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Sample records for mediator subunit dependence

  1. Subunit architecture and functional modular rearrangements of the transcriptional mediator complex.

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    Tsai, Kuang-Lei; Tomomori-Sato, Chieri; Sato, Shigeo; Conaway, Ronald C; Conaway, Joan W; Asturias, Francisco J

    2014-06-05

    The multisubunit Mediator, comprising ∼30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA-binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Therapeutic potential of Mediator complex subunits in metabolic diseases.

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    Ranjan, Amol; Ansari, Suraiya A

    2018-01-01

    The multisubunit Mediator is an evolutionary conserved transcriptional coregulatory complex in eukaryotes. It is needed for the transcriptional regulation of gene expression in general as well as in a gene specific manner. Mediator complex subunits interact with different transcription factors as well as components of RNA Pol II transcription initiation complex and in doing so act as a bridge between gene specific transcription factors and general Pol II transcription machinery. Specific interaction of various Mediator subunits with nuclear receptors (NRs) and other transcription factors involved in metabolism has been reported in different studies. Evidences indicate that ligand-activated NRs recruit Mediator complex for RNA Pol II-dependent gene transcription. These NRs have been explored as therapeutic targets in different metabolic diseases; however, they show side-effects as targets due to their overlapping involvement in different signaling pathways. Here we discuss the interaction of various Mediator subunits with transcription factors involved in metabolism and whether specific interaction of these transcription factors with Mediator subunits could be potentially utilized as therapeutic strategy in a variety of metabolic diseases. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  3. Mediator subunit18 controls flowering time and floral organ identity in Arabidopsis.

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    Zhengui Zheng

    Full Text Available Mediator is a conserved multi-protein complex that plays an important role in regulating transcription by mediating interactions between transcriptional activator proteins and RNA polymerase II. Much evidence exists that Mediator plays a constitutive role in the transcription of all genes transcribed by RNA polymerase II. However, evidence is mounting that specific Mediator subunits may control the developmental regulation of specific subsets of RNA polymerase II-dependent genes. Although the Mediator complex has been extensively studied in yeast and mammals, only a few reports on Mediator function in flowering time control of plants, little is known about Mediator function in floral organ identity. Here we show that in Arabidopsis thaliana, MEDIATOR SUBUNIT 18 (MED18 affects flowering time and floral organ formation through FLOWERING LOCUS C (FLC and AGAMOUS (AG. A MED18 loss-of-function mutant showed a remarkable syndrome of later flowering and altered floral organ number. We show that FLC and AG mRNA levels and AG expression patterns are altered in the mutant. Our results support parallels between the regulation of FLC and AG and demonstrate a developmental role for Mediator in plants.

  4. Depletion of Mediator Kinase Module Subunits Represses Superenhancer-Associated Genes in Colon Cancer Cells.

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    Kuuluvainen, Emilia; Domènech-Moreno, Eva; Niemelä, Elina H; Mäkelä, Tomi P

    2018-06-01

    In cancer, oncogene activation is partly mediated by acquired superenhancers, which therefore represent potential targets for inhibition. Superenhancers are enriched for BRD4 and Mediator, and both BRD4 and the Mediator MED12 subunit are disproportionally required for expression of superenhancer-associated genes in stem cells. Here we show that depletion of Mediator kinase module subunit MED12 or MED13 together with MED13L can be used to reduce expression of cancer-acquired superenhancer genes, such as the MYC gene, in colon cancer cells, with a concomitant decrease in proliferation. Whereas depletion of MED12 or MED13/MED13L caused a disproportional decrease of superenhancer gene expression, this was not seen with depletion of the kinases cyclin-dependent kinase 9 (CDK8) and CDK19. MED12-MED13/MED13L-dependent superenhancer genes were coregulated by β-catenin, which has previously been shown to associate with MED12. Importantly, β-catenin depletion caused reduced binding of MED12 at the MYC superenhancer. The effect of MED12 or MED13/MED13L depletion on cancer-acquired superenhancer gene expression was more specific than and partially distinct from that of BRD4 depletion, with the most efficient inhibition seen with combined targeting. These results identify a requirement of MED12 and MED13/MED13L for expression of acquired superenhancer genes in colon cancer, implicating these Mediator subunits as potential therapeutic targets for colon cancer, alone or together with BRD4. Copyright © 2018 American Society for Microbiology.

  5. PRDM16 enhances nuclear receptor-dependent transcription of the brown fat-specific Ucp1 gene through interactions with Mediator subunit MED1.

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    Iida, Satoshi; Chen, Wei; Nakadai, Tomoyoshi; Ohkuma, Yoshiaki; Roeder, Robert G

    2015-02-01

    PR domain-containing 16 (PRDM16) induces expression of brown fat-specific genes in brown and beige adipocytes, although the underlying transcription-related mechanisms remain largely unknown. Here, in vitro studies show that PRDM16, through its zinc finger domains, directly interacts with the MED1 subunit of the Mediator complex, is recruited to the enhancer of the brown fat-specific uncoupling protein 1 (Ucp1) gene through this interaction, and enhances thyroid hormone receptor (TR)-driven transcription in a biochemically defined system in a Mediator-dependent manner, thus providing a direct link to the general transcription machinery. Complementary cell-based studies show that upon forskolin treatment, PRDM16 induces Ucp1 expression in undifferentiated murine embryonic fibroblasts, that this induction depends on MED1 and TR, and, consistent with a direct effect, that PRDM16 is recruited to the Ucp1 enhancer. Related studies have defined MED1 and PRDM16 interaction domains important for Ucp1 versus Ppargc1a induction by PRDM16. These results reveal novel mechanisms for PRDM16 function through the Mediator complex. © 2015 Iida et al.; Published by Cold Spring Harbor Laboratory Press.

  6. Mediator subunit MED1 is a T3-dependent and T3-independent coactivator on the thyrotropin β gene promoter

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    Matsui, Keiji; Oda, Kasumi; Mizuta, Shumpei; Ishino, Ruri; Urahama, Norinaga; Hasegawa, Natsumi [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Roeder, Robert G. [Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Ito, Mitsuhiro, E-mail: itomi@med.kobe-u.ac.jp [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Department of Family and Community Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 654-0142 (Japan); Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan)

    2013-10-11

    Highlights: •MED1 is a bona fide T3-dependent coactivator on TSHB promoter. •Mice with LxxLL-mutant MED1 have attenuated TSHβ mRNA and thyroid hormone levels. •MED1 activates TSHB promoter T3-dependently in cultured cells. •T3-dependent MED1 action is enhanced when SRC1/SRC2 or HDAC2 is downregulated. •MED1 is also a T3-independent GATA2/Pit1 coactivator on TSHB promoter. -- Abstract: The MED1 subunit of the Mediator transcriptional coregulator complex is a nuclear receptor-specific coactivator. A negative feedback mechanism of thyroid-stimulating hormone (TSH, or thyrotropin) expression in the thyrotroph in the presence of triiodothyronine (T3) is employed by liganded thyroid hormone receptor β (TRβ) on the TSHβ gene promoter, where conventional histone-modifying coactivators act as corepressors. We now provide evidence that MED1 is a ligand-dependent positive cofactor on this promoter. TSHβ gene transcription was attenuated in MED1 mutant mice in which the nuclear receptor-binding ability of MED1 was specifically disrupted. MED1 stimulated GATA2- and Pit1-mediated TSHβ gene promoter activity in a ligand-independent manner in cultured cells. MED1 also stimulated transcription from the TSHβ gene promoter in a T3-dependent manner. The transcription was further enhanced when the T3-dependent corepressors SRC1, SRC2, and HDAC2 were downregulated. Hence, MED1 is a T3-dependent and -independent coactivator on the TSHβ gene promoter.

  7. Activity-dependent control of NMDA receptor subunit composition at hippocampal mossy fibre synapses.

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    Carta, Mario; Srikumar, Bettadapura N; Gorlewicz, Adam; Rebola, Nelson; Mulle, Christophe

    2018-02-15

    CA3 pyramidal cells display input-specific differences in the subunit composition of synaptic NMDA receptors (NMDARs). Although at low density, GluN2B contributes significantly to NMDAR-mediated EPSCs at mossy fibre synapses. Long-term potentiation (LTP) of NMDARs triggers a modification in the subunit composition of synaptic NMDARs by insertion of GluN2B. GluN2B subunits are essential for the expression of LTP of NMDARs at mossy fibre synapses. Single neurons express NMDA receptors (NMDARs) with distinct subunit composition and biophysical properties that can be segregated in an input-specific manner. The dynamic control of the heterogeneous distribution of synaptic NMDARs is crucial to control input-dependent synaptic integration and plasticity. In hippocampal CA3 pyramidal cells from mice of both sexes, we found that mossy fibre (MF) synapses display a markedly lower proportion of GluN2B-containing NMDARs than associative/commissural synapses. The mechanism involved in such heterogeneous distribution of GluN2B subunits is not known. Here we show that long-term potentiation (LTP) of NMDARs, which is selectively expressed at MF-CA3 pyramidal cell synapses, triggers a modification in the subunit composition of synaptic NMDARs by insertion of GluN2B. This activity-dependent recruitment of GluN2B at mature MF-CA3 pyramidal cell synapses contrasts with the removal of GluN2B subunits at other glutamatergic synapses during development and in response to activity. Furthermore, although expressed at low levels, GluN2B is necessary for the expression of LTP of NMDARs at MF-CA3 pyramidal cell synapses. Altogether, we reveal a previously unknown activity-dependent regulation and function of GluN2B subunits that may contribute to the heterogeneous plasticity induction rules in CA3 pyramidal cells. © 2017 Centre Nationnal de la Recherche Scientifique. The Journal of Physiology © 2017 The Physiological Society.

  8. Fungal mediator tail subunits contain classical transcriptional activation domains.

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    Liu, Zhongle; Myers, Lawrence C

    2015-04-01

    Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits of Saccharomyces cerevisiae, Candida albicans, and Candida dubliniensis Mediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains of S. cerevisiae Med2 and Med3, as well as C. dubliniensis Tlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  9. Mediator Recruitment to Heat Shock Genes Requires Dual Hsf1 Activation Domains and Mediator Tail Subunits Med15 and Med16*

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    Kim, Sunyoung; Gross, David S.

    2013-01-01

    The evolutionarily conserved Mediator complex is central to the regulation of gene transcription in eukaryotes because it serves as a physical and functional interface between upstream regulators and the Pol II transcriptional machinery. Nonetheless, its role appears to be context-dependent, and the detailed mechanism by which it governs the expression of most genes remains unknown. Here we investigate Mediator involvement in HSP (heat shock protein) gene regulation in the yeast Saccharomyces cerevisiae. We find that in response to thermal upshift, subunits representative of each of the four Mediator modules (Head, Middle, Tail, and Kinase) are rapidly, robustly, and selectively recruited to the promoter regions of HSP genes. Their residence is transient, returning to near-background levels within 90 min. Hsf1 (heat shock factor 1) plays a central role in recruiting Mediator, as indicated by the fact that truncation of either its N- or C-terminal activation domain significantly reduces Mediator occupancy, whereas removal of both activation domains abolishes it. Likewise, ablation of either of two Mediator Tail subunits, Med15 or Med16, reduces Mediator recruitment to HSP promoters, whereas deletion of both abolishes it. Accompanying the loss of Mediator, recruitment of RNA polymerase II is substantially diminished. Interestingly, Mediator antagonizes Hsf1 occupancy of non-induced promoters yet facilitates enhanced Hsf1 association with activated ones. Collectively, our observations indicate that Hsf1, via its dual activation domains, recruits holo-Mediator to HSP promoters in response to acute heat stress through cooperative physical and/or functional interactions with the Tail module. PMID:23447536

  10. Interaction of the regulatory subunit of the cAMP-dependent protein kinase with PATZ1 (ZNF278)

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    Yang, Weng-Lang; Ravatn, Roald; Kudoh, Kazuya; Alabanza, Leah; Chin, Khew-Voon

    2010-01-01

    The effects of cAMP in cell are predominantly mediated by the cAMP-dependent protein kinase (PKA), which is composed of two genetically distinct subunits, catalytic (C) and regulatory (R), forming a tetrameric holoenzyme R 2 C 2 . The only known function for the R subunit is that of inhibiting the activity of the C subunit kinase. It has been shown that overexpression of RIα, but not the C subunit kinase, is associated with neoplastic transformation. In addition, it has also been demonstrated that mutation in the RIα, but not the C subunit is associated with increased resistance to the DNA-damaging anticancer drug cisplatin, thus suggesting that the RIα subunit of PKA may have functions independent of the kinase. We show here that the RIα subunit interacts with a BTB/POZ domain zinc-finger transcription factor, PATZ1 (ZNF278), and co-expression with RIα results in its sequestration in the cytoplasm. The cytoplasmic/nuclear translocation is inducible by cAMP. C-terminus deletion abolishes PATZ1 interaction with RIα and results in its localization in the nucleus. PATZ1 transactivates the cMyc promoter and the presence of cAMP and co-expression with RIα modulates its transactivation. Moreover, PATZ1 is aberrantly expressed in cancer. Taken together, our results showed a potentially novel mechanism of cAMP signaling mediated through the interaction of RIα with PATZ1 that is independent of the kinase activity of PKA, and the aberrant expression of PATZ1 in cancer point to its role in cell growth regulation.

  11. The Arabidopsis mediator complex subunits MED16, MED14, and MED2 regulate mediator and RNA polymerase II recruitment to CBF-responsive cold-regulated genes.

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    Hemsley, Piers A; Hurst, Charlotte H; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R; De Cothi, Elizabeth A; Steele, John F; Knight, Heather

    2014-01-01

    The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation-induced freezing tolerance. In addition, these three subunits are required for low temperature-induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced.

  12. Double dissociation of spike timing-dependent potentiation and depression by subunit-preferring NMDA receptor antagonists in mouse barrel cortex.

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    Banerjee, Abhishek; Meredith, Rhiannon M; Rodríguez-Moreno, Antonio; Mierau, Susanna B; Auberson, Yves P; Paulsen, Ole

    2009-12-01

    Spike timing-dependent plasticity (STDP) is a strong candidate for an N-methyl-D-aspartate (NMDA) receptor-dependent form of synaptic plasticity that could underlie the development of receptive field properties in sensory neocortices. Whilst induction of timing-dependent long-term potentiation (t-LTP) requires postsynaptic NMDA receptors, timing-dependent long-term depression (t-LTD) requires the activation of presynaptic NMDA receptors at layer 4-to-layer 2/3 synapses in barrel cortex. Here we investigated the developmental profile of t-LTD at layer 4-to-layer 2/3 synapses of mouse barrel cortex and studied their NMDA receptor subunit dependence. Timing-dependent LTD emerged in the first postnatal week, was present during the second week and disappeared in the adult, whereas t-LTP persisted in adulthood. An antagonist at GluN2C/D subunit-containing NMDA receptors blocked t-LTD but not t-LTP. Conversely, a GluN2A subunit-preferring antagonist blocked t-LTP but not t-LTD. The GluN2C/D subunit requirement for t-LTD appears to be synapse specific, as GluN2C/D antagonists did not block t-LTD at horizontal cross-columnar layer 2/3-to-layer 2/3 synapses, which was blocked by a GluN2B antagonist instead. These data demonstrate an NMDA receptor subunit-dependent double dissociation of t-LTD and t-LTP mechanisms at layer 4-to-layer 2/3 synapses, and suggest that t-LTD is mediated by distinct molecular mechanisms at different synapses on the same postsynaptic neuron.

  13. Interaction of the regulatory subunit of the cAMP-dependent protein kinase with PATZ1 (ZNF278)

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    Yang, Weng-Lang [Long Island Jewish Medical Center, North Shore University Hospital, Manhasset, NY 11030 (United States); Ravatn, Roald [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Kudoh, Kazuya [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Department of Obstetrics and Gynecology, National Defense Medical College, Tokorozawa, Saitama (Japan); Alabanza, Leah [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Chin, Khew-Voon, E-mail: khew-voon.chin@utoledo.edu [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States)

    2010-01-15

    The effects of cAMP in cell are predominantly mediated by the cAMP-dependent protein kinase (PKA), which is composed of two genetically distinct subunits, catalytic (C) and regulatory (R), forming a tetrameric holoenzyme R{sub 2}C{sub 2}. The only known function for the R subunit is that of inhibiting the activity of the C subunit kinase. It has been shown that overexpression of RI{alpha}, but not the C subunit kinase, is associated with neoplastic transformation. In addition, it has also been demonstrated that mutation in the RI{alpha}, but not the C subunit is associated with increased resistance to the DNA-damaging anticancer drug cisplatin, thus suggesting that the RI{alpha} subunit of PKA may have functions independent of the kinase. We show here that the RI{alpha} subunit interacts with a BTB/POZ domain zinc-finger transcription factor, PATZ1 (ZNF278), and co-expression with RI{alpha} results in its sequestration in the cytoplasm. The cytoplasmic/nuclear translocation is inducible by cAMP. C-terminus deletion abolishes PATZ1 interaction with RI{alpha} and results in its localization in the nucleus. PATZ1 transactivates the cMyc promoter and the presence of cAMP and co-expression with RI{alpha} modulates its transactivation. Moreover, PATZ1 is aberrantly expressed in cancer. Taken together, our results showed a potentially novel mechanism of cAMP signaling mediated through the interaction of RI{alpha} with PATZ1 that is independent of the kinase activity of PKA, and the aberrant expression of PATZ1 in cancer point to its role in cell growth regulation.

  14. The Arabidopsis Mediator Complex Subunits MED16, MED14, and MED2 Regulate Mediator and RNA Polymerase II Recruitment to CBF-Responsive Cold-Regulated Genes[C][W][OPEN

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    Hemsley, Piers A.; Hurst, Charlotte H.; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R.; De Cothi, Elizabeth A.; Steele, John F.; Knight, Heather

    2014-01-01

    The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation–induced freezing tolerance. In addition, these three subunits are required for low temperature–induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced. PMID:24415770

  15. The Mediator subunit MDT-15 confers metabolic adaptation to ingested material.

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    Stefan Taubert

    2008-02-01

    Full Text Available In eukaryotes, RNA polymerase II (Pol(II dependent gene expression requires accessory factors termed transcriptional coregulators. One coregulator that universally contributes to Pol(II-dependent transcription is the Mediator, a multisubunit complex that is targeted by many transcriptional regulatory factors. For example, the Caenorhabditis elegans Mediator subunit MDT-15 confers the regulatory actions of the sterol response element binding protein SBP-1 and the nuclear hormone receptor NHR-49 on fatty acid metabolism. Here, we demonstrate that MDT-15 displays a broader spectrum of activities, and that it integrates metabolic responses to materials ingested by C. elegans. Depletion of MDT-15 protein or mutation of the mdt-15 gene abrogated induction of specific detoxification genes in response to certain xenobiotics or heavy metals, rendering these animals hypersensitive to toxin exposure. Intriguingly, MDT-15 appeared to selectively affect stress responses related to ingestion, as MDT-15 functional defects did not abrogate other stress responses, e.g., thermotolerance. Together with our previous finding that MDT-15:NHR-49 regulatory complexes coordinate a sector of the fasting response, we propose a model whereby MDT-15 integrates several transcriptional regulatory pathways to monitor both the availability and quality of ingested materials, including nutrients and xenobiotic compounds.

  16. Mediator Subunit Med28 Is Essential for Mouse Peri-Implantation Development and Pluripotency.

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    Lin Li

    Full Text Available The multi-subunit mammalian Mediator complex acts as an integrator of transcriptional regulation by RNA Polymerase II, and has emerged as a master coordinator of development and cell fate determination. We previously identified the Mediator subunit, MED28, as a cytosolic binding partner of merlin, the Neurofibromatosis 2 (NF2 tumor suppressor, and thus MED28 is distinct in having a cytosolic role as an NF2 interacting protein as well as a nuclear role as a Mediator complex subunit. Although limited in vitro studies have been performed on MED28, its in vivo function remains unknown. Employing a knockout mouse model, we describe for the first time the requirement for Med28 in the developing mouse embryo. Med28-deficiency causes peri-implantation lethality resulting from the loss of pluripotency of the inner cell mass accompanied by reduced expression of key pluripotency transcription factors Oct4 and Nanog. Further, overexpression of Med28 in mouse embryonic fibroblasts enhances the efficiency of their reprogramming to pluripotency. Cre-mediated inactivation of Med28 in induced pluripotent stem cells shows that Med28 is required for their survival. Intriguingly, heterozygous loss of Med28 results in differentiation of induced pluripotent stem cells into extraembryonic trophectoderm and primitive endoderm lineages. Our findings document the essential role of Med28 in the developing embryo as well as in acquisition and maintenance of pluripotency during reprogramming.

  17. Regulation of KV channel voltage-dependent activation by transmembrane β subunits

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    Xiaohui eSun

    2012-04-01

    Full Text Available Voltage-activated K+ (KV channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. KV channels contain a central pore-gate domain (PGD surrounded by four voltage-sensing domains (VSD. The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many KV channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the KV β subunits that contain transmembrane (TM segments including the KCNE family and the β subunits of large conductance, Ca2+- and voltage-activated K+ (BK channels. These TM β subunits affect the voltage-dependent activation of KV α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into KV channel modulation by TM β subunits.

  18. Mediator kinase module and human tumorigenesis.

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    Clark, Alison D; Oldenbroek, Marieke; Boyer, Thomas G

    2015-01-01

    Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit "kinase" module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways.

  19. Mining Protein Evolution for Insights into Mechanisms of Voltage-Dependent Sodium Channel Auxiliary Subunits.

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    Molinarolo, Steven; Granata, Daniele; Carnevale, Vincenzo; Ahern, Christopher A

    2018-02-21

    Voltage-gated sodium channel (VGSC) beta (β) subunits have been called the "overachieving" auxiliary ion channel subunit. Indeed, these subunits regulate the trafficking of the sodium channel complex at the plasma membrane and simultaneously tune the voltage-dependent properties of the pore-forming alpha-subunit. It is now known that VGSC β-subunits are capable of similar modulation of multiple isoforms of related voltage-gated potassium channels, suggesting that their abilities extend into the broader voltage-gated channels. The gene family for these single transmembrane immunoglobulin beta-fold proteins extends well beyond the traditional VGSC β1-β4 subunit designation, with deep roots into the cell adhesion protein family and myelin-related proteins - where inherited mutations result in a myriad of electrical signaling disorders. Yet, very little is known about how VGSC β-subunits support protein trafficking pathways, the basis for their modulation of voltage-dependent gating, and, ultimately, their role in shaping neuronal excitability. An evolutionary approach can be useful in yielding new clues to such functions as it provides an unbiased assessment of protein residues, folds, and functions. An approach is described here which indicates the greater emergence of the modern β-subunits roughly 400 million years ago in the early neurons of Bilateria and bony fish, and the unexpected presence of distant homologues in bacteriophages. Recent structural breakthroughs containing α and β eukaryotic sodium channels containing subunits suggest a novel role for a highly conserved polar contact that occurs within the transmembrane segments. Overall, a mixture of approaches will ultimately advance our understanding of the mechanism for β-subunit interactions with voltage-sensor containing ion channels and membrane proteins.

  20. Auxin-dependent compositional change in Mediator in ARF7- and ARF19-mediated transcription.

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    Ito, Jun; Fukaki, Hidehiro; Onoda, Makoto; Li, Lin; Li, Chuanyou; Tasaka, Masao; Furutani, Masahiko

    2016-06-07

    Mediator is a multiprotein complex that integrates the signals from transcription factors binding to the promoter and transmits them to achieve gene transcription. The subunits of Mediator complex reside in four modules: the head, middle, tail, and dissociable CDK8 kinase module (CKM). The head, middle, and tail modules form the core Mediator complex, and the association of CKM can modify the function of Mediator in transcription. Here, we show genetic and biochemical evidence that CKM-associated Mediator transmits auxin-dependent transcriptional repression in lateral root (LR) formation. The AUXIN/INDOLE 3-ACETIC ACID 14 (Aux/IAA14) transcriptional repressor inhibits the transcriptional activity of its binding partners AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 by making a complex with the CKM-associated Mediator. In addition, TOPLESS (TPL), a transcriptional corepressor, forms a bridge between IAA14 and the CKM component MED13 through the physical interaction. ChIP assays show that auxin induces the dissociation of MED13 but not the tail module component MED25 from the ARF7 binding region upstream of its target gene. These findings indicate that auxin-induced degradation of IAA14 changes the module composition of Mediator interacting with ARF7 and ARF19 in the upstream region of their target genes involved in LR formation. We suggest that this regulation leads to a quick switch of signal transmission from ARFs to target gene expression in response to auxin.

  1. The Mediator Complex MED15 Subunit Mediates Activation of Downstream Lipid-Related Genes by the WRINKLED1 Transcription Factor.

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    Kim, Mi Jung; Jang, In-Cheol; Chua, Nam-Hai

    2016-07-01

    The Mediator complex is known to be a master coordinator of transcription by RNA polymerase II, and this complex is recruited by transcription factors (TFs) to target promoters for gene activation or repression. The plant-specific TF WRINKLED1 (WRI1) activates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. However, no Mediator subunit has yet been identified that mediates WRI1 transcriptional activity. Promoter-β-glucuronidase fusion experiments showed that MEDIATOR15 (MED15) is expressed in the same cells in the embryo as WRI1. We found that the Arabidopsis (Arabidopsis thaliana) MED15 subunit of the Mediator complex interacts directly with WRI1 in the nucleus. Overexpression of MED15 or WRI1 increased transcript levels of WRI1 target genes involved in glycolysis and fatty acid biosynthesis; these genes were down-regulated in wild-type or WRI1-overexpressing plants by silencing of MED15 However, overexpression of MED15 in the wri1 mutant also increased transcript levels of WRI1 target genes, suggesting that MED15 also may act with other TFs to activate downstream lipid-related genes. Chromatin immunoprecipitation assays confirmed the association of MED15 with six WRI1 target gene promoters. Additionally, silencing of MED15 resulted in reduced fatty acid content in seedlings and mature seeds, whereas MED15 overexpression increased fatty acid content in both developmental stages. Similar results were found in wri1 mutant and WRI1 overexpression lines. Together, our results indicate that the WRI1/MED15 complex transcriptionally regulates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

  2. Silencing SlMED18, tomato Mediator subunit 18 gene, restricts internode elongation and leaf expansion.

    Science.gov (United States)

    Wang, Yunshu; Hu, Zongli; Zhang, Jianling; Yu, XiaoHui; Guo, Jun-E; Liang, Honglian; Liao, Changguang; Chen, Guoping

    2018-02-19

    Mediator complex, a conserved multi-protein, is necessary for controlling RNA polymerase II (Pol II) transcription in eukaryotes. Given little is known about them in tomato, a tomato Mediator subunit 18 gene was isolated and named SlMED18. To further explore the function of SlMED18, the transgenic tomato plants targeting SlMED18 by RNAi-mediated gene silencing were generated. The SlMED18-RNAi lines exhibited multiple developmental defects, including smaller size and slower growth rate of plant and significantly smaller compound leaves. The contents of endogenous bioactive GA 3 in SlMED18 silenced lines were slightly less than that in wild type. Furthermore, qRT-PCR analysis indicated that expression of gibberellins biosynthesis genes such as SlGACPS and SlGA20x2, auxin transport genes (PIN1, PIN4, LAX1 and LAX2) and several key regulators, KNOX1, KNOX2, PHAN and LANCEOLATE(LA), which involved in the leaf morphogenesis were significantly down-regulated in SlMED18-RNAi lines. These results illustrated that SlMED18 plays an essential role in regulating plant internode elongation and leaf expansion in tomato plants and it acts as a key positive regulator of gibberellins biosynthesis and signal transduction as well as auxin proper transport signalling. These findings are the basis for understanding the function of the individual Mediator subunits in tomato.

  3. Med1 subunit of the mediator complex in nuclear receptor-regulated energy metabolism, liver regeneration, and hepatocarcinogenesis.

    Science.gov (United States)

    Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K

    2014-01-01

    Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic

  4. Analysis of Schizosaccharomyces pombe mediator reveals a set of essential subunits conserved between yeast and metazoan cells

    DEFF Research Database (Denmark)

    Spåhr, H; Samuelsen, C O; Baraznenok, V

    2001-01-01

    . cerevisiae share an essential protein module, which associates with nonessential speciesspecific subunits. In support of this view, sequence analysis of the conserved yeast Mediator components Med4 and Med8 reveals sequence homology to the metazoan Mediator components Trap36 and Arc32. Therefore, 8 of 10...... essential genes conserved between S. pombe and S. cerevisiae also have a metazoan homolog, indicating that an evolutionary conserved Mediator core is present in all eukaryotic cells. Our data suggest a closer functional relationship between yeast and metazoan Mediator than previously anticipated....

  5. Mechanistic Exploration of Cancer Stem Cell Marker Voltage-Dependent Calcium Channel α2δ1 Subunit-mediated Chemotherapy Resistance in Small-Cell Lung Cancer.

    Science.gov (United States)

    Yu, Jiangyong; Wang, Shuhang; Zhao, Wei; Duan, Jianchun; Wang, Zhijie; Chen, Hanxiao; Tian, Yanhua; Wang, Di; Zhao, Jun; An, Tongtong; Bai, Hua; Wu, Meina; Wang, Jie

    2018-05-01

    Purpose: Chemoresistance in small-cell lung cancer (SCLC) is reportedly attributed to the existence of resistant cancer stem cells (CSC). Studies involving CSC-specific markers and related mechanisms in SCLC remain limited. This study explored the role of the voltage-dependent calcium channel α2δ1 subunit as a CSC marker in chemoresistance of SCLC, and explored the potential mechanisms of α2δ1-mediated chemoresistance and strategies of overcoming the resistance. Experimental Design: α2δ1-positive cells were identified and isolated from SCLC cell lines and patient-derived xenograft (PDX) models, and CSC-like properties were subsequently verified. Transcriptome sequencing and Western blotting were carried out to identify pathways involved in α2δ1-mediated chemoresistance in SCLC. In addition, possible interventions to overcome α2δ1-mediated chemoresistance were examined. Results: Different proportions of α2δ1 + cells were identified in SCLC cell lines and PDX models. α2δ1 + cells exhibited CSC-like properties (self-renewal, tumorigenic, differentiation potential, and high expression of genes related to CSCs and drug resistance). Chemotherapy induced the enrichment of α2δ1 + cells instead of CD133 + cells in PDXs, and an increased proportion of α2δ1 + cells corresponded to increased chemoresistance. Activation and overexpression of ERK in the α2δ1-positive H1048 cell line was identified at the protein level. mAb 1B50-1 was observed to improve the efficacy of chemotherapy and delay relapse as maintenance therapy in PDX models. Conclusions: SCLC cells expressing α2δ1 demonstrated CSC-like properties, and may contribute to chemoresistance. ERK may play a key role in α2δ1-mediated chemoresistance. mAb 1B50-1 may serve as a potential anti-SCLC drug. Clin Cancer Res; 24(9); 2148-58. ©2018 AACR . ©2018 American Association for Cancer Research.

  6. Oncogenic exon 2 mutations in Mediator subunit MED12 disrupt allosteric activation of cyclin C-CDK8/19.

    Science.gov (United States)

    Park, Min Ju; Shen, Hailian; Spaeth, Jason M; Tolvanen, Jaana H; Failor, Courtney; Knudtson, Jennifer F; McLaughlin, Jessica; Halder, Sunil K; Yang, Qiwei; Bulun, Serdar E; Al-Hendy, Ayman; Schenken, Robert S; Aaltonen, Lauri A; Boyer, Thomas G

    2018-03-30

    Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at high frequency in uterine fibroids (UFs) and breast fibroepithelial tumors as well as recurrently, albeit less frequently, in malignant uterine leimyosarcomas, chronic lymphocytic leukemias, and colorectal cancers. Previously, we reported that UF-linked mutations in MED12 disrupt its ability to activate cyclin C (CycC)-dependent kinase 8 (CDK8) in Mediator, implicating impaired Mediator-associated CDK8 activity in the molecular pathogenesis of these clinically significant lesions. Notably, the CDK8 paralog CDK19 is also expressed in myometrium, and both CDK8 and CDK19 assemble into Mediator in a mutually exclusive manner, suggesting that CDK19 activity may also be germane to the pathogenesis of MED12 mutation-induced UFs. However, whether and how UF-linked mutations in MED12 affect CDK19 activation is unknown. Herein, we show that MED12 allosterically activates CDK19 and that UF-linked exon 2 mutations in MED12 disrupt its CDK19 stimulatory activity. Furthermore, we find that within the Mediator kinase module, MED13 directly binds to the MED12 C terminus, thereby suppressing an apparent UF mutation-induced conformational change in MED12 that otherwise disrupts its association with CycC-CDK8/19. Thus, in the presence of MED13, mutant MED12 can bind, but cannot activate, CycC-CDK8/19. These findings indicate that MED12 binding is necessary but not sufficient for CycC-CDK8/19 activation and reveal an additional step in the MED12-dependent activation process, one critically dependent on MED12 residues altered by UF-linked exon 2 mutations. These findings confirm that UF-linked mutations in MED12 disrupt composite Mediator-associated kinase activity and identify CDK8/19 as prospective therapeutic targets in UFs. © 2018 Park et al.

  7. Amplification of TLO Mediator Subunit Genes Facilitate Filamentous Growth in Candida Spp.

    Science.gov (United States)

    Liu, Zhongle; Moran, Gary P.; Myers, Lawrence C.

    2016-01-01

    Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated) gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of ‘free,’ non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large ‘free’ pool of the C. dubliniensis Tlo2 (CdTlo2) protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which the

  8. Amplification of TLO Mediator Subunit Genes Facilitate Filamentous Growth in Candida Spp.

    Directory of Open Access Journals (Sweden)

    Zhongle Liu

    2016-10-01

    Full Text Available Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of 'free,' non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large 'free' pool of the C. dubliniensis Tlo2 (CdTlo2 protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which

  9. Age-dependent modifications of AMPA receptor subunit expression levels and related cognitive effects in 3xTg-AD mice

    Directory of Open Access Journals (Sweden)

    Pamela eCantanelli

    2014-08-01

    Full Text Available GluA1, GluA2, GluA3, and GluA4 are the constitutive subunits of AMPA receptors (AMPARs, the major mediators of fast excitatory transmission in the mammalian central nervous system. Most AMPARs are Ca2+-impermeable because of the presence of the GluA2 subunit. GluA2 mRNA undergoes an editing process that results in a Q to R substitution, a key factor in the regulation of AMPAR Ca2+-permeability. AMPARs lacking GluA2 or containing the unedited subunit are permeable to Ca2+ and Zn2+. The phenomenon physiologically modulates synaptic plasticity while, in pathologic conditions, leads to increased vulnerability to excitotoxic neuronal death. Given the importance of these subunits, we have therefore evaluated possible associations between changes in expression levels of AMPAR subunits and development of cognitive deficits in 3xTg-AD mice, a widely investigated transgenic mouse model of Alzheimer’s disease. With qRT-PCR, we assayed hippocampal mRNA expression levels of GluA1-4 subunits occurring in young [3 months of age (m.o.a.] and old (12 m.o.a Tg-AD mice and made comparisons with levels found in age-matched wild type (WT mice. Efficiency of GluA2 RNA editing was also analyzed. All animals were cognitively tested for short- and long-term spatial memory with the Morris Water Maze (MWM navigation task. 3xTg-AD mice showed age-dependent decreases of mRNA levels for all the AMPAR subunits, with the exception of GluA2. Editing remained fully efficient with aging in 3xTg-AD and WT mice. A one-to-one correlation analysis between MWM performances and GluA1-4 mRNA expression profiles showed negative correlations between GluA2 levels and MWM performances in young 3xTg-AD mice. On the contrary, positive correlations between GluA2 mRNA and MWM performances were found in young WT mice. Our data suggest that increases of AMPARs that contain GluA1, GluA3, and GluA4 subunits may help in maintaining cognition in pre-symptomatic 3xTg-AD mice.

  10. Mediator Complex Subunits MED2, MED5, MED16, and MED23 Genetically Interact in the Regulation of Phenylpropanoid Biosynthesis.

    Science.gov (United States)

    Dolan, Whitney L; Dilkes, Brian P; Stout, Jake M; Bonawitz, Nicholas D; Chapple, Clint

    2017-12-01

    The phenylpropanoid pathway is a major global carbon sink and is important for plant fitness and the engineering of bioenergy feedstocks. In Arabidopsis thaliana , disruption of two subunits of the transcriptional regulatory Mediator complex, MED5a and MED5b, results in an increase in phenylpropanoid accumulation. By contrast, the semidominant MED5b mutation reduced epidermal fluorescence4-3 ( ref4-3 ) results in dwarfism and constitutively repressed phenylpropanoid accumulation. Here, we report the results of a forward genetic screen for suppressors of ref4-3. We identified 13 independent lines that restore growth and/or phenylpropanoid accumulation in the ref4-3 background. Two of the suppressors restore growth without restoring soluble phenylpropanoid accumulation, indicating that the growth and metabolic phenotypes of the ref4-3 mutant can be genetically disentangled. Whole-genome sequencing revealed that all but one of the suppressors carry mutations in MED5b or other Mediator subunits. RNA-seq analysis showed that the ref4-3 mutation causes widespread changes in gene expression, including the upregulation of negative regulators of the phenylpropanoid pathway, and that the suppressors reverse many of these changes. Together, our data highlight the interdependence of individual Mediator subunits and provide greater insight into the transcriptional regulation of phenylpropanoid biosynthesis by the Mediator complex. © 2017 American Society of Plant Biologists. All rights reserved.

  11. G-protein signaling leverages subunit-dependent membrane affinity to differentially control βγ translocation to intracellular membranes.

    Science.gov (United States)

    O'Neill, Patrick R; Karunarathne, W K Ajith; Kalyanaraman, Vani; Silvius, John R; Gautam, N

    2012-12-18

    Activation of G-protein heterotrimers by receptors at the plasma membrane stimulates βγ-complex dissociation from the α-subunit and translocation to internal membranes. This intermembrane movement of lipid-modified proteins is a fundamental but poorly understood feature of cell signaling. The differential translocation of G-protein βγ-subunit types provides a valuable experimental model to examine the movement of signaling proteins between membranes in a living cell. We used live cell imaging, mathematical modeling, and in vitro measurements of lipidated fluorescent peptide dissociation from vesicles to determine the mechanistic basis of the intermembrane movement and identify the interactions responsible for differential translocation kinetics in this family of evolutionarily conserved proteins. We found that the reversible translocation is mediated by the limited affinity of the βγ-subunits for membranes. The differential kinetics of the βγ-subunit types are determined by variations among a set of basic and hydrophobic residues in the γ-subunit types. G-protein signaling thus leverages the wide variation in membrane dissociation rates among different γ-subunit types to differentially control βγ-translocation kinetics in response to receptor activation. The conservation of primary structures of γ-subunits across mammalian species suggests that there can be evolutionary selection for primary structures that confer specific membrane-binding affinities and consequent rates of intermembrane movement.

  12. Suppression of Mediator is regulated by Cdk8-dependent Grr1 turnover of the Med3 coactivator.

    Science.gov (United States)

    Gonzalez, Deyarina; Hamidi, Nurul; Del Sol, Ricardo; Benschop, Joris J; Nancy, Thomas; Li, Chao; Francis, Lewis; Tzouros, Manuel; Krijgsveld, Jeroen; Holstege, Frank C P; Conlan, R Steven

    2014-02-18

    Mediator, an evolutionary conserved large multisubunit protein complex with a central role in regulating RNA polymerase II-transcribed genes, serves as a molecular switchboard at the interface between DNA binding transcription factors and the general transcription machinery. Mediator subunits include the Cdk8 module, which has both positive and negative effects on activator-dependent transcription through the activity of the cyclin-dependent kinase Cdk8, and the tail module, which is required for positive and negative regulation of transcription, correct preinitiation complex formation in basal and activated transcription, and Mediator recruitment. Currently, the molecular mechanisms governing Mediator function remain largely undefined. Here we demonstrate an autoregulatory mechanism used by Mediator to repress transcription through the activity of distinct components of different modules. We show that the function of the tail module component Med3, which is required for transcription activation, is suppressed by the kinase activity of the Cdk8 module. Med3 interacts with, and is phosphorylated by, Cdk8; site-specific phosphorylation triggers interaction with and degradation by the Grr1 ubiquitin ligase, thereby preventing transcription activation. This active repression mechanism involving Grr1-dependent ubiquitination of Med3 offers a rationale for the substoichiometric levels of the tail module that are found in purified Mediator and the corresponding increase in tail components seen in cdk8 mutants.

  13. Oxidative inhibition of the vascular Na+-K+ pump via NADPH oxidase-dependent β1-subunit glutathionylation: implications for angiotensin II-induced vascular dysfunction.

    Science.gov (United States)

    Liu, Chia-Chi; Karimi Galougahi, Keyvan; Weisbrod, Robert M; Hansen, Thomas; Ravaie, Ramtin; Nunez, Andrea; Liu, Yi B; Fry, Natasha; Garcia, Alvaro; Hamilton, Elisha J; Sweadner, Kathleen J; Cohen, Richard A; Figtree, Gemma A

    2013-12-01

    Glutathionylation of the Na(+)-K(+) pump's β1-subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na(+)-K(+) pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific β-subunit isoform expression and receptor-coupled pathways. As Na(+)-K(+) pump activity is an important determinant of vascular tone through effects on [Ca(2+)]i, we have examined the role of oxidative regulation of the Na(+)-K(+) pump in mediating angiotensin II (Ang II)-induced increases in vascular reactivity. β1-subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs), and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na(+)-K(+)ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K(+)-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na(+)-K(+) ATPase and decrease in K(+)-induced relaxation were reversed by preincubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of β1-subunit. Knock-out of FXYD1 dramatically decreased K(+)-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8 mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased β1-subunit glutathionylation, and enhanced K(+)-induced vasorelaxation. Ang II inhibits the Na(+)-K(+) pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pump's β1-subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na(+)-K(+) pump and may have an

  14. Differential expression of Mediator complex subunit MED15 in testicular germ cell tumors.

    Science.gov (United States)

    Klümper, Niklas; Syring, Isabella; Offermann, Anne; Shaikhibrahim, Zaki; Vogel, Wenzel; Müller, Stefan C; Ellinger, Jörg; Strauß, Arne; Radzun, Heinz Joachim; Ströbel, Philipp; Brägelmann, Johannes; Perner, Sven; Bremmer, Felix

    2015-09-17

    Testicular germ cell tumors (TGCT) are the most common cancer entities in young men with increasing incidence observed in the last decades. For therapeutic management it is important, that TGCT are divided into several histological subtypes. MED15 is part of the multiprotein Mediator complex which presents an integrative hub for transcriptional regulation and is known to be deregulated in several malignancies, such as prostate cancer and bladder cancer role, whereas the role of the Mediator complex in TGCT has not been investigated so far. Aim of the study was to investigate the implication of MED15 in TGCT development and its stratification into histological subtypes. Immunohistochemical staining (IHC) against Mediator complex subunit MED15 was conducted on a TGCT cohort containing tumor-free testis (n = 35), intratubular germ cell neoplasia unclassified (IGCNU, n = 14), seminomas (SEM, n = 107) and non-seminomatous germ cell tumors (NSGCT, n = 42), further subdivided into embryonic carcinomas (EC, n = 30), yolk sac tumors (YST, n = 5), chorionic carcinomas (CC, n = 5) and teratomas (TER, n = 2). Quantification of MED15 protein expression was performed through IHC followed by semi-quantitative image analysis using the Definiens software. In tumor-free seminiferous tubules, MED15 protein expression was absent or only low expressed in spermatogonia. Interestingly, the precursor lesions IGCNU exhibited heterogeneous but partly very strong MED15 expression. SEM weakly express the Mediator complex subunit MED15, whereas NSGCT and especially EC show significantly enhanced expression compared to tumor-free testis. In conclusion, MED15 is differentially expressed in tumor-free testis and TGCT. While MED15 is absent or low in tumor-free testis and SEM, NSGCT highly express MED15, hinting at the diagnostic potential of this marker to distinguish between SEM and NSGCT. Further, the precursor lesion IGCNU showed increased nuclear MED15

  15. Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex.

    Science.gov (United States)

    Tsai, Ming-Feng; Phillips, Charles B; Ranaghan, Matthew; Tsai, Chen-Wei; Wu, Yujiao; Willliams, Carole; Miller, Christopher

    2016-04-21

    Mitochondrial Ca(2+) uptake, a process crucial for bioenergetics and Ca(2+) signaling, is catalyzed by the mitochondrial calcium uniporter. The uniporter is a multi-subunit Ca(2+)-activated Ca(2+) channel, with the Ca(2+) pore formed by the MCU protein and Ca(2+)-dependent activation mediated by MICU subunits. Recently, a mitochondrial inner membrane protein EMRE was identified as a uniporter subunit absolutely required for Ca(2+) permeation. However, the molecular mechanism and regulatory purpose of EMRE remain largely unexplored. Here, we determine the transmembrane orientation of EMRE, and show that its known MCU-activating function is mediated by the interaction of transmembrane helices from both proteins. We also reveal a second function of EMRE: to maintain tight MICU regulation of the MCU pore, a role that requires EMRE to bind MICU1 using its conserved C-terminal polyaspartate tail. This dual functionality of EMRE ensures that all transport-competent uniporters are tightly regulated, responding appropriately to a dynamic intracellular Ca(2+) landscape.

  16. The evolutionarily conserved mediator subunit MDT-15/MED15 links protective innate immune responses and xenobiotic detoxification.

    Directory of Open Access Journals (Sweden)

    Read Pukkila-Worley

    2014-05-01

    Full Text Available Metazoans protect themselves from environmental toxins and virulent pathogens through detoxification and immune responses. We previously identified a small molecule xenobiotic toxin that extends survival of Caenorhabditis elegans infected with human bacterial pathogens by activating the conserved p38 MAP kinase PMK-1 host defense pathway. Here we investigate the cellular mechanisms that couple activation of a detoxification response to innate immunity. From an RNAi screen of 1,420 genes expressed in the C. elegans intestine, we identified the conserved Mediator subunit MDT-15/MED15 and 28 other gene inactivations that abrogate the induction of PMK-1-dependent immune effectors by this small molecule. We demonstrate that MDT-15/MED15 is required for the xenobiotic-induced expression of p38 MAP kinase PMK-1-dependent immune genes and protection from Pseudomonas aeruginosa infection. We also show that MDT-15 controls the induction of detoxification genes and functions to protect the host from bacteria-derived phenazine toxins. These data define a central role for MDT-15/MED15 in the coordination of xenobiotic detoxification and innate immune responses.

  17. The evolutionarily conserved mediator subunit MDT-15/MED15 links protective innate immune responses and xenobiotic detoxification.

    Science.gov (United States)

    Pukkila-Worley, Read; Feinbaum, Rhonda L; McEwan, Deborah L; Conery, Annie L; Ausubel, Frederick M

    2014-05-01

    Metazoans protect themselves from environmental toxins and virulent pathogens through detoxification and immune responses. We previously identified a small molecule xenobiotic toxin that extends survival of Caenorhabditis elegans infected with human bacterial pathogens by activating the conserved p38 MAP kinase PMK-1 host defense pathway. Here we investigate the cellular mechanisms that couple activation of a detoxification response to innate immunity. From an RNAi screen of 1,420 genes expressed in the C. elegans intestine, we identified the conserved Mediator subunit MDT-15/MED15 and 28 other gene inactivations that abrogate the induction of PMK-1-dependent immune effectors by this small molecule. We demonstrate that MDT-15/MED15 is required for the xenobiotic-induced expression of p38 MAP kinase PMK-1-dependent immune genes and protection from Pseudomonas aeruginosa infection. We also show that MDT-15 controls the induction of detoxification genes and functions to protect the host from bacteria-derived phenazine toxins. These data define a central role for MDT-15/MED15 in the coordination of xenobiotic detoxification and innate immune responses.

  18. Reconstitution of active human core Mediator complex reveals a critical role of the MED14 subunit.

    Science.gov (United States)

    Cevher, Murat A; Shi, Yi; Li, Dan; Chait, Brian T; Malik, Sohail; Roeder, Robert G

    2014-12-01

    The evolutionarily conserved Mediator complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here we report the reconstitution of a functional 15-subunit human core Mediator complex and its characterization by functional assays and chemical cross-linking coupled to MS (CX-MS). Whereas the reconstituted head and middle modules can stably associate, basal and coactivator functions are acquired only after incorporation of MED14 into the bimodular complex. This results from a dramatically enhanced ability of MED14-containing complexes to associate with Pol II. Altogether, our analyses identify MED14 as both an architectural and a functional backbone of the Mediator complex. We further establish a conditional requirement for metazoan-specific MED26 that becomes evident in the presence of heterologous nuclear factors. This general approach paves the way for systematic dissection of the multiple layers of functionality associated with the Mediator complex.

  19. Redox regulation of the MED28 and MED32 mediator subunits is important for development and senescence.

    Science.gov (United States)

    Shaikhali, Jehad; Davoine, Céline; Björklund, Stefan; Wingsle, Gunnar

    2016-05-01

    Mediator is a conserved multi-protein complex that acts as a bridge between promoter-bound transcriptional regulators and RNA polymerase II. While redox signaling is important in adjusting plant metabolism and development, the involvement of Mediator in redox homeostasis and regulation only recently started to emerge. Our previous results show that the MED10a, MED28, and MED32 Mediator subunits form various types of covalent oligomers linked by intermolecular disulfide bonds in vitro. To link that with biological significance we have characterized Arabidopsis med32 and med28 mutants and found that they are affected in root development and senescence, phenotypes possibly associated to redox changes.

  20. The Contrasting Role of the Mediator Subunit MED30 in the Progression of Bladder Cancer.

    Science.gov (United States)

    Syring, Isabella; Weiten, Richard; Müller, Tim; Schmidt, Doris; Steiner, Susanne; Kristiansen, Glen; Müller, Stefan C; Ellinger, Jörg

    2017-12-01

    The Mediator complex is a key regulator of gene transcription, and several studies have demonstrated altered expression of particular subunits in diverse human diseases, especially cancer. To date, nothing is known about the role of MED30 in bladder cancer. We, therefore, performed an RNA expression and survival analysis of the subunit MED30 in 537 samples of bladder cancer by using the database cBioPortal. To validate these data on the protein level, we practiced immunohistochemical staining against MED30 on a tissue microarray containing 210 samples of all tumour stages and performed survival analyses. For functional analysis, the siRNA-mediated knockdown of MED30 was performed in the cell lines T24 and TCCSUP followed by proliferation, migration, and invasion assays. On the mRNA and protein levels, higher expression of MED30 is associated with better patient survival. In accordance with this, advanced T- and N-stages showed lower expression of MED30. In contrast, knockdown of MED30 led to reduction of the tumour parameters proliferation, migration, and invasion in the BCa cell lines. MED30 appears to be integrated in the progression of the urothelial tumour in the bladder. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  1. Reconstitution of active human core Mediator complex reveals a pivotal role of the MED14 subunit

    Science.gov (United States)

    Cevher, Murat A.; Shi, Yi; Li, Dan; Chait, Brian T.; Malik, Sohail; Roeder, Robert G.

