniform stoichiometry and high density. Here we compared the relative activities of a TCC-nic vaccine and two control KLH-nic vaccines using Alum as an adjuvant or GLA-SE, which contains a synthetic TLR4 agonist formulated in a stable oil-in-water emulsion. The results showed that the TCC's high hapten density correlated with a better immune response in mice as measured by anti-nicotine Ab titer, affinity, and specificity, and was responsible for a reduction in anti-carrier immunogenicity. The Ab responses achieved with this synthetic vaccine resulted in a nicotine binding capacity in serum that could prevent >90% of a nicotine dose equivalent to three smoked cigarettes (0.05 mg/kg) from reaching the brain. PMID:25494044

  18. Design and characterization of the anion-sensitive coiled-coil peptide.

    PubMed Central

    Hoshino, M.; Yumoto, N.; Yoshikawa, S.; Goto, Y.

    1997-01-01

    As a model for analyzing the role of charge repulsion in proteins and its shielding by the solvent, we designed a peptide of 27 amino acid residues that formed a homodimeric coiled-coil. The interface between the coils consisted of hydrophobic Leu and Val residues, and 10 Lys residues per monomer were incorporated into the positions exposed to solvent. During the preparation of a disulfide-linked dimer in which the two peptides were linked in parallel by the two disulfide bonds located at the N and C terminals, a cyclic monomer with an intramolecular disulfide bond was also obtained. On the basis of CD and 1H-NMR, the conformational stabilities of these isomers and several reference peptides were examined. Whereas all these peptides were unfolded in the absence of salt at pH 4.7 and 20 degrees C, the addition of NaClO4 cooperatively stabilized the alpha-helical conformation. The crosslinking of the peptides by disulfide bonds significantly decreased the midpoint salt concentration of the transition. The 1H-NMR spectra in the presence of NaClO4 suggested that, whereas the disulfide-bonded dimer assumed a native-like conformation, the cyclic monomer assumed a molten globule-like conformation with disordered side chains. However, the cyclic monomer exhibited cooperative transitions against temperature and Gdn-HCl that were only slightly less cooperative than those of the disulfide-bonded parallel dimer. These results indicate that the charge repulsion critically destabilizes the native-like state as well as the molten globule-like state, and that the solvent-dependent charge repulsion may be useful for controlling the conformation of designed peptides. PMID:9232640

  19. A Single Missense Mutation in a Coiled-Coil Domain of Escherichia coli Ribosomal Protein S2 Confers a Thermosensitive Phenotype That Can Be Suppressed by Ribosomal Protein S1

    PubMed Central

    Aseev, Leonid V.; Chugunov, Anton O.; Efremov, Roman G.

    2013-01-01

    Ribosomal protein S2 is an essential component of translation machinery, and its viable mutated variants conferring distinct phenotypes serve as a valuable tool in studying the role of S2 in translation regulation. One of a few available rpsB mutants, rpsB1, shows thermosensitivity and ensures enhanced expression of leaderless mRNAs. In this study, we identified the nature of the rpsB1 mutation. Sequencing of the rpsB1 allele revealed a G-to-A transition in the part of the rpsB gene which encodes a coiled-coil domain of S2. The resulting E132K substitution resides in a highly conserved site, TKKE, a so-called N-terminal capping box, at the beginning of the second alpha helix. The protruding coiled-coil domain of S2 is known to provide binding with 16S rRNA in the head of the 30S subunit and, in addition, to interact with a key mRNA binding protein, S1. Molecular dynamics simulations revealed a detrimental impact of the E132K mutation on the coiled-coil structure and thereby on the interactions between S2 and 16S rRNA, providing a clue for the thermosensitivity of the rpsB1 mutant. Using a strain producing a leaderless lacZ transcript from the chromosomal lac promoter, we demonstrated that not only the rpsB1 mutation generating S2/S1-deficient ribosomes but also the rpsA::IS10 mutation leading to partial deficiency in S1 alone increased translation efficiency of the leaderless mRNA by about 10-fold. Moderate overexpression of S1 relieved all these effects and, moreover, suppressed the thermosensitive phenotype of rpsB1, indicating the role of S1 as an extragenic suppressor of the E132K mutation. PMID:23104805

  20. Crystal Structure of the Signaling Helix Coiled-coil Domain of the b1 Subunit of the Soluble guanylyl Cyclase

    SciTech Connect

    Ma, X.; Beuve, A; van den Akker, F

    2010-01-01

    The soluble guanylyl cyclase (sGC) is a heterodimeric enzyme that, upon activation by nitric oxide, stimulates the production of the second messenger cGMP. Each sGC subunit harbor four domains three of which are used for heterodimerization: H-NOXA/H-NOBA domain, coiled-coil domain (CC), and catalytic guanylyl cyclase domain. The CC domain has previously been postulated to be part of a larger CC family termed the signaling helix (S-helix) family. Homodimers of sGC have also been observed but are not functionally active yet are likely transient awaiting their intended heterodimeric partner. To investigate the structure of the CC S-helix region, we crystallized and determined the structure of the CC domain of the sGC{beta}1 subunit comprising residues 348-409. The crystal structure was refined to 2.15 {angstrom} resolution. The CC structure of sGC{beta}1 revealed a tetrameric arrangement comprised of a dimer of CC dimers. Each monomer is comprised of a long a-helix, a turn near residue P399, and a short second a-helix. The CC structure also offers insights as to how sGC homodimers are not as stable as (functionally) active heterodimers via a possible role for inter-helix salt-bridge formation. The structure also yielded insights into the residues involved in dimerization. In addition, the CC region is also known to harbor a number of congenital and man-made mutations in both membrane and soluble guanylyl cyclases and those function-affecting mutations have been mapped onto the CC structure. This mutant analysis indicated an importance for not only certain dimerization residue positions, but also an important role for other faces of the CC dimer which might perhaps interact with adjacent domains. Our results also extend beyond guanylyl cyclases as the CC structure is, to our knowledge, the first S-helix structure and serves as a model for all S-helix containing family members.

  1. Adenovirus E4-ORF3-dependent relocalization of TIF1{alpha} and TIF1{gamma} relies on access to the Coiled-Coil motif

    SciTech Connect

    Vink, Elizabeth I.; Yondola, Mark A.; Wu, Kai; Hearing, Patrick

    2012-01-20

    The adenovirus E4-ORF3 protein promotes viral replication by relocalizing cellular proteins into nuclear track structures, interfering with potential anti-viral activities. E4-ORF3 targets transcriptional intermediary factor 1 alpha (TIF1{alpha}), but not homologous TIF1{beta}. Here, we introduce TIF1{gamma} as a novel E4-ORF3-interacting partner. E4-ORF3 relocalizes endogenous TIF1{gamma} in virus-infected cells in vivo and binds to TIF1{gamma} in vitro. We used the homologous nature, yet differing binding capabilities, of these proteins to study how E4-ORF3 targets proteins for track localization. We mapped the ability of E4-ORF3 to interact with specific TIF1 subdomains, demonstrating that E4-ORF3 interacts with the Coiled-Coil domains of TIF1{alpha}, TIF1{beta}, and TIF1{gamma}, and that the C-terminal half of TIF1{beta} interferes with this interaction. The results of E4-ORF3-directed TIF1 protein relocalization assays performed in vivo were verified using coimmunoprecipitation assays in vitro. These results suggest that E4-ORF3 targets proteins for relocalization through a loosely homologous sequence dependent on accessibility.

  2. A Coiled-Coil Enabled Split-Luciferase Three-Hybrid System: Applied Toward Profiling Inhibitors of Protein Kinases

    PubMed Central

    Jester, Benjamin W.; Cox, Kurt J.; Gaj, Alicia; Shomin, Carolyn D.; Porter, Jason R.; Ghosh, Indraneel

    2010-01-01

    The 518 protein kinases encoded in the human genome are exquisitely regulated and their aberrant function(s) are often associated with human disease. Thus, in order to advance therapeutics and to probe signal transduction cascades there is considerable interest in the development of inhibitors that can selectively target protein kinases. However, identifying specific compounds against such a large array of protein kinases is difficult to routinely achieve utilizing traditional activity assays, where purified protein kinases are necessary. Toward a simple, rapid, and practical method for identifying specific inhibitors, we describe the development and application of a split-protein methodology utilizing a coiled-coil assisted three-hybrid system. In this approach, a protein kinase of interest is attached to the C-terminal fragment of split-firefly luciferase and the coiled-coil Fos, which is specific for the coiled-coil Jun, is attached to the N-terminal fragment. Upon addition of Jun conjugated to a pan-kinase inhibitor such as staurosporine, a three-hybrid complex is established with concomitant reassembly of the split-luciferase enzyme. An inhibitor can be potentially identified by the commensurate loss in split-luciferase activity by displacement of the modified staurosporine. We demonstrate that this new three-hybrid approach is potentially general by testing protein kinases from the different kinase families. To interrogate whether this method allows for screening inhibitors, we tested six different protein kinases against a library of 80 known protein kinase inhibitors. Finally, we demonstrate that this three-hybrid system can potentially provide a rapid method for structure/function analysis as well as aid in the identification of allosteric inhibitors. PMID:20669947

  3. Second coiled-coil domain of KCNQ channel controls current expression and subfamily specific heteromultimerization by salt bridge networks.

    PubMed

    Nakajo, Koichi; Kubo, Yoshihiro

    2008-06-15

    KCNQ channels carry the slowly activating, voltage-dependent M-current in excitable cells such as neurons. Although the KCNQ2 homomultimer can form a functional voltage-gated K(+) channel, heteromultimerization with KCNQ3 produces a > 10-fold increase in current amplitude. All KCNQ channels contain double coiled-coil domains (TCC1 and TCC2, or A-domain Head and Tail), of which TCC2 (A-domain Tail) is thought to be important for subunit recognition, channel assembly and surface expression. The mechanism by which TCC2 recognizes and associates with its partner is not fully understood, however. Our aim in the present study was to elucidate the recognition mechanism by examining the phenotypes of TCC2-deletion mutants, TCC2-swapped chimeras and point mutants. Electrophysiological analysis using Xenopus oocytes under two-electrode voltage clamp revealed that homotetrameric KCNQ3 TCC2 is a negative regulator of current expression in the absence of KCNQ2 TCC2. Recent structural analysis of KCNQ4 TCC2 revealed the presence of intercoil salt bridge networks. We therefore swapped the sign of the charged residues reportedly involved in the salt bridge formation and functionally confirmed that the intercoil salt bridge network is responsible for the subunit recognition between KCNQ2 and KCNQ3. Finally, we constructed TCC2-swapped KCNQ2/KCNQ3 mutants with KCNQ1 TCC2 or GCN4-pLI, a coiled-coil domain from an unrelated protein, and found that TCC2 is substitutable and even GCN4-pLI can work as a substitute for TCC2. Our present data provide some new insights into the role played by TCC2 during current expression, and also provide functional evidence of the importance of the intercoil salt bridge network for subunit recognition and coiled-coil formation, as is suggested by recent crystallographic data. PMID:18440995

  4. Missense mutation in immunodeficient patients shows the multifunctional roles of coiled-coil domain 3 (CC3) in STIM1 activation.

    PubMed

    Maus, Mate; Jairaman, Amit; Stathopulos, Peter B; Muik, Martin; Fahrner, Marc; Weidinger, Carl; Benson, Melina; Fuchs, Sebastian; Ehl, Stephan; Romanin, Christoph; Ikura, Mitsuhiko; Prakriya, Murali; Feske, Stefan

    2015-05-12

    Store-operated Ca(2+) entry (SOCE) is a universal Ca(2+) influx pathway that is important for the function of many cell types. SOCE occurs upon depletion of endoplasmic reticulum (ER) Ca(2+) stores and relies on a complex molecular interplay between the plasma membrane (PM) Ca(2+) channel ORAI1 and the ER Ca(2+) sensor stromal interaction molecule (STIM) 1. Patients with null mutations in ORAI1 or STIM1 genes present with severe combined immunodeficiency (SCID)-like disease. Here, we describe the molecular mechanisms by which a loss-of-function STIM1 mutation (R429C) in human patients abolishes SOCE. R429 is located in the third coiled-coil (CC3) domain of the cytoplasmic C terminus of STIM1. Mutation of R429 destabilizes the CC3 structure and alters the conformation of the STIM1 C terminus, thereby releasing a polybasic domain that promotes STIM1 recruitment to ER-PM junctions. However, the mutation also impairs cytoplasmic STIM1 oligomerization and abolishes STIM1-ORAI1 interactions. Thus, despite its constitutive localization at ER-PM junctions, mutant STIM1 fails to activate SOCE. Our results demonstrate multifunctional roles of the CC3 domain in regulating intra- and intermolecular STIM1 interactions that control (i) transition of STIM1 from a quiescent to an active conformational state, (ii) cytoplasmic STIM1 oligomerization, and (iii) STIM1-ORAI1 binding required for ORAI1 activation. PMID:25918394

  5. Missense mutation in immunodeficient patients shows the multifunctional roles of coiled-coil domain 3 (CC3) in STIM1 activation

    PubMed Central

    Maus, Mate; Jairaman, Amit; Stathopulos, Peter B.; Muik, Martin; Fahrner, Marc; Weidinger, Carl; Benson, Melina; Fuchs, Sebastian; Ehl, Stephan; Romanin, Christoph; Ikura, Mitsuhiko; Prakriya, Murali; Feske, Stefan

    2015-01-01

    Store-operated Ca2+ entry (SOCE) is a universal Ca2+ influx pathway that is important for the function of many cell types. SOCE occurs upon depletion of endoplasmic reticulum (ER) Ca2+ stores and relies on a complex molecular interplay between the plasma membrane (PM) Ca2+ channel ORAI1 and the ER Ca2+ sensor stromal interaction molecule (STIM) 1. Patients with null mutations in ORAI1 or STIM1 genes present with severe combined immunodeficiency (SCID)-like disease. Here, we describe the molecular mechanisms by which a loss-of-function STIM1 mutation (R429C) in human patients abolishes SOCE. R429 is located in the third coiled-coil (CC3) domain of the cytoplasmic C terminus of STIM1. Mutation of R429 destabilizes the CC3 structure and alters the conformation of the STIM1 C terminus, thereby releasing a polybasic domain that promotes STIM1 recruitment to ER–PM junctions. However, the mutation also impairs cytoplasmic STIM1 oligomerization and abolishes STIM1–ORAI1 interactions. Thus, despite its constitutive localization at ER–PM junctions, mutant STIM1 fails to activate SOCE. Our results demonstrate multifunctional roles of the CC3 domain in regulating intra- and intermolecular STIM1 interactions that control (i) transition of STIM1 from a quiescent to an active conformational state, (ii) cytoplasmic STIM1 oligomerization, and (iii) STIM1–ORAI1 binding required for ORAI1 activation. PMID:25918394

  6. All-atom simulations and free-energy calculations of coiled-coil peptides with lipid bilayers: binding strength, structural transition, and effect on lipid dynamics.

    PubMed

    Woo, Sun Young; Lee, Hwankyu

    2016-01-01

    Peptides E and K, which are synthetic coiled-coil peptides for membrane fusion, were simulated with lipid bilayers composed of lipids and cholesterols at different ratios using all-atom models. We first calculated free energies of binding from umbrella sampling simulations, showing that both E and K peptides tend to adsorb onto the bilayer surface, which occurs more strongly in the bilayer composed of smaller lipid headgroups. Then, unrestrained simulations show that K peptides more deeply insert into the bilayer with partially retaining the helical structure, while E peptides less insert and predominantly become random coils, indicating the structural transition from helices to random coils, in quantitative agreement with experiments. This is because K peptides electrostatically interact with lipid phosphates, as well as because hydrocarbons of lysines of K peptide are longer than those of glutamic acids of E peptide and thus form stronger hydrophobic interactions with lipid tails. This deeper insertion of K peptide increases the bilayer dynamics and a vacancy below the peptide, leading to the rearrangement of smaller lipids. These findings help explain the experimentally observed or proposed differences in the insertion depth, binding strength, and structural transition of E and K peptides, and support the snorkeling effect. PMID:26926570

  7. All-atom simulations and free-energy calculations of coiled-coil peptides with lipid bilayers: binding strength, structural transition, and effect on lipid dynamics

    PubMed Central

    Woo, Sun Young; Lee, Hwankyu

    2016-01-01

    Peptides E and K, which are synthetic coiled-coil peptides for membrane fusion, were simulated with lipid bilayers composed of lipids and cholesterols at different ratios using all-atom models. We first calculated free energies of binding from umbrella sampling simulations, showing that both E and K peptides tend to adsorb onto the bilayer surface, which occurs more strongly in the bilayer composed of smaller lipid headgroups. Then, unrestrained simulations show that K peptides more deeply insert into the bilayer with partially retaining the helical structure, while E peptides less insert and predominantly become random coils, indicating the structural transition from helices to random coils, in quantitative agreement with experiments. This is because K peptides electrostatically interact with lipid phosphates, as well as because hydrocarbons of lysines of K peptide are longer than those of glutamic acids of E peptide and thus form stronger hydrophobic interactions with lipid tails. This deeper insertion of K peptide increases the bilayer dynamics and a vacancy below the peptide, leading to the rearrangement of smaller lipids. These findings help explain the experimentally observed or proposed differences in the insertion depth, binding strength, and structural transition of E and K peptides, and support the snorkeling effect. PMID:26926570

  8. All-atom simulations and free-energy calculations of coiled-coil peptides with lipid bilayers: binding strength, structural transition, and effect on lipid dynamics

    NASA Astrophysics Data System (ADS)

    Woo, Sun Young; Lee, Hwankyu

    2016-03-01

    Peptides E and K, which are synthetic coiled-coil peptides for membrane fusion, were simulated with lipid bilayers composed of lipids and cholesterols at different ratios using all-atom models. We first calculated free energies of binding from umbrella sampling simulations, showing that both E and K peptides tend to adsorb onto the bilayer surface, which occurs more strongly in the bilayer composed of smaller lipid headgroups. Then, unrestrained simulations show that K peptides more deeply insert into the bilayer with partially retaining the helical structure, while E peptides less insert and predominantly become random coils, indicating the structural transition from helices to random coils, in quantitative agreement with experiments. This is because K peptides electrostatically interact with lipid phosphates, as well as because hydrocarbons of lysines of K peptide are longer than those of glutamic acids of E peptide and thus form stronger hydrophobic interactions with lipid tails. This deeper insertion of K peptide increases the bilayer dynamics and a vacancy below the peptide, leading to the rearrangement of smaller lipids. These findings help explain the experimentally observed or proposed differences in the insertion depth, binding strength, and structural transition of E and K peptides, and support the snorkeling effect.

  9. Crystallographic characterization of the C-terminal coiled-coil region of mouse Bicaudal-D1 (BICD1)

    PubMed Central

    Terawaki, Shin-ichi; Ootsuka, Hiroki; Higuchi, Yoshiki; Wakamatsu, Kaori

    2014-01-01

    Bicaudal-D1 (BICD1) is an α-helical coiled-coil protein which is evolutionarily conserved from Drosophila to mammals and facilitates the attachment of specific cargo factors to the dynein motor complex. The C-terminal coiled-coil region (CC3) of BICD1 plays an important role in sorting cargo, linking proteins such as the small GTPase Rab6 and the nuclear pore complex component Ran-binding protein 2 (RanBP2) to the dynein motor complex. This report describes the crystallization and X-ray data collection of the BICD1 CC3 region, as well as the preparation of the complex of BICD1 CC3 with a constitutively active mutant of Rab6. The crystals of the BICD1 CC3 region belonged to space group C2, with unit-cell parameters a = 59.0, b = 36.8, c = 104.3 Å, α = γ = 90, β = 99.8°. The X-ray diffraction data set was collected to 1.50 Å resolution. PMID:25084392

  10. A coiled-coil motif in non-structural protein 3 (NS3) of bluetongue virus forms an oligomer.

    PubMed

    Chacko, Nirmal; Mohanty, Nihar Nalini; Biswas, Sanchay Kumar; Chand, Karam; Yogisharadhya, Revanaiah; Pandey, Awadh Bihari; Mondal, Bimalendu; Shivachandra, Sathish Bhadravati

    2015-10-01

    Bluetongue, an arthropod-borne non-contagious hemorrhagic disease of small ruminants, is caused by bluetongue virus (BTV). Several structural and non-structural proteins encoded by BTV have been associated with virulence mechanisms. In the present study, the NS3 protein sequences of bluetongue viral serotypes were analyzed for the presence of heptad regions and oligomer formation. Bioinformatic analysis of NS3 sequences of all 26 BTV serotypes revealed the presence of at least three coiled-coil motifs (CCMs). A conserved α-helical heptad sequence was identified at 14-26 aa (CCM-I), 185-198aa (CCM-II), and 94-116 aa (CCM-III). Among these, CCM-I occurs close to the N-terminus of NS3 and was presumed to be involved in oligomerization. Furthermore, the N-terminus of NS3 (1M-R117 aa) was over-expressed as a recombinant fusion protein in a prokaryotic expression system. Biochemical characterization of recombinant NS3Nt protein revealed that it forms SDS-resistant dimers and high-order oligomers (hexamer and/or octamer) under reducing or non-reducing conditions. Coiled-coil motifs are believed to be critical for NS protein oligomerization and have potential roles in the formation of viroporin ring/pore either with six/eight subunits and this is the first study toward characterization of CCMs in NS3 of bluetongue virus. PMID:26318174

  11. Alanine zipper-like coiled-coil domains are necessary for homotypic dimerization of plant GAGA-factors in the nucleus and nucleolus.

    PubMed

    Wanke, Dierk; Hohenstatt, Mareike L; Dynowski, Marek; Bloss, Ulrich; Hecker, Andreas; Elgass, Kirstin; Hummel, Sabine; Hahn, Achim; Caesar, Katharina; Schleifenbaum, Frank; Harter, Klaus; Berendzen, Kenneth W

    2011-01-01

    GAGA-motif binding proteins control transcriptional activation or repression of homeotic genes. Interestingly, there are no sequence similarities between animal and plant proteins. Plant BBR/BPC-proteins can be classified into two distinct groups: Previous studies have elaborated on group I members only and so little is known about group II proteins. Here, we focused on the initial characterization of AtBPC6, a group II protein from Arabidopsis thaliana. Comparison of orthologous BBR/BPC sequences disclosed two conserved signatures besides the DNA binding domain. A first peptide signature is essential and sufficient to target AtBPC6-GFP to the nucleus and nucleolus. A second domain is predicted to form a zipper-like coiled-coil structure. This novel type of domain is similar to Leucine zippers, but contains invariant alanine residues with a heptad spacing of 7 amino acids. By yeast-2-hybrid and BiFC-assays we could show that this Alanine zipper domain is essential for homotypic dimerization of group II proteins in vivo. Interhelical salt bridges and charge-stabilized hydrogen bonds between acidic and basic residues of the two monomers are predicted to form an interaction domain, which does not follow the classical knobs-into-holes zipper model. FRET-FLIM analysis of GFP/RFP-hybrid fusion proteins validates the formation of parallel dimers in planta. Sequence comparison uncovered that this type of domain is not restricted to BBR/BPC proteins, but is found in all kingdoms. PMID:21347358

  12. Alanine Zipper-Like Coiled-Coil Domains Are Necessary for Homotypic Dimerization of Plant GAGA-Factors in the Nucleus and Nucleolus

    PubMed Central

    Bloss, Ulrich; Hecker, Andreas; Elgass, Kirstin; Hummel, Sabine; Hahn, Achim; Caesar, Katharina; Schleifenbaum, Frank; Harter, Klaus; Berendzen, Kenneth W.

    2011-01-01

    GAGA-motif binding proteins control transcriptional activation or repression of homeotic genes. Interestingly, there are no sequence similarities between animal and plant proteins. Plant BBR/BPC-proteins can be classified into two distinct groups: Previous studies have elaborated on group I members only and so little is known about group II proteins. Here, we focused on the initial characterization of AtBPC6, a group II protein from Arabidopsis thaliana. Comparison of orthologous BBR/BPC sequences disclosed two conserved signatures besides the DNA binding domain. A first peptide signature is essential and sufficient to target AtBPC6-GFP to the nucleus and nucleolus. A second domain is predicted to form a zipper-like coiled-coil structure. This novel type of domain is similar to Leucine zippers, but contains invariant alanine residues with a heptad spacing of 7 amino acids. By yeast-2-hybrid and BiFC-assays we could show that this Alanine zipper domain is essential for homotypic dimerization of group II proteins in vivo. Interhelical salt bridges and charge-stabilized hydrogen bonds between acidic and basic residues of the two monomers are predicted to form an interaction domain, which does not follow the classical knobs-into-holes zipper model. FRET-FLIM analysis of GFP/RFP-hybrid fusion proteins validates the formation of parallel dimers in planta. Sequence comparison uncovered that this type of domain is not restricted to BBR/BPC proteins, but is found in all kingdoms. PMID:21347358

  13. Intermediate filament mechanics in vitro and in the cell: From coiled coils to filaments, fibers and networks

    PubMed Central

    Köster, Sarah; Weitz, David; Goldman, Robert D.; Aebi, Ueli; Herrmann, Harald

    2015-01-01

    Summary Intermediate filament proteins form filaments, fibers and networks both in the cytoplasm and the nucleus of metazoan cells. Their general structural building plan accommodates highly varying amino acid sequences to yield extended dimeric α-helical coiled coils of highly conserved design. These “rod” particles are the basic building blocks of intrinsically flexible, filamentous structures that are able to resist high mechanical stresses, i.e. bending and stretching to a considerable degree, both in vitro and in the cell. Biophysical and computer modeling studies are beginning to unfold detailed structural and mechanical insights into these major supramolecular assemblies of cell architecture, not only in the “test tube” but also in the cellular and tissue context. PMID:25621895

  14. Multimodality Imaging of Coiled-Coil Mediated Self-Assembly in a “Drug Free” Therapeutic System

    PubMed Central

    Zhang, Rui; Yang, Jiyuan; Chu, Te-Wei; Hartley, Jonathan M.; Kopeček, Jindřich

    2015-01-01

    We used two complementary coiled-coil peptides CCE/CCK to develop a “drug free” therapeutic system, which can specifically kill cancer cells without a drug. CCE was attached to the Fab’ fragment of anti-CD20 1F5 antibody (Fab’-CCE), and CCK was conjugated in multiple grafts to poly[N-(2-hydroxypropyl)methacrylamide] (P-(CCK)x). Two conjugates are consecutively administered: First, Fab’-CCE coats peptide CCE at CD20 antigen of lymphoma cell surface; second, CCE/CCK biorecognition between Fab’-CCE and P-(CCK)x leads to coiled-coil formation, CD20 crosslinking, membrane reorganization, and ultimately cell apoptosis. To prove that two conjugates can assemble at cell surface, multiple fluorescence imaging studies were performed, including 2-channel FMT, 3D confocal microscopy, and 4-color FACS. Confocal microscopy showed co-localization of two fluorescently labeled conjugates on non-Hodgkin's lymphoma (NHL) Raji cell surface, indicating “two-step” targeting specificity. The fluorescent images also revealed that these two conjugates could disrupt normal membrane lipid distribution and form lipid raft clusters, leading to cancer cell apoptosis. This “two-step” biorecognition capacity was further demonstrated in a NHL xenograft model, using fluorescent images at whole-body, tissue and cell levels. We also found that delaying injection of P-(CCK)x could significantly enhance targeting efficacy. This high-specificity therapeutics provide a safe option to treat NHL and other B cell malignancies. PMID:25612325

  15. Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils

    PubMed Central

    Tegoni, Matteo; Yu, Fangting; Bersellini, Manuela; Penner-Hahn, James E.; Pecoraro, Vincent L.

    2012-01-01

    One of the ultimate objectives of de novo protein design is to realize systems capable of catalyzing redox reactions on substrates. This goal is challenging as redox-active proteins require design considerations for both the reduced and oxidized states of the protein. In this paper, we describe the spectroscopic characterization and catalytic activity of a de novo designed metallopeptide Cu(I/II)(TRIL23H)3+/2+, where Cu(I/II) is embeded in α-helical coiled coils, as a model for the CuT2 center of copper nitrite reductase. In Cu(I/II)(TRIL23H)3+/2+, Cu(I) is coordinated to three histidines, as indicated by X-ray absorption data, and Cu(II) to three histidines and one or two water molecules. Both ions are bound in the interior of the three-stranded coiled coils with affinities that range from nano- to micromolar [Cu(II)], and picomolar [Cu(I)]. The Cu(His)3 active site is characterized in both oxidation states, revealing similarities to the CuT2 site in the natural enzyme. The species Cu(II)(TRIL23H)32+ in aqueous solution can be reduced to Cu(I)(TRIL23H)3+ using ascorbate, and reoxidized by nitrite with production of nitric oxide. At pH 5.8, with an excess of both the reductant (ascorbate) and the substrate (nitrite), the copper peptide Cu(II)(TRIL23H)32+ acts as a catalyst for the reduction of nitrite with at least five turnovers and no loss of catalytic efficiency after 3.7 h. The catalytic activity, which is first order in the concentration of the peptide, also shows a pH dependence that is described and discussed. PMID:23236170

  16. Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils.

    PubMed

    Tegoni, Matteo; Yu, Fangting; Bersellini, Manuela; Penner-Hahn, James E; Pecoraro, Vincent L

    2012-12-26

    One of the ultimate objectives of de novo protein design is to realize systems capable of catalyzing redox reactions on substrates. This goal is challenging as redox-active proteins require design considerations for both the reduced and oxidized states of the protein. In this paper, we describe the spectroscopic characterization and catalytic activity of a de novo designed metallopeptide Cu(I/II)(TRIL23H)(3)(+/2+), where Cu(I/II) is embeded in α-helical coiled coils, as a model for the Cu(T2) center of copper nitrite reductase. In Cu(I/II)(TRIL23H)(3)(+/2+), Cu(I) is coordinated to three histidines, as indicated by X-ray absorption data, and Cu(II) to three histidines and one or two water molecules. Both ions are bound in the interior of the three-stranded coiled coils with affinities that range from nano- to micromolar [Cu(II)], and picomolar [Cu(I)]. The Cu(His)(3) active site is characterized in both oxidation states, revealing similarities to the Cu(T2) site in the natural enzyme. The species Cu(II)(TRIL23H)(3)(2+) in aqueous solution can be reduced to Cu(I)(TRIL23H)(3)(+) using ascorbate, and reoxidized by nitrite with production of nitric oxide. At pH 5.8, with an excess of both the reductant (ascorbate) and the substrate (nitrite), the copper peptide Cu(II)(TRIL23H)(3)(2+) acts as a catalyst for the reduction of nitrite with at least five turnovers and no loss of catalytic efficiency after 3.7 h. The catalytic activity, which is first order in the concentration of the peptide, also shows a pH dependence that is described and discussed. PMID:23236170

  17. Insights on the structure and stability of Licanantase: a trimeric acid-stable coiled-coil lipoprotein from Acidithiobacillus thiooxidans

    PubMed Central

    Abarca, Fernando; Gutierrez-Maldonado, Sebastian E.; Parada, Pilar; Martinez, Patricio; Maass, Alejandro

    2014-01-01

    Licanantase (Lic) is the major component of the secretome of Acidithiobacillus thiooxidans when grown in elemental sulphur. When used as an additive, Lic improves copper recovery from bioleaching processes. However, this recovery enhancement is not fully understood. In this context, our aim is to predict the 3D structure of Lic, to shed light on its structure-function relationships. Bioinformatics analyses on the amino acid sequence of Lic showed a great similarity with Lpp, an Escherichia coli Lipoprotein that can form stable trimers in solution. Lic and Lpp share the secretion motif, intracellular processing and alpha helix structure, as well as the distribution of hydrophobic residues in heptads forming a hydrophobic core, typical of coiled-coil structures. Cross-linking experiments showed the presence of Lic trimers, supporting our predictions. Taking the in vitro and in silico evidence as a whole, we propose that the most probable structure for Lic is a trimeric coiled-coil. According to this prediction, a suitable model for Lic was produced using the de novo algorithm “Rosetta Fold-and-Dock”. To assess the structural stability of our model, Molecular Dynamics (MD) and Replica Exchange MD simulations were performed using the structure of Lpp and a 14-alanine Lpp mutant as controls, at both acidic and neutral pH. Our results suggest that Lic was the most stable structure among the studied proteins in both pH conditions. This increased stability can be explained by a higher number of both intermonomer hydrophobic contacts and hydrogen bonds, key elements for the stability of Lic’s secondary and tertiary structure. PMID:25165619

  18. The Structures of Coiled-Coil Domains from Type III Secretion System Translocators Reveal Homology to Pore-Forming Toxins

    SciTech Connect

    Barta, Michael L.; Dickenson, Nicholas E.; Patil, Mrinalini; Keightley, Andrew; Wyckoff, Gerald J.; Picking, William D.; Picking, Wendy L.; Geisbrecht, Brian V.

    2012-03-26

    Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SSs) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) that is responsible for over one million deaths per year. The Shigella type III secretion apparatus is composed of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized unidirectional conduit to promote bacterial invasion. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC. While the atomic structure of IpaD has been elucidated and studied, structural data on the hydrophobic translocators from the T3SS family remain elusive. We present here the crystal structures of a protease-stable fragment identified within the N-terminal regions of IpaB from S. flexneri and SipB from Salmonella enterica serovar Typhimurium determined at 2.1 {angstrom} and 2.8 {angstrom} limiting resolution, respectively. These newly identified domains are composed of extended-length (114 {angstrom} in IpaB and 71 {angstrom} in SipB) coiled-coil motifs that display a high degree of structural homology to one another despite the fact that they share only 21% sequence identity. Further structural comparisons also reveal substantial similarity to the coiled-coil regions of pore-forming proteins from other Gram-negative pathogens, notably, colicin Ia. This suggests that these mechanistically separate and functionally distinct membrane-targeting proteins may have diverged from a common ancestor during the course of pathogen-specific evolutionary events.

  19. Decorin induces rapid secretion of thrombospondin-1 in basal breast carcinoma cells via inhibition of Ras homolog gene family, member A/Rho-associated coiled-coil containing protein kinase 1.

    PubMed

    Neill, Thomas; Jones, Holly R; Crane-Smith, Zoe; Owens, Rick T; Schaefer, Liliana; Iozzo, Renato V

    2013-05-01

    Pathological neovascularization relies on an imbalance between potent proangiogenic agents and equally effective antiangiogenic cues. Collectively, these factors contribute to an angiogenic niche within the tumor microenvironment. Oncogenic events and hypoxia contribute to augmented levels of angiokines, and thereby activate the so-called angiogenic switch to promote aggressive tumorigenic and metastatic growth. Soluble decorin functions as a paracrine pan-inhibitor of receptor tyrosine kinases, such as Met and epidermal growth factor receptor, and thus is capable of suppressing angiogenesis under normoxia. This leads to noncanonical repression of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor A (VEGFA), and concurrent induction of thrombospondin-1. The substantial induction of endogenous tumor cell-derived thrombospondin-1, a potent antiangiogenic effector, led us to the discovery of an unexpected secretory phenotype occurring very rapidly (within 5 min) after decorin treatment of the triple-negative basal breast carcinoma cell line MDA-MB-231. Surprisingly, the effect was not mediated by Met receptor antagonism, as initially hypothesized, but required epidermal growth factor receptor signaling to achieve swift and robust thrombospondin-1 release. Furthermore, this effect was ultimately dependent on the prompt degradation of Ras homolog gene family member A, via the 26S proteasome, leading to direct inactivation of Rho-associated coiled-coil containing protein kinase 1. The latter led to derepression of thrombospondin-1 secretion. Collectively, these data provide a novel mechanistic role for Rho-associated coiled-coil containing protein kinase 1, in addition to providing the first conclusive evidence of decorin exclusively targeting a receptor tyrosine kinase to achieve a specific effect. The overall effects of soluble decorin on the tumor microenvironment would cause an immediately-early as well as a sustained antiangiogenic response

  20. Analysis of the coding sequence and expression of the coiled-coil α-helical rod protein 1 gene in normal and neoplastic epithelial cervical cells

    PubMed Central

    PACHOLSKA-BOGALSKA, JOANNA; MYGA-NOWAK, MAGDALENA; CIEPŁUCH, KATARZYNA; JÓZEFIAK, AGATA; KWAŒNIEWSKA, ANNA; GOźDZICKA-JÓZEFIAK, ANNA

    2012-01-01

    The role of the CCHCR1 (coiled-coil α-helical rod protein 1) protein in the cell is poorly understood. It is thought to be engaged in processes such as proliferation and differentiation of epithelial cells, tissue-specific gene transcription and steroidogenesis. It is supposed to participate in keratinocyte transformation. It has also been found that this protein interacts with the E2 protein of human papilloma virus type 16 (HPV16). The oncogenic HPV forms, such as HPV16, are known to be necessary but not sufficient agents in the development of cervical carcinoma. In the present study, the CCHCR1 gene coding sequence and its expression was analyzed in normal, precancerous and cervical cancer cells. Changes in the non-coding region were found in 20.3% of the examined probes from women with cervical cancer or precancerous lesions and in 16.67% of the control probes. Most of the detected changes were single nucleotide polymorphisms (SNPs). Changes in the coding region were found in 22.8% of the probes with cervical cancer and in 16.67% of the control probes and all of them were SNPs. The level of CCHCR1 transcripts was determined using the real-time PCR method and the highest gene expression was detected in the H-SIL group and slightly decreased in the cervical carcinoma cells, compared with the control probes. It suggests that CCHCR1 could have a role in the process of cervical epithelial cell transformation, but this suggestion must be confirmed experimentally. PMID:22218424

  1. The coiled-coil domain containing protein CCDC151 is required for the function of IFT-dependent motile cilia in animals.

    PubMed

    Jerber, Julie; Baas, Dominique; Soulavie, Fabien; Chhin, Brigitte; Cortier, Elisabeth; Vesque, Christine; Thomas, Joëlle; Durand, Bénédicte

    2014-02-01

    Cilia are evolutionarily conserved organelles endowed with essential physiological and developmental functions. In humans, disruption of cilia motility or signaling leads to complex pleiotropic genetic disorders called ciliopathies. Cilia motility requires the assembly of multi-subunit motile components such as dynein arms, but mechanisms underlying their assembly pathway and transport into the axoneme are still largely unknown. We identified a previously uncharacterized coiled-coil domain containing protein CCDC151, which is evolutionarily conserved in motile ciliated species and shares ancient features with the outer dynein arm-docking complex 2 of Chlamydomonas. In Drosophila, we show that CG14127/CCDC151 is associated with motile intraflagellar transport (IFT)-dependent cilia and required for geotaxis behavior of adult flies. In zebrafish, Ccdc151 is expressed in tissues with motile cilia, and morpholino-induced depletion of Ccdc151 leads to left-right asymmetry defects and kidney cysts. We demonstrate that Ccdc151 is required for proper motile function of cilia in the Kupffer's vesicle and in the pronephros by controlling dynein arm assembly, showing that Ccdc151 is a novel player in the control of IFT-dependent dynein arm assembly in animals. However, we observed that CCDC151 is also implicated in other cellular functions in vertebrates. In zebrafish, ccdc151 is involved in proper orientation of cell divisions in the pronephros and genetically interacts with prickle1 in this process. Furthermore, knockdown experiments in mammalian cells demonstrate that CCDC151 is implicated in the regulation of primary cilium length. Hence, CCDC151 is required for motile cilia function in animals but has acquired additional non-motile functions in vertebrates. PMID:24067530

  2. A systematic study of fundamentals in α-helical coiled coil mimicry by alternating sequences of β- and γ-amino acids.

    PubMed

    Rezaei Araghi, Raheleh; Baldauf, Carsten; Gerling, Ulla I M; Cadicamo, Cosimo Damiano; Koksch, Beate

    2011-08-01

    Aimed at understanding the crucially important structural features for the integrity of α-helical mimicry by βγ-sequences, an α-amino acid sequence in a native peptide was substituted by differently arranged βγ-sequences. The self- and hetero-assembly of a series of αβγ-chimeric sequences based on a 33-residue GCN4-derived peptide was investigated by means of molecular dynamics, circular dichroism, and a disulfide exchange assay. Despite the native-like behavior of βγ alternating sequences such as retention of α-helix dipole and the formation of 13-membered α-helix turns, the αβγ-chimeras with different βγ substitution patterns do not equally mimic the structural behavior of the native parent peptide in solution. The preservation of the key residue contacts such as van der Waals interactions and intrahelical H-bonding, which can be met only by particular substitution patterns, thermodynamically favor the adoption of coiled coil folding motif. In this study, we show how successfully the destabilizing structural consequences of α → βγ modification can be harnessed by reducing the solvent-exposed hydrophobic surface area and placing of suitably long and bulky helix-forming side chains at the hydrophobic core. The pairing of αβγ-chimeric sequences with the native wild-type are thermodynamically allowed in the case of ideal arrangement of β- and γ-residues. This indicates a similarity in local side chain packing of β- and γ-amino acids at the helical interface of αβγ-chimeras and the native α-peptide. Consequently, the backbone extended residues are able to participate in classical "knob-into-hole" packing with native α-peptide. PMID:21638022

  3. TACC2 (transforming acidic coiled-coil protein 2) in breast carcinoma as a potent prognostic predictor associated with cell proliferation.

    PubMed

    Onodera, Yoshiaki; Takagi, Kiyoshi; Miki, Yasuhiro; Takayama, Ken-Ichi; Shibahara, Yukiko; Watanabe, Mika; Ishida, Takanori; Inoue, Satoshi; Sasano, Hironobu; Suzuki, Takashi

    2016-08-01

    Transforming acidic coiled-coil protein 2 (TACC2) belongs to TACC family proteins and involved in a variety of cellular processes through interactions with some molecules involved in centrosomes/microtubules dynamics. Mounting evidence suggests that TACCs is implicated in the progression of some human malignancies, but significance of TACC2 protein in breast carcinoma is still unknown. Therefore, in this study, we examined the clinical significance of TACC2 in breast carcinoma and biological functions by immunohistochemistry and in vitro experiments. Immunohistochemistry for TACC2 was performed in 154 cases of invasive ductal carcinoma. MCF-7 and MDA-MB-453 breast carcinoma cell lines were transfected with small interfering RNA (siRNA) for TACC2, and subsequently, cell proliferation, 5-Bromo-2'-deoxyuridine (BrdU), and invasion assays were performed. TACC2 immunoreactivity was detected in 78 out of 154 (51%) breast carcinoma tissues, and it was significantly associated with Ki-67 LI. The immunohistochemical TACC2 status was significantly associated with increased incidence of recurrence and breast cancer-specific death of the patients, and multivariate analyses demonstrated TACC2 status as an independent prognostic factor for both disease-free and breast cancer-specific survival. Subsequent in vitro experiments showed that TACC2 significantly increased the proliferation activity of MCF-7 and MDA-MB-453. These results suggest that TACC2 plays an important role in the cell proliferation of breast carcinoma and therefore immunohistochemical TACC2 status is a candidate of worse prognostic factor in breast cancer cases. PMID:27333920

  4. Biogenesis of the Secretory Granule: Chromogranin a Coiled-Coil Structure Results in Unusual Physical Properties And Suggests a Mechanism for Granule Core Condensation

    SciTech Connect

    Mosley, C.A.; Taupenot, L.; Biswas, N.; Taulane, J.P.; Olson, N.H.; Vaingankar, S.M.; Wen, G.; Schork, N.J.; Ziegler, M.G.; Mahata, S.K.; O'Connor, D.T.

    2009-06-03

    The secretory pro-hormone chromogranin A (CHGA) is densely packed into storage granules along with catecholamines, playing a catalytic role in granule biogenesis. 3-Dimensional structural data on CHGA are lacking. We found a superfamily structural homology for CHGA in the tropomyosin family of alpha-helical coiled-coils, even in mid-molecule regions where primary sequence identity is only modest. The assignment was confirmed by an independent algorithm, suggesting approximately 6-7 such domains spanning CHGA. We provide additional physiochemical evidence (chromatographic, spectral, microscopic) consistent with this unusual structure. Alpha-helical secondary structure (at up to approximately 45%) was confirmed by circular dichroism. CHGA molecular mass was estimated by MALDI-TOF mass spectrometry at approximately 50 kDa and by denaturing gel filtration at approximately 50-61 kDa, while its native Stokes radius was approximately 84.8 A, as compared to an expected approximately 30 A; the increase gave rise to an apparent native molecular weight of approximately 578 kDa, also consistent with the extended conformation of a coiled-coil. Small-angle X-ray scattering (SAXS) on CHGA in solution best fit an elongated cylindrical conformation in the monodisperse region with a radius of gyration of the rod cross-section (Rt) of approximately 52 A, compatible with a coiled-coil in the hydrated, aqueous state, or a multimeric coiled-coil. Electron microscopy with negative staining revealed an extended, filamentous CHGA structure with a diameter of approximately 94 +/- 4.5 A. Extended, coiled-coil conformation is likely to permit protein 'packing' in the secretory granule at approximately 50% higher density than a globular/spherical conformation. Natural allelic variation in the catestatin region was predicted to disrupt the coiled-coil. Chromaffin granule ultrastructure revealed a approximately 108 +/- 6.3 A periodicity of electron density, suggesting nucleation of a binding

  5. Magnetic Field Alignment of PS-P4VP: a Non-Liquid Crystalline Coil-Coil Block Copolymer

    NASA Astrophysics Data System (ADS)

    Rokhlenko, Yekaterina; Zhang, Kai; Larson, Steven; Gopalan, Padma; O'Hern, Corey; Osuji, Chinedum

    2015-03-01

    Magnetic fields provide the ability to control alignment of self-assembled soft materials such as block copolymers. Most prior work in this area has relied on the presence of ordered assemblies of anisotropic liquid crystalline species to ensure sufficient magnetic anisotropy to drive alignment. Recent experiments with poly(styrene-b-4-vinylpyridine), a non-liquid crystalline BCP, however, show field-induced alignment of a lamellar microstructure during cooling across the order-disorder transition. Using in situ x-ray scattering, we examine the roles of field strength and cooling rate on the alignment response of this low MW coil-coil BCP. Alignment is first observed at field strengths as low as 1 Tesla and improves markedly with both increasing field strength and slower cooling. We present a geometric argument to illustrate the origin of a finite, non-trivial magnetic susceptibility anisotropy for highly stretched surface-tethered polymer chains and corroborate this using coarse-grained molecular dynamics simulations. We rationalize the magnetic field response of the system in terms of the mobility afforded by the absence of entanglements, the intrinsic anisotropy resulting from the stretched polymer chains and sterically constrained conjugated rings, and the large grain size in these low molecular weight materials.

  6. Rapid Covalent Fluorescence Labeling of Membrane Proteins on Live Cells via Coiled-Coil Templated Acyl Transfer.

    PubMed

    Reinhardt, Ulrike; Lotze, Jonathan; Mörl, Karin; Beck-Sickinger, Annette G; Seitz, Oliver

    2015-10-21

    Fluorescently labeled proteins enable the microscopic imaging of protein localization and function in live cells. In labeling reactions targeted against specific tag sequences, the size of the fluorophore-tag is of major concern. The tag should be small to prevent interference with protein function. Furthermore, rapid and covalent labeling methods are desired to enable the analysis of fast biological processes. Herein, we describe the development of a method in which the formation of a parallel coiled coil triggers the transfer of a fluorescence dye from a thioester-linked coil peptide conjugate onto a cysteine-modified coil peptide. This labeling method requires only small tag sequences (max 23 aa) and occurs with high tag specificity. We show that size matching of the coil peptides and a suitable thioester reactivity allow the acyl transfer reaction to proceed within minutes (rather than hours). We demonstrate the versatility of this method by applying it to the labeling of different G-protein coupled membrane receptors including the human neuropeptide Y receptors 1, 2, 4, 5, the neuropeptide FF receptors 1 and 2, and the dopamine receptor 1. The labeled receptors are fully functional and able to bind the respective ligand with high affinity. Activity is not impaired as demonstrated by activation, internalization, and recycling experiments. PMID:26367072

  7. Insights into the coiled-coil organization of the Hendra virus phosphoprotein from combined biochemical and SAXS studies.

    PubMed

    Beltrandi, Matilde; Blocquel, David; Erales, Jenny; Barbier, Pascale; Cavalli, Andrea; Longhi, Sonia

    2015-03-01

    Nipah and Hendra viruses are recently emerged paramyxoviruses belonging to the Henipavirus genus. The Henipavirus phosphoprotein (P) consists of a large intrinsically disordered domain and a C-terminal domain (PCT) containing alternating disordered and ordered regions. Among these latter is the P multimerization domain (PMD). Using biochemical, analytical ultracentrifugation and small-angle X-ray scattering (SAXS) studies, we show that Hendra virus (HeV) PMD forms an elongated coiled-coil homotrimer in solution, in agreement with our previous findings on Nipah virus (NiV) PMD. However, the orientation of the N-terminal region differs from that observed in solution for NiV PMD, consistent with the ability of this region to adopt different conformations. SAXS studies provided evidence for a trimeric organization also in the case of PCT, thus extending and strengthening our findings on PMD. The present results are discussed in light of conflicting reports in the literature pointing to a tetrameric organization of paramyxoviral P proteins. PMID:25637789

  8. Synthesis, morphology, and sensory applications of multifunctional rod-coil-coil triblock copolymers and their electrospun nanofibers.

    PubMed

    Chiu, Yu-Cheng; Chen, Yougen; Kuo, Chi-Ching; Tung, Shih-Huang; Kakuchi, Toyoji; Chen, Wen-Chang

    2012-07-25

    We report the synthesis, morphology, and applications of conjugated rod-coil-coil triblock copolymers, polyfluorene-block-poly(N-isopropylacrylamide)-block-poly(N-methylolacrylamide) (PF-b-PNIPAAm-b-PNMA), prepared by atom transfer radical polymerization first and followed by click coupling reaction. The blocks of PF, PNIPAAm, and PNMA were designed for fluorescent probing, hydrophilic thermo-responsive and chemically cross-linking, respectively. In the following, the electrospun (ES) nanofibers of PF-b-PNIPAAm-b-PNMA were prepared in pure water using a single-capillary spinneret. The SAXS and TEM results suggested the lamellar structure of the PF-b-PNIPAAm-b-PNMA along the fiber axis. These obtained nanofibers showed outstanding wettability and dimension stability in the aqueous solution, and resulted in a reversible on/off transition on photoluminescence as the temperatures varied. Furthermore, the high surface/volume ratio of the ES nanofibers efficiently enhanced the temperature-sensitivity and responsive speed compared to those of the drop-cast film. The results indicated that the ES nanofibers of the conjugated rod-coil block copolymers would have potential applications for multifunctional sensory devices. PMID:22712723

  9. Biochemical and structural studies of the oligomerization domain of the Nipah virus phosphoprotein: evidence for an elongated coiled-coil homotrimer.

    PubMed

    Blocquel, David; Beltrandi, Matilde; Erales, Jenny; Barbier, Pascale; Longhi, Sonia

    2013-11-01

    Nipah virus (NiV) is a recently emerged severe human pathogen that belongs to the Henipavirus genus within the Paramyxoviridae family. The NiV genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that is the substrate used by the polymerase for transcription and replication. The polymerase is recruited onto the nucleocapsid via its cofactor, the phosphoprotein (P). The NiV P protein has a modular organization, with alternating disordered and ordered domains. Among these latter, is the P multimerization domain (PMD) that was predicted to adopt a coiled-coil conformation. Using both biochemical and biophysical approaches, we show that NiV PMD forms a highly stable and elongated coiled-coil trimer, a finding in striking contrast with respect to the PMDs of Paramyxoviridae members investigated so far that were all found to tetramerize. The present results therefore represent the first report of a paramyxoviral P protein forming trimers. PMID:24074578

  10. The C-terminal region of the transcriptional regulator THAP11 forms a parallel coiled-coil domain involved in protein dimerization.

    PubMed

    Cukier, Cyprian D; Maveyraud, Laurent; Saurel, Olivier; Guillet, Valérie; Milon, Alain; Gervais, Virginie

    2016-06-01

    Thanatos associated protein 11 (THAP11) is a cell cycle and cell growth regulator differentially expressed in cancer cells. THAP11 belongs to a distinct family of transcription factors recognizing specific DNA sequences via an atypical zinc finger motif and regulating diverse cellular processes. Outside the extensively characterized DNA-binding domain, THAP proteins vary in size and predicted domains, for which structural data are still lacking. We report here the crystal structure of the C-terminal region of human THAP11 protein, providing the first 3D structure of a coiled-coil motif from a THAP family member. We further investigate the stability, dynamics and oligomeric properties of the determined structure combining molecular dynamics simulations and biophysical experiments. Our results show that the C-ter region of THAP11 forms a left-handed parallel homo-dimeric coiled-coil structure possessing several unusual features. PMID:26975212

  11. Heteronuclear NMR assignments and secondary structure of the coiled coil trimerization domain from cartilage matrix protein in oxidized and reduced forms.

    PubMed Central

    Wiltscheck, R.; Kammerer, R. A.; Dames, S. A.; Schulthess, T.; Blommers, M. J.; Engel, J.; Alexandrescu, A. T.

    1997-01-01

    The C-terminal oligomerization domain of chicken cartilage matrix protein is a trimeric coiled coil comprised of three identical 43-residue chains. NMR spectra of the protein show equivalent magnetic environments for each monomer, indicating a parallel coiled coil structure with complete threefold symmetry. Sequence-specific assignments for 1H-, 15N-, and 13C-NMR resonances have been obtained from 2D 1H NOESY and TOCSY spectra, and from 3D HNCA, 15N NOESY-HSQC, and HCCH-TOCSY spectra. A stretch of alpha-helix encompassing five heptad repeats (35 residues) has been identified from intra-chain HN-HN and HN-H alpha NOE connectivities. 3JHNH alpha coupling constants, and chemical shift indices. The alpha-helix begins immediately downstream of inter-chain disulfide bonds between residues Cys 5 and Cys 7, and extends to near the C-terminus of the molecule. The threefold symmetry of the molecule is maintained when the inter-chain disulfide bonds that flank the N-terminus of the coiled coil are reduced. Residues Ile 21 through Glu 36 show conserved chemical shifts and NOE connectivities, as well as strong protection from solvent exchange in the oxidized and reduced forms of the protein. By contrast, residues Ile 10 through Val 17 show pronounced chemical shift differences between the oxidized and reduced protein. Strong chemical exchange NOEs between HN resonances and water indicate solvent exchange on time scales faster than 10 s, and suggests a dynamic fraying of the N-terminus of the coiled coil upon reduction of the disulfide bonds. Possible roles for the disulfide crosslinks of the oligomerization domain in the function of cartilage matrix protein are proposed. PMID:9260286

  12. An alternative conformation of the gp41 heptad repeat 1 region coiled coil exists in the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor

    SciTech Connect

    Mische, Claudia C.; Yuan Wen; Strack, Bettina; Craig, Stewart; Farzan, Michael; Sodroski, Joseph . E-mail: joseph_sodroski@dfci.harvard.edu

    2005-07-20

    The human immunodeficiency virus (HIV-1) transmembrane envelope glycoprotein, gp41, which mediates virus-cell fusion, exists in at least three different conformations within the trimeric envelope glycoprotein complex. The structures of the prefusogenic and intermediate states are unknown; structures representing the postfusion state have been solved. In the postfusion conformation, three helical heptad repeat 2 (HR2) regions pack in an antiparallel fashion into the hydrophobic grooves on the surface of a triple-helical coiled coil formed by the heptad repeat 1 (HR1) regions. We studied the prefusogenic conformation of gp41 by mutagenic alteration of membrane-anchored and soluble forms of the HIV-1 envelope glycoproteins. Our results indicate that, in the HIV-1 envelope glycoprotein precursor, the gp41 HR1 region is in a conformation distinct from that of a trimeric coiled coil. Thus, the central gp41 coiled coil is formed during the transition of the HIV-1 envelope glycoproteins from the precursor state to the receptor-bound intermediate.

  13. Solid-supported polymer bilayers formed by coil-coil block copolymers.

    PubMed

    Yang, Yan-Ling; Tsao, Heng-Kwong; Sheng, Yu-Jane

    2016-08-14

    The formation and physical properties of solid-supported polymer bilayers (SPBs) on an adhesive substrate have been explored by dissipative particle dynamics simulations. A SPB is developed by the adsorption of vesicles formed by diblock copolymers in a selective solvent. The adsorbed vesicle can remain intact or become ruptured into a SPB, depending on the interaction between solvophobic blocks and solvent and the interaction between solvophilic blocks and the substrate. The morphological phase diagram of adsorbed vesicles is acquired. The influence of polymer adhesion strength and solvophobicity on the geometrical and mechanical properties of a SPB is systematically studied as well. It is found that vesicular disruption is easily triggered for strong adhesion strength. Moreover, for strong adhesion strength and weak solvophobicity, the fluctuation of membrane height is impeded while the area of fluctuation is enhanced. PMID:27418114

  14. Defining the minimum size of a hydrophobic cluster in two-stranded α-helical coiled-coils: Effects on protein stability

    PubMed Central

    Lu, Stephen M.; Hodges, Robert S.

