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Sample records for coiled-coil interactions contribute

  1. Analysis of Coiled-Coil Interactions between Core Proteins of the Spindle Pole Body

    SciTech Connect

    Zizlsperger, N.; Malashkevich, V; Pillay, S; Keating, A

    2008-01-01

    The spindle pole body (SPB) is a multiprotein complex that organizes microtubules in yeast. Due to its large size and association with the nuclear membrane, little is known about its detailed structure. In particular, although many SPB components and some of the interactions between them have been identified, the molecular details of how most of these interactions occur are not known. The prevalence of predicted coiled-coil regions in SPB proteins suggests that some interactions may occur via coiled coils. Here this hypothesis is supported by biochemical characterization of isolated coiled-coil peptides derived from SPB proteins. Formation of four strongly self-associating coiled-coil complexes from Spc29, Spc42, and Spc72 was demonstrated by circular dichroism (CD) spectroscopy and a fluorescence resonance energy transfer (FRET) assay. Many weaker self- and heteroassociations were also detected by CD, FRET, and/or cross-linking. The thermal stabilities of nine candidate homooligomers were assessed; six unfolded cooperatively with melting temperatures ranging from <11 to >50 C. Solution studies established that coiled-coil peptides derived from Spc42 and Spc72 form parallel dimers, and this was confirmed for Spc42 by a high-resolution crystal structure. These data contribute to a growing body of knowledge that will ultimately provide a detailed model of the SPB structure.

  2. 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.

  3. 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

  4. 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.

  5. Pharmacological interference with protein-protein interactions mediated by coiled-coil motifs.

    PubMed

    Strauss, H M; Keller, S

    2008-01-01

    Coiled coils are bundles of intertwined alpha-helices that provide protein-protein interaction sites for the dynamic assembly and disassembly of protein complexes. The coiled-coil motif combines structural versatility and adaptability with mechanical strength and specificity. Multimeric proteins that rely on coiled-coil interactions are structurally and functionally very diverse, ranging from simple homodimeric transcription factors to elaborate heteromultimeric scaffolding clusters. Several coiled-coil-bearing proteins are of outstanding pharmacological importance, most notably SNARE proteins involved in vesicular trafficking of neurotransmitters and viral fusion proteins. Together with their crucial roles in many physiological and pathological processes, the structural simplicity and reversible nature of coiled-coil associations render them a promising target for pharmacological interference, as successfully exemplified by botulinum toxins and viral fusion inhibitors. The alpha-helical coiled coil is a ubiquitous protein domain that mediates highly specific homo- and heteromeric protein-protein interactions among a wide range of proteins. The coiled-coil motif was first proposed by Crick on the basis of X-ray diffraction data on alpha-keratin more than 50 years ago (Crick 1952, 1953) and nowadays belongs to the best-characterized protein interaction modules. By definition, a coiled coil is an oligomeric protein assembly consisting of several right-handed amphipathic alpha-helices that wind around each other into a superhelix (or a supercoil) in which the hydrophobic surfaces of the constituent helices are in continuous contact, forming a hydrophobic core. Both homomeric and heteromeric coiled coils with different stoichiometries are possible, and the helices can be aligned in either a parallel or an antiparallel topology (Harbury et al. 1993, 1994). Stoichiometry and topology are governed by the primary structure, that is, the sequence of the polypeptide chains

  6. 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

  7. 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

  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. 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

  11. 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

  12. Interactions of HIV-1 inhibitory peptide T20 with the gp41 N-HR coiled coil.

    PubMed

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

    2009-02-01

    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 (K(D) = 30 nm), close to its IC50 concentration (approximately 3 nm) but much weaker than the affinity of a related inhibitory C-peptide C37 (K(D) = 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 (K(D) = 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

  13. SYNZIP Protein Interaction Toolbox: in Vitro and in Vivo Specifications of Heterospecific Coiled-Coil Interaction Domains

    PubMed Central

    2012-01-01

    The synthetic biology toolkit contains a growing number of parts for regulating transcription and translation, but very few that can be used to control protein association. Here we report characterization of 22 previously published heterospecific synthetic coiled-coil peptides called SYNZIPs. We present biophysical analysis of the oligomerization states, helix orientations, and affinities of 27 SYNZIP pairs. SYNZIP pairs were also tested for interaction in two cell-based assays. In a yeast two-hybrid screen, >85% of 253 comparable interactions were consistent with prior in vitro measurements made using coiled-coil microarrays. In a yeast-signaling assay controlled by coiled-coil mediated scaffolding, 12 SYNZIP pairs were successfully used to down-regulate the expression of a reporter gene following treatment with α-factor. Characterization of these interaction modules dramatically increases the number of available protein interaction parts for synthetic biology and should facilitate a wide range of molecular engineering applications. Summary characteristics of 27 SYNZIP peptide pairs are reported in specification sheets available in the Supporting Information and at the SYNZIP Web site [http://keatingweb.mit.edu/SYNZIP/]. PMID:22558529

  14. 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

  15. 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

  16. Salt effects on hydrophobic interaction and charge screening in the folding of a negatively charged peptide to a coiled coil (leucine zipper).

    PubMed

    Jelesarov, I; Dürr, E; Thomas, R M; Bosshard, H R

    1998-05-19

    The stability of a coiled coil or leucine zipper is controlled by hydrophobic interactions and electrostatic forces between the constituent helices. We have designed a 30-residue peptide with the repeating seven-residue pattern of a coiled coil, (abcdefg)n, and with Glu in positions e and g of each heptad. The glutamate side chains prevented folding at pH values above 6 because of electrostatic repulsion across the helix dimer interface as well as within the individual helices. Protonation of the carboxylates changed the conformation from a random coil monomer to a coiled coil dimer. Folding at alkaline pH where the peptide had a net charge of -7e was promoted by the addition of salts. The nature of the charge screening cation was less important than that of the anion. The high salt concentrations (>1 M) necessary to induce folding indicated that the salt-induced folding resulted from alterations in the protein-water interaction. Folding was promoted by the kosmotropic anions sulfate and fluoride and to a lesser extent by the weak kosmotrope formate, whereas chloride and the strong chaotrope perchlorate were ineffective. Kosmotropes are excluded from the protein surface, which is preferentially hydrated, and this promotes folding by strengthening hydrophobic interactions at the coiled coil interface. Although charge neutralization also contributed to folding, it was effective only when the screening cation was partnered by a good kosmotropic anion. Folding conformed to a two-state transition from random coil monomer to coiled coil dimer and was enthalpy driven and characterized by a change in the heat capacity of unfolding of 3.9 +/- 1.2 kJ mol-1 K-1. The rate of folding was analyzed by fluorescence stopped-flow measurements. Folding occurred in a biphasic reaction in which the rapid formation of an initial dimer (kf = 2 x 10(7) M-1 s-1) was followed by an equally rapid concentration-independent rearrangement to the folded dimer (k > 100 s-1).

  17. Salt effects on hydrophobic interaction and charge screening in the folding of a negatively charged peptide to a coiled coil (leucine zipper).

    PubMed

    Jelesarov, I; Dürr, E; Thomas, R M; Bosshard, H R

    1998-05-19

    The stability of a coiled coil or leucine zipper is controlled by hydrophobic interactions and electrostatic forces between the constituent helices. We have designed a 30-residue peptide with the repeating seven-residue pattern of a coiled coil, (abcdefg)n, and with Glu in positions e and g of each heptad. The glutamate side chains prevented folding at pH values above 6 because of electrostatic repulsion across the helix dimer interface as well as within the individual helices. Protonation of the carboxylates changed the conformation from a random coil monomer to a coiled coil dimer. Folding at alkaline pH where the peptide had a net charge of -7e was promoted by the addition of salts. The nature of the charge screening cation was less important than that of the anion. The high salt concentrations (>1 M) necessary to induce folding indicated that the salt-induced folding resulted from alterations in the protein-water interaction. Folding was promoted by the kosmotropic anions sulfate and fluoride and to a lesser extent by the weak kosmotrope formate, whereas chloride and the strong chaotrope perchlorate were ineffective. Kosmotropes are excluded from the protein surface, which is preferentially hydrated, and this promotes folding by strengthening hydrophobic interactions at the coiled coil interface. Although charge neutralization also contributed to folding, it was effective only when the screening cation was partnered by a good kosmotropic anion. Folding conformed to a two-state transition from random coil monomer to coiled coil dimer and was enthalpy driven and characterized by a change in the heat capacity of unfolding of 3.9 +/- 1.2 kJ mol-1 K-1. The rate of folding was analyzed by fluorescence stopped-flow measurements. Folding occurred in a biphasic reaction in which the rapid formation of an initial dimer (kf = 2 x 10(7) M-1 s-1) was followed by an equally rapid concentration-independent rearrangement to the folded dimer (k > 100 s-1). PMID:9585569

  18. 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

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

    PubMed

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

    2012-11-14

    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.

  20. 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

  1. 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.

  2. JunB potentiates function of BRCA1 activation domain 1 (AD1) through a coiled-coil-mediated interaction

    PubMed Central

    Hu, Yan-Fen; Li, Rong

    2002-01-01

    BRCA1 is involved in the regulation of multiple nuclear events including transcription. AD1, one of the two trans-activation domains in BRCA1, stimulates transcription in a cell context-dependent manner. Here, it is shown that BRCA1 interacts with Jun proteins via a coiled-coil motif in AD1 and the basic leucine zipper (bZIP) region of the Jun proteins. The Jun-interacting domain in BRCA1 is critical for AD1-mediated transcriptional activation. In particular, the strength of AD1 in transcriptional activation is limited by the JunB level and ectopic expression of JunB potentiates the transcriptional activity of AD1. Furthermore, JunB mRNA expression is down-regulated in many ovarian tumor tissues examined. Thus, the coiled-coil-mediated cooperation between BRCA1 and JunB may facilitate the function of these proteins in tissue-specific transcriptional regulation and tumor suppression. PMID:12080089

  3. 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

  4. 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.

  5. Phylogenetic occurrence of coiled coil proteins: implications for tissue structure in metazoa via a coiled coil tissue matrix.

    PubMed

    Odgren, P R; Harvie, L W; Fey, E G

    1996-04-01

    We examined GenBank sequence files with a heptad repeat analysis program to assess the phylogenetic occurrence of coiled coil proteins, how heptad repeat domains are organized within them, and what structural/functional categories they comprise. Of 102,007 proteins analyzed, 5.95% (6,074) contained coiled coil domains; 1.26% (1,289) contained "extended" (> 75 amino acid) domains. While the frequency of proteins containing coiled coils was surprisingly constant among all biota, extended coiled coil proteins were fourfold more frequent in the animal kingdom and may reflect early events in the divergence of plants and animals. Structure/function categories of extended coils also revealed phylogenetic differences. In pathogens and parasites, many extended coiled coil proteins are external and bind host proteins. In animals, the majority of extended coiled coil proteins were identified as constituents of two protein categories: 1) myosins and motors; or 2) components of the nuclear matrix-intermediate filament scaffold. This scaffold, produced by sequential extraction of epithelial monolayers in situ, contains only 1-2% of the cell mass while accurately retaining morphological features of living epithelium and is greatly enriched in proteins with extensive, interrupted coiled coil forming domains. The increased occurrence of this type of protein in metazoa compared with plants or protists leads us to hypothesize a tissue-wide matrix of coiled coil interactions underlying metazoan differentiated cell and tissue structure.

  6. 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

  7. α/β coiled coils.

    PubMed

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

    2016-01-15

    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.

  8. 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

  9. The evolution and structure prediction of coiled coils across all genomes.

    PubMed

    Rackham, Owen J L; Madera, Martin; Armstrong, Craig T; Vincent, Thomas L; Woolfson, Derek N; Gough, Julian

    2010-10-29

    Coiled coils are α-helical interactions found in many natural proteins. Various sequence-based coiled-coil predictors are available, but key issues remain: oligomeric state and protein-protein interface prediction and extension to all genomes. We present SpiriCoil (http://supfam.org/SUPERFAMILY/spiricoil), which is based on a novel approach to the coiled-coil prediction problem for coiled coils that fall into known superfamilies: hundreds of hidden Markov models representing coiled-coil-containing domain families. Using whole domains gives the advantage that sequences flanking the coiled coils help. SpiriCoil performs at least as well as existing methods at detecting coiled coils and significantly advances the state of the art for oligomer state prediction. SpiriCoil has been run on over 16 million sequences, including all completely sequenced genomes (more than 1200), and a resulting Web interface supplies data downloads, alignments, scores, oligomeric state classifications, three-dimensional homology models and visualisation. This has allowed, for the first time, a genomewide analysis of coiled-coil evolution. We found that coiled coils have arisen independently de novo well over a hundred times, and these are observed in 16 different oligomeric states. Coiled coils in almost all oligomeric states were present in the last universal common ancestor of life. The vast majority of occasions that individual coiled coils have arisen de novo were before the last universal common ancestor of life; we do, however, observe scattered instances throughout subsequent evolutionary history, mostly in the formation of the eukaryote superkingdom. Coiled coils do not change their oligomeric state over evolution and did not evolve from the rearrangement of existing helices in proteins; coiled coils were forged in unison with the fold of the whole protein.

  10. Structural analysis of intermolecular interactions in the kinesin adaptor complex fasciculation and elongation protein zeta 1/ short coiled-coil protein (FEZ1/SCOCO).

    PubMed

    Alborghetti, Marcos Rodrigo; Furlan, Ariane da Silva; da Silva, Júlio César; Sforça, Maurício Luís; Honorato, Rodrigo Vargas; Granato, Daniela Campos; dos Santos Migueleti, Deivid Lucas; Neves, Jorge L; de Oliveira, Paulo Sergio Lopes; Paes-Leme, Adriana Franco; Zeri, Ana Carolina de Mattos; de Torriani, Iris Concepcion Linares; Kobarg, Jörg

    2013-01-01

    Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.

  11. Head-to-tail interactions of the coiled-coil domains regulate ClpB activity and cooperation with Hsp70 in protein disaggregation

    PubMed Central

    Carroni, Marta; Kummer, Eva; Oguchi, Yuki; Wendler, Petra; Clare, Daniel K; Sinning, Irmgard; Kopp, Jürgen; Mogk, Axel; Bukau, Bernd; Saibil, Helen R

    2014-01-01

    The hexameric AAA+ chaperone ClpB reactivates aggregated proteins in cooperation with the Hsp70 system. Essential for disaggregation, the ClpB middle domain (MD) is a coiled-coil propeller that binds Hsp70. Although the ClpB subunit structure is known, positioning of the MD in the hexamer and its mechanism of action are unclear. We obtained electron microscopy (EM) structures of the BAP variant of ClpB that binds the protease ClpP, clearly revealing MD density on the surface of the ClpB ring. Mutant analysis and asymmetric reconstructions show that MDs adopt diverse positions in a single ClpB hexamer. Adjacent, horizontally oriented MDs form head-to-tail contacts and repress ClpB activity by preventing Hsp70 interaction. Tilting of the MD breaks this contact, allowing Hsp70 binding, and releasing the contact in adjacent subunits. Our data suggest a wavelike activation of ClpB subunits around the ring. DOI: http://dx.doi.org/10.7554/eLife.02481.001 PMID:24843029

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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

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

    PubMed

    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.

  19. 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

  20. Interaction of the transforming acidic coiled-coil 1 (TACC1) protein with ch-TOG and GAS41/NuBI1 suggests multiple TACC1-containing protein complexes in human cells.

    PubMed Central

    Lauffart, Brenda; Howell, Scott J; Tasch, Jason E; Cowell, John K; Still, Ivan H

    2002-01-01

    Dysregulation of the human transforming acidic coiled-coil (TACC) proteins is thought to be important in the evolution of breast cancer and multiple myeloma. However, the exact role of these proteins in the oncogenic process is currently unknown. Using the full-length TACC1 protein as bait to screen a human mammary epithelial cDNA library, we have identified two genes that are also amplified and overexpressed in tumours derived from different cellular origins. TACC1 interacts with the C-terminus of both the microtubule-associated colonic and hepatic tumour overexpressed (ch-TOG) protein, and the oncogenic transcription factor glioma amplified sequence 41/NuMA binding protein 1 (GAS41/NuBI1; where NuMA stands for nuclear mitotic apparatus protein 1). This suggests that the TACC proteins can form multiple complexes, dysregulation of which may be an important step during tumorigenesis. PMID:11903063

  1. Predicting coiled coils by use of pairwise residue correlations.

    PubMed Central

    Berger, B; Wilson, D B; Wolf, E; Tonchev, T; Milla, M; Kim, P S

    1995-01-01

    A method is presented that predicts coiled-coil domains in protein sequences by using pairwise residue correlations obtained from a (two-stranded) coiled-coil database of 58,217 amino acid residues. A program called PAIRCOIL implements this method and is significantly better than existing methods at distinguishing coiled coils from alpha-helices that are not coiled coils. The database of pairwise residue correlations suggests structural features that stabilize or destabilize coiled coils. Images Fig. 1 Fig. 2 PMID:7667278

  2. 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

  3. Routine phasing of coiled-coil protein crystal structures with AMPLE

    PubMed Central

    Thomas, Jens M. H.; Keegan, Ronan M.; Bibby, Jaclyn; Winn, Martyn D.; Mayans, Olga; Rigden, Daniel J.

    2015-01-01

    Coiled-coil protein folds are among the most abundant in nature. These folds consist of long wound α-helices and are architecturally simple, but paradoxically their crystallographic structures are notoriously difficult to solve with molecular-replacement techniques. The program AMPLE can solve crystal structures by molecular replacement using ab initio search models in the absence of an existent homologous protein structure. AMPLE has been benchmarked on a large and diverse test set of coiled-coil crystal structures and has been found to solve 80% of all cases. Successes included structures with chain lengths of up to 253 residues and resolutions down to 2.9 Å, considerably extending the limits on size and resolution that are typically tractable by ab initio methodologies. The structures of two macromolecular complexes, one including DNA, were also successfully solved using their coiled-coil components. It is demonstrated that both the ab initio modelling and the use of ensemble search models contribute to the success of AMPLE by comparison with phasing attempts using single structures or ideal polyalanine helices. These successes suggest that molecular replacement with AMPLE should be the method of choice for the crystallo­graphic elucidation of a coiled-coil structure. Furthermore, AMPLE may be able to exploit the presence of a coiled coil in a complex to provide a convenient route for phasing. PMID:25866657

  4. 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.

  5. Crystal structure of the central coiled-coil domain from human liprin-β2.

    PubMed

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

    2011-05-10

    Liprins are a conserved family of scaffolding proteins important for the proper regulation and development of neuronal synapses. Humans have four liprin-αs and two liprin-βs 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-β2 coiled-coil forms an extended dimer. Several protease-resistant subdomains within the liprin-β1 and liprin-β2 coiled-coils were also identified. A 2.0 Å crystal structure of the central, protease-resistant core of the liprin-β2 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.

  6. Cloning, overexpression, purification and crystallization of the CRN12 coiled-coil domain from Leishmania donovani

    PubMed Central

    Srivastava, Vijay Kumar; Rana, Ajay Kumar; Sahasrabuddhe, Amogh A.; Gupta, C. M; Pratap, J. V.

    2013-01-01

    Leishmania donovani coronin CRN12 is an actin-binding protein which consists of two domains: an N-terminal WD repeat domain and a C-terminal coiled-coil domain. The coiled-coil domain is 53 residues in length. Helix–helix interactions in general and coiled coils in particular are ubiquitous in the structure of proteins and play a significant role in the association among proteins, including supramolecular assemblies and transmembrane receptors that mediate cellular signalling, transport and actin dynamics. The L. donovani coronin CRN12 coiled-coil domain (5.8 kDa) was cloned, overexpressed, purified to homogeneity and the N-terminal 6×His tag was successfully removed by thrombin cleavage. Crystals of recombinant L. donovani coronin CRN12 coiled-coil domain were grown by vapour diffusion using a hanging-drop setup. Diffraction-quality crystals were obtained and data extending to 2.46 Å resolution were collected at 100 K on BM14, ESRF, Grenoble, France. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 118.0, b = 50.6, c = 46.0 Å, β = 111.0°. Matthews coefficient (V M) calculations suggested the presence of 4–6 molecules in the asymmetric unit, corresponding to a solvent content of ∼33–55%, and are consistent with self-rotation function calculations. PMID:23695571

  7. Cloning, overexpression, purification and crystallization of the CRN12 coiled-coil domain from Leishmania donovani.

    PubMed

    Srivastava, Vijay Kumar; Rana, Ajay Kumar; Sahasrabuddhe, Amogh A; Gupta, C M; Pratap, J V

    2013-05-01

    Leishmania donovani coronin CRN12 is an actin-binding protein which consists of two domains: an N-terminal WD repeat domain and a C-terminal coiled-coil domain. The coiled-coil domain is 53 residues in length. Helix-helix interactions in general and coiled coils in particular are ubiquitous in the structure of proteins and play a significant role in the association among proteins, including supramolecular assemblies and transmembrane receptors that mediate cellular signalling, transport and actin dynamics. The L. donovani coronin CRN12 coiled-coil domain (5.8 kDa) was cloned, overexpressed, purified to homogeneity and the N-terminal 6×His tag was successfully removed by thrombin cleavage. Crystals of recombinant L. donovani coronin CRN12 coiled-coil domain were grown by vapour diffusion using a hanging-drop setup. Diffraction-quality crystals were obtained and data extending to 2.46 Å resolution were collected at 100 K on BM14, ESRF, Grenoble, France. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 118.0, b = 50.6, c = 46.0 Å, β = 111.0°. Matthews coefficient (VM) calculations suggested the presence of 4-6 molecules in the asymmetric unit, corresponding to a solvent content of ∼33-55%, and are consistent with self-rotation function calculations. PMID:23695571

  8. Insertion of a coiled-coil peptide from influenza virus hemagglutinin into membranes

    SciTech Connect

    Yu, Y.G.; Shin, Y.K.; King, D.S.

    1994-10-14

    The trimeric protein hemagglutinin (HA) of the influenza viral envelope is essential for cell entry. To investigate the interaction of HA with membranes, two 40-residue, cysteine-substituted peptides comprising the loop region and the first part of the coiled-coil stem were synthesized and modified with a nitroxide spin label. Electron paramagnetic resonance analysis revealed that the peptide inserts reversibly into phospholipid vesicles under endosomal pH conditions. This result suggests that some or all of the long coiled-coil trimer of HA may insert into membranes, which could bring the viral and cell membranes closer together and facilitate fusion. 16 refs., 5 figs.

  9. A periodic table of coiled-coil protein structures.

    PubMed

    Moutevelis, Efrosini; Woolfson, Derek N

    2009-01-23

    Coiled coils are protein structure domains with two or more alpha-helices packed together via interlacing of side chains known as knob-into-hole packing. We analysed and classified a large set of coiled-coil structures using a combination of automated and manual methods. This led to a systematic classification that we termed a "periodic table of coiled coils," which we have made available at http://coiledcoils.chm.bris.ac.uk/ccplus/search/periodic_table. In this table, coiled-coil assemblies are arranged in columns with increasing numbers of alpha-helices and in rows of increased complexity. The table provides a framework for understanding possibilities in and limits on coiled-coil structures and a basis for future prediction, engineering and design studies.

  10. Subunit b-dimer of the Escherichia coli ATP synthase can form left-handed coiled-coils.

    PubMed

    Wise, John G; Vogel, Pia D

    2008-06-01

    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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. Inter-molecular coiled-coil formation in human apolipoprotein E C-terminal domain.

    PubMed

    Choy, Nicole; Raussens, Vincent; Narayanaswami, Vasanthy

    2003-11-28

    Human apolipoprotein E (apoE) is composed of an N-terminal (NT) domain (residues 1-191) that bears low-density lipoprotein receptor-binding sites, and a C-terminal (CT) domain (residues 210-299), which houses lipoprotein binding and apoE self-association sites. The NT domain is comprised of a four-helix bundle, while the structural organization of the CT domain is not known. Secondary structural algorithms predict that the apoE CT domain adopts an amphipathic alpha-helical conformation. On the basis of further sequence predictions, we identified a segment (residues 218-266) in the apoE CT domain that bears a high propensity to form a coiled-coil helix, which coincides with the putative lipoprotein-binding surface. An apoE construct bearing residues 201-299 that encompasses the entire CT domain was designed, expressed in Escherichia coli and purified by affinity chromatography. Circular dichroism (CD) spectroscopy of the apoE CT domain reveals spectra characteristic of coiled-coil helices, with the ratio of molar ellipticities at 222 nm and 208 nm ([theta](222)/[theta](208)) of 1.03. Trifluoroethanol (TFE) stabilized the secondary structure of the apoE CT domain and disrupted coiled-coil helix formation as determined by CD and tryptophan fluorescence analysis. Analytical ultracentrifugation and lysine-specific cross-linking analysis of the apoE CT domain revealed predominant formation of dimeric and tetrameric species in aqueous buffers, and monomeric forms in 50% TFE. Guanidine hydrochloride-induced denaturation studies reveal that, at low concentrations of denaturant, the apoE CT domain maintains the [theta](222)/[theta](208) ratio at approximately 1.0 and elicits an altered tertiary environment with a shift in oligomeric state towards a dimer, indicative of the role of coiled-coil helix formation in inter molecular interactions. Further, coiled-coil formation is disrupted by protonation below pH 6.0, with a corresponding decrease in Trp fluorescence emission

  16. Considerations in the design and optimization of coiled coil structures.

    PubMed

    Mason, Jody M; Müller, Kristian M; Arndt, Katja M

    2007-01-01

    Coiled coil motifs are, despite their apparent simplicity, highly specific, and play a significant role in the understanding of tertiary structure and its formation. The most commonly observed of the coiled coils, the parallel dimeric, is yet to be fully characterized for this structural class in general. Nonetheless, strict rules have emerged for the necessity of specific types of amino acids at specific positions. In this chapter, we discuss this system in light of existing coiled coil structures and in applying rules to coiled coils that are to be designed or optimized. Understanding and expanding on these rules is crucial in using these motifs, which play key roles in virtually every cellular process, to act as drug-delivery agents by sequestering other proteins that are not behaving natively or that have been upregulated (for example, by binding to coiled coil domains implicated in oncogenesis). The roles of the a and d "hydrophobic" core positions and the e and g "electrostatic" edge positions in directing oligomerization and pairing specificity are discussed. Also discussed is the role of these positions in concert with the b, c, and f positions in maintaining alpha-helical propensity, helix solubility, and dimer stability. PMID:17041258

  17. Considerations in the design and optimization of coiled coil structures.

    PubMed

    Mason, Jody M; Müller, Kristian M; Arndt, Katja M

    2007-01-01

    Coiled coil motifs are, despite their apparent simplicity, highly specific, and play a significant role in the understanding of tertiary structure and its formation. The most commonly observed of the coiled coils, the parallel dimeric, is yet to be fully characterized for this structural class in general. Nonetheless, strict rules have emerged for the necessity of specific types of amino acids at specific positions. In this chapter, we discuss this system in light of existing coiled coil structures and in applying rules to coiled coils that are to be designed or optimized. Understanding and expanding on these rules is crucial in using these motifs, which play key roles in virtually every cellular process, to act as drug-delivery agents by sequestering other proteins that are not behaving natively or that have been upregulated (for example, by binding to coiled coil domains implicated in oncogenesis). The roles of the a and d "hydrophobic" core positions and the e and g "electrostatic" edge positions in directing oligomerization and pairing specificity are discussed. Also discussed is the role of these positions in concert with the b, c, and f positions in maintaining alpha-helical propensity, helix solubility, and dimer stability.

  18. 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

  19. Crystallization of the coiled-coil domain of Atg16 essential for autophagy

    PubMed Central

    Fujioka, Yuko; Noda, Nobuo N.; Matsushita, Minako; Ohsumi, Yoshinori; Inagaki, Fuyuhiko

    2008-01-01

    Atg16 is a scaffold protein that interacts with Atg12–Atg5 protein conjugates via its N-terminal domain and self-assembles via its coiled-coil domain, thus forming a multimeric Atg12–Atg5–Atg16 complex that is essential for autophagy. The coiled-coil domain of Atg16 was expressed, purified and crystallized. The crystal belonged to space group P41212 or P43212, with unit-cell parameters a = 127.7, c = 77.8 Å. Self-rotation functions and volume-to-weight ratio values suggested that the crystal may contain six molecules per asymmetric unit. Since the domain does not contain a methionine residue, selenomethionine-labelled crystals were prepared with a leucine-to-methionine substitution in the coiled-coil domain and these crystals were used for the collection of single-wavelength anomalous dispersion data to 2.5 Å resolution. PMID:18997338

  20. Tailored Presentation of Carbohydrates on a Coiled Coil-Based Scaffold for Asialoglycoprotein Receptor Targeting.

    PubMed

    Zacco, Elsa; Hütter, Julia; Heier, Jason L; Mortier, Jérémie; Seeberger, Peter H; Lepenies, Bernd; Koksch, Beate

    2015-09-18

    The coiled-coil folding motif represents an ideal scaffold for the defined presentation of ligands due to the possibility of positioning them at specific distances along the axis. We created a coiled-coil glycopeptide library to characterize the distances between the carbohydrate-binding sites of the asialoglycoprotein receptors (ASGPR) on hepatocytes. The components of the glycopeptide library vary for the number of displayed ligands (galactose), their position on the peptide sequence, and the space between peptide backbone and carbohydrate. We determined the binding of the glycopeptides to the hepatocytes, and we established the optimal distance and orientation of the galactose moieties for interaction with the ASGPR using flow cytometry. We confirmed that the binding occurs through endocytosis mediated by ASGPR via inhibition studies with cytochalasin D; fluorescence microscopy studies display the uptake of the carrier peptides inside the cell. Thus, this study demonstrates that the coiled-coil motif can be used as reliable scaffold for the rational presentation of ligands.

  1. Coiled coils ensure the physiological ectodomain shedding of collagen XVII.

    PubMed

    Nishie, Wataru; Jackow, Joanna; Hofmann, Silke C; Franzke, Claus-Werner; Bruckner-Tuderman, Leena

    2012-08-24

    α-Helical coiled coils, frequent protein oligomerization motifs, are commonly observed in vital proteins. Here, using collagen XVII as an example, we provide evidence for a novel function of coiled coils in the regulation of ectodomain shedding. Transmembrane collagen XVII, an epithelial cell surface receptor, mediates dermal-epidermal adhesion in the skin, and its dysfunction is linked to human skin blistering diseases. The ectodomain of this collagen is constitutively shed from the cell surface by proteinases of a disintegrin and metalloprotease family; however, the mechanisms regulating shedding remain elusive. Here, we used site-specific mutagenesis to target the coiled-coil heptad repeats within the juxtamembranous, extracellular noncollagenous 16th A (NC16A) domain of collagen XVII. This resulted in a substantial increase of ectodomain shedding, which was not mediated by disintegrin and metalloproteases. Instead, conformational changes induced by the mutation(s) unmasked a furin recognition sequence that was used for cleavage. This study shows that apart from their functions in protein oligomerization, coiled coils can also act as regulators of ectodomain shedding depending on the biological context.

  2. 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.

  3. 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

  4. Infrared amide I' band of the coiled coil.

    PubMed

    Reisdorf, W C; Krimm, S

    1996-02-01

    The Fourier transform infrared (FTIR) spectra of several coiled-coil proteins have been shown to possess unusual features in the amide I' region. Band maxima occur in the vicinity of 1630 cm-1, with component bands at higher frequency. This is well below the observed band at 1650 cm-1 found in standard alpha-helical polypeptides such as poly-L-alanine. Normal mode calculations on models of the coiled-coil structure have been performed to investigate this issue. We find that the observed band profile can be reproduced with very small random variation on the phi, psi of tropomyosin. We believe that the shift to lower frequency is due to additional hydrogen bonding of the solvent accessible backbone CO groups to water.

  5. 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

  6. 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

  7. Molecular dynamics guided study of salt bridge length dependence in both fluorinated and non-fluorinated parallel dimeric coiled-coils

    PubMed Central

    Pendley, Scott S.; Yu, Yihua B.; Cheatham, Thomas E.

    2009-01-01

    The α-helical coiled-coil is one of the most common oligomerization motifs found in both native and engineered proteins. To better understand the stability and dynamics of coiled-coil motifs, including those modified by fluorination, several fluorinated and non-fluorinated parallel dimeric coiled-coil protein structures were designed and modeled. We also attempt to investigate how changing the length and geometry of the important stabilizing salt bridges influences the coiled-coil protein structure. Molecular dynamics (MD) and free energy simulations with AMBER employed a particle mesh Ewald treatment of the electrostatics in explicit TIP3P solvent with balanced force field treatments. Preliminary studies with legacy force fields (ff94, ff96, ff99) show a profound instability of the coiled-coil structures in short MD simulation. Significantly better behavior is evident with the more balanced ff99SB and ff03 protein force fields. Overall, the results suggest that the coiled-coil structures can readily accommodate the larger acidic arginine or S-2,7-diaminoheptanedoic acid mutants in the salt bridge, whereas substitution of the smaller L-ornithine residue leads to rapid disruption of the coiled-coil structure on the MD simulation time scale. This structural distortion of the secondary structure allows both the formation of large hydration pockets proximal to the charged groups and within the hydrophobic core. Moreover, the increased structural fluctuations and movement lead to a decrease in the water occupancy lifetimes in the hydration pockets. In contrast, analysis of the hydration in the stable dimeric coiled coils shows high occupancy water sites along the backbone residues with no water occupancy in the hydrophobic core, although transitory water interactions with the salt bridge residues are evident. The simulations of the fluorinated coiled-coils suggest that in some cases fluorination electrostatically stabilizes the intermolecular coiled-coil salt bridges

  8. The d'--d--d' vertical triad is less discriminating than the a'--a--a' vertical triad in the antiparallel coiled-coil dimer motif.

    PubMed

    Steinkruger, Jay D; Bartlett, Gail J; Hadley, Erik B; Fay, Lindsay; Woolfson, Derek N; Gellman, Samuel H

    2012-02-01

    Elucidating relationships between the amino-acid sequences of proteins and their three-dimensional structures, and uncovering non-covalent interactions that underlie polypeptide folding, are major goals in protein science. One approach toward these goals is to study interactions between selected residues, or among constellations of residues, in small folding motifs. The α-helical coiled coil has served as a platform for such studies because this folding unit is relatively simple in terms of both sequence and structure. Amino acid side chains at the helix-helix interface of a coiled coil participate in so-called "knobs-into-holes" (KIH) packing whereby a side chain (the knob) on one helix inserts into a space (the hole) generated by four side chains on a partner helix. The vast majority of sequence-stability studies on coiled-coil dimers have focused on lateral interactions within these KIH arrangements, for example, between an a position on one helix and an a' position of the partner in a parallel coiled-coil dimer, or between a--d' pairs in an antiparallel dimer. More recently, it has been shown that vertical triads (specifically, a'--a--a' triads) in antiparallel dimers exert a significant impact on pairing preferences. This observation provides impetus for analysis of other complex networks of side-chain interactions at the helix-helix interface. Here, we describe a combination of experimental and bioinformatics studies that show that d'--d--d' triads have much less impact on pairing preference than do a'--a--a' triads in a small, designed antiparallel coiled-coil dimer. However, the influence of the d'--d--d' triad depends on the lateral a'--d interaction. Taken together, these results strengthen the emerging understanding that simple pairwise interactions are not sufficient to describe side-chain interactions and overall stability in antiparallel coiled-coil dimers; higher-order interactions must be considered as well.

  9. 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.

  10. 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

  11. Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments.

    PubMed

    Gradišar, Helena; Božič, Sabina; Doles, Tibor; Vengust, Damjan; Hafner-Bratkovič, Iva; Mertelj, Alenka; Webb, Ben; Šali, Andrej; Klavžar, Sandi; Jerala, Roman

    2013-06-01

    Protein structures evolved through a complex interplay of cooperative interactions, and it is still very challenging to design new protein folds de novo. Here we present a strategy to design self-assembling polypeptide nanostructured polyhedra based on modularization using orthogonal dimerizing segments. We designed and experimentally demonstrated the formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled coil-forming segments separated by flexible peptide hinges. The path of the polypeptide chain is guided by a defined order of segments that traverse each of the six edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. The coincidence of the polypeptide termini in the same vertex is demonstrated by reconstituting a split fluorescent protein in the polypeptide with the correct tetrahedral topology. Polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides a foundation for constructing new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments.