    2014-01-01

    The evolutionarily conserved Mediator complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here, we report the reconstitution of a functional 15-subunit human core Mediator complex and its characterization by functional assays and chemical cross-linking coupled to mass spectrometry (CX-MS). Whereas the reconstituted head and middle modules can stably associate, only with incorporation of MED14 into the bi-modular complex does it acquire basal and coactivator functions. This results from a dramatically enhanced ability of MED14-containing complexes to associate with Pol II. Altogether, our analyses identify MED14 as both an architectural and a functional backbone of the Mediator complex. We further establish a conditional requirement for metazoan-specific MED26 that becomes evident in the presence of heterologous nuclear factors. This general approach paves the way for systematically dissecting the multiple layers of functionalities associated with the Mediator complex. PMID:25383669

  2. TRAP230/ARC240 and TRAP240/ARC250 Mediator subunits are functionally conserved through evolution

    DEFF Research Database (Denmark)

    Samuelsen, Camilla O; Baraznenok, Vera; Khorosjutina, Olga

    2003-01-01

    In Saccharomyces cerevisiae Mediator, a subgroup of proteins (Srb8, Srb9, Srb10, and Srb11) form a module, which is involved in negative regulation of transcription. Homologues of Srb10 and Srb11 are found in some mammalian Mediator preparations, whereas no clear homologues have been reported...... for Srb8 and Srb9. Here, we identify a TRAP240/ARC250 homologue in Schizosaccharomyces pombe and demonstrate that this protein, spTrap240, is stably associated with a larger form of Mediator, which also contains conserved homologues of Srb8, Srb10, and Srb11. We find that spTrap240 and Sch. pombe Srb8 (sp......Srb8) regulate the same distinct subset of genes and have indistinguishable phenotypic characteristics. Importantly, Mediator containing the spSrb8/spTrap240/spSrb10/spSrb11 subunits is isolated only in free form, devoid of RNA polymerase II. In contrast, Mediator lacking this module associates...

  3. Sequence analysis of dolphin ferritin H and L subunits and possible iron-dependent translational control of dolphin ferritin gene

    Directory of Open Access Journals (Sweden)

    Sasaki Yukako

    2008-10-01

    Full Text Available Abstract Background Iron-storage protein, ferritin plays a central role in iron metabolism. Ferritin has dual function to store iron and segregate iron for protection of iron-catalyzed reactive oxygen species. Tissue ferritin is composed of two kinds of subunits (H: heavy chain or heart-type subunit; L: light chain or liver-type subunit. Ferritin gene expression is controlled at translational level in iron-dependent manner or at transcriptional level in iron-independent manner. However, sequencing analysis of marine mammalian ferritin subunits has not yet been performed fully. The purpose of this study is to reveal cDNA-derived amino acid sequences of cetacean ferritin H and L subunits, and demonstrate the possibility of expression of these subunits, especially H subunit, by iron. Methods Sequence analyses of cetacean ferritin H and L subunits were performed by direct sequencing of polymerase chain reaction (PCR fragments from cDNAs generated via reverse transcription-PCR of leukocyte total RNA prepared from blood samples of six different dolphin species (Pseudorca crassidens, Lagenorhynchus obliquidens, Grampus griseus, Globicephala macrorhynchus, Tursiops truncatus, and Delphinapterus leucas. The putative iron-responsive element sequence in the 5'-untranslated region of the six different dolphin species was revealed by direct sequencing of PCR fragments obtained using leukocyte genomic DNA. Results Dolphin H and L subunits consist of 182 and 174 amino acids, respectively, and amino acid sequence identities of ferritin subunits among these dolphins are highly conserved (H: 99–100%, (99→98 ; L: 98–100%. The conserved 28 bp IRE sequence was located -144 bp upstream from the initiation codon in the six different dolphin species. Conclusion These results indicate that six different dolphin species have conserved ferritin sequences, and suggest that these genes are iron-dependently expressed.

  4. The nematode homologue of Mediator complex subunit 28, F28F8.5, is a critical regulator of C. elegans development.

    Science.gov (United States)

    Kostrouchová, Markéta; Kostrouch, David; Chughtai, Ahmed A; Kaššák, Filip; Novotný, Jan P; Kostrouchová, Veronika; Benda, Aleš; Krause, Michael W; Saudek, Vladimír; Kostrouchová, Marta; Kostrouch, Zdeněk

    2017-01-01

    The evolutionarily conserved Mediator complex is a critical player in regulating transcription. Comprised of approximately two dozen proteins, the Mediator integrates diverse regulatory signals through direct protein-protein interactions that, in turn, modulate the influence of Mediator on RNA Polymerase II activity. One Mediator subunit, MED28, is known to interact with cytoplasmic structural proteins, providing a potential direct link between cytoplasmic dynamics and the control of gene transcription. Although identified in many animals and plants, MED28 is not present in yeast; no bona fide MED28 has been described previously in Caenorhabditis elegans. Here, we identify bioinformatically F28F8.5, an uncharacterized predicted protein, as the nematode homologue of MED28. As in other Metazoa, F28F8.5 has dual nuclear and cytoplasmic localization and plays critical roles in the regulation of development. F28F8.5 is a vital gene and its null mutants have severely malformed gonads and do not reproduce. F28F8.5 interacts on the protein level with the Mediator subunits MDT-6 and MDT-30. Our results indicate that F28F8.5 is an orthologue of MED28 and suggest that the potential to link cytoplasmic and nuclear events is conserved between MED28 vertebrate and nematode orthologues.

  5. Genome-wide association of mediator and RNA polymerase II in wild-type and mediator mutant yeast.

    Science.gov (United States)

    Paul, Emily; Zhu, Z Iris; Landsman, David; Morse, Randall H

    2015-01-01

    Mediator is a large, multisubunit complex that is required for essentially all mRNA transcription in eukaryotes. In spite of the importance of Mediator, the range of its targets and how it is recruited to these is not well understood. Previous work showed that in Saccharomyces cerevisiae, Mediator contributes to transcriptional activation by two distinct mechanisms, one depending on the tail module triad and favoring SAGA-regulated genes, and the second occurring independently of the tail module and favoring TFIID-regulated genes. Here, we use chromatin immunoprecipitation sequencing (ChIP-seq) to show that dependence on tail module subunits for Mediator recruitment and polymerase II (Pol II) association occurs preferentially at SAGA-regulated over TFIID-regulated genes on a genome-wide scale. We also show that recruitment of tail module subunits to active gene promoters continues genome-wide when Mediator integrity is compromised in med17 temperature-sensitive (ts) yeast, demonstrating the modular nature of the Mediator complex in vivo. In addition, our data indicate that promoters exhibiting strong and stable occupancy by Mediator have a wide range of activity and are enriched for targets of the Tup1-Cyc8 repressor complex. We also identify a number of strong Mediator occupancy peaks that overlap dubious open reading frames (ORFs) and are likely to include previously unrecognized upstream activator sequences. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. Cardiomyocyte-Specific Ablation of Med1 Subunit of the Mediator Complex Causes Lethal Dilated Cardiomyopathy in Mice.

    Science.gov (United States)

    Jia, Yuzhi; Chang, Hsiang-Chun; Schipma, Matthew J; Liu, Jing; Shete, Varsha; Liu, Ning; Sato, Tatsuya; Thorp, Edward B; Barger, Philip M; Zhu, Yi-Jun; Viswakarma, Navin; Kanwar, Yashpal S; Ardehali, Hossein; Thimmapaya, Bayar; Reddy, Janardan K

    2016-01-01

    Mediator, an evolutionarily conserved multi-protein complex consisting of about 30 subunits, is a key component of the polymerase II mediated gene transcription. Germline deletion of the Mediator subunit 1 (Med1) of the Mediator in mice results in mid-gestational embryonic lethality with developmental impairment of multiple organs including heart. Here we show that cardiomyocyte-specific deletion of Med1 in mice (csMed1-/-) during late gestational and early postnatal development by intercrossing Med1fl/fl mice to α-MyHC-Cre transgenic mice results in lethality within 10 days after weaning due to dilated cardiomyopathy-related ventricular dilation and heart failure. The csMed1-/- mouse heart manifests mitochondrial damage, increased apoptosis and interstitial fibrosis. Global gene expression analysis revealed that loss of Med1 in heart down-regulates more than 200 genes including Acadm, Cacna1s, Atp2a2, Ryr2, Pde1c, Pln, PGC1α, and PGC1β that are critical for calcium signaling, cardiac muscle contraction, arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy and peroxisome proliferator-activated receptor regulated energy metabolism. Many genes essential for oxidative phosphorylation and proper mitochondrial function such as genes coding for the succinate dehydrogenase subunits of the mitochondrial complex II are also down-regulated in csMed1-/- heart contributing to myocardial injury. Data also showed up-regulation of about 180 genes including Tgfb2, Ace, Atf3, Ctgf, Angpt14, Col9a2, Wisp2, Nppa, Nppb, and Actn1 that are linked to cardiac muscle contraction, cardiac hypertrophy, cardiac fibrosis and myocardial injury. Furthermore, we demonstrate that cardiac specific deletion of Med1 in adult mice using tamoxifen-inducible Cre approach (TmcsMed1-/-), results in rapid development of cardiomyopathy and death within 4 weeks. We found that the key findings of the csMed1-/- studies described above are highly reproducible in TmcsMed1-/- mouse heart

  7. Subcellular compartmentation, interdependency and dynamics of the cyclic AMP-dependent PKA subunits during pathogenic differentiation in rice blast.

    Science.gov (United States)

    Selvaraj, Poonguzhali; Tham, Hong Fai; Ramanujam, Ravikrishna; Naqvi, Naweed I

    2017-08-01

    The cAMP-dependent PKA signalling plays a central role in growth, asexual development and pathogenesis in fungal pathogens. Here, we functionally characterised RPKA, the regulatory subunit of cAMP/PKA and studied the dynamics and organisation of the PKA subunits in the rice blast pathogen Magnaporthe oryzae. The RPKA subunit was essential for proper vegetative growth, asexual sporulation and surface hydrophobicity in M. oryzae. A spontaneous suppressor mutation, SMR19, that restored growth and conidiation in the RPKA deletion mutant was isolated and characterised. SMR19 enhanced conidiation and appressorium formation but failed to suppress the pathogenesis defects in rpkAΔ. The PKA activity was undetectable in the mycelial extracts of SMR19, which showed a single mutation (val242leu) in the highly conserved active site of the catalytic subunit (CPKA) of cAMP/PKA. The two subunits of cAMP/PKA showed different subcellular localisation patterns with RpkA being predominantly nucleocytoplasmic in conidia, while CpkA was largely cytosolic and/or vesicular. The CpkA anchored RpkA in cytoplasmic vesicles, and localisation of PKA in the cytoplasm was governed by CpkA in a cAMP-dependant or independent manner. We show that there exists a tight regulation of PKA subunits at the level of transcription, and the cAMP signalling is differentially compartmentalised in a stage-specific manner in rice blast. © 2017 John Wiley & Sons Ltd.

  8. Age-related changes in functional postsynaptic nicotinic acetylcholine receptor subunits in neurons of the laterodorsal tegmental nucleus, a nucleus important in drug addiction.

    Science.gov (United States)

    Christensen, Mark H; Kohlmeier, Kristi A

    2016-03-01

    The earlier an individual initiates cigarette smoking, the higher the likelihood of development of dependency to nicotine, the addictive ingredient in cigarettes. One possible mechanism underlying this higher addiction liability is an ontogenetically differential cellular response induced by nicotine in neurons mediating the reinforcing or euphoric effects of this drug, which could arise from age-related differences in the composition of nicotinic acetylcholine receptor (nAChR) subunits. In the current study, we examined whether the subunit composition of nAChRs differed between neurons within the laterodorsal tegmentum (LDT), a nucleus importantly involved in drug addiction associated behaviours, across two periods of ontogeny in which nicotine-mediated excitatory responses were shown to depend on age. To this end, whole-cell patch-clamp recordings in mouse brain slices from identified LDT neurons, in combination with nAChR subunit-specific receptor antagonists, were conducted. Comparison of the contribution of different nAChR subunits to acetylcholine (ACh)-induced inward currents indicated that the contributions of the β2 and/or β4 and α7 nAChR subunits alter across age. Taken together, we conclude that across a limited ontogenetic period, there is plasticity in the subunit composition of nAChRs in LDT neurons. In addition, our data indicate, for the first time, functional presence of α6 nAChR subunits in LDT neurons within the age ranges studied. Changes in subunit composition of nAChRs across ontogeny could contribute to the age-related differential excitability induced by nicotine. Differences in the subunit composition of nAChRs within the LDT would be expected to contribute to ontogenetic-dependent outflow from the LDT to target regions, which include reward-related circuitry. © 2014 Society for the Study of Addiction.

  9. The nematode homologue of Mediator complex subunit 28, F28F8.5, is a critical regulator of C. elegans development

    Directory of Open Access Journals (Sweden)

    Markéta Kostrouchová

    2017-06-01

    Full Text Available The evolutionarily conserved Mediator complex is a critical player in regulating transcription. Comprised of approximately two dozen proteins, the Mediator integrates diverse regulatory signals through direct protein-protein interactions that, in turn, modulate the influence of Mediator on RNA Polymerase II activity. One Mediator subunit, MED28, is known to interact with cytoplasmic structural proteins, providing a potential direct link between cytoplasmic dynamics and the control of gene transcription. Although identified in many animals and plants, MED28 is not present in yeast; no bona fide MED28 has been described previously in Caenorhabditis elegans. Here, we identify bioinformatically F28F8.5, an uncharacterized predicted protein, as the nematode homologue of MED28. As in other Metazoa, F28F8.5 has dual nuclear and cytoplasmic localization and plays critical roles in the regulation of development. F28F8.5 is a vital gene and its null mutants have severely malformed gonads and do not reproduce. F28F8.5 interacts on the protein level with the Mediator subunits MDT-6 and MDT-30. Our results indicate that F28F8.5 is an orthologue of MED28 and suggest that the potential to link cytoplasmic and nuclear events is conserved between MED28 vertebrate and nematode orthologues.

  10. Assessing subunit dependency of the Plasmodium proteasome using small molecule inhibitors and active site probes.

    Science.gov (United States)

    Li, Hao; van der Linden, Wouter A; Verdoes, Martijn; Florea, Bogdan I; McAllister, Fiona E; Govindaswamy, Kavitha; Elias, Joshua E; Bhanot, Purnima; Overkleeft, Herman S; Bogyo, Matthew

    2014-08-15

    The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

  11. Contribution of the major and minor subunits to fimbria-mediated adherence of Haemophilus influenzae to human epithelial cells and erythrocytes

    NARCIS (Netherlands)

    van Ham, S. M.; van Alphen, L.; Mooi, F. R.; van Putten, J. P.

    1995-01-01

    Fimbriae are colonization factors of the human pathogen Haemophilus influenzae in that they mediate bacterial adherence to human eukaryotic cells. The contribution of the major (HifA) and putative minor (HifD and HifE) subunits of H. influenzae fimbriae to fimbria-specific adherence was studied by

  12. Inhibition of Ku70 acetylation by INHAT subunit SET/TAF-Iβ regulates Ku70-mediated DNA damage response.

    Science.gov (United States)

    Kim, Kee-Beom; Kim, Dong-Wook; Park, Jin Woo; Jeon, Young-Joo; Kim, Daehwan; Rhee, Sangmyung; Chae, Jung-Il; Seo, Sang-Beom

    2014-07-01

    DNA double-strand breaks (DSBs) can cause either cell death or genomic instability. The Ku heterodimer Ku70/80 is required for the NHEJ (non-homologous end-joining) DNA DSB repair pathway. The INHAT (inhibitor of histone acetyltransferases) complex subunit, SET/TAF-Iβ, can inhibit p300- and PCAF-mediated acetylation of both histone and p53, thereby repressing general transcription and that of p53 target genes. Here, we show that SET/TAF-Iβ interacts with Ku70/80, and that this interaction inhibits CBP- and PCAF-mediated Ku70 acetylation in an INHAT domain-dependent manner. Notably, DNA damage by UV disrupted the interaction between SET/TAF-Iβ and Ku70. Furthermore, we demonstrate that overexpressed SET/TAF-Iβ inhibits recruitment of Ku70/80 to DNA damage sites. We propose that dysregulation of SET/TAF-Iβ expression prevents repair of damaged DNA and also contributes to cellular proliferation. All together, our findings indicate that SET/TAF-Iβ interacts with Ku70/80 in the nucleus and inhibits Ku70 acetylation. Upon DNA damage, SET/TAF-Iβ dissociates from the Ku complex and releases Ku70/Ku80, which are then recruited to DNA DSB sites via the NHEJ DNA repair pathway.

  13. Vascular smooth muscle cells express the alpha(1A) subunit of a P-/Q-type voltage-dependent Ca(2+)Channel, and It is functionally important in renal afferent arterioles

    DEFF Research Database (Denmark)

    Hansen, Pernille B. Lærkegaard; Jensen, Boye L.; Andreasen, D

    2000-01-01

    In the present study, we tested whether the alpha(1A) subunit, which encodes a neuronal isoform of voltage-dependent Ca(2+) channels (VDCCs) (P-/Q-type), was present and functional in vascular smooth muscle and renal resistance vessels. By reverse transcription-polymerase chain reaction...... preglomerular resistance vessels and aorta, as well as mesangial cells, and that P-type VDCCs contribute to Ca(2+) influx in aortic and renal VSMCs and are involved in depolarization-mediated contraction in renal afferent arterioles....

  14. Helicobacter pylori VacA toxin/subunit p34: targeting of an anion channel to the inner mitochondrial membrane.

    Directory of Open Access Journals (Sweden)

    Grazyna Domańska

    2010-04-01

    Full Text Available The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% beta-strands, similar to pore-forming beta-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylaminobenzoic acid (NPPB, a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal.

  15. Novel isoforms of the TFIID subunit TAF4 modulate nuclear receptor-mediated transcriptional activity

    International Nuclear Information System (INIS)

    Brunkhorst, Adrian; Neuman, Toomas; Hall, Anita; Arenas, Ernest; Bartfai, Tamas; Hermanson, Ola; Metsis, Madis

    2004-01-01

    The transcription factor TFIID consists of TATA-binding protein (TBP) and TBP-associated factors (TAFs). TAFs are essential for modulation of transcriptional activity but the regulation of TAFs is complex and many important aspects remain unclear. In this study, we have identified and characterized five novel truncated forms of the TFIID subunit TAF4 (TAF II 135). Analysis of the mouse gene structure revealed that all truncations were the results of alternative splicing and resulted in the loss of domains or parts of domains implicated in TAF4 functional interactions. Results from transcriptional assays showed that several of the TAF4 isoforms exerted dominant negative effects on TAF4 activity in nuclear receptor-mediated transcriptional activation. In addition, alternative TAF4 isoforms could be detected in specific cell types. Our results indicate an additional level of complexity in TAF4-mediated regulation of transcription and suggest context-specific roles for these new TAF4 isoforms in transcriptional regulation in vivo

  16. A dominant mutation in mediator of paramutation2, one of three second-largest subunits of a plant-specific RNA polymerase, disrupts multiple siRNA silencing processes.

    Science.gov (United States)

    Sidorenko, Lyudmila; Dorweiler, Jane E; Cigan, A Mark; Arteaga-Vazquez, Mario; Vyas, Meenal; Kermicle, Jerry; Jurcin, Diane; Brzeski, Jan; Cai, Yu; Chandler, Vicki L

    2009-11-01

    Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA-mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V-like complexes could provide maize with a greater diversification of RNA-mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state-a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV-like) and potentially processes downstream (Pol-V-like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2-1 dominance

  17. Identification of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel target of bisphenol A.

    Science.gov (United States)

    Ito, Yuki; Ito, Takumi; Karasawa, Satoki; Enomoto, Teruya; Nashimoto, Akihiro; Hase, Yasuyoshi; Sakamoto, Satoshi; Mimori, Tsuneyo; Matsumoto, Yoshihisa; Yamaguchi, Yuki; Handa, Hiroshi

    2012-01-01

    Bisphenol A (BPA) forms the backbone of plastics and epoxy resins used to produce packaging for various foods and beverages. BPA is also an estrogenic disruptor, interacting with human estrogen receptors (ER) and other related nuclear receptors. Nevertheless, the effects of BPA on human health remain unclear. The present study identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel BPA-binding protein. DNA-PKcs, in association with the Ku heterodimer (Ku70/80), is a critical enzyme involved in the repair of DNA double-strand breaks. Low levels of DNA-PK activity are previously reported to be associated with an increased risk of certain types of cancer. Although the Kd for the interaction between BPA and a drug-binding mutant of DNA-PKcs was comparatively low (137 nM), high doses of BPA were required before cellular effects were observed (100-300 μM). The results of an in vitro kinase assay showed that BPA inhibited DNA-PK kinase activity in a concentration-dependent manner. In M059K cells, BPA inhibited the phosphorylation of DNA-PKcs at Ser2056 and H2AX at Ser139 in response to ionizing radiation (IR)-irradiation. BPA also disrupted DNA-PKcs binding to Ku70/80 and increased the radiosensitivity of M059K cells, but not M059J cells (which are DNA-PKcs-deficient). Taken together, these results provide new evidence of the effects of BPA on DNA repair in mammalian cells, which are mediated via inhibition of DNA-PK activity. This study may warrant the consideration of the possible carcinogenic effects of high doses of BPA, which are mediated through its action on DNA-PK.

  18. Laccase/Mediator Systems

    NARCIS (Netherlands)

    Hilgers, Roelant; Vincken, Jean Paul; Gruppen, Harry; Kabel, Mirjam A.

    2018-01-01

    Laccase-mediator systems (LMS) have been widely studied for their capacity to oxidize the nonphenolic subunits of lignin (70-90% of the polymer). The phenolic subunits (10-30% of the polymer), which can also be oxidized without mediators, have received considerably less attention. Consequently, it

  19. Evolution of the cAMP-dependent protein kinase (PKA catalytic subunit isoforms.

    Directory of Open Access Journals (Sweden)

    Kristoffer Søberg

    Full Text Available The 3',5'-cyclic adenosine monophosphate (cAMP-dependent protein kinase, or protein kinase A (PKA, pathway is one of the most versatile and best studied signaling pathways in eukaryotic cells. The two paralogous PKA catalytic subunits Cα and Cβ, encoded by the genes PRKACA and PRKACB, respectively, are among the best understood model kinases in signal transduction research. In this work, we explore and elucidate the evolution of the alternative 5' exons and the splicing pattern giving rise to the numerous PKA catalytic subunit isoforms. In addition to the universally conserved Cα1/Cβ1 isoforms, we find kinase variants with short N-termini in all main vertebrate classes, including the sperm-specific Cα2 isoform found to be conserved in all mammals. We also describe, for the first time, a PKA Cα isoform with a long N-terminus, paralogous to the PKA Cβ2 N-terminus. An analysis of isoform-specific variation highlights residues and motifs that are likely to be of functional importance.

  20. The Tlo Proteins Are Stoichiometric Components of Candida albicans Mediator Anchored via the Med3 Subunit

    Science.gov (United States)

    Zhang, Anda; Petrov, Kostadin O.; Hyun, Emily R.; Liu, Zhongle; Gerber, Scott A.

    2012-01-01

    The amplification of the TLO (for telomere-associated) genes in Candida albicans, compared to its less pathogenic, close relative Candida dubliniensis, suggests a role in virulence. Little, however, is known about the function of the Tlo proteins. We have purified the Mediator coactivator complex from C. albicans (caMediator) and found that Tlo proteins are a stoichiometric component of caMediator. Many members of the Tlo family are expressed, and each is a unique member of caMediator. Protein expression analysis of individual Tlo proteins, as well as the purification of tagged Tlo proteins, demonstrate that there is a large free population of Tlo proteins in addition to the Mediator-associated population. Coexpression and copurification of Tloα12 and caMed3 in Escherichia coli established a direct physical interaction between the two proteins. We have also made a C. albicans med3Δ/Δ strain and purified an intact Mediator from this strain. The analysis of the composition of the med3Δ Mediator shows that it lacks a Tlo subunit. Regarding Mediator function, the med3Δ/Δ strain serves as a substitute for the difficult-to-make tloΔ/Δ C. albicans strain. A potential role of the TLO and MED3 genes in virulence is supported by the inability of the med3Δ/Δ strain to form normal germ tubes. This study of caMediator structure provides initial clues to the mechanism of action of the Tlo genes and a platform for further mechanistic studies of caMediator's involvement in gene regulatory patterns that underlie pathogenesis. PMID:22562472

  1. MC EMiNEM maps the interaction landscape of the Mediator.

    Directory of Open Access Journals (Sweden)

    Theresa Niederberger

    Full Text Available The Mediator is a highly conserved, large multiprotein complex that is involved essentially in the regulation of eukaryotic mRNA transcription. It acts as a general transcription factor by integrating regulatory signals from gene-specific activators or repressors to the RNA Polymerase II. The internal network of interactions between Mediator subunits that conveys these signals is largely unknown. Here, we introduce MC EMiNEM, a novel method for the retrieval of functional dependencies between proteins that have pleiotropic effects on mRNA transcription. MC EMiNEM is based on Nested Effects Models (NEMs, a class of probabilistic graphical models that extends the idea of hierarchical clustering. It combines mode-hopping Monte Carlo (MC sampling with an Expectation-Maximization (EM algorithm for NEMs to increase sensitivity compared to existing methods. A meta-analysis of four Mediator perturbation studies in Saccharomyces cerevisiae, three of which are unpublished, provides new insight into the Mediator signaling network. In addition to the known modular organization of the Mediator subunits, MC EMiNEM reveals a hierarchical ordering of its internal information flow, which is putatively transmitted through structural changes within the complex. We identify the N-terminus of Med7 as a peripheral entity, entailing only local structural changes upon perturbation, while the C-terminus of Med7 and Med19 appear to play a central role. MC EMiNEM associates Mediator subunits to most directly affected genes, which, in conjunction with gene set enrichment analysis, allows us to construct an interaction map of Mediator subunits and transcription factors.

  2. MC EMiNEM maps the interaction landscape of the Mediator.

    Science.gov (United States)

    Niederberger, Theresa; Etzold, Stefanie; Lidschreiber, Michael; Maier, Kerstin C; Martin, Dietmar E; Fröhlich, Holger; Cramer, Patrick; Tresch, Achim

    2012-01-01

    The Mediator is a highly conserved, large multiprotein complex that is involved essentially in the regulation of eukaryotic mRNA transcription. It acts as a general transcription factor by integrating regulatory signals from gene-specific activators or repressors to the RNA Polymerase II. The internal network of interactions between Mediator subunits that conveys these signals is largely unknown. Here, we introduce MC EMiNEM, a novel method for the retrieval of functional dependencies between proteins that have pleiotropic effects on mRNA transcription. MC EMiNEM is based on Nested Effects Models (NEMs), a class of probabilistic graphical models that extends the idea of hierarchical clustering. It combines mode-hopping Monte Carlo (MC) sampling with an Expectation-Maximization (EM) algorithm for NEMs to increase sensitivity compared to existing methods. A meta-analysis of four Mediator perturbation studies in Saccharomyces cerevisiae, three of which are unpublished, provides new insight into the Mediator signaling network. In addition to the known modular organization of the Mediator subunits, MC EMiNEM reveals a hierarchical ordering of its internal information flow, which is putatively transmitted through structural changes within the complex. We identify the N-terminus of Med7 as a peripheral entity, entailing only local structural changes upon perturbation, while the C-terminus of Med7 and Med19 appear to play a central role. MC EMiNEM associates Mediator subunits to most directly affected genes, which, in conjunction with gene set enrichment analysis, allows us to construct an interaction map of Mediator subunits and transcription factors.

  3. FXYD proteins reverse inhibition of the Na+-K+ pump mediated by glutathionylation of its beta1 subunit.

    Science.gov (United States)

    Bibert, Stéphanie; Liu, Chia-Chi; Figtree, Gemma A; Garcia, Alvaro; Hamilton, Elisha J; Marassi, Francesca M; Sweadner, Kathleen J; Cornelius, Flemming; Geering, Käthi; Rasmussen, Helge H

    2011-05-27

    The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump β(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the β(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.

  4. Regulation of metabolism by the Mediator complex.

    Science.gov (United States)

    Youn, Dou Yeon; Xiaoli, Alus M; Pessin, Jeffrey E; Yang, Fajun

    2016-01-01

    The Mediator complex was originally discovered in yeast, but it is conserved in all eukaryotes. Its best-known function is to regulate RNA polymerase II-dependent gene transcription. Although the mechanisms by which the Mediator complex regulates transcription are often complicated by the context-dependent regulation, this transcription cofactor complex plays a pivotal role in numerous biological pathways. Biochemical, molecular, and physiological studies using cancer cell lines or model organisms have established the current paradigm of the Mediator functions. However, the physiological roles of the mammalian Mediator complex remain poorly defined, but have attracted a great interest in recent years. In this short review, we will summarize some of the reported functions of selective Mediator subunits in the regulation of metabolism. These intriguing findings suggest that the Mediator complex may be an important player in nutrient sensing and energy balance in mammals.

  5. Functional characterization of the mammalian iAAA protease subunit, YME1L

    OpenAIRE

    Majczak, Joanna

    2008-01-01

    The iAAA protease is an ATP-dependent proteolytic complex in the mitochondrial inner membrane and belongs to the highly conserved family of AAA proteins. In the yeast Saccharomyces cerevisiae, the iAAA protease is a homo-oligomeric complex composed of Yme1p subunits which are active in the intermembrane space and mediate protein quality control. Yeast cells lacking Yme1p are characterized by pleiotropic phenotypes including a respiratory deficiency at elevated temperature and an aberrant mito...

  6. MEDIATOR18 and MEDIATOR20 confer susceptibility to Fusarium oxysporum in Arabidopsis thaliana

    OpenAIRE

    Fallath, Thorya; Kidd, Brendan N.; Stiller, Jiri; Davoine, Celine; Bj?rklund, Stefan; Manners, John M.; Kazan, Kemal; Schenk, Peer M.

    2017-01-01

    The conserved protein complex known as Mediator conveys transcriptional signals by acting as an intermediary between transcription factors and RNA polymerase II. As a result, Mediator subunits play multiple roles in regulating developmental as well as abiotic and biotic stress pathways. In this report we identify the head domain subunits MEDIATOR18 and MEDIATOR20 as important susceptibility factors for Fusarium oxysporum infection in Arabidopsis thaliana. Mutants of MED18 and MED20 display do...

  7. An in vivo requirement for the mediator subunit med14 in the maintenance of stem cell populations.

    Science.gov (United States)

    Burrows, Jeffrey T A; Pearson, Bret J; Scott, Ian C

    2015-04-14

    The Mediator complex has recently been shown to be a key player in the maintenance of embryonic and induced pluripotent stem cells. However, the in vivo consequences of loss of many Mediator subunits are unknown. We identified med14 as the gene affected in the zebrafish logelei (log) mutant, which displayed a morphological arrest by 2 days of development. Surprisingly, microarray analysis showed that transcription was not broadly affected in log mutants. Indeed, log cells transplanted into a wild-type environment were able to survive into adulthood. In planarians, RNAi knockdown demonstrated a requirement for med14 and many other Mediator components in adult stem cell maintenance and regeneration. Multiple stem/progenitor cell populations were observed to be reduced or absent in zebrafish med14 mutant embryos. Taken together, our results show a critical, evolutionarily conserved, in vivo function for Med14 (and Mediator) in stem cell maintenance, distinct from a general role in transcription. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Involvement of N-methyl-D-aspartate receptor subunits in zinc-mediated modification of CA1 long-term potentiation in the developing hippocampus.

    Science.gov (United States)

    Takeda, Atsushi; Itagaki, Kosuke; Ando, Masaki; Oku, Naoto

    2012-03-01

    Zinc is an endogenous N-methyl-D-aspartate (NMDA) receptor blocker. It is possible that zinc-mediated modification of hippocampal CA1 long-term potentiation (LTP) is linked to the expression of NMDA receptor subunits, which varies with postnatal development. In the present study, the effect of ZnCl(2) and CaEDTA, a membrane-impermeable zinc chelator, on CA1 LTP induction was examined in hippocampal slices from immature (3-week-old) and young (6-week-old) rats. Tetanus (10-100 Hz, 1 sec)-induced CA1 LTP was more greatly enhanced in 3-week-old rats. CA1 LTP was inhibited in the presence of 2-amino-5-phosphonovalerate (APV), an NMDA receptor antagonist, and CaEDTA in 3-week-old rats, as in the case of 6-week-old rats reported previously. In 3-week-old rats, on the other hand, 5 μM ZnCl(2) attenuated NMDA receptor-mediated EPSPs more than in 6-week-old rats and significantly attenuated CA1 LTP. Moreover, 5 μM ZnCl(2) significantly attenuated CA1 LTP in the presence of (2R,4S)-4-(3-phosphonopropyl)-2-piperidinecarboxylic acid (PPPA), an NR2A antagonist, in 3-week-old rats, but not that in the presence of ifenprodil, an NR2B antagonist, suggesting that zinc-mediated attenuation of CA1 LTP is associated with the preferential expression of NR2B subunit in 3-week-old rats. In 6-week-old rats, however, 5 μM ZnCl(2) significantly potentiated CA1 LTP and also CA1 LTP in the presence of PPPA. The present study demonstrates that endogenous zinc may participate in the induction of CA1 LTP. It is likely that the changes in expression of NMDA receptor subunits are involved in the zinc-mediated modification of CA1 LTP in the developing hippocampus. Copyright © 2011 Wiley Periodicals, Inc.

  9. Cloning, chromosomal localization, and functional expression of the alpha 1 subunit of the L-type voltage-dependent calcium channel from normal human heart

    NARCIS (Netherlands)

    Schultz, D; Mikala, G; Yatani, A; Engle, D B; Iles, D E; Segers, B; Sinke, R J; Weghuis, D O; Klöckner, U; Wakamori, M

    1993-01-01

    A unique structural variant of the cardiac L-type voltage-dependent calcium channel alpha 1 subunit cDNA was isolated from libraries derived from normal human heart mRNA. The deduced amino acid sequence shows significant homology to other calcium channel alpha 1 subunits. However, differences from

  10. NR2B subunit-dependent long-term potentiation enhancement in the rat cortical auditory system in vivo following masking of patterned auditory input by white noise exposure during early postnatal life.

    Science.gov (United States)

    Hogsden, Jennifer L; Dringenberg, Hans C

    2009-08-01

    The composition of N-methyl-D-aspartate (NMDA) receptor subunits influences the degree of synaptic plasticity expressed during development and into adulthood. Here, we show that theta-burst stimulation of the medial geniculate nucleus reliably induced NMDA receptor-dependent long-term potentiation (LTP) of field postsynaptic potentials recorded in the primary auditory cortex (A1) of urethane-anesthetized rats. Furthermore, substantially greater levels of LTP were elicited in juvenile animals (30-37 days old; approximately 55% maximal potentiation) than in adult animals (approximately 30% potentiation). Masking patterned sound via continuous white noise exposure during early postnatal life (from postnatal day 5 to postnatal day 50-60) resulted in enhanced, juvenile-like levels of LTP (approximately 70% maximal potentiation) relative to age-matched controls reared in unaltered acoustic environments (approximately 30%). Rats reared in white noise and then placed in unaltered acoustic environments for 40-50 days showed levels of LTP comparable to those of adult controls, indicating that white noise rearing results in a form of developmental arrest that can be overcome by subsequent patterned sound exposure. We explored the mechanisms mediating white noise-induced plasticity enhancements by local NR2B subunit antagonist application in A1. NR2B subunit antagonists (Ro 25-6981 or ifenprodil) completely reversed white noise-induced LTP enhancement at concentrations that did not affect LTP in adult or age-matched controls. We conclude that white noise exposure during early postnatal life results in the maintenance of juvenile-like, higher levels of plasticity in A1, an effect that appears to be critically dependent on NR2B subunit activation.

  11. The mediator complex in genomic and non-genomic signaling in cancer.

    Science.gov (United States)

    Weber, Hannah; Garabedian, Michael J

    2018-05-01

    Mediator is a conserved, multi-subunit macromolecular machine divided structurally into head, middle, and tail modules, along with a transiently associating kinase module. Mediator functions as an integrator of transcriptional regulatory activity by interacting with DNA-bound transcription factors and with RNA polymerase II (Pol II) to both activate and repress gene expression. Mediator has been shown to affect multiple steps in transcription, including chromatin looping between enhancers and promoters, pre-initiation complex formation, transcriptional elongation, and mRNA splicing. Individual Mediator subunits participate in regulation of gene expression by the estrogen and androgen receptors and are altered in a number of endocrine cancers, including breast and prostate cancer. In addition to its role in genomic signaling, MED12 has been implicated in non-genomic signaling by interacting with and activating TGF-beta receptor 2 in the cytoplasm. Recent structural studies have revealed extensive inter-domain interactions and complex architecture of the Mediator-Pol II complex, suggesting that Mediator is capable of reorganizing its conformation and composition to fit cellular needs. We propose that alterations in Mediator subunit expression that occur in various cancers could impact the organization and function of Mediator, resulting in changes in gene expression that promote malignancy. A better understanding of the role of Mediator in cancer could reveal new approaches to the diagnosis and treatment of Mediator-dependent endocrine cancers, especially in settings of therapy resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Two conserved modules of Schizosaccharomyces pombe Mediator regulate distinct cellular pathways

    DEFF Research Database (Denmark)

    Linder, Tomas; Rasmussen, Nina; Samuelsen, Camilla O

    2008-01-01

    Mediator is an evolutionary conserved coregulator complex required for transcription of almost all RNA polymerase II-dependent genes. The Schizosaccharomyces pombe Mediator consists of two dissociable components-a core complex organized into a head and middle domain as well as the Cdk8 regulatory...... subcomplex. In this work we describe a functional characterization of the S. pombe Mediator. We report the identification of the S. pombe Med20 head subunit and the isolation of ts alleles of the core head subunit encoding med17+. Biochemical analysis of med8(ts), med17(ts), Deltamed18, Deltamed20...... and Deltamed27 alleles revealed a stepwise head domain molecular architecture. Phenotypical analysis of Cdk8 and head module alleles including expression profiling classified the Mediator mutant alleles into one of two groups. Cdk8 module mutants flocculate due to overexpression of adhesive cell...

  13. Enhanced Long-Term and Impaired Short-Term Spatial Memory in GluA1 AMPA Receptor Subunit Knockout Mice: Evidence for a Dual-Process Memory Model

    Science.gov (United States)

    Sanderson, David J.; Good, Mark A.; Skelton, Kathryn; Sprengel, Rolf; Seeburg, Peter H.; Rawlins, J. Nicholas P.; Bannerman, David M.

    2009-01-01

    The GluA1 AMPA receptor subunit is a key mediator of hippocampal synaptic plasticity and is especially important for a rapidly-induced, short-lasting form of potentiation. GluA1 gene deletion impairs hippocampus-dependent, spatial working memory, but spares hippocampus-dependent spatial reference memory. These findings may reflect the necessity of…

  14. Role of the Na(+)/K(+)-ATPase beta-subunit in peptide-mediated transdermal drug delivery.

    Science.gov (United States)

    Wang, Changli; Ruan, Renquan; Zhang, Li; Zhang, Yunjiao; Zhou, Wei; Lin, Jun; Ding, Weiping; Wen, Longping

    2015-04-06

    In this work, we discovered that the Na(+)/K(+)-ATPase beta-subunit (ATP1B1) on epidermal cells plays a key role in the peptide-mediated transdermal delivery of macromolecular drugs. First, using a yeast two-hybrid assay, we screened candidate proteins that have specific affinity for the short peptide TD1 (ACSSSPSKHCG) identified in our previous work. Then, we verified the specific binding of TD1 to ATP1B1 in yeast and mammalian cells by a pull-down ELISA and an immunoprecipitation assay. Finally, we confirmed that TD1 mainly interacted with the C-terminus of ATP1B1. Our results showed that the interaction between TD1 and ATP1B1 affected not only the expression and localization of ATP1B1, but also the epidermal structure. In addition, this interaction could be antagonized by the exogenous competitor ATP1B1 or be inhibited by ouabain, which results in the decreased delivery of macromolecular drugs across the skin. The discovery of a critical role of ATP1B1 in the peptide-mediated transdermal drug delivery is of great significance for the future development of new transdermal peptide enhancers.

  15. The Mediator complex and transcription regulation

    Science.gov (United States)

    Poss, Zachary C.; Ebmeier, Christopher C.

    2013-01-01

    The Mediator complex is a multi-subunit assembly that appears to be required for regulating expression of most RNA polymerase II (pol II) transcripts, which include protein-coding and most non-coding RNA genes. Mediator and pol II function within the pre-initiation complex (PIC), which consists of Mediator, pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH and is approximately 4.0 MDa in size. Mediator serves as a central scaffold within the PIC and helps regulate pol II activity in ways that remain poorly understood. Mediator is also generally targeted by sequence-specific, DNA-binding transcription factors (TFs) that work to control gene expression programs in response to developmental or environmental cues. At a basic level, Mediator functions by relaying signals from TFs directly to the pol II enzyme, thereby facilitating TF-dependent regulation of gene expression. Thus, Mediator is essential for converting biological inputs (communicated by TFs) to physiological responses (via changes in gene expression). In this review, we summarize an expansive body of research on the Mediator complex, with an emphasis on yeast and mammalian complexes. We focus on the basics that underlie Mediator function, such as its structure and subunit composition, and describe its broad regulatory influence on gene expression, ranging from chromatin architecture to transcription initiation and elongation, to mRNA processing. We also describe factors that influence Mediator structure and activity, including TFs, non-coding RNAs and the CDK8 module. PMID:24088064

  16. Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage.

    Science.gov (United States)

    Lu, Chenggang; Fuller, Margaret T

    2015-12-01

    Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s). In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. Here we show that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC), a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs), spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. Our results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages.

  17. Adaptor protein complex 2-mediated, clathrin-dependent endocytosis, and related gene activities, are a prominent feature during maturation stage amelogenesis.

    Science.gov (United States)

    Lacruz, Rodrigo S; Brookes, Steven J; Wen, Xin; Jimenez, Jaime M; Vikman, Susanna; Hu, Ping; White, Shane N; Lyngstadaas, S Petter; Okamoto, Curtis T; Smith, Charles E; Paine, Michael L

    2013-03-01

    Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real-time PCR, we show that the expression of clathrin and adaptor protein subunits are upregulated in maturation stage rodent enamel organ cells. AP complex 2 (AP-2) is the most upregulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts, with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin-dependent endocytosis, thus implying the likelihood of specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also upregulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1); cluster of differentiation 63 and 68 (Cd63 and Cd68); ATPase, H(+) transporting, lysosomal V0 subunit D2 (Atp6v0d2); ATPase, H(+) transporting, lysosomal V1 subunit B2 (Atp6v1b2); chloride channel, voltage-sensitive 7 (Clcn7); and cathepsin K (Ctsk). Immunohistologic data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain showed upregulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor-regulated pathway for the endocytosis of enamel matrix proteins. These data

  18. The Apical Localization of Na+, K+-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 Subunit.

    Science.gov (United States)

    Lobato-Álvarez, Jorge A; Roldán, María L; López-Murillo, Teresa Del Carmen; González-Ramírez, Ricardo; Bonilla-Delgado, José; Shoshani, Liora

    2016-01-01

    Na + , K + -ATPase, or the Na + pump, is a key component in the maintenance of the epithelial phenotype. In most epithelia, the pump is located in the basolateral domain. Studies from our laboratory have shown that the β 1 subunit of Na + , K + -ATPase plays an important role in this mechanism because homotypic β 1 -β 1 interactions between neighboring cells stabilize the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE), the Na + pump is located in the apical domain. The mechanism of polarization in this epithelium is unclear. We hypothesized that the apical polarization of the pump in RPE cells depends on the expression of its β 2 subunit. ARPE-19 cells cultured for up to 8 weeks on inserts did not polarize, and Na + , K + -ATPase was expressed in the basolateral membrane. In the presence of insulin, transferrin and selenic acid (ITS), ARPE-19 cells cultured for 4 weeks acquired an RPE phenotype, and the Na + pump was visible in the apical domain. Under these conditions, Western blot analysis was employed to detect the β 2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the β 2 subunit. qPCR results showed a time-dependent increase in the level of β 2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the β 2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α 2 subunit in that domain. Our results demonstrate that the apical polarization of Na + , K + -ATPase in RPE cells depends on the expression of the β 2 subunit.

  19. Constitutively active signaling by the G protein βγ-subunit mediates intrinsically increased phosphodiesterase-4 activity in human asthmatic airway smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    Aihua Hu

    Full Text Available Signaling by the Gβγ subunit of Gi protein, leading to downstream c-Src-induced activation of the Ras/c-Raf1/MEK-ERK1/2 signaling pathway and its upregulation of phosphodiesterase-4 (PDE4 activity, was recently shown to mediate the heightened contractility in proasthmatic sensitized isolated airway smooth muscle (ASM, as well as allergen-induced airway hyperresponsiveness and inflammation in an in vivo animal model of allergic asthma. This study investigated whether cultured human ASM (HASM cells derived from asthmatic donor lungs exhibit constitutively increased PDE activity that is attributed to intrinsically upregulated Gβγ signaling coupled to c-Src activation of the Ras/MEK/ERK1/2 cascade. We show that, relative to normal cells, asthmatic HASM cells constitutively exhibit markedly increased intrinsic PDE4 activity coupled to heightened Gβγ-regulated phosphorylation of c-Src and ERK1/2, and direct co-localization of the latter with the PDE4D isoform. These signaling events and their induction of heightened PDE activity are acutely suppressed by treating asthmatic HASM cells with a Gβγ inhibitor. Importantly, along with increased Gβγ activation, asthmatic HASM cells also exhibit constitutively increased direct binding of the small Rap1 GTPase-activating protein, Rap1GAP, to the α-subunit of Gi protein, which serves to cooperatively facilitate Ras activation and, thereby, enable enhanced Gβγ-regulated ERK1/2-stimulated PDE activity. Collectively, these data are the first to identify that intrinsically increased signaling via the Gβγ subunit, facilitated by Rap1GAP recruitment to the α-subunit, mediates the constitutively increased PDE4 activity detected in asthmatic HASM cells. These new findings support the notion that interventions targeted at suppressing Gβγ signaling may lead to novel approaches to treat asthma.

  20. Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage.

    Directory of Open Access Journals (Sweden)

    Chenggang Lu

    2015-12-01

    Full Text Available Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s. In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. Here we show that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC, a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs, spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. Our results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages.

  1. The function of the Mediator complex in plant immunity.

    Science.gov (United States)

    An, Chuanfu; Mou, Zhonglin

    2013-03-01

    Upon pathogen infection, plants undergo dramatic transcriptome reprogramming to shift from normal growth and development to immune response. During this rapid process, the multiprotein Mediator complex has been recognized as an important player to fine-tune gene-specific and pathway-specific transcriptional reprogramming by acting as an adaptor/coregulator between sequence-specific transcription factor and RNA polymerase II (RNAPII). Here, we review current understanding of the role of five functionally characterized Mediator subunits (MED8, MED15, MED16, MED21 and MED25) in plant immunity. All these Mediator subunits positively regulate resistance against leaf-infecting biotrophic bacteria or necrotrophic fungi. While MED21 appears to regulate defense against fungal pathogens via relaying signals from upstream regulators and chromatin modification to RNAPII, the other four Mediator subunits locate at different positions of the defense network to convey phytohormone signal(s). Fully understanding the role of Mediator in plant immunity needs to characterize more Mediator subunits in both Arabidopsis and other plant species. Identification of interacting proteins of Mediator subunits will further help to reveal their specific regulatory mechanisms in plant immunity.

  2. Organization and alternative splicing of the Caenorhabditis elegans cAMP-dependent protein kinase catalytic-subunit gene (kin-1).

    Science.gov (United States)

    Tabish, M; Clegg, R A; Rees, H H; Fisher, M J

    1999-04-01

    The cAMP-dependent protein kinase (protein kinase A, PK-A) is multifunctional in nature, with key roles in the control of diverse aspects of eukaryotic cellular activity. In the case of the free-living nematode, Caenorhabditis elegans, a gene encoding the PK-A catalytic subunit has been identified and two isoforms of this subunit, arising from a C-terminal alternative-splicing event, have been characterized [Gross, Bagchi, Lu and Rubin (1990) J. Biol. Chem. 265, 6896-6907]. Here we report the occurrence of N-terminal alternative-splicing events that, in addition to generating a multiplicity of non-myristoylatable isoforms, also generate the myristoylated variant(s) of the catalytic subunit that we have recently characterized [Aspbury, Fisher, Rees and Clegg (1997) Biochem. Biophys. Res. Commun. 238, 523-527]. The gene spans more than 36 kb and is divided into a total of 13 exons. Each of the mature transcripts contains only 7 exons. In addition to the already characterized exon 1, the 5'-untranslated region and first intron actually contain 5 other exons, any one of which may be alternatively spliced on to exon 2 at the 5' end of the pre-mRNA. This N-terminal alternative splicing occurs in combination with either of the already characterized C-terminal alternative exons. Thus, C. elegans expresses at least 12 different isoforms of the catalytic subunit of PK-A. The significance of this unprecedented structural diversity in the family of PK-A catalytic subunits is discussed.

  3. Susceptibility of β1 Na+-K+ pump subunit to glutathionylation and oxidative inhibition depends on conformational state of pump.

    Science.gov (United States)

    Liu, Chia-Chi; Garcia, Alvaro; Mahmmoud, Yasser A; Hamilton, Elisha J; Galougahi, Keyvan Karimi; Fry, Natasha A S; Figtree, Gemma A; Cornelius, Flemming; Clarke, Ronald J; Rasmussen, Helge H

    2012-04-06

    Glutathionylation of cysteine 46 of the β1 subunit of the Na(+)-K(+) pump causes pump inhibition. However, the crystal structure, known in a state analogous to an E2·2K(+)·P(i) configuration, indicates that the side chain of cysteine 46 is exposed to the lipid bulk phase of the membrane and not expected to be accessible to the cytosolic glutathione. We have examined whether glutathionylation depends on the conformational changes in the Na(+)-K(+) pump cycle as described by the Albers-Post scheme. We measured β1 subunit glutathionylation and function of Na(+)-K(+)-ATPase in membrane fragments and in ventricular myocytes. Signals for glutathionylation in Na(+)-K(+)-ATPase-enriched membrane fragments suspended in solutions that preferentially induce E1ATP and E1Na(3) conformations were much larger than signals in solutions that induce the E2 conformation. Ouabain further reduced glutathionylation in E2 and eliminated an increase seen with exposure to the oxidant peroxynitrite (ONOO(-)). Inhibition of Na(+)-K(+)-ATPase activity after exposure to ONOO(-) was greater when the enzyme had been in the E1Na(3) than the E2 conformation. We exposed myocytes to different extracellular K(+) concentrations to vary the membrane potential and hence voltage-dependent conformational poise. K(+) concentrations expected to shift the poise toward E2 species reduced glutathionylation, and ouabain eliminated a ONOO(-)-induced increase. Angiotensin II-induced NADPH oxidase-dependent Na(+)-K(+) pump inhibition was eliminated by conditions expected to shift the poise toward the E2 species. We conclude that susceptibility of the β1 subunit to glutathionylation depends on the conformational poise of the Na(+)-K(+) pump.