    2004-01-01

    The α-helical coiled-coil motif is characterized by a heptad repeat pattern (abcdefg)n in which residues a and d form the hydrophobic core. Long coiled-coils (e.g., tropomyosin, 284 residues per polypeptide chain) typically do not have a continuous hydrophobic core of stabilizing residues, but rather one that consists of alternating clusters of stabilizing and destabilizing residues. We have arbitrarily defined a cluster as a minimum of three consecutive stabilizing or destabilizing residues in the hydrophobic core. We report here on a series of two-stranded, disulfide-bridged parallel α-helical coiled-coils that contain a central cassette of three consecutive hydrophobic core positions (d, a, and d) with a destabilizing cluster of three consecutive Ala residues in the hydrophobic core on each side of the cassette. The effect of adding one to three stabilizing hydrophobes in these positions (Leu or Ile; denoted as •) was investigated. Alanine residues (denoted as ○) are used to represent destabilizing residues. The peptide with three Ala residues in the d a d cassette positions (○○○) was among the least stable coiled-coil (Tm = 39.3°C and Urea1/2 = 1.9 M). Surprisingly, the addition of one stabilizing hydrophobe (Leu) to the cassette or two stabilizing hydrophobes (Leu), still interspersed by an Ala in the cassette (•○•), also did not lead to any gain in stability. However, peptides with two adjacent hydrophobes in the cassette (••○)(○••) did show a gain in stability of 0.9 kcal/mole over the peptide with two interspersed hydrophobes (•○•). Because the latter three peptides have the same inherent hydrophobicity, the juxtaposition of stabilizing hydrophobes leads to a synergistic effect, and thus a clustering effect. The addition of a third stabilizing hydrophobe to the cassette (•••) resulted in a further synergistic gain in stability of 1.7 kcal/mole (Tm = 54.1°C and Urea1/2 = 3.3M). Therefore, the role of hydrophobicity

  15. Identification of BECN1 and ATG14 Coiled-Coil Interface Residues That Are Important for Starvation-Induced Autophagy.

    PubMed

    Mei, Yang; Su, Minfei; Sanishvili, Ruslan; Chakravarthy, Srinivas; Colbert, Christopher L; Sinha, Sangita C

    2016-08-01

    Autophagy, an essential eukaryotic homeostasis pathway, allows the sequestration of unwanted, damaged, or harmful cytoplasmic components in vesicles called autophagosomes, permitting subsequent lysosomal degradation and nutrient recycling. Autophagosome nucleation is mediated by class III phosphatidylinositol-3-kinase complexes that include two key autophagy proteins, BECN1/Beclin 1 and ATG14/BARKOR, which form parallel heterodimers via their coiled-coil domains (CCDs). Here we present the 1.46 Å X-ray crystal structure of the antiparallel, human BECN1 CCD homodimer, which represents BECN1 oligomerization outside the autophagosome nucleation complex. We use circular dichroism and small-angle X-ray scattering (SAXS) to show that the ATG14 CCD is significantly disordered but becomes more helical in the BECN1:ATG14 heterodimer, although it is less well-folded than the BECN1 CCD homodimer. SAXS also indicates that the BECN1:ATG14 heterodimer is more curved than other BECN1-containing CCD dimers, which has important implications for the structure of the autophagosome nucleation complex. A model of the BECN1:ATG14 CCD heterodimer that agrees well with the SAXS data shows that BECN1 residues at the homodimer interface are also responsible for heterodimerization, allowing us to identify ATG14 interface residues. Finally, we verify the role of BECN1 and ATG14 interface residues in binding by assessing the impact of point mutations of these residues on co-immunoprecipitation of the partner and demonstrate that these mutations abrogate starvation-induced upregulation of autophagy but do not impact basal autophagy. Thus, this research provides insights into structures of the BECN1 CCD homodimer and the BECN1:ATG14 CCD heterodimer and identifies interface residues that are important for BECN1:ATG14 heterodimerization and for autophagy. PMID:27383850

  16. Stability and specificity of heterodimer formation for the coiled-coil neck regions of the motor proteins Kif3A and Kif3B: the role of unstructured oppositely charged regions

    PubMed Central

    Chana, M.S.; Tripet, B.P.; Mant, C.T.; Hodges, R.

    2005-01-01

    We investigated the folding, stability, and specificity of dimerization of the neck regions of the kinesin-like proteins Kif3A (residues 356–416) and Kif3B (residues 351–411). We showed that the complementary charged regions found in the hinge regions (which directly follow the neck regions) of these proteins do not adopt any secondary structure in solution. We then explored the ability of the complementary charged regions to specify heterodimer formation for the neck region coiled-coils found in Kif3A and Kif3B. Redox experiments demonstrated that oppositely charged regions specified the formation of a heterodimeric coiled-coil. Denaturation studies with urea demonstrated that the negatively charged region of Kif3A dramatically destabilized its neck coiled-coil (urea1/2 value of 3.9 m compared with 6.7 m for the coiled-coil alone). By comparison, the placement of a positively charged region C-terminal to the neck coiled-coil of Kif3B had little effect on stability (urea1/2 value of 8.2 m compared with 8.8 m for the coiled-coil alone). The pairing of complementary charged regions leads to specific heterodimer formation where the stability of the heterodimeric neck coiled-coil with charged regions had similar stability (urea1/2 value of 7.8 m) to the most stable homodimer (Kif3B) with charged regions (urea1/2 value of 8.0 m) and dramatically more stable than the Kif3A homodimer with charged regions (urea1/2, value of 3.9 m). The heterodimeric coiled-coil with charged extensions has essentially the same stability as the heterodimeric coiled-coil on its own (urea1/2 values of 7.8 and 8.1 m, respectively) suggesting that specificity of heterodimerization is driven by non-specific attraction of the oppositely unstructured charged regions without affecting stability of the heterodimeric coiled-coil. PMID:15705165

  17. Structural Comparisons of Apo- and Metalated Three-Stranded Coiled Coils Clarify Metal Binding Determinants in Thiolate Containing Designed Peptides

    SciTech Connect

    Chakraborty, Saumen; Touw, Debra S.; Peacock, Anna F.A.; Stuckey, Jeanne; Pecoraro, Vincent L.

    2010-11-05

    Over the past two decades, designed metallopeptides have held the promise for understanding a variety of fundamental questions in metallobiochemistry; however, these dreams have not yet been realized because of a lack of structural data to elaborate the protein scaffolds before metal complexation and the resultant metalated structures which ultimately exist. This is because there are few reports of structural characterization of such systems either in their metalated or nonmetalated forms and no examples where an apo structure and the corresponding metalated peptide assembly have both been defined by X-ray crystallography. Herein we present X-ray structures of two de novo designed parallel three-stranded coiled coils (designed using the heptad repeat (a {yields} g)) CSL9C (CS = Coil Ser) and CSL19C in their nonmetalated forms, determined to 1.36 and 2.15 {angstrom} resolutions, respectively. Leucines from either position 9 (a site) or 19 (d site) are replaced by cysteine to generate the constructs CSL9C and CSL19C, respectively, yielding thiol-rich pockets at the hydrophobic interior of these peptides, suitable to bind heavy metals such as As(III), Hg(II), Cd(II), and Pb(II). We use these structures to understand the inherent structural differences between a and d sites to clarify the basis of the observed differential spectroscopic behavior of metal binding in these types of peptides. Cys side chains of (CSL9C){sub 3} show alternate conformations and are partially preorganized for metal binding, whereas cysteines in (CSL19C){sub 3} are present as a single conformer. Zn(II) ions, which do not coordinate or influence Cys residues at the designed metal sites but are essential for forming X-ray quality crystals, are bound to His and Glu residues at the crystal packing interfaces of both structures. These 'apo' structures are used to clarify the changes in metal site organization between metalated As(CSL9C){sub 3} and to speculate on the differential basis of Hg

  18. Metal-binding properties and structural characterization of a self-assembled coiled coil: formation of a polynuclear Cd-thiolate cluster.

    PubMed

    Zaytsev, Daniil V; Morozov, Vasily A; Fan, Jiufeng; Zhu, Xianchun; Mukherjee, Madhumita; Ni, Shuisong; Kennedy, Michael A; Ogawa, Michael Y

    2013-02-01

    This paper describes the design, characterization, and metal-binding properties of a 32-residue polypeptide called AQ-C16C19. The sequence of this peptide is composed of four repeats of the seven residue sequence Ile-Ala-Ala-Leu-Glu-Gln-Lys but with a Cys-X-X-Cys metal-binding motif substituted at positions 16-19. Size exclusion chromatography with multiangle light scattering detection (SEC-MALS) and circular dichroism (CD) spectroscopy studies showed that the apo peptide exhibits a pH-dependent oligomerization state in which a three-stranded α-helical coiled coil is dominant between pH5.4 and 8.5. The Cd(2+)-binding properties of the AQ-C16C19 peptide were studied by ultraviolet-visible spectroscopy (UV-vis), electrospray ionization mass spectrometry (ESI MS), and (113)Cd NMR techniques. The holoprotein was found to contain a polynuclear cadmium-thiolate center formed within the hydrophobic core of the triple-stranded α-helical coiled-coil structure. The X-ray crystal structure of the Cd-loaded peptide, resolved at 1.85Å resolution, revealed an adamantane-like configuration of the polynuclear metal center consisting of four cadmium ions, six thiolate sulfur ligands from cysteine residues and four oxygen-donor ligands. Three of these are from glutamic acid residues and one is from an exogenous water molecule. Thus, each cadmium ion is coordinated in a distorted tetrahedral S(3)O geometry. The metal cluster was found to form cooperatively at pH5.4 but in a stepwise fashion at pH>7. The results demonstrate that synthetic coiled-coils can be designed to incorporate multinuclear metal clusters, a proof-of-concept for their potential use in developing synthetic metalloenzymes and multi-electron redox agents. PMID:23160144

  19. Methods for Solving Highly Symmetric De Novo Designed Metalloproteins: Crystallographic Examination of a Novel Three-Stranded Coiled-Coil Structure Containing d-Amino Acids.

    PubMed

    Ruckthong, L; Stuckey, J A; Pecoraro, V L

    2016-01-01

    The core objective of de novo metalloprotein design is to define metal-protein relationships that control the structure and function of metal centers by using simplified proteins. An essential requirement to achieve this goal is to obtain high resolution structural data using either NMR or crystallographic studies in order to evaluate successful design. X-ray crystal structures have proven that a four heptad repeat scaffold contained in the three-stranded coiled coil (3SCC), called CoilSer (CS), provides an excellent motif for modeling a three Cys binding environment capable of chelating metals into geometries that resemble heavy metal sites in metalloregulatory systems. However, new generations of more complicated designs that feature, for example, a d-amino acid or multiple metal ligand sites in the helical sequence require a more stable construct. In doing so, an extra heptad was introduced into the original CS sequence, yielding a GRAND-CoilSer (GRAND-CS) to retain the 3SCC folding. An apo-(GRAND-CSL12DLL16C)3 crystal structure, designed for Cd(II)S3 complexation, proved to be a well-folded parallel 3SCC. Because this structure is novel, protocols for crystallization, structural determination, and refinements of the apo-(GRAND-CSL12DLL16C)3 are described. This report should be generally useful for future crystallographic studies of related coiled-coil designs. PMID:27586331

  20. Cholera toxin B subunit-five-stranded α-helical coiled-coil fusion protein: "five-to-five" molecular chimera displays robust physicochemical stability.

    PubMed

    Arakawa, Takeshi; Harakuni, Tetsuya

    2014-09-01

    To create a physicochemically stable cholera toxin (CT) B subunit (CTB), it was fused to the five-stranded α-helical coiled-coil domain of cartilage oligomeric matrix protein (COMP). The chimeric fusion protein (CTB-COMP) was expressed in Pichia pastoris, predominantly as a pentamer, and retained its affinity for the monosialoganglioside GM1, a natural receptor of CT. The fusion protein displayed thermostability, tolerating the boiling temperature of water for 10min, whereas unfused CTB readily dissociated to its monomers and lost its affinity for GM1. The fusion protein also displayed resistance to strong acid at pHs as low as 0.1, and to the protein denaturant sodium dodecyl sulfate at concentrations up to 10%. Intranasal administration of the fusion protein to mice induced anti-B subunit serum IgG, even after the protein was boiled, whereas unfused CTB showed no thermostable mucosal immunogenicity. This study demonstrates that CTB fused to a pentameric α-helical coiled coil has a novel physicochemical phenotype, which may provide important insight into the molecular design of enterotoxin-B-subunit-based vaccines and vaccine delivery molecules. PMID:25045819

  1. An Evolutionarily Conserved Family of Virion Tail Needles Related to Bacteriophage P22 gp26: Correlation between Structural Stability and Length of the -Helical Trimeric Coiled Coil

    SciTech Connect

    Bhardwaj, A.; Walker-Kopp, N; Casjens, S; Cingolani, G

    2009-01-01

    Bacteriophages of the Podoviridae family use short noncontractile tails to inject their genetic material into Gram-negative bacteria. In phage P22, the tail contains a thin needle, encoded by the phage gene 26, which is essential both for stabilization and for ejection of the packaged viral genome. Bioinformatic analysis of the N-terminal domain of gp26 (residues 1-60) led us to identify a family of genes encoding putative homologues of the tail needle gp26. To validate this idea experimentally and to explore their diversity, we cloned the gp26-like gene from phages HK620, Sf6 and HS1, and characterized these gene products in solution. All gp26-like factors contain an elongated {alpha}-helical coiled-coil core consisting of repeating, adjacent trimerization heptads and form trimeric fibers with length ranging between about 240 to 300 {angstrom}. gp26 tail needles display a high level of structural stability in solution, with Tm (temperature of melting) between 85 and 95 C. To determine how the structural stability of these phage fibers correlates with the length of the {alpha}-helical core, we investigated the effect of insertions and deletions in the helical core. In the P22 tail needle, we identified an 85-residue-long helical domain, termed MiCRU (minimal coiled-coil repeat unit), that can be inserted in-frame inside the gp26 helical core, preserving the straight morphology of the fiber. Likewise, we were able to remove three quarters of the helical core of the HS1 tail needle, minimally decreasing the stability of the fiber. We conclude that in the gp26 family of tail needles, structural stability increases nonlinearly with the length of the {alpha}-helical core. Thus, the overall stability of these bacteriophage fibers is not solely dependent on the number of trimerization repeats in the {alpha}-helical core.

  2. Fiber knob domain lacking the shaft sequence but fused to a coiled coil is a candidate subunit vaccine against egg-drop syndrome.

    PubMed

    Harakuni, Tetsuya; Andoh, Kiyohiko; Sakamoto, Ryu-Ichi; Tamaki, Yukihiro; Miyata, Takeshi; Uefuji, Hirotaka; Yamazaki, Ken-Ichi; Arakawa, Takeshi

    2016-06-01

    Egg-drop syndrome (EDS) virus is an avian adenovirus that causes a sudden drop in egg production and in the quality of the eggs when it infects chickens, leading to substantial economic losses in the poultry industry. Inactivated EDS vaccines produced in embryonated duck eggs or cell culture systems are available for the prophylaxis of EDS. However, recombinant subunit vaccines that are efficacious and inexpensive are a desirable alternative. In this study, we engineered chimeric fusion proteins in which the trimeric fiber knob domain lacking the triple β-spiral motif in the fiber shaft region was genetically fused to trimeric coiled coils, such as those of the engineered form of the GCN4 leucine zipper peptide or chicken cartilage matrix protein (CMP). The fusion proteins were expressed predominantly as soluble trimeric proteins in Escherichia coli at levels of 15-80mg/L of bacterial culture. The single immunization of chickens with the purified fusion proteins, at a dose equivalent to 10μg of the knob moiety, elicited serum antibodies with high hemagglutination inhibition (HI) activities, similar to those induced by an inactivated EDS vaccine. A dose-response analysis indicated that a single immunization with as little as 1μg of the knob moiety of the CMP-knob fusion protein was as effective as the inactivated vaccine in inducing antibodies with HI activity. The immunization of laying hens had no apparent adverse effects on egg production and effectively prevented clinical symptoms of EDS when the chickens were challenged with pathogenic EDS virus. This study demonstrates that the knob domain lacking the shaft sequence but fused to a trimeric coiled coil is a promising candidate subunit vaccine for the prophylaxis of EDS in chickens. PMID:27105561

  3. Coiled-coil domain of PML is essential for the aberrant dynamics of PML-RAR{alpha}, resulting in sequestration and decreased mobility of SMRT

    SciTech Connect

    Huang Ying; Qiu Jihui; Chen Guoqiang; Dong Shuo

    2008-01-11

    Promyelocytic leukemia-retinoic acid receptor {alpha} (PML-RAR{alpha}) is the most frequent RAR{alpha} fusion protein in acute promyelocytic leukemia (APL). Our previous study has demonstrated that, compared with RAR{alpha}, PML-RAR{alpha} had reduced intranuclear mobility accompanied with mislocalization. To understand the molecular basis for the altered dynamics of PML-RAR{alpha} fusion protein, we performed FRAP analysis at a single cell level. Results indicated that three known sumoylation site mutated PML-RAR{alpha} had same intracellular localization and reduced mobility as wild-type counterpart. The coiled-coil domain of PML is responsible for the aberrant dynamics of PML-RAR{alpha}. In addition, we revealed that co-repressor SMRT co-localized with PML-RAR{alpha}, resulting in the immobilization of SMRT while ATRA treatment eliminated their association and reversed the immobile effect of SMRT. Furthermore, co-activator CBP, co-localized with PML-RAR{alpha} in an ATRA-independent way, was demonstrated as a high dynamic intranuclear molecule. These results would shed new insights for the molecular mechanisms of PML-RAR{alpha}-associated leukemogenesis.

  4. Control of Smc Coiled Coil Architecture by the ATPase Heads Facilitates Targeting to Chromosomal ParB/parS and Release onto Flanking DNA.

    PubMed

    Minnen, Anita; Bürmann, Frank; Wilhelm, Larissa; Anchimiuk, Anna; Diebold-Durand, Marie-Laure; Gruber, Stephan

    2016-03-01

    Smc/ScpAB promotes chromosome segregation in prokaryotes, presumably by compacting and resolving nascent sister chromosomes. The underlying mechanisms, however, are poorly understood. Here, we investigate the role of the Smc ATPase activity in the recruitment of Smc/ScpAB to the Bacillus subtilis chromosome. We demonstrate that targeting of Smc/ScpAB to ParB/parS loading sites is strictly dependent on engagement of Smc head domains and relies on an open organization of the Smc coiled coils. We find that dimerization of the Smc hinge domain stabilizes closed Smc rods and hinders head engagement as well as chromosomal targeting. Conversely, the ScpAB sub-complex promotes head engagement and Smc rod opening and thereby facilitates recruitment of Smc to parS sites. Upon ATP hydrolysis, Smc/ScpAB is released from loading sites and relocates within the chromosome-presumably through translocation along DNA double helices. Our findings define an intermediate state in the process of chromosome organization by Smc. PMID:26904953

  5. Control of Smc Coiled Coil Architecture by the ATPase Heads Facilitates Targeting to Chromosomal ParB/parS and Release onto Flanking DNA

    PubMed Central

    Minnen, Anita; Bürmann, Frank; Wilhelm, Larissa; Anchimiuk, Anna; Diebold-Durand, Marie-Laure; Gruber, Stephan

    2016-01-01

    Summary Smc/ScpAB promotes chromosome segregation in prokaryotes, presumably by compacting and resolving nascent sister chromosomes. The underlying mechanisms, however, are poorly understood. Here, we investigate the role of the Smc ATPase activity in the recruitment of Smc/ScpAB to the Bacillus subtilis chromosome. We demonstrate that targeting of Smc/ScpAB to ParB/parS loading sites is strictly dependent on engagement of Smc head domains and relies on an open organization of the Smc coiled coils. We find that dimerization of the Smc hinge domain stabilizes closed Smc rods and hinders head engagement as well as chromosomal targeting. Conversely, the ScpAB sub-complex promotes head engagement and Smc rod opening and thereby facilitates recruitment of Smc to parS sites. Upon ATP hydrolysis, Smc/ScpAB is released from loading sites and relocates within the chromosome—presumably through translocation along DNA double helices. Our findings define an intermediate state in the process of chromosome organization by Smc. PMID:26904953

  6. Forced expression of desmin and desmin mutants in cultured cells: impact of myopathic missense mutations in the central coiled-coil domain on network formation.

    PubMed

    Bär, Harald; Kostareva, Anna; Sjöberg, Gunnar; Sejersen, Thomas; Katus, Hugo A; Herrmann, Harald

    2006-05-15

    We recently demonstrated that inherited disease-causing mutations clustered in the alpha-helical coiled-coil "rod" domain of the muscle-specific intermediate filament (IF) protein desmin display a wide range of inhibitory effects on regular in vitro assembly. In these studies, we showed that individual mutations exhibited phenotypes that were not, with respect to the severity of interference, predictable by our current knowledge of the structural design of IF proteins. Moreover, the behavior of some mutated proteins in a standard tissue culture cell expression system was found to be even more complex. Here, we systematically investigate the behavior of these disease mutants in four different cell types: three not containing desmin or the related IF protein vimentin and the standard fibroblast line 3T3, which has an extensive vimentin system. The ability of the mutants to form filaments in the vimentin-free cells varies considerably, and only the mutants forming IFs in vitro generate extended filamentous networks. Furthermore, these latter mutants integrate into the 3T3 vimentin network but all the others do not. Instead, they cause the endogenous network of 3T3 vimentin to reorganize into perinuclear bundles. In addition, most of these assembly-deficient mutant desmins completely segregate from the vimentin system. Instead, the small round to fibrillar particles formed distribute independently throughout the cytoplasm as well as between the collapsed vimentin filament arrays in the perinuclear area. PMID:16519886

  7. The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus

    PubMed Central

    Kracklauer, Martin P.; Wiora, Heather M.; Deery, William J.; Chen, Xin; Bolival, Benjamin; Romanowicz, Dwight; Simonette, Rebecca A.; Fuller, Margaret T.; Fischer, Janice A.; Beckingham, Kathleen M.

    2010-01-01

    Maintaining the proximity of centrosomes to nuclei is important in several cellular contexts, and LINC complexes formed by SUN and KASH proteins are crucial in this process. Here, we characterize the presumed Drosophila ortholog of the mammalian SUN protein, sperm-associated antigen 4 (Spag4, previously named Giacomo), and demonstrate that Spag4 is required for centriole and nuclear attachment during spermatogenesis. Production of spag4 mRNA is limited to the testis, and Spag4 protein shows a dynamic pattern of association with the germline nuclei, including a concentration of protein at the site of attachment of the single spermatid centriole. In the absence of Spag4, nuclei and centrioles or basal bodies (BBs) dissociate from each other after meiosis. This role of Spag4 in centriolar attachment does not involve either of the two KASH proteins of the Drosophila genome (Klarsicht and MSP-300), but does require the coiled-coil protein Yuri Gagarin. Yuri shows an identical pattern of localization at the nuclear surface to Spag4 during spermatogenesis, and epistasis studies show that the activities of Yuri and dynein-dynactin are downstream of spag4 in this centriole attachment pathway. The later defects in spermatogenesis seen for yuri and spag4 mutants are similar, suggesting they could be secondary to initial disruption of events at the nuclear surface. PMID:20647369

  8. The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus.

    PubMed

    Kracklauer, Martin P; Wiora, Heather M; Deery, William J; Chen, Xin; Bolival, Benjamin; Romanowicz, Dwight; Simonette, Rebecca A; Fuller, Margaret T; Fischer, Janice A; Beckingham, Kathleen M

    2010-08-15

    Maintaining the proximity of centrosomes to nuclei is important in several cellular contexts, and LINC complexes formed by SUN and KASH proteins are crucial in this process. Here, we characterize the presumed Drosophila ortholog of the mammalian SUN protein, sperm-associated antigen 4 (Spag4, previously named Giacomo), and demonstrate that Spag4 is required for centriole and nuclear attachment during spermatogenesis. Production of spag4 mRNA is limited to the testis, and Spag4 protein shows a dynamic pattern of association with the germline nuclei, including a concentration of protein at the site of attachment of the single spermatid centriole. In the absence of Spag4, nuclei and centrioles or basal bodies (BBs) dissociate from each other after meiosis. This role of Spag4 in centriolar attachment does not involve either of the two KASH proteins of the Drosophila genome (Klarsicht and MSP-300), but does require the coiled-coil protein Yuri Gagarin. Yuri shows an identical pattern of localization at the nuclear surface to Spag4 during spermatogenesis, and epistasis studies show that the activities of Yuri and dynein-dynactin are downstream of spag4 in this centriole attachment pathway. The later defects in spermatogenesis seen for yuri and spag4 mutants are similar, suggesting they could be secondary to initial disruption of events at the nuclear surface. PMID:20647369

  9. Antibody elicited against the gp41 N-heptad repeat (NHR) coiled-coil can neutralize HIV-1 with modest potency but non-neutralizing antibodies also bind to NHR mimetics

    SciTech Connect

    Nelson, Josh D.; Kinkead, Heather; Brunel, Florence M.; Leaman, Dan; Jensen, Richard; Louis, John M.; Maruyama, Toshiaki; Bewley, Carole A.; Bowdish, Katherine; Clore, G. Marius; Dawson, Philip E.; Frederickson, Shana; Mage, Rose G.; Richman, Douglas D.; Burton, Dennis R.; Zwick, Michael B.

    2008-07-20

    Following CD4 receptor binding to the HIV-1 envelope spike (Env), the conserved N-heptad repeat (NHR) region of gp41 forms a coiled-coil that is a precursor to the fusion reaction. Although it has been a target of drug and vaccine design, there are few monoclonal antibody (mAb) tools with which to probe the antigenicity and immunogenicity specifically of the NHR coiled-coil. Here, we have rescued HIV-1-neutralizing anti-NHR mAbs from immune phage display libraries that were prepared (i) from b9 rabbits immunized with a previously described mimetic of the NHR coiled-coil, N35{sub CCG}-N13, and (ii) from an HIV-1 infected individual. We describe a rabbit single-chain Fv fragment (scFv), 8K8, and a human Fab, DN9, which specifically recognize NHR coiled-coils that are unoccupied by peptide corresponding to the C-heptad repeat or CHR region of gp41 (e.g. C34). The epitopes of 8K8 and DN9 were found to partially overlap with that of a previously described anti-NHR mAb, IgG D5; however, 8K8 and DN9 were much more specific than D5 for unoccupied NHR trimers. The mAbs, including a whole IgG 8K8 molecule, neutralized primary HIV-1 of clades B and C in a pseudotyped virus assay with comparable, albeit relatively modest potency. Finally, a human Fab T3 and a rabbit serum (both non-neutralizing) were able to block binding of D5 and 8K8 to a gp41 NHR mimetic, respectively, but not the neutralizing activity of these mAbs. We conclude from these results that NHR coiled-coil analogs of HIV-1 gp41 elicit many Abs during natural infection and through immunization, but that due to limited accessibility to the corresponding region on fusogenic gp41 few can neutralize. Caution is therefore required in targeting the NHR for vaccine design. Nevertheless, the mAb panel may be useful as tools for elucidating access restrictions to the NHR of gp41 and in designing potential improvements to mimetics of receptor-activated Env.

  10. The DNA rearrangement that generates the TRK-T3 oncogene involves a novel gene on chromosome 3 whose product has a potential coiled-coil domain.

    PubMed Central

    Greco, A; Mariani, C; Miranda, C; Lupas, A; Pagliardini, S; Pomati, M; Pierotti, M A

    1995-01-01

    Oncogenic rearrangements of the NTRK1 gene (also designated TRKA), encoding one of the receptors for the nerve growth factor, are frequently detected in thyroid carcinomas. Such rearrangements fuse the NTRK1 tyrosine kinase domain to 5'-end sequences belonging to different genes. In previously reported studies we have demonstrated that NTRK1 oncogenic activation involves two genes, TPM3 and TPR, both localized similarly to the receptor tyrosine kinase, on the q arm of chromosome 1. Here we report the characterization of a novel NTRK1-derived thyroid oncogene, named TRK-T3. A cDNA clone, capable of transforming activity, was isolated from a transformant cell line. Sequence analysis revealed that TRK-T3 contains 1,412 nucleotides of NTRK1 preceded by 598 nucleotides belonging to a novel gene that we have named TFG (TRK-fused gene). The TRK-T3 amino acid sequence displays, within the TFG region, a coiled-coil motif that could endow the oncoprotein with the capability to form complexes. The TRK-T3 oncogene encodes a 68-kDa cytoplasmic protein reacting with NTRK1-specific antibodies. By sedimentation gradient experiments the TRK-T3 oncoprotein was shown to form, in vivo, multimeric complexes, most likely trimers or tetramers. The TFG gene is ubiquitously expressed and is located on chromosome 3. The breakpoint producing the TRK-T3 oncogene occurs within exons of both the TFG gene and the NTRK1 gene and produces a chimeric exon that undergoes alternative splicing. Molecular analysis of the NTRK1 rearranged fragments indicated that the chromosomal rearrangement is reciprocal and balanced and involves loss of a few nucleotides of germ line sequences. PMID:7565764

  11. Functional and Mechanistic Analyses of Biomimetic Aminoacyl Transfer Reactions in de novo Designed Coiled Coil Peptides via Rational Active Site Engineering

    PubMed Central

    Leman, Luke J.; Weinberger, Dana A.; Huang, Zheng-Zheng; Wilcoxen, Keith M.; Ghadiri, M. Reza

    2008-01-01

    Ribosomes and nonribosomal peptide synthetases (NRPSs) carry out instructed peptide synthesis through a series of directed intermodular aminoacyl transfer reactions. We recently reported the design of coiled-coil assemblies that could functionally mimic the elementary aminoacyl loading and intermodular aminoacyl transfer steps of NRPSs. These peptides were designed initially to accelerate aminoacyl transfer mainly through catalysis by approximation by closely juxtaposing four active site moieties, two each from adjacent noncovalently-associated helical modules. In our designs peptide self-assembly positions a cysteine residue that is used to covalently capture substrates from solution via transthiolesterification (substrate loading step to generate the aminoacyl donor site) adjacent to an aminoacyl acceptor site provided by a covalently tethered amino acid or modeled by the ε-amine of an active site lysine. However, through systematic functional analyses of 48 rationally designed peptide sequences, we have now determined that the substrate loading and intermodular aminoacyl transfer steps can be significantly influenced (up to ~103-fold) by engineering changes in the active site microenvironment through amino acid substitutions and variations in the inter-residue distances and geometry. Mechanistic studies based on 15N-NMR and kinetic analysis further indicate that certain active site constellations furnish an unexpectedly large pKa depression (1.5 pH units) of the aminoacyl-acceptor moiety, helping to explain the observed high rates of aminoacyl transfer in those constructs. Taken together, our studies demonstrate the feasibility of engineering efficient de novo peptide sequences possessing active sites and functions reminiscent of those in natural enzymes. PMID:17302417

  12. Contribution of hydrophobic interactions to protein stability.

    PubMed

    Pace, C Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R; Shirley, Bret A; Hendricks, Marsha McNutt; Iimura, Satoshi; Gajiwala, Ketan; Scholtz, J Martin; Grimsley, Gerald R

    2011-05-01

    Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin headpiece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compared our results with those of previous studies and reached the following conclusions: (1) Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6±0.3 kcal/mol per -CH(2)- group), than to the stability of a large protein, VlsE (1.6±0.3 kcal/mol per -CH(2)- group). (2) Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kilocalories per mole) Phe18 (3.9), Met13 (3.1), Phe7 (2.9), Phe11 (2.7), and Leu21 (2.7). (3) Based on the Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a -CH(2)- group on folding contributes, on average, 1.1±0.5 kcal/mol to protein stability. (4) The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔG(tr) values from water to cyclohexane. (5) For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60±4% and hydrogen bonds contribute 40±4% to protein stability. (6) Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominantly by hydrophobic interactions. PMID:21377472

  13. Titins in C.elegans with unusual features: coiled-coil domains, novel regulation of kinase activity and two new possible elastic regions.

    PubMed

    Flaherty, Denise B; Gernert, Kim M; Shmeleva, Nataliya; Tang, Xuexin; Mercer, Kristina B; Borodovsky, Mark; Benian, Guy M

    2002-10-25

    We report that there are previously unrecognized proteins in Caenorhabditis elegans that are similar to the giant muscle proteins called titins, and these are encoded by a single approximately 90kb gene. The gene structure was predicted by GeneMark.hmm and then experimentally verified. The Ce titin gene encodes polypeptides of 2.2MDa, 1.2MDa and 301kDa. The 2.2MDa isoform resembles twitchin and UNC-89 in that it contains multiple Ig (56) and FnIII (11) domains, and a single protein kinase domain. In addition, however, the 2.2MDa isoform contains four classes of short, 14-51 residue, repeat motifs arranged mostly in many tandem copies. One of these tandem repeat regions is similar to the PEVK regions of vertebrate and fly titins. As the PEVK region is one of the main elastic elements of the titins and is also composed of short tandem repeats, this suggests that the repeat motifs in the Ce titins may have a similar elastic function. An interesting aspect of the two largest Ce titin isoforms, is that in contrast to other members of the twitchin/titin family, there are multiple regions which are likely to form coiled-coil structure. In transgenic animals, the first approximately 100 residues of the largest isoforms targets to dense bodies, the worm analogs of Z-discs. Anti-Ce titin antibodies show localization to muscle I-bands beginning at the L2-L3 larval stages and this pattern continues into adult muscle. Ce titins may not have a role in early myofibril assembly: (1) Ce titins are too short to span half a sarcomere, and the onset of their expression is well after the initial assembly of thick filaments. (2) Ce titins are not localized to I-bands in embryonic or L1 larval muscle. The Ce titin protein kinase domain is most similar to the kinase domains of the twitchins and projectin. The Ce titin kinase has protein kinase activity in vitro, and this activity is regulated by a novel mechanism. PMID:12381307

  14. Electrostatic Contributions to the Stability of the GCN4 Leucine Zipper Structure

    PubMed Central

    Matousek, William M.; Ciani, Barbara; Fitch, Carolyn A.; Bertrand García-Moreno, E.; Kammerer, Richard A.; Alexandrescu, Andrei T.

    2007-01-01

    Summary Ion pairs are ubiquitous in X-ray structures of coiled coils, and mutagenesis of charged residues can result in large stability losses. By contrast, pKa values determined by NMR in solution often predict only small contributions to stability from charge interactions. To help reconcile these results we used triple-resonance NMR to determine pKa values for all groups that ionize between pH 1 and 13 in the 33-residue leucine zipper fragment, GCN4p. In addition to the native state we also determined comprehensive pKa values for two models of the GCN4p denatured state: the protein in 6 M urea, and unfolded peptide fragments of the protein in water. Only residues that form ion pairs in multiple X-ray structures of GCN4p gave large pKa differences between the native and denatured states. Moreover, electrostatic contributions to stability were not equivalent for oppositely charged partners in ion pairs, suggesting that the interactions between a charge and its environment are as important as those within the ion pair. The pH dependence of protein stability calculated from NMR-derived pKa values agreed with the stability profile measured from equilibrium urea-unfolding experiments as a function of pH. The stability profile was also reproduced with structure-based continuum electrostatic calculations, although contributions to stability were overestimated at the extremes of pH. We consider potential sources of errors in the calculations, and how pKa predictions could be improved. Our results show that although hydrophobic packing and hydrogen bonding have dominant roles, electrostatic interactions also make significant contributions to the stability of the coiled coil. PMID:17920624

  15. Contribution of Hydrophobic Interactions to Protein Stability

    PubMed Central

    Pace, C. Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R.; Shirley, Bret A.; Hendricks, Marsha McNutt; Iimura, Satoshi; Gajiwala, Ketan; Scholtz, J. Martin; Grimsley, Gerald R.

    2011-01-01

    Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin head piece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compare our results with previous studies and reach the following conclusions. 1. Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6 ± 0.3 kcal/mole per –CH2– group), than to the stability of a large protein, VlsE (1.6 ± 0.3 kcal/mol per –CH2– group). 2. Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kcal/mol): Phe 18 (3.9), Met 13 (3.1), Phe 7 (2.9), Phe 11 (2.7), and Leu 21 (2.7). 3. Based on Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a –CH2– group on folding contributes, on average, 1.1 ± 0.5 kcal/mol to protein stability. 4. The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔGtr values from water to cyclohexane. 5. For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60 ± 4% and hydrogen bonds 40 ± 4% to protein stability. 6. Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominately by hydrophobic interactions. PMID:21377472

  16. Effect of coiled-coil peptides on the function of the type III secretion system-dependent activity of enterohemorragic Escherichia coli O157:H7 and Citrobacter rodentium.

    PubMed

    Larzábal, Mariano; Zotta, Elsa; Ibarra, Cristina; Rabinovitz, Bettina C; Vilte, Daniel A; Mercado, Elsa C; Cataldi, Ángel

    2013-01-01

    Many animal and human pathogenic Gram-negative bacteria such as Salmonella, Yersinia, enterohemorrhagic Escherichia coli (EHEC), and enteropathogenic Escherichia coli (EPEC) possess a type III secretion system (TTSS) that is used to deliver virulence proteins directly into the host cell. Recent evidence has suggested that CoilA and CoilB, two synthetic peptides corresponding to coiled-coil domains of the translocator protein EspA, are effective in inhibiting the action of TTSS from EPEC. In the current study, the action of these coiled-coil peptides on the TTSS of EHEC O157:H7 and Citrobacter rodentium was examined. CoilA and CoilB showed to be effective in reducing the red blood cell lysis mediated by EHEC O157:H7 and the in vitro secretion of translocator proteins EspB and EspD by EHEC O157:H7 and EspD by C. rodentium. Treatment of mice with CoilA and CoilB peptides prevented colon damage when the animals were inoculated with C. rodentium. Colon samples of the non-treated group showed areas with loss of superficial epithelium, damaged cells, and endoluminal mononuclear inflammatory infiltrate, consistent with histological lesions induced by C. rodentium, whereas mice treated with the synthetic peptides displayed normal surface epithelium showing a similar structure as the uninfected control group. These encouraging results prompt us to test coiled-coil peptides as treatment or vaccines in other models of bacterial infections in future work. PMID:23312797

  17. Crystal Structure of C-Terminal Coiled-Coil Domain of SYCP1 Reveals Non-Canonical Anti-Parallel Dimeric Structure of Transverse Filament at the Synaptonemal Complex

    PubMed Central

    Jeong, Jae-Hee; Kim, Yeon-Gil; Park, Hyun Ho

    2016-01-01

    The synaptonemal complex protein 1 (SYCP1) is the main structural element of transverse filaments (TFs) of the synaptonemal complex (SC), which is a meiosis-specific complex structure formed at the synapse of homologue chromosomes to hold them together. The N-terminal domain of SYCP1 is known to be located within the central elements (CEs), whereas the C-terminal domain is located toward lateral elements (LEs). SYCP1 is a well-known meiosis marker that is also known to be a prognostic marker in the early stage of several cancers including breast, gliomas, and ovarian cancers. The structure of SC, especially the TF structure formed mainly by SYCP1, remains unclear without any structural information. To elucidate a molecular basis of SC formation and function, we first solved the crystal structure of C-terminal coiled-coil domain of SYCP1. The coiled-coil domain of SYCP1 forms asymmetric, anti-parallel dimers in solution. PMID:27548613

  18. Crystal Structure of C-Terminal Coiled-Coil Domain of SYCP1 Reveals Non-Canonical Anti-Parallel Dimeric Structure of Transverse Filament at the Synaptonemal Complex.

    PubMed

    Seo, Eun Kyung; Choi, Jae Young; Jeong, Jae-Hee; Kim, Yeon-Gil; Park, Hyun Ho

    2016-01-01

    The synaptonemal complex protein 1 (SYCP1) is the main structural element of transverse filaments (TFs) of the synaptonemal complex (SC), which is a meiosis-specific complex structure formed at the synapse of homologue chromosomes to hold them together. The N-terminal domain of SYCP1 is known to be located within the central elements (CEs), whereas the C-terminal domain is located toward lateral elements (LEs). SYCP1 is a well-known meiosis marker that is also known to be a prognostic marker in the early stage of several cancers including breast, gliomas, and ovarian cancers. The structure of SC, especially the TF structure formed mainly by SYCP1, remains unclear without any structural information. To elucidate a molecular basis of SC formation and function, we first solved the crystal structure of C-terminal coiled-coil domain of SYCP1. The coiled-coil domain of SYCP1 forms asymmetric, anti-parallel dimers in solution. PMID:27548613

  19. Quantitative Analyses of Cryptochrome-mBMAL1 Interactions

    PubMed Central

    Czarna, Anna; Breitkreuz, Helena; Mahrenholz, Carsten C.; Arens, Julia; Strauss, Holger M.; Wolf, Eva

    2011-01-01

    The mammalian cryptochromes mCRY1 and mCRY2 act as transcriptional repressors within the 24-h transcription-translational feedback loop of the circadian clock. The C-terminal tail and a preceding predicted coiled coil (CC) of the mCRYs as well as the C-terminal region of the transcription factor mBMAL1 are involved in transcriptional feedback repression. Here we show by fluorescence polarization and isothermal titration calorimetry that purified mCRY1/2CCtail proteins form stable heterodimeric complexes with two C-terminal mBMAL1 fragments. The longer mBMAL1 fragment (BMAL490) includes Lys-537, which is rhythmically acetylated by mCLOCK in vivo. mCRY1 (but not mCRY2) has a lower affinity to BMAL490 than to the shorter mBMAL1 fragment (BMAL577) and a K537Q mutant version of BMAL490. Using peptide scan analysis we identify two mBMAL1 binding epitopes within the coiled coil and tail regions of mCRY1/2 and document the importance of positively charged mCRY1 residues for mBMAL1 binding. A synthetic mCRY coiled coil peptide binds equally well to the short and to the long (wild-type and K537Q mutant) mBMAL1 fragments. In contrast, a peptide including the mCRY1 tail epitope shows a lower affinity to BMAL490 compared with BMAL577 and BMAL490(K537Q). We propose that Lys-537mBMAL1 acetylation enhances mCRY1 binding by affecting electrostatic interactions predominantly with the mCRY1 tail. Our data reveal different molecular interactions of the mCRY1/2 tails with mBMAL1, which may contribute to the non-redundant clock functions of mCRY1 and mCRY2. Moreover, our study suggests the design of peptidic inhibitors targeting the interaction of the mCRY1 tail with mBMAL1. PMID:21521686

  20. Monoclonal anti-mouse laminin antibodies: AL-1 reacts with laminin alpha1 chain, AL-2 with laminin beta1 chain, and AL-4 with the coiled-coil domain of laminin beta1 chain.

    PubMed

    Schéele, Susanne; Sasaki, Takako; Arnal-Estapé, Anna; Durbeej, Madeleine; Ekblom, Peter

    2006-07-01

    We analyzed the reactivity of three different commercially available rat monoclonal antibodies raised against mouse laminin-alpha1beta1gamma1 (laminin-111), AL-1, AL-2, and AL-4. Using ELISA assays, Western blot analysis and immunostainings we present refined epitope maps for these three laminin monoclonals. AL-1 reacted, as predicted with laminin alpha1 chain. AL-4 has also been marketed as an alpha1 chain specific probe, but we show here that AL-4 detects mouse laminin beta1 chain, in the distal part of the coiled-coil region. AL-2 was predicted to react with all three chains near the cross-region, but seems to primarily react with laminin beta1 chain. PMID:16631359

  1. Full-length Gα(q)-phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain.

    PubMed

    Lyon, Angeline M; Dutta, Somnath; Boguth, Cassandra A; Skiniotis, Georgios; Tesmer, John J G

    2013-03-01

    Phospholipase C-β (PLCβ) is directly activated by Gαq, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM three-dimensional reconstructions of human full-length PLCβ3 in complex with mouse Gαq. The distal CTD forms an extended monomeric helical bundle consisting of three antiparallel segments with structural similarity to membrane-binding bin-amphiphysin-Rvs (BAR) domains. Sequence conservation of the distal CTD suggests putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of Gαq in both the crystal structure and cryo-EM reconstructions. Functional analysis suggests that the distal CTD has roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis. PMID:23377541

  2. Full-length Gαq-phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain

    SciTech Connect

    Lyon, Angeline M.; Dutta, Somnath; Boguth, Cassandra A.; Skiniotis, Georgios; Tesmer, John J.G.

    2014-08-21

    Phospholipase C-β (PLCβ) is directly activated by Gαq, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM three-dimensional reconstructions of human full-length PLCβ3 in complex with mouse Gαq. The distal CTD forms an extended monomeric helical bundle consisting of three antiparallel segments with structural similarity to membrane-binding bin-amphiphysin-Rvs (BAR) domains. Sequence conservation of the distal CTD suggests putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of Gαq in both the crystal structure and cryo-EM reconstructions. Functional analysis suggests that the distal CTD has roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis.

  3. F-actin asymmetry and the endoplasmic reticulum-associated TCC-1 protein contribute to stereotypic spindle movements in the Caenorhabditis elegans embryo.

    PubMed

    Berends, Christian W H; Muñoz, Javier; Portegijs, Vincent; Schmidt, Ruben; Grigoriev, Ilya; Boxem, Mike; Akhmanova, Anna; Heck, Albert J R; van den Heuvel, Sander

    2013-07-01

    The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During development, dividing cells control the position of the spindle to determine the size, location, and fate of daughter cells. Spindle positioning depends on pulling forces that act between the cell periphery and astral microtubules. This involves dynein recruitment to the cell cortex by a heterotrimeric G-protein α subunit in complex with a TPR-GoLoco motif protein (GPR-1/2, Pins, LGN) and coiled-coil protein (LIN-5, Mud, NuMA). In this study, we searched for additional factors that contribute to spindle positioning in the one-cell Caenorhabditis elegans embryo. We show that cortical actin is not needed for Gα-GPR-LIN-5 localization and pulling force generation. Instead, actin accumulation in the anterior actually reduces pulling forces, possibly by increasing cortical rigidity. Examining membrane-associated proteins that copurified with GOA-1 Gα, we found that the transmembrane and coiled-coil domain protein 1 (TCC-1) contributes to proper spindle movements. TCC-1 localizes to the endoplasmic reticulum membrane and interacts with UNC-116 kinesin-1 heavy chain in yeast two-hybrid assays. RNA interference of tcc-1 and unc-116 causes similar defects in meiotic spindle positioning, supporting the concept of TCC-1 acting with kinesin-1 in vivo. These results emphasize the contribution of membrane-associated and cortical proteins other than Gα-GPR-LIN-5 in balancing the pulling forces that position the spindle during asymmetric cell division. PMID:23699393

  4. F-actin asymmetry and the endoplasmic reticulum–associated TCC-1 protein contribute to stereotypic spindle movements in the Caenorhabditis elegans embryo

    PubMed Central

    Berends, Christian W. H.; Muñoz, Javier; Portegijs, Vincent; Schmidt, Ruben; Grigoriev, Ilya; Boxem, Mike; Akhmanova, Anna; Heck, Albert J. R.; van den Heuvel, Sander

    2013-01-01

    The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During development, dividing cells control the position of the spindle to determine the size, location, and fate of daughter cells. Spindle positioning depends on pulling forces that act between the cell periphery and astral microtubules. This involves dynein recruitment to the cell cortex by a heterotrimeric G-protein α subunit in complex with a TPR-GoLoco motif protein (GPR-1/2, Pins, LGN) and coiled-coil protein (LIN-5, Mud, NuMA). In this study, we searched for additional factors that contribute to spindle positioning in the one-cell Caenorhabditis elegans embryo. We show that cortical actin is not needed for Gα–GPR–LIN-5 localization and pulling force generation. Instead, actin accumulation in the anterior actually reduces pulling forces, possibly by increasing cortical rigidity. Examining membrane-associated proteins that copurified with GOA-1 Gα, we found that the transmembrane and coiled-coil domain protein 1 (TCC-1) contributes to proper spindle movements. TCC-1 localizes to the endoplasmic reticulum membrane and interacts with UNC-116 kinesin-1 heavy chain in yeast two-hybrid assays. RNA interference of tcc-1 and unc-116 causes similar defects in meiotic spindle positioning, supporting the concept of TCC-1 acting with kinesin-1 in vivo. These results emphasize the contribution of membrane-associated and cortical proteins other than Gα–GPR–LIN-5 in balancing the pulling forces that position the spindle during asymmetric cell division. PMID:23699393

  5. Modes of interaction among yeast Nej1, Lif1 and Dnl4 proteins and comparison to human XLF, XRCC4 and Lig4.

    PubMed

    Deshpande, Rajashree A; Wilson, Thomas E

    2007-10-01

    The nonhomologous end joining (NHEJ) pathway of double-strand break repair depends on DNA ligase IV and its interacting partner protein XRCC4 (Lif1 in yeast). A third yeast protein, Nej1, interacts with Lif1 and supports NHEJ, similar to the distantly related mammalian Nej1 orthologue XLF (also known as Cernunnos). XRCC4/Lif1 and XLF/Nej1 are themselves related and likely fold into similar coiled-coil structures, which suggests many possible modes of interaction between these proteins. Using yeast two-hybrid and co-precipitation methods we examined these interactions and the protein domains required to support them. Results suggest that stable coiled-coil homodimers are a predominant form of XLF/Nej1, just as for XRCC4/Lif1, but that similar heterodimers are not. XLF-XRCC4 and Nej1-Lif1 interactions were instead mediated independently of the coiled coil, and by different regions of XLF and Nej1. Specifically, the globular head of XRCC4/Lif1 interacted with N- and C-terminal domains of XLF and Nej1, respectively. Direct interactions between XLF/Nej1 and DNA ligase IV were also observed, but again appeared qualitatively different than the stable coiled-coil-mediated interaction between XRCC4/Lif1 and DNA ligase IV. The implications of these findings for DNA ligase IV function are considered in light of the evolutionary pattern in the XLF/XRCC4 and XLF/Nej1 family. PMID:17567543

  6. The conserved glycine-rich segment linking the N-terminal fusion peptide to the coiled coil of human T-cell leukemia virus type 1 transmembrane glycoprotein gp21 is a determinant of membrane fusion function.

    PubMed

    Wilson, Kirilee A; Bär, Séverine; Maerz, Anne L; Alizon, Marc; Poumbourios, Pantelis

    2005-04-01

    Retroviral transmembrane proteins (TMs) contain an N-terminal fusion peptide that initiates virus-cell membrane fusion. The fusion peptide is linked to the coiled-coil core through a conserved sequence that is often rich in glycines. We investigated the functional role of the glycine-rich segment, Met-326 to Ser-337, of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, by alanine and proline scanning mutagenesis. Alanine substitution for the hydrophobic residue Ile-334 caused an approximately 90% reduction in cell-cell fusion activity without detectable effects on the lipid-mixing and pore formation phases of fusion. Alanine substitutions at other positions had smaller effects (Gly-329, Val-330, and Gly-332) or no effect on fusion function. Proline substitution for glycine residues inhibited cell-cell fusion function with position-dependent effects on the three phases of fusion. Retroviral glycoprotein fusion function thus appears to require flexibility within the glycine-rich segment and hydrophobic contacts mediated by this segment. PMID:15767455

  7. The Conserved Glycine-Rich Segment Linking the N-Terminal Fusion Peptide to the Coiled Coil of Human T-Cell Leukemia Virus Type 1 Transmembrane Glycoprotein gp21 Is a Determinant of Membrane Fusion Function

    PubMed Central

    Wilson, Kirilee A.; Bär, Séverine; Maerz, Anne L.; Alizon, Marc; Poumbourios, Pantelis

    2005-01-01

    Retroviral transmembrane proteins (TMs) contain an N-terminal fusion peptide that initiates virus-cell membrane fusion. The fusion peptide is linked to the coiled-coil core through a conserved sequence that is often rich in glycines. We investigated the functional role of the glycine-rich segment, Met-326 to Ser-337, of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, by alanine and proline scanning mutagenesis. Alanine substitution for the hydrophobic residue Ile-334 caused an ∼90% reduction in cell-cell fusion activity without detectable effects on the lipid-mixing and pore formation phases of fusion. Alanine substitutions at other positions had smaller effects (Gly-329, Val-330, and Gly-332) or no effect on fusion function. Proline substitution for glycine residues inhibited cell-cell fusion function with position-dependent effects on the three phases of fusion. Retroviral glycoprotein fusion function thus appears to require flexibility within the glycine-rich segment and hydrophobic contacts mediated by this segment. PMID:15767455

  8. The WTX Tumor Suppressor Interacts with the Transcriptional Corepressor TRIM28*

    PubMed Central

    Kim, Woo Jae; Wittner, Ben S.; Amzallag, Arnaud; Brannigan, Brian W.; Ting, David T.; Ramaswamy, Sridhar; Maheswaran, Shyamala; Haber, Daniel A.

    2015-01-01

    WTX encodes a tumor suppressor implicated in the pediatric kidney cancer Wilms tumor and in mesenchymal differentiation with potentially distinct functions in the cytoplasm, at the plasma membrane, and in the nucleus. Although modulating components of the WNT signaling pathway is a proposed function for cytoplasmic and membrane-bound WTX, its nuclear properties are not well understood. Here we report that the transcriptional corepressor TRIM28 is the major binding partner for nuclear WTX. WTX interacted with the coiled coil domain of TRIM28 required for its binding to Krüppel-associated box domains of transcription factors and for its chromatin recruitment through its own coiled coil and proline-rich domains. Knockdown of endogenous WTX reduced the recruitment of TRIM28 to a chromatinized reporter sequence and its ability to repress a target transcript. In mouse embryonic stem cells where TRIM28 plays a major role in repressing endogenous retroviruses and long interspersed elements, knockdown of either TRIM28 or WTX combined with single molecule RNA sequencing revealed a highly significant shared set of differentially regulated transcripts, including derepression of non-coding repetitive sequences and their neighboring protein encoding genes (p < 1e−20). In mesenchymal precursor cells, depletion of WTX and TRIM28 resulted in analogous β-catenin-independent defects in adipogenic and osteogenic differentiation, and knockdown of WTX reduced TRIM28 binding to Pparγ promoter. Together, the physical and functional interaction between WTX and TRIM28 suggests that the nuclear fraction of WTX plays a role in epigenetic silencing, an effect that may contribute to its function as a regulator of cellular differentiation and tumorigenesis. PMID:25882849

  9. Structure-function analysis of the Anopheles gambiae LRIM1/APL1C complex and its interaction with complement C3-like protein TEP1.

    PubMed

    Povelones, Michael; Upton, Leanna M; Sala, Katarzyna A; Christophides, George K

    2011-04-01

    Malaria threatens half the world's population and exacts a devastating human toll. The principal malaria vector in Africa, the mosquito Anopheles gambiae, encodes 24 members of a recently identified family of leucine-rich repeat proteins named LRIMs. Two members of this family, LRIM1 and APL1C, are crucial components of the mosquito complement-like pathway that is important for immune defense against Plasmodium parasites. LRIM1 and APL1C circulate in the hemolymph exclusively as a disulfide-bonded complex that specifically interacts with the mature form of the complement C3-like protein, TEP1. We have investigated the specificity of LRIM1/APL1C complex formation and which regions of these proteins are required for interactions with TEP1. To address these questions, we have generated a set of LRIM1 and APL1C alleles altering key conserved structural elements and assayed them in cell culture for complex formation and interaction with TEP1. Our data indicate that heterocomplex formation is an intrinsic ability of LRIM1 and APL1C and identify key homologous cysteine residues forming the intermolecular disulfide bond. We also demonstrate that the coiled-coil domain is the binding site for TEP1 but also contributes to the specificity of LRIM1/APL1C complex formation. In addition, we show that the LRIM1/APL1C complex interacts with the mature forms of three other TEP proteins, one of which, TEP3, we have characterized as a Plasmodium antagonist. We conclude that LRIM1 and APL1C contain three distinct modules: a C-terminal coiled-coil domain that can carry different TEP protein cargoes, potentially with distinct functions, a central cysteine-rich region that controls complex formation and an N-terminal leucine-rich repeat with a putative role in pathogen recognition. PMID:21533217

  10. An aromatic amino acid in the coiled-coil 1 domain plays a crucial role in the auto-inhibitory mechanism of STIM1.

    PubMed

    Yu, Junwei; Zhang, Haining; Zhang, Mingshu; Deng, Yongqiang; Wang, Huiyu; Lu, Jingze; Xu, Tao; Xu, Pingyong

    2013-09-15

    STIM1 (stromal interaction molecule 1) is one of the key elements that mediate store-operated Ca²⁺ entry via CRAC (Ca²⁺- release-activated Ca²⁺) channels in immune and non-excitable cells. Under physiological conditions, the intramolecular auto-inhibitions in STIM1 C- and STIM1 N-termini play essential roles in keeping STIM1 in an inactive state. However, the auto-inhibitory mechanism of the STIM1 C-terminus is still unclear. In the present study, we first predicted a short inhibitory domain (residues 310-317) in human STIM1 that might determine the different localizations of human STIM1 from Caenorhabditis elegans STIM1 in resting cells. Next, we confirmed the prediction and further identified an aromatic amino acid residue, Tyr³¹⁶, that played a crucial role in maintaining STIM1 in a closed conformation in quiescent cells. Full-length STIM1-Y316A formed constitutive clusters near the plasma membrane and activated the CRAC channel in the resting state when co-expressed with Orai1. The introduction of a Y316A mutation caused the higher-order oligomerization of the in vitro purified STIM1 fragment containing both the auto-inhibitory domain and CAD(CRAC-activating domain).We propose that the Tyr³¹⁶ residue may be involved in the auto-inhibitory mechanism of the STIM1 C-terminus in the quiescent state. This inhibition could be achieved either by interacting with the CAD using hydrogen and/or hydrophobic bonds, or by an intermolecular interaction using repulsive forces, which maintained a dimeric STIM1. PMID:23795811

  11. Molecular cloning of a coiled-coil-nucleotide-binding-site-leucine-rich repeat gene from pearl millet and its expression pattern in response to the downy mildew pathogen.