  12. Dimeric Coiled-coil Structure of Saccharomyces cerevisiae Atg16 and Its Functional Significance in Autophagy*

    PubMed Central

    Fujioka, Yuko; Noda, Nobuo N.; Nakatogawa, Hitoshi; Ohsumi, Yoshinori; Inagaki, Fuyuhiko

    2010-01-01

    Atg16 interacts with the Atg12-Atg5 protein conjugate through its N-terminal domain and self-assembles through its coiled-coil domain (CCD). Formation of the Atg12-Atg5·Atg16 complex is essential for autophagy, the bulk degradation process conserved among most eukaryotes. Here, we report the crystal structures of full-length Saccharomyces cerevisiae Atg16 at 2.8 Å resolution and its CCD at 2.5 Å resolution. The CCD and full-length Atg16 each exhibit an extended α-helix, 90 and 130 Å, respectively, and form a parallel coiled-coil dimer in the crystals. Although the apparent molecular weight of Atg16 observed by gel-filtration chromatography suggests that Atg16 is tetrameric, an analytical ultracentrifugation study showed Atg16 as a dimer in solution, consistent with the crystal structure. Evolutionary conserved surface residues clustered at the C-terminal half of Atg16 CCD were shown to be crucial for autophagy. These results will give a structural basis for understanding the molecular functions and significance of Atg16 in autophagy. PMID:19889643

  13. Self assembly of coiled-coil peptide-porphyrin complexes.

    PubMed

    Kokona, Bashkim; Kim, Andrew M; Roden, R Claire; Daniels, Joshua P; Pepe-Mooney, Brian J; Kovaric, Brian C; de Paula, Julio C; Johnson, Karl A; Fairman, Robert

    2009-06-01

    We are interested in the controlled assembly of photoelectronic materials using peptides as scaffolds and porphyrins as the conducting material. We describe the integration of a peptide-based polymer strategy with the ability of designed basic peptides to bind anionic porphyrins in order to create regulated photoelectronically active biomaterials. We have described our peptide system in earlier work, which demonstrates the ability of a peptide to form filamentous materials made up of self-assembling coiled-coil structures. We have modified this peptide system to include lysine residues appropriately positioned to specifically bind meso-tetrakis(4-sulfonatophenyl)porphine (TPPS(4)), a porphyrin that contains four negatively charged sulfonate groups at neutral pH. We measure the binding of TPPS(4) to our peptide using UV--visible and fluorescence spectroscopies to follow the porphyrin signature. We determine the concomitant acquisition of helical secondary structure in the peptide upon TPPS(4) binding using circular dichroism spectropolarimetry. This binding fosters polymerization of the peptide, as shown by absorbance extinction effects in the peptide CD spectra. The morphologies of the peptide/porphyrin complexes, as imaged by atomic force microscopy, are consistent with the coiled-coil polymers that we had characterized earlier, except that the heights are slightly higher, consistent with porphyrin binding. Evidence for exciton coupling in the copolymers is shown by red-shifting in the UV--visible data, however, the coupling is weak based on a lack of fluorescence quenching in fluorescence experiments.

  14. 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

  15. 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

  16. 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

  17. KAP, the accessory subunit of kinesin-2, binds the predicted coiled-coil stalk of the motor subunits.

    PubMed

    Doodhi, Harinath; Ghosal, Debnath; Krishnamurthy, Mahalakshmi; Jana, Swadhin C; Shamala, Divya; Bhaduri, Anirban; Sowdhamini, R; Ray, Krishanu

    2009-03-17

    Kinesin-2 is an anterograde motor involved in intraflagellar transport and certain other intracellular transport processes. It consists of two different motor subunits and an accessory protein KAP (kinesin accessory protein). The motor subunits were shown to bind each other through the coiled-coil stalk domains, while KAP was proposed to bind the tail domains of the motor subunits. Although several genetic studies established that KAP plays an important role in kinesin-2 functions, its exact role remains unclear. Here, we report the results of a systematic analysis of the KAP binding sites by using recombinant Drosophila kinesin-2 subunits as well as the endogenous proteins. These show that at least one of the coiled-coil stalks is sufficient to bind the N-terminal region of DmKAP. The soluble complex involving the recombinant kinesin-2 fragments is reconstituted in vitro at high salt concentrations, suggesting that the interaction is primarily nonionic. Furthermore, independent distant homology modeling indicated that DmKAP may bind along the coiled-coil stalks through a combination of predominantly hydrophobic interactions and hydrogen bonds. These observations led us to propose that KAP would stabilize the motor subunit heterodimer and help assemble a greater kinesin-2 complex in vivo. PMID:19161286

  18. The coiled-coil domain of MURC/cavin-4 is involved in membrane trafficking of caveolin-3 in cardiomyocytes.

    PubMed

    Naito, Daisuke; Ogata, Takehiro; Hamaoka, Tetsuro; Nakanishi, Naohiko; Miyagawa, Kotaro; Maruyama, Naoki; Kasahara, Takeru; Taniguchi, Takuya; Nishi, Masahiro; Matoba, Satoaki; Ueyama, Tomomi

    2015-12-15

    Muscle-restricted coiled-coil protein (MURC), also referred to as cavin-4, is a member of the cavin family that works cooperatively with caveolins in caveola formation and function. Cavins are cytoplasmic proteins with coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, and its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/cavin-4 gene have been identified in patients with dilated cardiomyopathy. In the present study, we show the role of MURC/cavin-4 as a caveolar component in the heart. In H9c2 cells, MURC/cavin-4 was localized at the plasma membrane, whereas a MURC/cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. Additionally, although ΔCC did not alter Cav3 mRNA expression, ΔCC decreased the Cav3 protein level. MURC/cavin-4 and ΔCC similarly induced cardiomyocyte hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression than MURC/cavin-4. ΔCC induced ERK activation in cardiomyocytes. Transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) showed impaired cardiac function accompanied by cardiomyocyte hypertrophy and marked interstitial fibrosis. Hearts from ΔCC-Tg mice showed a reduction of the Cav3 protein level and activation of ERK. These results suggest that MURC/cavin-4 requires its coiled-coil domain to target the plasma membrane and to stabilize Cav3 at the plasma membrane of cardiomyocytes and that MURC/cavin-4 functions as a crucial caveolar component to regulate cardiac function. PMID:26497963

  19. The coiled-coil domain of MURC/cavin-4 is involved in membrane trafficking of caveolin-3 in cardiomyocytes.

    PubMed

    Naito, Daisuke; Ogata, Takehiro; Hamaoka, Tetsuro; Nakanishi, Naohiko; Miyagawa, Kotaro; Maruyama, Naoki; Kasahara, Takeru; Taniguchi, Takuya; Nishi, Masahiro; Matoba, Satoaki; Ueyama, Tomomi

    2015-12-15

    Muscle-restricted coiled-coil protein (MURC), also referred to as cavin-4, is a member of the cavin family that works cooperatively with caveolins in caveola formation and function. Cavins are cytoplasmic proteins with coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, and its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/cavin-4 gene have been identified in patients with dilated cardiomyopathy. In the present study, we show the role of MURC/cavin-4 as a caveolar component in the heart. In H9c2 cells, MURC/cavin-4 was localized at the plasma membrane, whereas a MURC/cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. Additionally, although ΔCC did not alter Cav3 mRNA expression, ΔCC decreased the Cav3 protein level. MURC/cavin-4 and ΔCC similarly induced cardiomyocyte hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression than MURC/cavin-4. ΔCC induced ERK activation in cardiomyocytes. Transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) showed impaired cardiac function accompanied by cardiomyocyte hypertrophy and marked interstitial fibrosis. Hearts from ΔCC-Tg mice showed a reduction of the Cav3 protein level and activation of ERK. These results suggest that MURC/cavin-4 requires its coiled-coil domain to target the plasma membrane and to stabilize Cav3 at the plasma membrane of cardiomyocytes and that MURC/cavin-4 functions as a crucial caveolar component to regulate cardiac function.

  20. 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.

  1. Isolation and cloning of Omp alpha, a coiled-coil protein spanning the periplasmic space of the ancestral eubacterium Thermotoga maritima.

    PubMed Central

    Engel, A M; Cejka, Z; Lupas, A; Lottspeich, F; Baumeister, W

    1992-01-01

    We have discovered a new oligomeric protein component associated with the outer membrane of the ancestral eubacterium Thermotoga maritima. In electron micrographs, the protein, Omp alpha, appears as a rod-shaped spacer that spans the periplasm, connecting the outer membrane to the inner cell body. Purification, biochemical characterization and sequencing of Omp alpha suggest that it is a homodimer composed of two subunits of 380 amino acids with a calculated M(r) of 43,000 and a pI of 4.54. The sequence of the omp alpha gene indicates a tripartite organization of the protein with a globular NH2-terminal domain of 64 residues followed by a putative coiled-coil segment of 300 residues and a COOH-terminal, membrane-spanning segment. The predicted length of the coiled-coil segment (45 nm) correlates closely with the spacing between the inner and outer membranes. Despite sequence similarity to a large number of coiled-coil proteins and high scores in a coiled-coil prediction algorithm, the sequence of the central rod-shaped domain of Omp alpha does not have the typical 3.5 periodicity of coiled-coil proteins but rather has a periodicity of 3.58 residues. Such a periodicity was also found in the central domain of staphylococcal M protein and beta-giardin and might be indicative of a subclass of fibrous proteins with packing interactions that are distinct from the ones seen in other two-stranded coiled-coils. Images PMID:1330536

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

    PubMed

    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

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. A basis set of de novo coiled-coil peptide oligomers for rational protein design and synthetic biology.

    PubMed

    Fletcher, Jordan M; Boyle, Aimee L; Bruning, Marc; Bartlett, Gail J; Vincent, Thomas L; Zaccai, Nathan R; Armstrong, Craig T; Bromley, Elizabeth H C; Booth, Paula J; Brady, R Leo; Thomson, Andrew R; Woolfson, Derek N

    2012-06-15

    Protein engineering, chemical biology, and synthetic biology would benefit from toolkits of peptide and protein components that could be exchanged reliably between systems while maintaining their structural and functional integrity. Ideally, such components should be highly defined and predictable in all respects of sequence, structure, stability, interactions, and function. To establish one such toolkit, here we present a basis set of de novo designed α-helical coiled-coil peptides that adopt defined and well-characterized parallel dimeric, trimeric, and tetrameric states. The designs are based on sequence-to-structure relationships both from the literature and analysis of a database of known coiled-coil X-ray crystal structures. These give foreground sequences to specify the targeted oligomer state. A key feature of the design process is that sequence positions outside of these sites are considered non-essential for structural specificity; as such, they are referred to as the background, are kept non-descript, and are available for mutation as required later. Synthetic peptides were characterized in solution by circular-dichroism spectroscopy and analytical ultracentrifugation, and their structures were determined by X-ray crystallography. Intriguingly, a hitherto widely used empirical rule-of-thumb for coiled-coil dimer specification does not hold in the designed system. However, the desired oligomeric state is achieved by database-informed redesign of that particular foreground and confirmed experimentally. We envisage that the basis set will be of use in directing and controlling protein assembly, with potential applications in chemical and synthetic biology. To help with such endeavors, we introduce Pcomp, an on-line registry of peptide components for protein-design and synthetic-biology applications. PMID:23651206

  11. Functional investigation of the plant-specific long coiled-coil proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana.

    PubMed

    Venkatakrishnan, Sowmya; Mackey, David; Meier, Iris

    2013-01-01

    We have identified and characterized two Arabidopsis long coiled-coil proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL). PICC (147 kDa) and PICL (87 kDa) are paralogs that consist predominantly of a long coiled-coil domain (expanded in PICC), with a predicted transmembrane domain at the immediate C-terminus. Orthologs of PICC and PICL were found exclusively in vascular plants. PICC and PICL GFP fusion proteins are anchored to the cytoplasmic surface of the endoplasmic reticulum (ER) membrane by a C-terminal transmembrane domain and a short tail domain, via a tail-anchoring mechanism. T-DNA-insertion mutants of PICC and PICL as well as the double mutant show an increased sensitivity to the plant abiotic stress hormone abscisic acid (ABA) in a post-germination growth response. PICC, but not PICL gene expression is induced by the bacterial pathogen-associated molecular pattern (PAMP) flg22. T-DNA insertion alleles of PICC, but not PICL, show increased susceptibility to the non-virulent strain P. syringae pv. tomato DC3000 hrcC, but not to the virulent strain P. syringae pv. tomato DC3000. This suggests that PICC mutants are compromised in PAMP-triggered immunity (PTI). The data presented here provide first evidence for the involvement of a plant long coiled-coil protein in a plant defense response.

  12. Functional Investigation of the Plant-Specific Long Coiled-Coil Proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana

    PubMed Central

    Venkatakrishnan, Sowmya; Mackey, David; Meier, Iris

    2013-01-01

    We have identified and characterized two Arabidopsis long coiled-coil proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL). PICC (147 kDa) and PICL (87 kDa) are paralogs that consist predominantly of a long coiled-coil domain (expanded in PICC), with a predicted transmembrane domain at the immediate C-terminus. Orthologs of PICC and PICL were found exclusively in vascular plants. PICC and PICL GFP fusion proteins are anchored to the cytoplasmic surface of the endoplasmic reticulum (ER) membrane by a C-terminal transmembrane domain and a short tail domain, via a tail-anchoring mechanism. T-DNA-insertion mutants of PICC and PICL as well as the double mutant show an increased sensitivity to the plant abiotic stress hormone abscisic acid (ABA) in a post-germination growth response. PICC, but not PICL gene expression is induced by the bacterial pathogen-associated molecular pattern (PAMP) flg22. T-DNA insertion alleles of PICC, but not PICL, show increased susceptibility to the non-virulent strain P. syringae pv. tomato DC3000 hrcC, but not to the virulent strain P. syringae pv. tomato DC3000. This suggests that PICC mutants are compromised in PAMP-triggered immunity (PTI). The data presented here provide first evidence for the involvement of a plant long coiled-coil protein in a plant defense response. PMID:23451199

  13. Functional investigation of the plant-specific long coiled-coil proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana.

    PubMed

    Venkatakrishnan, Sowmya; Mackey, David; Meier, Iris

    2013-01-01

    We have identified and characterized two Arabidopsis long coiled-coil proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL). PICC (147 kDa) and PICL (87 kDa) are paralogs that consist predominantly of a long coiled-coil domain (expanded in PICC), with a predicted transmembrane domain at the immediate C-terminus. Orthologs of PICC and PICL were found exclusively in vascular plants. PICC and PICL GFP fusion proteins are anchored to the cytoplasmic surface of the endoplasmic reticulum (ER) membrane by a C-terminal transmembrane domain and a short tail domain, via a tail-anchoring mechanism. T-DNA-insertion mutants of PICC and PICL as well as the double mutant show an increased sensitivity to the plant abiotic stress hormone abscisic acid (ABA) in a post-germination growth response. PICC, but not PICL gene expression is induced by the bacterial pathogen-associated molecular pattern (PAMP) flg22. T-DNA insertion alleles of PICC, but not PICL, show increased susceptibility to the non-virulent strain P. syringae pv. tomato DC3000 hrcC, but not to the virulent strain P. syringae pv. tomato DC3000. This suggests that PICC mutants are compromised in PAMP-triggered immunity (PTI). The data presented here provide first evidence for the involvement of a plant long coiled-coil protein in a plant defense response. PMID:23451199

  14. Crystal structure of a coiled-coil domain from human ROCK I.

    PubMed

    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

    2011-03-21

    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.

  15. 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

  16. A Reversible Association between Smc Coiled Coils Is Regulated by Lysine Acetylation and Is Required for Cohesin Association with the DNA.

    PubMed

    Kulemzina, Irina; Ang, Keven; Zhao, Xiaodan; Teh, Jun-Thing; Verma, Vikash; Suranthran, Sasikala; Chavda, Alap P; Huber, Roland G; Eisenhaber, Birgit; Eisenhaber, Frank; Yan, Jie; Ivanov, Dmitri

    2016-09-15

    Cohesin is a ring-shaped protein complex that is capable of embracing DNA. Most of the ring circumference is comprised of the anti-parallel intramolecular coiled coils of the Smc1 and Smc3 proteins, which connect globular head and hinge domains. Smc coiled coil arms contain multiple acetylated and ubiquitylated lysines. To investigate the role of these modifications, we substituted lysines for arginines to mimic the unmodified state and uncovered genetic interaction between the Smc arms. Using scanning force microscopy, we show that wild-type Smc arms associate with each other when the complex is not on DNA. Deacetylation of the Smc1/Smc3 dimers promotes arms' dissociation. Smc arginine mutants display loose packing of the Smc arms and, although they dimerize at the hinges, fail to connect the heads and associate with the DNA. Our findings highlight the importance of a "collapsed ring," or "rod," conformation of cohesin for its loading on the chromosomes. PMID:27618487

  17. X-ray scattering indicates that the leucine zipper is a coiled coil.

    PubMed Central

    Rasmussen, R; Benvegnu, D; O'Shea, E K; Kim, P S; Alber, T

    1991-01-01

    Dimerization of the bZIP class of eukaryotic transcriptional control proteins requires a sequence motif called the leucine zipper. We have grown two distinct crystal forms of a 33-amino acid peptide corresponding to the leucine zipper of the yeast transcriptional activator GCN4. This peptide is known to form a dimer of parallel helices in solution. X-ray scattering from both crystal forms shows reflections that are diagnostic of coiled coils. The most notable reflections occur at approximately 5.2 A resolution and correspond to the pitch of helices in coiled coils. There is no diffraction maximum near 5.4 A, the characteristic pitch of straight helices. Our results provide direct evidence that the leucine zipper of GCN4 is a coiled coil. Images PMID:1988953

  18. 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.

  19. Functional Analysis of the Bacteriophage T4 Rad50 Homolog (gp46) Coiled-coil Domain.

    PubMed

    Barfoot, Tasida; Herdendorf, Timothy J; Behning, Bryanna R; Stohr, Bradley A; Gao, Yang; Kreuzer, Kenneth N; Nelson, Scott W

    2015-09-25

    Rad50 and Mre11 form a complex involved in the detection and processing of DNA double strand breaks. Rad50 contains an anti-parallel coiled-coil with two absolutely conserved cysteine residues at its apex. These cysteine residues serve as a dimerization domain and bind a Zn(2+) cation in a tetrathiolate coordination complex known as the zinc-hook. Mutation of the zinc-hook in bacteriophage T4 is lethal, indicating the ability to bind Zn(2+) is critical for the functioning of the MR complex. In vitro, we found that complex formation between Rad50 and a peptide corresponding to the C-terminal domain of Mre11 enhances the ATPase activity of Rad50, supporting the hypothesis that the coiled-coil is a major conduit for communication between Mre11 and Rad50. We constructed mutations to perturb this domain in the bacteriophage T4 Rad50 homolog. Deletion of the Rad50 coiled-coil and zinc-hook eliminates Mre11 binding and ATPase activation but does not affect its basal activity. Mutation of the zinc-hook or disruption of the coiled-coil does not affect Mre11 or DNA binding, but their activation of Rad50 ATPase activity is abolished. Although these mutants excise a single nucleotide at a normal rate, they lack processivity and have reduced repetitive exonuclease rates. Restricting the mobility of the coiled-coil eliminates ATPase activation and repetitive exonuclease activity, but the ability to support single nucleotide excision is retained. These results suggest that the coiled-coiled domain adopts at least two conformations throughout the ATPase/nuclease cycle, with one conformation supporting enhanced ATPase activity and processivity and the other supporting nucleotide excision.

  20. Coiled-coils as assembly-directing domains in self-assembling structures

    NASA Astrophysics Data System (ADS)

    Sahin, Erinc

    2008-10-01

    The bioconjugation of proteins and peptides with synthetic polymers is a promising method to tailor chemical, biological and physical properties of both the polymeric and protein-based components. Here, we describe macromolecular assemblies of polyethylene glycol-coiled-coil alternating block copolymers guided by associations of coiled-coils. High molecular weight, alternating block copolymers of PEG and coiled-coil peptides were formed via facile reactions between N-hydroxysuccinimide esters and amines, leading to amide bond formation under anhydrous conditions. Confirmation of multiblock formation was assessed via a combination of NMR spectroscopy, size-exclusion chromatography, and electrophoretic analysis. Formation of the alternating block copolymers of PEG with coiled-coil peptides through the f-position on the heptads did not impair the ability of the coiled-coils to form heterodimers, as assessed via circular dichroic spectroscopy. Interestingly, the conjugation triggered homooligomer formation in one of the peptides that is monomeric in the absence of PEG. The macromolecular assembly of the homooligomer was characterized via circular dichroism, analytical ultracentrifugation, and dynamic and static light scattering. The copolymer structures were formed under physiological conditions, and exhibited controlled sizes relevant in applications such as drug delivery and controlled release. Our efforts toward synthesis of coiled-coils via recombinant expression have also lead to the discovery of an evolutionary phenomenon. We observed a strong inverse correlation between the pH of an organism's habitat and its proteomic pI, that is the isoelectric point of bulk of its proteome. This observation may be useful in informed selection of host organisms for recombinant expression of proteins with extreme isoelectric points.

  1. Dimerization of the ATRIP protein through the coiled-coil motif and its implication to the maintenance of stalled replication forks.

    PubMed

    Itakura, Eisuke; Sawada, Isao; Matsuura, Akira

    2005-12-01

    ATR (ATM and Rad3-related), a PI kinase-related kinase (PIKK), has been implicated in the DNA structure checkpoint in mammalian cells. ATR associates with its partner protein ATRIP to form a functional complex in the nucleus. In this study, we investigated the role of the ATRIP coiled-coil domain in ATR-mediated processes. The coiled-coil domain of human ATRIP contributes to self-dimerization in vivo, which is important for the stable translocation of the ATR-ATRIP complex to nuclear foci that are formed after exposure to genotoxic stress. The expression of dimerization-defective ATRIP diminishes the maintenance of replication forks during treatment with replication inhibitors. By contrast, it does not compromise the G2/M checkpoint after IR-induced DNA damage. These results show that there are two critical functions of ATR-ATRIP after the exposure to genotoxic stress: maintenance of the integrity of replication machinery and execution of cell cycle arrest, which are separable and are achieved via distinct mechanisms. The former function may involve the concentrated localization of ATR to damaged sites for which the ATRIP coiled-coil motif is critical.

  2. The predicted coiled-coil domain of myosin 10 forms a novel elongated domain that lengthens the head.

    PubMed

    Knight, Peter J; Thirumurugan, Kavitha; Xu, Yuhui; Wang, Fei; Kalverda, Arnout P; Stafford, Walter F; Sellers, James R; Peckham, Michelle

    2005-10-14

    Myosin 10 contains a region of predicted coiled coil 120 residues long. However, the highly charged nature and pattern of charges in the proximal 36 residues appear incompatible with coiled-coil formation. Circular dichroism, NMR, and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single alpha-helix (SAH) domain in solution and does not dimerize to form a coiled coil even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin 10 containing the motor, the three calmodulin binding domains, and the full-length predicted coiled coil showed that it was mostly monomeric at physiological protein concentration. In dimers the molecules were joined only at their extreme distal ends, and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin 5 heavy meromyosin obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus the predicted coiled coil of myosin 10 forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins 6 and 7a and MyoM and could function to increase the size of the working stroke. PMID:16030012

  3. Growth Factor Tethering to Protein Nanoparticles via Coiled-Coil Formation for Targeted Drug Delivery.

    PubMed

    Assal, Yasmine; Mizuguchi, Yoshinori; Mie, Masayasu; Kobatake, Eiry

    2015-08-19

    Protein-based nanoparticles are attractive carriers for drug delivery because they are biodegradable and can be genetically designed. Moreover, modification of protein-based nanoparticles with cell-specific ligands allows for active targeting abilities. Previously, we developed protein nanoparticles comprising genetically engineered elastin-like polypeptides (ELPs) with fused polyaspartic acid tails (ELP-D). Epidermal growth factor (EGF) was displayed on the surface of the ELP-D nanoparticles via genetic design to allow for active cell-targeting abilities. Herein, we focused on the coiled-coil structural motif as a means for noncovalent tethering of growth factor to ELP-D. Specifically, two peptides known to form a heterodimer via a coiled-coil structural motif were fused to ELP-D and single-chain vascular endothelial growth factor (scVEGF121), to facilitate noncovalent tethering upon formation of the heterodimer coiled-coil structure. Drug-loaded growth factor-tethered ELP-Ds were found to be effective against cancer cells by provoking cell apoptosis. These results demonstrate that tethering growth factor to protein nanoparticles through coiled-coil formation yields a promising biomaterial candidate for targeted drug delivery.

  4. 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

  5. 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

  6. Modulation of elasticity in functionally distinct domains of the tropomyosin coiled-coil

    PubMed Central

    Lakkaraju, Sirish Kaushik; Hwang, Wonmuk

    2009-01-01

    Alpha-helical coiled-coils are common protein structural motifs. Whereas vast information is available regarding their structure, folding, and stability, far less is known about their elastic properties, even though they play mechanical roles in many cases such as tropomyosin in muscle contraction or neck stalks of kinesin or myosin motor proteins. Using computer simulations, we characterized elastic properties of coiled-coils, either globally or locally. Global bending stiffness of standard leucine zipper coiled-coils was calculated using normal mode analysis. Mutations in hydrophobic residues involved in the knob-into-hole interface between the two α-helices affect elasticity significantly, whereas charged side chains forming inter-helical salt bridges do not. This suggests that coiled-coils with less regular heptad periodicity may have regional variations in flexibility. We show this by the flexibility map of tropomyosin, which was constructed by a local fluctuation analysis. Overall, flexibility varies by more than twofold and increases towards the C-terminal region of the molecule. Describing the coiled-coil as a twisted tape, it is generally more flexible in the splay bending than in the bending of the broad face. Actin binding sites in α zones show local rigidity minima. Broken core regions due to acidic residues at the hydrophobic face such as the Asp137 and the Glu218 are found to be the most labile with moduli for splay and broad face bending as 70 nm and 116 nm respectively. Such variation in flexibility could be relevant to the tropomyosin function, especially for moving across the non-uniform surface of F-actin to regulate myosin binding. PMID:19830262

  7. Membrane trafficking. The specificity of vesicle traffic to the Golgi is encoded in the golgin coiled-coil proteins.

    PubMed

    Wong, Mie; Munro, Sean

    2014-10-31

    The Golgi apparatus is a multicompartment central sorting station at the intersection of secretory and endocytic vesicular traffic. The mechanisms that permit cargo-loaded transport vesicles from different origins to selectively access different Golgi compartments are incompletely understood. We developed a rerouting and capture assay to investigate systematically the vesicle-tethering activities of 10 widely conserved golgin coiled-coil proteins. We find that subsets of golgins with distinct localizations on the Golgi surface have capture activities toward vesicles of different origins. These findings demonstrate that golgins act as tethers in vivo, and hence the specificity we find to be encoded in this tethering is likely to make a major contribution to the organization of membrane traffic at the Golgi apparatus.

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

    PubMed

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

    2016-06-02

    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.

  9. Intermediate filament-like network formed in vitro by a bacterial coiled coil protein.

    PubMed

    Hurme, R; Namork, E; Nurmiaho-Lassila, E L; Rhen, M

    1994-04-01

    The TlpA protein encoded by the virulence plasmid of Salmonella enterica is an alpha-helical 371-amino acid protein possessing characteristics similar to eukaryotic coiled coil proteins (Koski, P., Saarilahti, H., Sukupolvi, S., Taira, S., Rikkonen, P., Osterlund, K., Hurme, R., and Rhen, M. (1992) J. Biol. Chem. 267, 12258-12265). In this paper we have investigated inter- and intramolecular associations and the morphology of structures formed by TlpA. Dynamics and temperature stability of TlpA dimers were studied by examining the feasibility and conditions in which TlpA would form an artificial heterodimer with its truncated derivative. Formation of heterodimers, bridged by Cu(2+)-catalyzed air oxidation of adjacent Cys residues, showed that TlpA dimers are dynamic chain exchanging structures at 37 degrees C, whereas they were nonexchanging at room temperature or on ice. Chemical cross-linking suggested higher order interaction between TlpA dimers. Electron microscopy studies revealed two levels of TlpA organization in vitro: thin filaments and rods, 2-5 nm in diameter, and a higher ordered filament network consisting of tonofilament-like formations with a diameter of 8-15 nm. Electron microscopy of thin-sectioned Escherichia coli over-producing TlpA showed an extraordinary intracellular assembly of proteinacious lamellae with a striated appearance and a 38-nm periodicity. This study describes for the first time a bacterial protein capable of organizing itself into an ordered and suspectedly dynamic intermediate filament-like architecture. PMID:8144657

  10. Four-stranded coiled-coil elastic protein in the byssus of the giant clam, Tridacna maxima.

    PubMed

    Miserez, Ali; Li, Youli; Cagnon, Joel; Weaver, James C; Waite, J Herbert

    2012-02-13

    An elastic protein with a secondary structure distinct from all well-known load-bearing proteins is found in the byssus of the giant clam, Tridacna maxima . The byssus consists of a bundle of hundreds of individual threads, each measuring about about 100 μm in diameter, which exhibit a tendon-like mechanical response. The amino acid composition of Tridacna byssus, however, is unlike tendon collagen, lacking high glycine, proline, and hydroxyproline. Wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) measurements suggest that the constituent nanofibrils of the byssal threads are distinct from known secondary structure motifs previously reported for elastic proteins including the collagen triple-helix, the β-sheet nanocrystalline domains of silks, or the double-stranded coiled-coil regions of intermediate filaments. Instead, X-ray diffraction data indicate a structural organization in which four coiled-coil α-helices form a stable rope-like structure, which then further pack in a pseudohexagonal lattice to form nanofibrils. Amino acid composition analysis shows unusually high concentrations of acidic as well as basic residues, suggesting that the four-helix structure is stabilized by strong ionic interactions between oppositely charged residues in neighboring strands. The composition also suggests additional stabilization by disulfide cross-linking. On a larger scale, scanning and conventional transmission electron microscope (STEM and TEM) observations indicate that the nanofibrils exhibit an alternating periodicity of about 500 nm along the axial direction. A molecular model that combines the mechanical properties with the structural characteristics of the Tridacna byssal threads is proposed.

  11. 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.

  12. 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

  13. 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

  14. Plasmodium vivax antigen discovery based on alpha-helical coiled coil protein motif.

    PubMed

    Céspedes, Nora; Habel, Catherine; Lopez-Perez, Mary; Castellanos, Angélica; Kajava, Andrey V; Servis, Catherine; Felger, Ingrid; Moret, Remy; Arévalo-Herrera, Myriam; Corradin, Giampietro; Herrera, Sócrates

    2014-01-01

    Protein α-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with α-helical coiled coil motifs orthologous to Plasmodium falciparum were identified in the P. vivax genome. The peptides identified in silico were chemically synthesized; circular dichroism studies indicated partial or high α-helical content. Antigenicity was evaluated using human sera samples from malaria-endemic areas of Colombia and Papua New Guinea. Eight of these fragments were selected and used to assess immunogenicity in BALB/c mice. ELISA assays indicated strong reactivity of serum samples from individuals residing in malaria-endemic regions and sera of immunized mice, with the α-helical coiled coil structures. In addition, ex vivo production of IFN-γ by murine mononuclear cells confirmed the immunogenicity of these structures and the presence of T-cell epitopes in the peptide sequences. Moreover, sera of mice immunized with four of the eight antigens recognized native proteins on blood-stage P. vivax parasites, and antigenic cross-reactivity with three of the peptides was observed when reacted with both the P. falciparum orthologous fragments and whole parasites. Results here point to the α-helical coiled coil peptides as possible P. vivax malaria vaccine candidates as were observed for P. falciparum. Fragments selected here warrant further study in humans and non-human primate models to assess their protective efficacy as single components or assembled as hybrid linear epitopes.

  15. Historical review: another 50th anniversary--new periodicities in coiled coils.

    PubMed

    Gruber, Markus; Lupas, Andrei N

    2003-12-01

    In 1953, Francis Crick and Linus Pauling both proposed models of supercoiled alpha helices ('coiled coils') for the structure of keratin. These were the first attempts at modelling the tertiary structure of a protein. Crick emphasized the packing mode of the side-chains ('knobs-into-holes'), which required a periodicity of seven residues over two helical turns (7/2) and a supercoil in the opposite sense of the constituent helices. By contrast, Pauling envisaged a broader set of periodicities (4/1, 7/2, 18/5, 15/4, 11/3) and supercoils of both senses. Crick's model became canonical and the 'heptad repeat' essentially synonymous with coiled coils, but 50 years later new crystal structures and protein sequences show that the less common periodicities envisaged by Pauling also occur in coiled coils, adding a variant packing mode ('knobs-to-knobs') to the standard model. Pauling's laboratory notebooks suggest that he searched unsuccessfully for this packing mode in 1953.

  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. 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

  18. Targeting and import mechanism of coiled-coil helix coiled-coil helix domain-containing protein 3 (ChChd3) into the mitochondrial intermembrane space.

    PubMed

    Darshi, Manjula; Trinh, Kristina N; Murphy, Anne N; Taylor, Susan S

    2012-11-16

    Coiled-coil helix coiled-coil helix domain-containing protein 3 (ChChd3) is a mitochondrial inner membrane (IM) protein facing toward the intermembrane space (IMS). In the IMS, ChChd3 complexes with multiple proteins at the crista junctions and contact sites and plays a key role in maintaining crista integrity. ChChd3 is myristoylated at the N terminus and has a CHCH domain with twin CX(9)C motifs at its C terminus. The CHCH domain proteins are traditionally imported and trapped in the IMS by using a disulfide relay system mediated by Mia40 and Erv1. In this study, we systematically analyzed the role of the myristoylation and the CHCH domain in the import and mitochondrial localization of ChChd3. Based on our results, we predict that myristoylation promotes binding of ChChd3 to the outer membrane and that the CHCH domain translocates the protein across the outer membrane. By analysis of the CHCH domain cysteine mutants, we further show that they have distinct roles in binding to Mia40 in the IMS and proper folding of the protein. The transient disulfide-bonded intermediate with Mia40 is formed preferentially between the second cysteine in helix 1, Cys(193), and the active site cysteine in Mia40, Cys(55). Although each of the four cysteines is essential for folding of the protein and binding to mitofilin and Sam50, they are not involved in import. Together our results indicate that both the myristoylation and the CHCH domain are essential for the import and mitochondrial localization of ChChd3. Once imported, ChChd3 binds to Mia40 for further folding and assembly into macromolecular complexes.

  19. 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

  20. 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.

  1. The Heterotrimeric Laminin Coiled-Coil Domain Exerts Anti-Adhesive Effects and Induces a Pro-Invasive Phenotype

    PubMed Central

    Santos-Valle, Patricia; Guijarro-Muñoz, Irene; Cuesta, Ángel M.; Alonso-Camino, Vanesa; Villate, Maider; Álvarez-Cienfuegos, Ana; Blanco, Francisco J.; Sanz, Laura; Álvarez-Vallina, Luis

    2012-01-01

    Laminins are large heterotrimeric cross-shaped extracellular matrix glycoproteins with terminal globular domains and a coiled-coil region through which the three chains are assembled and covalently linked. Laminins are key components of basement membranes, and they serve as attachment sites for cell adhesion, migration and proliferation. In this work, we produced a recombinant fragment comprising the entire laminin coiled-coil of the α1-, β1-, and γ1-chains that assemble into a stable heterotrimeric coiled-coil structure independently of the rest of the molecule. This domain was biologically active and not only failed to serve as a substrate for cell attachment, spreading and focal adhesion formation but also inhibited cell adhesion to laminin when added to cells in a soluble form at the time of seeding. Furthermore, gene array expression profiling in cells cultured in the presence of the laminin coiled-coil domain revealed up-regulation of genes involved in cell motility and invasion. These findings were confirmed by real-time quantitative PCR and zymography assays. In conclusion, this study shows for the first time that the laminin coiled-coil domain displays anti-adhesive functions and has potential implications for cell migration during matrix remodeling. PMID:22723936

  2. 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

  3. Conformational consequences of cooperative binding of a coiled-coil peptide motif to poly(N-(2-hydroxypropyl) methacrylamide) HPMA copolymers.

    PubMed

    Griffiths, Peter C; Paul, Alison; Apostolovic, Bojana; Klok, Harm-Anton; de Luca, Edoardo; King, Stephen M; Heenan, Richard K

    2011-07-30

    Small-angle neutron scattering and pulsed-gradient spin-echo NMR have been used to examine the solution conformation of a series of water soluble poly(N-(2-hydroxypropyl) methacrylamide) P(HPMA) co-polymer drug delivery vehicles incorporating a coiled-coil peptide motif as a novel pH sensitive non-covalent linker. The conformation of the HPMA homopolymer is well-described by a Gaussian coil model and changing pH from pH 7 to pH 5 has little effect on the solution conformation, as quantified via the radius of gyration. Copolymerisation with 5-10mol% of the K3 peptide bearing methacrylate monomer (K3-MA), gave a series of copolymers that exhibited an increase in radius of gyration at both pH's, despite being typically 30% lower in molecular weight, indicating that the K3-MA causes a perturbation (expansion) of the copolymer conformation. Subsequent addition of an equimolar amount of the complementary peptide E3 makes little further difference to the conformation, indicative of the intimate binding (coiled-coil motif) between the two peptides. Again, the effects of pH are small. Only the addition of a large aromatic structure such as methotrexate causes a further perturbation of the structure - the hydrophobic interaction between the MTX units causes a significant collapse of the polymer coil. These findings further elaborate the understanding of those factors that determine the solution conformation of novel polymer therapeutics.

  4. Regulation of polysome assembly on the endoplasmic reticulum by a coiled-coil protein, p180.

    PubMed

    Ueno, Tomonori; Kaneko, Keiko; Sata, Tetsutaro; Hattori, Shunji; Ogawa-Goto, Kiyoko

    2012-04-01

    A coiled-coil microtubule-bundling protein, p180, was originally identified as one of the ribosome receptor candidates on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported that p180 plays crucial roles in upregulating collagen biosynthesis, mainly by facilitating ribosome association on the ER. Here, we provide evidence that p180 is required to form translationally active polysome/translocon complexes on the ER. Assembly of highly-developed polysomes on the ER was severely perturbed upon loss of p180. p180 associates with polysome/translocon complexes through multiple contact sites: it was coimmunoprecipitated with the translocon complex independently of ribosomes, while it can also bind to ribosomal large subunit specifically. The responsible domain of p180 for membrane polysome assembly was identified in the C-terminal coiled-coil region. The degree of ribosome occupation of collagen and fibronectin mRNAs was regulated in response to increased traffic demands. This effect appears to be exerted in a manner specific for a specified set of mRNAs. Collectively, our data suggest that p180 is required to form translationally active polysome/translocon complexes on the ER membrane, and plays a pivotal role in highly efficient biosynthesis on the ER membrane through facilitating polysome formation in professional secretory cells.