  4. Susceptibility of β1 Na+-K+ Pump Subunit to Glutathionylation and Oxidative Inhibition Depends on Conformational State of Pump*

    Science.gov (United States)

    Liu, Chia-Chi; Garcia, Alvaro; Mahmmoud, Yasser A.; Hamilton, Elisha J.; Galougahi, Keyvan Karimi; Fry, Natasha A. S.; Figtree, Gemma A.; Cornelius, Flemming; Clarke, Ronald J.; Rasmussen, Helge H.

    2012-01-01

    Glutathionylation of cysteine 46 of the β1 subunit of the Na+-K+ pump causes pump inhibition. However, the crystal structure, known in a state analogous to an E2·2K+·Pi configuration, indicates that the side chain of cysteine 46 is exposed to the lipid bulk phase of the membrane and not expected to be accessible to the cytosolic glutathione. We have examined whether glutathionylation depends on the conformational changes in the Na+-K+ pump cycle as described by the Albers-Post scheme. We measured β1 subunit glutathionylation and function of Na+-K+-ATPase in membrane fragments and in ventricular myocytes. Signals for glutathionylation in Na+-K+-ATPase-enriched membrane fragments suspended in solutions that preferentially induce E1ATP and E1Na3 conformations were much larger than signals in solutions that induce the E2 conformation. Ouabain further reduced glutathionylation in E2 and eliminated an increase seen with exposure to the oxidant peroxynitrite (ONOO−). Inhibition of Na+-K+-ATPase activity after exposure to ONOO− was greater when the enzyme had been in the E1Na3 than the E2 conformation. We exposed myocytes to different extracellular K+ concentrations to vary the membrane potential and hence voltage-dependent conformational poise. K+ concentrations expected to shift the poise toward E2 species reduced glutathionylation, and ouabain eliminated a ONOO−-induced increase. Angiotensin II-induced NADPH oxidase-dependent Na+-K+ pump inhibition was eliminated by conditions expected to shift the poise toward the E2 species. We conclude that susceptibility of the β1 subunit to glutathionylation depends on the conformational poise of the Na+-K+ pump. PMID:22354969

  5. Evolutionary Paths of the cAMP-Dependent Protein Kinase (PKA) Catalytic Subunits

    Science.gov (United States)

    Søberg, Kristoffer; Jahnsen, Tore; Rognes, Torbjørn; Skålhegg, Bjørn S.; Laerdahl, Jon K.

    2013-01-01

    3′,5′-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits Cα and Cβ, encoded by the two genes PRKACA and PRKACB, respectively, are among the best understood and characterized human kinases. Here we have studied the evolution of this gene family in chordates, arthropods, mollusks and other animals employing probabilistic methods and show that Cα and Cβ arose by duplication of an ancestral PKA catalytic subunit in a common ancestor of vertebrates. The two genes have subsequently been duplicated in teleost fishes. The evolution of the PRKACG retroposon in simians was also investigated. Although the degree of sequence conservation in the PKA Cα/Cβ kinase family is exceptionally high, a small set of signature residues defining Cα and Cβ subfamilies were identified. These conserved residues might be important for functions that are unique to the Cα or Cβ clades. This study also provides a good example of a seemingly simple phylogenetic problem which, due to a very high degree of sequence conservation and corresponding weak phylogenetic signals, combined with problematic nonphylogenetic signals, is nontrivial for state-of-the-art probabilistic phylogenetic methods. PMID:23593352

  6. Role for the MED21-MED7 Hinge in Assembly of the Mediator-RNA Polymerase II Holoenzyme*

    Science.gov (United States)

    Sato, Shigeo; Tomomori-Sato, Chieri; Tsai, Kuang-Lei; Yu, Xiaodi; Sardiu, Mihaela; Saraf, Anita; Washburn, Michael P.; Florens, Laurence; Asturias, Francisco J.; Conaway, Ronald C.

    2016-01-01

    Mediator plays an integral role in activation of RNA polymerase II (Pol II) transcription. A key step in activation is binding of Mediator to Pol II to form the Mediator-Pol II holoenzyme. Here, we exploit a combination of biochemistry and macromolecular EM to investigate holoenzyme assembly. We identify a subset of human Mediator head module subunits that bind Pol II independent of other subunits and thus probably contribute to a major Pol II binding site. In addition, we show that binding of human Mediator to Pol II depends on the integrity of a conserved “hinge” in the middle module MED21-MED7 heterodimer. Point mutations in the hinge region leave core Mediator intact but lead to increased disorder of the middle module and markedly reduced affinity for Pol II. These findings highlight the importance of Mediator conformation for holoenzyme assembly. PMID:27821593

  7. MC EMiNEM maps the interaction landscape of the Mediator

    OpenAIRE

    Niederberger, Theresa; Etzold, Stefanie; Lidschreiber, Michael; Maier, Kerstin C.; Martin, Dietmar E.; Fröhlich, Holger; Cramer, Patrick; Tresch, Achim

    2012-01-01

    The Mediator is a highly conserved, large multiprotein complex that is involved essentially in the regulation of eukaryotic mRNA transcription. It acts as a general transcription factor by integrating regulatory signals from gene-specific activators or repressors to the RNA Polymerase II. The internal network of interactions between Mediator subunits that conveys these signals is largely unknown. Here, we introduce MC EMiNEM, a novel method for the retrieval of functional dependencies between...

  8. Adaptor Protein Complex 2 (AP-2) Mediated, Clathrin Dependent Endocytosis, And Related Gene Activities, Are A Prominent Feature During Maturation Stage Amelogenesis

    Science.gov (United States)

    LACRUZ, Rodrigo S.; BROOKES, Steven J.; WEN, Xin; JIMENEZ, Jaime M.; VIKMAN, Susanna; HU, Ping; WHITE, Shane N.; LYNGSTADAAS, S. Petter; OKAMOTO, Curtis T.; SMITH, Charles E.; PAINE, Michael L.

    2012-01-01

    Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real time PCR, we show that the expression of clathrin and adaptor protein subunits are up-regulated in maturation stage rodent enamel organ cells. AP-2 is the most up-regulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin dependent endocytosis, thus implying the likelihood of a specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also up-regulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1), cluster of differentiation 63 and 68 (Cd63 and Cd68), ATPase, H+ transporting, lysosomal V0 subunit D2 (Atp6v0d2), ATPase, H+ transporting, lysosomal V1 subunit B2 (Atp6v1b2), chloride channel, voltage-sensitive 7 (Clcn7) and cathepsin K (Ctsk). Immunohistological data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain® showed up-regulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor regulated pathway for the endocytosis of enamel matrix proteins. These data together

  9. Subunit Stoichiometry of Human Muscle Chloride Channels

    OpenAIRE

    Fahlke, Christoph; Knittle, Timothy; Gurnett, Christina A.; Campbell, Kevin P.; George, Alfred L.

    1997-01-01

    Voltage-gated Cl? channels belonging to the ClC family appear to function as homomultimers, but the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl? channels in which one subunit was tagged by a mutation (D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their ...

  10. Advanced oxidation protein products induce chondrocyte apoptosis via receptor for advanced glycation end products-mediated, redox-dependent intrinsic apoptosis pathway.

    Science.gov (United States)

    Wu, Qian; Zhong, Zhao-Ming; Zhu, Si-Yuan; Liao, Cong-Rui; Pan, Ying; Zeng, Ji-Huan; Zheng, Shuai; Ding, Ruo-Ting; Lin, Qing-Song; Ye, Qing; Ye, Wen-Bin; Li, Wei; Chen, Jian-Ting

    2016-01-01

    Pro-inflammatory cytokine-induced chondrocyte apoptosis is a primary cause of cartilage destruction in the progression of rheumatoid arthritis (RA). Advanced oxidation protein products (AOPPs), a novel pro-inflammatory mediator, have been confirmed to accumulate in patients with RA. However, the effect of AOPPs accumulation on chondrocyte apoptosis and the associated cellular mechanisms remains unclear. The present study demonstrated that the plasma formation of AOPPs was enhanced in RA rats compared with normal. Then, chondrocyte were treated with AOPPs-modified rat serum albumin (AOPPs-RSA) in vitro. Exposure of chondrocyte to AOPPs activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and increased expression of NADPH oxidase subunits, which was mediated by receptor for advanced glycation end products (RAGE), but not scavenger receptor CD36. Moreover, AOPPs challenge triggered NADPH oxidase-dependent ROS generation which induced mitochondrial dysfunction and endoplasmic reticulum stress resulted in activation of caspase family that eventually lead to apoptosis. Lastly, blockade of RAGE, instead of CD36, largely attenuated these signals. Our study demonstrated first time that AOPPs induce chondrocyte apoptosis via RAGE-mediated and redox-dependent intrinsic apoptosis pathway in vitro. These data implicates that AOPPs may represent a novel pathogenic factor that contributes to RA progression. Targeting AOPPs-triggered cellular mechanisms might emerge as a promising therapeutic option for patients with RA.

  11. V-ATPase-mediated granular acidification is regulated by the V-ATPase accessory subunit Ac45 in POMC-producing cells.

    NARCIS (Netherlands)

    Jansen, E.J.S.; Hafmans, T.G.M.; Martens, G.J.

    2010-01-01

    The vacuolar (H(+))-ATPase (V-ATPase) is an important proton pump, and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive but has been hypothesized to involve an accessory subunit

  12. Disruption of Mediator rescues the stunted growth of a lignin-deficient Arabidopsis mutant.

    Science.gov (United States)

    Bonawitz, Nicholas D; Kim, Jeong Im; Tobimatsu, Yuki; Ciesielski, Peter N; Anderson, Nickolas A; Ximenes, Eduardo; Maeda, Junko; Ralph, John; Donohoe, Bryon S; Ladisch, Michael; Chapple, Clint

    2014-05-15

    Lignin is a phenylpropanoid-derived heteropolymer important for the strength and rigidity of the plant secondary cell wall. Genetic disruption of lignin biosynthesis has been proposed as a means to improve forage and bioenergy crops, but frequently results in stunted growth and developmental abnormalities, the mechanisms of which are poorly understood. Here we show that the phenotype of a lignin-deficient Arabidopsis mutant is dependent on the transcriptional co-regulatory complex, Mediator. Disruption of the Mediator complex subunits MED5a (also known as REF4) and MED5b (also known as RFR1) rescues the stunted growth, lignin deficiency and widespread changes in gene expression seen in the phenylpropanoid pathway mutant ref8, without restoring the synthesis of guaiacyl and syringyl lignin subunits. Cell walls of rescued med5a/5b ref8 plants instead contain a novel lignin consisting almost exclusively of p-hydroxyphenyl lignin subunits, and moreover exhibit substantially facilitated polysaccharide saccharification. These results demonstrate that guaiacyl and syringyl lignin subunits are largely dispensable for normal growth and development, implicate Mediator in an active transcriptional process responsible for dwarfing and inhibition of lignin biosynthesis, and suggest that the transcription machinery and signalling pathways responding to cell wall defects may be important targets to include in efforts to reduce biomass recalcitrance.

  13. Papillae formation on trichome cell walls requires the function of the mediator complex subunit Med25.

    Science.gov (United States)

    Fornero, Christy; Suo, Bangxia; Zahde, Mais; Juveland, Katelyn; Kirik, Viktor

    2017-11-01

    Glassy Hair 1 (GLH1) gene that promotes papillae formation on trichome cell walls was identified as a subunit of the transcriptional mediator complex MED25. The MED25 gene is shown to be expressed in trichomes. The expression of the trichome development marker genes GLABRA2 (GL2) and Ethylene Receptor2 (ETR2) is not affected in the glh1 mutant. Presented data suggest that Arabidopsis MED25 mediator component is likely involved in the transcription of genes promoting papillae deposition in trichomes. The plant cell wall plays an important role in communication, defense, organization and support. The importance of each of these functions varies by cell type. Specialized cells, such as Arabidopsis trichomes, exhibit distinct cell wall characteristics including papillae. To better understand the molecular processes important for papillae deposition on the cell wall surface, we identified the GLASSY HAIR 1 (GLH1) gene, which is necessary for papillae formation. We found that a splice-site mutation in the component of the transcriptional mediator complex MED25 gene is responsible for the near papillae-less phenotype of the glh1 mutant. The MED25 gene is expressed in trichomes. Reporters for trichome developmental marker genes GLABRA2 (GL2) and Ethylene Receptor2 (ETR2) were not affected in the glh1 mutant. Collectively, the presented results show that MED25 is necessary for papillae formation on the cell wall surface of leaf trichomes and suggest that the Arabidopsis MED25 mediator component is likely involved in the transcription of a subset of genes that promote papillae deposition in trichomes.

  14. Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

    DEFF Research Database (Denmark)

    Deshmukh, Atul S.; Treebak, Jonas Thue; Long, Yun Chau

    2008-01-01

    AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK...... activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from...... (Thr37/46) (P mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued...

  15. Neuron-specific regulation of class I PI3K catalytic subunits and their dysfunction in brain disorders

    Directory of Open Access Journals (Sweden)

    Christina eGross

    2014-02-01

    Full Text Available The PI3K complex plays important roles in virtually all cells of the body. The enzymatic activity of PI3K to phosphorylate phosphoinositides in the membrane is mediated by a group of catalytic and regulatory subunits. Among those, the class I catalytic subunits, p110α, p110β, p110γ and p110δ, have recently drawn attention in the neuroscience field due to their specific dysregulation in diverse brain disorders. While in non-neuronal cells these catalytic subunits may have partially redundant functions, there is increasing evidence that in neurons their roles are more specialized, and confined to distinct receptor-dependent pathways. This review will summarize the emerging role of class I PI3K catalytic subunits in neurotransmitter-regulated neuronal signaling, and their dysfunction in a variety of neurological diseases, including fragile X syndrome, schizophrenia and epilepsy. We will discuss recent literature describing the use of PI3K subunit-selective inhibitors to rescue brain disease-associated phenotypes in in vitro and animal models. These studies give rise to the exciting prospect that these drugs, originally designed for cancer treatment, may be repurposed as therapeutic drugs for brain disorders in the future.

  16. Prevalence and clinical significance of mediator complex subunit 12 mutations in 362 Han Chinese samples with uterine leiomyoma.

    Science.gov (United States)

    Wu, Juan; Zou, Yang; Luo, Yong; Guo, Jiu-Bai; Liu, Fa-Ying; Zhou, Jiang-Yan; Zhang, Zi-Yu; Wan, Lei; Huang, Ou-Ping

    2017-07-01

    Uterine leiomyomas (ULs) are the most common gynecological benign tumors originating from the myometrium. Prevalent mutations in the mediator complex subunit 12 (MED12) gene have been identified in ULs, and functional evidence has revealed that these mutations may promote the development of ULs. However, whether MED12 mutations are associated with certain clinical characteristics in ULs remains largely unknown. In the present study, the potential mutations of MED12 and its paralogous gene, mediator complex subunit 12-like (MED12L), were screened in 362 UL tumors from Han Chinese patients. A total of 158 out of 362 UL tumors (43.6%) were identified as harboring MED12 somatic mutations, and the majority of these mutations were restricted to the 44th residue. MED12 mutations were also observed in 2 out of 145 (1.4%) adjacent control myometrium. Furthermore, the mutation spectrum of MED12 in the concurrent leiomyomas was noticeably different. Correlation analysis of MED12 mutations with the available clinical features indicated that patients with mutated MED12 tended to have smaller cervical diameters. By contrast, no MED12L mutation was identified in the present samples. In summary, the present study demonstrated the presence of prevalent MED12 somatic mutations in UL samples, and the MED12 mutation was associated with smaller cervical diameters. The low mutation frequency of MED12 in adjacent control myometrium indicated that MED12 mutation may be an early event in the pathogenesis of ULs. Furthermore, MED12 mutation status in concurrent tumors from multiple leiomyomas supported several prior observations that the majority of these tumors arose independently.

  17. The Mediator Kinase Module Restrains Epidermal Growth Factor Receptor Signaling and Represses Vulval Cell Fate Specification in Caenorhabditis elegans.

    Science.gov (United States)

    Grants, Jennifer M; Ying, Lisa T L; Yoda, Akinori; You, Charlotte C; Okano, Hideyuki; Sawa, Hitoshi; Taubert, Stefan

    2016-02-01

    Cell signaling pathways that control proliferation and determine cell fates are tightly regulated to prevent developmental anomalies and cancer. Transcription factors and coregulators are important effectors of signaling pathway output, as they regulate downstream gene programs. In Caenorhabditis elegans, several subunits of the Mediator transcriptional coregulator complex promote or inhibit vulva development, but pertinent mechanisms are poorly defined. Here, we show that Mediator's dissociable cyclin dependent kinase 8 (CDK8) module (CKM), consisting of cdk-8, cic-1/Cyclin C, mdt-12/dpy-22, and mdt-13/let-19, is required to inhibit ectopic vulval cell fates downstream of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. cdk-8 inhibits ectopic vulva formation by acting downstream of mpk-1/ERK, cell autonomously in vulval cells, and in a kinase-dependent manner. We also provide evidence that the CKM acts as a corepressor for the Ets-family transcription factor LIN-1, as cdk-8 promotes transcriptional repression by LIN-1. In addition, we find that CKM mutation alters Mediator subunit requirements in vulva development: the mdt-23/sur-2 subunit, which is required for vulva development in wild-type worms, is dispensable for ectopic vulva formation in CKM mutants, which instead display hallmarks of unrestrained Mediator tail module activity. We propose a model whereby the CKM controls EGFR-Ras-ERK transcriptional output by corepressing LIN-1 and by fine tuning Mediator specificity, thus balancing transcriptional repression vs. activation in a critical developmental signaling pathway. Collectively, these data offer an explanation for CKM repression of EGFR signaling output and ectopic vulva formation and provide the first evidence of Mediator CKM-tail module subunit crosstalk in animals. Copyright © 2016 by the Genetics Society of America.

  18. Assembly of the Arp5 (Actin-related Protein) Subunit Involved in Distinct INO80 Chromatin Remodeling Activities*

    Science.gov (United States)

    Yao, Wei; Beckwith, Sean L.; Zheng, Tina; Young, Thomas; Dinh, Van T.; Ranjan, Anand; Morrison, Ashby J.

    2015-01-01

    ATP-dependent chromatin remodeling, which repositions and restructures nucleosomes, is essential to all DNA-templated processes. The INO80 chromatin remodeling complex is an evolutionarily conserved complex involved in diverse cellular processes, including transcription, DNA repair, and replication. The functional diversity of the INO80 complex can, in part, be attributed to specialized activities of distinct subunits that compose the complex. Furthermore, structural analyses have identified biochemically discrete subunit modules that assemble along the Ino80 ATPase scaffold. Of particular interest is the Saccharomyces cerevisiae Arp5-Ies6 module located proximal to the Ino80 ATPase and the Rvb1-Rvb2 helicase module needed for INO80-mediated in vitro activity. In this study we demonstrate that the previously uncharacterized Ies2 subunit is required for Arp5-Ies6 association with the catalytic components of the INO80 complex. In addition, Arp5-Ies6 module assembly with the INO80 complex is dependent on distinct conserved domains within Arp5, Ies6, and Ino80, including the spacer region within the Ino80 ATPase domain. Arp5-Ies6 interacts with chromatin via assembly with the INO80 complex, as IES2 and INO80 deletion results in loss of Arp5-Ies6 chromatin association. Interestingly, ectopic addition of the wild-type Arp5-Ies6 module stimulates INO80-mediated ATP hydrolysis and nucleosome sliding in vitro. However, the addition of mutant Arp5 lacking unique insertion domains facilitates ATP hydrolysis in the absence of nucleosome sliding. Collectively, these results define the requirements of Arp5-Ies6 assembly, which are needed to couple ATP hydrolysis to productive nucleosome movement. PMID:26306040

  19. MEDIATOR18 and MEDIATOR20 confer susceptibility to Fusarium oxysporum in Arabidopsis thaliana

    Science.gov (United States)

    Stiller, Jiri; Davoine, Celine; Björklund, Stefan; Manners, John M.; Kazan, Kemal; Schenk, Peer M.

    2017-01-01

    The conserved protein complex known as Mediator conveys transcriptional signals by acting as an intermediary between transcription factors and RNA polymerase II. As a result, Mediator subunits play multiple roles in regulating developmental as well as abiotic and biotic stress pathways. In this report we identify the head domain subunits MEDIATOR18 and MEDIATOR20 as important susceptibility factors for Fusarium oxysporum infection in Arabidopsis thaliana. Mutants of MED18 and MED20 display down-regulation of genes associated with jasmonate signaling and biosynthesis while up-regulation of salicylic acid associated pathogenesis related genes and reactive oxygen producing and scavenging genes. We propose that MED18 and MED20 form a sub-domain within Mediator that controls the balance of salicylic acid and jasmonate associated defense pathways. PMID:28441405

  20. The structure of the TFIIH p34 subunit reveals a von Willebrand factor A like fold.

    Directory of Open Access Journals (Sweden)

    Dominik R Schmitt

    Full Text Available RNA polymerase II dependent transcription and nucleotide excision repair are mediated by a multifaceted interplay of subunits within the general transcription factor II H (TFIIH. A better understanding of the molecular structure of TFIIH is the key to unravel the mechanism of action of this versatile protein complex within these vital cellular processes. The importance of this complex becomes further evident in the context of severe diseases like xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy, that arise from single point mutations in TFIIH subunits. Here we describe the structure of the p34 subunit of the TFIIH complex from the eukaryotic thermophilic fungus Chaetomium thermophilum. The structure revealed that p34 contains a von Willebrand Factor A (vWA like domain, a fold which is generally known to be involved in protein-protein interactions. Within TFIIH p34 strongly interacts with p44, a positive regulator of the helicase XPD. Putative protein-protein interfaces are analyzed and possible binding sites for the p34-p44 interaction suggested.

  1. F4+ enterotoxigenic Escherichia coli (ETEC) adhesion mediated by the major fimbrial subunit FaeG.

    Science.gov (United States)

    Xia, Pengpeng; Song, Yujie; Zou, Yajie; Yang, Ying; Zhu, Guoqiang

    2015-09-01

    The FaeG subunit is the major constituent of F4(+) fimbriae, associated with glycoprotein and/or glycolipid receptor recognition and majorly contributes to the pathogen attachment to the host cells. To investigate the key factor involved in the fimbrial binding of F4(+) Escherichia coli, both the recombinant E. coli SE5000 strains carrying the fae operon gene clusters that express the different types of fimbriae in vitro, named as rF4ab, rF4ac, and rF4ad, respectively, corresponding to the fimbrial types F4ab, F4ac, and F4ad, and the three isogenic in-frame faeG gene deletion mutants were constructed. The adhesion assays and adhesion inhibition assays showed that ΔfaeG mutants had a significant reduction in the binding to porcine brush border as well as the intestinal epithelial cell lines, while the complemented strain ΔfaeG/pfaeG restored the adhesion function. The recombinant bacterial strains rF4ab, rF4ac, and rF4ad have the same binding property as wild-type F4(+) E. coli strains do and improvement in terms of binding to porcine brush border and the intestinal epithelial cells, and the adherence was blocked by the monoclonal antibody anti-F4 fimbriae. These data demonstrate that the fimbrial binding of F4(+) E. coli is directly mediated by the major FaeG subunit. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Molecular architecture of the yeast Mediator complex

    Science.gov (United States)

    Robinson, Philip J; Trnka, Michael J; Pellarin, Riccardo; Greenberg, Charles H; Bushnell, David A; Davis, Ralph; Burlingame, Alma L; Sali, Andrej; Kornberg, Roger D

    2015-01-01

    The 21-subunit Mediator complex transduces regulatory information from enhancers to promoters, and performs an essential role in the initiation of transcription in all eukaryotes. Structural information on two-thirds of the complex has been limited to coarse subunit mapping onto 2-D images from electron micrographs. We have performed chemical cross-linking and mass spectrometry, and combined the results with information from X-ray crystallography, homology modeling, and cryo-electron microscopy by an integrative modeling approach to determine a 3-D model of the entire Mediator complex. The approach is validated by the use of X-ray crystal structures as internal controls and by consistency with previous results from electron microscopy and yeast two-hybrid screens. The model shows the locations and orientations of all Mediator subunits, as well as subunit interfaces and some secondary structural elements. Segments of 20–40 amino acid residues are placed with an average precision of 20 Å. The model reveals roles of individual subunits in the organization of the complex. DOI: http://dx.doi.org/10.7554/eLife.08719.001 PMID:26402457

  3. Light-dependent roles of the G-protein α subunit GNA1 of Hypocrea jecorina (anamorph Trichoderma reesei

    Directory of Open Access Journals (Sweden)

    Kubicek Christian P

    2009-09-01

    Full Text Available Abstract Background The filamentous ascomycete Hypocrea jecorina (anamorph Trichoderma reesei is primarily known for its efficient enzymatic machinery that it utilizes to decompose cellulosic substrates. Nevertheless, the nature and transmission of the signals initiating and modulating this machinery are largely unknown. Heterotrimeric G-protein signaling represents one of the best studied signal transduction pathways in fungi. Results Analysis of the regulatory targets of the G-protein α subunit GNA1 in H. jecorina revealed a carbon source and light-dependent role in signal transduction. Deletion of gna1 led to significantly decreased biomass formation in darkness in submersed culture but had only minor effects on morphology and hyphal apical extension rates on solid medium. Cellulase gene transcription was abolished in Δgna1 on cellulose in light and enhanced in darkness. However, analysis of strains expressing a constitutively activated GNA1 revealed that GNA1 does not transmit the essential inducing signal. Instead, it relates a modulating signal with light-dependent significance, since induction still required the presence of an inducer. We show that regulation of transcription and activity of GNA1 involves a carbon source-dependent feedback cycle. Additionally we found a function of GNA1 in hydrophobin regulation as well as effects on conidiation and tolerance of osmotic and oxidative stress. Conclusion We conclude that GNA1 transmits a signal the physiological relevance of which is dependent on both the carbon source as well as the light status. The widespread consequences of mutations in GNA1 indicate a broad function of this Gα subunit in appropriation of intracellular resources to environmental (especially nutritional conditions.

  4. Ectopic Expression of α6 and δ GABAA Receptor Subunits in Hilar Somatostatin Neurons Increases Tonic Inhibition and Alters Network Activity in the Dentate Gyrus

    Science.gov (United States)

    Tong, Xiaoping; Peng, Zechun; Zhang, Nianhui; Cetina, Yliana; Huang, Christine S.; Wallner, Martin; Otis, Thomas S.

    2015-01-01

    The role of GABAA receptor (GABAAR)-mediated tonic inhibition in interneurons remains unclear and may vary among subgroups. Somatostatin (SOM) interneurons in the hilus of the dentate gyrus show negligible expression of nonsynaptic GABAAR subunits and very low tonic inhibition. To determine the effects of ectopic expression of tonic GABAAR subtypes in these neurons, Cre-dependent viral vectors were used to express GFP-tagged GABAAR subunits (α6 and δ) selectively in hilar SOM neurons in SOM-Cre mice. In single-transfected animals, immunohistochemistry demonstrated strong expression of either the α6 or δ subunit; in cotransfected animals, both subunits were consistently expressed in the same neurons. Electrophysiology revealed a robust increase of tonic current, with progressively larger increases following transfection of δ, α6, and α6/δ subunits, respectively, indicating formation of functional receptors in all conditions and likely coassembly of the subunits in the same receptor following cotransfection. An in vitro model of repetitive bursting was used to determine the effects of increased tonic inhibition in hilar SOM interneurons on circuit activity in the dentate gyrus. Upon cotransfection, the frequency of GABAAR-mediated bursting in granule cells was reduced, consistent with a reduction in synchronous firing among hilar SOM interneurons. Moreover, in vivo studies of Fos expression demonstrated reduced activation of α6/δ-cotransfected neurons following acute seizure induction by pentylenetetrazole. The findings demonstrate that increasing tonic inhibition in hilar SOM interneurons can alter dentate gyrus circuit activity during strong stimulation and suggest that tonic inhibition of interneurons could play a role in regulating excessive synchrony within the network. SIGNIFICANCE STATEMENT In contrast to many hippocampal interneurons, somatostatin (SOM) neurons in the hilus of the dentate gyrus have very low levels of nonsynaptic GABAARs and exhibit

  5. Exercisers' identities and exercise dependence: the mediating effect of exercise commitment.

    Science.gov (United States)

    Lu, Frank Jing-Horng; Hsu, Eva Ya-Wen; Wang, Junn-Ming; Huang, Mei-Yao; Chang, Jo-Ning; Wang, Chien-Hsin

    2012-10-01

    The purpose of this study was to examine the associations of exercise identity, exercise commitment, exercise dependence, and, particularly, the mediating effects of exercise commitment on the relationship between exercise identity and exercise dependence. 253 Taiwanese regular exercisers completed measures, including the Exercise Dependence Scale-Revised, the Exercise Identity Scale, the Exercise Commitment Scale, and the Godin Leisure Time Exercise Questionnaire. Results showed that exercise identity, exercise dependence, and two types of exercise commitment were moderately to highly correlated. Furthermore, structural equation modelling indicated that a "have to" commitment partially mediated the relationship between exercise identity and exercise dependence. Based on the mediating role of a "have to" commitment, the findings are particularly informative to exercise instructors and for exercise program managers.

  6. Functional intersection of ATM and DNA-dependent protein kinase catalytic subunit in coding end joining during V(D)J recombination

    DEFF Research Database (Denmark)

    Lee, Baeck-Seung; Gapud, Eric J; Zhang, Shichuan

    2013-01-01

    V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) ar......V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA......-PKcs) are serine-threonine kinases that orchestrate the cellular responses to DNA DSBs. During V(D)J recombination, ATM and DNA-PKcs have unique functions in the repair of coding DNA ends. ATM deficiency leads to instability of postcleavage complexes and the loss of coding ends from these complexes. DNA...... when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA...

  7. Modulation of NMDA Receptor Properties and Synaptic Transmission by the NR3A Subunit in Mouse Hippocampal and Cerebrocortical Neurons

    Science.gov (United States)

    Tong, Gary; Takahashi, Hiroto; Tu, Shichun; Shin, Yeonsook; Talantova, Maria; Zago, Wagner; Xia, Peng; Nie, Zhiguo; Goetz, Thomas; Zhang, Dongxian; Lipton, Stuart A.; Nakanishi, Nobuki

    2015-01-01

    Expression of the NR3A subunit with NR1/NR2 in Xenopus oocytes or mammalian cell lines leads to a reduction in N-methyl-D-aspartate (NMDA)-induced currents and decreased Mg2+ sensitivity and Ca2+ permeability compared with NR1/NR2 receptors. Consistent with these findings, neurons from NR3A knockout (KO) mice exhibit enhanced NMDA-induced currents. Recombinant NR3A can also form excitatory glycine receptors with NR1 in the absence of NR2. However, the effects of NR3A on channel properties in neurons and synaptic transmission have not been fully elucidated. To study physiological roles of NR3A subunits, we generated NR3A transgenic (Tg) mice. Cultured NR3A Tg neurons exhibited two populations of NMDA receptor (NMDAR) channels, reduced Mg2+ sensitivity, and decreased Ca2+ permeability in response to NMDA/glycine, but glycine alone did not elicit excitatory currents. In addition, NMDAR-mediated excitatory postsynaptic currents (EPSCs) in NR3A Tg hippocampal slices showed reduced Mg2+ sensitivity, consistent with the notion that NR3A subunits incorporated into synaptic NMDARs. To study the function of endogenous NR3A subunits, we compared NMDAR-mediated EPSCs in NR3A KO and WT control mice. In NR3A KO mice, the ratio of the amplitudes of the NMDAR-mediated component to α-amino-3-hydroxy-5-methyl-4-isox-azolepropionic acid receptor-mediated component of the EPSC was significantly larger than that seen in WT littermates. This result suggests that NR3A subunits contributed to the NMDAR-mediated component of the EPSC in WT mice. Taken together, these results show that NR3A subunits contribute to NMDAR responses from both synaptic and extra-synaptic receptors, likely composed of NR1, NR2, and NR3 subunits. PMID:18003876

  8. Fast and Slow Inhibition in the Visual Thalamus Is Influenced by Allocating GABAA Receptors with Different γ Subunits

    Directory of Open Access Journals (Sweden)

    Zhiwen Ye

    2017-04-01

    Full Text Available Cell-type specific differences in the kinetics of inhibitory postsynaptic conductance changes (IPSCs are believed to impact upon network dynamics throughout the brain. Much attention has focused on how GABAA receptor (GABAAR α and β subunit diversity will influence IPSC kinetics, but less is known about the influence of the γ subunit. We have examined whether GABAAR γ subunit heterogeneity influences IPSC properties in the thalamus. The γ2 subunit gene was deleted from GABAARs selectively in the dorsal lateral geniculate nucleus (dLGN. The removal of the γ2 subunit from the dLGN reduced the overall spontaneous IPSC (sIPSC frequency across all relay cells and produced an absence of IPSCs in a subset of relay neurons. The remaining slower IPSCs were both insensitive to diazepam and zinc indicating the absence of the γ2 subunit. Because these slower IPSCs were potentiated by methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM, we propose these IPSCs involve γ1 subunit-containing GABAAR activation. Therefore, γ subunit heterogeneity appears to influence the kinetics of GABAAR-mediated synaptic transmission in the visual thalamus in a cell-selective manner. We suggest that activation of γ1 subunit-containing GABAARs give rise to slower IPSCs in general, while faster IPSCs tend to be mediated by γ2 subunit-containing GABAARs.

  9. The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

    Science.gov (United States)

    Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

    2012-01-01

    The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

  10. Evolution of disorder in Mediator complex and its functional relevance.

    Science.gov (United States)

    Nagulapalli, Malini; Maji, Sourobh; Dwivedi, Nidhi; Dahiya, Pradeep; Thakur, Jitendra K

    2016-02-29

    Mediator, an important component of eukaryotic transcriptional machinery, is a huge multisubunit complex. Though the complex is known to be conserved across all the eukaryotic kingdoms, the evolutionary topology of its subunits has never been studied. In this study, we profiled disorder in the Mediator subunits of 146 eukaryotes belonging to three kingdoms viz., metazoans, plants and fungi, and attempted to find correlation between the evolution of Mediator complex and its disorder. Our analysis suggests that disorder in Mediator complex have played a crucial role in the evolutionary diversification of complexity of eukaryotic organisms. Conserved intrinsic disordered regions (IDRs) were identified in only six subunits in the three kingdoms whereas unique patterns of IDRs were identified in other Mediator subunits. Acquisition of novel molecular recognition features (MoRFs) through evolution of new subunits or through elongation of the existing subunits was evident in metazoans and plants. A new concept of 'junction-MoRF' has been introduced. Evolutionary link between CBP and Med15 has been provided which explain the evolution of extended-IDR in CBP from Med15 KIX-IDR junction-MoRF suggesting role of junction-MoRF in evolution and modulation of protein-protein interaction repertoire. This study can be informative and helpful in understanding the conserved and flexible nature of Mediator complex across eukaryotic kingdoms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. The Mediator Complex and Lipid Metabolism.

    Science.gov (United States)

    Zhang, Yi; Xiaoli; Zhao, Xiaoping; Yang, Fajun

    2013-03-01

    The precise control of gene expression is essential for all biological processes. In addition to DNA-binding transcription factors, numerous transcription cofactors contribute another layer of regulation of gene transcription in eukaryotic cells. One of such transcription cofactors is the highly conserved Mediator complex, which has multiple subunits and is involved in various biological processes through directly interacting with relevant transcription factors. Although the current understanding on the biological functions of Mediator remains incomplete, research in the past decade has revealed an important role of Mediator in regulating lipid metabolism. Such function of Mediator is dependent on specific transcription factors, including peroxisome proliferator-activated receptor-gamma (PPARγ) and sterol regulatory element-binding proteins (SREBPs), which represent the master regulators of lipid metabolism. The medical significance of these findings is apparent, as aberrant lipid metabolism is intimately linked to major human diseases, such as type 2 diabetes and cardiovascular disease. Here, we briefly review the functions and molecular mechanisms of Mediator in regulation of lipid metabolism.

  12. Identification of the large subunit of Ribulose 1,5-bisphosphate carboxylase/oxygenase as a substrate for transglutaminase in Medicageo sativa L. (alfalfa)

    International Nuclear Information System (INIS)

    Margosiak, S.A.; Dharma, A.; Carver, M.R.B.; Gonzales, A.P.; Louie, D.; Kuehn, G.D.

    1990-01-01

    Extract prepared from floral meristematic tissue of alfalfa (Medicago sativa L.) were investigated for expression of the enzyme transglutaminase in order to identify the major protein substrate for transglutaminase-directed modifications among plant proteins. The large polymorphic subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase in alfalfa, with molecular weights of 52,700 and 57,600, are major substrates for transglutaminase in these extracts. This was established by: (a) covalent conjugation of monodansylcadaverine to the large subunit followed by fluorescent detection in SDS-polyacrylamide gels; (b) covalent conjugation of [ 14 C]putrescine to the large subunit with detection by autoradiography; (c) covalent conjugation of monodansylcadaverine to the large subunit and demonstration of immunocross-reactivity on nitrocellulose transblot of the modified large subunit with antibody prepared in rabbits against dansylated-ovalbumin; (d) demonstration of a direct dependence of the rate of transglutaminase-mediated, [ 14 C]putresciene incorporation upon the concentration of ribulose, 1,5-bisphosphate carboxylase/oxygenase from alfalfa or spinach; and (e) presumptive evidence from size exclusion chromatography that transglutaminase may cofractionate with native molecules of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts

  13. Effect of microinjections of subunits of cAMP-dependent protein kinase on development, proliferation, and RNA synthesis in early embryos of the loach Misgurnus fossilis L

    International Nuclear Information System (INIS)

    Glukhov, A.I.; Benyumov, A.O.; Nesterova, M.V.; Severin, E.S.; Gazaryan, K.G.

    1986-01-01

    The effect of the catalytic and regulatory subunits of cAMP-dependent protein kinase type II on development, proliferation, and RNA synthesis was studied in loach embryos. It was found that injection of the catalytic subunit in a physiological concentration leads to a disturbance in the course of development and inhibits proliferation and RNA synthesis in the embryos. An increase in the concentration of this protein above the physiological level leads to death of the embryos in the first hours of development. Injection of the regulatory subunit stimulated the incorporation of labeled uridine into the acid-insoluble fraction of the embryos, beginning with the gastrula stage. The cell nuclei of loach embryos injected with subunits of protein kinase type II were transplanted into activated loach egg cells: subunits of protein kinase type I had no effect on the ability of nuclei of undetermined loach embryo cells to provide de novo development and their effect was reversible

  14. [Beta]-Adrenergic Receptor Activation Rescues Theta Frequency Stimulation-Induced LTP Deficits in Mice Expressing C-Terminally Truncated NMDA Receptor GluN2A Subunits

    Science.gov (United States)

    Moody, Teena D.; Watabe, Ayako M.; Indersmitten, Tim; Komiyama, Noboru H.; Grant, Seth G. N.; O'Dell, Thomas J.

    2011-01-01

    Through protein interactions mediated by their cytoplasmic C termini the GluN2A and GluN2B subunits of NMDA receptors (NMDARs) have a key role in the formation of NMDAR signaling complexes at excitatory synapses. Although these signaling complexes are thought to have a crucial role in NMDAR-dependent forms of synaptic plasticity such as long-term…

  15. The δ subunit of RNA polymerase guides promoter selectivity and virulence in Staphylococcus aureus.

    Science.gov (United States)

    Weiss, Andy; Ibarra, J Antonio; Paoletti, Jessica; Carroll, Ronan K; Shaw, Lindsey N

    2014-04-01

    In Gram-positive bacteria, and particularly the Firmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the δ factor, or RpoE. This enigmatic protein has been studied for more than 30 years for various organisms, but its function is still not well understood. In this study, we investigated its role in the major human pathogen Staphylococcus aureus. We showed conservation of important structural regions of RpoE in S. aureus and other species and demonstrated binding to core RNAP that is mediated by the β and/or β' subunits. To identify the impact of the δ subunit on transcription, we performed transcriptome sequencing (RNA-seq) analysis and observed 191 differentially expressed genes in the rpoE mutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, while several mobile genetic elements (SaPI5 and prophage SA3usa) were strongly upregulated. Phenotypically, the rpoE mutant had decreased accumulation and/or activity of a number of key virulence factors, including alpha toxin, secreted proteases, and Panton-Valentine leukocidin (PVL). We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the δ subunit of RNAP is a critical component of the S. aureus transcription machinery and plays an important role during infection.

  16. Adaptor Protein Complex-2 (AP-2) and Epsin-1 Mediate Protease-activated Receptor-1 Internalization via Phosphorylation- and Ubiquitination-dependent Sorting Signals*

    Science.gov (United States)

    Chen, Buxin; Dores, Michael R.; Grimsey, Neil; Canto, Isabel; Barker, Breann L.; Trejo, JoAnn

    2011-01-01

    Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs. PMID:21965661

  17. Flexible Connectors between Capsomer Subunits that Regulate Capsid Assembly.

    Science.gov (United States)

    Hasek, Mary L; Maurer, Joshua B; Hendrix, Roger W; Duda, Robert L

    2017-08-04

    Viruses build icosahedral capsids of specific size and shape by regulating the spatial arrangement of the hexameric and pentameric protein capsomers in the growing shell during assembly. In the T=7 capsids of Escherichia coli bacteriophage HK97 and other phages, 60 capsomers are hexons, while the rest are pentons that are correctly positioned during assembly. Assembly of the HK97 capsid to the correct size and shape has been shown to depend on specific ionic contacts between capsomers. We now describe additional ionic interactions within capsomers that also regulate assembly. Each is between the long hairpin, the "E-loop," that extends from one subunit to the adjacent subunit within the same capsomer. Glutamate E153 on the E-loop and arginine R210 on the adjacent subunit's backbone alpha-helix form salt bridges in hexamers and pentamers. Mutations that disrupt these salt bridges were lethal for virus production, because the mutant proteins assembled into tubes or sheets instead of capsids. X-ray structures show that the E153-R210 links are flexible and maintained during maturation despite radical changes in capsomer shape. The E153-R210 links appear to form early in assembly to enable capsomers to make programmed changes in their shape during assembly. The links also prevent flattening of capsomers and premature maturation. Mutant phenotypes and modeling support an assembly model in which flexible E153-R210 links mediate capsomer shape changes that control where pentons are placed to create normal-sized capsids. The E-loop may be conserved in other systems in order to play similar roles in regulating assembly. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Architecture of the RNA polymerase II-Mediator core initiation complex.

    Science.gov (United States)

    Plaschka, C; Larivière, L; Wenzeck, L; Seizl, M; Hemann, M; Tegunov, D; Petrotchenko, E V; Borchers, C H; Baumeister, W; Herzog, F; Villa, E; Cramer, P

    2015-02-19

    The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.

  19. Loss of ferulate 5-hydroxylase leads to Mediator-dependent inhibition of soluble phenylpropanoid biosynthesis in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Nickolas; Bonawitz, Nicholas D.; Nyffeler, Kayleigh E.; Chapple, Clint

    2015-06-05

    Phenylpropanoids are phenylalanine-derived specialized metabolites and include important structural components of plant cell walls, such as lignin and hydroxycinnamic acids, as well as ultraviolet and visible light-absorbing pigments, such as hydroxycinnamate esters (HCEs) and anthocyanins. Previous work has revealed a remarkable degree of plasticity in HCE biosynthesis, such that most Arabidopsis (Arabidopsis thaliana) mutants with blockages in the pathway simply redirect carbon flux to atypical HCEs. In contrast, the ferulic acid hydroxylase1 (fah1) mutant accumulates greatly reduced levels of HCEs, suggesting that phenylpropanoid biosynthesis may be repressed in response to the loss of FERULATE 5-HYDROXYLASE (F5H) activity. Here, we show that in fah1 mutant plants, the activity of HCE biosynthetic enzymes is not limiting for HCE accumulation, nor is phenylpropanoid flux diverted to the synthesis of cell wall components or flavonol glycosides. We further show that anthocyanin accumulation is also repressed in fah1 mutants and that this repression is specific to tissues in which F5H is normally expressed. Finally, we show that repression of both HCE and anthocyanin biosynthesis in fah1 mutants is dependent on the MED5a/5b subunits of the transcriptional coregulatory complex Mediator, which are similarly required for the repression of lignin biosynthesis and the stunted growth of the phenylpropanoid pathway mutant reduced epidermal fluorescence8. Taken together, these observations show that the synthesis of HCEs and anthocyanins is actively repressed in a MEDIATOR-dependent manner in Arabidopsis fah1 mutants and support an emerging model in which MED5a/5b act as central players in the homeostatic repression of phenylpropanoid metabolism.

  20. The head module of Mediator directs activation of preloaded RNAPII in vivo.

    Science.gov (United States)

    Lee, Sarah K; Chen, Xu; Huang, Liangqun; Stargell, Laurie A

    2013-12-01

    The successful synthesis of a transcript by RNA polymerase II (RNAPII) is a multistage process with distinct rate-limiting steps that can vary depending on the particular gene. A growing number of genes in a variety of organisms are regulated at steps after the recruitment of RNAPII. The best-characterized Saccharomyces cerevisiae gene regulated in this manner is CYC1. This gene has high occupancy of RNAPII under non-inducing conditions, defining it as a poised gene. Here, we find that subunits of the head module of Mediator, Med18 and Med20, and Med19 are required for activation of transcription at the CYC1 promoter in response to environmental cues. These subunits of Mediator are required at the preloaded promoter for normal levels of recruitment and activity of the general transcription factor TFIIH. Strikingly, these Mediator components are dispensable for activation by the same activator at a different gene, which lacks a preloaded polymerase in the promoter region. Based on these results and other studies, we speculate that Mediator plays an essential role in triggering an inactive polymerase at CYC1 into a productively elongating form.

  1. Basic residues in the 74-83 and 191-198 segments of protein kinase CK2 catalytic subunit are implicated in negative but not in positive regulation by the beta-subunit

    DEFF Research Database (Denmark)

    Sarno, S; Vaglio, P; Marin, O

    1997-01-01

    by the beta-subunit many fold more than that of alpha wild type, while extrastimulation by beta mutant D55L56E57A, observable with alpha wild type, is abolished with these mutants. These data support the conclusion that down regulation by the acidic residues clustered in the N-terminal moiety of beta...... is mediated by basic residues in the 74-83 and in the 191-198 sequences of the alpha-subunit. These are also implicated in substrate recognition consistent with the concept that the N-terminal acidic region of the beta subunit operates as a pseudosubstrate. In contrast, another CK2alpha mutant, V66A, is more...

  2. The Mediator complex: a central integrator of transcription

    Science.gov (United States)

    Allen, Benjamin L.; Taatjes, Dylan J.

    2016-01-01

    The RNA polymerase II (pol II) enzyme transcribes all protein-coding and most non-coding RNA genes and is globally regulated by Mediator, a large, conformationally flexible protein complex with variable subunit composition (for example, a four-subunit CDK8 module can reversibly associate). These biochemical characteristics are fundamentally important for Mediator's ability to control various processes important for transcription, including organization of chromatin architecture and regulation of pol II pre-initiation, initiation, re-initiation, pausing, and elongation. Although Mediator exists in all eukaryotes, a variety of Mediator functions appear to be specific to metazoans, indicative of more diverse regulatory requirements. PMID:25693131

  3. Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

    Energy Technology Data Exchange (ETDEWEB)

    Lind, Genevieve E.; Mou, Tung-Chung; Tamborini, Lucia; Pomper, Martin G.; De Micheli, Carlo; Conti, Paola; Pinto, Andrea; Hansen, Kasper B. (JHU); (Milan); (Montana)

    2017-07-31

    NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity.

  4. Dis3- and exosome subunit-responsive 3′ mRNA instability elements

    International Nuclear Information System (INIS)

    Kiss, Daniel L.; Hou, Dezhi; Gross, Robert H.; Andrulis, Erik D.

    2012-01-01

    Highlights: ► Successful use of a novel RNA-specific bioinformatic tool, RNA SCOPE. ► Identified novel 3′ UTR cis-acting element that destabilizes a reporter mRNA. ► Show exosome subunits are required for cis-acting element-mediated mRNA instability. ► Define precise sequence requirements of novel cis-acting element. ► Show that microarray-defined exosome subunit-regulated mRNAs have novel element. -- Abstract: Eukaryotic RNA turnover is regulated in part by the exosome, a nuclear and cytoplasmic complex of ribonucleases (RNases) and RNA-binding proteins. The major RNase of the complex is thought to be Dis3, a multi-functional 3′–5′ exoribonuclease and endoribonuclease. Although it is known that Dis3 and core exosome subunits are recruited to transcriptionally active genes and to messenger RNA (mRNA) substrates, this recruitment is thought to occur indirectly. We sought to discover cis-acting elements that recruit Dis3 or other exosome subunits. Using a bioinformatic tool called RNA SCOPE to screen the 3′ untranslated regions of up-regulated transcripts from our published Dis3 depletion-derived transcriptomic data set, we identified several motifs as candidate instability elements. Secondary screening using a luciferase reporter system revealed that one cassette—harboring four elements—destabilized the reporter transcript. RNAi-based depletion of Dis3, Rrp6, Rrp4, Rrp40, or Rrp46 diminished the efficacy of cassette-mediated destabilization. Truncation analysis of the cassette showed that two exosome subunit-sensitive elements (ESSEs) destabilized the reporter. Point-directed mutagenesis of ESSE abrogated the destabilization effect. An examination of the transcriptomic data from exosome subunit depletion-based microarrays revealed that mRNAs with ESSEs are found in every up-regulated mRNA data set but are underrepresented or missing from the down-regulated data sets. Taken together, our findings imply a potentially novel mechanism of m

  5. Deletion of the GluA1 AMPA receptor subunit impairs recency-dependent object recognition memory

    Science.gov (United States)

    Sanderson, David J.; Hindley, Emma; Smeaton, Emily; Denny, Nick; Taylor, Amy; Barkus, Chris; Sprengel, Rolf; Seeburg, Peter H.; Bannerman, David M.