    PubMed

    Veena, Mariswamy; Melvin, Prasad; Prabhu, Sreedhara Ashok; Shailasree, Sekhar; Shetty, Hunthrike Shekar; Kini, Kukkundoor Ramachandra

    2016-03-01

    Downy mildew caused by Sclerospora graminicola is a devastating disease of pearl millet. Based on candidate gene approach, a set of 22 resistance gene analogues were identified. The clone RGPM 301 (AY117410) containing a partial sequence shared 83% similarity to rice R-proteins. A full-length R-gene RGA RGPM 301 of 3552 bp with 2979 bp open reading frame encoding 992 amino acids was isolated by the degenerate primers and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) approach. It had a molecular mass of 113.96 kDa and isoelectric point (pI) of 8.71. The sequence alignment and phylogenetic analysis grouped it to a non-TIR NBS LRR group. The quantitative real-time PCR (qRT-PCR) analysis revealed higher accumulation of the transcripts following inoculation with S. graminicola in the resistant cultivar (IP18296) compared to susceptible cultivar (7042S). Further, significant induction in the transcript levels were observed when treated with abiotic elicitor β-aminobutyric acid (BABA) and biotic elicitor Pseudomonas fluorescens. Exogenous application of phytohormones jasmonic acid or salicylic acid also up-regulated the expression levels of RGA RGPM 301. The treatment of cultivar IP18296 with mitogen-activated protein kinase (MPK) inhibitors (PD98059 and U0126) suppressed the levels of RGA RGPM 301. A 3.5 kb RGA RGPM 301 which is a non-TIR NBS-LRR protein was isolated from pearl millet and its up-regulation during downy mildew interaction was demonstrated by qRT-PCR. These studies indicate a role for this RGA in pearl millet downy mildew interaction. PMID:26842722

  12. Solving coiled-coil protein structures

    DOE PAGESBeta

    Dauter, Zbigniew

    2015-02-26

    With the availability of more than 100,000 entries stored in the Protein Data Bank (PDB) that can be used as search models, molecular replacement (MR) is currently the most popular method of solving crystal structures of macromolecules. Significant methodological efforts have been directed in recent years towards making this approach more powerful and practical. This resulted in the creation of several computer programs, highly automated and user friendly, that are able to successfully solve many structures even by researchers who, although interested in structures of biomolecules, are not very experienced in crystallography.

  13. Vibrational solvatochromism. III. Rigorous treatment of the dispersion interaction contribution.

    PubMed

    Błasiak, Bartosz; Cho, Minhaeng

    2015-10-28

    A rigorous first principles theory of vibrational solvatochromism including the intermolecular dispersion interaction, which is based on the effective fragment potential method, is developed. The present theory is an extended version of our previous vibrational solvatochromism model that took into account the Coulomb, exchange-repulsion, and induction interactions. We show that the frequency shifts of the amide I mode of N-methylacetamide in H2O and CDCl3, when combined with molecular dynamics simulations, can be quantitatively reproduced by the theory, which indicates that the dispersion interaction contribution to the vibrational frequency shift is not always negligibly small. Nonetheless, the reason that the purely Coulombic interaction model for vibrational solvatochromism works well for describing amide I mode frequency shifts in polar solvents is because the electrostatic contribution is strong and highly sensitive to the relative orientation of surrounding solvent molecules, which is in stark contrast with polarization, dispersion, and exchange-repulsion contributions. It is believed that the theory presented and discussed here will be of great use in quantitatively describing vibrational solvatochromism and electrochromism of infrared probes in not just polar solvent environments but also in biopolymers such as proteins. PMID:26520502

  14. Vibrational solvatochromism. III. Rigorous treatment of the dispersion interaction contribution

    NASA Astrophysics Data System (ADS)

    Błasiak, Bartosz; Cho, Minhaeng

    2015-10-01

    A rigorous first principles theory of vibrational solvatochromism including the intermolecular dispersion interaction, which is based on the effective fragment potential method, is developed. The present theory is an extended version of our previous vibrational solvatochromism model that took into account the Coulomb, exchange-repulsion, and induction interactions. We show that the frequency shifts of the amide I mode of N-methylacetamide in H2O and CDCl3, when combined with molecular dynamics simulations, can be quantitatively reproduced by the theory, which indicates that the dispersion interaction contribution to the vibrational frequency shift is not always negligibly small. Nonetheless, the reason that the purely Coulombic interaction model for vibrational solvatochromism works well for describing amide I mode frequency shifts in polar solvents is because the electrostatic contribution is strong and highly sensitive to the relative orientation of surrounding solvent molecules, which is in stark contrast with polarization, dispersion, and exchange-repulsion contributions. It is believed that the theory presented and discussed here will be of great use in quantitatively describing vibrational solvatochromism and electrochromism of infrared probes in not just polar solvent environments but also in biopolymers such as proteins.

  15. Magnetic interactions in strongly correlated systems: Spin and orbital contributions

    SciTech Connect

    Secchi, A.; Lichtenstein, A.I.; Katsnelson, M.I.

    2015-09-15

    We present a technique to map an electronic model with local interactions (a generalized multi-orbital Hubbard model) onto an effective model of interacting classical spins, by requiring that the thermodynamic potentials associated to spin rotations in the two systems are equivalent up to second order in the rotation angles, when the electronic system is in a symmetry-broken phase. This allows to determine the parameters of relativistic and non-relativistic magnetic interactions in the effective spin model in terms of equilibrium Green’s functions of the electronic model. The Hamiltonian of the electronic system includes, in addition to the non-relativistic part, relativistic single-particle terms such as the Zeeman coupling to an external magnetic field, spin–orbit coupling, and arbitrary magnetic anisotropies; the orbital degrees of freedom of the electrons are explicitly taken into account. We determine the complete relativistic exchange tensors, accounting for anisotropic exchange, Dzyaloshinskii–Moriya interactions, as well as additional non-diagonal symmetric terms (which may include dipole–dipole interaction). The expressions of all these magnetic interactions are determined in a unified framework, including previously disregarded features such as the vertices of two-particle Green’s functions and non-local self-energies. We do not assume any smallness in spin–orbit coupling, so our treatment is in this sense exact. Finally, we show how to distinguish and address separately the spin, orbital and spin–orbital contributions to magnetism, providing expressions that can be computed within a tight-binding Dynamical Mean Field Theory.

  16. A Bub1–Mad1 interaction targets the Mad1–Mad2 complex to unattached kinetochores to initiate the spindle checkpoint

    PubMed Central

    Moyle, Mark W.; Kim, Taekyung; Hattersley, Neil; Espeut, Julien; Cheerambathur, Dhanya K.; Oegema, Karen

    2014-01-01

    Recruitment of Mad1–Mad2 complexes to unattached kinetochores is a central event in spindle checkpoint signaling. Despite its importance, the mechanism that recruits Mad1–Mad2 to kinetochores is unclear. In this paper, we show that MAD-1 interacts with BUB-1 in Caenorhabditis elegans. Mutagenesis identified specific residues in a segment of the MAD-1 coiled coil that mediate the BUB-1 interaction. In addition to unattached kinetochores, MAD-1 localized between separating meiotic chromosomes and to the nuclear periphery. Mutations in the MAD-1 coiled coil that selectively disrupt interaction with BUB-1 eliminated MAD-1 localization to unattached kinetochores and between meiotic chromosomes, both of which require BUB-1, and abrogated checkpoint signaling. The identified MAD-1 coiled-coil segment interacted with a C-terminal region of BUB-1 that contains its kinase domain, and mutations in this region prevented MAD-1 kinetochore targeting independently of kinase activity. These results delineate an interaction between BUB-1 and MAD-1 that targets MAD-1–MAD-2 complexes to kinetochores and is essential for spindle checkpoint signaling. PMID:24567362

  17. Interaction of the Spo20 Membrane-Sensor Motif with Phosphatidic Acid and Other Anionic Lipids, and Influence of the Membrane Environment

    PubMed Central

    Horchani, Habib; de Saint-Jean, Maud; Barelli, Hélène; Antonny, Bruno

    2014-01-01

    The yeast protein Spo20 contains a regulatory amphipathic motif that has been suggested to recognize phosphatidic acid, a lipid involved in signal transduction, lipid metabolism and membrane fusion. We have investigated the interaction of the Spo20 amphipathic motif with lipid membranes using a bioprobe strategy that consists in appending this motif to the end of a long coiled-coil, which can be coupled to a GFP reporter for visualization in cells. The resulting construct is amenable to in vitro and in vivo experiments and allows unbiased comparison between amphipathic helices of different chemistry. In vitro, the Spo20 bioprobe responded to small variations in the amount of phosphatidic acid. However, this response was not specific. The membrane binding of the probe depended on the presence of phosphatidylethanolamine and also integrated the contribution of other anionic lipids, including phosphatidylserine and phosphatidyl-inositol-(4,5)bisphosphate. Inverting the sequence of the Spo20 motif neither affected the ability of the probe to interact with anionic liposomes nor did it modify its cellular localization, making a stereo-specific mode of phosphatidic acid recognition unlikely. Nevertheless, the lipid binding properties and the cellular localization of the Spo20 alpha-helix differed markedly from that of another amphipathic motif, Amphipathic Lipid Packing Sensor (ALPS), suggesting that even in the absence of stereo specific interactions, amphipathic helices can act as subcellular membrane targeting determinants in a cellular context. PMID:25426975

  18. Person-Environment Interactions Contributing to Nursing Home Resident Falls

    PubMed Central

    Hill, Elizabeth E.; Nguyen, Tam H.; Shaha, Maya; Wenzel, Jennifer A.; DeForge, Bruce R.; Spellbring, Ann Marie

    2011-01-01

    Although approximately 50% of nursing home residents fall annually, the surrounding circumstances remain inadequately understood. This study explored nursing staff perspectives of person, environment, and interactive circumstances surrounding nursing home falls. Focus groups were conducted at two nursing homes in the mid-Atlantic region with the highest and lowest fall rates among corporate facilities. Two focus groups were conducted per facility: one with licensed nurses and one with geriatric nursing assistants. Thematic and content analysis revealed three themes and 11 categories. Three categories under the Person theme were Change in Residents’ Health Status, Decline in Residents’ Abilities, and Residents’ Behaviors and Personality Characteristics. There were five Nursing Home Environment categories: Design Safety, Limited Space, Obstacles, Equipment Misuse and Malfunction, and Staff and Organization of Care. Three Interactions Leading to Falls categories were identified: Reasons for Falls, Time of Falls, and High-Risk Activities. Findings highlight interactions between person and environment factors as significant contributors to resident falls. PMID:20077985

  19. The Interactive Account of ventral occipitotemporal contributions to reading

    PubMed Central

    Price, Cathy J.; Devlin, Joseph T.

    2011-01-01

    The ventral occipitotemporal cortex (vOT) is involved in the perception of visually presented objects and written words. The Interactive Account of vOT function is based on the premise that perception involves the synthesis of bottom-up sensory input with top-down predictions that are generated automatically from prior experience. We propose that vOT integrates visuospatial features abstracted from sensory inputs with higher level associations such as speech sounds, actions and meanings. In this context, specialization for orthography emerges from regional interactions without assuming that vOT is selectively tuned to orthographic features. We discuss how the Interactive Account explains left vOT responses during normal reading and developmental dyslexia; and how it accounts for the behavioural consequences of left vOT damage. PMID:21549634

  20. Gene-Environment Interactions in Stress Response Contribute Additively to a Genotype-Environment Interaction

    PubMed Central

    Matsui, Takeshi; Ehrenreich, Ian M.

    2016-01-01

    How combinations of gene-environment interactions collectively give rise to genotype-environment interactions is not fully understood. To shed light on this problem, we genetically dissected an environment-specific poor growth phenotype in a cross of two budding yeast strains. This phenotype is detectable when certain segregants are grown on ethanol at 37°C (‘E37’), a condition that differs from the standard culturing environment in both its carbon source (ethanol as opposed to glucose) and temperature (37°C as opposed to 30°C). Using recurrent backcrossing with phenotypic selection, we identified 16 contributing loci. To examine how these loci interact with each other and the environment, we focused on a subset of four loci that together can lead to poor growth in E37. We measured the growth of all 16 haploid combinations of alleles at these loci in all four possible combinations of carbon source (ethanol or glucose) and temperature (30 or 37°C) in a nearly isogenic population. This revealed that the four loci act in an almost entirely additive manner in E37. However, we also found that these loci have weaker effects when only carbon source or temperature is altered, suggesting that their effect magnitudes depend on the severity of environmental perturbation. Consistent with such a possibility, cloning of three causal genes identified factors that have unrelated functions in stress response. Thus, our results indicate that polymorphisms in stress response can show effects that are intensified by environmental stress, thereby resulting in major genotype-environment interactions when multiple of these variants co-occur. PMID:27437938

  1. A testis-specific and testis developmentally regulated tumor protein D52 (TPD52)-like protein TPD52L3/hD55 interacts with TPD52 family proteins

    SciTech Connect

    Cao Qinhong; Chen Jie; Zhu Li; Liu Yun; Zhou Zuomin; Sha Jiahao; Wang Shui; Li Jianmin . E-mail: jianminli@njmu.edu.cn

    2006-06-09

    Tumor protein D52-like proteins (TPD52) are small coiled-coil motif bearing proteins that were first identified in breast cancer. TPD52 and related proteins have been implicated in cell proliferation, apoptosis, and vesicle trafficking. To date, three human TPD52 members had been identified, named hD52 (TPD52), hD53 (TPD52L1), and hD54 (TPD52L2). The most important characteristic of the protein family is a highly conserved coiled-coil motif that is required for homo- and heteromeric interaction with other TPD52-like proteins. Herein, we identified a novel TPD52-like sequence (TPD52L3, or hD55) in human testis using cDNA microarray. Sequence analysis of the deduced protein suggests that hD55 contains a coiled-coil motif and is highly conserved compared with other TPD52-like sequences. Yeast two-hybrid and GST pull-down assays revealed that hD55 interacts with hD52, hD53, hD54, and itself. cDNA microarray detection found that hD55 was expressed at 5.6-fold higher levels in adult testis than in fetal testis. Additionally, the expression profile shows that hD55 is testis-specific, indicating a potential role for hD55 in testis development and spermatogenesis.

  2. Electrostatic forces contribute to interactions between trp repressor dimers.

    PubMed Central

    Martin, K S; Royer, C A; Howard, K P; Carey, J; Liu, Y C; Matthews, K; Heyduk, E; Lee, J C

    1994-01-01

    The trp repressor of Escherichia coli (TR), although generally considered to be dimeric, has been shown by fluorescence anisotropy of extrinsically labeled protein to undergo oligomerization in solution at protein concentrations in the micromolar range (Fernando, T., and C. A. Royer 1992. Biochemistry. 31:3429-3441). Providing evidence that oligomerization is an intrinsic property of TR, the present studies using chemical cross-linking, analytical ultracentrifugation, and molecular sieve chromatography demonstrate that unmodified TR dimers form higher order aggregates. Tetramers and higher order species were observed in chemical cross-linking experiments at concentrations between 1 and 40 microM. Results from analytical ultracentrifugation and gel filtration chromatography were consistent with average molecular weight values between tetramer and dimer, although no plateaus in the association were evident over the concentration ranges studied, indicating that higher order species are populated. Analytical ultracentrifugation data in presence of corepressor imply that corepressor binding destabilizes the higher order aggregates, an observation that is consistent with the earlier fluorescence work. Through the investigation of the salt and pH dependence of oligomerization, the present studies have revealed an electrostatic component to the interactions between TR dimers. Images FIGURE 1 PMID:8038388

  3. Genetic interactions contribute less than additive effects to quantitative trait variation in yeast

    PubMed Central

    Bloom, Joshua S.; Kotenko, Iulia; Sadhu, Meru J.; Treusch, Sebastian; Albert, Frank W.; Kruglyak, Leonid

    2015-01-01

    Genetic mapping studies of quantitative traits typically focus on detecting loci that contribute additively to trait variation. Genetic interactions are often proposed as a contributing factor to trait variation, but the relative contribution of interactions to trait variation is a subject of debate. Here we use a very large cross between two yeast strains to accurately estimate the fraction of phenotypic variance due to pairwise QTL–QTL interactions for 20 quantitative traits. We find that this fraction is 9% on average, substantially less than the contribution of additive QTL (43%). Statistically significant QTL–QTL pairs typically have small individual effect sizes, but collectively explain 40% of the pairwise interaction variance. We show that pairwise interaction variance is largely explained by pairs of loci at least one of which has a significant additive effect. These results refine our understanding of the genetic architecture of quantitative traits and help guide future mapping studies. PMID:26537231

  4. Shaping Learner Contributions in an EFL Classroom: Implications for L2 Classroom Interactional Competence

    ERIC Educational Resources Information Center

    Can Daskin, Nilüfer

    2015-01-01

    This study investigated the interactional patterns for shaping learner contributions in an EFL classroom with reference to Walsh's classroom interactional competence (CIC). In doing so, an EFL class at an English preparatory school in a Turkish state university was both videotaped and audiotaped in the course of six classroom hours. Conversation…

  5. An N-terminal glycine-rich sequence contributes to retrovirus trimer of hairpins stability

    SciTech Connect

    Wilson, Kirilee A.; Maerz, Anne L.; Baer, Severine; Drummer, Heidi E.; Poumbourios, Pantelis . E-mail: apoumbourios@burnet.edu.au

    2007-08-10

    Retroviral transmembrane proteins (TMs) contain a glycine-rich segment linking the N-terminal fusion peptide and coiled coil core. Previously, we reported that the glycine-rich segment (Met-326-Ser-337) of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, is a determinant of membrane fusion function [K.A. Wilson, S. Baer, A.L. Maerz, M. Alizon, P. Poumbourios, The conserved glycine-rich segment linking the N-terminal fusion peptide to the coiled coil of human T-cell leukemia virus type 1 transmembrane glycoprotein gp21 is a determinant of membrane fusion function, J. Virol. 79 (2005) 4533-4539]. Here we show that the reduced fusion activity of an I334A mutant correlated with a decrease in stability of the gp21 trimer of hairpins conformation, in the context of a maltose-binding protein-gp21 chimera. The stabilizing influence of Ile-334 required the C-terminal membrane-proximal sequence Trp-431-Ser-436. Proline substitution of four of five Gly residues altered gp21 trimer of hairpins stability. Our data indicate that flexibility within and hydrophobic interactions mediated by this region are determinants of gp21 stability and membrane fusion function.

  6. An N-terminal glycine-rich sequence contributes to retrovirus trimer of hairpins stability.

    PubMed

    Wilson, Kirilee A; Maerz, Anne L; Bär, Séverine; Drummer, Heidi E; Poumbourios, Pantelis

    2007-08-10

    Retroviral transmembrane proteins (TMs) contain a glycine-rich segment linking the N-terminal fusion peptide and coiled coil core. Previously, we reported that the glycine-rich segment (Met-326-Ser-337) of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, is a determinant of membrane fusion function [K.A. Wilson, S. Bär, A.L. Maerz, M. Alizon, P. Poumbourios, The conserved glycine-rich segment linking the N-terminal fusion peptide to the coiled coil of human T-cell leukemia virus type 1 transmembrane glycoprotein gp21 is a determinant of membrane fusion function, J. Virol. 79 (2005) 4533-4539]. Here we show that the reduced fusion activity of an I334A mutant correlated with a decrease in stability of the gp21 trimer of hairpins conformation, in the context of a maltose-binding protein-gp21 chimera. The stabilizing influence of Ile-334 required the C-terminal membrane-proximal sequence Trp-431-Ser-436. Proline substitution of four of five Gly residues altered gp21 trimer of hairpins stability. Our data indicate that flexibility within and hydrophobic interactions mediated by this region are determinants of gp21 stability and membrane fusion function. PMID:17577584

  7. Professional Interaction, Relevant Practical Experience, and Intellectual Contributions at Nondoctoral AACSB-Accredited Accounting Programs

    ERIC Educational Resources Information Center

    Arlinghaus, Barry P.

    2008-01-01

    In this article, the author discusses a survey of faculty members at nondoctoral AACSB-accredited accounting programs in the United States. The purpose of the survey was to determine the environment for professional interaction and relevant experience in light of institutional demands for intellectual contributions. The findings show that the…

  8. Early Markers of Language and Attention: Mutual Contributions and the Impact of Parent-Infant Interactions

    ERIC Educational Resources Information Center

    Gartstein, Maria A.; Crawford, Jennifer; Robertson, Christopher D.

    2008-01-01

    This study was conducted to explore the contribution of attentional skills to early language, and the influence of early language markers on the development of attention, simultaneously examining the impact of parent-child interaction factors (reciprocity/synchrony and sensitivity/responsivity), including their potential moderator effects. All…

  9. Intersegment interactions and helix-coil transition within the generalized model of polypeptide chains approach

    NASA Astrophysics Data System (ADS)

    Badasyan, A. V.; Hayrapetyan, G. N.; Tonoyan, Sh. A.; Mamasakhlisov, Y. Sh.; Benight, A. S.; Morozov, V. F.

    2009-09-01

    The generalized model of polypeptide chains is extended to describe the helix-coil transition in a system comprised of two chains interacting side-by-side. The Hamiltonian of the model takes into account four possible types of interactions between repeated units of the two chains, i.e., helix-helix, helix-coil, coil-helix, and coil-coil. Analysis reveals when the energy Ihh+Icc of (h-h, c-c) interactions overwhelms the energy Ihc+Ich of mixed (h-c, c-h) interactions, the correlation length rises substantially, resulting in narrowing of the transition interval. In the opposite case, when Ihh+Iccinteractions from the same theoretical perspective.

  10. Assessing Energetic Contributions to Binding from a Disordered Region in a Protein-Protein Interaction

    SciTech Connect

    S Cho; C Swaminathan; D Bonsor; M Kerzic; R Guan; J Yang; C Kieke; P Anderson; D Kranz; et al.

    2011-12-31

    Many functional proteins are at least partially disordered prior to binding. Although the structural transitions upon binding of disordered protein regions can influence the affinity and specificity of protein complexes, their precise energetic contributions to binding are unknown. Here, we use a model protein-protein interaction system in which a locally disordered region has been modified by directed evolution to quantitatively assess the thermodynamic and structural contributions to binding of disorder-to-order transitions. Through X-ray structure determination of the protein binding partners before and after complex formation and isothermal titration calorimetry of the interactions, we observe a correlation between protein ordering and binding affinity for complexes along this affinity maturation pathway. Additionally, we show that discrepancies between observed and calculated heat capacities based on buried surface area changes in the protein complexes can be explained largely by heat capacity changes that would result solely from folding the locally disordered region. Previously developed algorithms for predicting binding energies of protein-protein interactions, however, are unable to correctly model the energetic contributions of the structural transitions in our model system. While this highlights the shortcomings of current computational methods in modeling conformational flexibility, it suggests that the experimental methods used here could provide training sets of molecular interactions for improving these algorithms and further rationalizing molecular recognition in protein-protein interactions.

  11. Disorder and structure in the Rab11 binding domain of Rab11 family interacting protein 2.

    PubMed

    Wei, Jie; Liu, Yuqi; Bose, Kakoli; Henry, Gillian D; Baleja, James D

    2009-01-27

    Rab11 plays a central role in plasma membrane recycling which returns cellular receptors for reuse at the cell surface. A recently identified family of Rab11 interacting proteins (FIP) includes FIP2. The C-terminal region of FIP2 is essential for colocalization with Rab11 on early endosomes and for enabling formation of higher-order oligomers. Rab11 binding and oligomerization of FIP2 are separable. Here we have determined the three-dimensional structure of the 40-residue coiled-coil oligomerization domain of FIP2 in the absence of Rab11 using NMR methods. The N-terminal half showed strong NOE cross-peaks and well-dispersed NMR resonances, whereas the C-terminal half had fewer NOE cross-peaks and less chemical shift dispersion. The 10 C-terminal residues were mostly disordered. The final structures of the dimer had favorable Ramachandran angles and a root-mean-square deviation of 0.59 +/- 0.13 A over superimposed backbone residues. The structure allows a comparison to a structure of FIP2 in complex with Rab11 that was determined crystallographically. In complex with Rab11, the C-terminal residues are not disordered but have a helical structure that predicts residual dipolar coupling constants that are incompatible with those measured on the unbound FIP2. In both structures, a histidine residue is found at the normally hydrophobic position of the heptad repeat of the coiled coil, and here we show its ionization destabilizes the coiled-coil structure. Together, these data allow us to build a model in which the binding of FIP family proteins to Rab11 can be described in terms of conformational changes and that suggests new modes of regulation. PMID:19119858

  12. A bivariate mann-whitney approach for unraveling genetic variants and interactions contributing to comorbidity.

    PubMed

    Wen, Yalu; Schaid, Daniel J; Lu, Qing

    2013-04-01

    Although comorbidity among complex diseases (e.g., drug dependence syndromes) is well documented, genetic variants contributing to the comorbidity are still largely unknown. The discovery of genetic variants and their interactions contributing to comorbidity will likely shed light on underlying pathophysiological and etiological processes, and promote effective treatments for comorbid conditions. For this reason, studies to discover genetic variants that foster the development of comorbidity represent high-priority research projects, as manifested in the behavioral genetics studies now underway. The yield from these studies can be enhanced by adopting novel statistical approaches, with the capacity of considering multiple genetic variants and possible interactions. For this purpose, we propose a bivariate Mann-Whitney (BMW) approach to unravel genetic variants and interactions contributing to comorbidity, as well as those unique to each comorbid condition. Through simulations, we found BMW outperformed two commonly adopted approaches in a variety of underlying disease and comorbidity models. We further applied BMW to datasets from the Study of Addiction: Genetics and Environment, investigating the contribution of 184 known nicotine dependence (ND) and alcohol dependence (AD) single nucleotide polymorphisms (SNPs) to the comorbidity of ND and AD. The analysis revealed a candidate SNP from CHRNA5, rs16969968, associated with both ND and AD, and replicated the findings in an independent dataset with a P-value of 1.06 × 10(-03) . PMID:23334941

  13. Seeds of strategic and interactional psychotherapies: seminal contributions of Milton H. Erickson.

    PubMed

    Zeig, J K; Geary, B B

    1990-10-01

    The life and work of Milton H. Erickson exerts a considerable influence upon the development of strategic and interactional psychotherapies. In this paper we trace the historical course of Erickson's impact in these areas from his early associations with Gregory Bateson and Margaret Mead through his contributions to the ideologies of Jay Haley and practitioners at the Mental Research Institute. We have identified seven philosophical and methodological realms which represent the incorporation of Ericksonian principles into strategic and interactional family therapy models. PMID:2270835

  14. XTACC3-XMAP215 association reveals an asymmetric interaction promoting microtubule elongation.

    PubMed

    Mortuza, Gulnahar B; Cavazza, Tommaso; Garcia-Mayoral, Maria Flor; Hermida, Dario; Peset, Isabel; Pedrero, Juan G; Merino, Nekane; Blanco, Francisco J; Lyngsø, Jeppe; Bruix, Marta; Pedersen, Jan Skov; Vernos, Isabelle; Montoya, Guillermo

    2014-01-01

    chTOG is a conserved microtubule polymerase that catalyses the addition of tubulin dimers to promote microtubule growth. chTOG interacts with TACC3, a member of the transforming acidic coiled-coil (TACC) family. Here we analyse their association using the Xenopus homologues, XTACC3 (TACC3) and XMAP215 (chTOG), dissecting the mechanism by which their interaction promotes microtubule elongation during spindle assembly. Using SAXS, we show that the TACC domain (TD) is an elongated structure that mediates the interaction with the C terminus of XMAP215. Our data suggest that one TD and two XMAP215 molecules associate to form a four-helix coiled-coil complex. A hybrid methods approach was used to define the precise regions of the TACC heptad repeat and the XMAP215 C terminus required for assembly and functioning of the complex. We show that XTACC3 can induce the recruitment of larger amounts of XMAP215 by increasing its local concentration, thereby promoting efficient microtubule elongation during mitosis. PMID:25262927

  15. Genetic Interactions Involving Five or More Genes Contribute to a Complex Trait in Yeast

    PubMed Central

    Taylor, Matthew B.; Ehrenreich, Ian M.

    2014-01-01

    Recent research suggests that genetic interactions involving more than two loci may influence a number of complex traits. How these ‘higher-order’ interactions arise at the genetic and molecular levels remains an open question. To provide insights into this problem, we dissected a colony morphology phenotype that segregates in a yeast cross and results from synthetic higher-order interactions. Using backcrossing and selective sequencing of progeny, we found five loci that collectively produce the trait. We fine-mapped these loci to 22 genes in total and identified a single gene at each locus that caused loss of the phenotype when deleted. Complementation tests or allele replacements provided support for functional variation in these genes, and revealed that pre-existing genetic variants and a spontaneous mutation interact to cause the trait. The causal genes have diverse functions in endocytosis (END3), oxidative stress response (TRR1), RAS-cAMP signalling (IRA2), and transcriptional regulation of multicellular growth (FLO8 and MSS11), and for the most part have not previously been shown to exhibit functional relationships. Further efforts uncovered two additional loci that together can complement the non-causal allele of END3, suggesting that multiple genotypes in the cross can specify the same phenotype. Our work sheds light on the complex genetic and molecular architecture of higher-order interactions, and raises questions about the broader contribution of such interactions to heritable trait variation. PMID:24784154

  16. β-arrestin-1 contributes to brown fat function and directly interacts with PPARα and PPARγ.

    PubMed

    Wang, Congcong; Zeng, Xianglu; Zhou, Zhaocai; Zhao, Jian; Pei, Gang

    2016-01-01

    The peroxisome proliferator-activated receptor (PPAR) family plays central roles in brown adipose tissue (BAT) adipogenesis and contributes to body temperature maintenance. The transcriptional activity of PPAR family has been shown to be tightly controlled by cellular signal networks. β-arrestins function as major secondary messengers of G protein-coupled receptors (GPCR) signaling by functional interactions with diverse proteins. Here, we report that β-arrestin-1 knock-out mice show enhanced cold tolerance. We found that β-arrestin-1 directly interacts with PPARα and PPARγ through a LXXXLXXXL motif, while D371 in PPARα and L311/N312/D380 in PPARγ are required for their interactions with β-arrestin-1. Further mechanistic studies showed that β-arrestin-1 promotes PPARα- but represses PPARγ-mediated transcriptional activities, providing potential regulatory pathway for BAT function. PMID:27301785

  17. β-arrestin-1 contributes to brown fat function and directly interacts with PPARα and PPARγ

    PubMed Central

    Wang, Congcong; Zeng, Xianglu; Zhou, Zhaocai; Zhao, Jian; Pei, Gang

    2016-01-01

    The peroxisome proliferator-activated receptor (PPAR) family plays central roles in brown adipose tissue (BAT) adipogenesis and contributes to body temperature maintenance. The transcriptional activity of PPAR family has been shown to be tightly controlled by cellular signal networks. β-arrestins function as major secondary messengers of G protein-coupled receptors (GPCR) signaling by functional interactions with diverse proteins. Here, we report that β-arrestin-1 knock-out mice show enhanced cold tolerance. We found that β-arrestin-1 directly interacts with PPARα and PPARγ through a LXXXLXXXL motif, while D371 in PPARα and L311/N312/D380 in PPARγ are required for their interactions with β-arrestin-1. Further mechanistic studies showed that β-arrestin-1 promotes PPARα- but represses PPARγ-mediated transcriptional activities, providing potential regulatory pathway for BAT function. PMID:27301785

  18. CFL3D Contribution to the AIAA Supersonic Shock Boundary Layer Interaction Workshop

    NASA Technical Reports Server (NTRS)

    Rumsey, Christopher L.

    2010-01-01

    This paper documents the CFL3D contribution to the AIAA Supersonic Shock Boundary Layer Interaction Workshop, held in Orlando, Florida in January 2010. CFL3D is a Reynolds-averaged Navier-Stokes code. Four shock boundary layer interaction cases are computed using a one-equation turbulence model widely used for other aerodynamic problems of interest. Two of the cases have experimental data available at the workshop, and two of the cases do not. The effect of grid, flux scheme, and thin-layer approximation are investigated. Comparisons are made to the available experimental data. All four cases exhibit strong three-dimensional behavior in and near the interaction regions, resulting from influences of the tunnel side-walls.

  19. Conformational Contribution to the Heat Capacity of Interacting System of Carbohydrate Polymer - Water.

    NASA Astrophysics Data System (ADS)

    Pyda, Marek; Wunderlich, Bernhard

    2001-03-01

    Based on the measured heat capacities of amorphous, dry starch and starch with low concentration of water above the partial glass transition of starch, the calculated Cp has been estimated from its vibrational, external, and conformational contributions. The conformational part is evaluated from a fit of the experimental Cp of starch and starch-water, decreased by the vibrational and the external Cp to a one-dimensional Ising-type model for two discrete states, and stiffness, cooperativity, and degeneracy parameters. These differences above the glass transition are interpreted as contributions of different conformational heat capacities from interacting chains of carbohydrate with water. The vibrational contribution was calculated as the heat capacity contributions from group and skeletal vibrations. The external contribution was computed based on thermal expansivity and compressibility as a function of temperature from experimental data of the partial liquid state of both dry starch and starch-water. The calculated and experimental heat capacities of starch-water and dry starch are compared over the whole range of temperatures measurements from 8 to 490 K. NSF, Polymers Program, DMR-9703692, and the Div. of Mat. Sci., BES, DOE at ORNL, managed UT-Batelle, LLC, for the U.S. Department of Energy, under contract number DOE- AC05-00OR22725.

  20. PMLRAR homodimers: distinct DNA binding properties and heteromeric interactions with RXR.

    PubMed Central

    Perez, A; Kastner, P; Sethi, S; Lutz, Y; Reibel, C; Chambon, P

    1993-01-01

    Fusion proteins (named PMLRAR) between PML and the retinoic acid receptor alpha (RAR alpha) are generated as a result of the t(15;17) chromosomal translocation found in acute promyelocytic leukemia (APL). We show here that PMLRAR proteins exist in solution as stable homodimers whose formation is mediated by a presumptive coiled coil in the PML moiety. In contrast to RAR alpha, which requires heterodimerization with RXR for efficient DNA binding, PMLRAR homodimers can bind to target sequences in the absence of RXR, and the binding pattern of PMLRAR homodimeric complexes to directly repeated motif (DR) response elements with 1-5 bp spacers is different from that of RAR/RXR heterodimeric complexes. We show that the presence of RXR induces the formation of PMLRAR/RXR heteromeric complexes which bind to DNA via one RAR DNA binding domain (DBD) and one RXR DBD, like 'classical' RAR/RXR heterodimers. PMLRAR interaction with RXR occurs in solution and in transfected cultured Cos cells, and PMLRAR is able to sequester RXR efficiently in the cytoplasm, suggesting that dominant 'inactivation' of RXR may be a possible mechanism of action for PMLRAR. Accordingly, we show that PMLRAR can both prevent the binding of the vitamin D3 receptor (VDR) to a target sequence in vitro and inhibit vitamin D3-dependent activation of a VDR-responsive reporter gene in transfected cells. These results suggest that both the distinct DNA binding properties of PMLRAR homodimers and the sequestration of RXR by PMLRARs may contribute to the molecular mechanisms which underlie the pathogenesis of APL. We also report that RXR alpha transcripts are down-regulated by RA-treatment in promyelocytic cells. Images PMID:8393784

  1. Conditioned place preference for social interaction in rats: contribution of sensory components

    PubMed Central

    Kummer, Kai; Klement, Sabine; Eggart, Vincent; Mayr, Michael J.; Saria, Alois; Zernig, Gerald

    2011-01-01

    A main challenge in the therapy of drug dependent individuals is to help them reactivate interest in non-drug-associated activities. We previously developed a rat experimental model based on the conditioned place preference (CPP) paradigm in which only four 15-min episodes of social interaction with a gender- and weight-matched male Sprague Dawley rat (1) reversed CPP from cocaine to social interaction despite continuing cocaine training and (2) prevented the reinstatement of cocaine CPP. In the present study, we investigated which of the sensory modalities of the composite stimulus “social interaction” contributes most to the rats' preference for it. If touch was limited by steel bars spaced at a distance of 2 cm and running across the whole length of a partitioning, CPP was still acquired, albeit to a lesser degree. If both rats were placed on the same side of a partitioning, rats did not develop CPP for social interaction. Thus, decreasing the available area for social interaction from 750 to 375 cm2 prevented the acquisition of CPP to social interaction despite the fact that animals could touch each other more intensely than through the bars of the partitioning. When touch was fully restricted by a glass screen dividing the conditioning chambers, and the only sensory modalities left were visual and olfactory cues, place preference shifted to place aversion. Overall, our findings indicate that the major rewarding sensory component of the composite stimulus “social interaction” is touch (taction). PMID:22232578

  2. ALDH16A1 is a novel non-catalytic enzyme that may be involved in the etiology of gout via protein–protein interactions with HPRT1

    PubMed Central

    Vasiliou, Vasilis; Sandoval, Monica; Backos, Donald S.; Jackson, Brian C.; Chen, Ying; Reigan, Philip; Lanaspa, Miguel A.; Johnson, Richard J.; Koppaka, Vindhya; Thompson, David C.

    2013-01-01

    Gout, a common form of inflammatory arthritis, is strongly associated with elevated uric acid concentrations in the blood (hyperuricemia). A recent study in Icelanders identified a rare missense single nucleotide polymorphism (SNP) in the ALDH16A1 gene, ALDH16A1*2, to be associated with gout and serum uric acid levels. ALDH16A1 is a novel and rather unique member of the ALDH superfamily in relation to its gene and protein structures. ALDH16 genes are present in fish, amphibians, protista, bacteria but absent from archaea, fungi and plants. In most mammalian species, two ALDH16A1 spliced variants (ALDH16A1, long form and ALDH16A1_v2, short form) have been identified and both are expressed in HepG-2, HK-2 and HK-293 human cell lines. The ALDH16 proteins contain two ALDH domains (as opposed to one in the other members of the superfamily), four transmembrane and one coiled-coil domains. The active site of ALDH16 proteins from bacterial, frog and lower animals contain the catalytically important cysteine residue (Cys-302); this residue is absent from the mammalian and fish orthologs. Molecular modeling predicts that both the short and long forms of human ALDH16A1 protein would lack catalytic activity but may interact with the hypoxanthine-guanine phosphoribosyltransferase (HPRT1) protein, a key enzyme involved in uric acid metabolism and gout. Interestingly, such protein-protein interactions with HPRT1 are predicted to be impaired for the long or short forms of ALDH16A1*2. These results lead to the intriguing possibility that association between ALDH16A1 and HPRT1 may be required for optimal HPRT activity with disruption of this interaction possibly contributing to the hyperuricemia seen in ALDH16A1*2 carriers. PMID:23348497

  3. Social inequalities in health: measuring the contribution of housing deprivation and social interactions for Spain

    PubMed Central

    2012-01-01

    Introduction Social factors have been proved to be main determinants of individuals’ health. Recent studies have also analyzed the contribution of some of those factors, such as education and job status, to socioeconomic inequalities in health. The aim of this paper is to provide new evidence about the factors driving socioeconomic inequalities in health for the Spanish population by including housing deprivation and social interactions as health determinants. Methods Cross-sectional study based on the Spanish sample of European Statistics on Income and Living Conditions (EU-SILC) for 2006. The concentration index measuring income-related inequality in health is decomposed into the contribution of each determinant. Several models are estimated to test the influence of different regressors for three proxies of ill-health. Results Health inequality favouring the better-off is observed in the distribution of self-assessed health, presence of chronic diseases and presence of limiting conditions. Inequality is mainly explained, besides age, by social factors such as labour status and financial deprivation. Housing deprivation contributes to pro-rich inequality in a percentage ranging from 7.17% to 13.85%, and social interactions from 6.16% to 10.19%. The contribution of some groups of determinants significantly differs depending on the ill-health variable used. Conclusions Health inequalities can be mostly reduced or shaped by policy, as they are mainly explained by social determinants such as labour status, education and other socioeconomic conditions. The major role played on health inequality by variables taking part in social exclusion points to the need to focus on the most vulnerable groups. JEL Codes H51, I14, I18 PMID:23241384

  4. Bacteria–bacteria interactions within the microbiota of the ancestral metazoan Hydra contribute to fungal resistance

    PubMed Central

    Fraune, Sebastian; Anton-Erxleben, Friederike; Augustin, René; Franzenburg, Sören; Knop, Mirjam; Schröder, Katja; Willoweit-Ohl, Doris; Bosch, Thomas CG

    2015-01-01

    Epithelial surfaces of most animals are colonized by diverse microbial communities. Although it is generally agreed that commensal bacteria can serve beneficial functions, the processes involved are poorly understood. Here we report that in the basal metazoan Hydra, ectodermal epithelial cells are covered with a multilayered glycocalyx that provides a habitat for a distinctive microbial community. Removing this epithelial microbiota results in lethal infection by the filamentous fungus Fusarium sp. Restoring the complex microbiota in gnotobiotic polyps prevents pathogen infection. Although mono-associations with distinct members of the microbiota fail to provide full protection, additive and synergistic interactions of commensal bacteria are contributing to full fungal resistance. Our results highlight the importance of resident microbiota diversity as a protective factor against pathogen infections. Besides revealing insights into the in vivo function of commensal microbes in Hydra, our findings indicate that interactions among commensal bacteria are essential to inhibit pathogen infection. PMID:25514534

  5. Contribution of excited states to stellar weak-interaction rates in odd-A nuclei

    NASA Astrophysics Data System (ADS)

    Sarriguren, P.

    2016-05-01

    Weak-interaction rates, including β decay and electron capture, are studied in several odd-A nuclei in the p f -shell region at various densities and temperatures of astrophysical interest. Special attention is paid to the relative contribution to these rates of thermally populated excited states in the decaying nucleus. The nuclear structure involved in the weak processes is studied within a quasiparticle random-phase approximation with residual interactions in both particle-hole and particle-particle channels on top of a deformed Skyrme Hartree-Fock mean field with pairing correlations. In the range of densities and temperatures considered, it is found that the total rates do not differ much from the rates of the ground state fully populated. In any case, the changes are not larger than the uncertainties due to the nuclear-model dependence of the rates.

  6. Bacteria-bacteria interactions within the microbiota of the ancestral metazoan Hydra contribute to fungal resistance.

    PubMed

    Fraune, Sebastian; Anton-Erxleben, Friederike; Augustin, René; Franzenburg, Sören; Knop, Mirjam; Schröder, Katja; Willoweit-Ohl, Doris; Bosch, Thomas C G

    2015-07-01

    Epithelial surfaces of most animals are colonized by diverse microbial communities. Although it is generally agreed that commensal bacteria can serve beneficial functions, the processes involved are poorly understood. Here we report that in the basal metazoan Hydra, ectodermal epithelial cells are covered with a multilayered glycocalyx that provides a habitat for a distinctive microbial community. Removing this epithelial microbiota results in lethal infection by the filamentous fungus Fusarium sp. Restoring the complex microbiota in gnotobiotic polyps prevents pathogen infection. Although mono-associations with distinct members of the microbiota fail to provide full protection, additive and synergistic interactions of commensal bacteria are contributing to full fungal resistance. Our results highlight the importance of resident microbiota diversity as a protective factor against pathogen infections. Besides revealing insights into the in vivo function of commensal microbes in Hydra, our findings indicate that interactions among commensal bacteria are essential to inhibit pathogen infection. PMID:25514534

  7. Regulation of COP1 nuclear localization by the COP9 signalosome via direct interaction with CSN1.

    PubMed

    Wang, Xiping; Li, Wenjun; Piqueras, Raquel; Cao, Kaiming; Deng, Xing Wang; Wei, Ning

    2009-05-01

    COP1 and COP9 signalosome (CSN) are key regulators of plant light responses and development. Deficiency in either COP1 or CSN causes a constitutive photomorphogenic phenotype. Through coordinated actions of nuclear- and cytoplasmic-localization signals, COP1 can respond to light signals by differentially partitions between nuclear and cytoplasmic compartments. Previous genetic analysis in Arabidopsis indicated that the nuclear localization of COP1 requires CSN, an eight-subunit heteromeric complex. However the mechanism underlying the functional relationship between COP1 and CSN is unknown. We report here that COP1 weakly associates with CSN in vivo. Furthermore, we report on the direct interaction involving the coiled-coil domain of COP1 and the N-terminal domain of the CSN1 subunit. In onion epidermal cells, expression of CSN1 can stimulate nuclear localization of GUS-COP1, and the N-terminal domain of CSN1 is necessary and sufficient for this function. Moreover, CSN1-induced COP1 nuclear localization requires the nuclear-localization sequences of COP1, as well as its coiled-coil domain, which contains both the cytoplasmic localization sequences and the CSN1 interacting domain. We also provide genetic evidence that the CSN1 N-terminal domain is specifically required for COP1 nuclear localization in Arabidopsis hypocotyl cells. This study advances our understanding of COP1 localization, and the molecular interactions between COP1 and CSN. PMID:19175768

  8. Analysis of the Contribution of Individual Substituents in 4,6-Aminoglycoside-Ribosome Interaction

    PubMed Central

    Hobbie, Sven N.; Pfister, Peter; Brüll, Christian; Westhof, Eric; Böttger, Erik C.

    2005-01-01

    The 4,6-disubstituted 2-deoxystreptamines interfere with protein biosynthesis by specifically targeting the ribosomal A site. These drugs show subtle variations in the chemical groups of rings I, II, and III. In the present study we used site-directed mutagenesis to generate mutant strains of Mycobacterium smegmatis mc2155 SMR5 ΔrrnB with mutations in its single rRNA allele, rrnA. This genetic procedure gives rise to strains carrying homogeneous populations of mutant ribosomes and was used to study the contribution of individual chemical substituents to the binding of 4,6-disubstituted aminoglycosides. X-ray crystal structures of geneticin and tobramycin complexed to oligonucleotides containing the minimal bacterial ribosomal A site were used for interpretation of MICs determined for a panel of 4,6-aminoglycosides, including tobramycin, kanamycin A, kanamycin B, amikacin, gentamicin, and geneticin. Surprisingly, the considerable differences present within ring III did not seem to alter the interaction of the drug with the ribosome, as determined by site-directed mutagenesis of the A site. In contrast, subtle variations in ring I significantly influenced binding: (i) a 4′-hydroxyl moiety participates in the proper drug target interaction; and (ii) a 2′-amino group contributes an additional positive charge to ring I, making the drug less susceptible to any kind of sequence alteration within the decoding site, most notably, to conformational changes induced by transversion of U1495 to 1495A. The 4-amino-2-hydroxyl-1-oxobutyl extension at position 1 of ring II of amikacin provides an additional anchor and renders amikacin less dependent on the structural conformation of nucleotide U1406 compared to the dependencies of other kanamycins. Overall, the set of interactions forming the complex between drug substituents and nucleotides of the A site constitutes a network in which the interactions can partly compensate for each other when they are disrupted. PMID:16304180

  9. Isolating the non-polar contributions to the intermolecular potential for water-alkane interactions

    NASA Astrophysics Data System (ADS)

    Ballal, Deepti; Venkataraman, Pradeep; Fouad, Wael A.; Cox, Kenneth R.; Chapman, Walter G.

    2014-08-01

    Intermolecular potential models for water and alkanes describe pure component properties fairly well, but fail to reproduce properties of water-alkane mixtures. Understanding interactions between water and non-polar molecules like alkanes is important not only for the hydrocarbon industry but has implications to biological processes as well. Although non-polar solutes in water have been widely studied, much less work has focused on water in non-polar solvents. In this study we calculate the solubility of water in different alkanes (methane to dodecane) at ambient conditions where the water content in alkanes is very low so that the non-polar water-alkane interactions determine solubility. Only the alkane-rich phase is simulated since the fugacity of water in the water rich phase is calculated from an accurate equation of state. Using the SPC/E model for water and TraPPE model for alkanes along with Lorentz-Berthelot mixing rules for the cross parameters produces a water solubility that is an order of magnitude lower than the experimental value. It is found that an effective water Lennard-Jones energy ɛW/k = 220 K is required to match the experimental water solubility in TraPPE alkanes. This number is much higher than used in most simulation water models (SPC/E—ɛW/k = 78.2 K). It is surprising that the interaction energy obtained here is also higher than the water-alkane interaction energy predicted by studies on solubility of alkanes in water. The reason for this high water-alkane interaction energy is not completely understood. Some factors that might contribute to the large interaction energy, such as polarizability of alkanes, octupole moment of methane, and clustering of water at low concentrations in alkanes, are examined. It is found that, though important, these factors do not completely explain the anomalously strong attraction between alkanes and water observed experimentally.

  10. Genetic variations and miRNA-target interactions contribute to natural phenotypic variations in Populus.

    PubMed

    Chen, Jinhui; Xie, Jianbo; Chen, Beibei; Quan, Mingyang; Li, Ying; Li, Bailian; Zhang, Deqiang

    2016-10-01

    Variation in regulatory factors, including microRNAs (miRNAs), contributes to variation in quantitative and complex traits. However, in plants, variants in miRNAs and their target genes that contribute to natural phenotypic variation, and the underlying regulatory networks, remain poorly characterized. We investigated the associations and interactions of single-nucleotide polymorphisms (SNPs) in miRNAs and their target genes with phenotypes in 435 individuals from a natural population of Populus. We used RNA-seq to identify 217 miRNAs differentially expressed in a tension wood system, and identified 1196 candidate target genes; degradome sequencing confirmed 60 of the target sites. In addition, 72 miRNA-target pairs showed significant co-expression. Gene ontology (GO) term analysis showed that most of the genes in the co-regulated pairs participate in biological regulation. Genome resequencing found 5383 common SNPs (frequency ≥ 0.05) in 139 miRNAs and 31 037 SNPs in 819 target genes. Single-SNP association analyses identified 232 significant associations between wood traits (P ≤ 0.05) and SNPs in 102 miRNAs and 1387 associations with 478 target genes. Among these, 102 miRNA-target pairs associated with the same traits. Multi-SNP associations found 102 epistatic pairs associated with traits. Furthermore, a reconstructed regulatory network contained 12 significantly co-expressed pairs, including eight miRNAs and nine targets associated with traits. Lastly, both expression and genetic association showed that miR156i, miR156j, miR396a and miR6445b were involved in the formation of tension wood. This study shows that variants in miRNAs and target genes contribute to natural phenotypic variation and annotated roles and interactions of miRNAs and their target genes by genetic association analysis. PMID:27265357

  11. Interaction with PI3-kinase contributes to the cytotoxic activity of Apoptin

    PubMed Central

    Maddika, S; Wiechec, E; Ande, SR; Poon, IK; Fischer, U; Wesselborg, S; Jans, DA; Schulze-Osthoff, K; Los, M

    2010-01-01

    Apoptin, a small protein from the chicken anemia virus, has attracted attention because of its specificity in killing tumor cells. Localization of apoptin in the nucleus of tumor cells has been shown to be vital for proapoptotic activity, however, targeted expression of apoptin in the nucleus of normal cells does not harm the cells, indicating that nuclear localization of apoptin is insufficient for its cytotoxicity. Here, we demonstrate for the first time that apoptin interacts with the SH3 domain of p85, the regulatory subunit of phosphoinositide 3-kinase (PI3-K), through its proline-rich region. Apoptin derivatives devoid of this proline-rich region do not interact with p85, are unable to activate PI3-K, and show impaired apoptosis induction. Moreover, apoptin mutants containing the proline-rich domain are sufficient to elevate PI3-K activity and to induce apoptosis in cancer cells. Downregulation of p85 leads to nuclear exclusion of apoptin and impairs cell death induction, indicating that interaction with the p85 PI3-K subunit essentially contributes to the cytotoxic activity of apoptin. PMID:18059340

  12. Contribution of temperament to eating disorder symptoms in emerging adulthood: Additive and interactive effects.

    PubMed

    Burt, Nicole M; Boddy, Lauren E; Bridgett, David J

    2015-08-01

    Temperament characteristics, such as higher negative emotionality (NE) and lower effortful control (EC), are individual difference risk factors for developmental psychopathology. Research has also noted relations between temperament and more specific manifestations of psychopathology, such as eating disorders (EDs). Although work is emerging that indicates that NE and EC may additively contribute to risk for ED symptoms, no studies have considered the interactive effects of NE and EC in relation to ED symptoms. In the current investigation, we hypothesized that (1) low EC would be associated with increased ED symptoms, (2) high NE would be associated with increased ED symptoms, and (3) these temperament traits would interact, such that the relationship between NE and ED symptoms would be strongest in the presence of low EC. After controlling for gender and child trauma history, emerging adults' (N=160) lower EC (i.e., more difficulties with self-regulation) was associated with more ED symptoms. NE did not emerge as a direct predictor of ED symptoms. However, the anticipated interaction of these temperament characteristics on ED symptoms was found. The association between NE and ED symptoms was only significant in the context of low EC. These findings provide evidence that elevated NE may only be a risk factor for the development of eating disorders when individuals also have self-regulation difficulties. The implications of these findings for research and interventions are discussed. PMID:25875113

  13. Contribution of Trimeric Autotransporter C-Terminal Domains of Oligomeric Coiled-Coil Adhesin (Oca) Family Members YadA, UspA1, EibA, and Hia to Translocation of the YadA Passenger Domain and Virulence of Yersinia enterocolitica▿

    PubMed Central

    Ackermann, Nikolaus; Tiller, Maximilian; Anding, Gisela; Roggenkamp, Andreas; Heesemann, Jürgen

    2008-01-01

    The Oca family is a novel class of autotransporter-adhesins with highest structural similarity in their C-terminal transmembrane region, which supposedly builds a beta-barrel pore in the outer membrane (OM). The prototype of the Oca family is YadA, an adhesin of Yersinia enterocolitica and Yersinia pseudotuberculosis. YadA forms a homotrimeric lollipop-like structure on the bacterial surface. The C-terminal regions of three YadA monomers form a barrel in the OM and translocate the trimeric N-terminal passenger domain, consisting of stalk, neck, and head region to the exterior. To elucidate the structural and functional role of the C-terminal translocator domain (TLD) and to assess its promiscuous capability with respect to transport of related passenger domains, we constructed chimeric YadA proteins, which consist of the N-terminal YadA passenger domain and C-terminal TLDs of Oca family members UspA1 (Moraxella catarrhalis), EibA (Escherichia coli), and Hia (Haemophilus influenzae). These constructs were expressed in Y. enterocolitica and compared for OM localization, surface exposure, oligomerization, adhesion properties, serum resistance, and mouse virulence. We demonstrate that all chimeric YadA proteins translocated the YadA passenger domain across the OM. Y. enterocolitica strains producing YadA chimeras or wild-type YadA showed comparable binding to collagen and epithelial cells. However, strains producing YadA chimeras were attenuated in serum resistance and mouse virulence. These results demonstrate for the first time that TLDs of Oca proteins of different origin are efficient translocators of the YadA passenger domain and that the cognate TLD of YadA is essential for bacterial survival in human serum and mouse virulence. PMID:18487327

  14. Interaction between sphingomyelin and oxysterols contributes to atherosclerosis and sudden death

    PubMed Central

    Kummerow, Fred A

    2013-01-01

    Despite major public health efforts, coronary heart disease continues to be the leading cause of death in the United States. Oxidized lipids contribute to heart disease both by increasing deposition of calcium on the arterial wall, a major hallmark of atherosclerosis, and by interrupting blood flow, a major contributor to heart attack and sudden death. Oxidized cholesterol (oxysterols) enhances the production of sphingomyelin, a phospholipid found in the cellular membranes of the coronary artery. This increases the sphingomyelin content in the cell membrane, which in turn enhances the interaction between the membrane and ionic calcium (Ca2+), thereby increasing the risk of arterial calcification. Patients undergoing bypass surgery had greater concentrations of oxysterols in their plasma than cardiac catheterized controls with no stenosis, and had five times more sphingomyelin in their arteries than in the artery of the placenta of a newborn. The oxysterols found in the plasma of these patients were also found in the plasma of rabbits that had been fed oxidized cholesterol and in frying fats and powdered egg yolk intended for human consumption. Together these findings suggest that oxysterols found in the diet are absorbed and contribute to arterial calcification. Oxidized low-density lipoprotein (OxLDL) further contributes to heart disease by increasing the synthesis of thromboxane in platelets, which increases blood clotting. Cigarette smoke and trans fatty acids, found in partially hydrogenated soybean oil, both inhibit the synthesis of prostacyclin, which inhibits blood clotting. By increasing the ratio of thromboxane to prostacyclin, these factors interact to interrupt blood flow, thereby contributing to heart attack and sudden death. Levels of oxysterols and OxLDL increase primarily as a result of three diet or lifestyle factors: the consumption of oxysterols from commercially fried foods such as fried chicken, fish, and french fries; oxidation of cholesterol

  15. A direct interaction between fascin and microtubules contributes to adhesion dynamics and cell migration

    PubMed Central

    Villari, Giulia; Jayo, Asier; Zanet, Jennifer; Fitch, Briana; Serrels, Bryan; Frame, Margaret; Stramer, Brian M.; Goult, Benjamin T.; Parsons, Maddy

    2015-01-01

    ABSTRACT Fascin is an actin-binding and bundling protein that is highly upregulated in most epithelial cancers. Fascin promotes cell migration and adhesion dynamics in vitro and tumour cell metastasis in vivo. However, potential non-actin bundling roles for fascin remain unknown. Here, we show for the first time that fascin can directly interact with the microtubule cytoskeleton and that this does not depend upon fascin-actin bundling. Microtubule binding contributes to fascin-dependent control of focal adhesion dynamics and cell migration speed. We also show that fascin forms a complex with focal adhesion kinase (FAK, also known as PTK2) and Src, and that this signalling pathway lies downstream of fascin–microtubule association in the control of adhesion stability. These findings shed light on new non actin-dependent roles for fascin and might have implications for the design of therapies to target fascin in metastatic disease. PMID:26542021

  16. Cell interactions and genetic regulation that contribute to testicular Leydig cell development and differentiation.

    PubMed

    Martin, Luc J

    2016-06-01

    Leydig cells, located within the interstitial compartment of the testis, are major contributors of androgen synthesis and secretion, which play an important role in testis development, normal masculinization, maintenance of spermatogenesis, and general male fertility. Accordingly, dysfunction of Leydig cells may lead to various male reproductive maladies, including primary hypogonadism, cryptorchidism, and hypospadias. A better understanding of how cell interactions and gene regulation contribute to testicular Leydig cell development and differentiation may therefore help limit the incidence of such male reproductive pathologies. Several hormones and signaling molecules have been identified as important regulators of Leydig cell differentiation and function. Recent work on the regulation of testis development, especially of Leydig cells, has focused on the Desert hedgehog and platelet-derived growth factor signaling pathways. This review outlines recent findings regarding cell interactions and gene regulation involved in the development and regulation of fetal and adult Leydig cell populations. Mol. Reprod. Dev. 83: 470-487, 2016. © 2016 Wiley Periodicals, Inc. PMID:27079813

  17. Biochemical and Genetic Evidence for a SAP-PKC-θ Interaction Contributing to IL-4 Regulation

    PubMed Central

    Cannons, Jennifer L.; Wu, Julie Z.; Gomez-Rodriguez, Julio; Zhang, Jinyi; Dong, Baoxia; Liu, Yin; Shaw, Stephen; Siminovitch, Katherine A.; Schwartzberg, Pamela L.