  5. 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

  6. 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.

  7. Roles of the novel coiled-coil protein Rng10 in septum formation during fission yeast cytokinesis

    PubMed Central

    Liu, Yajun; Lee, I-Ju; Sun, Mingzhai; Lower, Casey A.; Runge, Kurt W.; Ma, Jianjie; Wu, Jian-Qiu

    2016-01-01

    Rho GAPs are important regulators of Rho GTPases, which are involved in various steps of cytokinesis and other processes. However, regulation of Rho-GAP cellular localization and function is not fully understood. Here we report the characterization of a novel coiled-coil protein Rng10 and its relationship with the Rho-GAP Rga7 in fission yeast. Both rng10Δ and rga7Δ result in defective septum and cell lysis during cytokinesis. Rng10 and Rga7 colocalize on the plasma membrane at the cell tips during interphase and at the division site during cell division. Rng10 physically interacts with Rga7 in affinity purification and coimmunoprecipitation. Of interest, Rga7 localization is nearly abolished without Rng10. Moreover, Rng10 and Rga7 work together to regulate the accumulation and dynamics of glucan synthases for successful septum formation in cytokinesis. Our results show that cellular localization and function of the Rho-GAP Rga7 are regulated by a novel protein, Rng10, during cytokinesis in fission yeast. PMID:27385337

  8. 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

  9. 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

  10. Mapping of the Neisseria meningitidis NadA Cell-Binding Site: Relevance of Predicted α-Helices in the NH2-Terminal and Dimeric Coiled-Coil Regions▿

    PubMed Central

    Tavano, Regina; Capecchi, Barbara; Montanari, Paolo; Franzoso, Susanna; Marin, Oriano; Sztukowska, Maryta; Cecchini, Paola; Segat, Daniela; Scarselli, Maria; Aricò, Beatrice; Papini, Emanuele

    2011-01-01

    NadA is a trimeric autotransporter protein of Neisseria meningitidis belonging to the group of oligomeric coiled-coil adhesins. It is implicated in the colonization of the human upper respiratory tract by hypervirulent serogroup B N. meningitidis strains and is part of a multiantigen anti-serogroup B vaccine. Structure prediction indicates that NadA is made by a COOH-terminal membrane anchor (also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil-rich stalk, and an NH2-terminal region involved in cell interaction. Electron microscopy analysis and structure prediction suggest that the apical region of NadA forms a compact and globular domain. Deletion studies proved that the NH2-terminal sequence (residues 24 to 87) is necessary for cell adhesion. In this study, to better define the NadA cell binding site, we exploited (i) a panel of NadA mutants lacking sequences along the coiled-coil stalk and (ii) several oligoclonal rabbit antibodies, and their relative Fab fragments, directed to linear epitopes distributed along the NadA ectodomain. We identified two critical regions for the NadA-cell receptor interaction with Chang cells: the NH2 globular head domain and the NH2 dimeric intrachain coiled-coil α-helices stemming from the stalk. This raises the importance of different modules within the predicted NadA structure. The identification of linear epitopes involved in receptor binding that are able to induce interfering antibodies reinforces the importance of NadA as a vaccine antigen. PMID:20971901

  11. Mapping of the Neisseria meningitidis NadA cell-binding site: relevance of predicted {alpha}-helices in the NH2-terminal and dimeric coiled-coil regions.

    PubMed

    Tavano, Regina; Capecchi, Barbara; Montanari, Paolo; Franzoso, Susanna; Marin, Oriano; Sztukowska, Maryta; Cecchini, Paola; Segat, Daniela; Scarselli, Maria; Aricò, Beatrice; Papini, Emanuele

    2011-01-01

    NadA is a trimeric autotransporter protein of Neisseria meningitidis belonging to the group of oligomeric coiled-coil adhesins. It is implicated in the colonization of the human upper respiratory tract by hypervirulent serogroup B N. meningitidis strains and is part of a multiantigen anti-serogroup B vaccine. Structure prediction indicates that NadA is made by a COOH-terminal membrane anchor (also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil-rich stalk, and an NH(2)-terminal region involved in cell interaction. Electron microscopy analysis and structure prediction suggest that the apical region of NadA forms a compact and globular domain. Deletion studies proved that the NH(2)-terminal sequence (residues 24 to 87) is necessary for cell adhesion. In this study, to better define the NadA cell binding site, we exploited (i) a panel of NadA mutants lacking sequences along the coiled-coil stalk and (ii) several oligoclonal rabbit antibodies, and their relative Fab fragments, directed to linear epitopes distributed along the NadA ectodomain. We identified two critical regions for the NadA-cell receptor interaction with Chang cells: the NH(2) globular head domain and the NH(2) dimeric intrachain coiled-coil α-helices stemming from the stalk. This raises the importance of different modules within the predicted NadA structure. The identification of linear epitopes involved in receptor binding that are able to induce interfering antibodies reinforces the importance of NadA as a vaccine antigen.

  12. 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

  13. 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

  14. A coiled coil switch mediates cold sensing by the thermosensory protein DesK.

    PubMed

    Saita, Emilio; Abriata, Luciano A; Tsai, Yi Ting; Trajtenberg, Felipe; Lemmin, Thomas; Buschiazzo, Alejandro; Dal Peraro, Matteo; de Mendoza, Diego; Albanesi, Daniela

    2015-10-01

    The thermosensor histidine kinase DesK from Bacillus subtilis senses changes in membrane fluidity initiating an adaptive response. Structural changes in DesK have been implicated in transmembrane signaling, but direct evidence is still lacking. On the basis of structure-guided mutagenesis, we now propose a mechanism of DesK-mediated signal sensing and transduction. The data indicate that stabilization/destabilization of a 2-helix coiled coil, which connects the transmembrane sensory domain of DesK to its cytosolic catalytic region, is crucial to control its signaling state. Computational modeling and simulations reveal couplings between protein, water and membrane mechanics. We propose that membrane thickening is the main driving force for signal sensing and that it acts by inducing helix stretching and rotation prompting an asymmetric kinase-competent state. Overall, the known structural changes of the sensor kinase, as well as further dynamic rearrangements that we now predict, consistently link structure determinants to activity modulation.

  15. Bioproduction of highly charged designer peptide surfactants via a chemically cleavable coiled-coil heteroconcatemer.

    PubMed

    Fletcher, Nicholas L; Paquet, Nicolas; Dickinson, Ellyce L; Dexter, Annette F

    2015-02-01

    Designer peptides have recently attracted attention as self-assembling fibrils, hydrogelators and green surfactants with the potential for sustainable bioproduction. Carboxylate-rich peptides in particular have shown potential as salt-resistant emulsifiers; however the expression of highly charged peptides of this kind remains a challenge. To achieve expression of a strongly anionic helical surfactant peptide, we paired the peptide with a cationic helical partner in a coiled-coil miniprotein and optimized the polypeptide sequence for net charge, hydropathy and predicted protease resistance (via the Guruprasad instability index). Our design permitted expression of a soluble concatemer that accumulates to high levels (22% of total protein) in E. coli. The concatemer showed high stability to heat and proteases, allowing isolation by simple heat and pH precipitation steps that yield concatemer at 133 mg per gram of dry cell weight and >99% purity. Aspartate-proline sites were included in the concatemer to allow cleavage with heat and acid to give monomeric peptides. We characterized the acid cleavage pathway of the concatemer by coupled liquid chromatography-mass spectrometry and modeled the kinetic pathways involved. The outcome represents the first detailed kinetic characterization of protein cleavage at aspartate-proline sites, and reveals unexpected cleavage preferences, such as favored cleavage at the C-termini of peptide helices. Chemical denaturation of the concatemer showed an extremely high thermodynamic stability of 38.9 kcal mol(-1) , with cleavage decreasing the stability of the coiled coil to 32.8 kcal mol(-1) . We determined an interfacial pressure of 29 mN m(-1) for both intact and cleaved concatemer at the air-water interface, although adsorption was slightly more rapid for the cleaved peptides. The cleaved peptides could be used to prepare heat-stable emulsions with droplet sizes in the nanometer range.

  16. 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

  17. De-novo modeling and ESR validation of a cyanobacterial F(o)F(1)-ATP synthase subunit bb' left-handed coiled coil.

    PubMed

    Volkov, Oleg A; Zaida, Tarek M; Voeller, Petra; Lill, Holger; Wise, John G; Vogel, Pia D

    2009-03-01

    The structure and functional role of the dimeric external stalk of F(o)F(1)-ATP synthases have been very actively researched over the last years. To understand the function, detailed knowledge of the structure and protein packing interactions in the dimer is required. In this paper we describe the application of structural prediction and molecular modeling approaches to elucidate the structural packing interaction of the cyanobacterial ATP synthase external stalk. In addition we present biophysical evidence derived from ESR spectroscopy and site directed spin labeling of stalk proteins that supports the proposed structural model. The use of the heterodimeric bb' dimer from a cyanobacterial ATP synthase (Synechocystis sp. PCC 6803) allowed, by specific introduction of spin labels along each individual subunit, the evaluation of the overall tertiary structure of the subunits by calculating inter-spin distances. At defined positions in both b and b' subunits, reporter groups were inserted to determine and confirm inter-subunit packing. The experiments showed that an approximately 100 residue long section of the cytoplasmic part of the bb'-dimer exists mostly as an elongated alpha-helix. The distant C-terminal end of the dimer, which is thought to interact with the delta-subunit, seemed to be disordered in experiments using soluble bb' proteins. A left-handed coiled coil packing of the dimer suggested from structure prediction studies and shown to be feasible in molecular modeling experiments was used together with the measured inter-spin distances of the inserted reporter groups determined in ESR experiments to support the hypothesis that a significant portion of the bb' structure exists as a left-handed coiled coil.

  18. Rho-associated coiled-coil kinase (ROCK) protein controls microtubule dynamics in a novel signaling pathway that regulates cell migration.

    PubMed

    Schofield, Alice V; Steel, Rohan; Bernard, Ora

    2012-12-21

    The two members of the Rho-associated coiled-coil kinase (ROCK1 and 2) family are established regulators of actin dynamics that are involved in the regulation of the cell cycle as well as cell motility and invasion. Here, we discovered a novel signaling pathway whereby ROCK regulates microtubule (MT) acetylation via phosphorylation of the tubulin polymerization promoting protein 1 (TPPP1/p25). We show that ROCK phosphorylation of TPPP1 inhibits the interaction between TPPP1 and histone deacetylase 6 (HDAC6), which in turn results in increased HDAC6 activity followed by a decrease in MT acetylation. As a consequence, we show that TPPP1 phosphorylation by ROCK increases cell migration and invasion via modulation of cellular acetyl MT levels. We establish here that the ROCK-TPPP1-HDAC6 signaling pathway is important for the regulation of cell migration and invasion.

  19. 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.

  20. 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

  1. Human Enhancer of Invasion-Cluster, a Coiled-Coil Protein Required for Passage through Mitosis

    PubMed Central

    Einarson, Margret B.; Cukierman, Edna; Compton, Duane A.; Golemis, Erica A.

    2004-01-01

    In a cross-species overexpression approach, we used the pseudohyphal transition of Saccharomyces cerevisiae as a model screening system to identify human genes that regulate cell morphology and the cell cycle. Human enhancer of invasion-cluster (HEI-C), encoding a novel evolutionarily conserved coiled-coil protein, was isolated in a screen for human genes that induce agar invasion in S. cerevisiae. In human cells, HEI-C is primarily localized to the spindle during mitosis. Depletion of HEI-C in vivo with short interfering RNAs results in severe mitotic defects. Analysis by immunofluorescence, flow cytometry analysis, and videomicroscopy indicates that HEI-C-depleted cells form metaphase plates with normal timing after G2/M transition, although in many cases cells have disorganized mitotic spindles. Subsequently, severe defects occur at the metaphase-anaphase transition, characterized by a significant delay at this stage or, more commonly, cellular disintegration accompanied by the display of classic biochemical markers of apoptosis. These mitotic defects occur in spite of the fact that HEI-C-depleted cells retain functional cell cycle checkpoints, as these cells arrest normally following nocodazole or hydroxyurea treatment. These results place HEI-C as a novel regulator of spindle function and integrity during the metaphase-anaphase transition. PMID:15082789

  2. Repacking protein cores with backbone freedom: structure prediction for coiled coils.

    PubMed

    Harbury, P B; Tidor, B; Kim, P S

    1995-08-29

    Progress in homology modeling and protein design has generated considerable interest in methods for predicting side-chain packing in the hydrophobic cores of proteins. Present techniques are not practically useful, however, because they are unable to model protein main-chain flexibility. Parameterization of backbone motions may represent a general and efficient method to incorporate backbone relaxation into such fixed main-chain models. To test this notion, we introduce a method for treating explicitly the backbone motions of alpha-helical bundles based on an algebraic parameterization proposed by Francis Crick in 1953 [Crick, F. H. C. (1953) Acta Crystallogr. 6, 685-689]. Given only the core amino acid sequence, a simple calculation can rapidly reproduce the crystallographic main-chain and core side-chain structures of three coiled coils (one dimer, one trimer, and one tetramer) to within 0.6-A root-mean-square deviations. The speed of the predictive method [approximately 3 min per rotamer choice on a Silicon Graphics (Mountain View, CA) 4D/35 computer] permits it to be used as a design tool.

  3. Regulation of Stat1 protein expression by phenylalanine 172 in the coiled-coil domain

    SciTech Connect

    Hoshino, Akemi; Fleur, Shella Saint; Fujii, Hodaka . E-mail: hodaka@med.nyu.edu

    2006-08-04

    Stat1 plays an essential role in signal transduction and gene expression of various cytokines including interferons (IFNs). Although the mechanism of cytokine-induced activation of Stat1 and transcriptional regulation of Stat1 gene expression have been established, post-transcriptional regulation of Stat1 protein expression is not fully understood. Here, we report identification of a mutant of Stat1 that has decreased expression levels by using inducible translocation trap (ITT), a reporter gene-based detection system of nuclear translocation. The substitution of serine for phenylalanine 172 (F172S) in the coiled-coil domain causes marked decrease in Stat1 protein expression in various cell lines without decreasing its mRNA levels. Our results suggest that the decrease is caused by translational/post-translational mechanisms independent of proteasome machinery. These results suggest a novel potential mechanism of determination of specificity of Stat proteins and showed that the ITT system is a powerful technique to identify mutants of nuclear translocating signal transducers.

  4. Rationally designed coiled-coil DNA looping peptides control DNA topology.

    PubMed

    Gowetski, Daniel B; Kodis, Erin J; Kahn, Jason D

    2013-09-01

    Artificial DNA looping peptides were engineered to study the roles of protein and DNA flexibility in controlling the geometry and stability of protein-mediated DNA loops. These LZD (leucine zipper dual-binding) peptides were derived by fusing a second, C-terminal, DNA-binding region onto the GCN4 bZip peptide. Two variants with different coiled-coil lengths were designed to control the relative orientations of DNA bound at each end. Electrophoretic mobility shift assays verified formation of a sandwich complex containing two DNAs and one peptide. Ring closure experiments demonstrated that looping requires a DNA-binding site separation of 310 bp, much longer than the length needed for natural loops. Systematic variation of binding site separation over a series of 10 constructs that cyclize to form 862-bp minicircles yielded positive and negative topoisomers because of two possible writhed geometries. Periodic variation in topoisomer abundance could be modeled using canonical DNA persistence length and torsional modulus values. The results confirm that the LZD peptides are stiffer than natural DNA looping proteins, and they suggest that formation of short DNA loops requires protein flexibility, not unusual DNA bendability. Small, stable, tunable looping peptides may be useful as synthetic transcriptional regulators or components of protein-DNA nanostructures.

  5. 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

  6. The coiled-coil domain of zebrafish TRPM7 regulates Mg·nucleotide sensitivity.

    PubMed

    Jansen, Chad; Sahni, Jaya; Suzuki, Sayuri; Horgen, F David; Penner, Reinhold; Fleig, Andrea

    2016-01-01

    TRPM7 is a member of the Transient-Receptor-Potential Melastatin ion channel family. TRPM7 is a unique fusion protein of an ion channel and an α-kinase. Although mammalian TRPM7 is well characterized biophysically and its pivotal role in cancer, ischemia and cardiovascular disease is becoming increasingly evident, the study of TRPM7 in mouse models has been hampered by embryonic lethality of transgenic ablations. In zebrafish, functional loss of TRPM7 (drTRPM7) manifests itself in an array of non-lethal physiological malfunctions. Here, we investigate the regulation of wild type drTRPM7 and multiple C-terminal truncation mutants. We find that the biophysical properties of drTRPM7 are very similar to mammalian TRPM7. However, pharmacological profiling reveals that drTRPM7 is facilitated rather than inhibited by 2-APB, and that the TRPM7 inhibitor waixenicin A has no effect. This is reminiscent of the pharmacological profile of human TRPM6, the sister channel kinase of TRPM7. Furthermore, using truncation mutations, we show that the coiled-coil domain of drTRPM7 is involved in the channel's regulation by magnesium (Mg) and Mg·adenosine triphosphate (Mg·ATP). We propose that drTRPM7 has two protein domains that regulate inhibition by intracellular magnesium and nucleotides, and one domain that is concerned with sensing magnesium only. PMID:27628598

  7. The coiled-coil domain of zebrafish TRPM7 regulates Mg·nucleotide sensitivity

    PubMed Central

    Jansen, Chad; Sahni, Jaya; Suzuki, Sayuri; Horgen, F. David; Penner, Reinhold; Fleig, Andrea

    2016-01-01

    TRPM7 is a member of the Transient-Receptor-Potential Melastatin ion channel family. TRPM7 is a unique fusion protein of an ion channel and an α-kinase. Although mammalian TRPM7 is well characterized biophysically and its pivotal role in cancer, ischemia and cardiovascular disease is becoming increasingly evident, the study of TRPM7 in mouse models has been hampered by embryonic lethality of transgenic ablations. In zebrafish, functional loss of TRPM7 (drTRPM7) manifests itself in an array of non-lethal physiological malfunctions. Here, we investigate the regulation of wild type drTRPM7 and multiple C-terminal truncation mutants. We find that the biophysical properties of drTRPM7 are very similar to mammalian TRPM7. However, pharmacological profiling reveals that drTRPM7 is facilitated rather than inhibited by 2-APB, and that the TRPM7 inhibitor waixenicin A has no effect. This is reminiscent of the pharmacological profile of human TRPM6, the sister channel kinase of TRPM7. Furthermore, using truncation mutations, we show that the coiled-coil domain of drTRPM7 is involved in the channel’s regulation by magnesium (Mg) and Mg·adenosine triphosphate (Mg·ATP). We propose that drTRPM7 has two protein domains that regulate inhibition by intracellular magnesium and nucleotides, and one domain that is concerned with sensing magnesium only. PMID:27628598

  8. Hg(II) binding to a weakly associated coiled coil nucleates an encoded metalloprotein fold: a kinetic analysis.

    PubMed

    Farrer, Brian T; Pecoraro, Vincent L

    2003-04-01

    A detailed kinetic analysis of metal encapsulation by a de novo-designed protein is described. The kinetic mechanism of Hg(II) encapsulation in the three-stranded coiled coil formed by the peptide CH(3)CO-G LKALEEK CKALEEK LKALEEK G-NH(2) (Baby L9C) is derived by global analysis. The mechanism involves rapid initial collapse of two peptides by Hg(II) forming Hg(Baby L9C(-H))(2) with a linear thiolato Hg(II) bound to the cysteine sulfur atoms. Here, Baby L9C(-H) denotes Baby L9C with the cysteine thiol deprotonated. Addition of the third peptide, forming the three-stranded coiled coil, is the rate-determining step and results in an intermediate state involving two separate species. One of the species, termed the properly folded intermediate, undergoes rapid deprotonation of the third cysteine thiol, yielding the desired three-stranded coiled coil with an encapsulated trigonal thiolato Hg(II). The other species, termed the misfolded intermediate, rearranges in an experimentally distinguishable step to the properly folded intermediate. The order of the reaction involving the addition of the third peptide with respect to the concentration of Baby L9C indicates that addition of the third helix only occurs through reaction of Hg(Baby L9C(-H))(2) and Baby L9C that is unassociated with a coiled coil. Temperature dependence of the reaction afforded activation parameters for both the addition of the third helix (deltaH = 20(2) kcalmol; deltaS= 40(5) calmol K) and the rearrangement of the misfolded intermediate steps (deltaH = 23(2) kcalmol; deltaS= 27(5) calmol K). The mechanism is discussed with regard to metalloprotein folding and metalloprotein design.

  9. 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

  10. 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

  11. L{sup 565}M mutation in HIV-1 glycoprotein 41 stabilizes the coiled-coil structure

    SciTech Connect

    Yamamoto, Daisuke . E-Mail: center@art.osaka-med.ac.jp; Li, Gui-Mei; Ikuta, Kazuyoshi; Goto, Toshiyuki

    2005-09-16

    N-terminal and C-terminal heptad repeats (NHR and CHR) of HIV type 1 (HIV-1) glycoprotein 41 are known to be regions directly related to cell fusion during virus attack, and their complex core constructs a coiled-coil structure in the fusion process. In our recent studies, MT-4/17-3-6, a strain of HIV-1, showed the strong resistance to peptide fusion inhibitors compared with other strains such as MT-4/LAI, L-2 and CU98-26, and had a distinctive L{sup 565}M mutation in the central region of NHR. To investigate the relationship between the mutation and resistance, we performed a molecular modeling of the coiled-coil of MT-4/17-3-6 by using energy minimization and molecular dynamics simulation based on the MT-4/LAI X-ray structure. As a result, we found that H{sup 564} in the NHR was pushed to the outer side by this mutation, and three hydrogen bond bridges of Y{sup 638}-H{sup 564}-E{sup 560}-Q{sup 650} could be formed, enclosing the coiled-coil. The binding of peptide inhibitors would be disturbed by the structural stabilization of these bridges in MT-4/17-3-6.

  12. 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

  13. Coiled-coil unwinding at the smooth muscle myosin head-rod junction is required for optimal mechanical performance.

    PubMed Central

    Lauzon, A M; Fagnant, P M; Warshaw, D M; Trybus, K M

    2001-01-01

    Myosin II has two heads that are joined together by an alpha-helical coiled-coil rod, which can separate in the region adjacent to the head-rod junction (Trybus, K. M. 1994. J. Biol. Chem. 269:20819-20822). To test whether this flexibility at the head-rod junction is important for the mechanical performance of myosin, we used the optical trap to measure the unitary displacements of heavy meromyosin constructs in which a stable coiled-coil sequence derived from the leucine zipper was introduced into the myosin rod. The zipper was positioned either immediately after the heads (0-hep zip) or following 15 heptads of native sequence (15-hep zip). The unitary displacement (d) decreased from d = 9.7 +/- 0.6 nm for wild-type heavy meromyosin (WT HMM) to d = 0.1 +/- 0.3 nm for the 0-hep zip construct (mean +/- SE). Native values were restored in the 15-hep zip construct (d = 7.5 +/- 0.7 nm). We conclude that flexibility at the myosin head-rod junction, which is provided by an unstable coiled-coil region, is essential for optimal mechanical performance. PMID:11259302

  14. 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

  15. De novo design of a non-natural fold for an iron-sulfur protein: alpha-helical coiled-coil with a four-iron four-sulfur cluster binding site in its central core.

    PubMed

    Grzyb, Joanna; Xu, Fei; Weiner, Lev; Reijerse, Eduard J; Lubitz, Wolfgang; Nanda, Vikas; Noy, Dror

    2010-03-01

    Using a 'metal-first' approach, we computationally designed, prepared, and characterized a four-iron four-sulfur (Fe(4)S(4)) cluster protein with a non-natural alpha-helical coiled-coil fold. The novelty of this fold lies in the placement of a Fe(4)S(4) cluster within the hydrophobic core of a four-helix bundle, making it unique among previous iron-sulfur (FeS) protein designs, and different from known natural FeS proteins. The apoprotein, recombinantly expressed and purified from E. coli, readily self-assembles with Fe(4)S(4) clusters in vitro. UV-Vis absorption and CD spectroscopy, elemental analysis, gel filtration, and analytical ultracentrifugation confirm that the protein is folded and assembled as designed, namely, alpha-helical coiled-coil binding a single Fe(4)S(4) cluster. Dithionite-reduced holoprotein samples have characteristic rhombic EPR spectra, typical of low-potential, [Fe(4)S(4)](+) (S=1/2), with g values of g(zy)=(1.970, 1.975), and g(x)=2.053. The temperature, and power dependence of the signal intensity were also characteristic of [Fe(4)S(4)](+) clusters with very efficient spin relaxation, but almost without any interaction between adjacent clusters. The new design is very promising although optimization is required, particularly for preventing aggregation, and adding second shell interactions to stabilize the reduced state. Its main advantage is its extendibility into a multi-FeS cluster protein by simply duplicating and translating the binding site along the coiled-coil axis. This opens new possibilities for designing protein-embedded redox chains that may be used as "wires" for coupling any given set of redox enzymes. PMID:20035711

  16. 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

    Aseev, Leonid V; Chugunov, Anton O; Efremov, Roman G; Boni, Irina V

    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.

  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. 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.

  19. Simultaneous formation of right- and left-handed anti-parallel coiled-coil interfaces by a coil2 fragment of human lamin A.

    PubMed

    Kapinos, Larisa E; Burkhard, Peter; Herrmann, Harald; Aebi, Ueli; Strelkov, Sergei V

    2011-04-22

    The elementary building block of all intermediate filaments (IFs) is a dimer featuring a central α-helical rod domain flanked by the N- and C-terminal end domains. In nuclear IF proteins (lamins), the rod domain consists of two coiled-coil segments, coil1 and coil2, that are connected by a short non-helical linker. Coil1 and the C-terminal part of coil2 contain the two highly conserved IF consensus motifs involved in the longitudinal assembly of dimers. The previously solved crystal structure of a lamin A fragment (residues 305-387) corresponding to the second half of coil2 has yielded a parallel left-handed coiled coil. Here, we present the crystal structure and solution properties of another human lamin A fragment (residues 328-398), which is largely overlapping with fragment 305-387 but harbors a short segment of the tail domain. Unexpectedly, no parallel coiled coil forms within the crystal. Instead, the α-helices are arranged such that two anti-parallel coiled-coil interfaces are formed. The most significant interface has a right-handed geometry, which is accounted for by a characteristic 15-residue repeat pattern that overlays with the canonical heptad repeat pattern. The second interface is a left-handed anti-parallel coiled coil based on the predicted heptad repeat pattern. In solution, the fragment reveals only a weak dimerization propensity. We speculate that the C-terminus of coil2 might unzip, thereby allowing for a right-handed coiled-coil interface to form between two laterally aligned dimers. Such an interface might co-exist with a heterotetrameric left-handed coiled-coil assembly, which is expected to be responsible for the longitudinal A(CN) contact.

  20. 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.

  1. 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

  2. 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

  3. Novel coiled-coil proteins regulate exocyst association with cortical microtubules in xylem cells via the conserved oligomeric golgi-complex 2 protein.

    PubMed

    Oda, Yoshihisa; Iida, Yuki; Nagashima, Yoshinobu; Sugiyama, Yuki; Fukuda, Hiroo

    2015-02-01

    Xylem vessel cells develop secondary cell walls in distinct patterns. Cortical microtubules are rearranged into distinct patterns and regulate secondary cell wall deposition; however, it is unclear how exocytotic membrane trafficking is linked to cortical microtubules. Here, we show that the novel coiled-coil proteins vesicle tethering 1 (VETH1) and VETH2 recruit EXO70A1, an exocyst subunit essential for correct patterning of secondary cell wall deposition, to cortical microtubules via the conserved oligomeric Golgi complex (COG) 2 protein. VETH1 and VETH2 encode an uncharacterized domain of an unknown function designated DUF869, and were preferentially up-regulated in xylem cells. VETH1-green fluorescent protein (GFP) and VETH2-GFP co-localized at novel vesicle-like small compartments, which exhibited microtubule plus-end-directed and end-tracking dynamics. VETH1 and VETH2 interacted with COG2, and this interaction promoted the association between cortical microtubules and EXO70A1 These results suggest that the VETH-COG2 complex ensures the correct secondary cell wall deposition pattern by recruiting exocyst components to cortical microtubules.

  4. Stability of 100 homo and heterotypic coiled-coil a-a' pairs for ten amino acids (A, L, I, V, N, K, S, T, E, and R).

    PubMed

    Acharya, Asha; Rishi, Vikas; Vinson, Charles

    2006-09-26

    We present the thermal stability monitored by circular dichroism (CD) spectroscopy at 222 nm of 100 heterodimers that contain all possible coiled-coil a-a' pairs for 10 amino acids (I, V, L, N, A, K S, T, E, and R). This includes the stability of 36 heterodimers for 6 amino acids (I, V, L, N, A, and K) previously described and 64 new heterodimers including the 4 amino acids (S, T, E, and R). We have calculated a double mutant alanine thermodynamic cycle to determine a-a' pair coupling energies to evaluate which a-a' pairs encourage specific dimerization partners. The four new homotypic a-a' pairs (T-T, S-S, R-R, E-E) are repulsive relative to A-A and have destabilizing coupling energies. Among the 90 heterotypic a-a' pairs, the stabilizing coupling energies contain lysine or arginine paired with either an aliphatic or a polar amino acid. The range in coupling energies for each amino acid reveals its potential to regulate dimerization specificity. The a-a' pairs containing isoleucine and asparagine have the greatest range in coupling energies and thus contribute dramatically to dimerization specificity, which is to encourage homodimerization. In contrast, the a-a' pairs containing charged amino acids (K, R, and E) show the least range in coupling energies and promiscuously encourage heterodimerization.

  5. The coiled-coil domain containing protein Ccdc136b antagonizes maternal Wnt/β-catenin activity during zebrafish dorsoventral axial patterning.

    PubMed

    Wei, Shi; Shang, Hanqiao; Cao, Yu; Wang, Qiang

    2016-07-20

    The coiled-coil domain containing protein CCDC136 is a putative tumor suppressor and significantly down-regulated in gastric and colorectal cancer tissues. However, little is known about its biological functions during vertebrate embryo development. Zebrafish has two CCDC136 orthologs, ccdc136a and ccdc136b, but only ccdc136b is highly expressed during early embryonic development. In this study, we demonstrate that ccdc136b is required for dorsal-ventral axial patterning in zebrafish embryos. ccdc136b morphants display strongly dorsalized phenotypes. Loss- and gain-of-function experiments in zebrafish embryos and mammalian cells show that Ccdc136b is a crucial negative regulator of the Wnt/β-catenin signaling pathway, and plays a critical role in the establishment of the dorsal-ventral axis. We further find that Ccdc136b interacts with APC, promotes the binding affinity of APC with β-catenin and then facilitates the turnover of β-catenin. These results provide the first evidence that CCDC136 regulates zebrafish dorsal-ventral patterning by antagonizing Wnt/β-catenin signal transduction and suggest a potential mechanism underlying its suppressive activity in carcinogenesis.

  6. Coiled-coil domain of enteropathogenic Escherichia coli type III secreted protein EspD is involved in EspA filament-mediated cell attachment and hemolysis.

    PubMed

    Daniell, S J; Delahay, R M; Shaw, R K; Hartland, E L; Pallen, M J; Booy, F; Ebel, F; Knutton, S; Frankel, G

    2001-06-01

    Many animal and plant pathogens use type III secretion systems to secrete key virulence factors, some directly into the host cell cytosol. However, the basis for such protein translocation has yet to be fully elucidated for any type III secretion system. We have previously shown that in enteropathogenic and enterohemorrhagic Escherichia coli the type III secreted protein EspA is assembled into a filamentous organelle that attaches the bacterium to the plasma membrane of the host cell. Formation of EspA filaments is dependent on expression of another type III secreted protein, EspD. The carboxy terminus of EspD, a protein involved in formation of the translocation pore in the host cell membrane, is predicted to adopt a coiled-coil conformation with 99% probability. Here, we demonstrate EspD-EspD protein interaction using the yeast two-hybrid system and column overlays. Nonconservative triple amino acid substitutions of specific EspD carboxy-terminal residues generated an enteropathogenic E. coli mutant that was attenuated in its ability to induce attaching and effacing lesions on HEp-2 cells. Although the mutation had no effect on EspA filament biosynthesis, it also resulted in reduced binding to and reduced hemolysis of red blood cells. These results segregate, for the first time, functional domains of EspD that control EspA filament length from EspD-mediated cell attachment and pore formation.

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

    PubMed

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

    2015-02-01

    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, that is, 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.

  8. 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

  9. The coiled-coil protein VIG1 is essential for tethering vacuoles to mitochondria during vacuole inheritance of Cyanidioschyzon merolae.

    PubMed

    Fujiwara, Takayuki; Kuroiwa, Haruko; Yagisawa, Fumi; Ohnuma, Mio; Yoshida, Yamato; Yoshida, Masaki; Nishida, Keiji; Misumi, Osami; Watanabe, Satoru; Tanaka, Kan; Kuroiwa, Tsuneyoshi

    2010-03-01

    Vacuoles/lysosomes function in endocytosis and in storage and digestion of metabolites. These organelles are inherited by the daughter cells in eukaryotes. However, the mechanisms of this inheritance are poorly understood because the cells contain multiple vacuoles that behave randomly. The primitive red alga Cyanidioschyzon merolae has a minimum set of organelles. Here, we show that C. merolae contains about four vacuoles that are distributed equally between the daughter cells by binding to dividing mitochondria. Binding is mediated by VIG1, a 30-kD coiled-coil protein identified by microarray analyses and immunological assays. VIG1 appears on the surface of free vacuoles in the cytosol and then tethers the vacuoles to the mitochondria. The vacuoles are released from the mitochondrion in the daughter cells following VIG1 digestion. Suppression of VIG1 by antisense RNA disrupted the migration of vacuoles. Thus, VIG1 is essential for tethering vacuoles to mitochondria during vacuole inheritance in C. merolae.

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

    PubMed

    Abarca, Fernando; Gutierrez-Maldonado, Sebastian E; Parada, Pilar; Martinez, Patricio; Maass, Alejandro; Perez-Acle, Tomas

    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.

  11. 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

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

    PubMed

    Abarca, Fernando; Gutierrez-Maldonado, Sebastian E; Parada, Pilar; Martinez, Patricio; Maass, Alejandro; Perez-Acle, Tomas

    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

  13. 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.

  14. THE STRUCTURES OF COILED-COIL DOMAINS FROM TYPE THREE SECRETION SYSTEM TRANSLOCATORS REVEAL HOMOLOGY TO PORE-FORMING TOXINS

    PubMed Central

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

    2012-01-01

    Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SS) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) which is responsible for over one million deaths per year. The Shigella type III secretion apparatus (T3SA) is comprised 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 Å and 2.8 Å limiting resolution, respectively. These newly identified domains are comprised of extended length (114 Å in IpaB and 71 Å 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. PMID:22321794

  15. 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

  16. 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

  17. A suite of modular fluorescence assays interrogate the human immunodeficiency virus glycoprotein-41 coiled coil and assist in determining binding mechanism of low molecular weight fusion inhibitors.

    PubMed

    Gochin, Miriam

    2012-10-01

    Several different segments of the gp41 N-heptad repeat coiled coil have been constructed using N-terminal bipyridyl modification of composite peptides and inducing trimerization by adding ferrous ions. These metallopeptides act as receptors in fluorescence-binding assays with corresponding fluorescently labeled C-peptide probes. The Fe(II) coordination complex quenches C-peptide fluorescence upon binding, and reversal of quenching by a small molecule inhibitor can be used to obtain the inhibitor-binding constant. A total of 10 peptide pairs targeting 25-46 residue segments of the coiled coil were constructed, with C-peptide probes of different lengths and binding affinities. The result is a suite of assays for exploring binding in the mM to nM range to any desired region of the coiled coil, including the hydrophobic pocket (HP), extended regions on either side of the pocket, or a region associated with T20 resistance mutations. These assays are high-throughput ready, and could be used to discover novel compounds binding along various regions of the gp41 coiled coil groove. They were used to evaluate a sub-μM low molecular weight fusion inhibitor, resulting in the finding that the molecule bound specifically to the HP and attained its potency from a low off-rate.

  18. 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.

  19. Design of beta-domain swapping, alpha/beta-protein, environmentally sensitive coiled coil and peptide functionalized titania materials

    NASA Astrophysics Data System (ADS)

    Nagarkar, Radhika P.