    2011-01-01

    Deletion of the GluA1 AMPA receptor subunit impairs short-term spatial recognition memory. It has been suggested that short-term recognition depends upon memory caused by the recent presentation of a stimulus that is independent of contextual–retrieval processes. The aim of the present set of experiments was to test whether the role of GluA1 extends to nonspatial recognition memory. Wild-type and GluA1 knockout mice were tested on the standard object recognition task and a context-independent recognition task that required recency-dependent memory. In a first set of experiments it was found that GluA1 deletion failed to impair performance on either of the object recognition or recency-dependent tasks. However, GluA1 knockout mice displayed increased levels of exploration of the objects in both the sample and test phases compared to controls. In contrast, when the time that GluA1 knockout mice spent exploring the objects was yoked to control mice during the sample phase, it was found that GluA1 deletion now impaired performance on both the object recognition and the recency-dependent tasks. GluA1 deletion failed to impair performance on a context-dependent recognition task regardless of whether object exposure in knockout mice was yoked to controls or not. These results demonstrate that GluA1 is necessary for nonspatial as well as spatial recognition memory and plays an important role in recency-dependent memory processes. PMID:21378100

  6. The Ku80 carboxy terminus stimulates joining and artemis-mediated processing of DNA ends

    DEFF Research Database (Denmark)

    Weterings, Eric; Verkaik, Nicole S; Keijzers, Guido

    2008-01-01

    Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PK(CS)) an......Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA......-PK(CS)) and the XRCC4/ligase IV complex. Activation of the DNA-PK(CS) serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation...... was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PK(CS) autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis...

  7. Evidence for Multiple Mediator Complexes in Yeast Independently Recruited by Activated Heat Shock Factor.

    Science.gov (United States)

    Anandhakumar, Jayamani; Moustafa, Yara W; Chowdhary, Surabhi; Kainth, Amoldeep S; Gross, David S

    2016-07-15

    Mediator is an evolutionarily conserved coactivator complex essential for RNA polymerase II transcription. Although it has been generally assumed that in Saccharomyces cerevisiae, Mediator is a stable trimodular complex, its structural state in vivo remains unclear. Using the "anchor away" (AA) technique to conditionally deplete select subunits within Mediator and its reversibly associated Cdk8 kinase module (CKM), we provide evidence that Mediator's tail module is highly dynamic and that a subcomplex consisting of Med2, Med3, and Med15 can be independently recruited to the regulatory regions of heat shock factor 1 (Hsf1)-activated genes. Fluorescence microscopy of a scaffold subunit (Med14)-anchored strain confirmed parallel cytoplasmic sequestration of core subunits located outside the tail triad. In addition, and contrary to current models, we provide evidence that Hsf1 can recruit the CKM independently of core Mediator and that core Mediator has a role in regulating postinitiation events. Collectively, our results suggest that yeast Mediator is not monolithic but potentially has a dynamic complexity heretofore unappreciated. Multiple species, including CKM-Mediator, the 21-subunit core complex, the Med2-Med3-Med15 tail triad, and the four-subunit CKM, can be independently recruited by activated Hsf1 to its target genes in AA strains. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  8. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    Directory of Open Access Journals (Sweden)

    Signe Tandrup Schmidt

    2016-03-01

    Full Text Available The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI. Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs, which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the

  9. Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting.

    OpenAIRE

    Heyduk, T; Heyduk, E; Severinov, K; Tang, H; Ebright, R H

    1996-01-01

    Escherichia coli RNA polymerase (RNAP) alpha subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2 alpha-->alpha 2-->alpha 2 beta-->alpha 2 beta beta'-->alpha 2 beta beta' sigma. In this work, we have used hydroxyl-radical protein footprinting to define determinants of alpha for interaction with beta, beta', and sigma. Our results indicate that amino acids 30-75 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta ...

  10. The Mediator complex of Caenorhabditis elegans: insights into the developmental and physiological roles of a conserved transcriptional coregulator.

    Science.gov (United States)

    Grants, Jennifer M; Goh, Grace Y S; Taubert, Stefan

    2015-02-27

    The Mediator multiprotein complex ('Mediator') is an important transcriptional coregulator that is evolutionarily conserved throughout eukaryotes. Although some Mediator subunits are essential for the transcription of all protein-coding genes, others influence the expression of only subsets of genes and participate selectively in cellular signaling pathways. Here, we review the current knowledge of Mediator subunit function in the nematode Caenorhabditis elegans, a metazoan in which established and emerging genetic technologies facilitate the study of developmental and physiological regulation in vivo. In this nematode, unbiased genetic screens have revealed critical roles for Mediator components in core developmental pathways such as epidermal growth factor (EGF) and Wnt/β-catenin signaling. More recently, important roles for C. elegans Mediator subunits have emerged in the regulation of lipid metabolism and of systemic stress responses, engaging conserved transcription factors such as nuclear hormone receptors (NHRs). We emphasize instances where similar functions for individual Mediator subunits exist in mammals, highlighting parallels between Mediator subunit action in nematode development and in human cancer biology. We also discuss a parallel between the association of the Mediator subunit MED12 with several human disorders and the role of its C. elegans ortholog mdt-12 as a regulatory hub that interacts with numerous signaling pathways. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Two subunits of human ORC are dispensable for DNA replication and proliferation.

    Science.gov (United States)

    Shibata, Etsuko; Kiran, Manjari; Shibata, Yoshiyuki; Singh, Samarendra; Kiran, Shashi; Dutta, Anindya

    2016-12-01

    The six-subunit Origin Recognition Complex (ORC) is believed to be an essential eukaryotic ATPase that binds to origins of replication as a ring-shaped heterohexamer to load MCM2-7 and initiate DNA replication. We have discovered that human cell lines in culture proliferate with intact chromosomal origins of replication after disruption of both alleles of ORC2 or of the ATPase subunit, ORC1 . The ORC1 or ORC2 -depleted cells replicate with decreased chromatin loading of MCM2-7 and become critically dependent on another ATPase, CDC6, for survival and DNA replication. Thus, either the ORC ring lacking a subunit, even its ATPase subunit, can load enough MCM2-7 in partnership with CDC6 to initiate DNA replication, or cells have an ORC-independent, CDC6-dependent mechanism to load MCM2-7 on origins of replication.

  12. Protein kinase A regulatory subunit distribution in medulloblastoma

    International Nuclear Information System (INIS)

    Mucignat-Caretta, Carla; Denaro, Luca; Redaelli, Marco; D'Avella, Domenico; Caretta, Antonio

    2010-01-01

    Previous studies showed a differential distribution of the four regulatory subunits of cAMP-dependent protein kinases inside the brain, that changed in rodent gliomas: therefore, the distribution of these proteins inside the brain can give information on the functional state of the cells. Our goal was to examine human brain tumors to provide evidence for a differential distribution of protein kinase A in different tumors. The distribution of detergent insoluble regulatory (R1 and R2) and catalytic subunits of cAMP dependent kinases was examined in pediatric brain tumors by immunohistochemistry and fluorescent cAMP analogues binding. R2 is organized in large single dots in medulloblastomas, while it has a different appearance in other tumors. Fluorescent cAMP labelling was observed only in medulloblastoma. A different distribution of cAMP dependent protein kinases has been observed in medulloblastoma

  13. Heterologous desensitization of adenylate cyclase from pigeon erythrocytes under the action of the catalytic subunit of cAMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Popov, K.M.; Bulargina, T.V.; Severin, E.S.

    1985-01-01

    Preincubation of the plasma membranes from pigeon erythrocytes with the catalytic subunit of cAMP-dependent protein kinase leads to desensitization of adenylate cyclase of the erythrocytes. The adenylate cyclase activity, measured in the presence of 10 μM isoproterenol and 50 μM GTP-γ-S, is decreased by 40% in 10 min of incubation, while the activity in the presence of 50 μM GTP-γ-S is decreased by 35% in 20 min. The decrease in the adenylate cyclase activity is due to an increase in the lag phase of activation of the enzyme in the presence of a GTP analog stable to hydrolysis and a decrease in the activity in the steady-state phase of activation. Heterologous desensitization of adenylate cyclase under the action of cAMP-dependent protein kinase is coupled with a decrease in the number of β-adrenoreceptors capable of passing into a state of high affinity for antagonists in the absence of guanylic nucleotides. The influence of the catalytic subunit on adenylate cyclase entirely models the process of desensitization of the enzyme absorbed in the influence of isoproterenol or cAMP on erythrocytes

  14. Highly conserved small subunit residues influence rubisco large subunit catalysis.

    Science.gov (United States)

    Genkov, Todor; Spreitzer, Robert J

    2009-10-30

    The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

  15. CSNAP Is a Stoichiometric Subunit of the COP9 Signalosome

    Directory of Open Access Journals (Sweden)

    Shelly Rozen

    2015-10-01

    Full Text Available The highly conserved COP9 signalosome (CSN complex is a key regulator of all cullin-RING-ubiquitin ligases (CRLs, the largest family of E3 ubiquitin ligases. Until now, it was accepted that the CSN is composed of eight canonical components. Here, we report the discovery of an additional integral and stoichiometric subunit that had thus far evaded detection, and we named it CSNAP (CSN acidic protein. We show that CSNAP binds CSN3, CSN5, and CSN6, and its incorporation into the CSN complex is mediated through the C-terminal region involving conserved aromatic residues. Moreover, depletion of this small protein leads to reduced proliferation and a flattened and enlarged morphology. Finally, on the basis of sequence and structural properties shared by both CSNAP and DSS1, a component of the related 19S lid proteasome complex, we propose that CSNAP, the ninth CSN subunit, is the missing paralogous subunit of DSS1.

  16. Tuning of the Na,K-ATPase by the beta subunit

    Science.gov (United States)

    Hilbers, Florian; Kopec, Wojciech; Isaksen, Toke Jost; Holm, Thomas Hellesøe; Lykke-Hartmann, Karin; Nissen, Poul; Khandelia, Himanshu; Poulsen, Hanne

    2016-02-01

    The vital gradients of Na+ and K+ across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor physiology in mammals. Here, we show that compared to β1 and β3, β2 stabilizes the Na+-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the β transmembrane helix correlates with its functional effect, suggesting that the relative orientation of β modulates ion binding at the α subunit. β2 is primarily expressed in granule neurons and glomeruli in the cerebellum, and we propose that its unique functional characteristics are important to respond appropriately to the cerebellar Na+ and K+ gradients.

  17. Emerging functions of multi-protein complex Mediator with special emphasis on plants.

    Science.gov (United States)

    Malik, Naveen; Agarwal, Pinky; Tyagi, Akhilesh

    2017-10-01

    Mediator is a multi-subunit protein complex which is involved in transcriptional regulation in yeast and other eukaryotes. As a co-activator, it connects information from transcriptional activators/repressors to transcriptional machinery including RNA polymerase II and general transcription factors. It is not only involved in transcription initiation but also has important roles to play in transcription elongation and termination. Functional attributes of different Mediator subunits have been largely defined in yeast and mammalian systems earlier, while such studies in plants have gained momentum recently. Mediator regulates various processes related to plant development and is also involved in biotic and abiotic stress response. Thus, plant Mediator, like yeast and mammalian Mediator complex, is indispensable for plant growth and survival. Interaction of its multiple subunits with other regulatory proteins and their ectopic expression or knockdown in model plant like Arabidopsis and certain crop plants are paving the way to biochemical analysis and unravel molecular mechanisms of action of Mediator in plants.

  18. Mediator structure and rearrangements required for holoenzyme formation.

    Science.gov (United States)

    Tsai, Kuang-Lei; Yu, Xiaodi; Gopalan, Sneha; Chao, Ti-Chun; Zhang, Ying; Florens, Laurence; Washburn, Michael P; Murakami, Kenji; Conaway, Ronald C; Conaway, Joan W; Asturias, Francisco J

    2017-04-13

    The conserved Mediator co-activator complex has an essential role in the regulation of RNA polymerase II transcription in all eukaryotes. Understanding the structure and interactions of Mediator is crucial for determining how the complex influences transcription initiation and conveys regulatory information to the basal transcription machinery. Here we present a 4.4 Å resolution cryo-electron microscopy map of Schizosaccharomyces pombe Mediator in which conserved Mediator subunits are individually resolved. The essential Med14 subunit works as a central backbone that connects the Mediator head, middle and tail modules. Comparison with a 7.8 Å resolution cryo-electron microscopy map of a Mediator-RNA polymerase II holoenzyme reveals that changes in the structure of Med14 facilitate a large-scale Mediator rearrangement that is essential for holoenzyme formation. Our study suggests that access to different conformations and crosstalk between structural elements are essential for the Mediator regulation mechanism, and could explain the capacity of the complex to integrate multiple regulatory signals.

  19. Tuning of the Na,K-ATPase by the beta subunit

    DEFF Research Database (Denmark)

    Hilbers, Florian; Kopec, Wojciech; Isaksen, Toke Jost

    2016-01-01

    The vital gradients of Na(+) and K(+) across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor...... to the cerebellar Na(+) and K(+) gradients....... physiology in mammals. Here, we show that compared to β1 and β3, β2 stabilizes the Na(+)-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the β transmembrane helix...

  20. Probing the functional subunits of the tonoplast H+-ATPase

    International Nuclear Information System (INIS)

    Randall, S.K.; Lai, S.; Sze, H.

    1986-01-01

    The tonoplast ATPase of oat roots is composed of at least three polypeptides of 72, 60, and 16 kDa. The 16 kDA polypeptide covalently binds N,N'-dicyclohexylcarbodiimide and is postulated to be a component of the proton channel. Initial studies to identify other subunits indicate that both the 72 and 60 kDa subunits covalently bind 14 C]-7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and [ 14 C]N-ethylamleimide, inhibitors of the tonoplast ATPase. ATP prevents binding of these inhibitors suggesting that both the 72 and 60 kDa subunits are involved in substrate binding. Polyclonal antibody has been made to the 72 kDa subunit. Western blot analysis of tonoplast vesicles reveals single reactive polypeptide (72 kDa). The antibody shows no cross-reactivity towards either the mitochondrial F 1 -ATPase or the plasma membrane ATPase. This antibody specifically inhibits ATP hydrolysis and ATP-dependent H + pumping in native tonoplast vesicles. The authors conclude that the 72 kDa subunit is intimately associated with the catalytic (or ATP-binding) site

  1. High Affinity IgE-Fc Receptor alpha and gamma Subunit Interactions

    International Nuclear Information System (INIS)

    Rashid, A.; Housden, J. E. M.; Sabban, S.; Helm, B.

    2014-01-01

    Objective: To explore the relationships between the subunits (alpha, beta and gamma) of the high affinity IgE receptor (Fc and RI) and its ability to mediate transmembrane signaling. Study Design: Experimental study. Place and Duration of Study: Department of Molecular Biology and Biotechnology, University of Sheffield, UK, from 2008 to 2009. Methodology: The approach employed was to create a chimera (human alpha-gamma-gamma) using the extracellular (EC) domain of the human high affinity IgE receptor. The alpha subunit (huFc and RIalpha) of IgE receptor was spliced onto the rodent gamma TM and cytoplasmic domain (CD). This was transfected into the Rat Basophilic Leukemia cell line in order to assess the possibility of selectively activating cells transfected with this single pass construct for antigen induced mediator release. Results: The RBLs cell lines transfected with the huFc and RIalpha/gamma/gamma cDNA constructs were assessed for the cell surface expression of the huFc and RIalpha subunit and the response to the antigenic stimulus by looking for degranulation and intracellular Ca2+ mobilisation. The results obtained showed the absence of huFc and RIalpha subunit expression on the surface of transfected cells as seen by flowcytometric studies, beta-hexosaminidase assays and intracellular calcium mobilisation studies. Conclusion: In the present study the grounds for non-expression of huFc and RIalpha/gamma/gamma cDNA remains elusive but may be due to the fact that the human-rodent chimeric receptors are assembled differently than the endogenous rodent receptors as seen in study in which COS 7 cells were transfected with human/rat chimeric complexes. (author)

  2. Self-subunit swapping occurs in another gene type of cobalt nitrile hydratase.

    Directory of Open Access Journals (Sweden)

    Yi Liu

    Full Text Available Self-subunit swapping is one of the post-translational maturation of the cobalt-containing nitrile hydratase (Co-NHase family of enzymes. All of these NHases possess a gene organization of , which allows the activator protein to easily form a mediatory complex with the α-subunit of the NHase after translation. Here, we discovered that the incorporation of cobalt into another type of Co-NHase, with a gene organization of , was also dependent on self-subunit swapping. We successfully isolated a recombinant NHase activator protein (P14K of Pseudomonas putida NRRL-18668 by adding a Strep-tag N-terminal to the P14K gene. P14K was found to form a complex [α(StrepP14K(2] with the α-subunit of the NHase. The incorporation of cobalt into the NHase of P. putida was confirmed to be dependent on the α-subunit substitution between the cobalt-containing α(StrepP14K(2 and the cobalt-free NHase. Cobalt was inserted into cobalt-free α(StrepP14K(2 but not into cobalt-free NHase, suggesting that P14K functions not only as a self-subunit swapping chaperone but also as a metallochaperone. In addition, NHase from P. putida was also expressed by a mutant gene that was designed with a order. Our findings expand the general features of self-subunit swapping maturation.

  3. Immunochemical aspects of crotoxim and its subunits

    International Nuclear Information System (INIS)

    Nakazone, A.K.

    1979-01-01

    Crotamine and crotoxin with the subunits - phospholipase A and crotapotin - were obtained by purification from Crotalus durissus terrificus venom. Interaction studies of the subunits using crotalic antiserum, indicated that: crotoxin is formed of crotapotin and phospholipase A with the molar ratio of 1 to 1; using crotapotin 125 I the presence of a soluble complex was shown with the same antiserum. Immunological precipitation reactions demonstrated that crotapotin is antigenic: crotapotin and phospholipase A presented similar antigenic determinants; crotoxin antiserum reacted with each one of the submits; when the subunits are mixed to form synthetic crotoxin some antigenic determinants are masked in the process of interaction. Crotamine, interacted with crotapotin 1:1, without hidden antigenic determinants crotapotin antigenic site seems to be formed by, at least, one lysine. Enzimatical activity of phospholipase A apreared to be dependent on some reaction conditions when its arginine residues are blocked. Tyrosines of phospholipase A are more susceptible to labelling with 131 I than crotapotin. Gama irradiation of aqueous solutions of the subunits produced modifications in the ultraviolet spectra. A decrease of the enzymatic activity occured as a function of radiation dosis. Immunological activities of crotapotin and phospholipase A were not altered [pt

  4. The secreted form of the p40 subunit of interleukin (IL)-12 inhibits IL-23 functions and abrogates IL-23-mediated antitumour effects

    Science.gov (United States)

    Shimozato, Osamu; Ugai, Shin-ichi; Chiyo, Masako; Takenobu, Hisanori; Nagakawa, Hiroyasu; Wada, Akihiko; Kawamura, Kiyoko; Yamamoto, Hiroshi; Tagawa, Masatoshi

    2006-01-01

    Interleukin (IL)-23 is a heterodimeric cytokine consisting of a novel p19 molecule and the p40 subunit of IL-12. Since secreted p40 can act as an antagonist for IL-12, we investigated whether p40 also inhibited IL-23-mediated immunological functions. p40 did not induce interferon (IFN)-γ or IL-17 production from splenocytes but impaired IL-23-induced cytokine production by competitive binding to the IL-23 receptors. Furthermore, a mixed population of murine colon carcinoma Colon 26 cells transduced with the p40 gene and those transduced with the IL-23 gene developed tumours in syngenic mice, whereas the IL-23-expressing Colon 26 cells were completely rejected. p40 also suppressed IFN-γ production of antigen-stimulated splenocytes and IL-23-mediated cytotoxic T-lymphocyte activities in the mice that rejected Colon 26 cells expressing IL-23. p40 can thereby antagonize IL-23 and is a possible therapeutic agent for suppression of IL-23 functions. PMID:16423037

  5. NSs protein of rift valley fever virus promotes posttranslational downregulation of the TFIIH subunit p62.

    Science.gov (United States)

    Kalveram, Birte; Lihoradova, Olga; Ikegami, Tetsuro

    2011-07-01

    Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus.

  6. Med5(Nut1) and Med17(Srb4) Are Direct Targets of Mediator Histone H4 Tail Interactions

    Science.gov (United States)

    Liu, Zhongle; Myers, Lawrence C.

    2012-01-01

    The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. In addition to its canonical role in transcriptional activation, recent studies have demonstrated that S. cerevisiae Mediator can interact directly with nucleosomes, and their histone tails. Mutations in Mediator subunits have shown that Mediator and certain chromatin structures mutually impact each other structurally and functionally in vivo. We have taken a UV photo cross-linking approach to further delineate the molecular basis of Mediator chromatin interactions and help determine whether the impact of certain Mediator mutants on chromatin is direct. Specifically, by using histone tail peptides substituted with an amino acid analog that is a UV activatible crosslinker, we have identified specific subunits within Mediator that participate in histone tail interactions. Using Mediator purified from mutant yeast strains we have evaluated the impact of these subunits on histone tail binding. This analysis has identified the Med5 subunit of Mediator as a target for histone tail interactions and suggests that the previously observed effect of med5 mutations on telomeric heterochromatin and silencing is direct. PMID:22693636

  7. Inhibition of K+ Transport through Na+, K+-ATPase by Capsazepine: Role of Membrane Span 10 of the α-Subunit in the Modulation of Ion Gating

    OpenAIRE

    Mahmmoud, Yasser A.; Shattock, Michael; Cornelius, Flemming; Pavlovic, Davor

    2014-01-01

    Capsazepine (CPZ) inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity. In this study we have investigated the functional effects of CPZ on Na+,K+-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na+,K+-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na+,K+-ATPase current in the presence of extracellular K+. In contrast, CPZ stimulated pum...

  8. Identification of a conserved archaeal RNA polymerase subunit contacted by the basal transcription factor TFB.

    Science.gov (United States)

    Magill, C P; Jackson, S P; Bell, S D

    2001-12-14

    Archaea possess two general transcription factors that are required to recruit RNA polymerase (RNAP) to promoters in vitro. These are TBP, the TATA-box-binding protein and TFB, the archaeal homologue of TFIIB. Thus, the archaeal and eucaryal transcription machineries are fundamentally related. In both RNAP II and archaeal transcription systems, direct contacts between TFB/TFIIB and the RNAP have been demonstrated to mediate recruitment of the polymerase to the promoter. However the subunit(s) directly contacted by these factors has not been identified. Using systematic yeast two-hybrid and biochemical analyses we have identified an interaction between the N-terminal domain of TFB and an evolutionarily conserved subunit of the RNA polymerase, RpoK. Intriguingly, homologues of RpoK are found in all three nuclear RNA polymerases (Rpb6) and also in the bacterial RNA polymerase (omega-subunit).

  9. The metal-ion-dependent adhesion site in the Von Willebrand factor-A domain of α2δ subunits is key to trafficking voltage-gated Ca2+ channels

    Science.gov (United States)

    Cantí, C.; Nieto-Rostro, M.; Foucault, I.; Heblich, F.; Wratten, J.; Richards, M. W.; Hendrich, J.; Douglas, L.; Page, K. M.; Davies, A.; Dolphin, A. C.

    2005-01-01

    All auxiliary α2δ subunits of voltage-gated Ca2+ (CaV) channels contain an extracellular Von Willebrand factor-A (VWA) domain that, in α2δ-1 and -2, has a perfect metal-ion-dependent adhesion site (MIDAS). Modeling of the α2δ-2 VWA domain shows it to be highly likely to bind a divalent cation. Mutating the three key MIDAS residues responsible for divalent cation binding resulted in a MIDAS mutant α2δ-2 subunit that was still processed and trafficked normally when it was expressed alone. However, unlike WT α2δ-2, the MIDAS mutant α2δ-2 subunit did not enhance and, in some cases, further diminished CaV1.2, -2.1, and -2.2 currents coexpressed with β1b by using either Ba2+ or Na+ as a permeant ion. Furthermore, expression of the MIDAS mutant α2δ-2 reduced surface expression and strongly increased the perinuclear retention of CaVα1 subunits at the earliest time at which expression was observed in both Cos-7 and NG108–15 cells. Despite the presence of endogenous α2δ subunits, heterologous expression of α2δ-2 in differentiated NG108–15 cells further enhanced the endogenous high-threshold Ca2+ currents, whereas this enhancement was prevented by the MIDAS mutations. Our results indicate that α2δ subunits normally interact with the CaVα1 subunit early in their maturation, before the appearance of functional plasma membrane channels, and an intact MIDAS motif in the α2δ subunit is required to promote trafficking of the α1 subunit to the plasma membrane by an integrin-like switch. This finding provides evidence for a primary role of a VWA domain in intracellular trafficking of a multimeric complex, in contrast to the more usual roles in binding extracellular ligands in other exofacial VWA domains. PMID:16061813

  10. Hormonal regulation of Na+/K+-dependent ATPase activity and pump function in corneal endothelial cells.

    Science.gov (United States)

    Hatou, Shin

    2011-10-01

    Na- and K-dependent ATPase (Na,K-ATPase) in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. We investigated the role of dexamethasone in the regulation of Na,K-ATPase activity and pump function in these cells. Mouse corneal endothelial cells were exposed to dexamethasone or insulin. ATPase activity was evaluated by spectrophotometric measurement, and pump function was measured using an Ussing chamber. Western blotting and immunocytochemistry were performed to measure the expression of the Na,K-ATPase α1-subunit. Dexamethasone increased Na,K-ATPase activity and the pump function of endothelial cells. Western blot analysis indicated that dexamethasone increased the expression of the Na,K-ATPase α1-subunit but decreased the ratio of active to inactive Na,K-ATPase α1-subunit. Insulin increased Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were transient and blocked by protein kinase C inhibitors and inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A). Western blot analysis indicated that insulin decreased the amount of inactive Na,K-ATPase α1-subunit, but the expression of total Na,K-ATPase α1-subunit was unchanged. Immunocytochemistry showed that insulin increased cell surface expression of the Na,K-ATPase α1-subunit. Our results suggest that dexamethasone and insulin stimulate Na,K-ATPase activity in mouse corneal endothelial cells. The effect of dexamethasone activation in these cells was mediated by Na,K-ATPase synthesis and an increased enzymatic activity because of dephosphorylation of Na,K-ATPase α1-subunits. The effect of insulin is mediated by the protein kinase C, PP1, and/or PP2A pathways.

  11. Activation of cAMP-dependent signaling induces oxidative modification of the cardiac Na+-K+ pump and inhibits its activity.

    Science.gov (United States)

    White, Caroline N; Liu, Chia-Chi; Garcia, Alvaro; Hamilton, Elisha J; Chia, Karin K M; Figtree, Gemma A; Rasmussen, Helge H

    2010-04-30

    Cellular signaling can inhibit the membrane Na(+)-K(+) pump via protein kinase C (PKC)-dependent activation of NADPH oxidase and a downstream oxidative modification, glutathionylation, of the beta(1) subunit of the pump alpha/beta heterodimer. It is firmly established that cAMP-dependent signaling also regulates the pump, and we have now examined the hypothesis that such regulation can be mediated by glutathionylation. Exposure of rabbit cardiac myocytes to the adenylyl cyclase activator forskolin increased the co-immunoprecipitation of NADPH oxidase subunits p47(phox) and p22(phox), required for its activation, and increased superoxide-sensitive fluorescence. Forskolin also increased glutathionylation of the Na(+)-K(+) pump beta(1) subunit and decreased its co-immunoprecipitation with the alpha(1) subunit, findings similar to those already established for PKC-dependent signaling. The decrease in co-immunoprecipitation indicates a decrease in the alpha(1)/beta(1) subunit interaction known to be critical for pump function. In agreement with this, forskolin decreased ouabain-sensitive electrogenic Na(+)-K(+) pump current (arising from the 3:2 Na(+):K(+) exchange ratio) of voltage-clamped, internally perfused myocytes. The decrease was abolished by the inclusion of superoxide dismutase, the inhibitory peptide for the epsilon-isoform of PKC or inhibitory peptide for NADPH oxidase in patch pipette solutions that perfuse the intracellular compartment. Pump inhibition was also abolished by inhibitors of protein kinase A and phospholipase C. We conclude that cAMP- and PKC-dependent inhibition of the cardiac Na(+)-K(+) pump occurs via a shared downstream oxidative signaling pathway involving NADPH oxidase activation and glutathionylation of the pump beta(1) subunit.

  12. Importance of Mediator complex in the regulation and integration of diverse signaling pathways in plants.

    Science.gov (United States)

    Samanta, Subhasis; Thakur, Jitendra K

    2015-01-01

    Basic transcriptional machinery in eukaryotes is assisted by a number of cofactors, which either increase or decrease the rate of transcription. Mediator complex is one such cofactor, and recently has drawn a lot of interest because of its integrative power to converge different signaling pathways before channeling the transcription instructions to the RNA polymerase II machinery. Like yeast and metazoans, plants do possess the Mediator complex across the kingdom, and its isolation and subunit analyses have been reported from the model plant, Arabidopsis. Genetic, and molecular analyses have unraveled important regulatory roles of Mediator subunits at every stage of plant life cycle starting from flowering to embryo and organ development, to even size determination. It also contributes immensely to the survival of plants against different environmental vagaries by the timely activation of its resistance mechanisms. Here, we have provided an overview of plant Mediator complex starting from its discovery to regulation of stoichiometry of its subunits. We have also reviewed involvement of different Mediator subunits in different processes and pathways including defense response pathways evoked by diverse biotic cues. Wherever possible, attempts have been made to provide mechanistic insight of Mediator's involvement in these processes.

  13. Mediator: A key regulator of plant development.

    Science.gov (United States)

    Buendía-Monreal, Manuel; Gillmor, C Stewart

    2016-11-01

    Mediator is a multiprotein complex that regulates transcription at the level of RNA pol II assembly, as well as through regulation of chromatin architecture, RNA processing and recruitment of epigenetic marks. Though its modular structure is conserved in eukaryotes, its subunit composition has diverged during evolution and varies in response to environmental and tissue-specific inputs, suggesting different functions for each subunit and/or Mediator conformation. In animals, Mediator has been implicated in the control of differentiation and morphogenesis through modulation of numerous signaling pathways. In plants, studies have revealed roles for Mediator in regulation of cell division, cell fate and organogenesis, as well as developmental timing and hormone responses. We begin this review with an overview of biochemical mechanisms of yeast and animal Mediator that are likely to be conserved in all eukaryotes, as well as a brief discussion of the role of Mediator in animal development. We then present a comprehensive review of studies of the role of Mediator in plant development. Finally, we point to important questions for future research on the role of Mediator as a master coordinator of development. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. DNA binding properties of the small cascade subunit Csa5.

    Directory of Open Access Journals (Sweden)

    Michael Daume

    Full Text Available CRISPR-Cas systems provide immunity against viral attacks in archaeal and bacterial cells. Type I systems employ a Cas protein complex termed Cascade, which utilizes small CRISPR RNAs to detect and degrade the exogenic DNA. A small sequence motif, the PAM, marks the foreign substrates. Previously, a recombinant type I-A Cascade complex from the archaeon Thermoproteus tenax was shown to target and degrade DNA in vitro, dependent on a native PAM sequence. Here, we present the biochemical analysis of the small subunit, Csa5, of this Cascade complex. T. tenax Csa5 preferentially bound ssDNA and mutants that showed decreased ssDNA-binding and reduced Cascade-mediated DNA cleavage were identified. Csa5 oligomerization prevented DNA binding. Specific recognition of the PAM sequence was not observed. Phylogenetic analyses identified Csa5 as a universal member of type I-A systems and revealed three distinct groups. A potential role of Csa5 in R-loop stabilization is discussed.

  15. Off-pathway assembly of fimbria subunits is prevented by chaperone CfaA of CFA/I fimbriae from enterotoxigenic E. coli.

    Science.gov (United States)

    Bao, Rui; Liu, Yang; Savarino, Stephen J; Xia, Di

    2016-12-01

    The assembly of the class 5 colonization factor antigen I (CFA/I) fimbriae of enterotoxigenic E. coli was proposed to proceed via the alternate chaperone-usher pathway. Here, we show that in the absence of the chaperone CfaA, CfaB, the major pilin subunit of CFA/I fimbriae, is able to spontaneously refold and polymerize into cyclic trimers. CfaA kinetically traps CfaB to form a metastable complex that can be stabilized by mutations. Crystal structure of the stabilized complex reveals distinctive interactions provided by CfaA to trap CfaB in an assembly competent state through donor-strand complementation (DSC) and cleft-mediated anchorage. Mutagenesis indicated that DSC controls the stability of the chaperone-subunit complex and the cleft-mediated anchorage of the subunit C-terminus additionally assist in subunit refolding. Surprisingly, over-stabilization of the chaperone-subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA acts predominantly as a kinetic trap by stabilizing subunit to avoid its off-pathway self-polymerization that results in energetically favorable trimers and could serve as a driving force for CFA/I pilus assembly, representing an energetic landscape unique to class 5 fimbria assembly. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Molecular Microbiology published by John Wiley & Sons Ltd.

  16. Inter-subunit interactions across the upper voltage sensing-pore domain interface contribute to the concerted pore opening transition of Kv channels.

    Directory of Open Access Journals (Sweden)

    Tzilhav Shem-Ad

    Full Text Available The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.

  17. Inter-subunit interactions across the upper voltage sensing-pore domain interface contribute to the concerted pore opening transition of Kv channels.

    Science.gov (United States)

    Shem-Ad, Tzilhav; Irit, Orr; Yifrach, Ofer

    2013-01-01

    The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.

  18. Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment.

    Science.gov (United States)

    Eyboulet, Fanny; Cibot, Camille; Eychenne, Thomas; Neil, Helen; Alibert, Olivier; Werner, Michel; Soutourina, Julie

    2013-12-01

    Mediator is a large multiprotein complex conserved in all eukaryotes. The crucial function of Mediator in transcription is now largely established. However, we found that this complex also plays an important role by connecting transcription with DNA repair. We identified a functional contact between the Med17 Mediator subunit and Rad2/XPG, the 3' endonuclease involved in nucleotide excision DNA repair. Genome-wide location analyses revealed that Rad2 is associated with RNA polymerase II (Pol II)- and Pol III-transcribed genes and telomeric regions in the absence of exogenous genotoxic stress. Rad2 occupancy of Pol II-transcribed genes is transcription-dependent. Genome-wide Rad2 occupancy of class II gene promoters is well correlated with that of Mediator. Furthermore, UV sensitivity of med17 mutants is correlated with reduced Rad2 occupancy of class II genes and concomitant decrease of Mediator interaction with Rad2 protein. Our results suggest that Mediator is involved in DNA repair by facilitating Rad2 recruitment to transcribed genes.

  19. Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila.

    Science.gov (United States)

    Velentzas, Panagiotis D; Velentzas, Athanassios D; Pantazi, Asimina D; Mpakou, Vassiliki E; Zervas, Christos G; Papassideri, Issidora S; Stravopodis, Dimitrios J

    2013-01-01

    Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, we herein examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx(MS1096) genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, we proved that proteasomal integrity and ubiquitination proficiency essentially control fly's eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, α5, β5 and β6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing's, but not eye's, morphogenetic organization and architecture. However, Atg9 proved indispensable for

  20. Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila.

    Directory of Open Access Journals (Sweden)

    Panagiotis D Velentzas

    Full Text Available Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, we herein examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx(MS1096 genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, we proved that proteasomal integrity and ubiquitination proficiency essentially control fly's eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, α5, β5 and β6 or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4. Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18 autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing's, but not eye's, morphogenetic organization and architecture. However, Atg9 proved

  1. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

    Science.gov (United States)

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

    2016-01-01

    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  2. Med5(Nut1 and Med17(Srb4 are direct targets of mediator histone H4 tail interactions.

    Directory of Open Access Journals (Sweden)

    Zhongle Liu

    Full Text Available The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. In addition to its canonical role in transcriptional activation, recent studies have demonstrated that S. cerevisiae Mediator can interact directly with nucleosomes, and their histone tails. Mutations in Mediator subunits have shown that Mediator and certain chromatin structures mutually impact each other structurally and functionally in vivo. We have taken a UV photo cross-linking approach to further delineate the molecular basis of Mediator chromatin interactions and help determine whether the impact of certain Mediator mutants on chromatin is direct. Specifically, by using histone tail peptides substituted with an amino acid analog that is a UV activatible crosslinker, we have identified specific subunits within Mediator that participate in histone tail interactions. Using Mediator purified from mutant yeast strains we have evaluated the impact of these subunits on histone tail binding. This analysis has identified the Med5 subunit of Mediator as a target for histone tail interactions and suggests that the previously observed effect of med5 mutations on telomeric heterochromatin and silencing is direct.

  3. Mediator phosphorylation prevents stress response transcription during non-stress conditions.

    Science.gov (United States)

    Miller, Christian; Matic, Ivan; Maier, Kerstin C; Schwalb, Björn; Roether, Susanne; Strässer, Katja; Tresch, Achim; Mann, Matthias; Cramer, Patrick

    2012-12-28

    The multiprotein complex Mediator is a coactivator of RNA polymerase (Pol) II transcription that is required for the regulated expression of protein-coding genes. Mediator serves as an end point of signaling pathways and regulates Pol II transcription, but the mechanisms it uses are not well understood. Here, we used mass spectrometry and dynamic transcriptome analysis to investigate a functional role of Mediator phosphorylation in gene expression. Affinity purification and mass spectrometry revealed that Mediator from the yeast Saccharomyces cerevisiae is phosphorylated at multiple sites of 17 of its 25 subunits. Mediator phosphorylation levels change upon an external stimulus set by exposure of cells to high salt concentrations. Phosphorylated sites in the Mediator tail subunit Med15 are required for suppression of stress-induced changes in gene expression under non-stress conditions. Thus dynamic and differential Mediator phosphorylation contributes to gene regulation in eukaryotic cells.

  4. Maladaptive perfectionism as mediator among psychological control, eating disorders, and exercise dependence symptoms in habitual exerciser.

    Science.gov (United States)

    Costa, Sebastiano; Hausenblas, Heather A; Oliva, Patrizia; Cuzzocrea, Francesca; Larcan, Rosalba

    2016-03-01

    Background and aims The current study examined the mediating role of maladaptive perfectionism among parental psychological control, eating disorder symptoms, and exercise dependence symptoms by gender in habitual exercisers. Methods Participants were 348 Italian exercisers (n = 178 men and n = 170 women; M age = 20.57, SD = 1.13) who completed self-report questionnaires assessing their parental psychological control, maladaptive perfectionism, eating disorder symptoms, and exercise dependence symptoms. Results Results of the present study confirmed the mediating role of maladaptive perfectionism for eating disorder and exercise dependence symptoms for the male and female exercisers in the maternal data. In the paternal data, maladaptive perfectionism mediated the relationships between paternal psychological control and eating disorder and exercise dependence symptoms as full mediator for female participants and as partial mediator for male participants. Discussion Findings of the present study suggest that it may be beneficial to consider dimensions of maladaptive perfectionism and parental psychological control when studying eating disorder and exercise dependence symptoms in habitual exerciser.

  5. Characterization of the regulatory subunit from brain cyclic AMP-dependent protein kinase II

    International Nuclear Information System (INIS)

    Stein, J.C.

    1985-01-01

    Tryptic peptides derived from the regulatory subunits of brain and heart cAMP-dependent protein kinase II were mapped by reverse phase HPLC. At 280 nm, 15 unique peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. At 210 nm, 13 brain-RII specific and 15 heart-RII specific tryptic peptides were identified and resolved. Two-dimensional mapping analyses revealed that several 37 P-labeled tryptic fragments derived from the autophosphorylation and the photoaffinity labeled cAMP-binding sites of brain RII were separate and distinct from the 32 P-peptides isolated from similarly treated heart RII. The tryptic phosphopeptide containing the autophosphorylation site in brain RII was purified. The sequence and phosphorylation site is: Arg-Ala-Ser(P)-Val-Cys-Ala-Glu-Ala-Tyr-Asn-Pro-Asp-Glu-Glu-Glu-Asp-Asp-Ala-Glu. Astrocytes and neurons exhibit high levels of the brain RII enzyme, while oligodendrocytes contain the heart RII enzyme. Monoclonal antibodies to bovine cerebral cortex RII were made and characterized. The antibodies elucidated a subtle difference between membrane-associated and cytosolic RII from cerebral cortex

  6. Importance of Mediator complex in the regulation and integration of diverse signaling pathways in plants

    Directory of Open Access Journals (Sweden)

    Subhasis eSamanta

    2015-09-01

    Full Text Available Basic transcriptional machinery in eukaryotes is assisted by a number of cofactors, which either increase or decrease the rate of transcription. Mediator complex is one such cofactor, and recently has drawn a lot of interest because of its integrative power to converge different signaling pathways before channelling the transcription instructions to the RNA polymerase II machinery. Like yeast and metazoans, plants do possess the Mediator complex across the kingdom, and its isolation and subunit analyses have been reported from the model plant, Arabidopsis. Genetic and molecular analyses have unravelled important regulatory roles of Mediator subunits at every stage of plant life cycle starting from flowering to embryo and organ development, to even size determination. It also contributes immensely to the survival of plants against different environmental vagaries by the timely activation of its resistance mechanisms. Here, we have provided an overview of plant Mediator complex starting from its discovery to regulation of stoichiometry of its subunits. We have also reviewed involvement of different Mediator subunits in different processes and pathways including defense response pathways evoked by diverse biotic cues. Wherever possible, attempts have been made to provide mechanistic insight of Mediator’s involvement in these processes.

  7. SAD1, an RNA polymerase I subunit A34.5 of rice, interacts with Mediator and controls various aspects of plant development.

    Science.gov (United States)

    Li, Weiqiang; Yoshida, Akiko; Takahashi, Megumu; Maekawa, Masahiko; Kojima, Mikiko; Sakakibara, Hitoshi; Kyozuka, Junko

    2015-01-01

    The DWARF14 (D14) gene of rice functions within the signaling pathway of strigolactones, a group of plant hormones that inhibits shoot branching. We isolated a recessive mutant named super apical dormant (sad1-1) from a suppressor screen of d14-1. The growth of tillers (vegetative shoot branches) is suppressed in both the d14-1 sad1-1 double mutant and the sad1-1 single mutant. In addition, the sad1-1 mutant shows pleiotropic defects throughout development. SAD1 encodes an ortholog of RPA34.5, a subunit of RNA polymerase I (Pol I). Consequently, the level of ribosomal RNA (rRNA) is severely reduced in the sad1-1 mutant. These results indicate that proper ribosome function is a prerequisite for normal development in plants. The Arabidopsis ortholog of SAD1 was previously isolated as a Mediator-interacting protein. Here we show that SAD1 interacts physically with the Mediator complex through direct binding with OsMED4, a component of the middle module of the Mediator complex in rice. It is known that Mediator interacts with Pol II, which transcribes mRNAs and functions as a central regulator of transcription. This study indicates a novel aspect of Mediator function in Pol I-controlled rRNA transcription. TFIIF2 and RPC53 are the counterparts of RPA34.5 in Pol II and Pol III, respectively. We demonstrate that the rice orthologs of these proteins also interact with OsMED4. Our results suggest that interaction with MED4 in the Mediator complex is a common feature of the three types of RNA polymerases. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  8. H3K27 methylation and H3S28 phosphorylation-dependent transcriptional regulation by INHAT subunit SET/TAF-Iβ.

    Science.gov (United States)

    Kim, Ji-Young; Kim, Kee-Beom; Son, Hye-Ju; Chae, Yun-Cheol; Oh, Si-Taek; Kim, Dong-Wook; Pak, Jhang Ho; Seo, Sang-Beom

    2012-09-21

    Significant progress has been made in understanding the relationship between histone modifications and 'reader' molecules and their effects on transcriptional regulation. A previously identified INHAT complex subunit, SET/TAF-Iβ, binds to histones and inhibits histone acetylation. To investigate the binding specificities of SET/TAF-Iβ to various histone modifications, we employed modified histone tail peptide array analyses. SET/TAF-Iβ strongly recognized PRC2-mediated H3K27me1/2/3; however, the bindings were completely disrupted by H3S28 phosphorylation. We have demonstrated that SET/TAF-Iβ is sequentially recruited to the target gene promoter ATF3 after the PRC2 complex via H3K27me recognition and may offer additive effects in the repression of the target gene. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. The alpha2-delta protein: an auxiliary subunit of voltage-dependent calcium channels as a recognized drug target.

    Science.gov (United States)

    Thorpe, Andrew J; Offord, James

    2010-07-01

    Currently, there are two drugs on the market, gabapentin (Neurontin) and pregabalin (Lyrica), that are proposed to exert their therapeutic effect through binding to the alpha2-delta subunit of voltage-sensitive calcium channels. This activity was unexpected, as the alpha2-delta subunit had previously been considered not to be a pharmacological target. In this review, the role of the alpha2-delta subunits is discussed and the mechanism of action of the alpha2-delta ligands in vitro and in vivo is summarized. Finally, new insights into the mechanism of drugs that bind to this protein are discussed.

  10. Understanding large multiprotein complexes: applying a multiple allosteric networks model to explain the function of the Mediator transcription complex.

    Science.gov (United States)

    Lewis, Brian A

    2010-01-15

    The regulation of transcription and of many other cellular processes involves large multi-subunit protein complexes. In the context of transcription, it is known that these complexes serve as regulatory platforms that connect activator DNA-binding proteins to a target promoter. However, there is still a lack of understanding regarding the function of these complexes. Why do multi-subunit complexes exist? What is the molecular basis of the function of their constituent subunits, and how are these subunits organized within a complex? What is the reason for physical connections between certain subunits and not others? In this article, I address these issues through a model of network allostery and its application to the eukaryotic RNA polymerase II Mediator transcription complex. The multiple allosteric networks model (MANM) suggests that protein complexes such as Mediator exist not only as physical but also as functional networks of interconnected proteins through which information is transferred from subunit to subunit by the propagation of an allosteric state known as conformational spread. Additionally, there are multiple distinct sub-networks within the Mediator complex that can be defined by their connections to different subunits; these sub-networks have discrete functions that are activated when specific subunits interact with other activator proteins.

  11. Leptin reverses corticosterone-induced inhibition of neural stem cell proliferation through activating the NR2B subunits of NMDA receptors

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Wen-Zhu [Anesthesia and Operation Center, Hainan Branch of Chinese PLA General Hospital, Hainan 572013 (China); Anesthesia and Operation Center, Chinese PLA General Hospital, Beijing 100853 (China); Miao, Yu-Liang [Department of Anesthesiology, PLA No. 306 Hospital, Beijing 100101 (China); Guo, Wen-Zhi [Department of Anesthesiology, Beijing Military General Hospital of Chinese People’s Liberation Army, Beijing 100700 (China); Wu, Wei, E-mail: wwzwgk@163.com [Department of Head and Neck Surgery of Otolaryngology, PLA No. 306 Hospital, Beijing 100101 (China); Li, Bao-Wei [Department of Head and Neck Surgery of Otolaryngology, PLA No. 306 Hospital, Beijing 100101 (China); An, Li-Na [Department of Anesthesiology, Armed Police General Hospital, Beijing 100039 (China); Fang, Wei-Wu [Department of Anesthesiology, PLA No. 306 Hospital, Beijing 100101 (China); Mi, Wei-Dong, E-mail: elite2005gg@163.com [Anesthesia and Operation Center, Chinese PLA General Hospital, Beijing 100853 (China)

    2014-04-25

    Highlights: • Leptin promotes the proliferation of neural stem cells isolated from embryonic mouse hippocampus. • Leptin reverses corticosterone-induced inhibition of neural stem cell proliferation. • The effects of leptin are partially mediated by upregulating NR2B subunits. - Abstract: Corticosterone inhibits the proliferation of hippocampal neural stem cells (NSCs). The removal of corticosterone-induced inhibition of NSCs proliferation has been reported to contribute to neural regeneration. Leptin has been shown to regulate brain development, improve angiogenesis, and promote neural regeneration; however, its effects on corticosterone-induced inhibition of NSCs proliferation remain unclear. Here we reported that leptin significantly promoted the proliferation of hippocampal NSCs in a concentration-dependent pattern. Also, leptin efficiently reversed the inhibition of NSCs proliferation induced by corticosterone. Interestingly, pre-treatment with non-specific NMDA antagonist MK-801, specific NR2B antagonist Ro 25-6981, or small interfering RNA (siRNA) targeting NR2B, significantly blocked the effect of leptin on corticosterone-induced inhibition of NSCs proliferation. Furthermore, corticosterone significantly reduced the protein expression of NR2B, whereas pre-treatment with leptin greatly reversed the attenuation of NR2B expression caused by corticosterone in cultured hippocampal NSCs. Our findings demonstrate that leptin reverses the corticosterone-induced inhibition of NSCs proliferation. This process is, at least partially mediated by increased expression of NR2B subunits of NMDA receptors.

  12. Functional interplay between Mediator and TFIIB in preinitiation complex assembly in relation to promoter architecture.

    Science.gov (United States)

    Eychenne, Thomas; Novikova, Elizaveta; Barrault, Marie-Bénédicte; Alibert, Olivier; Boschiero, Claire; Peixeiro, Nuno; Cornu, David; Redeker, Virginie; Kuras, Laurent; Nicolas, Pierre; Werner, Michel; Soutourina, Julie

    2016-09-15

    Mediator is a large coregulator complex conserved from yeast to humans and involved in many human diseases, including cancers. Together with general transcription factors, it stimulates preinitiation complex (PIC) formation and activates RNA polymerase II (Pol II) transcription. In this study, we analyzed how Mediator acts in PIC assembly using in vivo, in vitro, and in silico approaches. We revealed an essential function of the Mediator middle module exerted through its Med10 subunit, implicating a key interaction between Mediator and TFIIB. We showed that this Mediator-TFIIB link has a global role on PIC assembly genome-wide. Moreover, the amplitude of Mediator's effect on PIC formation is gene-dependent and is related to the promoter architecture in terms of TATA elements, nucleosome occupancy, and dynamics. This study thus provides mechanistic insights into the coordinated function of Mediator and TFIIB in PIC assembly in different chromatin contexts. © 2016 Eychenne et al.; Published by Cold Spring Harbor Laboratory Press.

  13. The role of exercise dependence for the relationship between exercise behavior and eating pathology: mediator or moderator?

    Science.gov (United States)

    Cook, Brian J; Hausenblas, Heather A

    2008-05-01

    Our study examined the potential mediating or moderating effect of exercise dependence on the exercise-eating pathology relationship. Female university students (N = 330) completed Internet-based self-report measures of exercise behavior, exercise dependence, and eating pathology. Exercise dependence served as a mediator for the relationship between exercise and eating pathology. This unidirectional causal model suggests that an individual's pathological motivation or compulsion to exercise is the critical mediating component in the exercise-eating pathology relationship. The best target for removing the link between exercise behavior and eating pathology may be reformulating exercise dependence symptoms.