    2012-01-01

    SAP, an adaptor molecule that recruits Fyn to the SLAM-family of immunomodulatory receptors, is mutated in X-linked lymphoproliferative disease. CD4+ T cells from SAP-deficient mice have defective TCR-induced IL-4 production and impaired T cell-mediated help for germinal center formation; however, the downstream intermediates contributing to these defects remain unclear. We previously found that SAP-deficient CD4+ T cells exhibit decreased PKC-θ recruitment upon TCR stimulation. We demonstrate here using GST-pulldowns and co-immunoprecipitation studies that SAP constitutively associates with PKC-θ in T cells. SAP-PKC-θ interactions required R78 of SAP, a residue previously implicated in Fyn recruitment, yet SAP’s interactions with PKC-θ occurred independent of phosphotyrosine binding and Fyn. Overexpression of SAP in T cells increased and sustained PKC-θ recruitment to the immune synapse and elevated IL-4 production in response to TCR plus SLAM-mediated stimulation. Moreover, PKC-θ, like SAP, was required for SLAM-mediated increases in IL-4 production and conversely, membrane-targeted PKC-θ mutants rescued IL-4 expression in SAP−/− CD4+ T cells, providing genetic evidence that PKC-θ is a critical component of SLAM/SAP-mediated pathways that influence TCR-driven IL-4 production. PMID:20668219

  18. Maternal Dispositional Empathy and Electrodermal Reactivity: Interactive Contributions to Maternal Sensitivity with Toddler-Aged Children

    PubMed Central

    Emery, Helen T.; McElwain, Nancy L.; Groh, Ashley M.; Haydon, Katherine C.; Roisman, Glenn I.

    2015-01-01

    The present study investigated maternal dispositional empathy and skin conductance level (SCL) reactivity to infant emotional cues as joint predictors of maternal sensitivity. Sixty-four mother-toddler dyads (31 boys) were observed across a series of interaction tasks during a laboratory visit, and maternal sensitivity was coded from approximately 55 minutes of observation per family. In a second, mother-only laboratory visit, maternal SCL reactivity to infant cues was assessed using a cry-laugh audio paradigm. Mothers reported on their dispositional empathy via a questionnaire. As hypothesized, mothers with greater dispositional empathy exhibited more sensitive behavior at low, but not high, levels of SCL reactivity to infant cues. Analyses examining self-reported emotional reactivity to the cry-laugh audio paradigm yielded a similar finding: dispositional empathy was related to greater sensitivity when mothers reported low, but not high, negative emotional reactivity. Results provide support for Dix’s (1991) affective model of parenting that underscores the combined contribution of the parent’s empathic tendencies and his/her own emotional experience in response to child emotions. Specificity of the Empathy × Reactivity interaction is discussed with respect to the context in which reactivity was assessed (infant cry versus laugh) and the type of sensitivity examined (sensitivity to the child’s distress versus non-distress). PMID:24955589

  19. Contributions of host cellular trafficking and organization to the outcomes of plant-pathogen interactions.

    PubMed

    Underwood, William

    2016-08-01

    In recent years it has become increasingly apparent that dynamic changes in protein localization, membrane trafficking pathways, and cellular organization play a major role in determining the outcome of interactions between plants and pathogenic microorganisms. Plants have evolved sophisticated perception systems to recognize the presence of potentially pathogenic microorganisms via the detection of non-self or modified-self elicitor molecules, pathogen virulence factors, or the activities of such virulence factors. Dynamic changes to host cellular organization and membrane trafficking pathways play pivotal roles in detection and signaling by plant immune receptors and are vital for the execution of spatially targeted defense responses to thwart invasion by potential pathogens. Conversely, from the perspective of the pathogen, the ability to manipulate plant cellular organization and trafficking processes to establish infection structures and deliver virulence factors is a major determinant of pathogen success. This review summarizes selected topics that illustrate how dynamic changes in host cellular trafficking and organization shape the outcomes of diverse plant-pathogen interactions and addresses ways in which our rapidly expanding knowledge of the cell biological processes that contribute to immunity or infection may influence efforts to improve plant disease resistance. PMID:27216829

  20. Tumor loci and their interactions on mouse chromosome 19 that contribute to testicular germ cell tumors

    PubMed Central

    2014-01-01

    Background Complex genetic factors underlie testicular germ cell tumor (TGCT) development. One experimental approach to dissect the genetics of TGCT predisposition is to use chromosome substitution strains, such as the 129.MOLF-Chr 19 (M19). M19 carries chromosome (Chr) 19 from the MOLF whereas all other chromosomes are from the 129 strain. 71% of M19 males develop TGCTs in contrast to 5% in 129 strain. To identify and map tumor loci from M19 we generated congenic strains harboring MOLF chromosome 19 segments on 129 strain background and monitored their TGCT incidence. Results We found 3 congenic strains that each harbored tumor promoting loci that had high (14%-32%) whereas 2 other congenics had low (4%) TGCT incidences. To determine how multiple loci influence TGCT development, we created double and triple congenic strains. We found additive interactions were predominant when 2 loci were combined in double congenic strains. Surprisingly, we found an example where 2 loci, both which do not contribute significantly to TGCT, when combined in a double congenic strain resulted in greater than expected TGCT incidence (positive interaction). In an opposite example, when 2 loci with high TGCT incidences were combined, males of the double congenic showed lower than expected TGCT incidence (negative interaction). For the triple congenic strain, depending on the analysis, the overall TGCT incidence could be additive or could also be due to a positive interaction of one region with others. Additionally, we identified loci that promote bilateral tumors or testicular abnormalities. Conclusions The congenic strains each with their characteristic TGCT incidences, laterality of tumors and incidence of testicular abnormalities, are useful for identification of TGCT susceptibility modifier genes that map to Chr 19 and also for studies on the genetic and environmental causes of TGCT development. TGCTs are a consequence of aberrant germ cell and testis development. By defining

  1. Interactions of the Gasotransmitters Contribute to Microvascular Tone (Dys)regulation in the Preterm Neonate

    PubMed Central

    Dyson, Rebecca M.; Palliser, Hannah K.; Latter, Joanna L.; Kelly, Megan A.; Chwatko, Grazyna; Glowacki, Rafal; Wright, Ian M. R.

    2015-01-01

    Background & Aims Hydrogen sulphide (H2S), nitric oxide (NO), and carbon monoxide (CO) are involved in transitional microvascular tone dysregulation in the preterm infant; however there is conflicting evidence on the interaction of these gasotransmitters, and their overall contribution to the microcirculation in newborns is not known. The aim of this study was to measure the levels of all 3 gasotransmitters, characterise their interrelationships and elucidate their combined effects on microvascular blood flow. Methods 90 preterm neonates were studied at 24h postnatal age. Microvascular studies were performed by laser Doppler. Arterial COHb levels (a measure of CO) were determined through co-oximetry. NO was measured as nitrate and nitrite in urine. H2S was measured as thiosulphate by liquid chromatography. Relationships between levels of the gasotransmitters and microvascular blood flow were assessed through partial correlation controlling for the influence of gestational age. Structural equation modelling was used to examine the combination of these effects on microvascular blood flow and derive a theoretical model of their interactions. Results No relationship was observed between NO and CO (p = 0.18, r = 0.18). A positive relationship between NO and H2S (p = 0.008, r = 0.28) and an inverse relationship between CO and H2S (p = 0.01, r = -0.33) exists. Structural equation modelling was used to examine the combination of these effects on microvascular blood flow. The model with the best fit is presented. Conclusions The relationships between NO and H2S, and CO and H2S may be of importance in the preterm newborn, particularly as NO levels in males are associated with higher H2S levels and higher microvascular blood flow and CO in females appears to convey protection against vascular dysregulation. Here we present a theoretical model of these interactions and their overall effects on microvascular flow in the preterm newborn, upon which future mechanistic studies may

  2. Interaction of Mycoplasma gallisepticum with Chicken Tracheal Epithelial Cells Contributes to Macrophage Chemotaxis and Activation

    PubMed Central

    Majumder, Sanjukta

    2015-01-01

    Mycoplasma gallisepticum colonizes the chicken respiratory mucosa and mediates a severe inflammatory response hallmarked by subepithelial leukocyte infiltration. We recently reported that the interaction of M. gallisepticum with chicken tracheal epithelial cells (TECs) mediated the upregulation of chemokine and inflammatory cytokine genes in these cells (S. Majumder, F. Zappulla, and L. K. Silbart, PLoS One 9:e112796, http://dx.doi.org/10.1371/journal.pone.0112796). The current study extends these observations and sheds light on how this initial interaction may give rise to subsequent inflammatory events. Conditioned medium from TECs exposed to the virulent Rlow strain induced macrophage chemotaxis to a much higher degree than the nonvirulent Rhigh strain. Coculture of chicken macrophages (HD-11) with TECs exposed to live mycoplasma revealed the upregulation of several proinflammatory genes associated with macrophage activation, including interleukin-1β (IL-1β), IL-6, IL-8, CCL20, macrophage inflammatory protein 1β (MIP-1β), CXCL-13, and RANTES. The upregulation of these genes was similar to that observed upon direct contact of HD-11 cells with live M. gallisepticum. Coculture of macrophages with Rlow-exposed TECs also resulted in prolonged expression of chemokine genes, such as those encoding CXCL-13, MIP-1β, RANTES, and IL-8. Taken together, these studies support the notion that the initial interaction of M. gallisepticum with host respiratory epithelial cells contributes to macrophage chemotaxis and activation by virtue of robust upregulation of inflammatory cytokine and chemokine genes, thereby setting the stage for chronic tissue inflammation. PMID:26527215

  3. Interaction of Mycoplasma gallisepticum with Chicken Tracheal Epithelial Cells Contributes to Macrophage Chemotaxis and Activation.

    PubMed

    Majumder, Sanjukta; Silbart, Lawrence K

    2016-01-01

    Mycoplasma gallisepticum colonizes the chicken respiratory mucosa and mediates a severe inflammatory response hallmarked by subepithelial leukocyte infiltration. We recently reported that the interaction of M. gallisepticum with chicken tracheal epithelial cells (TECs) mediated the upregulation of chemokine and inflammatory cytokine genes in these cells (S. Majumder, F. Zappulla, and L. K. Silbart, PLoS One 9:e112796, http://dx.doi.org/10.1371/journal.pone.0112796). The current study extends these observations and sheds light on how this initial interaction may give rise to subsequent inflammatory events. Conditioned medium from TECs exposed to the virulent Rlow strain induced macrophage chemotaxis to a much higher degree than the nonvirulent Rhigh strain. Coculture of chicken macrophages (HD-11) with TECs exposed to live mycoplasma revealed the upregulation of several proinflammatory genes associated with macrophage activation, including interleukin-1β (IL-1β), IL-6, IL-8, CCL20, macrophage inflammatory protein 1β (MIP-1β), CXCL-13, and RANTES. The upregulation of these genes was similar to that observed upon direct contact of HD-11 cells with live M. gallisepticum. Coculture of macrophages with Rlow-exposed TECs also resulted in prolonged expression of chemokine genes, such as those encoding CXCL-13, MIP-1β, RANTES, and IL-8. Taken together, these studies support the notion that the initial interaction of M. gallisepticum with host respiratory epithelial cells contributes to macrophage chemotaxis and activation by virtue of robust upregulation of inflammatory cytokine and chemokine genes, thereby setting the stage for chronic tissue inflammation. PMID:26527215

  4. ARG1 (altered response to gravity) encodes a DnaJ-like protein that potentially interacts with the cytoskeleton

    NASA Technical Reports Server (NTRS)

    Sedbrook, J. C.; Chen, R.; Masson, P. H.

    1999-01-01

    Gravitropism allows plant organs to direct their growth at a specific angle from the gravity vector, promoting upward growth for shoots and downward growth for roots. Little is known about the mechanisms underlying gravitropic signal transduction. We found that mutations in the ARG1 locus of Arabidopsis thaliana alter root and hypocotyl gravitropism without affecting phototropism, root growth responses to phytohormones or inhibitors of auxin transport, or starch accumulation. The positional cloning of ARG1 revealed a DnaJ-like protein containing a coiled-coil region homologous to coiled coils found in cytoskeleton-interacting proteins. These data suggest that ARG1 participates in a gravity-signaling process involving the cytoskeleton. A combination of Northern blot studies and analysis of ARG1-GUS fusion-reporter expression in transgenic plants demonstrated that ARG1 is expressed in all organs. Ubiquitous ARG1 expression in Arabidopsis and the identification of an ortholog in Caenorhabditis elegans suggest that ARG1 is involved in other essential processes.

  5. Grain setting defect1 (GSD1) function in rice depends on S-acylation and interacts with actin 1 (OsACT1) at its C-terminal

    PubMed Central

    Gui, Jinshan; Zheng, Shuai; Shen, Junhui; Li, Laigeng

    2015-01-01

    Grain setting defect1 (GSD1), a plant-specific remorin protein specifically localized at the plasma membrane (PM) and plasmodesmata of phloem companion cells, affects grain setting in rice through regulating the transport of photoassimilates. Here, we show new evidence demonstrating that GSD1 is localized at the cytoplasmic face of the PM and a stretch of 45 amino acid residues at its C-terminal is required for its localization. Association with the PM is mediated by S-acylation of cysteine residues Cys-524 and Cys-527, in a sequence of 45 amino acid residues essential for GSD1 function in rice. Furthermore, the coiled-coil domain in GSD1 is necessary for sufficient interaction with OsACT1. Together, these results reveal that GSD1 attaches to the PM through S-acylation and interacts with OsACT1 through its coiled-coil domain structure to regulate plasmodesmata conductance for photoassimilate transport in rice. PMID:26483819

  6. Complex T cell interactions contribute to Helicobacter pylori gastritis in mice.

    PubMed

    Gray, Brian M; Fontaine, Clinton A; Poe, Sara A; Eaton, Kathryn A

    2013-03-01

    Disease due to the gastric pathogen Helicobacter pylori varies in severity from asymptomatic to peptic ulcer disease and cancer. Accumulating evidence suggests that one source of this variation is an abnormal host response. The goal of this study was to use a mouse model of H. pylori gastritis to investigate the roles of regulatory T cells (Treg) as well as proinflammatory T cells (Th1 and Th17) in gastritis, gastric T cell engraftment, and gastric cytokine production. Our results support published data indicating that severe gastritis in T cell recipient mice is due to failure of Treg engraftment, that Treg ameliorate gastritis, and that the proinflammatory response is attributable to interactions between several cell subsets and cytokines. We confirmed that gamma interferon (IFN-γ) is essential for induction of gastritis but showed that IFN-γ-producing CD4 T cells are not necessary. Interleukin 17A (IL-17A) also contributed to gastritis, but to a lesser extent than IFN-γ. Tumor necrosis factor alpha (TNF-α) and IL-17F were also elevated in association with disease. These results indicate that while H. pylori-specific CD4(+) T cells and IFN-γ are both essential for induction of gastritis due to H. pylori, IFN-γ production by T cells is not essential. It is likely that other proinflammatory cytokines, such as IL-17F and TNF-α, shown to be elevated in this model, also contribute to the induction of disease. We suggest that gastritis due to H. pylori is associated with loss of immunoregulation and alteration of several cytokines and cell subsets and cannot be attributed to a single immune pathway. PMID:23264048

  7. Parent-Child Interaction and Children's Peer Relationships: The Differential Contributions of Mothers and Fathers to Children's Dyadic Peer Interactions.

    ERIC Educational Resources Information Center

    Kahen, Vanessa J.

    This study examined how parents' engagement and affect during parent-child interaction related to children's ability to successfully interact with peers. Subjects were 56 families, each with a child between the ages of 4 and 6. Ten-minute interactions were videotaped as parents obtained information about a story previously heard by the child, and…

  8. Assessment of odor activity value coefficient and odor contribution based on binary interaction effects in waste disposal plant

    NASA Astrophysics Data System (ADS)

    Wu, Chuandong; Liu, Jiemin; Yan, Luchun; Chen, Haiying; Shao, Huiqi; Meng, Tian

    2015-02-01

    Odor activity value (OAV) has been widely used for the assessment of odor pollution from various sources. However, little attention has been paid to the extreme OAV variation and potential inaccuracies of odor contribution assessment caused by odor interaction effects. The objective of this study is to assess the odor interaction effect for precise assessment of odor contribution. In this paper, samples were collected from a food waste disposal plant, and analyzed by instrumental and olfactory method to conclude odorants' occurrence and OAV. Then odor activity value coefficient (γ) was first proposed to evaluate the type and the level of binary interaction effects based on determination of OAV variation. By multiplying OAV and γ, odor activity factor (OAF) was used to reflect the real OAV. Correlation between the sum of OAF and odor concentration reached 80.0 ± 5.7%, which was 10 times higher than the sum of OAV used before. Results showed that hydrogen sulfide contributed most (annual average 66.4 ± 15.8%) to odor pollution in the waste disposal plant. However, as odor intensity of samples in summer rising, odor contribution of trimethylamine increased to 48.3 ± 3.7% by the strong synergistic interaction effect, while odor contribution of phenol decreased to 0.1 ± 0.02% for the increasing antagonistic interaction effect.

  9. Interaction contributions in thermal conductivity of three-dimensional complex liquids

    NASA Astrophysics Data System (ADS)

    Shahzad, Aamir; He, Mao-Gang

    2013-07-01

    This work generalizes Evans' homogenous nonequilibrium molecular dynamics (HNEMD) algorithm for computing the thermal conductivity of strongly-coupled complex (dusty) plasma liquids (SCCDPLs) described by the Yukawa potential. The effects of external field strength along with different screening strengths on the conductivity of Yukawa liquids have investigated using HNEMD simulations. We have carried out some more linear and nonlinear molecular dynamics calculations of the thermal conductivity, and the obtained simulation results of SCCDPLs are presented for various plasma coupling and screening parameters. Our calculations show that Yukawa liquid exhibits a non-Newtonian behavior that the thermal conductivity increases with increasing field strength which explains interaction contributions in Yukawa conductivity, for the first time. The simulation results obtained with different external filed strengths are in reasonable agreement with earlier simulation results and with reference set of data showed deviations within less than ±10% for most of the present data point. It is shown that new simulations extended the range of field strength (0.001≤F*≤0.1) used in the earlier studies in order to find out the size of the linear regimes and to explain the nature of nonlinearity of SCCDPLs.

  10. Interacting vegetative and thermal contributions to water movement in desert soil

    USGS Publications Warehouse

    Garcia, C.A.; Andraski, B.J.; Stonestrom, D.A.; Cooper, C.A.; Simunek, J.; Wheatcraft, S.W.

    2011-01-01

    Thermally driven water-vapor flow can be an important component of total water movement in bare soil and in deep unsaturated zones, but this process is often neglected when considering the effects of soil-plant-atmosphere interactions on shallow water movement. The objectives of this study were to evaluate the coupled and separate effects of vegetative and thermal-gradient contributions to soil water movement in desert environments. The evaluation was done by comparing a series of simulations with and without vegetation and thermal forcing during a 4.7-yr period (May 2001-December 2005). For vegetated soil, evapotranspiration alone reduced root-zone (upper 1 m) moisture to a minimum value (25 mm) each year under both isothermal and nonisothermal conditions. Variations in the leaf area index altered the minimum storage values by up to 10 mm. For unvegetated isothermal and nonisothermal simulations, root-zone water storage nearly doubled during the simulation period and created a persistent driving force for downward liquid fluxes below the root zone (total net flux ~1 mm). Total soil water movement during the study period was dominated by thermally driven vapor fluxes. Thermally driven vapor flow and condensation supplemented moisture supplies to plant roots during the driest times of each year. The results show how nonisothermal flow is coupled with plant water uptake, potentially influencing ecohydrologic relations in desert environments. ?? Soil Science Society of America 5585 Guilford Rd., Madison, WI 53711 USA. All rights reserved.

  11. Interacting vegetative and thermal contributions to water movement in desert soil

    USGS Publications Warehouse

    Garcia, C.A.; Andraski, B.J.; Stonestrom, D.A.; Cooper, C.A.; Šimůnek, J.; Wheatcraft, S.W.

    2011-01-01

    Thermally driven water-vapor flow can be an important component of total water movement in bare soil and in deep unsaturated zones, but this process is often neglected when considering the effects of soil–plant–atmosphere interactions on shallow water movement. The objectives of this study were to evaluate the coupled and separate effects of vegetative and thermal-gradient contributions to soil water movement in desert environments. The evaluation was done by comparing a series of simulations with and without vegetation and thermal forcing during a 4.7-yr period (May 2001–December 2005). For vegetated soil, evapotranspiration alone reduced root-zone (upper 1 m) moisture to a minimum value (25 mm) each year under both isothermal and nonisothermal conditions. Variations in the leaf area index altered the minimum storage values by up to 10 mm. For unvegetated isothermal and nonisothermal simulations, root-zone water storage nearly doubled during the simulation period and created a persistent driving force for downward liquid fluxes below the root zone (total net flux ~1 mm). Total soil water movement during the study period was dominated by thermally driven vapor fluxes. Thermally driven vapor flow and condensation supplemented moisture supplies to plant roots during the driest times of each year. The results show how nonisothermal flow is coupled with plant water uptake, potentially influencing ecohydrologic relations in desert environments.

  12. Evaluating contributions of natural language parsers to protein–protein interaction extraction

    PubMed Central

    Miyao, Yusuke; Sagae, Kenji; Sætre, Rune; Matsuzaki, Takuya; Tsujii, Jun'ichi

    2009-01-01

    Motivation: While text mining technologies for biomedical research have gained popularity as a way to take advantage of the explosive growth of information in text form in biomedical papers, selecting appropriate natural language processing (NLP) tools is still difficult for researchers who are not familiar with recent advances in NLP. This article provides a comparative evaluation of several state-of-the-art natural language parsers, focusing on the task of extracting protein–protein interaction (PPI) from biomedical papers. We measure how each parser, and its output representation, contributes to accuracy improvement when the parser is used as a component in a PPI system. Results: All the parsers attained improvements in accuracy of PPI extraction. The levels of accuracy obtained with these different parsers vary slightly, while differences in parsing speed are larger. The best accuracy in this work was obtained when we combined Miyao and Tsujii's Enju parser and Charniak and Johnson's reranking parser, and the accuracy is better than the state-of-the-art results on the same data. Availability: The PPI extraction system used in this work (AkanePPI) is available online at http://www-tsujii.is.s.u-tokyo.ac.jp/-100downloads/downloads.cgi. The evaluated parsers are also available online from each developer's site. Contact: yusuke@is.s.u-tokyo.ac.jp PMID:19073593

  13. Intermonomer Interactions in Hemagglutinin Subunits HA1 and HA2 Affecting Hemagglutinin Stability and Influenza Virus Infectivity

    PubMed Central

    DeFeo, Christopher J.; Alvarado-Facundo, Esmeralda; Vassell, Russell

    2015-01-01

    ABSTRACT Influenza virus hemagglutinin (HA) mediates virus entry by binding to cell surface receptors and fusing the viral and endosomal membranes following uptake by endocytosis. The acidic environment of endosomes triggers a large-scale conformational change in the transmembrane subunit of HA (HA2) involving a loop (B loop)-to-helix transition, which releases the fusion peptide at the HA2 N terminus from an interior pocket within the HA trimer. Subsequent insertion of the fusion peptide into the endosomal membrane initiates fusion. The acid stability of HA is influenced by residues in the fusion peptide, fusion peptide pocket, coiled-coil regions of HA2, and interactions between the surface (HA1) and HA2 subunits, but details are not fully understood and vary among strains. Current evidence suggests that the HA from the circulating pandemic 2009 H1N1 influenza A virus [A(H1N1)pdm09] is less stable than the HAs from other seasonal influenza virus strains. Here we show that residue 205 in HA1 and residue 399 in the B loop of HA2 (residue 72, HA2 numbering) in different monomers of the trimeric A(H1N1)pdm09 HA are involved in functionally important intermolecular interactions and that a conserved histidine in this pair helps regulate HA stability. An arginine-lysine pair at this location destabilizes HA at acidic pH and mediates fusion at a higher pH, while a glutamate-lysine pair enhances HA stability and requires a lower pH to induce fusion. Our findings identify key residues in HA1 and HA2 that interact to help regulate H1N1 HA stability and virus infectivity. IMPORTANCE Influenza virus hemagglutinin (HA) is the principal antigen in inactivated influenza vaccines and the target of protective antibodies. However, the influenza A virus HA is highly variable, necessitating frequent vaccine changes to match circulating strains. Sequence changes in HA affect not only antigenicity but also HA stability, which has important implications for vaccine production, as well

  14. Predicting Children's Interactions with Unfamiliar Peers: Contributions of Parent-Child Interaction Style and Child Individual Behavior.

    ERIC Educational Resources Information Center

    Carrillo, Sonia; And Others

    This study examined children's play interaction styles with unfamiliar peers; used mother-child and father-child dyadic qualities independently to predict children's social behavior; determined the relationship between children's individual behaviors and peer dyadic characteristics; and compared mother-child and father-child interactions on both…

  15. Dimerization is required for SH3PX1 tyrosine phosphorylation in response to epidermal growth factor signalling and interaction with ACK2

    PubMed Central

    Childress, Chandra; Lin, Qiong; Yang, Wannian

    2005-01-01

    SH3PX1 [SNX9 (sorting nexin 9)] is a member of SNX super-family that is recognized by sharing a PX (phox homology) domain. We have previously shown that SH3PX1, phosphorylated by ACK2 (activated Cdc42-associated tyrosine kinase 2), regulates the degradation of EGF (epidermal growth factor) receptor. In mapping the tyrosine phosphorylation region, we found that the C-terminus of SH3PX1 is required for its tyrosine phosphorylation. Further analysis indicates that this region, known as the coiled-coil domain or the BAR (Bin–amphiphysin–Rvs homology) domain, is the dimerization domain of SH3PX1. Truncation of as little as 13 amino acid residues at the very C-terminus in the coiled-coil/BAR domain of SH3PX1 resulted in no dimerization, no ACK2-catalysed and EGF-stimulated tyrosine phosphorylation and no interaction with ACK2. The intracellular localization of SH3PX1 became dysfunctional upon truncation in the BAR domain. Taken together, our results indicate that the dimerization, which is mediated by the BAR domain, is essential for the intracellular function of SH3PX1. PMID:16316319

  16. Dimerization is required for SH3PX1 tyrosine phosphorylation in response to epidermal growth factor signalling and interaction with ACK2.

    PubMed

    Childress, Chandra; Lin, Qiong; Yang, Wannian

    2006-03-15

    SH3PX1 [SNX9 (sorting nexin 9)] is a member of SNX super-family that is recognized by sharing a PX (phox homology) domain. We have previously shown that SH3PX1, phosphorylated by ACK2 (activated Cdc42-associated tyrosine kinase 2), regulates the degradation of EGF (epidermal growth factor) receptor. In mapping the tyrosine phosphorylation region, we found that the C-terminus of SH3PX1 is required for its tyrosine phosphorylation. Further analysis indicates that this region, known as the coiled-coil domain or the BAR (Bin-amphiphysin-Rvs homology) domain, is the dimerization domain of SH3PX1. Truncation of as little as 13 amino acid residues at the very C-terminus in the coiled-coil/BAR domain of SH3PX1 resulted in no dimerization, no ACK2-catalysed and EGF-stimulated tyrosine phosphorylation and no interaction with ACK2. The intracellular localization of SH3PX1 became dysfunctional upon truncation in the BAR domain. Taken together, our results indicate that the dimerization, which is mediated by the BAR domain, is essential for the intracellular function of SH3PX1. PMID:16316319

  17. Mortality from desiccation contributes to a genotype–temperature interaction for cold survival in Drosophila melanogaster

    PubMed Central

    Kobey, Robert L.; Montooth, Kristi L.

    2013-01-01

    SUMMARY Survival at cold temperatures is a complex trait, primarily because of the fact that the physiological cause of injury may differ across degrees of cold exposure experienced within the lifetime of an ectothermic individual. In order to better understand how chill-sensitive insects experience and adapt to low temperatures, we investigated the physiological basis for cold survival across a range of temperature exposures from −4 to 6°C in five genetic lines of the fruit fly Drosophila melanogaster. Genetic effects on cold survival were temperature dependent and resulted in a significant genotype–temperature interaction for survival across cold temperature exposures that differ by as little as 2°C. We investigated desiccation as a potential mechanism of injury across these temperature exposures. Flies were dehydrated following exposures near 6°C, whereas flies were not dehydrated following exposures near −4°C. Furthermore, decreasing humidity during cold exposure decreased survival, and increasing humidity during cold exposure increased survival at 6°C, but not at −4°C. These results support the conclusion that in D. melanogaster there are multiple physiological mechanisms of cold-induced mortality across relatively small differences in temperature, and that desiccation contributes to mortality for exposures near 6°C but not for subzero temperatures. Because D. melanogaster has recently expanded its range from tropical to temperate latitudes, the complex physiologies underlying cold tolerance are likely to be important traits in the recent evolutionary history of this fruit fly. PMID:23197100

  18. Modular unfolding and dissociation of the human respiratory syncytial virus phosphoprotein p and its interaction with the m(2-1) antiterminator: a singular tetramer-tetramer interface arrangement.

    PubMed

    Esperante, Sebastián A; Paris, Gastón; de Prat-Gay, Gonzalo

    2012-10-16

    Paramyxoviruses share the essential RNA polymerase complex components, namely, the polymerase (L), phosphoprotein (P), and nucleoprotein (N). Human respiratory syncytial virus (RSV) P is the smallest polypeptide among the family, sharing a coiled coil tetramerization domain, which disruption renders the virus inactive. We show that unfolding of P displays a first transition with low cooperativity but substantial loss of α-helix content and accessibility to hydrophobic sites, indicative of loose chain packing and fluctuating tertiary structure, typical of molten globules. The lack of unfolding baseline indicates a native state in conformational exchange and metastable at 20 °C. The second transition starts from a true intermediate state, with only the tetramerization domain remaining folded. The tetramerization domain undergoes a two-state dissociation/unfolding reaction (37.3 kcal mol(-1)). The M(2-1) transcription antiterminator, unique to RSV and Metapneumovirus, forms a nonglobular P:M(2-1) complex with a 1:1 stoichiometry and a K(D) of 8.1 nM determined by fluorescence anisotropy, far from the strikingly coincident dissociation range of P and M(2-1) tetramers (10(-28) M(3)). The M(2-1) binding region has been previously mapped to the N-terminal module of P, strongly suggesting the latter as the metastable molten globule domain. Folding, oligomerization, and assembly events between proteins and with RNA are coupled in the RNA polymerase complex. Quantitative assessment of the hierarchy of these interactions and their mechanisms contribute to the general understanding of RNA replication and transcription in Paramyxoviruses. In particular, the unique P-M(2-1) interface present in RSV provides a valuable antiviral target for this worldwide spread human pathogen. PMID:22978633

  19. The kinesin-1 motor protein is regulated by a direct interaction of its head and tail

    PubMed Central

    Dietrich, Kristen A.; Sindelar, Charles V.; Brewer, Paul D.; Downing, Kenneth H.; Cremo, Christine R.; Rice, Sarah E.

    2008-01-01

    Kinesin-1 is a molecular motor protein that transports cargo along microtubules. Inside cells, the vast majority of kinesin-1 is regulated to conserve ATP and to ensure its proper intracellular distribution and coordination with other molecular motors. Regulated kinesin-1 folds in half at a hinge in its coiled-coil stalk. Interactions between coiled-coil regions near the enzymatically active heads at the N terminus and the regulatory tails at the C terminus bring these globular elements in proximity and stabilize the folded conformation. However, it has remained a mystery how kinesin-1's microtubule-stimulated ATPase activity is regulated in this folded conformation. Here, we present evidence for a direct interaction between the kinesin-1 head and tail. We photochemically cross-linked heads and tails and produced an 8-Å cryoEM reconstruction of the cross-linked head–tail complex on microtubules. These data demonstrate that a conserved essential regulatory element in the kinesin-1 tail interacts directly and specifically with the enzymatically critical Switch I region of the head. This interaction suggests a mechanism for tail-mediated regulation of the ATPase activity of kinesin-1. In our structure, the tail makes simultaneous contacts with the kinesin-1 head and the microtubule, suggesting the tail may both regulate kinesin-1 in solution and hold it in a paused state with high ADP affinity on microtubules. The interaction of the Switch I region of the kinesin-1 head with the tail is strikingly similar to the interactions of small GTPases with their regulators, indicating that other kinesin motors may share similar regulatory mechanisms. PMID:18579780

  20. The kinesin-1 motor protein is regulated by a direct interaction of its head and tail.

    PubMed

    Dietrich, Kristen A; Sindelar, Charles V; Brewer, Paul D; Downing, Kenneth H; Cremo, Christine R; Rice, Sarah E

    2008-07-01

    Kinesin-1 is a molecular motor protein that transports cargo along microtubules. Inside cells, the vast majority of kinesin-1 is regulated to conserve ATP and to ensure its proper intracellular distribution and coordination with other molecular motors. Regulated kinesin-1 folds in half at a hinge in its coiled-coil stalk. Interactions between coiled-coil regions near the enzymatically active heads at the N terminus and the regulatory tails at the C terminus bring these globular elements in proximity and stabilize the folded conformation. However, it has remained a mystery how kinesin-1's microtubule-stimulated ATPase activity is regulated in this folded conformation. Here, we present evidence for a direct interaction between the kinesin-1 head and tail. We photochemically cross-linked heads and tails and produced an 8-A cryoEM reconstruction of the cross-linked head-tail complex on microtubules. These data demonstrate that a conserved essential regulatory element in the kinesin-1 tail interacts directly and specifically with the enzymatically critical Switch I region of the head. This interaction suggests a mechanism for tail-mediated regulation of the ATPase activity of kinesin-1. In our structure, the tail makes simultaneous contacts with the kinesin-1 head and the microtubule, suggesting the tail may both regulate kinesin-1 in solution and hold it in a paused state with high ADP affinity on microtubules. The interaction of the Switch I region of the kinesin-1 head with the tail is strikingly similar to the interactions of small GTPases with their regulators, indicating that other kinesin motors may share similar regulatory mechanisms. PMID:18579780

  1. Separation of the attractive and repulsive contributions to the adsorbate-adsorbate interactions of polar adsorbates on Si(100)

    NASA Astrophysics Data System (ADS)

    Lin, Ying-Hsiu; Jeng, Horng-Tay; Lin, Deng-Sung

    2015-11-01

    Dissociative adsorption of H2O, NH3, CH3OH and CH3NH2 polar molecules on the Si(100) surface results in a 1:1 mixture of two adsorbates (H and multi-atomic fragment A = OH, NH2, CH3O, CH3NH, respectively) on the surface. By using density functional theory (DFT) calculations, the adsorption geometry, the total energies and the charge densities for various possible ordered structures of the mixed adsorbate layer have been found. Analyzing the systematic trends in the total energies unveils concurrently the nearest-neighbor interactions ENN and the next nearest-neighbor interactions ENNN between two polar adsorbates A. In going from small to large polar adsorbates, ENN's exhibit an attractive-to-repulsive crossover behavior, indicating that they include competing attractive and repulsive contributions. Exploration of the charge density distributions allows the estimation of the degree of charge overlapping between immediately neighboring A's, the resulting contribution of the steric repulsions, and that of the attractive interactions to the corresponding ENN's. The attractive contributions to nearest neighboring adsorbate-adsorbate interactions between the polar adsorbates under study are shown to result from hydrogen bonds or dipole-dipole interactions.

  2. Paramagnetic Effects on Nuclear Relaxation in Enzyme-Bound Co(II)-Adenine Nucleotide Complexes: Relative Contributions of Dipolar and Scalar Interactions

    NASA Astrophysics Data System (ADS)

    Ray, Bruce D.; Jarori, Gotam K.; Nageswara Rao, B. D.

    1999-01-01

    31P NMR measurements on CoADP bound to creatine kinase designed to estimate the relative contribution of scalar and dipolar interactions to31P spin relaxation rates show that these rates are primarily due to distance-dependent dipolar interactions and that the contribution of the scalar interaction is negligible.

  3. Automated calculation of anharmonic vibrational contributions to first hyperpolarizabilities: Quadratic response functions from vibrational configuration interaction wave functions

    NASA Astrophysics Data System (ADS)

    Hansen, Mikkel Bo; Christiansen, Ove; Hättig, Christof

    2009-10-01

    Quadratic response functions are derived and implemented for a vibrational configuration interaction state. Combined electronic and vibrational quadratic response functions are derived using Born-Oppenheimer vibronic product wave functions. Computational tractable expressions are derived for determining the total quadratic response contribution as a sum of contributions involving both electronic and vibrational linear and quadratic response functions. In the general frequency-dependent case this includes a new and more troublesome type of electronic linear response function. Pilot calculations for the FH, H2O, CH2O, and pyrrole molecules demonstrate the importance of vibrational contributions for accurate comparison to experiment and that the vibrational contributions in some cases can be very large. The calculation of transition properties between vibrational states is combined with sum-over-states expressions for analysis purposes. On the basis of this some simple analysis methods are suggested. Also, a preliminary study of the effect of finite lifetimes on quadratic response functions is presented.

  4. Sense of Community in Graduate Online Education: Contribution of Learner to Learner Interaction

    ERIC Educational Resources Information Center

    Shackelford, Jo L.; Maxwell, Marge

    2012-01-01

    Distance learning technologies offer a multitude of ways to build interaction into online courses to support learning. Based on social constructivism theory, this study explored which types of interaction are most predictive of students' sense of community in online graduate courses at a regional comprehensive university. Surveys were used to…

  5. Bem1p contributes to secretory pathway polarization through a direct interaction with Exo70p

    PubMed Central

    Liu, Dongmei

    2014-01-01

    The exocyst serves to tether secretory vesicles to cortical sites specified by polarity determinants, in preparation for fusion with the plasma membrane. Although most exocyst components are brought to these sites by riding on secretory vesicles as they are actively transported along actin cables, Exo70p displays actin-independent localization to these sites, implying an interaction with a polarity determinant. Here we show that Exo70p directly and specifically binds to the polarity determinant scaffold protein Bem1p. The interaction involves multiple domains of both Exo70p and Bem1p. Mutations in Exo70p that disrupt its interaction with Bem1, without impairing its interactions with other known binding partners, lead to the loss of actin-independent localization. Synthetic genetic interactions confirm the importance of the Exo70p–Bem1p interaction, although there is some possible redundancy with Sec3p and Sec15p, other exocyst components that also interact with polarity determinants. Similar to Sec3p, the actin-independent localization of Exo70p requires a synergistic interaction with the phosphoinositide PI(4,5)P2. PMID:25313406

  6. Derivative interactions and perturbative UV contributions in N Higgs doublet models

    NASA Astrophysics Data System (ADS)

    Kikuta, Yohei; Yamamoto, Yasuhiro

    2016-05-01

    We study the Higgs derivative interactions on models including arbitrary number of the Higgs doublets. These interactions are generated by two ways. One is higher order corrections of composite Higgs models, and the other is integration of heavy scalars and vectors. In the latter case, three point couplings between the Higgs doublets and these heavy states are the sources of the derivative interactions. Their representations are constrained to couple with the doublets. We explicitly calculate all derivative interactions generated by integrating out. Their degrees of freedom and conditions to impose the custodial symmetry are discussed. We also study the vector boson scattering processes with a couple of two Higgs doublet models to see experimental signals of the derivative interactions. They are differently affected by each heavy field.

  7. Species distribution models contribute to determine the effect of climate and interspecific interactions in moving hybrid zones.

    PubMed

    Engler, J O; Rödder, D; Elle, O; Hochkirch, A; Secondi, J

    2013-11-01

    Climate is a major factor delimiting species' distributions. However, biotic interactions may also be prominent in shaping geographical ranges, especially for parapatric species forming hybrid zones. Determining the relative effect of each factor and their interaction of the contact zone location has been difficult due to the lack of broad scale environmental data. Recent developments in species distribution modelling (SDM) now allow disentangling the relative contributions of climate and species' interactions in hybrid zones and their responses to future climate change. We investigated the moving hybrid zone between the breeding ranges of two parapatric passerines in Europe. We conducted SDMs representing the climatic conditions during the breeding season. Our results show a large mismatch between the realized and potential distributions of the two species, suggesting that interspecific interactions, not climate, account for the present location of the contact zone. The SDM scenarios show that the southerly distributed species, Hippolais polyglotta, might lose large parts of its southern distribution under climate change, but a similar gain of novel habitat along the hybrid zone seems unlikely, because interactions with the other species (H. icterina) constrain its range expansion. Thus, whenever biotic interactions limit range expansion, species may become 'trapped' if range loss due to climate change is faster than the movement of the contact zone. An increasing number of moving hybrid zones are being reported, but the proximate causes of movement often remain unclear. In a global context of climate change, we call for more interest in their interactions with climate change. PMID:24016292

  8. Causal functional contributions and interactions in the attention network of the brain: an objective multi-perturbation analysis.

    PubMed

    Zavaglia, Melissa; Hilgetag, Claus C

    2016-06-01

    Spatial attention is a prime example for the distributed network functions of the brain. Lesion studies in animal models have been used to investigate intact attentional mechanisms as well as perspectives for rehabilitation in the injured brain. Here, we systematically analyzed behavioral data from cooling deactivation and permanent lesion experiments in the cat, where unilateral deactivation of the posterior parietal cortex (in the vicinity of the posterior middle suprasylvian cortex, pMS) or the superior colliculus (SC) cause a severe neglect in the contralateral hemifield. Counterintuitively, additional deactivation of structures in the opposite hemisphere reverses the deficit. Using such lesion data, we employed a game-theoretical approach, multi-perturbation Shapley value analysis (MSA), for inferring functional contributions and network interactions of bilateral pMS and SC from behavioral performance in visual attention studies. The approach provides an objective theoretical strategy for lesion inferences and allows a unique quantitative characterization of regional functional contributions and interactions on the basis of multi-perturbations. The quantitative analysis demonstrated that right posterior parietal cortex and superior colliculus made the strongest positive contributions to left-field orienting, while left brain regions had negative contributions, implying that their perturbation may reverse the effects of contralateral lesions or improve normal function. An analysis of functional modulations and interactions among the regions revealed redundant interactions (implying functional overlap) between regions within each hemisphere, and synergistic interactions between bilateral regions. To assess the reliability of the MSA method in the face of variable and incomplete input data, we performed a sensitivity analysis, investigating how much the contribution values of the four regions depended on the performance of specific configurations and on the

  9. Contribution of Physical Interactions to Signaling Specificity between a Diguanylate Cyclase and Its Effector

    PubMed Central

    Dahlstrom, Kurt M.; Giglio, Krista M.; Collins, Alan J.; Sondermann, Holger

    2015-01-01

    ABSTRACT Cyclic diguanylate (c-di-GMP) is a bacterial second messenger that controls multiple cellular processes. c-di-GMP networks have up to dozens of diguanylate cyclases (DGCs) that synthesize c-di-GMP along with many c-di-GMP-responsive target proteins that can bind and respond to this signal. For such networks to have order, a mechanism(s) likely exists that allow DGCs to specifically signal their targets, and it has been suggested that physical interactions might provide such specificity. Our results show a DGC from Pseudomonas fluorescens physically interacting with its target protein at a conserved interface, and this interface can be predictive of DGC-target protein interactions. Furthermore, we demonstrate that physical interaction is necessary for the DGC to maximally signal its target. If such “local signaling” is a theme for even a fraction of the DGCs used by bacteria, it becomes possible to posit a model whereby physical interaction allows a DGC to directly signal its target protein, which in turn may help curtail undesired cross talk with other members of the network. PMID:26670387

  10. Gene-gene interactions contribute to eye colour variation in humans.

    PubMed

    Pośpiech, Ewelina; Draus-Barini, Jolanta; Kupiec, Tomasz; Wojas-Pelc, Anna; Branicki, Wojciech

    2011-06-01

    Prediction of phenotypes from genetic data is considered to be the first practical application of data gained from association studies, with potential importance for medicine and the forensic sciences. Multiple genes and polymorphisms have been found to be associated with variation in human pigmentation. Their analysis enables prediction of blue and brown eye colour with a reasonably high accuracy. More accurate prediction, especially in the case of intermediate eye colours, may require better understanding of gene-gene interactions affecting this polygenic trait. Using multifactor dimensionality reduction and logistic regression methods, a study of gene-gene interactions was conducted based on variation in 11 known pigmentation genes examined in a cohort of 718 individuals of European descent. The study revealed significant interactions of a redundant character between the HERC2 and OCA2 genes affecting determination of hazel eye colour and between HERC2 and SLC24A4 affecting determination of blue eye colour. Our research indicates interactive effects of a synergistic character between HERC2 and OCA2, and also provides evidence for a novel strong synergistic interaction between HERC2 and TYRP1, both affecting determination of green eye colour. PMID:21471978

  11. A Three-Way Interaction among Maternal and Fetal Variants Contributing to Congenital Heart Defects.

    PubMed

    Li, Ming; Li, Jingyun; Wei, Changshuai; Lu, Qing; Tang, Xinyu; Erickson, Stephen W; MacLeod, Stewart L; Hobbs, Charlotte A

    2016-01-01

    Congenital heart defects (CHDs) develop through a complex interplay between genetic variants, epigenetic modifications, and maternal environmental exposures. Genetic studies of CHDs have commonly tested single genetic variants for association with CHDs. Less attention has been given to complex gene-by-gene and gene-by-environment interactions. In this study, we applied a recently developed likelihood-ratio Mann-Whitney (LRMW) method to detect joint actions among maternal variants, fetal variants, and maternal environmental exposures, allowing for high-order statistical interactions. All subjects are participants from the National Birth Defect Prevention Study, including 623 mother-offspring pairs with CHD-affected pregnancies and 875 mother-offspring pairs with unaffected pregnancies. Each individual has 872 single nucleotide polymorphisms encoding for critical enzymes in the homocysteine, folate, and trans-sulfuration pathways. By using the LRMW method, three variants (fetal rs625879, maternal rs2169650, and maternal rs8177441) were identified with a joint association to CHD risk (nominal P-value = 1.13e-07). These three variants are located within genes BHMT2, GSTP1, and GPX3, respectively. Further examination indicated that maternal SNP rs2169650 may interact with both fetal SNP rs625879 and maternal SNP rs8177441. Our findings suggest that the risk of CHD may be influenced by both the intragenerational interaction within the maternal genome and the intergenerational interaction between maternal and fetal genomes. PMID:26612412

  12. Spin Relaxation in III-V Semiconductors in various systems: Contribution of Electron-Electron Interaction

    NASA Astrophysics Data System (ADS)

    Dogan, Fatih; Kesserwan, Hasan; Manchon, Aurelien

    2015-03-01

    In spintronics, most of the phenomena that we are interested happen at very fast time scales and are rich in structure in time domain. Our understanding, on the other hand, is mostly based on energy domain calculations. Many of the theoretical tools use approximations and simplifications that can be perceived as oversimplifications. We compare the structure, material, carrier density and temperature dependence of spin relaxation time in n-doped III-V semiconductors using Elliot-Yafet (EY) and D'yakanov-Perel'(DP) with real time analysis using kinetic spin Bloch equations (KSBE). The EY and DP theories fail to capture details as the system investigated is varied. KSBE, on the other hand, incorporates all relaxation sources as well as electron-electron interaction which modifies the spin relaxation time in a non-linear way. Since el-el interaction is very fast (~ fs) and spin-conserving, it is usually ignored in the analysis of spin relaxation. Our results indicate that electron-electron interaction cannot be neglected and its interplay with the other (spin and momentum) relaxation mechanisms (electron-impurity and electron-phonon scattering) dramatically alters the resulting spin dynamics. We use each interaction explicitly to investigate how, in the presence of others, each relaxation source behaves. We use GaAs and GaN for zinc-blend structure, and GaN and AlN for the wurtzite structure.

  13. Dispersion Interactions between Urea and Nucleobases Contribute to the Destabilization of RNA by Urea in Aqueous Solution

    PubMed Central

    Kasavajhala, Koushik; Bikkina, Swetha; Patil, Indrajit; MacKerell, Alexander D.; Priyakumar, U. Deva

    2015-01-01

    Urea has long been used to investigate protein folding and, more recently, RNA folding. Studies have proposed that urea denatures RNA by participating in stacking interactions and hydrogen bonds with nucleic acid bases. In this study, the ability of urea to form unconventional stacking interactions with RNA bases is investigated using ab initio calculations (RI-MP2 and CCSD(T) methods with the aug-cc-pVDZ basis set). A total of 29 stable nucleobase-urea stacked complexes are identified in which the intermolecular interaction energies (up to −14 kcal/mol) are dominated by dispersion effects. Natural bond orbital (NBO) and atoms in molecules (AIM) calculations further confirm strong interactions between urea and nucleobases. Calculations on model systems with multiple urea and water molecules interacting with a guanine base lead to a hypothesis that urea molecules along with water are able to form cage-like structures capable of trapping nucleic acid bases in extrahelical states by forming both hydrogen bonded and dispersion interactions, thereby contributing to the unfolding of RNA in the presence of urea in aqueous solution. PMID:25668757

  14. Plasmid replication initiator interactions with origin 13-mers and polymerase subunits contribute to strand-specific replisome assembly

    PubMed Central

    Wawrzycka, Aleksandra; Gross, Marta; Wasaznik, Anna; Konieczny, Igor

    2015-01-01

    Although the molecular basis for replisome activity has been extensively investigated, it is not clear what the exact mechanism for de novo assembly of the replication complex at the replication origin is, or how the directionality of replication is determined. Here, using the plasmid RK2 replicon, we analyze the protein interactions required for Escherichia coli polymerase III (Pol III) holoenzyme association at the replication origin. Our investigations revealed that in E. coli, replisome formation at the plasmid origin involves interactions of the RK2 plasmid replication initiation protein (TrfA) with both the polymerase β- and α-subunits. In the presence of other replication proteins, including DnaA, helicase, primase and the clamp loader, TrfA interaction with the β-clamp contributes to the formation of the β-clamp nucleoprotein complex on origin DNA. By reconstituting in vitro the replication reaction on ssDNA templates, we demonstrate that TrfA interaction with the β-clamp and sequence-specific TrfA interaction with one strand of the plasmid origin DNA unwinding element (DUE) contribute to strand-specific replisome assembly. Wild-type TrfA, but not the TrfA QLSLF mutant (which does not interact with the β-clamp), in the presence of primase, helicase, Pol III core, clamp loader, and β-clamp initiates DNA synthesis on ssDNA template containing 13-mers of the bottom strand, but not the top strand, of DUE. Results presented in this work uncovered requirements for anchoring polymerase at the plasmid replication origin and bring insights of how the directionality of DNA replication is determined. PMID:26195759

  15. Contributions of the RAD51 N-terminal domain to BRCA2-RAD51 interaction.

    PubMed

    Subramanyam, Shyamal; Jones, William T; Spies, Maria; Spies, M Ashley

    2013-10-01

    RAD51 DNA strand exchange protein catalyzes the central step in homologous recombination, a cellular process fundamentally important for accurate repair of damaged chromosomes, preservation of the genetic integrity, restart of collapsed replication forks and telomere maintenance. BRCA2 protein, a product of the breast cancer susceptibility gene, is a key recombination mediator that interacts with RAD51 and facilitates RAD51 nucleoprotein filament formation on single-stranded DNA generated at the sites of DNA damage. An accurate atomistic level description of this interaction, however, is limited to a partial crystal structure of the RAD51 core fused to BRC4 peptide. Here, by integrating homology modeling and molecular dynamics, we generated a structure of the full-length RAD51 in complex with BRC4 peptide. Our model predicted previously unknown hydrogen bonding patterns involving the N-terminal domain (NTD) of RAD51. These interactions guide positioning of the BRC4 peptide within a cavity between the core and the NTDs; the peptide binding separates the two domains and restricts internal dynamics of RAD51 protomers. The model's depiction of the RAD51-BRC4 complex was validated by free energy calculations and in vitro functional analysis of rationally designed mutants. All generated mutants, RAD51(E42A), RAD51(E59A), RAD51(E237A), RAD51(E59A/E237A) and RAD51(E42A/E59A/E237A) maintained basic biochemical activities of the wild-type RAD51, but displayed reduced affinities for the BRC4 peptide. Strong correlation between the calculated and experimental binding energies confirmed the predicted structure of the RAD51-BRC4 complex and highlighted the importance of RAD51 NTD in RAD51-BRCA2 interaction. PMID:23935068

  16. Kinetic contributions to gating by interactions unique to N-methyl-D-aspartate (NMDA) receptors.

    PubMed

    Borschel, William F; Cummings, Kirstie A; Tindell, LeeAnn K; Popescu, Gabriela K

    2015-10-30

    Among glutamate-gated channels, NMDA receptors produce currents that subside with unusually slow kinetics, and this feature is essential to the physiology of central excitatory synapses. Relative to the homologous AMPA and kainate receptors, NMDA receptors have additional intersubunit contacts in the ligand binding domain that occur at both conserved and non-conserved sites. We examined GluN1/GluN2A single-channel currents with kinetic analyses and modeling to probe these class-specific intersubunit interactions for their role in glutamate binding and receptor gating. We found that substitutions that eliminate such interactions at non-conserved sites reduced stationary gating, accelerated deactivation, and imparted sensitivity to aniracetam, an AMPA receptor-selective positive modulator. Abolishing unique contacts at conserved sites also reduced stationary gating and accelerated deactivation. These results show that contacts specific to NMDA receptors, which brace the heterodimer interface within the ligand binding domain, stabilize actively gating receptor conformations and result in longer bursts and slower deactivations. They support the view that the strength of the heterodimer interface modulates gating in both NMDA and non-NMDA receptors and that unique interactions at this interface are responsible in part for basic differences between the kinetics of NMDA and non-NMDA currents at glutamatergic synapses. PMID:26370091

  17. TatBC-Independent TatA/Tat Substrate Interactions Contribute to Transport Efficiency

    PubMed Central

    Taubert, Johannes; Hou, Bo; Risselada, H. Jelger; Mehner, Denise; Lünsdorf, Heinrich; Grubmüller, Helmut; Brüser, Thomas

    2015-01-01

    The Tat system can transport folded, signal peptide-containing proteins (Tat substrates) across energized membranes of prokaryotes and plant plastids. A twin-arginine motif in the signal peptide of Tat substrates is recognized by TatC-containing complexes, and TatA permits the membrane passage. Often, as in the model Tat systems of Escherichia coli and plant plastids, a third component – TatB – is involved that resembles TatA but has a higher affinity to TatC. It is not known why most TatA dissociates from TatBC complexes in vivo and distributes more evenly in the membrane. Here we show a TatBC-independent substrate-binding to TatA from Escherichia coli, and we provide evidence that this binding enhances Tat transport. First hints came from in vivo cross-linking data, which could be confirmed by affinity co-purification of TatA with the natural Tat substrates HiPIP and NrfC. Two positions on the surface of HiPIP could be identified that are important for the TatA interaction and transport efficiency, indicating physiological relevance of the interaction. Distributed TatA thus may serve to accompany membrane-interacting Tat substrates to the few TatBC spots in the cells. PMID:25774531

  18. Contribution of SUMO-interacting motifs and SUMOylation to the antiretroviral properties of TRIM5α

    PubMed Central

    Brandariz-Nuñez, Alberto; Roa, Amanda; Valle-Casuso, Jose Carlos; Biris, Nikolaos; Ivanov, Dmitri; Diaz-Griffero, Felipe

    2012-01-01

    Recent findings suggested that the SUMO-interacting motifs (SIMs) present in the human TRIM5α (TRIM5αhu) protein play an important role in the ability of TRIM5αhu to restrict N-MLV. Here we explored the role of SIMs in the ability of rhesus TRIM5α (TRIM5αrh) to restrict HIV-1, and found that TRIM5αrh SIM mutants IL376KK (SIM1mut) and VI405KK (SIM2mut) completely lost their ability to block HIV-1 infection. Interestingly, these mutants also lost the recently described property of TRIM5αrh to shuttle into the nucleus. Analysis of these variants revealed that they are unable to interact with the HIV-1 core, which might explain the reason that these variants are not active against HIV-1. Furthermore, NMR titration experiments to assay the binding between the PRYSPRY domain of TRIM5αrh and the small ubiquitin-like modifier 1(SUMO-1) revealed no interaction. In addition, we examined the role of SUMOylation in restriction, and find out that inhibition of SUMOylation by the adenoviral protein Gam1 did not altered the retroviral restriction ability of TRIM5α. Overall, our results do not support a role for SIMs or SUMOylation in the antiviral properties of TRIM5α. PMID:23084420

  19. Dopamine and oxytocin interactions underlying behaviors: potential contributions to behavioral disorders.

    PubMed

    Baskerville, Tracey A; Douglas, Alison J

    2010-06-01

    Dopamine is an important neuromodulator that exerts widespread effects on the central nervous system (CNS) function. Disruption in dopaminergic neurotransmission can have profound effects on mood and behavior and as such is known to be implicated in various neuropsychiatric behavioral disorders including autism and depression. The subsequent effects on other neurocircuitries due to dysregulated dopamine function have yet to be fully explored. Due to the marked social deficits observed in psychiatric patients, the neuropeptide, oxytocin is emerging as one particular neural substrate that may be influenced by the altered dopamine levels subserving neuropathologic-related behavioral diseases. Oxytocin has a substantial role in social attachment, affiliation and sexual behavior. More recently, it has emerged that disturbances in peripheral and central oxytocin levels have been detected in some patients with dopamine-dependent disorders. Thus, oxytocin is proposed to be a key neural substrate that interacts with central dopamine systems. In addition to psychosocial improvement, oxytocin has recently been implicated in mediating mesolimbic dopamine pathways during drug addiction and withdrawal. This bi-directional role of dopamine has also been implicated during some components of sexual behavior. This review will discuss evidence for the existence dopamine/oxytocin positive interaction in social behavioral paradigms and associated disorders such as sexual dysfunction, autism, addiction, anorexia/bulimia, and depression. Preliminary findings suggest that whilst further rigorous testing has to be conducted to establish a dopamine/oxytocin link in human disorders, animal models seem to indicate the existence of broad and integrated brain circuits where dopamine and oxytocin interactions at least in part mediate socio-affiliative behaviors. A profound disruption to these pathways is likely to underpin associated behavioral disorders. Central oxytocin pathways may serve as a

  20. The AMPTE program's contribution to studies of the solar wind-magnetosphere-ionosphere interaction

    SciTech Connect

    Sibeck, D.G. )

    1990-12-01

    The Active Magnetospheric Particle Tracer Explorers (AMPTE) program provided important information on the behavior of clouds of plasma artificially injected into the solar wind and the earth's magnetosphere. Now that the releases are over, data from the satellites are being analyzed to investigate the processes by which the ambient solar wind mass, momentum, and energy are transferred to the magnetosphere. Work in progress at APL indicates that the solar wind is much more inhomogeneous than previously believed, that the solar wind constantly buffets the magnetosphere, and that ground observers may remotely sense these interactions as geomagnetic pulsations. 8 refs.