    2009-12-01

    The objective of this dissertation is to apply rational peptide design to fabricate nanomaterials via self-assembly. This has been demonstrated in structurally diverse systems with an aim of deciphering the underlying principles governing how sequence affects the peptide's ability to adopt a specific secondary structure and ultimate material properties that are realized from the association of these secondary structural elements. Several amyloidogenic proteins have been shown to self-assemble into fibrils using a mechanism known as domain swapping. Here, discreet units of secondary structure are exchanged among discreet proteins during self-assembly to form extended networks with precise three dimensional organization. The possibility of using these mechanisms to design peptides capable of controlled assembly and fibril formation leading to materials with targeted properties is explored. By altering the placement of a beta-turn sequence that varies the size and location of the exchanged strand, twisting, non-twisting and laminated fibrillar nanostructures are obtained. Hydrogels prepared from these strand swapping beta-hairpins have varied rheological properties due to differences in their fibrillar nanostructures. In a second distinct design, alpha/beta-proteins are used to prepare environmentally sensitive hydrogels. Here, multiple distinct motifs for structural integrity and dynamic response within a single self-assembling peptide allow the amyloid-like fibrils formed to controllably alter their nano-topography in response to an external stimulus such as temperature. The development of these self-assembling alpha/beta-protein motifs also necessitated the design of pH sensitive antiparallel coiled coils. Exploring the basic principles responsible for pH dependent conformational changes in coiled coils can lead to new insights in the control of protein structure and function. Lastly, this dissertation discusses the interface between biomolecules and inorganic

  20. A Coiled-Coil Mimetic Intercepts BCR-ABL1 Dimerization in Native and Kinase-Mutant Chronic Myeloid Leukemia

    PubMed Central

    Woessner, David W.; Eiring, Anna M.; Bruno, Benjamin J.; Zabriskie, Matthew S.; Reynolds, Kimberly R.; Miller, Geoffrey D.; O’Hare, Thomas; Deininger, Michael W.; Lim, Carol S.

    2015-01-01

    Targeted therapy of chronic myeloid leukemia is currently based on small-molecule inhibitors that directly bind the tyrosine kinase domain of BCR-ABL1. This strategy has generally been successful, but is subject to drug resistance due to point mutations in the kinase domain. Kinase activity requires transactivation of BCR-ABL1 following an oligomerization event, which is mediated by the coiled-coil (CC) domain at the N-terminus of the protein. Here, we describe a rationally engineered mutant version of the CC domain, called CCmut3, which interferes with BCR-ABL1 oligomerization and promotes apoptosis in BCR-ABL1-expressing cells, regardless of kinase domain mutation status. CCmut3 exhibits strong pro-apoptotic and anti-proliferative activity in cell lines expressing native BCR-ABL1, single kinase domain mutant BCR-ABL1 (E255V and T315I) or compound mutant BCR-ABL1 (E255V/T315I). Moreover, CCmut3 inhibits colony formation by primary CML CD34+ cells ex vivo, including a sample expressing the T315I mutant. These data suggest that targeting BCR-ABL1 with CC mutants may provide a novel alternative strategy for treating patients with resistance to current targeted therapies. PMID:25721898

  1. Mcp4, a Meiotic Coiled-Coil Protein, Plays a Role in F-Actin Positioning during Schizosaccharomyces pombe Meiosis▿

    PubMed Central

    Ohtaka, Ayami; Okuzaki, Daisuke; Saito, Takamune T.; Nojima, Hiroshi

    2007-01-01

    Some meiosis-specific proteins of Schizosaccharomyces pombe harbor coiled-coil motifs and play essential roles in meiotic progression. Here we describe Mcp4, a novel meiosis-specific protein whose expression is abruptly induced at the horsetail phase and which remains expressed until sporulation is finished. Fluorescence microscopic analysis revealed that Mcp4 alters its subcellular localization during meiosis in a manner that partially resembles the movement of F-actin during meiosis. Mcp4 and F-actin never colocalize; rather, they are located in a side-by-side manner. When forespore membrane formation begins at metaphase II, the Mcp4 signals assemble at the lagging face of the dividing nuclei. At this stage, they are sandwiched between F-actin and the nucleus. Mcp4, in turn, appears to sandwich F-actin with Meu14. In mcp4Δ cells at anaphase II, the F-actin, which is normally dumbbell-shaped, adopts an abnormal balloon shape. Spores of mcp4Δ cells were sensitive to NaCl, although their shape and viability were normal. Taken together, we conclude that Mcp4 plays a role in the accurate positioning of F-actin during S. pombe meiosis. PMID:17435009

  2. Coiled-coil domain containing 42 (Ccdc42) is necessary for proper sperm development and male fertility in the mouse.

    PubMed

    Pasek, Raymond C; Malarkey, Erik; Berbari, Nicolas F; Sharma, Neeraj; Kesterson, Robert A; Tres, Laura L; Kierszenbaum, Abraham L; Yoder, Bradley K

    2016-04-15

    Spermiogenesis is the differentiation of spermatids into motile sperm consisting of a head and a tail. The head harbors a condensed elongated nucleus partially covered by the acrosome-acroplaxome complex. Defects in the acrosome-acroplaxome complex are associated with abnormalities in sperm head shaping. The head-tail coupling apparatus (HTCA), a complex structure consisting of two cylindrical microtubule-based centrioles and associated components, connects the tail or flagellum to the sperm head. Defects in the development of the HTCA cause sperm decapitation and disrupt sperm motility, two major contributors to male infertility. Here, we provide data indicating that mutations in the gene Coiled-coil domain containing 42 (Ccdc42) is associated with malformation of the mouse sperm flagella. In contrast to many other flagella and motile cilia genes, Ccdc42 expression is only observed in the brain and developing sperm. Male mice homozygous for a loss-of-function Ccdc42 allele (Ccdc42(KO)) display defects in the number and location of the HTCA, lack flagellated sperm, and are sterile. The testes enriched expression of Ccdc42 and lack of other phenotypes in mutant mice make it an ideal candidate for screening cases of azoospermia in humans. PMID:26945718

  3. 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.

  4. 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.

  5. 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

  6. A novel natural mutation AαPhe98Ile in the fibrinogen coiled-coil affects fibrinogen function.

    PubMed

    Riedelová-Reicheltová, Zuzana; Kotlín, Roman; Suttnar, Jiří; Geierová, Véra; Riedel, Tomáš; Májek, Pavel; Dyr, Jan Evangelista

    2014-01-01

    The aim of this study was to investigate the structure and function of fibrinogen obtained from a patient with normal coagulation times and idiopathic thrombophilia. This was done by SDS-PAGE and DNA sequence analyses, scanning electron microscopy, fibrinopeptide release, fibrin polymerisation initiated by thrombin and reptilase, fibrinolysis, and platelet aggregometry. A novel heterozygous point mutation in the fibrinogen Aα chain, Phe98 to Ile, was found and designated as fibrinogen Vizovice. The mutation, which is located in the RGDF sequence (Aα 95-98) of the fibrinogen coiled-coil region, significantly affected fibrin clot morphology. Namely, the clot formed by fibrinogen Vizovice contained thinner and curled fibrin fibers with reduced length. Lysis of the clots prepared from Vizovice plasma and isolated fibrinogen were found to be impaired. The lysis rate of Vizovice clots was almost four times slower than the lysis rate of control clots. In the presence of platelets agonists the mutant fibrinogen caused increased platelet aggregation. The data obtained show that natural mutation of Phe98 to Ile in the fibrinogen Aα chain influences lateral aggregation of fibrin protofibrils, fibrinolysis, and platelet aggregation. They also suggest that delayed fibrinolysis, together with the abnormal fibrin network morphology and increased platelet aggregation, may be the direct cause of thrombotic complications in the patient associated with pregnancy loss. PMID:24108601

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

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

    PubMed Central

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

    2010-01-01

    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 metallated structures which ultimately exist. This is because there are few reports of structural characterization of such systems either in their metallated or non-metallated forms and no examples where an apo structure and the corresponding metallated 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→g)) CSL9C (CS = Coil Ser) and CSL19C in their non-metallated forms, determined to 1.36 Å and 2.15 Å 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)3 show alternate conformations and are partially preorganized for metal binding, whereas cysteines in (CSL19C)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 metallated As(CSL9C)3 and to speculate on the differential basis of Hg(II) binding in a versus d

  13. Three novel mutations in KIF21A highlight the importance of the third coiled-coil stalk domain in the etiology of CFEOM1

    PubMed Central

    Chan, Wai-Man; Andrews, Caroline; Dragan, Laryssa; Fredrick, Douglas; Armstrong, Linlea; Lyons, Christopher; Geraghty, Michael T; Hunter, David G; Yazdani, Ahmad; Traboulsi, Elias I; Pott, Jan WR; Gutowski, Nicholas J; Ellard, Sian; Young, Elizabeth; Hanisch, Frank; Koc, Feray; Schnall, Bruce; Engle, Elizabeth C

    2007-01-01

    Background Congenital fibrosis of the extraocular muscles types 1 and 3 (CFEOM1/CFEOM3) are autosomal dominant strabismus disorders that appear to result from maldevelopment of ocular nuclei and nerves. We previously reported that most individuals with CFEOM1 and rare individuals with CFEOM3 harbor heterozygous mutations in KIF21A. KIF21A encodes a kinesin motor involved in anterograde axonal transport, and the familial and de novo mutations reported to date predictably alter one of only a few KIF21A amino acids – three within the third coiled-coil region of the stalk and one in the distal motor domain, suggesting they result in altered KIF21A function. To further define the spectrum of KIF21A mutations in CFEOM we have now identified all CFEOM probands newly enrolled in our study and determined if they harbor mutations in KIF21A. Results Sixteen CFEOM1 and 29 CFEOM3 probands were studied. Three previously unreported de novo KIF21A mutations were identified in three CFEOM1 probands, all located in the same coiled-coil region of the stalk that contains all but one of the previously reported mutations. Eight additional CFEOM1 probands harbored three of the mutations previously reported in KIF21A; seven had one of the two most common mutations, while one harbored the mutation in the distal motor domain. No mutation was detected in 5 CFEOM1 or any CFEOM3 probands. Conclusion Analysis of sixteen CFEOM1 probands revealed three novel KIF21A mutations and confirmed three reported mutations, bringing the total number of reported KIF21A mutations in CFEOM1 to 11 mutations among 70 mutation positive probands. All three new mutations alter amino acids in heptad repeats within the third coiled-coil region of the KIF21A stalk, further highlighting the importance of alterations in this domain in the etiology of CFEOM1. PMID:17511870

  14. Mitochondrial Hsp90 is a ligand-activated molecular chaperone coupling ATP binding to dimer closure through a coiled-coil intermediate

    PubMed Central

    Sung, Nuri; Lee, Jungsoon; Kim, Ji-Hyun; Chang, Changsoo; Joachimiak, Andrzej; Lee, Sukyeong; Tsai, Francis T. F.

    2016-01-01

    Heat-shock protein of 90 kDa (Hsp90) is an essential molecular chaperone that adopts different 3D structures associated with distinct nucleotide states: a wide-open, V-shaped dimer in the apo state and a twisted, N-terminally closed dimer with ATP. Although the N domain is known to mediate ATP binding, how Hsp90 senses the bound nucleotide and facilitates dimer closure remains unclear. Here we present atomic structures of human mitochondrial Hsp90N (TRAP1N) and a composite model of intact TRAP1 revealing a previously unobserved coiled-coil dimer conformation that may precede dimer closure and is conserved in intact TRAP1 in solution. Our structure suggests that TRAP1 normally exists in an autoinhibited state with the ATP lid bound to the nucleotide-binding pocket. ATP binding displaces the ATP lid that signals the cis-bound ATP status to the neighboring subunit in a highly cooperative manner compatible with the coiled-coil intermediate state. We propose that TRAP1 is a ligand-activated molecular chaperone, which couples ATP binding to dramatic changes in local structure required for protein folding. PMID:26929380

  15. 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.

  16. Mcp7, a meiosis-specific coiled-coil protein of fission yeast, associates with Meu13 and is required for meiotic recombination.

    PubMed

    Saito, Takamune T; Tougan, Takahiro; Kasama, Takashi; Okuzaki, Daisuke; Nojima, Hiroshi

    2004-01-01

    We previously showed that Meu13 of Schizosaccharomyces pombe functions in homologous pairing and recombination at meiosis I. Here we show that a meiosis-specific gene encodes a coiled-coil protein that complexes with Meu13 during meiosis in vivo. This gene denoted as mcp7+ (after meiotic coiled-coil protein) is an ortholog of Mnd1 of Saccharomyces cerevisiae. Mcp7 proteins are detected on meiotic chromatin. The phenotypes of mcp7Delta cells are similar to those of meu13Delta cells as they show reduced recombination rates and spore viability and produce spores with abnormal morphology. However, a delay in initiation of meiosis I chromosome segregation of mcp7Delta cells is not so conspicuous as meu13Delta cells, and no meiotic delay is observed in mcp7Deltameu13Delta cells. Mcp7 and Meu13 proteins depend on each other differently; Mcp7 becomes more stable in meu13Delta cells, whereas Meu13 becomes less stable in mcp7Delta cells. Genetic analysis shows that Mcp7 acts in the downstream of Dmc1, homologs of Escherichia coli RecA protein, for both recombination and subsequent sporulation. Taken together, we conclude that Mcp7 associates with Meu13 and together they play a key role in meiotic recombination. PMID:15210864

  17. Mcp7, a meiosis-specific coiled-coil protein of fission yeast, associates with Meu13 and is required for meiotic recombination

    PubMed Central

    Saito, Takamune T.; Tougan, Takahiro; Kasama, Takashi; Okuzaki, Daisuke; Nojima, Hiroshi

    2004-01-01

    We previously showed that Meu13 of Schizosaccharomyces pombe functions in homologous pairing and recombination at meiosis I. Here we show that a meiosis-specific gene encodes a coiled-coil protein that complexes with Meu13 during meiosis in vivo. This gene denoted as mcp7+ (after meiotic coiled-coil protein) is an ortholog of Mnd1 of Saccharomyces cerevisiae. Mcp7 proteins are detected on meiotic chromatin. The phenotypes of mcp7Δ cells are similar to those of meu13Δ cells as they show reduced recombination rates and spore viability and produce spores with abnormal morphology. However, a delay in initiation of meiosis I chromosome segregation of mcp7Δ cells is not so conspicuous as meu13Δ cells, and no meiotic delay is observed in mcp7Δmeu13Δ cells. Mcp7 and Meu13 proteins depend on each other differently; Mcp7 becomes more stable in meu13Δ cells, whereas Meu13 becomes less stable in mcp7Δ cells. Genetic analysis shows that Mcp7 acts in the downstream of Dmc1, homologs of Escherichia coli RecA protein, for both recombination and subsequent sporulation. Taken together, we conclude that Mcp7 associates with Meu13 and together they play a key role in meiotic recombination. PMID:15210864

  18. 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

  19. 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

  20. 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

  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. Conformational Transition Between Four and Five-Stranded Phenylalanine Zippers Determined by a Local Packing Interaction

    SciTech Connect

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

    2006-01-01

    Alpha-helical coiled coils play a crucial role in mediating specific protein-protein interactions. However, the rules and mechanisms that govern helix-helix association in coiled coils remain incompletely understood. Here we have engineered a seven heptad 'Phe-zipper' protein (Phe-14) with phenylalanine residues at all 14 hydrophobic a and d positions, and generated a further variant (Phe-14{sub M}) in which a single core Phe residue is substituted with Met. Phe-14 forms a discrete {alpha}-helical pentamer in aqueous solution, while Phe-14M folds into a tetrameric helical structure. X-ray crystal structures reveal that in both the tetramer and the pentamer the a and d side-chains interlock in a classical knobs-into-holes packing to produce parallel coiled-coil structures enclosing large tubular cavities. However, the presence of the Met residue in the apolar interface of the tetramer markedly alters its local coiled-coil conformation and superhelical geometry. Thus, short-range interactions involving the Met side-chain serve to preferentially select for tetramer formation, either by inhibiting a nucleation step essential for pentamer folding or by abrogating an intermediate required to form the pentamer. Although specific trigger sequences have not been clearly identified in dimeric coiled coils, higher-order coiled coils, as well as other oligomeric multi-protein complexes, may require such sequences to nucleate and direct their assembly.

  3. 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

  4. 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

  5. Hydrogen-Rich Medium Attenuated Lipopolysaccharide-Induced Monocyte-Endothelial Cell Adhesion and Vascular Endothelial Permeability via Rho-Associated Coiled-Coil Protein Kinase.

    PubMed

    Xie, Keliang; Wang, Weina; Chen, Hongguang; Han, Huanzhi; Liu, Daquan; Wang, Guolin; Yu, Yonghao

    2015-07-01

    Sepsis is the leading cause of death in critically ill patients. In recent years, molecular hydrogen, as an effective free radical scavenger, has been shown a selective antioxidant and anti-inflammatory effect, and it is beneficial in the treatment of sepsis. Rho-associated coiled-coil protein kinase (ROCK) participates in junction between normal cells, and regulates vascular endothelial permeability. In this study, we used lipopolysaccharide to stimulate vascular endothelial cells and explored the effects of hydrogen-rich medium on the regulation of adhesion of monocytes to endothelial cells and vascular endothelial permeability. We found that hydrogen-rich medium could inhibit adhesion of monocytes to endothelial cells and decrease levels of adhesion molecules, whereas the levels of transepithelial/endothelial electrical resistance values and the expression of vascular endothelial cadherin were increased after hydrogen-rich medium treatment. Moreover, hydrogen-rich medium could lessen the expression of ROCK, as a similar effect of its inhibitor Y-27632. In addition, hydrogen-rich medium could also inhibit adhesion of polymorphonuclear neutrophils to endothelial cells. In conclusion, hydrogen-rich medium could regulate adhesion of monocytes/polymorphonuclear neutrophils to endothelial cells and vascular endothelial permeability, and this effect might be related to the decreased expression of ROCK protein.

  6. 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.

  7. 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

  8. An Evolutionarily Conserved Coiled-Coil Protein Implicated in Polycystic Kidney Disease Is Involved in Basal Body Duplication and Flagellar Biogenesis in Trypanosoma brucei†

    PubMed Central

    Morgan, Gareth W.; Denny, Paul W.; Vaughan, Sue; Goulding, David; Jeffries, Tim R.; Smith, Deborah F.; Gull, Keith; Field, Mark C.

    2005-01-01

    Trypanosoma brucei is a flagellated protozoan with a highly polarized cellular structure. TbLRTP is a trypanosomal protein containing multiple SDS22-class leucine-rich repeats and a coiled-coil domain with high similarity to a mammalian testis-specific protein of unknown function. Homologues are present in a wide range of higher eukaryotes including zebra fish, where the gene product has been implicated in polycystic kidney disease. Western blot analysis and immunofluorescence with antibodies against recombinant TbLRTP indicate that the protein is expressed throughout the trypanosome life cycle and localizes to distal zones of the basal bodies. Overexpression and RNA interference demonstrate that TbLRTP is important for faithful basal body duplication and flagellum biogenesis. Expression of excess TbLRTP suppresses new flagellum assembly, while reduction of TbLRTP protein levels often results in the biogenesis of additional flagellar axonemes and paraflagellar rods that, most remarkably, are intracellular and fully contained within the cytoplasm. The mutant flagella are devoid of membrane and are often associated with four microtubules in an arrangement similar to that observed in the normal flagellar attachment zone. Aberrant basal body and flagellar biogenesis in TbLRTP mutants also influences cell size and cytokinesis. These findings demonstrate that TbLRTP suppresses basal body replication and subsequent flagellar biogenesis and indicate a critical role for the LRTP family of proteins in the control of the cell cycle. These data further underscore the role of aberrant flagellar biogenesis as a disease mechanism. PMID:15831481

  9. From coiled coils to small globular proteins: design of a native-like three-helix bundle.

    PubMed Central

    Bryson, J. W.; Desjarlais, J. R.; Handel, T. M.; DeGrado, W. F.

    1998-01-01

    A monomolecular native-like three-helix bundle has been designed in an iterative process, beginning with a peptide that noncooperatively assembled into an antiparallel three-helix bundle. Three versions of the protein were designed in which specific interactions were incrementally added. The hydrodynamic and spectroscopic properties of the proteins were examined by size exclusion chromatography, sedimentation equilibrium, fluorescence spectroscopy, and NMR. The thermodynamics of folding were evaluated by monitoring the thermal and guanidine-induced unfolding transitions using far UV circular dichroism spectroscopy. The attainment of a unique, native-like state was achieved through the introduction of: (1) helix capping interactions; (2) electrostatic interactions between partially exposed charged residues; (3) a diverse collection of apolar side chains within the hydrophobic core. PMID:9655345

  10. 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.

  11. A silk purse from a sow's ear-bioinspired materials based on α-helical coiled coils.

    PubMed

    Quinlan, Roy A; Bromley, Elizabeth H; Pohl, Ehmke

    2015-02-01

    This past few years have heralded remarkable times for intermediate filaments with new revelations of their structural properties that has included the first crystallographic-based model of vimentin to build on the experimental data of intra-filament interactions determined by chemical cross-linking. Now with these and other advances on their assembly, their biomechanical and their cell biological properties outlined in this review, the exploitation of the biomechanical and structural properties of intermediate filaments, their nanocomposites and biomimetic derivatives in the biomedical and private sectors has started.

  12. Mcp6, a meiosis-specific coiled-coil protein of Schizosaccharomyces pombe, localizes to the spindle pole body and is required for horsetail movement and recombination.

    PubMed

    Saito, Takamune T; Tougan, Takahiro; Okuzaki, Daisuke; Kasama, Takashi; Nojima, Hiroshi

    2005-01-15

    We report here that a meiosis-specific gene of Schizosaccharomyces pombe denoted mcp6+ (meiotic coiled-coil protein) encodes a protein that is required for the horsetail movement of chromosomes at meiosis I. The mcp6+ gene is specifically transcribed during the horsetail phase. Green fluorescent protein (GFP)-tagged Mcp6 appears at the start of karyogamy, localizes to the spindle-pole body (SPB) and then disappears before chromosome segregation at meiosis I. In the mcp6Delta strain, the horsetail movement was either hampered (zygotic meiosis) or abolished (azygotic meiosis) and the pairing of homologous chromosomes was impaired. Accordingly, the allelic recombination rates of the mcp6Delta strain were only 10-40% of the wild-type rates. By contrast, the ectopic recombination rate of the mcp6Delta strain was twice the wild-type rate. This is probably caused by abnormal homologous pairing in mcp6Delta cells because of aberrant horsetail movement. Fluorescent microscopy indicates that SPB components such as Sad1, Kms1 and Spo15 localize normally in mcp6Delta cells. Because Taz1 and Swi6 also localized with Sad1 in mcp6Delta cells, Mcp6 is not required for telomere clustering. In a taz1Delta strain, which does not display telomere clustering, and the dhc1-d3 mutant, which lacks horsetail movement, Mcp6 localized with Sad1 normally. However, we observed abnormal astral microtubule organization in mcp6Delta cells. From these results, we conclude that Mcp6 is necessary for neither SPB organization nor telomere clustering, but is required for proper astral microtubule positioning to maintain horsetail movement. PMID:15654021

  13. 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

  14. A C-Terminal Coiled-Coil Region of CagL is Responsible for Helicobacter Pylori-Induced Il-8 Expression

    PubMed Central

    Wiedemann, Tobias; Hofbaur, Stefan; Loell, Eva; Rieder, Gabriele

    2016-01-01

    Interleukin-8 (IL-8) is a potent neutrophil-activating chemokine which triggers the infiltration and migration of neutrophils into areas of bacterial infection. Helicobacter pylori-infected patient studies as well as animal models have revealed that H. pylori type I strains carrying an intact cytotoxin-associated gene pathogenicity island (cag-PAI) with a functional type IV secretion system (T4SS) induce IL-8 expression and secretion in gastric mucosa. This gastric mucosal IL-8 expression correlates with severe histological changes due to H. pylori infection. In the present study, we explored a new recognition pattern on the bacterial adhesion protein CagL inducing IL-8 expression in H. pylori-infected host cells. To analyze the secreted IL-8 concentration, we performed IL-8 enzyme-linked immunosorbent assay (ELISA). To investigate the H. pylori-induced IL-8 expression on the transcriptional level, we transiently transfected gastric epithelial cells (AGS) with a human IL-8 luciferase reporter construct. The results of this study demonstrate that specifically the C-terminal coiled-coil region of the H. pylori CagL protein, a protein described to be located on the tip of the T4SS-pilus, is responsible for several in vitro observations: 1) H. pylori-induced IL-8 secretion via the transforming growth factor (TGF)-α activated epidermal growth factor-receptor (EGF-R) signaling pathway; 2) H. pylori-induced elongation of the cells, a typical CagA-induced phenotype; and 3) the bridging of the T4SS to its human target cells. This novel bacterial-host recognition sequence allows a new insight into how H. pylori induces the inflammatory response in gastric epithelial cells and facilitates the development of precancerous conditions. PMID:27766167

  15. Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions.

    PubMed Central

    Monera, O. D.; Kay, C. M.; Hodges, R. S.

    1994-01-01

    The objective of this study was to address the question of whether or not urea and guanidine hydrochloride (GdnHCl) give the same estimates of the stability of a particular protein. We previously suspected that the estimates of protein stability from GdnHCl and urea denaturation data might differ depending on the electrostatic interactions stabilizing the proteins. Therefore, 4 coiled-coil analogs were designed, where the number of intrachain and interchain electrostatic attractions (A) were systematically changed to repulsions (R): 20A, 15A5R, 10A10R, and 20R. The GdnHCl denaturation data showed that the 4 coiled-coil analogs, which had electrostatic interactions ranging from 20 attractions to 20 repulsions, had very similar [GdnHCl]1/2 values (average of congruent to 3.5 M) and, as well, their delta delta Gu values were very close to 0 (0.2 kcal/mol). In contrast, urea denaturation showed that the [urea]1/2 values proportionately decreased with the stepwise change from 20 electrostatic attractions to 20 repulsions (20A, 7.4 M; 15A5R, 5.4 M; 10A10R, 3.2 M; and 20R, 1.4 M), and the delta delta Gu values correspondingly increased with the increasing differences in electrostatic interactions (20A-15A5R, 1.5 kcal/mol; 20A-10A10R, 3.7 kcal/mol; and 20A-20R, 5.8 kcal/mol). These results indicate that the ionic nature of GdnHCl masks electrostatic interactions in these model proteins, a phenomenon that was absent when the unchanged urea was used. Thus, GdnHCl and urea denaturations may give vastly different estimates of protein stability, depending on how important electrostatic interactions are to the protein. PMID:7703845

  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

    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

  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 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

  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. Direct interactions between the coiled-coil tip of DksA and the trigger loop of RNA polymerase mediate transcriptional regulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E. coli DksA is in a class of transcription factors that modify RNA polymerase (RNAP) in all three kingdoms of life. DksA potentiates the effects of the global regulator ppGpp and the initiating NTP, controlling transcription initiation without binding to DNA. Incorporating benzoyl-phenylalanine (Bp...

  1. An evolutionarily conserved family of virion tail needles related to bacteriophage P22 gp26: correlation between structural stability and length of the α-helical trimeric coiled-coil

    PubMed Central

    Bhardwaj, Anshul; Walker-Kopp, Nancy; Casjens, Sherwood R.; Cingolani, Gino

    2009-01-01

    Bacteriophages of the Podoviridae family use short non-contractile 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 ejection of the packaged viral genome. Bio-informatic 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, HS1, and characterized these gene products in solution. All gp26-like factors contain an elongated α-helical coiled-coil core consisting of repeating, adjacent trimerization heptads and form trimeric fibers with length ranging between about 240Å to 300Å. Gp26-tail needles display high structural stability in solution, with Tm (temperature of melting) between 85–95°C. To determine how the structural stability of these phage fibers correlates with the length of the α-helical core, we investigated the effect of insertions and deletions in the helical core. In 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 gp26 helical core, preserving the straight morphology of the fiber. Likewise, we were able to remove three quarters of the helical core of HS1 tail needle minimally decreasing the stability of the fiber. We conclude that in the gp26 family of tail needles, structural stability increases non-linearly with the length of the α-helical core. Thus, the overall stability of these bacteriophage fibers is not solely dependent on the number of trimerization repeats in the α-helical core. PMID:19482036

  2. Full-Length Gαq–Phospholipase C-β3 Structure Reveals Interfaces of the C-terminal Coiled-Coil Domain

    PubMed Central

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

    2013-01-01

    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 3D reconstructions of human full-length PLCβ3 in complex with murine Gαq. The distal CTD forms an extended, monomeric helical bundle consisting of three anti-parallel segments with structural similarity to membrane-binding bin–amphiphysin–Rvs (BAR) domains. Sequence conservation of the distal CTD identifies 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 the distal CTD plays roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis. PMID:23377541

  3. 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.

  4. 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

  5. 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.

  6. Tubulin polymerization promoting protein 1 (Tppp1) phosphorylation by Rho-associated coiled-coil kinase (rock) and cyclin-dependent kinase 1 (Cdk1) inhibits microtubule dynamics to increase cell proliferation.

    PubMed

    Schofield, Alice V; Gamell, Cristina; Suryadinata, Randy; Sarcevic, Boris; Bernard, Ora

    2013-03-15

    Tubulin polymerization promoting protein 1 (Tppp1) regulates microtubule (MT) dynamics via promoting MT polymerization and inhibiting histone deacetylase 6 (Hdac6) activity to increase MT acetylation. Our results reveal that as a consequence, Tppp1 inhibits cell proliferation by delaying the G1/S-phase and the mitosis to G1-phase transitions. We show that phosphorylation of Tppp1 by Rho-associated coiled-coil kinase (Rock) prevents its Hdac6 inhibitory activity to enable cells to enter S-phase. Whereas, our analysis of the role of Tppp1 during mitosis revealed that inhibition of its MT polymerizing and Hdac6 regulatory activities were necessary for cells to re-enter the G1-phase. During this investigation, we also discovered that Tppp1 is a novel Cyclin B/Cdk1 (cyclin-dependent kinase) substrate and that Cdk phosphorylation of Tppp1 inhibits its MT polymerizing activity. Overall, our results show that dual Rock and Cdk phosphorylation of Tppp1 inhibits its regulation of the cell cycle to increase cell proliferation.

  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

    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

  8. DIPA-family coiled-coils bind conserved isoform-specific head domain of p120-catenin family: potential roles in hydrocephalus and heterotopia

    PubMed Central

    Markham, Nicholas O.; Doll, Caleb A.; Dohn, Michael R.; Miller, Rachel K.; Yu, Huapeng; Coffey, Robert J.; McCrea, Pierre D.; Gamse, Joshua T.; Reynolds, Albert B.

    2014-01-01

    p120-catenin (p120) modulates adherens junction (AJ) dynamics by controlling the stability of classical cadherins. Among all p120 isoforms, p120-3A and p120-1A are the most prevalent. Both stabilize cadherins, but p120-3A is preferred in epithelia, whereas p120-1A takes precedence in neurons, fibroblasts, and macrophages. During epithelial-to-mesenchymal transition, E- to N-cadherin switching coincides with p120-3A to -1A alternative splicing. These isoforms differ by a 101–amino acid “head domain” comprising the p120-1A N-terminus. Although its exact role is unknown, the head domain likely mediates developmental and cancer-associated events linked to p120-1A expression (e.g., motility, invasion, metastasis). Here we identified delta-interacting protein A (DIPA) as the first head domain–specific binding partner and candidate mediator of isoform 1A activity. DIPA colocalizes with AJs in a p120-1A- but not 3A-dependent manner. Moreover, all DIPA family members (Ccdc85a, Ccdc85b/DIPA, and Ccdc85c) interact reciprocally with p120 family members (p120, δ-catenin, p0071, and ARVCF), suggesting significant functional overlap. During zebrafish neural tube development, both knockdown and overexpression of DIPA phenocopy N-cadherin mutations, an effect bearing functional ties to a reported mouse hydrocephalus phenotype associated with Ccdc85c. These studies identify a novel, highly conserved interaction between two protein families that may participate either individually or collectively in N-cadherin–mediated development. PMID:25009281

  9. 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.

  10. 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.

  11. Thermodynamic characterization of the binding interaction between the histone demethylase LSD1/KDM1 and CoREST.

    PubMed

    Hwang, Sunhee; Schmitt, Allison A; Luteran, Andrea E; Toone, Eric J; McCafferty, Dewey G

    2011-02-01

    Flavin-dependent histone demethylases catalyze the posttranslational oxidative demethylation of mono- and dimethylated lysine residues, producing formaldehyde and hydrogen peroxide in addition to the corresponding demethylated protein. In vivo, histone demethylase LSD1 (KDM1; BCH110) is a component of the multiprotein complex that includes histone deacetylases (HDAC 1 and 2) and the scaffolding protein CoREST. Although little is known about the affinities of or the structural basis for the interaction between CoREST and HDACs, the structure of CoREST(286-482) bound to an α-helical coiled-coil tower domain within LSD1 has recently been reported. Given the significance of CoREST in directing demethylation to specific nucleosomal substrates, insight into the molecular basis of the interaction between CoREST and LSD1 may suggest a new means of inhibiting LSD1 activity by misdirecting the enzyme away from nucleosomal substrates. Toward this end, isothermal titration calorimetry studies were conducted to determine the affinity and thermodynamic parameters characterizing the binding interaction between LSD1 and CoREST(286-482). The proteins tightly interact in a 1:1 stoichiometry with a dissociation constant (K(d)) of 15.9 ± 2.07 nM, and their binding interaction is characterized by a favorable enthalpic contribution near room temperature with a smaller entropic penalty at pH 7.4. Additionally, one proton is transferred from the buffer to the heterodimeric complex at pH 7.4. From the temperature dependence of the enthalpy change of interaction, a constant-pressure heat capacity change (ΔC(p)) of the interaction was determined to be -0.80 ± 0.01 kcal mol(-1) K(-1). Notably, structure-driven truncation of CoREST revealed that the central binding determinant lies within the segment of residues 293-380, also known as the CoREST "linker" region, which is a central isolated helix that interacts with the LSD1 coiled-coil tower domain to create a triple-helical bundle

  12. 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.

  13. Energy system interaction and relative contribution during maximal exercise.

    PubMed

    Gastin, P B

    2001-01-01

    There are 3 distinct yet closely integrated processes that operate together to satisfy the energy requirements of muscle. The anaerobic energy system is divided into alactic and lactic components, referring to the processes involved in the splitting of the stored phosphagens, ATP and phosphocreatine (PCr), and the nonaerobic breakdown of carbohydrate to lactic acid through glycolysis. The aerobic energy system refers to the combustion of carbohydrates and fats in the presence of oxygen. The anaerobic pathways are capable of regenerating ATP at high rates yet are limited by the amount of energy that can be released in a single bout of intense exercise. In contrast, the aerobic system has an enormous capacity yet is somewhat hampered in its ability to delivery energy quickly. The focus of this review is on the interaction and relative contribution of the energy systems during single bouts of maximal exercise. A particular emphasis has been placed on the role of the aerobic energy system during high intensity exercise. Attempts to depict the interaction and relative contribution of the energy systems during maximal exercise first appeared in the 1960s and 1970s. While insightful at the time, these representations were based on calculations of anaerobic energy release that now appear questionable. Given repeated reproduction over the years, these early attempts have lead to 2 common misconceptions in the exercise science and coaching professions. First, that the energy systems respond to the demands of intense exercise in an almost sequential manner, and secondly, that the aerobic system responds slowly to these energy demands, thereby playing little role in determining performance over short durations. More recent research suggests that energy is derived from each of the energy-producing pathways during almost all exercise activities. The duration of maximal exercise at which equal contributions are derived from the anaerobic and aerobic energy systems appears to

  14. Energy system interaction and relative contribution during maximal exercise.

    PubMed

    Gastin, P B

    2001-01-01

    There are 3 distinct yet closely integrated processes that operate together to satisfy the energy requirements of muscle. The anaerobic energy system is divided into alactic and lactic components, referring to the processes involved in the splitting of the stored phosphagens, ATP and phosphocreatine (PCr), and the nonaerobic breakdown of carbohydrate to lactic acid through glycolysis. The aerobic energy system refers to the combustion of carbohydrates and fats in the presence of oxygen. The anaerobic pathways are capable of regenerating ATP at high rates yet are limited by the amount of energy that can be released in a single bout of intense exercise. In contrast, the aerobic system has an enormous capacity yet is somewhat hampered in its ability to delivery energy quickly. The focus of this review is on the interaction and relative contribution of the energy systems during single bouts of maximal exercise. A particular emphasis has been placed on the role of the aerobic energy system during high intensity exercise. Attempts to depict the interaction and relative contribution of the energy systems during maximal exercise first appeared in the 1960s and 1970s. While insightful at the time, these representations were based on calculations of anaerobic energy release that now appear questionable. Given repeated reproduction over the years, these early attempts have lead to 2 common misconceptions in the exercise science and coaching professions. First, that the energy systems respond to the demands of intense exercise in an almost sequential manner, and secondly, that the aerobic system responds slowly to these energy demands, thereby playing little role in determining performance over short durations. More recent research suggests that energy is derived from each of the energy-producing pathways during almost all exercise activities. The duration of maximal exercise at which equal contributions are derived from the anaerobic and aerobic energy systems appears to

  15. The Switch-associated Protein 70 (SWAP-70) Bundles Actin Filaments and Contributes to the Regulation of F-actin Dynamics*

    PubMed Central

    Chacón-Martínez, Carlos Andrés; Kiessling, Nadine; Winterhoff, Moritz; Faix, Jan; Müller-Reichert, Thomas; Jessberger, Rolf

    2013-01-01

    Coordinated assembly and disassembly of actin into filaments and higher order structures such as stress fibers and lamellipodia are fundamental for cell migration and adhesion. However, the precise spatiotemporal regulation of F-actin structures is not completely understood. SWAP-70, a phosphatidylinositol 3,4,5-trisphosphate-interacting, F-actin-binding protein, participates in actin rearrangements through yet unknown mechanisms. Here, we show that SWAP-70 is an F-actin-bundling protein that oligomerizes through a Gln/Glu-rich stretch within a coiled-coil region. SWAP-70 bundles filaments in parallel and anti-parallel fashion through its C-terminal F-actin binding domain and delays dilution-induced F-actin depolymerization. We further demonstrate that SWAP-70 co-localizes and directly interacts with cofilin, an F-actin severing and depolymerization factor, and contributes to the regulation of cofilin activity in vivo. In line with these activities, upon stem cell factor stimulation, murine bone marrow-derived mast cells lacking SWAP-70 display aberrant regulation of F-actin and actin free barbed ends dynamics. Moreover, proper stem cell factor-dependent cofilin activation via dephosphorylation and subcellular redistribution into a detergent-resistant cytoskeletal compartment also require SWAP-70. Together, these findings reveal an important role of SWAP-70 in the dynamic spatiotemporal regulation of F-actin networks. PMID:23921380

  16. Intramolecular Interactions and Regulation of Cofactor Binding by the Four Repressive Elements in the Caspase Recruitment Domain-containing Protein 11 (CARD11) Inhibitory Domain.