  14. Basal Glutathionylation of Na,K-ATPase α-Subunit Depends on Redox Status of Cells during the Enzyme Biosynthesis

    Directory of Open Access Journals (Sweden)

    Vladimir A. Mitkevich

    2016-01-01

    Full Text Available Many viruses induce oxidative stress and cause S-glutathionylation of Cys residues of the host and viral proteins. Changes in cell functioning during viral infection may be associated with glutathionylation of a number of key proteins including Na,K-ATPase which creates a gradient of sodium and potassium ions. It was found that Na,K-ATPase α-subunit has a basal glutathionylation which is not abrogated by reducing agent. We have shown that acute hypoxia leads to increase of total glutathionylation level of Na,K-ATPase α-subunit; however, basal glutathionylation of α-subunit increases under prolonged hypoxia only. The role of basal glutathionylation in Na,K-ATPase function remains unclear. Understanding significance of basal glutathionylation is complicated by the fact that there are no X-ray structures of Na,K-ATPase with the identified glutathione molecules. We have analyzed all X-ray structures of the Na,K-ATPase α-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues. Analysis of the structures showed that this unresolved density in the structure can be occupied by glutathione associated with cysteine residues. Here, we discuss the role of basal glutathionylation of Na,K-ATPase α-subunit and provide evidence supporting the view that this modification is cotranslational.

  15. Basal Glutathionylation of Na,K-ATPase α-Subunit Depends on Redox Status of Cells during the Enzyme Biosynthesis.

    Science.gov (United States)

    Mitkevich, Vladimir A; Petrushanko, Irina Yu; Poluektov, Yuri M; Burnysheva, Ksenia M; Lakunina, Valentina A; Anashkina, Anastasia A; Makarov, Alexander A

    2016-01-01

    Many viruses induce oxidative stress and cause S-glutathionylation of Cys residues of the host and viral proteins. Changes in cell functioning during viral infection may be associated with glutathionylation of a number of key proteins including Na,K-ATPase which creates a gradient of sodium and potassium ions. It was found that Na,K-ATPase α-subunit has a basal glutathionylation which is not abrogated by reducing agent. We have shown that acute hypoxia leads to increase of total glutathionylation level of Na,K-ATPase α-subunit; however, basal glutathionylation of α-subunit increases under prolonged hypoxia only. The role of basal glutathionylation in Na,K-ATPase function remains unclear. Understanding significance of basal glutathionylation is complicated by the fact that there are no X-ray structures of Na,K-ATPase with the identified glutathione molecules. We have analyzed all X-ray structures of the Na,K-ATPase α-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues. Analysis of the structures showed that this unresolved density in the structure can be occupied by glutathione associated with cysteine residues. Here, we discuss the role of basal glutathionylation of Na,K-ATPase α-subunit and provide evidence supporting the view that this modification is cotranslational.

  16. Strain- and Substrate-Dependent Redox Mediator and Electricity Production by Pseudomonas aeruginosa.

    Science.gov (United States)

    Bosire, Erick M; Blank, Lars M; Rosenbaum, Miriam A

    2016-08-15

    Pseudomonas aeruginosa is an important, thriving member of microbial communities of microbial bioelectrochemical systems (BES) through the production of versatile phenazine redox mediators. Pure culture experiments with a model strain revealed synergistic interactions of P. aeruginosa with fermenting microorganisms whereby the synergism was mediated through the shared fermentation product 2,3-butanediol. Our work here shows that the behavior and efficiency of P. aeruginosa in mediated current production is strongly dependent on the strain of P. aeruginosa We compared levels of phenazine production by the previously investigated model strain P. aeruginosa PA14, the alternative model strain P. aeruginosa PAO1, and the BES isolate Pseudomonas sp. strain KRP1 with glucose and the fermentation products 2,3-butanediol and ethanol as carbon substrates. We found significant differences in substrate-dependent phenazine production and resulting anodic current generation for the three strains, with the BES isolate KRP1 being overall the best current producer and showing the highest electrochemical activity with glucose as a substrate (19 μA cm(-2) with ∼150 μg ml(-1) phenazine carboxylic acid as a redox mediator). Surprisingly, P. aeruginosa PAO1 showed very low phenazine production and electrochemical activity under all tested conditions. Microbial fuel cells and other microbial bioelectrochemical systems hold great promise for environmental technologies such as wastewater treatment and bioremediation. While there is much emphasis on the development of materials and devices to realize such systems, the investigation and a deeper understanding of the underlying microbiology and ecology are lagging behind. Physiological investigations focus on microorganisms exhibiting direct electron transfer in pure culture systems. Meanwhile, mediated electron transfer with natural redox compounds produced by, for example, Pseudomonas aeruginosa might enable an entire microbial

  17. Molecular dynamics studies of the P pilus rod subunit PapA.

    Science.gov (United States)

    Vitagliano, Luigi; Ruggiero, Alessia; Pedone, Carlo; Berisio, Rita

    2009-03-01

    Adhesion of uropathogenic Escherichia coli to host tissues is mediated by pili, which extend from the outer cell membrane of the bacterium. Here we report molecular dynamics (MD) characterizations of the major constituent of P pili from the uropathogenic E. coli, PapA, in unliganded state and in complex with the G1 strand of the chaperone PapD. To mimic the PapA response to the gradual dissociation of the PapD G1 strand and to evaluate the role of PapA chaperone recognition sites, we also carried out MD simulations of complexes of PapA with fragments of PapD G1 strand, that leave either the P4 or both P3 and P4 sites unoccupied. Data on the unbound form of PapA indicate that, upon release of the chaperone, PapA evolves toward compact states that are likely not prone to subunit-subunit association. In line with recent experimental reports, this finding implies that chaperone release and subunit-subunit association must be concerted. Our data also indicated that the gradual unbinding of the chaperone from the PapA groove has increasingly strong structural consequences. Indeed, the release of the chaperone from the site P4, which is closest to the initiation site (P5), does not have dramatic effects on the domain structure, whereas its release from both the P4 and the adjacent P3 sites induces a quick structural transition toward a collapsed state, where the subunit groove is obstructed.

  18. Direct evidence that FK506 inhibition of FcepsilonRI-mediated exocytosis from RBL mast cells involves calcineurin.

    Science.gov (United States)

    Hultsch, T; Brand, P; Lohmann, S; Saloga, J; Kincaid, R L; Knop, J

    1998-05-01

    FcepsilonRI-mediated exocytosis of preformed mediators from mast cells and basophils (e.g. histamine, serotonin, beta-hexosaminidase) is sensitive to the immunosuppressants cyclosporin A and FK506 (IC50 200 and 4 nM, respectively) but not rapamycin. The mechanism of inhibition does not appear to involve tyrosine phosphorylation, hydrolysis of inositol phosphates or calcium flux. Here we report experiments using a molecular approach to assess the role of calcineurin, a serine/threonine phosphatase thought to be the primary pharmacological target of these drugs. Calcineurin's activity requires association of its catalytic (A) subunit with an intrinsic regulatory (B) subunit. We hypothesized that calcineurin-sensitive signalling events should be affected by the depletion of calcineurin B subunits, thereby reducing the number of active A:B complexes. We therefore transfected rat basophilic leukemia (RBL) cells with an inhibitory (dominant negative) form of the calcineurin A subunit, which binds the calcineurin B subunit with high affinity but does not possess catalytic activity (B subunit knock-out, BKO). In these transfected cells, the dose-response curve for the inhibition of FcepsilonRI-mediated exocytosis by FK506 was shifted to the left, indicating an increased drug sensitivity of BKO-transfected cells. We conclude that FK506 inhibition of FcepsilonRI-mediated exocytosis in mast cells specifically targets calcineurin activity.

  19. NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit p62▿

    Science.gov (United States)

    Kalveram, Birte; Lihoradova, Olga; Ikegami, Tetsuro

    2011-01-01

    Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus. PMID:21543505

  20. BID-dependent release of mitochondrial SMAC dampens XIAP-mediated immunity against Shigella

    Science.gov (United States)

    Andree, Maria; Seeger, Jens M; Schüll, Stephan; Coutelle, Oliver; Wagner-Stippich, Diana; Wiegmann, Katja; Wunderlich, Claudia M; Brinkmann, Kerstin; Broxtermann, Pia; Witt, Axel; Fritsch, Melanie; Martinelli, Paola; Bielig, Harald; Lamkemeyer, Tobias; Rugarli, Elena I; Kaufmann, Thomas; Sterner-Kock, Anja; Wunderlich, F Thomas; Villunger, Andreas; Martins, L Miguel; Krönke, Martin; Kufer, Thomas A; Utermöhlen, Olaf; Kashkar, Hamid

    2014-01-01

    The X-linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti-apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase-mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP-mediated immune response by inducing the BID-dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain-dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization. PMID:25056906

  1. Acetylation-Mediated Proteasomal Degradation of Core Histones during DNA Repair and Spermatogenesis

    Science.gov (United States)

    Qian, Min-Xian; Pang, Ye; Liu, Cui Hua; Haratake, Kousuke; Du, Bo-Yu; Ji, Dan-Yang; Wang, Guang-Fei; Zhu, Qian-Qian; Song, Wei; Yu, Yadong; Zhang, Xiao-Xu; Huang, Hai-Tao; Miao, Shiying; Chen, Lian-Bin; Zhang, Zi-Hui; Liang, Ya-Nan; Liu, Shan; Cha, Hwangho; Yang, Dong; Zhai, Yonggong; Komatsu, Takuo; Tsuruta, Fuminori; Li, Haitao; Cao, Cheng; Li, Wei; Li, Guo-Hong; Cheng, Yifan; Chiba, Tomoki; Wang, Linfang; Goldberg, Alfred L.; Shen, Yan; Qiu, Xiao-Bo

    2013-01-01

    SUMMARY Histone acetylation plays critical roles in chromatin remodeling, DNA repair, and epigenetic regulation of gene expression, but the underlying mechanisms are unclear. Proteasomes usually catalyze ATP- and polyubiquitin-dependent proteolysis. Here we show that the proteasomes containing the activator PA200 catalyze the polyubiquitin-independent degradation of histones. Most proteasomes in mammalian testes (“spermatoproteasomes”) contain a spermatid/sperm-specific α-subunit α4s/PSMA8 and/or the catalytic β-subunits of immunoproteasomes in addition to PA200. Deletion of PA200 in mice abolishes acetylation-dependent degradation of somatic core histones during DNA double-strand breaks, and delays core histone disappearance in elongated spermatids. Purified PA200 greatly promotes ATP-independent proteasomal degradation of the acetylated core histones, but not polyubiquitinated proteins. Furthermore, acetylation on histones is required for their binding to the bromodomain-like regions in PA200 and its yeast ortholog, Blm10. Thus, PA200/Blm10 specifically targets the core histones for acetylation-mediated degradation by proteasomes, providing mechanisms by which acetylation regulates histone degradation, DNA repair, and spermatogenesis. PMID:23706739

  2. Laccase/Mediator Systems: Their Reactivity toward Phenolic Lignin Structures.

    Science.gov (United States)

    Hilgers, Roelant; Vincken, Jean-Paul; Gruppen, Harry; Kabel, Mirjam A

    2018-02-05

    Laccase-mediator systems (LMS) have been widely studied for their capacity to oxidize the nonphenolic subunits of lignin (70-90% of the polymer). The phenolic subunits (10-30% of the polymer), which can also be oxidized without mediators, have received considerably less attention. Consequently, it remains unclear to what extent the presence of a mediator influences the reactions of the phenolic subunits of lignin. To get more insight in this, UHPLC-MS was used to study the reactions of a phenolic lignin dimer (GBG), initiated by a laccase from Trametes versicolor , alone or in combination with the mediators HBT and ABTS. The role of HBT was negligible, as its oxidation by laccase occurred slowly in comparison to that of GBG. Laccase and laccase/HBT oxidized GBG at a comparable rate, resulting in extensive polymerization of GBG. In contrast, laccase/ABTS converted GBG at a higher rate, as GBG was oxidized both directly by laccase but also by ABTS radical cations, which were rapidly formed by laccase. The laccase/ABTS system resulted in Cα oxidation of GBG and coupling of ABTS to GBG, rather than polymerization of GBG. Based on these results, we propose reaction pathways of phenolic lignin model compounds with laccase/HBT and laccase/ABTS.

  3. Importance of GluA1 subunit-containing AMPA glutamate receptors for morphine state-dependency.

    Directory of Open Access Journals (Sweden)

    Teemu Aitta-aho

    Full Text Available In state-dependency, information retrieval is most efficient when the animal is in the same state as it was during the information acquisition. State-dependency has been implicated in a variety of learning and memory processes, but its mechanisms remain to be resolved. Here, mice deficient in AMPA-type glutamate receptor GluA1 subunits were first conditioned to morphine (10 or 20 mg/kg s.c. during eight sessions over four days using an unbiased procedure, followed by testing for conditioned place preference at morphine states that were the same as or different from the one the mice were conditioned to. In GluA1 wildtype littermate mice the same-state morphine dose produced the greatest expression of place preference, while in the knockout mice no place preference was then detected. Both wildtype and knockout mice expressed moderate morphine-induced place preference when not at the morphine state (saline treatment at the test; in this case, place preference was weaker than that in the same-state test in wildtype mice. No correlation between place preference scores and locomotor activity during testing was found. Additionally, as compared to the controls, the knockout mice showed unchanged sensitization to morphine, morphine drug discrimination and brain regional μ-opioid receptor signal transduction at the G-protein level. However, the knockout mice failed to show increased AMPA/NMDA receptor current ratios in the ventral tegmental area dopamine neurons of midbrain slices after a single injection of morphine (10 mg/kg, s.c., sliced prepared 24 h afterwards, in contrast to the wildtype mice. The results indicate impaired drug-induced state-dependency in GluA1 knockout mice, correlating with impaired opioid-induced glutamate receptor neuroplasticity.

  4. A Common Structural Component for β-Subunit Mediated Modulation of Slow Inactivation in Different KV Channels

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    Nathalie Strutz-Seebohm

    2013-06-01

    Full Text Available Background/Aims: Potassium channels are tetrameric proteins providing potassium selective passage through lipid embedded proteinaceous pores with highest fidelity. The selectivity results from binding to discrete potassium binding sites and stabilization of a hydrated potassium ion in a central internal cavity. The four potassium binding sites, generated by the conserved TTxGYGD signature sequence are formed by the backbone carbonyls of the amino acids TXGYG. Residues KV1.5-Val481, KV4.3-Leu368 and KV7.1- Ile 313 represent the amino acids in the X position of the respective channels. Methods: Here, we study the impact of these residues on ion selectivity, permeation and inactivation kinetics as well as the modulation by β-subunits using site-specific mutagenesis, electrophysiological analyses and molecular dynamics simulations. Results: We identify this position as key in modulation of slow inactivation by structurally dissimilar β-subunits in different KV channels. Conclusion: We propose a model in which structural changes accompanying activation and β-subunit modulation allosterically constrain the backbone carbonyl oxygen atoms via the side chain of the respective X-residue in the signature sequence to reduce conductance during slow inactivation.

  5. Decreased surface expression of the δ subunit of the GABAA receptor contributes to reduced tonic inhibition in dentate granule cells in a mouse model of fragile X syndrome.

    Science.gov (United States)

    Zhang, Nianhui; Peng, Zechun; Tong, Xiaoping; Lindemeyer, A Kerstin; Cetina, Yliana; Huang, Christine S; Olsen, Richard W; Otis, Thomas S; Houser, Carolyn R

    2017-11-01

    While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABA A receptors (GABA A Rs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABA A R, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABA A Rs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with

  6. The Schizosaccharomyces pombe Mediator

    DEFF Research Database (Denmark)

    Venturi, Michela

    , Schizosaccharomyces pombe and mammalian Mediator. In our study, we have taken the S. pombe Mediator into consideration and characterized genetically and biochemically two subunits already know in S. cerevisiae, Med9 and Med11, but still not identified in the S. pombe Mediator. Genetic analysis has shown that med9......In the past several years great attention has been dedicated to the characterization of the Mediator complex in a different range of model organisms. Mediator is a conserved co-activator complex involved in transcriptional regulation and it conveys signals from regulatory transcription factors...... to the basal transcription machinery. Mediator was initially isolated from Saccharomyces cerevisiae based on its ability to render a RNA polymerase II in vitro transcription system responsive to activators. Additionally, structural studies have revealed striking structural similarities between S. cerevisiae...

  7. Lipid composition of cAMP-dependent protein kinase mutants of Aspergillus niger.

    Science.gov (United States)

    Jernejc, Katarina; Bencina, Mojca

    2003-08-29

    Lipid composition of cAMP-dependent protein kinase (PKA) Aspergillus niger mutants with overexpressed or deleted genes for either regulatory and/or the catalytic subunit of PKA was analyzed. Disruption of the gene encoding the PKA regulatory subunit resulted in 20% less total lipids, 30% less neutral lipids, four times more glycolipids and two-fold higher triacylglycerol lipase activity compared to the control strain. Concomitantly a five-fold decrease in phosphatidylcholine, accompanied with 1.5-, 1.8- and 2.8-fold increases in phosphatidylethanolamine, lysophosphatidylethanolamine and phosphatidylinositol, was determined, respectively. The lack of PKA activity, due to the disruption of a gene encoding the PKA catalytic subunit, resulted in a 1.6-times increase in total lipids with two times more neutral lipids associated with lower triacylglycerol lipase activity and a decrease in phospholipids. The mutants with unrestricted PKA activity synthesized twice as much citric acid as the control strain and three times more than strains lacking PKA activity. The results indicate the involvement of cAMP-mediated PKA activity in regulation of lipid biosynthesis as well as citric acid synthesis.

  8. Exercise dependence as a mediator of the exercise and eating disorders relationship: a pilot study.

    Science.gov (United States)

    Cook, Brian; Hausenblas, Heather; Crosby, Ross D; Cao, Li; Wonderlich, Stephen A

    2015-01-01

    Excessive exercise is a common feature of eating disorders (ED) and is associated with earlier ED onset, more ED symptoms, and higher persistence of ED behavior. Research indicates that exercise amount alone is not associated with ED. The purpose of this study was to investigate pathological attitudes and behaviors related to exercise (e.g., exercise dependence) as a mediator of the exercise and ED relationship. Participants were 43 women with an ED who completed measures of ED symptoms, exercise behavior, and exercise dependence. Analyses were conducted using the indirect bootstrapping method for examining mediation. Exercise dependence mediated the relationship between exercise and ED. This mediation model accounted for 14.34% of the variance in the relationship. Our results extend the literature by offering preliminary evidence of a psychological variable that may be a candidate for future interventions on the exercise and ED relationship. Implications and suggestions for future research are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Binding of CFA/I Pili of Enterotoxigenic Escherichia coli to Asialo-GM1 Is Mediated by the Minor Pilin CfaE.

    Science.gov (United States)

    Madhavan, T P Vipin; Riches, James D; Scanlon, Martin J; Ulett, Glen C; Sakellaris, Harry

    2016-05-01

    CFA/I pili are representatives of a large family of related pili that mediate the adherence of enterotoxigenic Escherichia coli to intestinal epithelial cells. They are assembled via the alternate chaperone-usher pathway and consist of two subunits, CfaB, which makes up the pilus shaft and a single pilus tip-associated subunit, CfaE. The current model of pilus-mediated adherence proposes that CFA/I has two distinct binding activities; the CfaE subunit is responsible for binding to receptors of unknown structure on erythrocyte and intestinal epithelial cell surfaces, while CfaB binds to various glycosphingolipids, including asialo-GM1. In this report, we present two independent lines of evidence that, contrary to the existing model, CfaB does not bind to asialo-GM1 independently of CfaE. Neither purified CfaB subunits nor CfaB assembled into pili bind to asialo-GM1. Instead, we demonstrate that binding activity toward asialo-GM1 resides in CfaE and this is essential for pilus binding to Caco-2 intestinal epithelial cells. We conclude that the binding activities of CFA/I pili for asialo-GM1, erythrocytes, and intestinal cells are inseparable, require the same amino acid residues in CfaE, and therefore depend on the same or very similar binding mechanisms. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  10. N terminus of Swr1 binds to histone H2AZ and provides a platform for subunit assembly in the chromatin remodeling complex.

    Science.gov (United States)

    Wu, Wei-Hua; Wu, Chwen-Huey; Ladurner, Andreas; Mizuguchi, Gaku; Wei, Debbie; Xiao, Hua; Luk, Ed; Ranjan, Anand; Wu, Carl

    2009-03-06

    Variant histone H2AZ-containing nucleosomes are involved in the regulation of gene expression. In Saccharomyces cerevisiae, chromatin deposition of histone H2AZ is mediated by the fourteen-subunit SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Previous work defined the role of seven SWR1 subunits (Swr1 ATPase, Swc2, Swc3, Arp6, Swc5, Yaf9, and Swc6) in maintaining complex integrity and H2AZ histone replacement activity. Here we examined the function of three additional SWR1 subunits, bromodomain containing Bdf1, actin-related protein Arp4 and Swc7, by analyzing affinity-purified mutant SWR1 complexes. We observed that depletion of Arp4 (arp4-td) substantially impaired the association of Bdf1, Yaf9, and Swc4. In contrast, loss of either Bdf1 or Swc7 had minimal effects on overall complex integrity. Furthermore, the basic H2AZ histone replacement activity of SWR1 in vitro required Arp4, but not Bdf1 or Swc7. Thus, three out of fourteen SWR1 subunits, Bdf1, Swc7, and previously noted Swc3, appear to have roles auxiliary to the basic histone replacement activity. The N-terminal region of the Swr1 ATPase subunit is necessary and sufficient to direct association of Bdf1 and Swc7, as well as Arp4, Act1, Yaf9 and Swc4. This same region contains an additional H2AZ-H2B specific binding site, distinct from the previously identified Swc2 subunit. These findings suggest that one SWR1 enzyme might be capable of binding two H2AZ-H2B dimers, and provide further insight on the hierarchy and interdependency of molecular interactions within the SWR1 complex.

  11. Redefining the modular organization of the core Mediator complex.

    Science.gov (United States)

    Wang, Xuejuan; Sun, Qianqian; Ding, Zhenrui; Ji, Jinhua; Wang, Jianye; Kong, Xiao; Yang, Jianghong; Cai, Gang

    2014-07-01

    The Mediator complex plays an essential role in the regulation of eukaryotic transcription. The Saccharomyces cerevisiae core Mediator comprises 21 subunits, which are organized into Head, Middle and Tail modules. Previously, the Head module was assigned to a distinct dense domain at the base, and the Middle and Tail modules were identified to form a tight structure above the Head module, which apparently contradicted findings from many biochemical and functional studies. Here, we compared the structures of the core Mediator and its subcomplexes, especially the first 3D structure of the Head + Middle modules, which permitted an unambiguous assignment of the three modules. Furthermore, nanogold labeling pinpointing four Mediator subunits from different modules conclusively validated the modular assignment, in which the Head and Middle modules fold back on one another and form the upper portion of the core Mediator, while the Tail module forms a distinct dense domain at the base. The new modular model of the core Mediator has reconciled the previous inconsistencies between the structurally and functionally defined Mediator modules. Collectively, these analyses completely redefine the modular organization of the core Mediator, which allow us to integrate the structural and functional information into a coherent mechanism for the Mediator's modularity and regulation in transcription initiation.

  12. Mediator-dependent Nuclear Receptor Functions

    Science.gov (United States)

    Chen, Wei; Roeder, Robert

    2011-01-01

    As gene-specific transcription factors, nuclear hormone receptors are broadly involved in many important biological processes. Their function on target genes requires the stepwise assembly of different coactivator complexes that facilitate chromatin remodeling and subsequent preinitiation complex (PIC) formation and function. Mediator has proved to be a crucial, and general, nuclear receptor-interacting coactivator, with demonstrated functions in transcription steps ranging from chromatin remodeling to subsequent PIC formation and function. Here we discuss (i) our current understanding of pathways that nuclear receptors and other interacting cofactors employ to recruit Mediator to target gene enhancers and promoters, including conditional requirements for the strong NR-Mediator interactions mediated by the NR AF2 domain and the MED1 LXXLLL motifs and (ii) mechanisms by which Mediator acts to transmit signals from enhancer-bound nuclear receptors to the general transcription machinery at core promoters to effect PIC formation and function. PMID:21854863

  13. Autoinactivation of the stargazin-AMPA receptor complex: subunit-dependency and independence from physical dissociation.

    Directory of Open Access Journals (Sweden)

    Artur Semenov

    Full Text Available Agonist responses and channel kinetics of native α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA receptors are modulated by transmembrane accessory proteins. Stargazin, the prototypical accessory protein, decreases desensitization and increases agonist potency at AMPA receptors. Furthermore, in the presence of stargazin, the steady-state responses of AMPA receptors show a gradual decline at higher glutamate concentrations. This "autoinactivation" has been assigned to physical dissociation of the stargazin-AMPA receptor complex and suggested to serve as a protective mechanism against overactivation. Here, we analyzed autoinactivation of GluA1-A4 AMPA receptors (all flip isoform expressed in the presence of stargazin. Homomeric GluA1, GluA3, and GluA4 channels showed pronounced autoinactivation indicated by the bell-shaped steady-state dose response curves for glutamate. In contrast, homomeric GluA2i channels did not show significant autoinactivation. The resistance of GluA2 to autoinactivation showed striking dependence on the splice form as GluA2-flop receptors displayed clear autoinactivation. Interestingly, the resistance of GluA2-flip containing receptors to autoinactivation was transferred onto heteromeric receptors in a dominant fashion. To examine the relationship of autoinactivation to physical separation of stargazin from the AMPA receptor, we analyzed a GluA4-stargazin fusion protein. Notably, the covalently linked complex and separately expressed proteins expressed a similar level of autoinactivation. We conclude that autoinactivation is a subunit and splice form dependent property of AMPA receptor-stargazin complexes, which involves structural rearrangements within the complex rather than any physical dissociation.

  14. Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

    International Nuclear Information System (INIS)

    Fu Guo; Yang Huayan; Wang Chen; Zhang Feng; You Zhendong; Wang Guiying; He Cheng; Chen Yizhang; Xu Zhihan

    2006-01-01

    Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to β 2 subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively

  15. Sex-dependent anti-stress effect of an α5 subunit containing GABAA receptor positive allosteric modulator

    Directory of Open Access Journals (Sweden)

    Sean C. Piantadosi

    2016-11-01

    Full Text Available Rationale: Current first-line treatments for stress-related disorders such as Major Depressive Disorder (MDD act on monoaminergic systems and take weeks to achieve a therapeutic effect with poor response and low remission rates. Recent research has implicated the GABAergic system in the pathophysiology of depression, including deficits in interneurons targeting the dendritic compartment of cortical pyramidal cells. Objectives: The present study evaluates whether SH-053-2'F-R-CH3 (denoted α5-PAM, a positive allosteric modulator selective for α5-subunit containing GABAA receptors found predominantly on cortical pyramidal cell dendrites has anti-stress effects. Methods: Female and male C57BL6/J mice were exposed to unpredictable chronic mild stress (UCMS and treated with α5-PAM acutely (30 minutes prior to assessing behavior or chronically before being assessed behaviorally. Results: Acute and chronic α5-PAM treatments produce a pattern of decreased stress-induced behaviors (denoted as behavioral emotionality across various tests in female, but not in male mice. Behavioral Z-scores calculated across a panel of tests designed to best model the range and heterogeneity of human symptomatology confirmed that acute and chronic α5-PAM treatments consistently produce significant decreases in behavioral emotionality in several independent cohorts of females. The behavioral responses to α5-PAM could not be completely accounted for by differences in drug brain disposition between female and male mice. In mice exposed to UCMS, expression of the Gabra5 gene was increased in the frontal cortex after acute treatment and in hippocampus after chronic treatment with α5-PAM in females only, and these expression changes correlated with behavioral emotionality. Conclusions: We showed that acute and chronic positive modulation of α5 subunit-containing GABAA receptors elicit anti-stress effects in a sex-dependent manner, suggesting novel therapeutic modalities.

  16. The number and distribution of AMPA receptor channels containing fast kinetic GluA3 and GluA4 subunits at auditory nerve synapses depend on the target cells.

    Science.gov (United States)

    Rubio, María E; Matsui, Ko; Fukazawa, Yugo; Kamasawa, Naomi; Harada, Harumi; Itakura, Makoto; Molnár, Elek; Abe, Manabu; Sakimura, Kenji; Shigemoto, Ryuichi

    2017-11-01

    The neurotransmitter receptor subtype, number, density, and distribution relative to the location of transmitter release sites are key determinants of signal transmission. AMPA-type ionotropic glutamate receptors (AMPARs) containing GluA3 and GluA4 subunits are prominently expressed in subsets of neurons capable of firing action potentials at high frequencies, such as auditory relay neurons. The auditory nerve (AN) forms glutamatergic synapses on two types of relay neurons, bushy cells (BCs) and fusiform cells (FCs) of the cochlear nucleus. AN-BC and AN-FC synapses have distinct kinetics; thus, we investigated whether the number, density, and localization of GluA3 and GluA4 subunits in these synapses are differentially organized using quantitative freeze-fracture replica immunogold labeling. We identify a positive correlation between the number of AMPARs and the size of AN-BC and AN-FC synapses. Both types of AN synapses have similar numbers of AMPARs; however, the AN-BC have a higher density of AMPARs than AN-FC synapses, because the AN-BC synapses are smaller. A higher number and density of GluA3 subunits are observed at AN-BC synapses, whereas a higher number and density of GluA4 subunits are observed at AN-FC synapses. The intrasynaptic distribution of immunogold labeling revealed that AMPAR subunits, particularly GluA3, are concentrated at the center of the AN-BC synapses. The central distribution of AMPARs is absent in GluA3-knockout mice, and gold particles are evenly distributed along the postsynaptic density. GluA4 gold labeling was homogenously distributed along both synapse types. Thus, GluA3 and GluA4 subunits are distributed at AN synapses in a target-cell-dependent manner.

  17. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

    Science.gov (United States)

    Zhao, Lan-Xue; Ge, Yan-Hui; Xiong, Cai-Hong; Tang, Ling; Yan, Ying-Hui; Law, Ping-Yee; Qiu, Yu; Chen, Hong-Zhuan

    2018-03-06

    M1 muscarinic acetylcholine receptors (M1 mAChRs) are the most abundant muscarinic receptors in the hippocampus and have been shown to have procognitive effects. AMPA receptors (AMPARs), an important subtype of ionotropic glutamate receptors, are key components in neurocognitive networks. However, the role of AMPARs in procognitive effects of M1 mAChRs and how M1 mAChRs affect the function of AMPARs remain poorly understood. Here, we found that basal expression of GluA1, a subunit of AMPARs, and its phosphorylation at Ser845 were maintained by M1 mAChR activity. Activation of M1 mAChRs promoted membrane insertion of GluA1, especially to postsynaptic densities. Impairment of hippocampus-dependent learning and memory by antagonism of M1 mAChRs paralleled the reduction of GluA1 expression, and improvement of learning and memory by activation of M1 mAChRs was accompanied by the synaptic insertion of GluA1 and its increased phosphorylation at Ser845. Furthermore, abrogation of phosphorylation of Ser845 residue of GluA1 ablated M1 mAChR-mediated improvement of learning and memory. Taken together, these results show a functional correlation of M1 mAChRs and GluA1 and the essential role of GluA1 in M1 mAChR-mediated cognitive improvement.-Zhao, L.-X., Ge, Y.-H., Xiong, C.-H., Tang, L., Yan, Y.-H., Law, P.-Y., Qiu, Y., Chen, H.-Z. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

  18. The U-box E3 ubiquitin ligase TUD1 functions with a heterotrimeric G α subunit to regulate Brassinosteroid-mediated growth in rice.

    Directory of Open Access Journals (Sweden)

    Xingming Hu

    Full Text Available Heterotrimeric G proteins are an important group of signaling molecules found in eukaryotes. They function with G-protein-coupled-receptors (GPCRs to transduce various signals such as steroid hormones in animals. Nevertheless, their functions in plants are not well-defined. Previous studies suggested that the heterotrimeric G protein α subunit known as D1/RGA1 in rice is involved in a phytohormone gibberellin-mediated signaling pathway. Evidence also implicates D1 in the action of a second phytohormone Brassinosteroid (BR and its pathway. However, it is unclear how D1 functions in this pathway, because so far no partner has been identified to act with D1. In this study, we report a D1 genetic interactor Taihu Dwarf1 (TUD1 that encodes a functional U-box E3 ubiquitin ligase. Genetic, phenotypic, and physiological analyses have shown that tud1 is epistatic to d1 and is less sensitive to BR treatment. Histological observations showed that the dwarf phenotype of tud1 is mainly due to decreased cell proliferation and disorganized cell files in aerial organs. Furthermore, we found that D1 directly interacts with TUD1. Taken together, these results demonstrate that D1 and TUD1 act together to mediate a BR-signaling pathway. This supports the idea that a D1-mediated BR signaling pathway occurs in rice to affect plant growth and development.

  19. The U-Box E3 Ubiquitin Ligase TUD1 Functions with a Heterotrimeric G α Subunit to Regulate Brassinosteroid-Mediated Growth in Rice

    Science.gov (United States)

    Hu, Xingming; Qian, Qian; Xu, Ting; Zhang, Yu'e; Dong, Guojun; Gao, Ting; Xie, Qi; Xue, Yongbiao

    2013-01-01

    Heterotrimeric G proteins are an important group of signaling molecules found in eukaryotes. They function with G-protein-coupled-receptors (GPCRs) to transduce various signals such as steroid hormones in animals. Nevertheless, their functions in plants are not well-defined. Previous studies suggested that the heterotrimeric G protein α subunit known as D1/RGA1 in rice is involved in a phytohormone gibberellin-mediated signaling pathway. Evidence also implicates D1 in the action of a second phytohormone Brassinosteroid (BR) and its pathway. However, it is unclear how D1 functions in this pathway, because so far no partner has been identified to act with D1. In this study, we report a D1 genetic interactor Taihu Dwarf1 (TUD1) that encodes a functional U-box E3 ubiquitin ligase. Genetic, phenotypic, and physiological analyses have shown that tud1 is epistatic to d1 and is less sensitive to BR treatment. Histological observations showed that the dwarf phenotype of tud1 is mainly due to decreased cell proliferation and disorganized cell files in aerial organs. Furthermore, we found that D1 directly interacts with TUD1. Taken together, these results demonstrate that D1 and TUD1 act together to mediate a BR-signaling pathway. This supports the idea that a D1-mediated BR signaling pathway occurs in rice to affect plant growth and development. PMID:23526892

  20. ASIC subunit ratio and differential surface trafficking in the brain.

    Science.gov (United States)

    Wu, Junjun; Xu, Yuanyuan; Jiang, Yu-Qing; Xu, Jiangping; Hu, Youjia; Zha, Xiang-ming

    2016-01-08

    Acid-sensing ion channels (ASICs) are key mediators of acidosis-induced responses in neurons. However, little is known about the relative abundance of different ASIC subunits in the brain. Such data are fundamental for interpreting the relative contribution of ASIC1a homomers and 1a/2 heteromers to acid signaling, and essential for designing therapeutic interventions to target these channels. We used a simple biochemical approach and semi-quantitatively determined the molar ratio of ASIC1a and 2 subunits in mouse brain. Further, we investigated differential surface trafficking of ASIC1a, ASIC2a, and ASIC2b. ASIC1a subunits outnumber the sum of ASIC2a and ASIC2b. There is a region-specific variation in ASIC2a and 2b expression, with cerebellum and striatum expressing predominantly 2b and 2a, respectively. Further, we performed surface biotinylation and found that surface ASIC1a and ASIC2a ratio correlates with their total expression. In contrast, ASIC2b exhibits little surface presence in the brain. This result is consistent with increased co-localization of ASIC2b with an ER marker in 3T3 cells. Our data are the first semi-quantitative determination of relative subunit ratio of various ASICs in the brain. The differential surface trafficking of ASICs suggests that the main functional ASICs in the brain are ASIC1a homomers and 1a/2a heteromers. This finding provides important insights into the relative contribution of various ASIC complexes to acid signaling in neurons.

  1. The light subunit of system bo,+ is fully functional in the absence of the heavy subunit

    OpenAIRE

    Reig, Núria; Chillarón, Josep; Bartoccioni, Paola; Fernández, Esperanza; Bendahan, Annie; Zorzano, Antonio; Kanner, Baruch; Palacín, Manuel; Bertran, Joan

    2002-01-01

    The heteromeric amino acid transporters are composed of a type II glycoprotein and a non-glycosylated polytopic membrane protein. System bo,+ exchanges dibasic for neutral amino acids. It is composed of rBAT and bo,+AT, the latter being the polytopic membrane subunit. Mutations in either of them cause malfunction of the system, leading to cystinuria. bo,+AT-reconstituted systems from HeLa or MDCK cells catalysed transport of arginine that was totally dependent on the presence of one of the bo...

  2. AKAP18:PKA-RIIα structure reveals crucial anchor points for recognition of regulatory subunits of PKA.

    Science.gov (United States)

    Götz, Frank; Roske, Yvette; Schulz, Maike Svenja; Autenrieth, Karolin; Bertinetti, Daniela; Faelber, Katja; Zühlke, Kerstin; Kreuchwig, Annika; Kennedy, Eileen J; Krause, Gerd; Daumke, Oliver; Herberg, Friedrich W; Heinemann, Udo; Klussmann, Enno

    2016-07-01

    A-kinase anchoring proteins (AKAPs) interact with the dimerization/docking (D/D) domains of regulatory subunits of the ubiquitous protein kinase A (PKA). AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling. Here, we elucidated the structure of an extended PKA-binding domain of AKAP18β bound to the D/D domain of the regulatory RIIα subunits of PKA. We identified three hydrophilic anchor points in AKAP18β outside the core PKA-binding domain, which mediate contacts with the D/D domain. Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA. We derived a different set of anchor points in AKAPs binding regulatory RI subunits of PKA. In vitro and cell-based experiments confirm the relevance of these sites for the interaction of RII subunits with AKAP18 and of RI subunits with the RI-specific smAKAP. Thus we report a novel mechanism governing interactions of AKAPs with PKA. The sequence specificity of each AKAP around the anchor points and the requirement of these points for the tight binding of PKA allow the development of selective inhibitors to unequivocally ascribe cellular functions to the AKAP18-PKA and other AKAP-PKA interactions. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  3. Role of the Rubisco Small Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Spreitzer, Robert Joseph [Univ. of Nebraska, Lincoln, NE (United States)

    2016-11-05

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis. However, it is a slow enzyme, and O2 competes with CO2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO2. If carboxylation could be increased or oxygenation decreased, an increase in net CO2 fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants, and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO2/O2 specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a

  4. Progesterone modulation of alpha5 nAChR subunits influences anxiety-related behavior during estrus cycle.

    Science.gov (United States)

    Gangitano, D; Salas, R; Teng, Y; Perez, E; De Biasi, M

    2009-06-01

    Smokers often report an anxiolytic effect of cigarettes. In addition, stress-related disorders such as anxiety, post-traumatic stress syndrome and depression are often associated with chronic nicotine use. To study the role of the alpha5 nicotinic acetylcholine receptor subunit in anxiety-related responses, control and alpha5 subunit null mice (alpha5(-/-)) were subjected to the open field activity (OFA), light-dark box (LDB) and elevated plus maze (EPM) tests. In the OFA and LDB, alpha5(-/-) behaved like wild-type controls. In the EPM, female alpha5(-/-) mice displayed an anxiolytic-like phenotype, while male alpha5(-/-) mice were undistinguishable from littermate controls. We studied the hypothalamus-pituitary-adrenal axis by measuring plasma corticosterone and hypothalamic corticotropin-releasing factor. Consistent with an anxiolytic-like phenotype, female alpha5(-/-) mice displayed lower basal corticosterone levels. To test whether gonadal steroids regulate the expression of alpha5, we treated cultured NTera 2 cells with progesterone and found that alpha5 protein levels were upregulated. In addition, brain levels of alpha5 mRNA increased upon progesterone injection into ovariectomized wild-type females. Finally, we tested anxiety levels in the EPM during the estrous cycle. The estrus phase (when progesterone levels are low) is anxiolytic-like in wild-type mice, but no cycle-dependent fluctuations in anxiety levels were found in alpha5(-/-) females. Thus, alpha5-containing neuronal nicotinic acetylcholine receptors may be mediators of anxiogenic responses, and progesterone-dependent modulation of alpha5 expression may contribute to fluctuations in anxiety levels during the ovarian cycle.

  5. The Cac2 subunit is essential for productive histone binding and nucleosome assembly in CAF-1

    Energy Technology Data Exchange (ETDEWEB)

    Mattiroli, Francesca; Gu, Yajie; Balsbaugh, Jeremy L.; Ahn, Natalie G.; Luger, Karolin

    2017-04-18

    Nucleosome assembly following DNA replication controls epigenome maintenance and genome integrity. Chromatin assembly factor 1 (CAF-1) is the histone chaperone responsible for histone (H3-H4)2 deposition following DNA synthesis. Structural and functional details for this chaperone complex and its interaction with histones are slowly emerging. Using hydrogen-deuterium exchange coupled to mass spectrometry, combined with in vitro and in vivo mutagenesis studies, we identified the regions involved in the direct interaction between the yeast CAF-1 subunits, and mapped the CAF-1 domains responsible for H3-H4 binding. The large subunit, Cac1 organizes the assembly of CAF-1. Strikingly, H3-H4 binding is mediated by a composite interface, shaped by Cac1-bound Cac2 and the Cac1 acidic region. Cac2 is indispensable for productive histone binding, while deletion of Cac3 has only moderate effects on H3-H4 binding and nucleosome assembly. These results define direct structural roles for yeast CAF-1 subunits and uncover a previously unknown critical function of the middle subunit in CAF-1.

  6. Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition

    DEFF Research Database (Denmark)

    Kachel, Hamid S; Patel, Rohit N; Franzyk, Henrik

    2016-01-01

    -fold selectivity of PhTX-12 over PhTX-343 for embryonic muscle-type nicotinic acetylcholine receptors (nAChRs) in TE671 cells. We investigated their inhibition of different neuronal nAChR subunit combinations as well as of embryonic muscle receptors expressed in Xenopus oocytes. Whole-cell currents...

  7. The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II

    DEFF Research Database (Denmark)

    Elmlund, Hans; Baraznenok, Vera; Lindahl, Martin

    2006-01-01

    CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8......-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II....

  8. Propranolol decreases retention of fear memory by modulating the stability of surface glutamate receptor GluA1 subunits in the lateral amygdala.

    Science.gov (United States)

    Zhou, Jun; Luo, Yi; Zhang, Jie-Ting; Li, Ming-Xing; Wang, Can-Ming; Guan, Xin-Lei; Wu, Peng-Fei; Hu, Zhuang-Li; Jin, You; Ni, Lan; Wang, Fang; Chen, Jian-Guo

    2015-11-01

    Posttraumatic stress disorder (PTSD) is a mental disorder with enhanced retention of fear memory and has profound impact on quality of life for millions of people worldwide. The β-adrenoceptor antagonist propranolol has been used in preclinical and clinical studies for the treatment of PTSD, but the mechanisms underlying its potential efficacy on fear memory retention remain to be elucidated. We investigated the action of propranolol on the retention of conditioned fear memory, the surface expression of glutamate receptor GluA1 subunits of AMPA receptors and synaptic adaptation in the lateral amygdala (LA) of rats. Propranolol attenuated reactivation-induced strengthening of fear retention while reducing enhanced surface expression of GluA1 subunits and restoring the impaired long-term depression in LA. These effects of propranolol were mediated by antagonizing reactivation-induced enhancement of adrenergic signalling, which activates PKA and calcium/calmodulin-dependent protein kinase II and then regulates the trafficking of AMPA receptors via phosphorylation of GluA1 subunits at the C-terminus. Both i.p. injection and intra-amygdala infusion of propranolol attenuated reactivation-induced enhancement of fear retention. Reactivation strengthens fear retention by increasing the level of noradrenaline and promotes the surface expression of GluA1 subunits and the excitatory synaptic transmission in LA. These findings uncover one mechanism underlying the efficiency of propranolol on retention of fear memories and suggest that β-adrenoceptor antagonists, which act centrally, may be more suitable for the treatment of PTSD. © 2015 The British Pharmacological Society.

  9. Identification of aspartate-184 as an essential residue in the catalytic subunit of cAMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Buechler, J.A.; Taylor, S.S.

    1988-01-01

    The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) was previously shown to be an irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase, and MgATP protected against inactivation. This inhibition by DCCD indicated that an essential carboxyl group was present at the active site of the enzyme even though identification of that carboxyl group was not possible. This presumably was because a nucleophile on the protein cross-linked to the electrophilic intermediate formed when the carbodiimide reacted with the carboxyl group. To circumvent this problem, the catalytic subunit first was treated with acetic anhydride to block accessible lysine residues, thus preventing intramolecular cross-linking. The DCCD reaction then was carried out in the presence of [ 14 C]glycine ethyl ester in order to trap any electrophilic intermediates that were generated by DCCD. The modified protein was treated with trypsin, and the resulting peptides were separated by HPLC. Two major radioactive peptides were isolated as well as one minor peptide. MgATP protected all three peptides from covalent modification. The two major peaks contained the same modified carboxyl group, which corresponded to Asp-184. The minor peak contained a modified glutamic acid, Glu-91. Both of these acidic residues are conserved in all protein kinases, which is consistent with their playing essential roles. The positions of Asp-184 and Glu-91 have been correlated with the overall domain structure of the molecule. Asp-184 may participate as a general base catalyst at the active site. A third carboxyl group, Glu-230, also was identified

  10. Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons.

    Science.gov (United States)

    Johar, Kaid; Priya, Anusha; Dhar, Shilpa; Liu, Qiuli; Wong-Riley, Margaret T T

    2013-11-01

    Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1 that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. The present study discovered that among the specificity family of transcription factors, it is the less known neuron-specific Sp4 that regulates the expression of all 13 subunits of mitochondrial cytochrome c oxidase (COX) enzyme in primary neurons. Sp4 also regulates the three mitochondrial transcription factors (TFAM, TFB1M, and TFB2M) and a COX assembly protein SURF-1 in primary neurons. © 2013 International Society for Neurochemistry.

  11. Generation of functional inhibitory synapses incorporating defined combinations of GABA(A or glycine receptor subunits

    Directory of Open Access Journals (Sweden)

    Christine Laura Dixon

    2015-12-01

    Full Text Available Fast inhibitory neurotransmission in the brain is mediated by wide range of GABAA receptor (GABAAR and glycine receptor (GlyR isoforms, each with different physiological and pharmacological properties. Because multiple isoforms are expressed simultaneously in most neurons, it is difficult to define the properties of inhibitory postsynaptic currents mediated by individual isoforms in vivo. Although recombinant expression systems permit the expression of individual isoforms in isolation, they require exogenous agonist application which cannot mimic the dynamic neurotransmitter profile characteristic of native synapses. We describe a neuron-HEK293 cell co-culture technique for generating inhibitory synapses incorporating defined combinations of GABAAR or GlyR subunits. Primary neuronal cultures, prepared from embryonic rat cerebral cortex or spinal cord, are used to provide presynaptic GABAergic and glycinergic terminals, respectively. When the cultures are mature, HEK293 cells expressing the subunits of interest plus neuroligin 2A are plated onto the neurons, which rapidly form synapses onto HEK293 cells. Patch clamp electrophysiology is then used to analyze the physiological and pharmacological properties of the inhibitory postsynaptic currents mediated by the recombinant receptors. The method is suitable for investigating the kinetic properties or the effects of drugs on inhibitory postsynaptic currents mediated by defined GABAAR or GlyR isoforms of interest, the effects of hereditary disease mutations on the formation and function of both types of synapses, and synaptogenesis and synaptic clustering mechanisms. The entire cell preparation procedure takes 2 – 5 weeks.

  12. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, R.M.B. [Universidade Tecnica, Lisbon (Portugal). Inst. Superior de Agronomia]|[Universidade Nova de Lisboa, Oeiras (Portugal). Instituto de Tecnologia Quimica e Biologica; Franco, E.; Teixeira, A.R.N. [Universidade Tecnica, Lisbon (Portugal). Inst. Superior de Agronomia

    1996-08-15

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a 65 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of {sup 35}S-labelled ribulose biphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose biphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose biphosphate carboxylase. For short periods of time (<1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose biphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photosynthetic tissues. (Author).

  13. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation

    International Nuclear Information System (INIS)

    Ferreira, R.M.B.; Universidade Nova de Lisboa, Oeiras; Franco, E.; Teixeira, A.R.N.

    1996-01-01

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a 65 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35 S-labelled ribulose biphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose biphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose biphosphate carboxylase. For short periods of time (<1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose biphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photosynthetic tissues. (Author)

  14. PKC/CREB pathway mediates the expressions of GABAA receptor subunits in cultured hippocampal neurons after low-Mg2+ solution treatment.

    Science.gov (United States)

    Wu, Guofeng; Yu, Jinpeng; Wang, Likun; Ren, Siying; Zhang, Yixia

    2018-02-01

    To investigate the potential effects of the PKC/CREB pathway on the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons using a model of epilepsy that employed conditions of low magnesium (Mg 2+ ). A total of 108 embryonic rats at the age of 18 embryonic days (E18)prepared from adult female SD rats were used as experimental subjects. Primary rat hippocampal cultures were prepared from the embryonic 18 days rats. The cultured hippocampal neurons were then treated with artificial cerebrospinal fluid containing low Mg 2+ solutions to generate a low Mg 2+ model of epilepsy. The low Mg 2+ stimulation lasted for 3 h and then returned to in maintenance medium for 20 h. The changes of the GABA A receptor subunit α1, γ2, δ were observed by blocking or activating the function of the CREB. The quantification of the GABA A receptor subunit α1, γ2, δ and the CREB were determined by a qRT-PCR and a Western blot method. After the neurons were exposed to a low-Mg 2+ solution for 3 h, GABA A receptor mRNA expression markedly increased compared to the control, and then gradually decreased. In contrast, CREB mRNA levels exhibited a dramatic down-regulation 3 h after terminating low-Mg 2+ treatment, and then peaked at 9 h. Western blot analyses verified that staurosporine suppressed CREB phosphorylation (p-CREB). The mRNA expression of GABA A receptor subunit α1 increased only in the presence of staurosporine, whereas the expressions of subunits γ2 and δ significantly increased in the presence of either KG-501 or staurosporine. Furthermore, phorbol 12-myristate 13-acetate (PMA) decreased the expressions of GABA A subunits α1, γ2, and δ when administered alone. However, the administration of either KG-501 or staurosporine reversed the inhibitory effects of PMA. The PKC/CREB pathway may negatively regulate the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons in low Mg 2+ model of

  15. Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase.