  1. Water absorption in PEEK and PEI matrices. Contribution to the understanding of water-polar group interactions

    NASA Astrophysics Data System (ADS)

    Courvoisier, E.; Bicaba, Y.; Colin, X.

    2016-05-01

    The water absorption in two aromatic linear polymers (PEEK and PEI) was studied between 10% and 90% RH at 30, 50 and 70°C. It was found that these polymers display classical Henry and Fick's behaviors. Moreover, they have very close values of equilibrium water concentration C∞ and water diffusivity D presumably because their respective polar groups establish molecular interactions of the same nature with water. This assumption was checked from a literature compilation of values of C∞ and D for a large variety of linear and tridimensional polymers containing a single type of polar group. It was then evidenced that almost all types of carbonyl group (in particular, those belonging to imides, amides and ketones) have the same molar contribution to water absorption, except those belonging to esters which are much less hydrophilic. Furthermore, hydroxyl and sulfone groups are much more hydrophilic than carbonyl groups so that their molar contribution is located on another master curve. On this basis, semi-empirical structure/water transport property relationships were proposed. It was found that C∞ increases exponentially with the concentration of polar groups (presumably because water is doubly bonded), but also with the intensity of their molecular interactions with water. In contrast, D is inversely proportional to C∞, which means that polar group-water interactions slow down the rate of water diffusion.

  2. Wind-US Code Contributions to the First AIAA Shock Boundary Layer Interaction Prediction Workshop

    NASA Technical Reports Server (NTRS)

    Georgiadis, Nicholas J.; Vyas, Manan A.; Yoder, Dennis A.

    2013-01-01

    This report discusses the computations of a set of shock wave/turbulent boundary layer interaction (SWTBLI) test cases using the Wind-US code, as part of the 2010 American Institute of Aeronautics and Astronautics (AIAA) shock/boundary layer interaction workshop. The experiments involve supersonic flows in wind tunnels with a shock generator that directs an oblique shock wave toward the boundary layer along one of the walls of the wind tunnel. The Wind-US calculations utilized structured grid computations performed in Reynolds-averaged Navier-Stokes mode. Four turbulence models were investigated: the Spalart-Allmaras one-equation model, the Menter Baseline and Shear Stress Transport k-omega two-equation models, and an explicit algebraic stress k-omega formulation. Effects of grid resolution and upwinding scheme were also considered. The results from the CFD calculations are compared to particle image velocimetry (PIV) data from the experiments. As expected, turbulence model effects dominated the accuracy of the solutions with upwinding scheme selection indicating minimal effects.

  3. The importance of interacting climate modes on Australia's contribution to global carbon cycle extremes.

    PubMed

    Cleverly, James; Eamus, Derek; Luo, Qunying; Restrepo Coupe, Natalia; Kljun, Natascha; Ma, Xuanlong; Ewenz, Cacilia; Li, Longhui; Yu, Qiang; Huete, Alfredo

    2016-01-01

    The global carbon cycle is highly sensitive to climate-driven fluctuations of precipitation, especially in the Southern Hemisphere. This was clearly manifested by a 20% increase of the global terrestrial C sink in 2011 during the strongest sustained La Niña since 1917. However, inconsistencies exist between El Niño/La Niña (ENSO) cycles and precipitation in the historical record; for example, significant ENSO-precipitation correlations were present in only 31% of the last 100 years, and often absent in wet years. To resolve these inconsistencies, we used an advanced temporal scaling method for identifying interactions amongst three key climate modes (El Niño, the Indian Ocean dipole, and the southern annular mode). When these climate modes synchronised (1999-2012), drought and extreme precipitation were observed across Australia. The interaction amongst these climate modes, more than the effect of any single mode, was associated with large fluctuations in precipitation and productivity. The long-term exposure of vegetation to this arid environment has favoured a resilient flora capable of large fluctuations in photosynthetic productivity and explains why Australia was a major contributor not only to the 2011 global C sink anomaly but also to global reductions in photosynthetic C uptake during the previous decade of drought. PMID:26976754

  4. The importance of interacting climate modes on Australia’s contribution to global carbon cycle extremes

    NASA Astrophysics Data System (ADS)

    Cleverly, James; Eamus, Derek; Luo, Qunying; Restrepo Coupe, Natalia; Kljun, Natascha; Ma, Xuanlong; Ewenz, Cacilia; Li, Longhui; Yu, Qiang; Huete, Alfredo

    2016-03-01

    The global carbon cycle is highly sensitive to climate-driven fluctuations of precipitation, especially in the Southern Hemisphere. This was clearly manifested by a 20% increase of the global terrestrial C sink in 2011 during the strongest sustained La Niña since 1917. However, inconsistencies exist between El Niño/La Niña (ENSO) cycles and precipitation in the historical record; for example, significant ENSO–precipitation correlations were present in only 31% of the last 100 years, and often absent in wet years. To resolve these inconsistencies, we used an advanced temporal scaling method for identifying interactions amongst three key climate modes (El Niño, the Indian Ocean dipole, and the southern annular mode). When these climate modes synchronised (1999–2012), drought and extreme precipitation were observed across Australia. The interaction amongst these climate modes, more than the effect of any single mode, was associated with large fluctuations in precipitation and productivity. The long-term exposure of vegetation to this arid environment has favoured a resilient flora capable of large fluctuations in photosynthetic productivity and explains why Australia was a major contributor not only to the 2011 global C sink anomaly but also to global reductions in photosynthetic C uptake during the previous decade of drought.

  5. Observations of Ions in Comets: A Contribution Towards Understanding the Comet-Solar Wind Interaction

    NASA Astrophysics Data System (ADS)

    Jockers, K.; Bonev, T.; Credner, T.

    Since many years cometary ions have been observed by the authors and their coworkers in order to study the comet-solar wind interaction. Comets with water production rates ranging from 10^28 (46P/Wirtanen) to 6 10^30 molecules s^-1 (C/1995 O1 Hale-Bopp) have been observed. In this paper we briefly introduce the physics of the comet-solar wind interaction. New observations of comet C/1996 Q1 (Tabur) are presented, where for the first time H_2O^+ and CO^+ ions have been recorded exactly simultaneously with a two-channel system. They are compared with previous observations of comets C/1989 X1 (Austin), 46P (Wirtanen) and 109P (Swift-Tuttle). We use a new method of Wegmann et al. (1998), based on the MHD scaling law, to determine the water production of comet Tabur from its H_2O^+ column density map and obtain a value of 3.3 10^28 water molecules s^-1. Nonstationary phenomena like tail rays and so-called tail disconnections are very briefly reviewed. A movie of plasma envelopes observed in the light of OH^+ in comet 1995 O1 (Hale-Bopp) is presented on the attached CD-ROM.

  6. Intramolecular interactions contributing for the conformational preference of bioactive diphenhydramine: Manifestation of the gauche effect

    NASA Astrophysics Data System (ADS)

    de Rezende, Fátima M. P.; Andrade, Laize A. F.; Freitas, Matheus P.

    2015-08-01

    Diphenhydramine is an antihistamine used to treat some symptoms of allergies and the common cold. It is usually marketed as the hydrochloride salt, and both the neutral and cation forms have the O-C-C-N fragment. The gauche effect is well known in fluorine-containing chains, because its main origin is hyperconjugative and the σ∗C-F is a low-lying acceptor orbital, allowing electron delocalization in the conformation where F and an adjacent electronegative substituent in an ethane fragment are in the gauche orientation. Our experimental (NMR) and theoretical findings indicate that diphenhydramine exhibits the gauche effect, since the preferential conformations have the O-C-C-N moiety in this orientation due especially to antiperiplanar σC-H → σ∗C-O and σC-H → σ∗C-N interactions. This conformational preference is strengthened in the protonated form due to an incremental electrostatic gauche effect. Because the gauche conformation matches the bioactive structure of diphenhydramine complexed with histamine methyltransferase, it is suggested that intramolecular interactions, and not only induced fit, rule its bioactive form.

  7. The importance of interacting climate modes on Australia’s contribution to global carbon cycle extremes

    PubMed Central

    Cleverly, James; Eamus, Derek; Luo, Qunying; Restrepo Coupe, Natalia; Kljun, Natascha; Ma, Xuanlong; Ewenz, Cacilia; Li, Longhui; Yu, Qiang; Huete, Alfredo

    2016-01-01

    The global carbon cycle is highly sensitive to climate-driven fluctuations of precipitation, especially in the Southern Hemisphere. This was clearly manifested by a 20% increase of the global terrestrial C sink in 2011 during the strongest sustained La Niña since 1917. However, inconsistencies exist between El Niño/La Niña (ENSO) cycles and precipitation in the historical record; for example, significant ENSO–precipitation correlations were present in only 31% of the last 100 years, and often absent in wet years. To resolve these inconsistencies, we used an advanced temporal scaling method for identifying interactions amongst three key climate modes (El Niño, the Indian Ocean dipole, and the southern annular mode). When these climate modes synchronised (1999–2012), drought and extreme precipitation were observed across Australia. The interaction amongst these climate modes, more than the effect of any single mode, was associated with large fluctuations in precipitation and productivity. The long-term exposure of vegetation to this arid environment has favoured a resilient flora capable of large fluctuations in photosynthetic productivity and explains why Australia was a major contributor not only to the 2011 global C sink anomaly but also to global reductions in photosynthetic C uptake during the previous decade of drought. PMID:26976754

  8. Anomalously interacting Z* bosons: an example of JINR's contribution to physics at LHC

    NASA Astrophysics Data System (ADS)

    Bednyakov, V. A.; Yeletskikh, I. V.; Chizhov, M. V.; Boyko, I. R.

    2016-04-01

    Fundamental particle physics research at the Joint Institute for Nuclear Research (JINR) has always included the use of highest-energy accelerator machines, and it is only natural that from its very beginning, the institute played an active role in work on developing, assembling, and upgrading both the Large Hadron Collider itself and its detectors. Along with providing hardware and software support to secure the failure-free operation of detectors and the gathering and processing of experimental data, JINR sets as its primary goal to effectively participate in the unprecedentedly comprehensive and important LHC research program. As part of this program, the experimental search for new heavy chiral Z* and W* bosons is carried out by the ATLAS collaboration, an effort whose necessity was fully justified and strategy exhaustively developed by JINR physicists. The search results from the first run of the LHC are briefly discussed, together with the decisive contribution from JINR and future prospects.

  9. Lactate involvement in neuron-glia metabolic interaction: (13)C-NMR spectroscopy contribution.

    PubMed

    Bouzier-Sore, A-K; Serres, S; Canioni, P; Merle, M

    2003-09-01

    Glucose is commonly admitted to be the main substrate for brain energy requirement. However, it has been recently proposed that lactate, generated from glucose via glycolysis, would be the oxidative substrate for neurons, particularly during neuronal activation, according to a mechanism called the astrocyte-neuron lactate shuttle hypothesis (ANLSH). In that mechanism, glutamate released in the synaptic cleft during brain activation is taken up by astrocytes. This uptake, via the glutamate/Na(+) transporter, induces the entry of sodium, which is then excluded from the astrocytes via the Na(+)/K(+) ATPase. This exclusion consumes ATP, which stimulates glycolysis and thus lactate formation in astrocytes. This lactate is then transferred to neurons where it is utilized as oxidative substrate. This review tries to gather the recent evidences that support this hypothesis and presents the contribution of NMR to this matter. PMID:14652173

  10. The CHUVA Project Contributions to the Understanding of Anthropogenic Interactions Affecting the Atmospheric Physics over Amazonas.

    NASA Astrophysics Data System (ADS)

    Machado, L.; Cecchini, M. A.; Gonçalves, W.

    2014-12-01

    CHUVA, meaning "rain" in Portuguese, is the acronym for the Cloud processes of tHe main precipitation systems in Brazil: A contribUtion to cloud resolVing modeling and to the GPM (GlobAl Precipitation Measurement). The CHUVA project has conducted six field campaigns; the last campaign was held in Manaus in 2014 jointly with GoAmazon and ACRIDICON. CHUVA's main scientific motivation is to contribute to the understanding of cloud processes, which represent one of the least understood components of the weather and climate system. This study will briefly describe the CHUVA project and the main scientific results obtained in the Amazon region. Specifically, we will describe the results of one year radar observation of Manaus rainfall and the relationship with black carbon. The results indicate that the aerosol influence on precipitating systems is modulated by the atmospheric instability degree. For stable atmospheres, the higher the aerosol concentration, the lower the precipitation over the region. On the other hand, for unstable cases, higher concentrations of particulate material are associated with more precipitation, elevated presence of ice and larger rain cells, which suggests an association with long lived systems. Also we will describe some preliminary results obtained during GoAmazon describing the cloud and rainfall size distribution (DSD). The DSD was adjusted to the gamma function using the momentum method and disposed in the three-dimensional space of the gamma parameters: the intercept, the shape and the width. Each point in this three-dimensional space corresponds to a specific DSD and the ensemble of points describes all regimes of precipitation in Amazon. Based in this Gamma space we will discuss the characteristics of the rainfall regime and anthropogenic features.

  11. Probing the Solution Structure of IκB Kinase (IKK) Subunit γ and Its Interaction with Kaposi Sarcoma-associated Herpes Virus Flice-interacting Protein and IKK Subunit β by EPR Spectroscopy*

    PubMed Central

    Bagnéris, Claire; Rogala, Kacper B.; Baratchian, Mehdi; Zamfir, Vlad; Kunze, Micha B. A.; Dagless, Selina; Pirker, Katharina F.; Collins, Mary K.; Hall, Benjamin A.; Barrett, Tracey E.; Kay, Christopher W. M.

    2015-01-01

    Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Because IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/β subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance spectroscopy combined with molecular modeling and molecular dynamics simulations revealed that IKKγ is a parallel coiled-coil whose response to binding of vFLIP or IKKβ is localized twisting/stiffening and not large-scale rearrangements. The coiled-coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers, which is located directly C-terminal of the vFLIP binding site. PMID:25979343

  12. Probing the Solution Structure of IκB Kinase (IKK) Subunit γ and Its Interaction with Kaposi Sarcoma-associated Herpes Virus Flice-interacting Protein and IKK Subunit β by EPR Spectroscopy.

    PubMed

    Bagnéris, Claire; Rogala, Kacper B; Baratchian, Mehdi; Zamfir, Vlad; Kunze, Micha B A; Dagless, Selina; Pirker, Katharina F; Collins, Mary K; Hall, Benjamin A; Barrett, Tracey E; Kay, Christopher W M

    2015-07-01

    Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Because IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/β subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance spectroscopy combined with molecular modeling and molecular dynamics simulations revealed that IKKγ is a parallel coiled-coil whose response to binding of vFLIP or IKKβ is localized twisting/stiffening and not large-scale rearrangements. The coiled-coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers, which is located directly C-terminal of the vFLIP binding site. PMID:25979343

  13. Under the radar: how unexamined biases in decision-making processes in clinical interactions can contribute to health care disparities.

    PubMed

    Dovidio, John F; Fiske, Susan T

    2012-05-01

    Several aspects of social psychological science shed light on how unexamined racial/ethnic biases contribute to health care disparities. Biases are complex but systematic, differing by racial/ethnic group and not limited to love-hate polarities. Group images on the universal social cognitive dimensions of competence and warmth determine the content of each group's overall stereotype, distinct emotional prejudices (pity, envy, disgust, pride), and discriminatory tendencies. These biases are often unconscious and occur despite the best intentions. Such ambivalent and automatic biases can influence medical decisions and interactions, systematically producing discrimination in health care and ultimately disparities in health. Understanding how these processes may contribute to bias in health care can help guide interventions to address racial and ethnic disparities in health. PMID:22420809

  14. Under the Radar: How Unexamined Biases in Decision-Making Processes in Clinical Interactions Can Contribute to Health Care Disparities

    PubMed Central

    Fiske, Susan T.

    2012-01-01

    Several aspects of social psychological science shed light on how unexamined racial/ethnic biases contribute to health care disparities. Biases are complex but systematic, differing by racial/ethnic group and not limited to love–hate polarities. Group images on the universal social cognitive dimensions of competence and warmth determine the content of each group's overall stereotype, distinct emotional prejudices (pity, envy, disgust, pride), and discriminatory tendencies. These biases are often unconscious and occur despite the best intentions. Such ambivalent and automatic biases can influence medical decisions and interactions, systematically producing discrimination in health care and ultimately disparities in health. Understanding how these processes may contribute to bias in health care can help guide interventions to address racial and ethnic disparities in health. PMID:22420809

  15. Defining the contributions of permanent electrostatics, Pauli repulsion, and dispersion in density functional theory calculations of intermolecular interaction energies.

    PubMed

    Horn, Paul R; Mao, Yuezhi; Head-Gordon, Martin

    2016-03-21

    In energy decomposition analysis of Kohn-Sham density functional theory calculations, the so-called frozen (or pre-polarization) interaction energy contains contributions from permanent electrostatics, dispersion, and Pauli repulsion. The standard classical approach to separate them suffers from several well-known limitations. We introduce an alternative scheme that employs valid antisymmetric electronic wavefunctions throughout and is based on the identification of individual fragment contributions to the initial supersystem wavefunction as determined by an energetic optimality criterion. The density deformations identified with individual fragments upon formation of the initial supersystem wavefunction are analyzed along with the distance dependence of the new and classical terms for test cases that include the neon dimer, ammonia borane, water-Na(+), water-Cl(-), and the naphthalene dimer. PMID:27004862

  16. PRICKLE1 Contributes to Cancer Cell Dissemination through Its Interaction with mTORC2.

    PubMed

    Daulat, Avais M; Bertucci, François; Audebert, Stéphane; Sergé, Arnauld; Finetti, Pascal; Josselin, Emmanuelle; Castellano, Rémy; Birnbaum, Daniel; Angers, Stéphane; Borg, Jean-Paul

    2016-05-23

    Components of the evolutionarily conserved developmental planar cell polarity (PCP) pathway were recently described to play a prominent role in cancer cell dissemination. However, the molecular mechanisms by which PCP molecules drive the spread of cancer cells remain largely unknown. PRICKLE1 encodes a PCP protein bound to the promigratory serine/threonine kinase MINK1. We identify RICTOR, a member of the mTORC2 complex, as a PRICKLE1-binding partner and show that the integrity of the PRICKLE1-MINK1-RICTOR complex is required for activation of AKT, regulation of focal adhesions, and cancer cell migration. Disruption of the PRICKLE1-RICTOR interaction results in a strong impairment of breast cancer cell dissemination in xenograft assays. Finally, we show that upregulation of PRICKLE1 in basal breast cancers, a subtype characterized by high metastatic potential, is associated with poor metastasis-free survival. PMID:27184734

  17. Cation–π Interactions Contribute to Substrate Recognition in γ‐Butyrobetaine Hydroxylase Catalysis

    PubMed Central

    Kamps, Jos J. A. G.; Khan, Amjad; Choi, Hwanho; Lesniak, Robert K.; Brem, Jürgen; Rydzik, Anna M.; McDonough, Michael A.; Schofield, Christopher J.; Claridge, Timothy D. W.

    2015-01-01

    Abstract γ‐Butyrobetaine hydroxylase (BBOX) is a non‐heme FeII‐ and 2‐oxoglutarate‐dependent oxygenase that catalyzes the stereoselective hydroxylation of an unactivated C−H bond of γ‐butyrobetaine (γBB) in the final step of carnitine biosynthesis. BBOX contains an aromatic cage for the recognition of the positively charged trimethylammonium group of the γBB substrate. Enzyme binding and kinetic analyses on substrate analogues with P and As substituting for N in the trimethylammonium group show that the analogues are good BBOX substrates, which follow the efficiency trend N+>P+>As+. The results reveal that an uncharged carbon analogue of γBB is not a BBOX substrate, thus highlighting the importance of the energetically favorable cation–π interactions in productive substrate recognition. PMID:26660433

  18. Galaxies and Genes: Towards an Automatic Modeling of Interacting Galaxies (Oral Contribution)

    NASA Astrophysics Data System (ADS)

    Theis, Christian; Gerds, Christoph; Spinneker, Christian

    The main problems in modeling interacting galaxies are the extended parameter space and the fairly high CPU costs of self-consistent N-body simulations. Therefore, traditional modeling techniques suffer from either extreme CPU demands or trapping in local optima (or both). A very promising alternative approach are evolutionary algorithms which mimic natural adaptation in order to optimize the numerical models. One main advantage is their very weak dependence on starting points which makes them much less prone to trapping in local optima. We present a Genetic Algorithm (GA) coupled with a fast (but not self-consistent) restricted N-body solver. This combination allows us to identify interesting regions of parameter space within only a few CPU hours on a standard PC or a few CPU minutes on a parallel computer. Especially, we demonstrate here the ability of GA-based fitting procedures to analyse observational data automatically, provided the data are sufficiently accurate.

  19. Model for evaluating patterned charge regulation contribution to electrostatic interactions between proteins

    NASA Astrophysics Data System (ADS)

    Hollenbeck, Dawn; Martini, K. Michael; Langner, Andreas; Ross, David; Harkin, Anthony; Nelson, Edward; Thurston, George

    2010-03-01

    We study the pattern-specific work of charging for two spherical model proteins in close proximity in ionic solution, using a grand-canonical partition function together with a coarse-grained, linear Debye-Huckel model to calculate the needed work of charging for each possible proton occupancy configuration. We seek to delineate a parameter-space phase diagram to characterize the circumstances under which patterned charge regulation, attractions due to heterogeneous protein charging patterns, and screened net protein charge could individually dominate the electrostatic portion of the interaction between model particles. Within the model, we place titratable residues in accordance with the tertiary protein structure, as is done in the case of a single protein within the Tanford-Kirkwood protein electrostatics model. We use Monte-Carlo simulation and analytical work to evaluate how the local statistics of the charging patterns on each protein respond to close proximity and relative orientation of neighboring proteins.

  20. Fundamentals of Nanoscale Polymer–Protein Interactions and Potential Contributions to Solid-State Nanobioarrays

    PubMed Central

    2015-01-01

    Protein adsorption onto polymer surfaces is a very complex, ubiquitous, and integrated process, impacting essential areas of food processing and packaging, health devices, diagnostic tools, and medical products. The nature of protein–surface interactions is becoming much more complicated with continuous efforts toward miniaturization, especially for the development of highly compact protein detection and diagnostic devices. A large body of literature reports on protein adsorption from the perspective of ensemble-averaged behavior on macroscopic, chemically homogeneous, polymeric surfaces. However, protein–surface interactions governing the nanoscale size regime may not be effectively inferred from their macroscopic and microscopic characteristics. Recently, research efforts have been made to produce periodically arranged, nanoscopic protein patterns on diblock copolymer surfaces solely through self-assembly. Intriguing protein adsorption phenomena are directly probed on the individual biomolecule level for a fundamental understanding of protein adsorption on nanoscale surfaces exhibiting varying degrees of chemical heterogeneity. Insight gained from protein assembly on diblock copolymers can be effectively used to control the surface density, conformation, orientation, and biofunctionality of prebound proteins in highly miniaturized applications, now approaching the nanoscale. This feature article will highlight recent experimental and theoretical advances made on these fronts while focusing on single-biomolecule-level investigations of protein adsorption behavior combined with surface chemical heterogeneity on the length scale commensurate with a single protein. This article will also address advantages and challenges of the self-assembly-driven patterning technology used to produce protein nanoarrays and its implications for ultrahigh density, functional, and quantifiable protein detection in a highly miniaturized format. PMID:24456577

  1. Fundamentals of nanoscale polymer-protein interactions and potential contributions to solid-state nanobioarrays.

    PubMed

    Hahm, Jong-in

    2014-08-26

    Protein adsorption onto polymer surfaces is a very complex, ubiquitous, and integrated process, impacting essential areas of food processing and packaging, health devices, diagnostic tools, and medical products. The nature of protein-surface interactions is becoming much more complicated with continuous efforts toward miniaturization, especially for the development of highly compact protein detection and diagnostic devices. A large body of literature reports on protein adsorption from the perspective of ensemble-averaged behavior on macroscopic, chemically homogeneous, polymeric surfaces. However, protein-surface interactions governing the nanoscale size regime may not be effectively inferred from their macroscopic and microscopic characteristics. Recently, research efforts have been made to produce periodically arranged, nanoscopic protein patterns on diblock copolymer surfaces solely through self-assembly. Intriguing protein adsorption phenomena are directly probed on the individual biomolecule level for a fundamental understanding of protein adsorption on nanoscale surfaces exhibiting varying degrees of chemical heterogeneity. Insight gained from protein assembly on diblock copolymers can be effectively used to control the surface density, conformation, orientation, and biofunctionality of prebound proteins in highly miniaturized applications, now approaching the nanoscale. This feature article will highlight recent experimental and theoretical advances made on these fronts while focusing on single-biomolecule-level investigations of protein adsorption behavior combined with surface chemical heterogeneity on the length scale commensurate with a single protein. This article will also address advantages and challenges of the self-assembly-driven patterning technology used to produce protein nanoarrays and its implications for ultrahigh density, functional, and quantifiable protein detection in a highly miniaturized format. PMID:24456577

  2. Contributions of conserved serine residues to the interactions of ligands with dopamine D2 receptors.

    PubMed

    Cox, B A; Henningsen, R A; Spanoyannis, A; Neve, R L; Neve, K A

    1992-08-01

    Four dopamine D2 receptor mutants were constructed, in each of which an alanine residue was substituted for one of four conserved serine residues, i.e., Ser-193, Ser-194, Ser-197, and Ser-391. Wild-type and mutant receptors were expressed transiently in COS-7 cells and stably in C6 glioma cells for analysis of ligand-receptor interactions. In radioligand binding assays, the affinity of D2 receptors for dopamine was decreased 50-fold by substitution of alanine for Ser-193, implicating this residue in the binding of dopamine. Each mutant had smaller decreases in affinity for one or more of the ligands tested, with no apparent relationship between the class of ligand and the pattern of mutation-induced changes in affinity, except that the potency of agonists was decreased by substitution for Ser-193. The potency of dopamine for inhibition of adenylyl cyclase was reduced substantially by substitution of alanine for Ser-193 or Ser-197. Mutation of Ser-194 led to a complete loss of efficacy for dopamine and p-tyramine, which would be consistent with an interaction between Ser-194 and the p-hydroxyl substituent of dopamine that is necessary for activation of the receptors to occur. Because mutation of the corresponding residues of beta 2-adrenergic receptors has very different consequences, we conclude that although the position of these serine residues is highly conserved among catecholamine receptors, and the residues as a group are important in ligand binding and activation of receptors by agonists, the function of each of the residues considered separately varies among catecholamine receptors. PMID:1321233

  3. Contribution to the beam plasma material interactions during material processing with TEA CO2 laser radiation

    NASA Astrophysics Data System (ADS)

    Jaschek, Rainer; Konrad, Peter E.; Mayerhofer, Roland; Bergmann, Hans W.; Bickel, Peter G.; Kowalewicz, Roland; Kuttenberger, Alfred; Christiansen, Jens

    1995-03-01

    The TEA-CO2-laser (transversely excited atmospheric pressure) is a tool for the pulsed processing of materials with peak power densities up to 1010 W/cm2 and a FWHM of 70 ns. The interaction between the laser beam, the surface of the work piece and the surrounding atmosphere as well as gas pressure and the formation of an induced plasma influences the response of the target. It was found that depending on the power density and the atmosphere the response can take two forms. (1) No target modification due to optical break through of the atmosphere and therefore shielding of the target (air pressure above 10 mbar, depending on the material). (2) Processing of materials (air pressure below 10 mbar, depending on the material) with melting of metallic surfaces (power density above 0.5 109 W/cm2), hole formation (power density of 5 109 W/cm2) and shock hardening (power density of 3.5 1010 W/cm2). All those phenomena are usually linked with the occurrence of laser supported combustion waves and laser supported detonation waves, respectively for which the mechanism is still not completely understood. The present paper shows how short time photography and spatial and temporal resolved spectroscopy can be used to better understand the various processes that occur during laser beam interaction. The spectra of titanium and aluminum are observed and correlated with the modification of the target. If the power density is high enough and the gas pressure above a material and gas composition specific threshold, the plasma radiation shows only spectral lines of the background atmosphere. If the gas pressure is below this threshold, a modification of the target surface (melting, evaporation and solid state transformation) with TEA-CO2- laser pulses is possible and the material specific spectra is observed. In some cases spatial and temporal resolved spectroscopy of a plasma allows the calculation of electron temperatures by comparison of two spectral lines.

  4. Nonenzymatic domains of Kalirin7 contribute to spine morphogenesis through interactions with phosphoinositides and Abl

    PubMed Central

    Ma, Xin-Ming; Miller, Megan B.; Vishwanatha, K. S.; Gross, Maegan J.; Wang, Yanping; Abbott, Thomas; Lam, TuKiet T.; Mains, Richard E.; Eipper, Betty A.

    2014-01-01

    Like several Rho GDP/GTP exchange factors (GEFs), Kalirin7 (Kal7) contains an N-terminal Sec14 domain and multiple spectrin repeats. A natural splice variant of Kalrn lacking the Sec14 domain and four spectrin repeats is unable to increase spine formation; our goal was to understand the function of the Sec14 and spectrin repeat domains. Kal7 lacking its Sec14 domain still increased spine formation, but the spines were short. Strikingly, Kal7 truncation mutants containing only the Sec14 domain and several spectrin repeats increased spine formation. The Sec14 domain bound phosphoinositides, a minor but crucial component of cellular membranes, and binding was increased by a phosphomimetic mutation. Expression of KalSec14-GFP in nonneuronal cells impaired receptor-mediated endocytosis, linking Kal7 to membrane trafficking. Consistent with genetic studies placing Abl, a non–receptor tyrosine kinase, and the Drosophila orthologue of Kalrn into the same signaling pathway, Abl1 phosphorylated two sites in the fourth spectrin repeat of Kalirin, increasing its sensitivity to calpain-mediated degradation. Treating cortical neurons of the wild-type mouse, but not the Kal7KO mouse, with an Abl inhibitor caused an increase in linear spine density. Phosphorylation of multiple sites in the N-terminal Sec14/spectrin region of Kal7 may allow coordination of the many signaling pathways contributing to spine morphogenesis. PMID:24600045

  5. Neuron-Glia Interactions in Neural Plasticity: Contributions of Neural Extracellular Matrix and Perineuronal Nets

    PubMed Central

    Dzyubenko, Egor; Gottschling, Christine

    2016-01-01

    Synapses are specialized structures that mediate rapid and efficient signal transmission between neurons and are surrounded by glial cells. Astrocytes develop an intimate association with synapses in the central nervous system (CNS) and contribute to the regulation of ion and neurotransmitter concentrations. Together with neurons, they shape intercellular space to provide a stable milieu for neuronal activity. Extracellular matrix (ECM) components are synthesized by both neurons and astrocytes and play an important role in the formation, maintenance, and function of synapses in the CNS. The components of the ECM have been detected near glial processes, which abut onto the CNS synaptic unit, where they are part of the specialized macromolecular assemblies, termed perineuronal nets (PNNs). PNNs have originally been discovered by Golgi and represent a molecular scaffold deposited in the interface between the astrocyte and subsets of neurons in the vicinity of the synapse. Recent reports strongly suggest that PNNs are tightly involved in the regulation of synaptic plasticity. Moreover, several studies have implicated PNNs and the neural ECM in neuropsychiatric diseases. Here, we highlight current concepts relating to neural ECM and PNNs and describe an in vitro approach that allows for the investigation of ECM functions for synaptogenesis. PMID:26881114

  6. Contribution of the C-Terminal Region of a Group II Chaperonin to its Interaction with Prefoldin and Substrate Transfer.

    PubMed

    Zako, Tamotsu; Sahlan, Muhamad; Fujii, Sayaka; Yamamoto, Yohei Y; Tai, Phan The; Sakai, Kotaro; Maeda, Mizuo; Yohda, Masafumi

    2016-06-01

    Prefoldin is a molecular chaperone that captures an unfolded protein substrate and transfers it to a group II chaperonin. Previous studies have shown that the interaction sites for prefoldin are located in the helical protrusions of group II chaperonins. However, it does not exclude the possibility of the existence of other interaction sites. In this study, we constructed C-terminal truncation mutants of a group II chaperonin and examined the effects of these mutations on the chaperone's function and interaction with prefoldin. Whereas the mutants with up to 6 aa truncation from the C-terminus retained more than 90% chaperone activities for protecting citrate synthase from thermal aggregation and refolding of green fluorescent protein and isopropylmalate dehydrogenase, the truncation mutants showed decreased affinities for prefoldin. Consequently, the truncation mutants showed reduced transfer efficiency of the denatured substrate protein from prefoldin and subsequent chaperonin-dependent refolding. The results clearly show that the C-terminal region of group II chaperonins contributes to their interactions with prefoldin, the transfer of the substrate protein from prefoldin and its refolding. PMID:27079363

  7. Testing the relative contribution of positive and negative interactions in rocky intertidal communities

    SciTech Connect

    Bertness, M.D.; Leonard, G.H.; Levine, J.M.; Schmidt, P.R.; Ingraham, A.O.

    1999-12-01

    In contrast to many other biotic forces, such as competition and predation, the role played by habitat modification by plants and sessile animals in natural communities has not been given the experimental attention it deserves. To test the hypothesis that habitat modification by seaweed canopies can have direct positive effects on rocky intertidal communities, the authors quantified habitat amelioration by Ascophyllum nodosum canopies and its consequences on understory organisms in the Gulf of Maine, USA. At the upper and lower elevational borders of the algal canopy, the authors examined the recruitment, growth, and survivorship of common benthic organisms in canopy removal, and shaded canopy removal plots intended to mimic canopy habitat modifications. The algal canopy greatly reduced potential physical stresses, particularly at high tidal heights. Maximum daily rock temperatures were 5--10 C lower and evaporative water loss was in order of magnitude less under the canopy than in canopy removal plots. The response of understory organisms to canopy removal, however, was species specific and somewhat idiosyncratic. Nonetheless, in general, at the high intertidal border of the canopy the recruitment, growth, and survival of understory organisms were enhanced by the canopy, whereas at the low intertidal border canopy effects were negative or neutral. nearly half of the interactions the authors studied were positive in the high zone.

  8. Sensorimotor Decoupling Contributes to Triadic Attention: A Longitudinal Investigation of Mother-Infant-Object Interactions.

    PubMed

    de Barbaro, Kaya; Johnson, Christine M; Forster, Deborah; Deák, Gedeon O

    2016-03-01

    Previous developmental accounts of joint object activity identify a qualitative "shift" around 9-12 months. In a longitudinal study of 26 dyads, videos of joint object interactions at 4, 6, 9, and 12 months were coded for all targets of gaze and manual activity (at 10 Hz). At 12 months, infants distribute their sensorimotor modalities between objects handled by the parent and others controlled by the infant. Analyses reveal novel trajectories in distributed joint object activity across the 1st year. At 4 months, infants predominantly look at and manipulate a single object, typically held by their mothers. Between 6 and 9 months, infants increasingly decouple their visual and haptic modalities and distribute their attention between objects held by their mothers and by themselves. These previously unreported developments in the distribution of multimodal object activity might "bridge the gap" to coordinated joint activity between 6 and 12 months. PMID:26613383

  9. A neuron-glia interaction involving GABA Transaminase contributes to sleep loss in sleepless mutants

    PubMed Central

    Chen, Wen-Feng; Maguire, Sarah; Sowcik, Mallory; Luo, Wenyu; Koh, Kyunghee; Sehgal, Amita

    2014-01-01

    Sleep is an essential process and yet mechanisms underlying it are not well understood. Loss of the Drosophila quiver/sleepless (qvr/sss) gene increases neuronal excitability and diminishes daily sleep, providing an excellent model for exploring the underpinnings of sleep regulation. Here, we used a proteomic approach to identify proteins altered in sss brains. We report that loss of sleepless post-transcriptionally elevates the CG7433 protein, a mitochondrial γ-aminobutyric acid transaminase (GABAT), and reduces GABA in fly brains. Loss of GABAT increases daily sleep and improves sleep consolidation, indicating that GABAT promotes wakefulness. Importantly, disruption of the GABAT gene completely suppresses the sleep phenotype of sss mutants, demonstrating that GABAT is required for loss of sleep in sss mutants. While SSS acts in distinct populations of neurons, GABAT acts in glia to reduce sleep in sss flies. Our results identify a novel mechanism of interaction between neurons and glia that is important for the regulation of sleep. PMID:24637426

  10. PE_PGRS33 Contributes to Mycobacterium tuberculosis Entry in Macrophages through Interaction with TLR2

    PubMed Central

    Palucci, Ivana; Camassa, Serena; Cascioferro, Alessandro; Sali, Michela; Anoosheh, Saber; Zumbo, Antonella; Minerva, Mariachiara; Iantomasi, Raffaella; De Maio, Flavio; Di Sante, Gabriele; Ria, Francesco; Sanguinetti, Maurizio; Palù, Giorgio; Brennan, Michael J.; Manganelli, Riccardo; Delogu, Giovanni

    2016-01-01

    PE_PGRS represent a large family of proteins typical of pathogenic mycobacteria whose members are characterized by an N-terminal PE domain followed by a large Gly-Ala repeat-rich C-terminal domain. Despite the abundance of PE_PGRS-coding genes in the Mycobacterium tuberculosis (Mtb) genome their role and function in the biology and pathogenesis still remains elusive. In this study, we generated and characterized an Mtb H37Rv mutant (MtbΔ33) in which the structural gene of PE_PGRS33, a prototypical member of the protein family, was inactivated. We showed that this mutant entered macrophages with an efficiency up to ten times lower than parental or complemented strains, while its efficiency in infecting pneumocytes remained unaffected. Interestingly, the lack of PE_PGRS33 did not affect the intracellular growth of this mutant in macrophages. Using a series of functional deletion mutants of the PE_PGRS33 gene to complement the MtbΔ33 strain, we demonstrated that the PGRS domain is required to mediate cell entry into macrophages, with the key domain encompassing position 140–260 amino acids of PE_PGRS33. PE_PGRS33-mediated entry into macrophages was abolished in TLR2-deficient mice, as well as following treatment with wortmannin or an antibody against the complement receptor 3 (CR3), indicating that PE_PGRS33-mediated entry of Mtb in macrophages occurs through interaction with TLR2. PMID:26978522

  11. Regulation of primary plant metabolism during plant-pathogen interactions and its contribution to plant defense

    PubMed Central

    Rojas, Clemencia M.; Senthil-Kumar, Muthappa; Tzin, Vered; Mysore, Kirankumar S.

    2014-01-01

    Plants are constantly exposed to microorganisms in the environment and, as a result, have evolved intricate mechanisms to recognize and defend themselves against potential pathogens. One of these responses is the downregulation of photosynthesis and other processes associated with primary metabolism that are essential for plant growth. It has been suggested that the energy saved by downregulation of primary metabolism is diverted and used for defense responses. However, several studies have shown that upregulation of primary metabolism also occurs during plant-pathogen interactions. We propose that upregulation of primary metabolism modulates signal transduction cascades that lead to plant defense responses. In support of this thought, we here compile evidence from the literature to show that upon exposure to pathogens or elicitors, plants induce several genes associated with primary metabolic pathways, such as those involved in the synthesis or degradation of carbohydrates, amino acids and lipids. In addition, genetic studies have confirmed the involvement of these metabolic pathways in plant defense responses. This review provides a new perspective highlighting the relevance of primary metabolism in regulating plant defense against pathogens with the hope to stimulate further research in this area. PMID:24575102

  12. Polarization contributions to intermolecular interactions revisited with fragment electric-field response functions

    SciTech Connect

    Horn, Paul R. E-mail: mhg@cchem.berkeley.edu; Head-Gordon, Martin E-mail: mhg@cchem.berkeley.edu

    2015-09-21

    The polarization energy in intermolecular interactions treated by self-consistent field electronic structure theory is often evaluated using a constraint that the atomic orbital (AO) to molecular orbital transformation is blocked by fragments. This approach is tied to AO basis sets, overestimates polarization energies in the overlapping regime, particularly in large AO basis sets, and lacks a useful complete basis set limit. These problems are addressed by the construction of polarization subspaces based on the responses of isolated fragments to weak electric fields. These subspaces are spanned by fragment electric-field response functions, which can capture effects up to the dipole (D), or quadrupole (DQ) level, or beyond. Schemes are presented for the creation of both non-orthogonal and orthogonal fragment subspaces, and the basis set convergence of the polarization energies computed using these spaces is assessed. Numerical calculations for the water dimer, water–Na{sup +}, water–Mg{sup 2+}, water–F{sup −}, and water–Cl{sup −} show that the non-orthogonal DQ model is very satisfactory, with small differences relative to the orthogonalized model. Additionally, we prove a fundamental difference between the polarization degrees of freedom in the fragment-blocked approaches and in constrained density schemes. Only the former are capable of properly prohibiting charge delocalization during polarization.

  13. Polarization contributions to intermolecular interactions revisited with fragment electric-field response functions.

    PubMed

    Horn, Paul R; Head-Gordon, Martin

    2015-09-21

    The polarization energy in intermolecular interactions treated by self-consistent field electronic structure theory is often evaluated using a constraint that the atomic orbital (AO) to molecular orbital transformation is blocked by fragments. This approach is tied to AO basis sets, overestimates polarization energies in the overlapping regime, particularly in large AO basis sets, and lacks a useful complete basis set limit. These problems are addressed by the construction of polarization subspaces based on the responses of isolated fragments to weak electric fields. These subspaces are spanned by fragment electric-field response functions, which can capture effects up to the dipole (D), or quadrupole (DQ) level, or beyond. Schemes are presented for the creation of both non-orthogonal and orthogonal fragment subspaces, and the basis set convergence of the polarization energies computed using these spaces is assessed. Numerical calculations for the water dimer, water-Na(+), water-Mg(2+), water-F(-), and water-Cl(-) show that the non-orthogonal DQ model is very satisfactory, with small differences relative to the orthogonalized model. Additionally, we prove a fundamental difference between the polarization degrees of freedom in the fragment-blocked approaches and in constrained density schemes. Only the former are capable of properly prohibiting charge delocalization during polarization. PMID:26395691

  14. Affinity thermoprecipitatin: Contribution of the efficiency of ligand-protein interaction and access of the ligand.

    PubMed

    Galaev, I Y; Mattiasson, B

    1993-05-01

    Conjugates to two thermoprecipitating polymers, poly(N-vinyl caprolactam) and poly(N-isopropylacrylmide), with soybean trypsin inhibitor, Cibacron Blue 3GA, Cu-iminodiacetic acid, and p-aminobenzamidine were synthesized. The interaction of these conjugates with trypsin and lactate dehydrogenase was studied. Coupling of the ligand to a polymer resulted in a 100-1000-fold decrease in enzyme-affinity. Rough theoretical estimates revealed that a successful affinity precipitation required that the binding of a target protein and a ligand coupled to a polymer have binding constants on the order of 10(-7)-10(-8) M. Such strong affinity of low molecular weight ligands that can provide binding constants of 10(-9)-10(-11) M or alternatively multipoint attachment of the target protein molecule. The ligand in the ligand-polymer conjugate is still accessible to the protein after thermoprecipitation, and the latter can bind with the particle of the dispersion of thermoprecipitated ligand-polymer precipitate may result in stripping of enzyme molecules from the surface of the particles. PMID:18601296

  15. Do Interactions Between Gut Ecology and Environmental Chemicals Contribute to Obesity and Diabetes?

    PubMed Central

    Snedeker, Suzanne M.

    2011-01-01

    Background: Gut microbiota are important factors in obesity and diabetes, yet little is known about their role in the toxicodynamics of environmental chemicals, including those recently found to be obesogenic and diabetogenic. Objectives: We integrated evidence that independently links gut ecology and environmental chemicals to obesity and diabetes, providing a framework for suggesting how these environmental factors may interact with these diseases, and identified future research needs. Methods: We examined studies with germ-free or antibiotic-treated laboratory animals, and human studies that evaluated how dietary influences and microbial changes affected obesity and diabetes. Strengths and weaknesses of studies evaluating how environmental chemical exposures may affect obesity and diabetes were summarized, and research gaps on how gut ecology may affect the disposition of environmental chemicals were identified. Results: Mounting evidence indicates that gut microbiota composition affects obesity and diabetes, as does exposure to environmental chemicals. The toxicology and pharmacology literature also suggests that interindividual variations in gut microbiota may affect chemical metabolism via direct activation of chemicals, depletion of metabolites needed for biotransformation, alteration of host biotransformation enzyme activities, changes in enterohepatic circulation, altered bioavailability of environmental chemicals and/or antioxidants from food, and alterations in gut motility and barrier function. Conclusions: Variations in gut microbiota are likely to affect human toxicodynamics and increase individual exposure to obesogenic and diabetogenic chemicals. Combating the global obesity and diabetes epidemics requires a multifaceted approach that should include greater emphasis on understanding and controlling the impact of interindividual gut microbe variability on the disposition of environmental chemicals in humans. PMID:22042266

  16. Mothers as a resource in times of stress: interactive contributions of socialization of coping and stress to youth psychopathology.

    PubMed

    Abaied, Jamie L; Rudolph, Karen D

    2010-02-01

    This study examined the hypothesis that maternal socialization of coping would make a differential contribution to youth depression and externalizing psychopathology depending on youths' level of exposure to life stress. A sample of 155 youth (M age = 12.41, SD = 1.21) and their maternal caregivers completed semi-structured interviews and questionnaires in a two-wave longitudinal study over a 1-year period. Results provided evidence for two types of socialization x stress interactions-an amplification-effects model and a differential-effects model. In the context of interpersonal stress, findings supported an amplification-effects model wherein the risk and protective effects of engagement and disengagement socialization of coping emerged in youth exposed to high but not mild levels of stress. In the context of noninterpersonal stress, findings supported a differential-effects model wherein disengagement socialization of coping contributed to heightened risk among youth exposed to high stress but dampened risk among youth exposed to mild stress. This research identifies maternal socialization of coping as a noteworthy contributor to risk for youth psychopathology, and highlights the need to consider parenting x environment interactions when investigating parenting processes related to youth psychopathology. PMID:19908139

  17. Interparticle interactions and surface contribution to the effective anisotropy in biocompatible iron oxide nanoparticles used for contrast agents

    NASA Astrophysics Data System (ADS)

    Arelaro, A. D.; Brandl, A. L.; Lima, E.; Gamarra, L. F.; Brito, G. E. S.; Pontuschka, W. M.; Goya, G. F.

    2005-05-01

    We have investigated the dynamic magnetic properties of dextran-coated magnetite (Fe3O4) nanoparticles in the form of (a) particles suspended in a carrier liquid and (b) concentrated powder obtained from lyophilization. The blocking temperature was found to increase from TB=42(2)to52(2)K (@μ0H=10mT) after lyophilization, showing the effects of dipolar interactions in samples with identical size distributions. The temperature dependence of the hyperfine field Bhyp(T) reveals the effects of collective magnetic excitations at low temperature, and allowed us to obtain the magnetic anisotropy energy Ea=3.6×10-21J for noninteracting particles. The obtained values can be understood assuming only magnetocrystalline anisotropy, without any additional contributions from surface, shape, or exchange origin. Moreover, a magnetocrystalline anisotropy constant value K1=10kJ/m3 was obtained by assuming the cubic phase with easy magnetic direction [111] of the bulk material above the Verwey transition, supporting the idea that the Verwey transition is absent in nanosized particles. Accordingly, no indication of magnetic transition at TV could be observed in our measurements. From the dynamical parameters of ac susceptibility χ(f ,T) curves, the contribution of the dipolar interactions to the total anisotropy energy barrier could be estimated to be Ω =4.5×10-21J, larger than the single-particle value.

  18. Microtubule-Associated Protein 1 Light Chain 3 Interacts with and Contributes to Growth Inhibiting Effect of PML

    PubMed Central

    Hou, Jia-Kai; Fan, Li; Xu, Yi-Wei; Liu, Man-Hua; Yan, Shu-Yang; Chen, Guo-Qiang; Huang, Ying

    2014-01-01

    Previously we reported that the expression of promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARα) fusion gene, which is caused by specific translocation (15;17) in acute promyelocytic leukemia, can enhance constitutive autophagic activity in leukemic and nonleukemic cells, and PML overexpression can sequestrate part of microtubule-associated protein light chain 3 (LC3) protein in PML nuclear bodies, suggesting that LC3 protein also distributes into nuclei although it is currently thought to function primarily in the cytoplasm, the site of autophagosomal formation. However, its potential significance of nucleoplasmic localizations remains greatly elusive. Here we demonstrate that PML interacts with LC3 in a cell type-independent manner as assessed by Co-IP assay and co-localization observation. Overexpressed PML significantly coprecipitates with endogenous and nuclear LC3 protein. Furthermore, a fraction of endogenous PML protein is found to be co-localized with LC3 protein under steady state condition, which is further enhanced by IFNα induction, indicating that PML up-regulation potentiates this interaction. Additionally, DsRed-PML associates with EGFP-LC3 during telophase and G1 phase but not in metaphase and anaphase. Two potential LC3-interacting region (LIR) motifs in PML are required for interaction of PML with LC3 while this association is independent of autophagic activity. Finally, we show that interaction between PML and LC3 contributes to cell growth inhibition function of PML. Considering that PML is an important tumor suppressor, we propose that nuclear portion of LC3 protein may associate with PML to control cell growth for prevention and inhibition of cancer occurrence and development. PMID:25419843

  19. Microtubule-associated protein 1B interaction with tubulin tyrosine ligase contributes to the control of microtubule tyrosination.

    PubMed

    Utreras, Elías; Jiménez-Mateos, Eva Maria; Contreras-Vallejos, Erick; Tortosa, Elena; Pérez, Mar; Rojas, Sebastián; Saragoni, Lorena; Maccioni, Ricardo B; Avila, Jesús; González-Billault, Christian

    2008-01-01

    Microtubule-associated protein 1B (MAP1B) is the first microtubule-associated protein to be expressed during nervous system development. MAP1B belongs to a large family of proteins that contribute to the stabilization and/or enhancement of microtubule polymerization. These functions are related to the control of the dynamic properties of microtubules. The C-terminal domain of the neuronal alpha-tubulin isotype is characterized by the presence of an acidic polypeptide, with the last amino acid being tyrosine. This tyrosine residue may be enzymatically removed from the protein by an unknown carboxypeptidase activity. Subsequently, the tyrosine residue is again incorporated into this tubulin by another enzyme, tubulin tyrosine ligase, to yield tyrosinated tubulin. Because neurons lacking MAP1B have a reduced proportion of tyrosinated microtubules, we analyzed the possible interaction between MAP1B and tubulin tyrosine ligase. Our results show that these proteins indeed interact and that the interaction is not affected by MAP1B phosphorylation. Additionally, neurons lacking MAP1B, when exposed to drugs that reversibly depolymerize microtubules, do not fully recover tyrosinated microtubules upon drug removal. These results suggest that MAP1B regulates tyrosination of alpha-tubulin in neuronal microtubules. This regulation may be important for general processes involved in nervous system development such as axonal guidance and neuronal migration. PMID:18075266

  20. A Surface Enolase Participates in Borrelia burgdorferi-Plasminogen Interaction and Contributes to Pathogen Survival within Feeding Ticks

    PubMed Central

    Nogueira, Sarah Veloso; Smith, Alexis A.; Qin, Jin-Hong

    2012-01-01

    Borrelia burgdorferi, a tick-borne bacterial pathogen, causes a disseminated infection involving multiple organs known as Lyme disease. Surface proteins can directly participate in microbial virulence by facilitating pathogen dissemination via interaction with host factors. We show here that a fraction of the B. burgdorferi chromosomal gene product BB0337, annotated as enolase or phosphopyruvate dehydratase, is associated with spirochete outer membrane and is surface exposed. B. burgdorferi enolase, either in a recombinant form or as a membrane-bound native antigen, displays enzymatic activities intrinsic to the glycolytic pathway. However, the protein also interacts with host plasminogen, potentially leading to its activation and resulting in B. burgdorferi-induced fibrinolysis. As expected, enolase displayed consistent expression in vivo, however, with a variable temporal and spatial expression during spirochete infection in mice and ticks. Despite an extracellular exposure of the antigen and a potential role in host-pathogen interaction, active immunization of mice with recombinant enolase failed to evoke protective immunity against subsequent B. burgdorferi infection. In contrast, enolase immunization of murine hosts significantly reduced the acquisition of spirochetes by feeding ticks, suggesting that the protein could have a stage-specific role in B. burgdorferi survival in the feeding vector. Strategies to interfere with the function of surface enolase could contribute to the development of novel preventive measures to interrupt the spirochete infection cycle and reduce the incidences of Lyme disease. PMID:22025510

  1. Manufactured and Airborne Nanoparticle Cardiopulmonary Interactions: A Review of Mechanisms and the Possible Contribution of Mast Cells

    PubMed Central

    Shannahan, Jonathan H.; Kodavanti, Urmila P.; Brown, Jared M.