    PubMed

    Jattani, Rakhi P; Tritapoe, Julia M; Pomerantz, Joel L

    2016-04-15

    The CARD11 signaling scaffold transmits signaling between antigen receptors on B and T lymphocytes and the transcription factor NF-κB during the adaptive immune response. CARD11 activity is controlled by an inhibitory domain (ID), which participates in intramolecular interactions and prevents cofactor binding prior to receptor triggering. Oncogenic CARD11 mutations associated with the activated B cell-like subtype of diffuse large B cell lymphoma somehow perturb ID-mediated autoinhibition to confer CARD11 with the dysregulated spontaneous signaling to NF-κB that is required for the proliferation and survival of the lymphoma. Here, we investigate how the four repressive elements (REs) we have discovered in the CARD11 ID function to inhibit CARD11 activity with cooperativity and redundancy. We find that each RE contributes to the maintenance of the closed inactive state of CARD11 that predominates in the absence of receptor engagement. Each RE also contributes to the prevention of Bcl10 binding in the basal unstimulated state. RE1, RE2, and RE3 participate in intramolecular interactions with other CARD11 domains and share domain targets for binding. Remarkably, diffuse large B cell lymphoma-associated gain-of-function mutations in the caspase recruitment domain, LATCH, or coiled coil can perturb intramolecular interactions mediated by multiple REs, suggesting how single amino acid oncogenic CARD11 mutations can perturb or bypass the action of redundant inhibitory REs to achieve the level of hyperactive CARD11 signaling required to support lymphoma growth.

  17. 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.

  18. 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

  19. 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

  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. Neurobiological mechanisms contributing to alcohol-stress-anxiety interactions.

    PubMed

    Silberman, Yuval; Bajo, Michal; Chappell, Ann M; Christian, Daniel T; Cruz, Maureen; Diaz, Marvin R; Kash, Thomas; Lack, Anna K; Messing, Robert O; Siggins, George R; Winder, Danny; Roberto, Marisa; McCool, Brian A; Weiner, Jeff L

    2009-11-01

    This article summarizes the proceedings of a symposium that was presented at a conference entitled "Alcoholism and Stress: A Framework for Future Treatment Strategies." The conference was held in Volterra, Italy on May 6-9, 2008 and this symposium was chaired by Jeff L. Weiner. The overall goal of this session was to review recent findings that may shed new light on the neurobiological mechanisms that underlie the complex relationships between stress, anxiety, and alcoholism. Dr. Danny Winder described a novel interaction between D1 receptor activation and the corticotrophin-releasing factor (CRF) system that leads to an increase in glutamatergic synaptic transmission in the bed nucleus of the stria terminalis. Dr. Marisa Roberto presented recent data describing how protein kinase C epsilon, ethanol, and CRF interact to alter GABAergic inhibition in the central nucleus of the amygdala. Dr. Jeff Weiner presented recent advances in our understanding of inhibitory circuitry within the basolateral amygdala (BLA) and how acute ethanol exposure enhances GABAergic inhibition in these pathways. Finally, Dr. Brian McCool discussed recent findings on complementary glutamatergic and GABAergic adaptations to chronic ethanol exposure and withdrawal in the BLA. Collectively, these investigators have identified novel mechanisms through which neurotransmitter and neuropeptide systems interact to modulate synaptic activity in stress and anxiety circuits. Their studies have also begun to describe how acute and chronic ethanol exposure influence excitatory and inhibitory synaptic communication in these pathways. These findings point toward a number of novel neurobiological targets that may prove useful for the development of more effective treatment strategies for alcohol use disorders.

  2. 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.

  3. 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

  4. Interactive Contributions of Cumulative Peer Stress and Executive Function Deficits to Depression in Early Adolescence

    ERIC Educational Resources Information Center

    Agoston, Anna M.; Rudolph, Karen D.

    2016-01-01

    Exposure to peer stress contributes to adolescent depression, yet not all youth experience these effects. Thus, it is important to identify individual differences that shape the consequences of peer stress. This research investigated the interactive contribution of cumulative peer stress during childhood (second-fifth grades) and executive…

  5. Subject Line Preferences and Other Factors Contributing to Coherence and Interaction in Student Discussion Forums

    ERIC Educational Resources Information Center

    Skogs, Julie

    2013-01-01

    A number of factors may affect student interaction in an asynchronous online discussion forum used in learning. This study deals with student preferences for the subject line of messages and in what ways the choice of subject line contributes to coherence and interaction reflected in the textual and interpersonal functions of the linguistic items…

  6. 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…

  7. An estimate of the enthalpic contribution to the interaction between dextran and albumin.

    PubMed Central

    Comper, W D; Laurent, T C

    1978-01-01

    It is demonstrated that exclusion phenomena appear to dominate the interaction of dextran with albumin in aqueous solution. The enthalpic contribution to the interaction coefficient describing dextran/albumin mixtures is small, although its determination was subject to considerable error. These results support the earlier assumptions of the type of interaction between the two polymers. The conclusions are primarily based on the interpretation of the temperature-dependence of the interaction coefficient, as measured by light-scattering in the temperature range 6--33 degrees C. Enthalpy of dilution measurements of dextran/albumin mixtures by microcalorimetry were in qualitative agreement with the light-scattering data. PMID:743220

  8. 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.

  9. 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.

  10. Chibby interacts with NBPF1 and clusterin, two candidate tumor suppressors linked to neuroblastoma.

    PubMed

    Vandepoele, Karl; Staes, Katrien; Andries, Vanessa; van Roy, Frans

    2010-04-15

    The NBPF genes are members of a gene family that underwent a remarkable increase in their copy number during recent primate evolution. The NBPF proteins contain 5 to 40 copies of a domain known as the NBPF repeat or DUF1220. Very little is known about the function of these domains or about the NBPF proteins. We performed a yeast two-hybrid screening with the aminoterminal domain of NBPF11 and found that Chibby, a documented repressor of Wnt signaling, interacts with multiple NBPF proteins. More specifically, a coiled-coil region in the NBPF proteins interacts with the coiled-coil domain in the carboxyterminal region of Chibby. Nonetheless, this interaction did not influence the repressor function of Chibby in a TOPFLASH reporter assay. Using Chibby as bait in a new yeast two-hybrid screening, we identified clusterin as a binding protein. Chibby and clusterin were co-immunoprecipitated with NBPF1, suggesting the formation of a tri-molecular complex. Although we have not pinpointed the role of these mutual interactions, the possible formation of a macromolecular complex of three candidate tumor suppressor proteins, including the enigmatic NBPF1, points at important functional implications. PMID:20096688

  11. Role of MINOS in mitochondrial membrane architecture: cristae morphology and outer membrane interactions differentially depend on mitofilin domains.

    PubMed

    Zerbes, Ralf M; Bohnert, Maria; Stroud, David A; von der Malsburg, Karina; Kram, Anita; Oeljeklaus, Silke; Warscheid, Bettina; Becker, Thomas; Wiedemann, Nils; Veenhuis, Marten; van der Klei, Ida J; Pfanner, Nikolaus; van der Laan, Martin

    2012-09-14

    The mitochondrial inner membrane contains a large protein complex crucial for membrane architecture, the mitochondrial inner membrane organizing system (MINOS). MINOS is required for keeping cristae membranes attached to the inner boundary membrane via crista junctions and interacts with protein complexes of the mitochondrial outer membrane. To study if outer membrane interactions and maintenance of cristae morphology are directly coupled, we generated mutant forms of mitofilin/Fcj1 (formation of crista junction protein 1), a core component of MINOS. Mitofilin consists of a transmembrane anchor in the inner membrane and intermembrane space domains, including a coiled-coil domain and a conserved C-terminal domain. Deletion of the C-terminal domain disrupted the MINOS complex and led to release of cristae membranes from the inner boundary membrane, whereas the interaction of mitofilin with the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM) were enhanced. Deletion of the coiled-coil domain also disturbed the MINOS complex and cristae morphology; however, the interactions of mitofilin with TOM and SAM were differentially affected. Finally, deletion of both intermembrane space domains disturbed MINOS integrity as well as interactions with TOM and SAM. Thus, the intermembrane space domains of mitofilin play distinct roles in interactions with outer membrane complexes and maintenance of MINOS and cristae morphology, demonstrating that MINOS contacts to TOM and SAM are not sufficient for the maintenance of inner membrane architecture.

  12. 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…

  13. 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…

  14. Activities Contributing a Great Deal to the Students' Interactive Skills in Foreign Language Classes

    ERIC Educational Resources Information Center

    Asatryan, Susanna

    2016-01-01

    While teaching speaking it is desired to provide a rich environment in class for meaningful communication to take place. With this aim, various speaking activities can contribute a great deal to students in developing their interactive skills necessary for life. These activities make students active in the learning process and at the same time…

  15. 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.

  16. Genome-wide contribution of genotype by environment interaction to variation of diabetes-related traits.

    PubMed

    Zheng, Ju-Sheng; Arnett, Donna K; Lee, Yu-Chi; Shen, Jian; Parnell, Laurence D; Smith, Caren E; Richardson, Kris; Li, Duo; Borecki, Ingrid B; Ordovás, José M; Lai, Chao-Qiang

    2013-01-01

    While genome-wide association studies (GWAS) and candidate gene approaches have identified many genetic variants that contribute to disease risk as main effects, the impact of genotype by environment (GxE) interactions remains rather under-surveyed. To explore the importance of GxE interactions for diabetes-related traits, a tool for Genome-wide Complex Trait Analysis (GCTA) was used to examine GxE variance contribution of 15 macronutrients and lifestyle to the total phenotypic variance of diabetes-related traits at the genome-wide level in a European American population. GCTA identified two key environmental factors making significant contributions to the GxE variance for diabetes-related traits: carbohydrate for fasting insulin (25.1% of total variance, P-nominal = 0.032) and homeostasis model assessment of insulin resistance (HOMA-IR) (24.2% of total variance, P-nominal = 0.035), n-6 polyunsaturated fatty acid (PUFA) for HOMA-β-cell-function (39.0% of total variance, P-nominal = 0.005). To demonstrate and support the results from GCTA, a GxE GWAS was conducted with each of the significant dietary factors and a control E factor (dietary protein), which contributed a non-significant GxE variance. We observed that GxE GWAS for the environmental factor contributing a significant GxE variance yielded more significant SNPs than the control factor. For each trait, we selected all significant SNPs produced from GxE GWAS, and conducted anew the GCTA to estimate the variance they contributed. We noted the variance contributed by these SNPs is higher than that of the control. In conclusion, we utilized a novel method that demonstrates the importance of genome-wide GxE interactions in explaining the variance of diabetes-related traits.

  17. Bacterial beta-lactamase fragmentation complementation strategy can be used as a method for identifying interacting protein pairs.

    PubMed

    Park, Jong-Hwa; Back, Jung Ho; Hahm, Soo Hyun; Shim, Hye-Young; Park, Min Ju; Ko, Sung Il; Han, Ye Sun

    2007-10-01

    We investigated the applicability of the TEM-1 beta- lactamase fragment complementation (BFC) system to develop a strategy for the screening of protein-protein interactions in bacteria. A BFC system containing a human Fas-associated death domain (hFADD) and human Fas death domain (hFasDD) was generated. The hFADD-hFasDD interaction was verified by cell survivability in ampicillin-containing medium and the colorimetric change of nitrocefin. It was also confirmed by His pull-down assay using cell lysates obtained in selection steps. A coiled-coil helix coiled-coil domain-containing protein 5 (CHCH5) was identified as an interacting protein of human uracil DNA glycosylase (hUNG) from the bacterial BFC cDNA library strategy. The interaction between hUNG and CHCH5 was further confirmed with immunoprecipitation using a mammalian expression system. CHCH5 enhanced the DNA glycosylase activity of hUNG to remove uracil from DNA duplexes containing a U/G mismatch pair. These results suggest that the bacterial BFC cDNA library strategy can be effectively used to identify interacting protein pairs.

  18. Genetic Interactions between Chromosomes 11 and 18 Contribute to Airway Hyperresponsiveness in Mice

    PubMed Central

    Ferreira, Caroline M.; Chen, James L.; Li, Jianrong; Shimomura, Kazuhiro; Yang, Xinan

    2012-01-01

    We used two-dimensional quantitative trait locus analysis to identify interacting genetic loci that contribute to the native airway constrictor hyperresponsiveness to methacholine that characterizes A/J mice, relative to C57BL/6J mice. We quantified airway responsiveness to intravenous methacholine boluses in eighty-eight (C57BL/6J X A/J) F2 and twenty-seven (A/J X C57BL/6J) F2 mice as well as ten A/J mice and six C57BL/6J mice; all studies were performed in male mice. Mice were genotyped at 384 SNP markers, and from these data two-QTL analyses disclosed one pair of interacting loci on chromosomes 11 and 18; the homozygous A/J genotype at each locus constituted the genetic interaction linked to the hyperresponsive A/J phenotype. Bioinformatic network analysis of potential interactions among proteins encoded by genes in the linked regions disclosed two high priority subnetworks - Myl7, Rock1, Limk2; and Npc1, Npc1l1. Evidence in the literature supports the possibility that either or both networks could contribute to the regulation of airway constrictor responsiveness. Together, these results should stimulate evaluation of the genetic contribution of these networks in the regulation of airway responsiveness in humans. PMID:22253740

  19. Contribution of membrane permeability and unstirred layer diffusion to nitric oxide-red blood cell interaction

    PubMed Central

    Deonikar, Prabhakar; Kavdia, Mahendra

    2012-01-01

    Nitric oxide (NO) consumption by red blood cell (RBC) hemoglobin (Hb) in vasculature is critical in regulating the vascular tone. The paradox of NO production at endothelium in close proximity of an effective NO scavenger Hb in RBCs is mitigated by lower NO consumption by RBCs compared to that of free Hb due to transport resistances including membrane resistance, extra- and intra- cellular resistances for NO biotransport to the RBC. Relative contribution of each transport resistance on NO-RBC interactions is still not clear. We developed a mathematical model of NO transport to a single RBC to quantify the contributions from individual transport barriers by analyzing the effect of RBC membrane permeability (Pm), hematocrit (Hct) and NO-Hb reaction rate constants on NO-RBC interactions. Our results indicated that intracellular diffusion of NO was not a rate limiting step for NO-RBC interactions. The extracellular diffusion contributed 70–90% of total transport resistance for Pm >1 cm/s whereas membrane resistance accounts for 50–75% of total transport resistance for Pm < 0.1 cm/s. We propose a narrow Pm range of 0.21–0.44 cm/s for 10–45% Hct, respectively, below which membrane resistance is more significant and above which extracellular diffusion is a dominating transport resistance for NO-RBC interactions. PMID:23116664

  20. 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

  1. Assessment of hydrophobic interactions and their contributions through the analysis of the methane dimer.

    PubMed

    Alaniz, Víctor Duarte; Rocha-Rinza, Tomás; Cuevas, Gabriel

    2015-03-01

    Hydrophobic Interactions (HIs) are important in many phenomena of molecular recognition in chemistry and biology. Still, the relevance of HIs is sometimes difficult to evaluate particularly in large systems and intramolecular interactions. We put forward a method to estimate the magnitude and the different contributions of a given HI of the C···C, H-C···H, and H···H type through (i) the analysis of the electron density in the intermolecular region for eleven relative orientations of the methane dimer and (ii) the subsequent decomposition of the corresponding interaction energy in physically significant contributions using Symmetry Adapted Perturbation Theory (SAPT). Strong correlations were found between the topological properties of ρ(r) calculated at intermolecular bond critical points and Eint(SAPT) plus its different contributions with the C···C distance of the considered orientations of (CH4 )2 . These correlations were used to construct Mollier-like diagrams of Eint(SAPT) and its components as a function of the separation between two carbons and the orientation of the groups bonded to these atoms. The ethane dimer and tert-butylcyclohexane are used as representative examples of this new approach. Overall, we anticipate that this new method might prove useful in the study of both intramolecular and intermolecular HIs particularly of those within large systems wherein SAPT or electronic structure calculations are computationally expensive or even prohibitive.

  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. 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

  4. 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

  5. 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.

  6. Breit interaction contribution to parity violating potentials in chiral molecules containing light nuclei.

    PubMed

    Berger, Robert

    2008-10-21

    The importance of the Breit interaction for an accurate prediction of parity violating energy differences between enantiomers is studied within electroweak quantum chemical frameworks. Besides two-electron orbit-orbit and spin-spin coupling contributions, the Breit interaction gives rise to the spin-other-orbit coupling term of the Breit-Pauli Hamiltonian. The present numerical study demonstrates that neglect of this latter term leads in hydrogen peroxide (H(2)O(2)) to relative deviations in the parity violating potential (V(pv)) by about 10%, whereas further relativistic corrections accounted for within a four-component Dirac-Hartree-Fock-Coulomb (DHFC) framework remain smaller, below 5%. Thus, the main source of discrepancy between previous one-component based (coupled perturbed) Hartree-Fock (HF) and four-component Dirac-Hartree-Fock results for parity violating potentials in H(2)O(2) is the neglect of the Breit contribution in DHFC. In heavier homologs of hydrogen peroxide the relative contribution of the spin-other-orbit coupling term to V(pv) decreases with increasing nuclear charge, whereas other relativistic effects become increasingly important. As shown for the H(2)X(2) (X = O,S,Se,Te,Po) series of molecules and for CHBrClF, to a good approximation these other relativistic influences on V(pv) can be accounted for in one-component based HF calculations with the help of relativistic enhancement factors proposed earlier in the theory of atomic parity violation.

  7. Structural Basis of the Interaction between Tuberous Sclerosis Complex 1 (TSC1) and Tre2-Bub2-Cdc16 Domain Family Member 7 (TBC1D7).

    PubMed

    Qin, Jiayue; Wang, Zhizhi; Hoogeveen-Westerveld, Marianne; Shen, Guobo; Gong, Weimin; Nellist, Mark; Xu, Wenqing

    2016-04-15

    Mutations in TSC1 or TSC2 cause tuberous sclerosis complex (TSC), an autosomal dominant disorder characterized by the occurrence of benign tumors in various vital organs and tissues. TSC1 and TSC2, the TSC1 and TSC2 gene products, form the TSC protein complex that senses specific cellular growth conditions to control mTORC1 signaling. TBC1D7 is the third subunit of the TSC complex, and helps to stabilize the TSC1-TSC2 complex through its direct interaction with TSC1. Homozygous inactivation of TBC1D7 causes intellectual disability and megaencephaly. Here we report the crystal structure of a TSC1-TBC1D7 complex and biochemical characterization of the TSC1-TBC1D7 interaction. TBC1D7 interacts with the C-terminal region of the predicted coiled-coil domain of TSC1. The TSC1-TBC1D7 interface is largely hydrophobic, involving the α4 helix of TBC1D7. Each TBC1D7 molecule interacts simultaneously with two parallel TSC1 helices from two TSC1 molecules, suggesting that TBC1D7 may stabilize the TSC complex by tethering the C-terminal ends of two TSC1 coiled-coils.

  8. 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

  9. 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

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

    PubMed

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

    2014-08-14

    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 ε(W)/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-ε(W)/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. PMID:25134597

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

    PubMed

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

    2014-08-14

    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 ε(W)/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-ε(W)/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.

  12. Can interaction of materials with the dentin-pulp complex contribute to dentin regeneration?

    PubMed

    Ferracane, Jack L; Cooper, Paul R; Smith, Anthony J

    2010-02-01

    Understanding outcomes of the interaction between a dental material and tooth tissue is important in terms not only of biocompatibility but also of the potential for the material to modulate the response of the tissue. This interaction is influenced by many factors, including the chemistry of the material and any of its eluted components or degradation products, and the manner in which the tissue responds to these agents. Past studies of this interaction have primarily been aimed at identifying cytotoxic effects. More recently, investigations have focused on specific cellular responses, and in particular, on understanding how the materials themselves actually may contribute to regenerative processes in the tooth. Recent work has demonstrated the solubilization of proteins from dentin exposed to certain materials, such as calcium hydroxide, mineral trioxide aggregate, and acidic solutions that relate to those used in dentin bonding agents, with the subsequent modulation by these proteins of gene expression in odontoblast-like cells. This work suggests that dentin bridge formation under such materials may be stimulated through this process. Thus, there is much merit in examining both how new dental materials can be developed and how more traditional ones can be modified to preferentially stimulate regenerative processes when preferred. This review summarizes current knowledge about the potential beneficial effects derived from the interaction of dental materials with the dentin-pulp complex, as well as potential future developments in this exciting field.

  13. 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

  14. 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.

  15. 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.

  16. RET finger protein is a transcriptional repressor and interacts with enhancer of polycomb that has dual transcriptional functions.

    PubMed

    Shimono, Y; Murakami, H; Hasegawa, Y; Takahashi, M

    2000-12-15

    RET finger protein (RFP) belongs to the large B-box RING finger protein family and is known to become oncogenic by fusion with RET tyrosine kinase. Although RFP is reported to be a nuclear protein that is present in the nuclear matrix, its function is largely unknown. Here we show that RFP interacts with Enhancer of Polycomb (EPC) and strongly represses the gene transcription. Yeast two-hybrid assays revealed that the coiled-coil domain of RFP was associated with the EPcA domain and the carboxyl-terminal region of EPC. In addition, both proteins were co-precipitated from the lysates of human cells and mostly colocalized in the nucleus. Using the luciferase reporter-gene assay, we found that they repress the gene transcription activity independent of the differences of enhancers and promoters used, although the repressive activity of RFP was much stronger than that of EPC. The coiled-coil domain of RFP and the carboxyl-terminal region of EPC were most important for the repressive activity of each protein, whereas the EPcA domain had the transcription activating ability that is unique as the Polycomb group protein function. These results suggested that RFP may be involved in the epigenetic gene silencing mechanism cooperating with Polycomb group proteins and that EPC is a unique molecule with both repressive and transactivating activities.

  17. 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.

  18. 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

  19. 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

  20. Characterization of the Interaction between the Salmonella Type III Secretion System Tip Protein SipD and the Needle Protein PrgI by Paramagnetic Relaxation Enhancement*

    PubMed Central

    Rathinavelan, Thenmalarchelvi; Tang, Chun; De Guzman, Roberto N.

    2011-01-01

    Many Gram-negative bacteria that cause major diseases and mortality worldwide require the type III secretion system (T3SS) to inject virulence proteins into their hosts and cause infections. A structural component of the T3SS is the needle apparatus, which consists of a base, an external needle, and a tip complex. In Salmonella typhimurium, the external needle is assembled by the polymerization of the needle protein PrgI. On top of this needle sits a tip complex, which is partly formed by the tip protein SipD. How SipD interacts with PrgI during the assembly of the T3SS needle apparatus remains unknown. The central region of PrgI forms an α-helical hairpin, whereas SipD has a long central coiled-coil, which is a defining structural feature of other T3SS tip proteins as well. Using NMR paramagnetic relaxation enhancement, we have identified a specific region on the SipD coiled-coil that interacts directly with PrgI. We present a model of how SipD might dock at the tip of the needle based on our paramagnetic relaxation enhancement results, thus offering new insight about the mechanism of assembly of the T3SS needle apparatus. PMID:21138848

  1. 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

  2. Universal Repulsive Contribution to the Solvent-Induced Interaction Between Sizable, Curved Hydrophobes.

    PubMed

    Jabes, B Shadrack; Bratko, Dusan; Luzar, Alenka

    2016-08-18

    In addition to the direct attraction, sizable hydrophobes in water experience an attractive force mediated by interfacial water. Using simple geometric arguments, we identify the conditions at which the water-induced interaction between curved hydrocarbon surfaces becomes repulsive. The repulsive contribution arises from the thermodynamic penalty due to the emergence of the liquid/vapor boundary created as water gets expelled between curved hydrophobes. By augmenting the mean field approach with atomistic simulations of pristine and alkyl-coated graphitic nanoparticles in three distinct geometries, spherical, cylindrical and planar, immersed in water, we show the macroscopic thermodynamics remarkably works down to the molecular scale. The new insights improve the prediction and control of wetting and dispersion properties for a broad class of nonpolar nanoparticles.

  3. 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

  4. Maternal dispositional empathy and electrodermal reactivity: Interactive contributions to maternal sensitivity with toddler-aged children.

    PubMed

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

    2014-08-01

    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 or 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 vs. laugh) and the type of sensitivity examined (sensitivity to the child's distress vs. nondistress). PMID:24955589

  5. 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.

  6. 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

  7. Interaction of thrombospondin1 and CD36 contributes to obesity-associated podocytopathy.

    PubMed

    Cui, Wenpeng; Maimaitiyiming, Hasiyeti; Zhou, Qi; Norman, Heather; Zhou, Changcheng; Wang, Shuxia

    2015-07-01

    Obesity is associated with podocyte injury and the development of proteinuria. Elevated plasma free fatty acid is one of the characteristics of obesity and has been linked to podocyte dysfunction. However, the mechanisms remain unclear. In the current study, we examined the effect of saturated free fatty acid (FFA) on human podocyte apoptosis and function in vitro. The mechanism and its in vivo relevance were also determined. We found that FFA treatment induced human podocyte apoptosis and dysfunction, which was associated with increased expression of a matricellular protein-thrombospondin1 (TSP1). FFA stimulated TSP1 expression in podocytes at the transcriptional levels through activation of MAPK pathway. Addition of purified TSP1 to cell culture media induced podocyte apoptosis and dysfunction. Tis effect is though a TGF-β independent mechanism. Moreover, peptide treatment to block TSP1 binding to its receptor-CD36 attenuated FFA induced podocyte apoptosis, suggesting that TSP1/CD36 interaction mediates FFA-induced podocyte apoptosis. Importantly, using a diet-induced obese mouse model, in vivo data demonstrated that obesity-associated podocyte apoptosis and dysfunction were attenuated in TSP1 deficient mice as well as in CD36 deficient mice. Taken together, these studies provide novel evidence that the interaction of TSP1 with its receptor CD36 contributes to obesity--associated podocytopathy.

  8. 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

  9. The 68-kDa Telomeric Repeat Binding Factor 1 (TRF1)-associated Protein (TAP68) Interacts with and Recruits TRF1 to the Spindle Pole during Mitosis*

    PubMed Central

    Lan, Jianping; Zhu, Yuanyuan; Xu, Leilei; Yu, Huijuan; Yu, Jian; Liu, Xing; Fu, Chuanhai; Wang, Xiaogang; Ke, Yuwen; Huang, He; Dou, Zhen

    2014-01-01

    The telomere capping protein TRF1 is a component of the multiprotein complex “shelterin,” which organizes the telomere into a high order structure. Besides telomere maintenance, telomere-associated proteins also have nontelomeric functions. For example, tankyrase 1 and TRF1 are required for the maintenance of faithful mitotic progression. However, the functional relevance of their centrosomal localization has not been established. Here, we report the identification of a TRF1-binding protein, TAP68, that interacts with TRF1 in mitotic cells. TAP68 contains two coiled-coil domains and a structural maintenance of chromosome motifs and co-localizes with TRF1 to telomeres during interphase. Immediately after nuclear envelope breakdown, TAP68 translocates toward the spindle poles followed by TRF1. Dissociation of TAP68 from the telomere is concurrent with the Nek2A-dependent phosphorylation at Thr-221. Biochemical characterization demonstrated that the first coiled-coil domain of TAP68 binds and recruits TRF1 to the centrosome. Inhibition of TAP68 expression by siRNA blocked the localization of TRF1 and tankyrase 1 to the centrosome. Furthermore, siRNA-mediated depletion of TAP68 perturbed faithful chromosome segregation and genomic stability. These findings suggest that TAP68 functions in mediating TRF1-tankyrase 1 localization to the centrosome and in mitotic regulation. PMID:24692559

  10. 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

  11. The Contribution of Pin End-Cup Interactions to Clot Strength Assessed with Thrombelastography.

    PubMed

    Nielsen, Vance G

    2016-01-01

    Viscoelastic methods have been developed to assess the contribution of plasma proteins and platelets to coagulation in vitro to guide clinical transfusion therapy. One of the cardinal precepts of determining clot strength is making sure that the viscoelastic technique includes complete exposure of the plastic pin in the testing chamber with the fluid analyzed so as to assure maximal interaction of the cup wall with the pin surface. However, the various contributions of the pin surface area to final clot strength have not been investigated. That is, it is not clear what is more important in the in vitro determination of clot strength, the surface area shared between the cup and pin filled with fluid or the final viscoelastic resistance of the gel matrix formed. Thus, the purpose of this investigation was to determine the clot strength when only the tip of the pin was engaged with plasma thrombus and to compare these values with clot strength values obtained when the pin was completely in plasma. After determining the minimal amount of plasma required to cover a pin tip in a thrombelastographic system (30 μL), clot strength (elastic modulus, G) was determined in plasma samples of 30 or 360 μL final volume (n = 12 per condition) after tissue factor activation. The G value with 30 μL volume was 1057 ± 601 dynes/cm (mean ± SD; 95% confidence interval, 675-1439 dynes/cm), which was (P = 0.0015) smaller than the G value associated with 360-μL sample volumes, that was 1712 ± 48 dynes/cm (confidence interval, 1681-1742 dynes/cm). In conclusion, these data demonstrate that clot strength is not determined by a simple ratio of surface area of pin and cup to volume of sample, but rather strength is importantly influenced by the viscoelastic resistance of the fluid assessed.

  12. 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…

  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. A protein interaction node at the neurotransmitter release site: domains of Aczonin/Piccolo, Bassoon, CAST, and rim converge on the N-terminal domain of Munc13-1.

    PubMed

    Wang, Xiaolu; Hu, Bin; Zieba, Agata; Neumann, Nicole G; Kasper-Sonnenberg, Monika; Honsbein, Annegret; Hultqvist, Greta; Conze, Tim; Witt, Wolfgang; Limbach, Christoph; Geitmann, Matthis; Danielson, Helena; Kolarow, Richard; Niemann, Gesa; Lessmann, Volkmar; Kilimann, Manfred W

    2009-10-01

    Multidomain scaffolding proteins organize the molecular machinery of neurotransmitter vesicle dynamics during synaptogenesis and synaptic activity. We find that domains of five active zone proteins converge on an interaction node that centers on the N-terminal region of Munc13-1 and includes the zinc-finger domain of Rim1, the C-terminal region of Bassoon, a segment of CAST1/ELKS2, and the third coiled-coil domain (CC3) of either Aczonin/Piccolo or Bassoon. This multidomain complex may constitute a center for the physical and functional integration of the protein machinery at the active zone. An additional connection between Aczonin and Bassoon is mediated by the second coiled-coil domain of Aczonin. Recombinant Aczonin-CC3, expressed in cultured neurons as a green fluorescent protein fusion protein, is targeted to synapses and suppresses vesicle turnover, suggesting involvements in synaptic assembly as well as activity. Our findings show that Aczonin, Bassoon, CAST1, Munc13, and Rim are closely and multiply interconnected, they indicate that Aczonin-CC3 can actively participate in neurotransmitter vesicle dynamics, and they highlight the N-terminal region of Munc13-1 as a hub of protein interactions by adding three new binding partners to its mechanistic potential in the control of synaptic vesicle priming.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. Separate and interactive contributions of weak inhibitory control and threat sensitivity to prediction of suicide risk

    PubMed Central

    Venables, Noah C.; Sellbom, Martin; Sourander, Andre; Kendler, Kenneth S.; Joiner, Thomas E.; Drislane, Laura E.; Sillanmäki, Lauri; Elonheimo, Henrik; Parkkola, Kai; Multimaki, Petteri; Patrick, Christopher J.

    2015-01-01

    Biobehavioral dispositions can serve as valuable referents for biologically-oriented research on core processes with relevance to many psychiatric conditions. The present study examined two such dispositional variables—weak response inhibition (or disinhibition; INH−) and threat sensitivity (or fearfulness; THT+)—as predictors of the serious transdiagnostic problem of suicide risk in two samples: male and female outpatients from a U.S. clinic (N=1,078), and a population-based male military cohort from Finland (N=3,855). INH− and THT+ were operationalized through scores on scale measures of disinhibition and fear/fearlessness, known to be related to DSM-defined clinical conditions and brain biomarkers. Suicide risk was assessed by clinician ratings (clinic sample) and questionnaires (both samples). Across samples and alternative suicide indices, INH− and THT+ each contributed uniquely to prediction of suicide risk—beyond internalizing and externalizing problems in the case of the clinic sample where diagnostic data were available. Further, in both samples, INH− and THT+ interactively predicted suicide risk, with individuals scoring concurrently high on both dispositions exhibiting markedly augmented risk. Findings demonstrate that dispositional constructs of INH− and THT+ are predictive of suicide risk, and hold potential as referents for biological research on suicidal behavior. PMID:25712631

  20. Unsteady Contribution of Water Vapor Condensation to Heat Losses at Flame-Wall Interaction

    NASA Astrophysics Data System (ADS)

    Boust, B.; Sotton, J.; Bellenoue, M.

    2012-11-01

    This study addresses experimentally the influence of a second-order phenomenon concerning wall heat losses in combustion chambers, namely water vapour phase change, that is likely to occur subsequently to flame-wall interaction. For this purpose, flame quenching experiments are carried out on methane-air mixtures in a constant volume combustion chamber, using heat gauges based on surface thermometry. The transient effect of condensation on wall heat flux, that was already predicted using numerical simulation, is evidenced in our experiments. Its evolution and magnitude are quantified and compared to the relevant literature. Although the time evolution of condensation heat flux was poorly predicted by numerical simulation, its magnitude is well estimated thanks to mass diffusion across the quenched layer at the wall. In the conditions investigated, the contribution of condensation to wall heat flux reaches about 0.1 MW/m2, which represents around 6% of conductive wall heat flux at quenching and up to 23% during the cooling phase after combustion.

  1. Separate and interactive contributions of weak inhibitory control and threat sensitivity to prediction of suicide risk.

    PubMed

    Venables, Noah C; Sellbom, Martin; Sourander, Andre; Kendler, Kenneth S; Joiner, Thomas E; Drislane, Laura E; Sillanmäki, Lauri; Elonheimo, Henrik; Parkkola, Kai; Multimaki, Petteri; Patrick, Christopher J

    2015-04-30

    Biobehavioral dispositions can serve as valuable referents for biologically oriented research on core processes with relevance to many psychiatric conditions. The present study examined two such dispositional variables-weak response inhibition (or disinhibition; INH-) and threat sensitivity (or fearfulness; THT+)-as predictors of the serious transdiagnostic problem of suicide risk in two samples: male and female outpatients from a U.S. clinic (N=1078), and a population-based male military cohort from Finland (N=3855). INH- and THT+ were operationalized through scores on scale measures of disinhibition and fear/fearlessness, known to be related to DSM-defined clinical conditions and brain biomarkers. Suicide risk was assessed by clinician ratings (clinic sample) and questionnaires (both samples). Across samples and alternative suicide indices, INH- and THT+ each contributed uniquely to prediction of suicide risk-beyond internalizing and externalizing problems in the case of the clinic sample where diagnostic data were available. Further, in both samples, INH- and THT+ interactively predicted suicide risk, with individuals scoring concurrently high on both dispositions exhibiting markedly augmented risk. Findings demonstrate that dispositional constructs of INH- and THT+ are predictive of suicide risk, and hold potential as referents for biological research on suicidal behavior. PMID:25712631

  2. Nature of aryl-tyrosine interactions contribute to β-hairpin scaffold stability: NMR evidence for alternate ring geometry.

    PubMed

    Makwana, Kamlesh Madhusudan; Mahalakshmi, Radhakrishnan

    2015-02-14

    The specific contribution of the acidic-aromatic β-sheet favouring amino acid tyrosine to the stability of short octapeptide β-hairpin structures is presented here. Solution NMR analysis in near-apolar environments suggests the energetically favourable mode of interaction to be T-shaped face-to-edge (FtE) and that a Trp-Tyr interacting pair is the most stabilizing. Alternate aryl geometries also exist in solution, which readily equilibrate between a preferred π···π conformation to an aromatic-amide conformation, without any change in the backbone structure. While the phenolic ring is readily accommodated at the "edge" of FtE aryl interactions, it exhibits an overall lowered contribution to scaffold stability in the "face" orientation. Such differential tyrosine interactions are key to its dual nature in proteins.