    Science.gov (United States)

    White, Caroline N; Figtree, Gemma A; Liu, Chia-Chi; Garcia, Alvaro; Hamilton, Elisha J; Chia, Karin K M; Rasmussen, Helge H

    2009-04-01

    The sarcolemmal Na(+)-K(+) pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to epsilon-protein kinase C (epsilonPKC), previously implicated in ANG II-induced Na(+)-K(+) pump inhibition. A role for epsilonPKC was also supported by an ANG II-induced increase in coimmunoprecipitation of epsilonPKC with the receptor for the activated kinase and with the cytosolic p47(phox) subunit of NADPH oxidase. ANG II decreased electrogenic Na(+)-K(+) pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by epsilonPKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the Na(+)-K(+) pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The alpha(1)-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated p22(phox) and cytosolic p47(phox) NADPH oxidase subunits at baseline. ANG II had no effect on alpha(1)/caveolin 3 or alpha(1)/p22(phox) interaction, but it increased alpha(1)/p47(phox) coimmunoprecipitation. We conclude that ANG II inhibits the Na(+)-K(+) pump via PKC-dependent NADPH oxidase activation.

  16. Mammalian mediator 19 mediates H1299 lung adenocarcinoma cell clone conformation, growth, and metastasis.

    Science.gov (United States)

    Xu, Lu-Lu; Guo, Shu-Liang; Ma, Su-Ren; Luo, Yong-Ai

    2012-01-01

    Mammalian mediator (MED) is a multi-protein coactivator that has been identified by several research groups. The involvement of the MED complex subunit 19 (MED 19) in the metastasis of lung adenocarcinoma cell line (H1299), which expresses the MED 19 subunit, was here investigated. When MED 19 expression was decreased by RNA interference H1299 cells demonstrated reduced clone formation, arrest in the S phase of the cell cycle, and lowered metastatic capacity. Thus, MED 19 appears to play important roles in the biological behavior of non-small cell lung carcinoma cells. These findings may be important for the development of novel lung carcinoma treatments.

  17. Rosmarinic acid counteracts activation of hepatic stellate cells via inhibiting the ROS-dependent MMP-2 activity: Involvement of Nrf2 antioxidant system

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Changfang; Zou, Yu; Liu, Yuzhang; Niu, Yingcai, E-mail: nyc1968@126.com

    2017-03-01

    Recently, oxidative stress is involved in hepatofibrogenesis. Matrix metalloproteinase-2 (MMP-2) is required for activation of hepatic stellate cells (HSCs) in response to reactive oxygen species (ROS). This study was designed to explore the hypothesis that the inhibitory effect of rosmarinic acid (RA) on HSCs activation might mainly result from its antioxidant capability by increasing the synthesis of glutathione (GSH) involved in nuclear factor kappa B (NF-κB)-dependent inhibition of MMP-2 activity. Here, we demonstrate that RA reverses activated HSCs to quiescent cells. Concomitantly, RA inhibits MMP-2 activity. RNA interference-imposed knockdown of NF-κB abolished down-regulation of MMP-2 by RA. RA-mediated inactivation of NF-κB could be blocked by the diphenyleneiodonium chloride (DPI; a ROS inhibitor). Conversely, transfection of dominant-negative (DN) mutant of extracellular signal-regulated kinases 2 (ERK2), c-Jun N-terminal kinase 1 (JNK1), or p38α kinase had no such effect. Simultaneously, RA suppresses ROS generation and lipid peroxidation (LPO) whereas increases cellular GSH in HSC-T6 cells. Furthermore, RA significantly increased antioxidant response element (ARE)-mediated luciferase activity, nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and catalytic subunits from glutamate cysteine ligase (GCLc) expression, but not modulatory subunits from GCL (GCLm). RA-mediated up-regulation of GClc is inhibited by the shRNA-induced Nrf2 knockdown. The knocking down of Nrf2 or buthionine sulfoximine (a GCL inhibitor) abolished RA-mediated inhibition of ROS. Collectively, these results provide novel insights into the mechanisms of RA as an antifibrogenic candidate in the prevention and treatment of liver fibrosis. - Highlights: • RA reverses activated HSCs to quiescent cells. • RA suppresses MMP-2 activity through a NF-κB-dependent pathway. • Inhibition of oxidative stress by RA is dependent on nuclear translocation of Nrf2

  18. Rosmarinic acid counteracts activation of hepatic stellate cells via inhibiting the ROS-dependent MMP-2 activity: Involvement of Nrf2 antioxidant system

    International Nuclear Information System (INIS)

    Lu, Changfang; Zou, Yu; Liu, Yuzhang; Niu, Yingcai

    2017-01-01

    Recently, oxidative stress is involved in hepatofibrogenesis. Matrix metalloproteinase-2 (MMP-2) is required for activation of hepatic stellate cells (HSCs) in response to reactive oxygen species (ROS). This study was designed to explore the hypothesis that the inhibitory effect of rosmarinic acid (RA) on HSCs activation might mainly result from its antioxidant capability by increasing the synthesis of glutathione (GSH) involved in nuclear factor kappa B (NF-κB)-dependent inhibition of MMP-2 activity. Here, we demonstrate that RA reverses activated HSCs to quiescent cells. Concomitantly, RA inhibits MMP-2 activity. RNA interference-imposed knockdown of NF-κB abolished down-regulation of MMP-2 by RA. RA-mediated inactivation of NF-κB could be blocked by the diphenyleneiodonium chloride (DPI; a ROS inhibitor). Conversely, transfection of dominant-negative (DN) mutant of extracellular signal-regulated kinases 2 (ERK2), c-Jun N-terminal kinase 1 (JNK1), or p38α kinase had no such effect. Simultaneously, RA suppresses ROS generation and lipid peroxidation (LPO) whereas increases cellular GSH in HSC-T6 cells. Furthermore, RA significantly increased antioxidant response element (ARE)-mediated luciferase activity, nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and catalytic subunits from glutamate cysteine ligase (GCLc) expression, but not modulatory subunits from GCL (GCLm). RA-mediated up-regulation of GClc is inhibited by the shRNA-induced Nrf2 knockdown. The knocking down of Nrf2 or buthionine sulfoximine (a GCL inhibitor) abolished RA-mediated inhibition of ROS. Collectively, these results provide novel insights into the mechanisms of RA as an antifibrogenic candidate in the prevention and treatment of liver fibrosis. - Highlights: • RA reverses activated HSCs to quiescent cells. • RA suppresses MMP-2 activity through a NF-κB-dependent pathway. • Inhibition of oxidative stress by RA is dependent on nuclear translocation of Nrf2

  19. Ebselen and congeners inhibit NADPH oxidase 2-dependent superoxide generation by interrupting the binding of regulatory subunits.

    Science.gov (United States)

    Smith, Susan M E; Min, Jaeki; Ganesh, Thota; Diebold, Becky; Kawahara, Tsukasa; Zhu, Yerun; McCoy, James; Sun, Aiming; Snyder, James P; Fu, Haian; Du, Yuhong; Lewis, Iestyn; Lambeth, J David

    2012-06-22

    NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS), which function in normal physiology and, when overproduced, in pathophysiology. Recent studies using mice deficient in Nox2 identify this isoform as a novel target against Nox2-implicated inflammatory diseases. Nox2 activation depends on the binding of the proline-rich domain of its heterodimeric partner p22phox to p47phox. A high-throughput screen that monitored this interaction via fluorescence polarization identified ebselen and several of its analogs as inhibitors. Medicinal chemistry was performed to explore structure-activity relationships and to optimize potency. Ebselen and analogs potently inhibited Nox1 and Nox2 activity but were less effective against other isoforms. Ebselen also blocked translocation of p47phox to neutrophil membranes. Thus, ebselen and its analogs represent a class of compounds that inhibit ROS generation by interrupting the assembly of Nox2-activating regulatory subunits. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo.

    Science.gov (United States)

    Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

    2017-07-12

    The Mediator complex has been described as a general transcription factor, but it is unclear if it is essential for Pol II transcription and/or is a required component of the preinitiation complex (PIC) in vivo. Here, we show that depletion of individual subunits, even those essential for cell growth, causes a general but only modest decrease in transcription. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in transcription. Depletion of head or middle subunits, but not tail subunits, causes a downstream shift in the Pol II occupancy profile, suggesting that Mediator at the core promoter inhibits promoter escape. Interestingly, a functional PIC and Pol II transcription can occur when Mediator is not detected at core promoters. These results provide strong evidence that Mediator is essential for Pol II transcription and stimulates PIC formation, but it is not a required component of the PIC in vivo.

  1. Submitochondrial distributions and stabilities of subunits 4, 5, and 6 of yeast cytochrome oxidase in assembly defective mutants.

    Science.gov (United States)

    Glerum, D M; Tzagoloff, A

    1997-08-04

    The concentration and submitochondrial distribution of the subunit polypeptides of cytochrome oxidase have been studied in wild type yeast and in different mutants impaired in assembly of this respiratory complex. All the subunit polypeptides of the enzyme are associated with mitochondrial membranes of wild type cells, except for a small fraction of subunits 4 and 6 that is recovered in the soluble protein fraction of mitochondria. Cytochrome oxidase mutants consistently display a severe reduction in the steady-state concentration of subunit 1 due to its increased turnover. As a consequence, most of subunit 4, which normally is associated with subunit 1, is found in the soluble fraction. A similar shift from membrane-bound to soluble subunit 6 is seen in mutants blocked in expression of subunit 5a. In contrast, null mutations in COX6 coding for subunit 6 promote loss of subunit 5a. The absence of subunit 5a in the cox6 mutant is the result of proteolytic degradation rather than regulation of its expression by subunit 6. The possible role of the ATP-dependent proteases Rca1p and Afg3p in proteolysis of subunits 1 and 5a has been assessed in strains with combined mutations in COX6, RCA1, and/or AFG3. Immunochemical assays indicate that another protease(s) must be responsible for most of the proteolytic loss of these proteins.

  2. Leptin and insulin engage specific PI3K subunits in hypothalamic SF1 neurons

    Directory of Open Access Journals (Sweden)

    Jong-Woo Sohn

    2016-08-01

    Full Text Available Objective: The ventromedial hypothalamic nucleus (VMH regulates energy balance and glucose homeostasis. Leptin and insulin exert metabolic effects via their cognate receptors expressed by the steroidogenic factor 1 (SF1 neurons within the VMH. However, detailed cellular mechanisms involved in the regulation of these neurons by leptin and insulin remain to be identified. Methods: We utilized genetically-modified mouse models and performed patch-clamp electrophysiology experiments to resolve this issue. Results: We identified distinct populations of leptin-activated and leptin-inhibited SF1 neurons. In contrast, insulin uniformly inhibited SF1 neurons. Notably, we found that leptin-activated, leptin-inhibited, and insulin-inhibited SF1 neurons are distinct subpopulations within the VMH. Leptin depolarization of SF1 neuron also required the PI3K p110β catalytic subunit. This effect was mediated by the putative transient receptor potential C (TRPC channel. On the other hand, hyperpolarizing responses of SF1 neurons by leptin and insulin required either of the p110α or p110β catalytic subunits, and were mediated by the putative ATP-sensitive K+ (KATP channel. Conclusions: Our results demonstrate that specific PI3K catalytic subunits are responsible for the acute effects of leptin and insulin on VMH SF1 neurons, and provide insights into the cellular mechanisms of leptin and insulin action on VMH SF1 neurons that regulate energy balance and glucose homeostasis. Author Video: Author Video Watch what authors say about their articles Keywords: Cellular mechanism, Conditional knockout mouse, Patch clamp technique, Functional heterogeneity, Homeostasis

  3. Mediator MED23 regulates basal transcription in vivo via an interaction with P-TEFb.

    Science.gov (United States)

    Wang, Wei; Yao, Xiao; Huang, Yan; Hu, Xiangming; Liu, Runzhong; Hou, Dongming; Chen, Ruichuan; Wang, Gang

    2013-01-01

    The Mediator is a multi-subunit complex that transduces regulatory information from transcription regulators to the RNA polymerase II apparatus. Growing evidence suggests that Mediator plays roles in multiple stages of eukaryotic transcription, including elongation. However, the detailed mechanism by which Mediator regulates elongation remains elusive. In this study, we demonstrate that Mediator MED23 subunit controls a basal level of transcription by recruiting elongation factor P-TEFb, via an interaction with its CDK9 subunit. The mRNA level of Egr1, a MED23-controlled model gene, is reduced 4-5 fold in Med23 (-/-) ES cells under an unstimulated condition, but Med23-deficiency does not alter the occupancies of RNAP II, GTFs, Mediator complex, or activator ELK1 at the Egr1 promoter. Instead, Med23 depletion results in a significant decrease in P-TEFb and RNAP II (Ser2P) binding at the coding region, but no changes for several other elongation regulators, such as DSIF and NELF. ChIP-seq revealed that Med23-deficiency partially reduced the P-TEFb occupancy at a set of MED23-regulated gene promoters. Further, we demonstrate that MED23 interacts with CDK9 in vivo and in vitro. Collectively, these results provide the mechanistic insight into how Mediator promotes RNAP II into transcription elongation.

  4. A functional portrait of Med7 and the mediator complex in Candida albicans.

    Science.gov (United States)

    Tebbji, Faiza; Chen, Yaolin; Richard Albert, Julien; Gunsalus, Kearney T W; Kumamoto, Carol A; Nantel, André; Sellam, Adnane; Whiteway, Malcolm

    2014-11-01

    Mediator is a multi-subunit protein complex that regulates gene expression in eukaryotes by integrating physiological and developmental signals and transmitting them to the general RNA polymerase II machinery. We examined, in the fungal pathogen Candida albicans, a set of conditional alleles of genes encoding Mediator subunits of the head, middle, and tail modules that were found to be essential in the related ascomycete Saccharomyces cerevisiae. Intriguingly, while the Med4, 8, 10, 11, 14, 17, 21 and 22 subunits were essential in both fungi, the structurally highly conserved Med7 subunit was apparently non-essential in C. albicans. While loss of CaMed7 did not lead to loss of viability under normal growth conditions, it dramatically influenced the pathogen's ability to grow in different carbon sources, to form hyphae and biofilms, and to colonize the gastrointestinal tracts of mice. We used epitope tagging and location profiling of the Med7 subunit to examine the distribution of the DNA sites bound by Mediator during growth in either the yeast or the hyphal form, two distinct morphologies characterized by different transcription profiles. We observed a core set of 200 genes bound by Med7 under both conditions; this core set is expanded moderately during yeast growth, but is expanded considerably during hyphal growth, supporting the idea that Mediator binding correlates with changes in transcriptional activity and that this binding is condition specific. Med7 bound not only in the promoter regions of active genes but also within coding regions and at the 3' ends of genes. By combining genome-wide location profiling, expression analyses and phenotyping, we have identified different Med7p-influenced regulons including genes related to glycolysis and the Filamentous Growth Regulator family. In the absence of Med7, the ribosomal regulon is de-repressed, suggesting Med7 is involved in central aspects of growth control.

  5. Divergence of RNA polymerase ? subunits in angiosperm plastid genomes is mediated by genomic rearrangement

    OpenAIRE

    Blazier, J. Chris; Ruhlman, Tracey A.; Weng, Mao-Lun; Rehman, Sumaiyah K.; Sabir, Jamal S. M.; Jansen, Robert K.

    2016-01-01

    Genes for the plastid-encoded RNA polymerase (PEP) persist in the plastid genomes of all photosynthetic angiosperms. However, three unrelated lineages (Annonaceae, Passifloraceae and Geraniaceae) have been identified with unusually divergent open reading frames (ORFs) in the conserved region of rpoA, the gene encoding the PEP ? subunit. We used sequence-based approaches to evaluate whether these genes retain function. Both gene sequences and complete plastid genome sequences were assembled an...

  6. Protein Kinase A Regulatory Subunits in Human Adipose Tissue

    Science.gov (United States)

    Mantovani, Giovanna; Bondioni, Sara; Alberti, Luisella; Gilardini, Luisa; Invitti, Cecilia; Corbetta, Sabrina; Zappa, Marco A.; Ferrero, Stefano; Lania, Andrea G.; Bosari, Silvano; Beck-Peccoz, Paolo; Spada, Anna

    2009-01-01

    OBJECTIVE—In human adipocytes, the cAMP-dependent pathway mediates signals originating from β-adrenergic activation, thus playing a key role in the regulation of important metabolic processes, i.e., lipolysis and thermogenesis. Cyclic AMP effects are mainly mediated by protein kinase A (PKA), whose R2B regulatory isoform is the most expressed in mouse adipose tissue, where it protects against diet-induced obesity and fatty liver development. The aim of the study was to investigate possible differences in R2B expression, PKA activity, and lipolysis in adipose tissues from obese and nonobese subjects. RESEARCH DESIGN AND METHODS—The expression of the different PKA regulatory subunits was evaluated by immunohistochemistry, Western blot, and real-time PCR in subcutaneous and visceral adipose tissue samples from 20 nonobese and 67 obese patients. PKA activity and glycerol release were evaluated in total protein extract and adipocytes isolated from fresh tissue samples, respectively. RESULTS—Expression techniques showed that R2B was the most abundant regulatory protein, both at mRNA and protein level. Interestingly, R2B mRNA levels were significantly lower in both subcutaneous and visceral adipose tissues from obese than nonobese patients and negatively correlated with BMI, waist circumference, insulin levels, and homeostasis model assessment of insulin resistance. Moreover, both basal and stimulated PKA activity and glycerol release were significantly lower in visceral adipose tissue from obese patients then nonobese subjects. CONCLUSIONS—Our results first indicate that, in human adipose tissue, there are important BMI-related differences in R2B expression and PKA activation, which might be included among the multiple determinants involved in the different lipolytic response to β-adrenergic activation in obesity. PMID:19095761

  7. Permeability transition in human mitochondria persists in the absence of peripheral stalk subunits of ATP synthase.

    Science.gov (United States)

    He, Jiuya; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2017-08-22

    The opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membranes of mitochondria can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane and ATP synthesis, and cell death. Pore opening can be inhibited by cyclosporin A mediated via cyclophilin D. It has been proposed that the pore is associated with the dimeric ATP synthase and the oligomycin sensitivity conferral protein (OSCP), a component of the enzyme's peripheral stalk, provides the site at which cyclophilin D interacts. Subunit b contributes a central α-helical structure to the peripheral stalk, extending from near the top of the enzyme's catalytic domain and crossing the membrane domain of the enzyme via two α-helices. We investigated the possible involvement of the subunit b and the OSCP in the PTP by generating clonal cells, HAP1-Δb and HAP1-ΔOSCP, lacking the membrane domain of subunit b or the OSCP, respectively, in which the corresponding genes, ATP5F1 and ATP5O , had been disrupted. Both cell lines preserve the characteristic properties of the PTP; therefore, the membrane domain of subunit b does not contribute to the PTP, and the OSCP does not provide the site of interaction with cyclophilin D. The membrane subunits ATP6, ATP8, and subunit c have been eliminated previously from possible participation in the PTP; thus, the only subunits of ATP synthase that could participate in pore formation are e, f, g, diabetes-associated protein in insulin-sensitive tissues (DAPIT), and the 6.8-kDa proteolipid.

  8. Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators

    Science.gov (United States)

    Liu, Yangfan P.; Tsai, I-Chun; Morleo, Manuela; Oh, Edwin C.; Leitch, Carmen C.; Massa, Filomena; Lee, Byung-Hoon; Parker, David S.; Finley, Daniel; Zaghloul, Norann A.; Franco, Brunella; Katsanis, Nicholas

    2014-01-01

    Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. Here, we show that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice. Finally, we tested the hypothesis that other proteasome-dependent pathways not known to be associated with ciliopathies are defective in the absence of ciliopathy proteins. We found that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBβ accumulation and that these defects were ameliorated with SFN treatment. Taken together, our data indicate that basal body proteasomal regulation governs paracrine signaling pathways and suggest that augmenting proteasomal function might benefit ciliopathy patients. PMID:24691443

  9. Mediator, SWI/SNF and SAGA complexes regulate Yap8-dependent transcriptional activation of ACR2 in response to arsenate.

    Science.gov (United States)

    Menezes, Regina Andrade; Pimentel, Catarina; Silva, Ana Rita Courelas; Amaral, Catarina; Merhej, Jawad; Devaux, Frédéric; Rodrigues-Pousada, Claudina

    2017-04-01

    Response to arsenic stress in Saccharomyces cerevisiae is orchestrated by the regulatory protein Yap8, which mediates transcriptional activation of ACR2 and ACR3. This study contributes to the state of art knowledge of the molecular mechanisms underlying yeast stress response to arsenate as it provides the genetic and biochemical evidences that Yap8, through cysteine residues 132, 137, and 274, is the sensor of presence of arsenate in the cytosol. Moreover, it is here reported for the first time the essential role of the Mediator complex in the transcriptional activation of ACR2 by Yap8. Based on our data, we propose an order-of-function map to recapitulate the sequence of events taking place in cells injured with arsenate. Modification of the sulfhydryl state of these cysteines converts Yap8 in its activated form, triggering the recruitment of the Mediator complex to the ACR2/ACR3 promoter, through the interaction with the tail subunit Med2. The Mediator complex then transfers the regulatory signals conveyed by Yap8 to the core transcriptional machinery, which culminates with TBP occupancy, ACR2 upregulation and cell adaptation to arsenate stress. Additional co-factors are required for the transcriptional activation of ACR2 by Yap8, particularly the nucleosome remodeling activity of SWI/SNF and SAGA complexes. Copyright © 2017. Published by Elsevier B.V.

  10. Pharmacological consequences of the coexpression of BK channel α and auxiliary β subunits

    Directory of Open Access Journals (Sweden)

    Yolima P. Torres

    2014-10-01

    Full Text Available Coded by a single gene (Slo1, KCM and activated by depolarizing potentials and by a rise in intracellular Ca2+ concentration, the large conductance voltage- and Ca+2-activated K+ channel (BK is unique among the superfamily of K+ channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K+ channels and a large C terminus composed of two regulators of K+ conductance domains (RCK domains, where the Ca2+-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified (i.e., β1, β2, β3 & β4 and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca+2 sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above.

  11. Pharmacological consequences of the coexpression of BK channel α and auxiliary β subunits.

    Science.gov (United States)

    Torres, Yolima P; Granados, Sara T; Latorre, Ramón

    2014-01-01

    Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca(2+) concentration, the large conductance voltage- and Ca(2+)-activated K(+) channel (BK) is unique among the superfamily of K(+) channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K(+) channels) and a large C terminus composed of two regulators of K(+) conductance domains (RCK domains), where the Ca(2+)-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified (i.e., β1, β2, β3, and β4) and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca(2+) sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above.

  12. Pharmacological consequences of the coexpression of BK channel α and auxiliary β subunits

    Science.gov (United States)

    Torres, Yolima P.; Granados, Sara T.; Latorre, Ramón

    2014-01-01

    Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca2+ concentration, the large conductance voltage- and Ca2+-activated K+ channel (BK) is unique among the superfamily of K+ channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K+ channels) and a large C terminus composed of two regulators of K+ conductance domains (RCK domains), where the Ca2+-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified (i.e., β1, β2, β3, and β4) and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca2+ sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above. PMID:25346693

  13. Role of Fyn-mediated NMDA receptor function in prediabetic neuropathy in mice

    Science.gov (United States)

    Suo, Meng; Wang, Ping

    2016-01-01

    Diabetic neuropathy is a common complication of diabetes. This study evaluated the role of Fyn kinase and N-methyl-d-aspartate receptors (NMDARs) in the spinal cord in diabetic neuropathy using an animal model of high-fat diet-induced prediabetes. We found that prediabetic wild-type mice exhibited tactile allodynia and thermal hypoalgesia after a 16-wk high-fat diet, relative to normal diet-fed wild-type mice. Furthermore, prediabetic wild-type mice exhibited increased tactile allodynia and thermal hypoalgesia at 24 wk relative to 16 wk. Such phenomena were correlated with increased expression and activation of NR2B subunit of NMDARs, as well as Fyn-NR2B interaction in the spinal cord. Fyn−/− mice developed prediabetes after 16-wk high-fat diet treatment and exhibited thermal hypoalgesia, without showing tactile allodynia or altered expression and activation of NR2B subunit, relative to normal diet-fed Fyn−/− mice. Finally, intrathecal administrations of Ro 25-6981 (selective NR2B subunit-containing NMDAR antagonist) dose-dependently alleviated tactile allodynia, but not thermal hypoalgesia, at 16 and 24 wk in prediabetic wild-type mice. Our results suggested that Fyn-mediated NR2B signaling plays a critical role in regulation of prediabetic neuropathy and that the increased expression/function of NR2B subunit-containing NMDARs may contribute to the progression of neuropathy in type 2 diabetes. PMID:27146985

  14. Overexpression of PLK3 Mediates the Degradation of Abnormal Prion Proteins Dependent on Chaperone-Mediated Autophagy.

    Science.gov (United States)

    Wang, Hui; Tian, Chan; Sun, Jing; Chen, Li-Na; Lv, Yan; Yang, Xiao-Dong; Xiao, Kang; Wang, Jing; Chen, Cao; Shi, Qi; Shao, Qi-Xiang; Dong, Xiao-Ping

    2017-08-01

    Polo-like kinase 3 (PLK3) is the main cause of cell cycle reentry-related neuronal apoptosis which has been implicated in the pathogenesis of prion diseases. Previous work also showed the regulatory activity of exogenous PLK3 on the degradation of PrP (prion protein) mutants and pathogenic PrP Sc ; however, the precise mechanisms remain unknown. In this study, we identified that the overexpression of PLK3-mediated degradation of PrP mutant and PrP Sc was repressed by lysosome rather than by proteasomal and macroautophagy inhibitors. Core components of chaperone-mediated autophagy (CMA) effectors, lysosome-associated membrane protein type 2A (LAMP2a), and heat shock cognate protein 70 (Hsc70) are markedly decreased in the HEK293T cells expressing PrP mutant and scrapie-infected cell line SMB-S15. Meanwhile, PrP mutant showed ability to interact with LAMP2a and Hsc70. Overexpression of PLK3 sufficiently increased the cellular levels of LAMP2a and Hsc70, accompanying with declining the accumulations of PrP mutant and PrP Sc . The kinase domain (KD) of PLK3 was responsible for elevating LAMP2a and Hsc70. Knockdown of endogenous PLK3 enhanced the activity of macroautophagy in the cultured cells. Moreover, time-dependent reductions of LAMP2a and Hsc70 were also observed in the brain tissues of hamster-adapted scrapie agent 263K-infected hamsters, indicating an impairment of CMA during prion infection. Those data indicate that the overexpression of PLK3-mediated degradation of abnormal PrP is largely dependent on CMA pathway.

  15. Reexamination of mood-mediation hypothesis of background-music-dependent effects in free recall.

    Science.gov (United States)

    Isarida, Toshiko K; Kubota, Takayuki; Nakajima, Saki; Isarida, Takeo

    2017-03-01

    The present study reexamined the mood-mediation hypothesis for explaining background-music-dependent effects in free recall. Experiments 1 and 2 respectively examined tempo- and tonality-dependent effects in free recall, which had been used as evidence for the mood-mediation hypothesis. In Experiments 1 and 2, undergraduates (n = 75 per experiment) incidentally learned a list of 20 unrelated words presented one by one at a rate of 5 s per word and then received a 30-s delayed oral free-recall test. Throughout the study and test sessions, a piece of music was played. At the time of test, one third of the participants received the same piece of music with the same tempo or tonality as at study, one third heard a different piece with the same tempo or tonality, and one third heard a different piece with a different tempo or tonality. Note that the condition of the same piece with a different tempo or tonality was excluded. Furthermore, the number of sampled pieces of background music was increased compared with previous studies. The results showed neither tempo- nor tonality-dependent effects, but only a background-music-dependent effect. Experiment 3 (n = 40) compared the effects of background music with a verbal association task and focal music (only listening to musical selections) on the participants' moods. The results showed that both the music tempo and tonality influenced the corresponding mood dimensions (arousal and pleasantness). These results are taken as evidence against the mood-mediation hypothesis. Theoretical implications are discussed.

  16. Mice Lacking the Alpha9 Subunit of the Nicotinic Acetylcholine Receptor Exhibit Deficits in Frequency Difference Limens and Sound Localization

    Directory of Open Access Journals (Sweden)

    Amanda Clause

    2017-06-01

    Full Text Available Sound processing in the cochlea is modulated by cholinergic efferent axons arising from medial olivocochlear neurons in the brainstem. These axons contact outer hair cells in the mature cochlea and inner hair cells during development and activate nicotinic acetylcholine receptors composed of α9 and α10 subunits. The α9 subunit is necessary for mediating the effects of acetylcholine on hair cells as genetic deletion of the α9 subunit results in functional cholinergic de-efferentation of the cochlea. Cholinergic modulation of spontaneous cochlear activity before hearing onset is important for the maturation of central auditory circuits. In α9KO mice, the developmental refinement of inhibitory afferents to the lateral superior olive is disturbed, resulting in decreased tonotopic organization of this sound localization nucleus. In this study, we used behavioral tests to investigate whether the circuit anomalies in α9KO mice correlate with sound localization or sound frequency processing. Using a conditioned lick suppression task to measure sound localization, we found that three out of four α9KO mice showed impaired minimum audible angles. Using a prepulse inhibition of the acoustic startle response paradigm, we found that the ability of α9KO mice to detect sound frequency changes was impaired, whereas their ability to detect sound intensity changes was not. These results demonstrate that cholinergic, nicotinic α9 subunit mediated transmission in the developing cochlear plays an important role in the maturation of hearing.

  17. Cyclic AMP regulation of the human glycoprotein hormone α-subunit gene is mediated by an 18-base-pair element

    International Nuclear Information System (INIS)

    Silver, B.J.; Bokar, J.A.; Virgin, J.B.; Vallen, E.A.; Milsted, A.; Nilson, J.H.

    1987-01-01

    cAMP regulates transcription of the gene encoding the α-subunit of human chorionic gonadotropin (hCG) in the choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, the authors inserted fragments from the 5' flanking region of the α-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between position -146 and -111. In the absence of cAMP, the α-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. They localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element. The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the α-subunit gene

  18. The MDT-15 subunit of mediator interacts with dietary restriction to modulate longevity and fluoranthene toxicity in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Jennifer Schleit

    Full Text Available Dietary restriction (DR, the limitation of calorie intake while maintaining proper nutrition, has been found to extend life span and delay the onset of age-associated disease in a wide range of species. Previous studies have suggested that DR can reduce the lethality of environmental toxins. To further examine the role of DR in toxin response, we measured life spans of the nematode Caenorhabditis elegans treated with the mutagenic polyaromatic hydrocarbon, fluoranthene (FLA. FLA is a direct byproduct of combustion, and is one of U.S. Environmental Protection Agency's sixteen priority environmental toxins. Treatment with 5 µg/ml FLA shortened the life spans of ad libitum fed nematodes, and DR resulted in increased sensitivity to FLA. To determine the role of detoxifying enzymes in the toxicity of FLA, we tested nematodes with mutations in the gene encoding the MDT-15 subunit of mediator, a transcriptional coactivator that regulates genes involved in fatty acid metabolism and detoxification. Mutation of mdt-15 increased the life span of FLA treated animals compared to wild-type animals with no difference observed between DR and ad libitum fed mdt-15 animals. We also examined mutants with altered insulin-IGF-1-like signaling (IIS, which is known to modulate life span and stress resistance in C. elegans independently of DR. Mutation of the genes coding for the insulin-like receptor DAF-2 or the FOXO-family transcription factor DAF16 did not alter the animals' susceptibility to FLA compared to wild type. Taken together, our results suggest that certain compounds have increased toxicity when combined with a DR regimen through increased metabolic activation. This increased metabolic activation appears to be mediated through the MDT-15 transcription factor and is independent of the IIS pathway.

  19. Behavioural endophenotypes in mice lacking the auxiliary GABAB receptor subunit KCTD16.

    Science.gov (United States)

    Cathomas, Flurin; Sigrist, Hannes; Schmid, Luca; Seifritz, Erich; Gassmann, Martin; Bettler, Bernhard; Pryce, Christopher R

    2017-01-15

    Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain and is implicated in the pathophysiology of a number of neuropsychiatric disorders. The GABA B receptors are G-protein coupled receptors consisting of principle subunits and auxiliary potassium channel tetramerization domain (KCTD) subunits. The KCTD subunits 8, 12, 12b and 16 are cytosolic proteins that determine the kinetics of the GABA B receptor response. Previously, we demonstrated that Kctd12 null mutant mice (Kctd12 -/- ) exhibit increased auditory fear learning and that Kctd12 +/- mice show altered circadian activity, as well as increased intrinsic excitability in hippocampal pyramidal neurons. KCTD16 has been demonstrated to influence neuronal excitability by regulating GABA B receptor-mediated gating of postsynaptic ion channels. In the present study we investigated for behavioural endophenotypes in Kctd16 -/- and Kctd16 +/- mice. Compared with wild-type (WT) littermates, auditory and contextual fear conditioning were normal in both Kctd16 -/- and Kctd16 +/- mice. When fear memory was tested on the following day, Kctd16 -/- mice exhibited less extinction of auditory fear memory relative to WT and Kctd16 +/- mice, as well as more contextual fear memory relative to WT and, in particular, Kctd16 +/- mice. Relative to WT, both Kctd16 +/- and Kctd16 -/- mice exhibited normal circadian activity. This study adds to the evidence that auxillary KCTD subunits of GABA B receptors contribute to the regulation of behaviours that could constitute endophenotypes for hyper-reactivity to aversive stimuli in neuropsychiatric disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Structures of transcription pre-initiation complex with TFIIH and Mediator.

    Science.gov (United States)

    Schilbach, S; Hantsche, M; Tegunov, D; Dienemann, C; Wigge, C; Urlaub, H; Cramer, P

    2017-11-09

    For the initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC-core Mediator complex at nominal resolutions of 4.7 Å and 5.8 Å, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively. The TFIIH core subunit Ssl2 (a homologue of human XPB) is positioned on downstream DNA by the 'E-bridge' helix in TFIIE, consistent with TFIIE-stimulated DNA opening. The TFIIH kinase module subunit Tfb3 (MAT1 in human) anchors the kinase Kin28 (CDK7), which is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC-core Mediator complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain of Pol II.

  1. The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis

    DEFF Research Database (Denmark)

    Yde, C W; Olsen, B B; Meek, D

    2008-01-01

    25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory beta-subunit of protein kinase CK2 by RNA...

  2. Identification of a GTP-binding protein α subunit that lacks an apparent ADP-ribosylation site for pertussis toxin

    International Nuclear Information System (INIS)

    Fong, H.K.W.; Yoshimoto, K.K.; Eversole-Cire, P.; Simon, M.I.

    1988-01-01

    Recent molecular cloning of cDNA for the α subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called T/sub r/ and T/sub c/, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, the authors have identified a G-protein α subunit, which they refer to as G/sub z/α, by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine T/sub r/ α-subunit cDNA. The deduced amino acid sequence of G/sub z/α is 41-67% identical with those of other known G-protein α subunits. However, the 355-residue G/sub z/α lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein α subunits. They suggest that G/sub z/α, which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems

  3. Ethanol activation of protein kinase A regulates GABA-A receptor subunit expression in the cerebral cortex and contributes to ethanol-induced hypnosis

    Directory of Open Access Journals (Sweden)

    Sandeep eKumar

    2012-04-01

    Full Text Available Protein kinases are implicated in neuronal cell functions such as modulation of ion channel function, trafficking and synaptic excitability. Both protein kinase C (PKC and A (PKA are involved in regulation of γ-aminobutyric acid type A (GABA-A receptors through phosphorylation. However, the role of PKA in regulating GABA-A receptors following acute ethanol exposure is not known. The present study investigated the role of PKA in ethanol effects on GABA-A receptor α1 subunit expression in the P2 synaptosomal fraction of the rat cerebral cortex. Additionally, GABA-related behaviors were also examined. Rats were administered ethanol (2.0 – 3.5 g/kg or saline and PKC, PKA and GABA-A receptor α1 subunit levels were measured by Western blot analysis. Ethanol (3.5 g/kg transiently increased GABA-A receptor α1 subunit expression and PKA RIIβ subunit expression at similar time points whereas PKA RIIα was increased at later time points. In contrast, PKC isoform expression remained unchanged. Notably, the moderate ethanol dose (2.0g/kg had no effect on GABA-A α1 subunit levels although PKA RIIα and RIIβ were increased at 10 and 60 minutes, when PKC isozymes are also known to be elevated. To determine if PKA activation was responsible for the ethanol-induced elevation of GABA-A α1 subunits, the PKA antagonist H89 was administered to rats prior to ethanol exposure. H89 administration prevented ethanol-induced increases in GABA-A receptor α1 subunit expression. Moreover, increasing PKA activity intracerebroventricularly with Sp-cAMP prior to a hypnotic dose of ethanol increased ethanol-induced loss of righting reflex duration. This effect appears to be mediated in part by GABA-A receptors as increasing PKA activity also increased the duration of muscimol-induced loss of righting reflex. Overall these data suggest that PKA mediates ethanol-induced GABA-A receptor expression and contributes to ethanol behavioral effects involving GABA-A receptors.

  4. Fragile X Mental Retardation Protein and Dendritic Local Translation of the Alpha Subunit of the Calcium/Calmodulin-Dependent Kinase II Messenger RNA Are Required for the Structural Plasticity Underlying Olfactory Learning.

    Science.gov (United States)

    Daroles, Laura; Gribaudo, Simona; Doulazmi, Mohamed; Scotto-Lomassese, Sophie; Dubacq, Caroline; Mandairon, Nathalie; Greer, Charles August; Didier, Anne; Trembleau, Alain; Caillé, Isabelle

    2016-07-15

    In the adult brain, structural plasticity allowing gain or loss of synapses remodels circuits to support learning. In fragile X syndrome, the absence of fragile X mental retardation protein (FMRP) leads to defects in plasticity and learning deficits. FMRP is a master regulator of local translation but its implication in learning-induced structural plasticity is unknown. Using an olfactory learning task requiring adult-born olfactory bulb neurons and cell-specific ablation of FMRP, we investigated whether learning shapes adult-born neuron morphology during their synaptic integration and its dependence on FMRP. We used alpha subunit of the calcium/calmodulin-dependent kinase II (αCaMKII) mutant mice with altered dendritic localization of αCaMKII messenger RNA, as well as a reporter of αCaMKII local translation to investigate the role of this FMRP messenger RNA target in learning-dependent structural plasticity. Learning induces profound changes in dendritic architecture and spine morphology of adult-born neurons that are prevented by ablation of FMRP in adult-born neurons and rescued by an metabotropic glutamate receptor 5 antagonist. Moreover, dendritically translated αCaMKII is necessary for learning and associated structural modifications and learning triggers an FMRP-dependent increase of αCaMKII dendritic translation in adult-born neurons. Our results strongly suggest that FMRP mediates structural plasticity of olfactory bulb adult-born neurons to support olfactory learning through αCaMKII local translation. This reveals a new role for FMRP-regulated dendritic local translation in learning-induced structural plasticity. This might be of clinical relevance for the understanding of critical periods disruption in autism spectrum disorder patients, among which fragile X syndrome is the primary monogenic cause. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  5. Positive modulation of delta-subunit containing GABAA receptors in mouse neurons

    DEFF Research Database (Denmark)

    Vardya, Irina; Hoestgaard-Jensen, Kirsten; Nieto-Gonzalez, Jose Luis

    2012-01-01

    δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA(A) recep......δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA......(A) receptors in mouse neurons in vitro and in vivo. Whole-cell patch-clamp recordings were carried out in the dentate gyrus in mouse brain slices. In granule cells, AA29504 (1 μM) caused a 4.2-fold potentiation of a tonic current induced by THIP (1 μM), while interneurons showed a potentiation of 2.6-fold......-free environment using Ca²⁺ imaging in cultured neurons, AA29504 showed GABA(A) receptor agonism in the absence of agonist. Finally, AA29504 exerted dose-dependent stress-reducing and anxiolytic effects in mice in vivo. We propose that AA29504 potentiates δ-containing GABA(A) receptors to enhance tonic inhibition...

  6. Decreased phosphorylation of δ and ε subunits of the acetylcholine receptor coincides with delayed postsynaptic maturation in PKC θ deficient mouse.

    Science.gov (United States)

    Lanuza, Maria A; Besalduch, Núria; González, Carmen; Santafé, Manel M; Garcia, Neus; Tomàs, Marta; Nelson, Phillip G; Tomàs, Josep

    2010-09-01

    Protein kinase C (PKC) activity is involved in the nicotinic acetylcholine receptor (nAChR) redistribution at the neuromuscular junction in vivo during postnatal maturation. Here we studied, in PKC theta (PKCtheta) deficient mice (KO), how the theta isoform of PKC is involved in the nAChR cluster maturation that is accompanied by the developmental activity-dependent neuromuscular synapse elimination process. We found that axonal elimination and dispersion of nAChR from the postsynaptic plaques and its redistribution to form the mature postsynaptic apparatus were delayed but not totally suppressed in PKCtheta deficient mice. Moreover, the delay in the maturation of the morphology of the nAChR clusters during the early postnatal synapse elimination period in the PKCtheta deficient mice coincides with a reduction in the PKCtheta-mediated phosphorylation on the delta subunit of the nAChR. In addition, we show evidence for PKCtheta regulation of PKA in normally phosphorylating the epsilon subunit of nAChR. We have also found that the theta isoform of PKC is located on the postsynaptic component of the neuromuscular junction but is also expressed by motoneurons in the spinal cord and in the motor nerve terminals. The results allow us to hypothesize that a spatially specific and opposing action of PKCtheta and PKA may result in activity-dependent alterations to synaptic connectivity at both the nerve inputs and the postsynaptic nAChR clusters. Copyright 2010 Elsevier Inc. All rights reserved.

  7. IGF-1-dependent subunit communication of the IGF-1 holoreceptor: Interactions between αβ heterodimeric receptor halves

    International Nuclear Information System (INIS)

    Wilden, P.A.; Treadway, J.L.; Morrison, B.D.; Pessin, J.E.

    1989-01-01

    Examination of 125 I-IGF-1 affinity cross-linking and β-subunit autophosphorylation has indicated that IGF-1 induces a covalent association of isolated αβ heterodimeric IGF-1 receptors into an α 2 β 2 heterotetrameric state, in a similar manner to that observed for the insulin receptor. The formation of the α 2 β 2 heterotetrameric IGF-1 receptor complex from the partially purified αβ heterodimers was time dependent with half-maximal formation in approximately 30 min at saturating IGF-1 concentrations. The IGF-1-dependent association of the partially purified αβ heterodimers into an α 2 β 2 heterotetrameric state was specific for the IGF-1 receptors since IGF-1 was unable to stimulate the protein kinase activity of the purified αβ heterodimeric insulin receptor complex. Incubation of the α 2 β 2 heterotetrameric IGF-1 holoreceptor with the specific sulfhydryl agent iodoacetamide (IAN) did not alter 125 I-IGF-1 binding or IGF-1 stimulation of protein kinase activity. However, IAN treatment of the αβ heterodimeric IGF-1 receptors inhibited the IGF-1 dependent covalent formation of the disulfide-linked α 2 β 2 heterotetrameric complex. These data indicate that IGF-1 induces the covalent association of isolated αβ heterodimeric IGF-1 receptor complexes into a disulfide-linked α 2 β 2 heterotetrameric state whereas Mn/MgATP induces a noncovalent association. Therefore, unlike the insulin receptor in which noncovalent association is sufficient for kinase activation, only the covalent assembly of the IGF-1 receptor αβ heterodimers into the α 2 β 2 heterotetrameric holoreceptor complex is associated with ligand-stimulated protein kinase activation

  8. The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin

    DEFF Research Database (Denmark)

    Mukherjee, Jayanta; Kretschmannova, Karla; Gouzer, Geraldine

    2011-01-01

    The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABA(A)Rs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we....... Mutating residues 360-375 decreases both the accumulation of α1-containing GABA(A)Rs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site....... Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit...

  9. The role of HSP70 in mediating age-dependent mortality in sepsis

    Science.gov (United States)

    McConnell, Kevin W.; Fox, Amy C.; Clark, Andrew T.; Chang, Nai-Yuan Nicholas; Dominguez, Jessica A.; Farris, Alton B.; Buchman, Timothy G.; Hunt, Clayton R.; Coopersmith, Craig M.

    2011-01-01

    Sepsis is primarily a disease of the aged, with increased incidence and mortality occurring in aged hosts. Heat shock protein (HSP) 70 plays an important role in both healthy aging and the stress response to injury. The purpose of this study was to determine the role of HSP70 in mediating mortality and the host inflammatory response in aged septic hosts. Sepsis was induced in both young (6–12week old) and aged (16–17 month old) HSP70−/− and wild type (WT) mice to determine if HSP70 modulated outcome in an age-dependent fashion. Young HSP70−/− and WT mice subjected to cecal ligation and puncture (CLP), Pseudomonas aeruginosa pneumonia or Streptococcus pneumoniae pneumonia had no differences in mortality, suggesting HSP70 does not mediate survival in young septic hosts. In contrast, mortality was higher in aged HSP70−/− mice than aged WT mice subjected to CLP (p=0.01), suggesting HSP70 mediates mortality in sepsis in an age-dependent fashion. Compared to WT mice, aged septic HSP70−/− mice had increased gut epithelial apoptosis and pulmonary inflammation. In addition, HSP70−/−mice had increased systemic levels of TNF-α, IL-6, IL-10 and IL-1β compared to WT mice. These data demonstrate that HSP70 is a key determinant of mortality in aged but not young hosts in sepsis. HSP70 may play a protective role in an age-dependent response to sepsis by preventing excessive gut apoptosis and both pulmonary and systemic inflammation. PMID:21296977

  10. Dose- and time-dependence of the host-mediated response to paclitaxel therapy: a mathematical modeling approach.

    Science.gov (United States)

    Benguigui, Madeleine; Alishekevitz, Dror; Timaner, Michael; Shechter, Dvir; Raviv, Ziv; Benzekry, Sebastien; Shaked, Yuval

    2018-01-05

    It has recently been suggested that pro-tumorigenic host-mediated processes induced in response to chemotherapy counteract the anti-tumor activity of therapy, and thereby decrease net therapeutic outcome. Here we use experimental data to formulate a mathematical model describing the host response to different doses of paclitaxel (PTX) chemotherapy as well as the duration of the response. Three previously described host-mediated effects are used as readouts for the host response to therapy. These include the levels of circulating endothelial progenitor cells in peripheral blood and the effect of plasma derived from PTX-treated mice on migratory and invasive properties of tumor cells in vitro . A first set of mathematical models, based on basic principles of pharmacokinetics/pharmacodynamics, did not appropriately describe the dose-dependence and duration of the host response regarding the effects on invasion. We therefore provide an alternative mathematical model with a dose-dependent threshold, instead of a concentration-dependent one, that describes better the data. This model is integrated into a global model defining all three host-mediated effects. It not only precisely describes the data, but also correctly predicts host-mediated effects at different doses as well as the duration of the host response. This mathematical model may serve as a tool to predict the host response to chemotherapy in cancer patients, and therefore may be used to design chemotherapy regimens with improved therapeutic outcome by minimizing host mediated effects.

  11. SGLT1-mediated transport in Caco-2 cells is highly dependent on cell bank origin

    DEFF Research Database (Denmark)

    Steffansen, B; Pedersen, Maria; Laghmoch, A M

    2017-01-01

    The Caco-2 cell line is a well-established in vitro model for studying transport phenomena for prediction of intestinal nutrient and drug absorption. However, for substances depending on transporters such predictions are complicated due to variable transporter expression and limited knowledge about...... transporter function during multiple cell passaging and cell thawings. In the case of SGLT1, a key transporter of oral absorption of D-glucose, one reason for compromised prediction could be inadequate expression of SGLT1 in Caco-2 cells and thereby limited sensitivity in the determination of SGLT1-mediated...... permeability (PSGLT1). Here, the objective was to characterize and compare SGLT1-mediated uptake in Caco-2 cells obtained from different cell banks. SGLT1-mediated uptake of the standard SGLT1 substrate, α-MDG, in Caco-2 cells was shown to be highly dependent on cell bank origin. The most robust and reliable...

  12. Piroxicam inhibits NMDA receptor-mediated excitotoxicity through allosteric inhibition of the GluN2B subunit: an in silico study elucidating a novel mechanism of action of the drug.

    Science.gov (United States)

    Mazumder, Muhammed Khairujjaman; Borah, Anupom

    2014-12-01

    Hyperactivation of GluN2B subunit containing N-methyl-d-aspartate receptors (NMDARs) significantly contributes to the development of several neurodegenerative diseases through a process called excitotoxicity. NMDARs are voltage-gated Ca2+ channels which when activated lead to excessive influx of Ca2+ into neurons thereby exacerbating several calcium-dependent pathways that cause oxidative stress and apoptosis. Several drugs are presently in use to counter the NMDAR-mediated excitotoxic events among which Ifenprodil and its derivatives are GluN2B selective allosteric antagonists. Certain non-steroidal anti-inflammatory drugs (NSAIDs) have also been reported to inhibit NMDARs and the resultant pathologies. Meanwhile, Piroxicam, which is a NSAID, has been reported to be protective in cerebral ischemia-induced neurodegeneration through various pathways. Since Piroxicam has more number of interacting groups as compared to other NSAIDs and also has structural similarities with Ifenprodil, we thought it prudent that Piroxicam may inhibit NMDARs similar to Ifenprodil. By using molecular docking as a tool, we validated the hypothesis and hereby report for the first time that Piroxicam can inhibit GluN2B containing NMDARs through allosteric mode similar to the well known selective antagonist--Ifenprodil; and thus can be a therapeutic drug for the prevention of excitotoxic neurodegeneration. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. The wavelength dependence of gold nanorod-mediated optical breakdown during infrared ultrashort pulses

    Energy Technology Data Exchange (ETDEWEB)

    Davletshin, Yevgeniy R.; Kumaradas, J. Carl [Department of Physics, Ryerson University, Toronto, ON (Canada)

    2017-04-15

    This paper investigates the wavelength dependence of the threshold of gold nanorod-mediated optical breakdown during picosecond and femtosecond near infrared optical pulses. It was found that the wavelength dependence in the picosecond regime is governed solely by the changes of a nanorod's optical properties. On the other hand, the optical breakdown threshold during femtosecond pulse exposure falls within one of two regimes. When the ratio of the maximum electric field from the outside to the inside of the nanorod is less then 7 (the absorption regime) the seed electrons are initiated by photo-thermal emission, and the wavelength dependence in the threshold of optical breakdown is the result of optical properties of the nanoparticle. When the ratio is greater than 7 (the near-field regime) more seed electrons are initiated by multiphoton ionization, and the wavelength dependence of the threshold of optical breakdown results from a combination of nanorod's optical properties and transitions in the order of multiphoton ionization. The findings of this study can guide the design of nanoparticle based optical breakdown applications. This analysis also deepens the understanding of nanoparticle-mediated laser induced breakdown for picosecond and femtosecond pulses at near infrared wavelengths. (copyright 2017 by WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  14. The Mediator subunit MED23 couples H2B mono-ubiquitination to transcriptional control and cell fate determination.