    2013-01-01

    Human inhalation exposures to manufactured nanoparticles (NP) and airborne ultrafine particles (UFP) continues to increase in both occupational and environmental settings. UFP exposures have been associated with increased cardiovascular mortality and morbidity, while ongoing research supports adverse systemic and cardiovascular health effects after NP exposures. Adverse cardiovascular health effects include alterations in heart rate variability, hypertension, thrombosis, arrhythmias, increased myocardial infarction, and atherosclerosis. Exactly how UFP and NP cause these negative cardiovascular effects is poorly understood, however a variety of mediators and mechanisms have been proposed. UFP and NP, as well as their soluble components, are known to systemically translocate from the lung. Translocated particles could mediate cardiovascular toxicity through direct interactions with the vasculature, blood, and heart. Recent study suggests that sensory nerve stimulation within the lung may also contribute to UFP- and NP-induced acute cardiovascular alterations. Activation of sensory nerves, such as C-fibers, within the lung may result in altered cardiac rhythm and function. Lastly, release of pulmonary-derived mediators into systemic circulation has been proposed to facilitate cardiovascular effects. In general, these proposed pulmonary-derived mediators include pro-inflammatory cytokines, oxidatively-modified macromolecules, vasoactive proteins, and prothrombotic factors. These pulmonary-derived mediators have been postulated to contribute to the subsequent prothrombotic, atherogenic, and inflammatory effects after exposure. This review will evaluate the potential contribution of individual mediators and mechanisms in facilitating cardiopulmonary toxicity following inhalation of UFP and NP. Lastly we will appraise the literature and propose a hypothesis regarding the possible role of mast cells in contributing to these systemic effects. PMID:22486349

  2. Direct Ca2+-dependent Heterophilic Interaction between Desmosomal Cadherins, Desmoglein and Desmocollin, Contributes to Cell–Cell Adhesion

    PubMed Central

    Chitaev, Nikolai A.; Troyanovsky, Sergey M.

    1997-01-01

    Human fibrosarcoma cells, HT-1080, feature extensive adherens junctions, lack mature desmosomes, and express a single known desmosomal protein, Desmoglein 2 (Dsg2). Transfection of these cells with bovine Desmocollin 1a (Dsc1a) caused dramatic changes in the subcellular distribution of endogenous Dsg2. Both cadherins clustered in the areas of the adherens junctions, whereas only a minor portion of Dsg2 was seen in these areas in the parental cells. Deletion mapping showed that intact extracellular cadherin-like repeats of Dsc1a (Arg1-Thr170) are required for the translocation of Dsg2. Deletion of the intracellular C-domain that mediates the interaction of Dsc1a with plakoglobin, or the CSI region that is involved in the binding to desmoplakin, had no effect. Coimmunoprecipitation experiments of cell lysates stably expressing Dsc1a with anti-Dsc or -Dsg antibodies demonstrate that the desmosomal cadherins, Dsg2 and Dsc1a, are involved in a direct Ca2+-dependent interaction. This conclusion was further supported by the results of solid phase binding experiments. These showed that the Dsc1a fragment containing cadherin-like repeats 1 and 2 binds directly to the extracellular portion of Dsg in a Ca2+-dependent manner. The contribution of the Dsg/ Dsc interaction to cell–cell adhesion was tested by coculturing HT-1080 cells expressing Dsc1a with HT-1080 cells lacking Dsc but expressing myc-tagged plakoglobin (MPg). In the latter cells, MPg and the endogenous Dsg form stable complexes. The observed specific coimmunoprecipitation of MPg by anti-Dsc antibodies in coculture indicates that an intercellular interaction between Dsc1 and Dsg is involved in cell–cell adhesion. PMID:9214392

  3. Activation of focal adhesion kinase through an interaction with β4 integrin contributes to tumorigenicity of colon cancer.

    PubMed

    Tai, Yu-Ling; Lai, I-Rue; Peng, Yu-Ju; Ding, Shih-Torng; Shen, Tang-Long

    2016-06-01

    High expression of either β4 integrin or focal adhesion kinase (FAK) has been reported in human colon cancer. However, it remains unclear how β4 integrin together with FAK contributes to the tumorigenicity of colon cancer. Here, we demonstrate that the co-overexpression of β4 integrin and FAK positively correlates with advanced stages of human colon cancer. Activated β4 integrin interacts with FAK and subsequently induces FAK phosphorylation at Tyr397. Furthermore, ablation of the β4 integrin/FAK complex and/or FAK activation impair colon cancer cell proliferation, anchorage-independent growth, and tumorigenicity. Our data indicate that the β4 integrin/FAK complex and subsequent FAK activation are essential regulators during the tumorigenicity of colon cancer, and we suggest an alternative strategy for colon cancer therapy. PMID:27178753

  4. Structure of a Highly Conserved Domain of Rock1 Required for Shroom-Mediated Regulation of Cell Morphology

    PubMed Central

    Bauer, Robert J.; Heroux, Annie; Zalewski, Jenna K.; Heber, Simone; Dosunmu-Ogunbi, Atinuke M.; Trakselis, Michael A.; Hildebrand, Jeffrey D.; VanDemark, Andrew P.

    2013-01-01

    Rho-associated coiled coil containing protein kinase (Rho-kinase or Rock) is a well-defined determinant of actin organization and dynamics in most animal cells characterized to date. One of the primary effectors of Rock is non-muscle myosin II. Activation of Rock results in increased contractility of myosin II and subsequent changes in actin architecture and cell morphology. The regulation of Rock is thought to occur via autoinhibition of the kinase domain via intramolecular interactions between the N-terminus and the C-terminus of the kinase. This autoinhibited state can be relieved via proteolytic cleavage, binding of lipids to a Pleckstrin Homology domain near the C-terminus, or binding of GTP-bound RhoA to the central coiled-coil region of Rock. Recent work has identified the Shroom family of proteins as an additional regulator of Rock either at the level of cellular distribution or catalytic activity or both. The Shroom-Rock complex is conserved in most animals and is essential for the formation of the neural tube, eye, and gut in vertebrates. To address the mechanism by which Shroom and Rock interact, we have solved the structure of the coiled-coil region of Rock that binds to Shroom proteins. Consistent with other observations, the Shroom binding domain is a parallel coiled-coil dimer. Using biochemical approaches, we have identified a large patch of residues that contribute to Shrm binding. Their orientation suggests that there may be two independent Shrm binding sites on opposing faces of the coiled-coil region of Rock. Finally, we show that the binding surface is essential for Rock colocalization with Shroom and for Shroom-mediated changes in cell morphology. PMID:24349032

  5. Genomic distance entrained clustering and regression modelling highlights interacting genomic regions contributing to proliferation in breast cancer

    PubMed Central

    2010-01-01

    Background Genomic copy number changes and regional alterations in epigenetic states have been linked to grade in breast cancer. However, the relative contribution of specific alterations to the pathology of different breast cancer subtypes remains unclear. The heterogeneity and interplay of genomic and epigenetic variations means that large datasets and statistical data mining methods are required to uncover recurrent patterns that are likely to be important in cancer progression. Results We employed ridge regression to model the relationship between regional changes in gene expression and proliferation. Regional features were extracted from tumour gene expression data using a novel clustering method, called genomic distance entrained agglomerative (GDEC) clustering. Using gene expression data in this way provides a simple means of integrating the phenotypic effects of both copy number aberrations and alterations in chromatin state. We show that regional metagenes derived from GDEC clustering are representative of recurrent regions of epigenetic regulation or copy number aberrations in breast cancer. Furthermore, detected patterns of genomic alterations are conserved across independent oestrogen receptor positive breast cancer datasets. Sequential competitive metagene selection was used to reveal the relative importance of genomic regions in predicting proliferation rate. The predictive model suggested additive interactions between the most informative regions such as 8p22-12 and 8q13-22. Conclusions Data-mining of large-scale microarray gene expression datasets can reveal regional clusters of co-ordinate gene expression, independent of cause. By correlating these clusters with tumour proliferation we have identified a number of genomic regions that act together to promote proliferation in ER+ breast cancer. Identification of such regions should enable prioritisation of genomic regions for combinatorial functional studies to pinpoint the key genes and interactions

  6. RNA polymerase III drives alternative splicing of the potassium channel–interacting protein contributing to brain complexity and neurodegeneration

    PubMed Central

    Massone, Sara; Vassallo, Irene; Castelnuovo, Manuele; Fiorino, Gloria; Gatta, Elena; Robello, Mauro; Borghi, Roberta; Tabaton, Massimo; Russo, Claudio; Dieci, Giorgio; Cancedda, Ranieri

    2011-01-01

    Alternative splicing generates protein isoforms that are conditionally or differentially expressed in specific tissues. The discovery of factors that control alternative splicing might clarify the molecular basis of biological and pathological processes. We found that IL1-α−dependent up-regulation of 38A, a small ribonucleic acid (RNA) polymerase III–transcribed RNA, drives the synthesis of an alternatively spliced form of the potassium channel–interacting protein (KCNIP4). The alternative KCNIP4 isoform cannot interact with the γ-secretase complex, resulting in modification of γ-secretase activity, amyloid precursor protein processing, and increased secretion of β-amyloid enriched in the more toxic Aβ x-42 species. Notably, synthesis of the variant KCNIP4 isoform is also detrimental to brain physiology, as it results in the concomitant blockade of the fast kinetics of potassium channels. This alternative splicing shift is observed at high frequency in tissue samples from Alzheimer’s disease patients, suggesting that RNA polymerase III cogenes may be upstream determinants of alternative splicing that significantly contribute to homeostasis and pathogenesis in the brain. PMID:21624954

  7. Partial Dominance, Overdominance, Epistasis and QTL by Environment Interactions Contribute to Heterosis in Two Upland Cotton Hybrids.

    PubMed

    Shang, Lianguang; Wang, Yumei; Cai, Shihu; Wang, Xiaocui; Li, Yuhua; Abduweli, Abdugheni; Hua, Jinping

    2016-03-01

    Based on two recombinant inbred line (RIL) populations, two corresponding backcross (BC) populations were constructed to elucidate the genetic basis of heterosis in Upland cotton (Gossypium hirsutum L.). The yield, and yield components, of these populations were evaluated in three environments. At the single-locus level, 78 and 66 quantitative trait loci (QTL) were detected using composite interval mapping in RIL and BC populations, respectively, and 29 QTL were identified based on mid-parental heterosis (MPH) data of two hybrids. Considering all traits together, a total of 50 (64.9%) QTL with partial dominance effect, and 27 (35.1%) QTL for overdominance effect were identified in two BC populations. At the two-locus level, 120 and 88 QTL with main effects (M-QTL), and 335 and 99 QTL involved in digenic interactions (E-QTL), were detected by inclusive composite interval mapping in RIL and BC populations, respectively. A large number of QTL by environment interactions (QEs) for M-QTL and E-QTL were detected in three environments. For most traits, average E-QTL explained a larger proportion of phenotypic variation than did M-QTL in two RIL populations and two BC populations. It was concluded that partial dominance, overdominance, epistasis, and QEs all contribute to heterosis in Upland cotton, and that partial dominance resulting from single loci and epistasis play a relatively more important role than other genetic effects in heterosis in Upland cotton. PMID:26715091

  8. Functional interactions between ubiquitin E2 enzymes and TRIM proteins.

    PubMed

    Napolitano, Luisa M; Jaffray, Ellis G; Hay, Ronald T; Meroni, Germana

    2011-03-01

    The TRIM (tripartite motif) family of proteins is characterized by the presence of the tripartite motif module, composed of a RING domain, one or two B-box domains and a coiled-coil region. TRIM proteins are involved in many cellular processes and represent the largest subfamily of RING-containing putative ubiquitin E3 ligases. Whereas their role as E3 ubiquitin ligases has been presumed, and in several cases established, little is known about their specific interactions with the ubiquitin-conjugating E2 enzymes or UBE2s. In the present paper, we report a thorough screening of interactions between the TRIM and UBE2 families. We found a general preference of the TRIM proteins for the D and E classes of UBE2 enzymes, but we also revealed very specific interactions between TRIM9 and UBE2G2, and TRIM32 and UBE2V1/2. Furthermore, we demonstrated that the TRIM E3 activity is only manifest with the UBE2 with which they interact. For most specific interactions, we could also observe subcellular co-localization of the TRIM involved and its cognate UBE2 enzyme, suggesting that the specific selection of TRIM-UBE2 pairs has physiological relevance. Our findings represent the basis for future studies on the specific reactions catalysed by the TRIM E3 ligases to determine the fate of their targets. PMID:21143188

  9. Abnormal Interactions between Perifollicular Mast Cells and CD8+ T-Cells May Contribute to the Pathogenesis of Alopecia Areata

    PubMed Central

    Bertolini, Marta; Zilio, Federica; Rossi, Alfredo; Gilhar, Amos; Keren, Aviad; Meyer, Katja C.; Wang, Eddy; Funk, Wolfgang; McElwee, Kevin; Paus, Ralf

    2014-01-01

    Alopecia areata (AA) is a CD8+ T-cell dependent autoimmune disease of the hair follicle (HF) in which the collapse of HF immune privilege (IP) plays a key role. Mast cells (MCs) are crucial immunomodulatory cells implicated in the regulation of T cell-dependent immunity, IP, and hair growth. Therefore, we explored the role of MCs in AA pathogenesis, focusing on MC interactions with CD8+ T-cells in vivo, in both human and mouse skin with AA lesions. Quantitative (immuno-)histomorphometry revealed that the number, degranulation and proliferation of perifollicular MCs are significantly increased in human AA lesions compared to healthy or non-lesional control skin, most prominently in subacute AA. In AA patients, perifollicular MCs showed decreased TGFβ1 and IL-10 but increased tryptase immunoreactivity, suggesting that MCs switch from an immuno-inhibitory to a pro-inflammatory phenotype. This concept was supported by a decreased number of IL-10+ and PD-L1+ MCs, while OX40L+, CD30L+, 4–1BBL+ or ICAM-1+ MCs were increased in AA. Lesional AA-HFs also displayed significantly more peri- and intrafollicular- CD8+ T-cells as well as more physical MC/CD8+ T-cell contacts than healthy or non-lesional human control skin. During the interaction with CD8+ T-cells, AA MCs prominently expressed MHC class I and OX40L, and sometimes 4–1BBL or ICAM-1, suggesting that MC may present autoantigens to CD8+ T-cells and/or co-stimulatory signals. Abnormal MC numbers, activities, and interactions with CD8+ T-cells were also seen in the grafted C3H/HeJ mouse model of AA and in a new humanized mouse model for AA. These phenomenological in vivo data suggest the novel AA pathobiology concept that perifollicular MCs are skewed towards pro-inflammatory activities that facilitate cross-talk with CD8+ T-cells in this disease, thus contributing to triggering HF-IP collapse in AA. If confirmed, MCs and their CD8+ T-cell interactions could become a promising new therapeutic target in the future

  10. Network of protein interactions within the Drosophila inner kinetochore

    PubMed Central

    Richter, Magdalena M.; Poznanski, Jaroslaw; Zdziarska, Anna; Czarnocki-Cieciura, Mariusz; Dadlez, Michal; Glover, David M.

    2016-01-01

    The kinetochore provides a physical connection between microtubules and the centromeric regions of chromosomes that is critical for their equitable segregation. The trimeric Mis12 sub-complex of the Drosophila kinetochore binds to the mitotic centromere using CENP-C as a platform. However, knowledge of the precise connections between Mis12 complex components and CENP-C has remained elusive despite the fundamental importance of this part of the cell division machinery. Here, we employ hydrogen–deuterium exchange coupled with mass spectrometry to reveal that Mis12 and Nnf1 form a dimer maintained by interacting coiled-coil (CC) domains within the carboxy-terminal parts of both proteins. Adjacent to these interacting CCs is a carboxy-terminal domain that also interacts with Nsl1. The amino-terminal parts of Mis12 and Nnf1 form a CENP-C-binding surface, which docks the complex and thus the entire kinetochore to mitotic centromeres. Mutational analysis confirms these precise interactions are critical for both structure and function of the complex. Thus, we conclude the organization of the Mis12–Nnf1 dimer confers upon the Mis12 complex a bipolar, elongated structure that is critical for kinetochore function. PMID:26911623

  11. Contributions of soil moisture interactions to climate change in the tropics in the GLACE-CMIP5 experiment

    NASA Astrophysics Data System (ADS)

    May, Wilhelm; Meier, Arndt; Rummukainen, Markku; Berg, Alexis; Chéruy, Frederique; Hagemann, Stefan

    2015-12-01

    Contributions of changes in soil moisture to the projected climate change in the tropics at the end of the twenty first century are quantified using the simulations from five different global climate models, which contributed to the GLACE-CMIP5 experiment. "GLACE" refers to the Global Land Atmosphere Coupling Experiment and "CMIP5" to the fifth phase of the Coupled Model Intercomparison Project. This is done by relating the overall projected changes in climate to those changes in climate that are related to the projected changes in soil moisture. The study focusses on two particular aspects of the interactions of the soil moisture with climate, the soil moisture-temperature coupling and the soil moisture-precipitation coupling. The simulations show distinct future changes in soil moisture content in the tropics, with a general tendency of increases in the central parts of the tropics and decreases in the subtropics. These changes are associated with corresponding changes in precipitation, with an overall tendency of an approximate 5 % change in soil moisture in response to a precipitation change of 1 mm/day. All five individual models are characterized by the same qualitative behaviour, despite differences in the strength and in the robustness of the coupling between soil moisture and precipitation. The changes in soil moisture content are found to give important contributions to the overall climate change in the tropics. This is in particularly the case for latent and sensible heat flux, for which about 80 % of the overall changes are related to soil moisture changes. Similarly, about 80 % of the overall near-surface temperature changes (with the mean temperature changes in the tropics removed) are associated with soil moisture changes. For precipitation, on the other hand, about 30-40 % of the overall change can be attributed to soil moisture changes. The robustness of the contributions of the soil moisture changes to the overall climate change varies between the

  12. Quercetin, an in vitro inhibitor of CYP3A, does not contribute to the interaction between nifedipine and grapefruit juice.

    PubMed Central

    Rashid, J; McKinstry, C; Renwick, A G; Dirnhuber, M; Waller, D G; George, C F

    1993-01-01

    Quercetin, a flavonoid present in various fruits, is a potent in vitro inhibitor of CYP3A. Its role in the reported interaction between grapefruit juice and nifedipine has been determined in vivo in humans. Eight healthy volunteers were given in random order 10 mg nifedipine orally, either alone or with 200 ml double strength grapefruit juice, or with 400 mg quercetin. The area under the plasma concentration-time curve (AUC) for nifedipine with grapefruit juice (mean 320 ng ml(-1) h) was increased significantly (P < 0.01) compared with the AUC when nifedipine was given alone (mean 218 ng ml(-1) h). The time to peak plasma concentration for nifedipine with grapefruit juice (1.5 h) was also increased (P < 0.05) compared with control (0.5 h) suggesting delayed absorption. Although quercetin delayed the time to peak nifedipine concentration (1.3 h) it did not alter the AUC of either the parent drug (mean 209 ng ml(-1) h) or its first-pass metabolite. The results suggest that quercetin does not contribute to the effects of grapefruit juice (which contains <10 mg of quercetin 200 ml(-1)) on the metabolism of nifedipine. Oral doses of quercetin, similar to those possible from the ingestion of other fruits such as strawberries, do not produce in vivo inhibition of CYP3A mediated metabolism of nifedipine. PMID:12959295

  13. Lipid solvation effects contribute to the affinity of Gly-xxx-Gly motif-mediated helix-helix interactions.

    PubMed

    Johnson, Rachel M; Rath, Arianna; Melnyk, Roman A; Deber, Charles M

    2006-07-18

    Interactions between transmembrane helices are mediated by the concave Gly-xxx-Gly motif surface. Whether Gly residues per se are sufficient for selection of this motif has not been established. Here, we used the in vivo TOXCAT assay to measure the relative affinities of all 18 combinations of Gly, Ala, and Ser "small-xxx-small" mutations in glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP) homodimers. Affinity values were compared with the accessibility to a methylene-sized probe of the total surface area of each helix monomer as a measure of solvation by membrane components. A strong inverse correlation was found between nonpolar-group lipid accessibility and dimer affinity (R = 0.75 for GpA, p = 0.013, and R = 0.81 for MCP, p = 0.004), suggesting that lipid as a poor membrane protein solvent, conceptually analogous to water in soluble protein folding, can contribute to dimer stability and help to define helix-helix interfaces. PMID:16834324

  14. Contributions of gas-phase plasma chemistry to surface modifications and gas-surface interactions: investigations of fluorocarbon rf plasmas

    NASA Astrophysics Data System (ADS)

    Cuddy, Michael F., II

    The fundamental aspects of inductively coupled fluorocarbon (FC) plasma chemistry were examined, with special emphasis on the contributions of gas-phase species to surface modifications. Characterization of the gas-phase constituents of single-source CF4-, C2F6-, C3F 8-, and C3F6-based plasmas was performed using spectroscopic and mass spectrometric techniques. The effects of varying plasma parameters, including applied rf power (P) and system pressure (p) were examined. Optical emission spectroscopy (OES) and laser-induced fluorescence (LIF) spectroscopy were employed to monitor the behavior of excited and ground CFx (x = 1,2) radicals, respectively. Mass spectrometric techniques, including ion energy analyses, elucidated behaviors of nascent ions in the FC plasmas. These gas-phase data were correlated with the net effect of substrate processing for Si and ZrO2 surfaces. Surface-specific analyses were performed for post-processed substrates via x-ray photoelectron spectroscopy (XPS) and contact angle goniometry. Generally, precursors with lower F/C ratios tended to deposit robust FC films of high surface energy. Precursors of higher F/C ratio, such as CF4, were associated with etching or removal of material from surfaces. Nonetheless, a net balance between deposition of FC moieties and etching of material exists for each plasma system. The imaging of radicals interacting with surfaces (IRIS) technique provided insight into the phenomena occurring at the interface of the plasma gas-phase and substrate of interest. IRIS results demonstrate that CFx radicals scatter copiously, with surface scatter coefficients, S, generally greater than unity under most experimental conditions. Such considerable S values imply surface-mediated production of the CFx radicals at FC-passivated sites. It is inferred that the primary route to surface production of CFx arises from energetic ion bombardment and ablation of surface FC films. Other factors which may influence the observed CFx

  15. Architecture of the Smc5/6 Complex of Saccharomyces cerevisiae Reveals a Unique Interaction between the Nse5-6 Subcomplex and the Hinge Regions of Smc5 and Smc6.

    PubMed

    Duan, Xinyuan; Yang, Yan; Chen, Yu-Hung; Arenz, Jacqueline; Rangi, Gurdish K; Zhao, Xiaolan; Ye, Hong

    2009-03-27

    The evolutionarily conserved structural maintenance of chromosome (SMC) proteins forms the core structures of three multisubunit complexes as follows: cohesin, condensin, and the Smc5/6 complex. These complexes play crucial roles in different aspects of chromosomal organization, duplication, and segregation. Although the architectures of cohesin and condensin are better understood, that of the more recently identified Smc5/6 complex remains to be elucidated. We have previously shown that the Smc5/6 complex of Saccharomyces cerevisiae contains Smc5, Smc6, and six non-SMC elements (Nse1-6). In this study, we investigated the architecture of the budding yeast Smc5/6 complex employing the yeast two-hybrid assay as well as in vitro biochemical approaches using purified recombinant proteins. These analyses revealed that Smc5 and Smc6 associate with each other at their hinge regions and constitute the backbone of the complex, whereas the Nse1-6 subunits form three distinct subcomplexes/entities that interact with different regions of Smc5 and Smc6. The Nse1, -3, and -4 subunits form a stable subcomplex that binds to the head and the adjacent coiled-coil region of Smc5. Nse2 binds to the middle of the coiled-coil region of Smc5. Nse5 and Nse6 interact with each other and, as a heterodimer, bind to the hinge regions of Smc5 and Smc6. These findings provide new insights into the structures of the Smc5/6 complex and lay the foundation for further investigations into the mechanism of its functions. PMID:19141609

  16. Npn-1 Contributes to Axon-Axon Interactions That Differentially Control Sensory and Motor Innervation of the Limb

    PubMed Central

    Bianchi, Elisa; Novitch, Bennett G.; Huber, Andrea B.

    2011-01-01

    The initiation, execution, and completion of complex locomotor behaviors are depending on precisely integrated neural circuitries consisting of motor pathways that activate muscles in the extremities and sensory afferents that deliver feedback to motoneurons. These projections form in tight temporal and spatial vicinities during development, yet the molecular mechanisms and cues coordinating these processes are not well understood. Using cell-type specific ablation of the axon guidance receptor Neuropilin-1 (Npn-1) in spinal motoneurons or in sensory neurons in the dorsal root ganglia (DRG), we have explored the contribution of this signaling pathway to correct innervation of the limb. We show that Npn-1 controls the fasciculation of both projections and mediates inter-axonal communication. Removal of Npn-1 from sensory neurons results in defasciculation of sensory axons and, surprisingly, also of motor axons. In addition, the tight coupling between these two heterotypic axonal populations is lifted with sensory fibers now leading the spinal nerve projection. These findings are corroborated by partial genetic elimination of sensory neurons, which causes defasciculation of motor projections to the limb. Deletion of Npn-1 from motoneurons leads to severe defasciculation of motor axons in the distal limb and dorsal-ventral pathfinding errors, while outgrowth and fasciculation of sensory trajectories into the limb remain unaffected. Genetic elimination of motoneurons, however, revealed that sensory axons need only minimal scaffolding by motor axons to establish their projections in the distal limb. Thus, motor and sensory axons are mutually dependent on each other for the generation of their trajectories and interact in part through Npn-1-mediated fasciculation before and within the plexus region of the limbs. PMID:21364975

  17. Progesterone receptor isoforms PRA and PRB differentially contribute to breast cancer cell migration through interaction with focal adhesion kinase complexes

    PubMed Central

    Bellance, Catherine; Khan, Junaid A.; Meduri, Geri; Guiochon-Mantel, Anne; Lombès, Marc; Loosfelt, Hugues

    2013-01-01

    Progesterone receptor (PR) and progestins affect mammary tumorigenesis; however, the relative contributions of PR isoforms A and B (PRA and PRB, respectively) in cancer cell migration remains elusive. By using a bi-inducible MDA-MB-231 breast cancer cell line expressing PRA and/or PRB, we analyzed the effect of conditional PR isoform expression. Surprisingly, unliganded PRB but not PRA strongly enhanced cell migration as compared with PR(–) cells. 17,21-Dimethyl-19-norpregna-4,9-dien-3,20-dione (R5020) progestin limited this effect and was counteracted by the antagonist 11β-(4-dimethyl­amino)­phenyl-17β-hydroxy-17-(1-propynyl)­estra-4,9-dien-3-one (RU486). Of importance, PRA coexpression potentiated PRB-mediated migration, whereas PRA alone was ineffective. PR isoforms differentially regulated expressions of major players of cell migration, such as urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type 1, uPA receptor (uPAR), and β1-integrin, which affect focal adhesion kinase (FAK) signaling. Moreover, unliganded PRB but not PRA enhanced FAK Tyr397 phosphorylation and colocalized with activated FAK in cell protrusions. Because PRB, as well as PRA, coimmunoprecipitated with FAK, both isoforms can interact with FAK complexes, depending on their respective nucleocytoplasmic trafficking. In addition, FAK degradation was coupled to R5020-dependent turnovers of PRA and PRB. Such an effect of PRB/PRA expression on FAK signaling might thus affect adhesion/motility, underscoring the implication of PR isoforms in breast cancer invasiveness and metastatic evolution with underlying therapeutic outcomes. PMID:23485561

  18. Analysis of Van der Waals interactions between nanoparticles with different geometries, with accounting for three-particle contributions to the total energy

    NASA Astrophysics Data System (ADS)

    Emelyanenko, K. A.

    2016-05-01

    The Axilrod-Teller-Muto method with corrections for triple interactions is used to calculate the energies of Van der Waals interaction for nanosystems containing particles with different geometries. Results are presented for symmetric systems with identical cubic particles of different sizes, for film and cubic particle systems, and for the systems with differently oriented nanorods. Boundary and particle arrangement effects are studied. The fundamental importance of allowing for nonadditive contributions to obtain a reliable quantitative description of interaction processes inside nanosystems is demonstrated. The results are compared to ones obtained using analytical macroscopic methods and the limits of the applicability of macroscopic approximations are estimated.

  19. Importance of Hydrodynamic Interactions in the Stepping Kinetics of Kinesin.

    PubMed

    Goldtzvik, Yonathan; Zhang, Zhechun; Thirumalai, D

    2016-03-01

    Conventional kinesin walks by a hand-over-hand mechanism on the microtubule (MT) by taking ∼8 nm discrete steps and consumes one ATP molecule per step. The time needed to complete a single step is on the order of 20 μs. We show, using simulations of a coarse-grained model of the complex containing the two motor heads, the MT and the coiled coil, that to obtain quantitative agreement with experiments for the stepping kinetics hydrodynamic interactions (HIs) have to be included. In simulations without hydrodynamic interactions, spanning nearly 20 μs, not a single step was completed in one hundred trajectories. In sharp contrast, nearly 14% of the steps reached the target binding site within 6 μs when HIs were included. Somewhat surprisingly, there are qualitative differences in the diffusion pathways in simulations with and without HI. The extent of movement of the trailing head of kinesin on the MT during the diffusion stage of stepping is considerably greater in simulations with HI than in those without HI. It is likely that inclusion of HI is crucial in the accurate description of motility of other motors as well. PMID:26702870

  20. Quantification of cytosolic interactions identifies Ede1 oligomers as key organizers of endocytosis

    PubMed Central

    Boeke, Dominik; Trautmann, Susanne; Meurer, Matthias; Wachsmuth, Malte; Godlee, Camilla; Knop, Michael; Kaksonen, Marko

    2014-01-01

    Clathrin-mediated endocytosis is a highly conserved intracellular trafficking pathway that depends on dynamic protein–protein interactions between up to 60 different proteins. However, little is known about the spatio-temporal regulation of these interactions. Using fluorescence (cross)-correlation spectroscopy in yeast, we tested 41 previously reported interactions in vivo and found 16 to exist in the cytoplasm. These detected cytoplasmic interactions included the self-interaction of Ede1, homolog of mammalian Eps15. Ede1 is the crucial scaffold for the organization of the early stages of endocytosis. We show that oligomerization of Ede1 through its central coiled coil domain is necessary for its localization to the endocytic site and we link the oligomerization of Ede1 to its function in locally concentrating endocytic adaptors and organizing the endocytic machinery. Our study sheds light on the importance of the regulation of protein–protein interactions in the cytoplasm for the assembly of the endocytic machinery in vivo. PMID:25366307

  1. Inter-Subunit Interactions across the Upper Voltage Sensing-Pore Domain Interface Contribute to the Concerted Pore Opening Transition of Kv Channels

    PubMed Central

    Shem-Ad, Tzilhav; Irit, Orr; Yifrach, Ofer

    2013-01-01

    The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels. PMID:24340010

  2. The Contribution of High-Order Metabolic Interactions to the Global Activity of a Four-Species Microbial Community.

    PubMed

    Guo, Xiaokan; Boedicker, James Q

    2016-09-01

    The activity of a biological community is the outcome of complex processes involving interactions between community members. It is often unclear how to accurately incorporate these interactions into predictive models. Previous work has shown a range of positive and negative metabolic pairwise interactions between species. Here we examine the ability of a modified general Lotka-Volterra model with cell-cell interaction coefficients to predict the overall metabolic rate of a well-mixed microbial community comprised of four heterotrophic natural isolates, experimentally quantifying the strengths of two, three, and four-species interactions. Within this community, interactions between any pair of microbial species were positive, while higher-order interactions, between 3 or more microbial species, slightly modulated community metabolism. For this simple community, the metabolic rate of can be well predicted only with taking into account pairwise interactions. Simulations using the experimentally determined interaction parameters revealed that spatial heterogeneity in the distribution of cells increased the importance of multispecies interactions in dictating function at both the local and global scales. PMID:27623159

  3. A simple retinal mechanism contributes to perceptual interactions between rod- and cone-mediated responses in primates.

    PubMed

    Grimes, William N; Graves, Logan R; Summers, Mathew T; Rieke, Fred

    2015-01-01

    Visual perception across a broad range of light levels is shaped by interactions between rod- and cone-mediated signals. Because responses of retinal ganglion cells, the output cells of the retina, depend on signals from both rod and cone photoreceptors, interactions occurring in retinal circuits provide an opportunity to link the mechanistic operation of parallel pathways and perception. Here we show that rod- and cone-mediated responses interact nonlinearly to control the responses of primate retinal ganglion cells; these nonlinear interactions, surprisingly, were asymmetric, with rod responses strongly suppressing subsequent cone responses but not vice-versa. Human psychophysical experiments revealed a similar perceptual asymmetry. Nonlinear interactions in the retinal output cells were well-predicted by linear summation of kinetically-distinct rod- and cone-mediated signals followed by a synaptic nonlinearity. These experiments thus reveal how a simple mechanism controlling interactions between parallel pathways shapes circuit output and perception. PMID:26098124

  4. The Contribution of the Dyadic Parent-Child Interaction Coding System (DPICS) Warm-Up Segments in Assessing Parent-Child Interactions

    ERIC Educational Resources Information Center

    Shanley, Jenelle R.; Niec, Larissa N.

    2011-01-01

    This study evaluated the inclusion of uncoded segments in the Dyadic Parent-Child Interaction Coding System, an analogue observation of parent-child interactions. The relationships between warm-up and coded segments were assessed, as well as the segments' associations with parent ratings of parent and child behaviors. Sixty-nine non-referred…

  5. To What Extent Do Teacher-Student Interaction Quality and Student Gender Contribute to Fifth Graders' Engagement in Mathematics Learning?

    ERIC Educational Resources Information Center

    Rimm-Kaufman, Sara E.; Baroody, Alison E.; Larsen, Ross A. A.; Curby, Timothy W.; Abry, Tashia

    2015-01-01

    This study examines concurrent teacher-student interaction quality and 5th graders' (n = 387) engagement in mathematics classrooms (n = 63) and considers how teacher-student interaction quality relates to engagement differently for boys and girls. Three approaches were used to measure student engagement in mathematics: Research assistants observed…

  6. Deriving Heterospecific Self-Assembling Protein–Protein Interactions Using a Computational Interactome Screen

    PubMed Central

    Crooks, Richard O.; Baxter, Daniel; Panek, Anna S.; Lubben, Anneke T.; Mason, Jody M.

    2016-01-01

    Interactions between naturally occurring proteins are highly specific, with protein-network imbalances associated with numerous diseases. For designed protein–protein interactions (PPIs), required specificity can be notoriously difficult to engineer. To accelerate this process, we have derived peptides that form heterospecific PPIs when combined. This is achieved using software that generates large virtual libraries of peptide sequences and searches within the resulting interactome for preferentially interacting peptides. To demonstrate feasibility, we have (i) generated 1536 peptide sequences based on the parallel dimeric coiled-coil motif and varied residues known to be important for stability and specificity, (ii) screened the 1,180,416 member interactome for predicted Tm values and (iii) used predicted Tm cutoff points to isolate eight peptides that form four heterospecific PPIs when combined. This required that all 32 hypothetical off-target interactions within the eight-peptide interactome be disfavoured and that the four desired interactions pair correctly. Lastly, we have verified the approach by characterising all 36 pairs within the interactome. In analysing the output, we hypothesised that several sequences are capable of adopting antiparallel orientations. We subsequently improved the software by removing sequences where doing so led to fully complementary electrostatic pairings. Our approach can be used to derive increasingly large and therefore complex sets of heterospecific PPIs with a wide range of potential downstream applications from disease modulation to the design of biomaterials and peptides in synthetic biology. PMID:26655848

  7. Deriving Heterospecific Self-Assembling Protein-Protein Interactions Using a Computational Interactome Screen.

    PubMed

    Crooks, Richard O; Baxter, Daniel; Panek, Anna S; Lubben, Anneke T; Mason, Jody M

    2016-01-29

    Interactions between naturally occurring proteins are highly specific, with protein-network imbalances associated with numerous diseases. For designed protein-protein interactions (PPIs), required specificity can be notoriously difficult to engineer. To accelerate this process, we have derived peptides that form heterospecific PPIs when combined. This is achieved using software that generates large virtual libraries of peptide sequences and searches within the resulting interactome for preferentially interacting peptides. To demonstrate feasibility, we have (i) generated 1536 peptide sequences based on the parallel dimeric coiled-coil motif and varied residues known to be important for stability and specificity, (ii) screened the 1,180,416 member interactome for predicted Tm values and (iii) used predicted Tm cutoff points to isolate eight peptides that form four heterospecific PPIs when combined. This required that all 32 hypothetical off-target interactions within the eight-peptide interactome be disfavoured and that the four desired interactions pair correctly. Lastly, we have verified the approach by characterising all 36 pairs within the interactome. In analysing the output, we hypothesised that several sequences are capable of adopting antiparallel orientations. We subsequently improved the software by removing sequences where doing so led to fully complementary electrostatic pairings. Our approach can be used to derive increasingly large and therefore complex sets of heterospecific PPIs with a wide range of potential downstream applications from disease modulation to the design of biomaterials and peptides in synthetic biology. PMID:26655848

  8. Interaction of Golgin-84 with the COG complex mediates the intra-Golgi retrograde transport.

    PubMed

    Sohda, Miwa; Misumi, Yoshio; Yamamoto, Akitsugu; Nakamura, Nobuhiro; Ogata, Shigenori; Sakisaka, Shotaro; Hirose, Shinichi; Ikehara, Yukio; Oda, Kimimitsu

    2010-12-01

    The coiled-coil Golgi membrane protein golgin-84 functions as a tethering factor for coat protein I (COPI) vesicles. Protein interaction analyses have revealed that golgin-84 interacts with another tether, the conserved oligomeric Golgi (COG) complex, through its subunit Cog7. Therefore, we explored the function of golgin-84 as the tether for COPI vesicles of intra-Golgi retrograde traffic. First, glycosylic maturation of both plasma membrane (CD44) and lysosomal (lamp1) glycoproteins was distorted in golgin-84 knockdown (KD) cells. The depletion of golgin-84 caused fragmentation of the Golgi with the mislocalization of Golgi resident proteins, resulting in the accumulation of vesicles carrying intra-Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and cis-Golgi membrane protein GPP130. Similar observations were obtained by diminution of the COG complex, suggesting a strong correlation between the two tethers. Indeed, COG complex-dependent (CCD) vesicles that accumulate in Cog3 or Cog7 KD cells carried golgin-84. Surprisingly, the interaction between golgin-84 and another candidate tethering partner CASP (CDP/cut alternatively spliced product) decreased in Cog3 KD cells. These results indicate that golgin-84 on COPI vesicles interact with the COG complex before SNARE assembly, suggesting that the interaction of golgin-84 with COG plays an important role in the tethering process of intra-Golgi retrograde vesicle traffic. PMID:20874812

  9. Constituent quark masses obtained from hadron masses with contributions of Fermi-Breit and Glozman-Riska hyperfine interactions

    SciTech Connect

    Borka Jovanovic, V.; Borka, D.; Ignjatovic, S. R.; Jovanovic, P.

    2010-12-01

    We use the color-spin and flavor-spin interaction Hamiltonians with SU(3) flavor symmetry breaking to obtain meson and baryon mass formulas. Adjusting these masses with experimental masses we determine the constituent quark masses. We discuss the constituent quark masses obtained from meson and baryon mass fits. The results for constituent quark masses are very similar in the case of two different phenomenological models: Fermi-Breit and Glozman-Riska hyperfine interactions.

  10. A simple retinal mechanism contributes to perceptual interactions between rod- and cone-mediated responses in primates

    PubMed Central

    Grimes, William N; Graves, Logan R; Summers, Mathew T; Rieke, Fred

    2015-01-01

    Visual perception across a broad range of light levels is shaped by interactions between rod- and cone-mediated signals. Because responses of retinal ganglion cells, the output cells of the retina, depend on signals from both rod and cone photoreceptors, interactions occurring in retinal circuits provide an opportunity to link the mechanistic operation of parallel pathways and perception. Here we show that rod- and cone-mediated responses interact nonlinearly to control the responses of primate retinal ganglion cells; these nonlinear interactions, surprisingly, were asymmetric, with rod responses strongly suppressing subsequent cone responses but not vice-versa. Human psychophysical experiments revealed a similar perceptual asymmetry. Nonlinear interactions in the retinal output cells were well-predicted by linear summation of kinetically-distinct rod- and cone-mediated signals followed by a synaptic nonlinearity. These experiments thus reveal how a simple mechanism controlling interactions between parallel pathways shapes circuit output and perception. DOI: http://dx.doi.org/10.7554/eLife.08033.001 PMID:26098124

  11. Affinity Enhancement by Ligand Clustering Effect Inspired by Peptide Dendrimers−Shank PDZ Proteins Interactions

    PubMed Central

    Liu, Jiahui; Liu, Miao; Zheng, Bo; Yao, Zhongping; Xia, Jiang

    2016-01-01

    High-affinity binders are desirable tools to probe the function that specific protein−protein interactions play in cell. In the process of seeking a general strategy to design high-affinity binders, we found a clue from the βPIX (p21-activated kinase interacting exchange factor)−Shank PDZ interaction in synaptic assembly: three PDZ-binding sites are clustered by a parallel coiled-coil trimer but bind to Shank PDZ protein with 1:1 stoichiometry (1 trimer/1 PDZ). Inspired by this architecture, we proposed that peptide dendrimer, mimicking the ligand clustering in βPIX, will also show enhanced binding affinity, yet with 1:1 stoichiometry. This postulation has been proven here, as we synthesized a set of monomeric, dimeric and trimeric peptides and measured their binding affinity and stoichiometry with Shank PDZ domains by isothermal titration calorimetry, native mass spectrometry and surface plasmon resonance. This affinity enhancement, best explained by proximity effect, will be useful to guide the design of high-affinity blockers for protein−protein interactions. PMID:26918521

  12. RBCK1, an E3 Ubiquitin Ligase, Interacts with and Ubiquinates the Human Pregnane X Receptor

    PubMed Central

    Rana, Ritu; Coulter, Sherry; Kinyamu, Harriet

    2013-01-01

    The pregnane X receptor (PXR, NR1I2) plays a pivotal role in the disposition and detoxification of numerous foreign and endogenous chemicals by increasing transcription of numerous target genes, including phase I and II drug-metabolizing enzymes and transporters. In the present study, yeast two-hybrid screening identified an E3 ubiquitin ligase, RBCK1 (Ring-B-box-coiled-coil protein interacting with protein kinase C-1), as a human pregnane X receptor (hPXR)–interacting protein. Coimmunoprecipitation studies confirmed the interaction between RBCK1 and hPXR when both were ectopically expressed in AD-293 cells. Domain mapping studies showed that the interaction between RBCK1 and hPXR involves all RBCK1 domains. We further demonstrate that RBCK1 ubiquitinates hPXR, and this may target hPXR for degradation by the ubiquitin-proteasome pathway. Simultaneous ectopic overexpression of RBCK1 and PXR decreased PXR levels in AD-293 cells, and this decrease was inhibited by the proteasomal inhibitor MG-132 (carbobenzoxy-Leu-Leu-leucinal). Furthermore, overexpression of RBCK1 decreased endogenous levels of PXR in HepG2 cells. Of importance, ectopic overexpression and silencing of endogenous RBCK1 in primary human hepatocytes resulted in a decrease and increase, respectively, in endogenous PXR protein levels and in the induction of PXR target genes by rifampicin. These results suggest that RBCK1 is important for the ubiquitination of PXR and may play a role in its proteasomal degradation. PMID:23160820

  13. Filamin A Protein Interacts with Human Immunodeficiency Virus Type 1 Gag Protein and Contributes to Productive Particle Assembly*

    PubMed Central

    Cooper, JoAnn; Liu, Ling; Woodruff, Elvin A.; Taylor, Harry E.; Goodwin, J. Shawn; D'Aquila, Richard T.; Spearman, Paul; Hildreth, James E. K.; Dong, Xinhong

    2011-01-01

    HIV-1 Gag precursor directs virus particle assembly and release. In a search for Gag-interacting proteins that are involved in late stages of the HIV-1 replication cycle, we performed yeast two-hybrid screening against a human cDNA library and identified the non-muscle actin filament cross-linking protein filamin A as a novel Gag binding partner. The 280-kDa filamin A regulates cortical actin network dynamics and participates in the anchoring of membrane proteins to the actin cytoskeleton. Recent studies have shown that filamin A facilitates HIV-1 cell-to-cell transmission by binding to HIV receptors and coreceptors and regulating their clustering on the target cell surface. Here we report a novel role for filamin A in HIV-1 Gag intracellular trafficking. We demonstrate that filamin A interacts with the capsid domain of HIV-1 Gag and that this interaction is involved in particle release in a productive manner. Disruption of this interaction eliminated Gag localization at the plasma membrane and induced Gag accumulation within internal compartments. Moreover, blocking clathrin-dependent endocytic pathways did not relieve the restriction to particle release induced by filamin A depletion. These results suggest that filamin A is involved in the distinct step of the Gag trafficking pathway. The discovery of the Gag-filamin A interaction may provide a new therapeutic target for the treatment of HIV infection. PMID:21705339

  14. Functional Contribution of Chorismate Synthase, Anthranilate Synthase, and Chorismate Mutase to Penetration Resistance in Barley-Powdery Mildew Interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant processes resulting from primary or secondary metabolism have been hypothesized to contribute to defense against microbial attack. Barley chorismate synthase (HvCS), anthranilate synthase alpha subunit 2 (HvASa2) and chorismate mutase 1 (HvCM1) occupy pivotal branch-points downstream of the s...

  15. Mothers as a Resource in Times of Stress: Interactive Contributions of Socialization of Coping and Stress to Youth Psychopathology

    ERIC Educational Resources Information Center

    Abaied, Jamie L.; Rudolph, Karen D.

    2010-01-01

    This study examined the hypothesis that maternal socialization of coping would make a differential contribution to youth depression and externalizing psychopathology depending on youths' level of exposure to life stress. A sample of 155 youth (M age = 12.41, SD = 1.21) and their maternal caregivers completed semi-structured interviews and…

  16. What Are the Unique and Interacting Contributions of School and Family Factors to Early Adolescents' Empathic Concern and Perspective Taking?

    ERIC Educational Resources Information Center

    Batanova, Milena D.; Loukas, Alexandra

    2012-01-01

    Empathy in children has received considerable attention in the literature, but limited research has investigated the contributions of various socializing factors on both affective (e.g., empathic concern) and cognitive (e.g., perspective taking) components of empathy in early adolescents. Guided by socialization theories, this study examined the…

  17. Experimental assessment of the contribution of electrodynamic interactions to long-distance recruitment of biomolecular partners: Theoretical basis.

    PubMed

    Preto, Jordane; Floriani, Elena; Nardecchia, Ilaria; Ferrier, Pierre; Pettini, Marco

    2012-04-01

    Highly specific spatiotemporal interactions between cognate molecular partners essentially sustain all biochemical transactions in living matter. That such an exquisite level of accuracy may result from encountering forces solely driven by thermal diffusive processes is unlikely. Here we propose a yet unexplored strategy to experimentally tackle the long-standing question of a possibly active recruitment at a distance of cognate partners of biomolecular reactions via the action of resonant electrodynamic interactions. We considered two simplified models for a preliminary feasibility investigation of the devised methodology. By taking advantage of advanced experimental techniques nowadays available, we propose to measure the characteristic encounter time scales of dually interacting biopartners and to compare them with theoretical predictions worked out in both the presence and absence of putative long-range electromagnetic forces. PMID:22680495

  18. Deformation-corrosion interactions for Zr alloys during I-SCC crack initiation. Part I: Chemical contributions

    NASA Astrophysics Data System (ADS)

    Jacques, Patrick; Lefebvre, Florence; Lemaignan, Clément

    1999-01-01

    For a better understanding of the initiation step of iodine-induced stress corrosion cracking in Zr alloys, responsible for pellet-cladding interaction (PCI) fuel rod failures, an analytical study has been undertaken, the aim of which being focused on the respective roles of local chemistry and stress/strain state on the crack nucleation. This first part is mostly related to the chemical environment. From the tensile tests performed under iodine rich and inert environments, it was concluded that no crack initiation could be detected following the tests in an inert atmosphere. The iodine induced stress corrosion initiation mechanism must therefore be analysed as a corrosion-strain interaction.

  19. The Contribution of Electrostatic and van der Waals Interactions to the Stereospecificity of the Reaction Catalyzed by Lactate Dehydrogenase

    PubMed Central

    van Beek, Jeroen; Callender, Robert; Gunner, M. R.

    1997-01-01

    Continuum electrostatic calculations in conjunction with molecular dynamics simulations have been used to investigate the source of the stereospecificity in the hydride transfer reaction catalyzed by lactate dehydrogenase (LDH). These studies show that favorable electrostatic interactions between the carboxamide group of the reduced nicotinamide adenine dinucleotide coenzyme and protein residues of the active site of LDH can account for much if not all of the stereospecificity of the LDH-catalyzed reaction, with A-side hydride transfer more than 107 times greater than B-side transfer. Unfavorable steric interactions within the binding complex for B-side transfer are not found. ImagesFIGURE 2 PMID:9017191

  20. Scaffold-forming and Adhesive Contributions of Synthetic Laminin-binding Proteins to Basement Membrane Assembly.

    PubMed

    McKee, Karen K; Capizzi, Stephanie; Yurchenco, Peter D

    2009-03-27

    Laminins that possess three short arms contribute to basement membrane assembly by anchoring to cell surfaces, polymerizing, and binding to nidogen and collagen IV. Although laminins containing the alpha4 and alpha5 subunits are expressed in alpha2-deficient congenital muscular dystrophy, they may be ineffective substitutes because they bind weakly to cell surfaces and/or because they lack the third arm needed for polymerization. We asked whether linker proteins engineered to bind to deficient laminins that provide such missing activities would promote basement membrane assembly in a Schwann cell model. A chimeric fusion protein (alphaLNNd) that adds a short arm terminus to laminin through the nidogen binding locus was generated and compared with the dystrophy-ameliorating protein miniagrin (mAgrin) that binds to the laminin coiled-coil dystroglycan and sulfatides. alphaLNNd was found to mediate laminin binding to collagen IV, to bind to galactosyl sulfatide, and to selectively convert alpha-short arm deletion-mutant laminins LmDeltaalphaLN and LmDeltaalphaLN-L4b into polymerizing laminins. This protein enabled polymerization-deficient laminin but not an adhesion-deficient laminin lacking LG domains (LmDeltaLG) to assemble an extracellular matrix on Schwann cell surfaces. mAgrin, on the other hand, enabled LmDeltaLG to form an extracellular matrix on cell surfaces without increasing accumulation of non-polymerizing laminins. These gain-of-function studies reveal distinct polymerization and anchorage contributions to basement membrane assembly in which the three different LN domains mediate the former, and the LG domains provide primary anchorage with secondary contributions from the alphaLN domain. These findings may be relevant for an understanding of the pathogenesis and treatment of laminin deficiency states. PMID:19189961

  1. What Motivates Students to Learn? Contribution of Student-to-Student Relations, Student-Faculty Interaction and Critical Thinking Skills

    ERIC Educational Resources Information Center

    Rugutt, John; Chemosit, Caroline C.

    2009-01-01

    This study used standard multiple linear regression to investigate relationships between student motivation, critical thinking skills, student-to-student relations, and student-faculty interaction with a sample of 2,190 undergraduate students. The study used measures first developed by Ellett, Culross, McMullen, and Rugutt, (1996), and later…

  2. Characteristics of current tasks that contribute to mentalizing judgments: does the engagement of the participants in the social interaction matter? Comment on Achim et al. (2013).

    PubMed

    Champagne-Lavau, Maud; Moreau, Noémie

    2013-12-01

    In a recent article, Achim et al. (2013) discussed the different sources of information that contribute to mentalizing judgments in current theory-of-mind (ToM) tasks. The authors rightly emphasized the dynamic aspect of real-life social interaction, suggesting that taking account of the ongoing changes occurring during social interaction would make ToM tasks more ecological. They proposed a framework (i.e., the Eight Sources of Information Framework) that specifies the 8 sources of information we get from the environment and/or from our memories to attribute mental states to others. Nevertheless, we believe that a central aspect of ToM is missing in this framework: the engagement (or not) of the participant in the social interaction during ToM assessment. Indeed, this framework fails to consider how the participant who takes part in the ToM task manages this information, depending on the fact that he or she is involved in the interaction or not and how the information concerning the agent may impact the participant attribution of mental states. We reviewed several arguments and results from the ToM literature suggesting that merely observing a social interaction is not equivalent to participating in an interaction in terms of cognitive processes involved in the attribution of mental states to others. PMID:24320766

  3. Genetic interactions with sex make a relatively small contribution to the heritability of complex traits in mice.

    PubMed

    Krohn, Jon; Speed, Doug; Palme, Rupert; Touma, Chadi; Mott, Richard; Flint, Jonathan

    2014-01-01

    The extent to which sex-specific genetic effects contribute to phenotypic variation is largely unknown. We applied a novel Bayesian method, sparse partitioning, to detect gene by sex (GxS) and gene by gene (GxG) quantitative loci (QTLs) in 1,900 outbred heterogeneous stock mice. In an analysis of 55 phenotypes, we detected 16 GxS and 6 GxG QTLs. The increase in the amount of phenotypic variance explained by models including GxS was small, ranging from 0.14% to 4.30%. We conclude that GxS rarely make a large overall contribution to the heritability of phenotypes, however there are cases where these will be individually important. PMID:24811081

  4. Interactions between N-Ethylmaleimide-sensitive factor and GluA2 contribute to effects of glucocorticoid hormones on AMPA receptor function in the rodent hippocampus.

    PubMed

    Xiong, Hui; Cassé, Frédéric; Zhou, Ming; Xiong, Zhi-Qi; Joels, Marian; Martin, Stéphane; Krugers, Harm J

    2016-07-01

    Glucocorticoid hormones, via activation of their receptors, promote memory consolidation, but the exact underlying mechanisms remain elusive. We examined how corticosterone regulates AMPA receptor (AMPAR) availability in the synapse, which is important for synaptic plasticity and memory formation. Peptides which specifically block the interaction between N-Ethylmaleimide-Sensitive Factor (NSF) and the AMPAR-subunit GluA2 prevented the increase in synaptic transmission and surface expression of AMPARs known to occur after corticosterone application to hippocampal neurons. Combining a live imaging Fluorescence Recovery After Photobleaching (FRAP) approach with the use of the pH-sensitive GFP-AMPAR tagging revealed that this NSF/GluA2 interaction was also essential for the increase of the mobile fraction and reduction of the diffusion of AMPARs after treating hippocampal neurons with corticosterone. We conclude that the interaction between NSF and GluA2 contributes to the effects of corticosterone on AMPAR function. © 2016 Wiley Periodicals, Inc. PMID:26766634

  5. Sliding Clamp–DNA Interactions Are Required for Viability and Contribute to DNA Polymerase Management in Escherichia coli

    SciTech Connect

    Heltzel, J.; Scouten Ponticelli, S; Sanders, L; Duzen, J; Cody, V; Pace, J; Snell, E; Sutton, M

    2009-01-01

    Sliding clamp proteins topologically encircle DNA and play vital roles in coordinating the actions of various DNA replication, repair, and damage tolerance proteins. At least three distinct surfaces of the Escherichia coli {beta} clamp interact physically with the DNA that it topologically encircles. We utilized mutant {beta} clamp proteins bearing G66E and G174A substitutions ({beta}159), affecting the single-stranded DNA-binding region, or poly-Ala substitutions in place of residues 148-HQDVR-152 ({beta}148-152), affecting the double-stranded DNA binding region, to determine the biological relevance of clamp-DNA interactions. As part of this work, we solved the X-ray crystal structure of {beta}148-152, which verified that the poly-Ala substitutions failed to significantly alter the tertiary structure of the clamp. Based on functional assays, both {beta}159 and {beta}148-152 were impaired for loading and retention on a linear primed DNA in vitro. In the case of {beta}148-152, this defect was not due to altered interactions with the DnaX clamp loader, but rather was the result of impaired {beta}148-152-DNA interactions. Once loaded, {beta}148-152 was proficient for DNA polymerase III (Pol III) replication in vitro. In contrast, {beta}148-152 was severely impaired for Pol II and Pol IV replication and was similarly impaired for direct physical interactions with these Pols. Despite its ability to support Pol III replication in vitro, {beta}148-152 was unable to support viability of E. coli. Nevertheless, physiological levels of {beta}148-152 expressed from a plasmid efficiently complemented the temperature-sensitive growth phenotype of a strain expressing {beta}159 (dnaN159), provided that Pol II and Pol IV were inactivated. Although this strain was impaired for Pol V-dependent mutagenesis, inactivation of Pol II and Pol IV restored the Pol V mutator phenotype. Taken together, these results support a model in which a sophisticated combination of competitive clamp

  6. Nucleolin interacts with influenza A nucleoprotein and contributes to viral ribonucleoprotein complexes nuclear trafficking and efficient influenza viral replication

    PubMed Central

    Terrier, Olivier; Carron, Coralie; De Chassey, Benoît; Dubois, Julia; Traversier, Aurélien; Julien, Thomas; Cartet, Gaëlle; Proust, Anaïs; Hacot, Sabine; Ressnikoff, Denis; Lotteau, Vincent; Lina, Bruno; Diaz, Jean-Jacques; Moules, Vincent; Rosa-Calatrava, Manuel

    2016-01-01

    Influenza viruses replicate their single-stranded RNA genomes in the nucleus of infected cells and these replicated genomes (vRNPs) are then exported from the nucleus to the cytoplasm and plasma membrane before budding. To achieve this export, influenza viruses hijack the host cell export machinery. However, the complete mechanisms underlying this hijacking remain not fully understood. We have previously shown that influenza viruses induce a marked alteration of the nucleus during the time-course of infection and notably in the nucleolar compartment. In this study, we discovered that a major nucleolar component, called nucleolin, is required for an efficient export of vRNPs and viral replication. We have notably shown that nucleolin interacts with the viral nucleoprotein (NP) that mainly constitutes vRNPs. Our results suggest that this interaction could allow vRNPs to “catch” the host cell export machinery, a necessary step for viral replication. PMID:27373907

  7. Nucleolin interacts with influenza A nucleoprotein and contributes to viral ribonucleoprotein complexes nuclear trafficking and efficient influenza viral replication.