  3. 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

  4. CONTRIBUTION TO FAMILIAL BREAST CANCER OF INHERITED MUTATIONS IN THE BRCA2-INTERACTING PROTEIN PALB2

    PubMed Central

    Casadei, Silvia; Norquist, Barbara M.; Walsh, Tom; Stray, Sunday; Mandell, Jessica B.; Lee, Ming K.; Stamatoyannopoulos, John A.; King, Mary-Claire

    2011-01-01

    Inherited mutations in the BRCA2-interacting protein PALB2 are known to be associated with increased risks of breast cancer. In order to evaluate the contribution of PALB2 to familial breast cancer in the United States, we sequenced the coding sequences and flanking regulatory regions of the gene from constitutional genomic DNA of 1144 familial breast cancer patients with wildtype sequences at BRCA1 and BRCA2. Overall, 3.4% (33/972) of patients not selected by ancestry and 0% (0/172) of patients specifically of Ashkenazi Jewish ancestry were heterozygous for a nonsense, frameshift, or frameshift-associated splice mutation in PALB2. Mutations were detected in both male and female breast cancer patients. All mutations were individually rare: the 33 heterozygotes harbored 13 different mutations, 5 previously reported and 8 novel. PALB2 heterozygotes were 4-fold more likely to have a male relative with breast cancer (P=0.0003) and 6-fold more likely to have a relative with pancreatic cancer (P=0.002), and 1.3-fold more likely to have a relative with ovarian cancer (P=0.18). Compared to their female relatives without mutations, increased risk of breast cancer for female PALB2 heterozygotes was 2.3-fold (95%CI [1.5–4.2]) by age 55 and 3.4-fold (95%CI [2.4–5.9]) by age 85. Loss of the wildtype PALB2 allele was observed in laser dissected tumor specimens from heterozygous patients. Given this mutation prevalence and risk, consideration might be given to clinical testing of PALB2 by complete genomic sequencing for familial breast cancer patients with wildtype sequences at BRCA1 and BRCA2. PMID:21285249

  5. Calculation of the first nonlinear contribution to the general-relativistic spin-spin interaction for binary systems.

    PubMed

    Porto, Rafael A; Rothstein, Ira Z

    2006-07-14

    We use recently developed effective field theory techniques to calculate the third order post-Newtonian correction to the spin-spin potential between two spinning objects. This correction represents the first contribution to the spin-spin interaction due to the nonlinear nature of general relativity and will play an important role in forthcoming gravity wave experiments.

  6. The nature of interactions that contribute to postzygotic reproductive isolation in hybrid copepods.

    PubMed

    Willett, Christopher S

    2011-05-01

    Deleterious interactions within the genome of hybrids can lower fitness and result in postzygotic reproductive isolation. Understanding the genetic basis of these deleterious interactions, known as Dobzhansky-Muller incompatibilities, is the subject of intense current study that seeks to elucidate the nature of these deleterious interactions. Hybrids from crosses of individuals from genetically divergent populations of the intertidal copepod Tigriopus californicus provide a useful model in which to study Dobzhansky-Muller incompatibilities. Studies of the basis of postzygotic reproductive isolation in this species have revealed a number of patterns. First, there is evidence for a breakdown in genomic coadaptation between mtDNA-encoded and nuclear-encoded proteins that can result in a reduction in hybrid fitness in some crosses. It appears from studies of the individual genes involved in these interactions that although this coadaptation could lead to asymmetries between crosses, patterns of genotypic viabilities are not often consistent with simple models of genomic coadaptation. Second, there is a large impact of environmental factors on these deleterious interactions suggesting that they are not strictly intrinsic in nature. Temperature in particular appears to play an important role in determining the nature of these interactions. Finally, deleterious interactions in these hybrid copepods appear to be complex in terms of the number of genetic factors that interact to lead to reductions in hybrid fitness. This complexity may stem from three or more factors that all interact to cause a single incompatibility or the same factor interacting with multiple other factors independently leading to multiple incompatibilities.

  7. Contributions of soil moisture interactions to future precipitation changes in the GLACE-CMIP5 experiment

    NASA Astrophysics Data System (ADS)

    May, Wilhelm; Rummukainen, Markku; Chéruy, Frederique; Hagemann, Stefan; Meier, Arndt

    2016-10-01

    Changes in soil moisture are likely to contribute to future changes in latent heat flux and various characteristics of daily precipitation. Such contributions during the second half of the twenty-first century are assessed using the simulations from the GLACE-CMIP5 experiment, applying a linear regression analysis to determine the magnitude of these contributions. As characteristics of daily precipitation, mean daily precipitation, the frequency of wet days and the intensity of precipitation on wet days are considered. Also, the frequency and length of extended wet and dry spells are studied. Particular focus is on the regional (for nine selected regions) as well as seasonal variations in the magnitude of the contributions of the projected differences in soil moisture to the future changes in latent heat flux and in the characteristics of daily precipitation. The results reveal the overall tendency that the projected differences in soil moisture contribute to the future changes in response to the anthropogenic climate forcing for all the meteorological variables considered here. These contributions are stronger and more robust (i.e., there are smaller deviations between individual climate models) for the latent heat flux than for the characteristics of daily precipitation. It is also found that the contributions of the differences in soil moisture to the future changes are generally stronger and more robust for the frequency of wet days than for the intensity of daily precipitation. Consistent with the contributions of the projected differences in soil moisture to the future changes in the frequency of wet days, soil moisture generally contributes to the future changes in the characteristics of wet and dry spells. The magnitude of these contributions does not differ systematically between the frequency and the length of such extended spells, but the contributions are generally slightly stronger for dry spells than for wet spells. Distinguishing between the nine

  8. Exploring the Contribution of a Content-Infused Interactive Whiteboard for School Readiness

    ERIC Educational Resources Information Center

    McManis, Lilla D.; Gunnewig, Susan B.; McManis, Mark H.

    2010-01-01

    This exploratory study examined the relationship between use of an interactive (touchscreen) whiteboard and development of school readiness skills. Over one school year, public school regular education prekindergarten classrooms used an interactive whiteboard with preloaded literacy and math activities. The children were low-income and English…

  9. 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.

  10. 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.

  11. Intraspecies Interaction of Fusarium graminearum Contributes to Reduced Toxin Production and Virulence.

    PubMed

    Walkowiak, Sean; Bonner, Christopher T; Wang, Li; Blackwell, Barbara; Rowland, Owen; Subramaniam, Rajagopal

    2015-11-01

    Fusarium graminearum is a pathogenic fungus that causes Fusarium head blight in wheat and lowers the yield and quality of grains by contamination with the trichothecene mycotoxin deoxynivalenol. The fungi coexist and interact with several different fusaria as well as other plant pathogenic fungi and bacteria in the field. In Canada, F. graminearum exists as two main trichothecene chemotypes: 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol. To understand the potential interactions between two isolates of these chemotypes, we conducted coinoculation studies both in culture and in planta. The studies showed that intraspecies interaction reduces trichothecene yield in culture and disease symptoms in wheat. To elucidate the genes involved in the intraspecies interaction, expression profiling was performed on RNA samples isolated from coinoculated cultures, and potential genes were identified by using the genome sequences of the respective isolates.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 perc...

  13. Contribution from the interaction Hamiltonian to the expectation value of particle number with the non-equilibrium quantum field theory

    SciTech Connect

    Hotta, Ryuuichi; Morozumi, Takuya; Takata, Hiroyuki

    2012-07-27

    We develop the method analyzing particle number non-conserving phenomena with non-equilibrium quantum field-theory. In this study, we consider a CP violating model with interaction Hamiltonian that breaks particle number conservation. To derive the quantum Boltzmann equation for the particle number, we solve Schwinger-Dyson equation, which are obtained from two particle irreducible closed-time-path (2PI CTP) effective action. In this calculation, we show the contribution from interaction Hamiltonian to the time evolution of expectation value of particle number.

  14. 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.

  15. 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

  16. 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

  17. Co-Constructing Mother-Infant Distress in Face-to-Face Interactions: Contributions of Microanalysis

    ERIC Educational Resources Information Center

    Beebe, Beatrice

    2004-01-01

    This article describes the use of microanalysis of videotapes to look at nonverbal elements (such as smiles, facial expressions, eye gaze, touch, and head and body movements toward and away from the other) to determine the caregiver-infant interactive (regulatory) experience. Video microanalysis of face-to-face play by infants and parents…

  18. 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

  19. Dispersion interactions between urea and nucleobases contribute to the destabilization of RNA by urea in aqueous solution.

    PubMed

    Kasavajhala, Koushik; Bikkina, Swetha; Patil, Indrajit; MacKerell, Alexander D; Priyakumar, U Deva

    2015-03-01

    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.

  20. A DNA-centric protein interaction map of ultraconserved elements reveals contribution of transcription factor binding hubs to conservation.

    PubMed

    Viturawong, Tar; Meissner, Felix; Butter, Falk; Mann, Matthias

    2013-10-31

    Ultraconserved elements (UCEs) have been the subject of great interest because of their extreme sequence identity and their seemingly cryptic and largely uncharacterized functions. Although in vivo studies of UCE sequences have demonstrated regulatory activity, protein interactors at UCEs have not been systematically identified. Here, we combined high-throughput affinity purification, high-resolution mass spectrometry, and SILAC quantification to map intrinsic protein interactions for 193 UCE sequences. The interactome contains over 400 proteins, including transcription factors with known developmental roles. We demonstrate based on our data that UCEs consist of strongly conserved overlapping binding sites. We also generated a fine-resolution interactome of a UCE, confirming the hub-like nature of the element. The intrinsic interactions mapped here are reflected in open chromatin, as indicated by comparison with existing ChIP data. Our study argues for a strong contribution of protein-DNA interactions to UCE conservation and provides a basis for further functional characterization of UCEs.

  1. 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

  2. Contribution of SUMO-interacting motifs and SUMOylation to the antiretroviral properties of TRIM5α.

    PubMed

    Brandariz-Nuñez, Alberto; Roa, Amanda; Valle-Casuso, Jose Carlos; Biris, Nikolaos; Ivanov, Dmitri; Diaz-Griffero, Felipe

    2013-01-20

    Recent findings suggested that the SUMO-interacting motifs (SIMs) present in the human TRIM5α (TRIM5α(hu)) protein play an important role in the ability of TRIM5α(hu) to restrict N-MLV. Here we explored the role of SIMs in the ability of rhesus TRIM5α (TRIM5α(rh)) to restrict HIV-1, and found that TRIM5α(rh) 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 TRIM5α(rh) 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 TRIM5α(rh) 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 alter the retroviral restriction ability of TRIM5α. Overall, our results do not support a role for SIMs or SUMOylation in the antiviral properties of TRIM5α.

  3. NAC1, A POZ/BTB protein interacts with Parkin and may contribute to Parkinson's disease.

    PubMed

    Korutla, L; Furlong, H A; Mackler, S A

    2014-01-17

    Loss-of-function in the Parkin protein is thought to play a part in causing neuronal cell death in patients with Parkinson's disease. This study explores the effect of Parkin degradation, via the overexpression of nucleus accumbens 1 (NAC1), on cell viability. It was found that NAC1 and Parkin are co-localized within the cell and interact with one another, leading to a decrease in Parkin levels. Moreover, NAC1 down-regulates Parkin by presenting it for ubiquitin-dependent proteasome degradation, which causes a decrease in proteasomal activity in neuronal cells. Consequently, this decrease in proteasomal activity leads to an increase in the cells' susceptibility to proteasome inhibition-induced toxicity. It was also found that Parkin and NAC1 are key proteins found to be present mainly in the cytoplasm and are co-localized in neurons of Parkinson's disease patients. Interestingly, mutation in the POZ/BTB domain (Q23L) of NAC1 disrupts the co-localization and interaction of NAC1 with Parkin and it further abrogates the proteasome inhibition-induced toxicity. We further observed that co-transfection of the mutant form of NAC1 with Parkin reversed the proteasome activity and 20S proteasome protein levels. These results indicate a novel interaction between NAC1 and Parkin that leads to neuronal cell death, a main characteristic in Parkinson's disease.

  4. N-terminal domains of ARC1 are essential for interaction with the N-terminal region of Exo70A1 in transducing self-incompatibility of Brassica oleracea.

    PubMed

    Shi, Songmei; Gao, Qiguo; Zeng, Jing; Liu, Xiaohuan; Pu, Quanming; Liu, Guixi; Zhang, Hecui; Yang, Xiaohong; Zhu, Liquan

    2016-09-01

    Self-incompatibility (SI) is an important mating system to prevent inbreeding and promote outcrossing. ARC1 and Exo70A1 function as the downstream targets of the S-locus receptor kinase and play conservative roles in Brassica SI signaling. Based on the sequence homology, Exo70A1 is divided into four subdomains: leucine zipper (Leu(128)-Leu(149)), hypervariable region (Ser(172)-Leu(197)), SUMO modification motif (Glu(260)-Ile(275)), and pfamExo70 domain (His(271)-Phe(627)). ARC1 contains four domains as follows: leucine zipper (Leu(116)-Leu(137)), coiled-coil domain (Thr(210)-Val(236)), U-box (Asp(282)-Trp(347)) motif, and ARM (Ala(415)-Thr(611)) domain. Bioinformatics analysis, yeast two-hybrid screening and pull-down assays show that leucine zipper and coiled-coil motifs of ARC1116-236 are required for the interaction with Exo70A1, while the addition of ARM motif results in loss of the interaction with Exo70A1. Meanwhile, the N-terminal of Exo70A1 without any domains shows a weak interaction with ARC1, and the level of LacZ expression increases with addition of leucine zipper and reaches the maximum value with hypervariable region and SUMO modification motif, indicating that hypervariable region and SUMO modification motif of Exo70A1172-275 is mainly responsible for the binding with ARC1, whereas pfamExo70 domain has little affinity for ARC1. Lys(181) located in the Exo70A1 hypervariable region may be the ubiquitination site mediating the interaction between ARC1 and Exo70A1. Therefore, both the leucine zipper with coiled-coil structure of ARC1116-236, and the hypervariable region and SUMO modification motif of Exo70A1172-275 are the core interaction domains between ARC1 and Exo70A1. Any factors affecting these core domains would be the regulators of ARC1 mediating ubiquitin degradation in self-incompatible system. PMID:27590064

  5. N-terminal domains of ARC1 are essential for interaction with the N-terminal region of Exo70A1 in transducing self-incompatibility of Brassica oleracea.

    PubMed

    Shi, Songmei; Gao, Qiguo; Zeng, Jing; Liu, Xiaohuan; Pu, Quanming; Liu, Guixi; Zhang, Hecui; Yang, Xiaohong; Zhu, Liquan

    2016-09-01

    Self-incompatibility (SI) is an important mating system to prevent inbreeding and promote outcrossing. ARC1 and Exo70A1 function as the downstream targets of the S-locus receptor kinase and play conservative roles in Brassica SI signaling. Based on the sequence homology, Exo70A1 is divided into four subdomains: leucine zipper (Leu(128)-Leu(149)), hypervariable region (Ser(172)-Leu(197)), SUMO modification motif (Glu(260)-Ile(275)), and pfamExo70 domain (His(271)-Phe(627)). ARC1 contains four domains as follows: leucine zipper (Leu(116)-Leu(137)), coiled-coil domain (Thr(210)-Val(236)), U-box (Asp(282)-Trp(347)) motif, and ARM (Ala(415)-Thr(611)) domain. Bioinformatics analysis, yeast two-hybrid screening and pull-down assays show that leucine zipper and coiled-coil motifs of ARC1116-236 are required for the interaction with Exo70A1, while the addition of ARM motif results in loss of the interaction with Exo70A1. Meanwhile, the N-terminal of Exo70A1 without any domains shows a weak interaction with ARC1, and the level of LacZ expression increases with addition of leucine zipper and reaches the maximum value with hypervariable region and SUMO modification motif, indicating that hypervariable region and SUMO modification motif of Exo70A1172-275 is mainly responsible for the binding with ARC1, whereas pfamExo70 domain has little affinity for ARC1. Lys(181) located in the Exo70A1 hypervariable region may be the ubiquitination site mediating the interaction between ARC1 and Exo70A1. Therefore, both the leucine zipper with coiled-coil structure of ARC1116-236, and the hypervariable region and SUMO modification motif of Exo70A1172-275 are the core interaction domains between ARC1 and Exo70A1. Any factors affecting these core domains would be the regulators of ARC1 mediating ubiquitin degradation in self-incompatible system.

  6. Glycation Contributes to Interaction Between Human Bone Alkaline Phosphatase and Collagen Type I.

    PubMed

    Halling Linder, Cecilia; Enander, Karin; Magnusson, Per

    2016-03-01

    Bone is a biological composite material comprised primarily of collagen type I and mineral crystals of calcium and phosphate in the form of hydroxyapatite (HA), which together provide its mechanical properties. Bone alkaline phosphatase (ALP), produced by osteoblasts, plays a pivotal role in the mineralization process. Affinity contacts between collagen, mainly type II, and the crown domain of various ALP isozymes were reported in a few in vitro studies in the 1980s and 1990s, but have not attracted much attention since, although such interactions may have important implications for the bone mineralization process. The objective of this study was to investigate the binding properties of human collagen type I to human bone ALP, including the two bone ALP isoforms B1 and B2. ALP from human liver, human placenta and E. coli were also studied. A surface plasmon resonance-based analysis, supported by electrophoresis and blotting, showed that bone ALP binds stronger to collagen type I in comparison with ALPs expressed in non-mineralizing tissues. Further, the B2 isoform binds significantly stronger to collagen type I in comparison with the B1 isoform. Human bone and liver ALP (with identical amino acid composition) displayed pronounced differences in binding, revealing that post-translational glycosylation properties govern these interactions to a large extent. In conclusion, this study presents the first evidence that glycosylation differences in human ALPs are of crucial importance for protein-protein interactions with collagen type I, although the presence of the ALP crown domain may also be necessary. Different binding affinities among the bone ALP isoforms may influence the mineral-collagen interface, mineralization kinetics, and degree of bone matrix mineralization, which are important factors determining the material properties of bone. PMID:26645431

  7. Contributed Review: The novel gas puff targets for laser-matter interaction experiments

    NASA Astrophysics Data System (ADS)

    Wachulak, Przemyslaw W.

    2016-09-01

    Various types of targetry are used nowadays in laser matter interaction experiments. Such targets are characterized using different methods capable of acquiring information about the targets such as density, spatial distribution, and temporal behavior. In this mini-review paper, a particular type of target will be presented. The targets under consideration are gas puff targets of various and novel geometries. Those targets were investigated using extreme ultraviolet (EUV) and soft X-ray (SXR) imaging techniques, such as shadowgraphy, tomography, and pinhole camera imaging. Details about characterization of those targets in the EUV and SXR spectral regions will be presented.

  8. The chiral S = -1 meson-baryon interaction with new constraints on the NLO contributions

    NASA Astrophysics Data System (ADS)

    Ramos, A.; Feijoo, A.; Magas, V. K.

    2016-10-01

    We present a study of the S = - 1 meson-baryon interaction, employing a chiral SU(3) Lagrangian up to next-to-leading order (NLO) and implementing unitarization in coupled channels. The parameters of the model have been fitted to a large set of experimental scattering data in different two-body channels, to threshold branching ratios, and to the precise SIDDHARTA value of the energy shift and width of kaonic hidrogen. In contrast to other groups, we have taken into consideration the K- p →K+Ξ- ,K0Ξ0 reaction data, since we found in a previous work to be especially sensitive to the NLO parameters of the chiral Lagrangian. In the present work we also include the Born terms, which usually have very little effect, and find them to be non-negligible in the K- p → KΞ channels, correspondingly causing significant modifications to the NLO parameters. We furthermore show that the importance of the Born terms becomes more visible in the isospin projected amplitudes of the K- p → KΞ reactions. The measurement of processes that filter single isospin components, like the KL0 p →K+Ξ0 reaction that could be measured at the proposed secondary KL0 beam at Jlab, would put valuable constraints on the chiral models describing the meson-baryon interaction in the S = - 1 sector.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. How can an understanding of plant-pollinator interactions contribute to global food security?

    PubMed

    Bailes, Emily J; Ollerton, Jeff; Pattrick, Jonathan G; Glover, Beverley J

    2015-08-01

    Pollination of crops by animals is an essential part of global food production, but evidence suggests that wild pollinator populations may be declining while a number of problems are besetting managed honey bee colonies. Animal-pollinated crops grown today, bred in an environment where pollination was less likely to limit fruit set, are often suboptimal in attracting and sustaining their pollinator populations. Research into plant-pollinator interactions is often conducted in a curiosity-driven, ecological framework, but may inform breeding and biotechnological approaches to enhance pollinator attraction and crop yield. In this article we review key topics in current plant-pollinator research that have potential roles in future crop breeding for enhanced global food security. PMID:26116979

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. Defining the contributions of permanent electrostatics, Pauli repulsion, and dispersion in density functional theory calculations of intermolecular interaction energies

    NASA Astrophysics Data System (ADS)

    Horn, Paul R.; Mao, Yuezhi; Head-Gordon, Martin

    2016-03-01

    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.

  2. 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

  3. Interaction between IRF6 and TGFA Genes Contribute to the Risk of Nonsyndromic Cleft Lip/Palate

    PubMed Central

    Letra, Ariadne; Fakhouri, Walid; Fonseca, Renata F.; Menezes, Renato; Kempa, Inga; Prasad, Joanne L.; McHenry, Toby G.; Lidral, Andrew C.; Moreno, Lina; Murray, Jeffrey C.; Daack-Hirsch, Sandra; Marazita, Mary L.; Castilla, Eduardo E.; Lace, Baiba; Orioli, Ieda M.; Granjeiro, Jose M.; Schutte, Brian C.; Vieira, Alexandre R.

    2012-01-01

    Previous evidence from tooth agenesis studies suggested IRF6 and TGFA interact. Since tooth agenesis is commonly found in individuals with cleft lip/palate (CL/P), we used four large cohorts to evaluate if IRF6 and TGFA interaction contributes to CL/P. Markers within and flanking IRF6 and TGFA genes were tested using Taqman or SYBR green chemistries for case-control analyses in 1,000 Brazilian individuals. We looked for evidence of gene-gene interaction between IRF6 and TGFA by testing if markers associated with CL/P were overtransmitted together in the case-control Brazilian dataset and in the additional family datasets. Genotypes for an additional 142 case-parent trios from South America drawn from the Latin American Collaborative Study of Congenital Malformations (ECLAMC), 154 cases from Latvia, and 8,717 individuals from several cohorts were available for replication of tests for interaction. Tgfa and Irf6 expression at critical stages during palatogenesis was analyzed in wild type and Irf6 knockout mice. Markers in and near IRF6 and TGFA were associated with CL/P in the Brazilian cohort (p<10−6). IRF6 was also associated with cleft palate (CP) with impaction of permanent teeth (p<10−6). Statistical evidence of interaction between IRF6 and TGFA was found in all data sets (p = 0.013 for Brazilians; p = 0.046 for ECLAMC; p = 10−6 for Latvians, and p = 0.003 for the 8,717 individuals). Tgfa was not expressed in the palatal tissues of Irf6 knockout mice. IRF6 and TGFA contribute to subsets of CL/P with specific dental anomalies. Moreover, this potential IRF6-TGFA interaction may account for as much as 1% to 10% of CL/P cases. The Irf6-knockout model further supports the evidence of IRF6-TGFA interaction found in humans. PMID:23029012

  4. Neuron-Glia Interactions in Neural Plasticity: Contributions of Neural Extracellular Matrix and Perineuronal Nets.

    PubMed

    Dzyubenko, Egor; Gottschling, Christine; Faissner, Andreas

    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.

  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. Protein-protein interactions in the synaptonemal complex.

    PubMed Central

    Tarsounas, M; Pearlman, R E; Gasser, P J; Park, M S; Moens, P B

    1997-01-01

    In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II. Images PMID:9285814

  7. The contribution of plant-soil interactions to biogeochemical cycles in a changing world

    NASA Astrophysics Data System (ADS)

    Pregitzer, K.

    2005-12-01

    -induced changes to the Earth's atmosphere will cascade through plants into the soil, where microbial communities mediate the ecosystem functions that regulate biogeochemical cycles. There are several key research opportunities as we attempt to understand how changes in the Earth's atmosphere cascade through terrestrial ecosystems to alter biogeochemical cycles. For example, far too little attention has been given to how the interactions between changes in atmospheric chemistry (e.g. carbon dioxide and nitrogen, or carbon dioxide and ozone) will impact C transformations in the soil. If we deliberately set out to understand how variable plant and microbial physiology are to interactive changes in atmospheric chemistry, it should be possible to build a deeper understanding of the fundamental processes controlling ecosystem response to global change.

  8. 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.

  9. 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.

  10. 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

  11. PE_PGRS33 Contributes to Mycobacterium tuberculosis Entry in Macrophages through Interaction with TLR2.

    PubMed

    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.

  12. 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

  13. 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

  14. Cocaine and alcohol interactions in the rat: contribution of cocaine metabolites to the pharmacological effects.

    PubMed

    Pan, W J; Hedaya, M A

    1999-04-01

    The pharmacokinetics and pharmacodynamics of cocaine and its three metabolites, benzoylecgonine, norcocaine, and cocaethylene, were investigated in awake, freely moving rats. This work was performed to examine the effect of alcohol coadministration on the metabolic profile of cocaine and to determine the contribution of cocaine metabolites to the pharmacological responses observed after cocaine administration. The plasma and brain extracellular fluid concentration-time profiles were characterized after intravenous (iv) administration of cocaine and the three metabolites in a crossover experimental design. The neurochemical response, measured as the change in dopamine concentration in the nucleus accumbens, and the cardiovascular responses, measured as the change in the mean arterial blood pressure, heart rate, and QRS interval, were monitored simultaneously. Cocaethylene had the highest brain-to-plasma distribution ratio, followed by cocaine, norcocaine, and benzoylecgonine. The estimated total body clearances for cocaine, benzoylecgonine, norcocaine, and cocaethylene were 140 +/- 19, 14.7 +/- 1.2, 130 +/- 19, and 111 +/- 16 mL/min/kg, respectively. Alcohol coadministration increased the formation of norcocaine, decreased the formation of benzoylecgonine, and resulted in the formation of the pharmacologically active metabolite cocaethylene. When cocaine was administered with alcohol, 12.9 +/- 3.1% to 15.3 +/- 2.9% of the cocaine dose was converted to cocaethylene. Benzoylecgonine did not have any central nervous system or cardiovascular activities after iv administration. Compared with cocaine, norcocaine and cocaethylene had more potent and prolonged effects on the neurochemical, heart rate, and QRS interval responses, and were equipotent in increasing the mean arterial blood pressure. These results indicate that changes in the cocaine metabolic profile and the formation of the pharmacologically active metabolite cocaethylene are, at least partially, responsible

  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. Rip1 (receptor-interacting protein kinase 1) mediates necroptosis and contributes to renal ischemia/reperfusion injury.

    PubMed

    Linkermann, Andreas; Bräsen, Jan H; Himmerkus, Nina; Liu, Shuya; Huber, Tobias B; Kunzendorf, Ulrich; Krautwald, Stefan

    2012-04-01

    Loss of kidney function in renal ischemia/reperfusion injury is due to programmed cell death, but the contribution of necroptosis, a newly discovered form of programmed necrosis, has not been evaluated. Here, we identified the presence of death receptor-mediated but caspase-independent cell death in murine tubular cells and characterized it as necroptosis by the addition of necrostatin-1, a highly specific receptor-interacting protein kinase 1 inhibitor. The detection of receptor-interacting protein kinase 1 and 3 in whole-kidney lysates and freshly isolated murine proximal tubules led us to investigate the contribution of necroptosis in a mouse model of renal ischemia/reperfusion injury. Treatment with necrostatin-1 reduced organ damage and renal failure, even when administered after reperfusion, resulting in a significant survival benefit in a model of lethal renal ischemia/reperfusion injury. Unexpectedly, specific blockade of apoptosis by zVAD, a pan-caspase inhibitor, did not prevent the organ damage or the increase in urea and creatinine in vivo in renal ischemia/reperfusion injury. Thus, necroptosis is present and has functional relevance in the pathophysiological course of ischemic kidney injury and shows the predominance of necroptosis over apoptosis in this setting. Necrostatin-1 may have therapeutic potential to prevent and treat renal ischemia/reperfusion injury. PMID:22237751

  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. Interaction among Btn1p, Btn2p, and Ist2p reveals potential interplay among the vacuole, amino acid levels, and ion homeostasis in the yeast Saccharomyces cerevisiae.

    PubMed

    Kim, Yoojin; Chattopadhyay, Subrata; Locke, Sarahjane; Pearce, David A

    2005-02-01

    Btn2p, a novel cytosolic coiled-coil protein in Saccharomyces cerevisiae, was previously shown to interact with and to be necessary for the correct localization of Rhb1p, a regulator of arginine uptake, and Yif1p, a Golgi protein. We now report the biochemical and physical interactions of Btn2p with Ist2p, a plasma membrane protein that is thought to have a function in salt tolerance. A deletion in Btn2p (btn2Delta strains) results in a failure to correctly localize Ist2p, and strains lacking Btn2p and Ist2p (btn2Delta ist2Delta strains) are unable to grow in the presence of 0.5 or 1.0 M NaCl. Btn2p was originally identified as being up-regulated in a btn1Delta strain, which lacks the vacuolar-lysosomal membrane protein, Btn1p, and serves as a model for Batten disease. This up-regulation of Btn2p was shown to contribute to the maintenance of a stable vacuolar pH in the btn1Delta strain. Btn1p was subsequently shown to be required for the optimal transport of arginine into the vacuole. Interestingly, btn1Delta ist2Delta strains are also unable to grow in the presence of 0.5 or 1.0 M NaCl, and ist2Delta suppresses the vacuolar arginine transport defect in btn1Delta strains. Although further investigation is required, we speculate that altered vacuolar arginine transport in btn1Delta strains represents a mechanism for maintaining or balancing cellular ion homeostasis. Btn2p interacts with at least three proteins that are seemingly involved in different biological functions in different subcellular locations. Due to these multiple interactions, we conclude that Btn2p may play a regulatory role across the cell in response to alterations in the intracellular environment that may be caused by changes in amino acid levels or pH, a disruption in protein trafficking, or imbalances in ion homeostasis resulting from either genetic or environmental manipulation.

  19. Apolipoprotein E Likely Contributes to a Maturation Step of Infectious Hepatitis C Virus Particles and Interacts with Viral Envelope Glycoproteins

    PubMed Central

    Lee, Ji-Young; Acosta, Eliana G.; Stoeck, Ina Karen; Long, Gang; Hiet, Marie-Sophie; Mueller, Birthe; Fackler, Oliver T.; Kallis, Stephanie

    2014-01-01

    ABSTRACT The assembly of infectious hepatitis C virus (HCV) particles is tightly linked to components of the very-low-density lipoprotein (VLDL) pathway. We and others have shown that apolipoprotein E (ApoE) plays a major role in production of infectious HCV particles. However, the mechanism by which ApoE contributes to virion assembly/release and how it gets associated with the HCV particle is poorly understood. We found that knockdown of ApoE reduces titers of infectious intra- and extracellular HCV but not of the related dengue virus. ApoE depletion also reduced amounts of extracellular HCV core protein without affecting intracellular core amounts. Moreover, we found that ApoE depletion affected neither formation of nucleocapsids nor their envelopment, suggesting that ApoE acts at a late step of assembly, such as particle maturation and infectivity. Importantly, we demonstrate that ApoE interacts with the HCV envelope glycoproteins, most notably E2. This interaction did not require any other viral proteins and depended on the transmembrane domain of E2 that also was required for recruitment of HCV envelope glycoproteins to detergent-resistant membrane fractions. These results suggest that ApoE plays an important role in HCV particle maturation, presumably by direct interaction with viral envelope glycoproteins. IMPORTANCE The HCV replication cycle is tightly linked to host cell lipid pathways and components. This is best illustrated by the dependency of HCV assembly on lipid droplets and the VLDL component ApoE. Although the role of ApoE for production of infectious HCV particles is well established, it is still poorly understood how ApoE contributes to virion formation and how it gets associated with HCV particles. Here, we provide experimental evidence that ApoE likely is required for an intracellular maturation step of HCV particles. Moreover, we demonstrate that ApoE associates with the viral envelope glycoproteins. This interaction appears to be dispensable

  20. Interaction of Epstein-Barr Virus BZLF1 C-Terminal Tail Structure and Core Zipper Is Required for DNA Replication but Not for Promoter Transactivation ▿

    PubMed Central

    McDonald, Carol M.; Petosa, Carlo; Farrell, Paul J.

    2009-01-01

    The Epstein-Barr virus (EBV) protein BZLF1 contains a bZIP DNA binding domain in which C-terminal tail residues fold back against a zipper region that forms a coiled coil and mediates dimerization. Point mutagenesis in the zipper region reveals the importance of individual residues within the 208SSENDRLR215 sequence that is conserved in C/EBP for transactivation and EBV DNA replication. The restoration of BZLF1 DNA replication activity by the complementation of two deleterious mutations (S208E and D236K) indicates that the interaction of the C-terminal tail and the core zipper is required for DNA replication, identifying a functional role for this structural feature unique to BZLF1. PMID:19144704

  1. Structural and motional contributions of the Bacillus subtilis ClpC N-domain in adaptor protein interactions

    PubMed Central

    Kojetin, Douglas J.; McLaughlin, Patrick D.; Thompson, Richele J.; Dubnau, David; Prepiak, Peter; Rance, Mark; Cavanagh, John

    2009-01-01

    Summary The AAA+ superfamily protein ClpC is a key regulator of cell development in Bacillus subtilis. As part of a large oligomeric complex, ClpC controls an array of cellular processes by recognizing, unfolding, and providing misfolded and aggregated proteins as substrates for the ClpP peptidase. ClpC is unique compared to other HSP100/Clp proteins, as it requires an adaptor protein for all fundamental activities. The NMR solution structure of the N-terminal repeat domain of ClpC (N-ClpCR) comprises two structural repeats of a four-helix motif. NMR experiments used to map the MecA adaptor protein interaction surface of N-ClpCR reveal that regions involved in the interaction possess conformational flexibility, as well as conformational exchange on the μs-ms time-scale. The electrostatic surface of N-ClpCR differs substantially compared to the N-domain of Escherichia coli ClpA and ClpB, suggesting that the electrostatic surface characteristics of HSP100/Clp N-domains may play a role in adaptor protein and substrate interaction specificity, and perhaps contribute to the unique adaptor protein requirement of ClpC. PMID:19361434

  2. Binding of acetylcholine and tetramethylammonium to a cyclophane receptor: anion's contribution to the cation-pi interaction.

    PubMed

    Bartoli, Sandra; Roelens, Stefano

    2002-07-17

    The interaction of the lipophilic cyclophane 1 with several acetylcholine (ACh) and tetramethylammonium (TMA) salts has been investigated in deuteriochloroform to ascertain the influence of the counterion on the cation-pi interaction. Reliable association constants have been measured for 17 salts of commonly used anions; corresponding binding free energies -DeltaG degrees ranged from over 8 kJ mol(-1) down to the limit of detection. The dramatic dependence of the binding energy on the anion showed that the latter takes part in the process with a passive and adverse contribution, which inhibits cation binding even to complete suppression in unfavorable cases. Thermodynamic parameters for the association of 1 with TMA picrate demonstrate that binding is enthalpic in origin, showing a substantial enthalpy gain (DeltaH degrees = -16.7 kJ mol(-1)) and an adverse entropic contribution (DeltaS degrees = -27.9 J mol(-1) K(-1)). A correlation has been found between the "goodness" of anions as binding partners and the solubility of their salts. Conversion of the anion into a more charge-dispersed species, for example, conversion of chloride into dialkyltrichlorostannate, improves cation binding substantially, indicating that charge dispersion is a main factor determining the influence of the anion on the cation-pi interaction. DFT computational studies show that the variation of the binding free energy of TMA with the counterion is closely accounted for by the electrostatic potential (EP) of the ion pair: guest binding appears to respond to the cation's charge density exposed to the receptor, which is determined by the anion's charge density through a polarization mechanism. A value of -DeltaG degrees = 38.6 kJ mol(-1) has been extrapolated for the free energy of binding of TMA to 1 in chloroform but in the absence of a counterion. The transmission of electrostatic effects from the ion pair to the cation-pi interaction demonstrates that host-guest association is governed by

  3. Partial Dominance, Overdominance, Epistasis and QTL by Environment Interactions Contribute to Heterosis in Two Upland Cotton Hybrids

    PubMed Central

    Shang, Lianguang; Wang, Yumei; Cai, Shihu; Wang, Xiaocui; Li, Yuhua; Abduweli, Abdugheni; Hua, Jinping

    2015-01-01

    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

  4. 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

  5. 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

  6. Resolving the infection process reveals striking differences in the contribution of environment, genetics and phylogeny to host-parasite interactions

    PubMed Central

    2011-01-01

    Background Infection processes consist of a sequence of steps, each critical for the interaction between host and parasite. Studies of host-parasite interactions rarely take into account the fact that different steps might be influenced by different factors and might, therefore, make different contributions to shaping coevolution. We designed a new method using the Daphnia magna - Pasteuria ramosa system, one of the rare examples where coevolution has been documented, in order to resolve the steps of the infection and analyse the factors that influence each of them. Results Using the transparent Daphnia hosts and fluorescently-labelled spores of the bacterium P. ramosa, we identified a sequence of infection steps: encounter between parasite and host; activation of parasite dormant spores; attachment of spores to the host; and parasite proliferation inside the host. The chances of encounter had been shown to depend on host genotype and environment. We tested the role of genetic and environmental factors in the newly described activation and attachment steps. Hosts of different genotypes, gender and species were all able to activate endospores of all parasite clones tested in different environments; suggesting that the activation cue is phylogenetically conserved. We next established that parasite attachment occurs onto the host oesophagus independently of host species, gender and environmental conditions. In contrast to spore activation, attachment depended strongly on the combination of host and parasite genotypes. Conclusions Our results show that different steps are influenced by different factors. Host-type-independent spore activation suggests that this step can be ruled out as a major factor in Daphnia-Pasteuria coevolution. On the other hand, we show that the attachment step is crucial for the pronounced genetic specificities of this system. We suggest that this one step can explain host population structure and could be a key force behind coevolutionary

  7. Contributions of phenylalanine 335 to ligand recognition by human surfactant protein D: ring interactions with SP-D ligands.