    Science.gov (United States)

    Yao, Xiao; Tang, Zhanyun; Fu, Xing; Yin, Jingwen; Liang, Yan; Li, Chonghui; Li, Huayun; Tian, Qing; Roeder, Robert G; Wang, Gang

    2015-12-02

    The Mediator complex orchestrates multiple transcription factors with the Pol II apparatus for precise transcriptional control. However, its interplay with the surrounding chromatin remains poorly understood. Here, we analyze differential histone modifications between WT and MED23(-/-) (KO) cells and identify H2B mono-ubiquitination at lysine 120 (H2Bub) as a MED23-dependent histone modification. Using tandem affinity purification and mass spectrometry, we find that MED23 associates with the RNF20/40 complex, the enzyme for H2Bub, and show that this association is critical for the recruitment of RNF20/40 to chromatin. In a cell-free system, Mediator directly and substantially increases H2Bub on recombinant chromatin through its cooperation with RNF20/40 and the PAF complex. Integrative genome-wide analyses show that MED23 depletion specifically reduces H2Bub on a subset of MED23-controlled genes. Importantly, MED23-coupled H2Bub levels are oppositely regulated during myogenesis and lung carcinogenesis. In sum, these results establish a mechanistic link between the Mediator complex and a critical chromatin modification in coordinating transcription with cell growth and differentiation. © 2015 The Authors.

  15. Genome-wide occupancy profile of mediator and the Srb8-11 module reveals interactions with coding regions

    DEFF Research Database (Denmark)

    Zhu, Xuefeng; Wirén, Marianna; Sinha, Indranil

    2006-01-01

    Mediator exists in a free form containing the Med12, Med13, CDK8, and CycC subunits (the Srb8-11 module) and a smaller form, which lacks these four subunits and associates with RNA polymerase II (Pol II), forming a holoenzyme. We use chromatin immunoprecipitation (ChIP) and DNA microarrays...... to investigate genome-wide localization of Mediator and the Srb8-11 module in fission yeast. Mediator and the Srb8-11 module display similar binding patterns, and interactions with promoters and upstream activating sequences correlate with increased transcription activity. Unexpectedly, Mediator also interacts...... with the downstream coding region of many genes. These interactions display a negative bias for positions closer to the 5' ends of open reading frames (ORFs) and appear functionally important, because downregulation of transcription in a temperature-sensitive med17 mutant strain correlates with increased Mediator...

  16. Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

    Science.gov (United States)

    Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

    1999-03-05

    Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

  17. Acetylcholine Receptor: Complex of Homologous Subunits

    Science.gov (United States)

    Raftery, Michael A.; Hunkapiller, Michael W.; Strader, Catherine D.; Hood, Leroy E.

    1980-06-01

    The acetylcholine receptor from the electric ray Torpedo californica is composed of five subunits; two are identical and the other three are structurally related to them. Microsequence analysis of the four polypeptides demonstrates amino acid homology among the subunits. Further sequence analysis of both membrane-bound and Triton-solubilized, chromatographically purified receptor gave the stoichiometry of the four subunits (40,000:50,000:60,000:65,000 daltons) as 2:1:1:1, indicating that this protein is a pentameric complex with a molecular weight of 255,000 daltons. Genealogical analysis suggests that divergence from a common ancestral gene occurred early in the evolution of the receptor. This shared ancestry argues that each of the four subunits plays a functional role in the receptor's physiological action.

  18. Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase {alpha} subunit.

    Science.gov (United States)

    Meier, Susan; Tavraz, Neslihan N; Dürr, Katharina L; Friedrich, Thomas

    2010-02-01

    The Na(+)/K(+)-ATPase mediates electrogenic transport by exporting three Na(+) ions in exchange for two K(+) ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb(+) ions in the crystal structures of the Na(+)/K(+)-ATPase has defined two "common" cation binding sites, I and II, which accommodate Na(+) or K(+) ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this "unique" Na(+) binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na(+)/K(+)-ATPase alpha(2) subunit decreases the affinity for extracellular and intracellular Na(+), in agreement with previous biochemical studies. Apparently, the DeltaYY deletion changes Na(+) affinity at site III but leaves the common sites unaffected, whereas the more extensive DeltaKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K(+), the DeltaYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na(+) dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na(+)/Na(+) exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na(+) release/binding mechanism. In analogy to the mechanism proposed for the H(+) leak currents of the wild-type Na(+)/K(+)-ATPase, we suggest that the DeltaYY deletion lowers the energy barrier for the intracellular Na(+) occlusion reaction, thus destabilizing the Na(+)-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the

  19. Candida albicans Swi/Snf and Mediator Complexes Differentially Regulate Mrr1-Induced MDR1 Expression and Fluconazole Resistance.

    Science.gov (United States)

    Liu, Zhongle; Myers, Lawrence C

    2017-11-01

    Long-term azole treatment of patients with chronic Candida albicans infections can lead to drug resistance. Gain-of-function (GOF) mutations in the transcription factor Mrr1 and the consequent transcriptional activation of MDR1 , a drug efflux coding gene, is a common pathway by which this human fungal pathogen acquires fluconazole resistance. This work elucidates the previously unknown downstream transcription mechanisms utilized by hyperactive Mrr1. We identified the Swi/Snf chromatin remodeling complex as a key coactivator for Mrr1, which is required to maintain basal and induced open chromatin, and Mrr1 occupancy, at the MDR1 promoter. Deletion of snf2 , the catalytic subunit of Swi/Snf, largely abrogates the increases in MDR1 expression and fluconazole MIC observed in MRR1 GOF mutant strains. Mediator positively and negatively regulates key Mrr1 target promoters. Deletion of the Mediator tail module med3 subunit reduces, but does not eliminate, the increased MDR1 expression and fluconazole MIC conferred by MRR1 GOF mutations. Eliminating the kinase activity of the Mediator Ssn3 subunit suppresses the decreased MDR1 expression and fluconazole MIC of the snf2 null mutation in MRR1 GOF strains. Ssn3 deletion also suppresses MDR1 promoter histone displacement defects in snf2 null mutants. The combination of this work with studies on other hyperactive zinc cluster transcription factors that confer azole resistance in fungal pathogens reveals a complex picture where the induction of drug efflux pump expression requires the coordination of multiple coactivators. The observed variations in transcription factor and target promoter dependence of this process may make the search for azole sensitivity-restoring small molecules more complicated. Copyright © 2017 American Society for Microbiology.

  20. Inhibiting fungal multidrug resistance by disrupting an activator-Mediator interaction.

    Science.gov (United States)

    Nishikawa, Joy L; Boeszoermenyi, Andras; Vale-Silva, Luis A; Torelli, Riccardo; Posteraro, Brunella; Sohn, Yoo-Jin; Ji, Fei; Gelev, Vladimir; Sanglard, Dominique; Sanguinetti, Maurizio; Sadreyev, Ruslan I; Mukherjee, Goutam; Bhyravabhotla, Jayaram; Buhrlage, Sara J; Gray, Nathanael S; Wagner, Gerhard; Näär, Anders M; Arthanari, Haribabu

    2016-02-25

    Eukaryotic transcription activators stimulate the expression of specific sets of target genes through recruitment of co-activators such as the RNA polymerase II-interacting Mediator complex. Aberrant function of transcription activators has been implicated in several diseases. However, therapeutic targeting efforts have been hampered by a lack of detailed molecular knowledge of the mechanisms of gene activation by disease-associated transcription activators. We previously identified an activator-targeted three-helix bundle KIX domain in the human MED15 Mediator subunit that is structurally conserved in Gal11/Med15 Mediator subunits in fungi. The Gal11/Med15 KIX domain engages pleiotropic drug resistance transcription factor (Pdr1) orthologues, which are key regulators of the multidrug resistance pathway in Saccharomyces cerevisiae and in the clinically important human pathogen Candida glabrata. The prevalence of C. glabrata is rising, partly owing to its low intrinsic susceptibility to azoles, the most widely used antifungal agent. Drug-resistant clinical isolates of C. glabrata most commonly contain point mutations in Pdr1 that render it constitutively active, suggesting that this transcriptional activation pathway represents a linchpin in C. glabrata multidrug resistance. Here we perform sequential biochemical and in vivo high-throughput screens to identify small-molecule inhibitors of the interaction of the C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation and re-sensitizes drug-resistant C. glabrata to azole antifungals in vitro and in animal models for disseminated and urinary tract C. glabrata infection. Determining the NMR structure of the C. glabrata Gal11A KIX domain provides a detailed understanding of the molecular mechanism of Pdr1 gene activation and multidrug resistance inhibition by iKIX1. We have demonstrated the feasibility of small-molecule targeting of a

  1. NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

    International Nuclear Information System (INIS)

    Liu Qing; He Xiaoqing; Liu Yongsheng; Du Bingbing; Wang Xiaoyan; Zhang Weisheng; Jia Pengfei; Dong Jingmei; Ma Jianxiu; Wang Xiaohu; Li Sha; Zhang Hong

    2008-01-01

    Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane subunit gp91 phox was dose-dependent. Meanwhile, the cytoplasmic subunit p47 phox was translocated to the cell membrane and localized with p22 phox and gp91 phox to form reactive NADPH oxidase. Our data suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.

  2. Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion

    International Nuclear Information System (INIS)

    Nishikado, Hideto; Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko; Ogawa, Hideoki; Okumura, Ko; Takai, Toshiro

    2015-01-01

    Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils. - Highlights: • We identified the murine IL3R as a novel target of papain-family cysteine proteases. • Papain-family cysteine proteases cleaved IL3Rα/CD123 on murine basophils. • Papain suppressed IL3- but not TSLP-induced expansion of murine basophils. • The inactivation of IL3R might be a strategy for pathogens to suppress host immunity

  3. Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion

    Energy Technology Data Exchange (ETDEWEB)

    Nishikado, Hideto [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko [Laboratory of Proteomics and Biomolecular Science, BioMedical Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Ogawa, Hideoki; Okumura, Ko [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Takai, Toshiro, E-mail: t-takai@juntendo.ac.jp [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan)

    2015-05-01

    Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils. - Highlights: • We identified the murine IL3R as a novel target of papain-family cysteine proteases. • Papain-family cysteine proteases cleaved IL3Rα/CD123 on murine basophils. • Papain suppressed IL3- but not TSLP-induced expansion of murine basophils. • The inactivation of IL3R might be a strategy for pathogens to suppress host immunity.

  4. Identification of novel transcriptional regulators of PKA subunits in Saccharomyces cerevisiae by quantitative promoter-reporter screening.

    Science.gov (United States)

    Pautasso, Constanza; Reca, Sol; Chatfield-Reed, Kate; Chua, Gordon; Galello, Fiorella; Portela, Paula; Zaremberg, Vanina; Rossi, Silvia

    2016-08-01

    The cAMP-dependent protein kinase (PKA) signaling is a broad pathway that plays important roles in the transduction of environmental signals triggering precise physiological responses. However, how PKA achieves the cAMP-signal transduction specificity is still in study. The regulation of expression of subunits of PKA should contribute to the signal specificity. Saccharomyces cerevisiae PKA holoenzyme contains two catalytic subunits encoded by TPK1, TPK2 and TPK3 genes, and two regulatory subunits encoded by BCY1 gene. We studied the activity of these gene promoters using a fluorescent reporter synthetic genetic array screen, with the goal of systematically identifying novel regulators of expression of PKA subunits. Gene ontology analysis of the identified modulators showed enrichment not only in the category of transcriptional regulators, but also in less expected categories such as lipid and phosphate metabolism. Inositol, choline and phosphate were identified as novel upstream signals that regulate transcription of PKA subunit genes. The results support the role of transcription regulation of PKA subunits in cAMP specificity signaling. Interestingly, known targets of PKA phosphorylation are associated with the identified pathways opening the possibility of a reciprocal regulation. PKA would be coordinating different metabolic pathways and these processes would in turn regulate expression of the kinase subunits. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Ferroxidase-Mediated Iron Oxide Biomineralization

    DEFF Research Database (Denmark)

    Zeth, Kornelius; Hoiczyk, Egbert; Okuda, Mitsuhiro

    2016-01-01

    Iron oxide biomineralization occurs in all living organisms and typically involves protein compartments ranging from 5 to 100nm in size. The smallest iron-oxo particles are formed inside dodecameric Dps protein cages, while the structurally related ferritin compartments consist of twice as many......, translocation, oxidation, nucleation, and storage, that are mediated by ferroxidase centers. Thus, compartmentalized iron oxide biomineralization yields uniform nanoparticles strictly determined by the sizes of the compartments, allowing customization for highly diverse nanotechnological applications....... identical protein subunits. The largest known compartments are encapsulins, icosahedra made of up to 180 protein subunits that harbor additional ferritin-like proteins in their interior. The formation of iron-oxo particles in all these compartments requires a series of steps including recruitment of iron...

  6. A risk allele for nicotine dependence in CHRNA5 is a protective allele for cocaine dependence.

    Science.gov (United States)

    Grucza, Richard A; Wang, Jen C; Stitzel, Jerry A; Hinrichs, Anthony L; Saccone, Scott F; Saccone, Nancy L; Bucholz, Kathleen K; Cloninger, C Robert; Neuman, Rosalind J; Budde, John P; Fox, Louis; Bertelsen, Sarah; Kramer, John; Hesselbrock, Victor; Tischfield, Jay; Nurnberger, John I; Almasy, Laura; Porjesz, Bernice; Kuperman, Samuel; Schuckit, Marc A; Edenberg, Howard J; Rice, John P; Goate, Alison M; Bierut, Laura J

    2008-12-01

    A nonsynonymous coding polymorphism, rs16969968, of the CHRNA5 gene that encodes the alpha-5 subunit of the nicotinic acetylcholine receptor (nAChR) has been found to be associated with nicotine dependence. The goal of this study was to examine the association of this variant with cocaine dependence. Genetic association analysis was performed in two independent samples of unrelated case and control subjects: 1) 504 European Americans participating in the Family Study on Cocaine Dependence (FSCD) and 2) 814 European Americans participating in the Collaborative Study on the Genetics of Alcoholism (COGA). In the FSCD, there was a significant association between the CHRNA5 variant and cocaine dependence (odds ratio = .67 per allele, p = .0045, assuming an additive genetic model), but in the reverse direction compared with that previously observed for nicotine dependence. In multivariate analyses that controlled for the effects of nicotine dependence, both the protective effect for cocaine dependence and the previously documented risk effect for nicotine dependence were statistically significant. The protective effect for cocaine dependence was replicated in the COGA sample. In COGA, effect sizes for habitual smoking, a proxy phenotype for nicotine dependence, were consistent with those observed in FSCD. The minor (A) allele of rs16969968, relative to the major G allele, appears to be both a risk factor for nicotine dependence and a protective factor for cocaine dependence. The biological plausibility of such a bidirectional association stems from the involvement of nAChRs with both excitatory and inhibitory modulation of dopamine-mediated reward pathways.

  7. Characterization of cyclin-dependent kinases and Cdc2/Cdc28 kinase subunits in Trichomonas vaginalis.

    Science.gov (United States)

    Amador, Erick; López-Pacheco, Karla; Morales, Nataly; Coria, Roberto; López-Villaseñor, Imelda

    2017-04-01

    Cyclin-dependent kinases (CDKs) have important roles in regulating key checkpoints between stages of the cell cycle. Their activity is tightly regulated through a variety of mechanisms, including through binding with cyclin proteins and the Cdc2/Cdc28 kinase subunit (CKS), and their phosphorylation at specific amino acids. Studies of the components involved in cell cycle control in parasitic protozoa are limited. Trichomonas vaginalis is the causative agent of trichomoniasis in humans and is therefore important in public health; however, some of the basic biological processes used by this organism have not been defined. Here, we characterized proteins potentially involved in cell cycle regulation in T. vaginalis. Three genes encoding protein kinases were identified in the T. vaginalis genome, and the corresponding recombinant proteins (TvCRK1, TvCRK2, TvCRK5) were studied. These proteins displayed similar sequence features to CDKs. Two genes encoding CKSs were also identified, and the corresponding recombinant proteins were found to interact with TvCRK1 and TvCRK2 by a yeast two-hybrid system. One putative cyclin B protein from T. vaginalis was found to bind to and activate the kinase activities of TvCRK1 and TvCRK5, but not TvCRK2. This work is the first characterization of proteins involved in cell cycle control in T. vaginalis.

  8. Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking

    International Nuclear Information System (INIS)

    Barrington, W.W.; Jacobson, K.A.; Hutchison, A.J.; Williams, M.; Stiles, G.L.

    1989-01-01

    A high-affinity iodinated agonist radioligand for the A2 adenosine receptor has been synthesized to facilitate studies of the A2 adenosine receptor binding subunit. The radioligand 125I-labeled PAPA-APEC (125I-labeled 2-[4-(2-[2-[(4- aminophenyl)methylcarbonylamino]ethylaminocarbonyl]- ethyl)phenyl]ethylamino-5'-N-ethylcarboxamidoadenosine) was synthesized and found to bind to the A2 adenosine receptor in bovine striatal membranes with high affinity (Kd = 1.5 nM) and A2 receptor selectivity. Competitive binding studies reveal the appropriate A2 receptor pharmacologic potency order with 5'-N-ethylcarboxamidoadenosine (NECA) greater than (-)-N6-[(R)-1-methyl- 2-phenylethyl]adenosine (R-PIA) greater than (+)-N6-[(S)-1-methyl-2- phenylethyl]adenosine (S-PIA). Adenylate cyclase assays, in human platelet membranes, demonstrate a dose-dependent stimulation of cAMP production. PAPA-APEC (1 microM) produces a 43% increase in cAMP production, which is essentially the same degree of increase produced by 5'-N- ethylcarboxamidoadenosine (the prototypic A2 receptor agonist). These findings combined with the observed guanine nucleotide-mediated decrease in binding suggest that PAPA-APEC is a full A2 agonist. The A2 receptor binding subunit was identified by photoaffinity-crosslinking studies using 125I-labeled PAPA-APEC and the heterobifunctional crosslinking agent N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH). After covalent incorporation, a single specifically radiolabeled protein with an apparent molecular mass of 45 kDa was observed on NaDodSO4/PAGE/autoradiography. Incorporation of 125I-labeled PAPA-APEC into this polypeptide is blocked by agonists and antagonists with the expected potency for A2 receptors and is decreased in the presence of 10(-4) M guanosine 5'-[beta, gamma-imido]triphosphate

  9. Hippocampal GluA1-containing AMPA receptors mediate context-dependent sensitization to morphine.

    Science.gov (United States)

    Xia, Yan; Portugal, George S; Fakira, Amanda K; Melyan, Zara; Neve, Rachael; Lee, H Thomas; Russo, Scott J; Liu, Jie; Morón, Jose A

    2011-11-09

    Glutamatergic systems, including AMPA receptors (AMPARs), are involved in opiate-induced neuronal and behavioral plasticity, although the mechanisms underlying these effects are not fully understood. In the present study, we investigated the effects of repeated morphine administration on AMPAR expression, synaptic plasticity, and context-dependent behavioral sensitization to morphine. We found that morphine treatment produced changes of synaptic AMPAR expression in the hippocampus, a brain area that is critically involved in learning and memory. These changes could be observed 1 week after the treatment, but only when mice developed context-dependent behavioral sensitization to morphine in which morphine treatment was associated with drug administration environment. Context-dependent behavioral sensitization to morphine was also associated with increased basal synaptic transmission and disrupted hippocampal long-term potentiation (LTP), whereas these effects were less robust when morphine administration was not paired with the drug administration environment. Interestingly, some effects may be related to the prior history of morphine exposure in the drug-associated environment, since alterations of AMPAR expression, basal synaptic transmission, and LTP were observed in mice that received a saline challenge 1 week after discontinuation of morphine treatment. Furthermore, we demonstrated that phosphorylation of GluA1 AMPAR subunit plays a critical role in the acquisition and expression of context-dependent behavioral sensitization, as this behavior is blocked by a viral vector that disrupts GluA1 phosphorylation. These data provide evidence that glutamatergic signaling in the hippocampus plays an important role in context-dependent sensitization to morphine and supports further investigation of glutamate-based strategies for treating opiate addiction.

  10. Basal Levels of AMPA Receptor GluA1 Subunit Phosphorylation at Threonine 840 and Serine 845 in Hippocampal Neurons

    Science.gov (United States)

    Babiec, Walter E.; Guglietta, Ryan; O'Dell, Thomas J.

    2016-01-01

    Dephosphorylation of AMPA receptor (AMPAR) GluA1 subunits at two sites, serine 845 (S845) and threonine 840 (T840), is thought to be involved in NMDA receptor-dependent forms of long-term depression (LTD). Importantly, the notion that dephosphorylation of these sites contributes to LTD assumes that a significant fraction of GluA1 subunits are…

  11. LINKING GABAA RECEPTOR SUBUNITS TO ALCOHOL-INDUCED CONDITIONED TASTE AVERSION AND RECOVERY FROM ACUTE ALCOHOL INTOXICATION

    Science.gov (United States)

    Blednov, Y.A.; Benavidez, J.M.; Black, M.; Chandra, D.; Homanics, G.E.; Rudolph, U.; Harris, R.A.

    2012-01-01

    GABA type A receptors (GABAA-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABAA-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011) and indicate this aversive property of ethanol is dependent on ethanol action on α2-containing GABAA-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor-incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABAA-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 and α3 (-/-) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism. PMID:23147414

  12. Calcium-dependent regulation of SNARE-mediated membrane fusion by calmodulin.

    Science.gov (United States)

    Di Giovanni, Jerome; Iborra, Cécile; Maulet, Yves; Lévêque, Christian; El Far, Oussama; Seagar, Michael

    2010-07-30

    Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca(2+) sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca(2+)-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca(2+)/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K(D) = 500 nm) and syntaxin 1 (K(D) = 2 microm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca(2+) sensors act antagonistically in SNARE-mediated fusion.

  13. Subunit stoichiometry of the chloroplast photosystem I complex

    International Nuclear Information System (INIS)

    Bruce, B.D.; Malkin, R.

    1988-01-01

    A native photosystem I (PS I) complex and a PS I core complex depleted of antenna subunits has been isolated from the uniformly 14 C-labeled aquatic higher plant, Lemna. These complexes have been analyzed for their subunit stoichiometry by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods. The results for both preparations indicate that one copy of each high molecular mass subunit is present per PS I complex and that a single copy of most low molecular mass subunits is also present. These results suggest that iron-sulfur center X, an early PS I electron acceptor proposed to bind to the high molecular mass subunits, contains a single [4Fe-4S] cluster which is bound to a dimeric structure of high molecular mass subunits, each providing 2 cysteine residues to coordinate this cluster

  14. Regulation of synaptic inhibition by phospho-dependent binding of the AP2 complex to a YECL motif in the GABAA receptor γ2 subunit

    Science.gov (United States)

    Kittler, Josef T.; Chen, Guojun; Kukhtina, Viktoria; Vahedi-Faridi, Ardeschir; Gu, Zhenglin; Tretter, Verena; Smith, Katharine R.; McAinsh, Kristina; Arancibia-Carcamo, I. Lorena; Saenger, Wolfram; Haucke, Volker; Yan, Zhen; Moss, Stephen J.

    2008-01-01

    The regulation of the number of γ2-subunit-containing GABAA receptors (GABAARs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface γ2-subunit-containing GABAARs is regulated. Here, we identify a γ2-subunit-specific Yxxφ-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for γ2-subunit tyrosine phosphorylation. Blocking GABAAR-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxxφ motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that γ2-subunit-containing heteromeric GABAARs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABAAR surface levels and synaptic inhibition. PMID:18305175

  15. SAHA (Vorinostat Corrects Inhibitory Synaptic Deficits Caused by Missense Epilepsy Mutations to the GABAA Receptor γ2 Subunit

    Directory of Open Access Journals (Sweden)

    Nela Durisic

    2018-03-01

    Full Text Available The GABAA receptor (GABAAR α1 subunit A295D epilepsy mutation reduces the surface expression of α1A295Dβ2γ2 GABAARs via ER-associated protein degradation. Suberanilohydroxamic acid (SAHA, also known as Vorinostat was recently shown to correct the misfolding of α1A295D subunits and thereby enhance the functional surface expression of α1A295Dβ2γ2 GABAARs. Here we investigated whether SAHA can also restore the surface expression of γ2 GABAAR subunits that incorporate epilepsy mutations (N40S, R43Q, P44S, R138G known to reduce surface expression via ER-associated protein degradation. As a control, we also investigated the γ2K289M epilepsy mutation that impairs gating without reducing surface expression. Effects of mutations were evaluated on inhibitory postsynaptic currents (IPSCs mediated by the major synaptic α1β2γ2 GABAAR isoform. Recordings were performed in neuron-HEK293 cell artificial synapses to minimise contamination by GABAARs of undefined subunit composition. Transfection with α1β2γ2N40S, α1β2γ2R43Q, α1β2γ2P44S and α1β2γ2R138G subunits produced IPSCs with decay times slower than those of unmutated α1β2γ2 GABAARs due to the low expression of mutant γ2 subunits and the correspondingly high expression of slow-decaying α1β2 GABAARs. SAHA pre-treatment significantly accelerated the decay time constants of IPSCs consistent with the upregulation of mutant γ2 subunit expression. This increase in surface expression was confirmed by immunohistochemistry. SAHA had no effect on either the IPSC kinetics or surface expression levels of α1β2γ2K289M GABAARs, confirming its specificity for ER-retained mutant γ2 subunits. We also found that α1β2γ2K289M GABAARs and SAHA-treated α1β2γ2R43Q, α1β2γ2P44S and α1β2γ2R138G GABAARs all mediated IPSCs that decayed at significantly faster rates than wild type receptors as temperature was increased from 22 to 40°C. This may help explain why these mutations cause febrile

  16. Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways

    International Nuclear Information System (INIS)

    Nguyen Ngoc, Tam Dan; Son, Young-Ok; Lim, Shin-Saeng; Shi, Xianglin; Kim, Jong-Ghee; Heo, Jung Sun; Choe, Youngji; Jeon, Young-Mi; Lee, Jeong-Chae

    2012-01-01

    Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G 2 /M phase. The addition of NaF induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. -- Highlights: ► The mode of NaF-induced cell death and the mechanisms involved were examined. ► NaF induced mainly apoptotic death of mouse embryonic stem cells (mESCs). ► NaF induced mitochondrial-mediated and caspase-dependent apoptosis. ► JNK- and p53-mediated pathways are involved in NaF-mediated apoptosis in the cells. ► ROS are the up-stream effector in NaF-mediated activation of JNK and p53 in mESCs.

  17. Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen Ngoc, Tam Dan [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Son, Young-Ok [Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Lim, Shin-Saeng [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Shi, Xianglin [Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Kim, Jong-Ghee [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Heo, Jung Sun [Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Choe, Youngji [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Jeon, Young-Mi, E-mail: young@jbnu.ac.kr [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Lee, Jeong-Chae, E-mail: leejc88@jbnu.ac.kr [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of)

    2012-03-15

    Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G{sub 2}/M phase. The addition of NaF induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. -- Highlights: ► The mode of NaF-induced cell death and the mechanisms involved were examined. ► NaF induced mainly apoptotic death of mouse embryonic stem cells (mESCs). ► NaF induced mitochondrial-mediated and caspase-dependent apoptosis. ► JNK- and p53-mediated pathways are involved in NaF-mediated apoptosis in the cells. ► ROS are the up-stream effector in NaF-mediated activation of JNK and p53 in mESCs.

  18. Ghrelin upregulates the phosphorylation of the GluN2B subunit of the NMDA receptor by activating GHSR1a and Fyn in the rat hippocampus.

    Science.gov (United States)

    Berrout, Liza; Isokawa, Masako

    2018-01-01

    Ghrelin and its receptor GHSR1a have been shown to exert numerous physiological functions in the brain, in addition to the well-established orexigenic role in the hypothalamus. Earlier work indicated that ghrelin stimulated the phosphorylation of the GluN1 subunit of the NMDA receptor (NMDAR) and enhanced synaptic transmission in the hippocampus. In the present study, we report that the exogenous application of ghrelin increased GluN2B phosphorylation. This increase was independent of GluN2B subunit activity or NMDAR channel activity. However, it depended on the activation of GHSR1a and Fyn as it was blocked by D-Lys3-GHRP-6 and PP2, respectively. Inhibitors for G-protein-regulated second messengers, such as Rp-cAMP, H89, TBB, ryanodine, and thapsigargin, unexpectedly enhanced GluN2B phosphorylation, suggesting that cAMP, PKA, casein kinase II, and cytosolic calcium signaling may oppose to the effect of ghrelin on the phosphorylation of GluN2B. Our findings suggest that 1) GluN2B is likely a molecular target of ghrelin and GHSR1a-driven signaling cascades, and 2) the ghrelin-mediated phosphorylation of GluN2B depends on Fyn activation under complex negative regulation by other second messengers. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Nicotine impairs cyclooxygenase-2-dependent kinin-receptor-mediated murine airway relaxations

    International Nuclear Information System (INIS)

    Xu, Yuan; Cardell, Lars-Olaf

    2014-01-01

    Introduction: Cigarette smoke induces local inflammation and airway hyperreactivity. In asthmatics, it worsens the symptoms and increases the risk for exacerbation. The present study investigates the effects of nicotine on airway relaxations in isolated murine tracheal segments. Methods: Segments were cultured for 24 h in the presence of vehicle, nicotine (10 μM) and/or dexamethasone (1 μM). Airway relaxations were assessed in myographs after pre-contraction with carbachol (1 μM). Kinin receptors, cyclooxygenase (COX) and inflammatory mediator expressions were assessed by real-time PCR and confocal-microscopy-based immunohistochemistry. Results: The organ culture procedure markedly increased bradykinin- (selective B 2 receptor agonist) and des-Arg 9 -bradykinin- (selective B 1 receptor agonist) induced relaxations, and slightly increased relaxation induced by isoprenaline, but not that induced by PGE 2 . The kinin receptor mediated relaxations were epithelium-, COX-2- and EP2-receptor-dependent and accompanied by drastically enhanced mRNA levels of kinin receptors, as well as inflammatory mediators MCP-1 and iNOS. Increase in COX-2 and mPGES-1 was verified both at mRNA and protein levels. Nicotine selectively suppressed the organ-culture-enhanced relaxations induced by des-Arg 9 -bradykinin and bradykinin, at the same time reducing mPGES-1 mRNA and protein expressions. α7-nicotinic acetylcholine receptor inhibitors α-bungarotoxin and MG624 both blocked the nicotine effects on kinin B 2 receptors, but not those on B 1 . Dexamethasone completely abolished kinin-induced relaxations. Conclusion: It is tempting to conclude that a local inflammatory process per se could have a bronchoprotective component by increasing COX-2 mediated airway relaxations and that nicotine could impede this safety mechanism. Dexamethasone further reduced airway inflammation together with relaxations. This might contribute to the steroid resistance seen in some patients with asthma

  20. Nicotine impairs cyclooxygenase-2-dependent kinin-receptor-mediated murine airway relaxations

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yuan, E-mail: yuan.xu@ki.se; Cardell, Lars-Olaf

    2014-02-15

    Introduction: Cigarette smoke induces local inflammation and airway hyperreactivity. In asthmatics, it worsens the symptoms and increases the risk for exacerbation. The present study investigates the effects of nicotine on airway relaxations in isolated murine tracheal segments. Methods: Segments were cultured for 24 h in the presence of vehicle, nicotine (10 μM) and/or dexamethasone (1 μM). Airway relaxations were assessed in myographs after pre-contraction with carbachol (1 μM). Kinin receptors, cyclooxygenase (COX) and inflammatory mediator expressions were assessed by real-time PCR and confocal-microscopy-based immunohistochemistry. Results: The organ culture procedure markedly increased bradykinin- (selective B{sub 2} receptor agonist) and des-Arg{sup 9}-bradykinin- (selective B{sub 1} receptor agonist) induced relaxations, and slightly increased relaxation induced by isoprenaline, but not that induced by PGE{sub 2}. The kinin receptor mediated relaxations were epithelium-, COX-2- and EP2-receptor-dependent and accompanied by drastically enhanced mRNA levels of kinin receptors, as well as inflammatory mediators MCP-1 and iNOS. Increase in COX-2 and mPGES-1 was verified both at mRNA and protein levels. Nicotine selectively suppressed the organ-culture-enhanced relaxations induced by des-Arg{sup 9}-bradykinin and bradykinin, at the same time reducing mPGES-1 mRNA and protein expressions. α7-nicotinic acetylcholine receptor inhibitors α-bungarotoxin and MG624 both blocked the nicotine effects on kinin B{sub 2} receptors, but not those on B{sub 1}. Dexamethasone completely abolished kinin-induced relaxations. Conclusion: It is tempting to conclude that a local inflammatory process per se could have a bronchoprotective component by increasing COX-2 mediated airway relaxations and that nicotine could impede this safety mechanism. Dexamethasone further reduced airway inflammation together with relaxations. This might contribute to the steroid resistance seen in

  1. The NDUFB6 subunit of the mitochondrial respiratory chain complex I is required for electron transfer activity: A proof of principle study on stable and controlled RNA interference in human cell lines

    International Nuclear Information System (INIS)

    Loublier, Sandrine; Bayot, Aurelien; Rak, Malgorzata; El-Khoury, Riyad; Benit, Paule; Rustin, Pierre

    2011-01-01

    Highlights: → NDUFB6 is required for activity of mitochondrial complex I in human cell lines. → Lentivirus based RNA interference results in frequent off target insertions. → Flp-In recombinase mediated miRNA insertion allows gene-specific extinction. -- Abstract: Molecular bases of inherited deficiencies of mitochondrial respiratory chain complex I are still unknown in a high proportion of patients. Among 45 subunits making up this large complex, more than half has unknown function(s). Understanding the function of these subunits would contribute to our knowledge on mitochondrial physiology but might also reveal that some of these subunits are not required for the catalytic activity of the complex. A direct consequence of this finding would be the reduction of the number of candidate genes to be sequenced in patients with decreased complex I activity. In this study, we tested two different methods to stably extinct complex I subunits in cultured cells. We first found that lentivirus-mediated shRNA expression frequently resulted in the unpredicted extinction of additional gene(s) beside targeted ones. This can be ascribed to uncontrolled genetic material insertions in the genome of the host cell. This approach thus appeared inappropriate to study unknown functions of a gene. Next, we found it possible to specifically extinct a CI subunit gene by direct insertion of a miR targeting CI subunits in a Flp site (HEK293 Flp-In cells). By using this strategy we unambiguously demonstrated that the NDUFB6 subunit is required for complex I activity, and defined conditions suitable to undertake a systematic and stable extinction of the different supernumerary subunits in human cells.

  2. The NDUFB6 subunit of the mitochondrial respiratory chain complex I is required for electron transfer activity: A proof of principle study on stable and controlled RNA interference in human cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Loublier, Sandrine; Bayot, Aurelien; Rak, Malgorzata; El-Khoury, Riyad; Benit, Paule [Inserm U676, Hopital Robert Debre, F-75019 Paris (France); Universite Paris 7, Faculte de medecine Denis Diderot, IFR02 Paris (France); Rustin, Pierre, E-mail: pierre.rustin@inserm.fr [Inserm U676, Hopital Robert Debre, F-75019 Paris (France); Universite Paris 7, Faculte de medecine Denis Diderot, IFR02 Paris (France)

    2011-10-22

    Highlights: {yields} NDUFB6 is required for activity of mitochondrial complex I in human cell lines. {yields} Lentivirus based RNA interference results in frequent off target insertions. {yields} Flp-In recombinase mediated miRNA insertion allows gene-specific extinction. -- Abstract: Molecular bases of inherited deficiencies of mitochondrial respiratory chain complex I are still unknown in a high proportion of patients. Among 45 subunits making up this large complex, more than half has unknown function(s). Understanding the function of these subunits would contribute to our knowledge on mitochondrial physiology but might also reveal that some of these subunits are not required for the catalytic activity of the complex. A direct consequence of this finding would be the reduction of the number of candidate genes to be sequenced in patients with decreased complex I activity. In this study, we tested two different methods to stably extinct complex I subunits in cultured cells. We first found that lentivirus-mediated shRNA expression frequently resulted in the unpredicted extinction of additional gene(s) beside targeted ones. This can be ascribed to uncontrolled genetic material insertions in the genome of the host cell. This approach thus appeared inappropriate to study unknown functions of a gene. Next, we found it possible to specifically extinct a CI subunit gene by direct insertion of a miR targeting CI subunits in a Flp site (HEK293 Flp-In cells). By using this strategy we unambiguously demonstrated that the NDUFB6 subunit is required for complex I activity, and defined conditions suitable to undertake a systematic and stable extinction of the different supernumerary subunits in human cells.

  3. β-Arrestin-2-Dependent Signaling Promotes CCR4-mediated Chemotaxis of Murine T-Helper Type 2 Cells.

    Science.gov (United States)

    Lin, Rui; Choi, Yeon Ho; Zidar, David A; Walker, Julia K L

    2018-06-01

    Allergic asthma is a complex inflammatory disease that leads to significant healthcare costs and reduction in quality of life. Although many cell types are implicated in the pathogenesis of asthma, CD4 + T-helper cell type 2 (Th2) cells are centrally involved. We previously reported that the asthma phenotype is virtually absent in ovalbumin-sensitized and -challenged mice that lack global expression of β-arrestin (β-arr)-2 and that CD4 + T cells from these mice displayed significantly reduced CCL22-mediated chemotaxis. Because CCL22-mediated activation of CCR4 plays a role in Th2 cell regulation in asthmatic inflammation, we hypothesized that CCR4-mediated migration of CD4 + Th2 cells to the lung in asthma may use β-arr-dependent signaling. To test this hypothesis, we assessed the effect of various signaling inhibitors on CCL22-induced chemotaxis using in vitro-polarized primary CD4 + Th2 cells from β-arr2-knockout and wild-type mice. Our results show, for the first time, that CCL22-induced, CCR4-mediated Th2 cell chemotaxis is dependent, in part, on a β-arr2-dependent signaling pathway. In addition, we show that this chemotactic signaling mechanism involves activation of P-p38 and Rho-associated protein kinase. These findings point to a proinflammatory role for β-arr2-dependent signaling and support β-arr2 as a novel therapeutic target in asthma.

  4. Soybean glycinin subunits: Characterization of physicochemical and adhesion properties.

    Science.gov (United States)

    Mo, Xiaoqun; Zhong, Zhikai; Wang, Donghai; Sun, Xiuzhi

    2006-10-04

    Soybean proteins have shown great potential for applications as renewable and environmentally friendly adhesives. The objective of this work was to study physicochemical and adhesion properties of soy glycinin subunits. Soybean glycinin was extracted from soybean flour and then fractionated into acidic and basic subunits with an estimated purity of 90 and 85%, respectively. Amino acid composition of glycinin subunits was determined. The high hydrophobic amino acid content is a major contributor to the solubility behavior and water resistance of the basic subunits. Acidic subunits and glycinin had similar solubility profiles, showing more than 80% solubility at pH 2.0-4.0 or 6.5-12.0, whereas basic subunits had considerably lower solubility with the minimum at pH 4.5-8.0. Thermal analysis using a differential scanning calorimeter suggested that basic subunits form new oligomeric structures with higher thermal stability than glycinin but no highly ordered structures present in isolated acidic subunits. The wet strength of basic subunits was 160% more than that of acidic subunits prepared at their respective isoelectric points (pI) and cured at 130 degrees C. Both pH and the curing temperature significantly affected adhesive performance. High-adhesion water resistance was usually observed for adhesives from protein prepared at their pI values and cured at elevated temperatures. Basic subunits are responsible for the water resistance of glycinin and are a good starting material for the development of water-resistant adhesives.

  5. Mediator complex cooperatively regulates transcription of retinoic acid target genes with Polycomb Repressive Complex 2 during neuronal differentiation.

    Science.gov (United States)

    Fukasawa, Rikiya; Iida, Satoshi; Tsutsui, Taiki; Hirose, Yutaka; Ohkuma, Yoshiaki

    2015-11-01

    The Mediator complex (Mediator) plays key roles in transcription and functions as the nexus for integration of various transcriptional signals. Previously, we screened for Mediator cyclin-dependent kinase (CDK)-interacting factors and identified three proteins related to chromatin regulation. One of them, SUZ12 is required for both stability and activity of Polycomb Repressive Complex 2 (PRC2). PRC2 primarily suppresses gene expression through histone H3 lysine 27 trimethylation, resulting in stem cell maintenance and differentiation; perturbation of this process leads to oncogenesis. Recent work showed that Mediator contributes to the embryonic stem cell state through DNA loop formation, which is strongly associated with chromatin architecture; however, it remains unclear how Mediator regulates gene expression in cooperation with chromatin regulators (i.e. writers, readers and remodelers). We found that Mediator CDKs interact directly with the PRC2 subunit EZH2, as well as SUZ12. Known PRC2 target genes were deregulated by Mediator CDK knockdown during neuronal differentiation, and both Mediator and PRC2 complexes co-occupied the promoters of developmental genes regulated by retinoic acid. Our results provide a mechanistic link between Mediator and PRC2 during neuronal differentiation. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  6. Nuclear DAMP complex-mediated RAGE-dependent macrophage cell death

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ruochan [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Department of Infectious Diseases and State Key Lab of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); Fu, Sha; Fan, Xue-Gong [Department of Infectious Diseases and State Key Lab of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); Lotze, Michael T.; Zeh, Herbert J. [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Tang, Daolin, E-mail: tangd2@upmc.edu [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Kang, Rui, E-mail: kangr@upmc.edu [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States)

    2015-03-13

    High mobility group box 1 (HMGB1), histone, and DNA are essential nuclear components involved in the regulation of chromosome structure and function. In addition to their nuclear function, these molecules act as damage-associated molecular patterns (DAMPs) alone or together when released extracellularly. The synergistic effect of these nuclear DNA-HMGB1-histone complexes as DAMP complexes (nDCs) on immune cells remains largely unexplored. Here, we demonstrate that nDCs limit survival of macrophages (e.g., RAW264.7 and peritoneal macrophages) but not cancer cells (e.g., HCT116, HepG2 and Hepa1-6). nDCs promote production of inflammatory tumor necrosis factor α (TNFα) release, triggering reactive oxygen species-dependent apoptosis and necrosis. Moreover, the receptor for advanced glycation end products (RAGE), but not toll-like receptor (TLR)-4 and TLR-2, was required for Akt-dependent TNFα release and subsequent cell death following treatment with nDCs. Genetic depletion of RAGE by RNAi, antioxidant N-Acetyl-L-cysteine, and TNFα neutralizing antibody significantly attenuated nDC-induced cell death. These findings provide evidence supporting novel signaling mechanisms linking nDCs and inflammation in macrophage cell death. - Highlights: • Nuclear DAMP complexes (nDCs) selectively induce cell death in macrophages, but not cancer cells. • TNFα-mediated oxidative stress is required for nDC-induced death. • RAGE-mediated Akt activation is required for nDC-induced TNFα release. • Blocking RAGE and TNFα inhibits nDC-induced macrophage cell death.

  7. Extricating Manual and Non-Manual Features for Subunit Level Medical Sign Modelling in Automatic Sign Language Classification and Recognition.

    Science.gov (United States)

    R, Elakkiya; K, Selvamani

    2017-09-22

    Subunit segmenting and modelling in medical sign language is one of the important studies in linguistic-oriented and vision-based Sign Language Recognition (SLR). Many efforts were made in the precedent to focus the functional subunits from the view of linguistic syllables but the problem is implementing such subunit extraction using syllables is not feasible in real-world computer vision techniques. And also, the present recognition systems are designed in such a way that it can detect the signer dependent actions under restricted and laboratory conditions. This research paper aims at solving these two important issues (1) Subunit extraction and (2) Signer independent action on visual sign language recognition. Subunit extraction involved in the sequential and parallel breakdown of sign gestures without any prior knowledge on syllables and number of subunits. A novel Bayesian Parallel Hidden Markov Model (BPaHMM) is introduced for subunit extraction to combine the features of manual and non-manual parameters to yield better results in classification and recognition of signs. Signer independent action aims in using a single web camera for different signer behaviour patterns and for cross-signer validation. Experimental results have proved that the proposed signer independent subunit level modelling for sign language classification and recognition has shown improvement and variations when compared with other existing works.

  8. Solution Structure of the N-Terminal Domain of Mediator Subunit MED26 and Molecular Characterization of Its Interaction with EAF1 and TAF7.

    Science.gov (United States)

    Lens, Zoé; Cantrelle, François-Xavier; Peruzzini, Riccardo; Hanoulle, Xavier; Dewitte, Frédérique; Ferreira, Elisabeth; Baert, Jean-Luc; Monté, Didier; Aumercier, Marc; Villeret, Vincent; Verger, Alexis; Landrieu, Isabelle

    2017-10-13

    MED26 is a subunit of Mediator, a large complex central to the regulation of gene transcription by RNA Polymerase II. MED26 plays a role in the switch between the initiation and elongation phases of RNA Polymerase II-mediated transcription process. Regulation of these steps requires successive binding of MED26 N-terminal domain (NTD) to TATA-binding protein-associated factor 7 (TAF7) and Eleven-nineteen lysine-rich in leukemia-Associated Factor 1 (EAF1). In order to investigate the mechanism of regulation by MED26, MED26-NTD structure was solved by NMR, revealing a 4-helix bundle. EAF1 (239-268) and TAF7 (205-235) peptide interactions were both mapped to the same groove formed by H3 and H4 helices of MED26-NTD. Both interactions are characterized by dissociation constants in the 10-μM range. Further experiments revealed a folding-upon-binding mechanism that leads to the formation of EAF1 (N247-S260) and TAF7 (L214-S227) helices. Chemical shift perturbations and nuclear Overhauser enhancement contacts support the involvement of residues I222/F223 in anchoring TAF7 helix to a hydrophobic pocket of MED26-NTD, including residues L48, W80 and I84. In addition, Ala mutations of charged residues located in the C-terminal disordered part of TAF7 and EAF1 peptides affected the binding, with a loss of affinity characterized by a 10-time increase of dissociation constants. A structural model of MED26-NTD/TAF7 complex shows bi-partite components, combining ordered and disordered segments, as well as hydrophobic and electrostatic contributions to the binding. This study provides molecular detail that will help to decipher the mechanistic basis for the initiation to elongation switch-function mediated by MED26-NTD. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like domain-dependent manner

    DEFF Research Database (Denmark)

    Kny, Melanie; Standera, Sybille; Hartmann-Petersen, Rasmus

    2011-01-01

    in ER-associated protein degradation (ERAD) and interacts directly with the ubiquitin ligase Hrd1, which is found in high molecular mass complexes of the ER membrane. Here we present the first evidence that Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like (UBL) domain-dependent manner. We...

  10. Regulation of synaptic inhibition by phospho-dependent binding of the AP2 complex to a YECL motif in the GABAA receptor gamma2 subunit.

    Science.gov (United States)

    Kittler, Josef T; Chen, Guojun; Kukhtina, Viktoria; Vahedi-Faridi, Ardeschir; Gu, Zhenglin; Tretter, Verena; Smith, Katharine R; McAinsh, Kristina; Arancibia-Carcamo, I Lorena; Saenger, Wolfram; Haucke, Volker; Yan, Zhen; Moss, Stephen J

    2008-03-04

    The regulation of the number of gamma2-subunit-containing GABA(A) receptors (GABA(A)Rs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface gamma2-subunit-containing GABA(A)Rs is regulated. Here, we identify a gamma2-subunit-specific Yxxvarphi-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for gamma2-subunit tyrosine phosphorylation. Blocking GABA(A)R-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxxvarphi motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that gamma2-subunit-containing heteromeric GABA(A)Rs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABA(A)R surface levels and synaptic inhibition.

  11. Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site.

    Science.gov (United States)

    Adams, Julian; Chen, Zhi-Ping; Van Denderen, Bryce J W; Morton, Craig J; Parker, Michael W; Witters, Lee A; Stapleton, David; Kemp, Bruce E

    2004-01-01

    AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.

  12. Tyrosine kinase inhibitors as modulators of trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity in breast cancer cell lines.

    Science.gov (United States)

    Collins, Denis M; Gately, Kathy; Hughes, Clare; Edwards, Connla; Davies, Anthony; Madden, Stephen F; O'Byrne, Kenneth J; O'Donovan, Norma; Crown, John

    2017-09-01

    Trastuzumab is an anti-HER2 monoclonal antibody (mAb) therapy capable of antibody-dependent cell-mediated cytotoxicity (ADCC) and used in the treatment of HER2+ breast cancer. Through interactions with FcƴR+ immune cell subsets, trastuzumab functions as a passive immunotherapy. The EGFR/HER2-targeting tyrosine kinase inhibitor (TKI) lapatinib and the next generation TKIs afatinib and neratinib, can alter HER2 levels, potentially modulating the ADCC response to trastuzumab. Using LDH-release assays, we investigated the impact of antigen modulation, assay duration and peripheral blood mononuclear cell (PBMC) activity on trastuzumab-mediated ADCC in breast cancer models of maximal (SKBR3) and minimal (MCF-7) target antigen expression to determine if modulating the ADCC response to trastuzumab using TKIs may be a viable approach for enhancing tumor immune reactivity. HER2 levels were determined in lapatinib, afatinib and neratinib-treated SKBR3 and MCF-7 using high content analysis (HCA). Trastuzumab-mediated ADCC was assessed following treatment with TKIs utilising a colorimetric LDH release-based protocol at 4 and 12h timepoints. PBMC activity was assessed against non-MHC-restricted K562 cells. A flow cytometry-based method (CFSE/7-AAD) was also used to measure trastuzumab-mediated ADCC in medium-treated SKBR3 and MCF-7. HER2 antigen levels were significantly altered by the three TKIs in both cell line models. The TKIs significantly reduced LDH levels directly in SKBR3 cells but not MCF-7. Lapatinib and neratinib augment trastuzumab-related ADCC in SKBR3 but the effect was not consistent with antigen expression levels and was dependent on volunteer PBMC activity (vs. K562). A 12h assay timepoint produced more consistent results. Trastuzumab-mediated ADCC (PBMC:target cell ratio of 10:1) was measured at 7.6±4.7% (T12) by LDH assay and 19±3.2 % (T12) using the flow cytometry-based method in the antigen-low model MCF-7. In the presence of effector cells with high

  13. Function and regulation of the Mediator complex.

    Science.gov (United States)

    Conaway, Ronald C; Conaway, Joan Weliky

    2011-04-01

    Over the past few years, advances in biochemical and genetic studies of the structure and function of the Mediator complex have shed new light on its subunit architecture and its mechanism of action in transcription by RNA polymerase II (pol II). The development of improved methods for reconstitution of recombinant Mediator subassemblies is enabling more in-depth analyses of basic features of the mechanisms by which Mediator interacts with and controls the activity of pol II and the general initiation factors. The discovery and characterization of multiple, functionally distinct forms of Mediator characterized by the presence or absence of the Cdk8 kinase module have led to new insights into how Mediator functions in both Pol II transcription activation and repression. Finally, progress in studies of the mechanisms by which the transcriptional activation domains (ADs) of DNA binding transcription factors target Mediator have brought to light unexpected complexities in the way Mediator participates in signal transduction. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. A CK2 site is reversibly phosphorylated in the photosystem II subunit CP29

    NARCIS (Netherlands)

    Testi, Maria Grazia; Croce, Roberta; Polverino-De Laureto, Patrizia; Bassi, Roberto

    1996-01-01

    Protein phosphorylation is a major mechanism in the regulation of protein function. In chloroplast thylakoids several photosystem II subunits, including the major antenna light-harvesting complex II and several core complex components, are reversibly phosphorylated depending on the redox state of

  15. Mediator Undergoes a Compositional Change during Transcriptional Activation.

    Science.gov (United States)

    Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

    2016-11-03

    Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Caspase-dependant activation of chymotrypsin-like proteases mediates nuclear events during Jurkat T cell apoptosis

    International Nuclear Information System (INIS)

    O'Connell, A.R.; Lee, B.W.; Stenson-Cox, C.