    PubMed

    Terrier, Olivier; Carron, Coralie; De Chassey, Benoît; Dubois, Julia; Traversier, Aurélien; Julien, Thomas; Cartet, Gaëlle; Proust, Anaïs; Hacot, Sabine; Ressnikoff, Denis; Lotteau, Vincent; Lina, Bruno; Diaz, Jean-Jacques; Moules, Vincent; Rosa-Calatrava, Manuel

    2016-01-01

    Influenza viruses replicate their single-stranded RNA genomes in the nucleus of infected cells and these replicated genomes (vRNPs) are then exported from the nucleus to the cytoplasm and plasma membrane before budding. To achieve this export, influenza viruses hijack the host cell export machinery. However, the complete mechanisms underlying this hijacking remain not fully understood. We have previously shown that influenza viruses induce a marked alteration of the nucleus during the time-course of infection and notably in the nucleolar compartment. In this study, we discovered that a major nucleolar component, called nucleolin, is required for an efficient export of vRNPs and viral replication. We have notably shown that nucleolin interacts with the viral nucleoprotein (NP) that mainly constitutes vRNPs. Our results suggest that this interaction could allow vRNPs to "catch" the host cell export machinery, a necessary step for viral replication. PMID:27373907

  8. Cloning and characterization of a novel RING finger protein that interacts with class V myosins.

    PubMed

    El-Husseini, A E; Vincent, S R

    1999-07-01

    We have identified a novel protein (BERP) that is a specific partner for the tail domain of myosin V. Class V myosins are a family of molecular motors thought to interact via their unique C-terminal tails with specific proteins for the targeted transport of organelles. BERP is highly expressed in brain and contains an N-terminal RING finger, followed by a B-box zinc finger, a coiled-coil (RBCC domain), and a unique C-terminal beta-propeller domain. A yeast two-hybrid screening indicated that the C-terminal beta-propeller domain mediates binding to the tail of the class V myosin myr6 (myosin Vb). This interaction was confirmed by immunoprecipitation, which also demonstrated that BERP could associate with myosin Va, the product of the dilute gene. Like myosin Va, BERP is expressed in a punctate pattern in the cytoplasm as well as in the neurites and growth cones of PC12 cells. We also found that the RBCC domain of BERP is involved in protein dimerization. Stable expression of a mutant form of BERP lacking the myosin-binding domain but containing the dimerization domain resulted in defective PC12 cell spreading and prevented neurite outgrowth in response to nerve growth factor. Our studies present a novel interaction for the beta-propeller domain and provide evidence for a role for BERP in myosin V-mediated cargo transport. PMID:10391919

  9. ZNF70, a novel ILDR2-interacting protein, contributes to the regulation of HES1 gene expression.

    PubMed

    Watanabe, Kazuhisa; Nakayama, Kazuhiro; Ohta, Satoshi; Tago, Kenji; Boonvisut, Supichaya; Millings, Elizabeth J; Fischer, Stuart G; LeDuc, Charles A; Leibel, Rudolph L; Iwamoto, Sadahiko

    2016-09-01

    A diabetes susceptibility gene, immunoglobulin-like domain containing receptor 2 (Ildr2), encodes a transmembrane protein localized to the endoplasmic reticulum membrane that is closely related to hepatic lipid metabolism. The livers of ob/ob mice in which Ildr2 is transiently overexpressed are relieved of hepatic steatosis. However, the molecular mechanisms through which ILDR2 affects these changes in hepatic lipid metabolism remain unknown. This study aimed to identify ILDR2-interacting proteins to further elucidate the molecular mechanisms underlying the role of ILDR2 in lipid homeostasis. We purified ILDR2-containing protein complexes using tandem affinity purification tagging and identified ZNF70, a member of the Kruppel C2H2-type zinc finger protein family, as a novel ILDR2-interacting protein. We demonstrated that ZNF70 interacts with ZFP64 and activates HES1 transcription by binding to the HES1 promoter. In addition, HES1 gene expression is increased in ILDR2-knockdown HepG2 cells, in which ZNF70 is translocated from the cytoplasm to the nucleus, suggesting that ZNF70 migration to the nucleus after dissociating from the ILDR2-ZNF70 complex activates HES1 transcription. These results support a novel link between ILDR2 and HES1 gene expression and suggest that ILDR2 is involved in a novel pathway in hepatic steatosis. PMID:27353377

  10. The self-interaction of native TDP-43 C terminus inhibits its degradation and contributes to early proteinopathies.

    PubMed

    Wang, I-Fan; Chang, Hsiang-Yu; Hou, Shin-Chen; Liou, Gunn-Guang; Way, Tzong-Der; James Shen, C-K

    2012-01-01

    The degraded, misfolded C terminus of TAR DNA-binding protein-43 is associated with a wide spectrum of neurodegenerative diseases, particularly frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis. However, the precise mechanism of pathological cleavage of the TAR DNA-binding protein-43 remains unknown. Here we show that the TAR DNA-binding protein-43 C-terminal protein physically interacts with itself or with the cellular-folded yeast prion domain of Sup35 forming dynamic aggregates. This prion-like nature governs known cellular functions of the TAR DNA-binding protein-43, including subcellular localisation and exon skipping of the cystic fibrosis transmembrane conductance regulator. Significantly, mutants with a failure to engage in prion-like interactions are processed into an ~24-kDa C-terminal fragment of the TAR DNA-binding protein-43. The estimated cleavage site of degraded TAR DNA-binding protein-43 fragments corresponds to the pathological cleavage site identified in patients with the TAR DNA-binding protein-43 proteinopathies. Consistently, epigallocatechin gallate constrains prion-like interactions, attenuating pathological-like degradation. Thus, the native folding of TAR DNA-binding protein-43 C terminus acts as a guardian of pathogenesis, which is directly associated with loss-of-function. PMID:22473010

  11. Electrostatic Contributions Drive the Interaction Between Staphylococcus aureus Protein Efb-C and its Complement Target C3d

    SciTech Connect

    Haspel, N.; Ricklin, D.; Geisbrecht, B.V.; Kavraki, L.E.; Lambris, J.D.

    2008-11-13

    The C3-inhibitory domain of Staphylococcus aureus extracellular fibrinogen-binding protein (Efb-C) defines a novel three-helix bundle motif that regulates complement activation. Previous crystallographic studies of Efb-C bound to its cognate subdomain of human C3 (C3d) identified Arg-131 and Asn-138 of Efb-C as key residues for its activity. In order to characterize more completely the physical and chemical driving forces behind this important interaction, we employed in this study a combination of structural, biophysical, and computational methods to analyze the interaction of C3d with Efb-C and the single-point mutants R131A and N138A. Our results show that while these mutations do not drastically affect the structure of the Efb-C/C3d recognition complex, they have significant adverse effects on both the thermodynamic and kinetic profiles of the resulting complexes. We also characterized other key interactions along the Efb-C/C3d binding interface and found an intricate network of salt bridges and hydrogen bonds that anchor Efb-C to C3d, resulting in its potent complement inhibitory properties.

  12. HCI and mobile health interventions: How human-computer interaction can contribute to successful mobile health interventions.

    PubMed

    Poole, Erika S

    2013-12-01

    Advances in mobile computing offer the potential to change when, where, and how health interventions are delivered. Rather than relying on occasional in-clinic interactions, mobile health (mHealth) interventions may overcome constraints due to limited clinician time, poor patient adherence, and inability to provide meaningful interventions at the most appropriate time. Technological capability, however, does not equate with user acceptance and adoption. How then can we ensure that mobile technologies for behavior change meet the needs of their target audience? In this paper, we argue that overcoming acceptance and adoption barriers requires interdisciplinary collaborations, bringing together not only technologists and health researchers but also human-computer interaction (HCI) experts. We discuss the value of human-computer interaction research to the nascent field of mHealth and demonstrate how research from HCI can offer complementary insights on the creation of mobile health interventions. We conclude with a discussion of barriers to interdisciplinary collaborations in mobile health and suggest ways to overcome them. PMID:24294328

  13. Electrostatic contributions drive the interaction between Staphylococcus aureus protein Efb-C and its complement target C3d

    PubMed Central

    Haspel, Nurit; Ricklin, Daniel; Geisbrecht, Brian V.; Kavraki, Lydia E.; Lambris, John D.

    2008-01-01

    The C3–inhibitory domain of Staphylococcus aureus extracellular fibrinogen-binding protein (Efb-C) defines a novel three-helix bundle motif that regulates complement activation. Previous crystallographic studies of Efb-C bound to its cognate subdomain of human C3 (C3d) identified Arg-131 and Asn-138 of Efb-C as key residues for its activity. In order to characterize more completely the physical and chemical driving forces behind this important interaction, we employed in this study a combination of structural, biophysical, and computational methods to analyze the interaction of C3d with Efb-C and the single-point mutants R131A and N138A. Our results show that while these mutations do not drastically affect the structure of the Efb-C/C3d recognition complex, they have significant adverse effects on both the thermodynamic and kinetic profiles of the resulting complexes. We also characterized other key interactions along the Efb-C/C3d binding interface and found an intricate network of salt bridges and hydrogen bonds that anchor Efb-C to C3d, resulting in its potent complement inhibitory properties. PMID:18687868

  14. PARK2 and proinflammatory/anti-inflammatory cytokine gene interactions contribute to the susceptibility to leprosy: a case–control study of North Indian population

    PubMed Central

    Chopra, Rupali; Kalaiarasan, Ponnusamy; Ali, Shafat; Srivastava, Amit K; Aggarwal, Shweta; Garg, Vijay K; Bhattacharya, Sambit N; Bamezai, Rameshwar N K

    2014-01-01

    Objectives Cytokines and related molecules in immune-response pathways seem important in deciding the outcome of the host–pathogen interactions towards different polar forms in leprosy. We studied the role of significant and functionally important single-nucleotide polymorphisms (SNPs) in these genes, published independently from our research group, through combined interaction with an additional analysis of the in silico network outcome, to understand how these impact the susceptibility towards the disease, leprosy. Design The study was designed to assess an overall combined contribution of significantly associated individual SNPs to reflect on epistatic interactions and their outcome in the form of the disease, leprosy. Furthermore, in silico approach was adopted to carry out protein–protein interaction study between PARK2 and proinflammatory/anti-inflammatory cytokines. Setting Population-based case–control study involved the data of North India. Protein–protein interaction networks were constructed using cytoscape. Participants Study included the data available from 2305 Northern Indians samples (829 patients with leprosy; 1476 healthy controls), generated by our research group. Primary and secondary outcome measures For genotype interaction analysis, all possible genotype combinations between selected SNPs were used as an independent variable, using binary logistic regression with the forward likelihood ratio method, keeping the gender as a covariate. Results Interaction analysis between PARK2 and significant SNPs of anti-inflammatory/proinflammatory cytokine genes, including BAT1 to BTNL2-DR spanning the HLA (6p21.3) region in a case–control comparison, showed that the combined analysis of: (1) PARK2, tumour necrosis factor (TNF), BTNL2-DR, interleukin (IL)-10, IL-6 and TGFBR2 increased the risk towards leprosy (OR=2.54); (2) PARK2, BAT1, NFKBIL1, LTA, TNF-LTB, IL12B and IL10RB provided increased protection (OR=0.26) in comparison with their

  15. Quantitative modeling assesses the contribution of bond strengthening, rebinding and force sharing to the avidity of biomolecule interactions.

    PubMed

    Lo Schiavo, Valentina; Robert, Philippe; Limozin, Laurent; Bongrand, Pierre

    2012-01-01

    Cell adhesion is mediated by numerous membrane receptors. It is desirable to derive the outcome of a cell-surface encounter from the molecular properties of interacting receptors and ligands. However, conventional parameters such as affinity or kinetic constants are often insufficient to account for receptor efficiency. Avidity is a qualitative concept frequently used to describe biomolecule interactions: this includes incompletely defined properties such as the capacity to form multivalent attachments. The aim of this study is to produce a working description of monovalent attachments formed by a model system, then to measure and interpret the behavior of divalent attachments under force. We investigated attachments between antibody-coated microspheres and surfaces coated with sparse monomeric or dimeric ligands. When bonds were subjected to a pulling force, they exhibited both a force-dependent dissociation consistent with Bell's empirical formula and a force- and time-dependent strengthening well described by a single parameter. Divalent attachments were stronger and less dependent on forces than monovalent ones. The proportion of divalent attachments resisting a force of 30 piconewtons for at least 5 s was 3.7 fold higher than that of monovalent attachments. Quantitative modeling showed that this required rebinding, i.e. additional bond formation between surfaces linked by divalent receptors forming only one bond. Further, experimental data were compatible with but did not require stress sharing between bonds within divalent attachments. Thus many ligand-receptor interactions do not behave as single-step reactions in the millisecond to second timescale. Rather, they exhibit progressive stabilization. This explains the high efficiency of multimerized or clustered receptors even when bonds are only subjected to moderate forces. Our approach provides a quantitative way of relating binding avidity to measurable parameters including bond maturation, rebinding and

  16. Higher-order electric multipole contributions to retarded non-additive three-body dispersion interaction energies between atoms: Equilateral triangle and collinear configurations

    SciTech Connect

    Salam, A.

    2013-12-28

    The theory of molecular quantum electrodynamics (QED) is used to calculate higher electric multipole contributions to the dispersion energy shift between three atoms or molecules arranged in a straight line or in an equilateral triangle configuration. As in two-body potentials, three-body dispersion interactions are viewed in the QED formalism to arise from exchange of virtual photons between coupled pairs of particles. By employing an interaction Hamiltonian that is quadratic in the electric displacement field means that third-order perturbation theory can be used to yield the energy shift for a particular combination of electric multipole polarizable species, with only six time-ordered diagrams needing to be summed over. Specific potentials evaluated include dipole-dipole-quadrupole (DDQ), dipole-quadrupole-quadrupole (DQQ), and dipole-dipole-octupole (DDO) terms. For the geometries of interest, near-zone limiting forms are found to exhibit an R{sup −11} dependence on separation distance for the DDQ interaction, and an R{sup −13} behaviour for DQQ and DDO shifts, agreeing with an earlier semi-classical computation. Retardation weakens the potential in each case by R{sup −1} in the far-zone. It is found that by decomposing the octupole moment into its irreducible components of weights-1 and -3 that the former contribution to the DDO potential may be taken to be a higher-order correction to the leading triple dipole energy shift.

  17. New insights into the interactions between cork chemical components and pesticides. The contribution of π-π interactions, hydrogen bonding and hydrophobic effect.

    PubMed

    Olivella, M À; Bazzicalupi, C; Bianchi, A; Fiol, N; Villaescusa, I

    2015-01-01

    The role of chemical components of cork in the sorption of several pesticides has been investigated. For this purpose raw cork and three cork extracted fractions (i.e. cork free of aliphatic extractives, cork free of all extractives and cork free of all extractives and suberin) were used as sorbent of three ionic pesticides (propazine, 2,4-dichlorophenoxy acetic acid (2,4-D) and alachlor) and five non-ionic pesticides (chlorpyrifos, isoproturon, metamitron, methomyl and oxamyl) with a logKow within the range -0.47 to 4.92. The effect of cations on the ionic pesticides, propazine and 2,4-D sorption was also analyzed. Results indicated that the highest yields were obtained for chlorpyrifos and alachlor sorption onto raw cork (>55%). After removal of aliphatic extractives sorption of all pesticides increased that ranged from 3% for propazine to 31% for alachlor. In contrast, removal of phenolic extractives caused a sorption decrease. Low sorption yields were obtained for hydrophobic pesticides such as metamitron, oxamyl and methomyl (<11%) by using all cork fractions and extremely low when using raw cork (<1%). FTIR analysis was useful to indicate that lignin moieties were the main components involved on the sorption process. Modelling calculations evidenced that π-stacking interactions with the aromatic groups of lignin play a major role in determining the adsorption properties of cork toward aromatic pesticides. Results presented in this paper gain insights into the cork affinities for pesticides and the interactions involved in the sorption process and also enables to envisage sorption affinity of cork for other organic pollutants. PMID:25240950

  18. Comparison of MCNPX and GEANT4 to Predict the Contribution of Non-elastic Nuclear Interactions to Absorbed Dose in Water, PMMA and A150

    NASA Astrophysics Data System (ADS)

    Shtejer, K.; Arruda-Neto, J. D. T.; Schulte, R.; Wroe, A.; Rodrigues, T. E.; de Menezes, M. O.; Moralles, M.; Guzmán, F.; Manso, M. V.

    2008-08-01

    Proton induced non-elastic nuclear reactions play an important role in the dose distribution of clinically used proton beams as they deposit dose of high biological effectiveness both within the primary beam path as well as outside the beam to untargeted tissues. Non-elastic nuclear reactions can be evaluated using transport codes based on the Monte Carlo method. In this work, we have utilized the Los Alamos code MCNPX and the CERN GEANT4 toolkit, which are currently the most widely used Monte Carlo programs for proton radiation transport simulations in medical physics, to study the contribution of non-elastic nuclear interactions to the absorbed dose of proton beams in the therapeutic energy range. The impact of different available theoretical models to address the nuclear reaction process was investigated. The contribution of secondary particles from non-elastic nuclear reactions was calculated in three materials relevant in radiotherapy applications: water, PMMA and A150. The results evidence that there are differences in the calculated contribution of the secondary particles heavier than protons to the absorbed dose, with different approaches to model the nuclear reactions. The MCNPX calculation give rise to a larger contribution of d, t, α3He to the total dose compared to the GEANT4 physical models chosen in this work.

  19. Comparison of MCNPX and GEANT4 to Predict the Contribution of Non-elastic Nuclear Interactions to Absorbed Dose in Water, PMMA and A150

    SciTech Connect

    Shtejer, K.; Arruda-Neto, J. D. T.; Rodrigues, T. E.; Schulte, R.; Wroe, A.; Menezes, M. O. de; Moralles, M.

    2008-08-11

    Proton induced non-elastic nuclear reactions play an important role in the dose distribution of clinically used proton beams as they deposit dose of high biological effectiveness both within the primary beam path as well as outside the beam to untargeted tissues. Non-elastic nuclear reactions can be evaluated using transport codes based on the Monte Carlo method. In this work, we have utilized the Los Alamos code MCNPX and the CERN GEANT4 toolkit, which are currently the most widely used Monte Carlo programs for proton radiation transport simulations in medical physics, to study the contribution of non-elastic nuclear interactions to the absorbed dose of proton beams in the therapeutic energy range. The impact of different available theoretical models to address the nuclear reaction process was investigated. The contribution of secondary particles from non-elastic nuclear reactions was calculated in three materials relevant in radiotherapy applications: water, PMMA and A150. The results evidence that there are differences in the calculated contribution of the secondary particles heavier than protons to the absorbed dose, with different approaches to model the nuclear reactions. The MCNPX calculation give rise to a larger contribution of d, t, {alpha}{sup 3}He to the total dose compared to the GEANT4 physical models chosen in this work.

  20. Deformation-corrosion interactions for Zr alloys during I-SCC crack initiation. Part II: Localised stress and strain contributions

    NASA Astrophysics Data System (ADS)

    Jacques, Patrick; Lefebvre, Florence; Lemaignan, Clément

    1999-01-01

    For a better understanding of the initiation step of iodine induced stress corrosion cracking (SCC) in Zr alloys, responsible for pellet-cladding interaction (PCI) fuel rod failures, an analytical study has been undertaken, the aim of which being focused on the respective roles of local chemistry and stress/strain state on the crack nucleation. This second part is mostly related to the local stress induced by strain incompatibilities between grains. Using EBSP (electron back-scattering pattern) to analyze the crystallographic orientation of all the grains of the samples tested in SCC, it was possible to conclude that the major parameter controlling the nucleation of the intergranular cracks is not related to grain to grain strain incompatibilities, but to the orientation of the grain boundary planes with respect to the tensile stress.

  1. Cutting edge: Bcl6-interacting corepressor contributes to germinal center T follicular helper cell formation and B cell helper function.

    PubMed

    Yang, Jessica A; Tubo, Noah J; Gearhart, Micah D; Bardwell, Vivian J; Jenkins, Marc K

    2015-06-15

    CD4(+) germinal center (GC)-T follicular helper (Tfh) cells help B cells become long-lived plasma cells and memory cells. The transcriptional repressor Bcl6 plays a key role in GC-Tfh formation by inhibiting the expression of genes that promote differentiation into other lineages. We determined whether BCOR, a component of a Polycomb repressive complex that interacts with the Bcl6 BTB domain, influences GC-Tfh differentiation. T cell-targeted BCOR deficiency led to a substantial loss of peptide:MHC class II-specific GC-Tfh cells following Listeria monocytogenes infection and a 2-fold decrease following immunization with a peptide in CFA. The reduction in GC-Tfh cells was associated with diminished plasma cell and GC B cell formation. Thus, T cell-expressed BCOR is critical for optimal GC-Tfh cell differentiation and humoral immunity. PMID:25964495

  2. Functional and physical interaction between p53 and BZLF1: implications for Epstein-Barr virus latency.

    PubMed Central

    Zhang, Q; Gutsch, D; Kenney, S

    1994-01-01

    The p53 tumor suppressor protein, which is commonly mutated in human cancers, has been shown to interact directly with virally encoded from papillomavirus, adenovirus, and simian virus 40. The disruption of p53 function may be required for efficient replication of certain viruses and may also play a role in the development of virally induced malignancies. Infection with Epstein-Barr virus (EBV) has been associated with the development of B-cell lymphomas and nasopharyngeal carcinoma. Here we show that the EBV immediate-early protein, BZLF1 (Z), which is responsible for initiating the switch from latent to lytic infection, can interact directly in vitro and in vivo with the tumor suppressor protein, p53. This interaction requires the coiled-coil dimerization domain of the Z protein and the carboxy-terminal portion of p53. Overexpression of wild-type p53 inhibits the ability of Z to disrupt viral latency. Likewise, Z inhibits p53-dependent transactivation in lymphoid cells. The direct interaction between Z and p53 may play a role in regulating the switch from latent to lytic viral infection. Images PMID:8114724

  3. Dynamin-related Protein 1 Oligomerization in Solution Impairs Functional Interactions with Membrane-anchored Mitochondrial Fission Factor.

    PubMed

    Clinton, Ryan W; Francy, Christopher A; Ramachandran, Rajesh; Qi, Xin; Mears, Jason A

    2016-01-01

    Mitochondrial fission is a crucial cellular process mediated by the mechanoenzymatic GTPase, dynamin-related protein 1 (Drp1). During mitochondrial division, Drp1 is recruited from the cytosol to the outer mitochondrial membrane by one, or several, integral membrane proteins. One such Drp1 partner protein, mitochondrial fission factor (Mff), is essential for mitochondrial division, but its mechanism of action remains unexplored. Previous studies have been limited by a weak interaction between Drp1 and Mff in vitro. Through refined in vitro reconstitution approaches and multiple independent assays, we show that removal of the regulatory variable domain (VD) in Drp1 enhances formation of a functional Drp1-Mff copolymer. This protein assembly exhibits greatly stimulated cooperative GTPase activity in solution. Moreover, when Mff was anchored to a lipid template, to mimic a more physiologic environment, significant stimulation of GTPase activity was observed with both WT and ΔVD Drp1. Contrary to recent findings, we show that premature Drp1 self-assembly in solution impairs functional interactions with membrane-anchored Mff. Instead, dimeric Drp1 species are selectively recruited by Mff to initiate assembly of a functional fission complex. Correspondingly, we also found that the coiled-coil motif in Mff is not essential for Drp1 interactions, but rather serves to augment cooperative self-assembly of Drp1 proximal to the membrane. Taken together, our findings provide a mechanism wherein the multimeric states of both Mff and Drp1 regulate their collaborative interaction. PMID:26578514

  4. Therapeutic potential of mitotic interaction between the nucleoporin Tpr and aurora kinase A

    PubMed Central

    Kobayashi, Akiko; Hashizume, Chieko; Dowaki, Takayuki; Wong, Richard W

    2015-01-01

    Spindle poles are defined by centrosomes; therefore, an abnormal number or defective structural organization of centrosomes can lead to loss of spindle bipolarity and genetic integrity. Previously, we showed that Tpr (translocated promoter region), a component of the nuclear pore complex (NPC), interacts with Mad1 and dynein to promote proper chromosome segregation during mitosis. Tpr also associates with p53 to induce autophagy. Here, we report that Tpr depletion induces mitotic catastrophe and enhances the rate of tetraploidy and polyploidy. Mechanistically, Tpr interacts, via its central domain, with Aurora A but not Aurora B kinase. In Tpr-depleted cells, the expression levels, centrosomal localization and phosphorylation of Aurora A were all reduced. Surprisingly, an Aurora A inhibitor, Alisertib (MLN8237), also disrupted centrosomal localization of Tpr and induced mitotic catastrophe and cell death in a time- and dose-dependent manner. Strikingly, over-expression of Aurora A disrupted Tpr centrosomal localization only in cells with supernumerary centrosomes but not in bipolar cells. Our results highlight the mutual regulation between Tpr and Aurora A and further confirm the importance of nucleoporin function in spindle pole organization, bipolar spindle assembly, and mitosis; functions that are beyond the conventional nucleocytoplasmic transport and NPC structural roles of nucleoporins. Furthermore, the central coiled-coil domain of Tpr binds to and sequesters extra Aurora A to safeguard bipolarity. This Tpr domain merits further investigation for its ability to inhibit Aurora kinase and as a potential therapeutic agent in cancer treatment. PMID:25789545

  5. Interactions between SNP alleles at multiple loci contribute to skin color differences between caucasoid and mongoloid subjects.

    PubMed

    Anno, Sumiko; Abe, Takashi; Yamamoto, Takushi

    2008-01-01

    This study aimed to identify single nucleotide polymorphism (SNP) alleles at multiple loci associated with racial differences in skin color using SNP genotyping. A total of 122 Caucasians in Toledo, Ohio and 100 Mongoloids in Japan were genotyped for 20 SNPs in 7 candidate genes, encoding the Agouti signaling protein (ASIP), tyrosinase-related protein 1 (TYRP1), tyrosinase (TYR), melanocortin 1 receptor (MC1R), oculocutaneous albinism II (OCA2), microphthalmia-associated transcription factor (MITF), and myosin VA (MYO5A). Data were used to analyze associations between the 20 SNP alleles using linkage disequilibrium (LD). Combinations of SNP alleles were jointly tested under LD for associations with racial groups by performing a chi(2) test for independence. Results showed that SNP alleles at multiple loci can be considered the haplotype that contributes to significant differences between the two population groups and suggest a high probability of LD. Confirmation of these findings requires further study with other ethnic groups to analyze the associations between SNP alleles at multiple loci and skin color variation among races. PMID:18392143

  6. NMR identification of the binding surfaces involved in the Salmonella and Shigella Type III secretion tip-translocon protein-protein interactions.

    PubMed

    McShan, Andrew C; Kaur, Kawaljit; Chatterjee, Srirupa; Knight, Kevin M; De Guzman, Roberto N

    2016-08-01

    The type III secretion system (T3SS) is essential for the pathogenesis of many bacteria including Salmonella and Shigella, which together are responsible for millions of deaths worldwide each year. The structural component of the T3SS consists of the needle apparatus, which is assembled in part by the protein-protein interaction between the tip and the translocon. The atomic detail of the interaction between the tip and the translocon proteins is currently unknown. Here, we used NMR methods to identify that the N-terminal domain of the Salmonella SipB translocon protein interacts with the SipD tip protein at a surface at the distal region of the tip formed by the mixed α/β domain and a portion of its coiled-coil domain. Likewise, the Shigella IpaB translocon protein and the IpaD tip protein interact with each other using similar surfaces identified for the Salmonella homologs. Furthermore, removal of the extreme N-terminal residues of the translocon protein, previously thought to be important for the interaction, had little change on the binding surface. Finally, mutations at the binding surface of SipD reduced invasion of Salmonella into human intestinal epithelial cells. Together, these results reveal the binding surfaces involved in the tip-translocon protein-protein interaction and advance our understanding of the assembly of the T3SS needle apparatus. Proteins 2016; 84:1097-1107. © 2016 Wiley Periodicals, Inc. PMID:27093649

  7. Chondroitin sulfate N-acetylgalactosaminyltransferase-2 contributes to the replication of infectious bursal disease virus via interaction with the capsid protein VP2.

    PubMed

    Zhang, Lizhou; Ren, Xiangang; Chen, Yuming; Gao, Yulong; Wang, Nian; Lu, Zhen; Gao, Li; Qin, Liting; Wang, Yongqiang; Gao, Honglei; Li, Kai; Jiang, Lili; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Qi, Xiaole; Wang, Xiaomei

    2015-03-01

    Infectious bursal disease virus (IBDV) is a birnavirus that causes a highly contagious immunosuppressive disease in young chickens. The capsid protein VP2 of IBDV plays multiple roles in its life cycle. To more comprehensively understand the functions of VP2 involved in the communication between virus and host, we used yeast two-hybrid screening to identify the cellular factors that interact with this protein. We found that chondroitin sulfate N-acetylgalactosaminyltransferase-2 (CSGalNAcT2), a typical type II transmembrane protein located in Golgi apparatus, could interact with VP2, and we confirmed this interaction by co-immunoprecipitation and confocal laser scanning microscopy assays. Additionally, up-regulation of CSGalNAcT2 during IBDV infection was observed. Overexpression and siRNA-mediated knockdown of CSGalNAcT2 assays suggested that CSGalNAcT2 promoted IBDV replication. Moreover, this enhancing effect of CSGalNAcT2 could be inhibited by Brefeldin A, which is a Golgi-disturbing agent. This indicated that the integrity of Golgi apparatus structure was involved in the function of CSGalNAcT2. Taken together, we concluded that CSGalNAcT2, located in the Golgi apparatus, contributed to the replication of IBDV via interaction with VP2. PMID:25807054

  8. An evolutionarily conserved interaction of tumor suppressor protein Pdcd4 with the poly(A)-binding protein contributes to translation suppression by Pdcd4.

    PubMed

    Fehler, Olesja; Singh, Priyanka; Haas, Astrid; Ulrich, Diana; Müller, Jan P; Ohnheiser, Johanna; Klempnauer, Karl-Heinz

    2014-01-01

    The tumor suppressor protein programmed cell death 4 (Pdcd4) has been implicated in the translational regulation of specific mRNAs, however, the identities of the natural Pdcd4 target mRNAs and the mechanisms by which Pdcd4 affects their translation are not well understood. Pdcd4 binds to the eukaryotic translation initiation factor eIF4A and inhibits its helicase activity, which has suggested that Pdcd4 suppresses translation initiation of mRNAs containing structured 5'-untranslated regions. Recent work has revealed a second inhibitory mechanism, which is eIF4A-independent and involves direct RNA-binding of Pdcd4 to the target mRNAs. We have now identified the poly(A)-binding protein (PABP) as a novel direct interaction partner of Pdcd4. The ability to interact with PABP is shared between human and Drosophila Pdcd4, indicating that it has been highly conserved during evolution. Mutants of Pdcd4 that have lost the ability to interact with PABP fail to stably associate with ribosomal complexes in sucrose density gradients and to suppress translation, as exemplified by c-myb mRNA. Overall, our work identifies PABP as a novel functionally relevant Pdcd4 interaction partner that contributes to the regulation of translation by Pdcd4. PMID:25190455

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

    PubMed Central

    Chun, Younghwa; Kim, Raehyung; Lee, Soojin

    2016-01-01

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

  10. Chondroitin Sulfate N-acetylgalactosaminyltransferase-2 Contributes to the Replication of Infectious Bursal Disease Virus via Interaction with the Capsid Protein VP2

    PubMed Central

    Zhang, Lizhou; Ren, Xiangang; Chen, Yuming; Gao, Yulong; Wang, Nian; Lu, Zhen; Gao, Li; Qin, Liting; Wang, Yongqiang; Gao, Honglei; Li, Kai; Jiang, Lili; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Qi, Xiaole; Wang, Xiaomei

    2015-01-01

    Infectious bursal disease virus (IBDV) is a birnavirus that causes a highly contagious immunosuppressive disease in young chickens. The capsid protein VP2 of IBDV plays multiple roles in its life cycle. To more comprehensively understand the functions of VP2 involved in the communication between virus and host, we used yeast two-hybrid screening to identify the cellular factors that interact with this protein. We found that chondroitin sulfate N-acetylgalactosaminyltransferase-2 (CSGalNAcT2), a typical type II transmembrane protein located in Golgi apparatus, could interact with VP2, and we confirmed this interaction by co-immunoprecipitation and confocal laser scanning microscopy assays. Additionally, up-regulation of CSGalNAcT2 during IBDV infection was observed. Overexpression and siRNA-mediated knockdown of CSGalNAcT2 assays suggested that CSGalNAcT2 promoted IBDV replication. Moreover, this enhancing effect of CSGalNAcT2 could be inhibited by Brefeldin A, which is a Golgi-disturbing agent. This indicated that the integrity of Golgi apparatus structure was involved in the function of CSGalNAcT2. Taken together, we concluded that CSGalNAcT2, located in the Golgi apparatus, contributed to the replication of IBDV via interaction with VP2. PMID:25807054

  11. An evolutionarily conserved interaction of tumor suppressor protein Pdcd4 with the poly(A)-binding protein contributes to translation suppression by Pdcd4

    PubMed Central

    Fehler, Olesja; Singh, Priyanka; Haas, Astrid; Ulrich, Diana; Müller, Jan P.; Ohnheiser, Johanna; Klempnauer, Karl-Heinz

    2014-01-01

    The tumor suppressor protein programmed cell death 4 (Pdcd4) has been implicated in the translational regulation of specific mRNAs, however, the identities of the natural Pdcd4 target mRNAs and the mechanisms by which Pdcd4 affects their translation are not well understood. Pdcd4 binds to the eukaryotic translation initiation factor eIF4A and inhibits its helicase activity, which has suggested that Pdcd4 suppresses translation initiation of mRNAs containing structured 5′-untranslated regions. Recent work has revealed a second inhibitory mechanism, which is eIF4A-independent and involves direct RNA-binding of Pdcd4 to the target mRNAs. We have now identified the poly(A)-binding protein (PABP) as a novel direct interaction partner of Pdcd4. The ability to interact with PABP is shared between human and Drosophila Pdcd4, indicating that it has been highly conserved during evolution. Mutants of Pdcd4 that have lost the ability to interact with PABP fail to stably associate with ribosomal complexes in sucrose density gradients and to suppress translation, as exemplified by c-myb mRNA. Overall, our work identifies PABP as a novel functionally relevant Pdcd4 interaction partner that contributes to the regulation of translation by Pdcd4. PMID:25190455

  12. BMSCs Interactions with Adventitial Fibroblasts Display Smooth Muscle Cell Lineage Potential in Differentiation and Migration That Contributes to Neointimal Formation.

    PubMed

    Wendan, Y; Changzhu, J; Xuhong, S; Hongjing, C; Hong, S; Dongxia, Y; Fang, X

    2016-01-01

    In this study a model of simulated vascular injury in vitro was used to study the characterization of bone-marrow-derived mesenchymal stem cells (BMSCs) morphology and to investigate the differentiation and migration of BMSCs in the presence of adventitial fibroblasts. BMSCs from rats were indirectly cocultured with adventitial fibroblasts in a transwell chamber apparatus for 7 days, and clonogenic assays demonstrated that BMSCs could be differentiated into smooth muscle-like cells with this process, including smooth muscle α-actin (α-SMA) expression by immunofluorescence staining. Cell morphology of BMSCs was assessed by inverted microscope, while cell proliferation was assessed by MTT assay. The expressions of TGF-β1, MMP-1, and NF-κB were detected by immunofluorescence staining and Smad3 mRNA was measured by reverse transcription PCR. Migration ability of BMSCs with DAPI-labeled nuclei was measured by laser confocal microscopy. Our results demonstrate that indirect interactions with adventitial fibroblasts can induce proliferation, differentiation, and migration of BMSCs that can actively participate in neointimal formation. Our results indicate that the pathogenesis of vascular remodeling might perform via TGF-β1/Smad3 signal transduction pathways. PMID:26880952

  13. MRNIP/C5orf45 Interacts with the MRN Complex and Contributes to the DNA Damage Response.

    PubMed

    Staples, Christopher J; Barone, Giancarlo; Myers, Katie N; Ganesh, Anil; Gibbs-Seymour, Ian; Patil, Abhijit A; Beveridge, Ryan D; Daye, Caroline; Beniston, Richard; Maslen, Sarah; Ahel, Ivan; Skehel, J Mark; Collis, Spencer J

    2016-09-01

    Through an RNAi-based screen for previously uncharacterized regulators of genome stability, we have identified the human protein C5orf45 as an important factor in preventing the accumulation of DNA damage in human cells. Here, we functionally characterize C5orf45 as a binding partner of the MRE11-RAD50-NBS1 (MRN) damage-sensing complex. Hence, we rename C5orf45 as MRNIP for MRN-interacting protein (MRNIP). We find that MRNIP is rapidly recruited to sites of DNA damage. Cells depleted of MRNIP display impaired chromatin loading of the MRN complex, resulting in reduced DNA end resection and defective ATM-mediated DNA damage signaling, a reduced ability to repair DNA breaks, and radiation sensitivity. Finally, we show that MRNIP phosphorylation on serine 115 leads to its nuclear localization, and this modification is required for MRNIP's role in promoting genome stability. Collectively, these data reveal that MRNIP is an important component of the human DNA damage response. PMID:27568553

  14. Plasminogen Binding Proteins and Plasmin Generation on the Surface of Leptospira spp.: The Contribution to the Bacteria-Host Interactions

    PubMed Central

    Vieira, Monica L.; Atzingen, Marina V.; Oliveira, Rosane; Mendes, Renata S.; Domingos, Renan F.; Vasconcellos, Silvio A.; Nascimento, Ana L. T. O.

    2012-01-01

    Leptospirosis is considered a neglected infectious disease of human and veterinary concern. Although extensive investigations on host-pathogen interactions have been pursued by several research groups, mechanisms of infection, invasion and persistence of pathogenic Leptospira spp. remain to be elucidated. We have reported the ability of leptospires to bind human plasminogen (PLG) and to generate enzimatically active plasmin (PLA) on the bacteria surface. PLA-coated Leptospira can degrade immobilized ECM molecules, an activity with implications in host tissue penetration. Moreover, we have identified and characterized several proteins that may act as PLG-binding receptors, each of them competent to generate active plasmin. The PLA activity associated to the outer surface of Leptospira could hamper the host immune attack by conferring the bacteria some benefit during infection. The PLA-coated leptospires obstruct complement C3b and IgG depositions on the bacterial surface, most probably through degradation. The decrease of leptospiral opsonization might be an important aspect of the immune evasion strategy. We believe that the presence of PLA on the leptospiral surface may (i) facilitate host tissue penetration, (ii) help the bacteria to evade the immune system and, as a consequence, (iii) permit Leptospira to reach secondary sites of infection. PMID:23118516

  15. Methyl-CpG-binding protein 2 is phosphorylated by homeodomain-interacting protein kinase 2 and contributes to apoptosis.

    PubMed

    Bracaglia, Giorgia; Conca, Barbara; Bergo, Anna; Rusconi, Laura; Zhou, Zhaolan; Greenberg, Michael E; Landsberger, Nicoletta; Soddu, Silvia; Kilstrup-Nielsen, Charlotte

    2009-12-01

    Mutations in the methyl-CpG-binding protein 2 (MeCP2) are associated with Rett syndrome and other neurological disorders. MeCP2 represses transcription mainly by recruiting various co-repressor complexes. Recently, MeCP2 phosphorylation at Ser 80, Ser 229 and Ser 421 was shown to occur in the brain and modulate MeCP2 silencing activities. However, the kinases directly responsible for this are largely unknown. Here, we identify the homeodomain-interacting protein kinase 2 (HIPK2) as a kinase that binds MeCP2 and phosphorylates it at Ser 80 in vitro and in vivo. HIPK2 modulates cell proliferation and apoptosis, and the neurological defects of Hipk2-null mice indicate its role in proper brain functions. We show that MeCP2 cooperates with HIPK2 in induction of apoptosis and that Ser 80 phosphorylation is required together with the DNA binding of MeCP2. These data are, to our knowledge, the first that describe a kinase associating with MeCP2, causing its specific phosphorylation in vivo and, furthermore, they reinforce the role of MeCP2 in regulating cell growth. PMID:19820693

  16. Methyl-CpG-binding protein 2 is phosphorylated by homeodomain-interacting protein kinase 2 and contributes to apoptosis

    PubMed Central

    Bracaglia, Giorgia; Conca, Barbara; Bergo, Anna; Rusconi, Laura; Zhou, Zhaolan; Greenberg, Michael E; Landsberger, Nicoletta; Soddu, Silvia; Kilstrup-Nielsen, Charlotte

    2009-01-01

    Mutations in the methyl-CpG-binding protein 2 (MeCP2) are associated with Rett syndrome and other neurological disorders. MeCP2 represses transcription mainly by recruiting various co-repressor complexes. Recently, MeCP2 phosphorylation at Ser 80, Ser 229 and Ser 421 was shown to occur in the brain and modulate MeCP2 silencing activities. However, the kinases directly responsible for this are largely unknown. Here, we identify the homeodomain-interacting protein kinase 2 (HIPK2) as a kinase that binds MeCP2 and phosphorylates it at Ser 80 in vitro and in vivo. HIPK2 modulates cell proliferation and apoptosis, and the neurological defects of Hipk2-null mice indicate its role in proper brain functions. We show that MeCP2 cooperates with HIPK2 in induction of apoptosis and that Ser 80 phosphorylation is required together with the DNA binding of MeCP2. These data are, to our knowledge, the first that describe a kinase associating with MeCP2, causing its specific phosphorylation in vivo and, furthermore, they reinforce the role of MeCP2 in regulating cell growth. PMID:19820693

  17. BMSCs Interactions with Adventitial Fibroblasts Display Smooth Muscle Cell Lineage Potential in Differentiation and Migration That Contributes to Neointimal Formation

    PubMed Central

    Wendan, Y.; Changzhu, J.; Xuhong, S.; Hongjing, C.; Hong, S.; Dongxia, Y.; Fang, X.

    2016-01-01

    In this study a model of simulated vascular injury in vitro was used to study the characterization of bone-marrow-derived mesenchymal stem cells (BMSCs) morphology and to investigate the differentiation and migration of BMSCs in the presence of adventitial fibroblasts. BMSCs from rats were indirectly cocultured with adventitial fibroblasts in a transwell chamber apparatus for 7 days, and clonogenic assays demonstrated that BMSCs could be differentiated into smooth muscle-like cells with this process, including smooth muscle α-actin (α-SMA) expression by immunofluorescence staining. Cell morphology of BMSCs was assessed by inverted microscope, while cell proliferation was assessed by MTT assay. The expressions of TGF-β1, MMP-1, and NF-κB were detected by immunofluorescence staining and Smad3 mRNA was measured by reverse transcription PCR. Migration ability of BMSCs with DAPI-labeled nuclei was measured by laser confocal microscopy. Our results demonstrate that indirect interactions with adventitial fibroblasts can induce proliferation, differentiation, and migration of BMSCs that can actively participate in neointimal formation. Our results indicate that the pathogenesis of vascular remodeling might perform via TGF-β1/Smad3 signal transduction pathways. PMID:26880952

  18. Uracil DNA Glycosylase BKRF3 Contributes to Epstein-Barr Virus DNA Replication through Physical Interactions with Proteins in Viral DNA Replication Complex

    PubMed Central

    Su, Mei-Tzu; Liu, I-Hua; Wu, Chia-Wei; Chang, Shu-Ming; Tsai, Ching-Hwa; Yang, Pei-Wen; Chuang, Yu-Chia; Lee, Chung-Pei

    2014-01-01

    ABSTRACT Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmunoprecipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication. IMPORTANCE Catalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme

  19. Do student self-efficacy and teacher-student interaction quality contribute to emotional and social engagement in fifth grade math?

    PubMed

    Martin, Daniel P; Rimm-Kaufman, Sara E

    2015-10-01

    This study examined (a) the contribution of math self-efficacy to students' perception of their emotional and social engagement in fifth grade math classes, and (b) the extent to which high quality teacher-student interactions compensated for students' low math self-efficacy in contributing to engagement. Teachers (n = 73) were observed three times during the year during math to measure the quality of teacher-student interactions (emotional, organizational, and instructional support). Fifth graders (n = 387) reported on their math self-efficacy at the beginning of the school year and then were surveyed about their feelings of engagement in math class three times during the year immediately after the lessons during which teachers were observed. Results of multi-level models indicated that students initially lower in math self-efficacy reported lower emotional and social engagement during math class than students with higher self-efficacy. However, in classrooms with high levels of teacher emotional support, students reported similar levels of both emotional and social engagement, regardless of their self-efficacy. No comparable findings emerged for organizational and instructional support. The discussion considers the significance of students' own feelings about math in relation to their engagement, as well as the ways in which teacher and classroom supports can compensate for students lack of agency. The work has implications for school psychologists and teachers eager to boost students' engagement in math class. PMID:26407834

  20. The interaction domains of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heteromultimeric channels.

    PubMed

    Myeong, Jongyun; Ko, Juyeon; Hong, Chansik; Yang, Dongki; Lee, Kyu Pil; Jeon, Ju-Hong; So, Insuk

    2016-06-01

    Transient receptor potential canonical (TRPC) family contains a non-selective cation channel, and four TRPC subunits form a functional tetrameric channel. TRPC4/5 channels form not only the homotetrameric channel but also a heterotetrameric channel with TRPC1. We investigated the interaction domain required for TRPC1/4 or TRPC1/5 heteromultimeric channels using FRET and the patch-clamp technique. TRPC1 only localized at the plasma membrane (PM) when it was coexpressed with TRPC4 or TRPC5. The TRPC1/4 or TRPC1/5 heteromultimeric showed the typical outward rectifying I/V curve. When TRPC1 and TRPC4 form a heteromeric channel, the N-terminal coiled-coil domain (CCD) and C-terminal 725-745 region of TRPC1 interact with the N-terminal CCD and C-terminal 700-728 region of TRPC4. However, when TRPC1 and TRPC5 form a heteromeric channel, the N-terminal CCD and C-terminal 673-725 region of TRPC1 interact with the N-terminal CCD and C-terminal 707-735 region of TRPC5. In conclusion, the N-terminal CCD of TRPC channels is essential for the heteromultimeric structure of TRPC channels, whereas specific C-terminal regions are required for unique heteromerization between subgroups of TRPC channels. PMID:27131740

  1. The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance

    PubMed Central

    Césari, Stella; Kanzaki, Hiroyuki; Fujiwara, Tadashi; Bernoux, Maud; Chalvon, Véronique; Kawano, Yoji; Shimamoto, Ko; Dodds, Peter; Terauchi, Ryohei; Kroj, Thomas

    2014-01-01

    Plant resistance proteins of the class of nucleotide-binding and leucine-rich repeat domain proteins (NB-LRRs) are immune sensors which recognize pathogen-derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB-LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR-independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. Upon recognition of the pathogen effector AVR-Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo- and hetero-complexes and interact through their coiled-coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR-Pia, neither RGA4 nor RGA5 is re-localized to the nucleus. These results establish a model for the interaction of hetero-pairs of NB-LRRs in plants: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor. PMID:25024433

  2. Multiple metasomatic events recorded in Kilbourne Hole peridotite xenoliths: the relative contribution of host basalt interaction vs. silicate metasomatic glass

    NASA Astrophysics Data System (ADS)

    Hammond, S. J.; Yoshikawa, M.; Harvey, J.; Burton, K. W.

    2010-12-01

    Stark differences between bulk-rock lithophile trace element budgets and the sum of the contributions from their constituent minerals are common, if not ubiquitous in peridotite xenoliths [1]. In the absence of modal metasomatism this discrepancy is often attributed to the “catch-all”, yet often vague process of cryptic metasomatism. This study presents comprehensive Sr-Nd isotope ratios for variably metasomatized bulk-rock peridotites, host basalts, constituent peridotite mineral phases and interstitial glass from 13 spinel lherzolite and harzburgite xenoliths from the Kilbourne Hole volcanic maar, New Mexico, USA. Similar measurements were also made on hand-picked interstitial glass from one of the most highly metasomatized samples (KH03-16) in an attempt to unravel the effects of multiple metasomatic events. In all Kilbourne Hole peridotites analysed, hand-picked, optically clean clinopyroxenes preserve a more primitive Sr isotope signature than the corresponding bulk-rock; a pattern preserved in all but one sample for Nd isotope measurements. Reaction textures, avoided during hand-picking, around clinopyroxene grains are evident in the most metasomatized samples and accompanied by films of high-SiO2 interstitial glass. The margins of primary minerals appear partially resorbed and trails of glassy melt inclusions similar in appearance to those previously reported from the same locality [2], terminate in these films. Hand-picked glass from KH03-16 reveals the most enriched 87Sr/86Sr of any component recovered from these xenoliths (87Sr/86Sr = 0.708043 ± 0.00009; [Sr] = 81 ppm). Similarly, the 143Nd/144Nd of the glass is amongst the most enriched of the peridotite components (143Nd/144Nd = 0.512893 ± 0.000012; [Nd] = 10 ppm). However, the host basalt (87Sr/86Sr = 0.703953 ± 0.00012; 143Nd/144Nd = 0.512873 ± 0.000013), similar in composition to nearby contemporaneous Potrillo Volcanic Field basalts [3], contains nearly an order of magnitude more Sr and more

  3. Structural basis of FYCO1 and MAP1LC3A interaction reveals a novel binding mode for Atg8-family proteins.

    PubMed

    Cheng, Xiaofang; Wang, Yingli; Gong, Yukang; Li, Faxiang; Guo, Yujiao; Hu, Shichen; Liu, Jianping; Pan, Lifeng

    2016-08-01

    FYCO1 (FYVE and coiled-coil domain containing 1) functions as an autophagy adaptor in directly linking autophagosomes with the microtubule-based kinesin motor, and plays an essential role in the microtubule plus end-directed transport of autophagic vesicles. The specific association of FYCO1 with autophagosomes is mediated by its interaction with Atg8-family proteins decorated on the outer surface of autophagosome. However, the mechanistic basis governing the interaction between FYCO1 and Atg8-family proteins is largely unknown. Here, using biochemical and structural analyses, we demonstrated that FYCO1 contains a unique LC3-interacting region (LIR), which discriminately binds to mammalian Atg8 orthologs and preferentially binds to the MAP1LC3A and MAP1LC3B. In addition to uncovering the detailed molecular mechanism underlying the FYCO1 LIR and MAP1LC3A interaction, the determined FYCO1-LIR-MAP1LC3A complex structure also reveals a unique LIR binding mode for Atg8-family proteins, and demonstrates, first, the functional relevance of adjacent sequences C-terminal to the LIR core motif for binding to Atg8-family proteins. Taken together, our findings not only provide new mechanistic insight into FYCO1-mediated transport of autophagosomes, but also expand our understanding of the interaction modes between LIR motifs and Atg8-family proteins in general. PMID:27246247

  4. CK2-regulated schwannomin-interacting protein IQCJ-SCHIP-1 association with AnkG contributes to the maintenance of the axon initial segment.

    PubMed

    Papandréou, Marie-Jeanne; Vacher, Hélène; Fache, Marie-Pierre; Klingler, Esther; Rueda-Boroni, Fanny; Ferracci, Géraldine; Debarnot, Claire; Pipéroglou, Christelle; Garcia Del Caño, Gontzal; Goutebroze, Laurence; Dargent, Bénédicte

    2015-08-01

    The axon initial segment (AIS) plays a central role in electrogenesis and in the maintenance of neuronal polarity. Its molecular organization is dependent on the scaffolding protein ankyrin (Ank) G and is regulated by kinases. For example, the phosphorylation of voltage-gated sodium channels by the protein kinase CK2 regulates their interaction with AnkG and, consequently, their accumulation at the AIS. We previously showed that IQ motif containing J-Schwannomin-Interacting Protein 1 (IQCJ-SCHIP-1), an isoform of the SCHIP-1, accumulated at the AIS in vivo. Here, we analyzed the molecular mechanisms involved in IQCJ-SCHIP-1-specific axonal location. We showed that IQCJ-SCHIP-1 accumulation in the AIS of cultured hippocampal neurons depended on AnkG expression. Pull-down assays and surface plasmon resonance analysis demonstrated that AnkG binds to CK2-phosphorylated IQCJ-SCHIP-1 but not to the non-phosphorylated protein. Surface plasmon resonance approaches using IQCJ-SCHIP-1, SCHIP-1a, another SCHIP-1 isoform, and their C-terminus tail mutants revealed that a segment including multiple CK2-phosphorylatable sites was directly involved in the interaction with AnkG. Pharmacological inhibition of CK2 diminished both IQCJ-SCHIP-1 and AnkG accumulation in the AIS. Silencing SCHIP-1 expression reduced AnkG cluster at the AIS. Finally, over-expression of IQCJ-SCHIP-1 decreased AnkG concentration at the AIS, whereas a mutant deleted of the CK2-regulated AnkG interaction site did not. Our study reveals that CK2-regulated IQJC-SCHIP-1 association with AnkG contributes to AIS maintenance. The axon initial segment (AIS) organization depends on ankyrin (Ank) G and kinases. Here we showed that AnkG binds to CK2-phosphorylated IQCJ-SCHIP-1, in a segment including 12 CK2-phosphorylatable sites. In cultured neurons, either pharmacological inhibition of CK2 or IQCJ-SCHIP-1 silencing reduced AnkG clustering. Overexpressed IQCJ-SCHIP-1 decreased AnkG concentration at the AIS whereas a

  5. Structural basis for recruitment of Rab6-interacting protein 1 to Golgi via a RUN domain.

    PubMed

    Recacha, Rosario; Boulet, Annick; Jollivet, Florence; Monier, Solange; Houdusse, Anne; Goud, Bruno; Khan, Amir R

    2009-01-14

    Small GTPase Rab6 regulates vesicle trafficking at the level of Golgi via recruitment of numerous and unrelated effectors. The crystal structure of Rab6a(GTP) in complex with a 378-residue internal fragment of the effector Rab6IP1 was solved at 3.2 angstroms resolution. This Rab6IP1 region encompasses an all alpha-helical RUN domain followed in tandem by a PLAT domain that adopts a beta sandwich fold. The structure reveals that the first and last alpha helices of the RUN domain mediate binding to switch I, switch II, and the interswitch region of Rab6. It represents the largest Rab-effector complex determined to date. Comparisons with the recent structure of Rab6 in complex with an unrelated effector, human golgin GCC185, reveals significant conformational changes in the conserved hydrophobic triad of Rab6. Flexibility in the switch and interswitch regions of Rab6 mediates recognition of compositionally distinct alpha-helical coiled coils, thereby contributing to Rab6 promiscuity in effector recruitment. PMID:19141279

  6. Contributions of primary and secondary biogenic VOC tototal OH reactivity during the CABINEX (Community Atmosphere-Biosphere INteractions Experiments)-09 field campaign

    NASA Astrophysics Data System (ADS)

    Kim, S.; Guenther, A.; Karl, T.; Greenberg, J.