    PubMed

    Crouch, Erika; McDonald, Barbara; Smith, Kelly; Cafarella, Tanya; Seaton, Barbara; Head, James

    2006-06-30

    Surfactant protein D (SP-D) is an innate immune effector that contributes to antimicrobial host defense and immune regulation. Interactions of SP-D with microorganisms and organic antigens involve binding of glycoconjugates to the C-type lectin carbohydrate recognition domain (CRD). A trimeric fusion protein encoding the human neck+CRD bound to the aromatic glycoside p-nitrophenyl-alpha-D-maltoside with nearly a log-fold higher affinity than maltose, the prototypical competitor. Maltotriose, which has the same linkage pattern as the maltoside, bound with intermediate affinity. Site-directed substitution of leucine for phenylalanine 335 (Phe-335) decreased affinities for the maltoside and maltotriose without significantly altering the affinity for maltose or glucose, and substitution of tyrosine or tryptophan for leucine restored preferential binding to maltotriose and the maltoside. A mutant with alanine at this position failed to bind to mannan or maltose-substituted solid supports. Crystallographic analysis of the human neck+CRD complexed with maltotriose or p-nitrophenyl-maltoside showed stacking of the terminal glucose or nitrophenyl ring with the aromatic ring of Phe-335. Our studies indicate that Phe-335, which is evolutionarily conserved in all known SP-Ds, plays important, if not critical, roles in SP-D function. PMID:16636058

  8. Interactions across the interface contribute the stability of homodimeric 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase.

    PubMed

    Hwang, Chi-Ching; Hsu, Chao-Nan; Huang, Tzu-Jung; Chiou, Shean-Jaw; Hong, Yi-Ren

    2009-10-01

    The dimerization of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase was studied by interrupting the salt bridge interactions between D249 and R167 in the dimeric interface. Substitution of alanine, lysine and serine for D249 decreased catalytic efficiency 30, 1400 and 1.4-fold, and lowered the melting temperature 6.9, 5.4 and 7.6 degrees C, respectively. The mutated enzymes have the dimeric species but the equilibrium between monomer and dimer for these mutants varies from each other, implying that these residues might contribute differently to the dimer stability. Thermal and urea-induced unfolding profiles for wild-type and mutant enzymes appeared as a two-state transition and three-state transition, respectively. In addition, mutation on D249 breaks the salt bridges and causes different effects on the loss of enzymatic activity for D249A, D249K and D249S mutants in the urea-induced unfolding profiles. Hence, D249 at the dimeric interface in 3alpha-HSD/CR is essential for conformational stability, oligomeric integrity and enzymatic activity. PMID:19683506

  9. 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.

  10. Contributions of Phenylalanine 335 to Ligand Recognition by Human Surfactant Protein D: Ring Interactions with SP-D Ligands

    SciTech Connect

    Crouch,E.; McDonald, B.; Smith, K.; Cararella, T.; Seaton, B.; Head, J.

    2006-01-01

    Surfactant Protein D (SP-D) is an innate immune effector that contributes to antimicrobial host defense and immune regulation. Interactions of SP-D with microorganisms and organic antigens involve binding of glycoconjugates to the C-type lectin carbohydrate recognition domain (CRD). A trimeric fusion protein encoding the human neck+CRD (hNCRD) bound to the aromatic glycoside, p-nitrophenyl-alpha-D-maltoside, with nearly a log-fold higher affinity than maltose, the prototypical competitor. Maltotriose, which has the same linkage pattern as the maltoside, bound with intermediate affinity. Site-directed substitution of leucine for phenylalanine 335 (Phe335) decreased affinities for the maltoside and maltotriose without significantly altering the affinity for maltose or glucose, and substitution of tyrosine or tryptophan for leucine restored preferential binding to maltotriose and the maltoside. A mutant with alanine at this position failed to bind to mannan or maltose-substituted solid supports. Crystallographic analysis of the hNCRD complexed with maltotriose or p-nitrophenyl-maltoside showed stacking of the terminal glucose or nitrophenyl ring with the aromatic ring of Phe335. Our studies indicate that Phe335, which is evolutionarily conserved in all known SP-Ds, plays important - if not critical roles - in SP-D function.

  11. 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

  12. 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

  13. Structural insights into the EB1–APC interaction

    PubMed Central

    Honnappa, Srinivas; John, Corinne M; Kostrewa, Dirk; Winkler, Fritz K; Steinmetz, Michel O

    2005-01-01

    EB1 proteins bind to microtubule ends where they act in concert with other components, including the adenomatous polyposis coli (APC) tumor suppressor, to regulate the microtubule filament system. We find that EB1 is a stable dimer with a parallel coiled coil and show that dimerization is essential for the formation of its C-terminal domain (EB1-C). The crystal structure of EB1-C reveals a highly conserved surface patch with a deep hydrophobic cavity at its center. EB1-C binds two copies of an APC-derived C-terminal peptide (C-APCp1) with equal 5 μM affinity. The conserved APC Ile2805–Pro2806 sequence motif serves as an anchor for the interaction of C-APCp1 with the hydrophobic cavity of EB1-C. Phosphorylation of the conserved Cdc2 site Ser2789–Lys2792 in C-APCp1 reduces binding four-fold, indicating that the interaction APC–EB1 is post-translationally regulated in cells. Our findings provide a basis for understanding the dynamic crosstalk of EB1 proteins with their molecular targets in eukaryotic organisms. PMID:15616574

  14. 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

  15. 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

  16. Progesterone receptor isoforms PRA and PRB differentially contribute to breast cancer cell migration through interaction with focal adhesion kinase complexes.

    PubMed

    Bellance, Catherine; Khan, Junaid A; Meduri, Geri; Guiochon-Mantel, Anne; Lombès, Marc; Loosfelt, Hugues

    2013-05-01

    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-dimethyl-amino)-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.

  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. The Contribution of High-Order Metabolic Interactions to the Global Activity of a Four-Species Microbial Community

    PubMed Central

    Guo, Xiaokan

    2016-01-01

    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

  20. 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

  1. 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

  2. Pan1 is an intrinsically disordered protein with homotypic interactions

    PubMed Central

    Pierce, B. D.; Toptygin, D.; Wendland, B.

    2013-01-01

    The yeast scaffold protein Pan1 contains two EH domains at its N-terminus, a predicted coiled-coil central region, and a C-terminal proline-rich domain. Pan1 is also predicted to contain regions of intrinsic disorder, characteristic of proteins that have many binding partners. In vitro biochemical data suggest that Pan1 exists as a dimer, and we have identified amino acids 705–848 as critical for this homotypic interaction. Tryptophan fluorescence was used to further characterize Pan1 conformational states. Pan1 contains four endogenous tryptophans, each in a distinct region of the protein: Trp312 and Trp642 are each in an EH domain, Trp957 is in the central region, and Trp1280 is a critical residue in the Arp2/3 activation domain. To examine the local environment of each of these tryptophans, three of the four tryptophans were mutagenized to phenylalanine to create four proteins, each with only one tryptophan residue. When quenched with acrylamide, these single tryptophan mutants appeared to undergo collisional quenching exclusively and were moderately accessible to the acrylamide molecule. Quenching with iodide or cesium, however, revealed different Stern-Volmer constants due to unique electrostatic environments of the tryptophan residues. Time-resolved fluorescence anisotropy data confirmed structural and disorder predictions of Pan1. Further experimentation to fully develop a model of Pan1 conformational dynamics will assist in a deeper understanding of the mechanisms of endocytosis. PMID:23801378

  3. 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…

  4. Identification of Tpr and α-actinin-4 as two novel SLK-interacting proteins.

    PubMed

    Jaberi, Aala; Hooker, Erika; Guillemette, Julie; Papillon, Joan; Kristof, Arnold S; Cybulsky, Andrey V

    2015-10-01

    Expression and activity of the Ste20-like kinase, SLK, are increased during kidney development and recovery from ischemia-reperfusion injury. SLK mediates apoptosis in various cells, and can regulate cell cycle progression and cytoskeletal remodeling. In cells, SLK is detected in a high molecular mass complex, suggesting that SLK is a dimer/oligomer, or is in tight association with other proteins. To better understand the regulation, localization and function of SLK, we sought to identify proteins in this high molecular mass complex. Analysis by mass spectroscopy identified the nucleoporin, translocated promoter region (Tpr), and the cytoskeletal protein, α-actinin-4, as potential SLK-interacting proteins. Using a protein complementation assay, we showed that the 350 amino acid C-terminal, coiled-coil domain of SLK was responsible for homodimerization, as well as interaction with Tpr and α-actinin-4. The association of SLK with Tpr and α-actinin-4, respectively, was confirmed by co-immunoprecipitation. Subsets of total cellular SLK colocalized with Tpr at the nuclear envelope, and α-actinin-4 in the cytoplasm. Expression of Tpr attenuated activation-specific autophosphorylation of SLK, and blocked SLK-induced apoptosis and AP-1 activity. In contrast to the effect of Tpr, autophosphorylation of SLK was not affected by α-actinin-4. Thus, SLK interacts with Tpr and α-actinin-4 in cells, and these protein-protein interactions may control the subcellular localization and the biological activity of SLK.

  5. 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

  6. A Peek into Tropomyosin Binding and Unfolding on the Actin Filament

    PubMed Central

    Singh, Abhishek; Hitchcock-DeGregori, Sarah E.

    2009-01-01

    Background Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin. Principal Findings Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle α-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven) increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering), and chain dissociation (analyzed using circular dichroism). Conclusions This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest that local disorder is a

  7. 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.

  8. The Implicit Contribution of Slab Modes to the Perpendicular Diffusion Coefficient of Particles Interacting with Two-component Turbulence

    NASA Astrophysics Data System (ADS)

    Shalchi, A.

    2016-10-01

    We explore the transport of energetic particles in two-component turbulence in which the stochastic magnetic field is assumed to be a superposition of slab and two-dimensional modes. It is known that in magnetostatic slab turbulence, the motion of particles across the mean magnetic field is subdiffusive. If a two-dimensional component is added, diffusion is recovered. It was also shown before that in two-component turbulence, the slab modes do not explicitly contribute to the perpendicular diffusion coefficient. In the current paper, the implicit contribution of slab modes is explored and it is shown that this contribution leads to a reduction of the perpendicular diffusion coefficient. This effect improves the agreement between simulations and analytical theory. Furthermore, the obtained results are relevant for investigations of diffusive shock acceleration.

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

    PubMed

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

    2013-07-10

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

  10. 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

  11. 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...

  12. 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…

  13. 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

  14. 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…

  15. The Contribution of Different Patterns of Teachers' Interactions to Young Children's Experiences of Democratic Values during Play

    ERIC Educational Resources Information Center

    Zachrisen, Berit

    2016-01-01

    Developing a sense of belonging and experiences about the value of community are important democratic values that children may learn during play in preschool. Through the different ways that teachers' interact with children during play, children can learn about democratic values. This study is part of a Nordic project on values education in early…

  16. Regional aspects of the North American land surface: Atmosphere interactions and their contributions to the variability and predictability of the regional hydrologic cycle

    NASA Astrophysics Data System (ADS)

    Luo, Yan

    In this study, we investigate the pathways responsible for soil moisture-precipitation interactions and the mechanisms for soil moisture memory at regional scales through analysis of NCEP's North American Regional Reanalysis dataset, which is derived from a system using the mesoscale Eta model coupled with Noah land surface model. The consideration of the relative availability of water and energy leads to the relative strengths of land-atmosphere interaction and soil moisture memory, which are related to the predictability of the regional hydrologic cycle. The seasonal and geographical variations in estimated interaction and memory may establish the relative predictability among the North American basins. The potential for seasonal predictability of the regional hydrologic cycle is conditioned by the foreknowledge of the land surface soil state, which contributes significantly to summer precipitation: (i) The precipitation variability and predictability by strong land-atmosphere interactions are most important in the monsoon regions of Mexico; (ii) Although strong in interactions, the poor soil moisture memory in the Colorado basin and the western part of the Mississippi basin lowers the predictability; (iii) The Columbia basin and the eastern part of the Mississippi basin also stand out as low predictability basins, in that they have good soil moisture memory, but weak strength in interactions, limiting their predictabilities. Our analysis has revealed a highly physically and statistically consistent picture, providing solid support to studies of predictability based on model simulations.

  17. 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

  18. 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

  19. 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

  20. Direct interaction of the N-terminal domain of ribosomal protein S1 with protein S2 in Escherichia coli.

    PubMed

    Byrgazov, Konstantin; Manoharadas, Salim; Kaberdina, Anna C; Vesper, Oliver; Moll, Isabella

    2012-01-01

    Despite of the high resolution structure available for the E. coli ribosome, hitherto the structure and localization of the essential ribosomal protein S1 on the 30 S subunit still remains to be elucidated. It was previously reported that protein S1 binds to the ribosome via protein-protein interaction at the two N-terminal domains. Moreover, protein S2 was shown to be required for binding of protein S1 to the ribosome. Here, we present evidence that the N-terminal domain of S1 (amino acids 1-106; S1(106)) is necessary and sufficient for the interaction with protein S2 as well as for ribosome binding. We show that over production of protein S1(106) affects E. coli growth by displacing native protein S1 from its binding pocket on the ribosome. In addition, our data reveal that the coiled-coil domain of protein S2 (S2α(2)) is sufficient to allow protein S1 to bind to the ribosome. Taken together, these data uncover the crucial elements required for the S1/S2 interaction, which is pivotal for translation initiation on canonical mRNAs in gram-negative bacteria. The results are discussed in terms of a model wherein the S1/S2 interaction surface could represent a possible target to modulate the selectivity of the translational machinery and thereby alter the translational program under distinct conditions.

  1. Proton-linked contributions to site-specific interactions of lambda cI repressor and OR.

    PubMed

    Senear, D F; Ackers, G K

    1990-07-17

    The effects of proton activity on the site-specific interactions of cI repressors with operator sites OR were studied by using DNase I footprint titration. Individual-site binding isotherms were obtained for the binding of repressor to each site of wild-type OR and of mutant operators in which binding to some sites is eliminated. The Gibbs energies for binding and for cooperativity (in every operator configuration) were determined at each pH (range 5-8). The proton-linked effects clearly account for a significant fraction of the difference in affinities for the three operator sites. The most dramatic effects on the repressor-operator binding interactions are at acid pH, and therefore do not involve the basic groups in the repressor N-terminal arm known to contact the DNA. Also, the proton-linked effects are different at the three operator sites as indicated by significantly different derivative relationships, partial derivative of ln k versus partial derivative of ln aH = net proton absorption (delta nu bar(H)). These results implicate ionizable repressor groups which may not contact the DNA and conformational differences between the three repressor-operator site complexes as being important components to the mechanism of site specificity. The extensive data base generated by these studies was also used to reevaluate the traditional models used to describe cooperativity in this system. The results confirm the lack of significant cooperative interaction between OR1 and OR3 at all conditions. However, the data for some experimental conditions are clearly inconsistent with the (selection) rule, that cooperative interaction between OR2 and OR3 is eliminated by ligation at OR1.

  2. 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

  3. 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.

  4. Therapeutic potential of mitotic interaction between the nucleoporin Tpr and aurora kinase A.

    PubMed

    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.

  5. Intracellular domains interactions and gated motions of IKS potassium channel subunits

    PubMed Central

    Haitin, Yoni; Wiener, Reuven; Shaham, Dana; Peretz, Asher; Cohen, Enbal Ben-Tal; Shamgar, Liora; Pongs, Olaf; Hirsch, Joel A; Attali, Bernard

    2009-01-01

    Voltage-gated K+ channels co-assemble with auxiliary β subunits to form macromolecular complexes. In heart, assembly of Kv7.1 pore-forming subunits with KCNE1 β subunits generates the repolarizing K+ current IKS. However, the detailed nature of their interface remains unknown. Mutations in either Kv7.1 or KCNE1 produce the life-threatening long or short QT syndromes. Here, we studied the interactions and voltage-dependent motions of IKS channel intracellular domains, using fluorescence resonance energy transfer combined with voltage-clamp recording and in vitro binding of purified proteins. The results indicate that the KCNE1 distal C-terminus interacts with the coiled-coil helix C of the Kv7.1 tetramerization domain. This association is important for IKS channel assembly rules as underscored by Kv7.1 current inhibition produced by a dominant-negative C-terminal domain. On channel opening, the C-termini of Kv7.1 and KCNE1 come close together. Co-expression of Kv7.1 with the KCNE1 long QT mutant D76N abolished the K+ currents and gated motions. Thus, during channel gating KCNE1 is not static. Instead, the C-termini of both subunits experience molecular motions, which are disrupted by the D76N causing disease mutation. PMID:19521339

  6. Modes of interaction between the Arabidopsis Rab protein, Ara4, and its putative regulator molecules revealed by a yeast expression system.

    PubMed

    Ueda, T; Matsuda, N; Uchimiya, H; Nakano, A

    2000-02-01

    Ara4, a member of the Rab/Ypt GTPase family derived from Arabidopsis thaliana, causes severe growth inhibition when expressed in several yeast ypt mutants. Mutational analysis of ARA4 indicated that the Ara4 protein titrates at least three factors in yeast, including the GDP dissociation inhibitor (GDI). The coexpression of AtGDI1 (Arabidopsis GDI) suppressed the growth defect caused by Ara4 in yeast ypt1, suggesting that Ara4 and AtGDI1 interact in yeast to compensate for the titration of yeast GDI. We screened an Arabidopsis cDNA library for other suppressors that may also interact with Ara4 physiologically. A novel suppressor, SAY1, encoded a hydrophilic protein with two putative coiled-coil regions, which showed partial similarity to the yeast Vps27 protein. To understand the structural requirements of Ara4 for interacting with these molecules, we examined whether AtGDI1 and SAY1 could suppress the growth defect of ypt1 caused by various mutant versions of ARA4. The results indicated that the interaction between Ara4 and AtGDI1 depends on the conserved C-terminal Cys-motif and Thr44 in the effector domain of Ara4. In contrast, neither of these motifs is necessary for the interaction between Say1 and Ara4. This approach provides a powerful method to dissect complex interactions between a GTPase and its regulators.

  7. 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

  8. Higher-order electric multipole contributions to retarded non-additive three-body dispersion interaction energies between atoms: equilateral triangle and collinear configurations.

    PubMed

    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(-11) dependence on separation distance for the DDQ interaction, and an R(-13) behaviour for DQQ and DDO shifts, agreeing with an earlier semi-classical computation. Retardation weakens the potential in each case by R(-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. PMID:24387355

  9. 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.

  10. 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.

  11. 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

  12. Interactions among Vascular-Tone Modulators Contribute to High Altitude Pulmonary Edema and Augmented Vasoreactivity in Highlanders

    PubMed Central

    Ali, Zahara; Mishra, Aastha; Kumar, Rahul; Alam, Perwez; Pandey, Priyanka; Ram, Rekhbala; Thinlas, Tashi; Mohammad, Ghulam; Pasha, M. A. Qadar

    2012-01-01

    Background The interactions among various biomarkers remained unexplored under the stressful environment of high-altitude. Present study evaluated interactions among biomarkers to study susceptibility for high altitude pulmonary edema (HAPE) in HAPE-patients (HAPE-p) and adaptation in highland natives (HLs); both in comparison to HAPE-free sojourners (HAPE-f). Methodology/Principal Findings All the subjects were recruited at 3500 m. We measured clinical parameters, biochemical levels in plasma and gene expression using RNA from blood; analyzed various correlations between and among the clinical parameters, especially arterial oxygen saturation (SaO2) and mean arterial pressure (MAP) and biochemical parameters like, asymmetric dimethylarginine (ADMA), serotonin (5-HT), 8-iso-prostaglandin F2α (8-isoPGF2α), endothelin-1 (ET-1), plasma renin activity (PRA), plasma aldosterone concentration (PAC), superoxide dismutase (SOD) and nitric oxide (NO) in HAPE-p, HAPE-f and HLs. ADMA, 5-HT, 8-isoPGF2α, ET-1 levels, and PAC were significantly higher (p<0.0001, each), whereas SOD activity and NO level were significantly lower in HAPE-p than HAPE-f (p≤0.001). Furthermore, ADMA, 5-HT, 8-isoPGF2α, NO levels and PAC were significantly higher (p<0.0001), whereas ET-1 level significantly (p<0.0001) and SOD activity non-significantly (p>0.05) lower in HLs than HAPE-f. The expression of respective genes differed in the three groups. In the correlations, SaO2 inversely correlated with ADMA, 5-HT and 8-isoPGF2α and positively with SOD in HAPE-p (p≤0.009). MAP correlated positively with 5-HT and 8-isoPGF2α in HAPE-p and HLs (p≤0.004). A strong positive correlation was observed between ADMA and 5-HT, 5-HT and 8-isoPGF2α (p≤0.001), whereas inverse correlation of SOD with ET-1 in HAPE-p and HLs (p≤0.004), with 5-HT and 8-isoPGF2α in HAPE-p (p = 0.01) and with 5-HT in HLs (p = 0.05). Conclusions/Significance The interactions among these markers confer enhanced

  13. BCL6 interacting corepressor contributes to germinal center T follicular helper cell formation and B cell helper function

    PubMed Central

    Yang, Jessica A.; Tubo, Noah J.; Gearhart, Micah D.; Bardwell, Vivian J.; Jenkins, Marc K.

    2015-01-01

    CD4+ germinal center (GC) T follicular helper (GC-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:MHCII-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 differentiation and humoral immunity. PMID:25964495

  14. 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.

  15. Charge-surrounded pockets and electrostatic interactions with small ions modulate the activity of retroviral fusion proteins.

    PubMed

    Lamb, Daniel; Schüttelkopf, Alexander W; van Aalten, Daan M F; Brighty, David W

    2011-02-03

    Refolding of viral class-1 membrane fusion proteins from a native state to a trimer-of-hairpins structure promotes entry of viruses into cells. Here we present the structure of the bovine leukaemia virus transmembrane glycoprotein (TM) and identify a group of asparagine residues at the membrane-distal end of the trimer-of-hairpins that is strikingly conserved among divergent viruses. These asparagines are not essential for surface display of pre-fusogenic envelope. Instead, substitution of these residues dramatically disrupts membrane fusion. Our data indicate that, through electrostatic interactions with a chloride ion, the asparagine residues promote assembly and profoundly stabilize the fusion-active structures that are required for viral envelope-mediated membrane fusion. Moreover, the BLV TM structure also reveals a charge-surrounded hydrophobic pocket on the central coiled coil and interactions with basic residues that cluster around this pocket are critical to membrane fusion and form a target for peptide inhibitors of envelope function. Charge-surrounded pockets and electrostatic interactions with small ions are common among class-1 fusion proteins, suggesting that small molecules that specifically target such motifs should prevent assembly of the trimer-of-hairpins and be of value as therapeutic inhibitors of viral entry.

  16. 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

  17. NMR characterization of the interaction of the Salmonella type III secretion system protein SipD and bile salts.

    PubMed

    Wang, Yu; Nordhues, Bryce A; Zhong, Dalian; De Guzman, Roberto N

    2010-05-18

    Salmonella and Shigella bacteria require the type III secretion system (T3SS) to inject virulence proteins into their hosts and initiate infections. The tip proteins SipD and IpaD are critical components of the Salmonella and Shigella T3SS, respectively. Recently, SipD and IpaD have been shown to interact with bile salts, which are enriched in the intestines, and are hypothesized to act as environmental sensors for these enteric pathogens. Bile salts activate the Shigella T3SS but repress the Salmonella T3SS, and the mechanism of this differing response to bile salts is poorly understood. Further, how SipD binds to bile salts is currently unknown. Computer modeling predicted that IpaD binds the bile salt deoxycholate in a cleft formed by the N-terminal domain and the long central coiled coil of IpaD. Here, we used NMR methods to determine which SipD residues are affected by the interaction with the bile salts deoxycholate, chenodeoxycholate, and taurodeoxcholate. The bile salts perturbed nearly the same set of SipD residues; however, the largest chemical shift perturbations occurred away from what was predicted for the bile salt binding site in IpaD. Our NMR results indicate that that bile salt interaction of SipD will be different from what was predicted for IpaD, suggesting a possible mechanism for the differing response of Salmonella and Shigella to bile salts.

  18. Inhibition of CYP2C19 and CYP3A4 by Omeprazole Metabolites and Their Contribution to Drug-Drug Interactions

    PubMed Central

    Shirasaka, Yoshiyuki; Sager, Jennifer E.; Lutz, Justin D.; Davis, Connie

    2013-01-01

    The aim of this study was to evaluate the contribution of metabolites to drug-drug interactions (DDI) using the inhibition of CYP2C19 and CYP3A4 by omeprazole and its metabolites as a model. Of the metabolites identified in vivo, 5-hydroxyomeprazole, 5′-O-desmethylomeprazole, omeprazole sulfone, and carboxyomeprazole had a metabolite to parent area under the plasma concentration–time curve (AUCm/AUCp) ratio ≥ 0.25 when either total or unbound concentrations were measured after a single 20-mg dose of omeprazole in a cocktail. All of the metabolites inhibited CYP2C19 and CYP3A4 reversibly. In addition omeprazole, omeprazole sulfone, and 5′-O-desmethylomeprazole were time dependent inhibitors (TDI) of CYP2C19, whereas omeprazole and 5′-O-desmethylomeprazole were found to be TDIs of CYP3A4. The in vitro inhibition constants and in vivo plasma concentrations were used to evaluate whether characterization of the metabolites affected DDI risk assessment. Identifying omeprazole as a TDI of both CYP2C19 and CYP3A4 was the most important factor in DDI risk assessment. Consideration of reversible inhibition by omeprazole and its metabolites would not identify DDI risk with CYP3A4, and with CYP2C19, reversible inhibition values would only identify DDI risk if the metabolites were included in the assessment. On the basis of inactivation data, CYP2C19 and CYP3A4 inhibition by omeprazole would be sufficient to identify risk, but metabolites were predicted to contribute 30–63% to the in vivo hepatic interactions. Therefore, consideration of metabolites may be important in quantitative predictions of in vivo DDIs. The results of this study show that, although metabolites contribute to in vivo DDIs, their relative abundance in circulation or logP values do not predict their contribution to in vivo DDI risk. PMID:23620487

  19. Low level of LAT-PLC-γ1 interaction is associated with Th2 polarized differentiation: a contributing factor to the etiology of asthma.

    PubMed

    Peng, Xiaohua; Cui, Zhilei; Gu, Wen; Xu, Weiguo; Guo, Xuejun

    2014-07-01

    Linker for activation of T cells (LAT) is a key adaptor in the T cell receptor (TCR) signaling pathway. The expression of LAT is lower in asthmatic patients than that in healthy people, but there is little knowledge about the mechanism underlying this phenomenon. This study was aimed to determine whether LAT-PLC-γ1 interaction was involved in the development of asthma. It was shown that the phosphorylation of PLC-γ1 decreased in the asthmatic mouse model and Th2 cell differentiated CD4(+) T cells. In addition, depleted endogenous PLC-γ1 promoted CD4(+) T cells to differentiate into IL-4-Productor. It was therefore concluded that the low level of LAT-PLC-γ1 interaction was associated with Th2 polarized differentiation, and this may contribute to the etiology of asthma.

  20. The Interaction of the Cellular Export Adaptor Protein Aly/REF with ICP27 Contributes to the Efficiency of Herpes Simplex Virus 1 mRNA Export

    PubMed Central

    Tian, Xiaochen; Devi-Rao, Gayathri; Golovanov, Alexander P.

    2013-01-01

    Herpes simplex virus 1 (HSV-1) protein ICP27 enables viral mRNA export by accessing the cellular mRNA export receptor TAP/NXF, which guides mRNA through the nuclear pore complex. ICP27 binds viral mRNAs and interacts with TAP/NXF, providing a link to the cellular mRNA export pathway. ICP27 also interacts with the mRNA export adaptor protein Aly/REF, which binds cellular mRNAs and also interacts with TAP/NXF. Studies using small interfering RNA (siRNA) knockdown indicated that Aly/REF is not required for cellular mRNA export, and similar knockdown studies during HSV-1 infection led us to conclude that Aly/REF may be dispensable for viral RNA export. Recently, the structural basis of the interaction of ICP27 with Aly/REF was elucidated at atomic resolution, and it was shown that three ICP27 residues, W105, R107, and L108, interface with the RNA recognition motif (RRM) domain of Aly/REF. Here, to determine the role the interaction of ICP27 and Aly/REF plays during infection, these residues were mutated to alanine, and a recombinant virus, WRL-A, was constructed. Virus production was reduced about 10-fold during WRL-A infection, and export of ICP27 protein and most viral mRNAs was less efficient. We conclude that interaction of ICP27 with Aly/REF contributes to efficient viral mRNA export. PMID:23637401

  1. The interaction of the cellular export adaptor protein Aly/REF with ICP27 contributes to the efficiency of herpes simplex virus 1 mRNA export.

    PubMed

    Tian, Xiaochen; Devi-Rao, Gayathri; Golovanov, Alexander P; Sandri-Goldin, Rozanne M

    2013-07-01

    Herpes simplex virus 1 (HSV-1) protein ICP27 enables viral mRNA export by accessing the cellular mRNA export receptor TAP/NXF, which guides mRNA through the nuclear pore complex. ICP27 binds viral mRNAs and interacts with TAP/NXF, providing a link to the cellular mRNA export pathway. ICP27 also interacts with the mRNA export adaptor protein Aly/REF, which binds cellular mRNAs and also interacts with TAP/NXF. Studies using small interfering RNA (siRNA) knockdown indicated that Aly/REF is not required for cellular mRNA export, and similar knockdown studies during HSV-1 infection led us to conclude that Aly/REF may be dispensable for viral RNA export. Recently, the structural basis of the interaction of ICP27 with Aly/REF was elucidated at atomic resolution, and it was shown that three ICP27 residues, W105, R107, and L108, interface with the RNA recognition motif (RRM) domain of Aly/REF. Here, to determine the role the interaction of ICP27 and Aly/REF plays during infection, these residues were mutated to alanine, and a recombinant virus, WRL-A, was constructed. Virus production was reduced about 10-fold during WRL-A infection, and export of ICP27 protein and most viral mRNAs was less efficient. We conclude that interaction of ICP27 with Aly/REF contributes to efficient viral mRNA export.

  2. Non-bonded interactions and its contribution to the NLO activity of Glycine Sodium Nitrate A vibrational approach

    NASA Astrophysics Data System (ADS)

    Vijayakumar, T.; Hubert Joe, I.; Reghunadhan Nair, C. P.; Jayakumar, V. S.

    2008-04-01

    Vibrational spectral analysis of the novel nonlinear optical (NLO) material, Glycine Sodium Nitrate (GSN) is carried out using NIR FT-Raman and FT-IR spectroscopy, supported by Density Functional Theoretical (DFT) computations to derive equilibrium geometry, vibrational wave numbers and first hyperpolarizability. The reasonable NLO efficiency, predicted for the first time in this novel compound, has been confirmed by Kurtz-Perry powder SHG experiments. The influence of Twisted Intramolecular Charge Transfer (TICT) caused by the strong ionic ground state hydrogen bonding between charged species making GSN crystal to have the non-centrosymmetric structure has been discussed. The shortening of C sbnd H bond lengths, blue-shifting of the stretching frequencies and intensity variation indicating the existence of 'blue-shift or improper' C sbnd H⋯O hydrogen bonding. The intense low wavenumber H-bond Raman vibrations due to electron-phonon coupling and non-bonded interactions in making the molecule NLO active have been analyzed based on the vibrational spectral features. The Natural Bond Orbital (NBO) analysis confirms the occurrence of a strong intra- and intermolecular N sbnd H⋯O and C sbnd H⋯O hydrogen bonds.

  3. 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

  4. 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

  5. Viral Interactions with B-Cells Contribute to Increased Regulatory T-Cells During Chronic HCV Infection

    PubMed Central

    Ayers, Chris L.; Firan, Mihail; Pillai, Vinodh; Lee, William M.

    2011-01-01

    Abstract Hepatitis C virus (HCV) has a propensity to establish chronic infection that is characterized by attenuated virus-specific T-cell responses. Mechanisms leading to T-cell attenuation are poorly understood and likely involve dysfunctional interactions between antigen-presenting cells (APC) and effector/regulatory T-cells. Reports on dendritic cells (DC) have described only minor dysfunction during HCV infection. However, there is a paucity of reports regarding B-cell function, despite clear associations with B-cell-related secondary sequelae. In this study we evaluated the state of B-cells during chronic HCV infection, and observed a diminished ability to respond to mitogenic stimuli, correlating with increased apoptosis. This was in contrast to their ex vivo phenotype, which indicated ongoing chronic activation in vivo. There was a high association of HCV-positive strand RNA with B-cells in a subset of HCV patients. Interestingly, ex-vivo-derived HCV RNA-positive B-cells induced significantly greater proliferation in allogeneic T-cells than in HCV-negative B-cells, correlating with an increased generation of CD4+CD25+FOXP3+ regulatory T-cells (Tregs). In-vitro exposure of healthy peripheral blood mononuclear cells (PBMC) to HCV resulted in robust activation of resting B-cells. These HCV-exposed B-cells also showed an enhanced ability to generate Tregs. Our results provide strong evidence for a novel and paradoxical link between HCV-induced enhanced APC function and the generation of Tregs. PMID:21449722

  6. Quantitative and temporal definition of the Mla transcriptional regulon during barley-powdery mildew interactions.

    PubMed

    Moscou, Matthew J; Lauter, Nick; Caldo, Rico A; Nettleton, Dan; Wise, Roger P

    2011-06-01

    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 complementary avirulence effectors (AVR(a)) are present in the pathogen. Presence of an appropriate AVR(a) protein triggers nuclear relocalization of MLA, in which MLA binds repressing host transcription factors. Timecourse expression profiles of plants harboring Mla1, Mla6, and Mla12 wild-type alleles versus paired loss-of-function mutants were compared to discover conserved transcriptional targets of MLA and downstream signaling cascades. Pathogen-dependent gene expression was equivalent or stronger in susceptible plants at 20 h after inoculation (HAI) and was attenuated at later timepoints, whereas resistant plants exhibited a time-dependent strengthening of the transcriptional response, increasing in both fold change and the number of genes differentially expressed. Deregulation at 20 HAI implicated 16 HAI as a crucial point in determining the future trajectory of this interaction and was interrogated by quantitative analysis. In total, 28 potential transcriptional targets of the MLA regulon were identified. These candidate targets possess a diverse set of predicted functions, suggesting that multiple pathways are required to mediate the hypersensitive reaction.

  7. PprA contributes to Deinococcus radiodurans resistance to nalidixic acid, genome maintenance after DNA damage and interacts with deinococcal topoisomerases.

    PubMed

    Kota, Swathi; Charaka, Vijaya K; Ringgaard, Simon; Waldor, Matthew K; Misra, Hari S

    2014-01-01

    PprA is known to contribute to Deinococcus radiodurans' remarkable capacity to survive a variety of genotoxic assaults. The molecular bases for PprA's role(s) in the maintenance of the damaged D. radiodurans genome are incompletely understood, but PprA is thought to promote D. radiodurans's capacity for DSB repair. PprA is found in a multiprotein DNA processing complex along with an ATP type DNA ligase, and the D. radiodurans toposiomerase IB (DraTopoIB) as well as other proteins. Here, we show that PprA is a key contributor to D. radiodurans resistance to nalidixic acid (Nal), an inhibitor of topoisomerase II. Growth of wild type D. radiodurans and a pprA mutant were similar in the absence of exogenous genotoxic insults; however, the pprA mutant exhibited marked growth delay and a higher frequency of anucleate cells following treatment with DNA-damaging agents. We show that PprA interacts with both DraTopoIB and the Gyrase A subunit (DraGyrA) in vivo and that purified PprA enhances DraTopoIB catalysed relaxation of supercoiled DNA. Thus, besides promoting DNA repair, our findings suggest that PprA also contributes to preserving the integrity of the D. radiodurans genome following DNA damage by interacting with DNA topoisomerases and by facilitating the actions of DraTopoIB.

  8. 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.

  9. 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

  10. Interactions between residues 2228-2240 within factor VIIIa C2 domain and factor IXa Gla domain contribute to propagation of clot formation.