    2011-08-01

    We present OH reactivity measurements using the comparative reactivity method with a branch enclosure technique for four different tree species (red oak, white pine, beech and red maple) in the UMBS PROPHET tower footprint during the Community Atmosphere Biosphere INteraction EXperiment (CABINEX) field campaign in July of 2009. Proton Transfer Reaction-Mass Spectrometry (PTR-MS) was sequentially used as a detector for OH reactivity and BVOC concentrations including isoprene and monoterpenes (MT) for enclosure air. Therefore, the measurement dataset contains both measured and calculated OH reactivity from well-known BVOC. The results indicate that isoprene and MT, and in one case a sesquiterpene, can account for the measured OH reactivity. Significant discrepancy between measured OH reactivity and calculated OH reactivity from isoprene and MT is found for the red maple enclosure dataset but it can be reconciled by adding reactivity from emission of a sesquiterpene, α-farnesene, detected by GC-MS. This leads us to conclude that no significant unknown BVOC emission contributed to ambient OH reactivity from these trees at least during the study period. However, this conclusion should be followed up by more comprehensive side-by-side intercomparison between measured and calculated OH reactivity and laboratory experiments with controlled temperature and light environments to verify effects of those essential parameters towards unknown/unmeasured reactive BVOC emissions. This conclusion leads us to explore the contribution towards ambient OH reactivity (the dominant OH sink in this ecosystem) oxidation products such as hydroxyacetone, glyoxal, methylglyoxal and C4 and C5-hydroxycarbonyl using recently published isoprene oxidation mechanisms (Mainz Isoprene Mechanism II and Leuven Isoprene Mechanism). Evaluation of conventionally unmeasured first generation oxidation products of isoprene and their possible contribution to ambient missing OH reactivity indicates that the

  7. The formation of vault-tubes: a dynamic interaction between vaults and vault PARP.

    PubMed

    van Zon, Arend; Mossink, Marieke H; Schoester, Martijn; Houtsmuller, Adriaan B; Scheffer, George L; Scheper, Rik J; Sonneveld, Pieter; Wiemer, Erik A C

    2003-11-01

    Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles that are composed of a major vault protein (MVP), two minor vault proteins [telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase (VPARP)] and small untranslated RNA molecules. Not all expressed TEP1 and VPARP in cells is bound to vaults. TEP1 is known to associate with the telomerase complex, whereas VPARP is also present in the nuclear matrix and in cytoplasmic clusters (VPARP-rods). We examined the subcellular localization and the dynamics of the vault complex in a non-small cell lung cancer cell line expressing MVP tagged with green fluorescent protein. Using quantitative fluorescence recovery after photobleaching (FRAP) it was shown that vaults move temperature independently by diffusion. However, incubation at room temperature (21 degrees C) resulted in the formation of distinct tube-like structures in the cytoplasm. Raising the temperature could reverse this process. When the vault-tubes were formed, there were fewer or no VPARP-rods present in the cytoplasm, suggesting an incorporation of the VPARP into the vault-tubes. MVP molecules have to interact with each other via their coiled-coil domain in order to form vault-tubes. Furthermore, the stability of microtubules influenced the efficiency of vault-tube formation at 21 degrees C. The dynamics and structure of the tubes were examined using confocal microscopy. Our data indicate a direct and dynamic relationship between vaults and VPARP, providing further clues to unravel the function of vaults. PMID:13130096

  8. TRIP: a novel double stranded RNA binding protein which interacts with the leucine rich repeat of flightless I.

    PubMed Central

    Wilson, S A; Brown, E C; Kingsman, A J; Kingsman, S M

    1998-01-01

    A northwestern screen of a CHO-K1 cell line cDNA library with radiolabelled HIV-1 TAR RNA identified a novel TAR RNA interacting protein, TRIP. The human trip cDNA was also cloned and its expression is induced by phorbol esters. The N-terminus of TRIP shows high homology to the coiled coil domain of FLAP, a protein which binds the leucine-rich repeat (LRR) of Flightless I (FLI) and the interaction of TRIP with the FLI LRR has been confirmed in vitro . TRIP does not bind single stranded DNA or RNA significantly and binds double stranded DNA weakly. In contrast, TRIP binds double stranded RNA with high affinity and two molecules of TRIP bind the TAR stem. The RNA binding domain has been identified and encompasses a lysine-rich motif. A TRIP-GFP fusion is localised in the cytoplasm and excluded from the nucleus. FLI has a C-terminal gelsolin-like domain which binds actin and therefore the association of TRIP with the FLI LRR may provide a link between the actin cytoskeleton and RNA in mammalian cells. PMID:9671805

  9. TRIP: a novel double stranded RNA binding protein which interacts with the leucine rich repeat of flightless I.

    PubMed

    Wilson, S A; Brown, E C; Kingsman, A J; Kingsman, S M

    1998-08-01

    A northwestern screen of a CHO-K1 cell line cDNA library with radiolabelled HIV-1 TAR RNA identified a novel TAR RNA interacting protein, TRIP. The human trip cDNA was also cloned and its expression is induced by phorbol esters. The N-terminus of TRIP shows high homology to the coiled coil domain of FLAP, a protein which binds the leucine-rich repeat (LRR) of Flightless I (FLI) and the interaction of TRIP with the FLI LRR has been confirmed in vitro . TRIP does not bind single stranded DNA or RNA significantly and binds double stranded DNA weakly. In contrast, TRIP binds double stranded RNA with high affinity and two molecules of TRIP bind the TAR stem. The RNA binding domain has been identified and encompasses a lysine-rich motif. A TRIP-GFP fusion is localised in the cytoplasm and excluded from the nucleus. FLI has a C-terminal gelsolin-like domain which binds actin and therefore the association of TRIP with the FLI LRR may provide a link between the actin cytoskeleton and RNA in mammalian cells. PMID:9671805

  10. Recent Progresses in Studying Helix-Helix Interactions in Proteins by Incorporating the Wenxiang Diagram into the NMR Spectroscopy.

    PubMed

    Zhou, Guo-Ping; Chen, Dong; Liao, Siming; Huang, Ri-Bo

    2016-01-01

    All residues in an alpha helix can be characterized and dispositioned on a 2D the wenxiang diagram, which possesses the following features: (1) the relative locations of the amino acids in the α-helix can be clearly displayed regardless how long it is; (2) direction of an alphahelix can be indicated; and (3) more information regarding each of the constituent amino acid residues in an alpha helix. Owing to its intuitionism and easy visibility, wenxiang diagrams have had an immense influence on our understanding of protein structure, protein-protein interactions, and the effect of helical structural stability on protein conformational transitions. In this review, we summarize two recent applications of wenxiang diagrams incorporating NMR spectroscopy in the researches of the coiled-coil protein interactions related to the regulation of contraction or relaxation states of vascular smooth muscle cells, and the effects of α-helical stability on the protein misfolding in prion disease, in hopes that the gained valuable information through these studies can stimulate more and more widely applications of wenxiang diagrams in structural biology. PMID:26286215

  11. Starch synthase 4 is located in the thylakoid membrane and interacts with plastoglobule-associated proteins in Arabidopsis.

    PubMed

    Gámez-Arjona, Francisco M; Raynaud, Sandy; Ragel, Paula; Mérida, Angel

    2014-10-01

    Starch synthesis requires the formation of a primer that can be subsequently elongated and branched. How this primer is produced, however, remains unknown. The control of the number of starch granules produced per chloroplast is also a matter of debate. We previously showed starch synthase 4 (SS4) to be involved in both processes, although the mechanisms involved are yet to be fully characterised. The present work shows that SS4 displays a specific localization different from other starch synthases. Thus, this protein is located in specific areas of the thylakoid membrane and interacts with the proteins fibrillin 1a (FBN1a) and 1b (FBN1b), which are mainly located in plastoglobules. SS4 would seem to be associated with plastoglobules attached to the thylakoids (or to that portion of the thylakoids where plastoglobules have originated), forming a complex that includes the FBN1s and other as-yet unidentified proteins. The present results also indicate that the localization pattern of SS4, and its interactions with the FBN1 proteins, are mediated through its N-terminal region, which contains two long coiled-coil motifs. The localization of SS4 in specific areas of the thylakoid membrane suggests that starch granules are originated at specific regions of the chloroplast. PMID:25088399

  12. Investigation of the electronic structures of organolanthanide sandwich complex anions by photoelectron spectroscopy: 4f orbital contribution in the metal-ligand interaction.

    PubMed

    Hosoya, Natsuki; Yada, Keizo; Masuda, Tomohide; Nakajo, Erika; Yabushita, Satoshi; Nakajima, Atsushi

    2014-05-01

    The electronic structures of lanthanide (Ln) ions sandwiched between 1,3,5,7-cyclooctatetraene (COT), Ln(COT)2(-), have been investigated by anion photoelectron spectroscopy. Complexes of 12 Ln atoms were investigated (excluding promethium (Pm), europium (Eu), and ytterbium (Yb)). The 213 nm photoelectron (PE) spectra of Ln(COT)2(-) exhibit two peaks assignable to the highest occupied molecular orbital (HOMO; e2u) and the next HOMO (HOMO-1; e2g) approximately at 2.6 and 3.6 eV, respectively, and their energy gap increases as the central metal atom progresses from lanthanum (La) to lutetium (Lu). Since lanthanide contraction shortens the distance between the Ln atom and the COT ligands, the widening energy gap represents the destabilization of the e2u orbital as well as the stabilization of the e2g orbital. Evidence for 4f orbital contribution in the metal-ligand interaction has been revealed by the Ln atom dependence in which the same e2u orbital symmetry enables an interaction between the 4f orbital of Ln atoms and the π orbital of COT. PMID:24742246

  13. Interaction of a putative BH3 domain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

    SciTech Connect

    Lee, Dong-Hwa; Ha, Ji-Hyang; Kim, Yul; Bae, Kwang-Hee; Park, Jae-Yong; Choi, Wan Sung; Yoon, Ho Sup; Park, Sung Goo; Park, Byoung Chul; Yi, Gwan-Su; Chi, Seung-Wook

    2011-05-20

    Highlights: {yields} Identification of a conserved BH3 motif in C-terminal coiled coil region of nCLU. {yields} The nCLU BH3 domain binds to BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. {yields} A conserved binding mechanism of nCLU BH3 and the other pro-apoptotic BH3 peptides with Bcl-X{sub L}. {yields} The absolutely conserved Leu323 and Asp328 of nCLU BH3 domain are critical for binding to Bcl-X{sub L.} {yields} Molecular understanding of the pro-apoptotic function of nCLU as a novel BH3-only protein. -- Abstract: Clusterin (CLU) is a multifunctional glycoprotein that is overexpressed in prostate and breast cancers. Although CLU is known to be involved in the regulation of apoptosis and cell survival, the precise molecular mechanism underlying the pro-apoptotic function of nuclear CLU (nCLU) remains unclear. In this study, we identified a conserved BH3 motif in C-terminal coiled coil (CC2) region of nCLU by sequence analysis and characterized the molecular interaction of the putative nCLU BH3 domain with anti-apoptotic Bcl-2 family proteins by nuclear magnetic resonance (NMR) spectroscopy. The chemical shift perturbation data demonstrated that the nCLU BH3 domain binds to pro-apoptotic BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. A structural model of the Bcl-X{sub L}/nCLU BH3 peptide complex reveals that the binding mode is remarkably similar to those of other Bcl-X{sub L}/BH3 peptide complexes. In addition, mutational analysis confirmed that Leu323 and Asp328 of nCLU BH3 domain, absolutely conserved in the BH3 motifs of BH3-only protein family, are critical for binding to Bcl-X{sub L}. Taken altogether, our results suggest a molecular basis for the pro-apoptotic function of nCLU by elucidating the residue specific interactions of the BH3 motif in nCLU with anti-apoptotic Bcl-2 family proteins.

  14. The contributions of soil-moisture interactions to climate change in the tropics in CMIP5 projections from the GLACE-CMIP5 experiment

    NASA Astrophysics Data System (ADS)

    May, Wilhelm; Rummukainen, Markku; Meier, Arndt

    2015-04-01

    The contributions of the projected changes in soil moisture to the overall climate change in the tropics at the end of the 21st century are quantified using the simulations from the GLACE-CMIP5 experiment. This is done by directly comparing the overall projected future changes in climate, which are partly related to changes in soil moisture, to the changes in climate that are not affected by any changes in soil moisture. As the five different climate models contributing to the experiment, i.e., CESM, EC-EARTH, GDFL, IPSL and MPI-ESM show quite different geographical distributions of the future changes in soil moisture in the tropics as well as different magnitudes, we do not consider ensemble mean values based on the corresponding simulations with these models but rather analyse the simulations from the different models separately. This allows for quantifying the contributions of the projected changes in soil moisture to climate change in the tropics for each climate model despite the different characteristics of the soil moisture changes themselves. We focus on two aspects of the interactions of the soil moisture with climate, i.e., the soil moisture-temperature coupling and the soil moisture-precipitation coupling/feedback. The simulations show marked future changes in soil moisture content in the tropics, with a general tendency of increases in the central parts of the tropics and decreases in the subtropics. These changes are associated with corresponding changes in precipitation, with an overall tendency of a 5change in soil moisture in response to a precipitation change of 1 mm/d. The changes in soil moisture content are found to give major contributions to the overall climate change in the tropics. This is particularly the case for the latent and sensible heat fluxes as well as near-surface temperature, where more than 80moisture changes. For precipitation, on the other hand, 30-40overall future changes are induced by the changes in soil moisture. The

  15. Deoxycholate Interacts with IpaD of Shigella flexneri in Inducing the Recruitment of IpaB to the Type III Secretion Apparatus Needle Tip*S⃞

    PubMed Central

    Stensrud, Kenneth F.; Adam, Philip R.; La Mar, Cassandra D.; Olive, Andrew J.; Lushington, Gerald H.; Sudharsan, Raghavi; Shelton, Naomi L.; Givens, Richard S.; Picking, Wendy L.; Picking, William D.

    2008-01-01

    Type III secretion (TTS) is an essential virulence function for Shigella flexneri that delivers effector proteins that are responsible for bacterial invasion of intestinal epithelial cells. The Shigella TTS apparatus (TTSA) consists of a basal body that spans the bacterial inner and outer membranes and a needle exposed at the pathogen surface. At the distal end of the needle is a “tip complex” composed of invasion plasmid antigen D (IpaD). IpaD not only regulates TTS, but is required for the recruitment and stable association of the translocator protein IpaB at the TTSA needle tip in the presence of deoxycholate or other bile salts. This phenomenon is not accompanied by induction of TTS or the recruitment of IpaC to the Shigella surface. We now show that IpaD specifically binds fluorescein-labeled deoxycholate and, based on energy transfer measurements and docking simulations, this interaction appears to occur where the N-terminal domain of IpaD meets its central coiled-coil, a region that may also be involved in needle-tip interactions. TTS is initiated as a series of distinct steps and that small molecules present in the bacterial milieu are capable of inducing the first step of TSS through interactions with the needle tip protein IpaD. Furthermore, the amino acids proposed to be important for deoxycholate binding by IpaD appear to have significant roles in regulating tip complex composition and pathogen entry into host cells. PMID:18450744

  16. PM-IRRAS investigation of the interaction of serum albumin and fibrinogen with a biomedical-grade stainless steel 316LVM surface.

    PubMed

    Desroches, Marie J; Chaudhary, Nida; Omanovic, Sasha

    2007-09-01

    Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was applied to investigate the interaction of bovine serum albumin (BSA) and fibrinogen with a biomedical-grade 316LVM stainless steel surface, in terms of the adsorption thermodynamics and adsorption-induced secondary structure changes of the proteins. Highly negative apparent Gibbs energy of adsorption values revealed a spontaneous adsorption of both proteins onto the surface, accompanied by significant changes in their secondary structure. It was determined that, at saturated surface coverages, lateral interactions between the adsorbed BSA molecules induced rather extensive secondary structure changes. Fibrinogen's two coiled coils appeared to undergo negligible secondary structure changes upon adsorption of the protein, while large structural rearrangements of the protein's globular domains occurred upon adsorption. The secondary structure of adsorbed fibrinogen was not influenced by lateral interactions between the adsorbed fibrinogen molecules. PM-IRRAS was deemed to be viable for investigating protein adsorption and for obtaining information on adsorption-induced changes in their secondary structures. PMID:17715960

  17. On the necessity of dissecting sequence similarity scores into segment-specific contributions for inferring protein homology, function prediction and annotation

    PubMed Central

    2014-01-01

    Background Protein sequence similarities to any types of non-globular segments (coiled coils, low complexity regions, transmembrane regions, long loops, etc. where either positional sequence conservation is the result of a very simple, physically induced pattern or rather integral sequence properties are critical) are pertinent sources for mistaken homologies. Regretfully, these considerations regularly escape attention in large-scale annotation studies since, often, there is no substitute to manual handling of these cases. Quantitative criteria are required to suppress events of function annotation transfer as a result of false homology assignments. Results The sequence homology concept is based on the similarity comparison between the structural elements, the basic building blocks for conferring the overall fold of a protein. We propose to dissect the total similarity score into fold-critical and other, remaining contributions and suggest that, for a valid homology statement, the fold-relevant score contribution should at least be significant on its own. As part of the article, we provide the DissectHMMER software program for dissecting HMMER2/3 scores into segment-specific contributions. We show that DissectHMMER reproduces HMMER2/3 scores with sufficient accuracy and that it is useful in automated decisions about homology for instructive sequence examples. To generalize the dissection concept for cases without 3D structural information, we find that a dissection based on alignment quality is an appropriate surrogate. The approach was applied to a large-scale study of SMART and PFAM domains in the space of seed sequences and in the space of UniProt/SwissProt. Conclusions Sequence similarity core dissection with regard to fold-critical and other contributions systematically suppresses false hits and, additionally, recovers previously obscured homology relationships such as the one between aquaporins and formate/nitrite transporters that, so far, was only

  18. Concluding the amyloid formation pathway of a coiled-coil-based peptide from the size of the critical nucleus.

    PubMed

    Gerling, Ulla I M; Miettinen, Markus S; Koksch, Beate

    2015-01-12

    The size of the critical nucleus acting as intermediate in the amyloid formation of a model peptide is calculated. The theoretical approach is based on experimentally determined amyloid formation rates and gives new insights into the amyloid formation pathway. PMID:25257178

  19. Novel nuclear targeting coiled-coil protein of Helicobacter pylori showing Ca(2+)-independent, Mg(2+)-dependent DNase I activity.

    PubMed

    Kwon, Young Chul; Kim, Sinil; Lee, Yong Seok; Lee, Je Chul; Cho, Myung-Je; Lee, Woo-Kon; Kang, Hyung-Lyun; Song, Jae-Young; Baik, Seung Chul; Ro, Hyeon Su

    2016-05-01

    HP0059, an uncharacterized gene of Helicobacter pylori, encodes a 284-aa-long protein containing a nuclear localization sequence (NLS) and multiple leucine-rich heptad repeats. Effects of HP0059 proteins in human stomach cells were assessed by incubation of recombinant HP0059 proteins with the AGS human gastric carcinoma cell line. Wild-type HP0059 proteins showed cytotoxicity in AGS cells in a concentration-dependent manner, whereas NLS mutant protein showed no effect, suggesting that the cytotoxicity is attributed to host nuclear localization. AGS cells transfected with pEGFP-HP0059 plasmid showed strong GFP signal merged to the chromosomal DNA region. The chromosome was fragmented into multiple distinct dots merged with the GFP signal after 12 h of incubation. The chromosome fragmentation was further explored by incubation of AGS chromosomal DNA with recombinant HP0059 proteins, which leaded to complete degradation of the chromosomal DNA. HP0059 protein also degraded circular plasmid DNA without consensus, being an indication of DNase I activity. The DNase was activated by MgCl2, but not by CaCl2. The activity was completely blocked by EDTA. The optimal pH and temperature for DNase activity were 7.0-8.0 and 55°C, respectively. These results indicate that HP0059 possesses a novel DNase I activity along with a role in the genomic instability of human gastric cells, which may result in the transformation of gastric cells. PMID:27095458

  20. Addition of magnesium chloride to enhance mono-dispersity of a coiled-coil recombinant mouse macrophage protein.

    PubMed

    Pahuja, Parveen; Srinivasan, Alagiri; Puri, Munish

    2014-04-01

    X-ray crystallography for the determination of three-dimensional structures of protein macromolecules represents an important tool in function assignment of uncharacterized proteins. However, crystallisation is often difficult to achieve. A protein sample fully characterized in terms of dispersity may increase the likelihood of successful crystallisation by improving the predictability of the crystallisation process. To maximize the probability of crystallisation of a novel mouse macrophage protein (rMMP), target molecule was characterized and refined to improve monodispersity. Addition of MgCl2 at low concentrations resolves the rMMP into a monodisperse solution, and finally successful crystallization of rMMP was achieved. The effect of MgCl2 was studied using gel filtration chromatography and dynamic light scattering. PMID:24385107

  1. Isolation and Characterization of Kinase Interacting Protein 1, a Pollen Protein That Interacts with the Kinase Domain of PRK1, a Receptor-Like Kinase of Petunia1

    PubMed Central

    Skirpan, Andrea L.; McCubbin, Andrew G.; Ishimizu, Takeshi; Wang, Xi; Hu, Yi; Dowd, Peter E.; Ma, Hong; Kao, Teh-hui

    2001-01-01

    Many receptor-like kinases have been identified in plants and have been shown by genetic or transgenic knockouts to play diverse physiological roles; however, to date, the cytosolic interacting proteins of relatively few of these kinases have been identified. We have previously identified a predominantly pollen-expressed receptor-like kinase of petunia (Petunia inflata), named PRK1, and we have shown by the antisense RNA approach that it is required for microspores to progress from the unicellular to bicellular stage. To investigate the PRK1-mediated signal transduction pathway, PRK1-K cDNA, encoding most of the cytoplasmic domain of PRK1, was used as bait in yeast (Saccharomyces cerevisiae) two-hybrid screens of pollen/pollen tube cDNA libraries of petunia. A protein named kinase interacting protein 1 (KIP1) was found to interact very strongly with PRK1-K. This interaction was greatly reduced when lysine-462 of PRK1-K, believed to be essential for kinase activity, was replaced with arginine (the resulting protein is named PRK1-K462R). The amino acid sequence of KIP1 deduced from full-length cDNA contains an EF-hand Ca2+-binding motif and nine predicted coiled-coil regions. The yeast two-hybrid assay and affinity chromatography showed that KIP1 interacts with itself to form a dimer or higher multimer. KIP1 is present in a single copy in the genome, and is expressed predominantly in pollen with a similar temporal pattern to PRK1. In situ hybridization showed that PRK1 and KIP1 transcripts were localized in the cytoplasm of pollen. PRK1-K phosphorylated KIP1-NT (amino acids 1–716), whereas PRK1-K462R only weakly phosphorylated KIP1-NT in vitro. PMID:11500547

  2. A Contribution to Identification of Novel Regulators of Plant Response to Sulfur Deficiency: Characteristics of a Tobacco Gene UP9C, Its Protein Product and the Effects of UP9C Silencing

    PubMed Central

    Lewandowska, Małgorzata; Wawrzyńska, Anna; Moniuszko, Grzegorz; Łukomska, Jolanta; Zientara, Katarzyna; Piecho, Marta; Hodurek, Paweł; Zhukov, Igor; Liszewska, Frantz; Nikiforova, Victoria; Sirko, Agnieszka

    2010-01-01

    Extensive changes in plant transcriptome and metabolome have been observed by numerous research groups after transferring plants from optimal conditions to sulfur (S) deficiency. Despite intensive studies and recent important achievements, like identification of SLIM1/EIL3 as a major transcriptional regulator of the response to S-deficiency, many questions concerning other elements of the regulatory network remain unanswered. Investigations of genes with expression regulated by S-deficiency stress encoding proteins of unknown function might help to clarify these problems. This study is focused on the UP9C gene and the UP9-like family in tobacco. Homologs of these genes exist in other plant species, including a family of four genes of unknown function in Arabidopsis thaliana (LSU1-4), of which two were reported as strongly induced by S-deficit and to a lesser extent by salt stress and nitrate limitation. Conservation of the predicted structural features, such as coiled coil region or nuclear localization signal, suggests that these proteins might have important functions possibly mediated by interactions with other proteins. Analysis of transgenic tobacco plants with silenced expression of UP9-like genes strongly argues for their significant role in regulation of plant response to S-deficit. Although our study shows that the UP9-like proteins are important components of such response and they might be also required during other stresses, their molecular functions remain a mystery. PMID:20147370

  3. Contribution of the interaction between the rabies virus P protein and I-kappa B kinase ϵ to the inhibition of type I IFN induction signalling.

    PubMed

    Masatani, Tatsunori; Ozawa, Makoto; Yamada, Kentaro; Ito, Naoto; Horie, Masayuki; Matsuu, Aya; Okuya, Kosuke; Tsukiyama-Kohara, Kyoko; Sugiyama, Makoto; Nishizono, Akira

    2016-02-01

    The P protein of rabies virus (RABV) is known to interfere with the phosphorylation of the host IFN regulatory factor 3 (IRF-3) and to consequently inhibit type I IFN induction. Previous studies, however, have only tested P proteins from laboratory-adapted fixed virus strains, and to the best of our knowledge there is no report about the effect of P proteins from street RABV strains or other lyssaviruses on the IRF-3-mediated type I IFN induction system. In this study, we evaluated the inhibitory effect of P proteins from several RABV strains, including fixed and street virus strains and other lyssaviruses (Lagos bat, Mokola and Duvenhage viruses), on IRF-3 signalling. All P proteins tested inhibited retinoic acid-inducible gene-1 (RIG-I)- and TANK binding kinase 1 (TBK1)-mediated IRF-3-dependent IFN-β promoter activities. On the other hand, the P proteins from the RABV street strains 1088 and HCM-9, but not from fixed strains Nishigahara (Ni) and CVS-11 and other lyssaviruses tested, significantly inhibited I-kappa B kinase ϵ (IKKϵ)-inducible IRF-3-dependent IFN-β promoter activity. Importantly, we revealed that the P proteins from the 1088 and HCM-9 strains, but not from the remaining viruses, interacted with IKKϵ. By using expression plasmids encoding chimeric P proteins from the 1088 strain and Ni strain, we found that the C-terminal region of the P protein is important for the interaction with IKKϵ. These findings suggest that the P protein of RABV street strains may contribute to efficient evasion of host innate immunity. PMID:26647356

  4. Relative contributions of sea surface salinity and temperature to density gradient and tropical instability waves: implications to eddy-mean flow interaction

    NASA Astrophysics Data System (ADS)

    Hasson, Audrey; Lee, Tong

    2015-04-01

    With their relatively uniform spatial and temporal sampling, satellite observations have revolutionized the estimates of the spatial derivative fields of various oceanic parameters that are not possible to derive from in-situ measurements on a global scale with sufficient spatial resolutions. For examples, the spatial gradients of sea surface height measurements from altimetry provide information about surface geostrophic currents; those of wind stress make possible the estimates of wind stress curl and divergence; those of sea surface temperature and salinity allow detections of thermal and haline fronts. These spatial derivatives fields are critical to the studies of ocean circulation and air-sea interaction. In particular, the spatial gradients of satellite-derived sea surface temperature and salinity (SST and SSS) have provided an unprecedented opportunity to study density gradient that is important to energy conversion between the background ocean state and the fluctuating flow field such as eddies and waves through baroclinic instability. In this study, we examine eddy-mean flow interaction in tropical oceans by studying the relations between background density gradient and tropical instability wave (TIW) variability using various satellite-derived SSS and SST products. In the equatorial Pacific and Atlantic Oceans, SSS is found to have equal or larger contribution to the background meridional density gradient. This has important consequence to the density variance associated with the TIWs (a proxy for the extraction of available potential energy from the background ocean state to the TIWs). Not accounting for salinity effect would under-estimate the TIW-related density variance by at least a factor of three.

  5. Transcriptional repression by RING finger protein TIF1 beta that interacts with the KRAB repressor domain of KOX1.

    PubMed Central

    Moosmann, P; Georgiev, O; Le Douarin, B; Bourquin, J P; Schaffner, W

    1996-01-01

    Many of the vertebrate zinc finger factors of the Kruppel type (C2H2 zinc fingers) contain in their N-terminus a conserved sequence referred to as the KRAB (Kruppel-associated box) domain that, when tethered to DNA, efficiently represses transcription. Using the yeast two-hybrid system, we have isolated an 835 amino acid RING finger (C3HC4 zinc finger) protein, TIF1 beta (also named KAP-1), that specifically interacts with the KRAB domain of the human zinc finger factor KOX1/ZNF10. TIF1 beta, TIF1 alpha, PML and efp belong to a characteristic subgroup of RING finger proteins that contain one or two other Cys/His-rich clusters (B boxes) and a putative coiled-coil in addition to the classical C3HC4 RING finger motif (RBCC configuration). Like TIF1 alpha, TIF1 beta also contains an additional Cys/His cluster (PHD finger) and a bromo-related domain. When tethered to DNA, TIF1 beta can repress transcription in transiently transfected mammalian cells both from promoter-proximal and remote (enhancer) positions, similarly to the KRAB domain itself. We propose that TIF1 beta is a mediator of the transcriptional repression exerted by the KRAB domain. PMID:9016654

  6. An integrated, peptide-based approach to site-specific protein immobilization for detection of biomolecular interactions.

    PubMed

    Kruis, Ilmar C; Löwik, Dennis W P M; Boelens, Wilbert C; van Hest, Jan C M; Pruijn, Ger J M

    2016-09-21

    We have developed an integrated solution for the site-specific immobilization of proteins on a biosensor surface, which may be widely applicable for high throughput analytical purposes. The gold surface of a biosensor was coated with an anti-fouling layer of zwitterionic peptide molecules from which leucine zipper peptides protrude. Proteins of interest, the autoantigenic proteins La and U1A, were immobilized via a simple incubation procedure by using the complementary leucine zipper sequence as a genetically fused binding tag. This tag forms a strong coiled-coil interaction that is stable during multiple consecutive measurements and under common regeneration conditions. Visualization of the immobilized proteins of interest via antibody binding with multiplex surface plasmon resonance imaging demonstrated 2.5 times higher binding responses than when these proteins were randomly attached to the surface via the commonly applied activated ester-mediated coupling. The proteins could also be immobilized in a leucine zipper-dependent manner directly from complex mixtures like bacterial lysates, eliminating the need for laborious purification steps. This method allows the production of uniform functional protein arrays by control over immobilized protein orientation and geometry and is compatible with high-throughput procedures. PMID:27328408

  7. End4p/Sla2p interacts with actin-associated proteins for endocytosis in Saccharomyces cerevisiae.

    PubMed

    Wesp, A; Hicke, L; Palecek, J; Lombardi, R; Aust, T; Munn, A L; Riezman, H

    1997-11-01

    end4-1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) of ABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis. PMID:9362070

  8. An Interaction between RRP6 and SU(VAR)3-9 Targets RRP6 to Heterochromatin and Contributes to Heterochromatin Maintenance in Drosophila melanogaster.

    PubMed

    Eberle, Andrea B; Jordán-Pla, Antonio; Gañez-Zapater, Antoni; Hessle, Viktoria; Silberberg, Gilad; von Euler, Anne; Silverstein, Rebecca A; Visa, Neus

    2015-09-01

    RNA surveillance factors are involved in heterochromatin regulation in yeast and plants, but less is known about the possible roles of ribonucleases in the heterochromatin of animal cells. Here we show that RRP6, one of the catalytic subunits of the exosome, is necessary for silencing heterochromatic repeats in the genome of Drosophila melanogaster. We show that a fraction of RRP6 is associated with heterochromatin, and the analysis of the RRP6 interaction network revealed physical links between RRP6 and the heterochromatin factors HP1a, SU(VAR)3-9 and RPD3. Moreover, genome-wide studies of RRP6 occupancy in cells depleted of SU(VAR)3-9 demonstrated that SU(VAR)3-9 contributes to the tethering of RRP6 to a subset of heterochromatic loci. Depletion of the exosome ribonucleases RRP6 and DIS3 stabilizes heterochromatic transcripts derived from transposons and repetitive sequences, and renders the heterochromatin less compact, as shown by micrococcal nuclease and proximity-ligation assays. Such depletion also increases the amount of HP1a bound to heterochromatic transcripts. Taken together, our results suggest that SU(VAR)3-9 targets RRP6 to a subset of heterochromatic loci where RRP6 degrades chromatin-associated non-coding RNAs in a process that is necessary to maintain the packaging of the heterochromatin. PMID:26389589

  9. An Interaction between RRP6 and SU(VAR)3-9 Targets RRP6 to Heterochromatin and Contributes to Heterochromatin Maintenance in Drosophila melanogaster

    PubMed Central

    Eberle, Andrea B.; Jordán-Pla, Antonio; Gañez-Zapater, Antoni; Hessle, Viktoria; Silberberg, Gilad; von Euler, Anne; Silverstein, Rebecca A.; Visa, Neus

    2015-01-01

    RNA surveillance factors are involved in heterochromatin regulation in yeast and plants, but less is known about the possible roles of ribonucleases in the heterochromatin of animal cells. Here we show that RRP6, one of the catalytic subunits of the exosome, is necessary for silencing heterochromatic repeats in the genome of Drosophila melanogaster. We show that a fraction of RRP6 is associated with heterochromatin, and the analysis of the RRP6 interaction network revealed physical links between RRP6 and the heterochromatin factors HP1a, SU(VAR)3-9 and RPD3. Moreover, genome-wide studies of RRP6 occupancy in cells depleted of SU(VAR)3-9 demonstrated that SU(VAR)3-9 contributes to the tethering of RRP6 to a subset of heterochromatic loci. Depletion of the exosome ribonucleases RRP6 and DIS3 stabilizes heterochromatic transcripts derived from transposons and repetitive sequences, and renders the heterochromatin less compact, as shown by micrococcal nuclease and proximity-ligation assays. Such depletion also increases the amount of HP1a bound to heterochromatic transcripts. Taken together, our results suggest that SU(VAR)3-9 targets RRP6 to a subset of heterochromatic loci where RRP6 degrades chromatin-associated non-coding RNAs in a process that is necessary to maintain the packaging of the heterochromatin. PMID:26389589

  10. The histone H2A deubiquitinase USP16 interacts with HERC2 and fine-tunes cellular response to DNA damage.

    PubMed

    Zhang, Zhuo; Yang, Huirong; Wang, Hengbin

    2014-11-21

    Histone ubiquitination at DNA double strand breaks facilitates the recruitment of downstream repair proteins; however, how the ubiquitination is dynamically regulated during repair and terminated after repair is not well understood. Here we report that the histone H2A deubiquitinase USP16 interacts with HERC2, fine-tunes the ubiquitin signal during repair, and importantly, is required for terminating the ubiquitination signal after repair. HERC2 interacts with the coiled-coil domain of USP16 through its C-terminal HECT domain. HERC2 knockdown affects the levels of ubiquitinated H2A through the action of USP16. In response to DNA damage, USP16 levels increase, and this increase is dependent on HERC2. Increased USP16 serves as a negative regulator for DNA damage-induced ubiquitin foci formation and affects downstream factor recruitment and DNA damage response. The functional significance of USP16 is further manifested in human Down syndrome patient cells, which contain three copies of USP16 genes and have altered cellular response to DNA damage. Finally, we demonstrated that USP16 could deubiquitinate both H2A Lys-119 and H2A Lys-15 ubiquitination in vitro. Therefore, this study identifies USP16 as a critical regulator of DNA damage response and H2A Lys-15 ubiquitination as a potential target of USP16. PMID:25305019

  11. Functional Characterization of the OFD1 Protein Reveals a Nuclear Localization and Physical Interaction with Subunits of a Chromatin Remodeling Complex

    PubMed Central

    Giorgio, Giovanna; Alfieri, Mariaevelina; Prattichizzo, Clelia; Zullo, Alessandro; Cairo, Stefano

    2007-01-01

    Oral-facial-digital (OFD) type I syndrome is an X-linked dominant disease (MIM311200) characterized by malformations of oral cavity, face, and digits and by cystic kidneys. We previously identified OFD1, the gene responsible for this disorder, which encodes for a centrosomal protein with an unknown function. We now report that OFD1 localizes both to the primary cilium and to the nucleus. Moreover, we demonstrate that the OFD1 protein is able to self-associate and that this interaction is mediated by its coiled-coil rich region. Interestingly, we identify an OFD1-interacting protein RuvBl1, a protein belonging to the AAA+-family of ATPases, which has been recently associated to cystic kidney in zebrafish and to ciliary assembly and function in Chlamydomonas reinhardtii. We also provide experimental evidence that OFD1, together with RuvBl1, is able to coimmunoprecipitate with subunits of the human TIP60 histone acetyltransferase (HAT) multisubunit complex. On the basis of these results, we hypothesize that OFD1 may be part of a multi-protein complex and could play different biological functions in the centrosome-primary cilium organelles as well as in the nuclear compartment. PMID:17761535

  12. The Bardet–Biedl syndrome-related protein CCDC28B modulates mTORC2 function and interacts with SIN1 to control cilia length independently of the mTOR complex

    PubMed Central

    Cardenas-Rodriguez, Magdalena; Irigoín, Florencia; Osborn, Daniel P.S.; Gascue, Cecilia; Katsanis, Nicholas; Beales, Philip L.; Badano, Jose L.

    2013-01-01

    CCDC28B encodes a coiled coil domain-containing protein involved in ciliogenesis that was originally identified as a second site modifier of the ciliopathy Bardet–Biedl syndrome. We have previously shown that the depletion of CCDC28B leads to shortened cilia; however, the mechanism underlying how this protein controls ciliary length is unknown. Here, we show that CCDC28B interacts with SIN1, a component of the mTOR complex 2 (mTORC2), and that this interaction is important both in the context of mTOR signaling and in a hitherto unknown, mTORC-independent role of SIN1 in cilia biology. We show that CCDC28B is a positive regulator of mTORC2, participating in its assembly/stability and modulating its activity, while not affecting mTORC1 function. Further, we show that Ccdc28b regulates cilia length in vivo, at least in part, through its interaction with Sin1. Importantly, depletion of Rictor, another core component of mTORC2, does not result in shortened cilia. Taken together, our findings implicate CCDC28B in the regulation of mTORC2, and uncover a novel function of SIN1 regulating cilia length that is likely independent of mTOR signaling. PMID:23727834

  13. Promoted Interaction of Nuclear Factor-κB With Demethylated Purinergic P2X3 Receptor Gene Contributes to Neuropathic Pain in Rats With Diabetes.

    PubMed

    Zhang, Hong-Hong; Hu, Ji; Zhou, You-Lang; Qin, Xin; Song, Zhen-Yuan; Yang, Pan-Pan; Hu, Shufen; Jiang, Xinghong; Xu, Guang-Yin

    2015-12-01

    Painful diabetic neuropathy is a common complication of diabetes produced by mechanisms that as yet are incompletely defined. The aim of this study was to investigate the roles of nuclear factor-κB (NF-κB) in the regulation of purinergic receptor P2X ligand-gated ion channel 3 (P2X3R) plasticity in dorsal root ganglion (DRG) neurons of rats with painful diabetes. Here, we showed that hindpaw pain hypersensitivity in streptozocin-induced diabetic rats was attenuated by treatment with purinergic receptor antagonist suramin or A-317491. The expression and function of P2X3Rs was markedly enhanced in hindpaw-innervated DRG neurons in diabetic rats. The CpG (cytosine guanine dinucleotide) island in the p2x3r gene promoter region was significantly demethylated, and the expression of DNA methyltransferase 3b was remarkably downregulated in DRGs in diabetic rats. The binding ability of p65 (an active form of NF-κB) with the p2x3r gene promoter region and p65 expression were enhanced significantly in diabetes. The inhibition of p65 signaling using the NF-κB inhibitor pyrrolidine dithiocarbamate or recombinant lentiviral vectors designated as lentiviral vector-p65 small interfering RNA remarkably suppressed P2X3R activities and attenuated diabetic pain hypersensitivity. Insulin treatment significantly attenuated pain hypersensitivity and suppressed the expression of p65 and P2X3Rs. Our findings suggest that the p2x3r gene promoter DNA demethylation and enhanced interaction with p65 contributes to P2X3R sensitization and diabetic pain hypersensitivity. PMID:26130762

  14. Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury

    PubMed Central

    Wang, Shaogui; Ni, Hong-Min; Dorko, Kenneth; Kumer, Sean C.; Schmitt, Timothy M.; Nawabi, Atta; Komatsu, Masaaki; Huang, Heqing; Ding, Wen-Xing

    2016-01-01

    Chronic alcohol exposure increased hepatic receptor-interacting protein kinase (RIP) 3 expression and necroptosis in the liver but its mechanisms are unclear. In the present study, we demonstrated that chronic alcohol feeding plus binge (Gao-binge) increased RIP3 but not RIP1 protein levels in mouse livers. RIP3 knockout mice had decreased serum alanine amino transferase activity and hepatic steatosis but had no effect on hepatic neutrophil infiltration compared with wild type mice after Gao-binge alcohol treatment. The hepatic mRNA levels of RIP3 did not change between Gao-binge and control mice, suggesting that alcohol-induced hepatic RIP3 proteins are regulated at the posttranslational level. We found that Gao-binge treatment decreased the levels of proteasome subunit alpha type-2 (PSMA2) and proteasome 26S subunit, ATPase 1 (PSMC1) and impaired hepatic proteasome function. Pharmacological or genetic inhibition of proteasome resulted in the accumulation of RIP3 in mouse livers. More importantly, human alcoholics had decreased expression of PSMA2 and PSMC1 but increased protein levels of RIP3 compared with healthy human livers. Moreover, pharmacological inhibition of RIP1 decreased Gao-binge-induced hepatic inflammation, neutrophil infiltration and NF-κB subunit (p65) nuclear translocation but failed to protect against steatosis and liver injury induced by Gao-binge alcohol. In conclusion, results from this study suggest that impaired hepatic proteasome function by alcohol exposure may contribute to hepatic accumulation of RIP3 resulting in necroptosis and steatosis while RIP1 kinase activity is important for alcohol-induced inflammation. PMID:26769846

  15. Quantitative and temporal definition of the Mla transcriptional regulon during barley-powdery mildew interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Barley Mildew resistance locus a (Mla) is a major determinant of immunity to the powdery mildew pathogen, Blumeria graminis f. sp. hordei. Alleles of Mla encode cytoplasmic- and membrane-localized coiled-coil, nucleotide binding site, leucine-rich repeat proteins that mediate resistance when complem...

  16. Protein 4.1 R-135 Interacts with a Novel Centrosomal Protein (CPAP) Which Is Associated with the γ-Tubulin Complex

    PubMed Central

    Hung, Liang-Yi; Tang, Chieh-Ju C.; Tang, Tang K.

    2000-01-01

    Using a yeast two-hybrid system, we isolated a novel human centrosomal protein, CPAP (centrosomal P4.1-associated protein), which specifically interacts with the head domain of the 135-kDa protein 4.1R isoform (4.1R-135). Sequence analysis revealed that the carboxyl terminus of CPAP has 31.3% amino acid identity with human Tcp-10 (a t-complex responder gene product). Interestingly, most of the sequence identity is restricted to two conserved regions. One carries a leucine zipper, which may form a series of heptad repeats involved in coiled-coil formation; the other contains unusual glycine repeats with unknown function. Immunofluorescence analysis revealed that CPAP and γ-tubulin are localized within the centrosome throughout the cell cycle. CPAP cosediments with γ-tubulin in sucrose gradients and coimmunoprecipitates with γ-tubulin, indicating that CPAP is a part of the γ-tubulin complex. Furthermore, functional analysis revealed that CPAP is localized within the center of microtubule asters and may participate in microtubule nucleation. The formation of microtubule asters was significantly inhibited by anti-CPAP antibody. Together, these observations indicate that CPAP may play an important role in cell division and centrosome function. PMID:11003675

  17. Protein 4.1 R-135 interacts with a novel centrosomal protein (CPAP) which is associated with the gamma-tubulin complex.

    PubMed

    Hung, L Y; Tang, C J; Tang, T K

    2000-10-01

    Using a yeast two-hybrid system, we isolated a novel human centrosomal protein, CPAP (centrosomal P4.1-associated protein), which specifically interacts with the head domain of the 135-kDa protein 4.1R isoform (4.1R-135). Sequence analysis revealed that the carboxyl terminus of CPAP has 31.3% amino acid identity with human Tcp-10 (a t-complex responder gene product). Interestingly, most of the sequence identity is restricted to two conserved regions. One carries a leucine zipper, which may form a series of heptad repeats involved in coiled-coil formation; the other contains unusual glycine repeats with unknown function. Immunofluorescence analysis revealed that CPAP and gamma-tubulin are localized within the centrosome throughout the cell cycle. CPAP cosediments with gamma-tubulin in sucrose gradients and coimmunoprecipitates with gamma-tubulin, indicating that CPAP is a part of the gamma-tubulin complex. Furthermore, functional analysis revealed that CPAP is localized within the center of microtubule asters and may participate in microtubule nucleation. The formation of microtubule asters was significantly inhibited by anti-CPAP antibody. Together, these observations indicate that CPAP may play an important role in cell division and centrosome function. PMID:11003675

  18. Crystal structure of a plectonemic RNA supercoil

    SciTech Connect

    Stagno, Jason R.; Ma, Buyong; Li, Jess; Altieri, Amanda S.; Byrd, R. Andrew; Ji, Xinhua

    2012-12-14

    Genome packaging is an essential housekeeping process in virtually all organisms for proper storage and maintenance of genetic information. Although the extent and mechanisms of packaging vary, the process involves the formation of nucleic-acid superstructures. Crystal structures of DNA coiled coils indicate that their geometries can vary according to sequence and/or the presence of stabilizers such as proteins or small molecules. However, such superstructures have not been revealed for RNA. Here we report the crystal structure of an RNA supercoil, which displays one level higher molecular organization than previously reported structures of DNA coiled coils. In the presence of an RNA-binding protein, two interlocking RNA coiled coils of double-stranded RNA, a 'coil of coiled coils', form a plectonemic supercoil. Molecular dynamics simulations suggest that protein-RNA interaction is required for the stability of the supercoiled RNA. This study provides structural insight into higher order packaging mechanisms of nucleic acids.

  19. DNA Topoisomerase I Domain Interactions Impact Enzyme Activity and Sensitivity to Camptothecin*

    PubMed Central

    Wright, Christine M.; van der Merwe, Marié; DeBrot, Amanda H.; Bjornsti, Mary-Ann

    2015-01-01

    During processes such as DNA replication and transcription, DNA topoisomerase I (Top1) catalyzes the relaxation of DNA supercoils. The nuclear enzyme is also the cellular target of camptothecin (CPT) chemotherapeutics. Top1 contains four domains: the highly conserved core and C-terminal domains involved in catalysis, a coiled-coil linker domain of variable length, and a poorly conserved N-terminal domain. Yeast and human Top1 share a common reaction mechanism and domain structure. However, the human Top1 is ∼100-fold more sensitive to CPT. Moreover, substitutions of a conserved Gly717 residue, which alter intrinsic enzyme sensitivity to CPT, induce distinct phenotypes in yeast. To address the structural basis for these differences, reciprocal swaps of yeast and human Top1 domains were engineered in chimeric enzymes. Here we report that intrinsic Top1 sensitivity to CPT is dictated by the composition of the conserved core and C-terminal domains. However, independent of CPT, biochemically similar chimeric enzymes produced strikingly distinct phenotypes in yeast. Expression of a human Top1 chimera containing the yeast linker domain proved toxic, even in the context of a catalytically inactive Y723F enzyme. Lethality was suppressed either by splicing the yeast N-terminal domain into the chimera, deleting the human N-terminal residues, or in enzymes reconstituted by polypeptide complementation. These data demonstrate a functional interaction between the N-terminal and linker domains, which, when mispaired between yeast and human enzymes, induces cell lethality. Because toxicity was independent of enzyme catalysis, the inappropriate coordination of N-terminal and linker domains may induce aberrant Top1-protein interactions to impair cell growth. PMID:25795777

  20. Biochemistry and Biophysics of HIV-1 gp41 – membrane interactions

    PubMed Central

    Cai, Lifeng; Gochin, Miriam; Liu, Keliang

    2011-01-01

    Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein – mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), N-terminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors. PMID:22044229

  1. Epstein-Barr virus latent membrane protein 1 (LMP1) C-terminal-activating region 3 contributes to LMP1-mediated cellular migration via its interaction with Ubc9.

    PubMed

    Bentz, Gretchen L; Whitehurst, Christopher B; Pagano, Joseph S

    2011-10-01

    Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), the principal viral oncoprotein and a member of the tumor necrosis factor receptor superfamily, is a constitutively active membrane signaling protein that regulates multiple signal transduction pathways via its C-terminal-activating region 1 (CTAR1) and CTAR2, and also the less-studied CTAR3. Because protein sumoylation among other posttranslational modifications may regulate many signaling pathways induced by LMP1, we investigated whether during EBV latency LMP1 regulates sumoylation processes that control cellular activation and cellular responses. By immunoprecipitation experiments, we show that LMP1 interacts with Ubc9, the single reported SUMO-conjugating enzyme. Requirements for LMP1-Ubc9 interactions include enzymatically active Ubc9: expression of inactive Ubc9 (Ubc9 C93S) inhibited the LMP1-Ubc9 interaction. LMP1 CTAR3, but not CTAR1 and CTAR2, participated in the LMP1-Ubc9 interaction, and amino acid sequences found in CTAR3, including the JAK-interacting motif, contributed to this interaction. Furthermore, LMP1 expression coincided with increased sumoylation of cellular proteins, and disruption of the Ubc9-LMP1 CTAR3 interaction almost completely abrogated LMP1-induced protein sumoylation, suggesting that this interaction promotes the sumoylation of downstream targets. Additional consequences of the disruption of the LMP1 CTAR3-Ubc9 interaction revealed effects on cellular migration, a hallmark of oncogenesis. Together, these data demonstrate that LMP1 CTAR3 does in fact function in intracellular signaling and leads to biological effects. We propose that LMP1, by interaction with Ubc9, modulates sumoylation processes, which regulate signal transduction pathways that affect phenotypic changes associated with oncogenesis. PMID:21795333

  2. Factoring the contribution of through-space and through-bond interactions to rates of photoinduced electron transfer in donor- spacer-acceptor molecules using ultrafast transient absorption spectroscopy

    SciTech Connect

    Gosztola, D.; Wang, Bing; Wasielewski, M.R. |

    1996-06-01

    Contributions from through-space and through-bond interactions to the electronic coupling matrix elements for photoinduced charge separation and recombination in linked donor-spacer-acceptor molecules were studied. The molecules consisted of a 4-piperidinyl-naphthalene-1,8-dicarboximide electron donor and a N-(n-octyl)pyromellitimide electron acceptor attached to the 1,5- and 1,8-positions of either anthracene or dibenzobicyclo(2.2.2)octatriene spacers.

  3. Solution NMR structures of Immunoglobulin-like domains 7 and 12 from Obscurin-like protein 1 contribute to the structural coverage of the human cancer protein interaction network

    PubMed Central

    Pulavarti, Surya VSRK; Huang, Yuanpeng J.; Pederson, Kari; Acton, Thomas B.; Xiao, Rong; Everett, John K.; Prestegard, James H.; Montelione, Gaetano T.

    2016-01-01

    High-quality solution NMR structures of immunoglobulin-like domains 7 and 12 from human Obscurin-like protein 1 were solved. The two domains share 30 % sequence identity and their structures are, as expected, rather similar. The new structures contribute to structural coverage of human cancer associated proteins. Mutations of Arg 812 in domain 7 cause the rare 3-M syndrome, and this site is located in a surface area predicted to be involved in protein-protein interactions. PMID:24989974

  4. Mitochondrial-Nuclear DNA Interactions Contribute to the Regulation of Nuclear Transcript Levels as Part of the Inter-Organelle Communication System

    PubMed Central

    Rodley, Chris D. M.; Grand, Ralph S.; Gehlen, Lutz R.; Greyling, Gary; Jones, M. Beatrix; O'Sullivan, Justin M.

    2012-01-01

    Nuclear and mitochondrial organelles must maintain a communication system. Loci on the mitochondrial genome were recently reported to interact with nuclear loci. To determine whether this is part of a DNA based communication system we used genome conformation capture to map the global network of DNA-DNA interactions between the mitochondrial and nuclear genomes (Mito-nDNA) in Saccharomyces cerevisiae cells grown under three different metabolic conditions. The interactions that form between mitochondrial and nuclear loci are dependent on the metabolic state of the yeast. Moreover, the frequency of specific mitochondrial - nuclear interactions (i.e. COX1-MSY1 and Q0182-RSM7) showed significant reductions in the absence of mitochondrial encoded reverse transcriptase machinery. Furthermore, these reductions correlated with increases in the transcript levels of the nuclear loci (MSY1 and RSM7). We propose that these interactions represent an inter-organelle DNA mediated communication system and that reverse transcription of mitochondrial RNA plays a role in this process. PMID:22292080

  5. The Protein Interaction of RNA Helicase B (RhlB) and Polynucleotide Phosphorylase (PNPase) Contributes to the Homeostatic Control of Cysteine in Escherichia coli*

    PubMed Central

    Tseng, Yi-Ting; Chiou, Ni-Ting; Gogiraju, Rajinikanth; Lin-Chao, Sue

    2015-01-01

    PNPase, one of the major enzymes with 3′ to 5′ single-stranded RNA degradation and processing activities, can interact with the RNA helicase RhlB independently of RNA degradosome formation in Escherichia coli. Here, we report that loss of interaction between RhlB and PNPase impacts cysteine homeostasis in E. coli. By random mutagenesis, we identified a mutant RhlBP238L that loses 75% of its ability to interact with PNPase but retains normal interaction with RNase E and RNA, in addition to exhibiting normal helicase activity. Applying microarray analyses to an E. coli strain with impaired RNA degradosome formation, we investigated the biological consequences of a weakened interaction between RhlB and PNPase. We found significant increases in 11 of 14 genes involved in cysteine biosynthesis. Subsequent Northern blot analyses showed that the up-regulated transcripts were the result of stabilization of the cysB transcript encoding a transcriptional activator for the cys operons. Furthermore, Northern blots of PNPase or RhlB mutants showed that RhlB-PNPase plays both a catalytic and structural role in regulating cysB degradation. Cells expressing the RhlBP238L mutant exhibited an increase in intracellular cysteine and an enhanced anti-oxidative response. Collectively, this study suggests a mechanism by which bacteria use the PNPase-RhlB exosome-like complex to combat oxidative stress by modulating cysB mRNA degradation. PMID:26494621

  6. Structure and Protein-Protein Interaction Studies on Chlamydia trachomatis Protein CT670 (YscO Homolog)

    SciTech Connect

    Lorenzini, Emily; Singer, Alexander; Singh, Bhag; Lam, Robert; Skarina, Tatiana; Chirgadze, Nickolay Y.; Savchenko, Alexei; Gupta, Radhey S.

    2010-07-28

    Comparative genomic studies have identified many proteins that are found only in various Chlamydiae species and exhibit no significant sequence similarity to any protein in organisms that do not belong to this group. The CT670 protein of Chlamydia trachomatis is one of the proteins whose genes are in one of the type III secretion gene clusters but whose cellular functions are not known. CT670 shares several characteristics with the YscO protein of Yersinia pestis, including the neighboring genes, size, charge, and secondary structure, but the structures and/or functions of these proteins remain to be determined. Although a BLAST search with CT670 did not identify YscO as a related protein, our analysis indicated that these two proteins exhibit significant sequence similarity. In this paper, we report that the CT670 crystal, solved at a resolution of 2 {angstrom}, consists of a single coiled coil containing just two long helices. Gel filtration and analytical ultracentrifugation studies showed that in solution CT670 exists in both monomeric and dimeric forms and that the monomer predominates at lower protein concentrations. We examined the interaction of CT670 with many type III secretion system-related proteins (viz., CT091, CT665, CT666, CT667, CT668, CT669, CT671, CT672, and CT673) by performing bacterial two-hybrid assays. In these experiments, CT670 was found to interact only with the CT671 protein (YscP homolog), whose gene is immediately downstream of ct670. A specific interaction between CT670 and CT671 was also observed when affinity chromatography pull-down experiments were performed. These results suggest that CT670 and CT671 are putative homologs of the YcoO and YscP proteins, respectively, and that they likely form a chaperone-effector pair.

  7. A mitotic function for the high-mobility group protein HMG20b regulated by its interaction with the BRC repeats of the BRCA2 tumor suppressor.

    PubMed

    Lee, M; Daniels, M J; Garnett, M J; Venkitaraman, A R

    2011-07-28

    The inactivation of BRCA2, a suppressor of breast, ovarian and other epithelial cancers, triggers instability in chromosome structure and number, which are thought to arise from defects in DNA recombination and mitotic cell division, respectively. Human BRCA2 controls DNA recombination via eight BRC repeats, evolutionarily conserved motifs of ∼35 residues, that