    PubMed

    Soeda, T; Nogami, K; Ogiwara, K; Shima, M

    2011-11-01

    Factor (F)VIII functions as a cofactor in the tenase complex responsible for phospholipid (PL)-dependent FXa generation by FIXa. We have recently reported that the FVIIIa C2 domain (residues 2228-2240) interacts with the FIXa Gla domain in this complex. We examined the role of this interaction in the generation of tenase activity during the process of clot formation, using a synthetic peptide corresponding to residues 2228-2240. The peptide 2228-2240 inhibited FVIIIa/FIXa-mediated FX activation dose-dependently in the presence of PL by >95% (IC50; ~10 μM). This effect was significantly greater than that obtained by peptide 1804-1818 (IC50; ~180 μM) which corresponds to another FIXa-interactive site in the light chain that provides the majority of binding energy for FIXa interaction. Peptide 2228-2240 had little effect on the prothrombin time and did not inhibit FIX activation in the coagulation process mediated by FVIIa/tissue factor or FXIa, suggesting specific inhibition of the intrinsic tenase complex. Clot waveform analysis, a plasma based-assay used to evaluate the process of intrinsic coagulation, demonstrated that peptide 2228-2240 significantly depressed both maximum coagulation velocity (|min1|) and acceleration (|min2|), reflecting the propagation of clot formation, although the clotting time was only marginally prolonged. Thromboelastography, an alternative whole blood based-assay, demonstrated that the peptide inhibited clot formation time, α-angle and maximal clot firmness, but had little effect on the clotting time. Interactions of the FVIIIa C2 domain (residues 2228-2240) with the FIXa Gla domain in the tenase complex appeared to contribute essentially to the propagation of clot formation.

  11. Interface contributions to the spin-orbit interaction parameters of electrons at the (001) GaAs/AlGaAs interface

    NASA Astrophysics Data System (ADS)

    Devizorova, Zh. A.; Shchepetilnikov, A. V.; Nefyodov, Yu. A.; Volkov, V. A.; Kukushkin, I. V.

    2014-09-01

    One-body mechanisms of spin splitting of the energy spectrum of 2D electrons in a one-side doped (001) GaAs/Al x Ga1 - x As quantum well have been studied theoretically and experimentally. The interfacial spin splitting has been shown to compensate (enhance) considerably the contribution of the bulk Dresselhaus (Bychkov-Rashba) mechanism. The theoretical approach is based on the solution of the effective mass equation in a quasi-triangular well supplemented by a new boundary condition at a high and atomically sharp hetero-barrier. The model takes into account the spin-orbit interaction of electrons with both bulk and interfacial crystal potential having C 2 v symmetry, as well as the lack of inversion symmetry and nonparabolicity of the conduction band in GaAs. The effective 2D spin Hamiltonian including both bulk and interface contributions to the Dresselhaus (αBIA) and Rashba (αSIA) constants has been derived. The analytical relation between these constants and the components of the anisotropic nonlinear g-factor tensor in an oblique quantizing magnetic field has been found. The experimental approach is based, on one hand, on the detection of electron spin resonance in the microwave range and, on the other hand, on photoluminescence measurements of the nonparabolicity parameter. The interface contributions to αBIA and αSIA have been found from comparison with the theory.

  12. 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

  13. 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.

  14. Myosin Vc Interacts with Rab32 and Rab38 Proteins and Works in the Biogenesis and Secretion of Melanosomes*

    PubMed Central

    Bultema, Jarred J.; Boyle, Judith A.; Malenke, Parker B.; Martin, Faye E.; Dell'Angelica, Esteban C.; Cheney, Richard E.; Di Pietro, Santiago M.

    2014-01-01

    Class V myosins are actin-based motors with conserved functions in vesicle and organelle trafficking. Herein we report the discovery of a function for Myosin Vc in melanosome biogenesis as an effector of melanosome-associated Rab GTPases. We isolated Myosin Vc in a yeast two-hybrid screening for proteins that interact with Rab38, a Rab protein involved in the biogenesis of melanosomes and other lysosome-related organelles. Rab38 and its close homolog Rab32 bind to Myosin Vc but not to Myosin Va or Myosin Vb. Binding depends on residues in the switch II region of Rab32 and Rab38 and regions of the Myosin Vc coiled-coil tail domain. Myosin Vc also interacts with Rab7a and Rab8a but not with Rab11, Rab17, and Rab27. Although Myosin Vc is not particularly abundant on pigmented melanosomes, its knockdown in MNT-1 melanocytes caused defects in the trafficking of integral membrane proteins to melanosomes with substantially increased surface expression of Tyrp1, nearly complete loss of Tyrp2, and significant Vamp7 mislocalization. Knockdown of Myosin Vc in MNT-1 cells more than doubled the abundance of pigmented melanosomes but did not change the number of unpigmented melanosomes. Together the data demonstrate a novel role for Myosin Vc in melanosome biogenesis and secretion. PMID:25324551

  15. 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

  16. 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.

  17. 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

  18. Deoxycholate interacts with IpaD of Shigella flexneri in inducing the recruitment of IpaB to the type III secretion apparatus needle tip.

    PubMed

    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

    2008-07-01

    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.

  19. Numerical solution of Bloch-Gruneisen function to determine the contribution of electron-phonon interaction in polycrystalline MgB 2 superconductor

    NASA Astrophysics Data System (ADS)

    Ansari, Intikhab A.

    2010-06-01

    Here, we report the efficient and feasible analytical method for the generalized Bloch-Gruneisen law in association with Debye temperature and various temperatures range in terms of incomplete gamma function. In addition, our results are in agreement with previous reports as shown in this letter. Bloch-Gruneisen function describes the contribution of electron-phonon interaction to the results of temperature dependence behavior of resistivity for integer and noninteger values of index m. In conclusion, the algorithm is constructed in Fortran 90 language for replicate the variation of temperature dependence of resistivity for pristine MgB 2 sample. Moreover, the comparison of numerical results with the proposed method reveals the validity and precision of the method.

  20. Oviduct-specific glycoprotein and heparin modulate sperm–zona pellucida interaction during fertilization and contribute to the control of polyspermy

    PubMed Central

    Coy, Pilar; Cánovas, Sebastián; Mondéjar, Irene; Saavedra, Maria Dolores; Romar, Raquel; Grullón, Luis; Matás, Carmen; Avilés, Manuel

    2008-01-01

    Polyspermy is an important anomaly of fertilization in placental mammals, causing premature death of the embryo. It is especially frequent under in vitro conditions, complicating the successful generation of viable embryos. A block to polyspermy develops as a result of changes after sperm entry (i.e., cortical granule exocytosis). However, additional factors may play an important role in regulating polyspermy by acting on gametes before sperm–oocyte interaction. Most studies have used rodents as models, but ungulates may differ in mechanisms preventing polyspermy. We hypothesize that zona pellucida (ZP) changes during transit of the oocyte along the oviductal ampulla modulate the interaction with spermatozoa, contributing to the regulation of polyspermy. We report here that periovulatory oviductal fluid (OF) from sows and heifers increases (both, con- and heterospecifically) ZP resistance to digestion with pronase (a parameter commonly used to measure the block to polyspermy), changing from digestion times of ≈1 min (pig) or 2 min (cattle) to 45 min (pig) or several hours (cattle). Exposure of oocytes to OF increases monospermy after in vitro fertilization in both species, and in pigs, sperm–ZP binding decreases. The resistance of OF-exposed oocytes to pronase was abolished by exposure to heparin-depleted medium; in a medium with heparin it was not altered. Proteomic analysis of the content released in the heparin-depleted medium after removal of OF-exposed oocytes allowed the isolation and identification of oviduct-specific glycoprotein. Thus, an oviduct-specific glycoprotein–heparin protein complex seems to be responsible for ZP changes in the oviduct before fertilization, affecting sperm binding and contributing to the regulation of polyspermy. PMID:18838686

  1. 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

  2. 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.

  3. NUP-1 Is a Large Coiled-Coil Nucleoskeletal Protein in Trypanosomes with Lamin-Like Functions

    PubMed Central

    DuBois, Kelly N.; Alsford, Sam; Holden, Jennifer M.; Buisson, Johanna; Swiderski, Michal; Bart, Jean-Mathieu; Ratushny, Alexander V.; Wan, Yakun; Bastin, Philippe; Barry, J. David; Navarro, Miguel; Horn, David; Aitchison, John D.; Rout, Michael P.; Field, Mark C.

    2012-01-01

    A unifying feature of eukaryotic nuclear organization is genome segregation into transcriptionally active euchromatin and transcriptionally repressed heterochromatin. In metazoa, lamin proteins preserve nuclear integrity and higher order heterochromatin organization at the nuclear periphery, but no non-metazoan lamin orthologues have been identified, despite the likely presence of nucleoskeletal elements in many lineages. This suggests a metazoan-specific origin for lamins, and therefore that distinct protein elements must compose the nucleoskeleton in other lineages. The trypanosomatids are highly divergent organisms and possess well-documented but remarkably distinct mechanisms for control of gene expression, including polycistronic transcription and trans-splicing. NUP-1 is a large protein localizing to the nuclear periphery of Trypanosoma brucei and a candidate nucleoskeletal component. We sought to determine if NUP-1 mediates heterochromatin organization and gene regulation at the nuclear periphery by examining the influence of NUP-1 knockdown on morphology, chromatin positioning, and transcription. We demonstrate that NUP-1 is essential and part of a stable network at the inner face of the trypanosome nuclear envelope, since knockdown cells have abnormally shaped nuclei with compromised structural integrity. NUP-1 knockdown also disrupts organization of nuclear pore complexes and chromosomes. Most significantly, we find that NUP-1 is required to maintain the silenced state of developmentally regulated genes at the nuclear periphery; NUP-1 knockdown results in highly specific mis-regulation of telomere-proximal silenced variant surface glycoprotein (VSG) expression sites and procyclin loci, indicating a disruption to normal chromatin organization essential to life-cycle progression. Further, NUP-1 depletion leads to increased VSG switching and therefore appears to have a role in control of antigenic variation. Thus, analogous to vertebrate lamins, NUP-1 is a major component of the nucleoskeleton with key roles in organization of the nuclear periphery, heterochromatin, and epigenetic control of developmentally regulated loci. PMID:22479148

  4. 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.

  5. TCS1, a Microtubule-Binding Protein, Interacts with KCBP/ZWICHEL to Regulate Trichome Cell Shape in Arabidopsis thaliana

    PubMed Central

    Tian, Juan; Wang, Xiaohong; Mao, Tonglin; Yuan, Ming; Li, Yunhai

    2016-01-01

    How cell shape is controlled is a fundamental question in developmental biology, but the genetic and molecular mechanisms that determine cell shape are largely unknown. Arabidopsis trichomes have been used as a good model system to investigate cell shape at the single-cell level. Here we describe the trichome cell shape 1 (tcs1) mutants with the reduced trichome branch number in Arabidopsis. TCS1 encodes a coiled-coil domain-containing protein. Pharmacological analyses and observations of microtubule dynamics show that TCS1 influences the stability of microtubules. Biochemical analyses and live-cell imaging indicate that TCS1 binds to microtubules and promotes the assembly of microtubules. Further results reveal that TCS1 physically associates with KCBP/ZWICHEL, a microtubule motor involved in the regulation of trichome branch number. Genetic analyses indicate that kcbp/zwi is epistatic to tcs1 with respect to trichome branch number. Thus, our findings define a novel genetic and molecular mechanism by which TCS1 interacts with KCBP to regulate trichome cell shape by influencing the stability of microtubules. PMID:27768706

  6. Putative members of the Arabidopsis Nup107-160 nuclear pore sub-complex contribute to pathogen defense.

    PubMed

    Wiermer, Marcel; Cheng, Yu Ti; Imkampe, Julia; Li, Meilan; Wang, Dongmei; Lipka, Volker; Li, Xin

    2012-06-01

    In eukaryotic cells, transduction of external stimuli into the nucleus to induce transcription and export of mRNAs for translation in the cytoplasm is mediated by nuclear pore complexes (NPCs) composed of nucleoporin proteins (Nups). We previously reported that Arabidopsis MOS3, encoding the homolog of vertebrate Nup96, is required for plant immunity and constitutive resistance mediated by the de-regulated Toll interleukin 1 receptor/nucleotide-binding/leucine-rich repeat (TNL)-type R gene snc1. In vertebrates, Nup96 is a component of the conserved Nup107-160 nuclear pore sub-complex, and implicated in immunity-related mRNA export. Here, we used a reverse genetics approach to examine the requirement for additional subunits of the predicted Arabidopsis Nup107-160 complex in plant immunity. We show that, among eight putative complex members, beside MOS3, only plants with defects in Nup160 or Seh1 are impaired in basal resistance. Constitutive resistance in the snc1 mutant and immunity mediated by TNL-type R genes also depend on functional Nup160 and have a partial requirement for Seh1. Conversely, resistance conferred by coiled coil-type immune receptors operates largely independently of both genes, demonstrating specific contributions to plant defense signaling. Our functional analysis further revealed that defects in nup160 and seh1 result in nuclear accumulation of poly(A) mRNA, and, in the case of nup160, considerable depletion of EDS1, a key positive regulator of basal and TNL-triggered resistance. These findings suggest that Nup160 is required for nuclear mRNA export and full expression of EDS1-conditioned resistance pathways in Arabidopsis.

  7. Coactivator TIF1β Interacts with Transcription Factor C/EBPβ and Glucocorticoid Receptor To Induce α1-Acid Glycoprotein Gene Expression

    PubMed Central

    Chang, Ching-Jin; Chen, Ya-Ling; Lee, Sheng-Chung

    1998-01-01

    The transcription of the α1-acid glycoprotein gene is induced by inflammatory cytokines and glucocorticoids. C/EBPβ is a major transcription factor involved in the induction of the agp gene by some cytokines. In this report, we have identified a novel transcriptional intermediary factor, TIF1β, which could enhance the transcription of the agp gene by the glucocorticoid receptor (GR) and C/EBPβ. TIF1β belongs to a subgroup of RING (really interesting new gene) finger proteins that contain a RING finger preceding two B box-type fingers and a putative coiled-coil domain (RBCC domain). Immunoprecipitation experiments showed that the interaction between GR and TIF1β is ligand independent. The overexpression of the TIF1β gene enhances GR-regulated expression in a ligand- and glucocorticoid-responsive element (GRE)-dependent manner. TIF1β can also augment C/EBPβ-mediated activity on wild-type and GRE-mutated agp genes, but this augmentation is diminished when all three C/EBPβ-binding elements are mutated. Functional and biochemical characterizations indicated that the bZIP domain of C/EBPβ and the RBCC domain, plant homeodomain finger, and bromodomain of TIF1β are crucial for the interactions of these proteins. Taken together, these results suggest that TIF1β serves as a converging mediator of signal transduction pathways of glucocorticoids and some inflammatory cytokines. PMID:9742105

  8. Role of the catalytic serine in the interactions of serine proteinases with protein inhibitors of the serpin family. Contribution of a covalent interaction to the binding energy of serpin-proteinase complexes.

    PubMed

    Olson, S T; Bock, P E; Kvassman, J; Shore, J D; Lawrence, D A; Ginsburg, D; Björk, I

    1995-12-15

    The contribution of a covalent bond to the stability of complexes of serine proteinases with inhibitors of the serpin family was evaluated by comparing the affinities of beta-trypsin and the catalytic serine-modified derivative, beta-anhydrotrypsin, for several serpin and non-serpin (Kunitz) inhibitors. Kinetic analyses showed that anhydrotrypsin had little or no ability to compete with trypsin for binding to alpha 1-proteinase inhibitor (alpha 1PI), plasminogen activator inhibitor 1 (PAI-1), antithrombin (AT), or AT-heparin complex when present at up to a 100-fold molar excess over trypsin. By contrast, equimolar levels of anhydrotrypsin blocked trypsin binding to non-serpin inhibitors. Equilibrium binding studies of inhibitor-enzyme interactions monitored by inhibitor displacement of the fluorescence probe, p-aminobenzamidine, from the enzyme active site, confirmed that the binding of serpins to anhydrotrypsin was undetectable in the case of alpha 1PI or AT (KI > 10(-5) M), of low affinity in the case of AT-heparin complex (KI 7-9 x 10(-6) M), and of moderate affinity in the case of PAI-1 (KI 2 x 10(-7) M). This contrasted with the stoichiometric high affinity binding of the serpins to trypsin as well as of the non-serpin inhibitors to both trypsin and anhydrotrypsin. Maximal KI values for serpin-trypsin interactions of 1 to 8 x 10(-11) M, obtained from kinetic analyses of association and dissociation rate constants, indicated that the affinity of serpins for trypsin was minimally 4 to 6 orders of magnitude greater than that of anhydrotrypsin. Anhydrotrypsin, unlike trypsin, failed to induce the characteristic fluorescence changes in a P9 Ser-->Cys PAI-1 variant labeled with a nitrobenzofuran fluorescent probe (NBD) which were shown previously to report the serpin conformational change associated with active enzyme binding. These results demonstrate that a covalent interaction involving the proteinase catalytic serine contributes a major fraction of the binding

  9. 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.

  10. Contribution of metabolites to P450 inhibition-based drug-drug interactions: scholarship from the drug metabolism leadership group of the innovation and quality consortium metabolite group.

    PubMed

    Yu, Hongbin; Balani, Suresh K; Chen, Weichao; Cui, Donghui; He, Ling; Humphreys, W Griffith; Mao, Jialin; Lai, W George; Lee, Anthony J; Lim, Heng-Keang; MacLauchlin, Christopher; Prakash, Chandra; Surapaneni, Sekhar; Tse, Susanna; Upthagrove, Alana; Walsky, Robert L; Wen, Bo; Zeng, Zhaopie

    2015-04-01

    Recent European Medicines Agency (final) and US Food and Drug Administration (draft) drug interaction guidances proposed that human circulating metabolites should be investigated in vitro for their drug-drug interaction (DDI) potential if present at ≥ 25% of the parent area under the time-concentration curve (AUC) (US Food and Drug Administration) or ≥ 25% of the parent and ≥ 10% of the total drug-related AUC (European Medicines Agency). To examine the application of these regulatory recommendations, a group of scientists, representing 18 pharmaceutical companies of the Drug Metabolism Leadership Group of the Innovation and Quality Consortium, conducted a scholarship to assess the risk of contributions by metabolites to cytochrome P450 (P450) inhibition-based DDIs. The group assessed the risk of having a metabolite as the sole contributor to DDI based on literature data and analysis of the 137 most frequently prescribed drugs, defined structural alerts associated with P450 inhibition/inactivation by metabolites, and analyzed current approaches to trigger in vitro DDI studies for metabolites. The group concluded that the risk of P450 inhibition caused by a metabolite alone is low. Only metabolites from 5 of 137 drugs were likely the sole contributor to the in vivo P450 inhibition-based DDIs. Two recommendations were provided when assessing the need to conduct in vitro P450 inhibition studies for metabolites: 1) consider structural alerts that suggest P450 inhibition potential, and 2) use multiple approaches (e.g., a metabolite cut-off value of 100% of the parent AUC and the R(met) strategy) to predict P450 inhibition-based DDIs caused by metabolites in the clinic.

  11. 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

  12. Contribution of metabolites to P450 inhibition-based drug-drug interactions: scholarship from the drug metabolism leadership group of the innovation and quality consortium metabolite group.

    PubMed

    Yu, Hongbin; Balani, Suresh K; Chen, Weichao; Cui, Donghui; He, Ling; Humphreys, W Griffith; Mao, Jialin; Lai, W George; Lee, Anthony J; Lim, Heng-Keang; MacLauchlin, Christopher; Prakash, Chandra; Surapaneni, Sekhar; Tse, Susanna; Upthagrove, Alana; Walsky, Robert L; Wen, Bo; Zeng, Zhaopie

    2015-04-01

    Recent European Medicines Agency (final) and US Food and Drug Administration (draft) drug interaction guidances proposed that human circulating metabolites should be investigated in vitro for their drug-drug interaction (DDI) potential if present at ≥ 25% of the parent area under the time-concentration curve (AUC) (US Food and Drug Administration) or ≥ 25% of the parent and ≥ 10% of the total drug-related AUC (European Medicines Agency). To examine the application of these regulatory recommendations, a group of scientists, representing 18 pharmaceutical companies of the Drug Metabolism Leadership Group of the Innovation and Quality Consortium, conducted a scholarship to assess the risk of contributions by metabolites to cytochrome P450 (P450) inhibition-based DDIs. The group assessed the risk of having a metabolite as the sole contributor to DDI based on literature data and analysis of the 137 most frequently prescribed drugs, defined structural alerts associated with P450 inhibition/inactivation by metabolites, and analyzed current approaches to trigger in vitro DDI studies for metabolites. The group concluded that the risk of P450 inhibition caused by a metabolite alone is low. Only metabolites from 5 of 137 drugs were likely the sole contributor to the in vivo P450 inhibition-based DDIs. Two recommendations were provided when assessing the need to conduct in vitro P450 inhibition studies for metabolites: 1) consider structural alerts that suggest P450 inhibition potential, and 2) use multiple approaches (e.g., a metabolite cut-off value of 100% of the parent AUC and the R(met) strategy) to predict P450 inhibition-based DDIs caused by metabolites in the clinic. PMID:25655830

  13. Deregulated G1–S control and energy stress contribute to the synthetic-lethal interactions between inactivation of RB and TSC1 or TSC2

    PubMed Central

    Gordon, Gabriel M.; Zhang, Tianyi; Zhao, Jiong; Du, Wei

    2013-01-01

    Summary Synthetic lethality is a potential strategy for cancer treatment by specifically promoting the death of cancer cells with particular defects such as the loss of the RB (RB1) tumor suppressor. We previously showed that inactivation of both RB and TSC2 induces synergistic apoptosis during the development of Drosophila melanogaster and in cancer cells. However, the in vivo mechanism of this synthetic-lethal interaction is not clear. Here, we show that synergistic cell death in tissues that have lost the RB and TSC orthologs rbf and dtsc1/gig, respectively, or overexpress Rheb and dE2F1, are correlated with synergistic defects in G1–S control, which causes cells to accumulate DNA damage. Coexpression of the G1–S inhibitor Dap, but not the G2–M inhibitor dWee1, decreases DNA damage and reduces cell death. In addition, we show that rbf and dtsc1 mutant cells are under energy stress, are sensitive to decreased energy levels and depend on the cellular energy stress-response pathway for survival. Decreasing mitochondrial ATP synthesis by inactivating cova or abrogating the energy-stress response by removing the metabolic regulator LKB1 both enhance the elimination of cells lacking either rbf or dtsc1. These observations, in conjunction with the finding that deregulation of TORC1 induces activation of JNK, indicate that multiple cellular stresses are induced and contribute to the synthetic-lethal interactions between RB and TSC1/TSC2 inactivation. The insights gained from this study suggest new approaches for targeting RB-deficient cancers. PMID:23447678

  14. 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.

  15. An Interaction of Renin-Angiotensin and Kallikrein-Kinin Systems Contributes to Vascular Hypertrophy in Angiotensin II-Induced Hypertension: In Vivo and In Vitro Studies

    PubMed Central

    Ceravolo, Graziela S.; Montezano, Augusto C.; Jordão, Maria T.; Akamine, Eliana H.; Costa, Tiago J.; Takano, Ana P.; Fernandes, Denise C.; Barreto-Chaves, Maria L.; Laurindo, Francisco R.; Tostes, Rita C.; Fortes, Zuleica B.; Chopard, Renato P.; Touyz, Rhian M.; Carvalho, Maria Helena C.

    2014-01-01

    The kallikrein-kinin and renin-angiotensin systems interact at multiple levels. In the present study, we tested the hypothesis that the B1 kinin receptor (B1R) contributes to vascular hypertrophy in angiotensin II (ANG II)–induced hypertension, through a mechanism involving reactive oxygen species (ROS) generation and extracellular signal-regulated kinase (ERK1/2) activation. Male Wistar rats were infused with vehicle (control rats), 400 ng/Kg/min ANG II (ANG II rats) or 400 ng/Kg/min ANG II plus B1 receptor antagonist, 350 ng/Kg/min des-Arg9-Leu8-bradykinin (ANGII+DAL rats), via osmotic mini-pumps (14 days) or received ANG II plus losartan (10 mg/Kg, 14 days, gavage - ANG II+LOS rats). After 14 days, ANG II rats exhibited increased systolic arterial pressure [(mmHg) 184±5.9 vs 115±2.3], aortic hypertrophy; increased ROS generation [2-hydroxyethidium/dihydroethidium (EOH/DHE): 21.8±2.7 vs 6.0±1.8] and ERK1/2 phosphorylation (% of control: 218.3±29.4 vs 100±0.25]. B1R expression was increased in aortas from ANG II and ANG II+DAL rats than in aortas from the ANG II+LOS and control groups. B1R antagonism reduced aorta hypertrophy, prevented ROS generation (EOH/DHE: 9.17±3.1) and ERK1/2 phosphorylation (137±20.7%) in ANG II rats. Cultured aortic vascular smooth muscle cells (VSMC) stimulated with low concentrations (0.1 nM) of ANG II plus B1R agonist exhibited increased ROS generation, ERK1/2 phosphorylation, proliferating-cell nuclear antigen expression and [H3]leucine incorporation. At this concentration, neither ANG II nor the B1R agonist produced any effects when tested individually. The ANG II/B1R agonist synergism was inhibited by losartan (AT1 blocker, 10 µM), B1R antagonist (10 µM) and Tiron (superoxide anion scavenger, 10 mM). These data suggest that B1R activation contributes to ANG II-induced aortic hypertrophy. This is associated with activation of redox-regulated ERK1/2 pathway that controls aortic smooth muscle cells growth. Our findings

  16. 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.

  17. 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

  18. Characterization of a 65 kDa NIF in the nuclear matrix of the monocot Allium cepa that interacts with nuclear spectrin-like proteins.

    PubMed

    Pérez-Munive, Clara; Blumenthal, Sonal S D; de la Espina, Susana Moreno Díaz

    2012-01-01

    Plant cells have a well organized nucleus and nuclear matrix, but lack orthologues of the main structural components of the metazoan nuclear matrix. Although data is limited, most plant nuclear structural proteins are coiled-coil proteins, such as the NIFs (nuclear intermediate filaments) in Pisum sativum that cross-react with anti-intermediate filament and anti-lamin antibodies, form filaments 6-12 nm in diameter in vitro, and may play the role of lamins. We have investigated the conservation and features of NIFs in a monocot species, Allium cepa, and compared them with onion lamin-like proteins. Polyclonal antisera against the pea 65 kDa NIF were used in 1D and 2D Western blots, ICM (imunofluorescence confocal microscopy) and IEM (immunoelectron microscopy). Their presence in the nuclear matrix was analysed by differential extraction of nuclei, and their association with structural spectrin-like proteins by co-immunoprecipitation and co-localization in ICM. NIF is a conserved structural component of the nucleus and its matrix in monocots with Mr and pI values similar to those of pea 65 kDa NIF, which localized to the nuclear envelope, perichromatin domains and foci, and to the nuclear matrix, interacting directly with structural nuclear spectrin-like proteins. Its similarities with some of the proteins described as onion lamin-like proteins suggest that they are highly related or perhaps the same proteins.

  19. 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

  20. Promoted interaction of nuclear factor-κB with demethylated cystathionine-β-synthetase gene contributes to gastric hypersensitivity in diabetic rats.

    PubMed

    Zhang, Hong-Hong; Hu, Ji; Zhou, You-Lang; Hu, Shufen; Wang, Yong-Meng; Chen, Wei; Xiao, Ying; Huang, Li-Yen Mae; Jiang, Xinghong; Xu, Guang-Yin

    2013-05-22

    Patients with long-standing diabetes frequently demonstrate gastric hypersensitivity with an unknown mechanism. The present study was designed to investigate roles for nuclear factor-κB (NF-κB) and the endogenous H2S-producing enzyme cystathionine-β-synthetase (CBS) signaling pathways by examining cbs gene methylation status in adult rats with diabetes. Intraperitoneal injection of streptozotocin (STZ) produced gastric hypersensitivity in female rats in response to gastric balloon distention. Treatment with the CBS inhibitor aminooxyacetic acid significantly attenuated STZ-induced gastric hypersensitivity in a dose-dependent fashion. Aminooxyacetic acid treatment also reversed hyperexcitability of gastric-specific dorsal root ganglion (DRG) neurons labeled by the dye DiI in diabetic rats. Conversely, the H2S donor NaHS enhanced neuronal excitability of gastric DRG neurons. Expression of CBS and p65 were markedly enhanced in gastric DRGs in diabetic rats. Blockade of NF-κB signaling using pyrrolidine dithiocarbamate reversed the upregulation of CBS expression. Interestingly, STZ treatment led to a significant demethylation of CpG islands in the cbs gene promoter region, as determined by methylation-specific PCR and bisulfite sequencing. STZ treatment also remarkably downregulated the expression of DNA methyltransferase 3a and 3b. More importantly, STZ treatment significantly enhanced the ability of cbs to bind DNA at the p65 consensus site, as shown by chromatin immunoprecipitation assays. Our findings suggest that upregulation of cbs expression is attributed to cbs promoter DNA demethylation and p65 activation and that the enhanced interaction of the cbs gene and p65 contributes to gastric hypersensitivity in diabetes. This finding may guide the development and evaluation of new treatment modalities for patients with diabetic gastric hypersensitivity.

  1. Contribution of Partial Charge Interactions and Base Stacking to the Efficiency of Primer Extension at and beyond Abasic Sites in DNA

    SciTech Connect

    Xia, Shuangluo; Vashishtha, Ashwani; Bulkley, David; Eom, Soo Hyun; Wang, Jimin; Konigsberg, William H.

    2012-08-31

    During DNA synthesis, base stacking and Watson-Crick (WC) hydrogen bonding increase the stability of nascent base pairs when they are in a ternary complex. To evaluate the contribution of base stacking to the incorporation efficiency of dNTPs when a DNA polymerase encounters an abasic site, we varied the penultimate base pairs (PBs) adjacent to the abasic site using all 16 possible combinations. We then determined pre-steady-state kinetic parameters with an RB69 DNA polymerase variant and solved nine structures of the corresponding ternary complexes. The efficiency of incorporation for incoming dNTPs opposite an abasic site varied between 2- and 210-fold depending on the identity of the PB. We propose that the A rule can be extended to encompass the fact that DNA polymerase can bypass dA/abasic sites more efficiently than other dN/abasic sites. Crystal structures of the ternary complexes show that the surface of the incoming base was stacked against the PB's interface and that the kinetic parameters for dNMP incorporation were consistent with specific features of base stacking, such as surface area and partial charge-charge interactions between the incoming base and the PB. Without a templating nucleotide residue, an incoming dNTP has no base with which it can hydrogen bond and cannot be desolvated, so that these surrounding water molecules become ordered and remain on the PB's surface in the ternary complex. When these water molecules are on top of a hydrophobic patch on the PB, they destabilize the ternary complex, and the incorporation efficiency of incoming dNTPs is reduced.

  2. Cadaverine covalently linked to peptidoglycan is required for interaction between the peptidoglycan and the periplasm-exposed S-layer-homologous domain of major outer membrane protein Mep45 in Selenomonas ruminantium.

    PubMed

    Kojima, Seiji; Ko, Kyong-Cheol; Takatsuka, Yumiko; Abe, Naoki; Kaneko, Jun; Itoh, Yoshifumi; Kamio, Yoshiyuki

    2010-11-01

    The peptidoglycan of Selenomonas ruminantium is covalently bound to cadaverine (PG-cadaverine), which likely plays a significant role in maintaining the integrity of the cell surface structure. The outer membrane of this bacterium contains a 45-kDa major protein (Mep45) that is a putative peptidoglycan-associated protein. In this report, we determined the nucleotide sequence of the mep45 gene and investigated the relationship between PG-cadaverine, Mep45, and the cell surface structure. Amino acid sequence analysis showed that Mep45 is comprised of an N-terminal S-layer-homologous (SLH) domain followed by α-helical coiled-coil region and a C-terminal β-strand-rich region. The N-terminal SLH domain was found to be protruding into the periplasmic space and was responsible for binding to peptidoglycan. It was determined that Mep45 binds to the peptidoglycan in a manner dependent on the presence of PG-cadaverine. Electron microscopy revealed that defective PG-cadaverine decreased the structural interactions between peptidoglycan and the outer membrane, consistent with the proposed role for PG-cadaverine. The C-terminal β-strand-rich region of Mep45 was predicted to be a membrane-bound unit of the 14-stranded β-barrel structure. Here we propose that PG-cadaverine possesses functional importance to facilitate the structural linkage between peptidoglycan and the outer membrane via specific interaction with the SLH domain of Mep45.

  3. Disruption of ionic interactions between the nucleotide binding domain 1 (NBD1) and middle (M) domain in Hsp100 disaggregase unleashes toxic hyperactivity and partial independence from Hsp70.

    PubMed

    Lipińska, Natalia; Ziętkiewicz, Szymon; Sobczak, Alicja; Jurczyk, Agnieszka; Potocki, Wojciech; Morawiec, Ewa; Wawrzycka, Aleksandra; Gumowski, Krzysztof; Ślusarz, Magdalena; Rodziewicz-Motowidło, Sylwia; Chruściel, Elżbieta; Liberek, Krzysztof

    2013-01-25

    Hsp100 chaperones cooperate with the Hsp70 chaperone system to disaggregate and reactivate heat-denatured aggregated proteins to promote cell survival after heat stress. The homology models of Hsp100 disaggregases suggest the presence of a conserved network of ionic interactions between the first nucleotide binding domain (NBD1) and the coiled-coil middle subdomain, the signature domain of disaggregating chaperones. Mutations intended to disrupt the putative ionic interactions in yeast Hsp104 and bacterial ClpB disaggregases resulted in remarkable changes of their biochemical properties. These included an increase in ATPase activity, a significant increase in the rate of in vitro substrate renaturation, and partial independence from the Hsp70 chaperone in disaggregation. Paradoxically, the increased activities resulted in serious growth impediments in yeast and bacterial cells instead of improvement of their thermotolerance. Our results suggest that this toxic activity is due to the ability of the mutated disaggregases to unfold independently from Hsp70, native folded proteins. Complementary changes that restore particular salt bridges within the suggested network suppressed the toxic effects. We propose a novel structural aspect of Hsp100 chaperones crucial for specificity and efficiency of the disaggregation reaction. PMID:23233670

  4. Intermolecular and Intramolecular Interactions Regulate Catalytic Activity of Myotonic Dystrophy Kinase-Related Cdc42-Binding Kinase α

    PubMed Central

    Tan, Ivan; Seow, Kah Tong; Lim, Louis; Leung, Thomas

    2001-01-01

    Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) is a Cdc42-binding serine/threonine kinase with multiple functional domains. We had previously shown MRCKα to be implicated in Cdc42-mediated peripheral actin formation and neurite outgrowth in HeLa and PC12 cells, respectively. Here we demonstrate that native MRCK exists in high-molecular-weight complexes. We further show that the three independent coiled-coil (CC) domains and the N-terminal region preceding the kinase domain are responsible for intermolecular interactions leading to MRCKα multimerization. N terminus-mediated dimerization and consequent transautophosphorylation are critical processes regulating MRCKα catalytic activities. A region containing the two distal CC domains (CC2 and CC3; residues 658 to 930) was found to interact intramolecularly with the kinase domain and negatively regulates its activity. Its deletion also resulted in an active kinase, confirming a negative autoregulatory role. We provide evidence that the N terminus-mediated dimerization and activation of MRCK and the negative autoregulatory kinase–distal CC interaction are two mutually exclusive events that tightly regulate the catalytic state of the kinase. Disruption of this interaction by a mutant kinase domain resulted in increased kinase activity. MRCK kinase activity was also elevated when cells were treated with phorbol ester, which can interact directly with a cysteine-rich domain next to the distal CC domain. We therefore suggest that binding of phorbol ester to MRCK releases its autoinhibition, allowing N-terminal dimerization and subsequent kinase activation. PMID:11283256

  5. 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.

  6. Pro178 and Pro183 of Selenoprotein S Are Essential Residues for Interaction with p97(VCP) during Endoplasmic Reticulum-associated Degradation*

    PubMed Central

    Lee, Jea Hwang; Kwon, Joon Hyun; Jeon, Yeong Ha; Ko, Kwan Young; Lee, Seung-Rock; Kim, Ick Young

    2014-01-01

    During endoplasmic reticulum (ER)-associated degradation, p97(VCP) is recruited to the ER membrane through interactions with transmembrane proteins, such as selenoprotein S (SelS), selenoprotein K (SelK), hrd1, and gp78. SelS has a single-spanning transmembrane domain and protects cells from ER stress-induced apoptosis through interaction with p97(VCP). The cytosolic tail of SelS consists of a coiled-coil domain, a putative VCP-interacting motif (VIM), and an unpronounced glycine- and proline-rich secondary structure. To understand the regulatory mechanism of SelS during ER stress, we investigated the interaction of the protein with p97(VCP) using mouse neuroblastoma cells and human embryonic kidney 293 cells. The SelS expression level increased when ER stress was induced. In addition, the effect of ER stress was enhanced, and recruitment of p97(VCP) to the ER membrane was inhibited in SelS knockdown cells. The effect of SelS knockdown was rescued by ectopic expression of SelS U188C. p97(VCP) interacted with SelS U188C and was recruited to the ER membrane. The expression of SelS[ΔVIM], which is a VIM deletion mutant of SelS, also showed both a recovery effect and an interaction with p97(VCP) in cells. However, mutants in which the proline residue positions 178 or 183 of SelS were changed to alanine or were deleted did not interact with p97(VCP). The proline mutants did not rescue ER stress in SelS knockdown cells. These results suggest that both Pro178 and Pro183 of SelS play important roles in the translocation of p97(VCP) to the ER membrane and protect cells from ER stress. PMID:24700463

  7. 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

  8. 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.

  9. Importance of the gamma-aminobutyric acid(B) receptor C-termini for G-protein coupling.