    2006-01-01

    Apoptosis involves a cascade of biochemical and morphological changes resulting in the systematic disintegration of the cell. Caspases are central mediators of this process. Supporting and primary roles for serine proteases as pro-apoptotic mediators have also been highlighted. Evidence for such roles comes largely from the use of pharmacological inhibitors; as a consequence information regarding their apoptotic function and biochemical properties has been limited. Here, we circumvented limitations associated with traditional serine protease inhibitors through use of a fluorescently labelled inhibitor of serine proteases (FLISP) that allowed for analysis of the specificity, regulation and positioning of apoptotic serine proteases within a classical apoptotic cascade. We demonstrate that staurosporine triggers a caspase-dependant induction of chymotrypsin-like activity in the nucleus of apoptotic Jurkat T cells. We show that serine protease activity is required for the generation of late stage nuclear events including condensation, fragmentation and DNA degradation. Furthermore, we reveal caspase-dependant activation of two chymotrypsin-like protein species that we hypothesize mediate cell death-associated nuclear events

  17. Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

    Science.gov (United States)

    Toubiana, Julie; Rossi, Anne-Lise; Belaidouni, Nadia; Grimaldi, David; Pene, Frederic; Chafey, Philippe; Comba, Béatrice; Camoin, Luc; Bismuth, Georges; Claessens, Yann-Erick; Mira, Jean-Paul; Chiche, Jean-Daniel

    2015-10-01

    TLR2 has a prominent role in host defense against a wide variety of pathogens. Stimulation of TLR2 triggers MyD88-dependent signaling to induce NF-κB translocation, and activates a Rac1-PI 3-kinase dependent pathway that leads to transactivation of NF-κB through phosphorylation of the P65 NF-κB subunit. This transactivation pathway involves tyrosine phosphorylations. The role of the tyrosine kinases in TLR signaling is controversial, with discrepancies between studies using only chemical inhibitors and knockout mice. Here, we show the involvement of the tyrosine-kinase Lyn in TLR2-dependent activation of NF-κB in human cellular models, by using complementary inhibition strategies. Stimulation of TLR2 induces the formation of an activation cluster involving TLR2, CD14, PI 3-kinase and Lyn, and leads to the activation of AKT. Lyn-dependent phosphorylation of the p110 catalytic subunit of PI 3-kinase is essential to the control of PI 3-kinase biological activity upstream of AKT and thereby to the transactivation of NF-κB. Thus, Lyn kinase activity is crucial in TLR2-mediated activation of the innate immune response in human mononuclear cells. © The Author(s) 2015.

  18. Linking GABA(A) receptor subunits to alcohol-induced conditioned taste aversion and recovery from acute alcohol intoxication.

    Science.gov (United States)

    Blednov, Y A; Benavidez, J M; Black, M; Chandra, D; Homanics, G E; Rudolph, U; Harris, R A

    2013-04-01

    GABA type A receptors (GABA(A)-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABA(A)-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011). All together, they indicate that aversive property of ethanol is dependent on ethanol action on α2-containing GABA(A)-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABA(A)-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 (-/-) and α3 (-/Y) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Transcriptional regulators of Na, K-ATPase subunits

    Directory of Open Access Journals (Sweden)

    Zhiqin eLi

    2015-10-01

    Full Text Available The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits have been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-to-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.

  20. Association of ω with the C-terminal region of β' subunit is essential for assembly of RNA polymerase in Mycobacterium tuberculosis.

    Science.gov (United States)

    Mao, Chunyou; Zhu, Yan; Lu, Pei; Feng, Lipeng; Chen, Shiyun; Hu, Yangbo

    2018-04-09

    The ω subunit is the smallest subunit of bacterial RNA polymerase (RNAP). Although homologs of ω are essential in both eukaryotes and archaea, this subunit has been known to be dispensable for RNAP in Escherichia coli ( Eco ) and in other bacteria. In this study, we characterized an indispensable role of the ω subunit in Mycobacterium tuberculosis ( Mtb ). Unlike the well-studied Eco RNAP, the Mtb RNAP core enzyme cannot be functionally assembled in the absence of the ω subunit. Importantly, substitution of Mtb ω with ω subunits from Eco or Thermus thermophiles ( Tth ) cannot restore the assembly of Mtb RNAP. Furthermore, by replacing different regions in Mtb ω with the corresponding regions from Eco ω, we found a non-conserved loop region in Mtb ω essential for its function in RNAP assembly. From RNAP structures, we noticed that the location of the C-terminal region of the β' subunit (β'CTD) in Mtb RNAP but not in Eco or Tth RNAP is close to the ω loop region. Deletion of this β'CTD in Mtb RNAP destabilized the binding of Mtb ω on RNAP and compromised Mtb core assembly, suggesting that these two regions may function together to play a role in ω-dependent RNAP assembly in Mtb Sequence alignment of the ω loop and the β'CTD regions suggests that the essential role of ω is probably restricted to mycobacteria. Together, our study characterized an essential role of Mtb ω and highlighted the importance of the ω loop region in Mtb RNAP assembly. Importance DNA-dependent RNA polymerase (RNAP), which is consisted of a multi-subunit core enzyme (α 2 ββ'ω) and a dissociable σ subunit, is the only enzyme in charge of transcription in bacteria. As the smallest subunit, the roles of ω remain the least well-studied. In Escherichia coli ( Eco ) and some other bacteria, the ω subunit is known to be non-essential for RNAP. In this study, we revealed an essential role of the ω subunit for RNAP assembly in the human pathogen Mycobacterium tuberculosis , and

  1. Alteration of skin wound healing in keratinocyte-specific mediator complex subunit 1 null mice.

    Science.gov (United States)

    Noguchi, Fumihito; Nakajima, Takeshi; Inui, Shigeki; Reddy, Janardan K; Itami, Satoshi

    2014-01-01

    MED1 (Mediator complex subunit 1) is a co-activator of various transcription factors that function in multiple transcriptional pathways. We have already established keratinocyte-specific MED1 null mice (Med1(epi-/-)) that develop epidermal hyperplasia. Herein, to investigate the function(s) of MED1 in skin wound healing, full-thickness skin wounds were generated in Med1(epi-/-) and age-matched wild-type mice and the healing process was analyzed. Macroscopic wound closure and the re-epithelialization rate were accelerated in 8-week-old Med1(epi-/-) mice compared with age-matched wild-type mice. Increased lengths of migrating epithelial tongues and numbers of Ki67-positive cells at the wounded epidermis were observed in 8-week-old Med1(epi-/-) mice, whereas wound contraction and the area of α-SMA-positive myofibroblasts in the granulation tissue were unaffected. Migration was enhanced in Med1(epi-/-) keratinocytes compared with wild-type keratinocytes in vitro. Immunoblotting revealed that the expression of follistatin was significantly decreased in Med1(epi-/-) keratinocytes. Moreover, the mitogen-activated protein kinase pathway was enhanced before and after treatment of Med1(epi-/-) keratinocytes with activin A in vitro. Cell-cycle analysis showed an increased ratio of S phase cells after activin A treatment of Med1(epi-/-) keratinocytes compared with wild-type keratinocytes. These findings indicate that the activin-follistatin system is involved in this acceleration of skin wound healing in 8-week-old Med1(epi-/-) mice. On the other hand, skin wound healing in 6-month-old Med1(epi-/-) mice was significantly delayed with decreased numbers of Ki67-positive cells at the wounded epidermis as well as BrdU-positive label retaining cells in hair follicles compared with age-matched wild-type mice. These results agree with our previous observation that hair follicle bulge stem cells are reduced in older Med1(epi-/-) mice, indicating a decreased contribution of hair

  2. Alteration of skin wound healing in keratinocyte-specific mediator complex subunit 1 null mice.

    Directory of Open Access Journals (Sweden)

    Fumihito Noguchi

    Full Text Available MED1 (Mediator complex subunit 1 is a co-activator of various transcription factors that function in multiple transcriptional pathways. We have already established keratinocyte-specific MED1 null mice (Med1(epi-/- that develop epidermal hyperplasia. Herein, to investigate the function(s of MED1 in skin wound healing, full-thickness skin wounds were generated in Med1(epi-/- and age-matched wild-type mice and the healing process was analyzed. Macroscopic wound closure and the re-epithelialization rate were accelerated in 8-week-old Med1(epi-/- mice compared with age-matched wild-type mice. Increased lengths of migrating epithelial tongues and numbers of Ki67-positive cells at the wounded epidermis were observed in 8-week-old Med1(epi-/- mice, whereas wound contraction and the area of α-SMA-positive myofibroblasts in the granulation tissue were unaffected. Migration was enhanced in Med1(epi-/- keratinocytes compared with wild-type keratinocytes in vitro. Immunoblotting revealed that the expression of follistatin was significantly decreased in Med1(epi-/- keratinocytes. Moreover, the mitogen-activated protein kinase pathway was enhanced before and after treatment of Med1(epi-/- keratinocytes with activin A in vitro. Cell-cycle analysis showed an increased ratio of S phase cells after activin A treatment of Med1(epi-/- keratinocytes compared with wild-type keratinocytes. These findings indicate that the activin-follistatin system is involved in this acceleration of skin wound healing in 8-week-old Med1(epi-/- mice. On the other hand, skin wound healing in 6-month-old Med1(epi-/- mice was significantly delayed with decreased numbers of Ki67-positive cells at the wounded epidermis as well as BrdU-positive label retaining cells in hair follicles compared with age-matched wild-type mice. These results agree with our previous observation that hair follicle bulge stem cells are reduced in older Med1(epi-/- mice, indicating a decreased contribution of hair

  3. Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.

    Directory of Open Access Journals (Sweden)

    Sujatha P Koduvayur

    Full Text Available The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.

  4. K-Cl Cotransporter 2-mediated Cl- Extrusion Determines Developmental Stage-dependent Impact of Propofol Anesthesia on Dendritic Spines.

    Science.gov (United States)

    Puskarjov, Martin; Fiumelli, Hubert; Briner, Adrian; Bodogan, Timea; Demeter, Kornel; Lacoh, Claudia-Marvine; Mavrovic, Martina; Blaesse, Peter; Kaila, Kai; Vutskits, Laszlo

    2017-05-01

    General anesthetics potentiating γ-aminobutyric acid (GABA)-mediated signaling are known to induce a persistent decrement in excitatory synapse number in the cerebral cortex when applied during early postnatal development, while an opposite action is produced at later stages. Here, the authors test the hypothesis that the effect of general anesthetics on synaptogenesis depends upon the efficacy of GABA receptor type A (GABAA)-mediated inhibition controlled by the developmental up-regulation of the potassium-chloride (K-Cl) cotransporter 2 (KCC2). In utero electroporation of KCC2 was used to prematurely increase the efficacy of (GABAA)-mediated inhibition in layer 2/3 pyramidal neurons in the immature rat somatosensory cortex. Parallel experiments with expression of the inward-rectifier potassium channel Kir2.1 were done to reduce intrinsic neuronal excitability. The effects of these genetic manipulations (n = 3 to 4 animals per experimental group) were evaluated using iontophoretic injection of Lucifer Yellow (n = 8 to 12 cells per animal). The total number of spines analyzed per group ranged between 907 and 3,371. The authors found a robust effect of the developmental up-regulation of KCC2-mediated Cl transport on the age-dependent action of propofol on dendritic spines. Premature expression of KCC2, unlike expression of a transport-inactive KCC2 variant, prevented a propofol-induced decrease in spine density. In line with a reduction in neuronal excitability, the above result was qualitatively replicated by overexpression of Kir2.1. The KCC2-dependent developmental increase in the efficacy of GABAA-mediated inhibition is a major determinant of the age-dependent actions of propofol on dendritic spinogenesis.

  5. 28 CFR 51.6 - Political subunits.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All political...

  6. The role of heat shock protein 70 in mediating age-dependent mortality in sepsis.

    Science.gov (United States)

    McConnell, Kevin W; Fox, Amy C; Clark, Andrew T; Chang, Nai-Yuan Nicholas; Dominguez, Jessica A; Farris, Alton B; Buchman, Timothy G; Hunt, Clayton R; Coopersmith, Craig M

    2011-03-15

    Sepsis is primarily a disease of the aged, with increased incidence and mortality occurring in aged hosts. Heat shock protein (HSP) 70 plays an important role in both healthy aging and the stress response to injury. The purpose of this study was to determine the role of HSP70 in mediating mortality and the host inflammatory response in aged septic hosts. Sepsis was induced in both young (6- to 12-wk-old) and aged (16- to 17-mo-old) HSP70(-/-) and wild-type (WT) mice to determine whether HSP70 modulated outcome in an age-dependent fashion. Young HSP70(-/-) and WT mice subjected to cecal ligation and puncture, Pseudomonas aeruginosa pneumonia, or Streptococcus pneumoniae pneumonia had no differences in mortality, suggesting HSP70 does not mediate survival in young septic hosts. In contrast, mortality was higher in aged HSP70(-/-) mice than aged WT mice subjected to cecal ligation and puncture (p = 0.01), suggesting HSP70 mediates mortality in sepsis in an age-dependent fashion. Compared with WT mice, aged septic HSP70(-/-) mice had increased gut epithelial apoptosis and pulmonary inflammation. In addition, HSP70(-/-) mice had increased systemic levels of TNF-α, IL-6, IL-10, and IL-1β compared with WT mice. These data demonstrate that HSP70 is a key determinant of mortality in aged, but not young hosts in sepsis. HSP70 may play a protective role in an age-dependent response to sepsis by preventing excessive gut apoptosis and both pulmonary and systemic inflammation.

  7. Melanopsin mediates light-dependent relaxation in blood vessels.

    Science.gov (United States)

    Sikka, Gautam; Hussmann, G Patrick; Pandey, Deepesh; Cao, Suyi; Hori, Daijiro; Park, Jong Taek; Steppan, Jochen; Kim, Jae Hyung; Barodka, Viachaslau; Myers, Allen C; Santhanam, Lakshmi; Nyhan, Daniel; Halushka, Marc K; Koehler, Raymond C; Snyder, Solomon H; Shimoda, Larissa A; Berkowitz, Dan E

    2014-12-16

    Melanopsin (opsin4; Opn4), a non-image-forming opsin, has been linked to a number of behavioral responses to light, including circadian photo-entrainment, light suppression of activity in nocturnal animals, and alertness in diurnal animals. We report a physiological role for Opn4 in regulating blood vessel function, particularly in the context of photorelaxation. Using PCR, we demonstrate that Opn4 (a classic G protein-coupled receptor) is expressed in blood vessels. Force-tension myography demonstrates that vessels from Opn4(-/-) mice fail to display photorelaxation, which is also inhibited by an Opn4-specific small-molecule inhibitor. The vasorelaxation is wavelength-specific, with a maximal response at ∼430-460 nm. Photorelaxation does not involve endothelial-, nitric oxide-, carbon monoxide-, or cytochrome p450-derived vasoactive prostanoid signaling but is associated with vascular hyperpolarization, as shown by intracellular membrane potential measurements. Signaling is both soluble guanylyl cyclase- and phosphodiesterase 6-dependent but protein kinase G-independent. β-Adrenergic receptor kinase 1 (βARK 1 or GRK2) mediates desensitization of photorelaxation, which is greatly reduced by GRK2 inhibitors. Blue light (455 nM) regulates tail artery vasoreactivity ex vivo and tail blood blood flow in vivo, supporting a potential physiological role for this signaling system. This endogenous opsin-mediated, light-activated molecular switch for vasorelaxation might be harnessed for therapy in diseases in which altered vasoreactivity is a significant pathophysiologic contributor.

  8. Plant Mediator complex and its critical functions in transcription regulation.

    Science.gov (United States)

    Yang, Yan; Li, Ling; Qu, Li-Jia

    2016-02-01

    The Mediator complex is an important component of the eukaryotic transcriptional machinery. As an essential link between transcription factors and RNA polymerase II, the Mediator complex transduces diverse signals to genes involved in different pathways. The plant Mediator complex was recently purified and comprises conserved and specific subunits. It functions in concert with transcription factors to modulate various responses. In this review, we summarize the recent advances in understanding the plant Mediator complex and its diverse roles in plant growth, development, defense, non-coding RNA production, response to abiotic stresses, flowering, genomic stability and metabolic homeostasis. In addition, the transcription factors interacting with the Mediator complex are also highlighted. © 2015 Institute of Botany, Chinese Academy of Sciences.

  9. Involvement of proteasomal subunits zeta and iota in RNA degradation.

    Science.gov (United States)

    Petit, F; Jarrousse, A S; Dahlmann, B; Sobek, A; Hendil, K B; Buri, J; Briand, Y; Schmid, H P

    1997-01-01

    We have identified two distinct subunits of 20 S proteasomes that are associated with RNase activity. Proteasome subunits zeta and iota, eluted from two-dimensional Western blots, hydrolysed tobacco mosaic virus RNA, whereas none of the other subunits degraded this substrate under the same conditions. Additionally, proteasomes were dissociated by 6 M urea, and subunit zeta, containing the highest RNase activity, was isolated by anion-exchange chromatography and gel filtration. Purified subunit zeta migrated as a single spot on two-dimensional PAGE with a molecular mass of approx. 28 kDa. Addition of anti-(subunit zeta) antibodies led to the co-precipitation of this proteasome subunit and nuclease activity. This is the first evidence that proteasomal alpha-type subunits are associated with an enzymic activity, and our results provide further evidence that proteasomes may be involved in cellular RNA metabolism. PMID:9337855

  10. Transcriptional regulators of Na, K-ATPase subunits

    OpenAIRE

    Zhiqin eLi; Sigrid A Langhans

    2015-01-01

    The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during developme...

  11. The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation

    OpenAIRE

    Malik, Sohail; Roeder, Robert G.

    2010-01-01

    The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action ...

  12. Deletion of P2X2 and P2X3 receptor subunits does not alter motility of the mouse colon

    Directory of Open Access Journals (Sweden)

    Matthew DeVries

    2010-03-01

    Full Text Available Purinergic P2X receptors contribute to neurotransmission in the gut. P2X receptors are ligand-gated cation channels that mediate synaptic excitation in subsets of enteric neurons. The present study evaluated colonic motility in vitro and in vivo in wild type (WT and P2X2 and P2X3 subunit knockout (KO mice. The muscarinic receptor agonist, bethanechol (0.3-3 micromolar, caused similar contractions of the longitudinal muscle in colon segments from WT, P2X2 and P2X3 subunit KO mice. Nicotine (1-300 micromolar, acting at neuronal nicotinic receptors, caused similar longitudinal muscle relaxations in colonic segments from WT and P2X2 and P2X3 subunit KO mice. Nicotine-induced relaxations were inhibited by nitro-L-arginine (NLA, 100 micromolar and apamin (0.1 micromolar which block inhibitory neuromuscular transmission. ATP (1-1000 micromolar caused contractions only in the presence of NLA and apamin. ATP-induced contractions were similar in colon segments from WT, P2X2 and P2X3 KO mice. The mouse colon generates spontaneous migrating motor complexes (MMCs in vitro. The MMC frequency was higher in P2X2 KO compared to WT tissues; other parameters of the MMC were similar in colon segments from WT, P2X2 and P2X3 KO mice. 5-Hydroxytryptophan-induced fecal output was similar in WT, P2X2 and P2X3 KO mice. These data indicate that nicotinic receptors are located predominately on inhibitory motor neurons supplying the longitudinal muscle in the mouse colon. P2X2 or P2X3 subunit containing receptors are not localized to motorneurons supplying the longitudinal muscle. Synaptic transmission mediated by P2X2 or P2X3 subunit containing receptors is not required for propulsive motility in the mouse colon.

  13. Olfactory bulb glomerular NMDA receptors mediate olfactory nerve potentiation and odor preference learning in the neonate rat.

    Directory of Open Access Journals (Sweden)

    Rebecca Lethbridge

    Full Text Available Rat pup odor preference learning follows pairing of bulbar beta-adrenoceptor activation with olfactory input. We hypothesize that NMDA receptor (NMDAR-mediated olfactory input to mitral cells is enhanced during training, such that increased calcium facilitates and shapes the critical cAMP pattern. Here, we demonstrate, in vitro, that olfactory nerve stimulation, at sniffing frequencies, paired with beta-adrenoceptor activation, potentiates olfactory nerve-evoked mitral cell firing. This potentiation is blocked by a NMDAR antagonist and by increased inhibition. Glomerular disinhibition also induces NMDAR-sensitive potentiation. In vivo, in parallel, behavioral learning is prevented by glomerular infusion of an NMDAR antagonist or a GABA(A receptor agonist. A glomerular GABA(A receptor antagonist paired with odor can induce NMDAR-dependent learning. The NMDA GluN1 subunit is phosphorylated in odor-specific glomeruli within 5 min of training suggesting early activation, and enhanced calcium entry, during acquisition. The GluN1 subunit is down-regulated 3 h after learning; and at 24 h post-training the GluN2B subunit is down-regulated. These events may assist memory stability. Ex vivo experiments using bulbs from trained rat pups reveal an increase in the AMPA/NMDA EPSC ratio post-training, consistent with an increase in AMPA receptor insertion and/or the decrease in NMDAR subunits. These results support a model of a cAMP/NMDA interaction in generating rat pup odor preference learning.

  14. Experience-Dependent Equilibration of AMPAR-Mediated Synaptic Transmission during the Critical Period

    Directory of Open Access Journals (Sweden)

    Kyung-Seok Han

    2017-01-01

    Full Text Available Experience-dependent synapse refinement is essential for functional optimization of neural circuits. However, how sensory experience sculpts excitatory synaptic transmission is poorly understood. Here, we show that despite substantial remodeling of synaptic connectivity, AMPAR-mediated synaptic transmission remains at equilibrium during the critical period in the mouse primary visual cortex. The maintenance of this equilibrium requires neurogranin (Ng, a postsynaptic calmodulin-binding protein important for synaptic plasticity. With normal visual experience, loss of Ng decreased AMPAR-positive synapse numbers, prevented AMPAR-silent synapse maturation, and increased spine elimination. Importantly, visual deprivation halted synapse loss caused by loss of Ng, revealing that Ng coordinates experience-dependent AMPAR-silent synapse conversion to AMPAR-active synapses and synapse elimination. Loss of Ng also led to sensitized long-term synaptic depression (LTD and impaired visually guided behavior. Our synaptic interrogation reveals that experience-dependent coordination of AMPAR-silent synapse conversion and synapse elimination hinges upon Ng-dependent mechanisms for constructive synaptic refinement during the critical period.

  15. Mechanism of the modulation of BK potassium channel complexes with different auxiliary subunit compositions by the omega-3 fatty acid DHA.

    Science.gov (United States)

    Hoshi, Toshinori; Tian, Yutao; Xu, Rong; Heinemann, Stefan H; Hou, Shangwei

    2013-03-19

    Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are well known for their functional versatility, which is bestowed in part by their rich modulatory repertoire. We recently showed that long-chain omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) found in oily fish lower blood pressure by activating vascular BK channels made of Slo1+β1 subunits. Here we examined the action of DHA on BK channels with different auxiliary subunit compositions. Neuronal Slo1+β4 channels were just as well activated by DHA as vascular Slo1+β1 channels. In contrast, the stimulatory effect of DHA was much smaller in Slo1+β2, Slo1+LRRC26 (γ1), and Slo1 channels without auxiliary subunits. Mutagenesis of β1, β2, and β4 showed that the large effect of DHA in Slo1+β1 and Slo1+β4 is conferred by the presence of two residues, one in the N terminus and the other in the first transmembrane segment of the β1 and β4 subunits. Transfer of this amino acid pair from β1 or β4 to β2 introduces a large response to DHA in Slo1+β2. The presence of a pair of oppositely charged residues at the aforementioned positions in β subunits is associated with a large response to DHA. The Slo1 auxiliary subunits are expressed in a highly tissue-dependent fashion. Thus, the subunit composition-dependent stimulation by DHA demonstrates that BK channels are effectors of omega-3 fatty acids with marked tissue specificity.

  16. Three alpha-subunits of heterotrimeric G proteins and an adenylyl cyclase have distinct roles in fruiting body development in the homothallic fungus Sordaria macrospora.

    Science.gov (United States)

    Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich

    2008-09-01

    Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different alpha-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Deltagsa1, Deltagsa2, and Deltagsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Galpha-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Deltagsa1Deltagsa2 and Deltagsa1Deltagsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Galpha-subunits, two recently generated Deltapre strains were crossed with all Deltagsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three DeltagsaDeltasac1 double mutants and one Deltagsa2Deltagsa3Deltasac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1-GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Galpha-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora.

  17. Direct and indirect associations between social anxiety and nicotine dependence and cessation problems: multiple mediator analyses.

    Science.gov (United States)

    Buckner, Julia D; Farris, Samantha G; Schmidt, Norman B; Zvolensky, Michael J

    2014-06-01

    Little empirical work has evaluated why socially anxious smokers are especially vulnerable to more severe nicotine dependence and cessation failure. Presumably, these smokers rely on cigarettes to help them manage their chronically elevated negative affect elicited by a wide array of social contexts. The current study examined the direct and indirect effects of social anxiety cross-sectionally in regard to a range of smoking processes among 466 treatment-seeking smokers. Negative affect and negative affect reduction motives were examined as mediators of the relations of social anxiety with nicotine dependence and cessation problems. Social anxiety was directly and robustly associated with perceived barriers to smoking cessation and problems experienced during past quit attempts. Social anxiety was also associated with greater nicotine dependence and smoking inflexibility indirectly through negative affect and negative affect smoking motives. Negative affect and smoking to reduce negative affect mediated these relations. These findings document the important role of negative affect and negative affect reduction motives in the relationships of social anxiety with nicotine dependence and cessation problems.

  18. Valence and atomic size dependent exchange barriers in vacancy-mediated dopant diffusion

    International Nuclear Information System (INIS)

    Nelson, J.S.; Schultz, P.A.; Wright, A.F.

    1998-01-01

    First-principles pseudopotential calculations of dopant-vacancy exchange barriers indicate a strong dependency on dopant valence and atomic size, in contrast to current models of vacancy-mediated dopant diffusion. First-row elements (B, C, N) are found to have exchange barriers which are an order of magnitude larger than the assumed value of 0.3 eV (the Si vacancy migration energy). copyright 1998 American Institute of Physics

  19. The Mediator Complex and Lipid Metabolism

    OpenAIRE

    Zhang, Yi; Xiaoli,; Zhao, Xiaoping; Yang, Fajun

    2013-01-01

    The precise control of gene expression is essential for all biological processes. In addition to DNA-binding transcription factors, numerous transcription cofactors contribute another layer of regulation of gene transcription in eukaryotic cells. One of such transcription cofactors is the highly conserved Mediator complex, which has multiple subunits and is involved in various biological processes through directly interacting with relevant transcription factors. Although the current understan...

  20. Activating RNAs associate with Mediator to enhance chromatin architecture and transcription.

    Science.gov (United States)

    Lai, Fan; Orom, Ulf A; Cesaroni, Matteo; Beringer, Malte; Taatjes, Dylan J; Blobel, Gerd A; Shiekhattar, Ramin

    2013-02-28

    Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms. Although the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X-chromosome inactivation and imprinting, different classes of lncRNAs may have varied biological functions. We and others have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their neighbouring genes using a cis-mediated mechanism. To define the precise mode by which such enhancer-like RNAs function, we depleted factors with known roles in transcriptional activation and assessed their role in RNA-dependent activation. Here we report that depletion of the components of the co-activator complex, Mediator, specifically and potently diminished the ncRNA-induced activation of transcription in a heterologous reporter assay using human HEK293 cells. In vivo, Mediator is recruited to ncRNA-a target genes and regulates their expression. We show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10. The Mediator complex harbouring disease- displays diminished ability to associate with activating ncRNAs. Chromosome conformation capture confirmed the presence of DNA looping between the ncRNA-a loci and its targets. Importantly, depletion of Mediator subunits or ncRNA-a reduced the chromatin looping between the two loci. Our results identify the human Mediator complex as the transducer of activating ncRNAs and highlight the importance of Mediator and activating ncRNA association in human disease.

  1. Molecular cloning and expression of heteromeric ACCase subunit genes from Jatropha curcas.

    Science.gov (United States)

    Gu, Keyu; Chiam, Huihui; Tian, Dongsheng; Yin, Zhongchao

    2011-04-01

    Acetyl-CoA carboxylase (ACCase) catalyzes the biotin-dependent carboxylation of acetyl-CoA to produce malonyl-CoA, which is the essential first step in the biosynthesis of long-chain fatty acids. ACCase exists as a multi-subunit enzyme in most prokaryotes and the chloroplasts of most plants and algae, while it is present as a multi-domain enzyme in the endoplasmic reticulum of most eukaryotes. The heteromeric ACCase of higher plants consists of four subunits: an α-subunit of carboxyltransferase (α-CT, encoded by accA gene), a biotin carboxyl carrier protein (BCCP, encoded by accB gene), a biotin carboxylase (BC, encoded by accC gene) and a β-subunit of carboxyltransferase (β-CT, encoded by accD gene). In this study, we cloned and characterized the genes accA, accB1, accC and accD that encode the subunits of heteromeric ACCase in Jatropha (Jatropha curcas), a potential biofuel plant. The full-length cDNAs of the four subunit genes were isolated from a Jatropha cDNA library and by using 5' RACE, whereas the genomic clones were obtained from a Jatropha BAC library. They encode a 771 amino acid (aa) α-CT, a 286-aa BCCP1, a 537-aa BC and a 494-aa β-CT, respectively. The single-copy accA, accB1 and accC genes are nuclear genes, while the accD gene is located in chloroplast genome. Jatropha α-CT, BCCP1, BC and β-CT show high identity to their homologues in other higher plants at amino acid level and contain all conserved domains for ACCase activity. The accA, accB1, accC and accD genes are temporally and spatially expressed in the leaves and endosperm of Jatropha plants, which are regulated by plant development and environmental factors. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  2. Comprehensive analysis of the transcriptional profile of the Mediator complex across human cancer types.

    Science.gov (United States)

    Syring, Isabella; Klümper, Niklas; Offermann, Anne; Braun, Martin; Deng, Mario; Boehm, Diana; Queisser, Angela; von Mässenhausen, Anne; Brägelmann, Johannes; Vogel, Wenzel; Schmidt, Doris; Majores, Michael; Schindler, Anne; Kristiansen, Glen; Müller, Stefan C; Ellinger, Jörg; Shaikhibrahim, Zaki; Perner, Sven

    2016-04-26

    The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking.We therefore performed a comprehensive in silico cancer vs. benign analysis of the Mediator complex subunits (MEDs) for 20 tumor entities using Oncomine datasets. The transcriptional expression profiles across almost all cancer entities showed differentially expressed MEDs as compared to benign tissue. Differential expression of MED8 in renal cell carcinoma (RCC) and MED12 in lung cancer (LCa) were validated and further investigated by immunohistochemical staining on tissue microarrays containing large numbers of specimen. MED8 in clear cell RCC (ccRCC) associated with shorter survival and advanced TNM stage and showed higher expression in metastatic than primary tumors. In vitro, siRNA mediated MED8 knockdown significantly impaired proliferation and motility in ccRCC cell lines, hinting at a role for MED8 to serve as a novel therapeutic target in ccRCC. Taken together, our Mediator complex transcriptome proved to be a valid tool for identifying cancer-related shifts in Mediator complex composition, revealing that MEDs do exhibit cancer specific transcriptional expression profiles.

  3. Phosphorylation of the protein kinase A catalytic subunit is induced by cyclic AMP deficiency and physiological stresses in the fission yeast, Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    McInnis, Brittney; Mitchell, Jessica; Marcus, Stevan

    2010-01-01

    Research highlights: → cAMP deficiency induces phosphorylation of PKA catalytic subunit (Pka1) in S. pombe. → Pka1 phosphorylation is further induced by physiological stresses. → Pka1 phosphorylation is not induced in cells lacking the PKA regulatory subunit. → Results suggest that cAMP-independent Pka1 phosphorylation is stimulatory in nature. -- Abstract: In the fission yeast, Schizosaccharomyces pombe, cyclic AMP (cAMP)-dependent protein kinase (PKA) is not essential for viability under normal culturing conditions, making this organism attractive for investigating mechanisms of PKA regulation. Here we show that S. pombe cells carrying a deletion in the adenylate cyclase gene, cyr1, express markedly higher levels of the PKA catalytic subunit, Pka1, than wild type cells. Significantly, in cyr1Δ cells, but not wild type cells, a substantial proportion of Pka1 protein is hyperphosphorylated. Pka1 hyperphosphorylation is strongly induced in cyr1Δ cells, and to varying degrees in wild type cells, by both glucose starvation and stationary phase stresses, which are associated with reduced cAMP-dependent PKA activity, and by KCl stress, the cellular adaptation to which is dependent on PKA activity. Interestingly, hyperphosphorylation of Pka1 was not detected in either cyr1 + or cyr1Δ S. pombe strains carrying a deletion in the PKA regulatory subunit gene, cgs1, under any of the tested conditions. Our results demonstrate the existence of a cAMP-independent mechanism of PKA catalytic subunit phosphorylation, which we propose could serve as a mechanism for inducing or maintaining specific PKA functions under conditions in which its cAMP-dependent activity is downregulated.

  4. Radioimmunoassay of TSH subunits in thyroid diseases and endocrine opthalmopahty

    International Nuclear Information System (INIS)

    Eder, W.

    1982-01-01

    Highly sensitive radioimmunoassays of hTSH sub-units were developed. The hormone preparations were labelled with 125-iodine according to a modified chloramine -T method, and purified by chromatography using biogel P6 and P60. Rabbit antisera were used as antibodies. Separation of the antibody-bound and of the free antigens was carried out via the double antibody method. The antiserum required for this purpose was obtained from a goat. The sensitivity of the assay was influenced by changing the protein content of the buffer, the incubation volume, the tracer amounts, the incubation time and the incubation temperature. For hTSH-α, the lowest detectable limit was found to be 50 pg/ml, for hTSH-#betta# 20 pg/ml. Thus, the sub-units could be determined for 98% of the patients under review. The #betta#-TSH radioimmunoassay is largely specific, TSH cross-reacts to a degree of 5%. The computerized evoluation was carried out by means of Spline approximation using the Siemens 4004 computer. Precision and accurateness are in compliance with generally accpted criteria. The serum levels of α and #betta# sub-units showed no discordancy with regard to TSH. In all groups of patients examined, the levels of the hormone-specific #betta#-chain were found to be exclusively dependent upon the actual thyroid activity. (orig.) [de

  5. Chemotaxis to cyclic AMP and folic acid is mediated by different G proteins in Dictyostelium discoideum

    NARCIS (Netherlands)

    Kesbeke, Fanja; Haastert, Peter J.M. van; Wit, René J.W. de; Snaar-Jagalska, B. Ewa

    1990-01-01

    Mutant Frigid A (fgdA) of Dictyostelium discoideum is defective in a functional Gα2 subunit of a G protein and is characterized by a complete blockade of the cyclic AMP-mediated sensory transduction steps, including cyclic AMP relay, chemotaxis and the cyclic GMP response. Folic acid-mediated

  6. Toxicity of isoproturon on Saccharomyces cerevisiae growing in mineral medium depends on glutathione-mediated antioxidant capacity.

    OpenAIRE

    Candeias, M; Alves-Pereira, I; Ferreira, Rui

    2011-01-01

    The results revealed an increase of viable cells, after 72 h of culture and an increase of antioxidant power mediated by GSH and GR activity in S. cerevisiae UE-ME3. The adaptive response of UE-ME3 strain to isoproturon, determined in MB, was clearly higher than observed in IGC-3507 strain. So, we presume that the extent of the toxic effect of isoproturon in both yeast strains depends on glutathione-mediated antioxidant capacity.

  7. The beta subunit of casein kinase II

    DEFF Research Database (Denmark)

    Boldyreff, B; Piontek, K; Schmidt-Spaniol, I

    1991-01-01

    cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies....

  8. Characterization of fimbrial subunits from Bordetella species

    NARCIS (Netherlands)

    Mooi, F.R.; Heide, H.G.J. van der; Avest, A.R. ter; Welinder, K.G.; Livey, I.; Zeijst, B.A.M. van der; Gaastra, W.

    Using antisera raised against serotype 2 and 3 fimbrial subunits from Bordetella pertussis, serologically related polypeptides were detected in Bordetella bronchiseptica, Bordetella parapertussis and Bordetella avium strains. The two B. pertussis fimbrial subunits, and three of the serologically

  9. The PKA-C3 catalytic subunit is required in two pairs of interneurons for successful mating of Drosophila.

    Science.gov (United States)

    Cassar, Marlène; Sunderhaus, Elizabeth; Wentzell, Jill S; Kuntz, Sara; Strauss, Roland; Kretzschmar, Doris

    2018-02-06

    Protein kinase A (PKA) has been shown to play a role in a plethora of cellular processes ranging from development to memory formation. Its activity is mediated by the catalytic subunits whereby many species express several paralogs. Drosophila encodes three catalytic subunits (PKA-C1-3) and whereas PKA-C1 has been well studied, the functions of the other two subunits were unknown. PKA-C3 is the orthologue of mammalian PRKX/Pkare and they are structurally more closely related to each other than to other catalytic subunits within their species. PRKX is expressed in the nervous system in mice but its function is also unknown. We now show that the loss of PKA-C3 in Drosophila causes copulation defects, though the flies are active and show no defects in other courtship behaviours. This phenotype is specifically due to the loss of PKA-C3 because PKA-C1 cannot replace PKA-C3. PKA-C3 is expressed in two pairs of interneurons that send projections to the ventro-lateral protocerebrum and the mushroom bodies and that synapse onto motor neurons in the ventral nerve cord. Rescue experiments show that expression of PKA-C3 in these interneurons is sufficient for copulation, suggesting a role in relaying information from the sensory system to motor neurons to initiate copulation.

  10. A multi-subunit Chlamydia vaccine inducing neutralizing antibodies and strong IFN-γ(+) CMI responses protects against a genital infection in minipigs

    DEFF Research Database (Denmark)

    Bøje, Sarah; Olsen, Anja Weinreich; Erneholm, Karin

    2016-01-01

    Chlamydia is the most widespread sexually transmitted bacterial disease and a prophylactic vaccine is highly needed. Ideally, this vaccine is required to induce a combined response of Th1 cell-mediated immune (CMI) response in concert with neutralizing antibodies. Using a novel Göttingen minipig...... animal model, we evaluated the immunogenicity and efficacy of a multi-subunit vaccine formulated in the strong Th1-inducing adjuvant CAF01. We evaluated a mixture of two fusion proteins (Hirep1 and CTH93) designed to promote either neutralizing antibodies or cell-mediated immunity, respectively. Hirep1...

  11. Cr(VI) induces mitochondrial-mediated and caspase-dependent apoptosis through reactive oxygen species-mediated p53 activation in JB6 Cl41 cells

    International Nuclear Information System (INIS)

    Son, Young-Ok; Hitron, J. Andrew; Wang Xin; Chang Qingshan; Pan Jingju; Zhang Zhuo; Liu Jiankang; Wang Shuxia; Lee, Jeong-Chae; Shi Xianglin

    2010-01-01

    Cr(VI) compounds are known to cause serious toxic and carcinogenic effects. Cr(VI) exposure can lead to a severe damage to the skin, but the mechanisms involved in the Cr(VI)-mediated toxicity in the skin are unclear. The present study examined whether Cr(VI) induces cell death by apoptosis or necrosis using mouse skin epidermal cell line, JB6 Cl41 cells. We also investigated the cellular mechanisms of Cr(VI)-induced cell death. This study showed that Cr(VI) induced apoptotic cell death in a dose-dependent manner, as demonstrated by the appearance of cell shrinkage, the migration of cells into the sub-G1 phase, the increase of Annexin V positively stained cells, and the formation of nuclear DNA ladders. Cr(VI) treatment resulted in the increases of mitochondrial membrane depolarization and caspases activation. Electron spin resonance (ESR) and fluorescence analysis revealed that Cr(VI) increased intracellular levels of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion radical in dose-dependent manner. Blockage of p53 by si-RNA transfection suppressed mitochondrial changes of Bcl-2 family composition, mitochondrial membrane depolarization, caspase activation and PARP cleavage, leading to the inhibition of Cr(VI)-induced apoptosis. Further, catalase treatment prevented p53 phosphorylation stimulated by Cr(VI) with the concomitant inhibition of caspase activation. These results suggest that Cr(VI) induced a mitochondrial-mediated and caspase-dependent apoptosis in skin epidermal cells through activation of p53, which are mainly mediated by reactive oxidants generated by the chemical.

  12. Malleable machines in transcription regulation: the mediator complex.

    Directory of Open Access Journals (Sweden)

    Agnes Tóth-Petróczy

    2008-12-01

    Full Text Available The Mediator complex provides an interface between gene-specific regulatory proteins and the general transcription machinery including RNA polymerase II (RNAP II. The complex has a modular architecture (Head, Middle, and Tail and cryoelectron microscopy analysis suggested that it undergoes dramatic conformational changes upon interactions with activators and RNAP II. These rearrangements have been proposed to play a role in the assembly of the preinitiation complex and also to contribute to the regulatory mechanism of Mediator. In analogy to many regulatory and transcriptional proteins, we reasoned that Mediator might also utilize intrinsically disordered regions (IDRs to facilitate structural transitions and transmit transcriptional signals. Indeed, a high prevalence of IDRs was found in various subunits of Mediator from both Saccharomyces cerevisiae and Homo sapiens, especially in the Tail and the Middle modules. The level of disorder increases from yeast to man, although in both organisms it significantly exceeds that of multiprotein complexes of a similar size. IDRs can contribute to Mediator's function in three different ways: they can individually serve as target sites for multiple partners having distinctive structures; they can act as malleable linkers connecting globular domains that impart modular functionality on the complex; and they can also facilitate assembly and disassembly of complexes in response to regulatory signals. Short segments of IDRs, termed molecular recognition features (MoRFs distinguished by a high protein-protein interaction propensity, were identified in 16 and 19 subunits of the yeast and human Mediator, respectively. In Saccharomyces cerevisiae, the functional roles of 11 MoRFs have been experimentally verified, and those in the Med8/Med18/Med20 and Med7/Med21 complexes were structurally confirmed. Although the Saccharomyces cerevisiae and Homo sapiens Mediator sequences are only weakly conserved, the

  13. RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism

    International Nuclear Information System (INIS)

    Yamaki, Nao; Negishi, Manabu; Katoh, Hironori

    2007-01-01

    In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as a Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85α and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1

  14. Kin28 regulates the transient association of Mediator with core promoters.

    Science.gov (United States)

    Jeronimo, Célia; Robert, François

    2014-05-01

    Mediator is an essential, broadly used eukaryotic transcriptional coactivator. How and what Mediator communicates from activators to RNA polymerase II (RNAPII) remains an open question. Here we performed genome-wide location profiling of Saccharomyces cerevisiae Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence, upstream of the pre-initiation complex. In the absence of Kin28 (CDK7) kinase activity or in cells in which the RNAPII C-terminal domain is mutated to replace Ser5 with alanine, however, Mediator accumulates at core promoters together with RNAPII. We propose that Mediator is released quickly from promoters after phosphorylation of Ser5 by Kin28 (CDK7), which also allows for RNAPII to escape from the promoter.

  15. The functions of Mediator in Candida albicans support a role in shaping species-specific gene expression.

    Directory of Open Access Journals (Sweden)

    Nathalie Uwamahoro

    Full Text Available The Mediator complex is an essential co-regulator of RNA polymerase II that is conserved throughout eukaryotes. Here we present the first study of Mediator in the pathogenic fungus Candida albicans. We focused on the Middle domain subunit Med31, the Head domain subunit Med20, and Srb9/Med13 from the Kinase domain. The C. albicans Mediator shares some roles with model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, such as functions in the response to certain stresses and the role of Med31 in the expression of genes regulated by the activator Ace2. The C. albicans Mediator also has additional roles in the transcription of genes associated with virulence, for example genes related to morphogenesis and gene families enriched in pathogens, such as the ALS adhesins. Consistently, Med31, Med20, and Srb9/Med13 contribute to key virulence attributes of C. albicans, filamentation, and biofilm formation; and ALS1 is a biologically relevant target of Med31 for development of biofilms. Furthermore, Med31 affects virulence of C. albicans in the worm infection model. We present evidence that the roles of Med31 and Srb9/Med13 in the expression of the genes encoding cell wall adhesins are different between S. cerevisiae and C. albicans: they are repressors of the FLO genes in S. cerevisiae and are activators of the ALS genes in C. albicans. This suggests that Mediator subunits regulate adhesion in a distinct manner between these two distantly related fungal species.

  16. Grb2 mediates semaphorin-4D-dependent RhoA inactivation.

    Science.gov (United States)

    Sun, Tianliang; Krishnan, Rameshkumar; Swiercz, Jakub M

    2012-08-01

    Signaling through the semaphorin 4D (Sema4D) receptor plexin-B1 is modulated by its interaction with tyrosine kinases ErbB-2 and Met. In cells expressing the plexin-B1-ErbB-2 receptor complex, ligand stimulation results in the activation of small GTPase RhoA and stimulation of cellular migration. By contrast, in cells expressing plexin-B1 and Met, ligand stimulation results in an association with the RhoGTPase-activating protein p190 RhoGAP and subsequent RhoA inactivation--a process that involves the tyrosine phosphorylation of plexin-B1 by Met. Inactivation of RhoA is necessary for Sema4D-mediated inhibition of cellular migration. It is, however, unknown how plexin-B1 phosphorylation regulates RhoGAP interaction and activity. Here we show that the activation of plexin-B1 by Sema4D and its subsequent tyrosine phosphorylation by Met creates a docking site for the SH2 domain of growth factor receptor bound-2 (Grb2). Grb2 is thereby recruited into the plexin-B1 receptor complex and, through its SH3 domain, interacts with p190 RhoGAP and mediates RhoA deactivation. Phosphorylation of plexin-B1 by Met and the recruitment of Grb2 have no effect on the R-RasGAP activity of plexin-B1, but are required for Sema4D-induced, RhoA-dependent antimigratory effects of Sema4D on breast cancer cells. These data show Grb2 as a direct link between plexin and p190-RhoGAP-mediated downstream signaling.

  17. Mediator Tail Module Is Required for Tac1-Activated CDR1 Expression and Azole Resistance in Candida albicans.

    Science.gov (United States)

    Liu, Zhongle; Myers, Lawrence C

    2017-11-01

    The human fungal pathogen Candida albicans develops drug resistance after long-term exposure to azole drugs in the treatment of chronic candidiasis. Gain-of-function (GOF) mutations in the transcription factor Tac1 and the consequent expression of its targets, drug efflux pumps Cdr1 and Cdr2, are a common mechanism by which C. albicans acquires fluconazole resistance. The mechanism by which GOF mutations hyperactivate Tac1 is currently unknown. Here, we define a transcriptional activation domain (TAD) at the C terminus of Tac1. GOF mutations within the Tac1 TAD, outside the context of full-length Tac1, generally do not enhance its absolute potential as a transcriptional activator. Negative regulation of the Tac1 TAD by the Tac1 middle region is necessary for the activating effect of GOF mutations or fluphenazine to be realized. We have found that full-length Tac1, when hyperactivated by xenobiotics or GOF mutations, facilitates the recruitment of the Mediator coactivator complex to the CDR1 promoter. Azole resistance and the activation of Tac1 target genes, such as CDR1 , are dependent on the Tac1 TAD and subunits of the Mediator tail module. The dependence of different Tac1 target promoters on the Mediator tail module, however, varies widely. Lastly, we show that hyperactivation of Tac1 is correlated with its Mediator-dependent phosphorylation, a potentially useful biomarker for Tac1 hyperactivation. The role of Mediator in events downstream of Tac1 hyperactivation in fluconazole-resistant clinical isolates is complex and provides opportunities and challenges for therapeutic intervention. Copyright © 2017 American Society for Microbiology.

  18. Transcription regulation by the Mediator complex.

    Science.gov (United States)

    Soutourina, Julie

    2018-04-01

    Alterations in the regulation of gene expression are frequently associated with developmental diseases or cancer. Transcription activation is a key phenomenon in the regulation of gene expression. In all eukaryotes, mediator of RNA polymerase II transcription (Mediator), a large complex with modular organization, is generally required for transcription by RNA polymerase II, and it regulates various steps of this process. The main function of Mediator is to transduce signals from the transcription activators bound to enhancer regions to the transcription machinery, which is assembled at promoters as the preinitiation complex (PIC) to control transcription initiation. Recent functional studies of Mediator with the use of structural biology approaches and functional genomics have revealed new insights into Mediator activity and its regulation during transcription initiation, including how Mediator is recruited to transcription regulatory regions and how it interacts and cooperates with PIC components to assist in PIC assembly. Novel roles of Mediator in the control of gene expression have also been revealed by showing its connection to the nuclear pore and linking Mediator to the regulation of gene positioning in the nuclear space. Clear links between Mediator subunits and disease have also encouraged studies to explore targeting of this complex as a potential therapeutic approach in cancer and fungal infections.

  19. K-Cl Cotransporter 2–mediated Cl− Extrusion Determines Developmental Stage–dependent Impact of Propofol Anesthesia on Dendritic Spines

    KAUST Repository

    Puskarjov, Martin; Fiumelli, Hubert; Briner, Adrian; Bodogan, Timea; Demeter, Kornel; Lacoh, Claudia Marvine; Mavrovic, Martina; Blaesse, Peter; Kaila, Kai; Vutskits, Laszlo

    2017-01-01

    Background: General anesthetics potentiating γ-aminobutyric acid (GABA)-mediated signaling are known to induce a persistent decrement in excitatory synapse number in the cerebral cortex when applied during early postnatal development, while an opposite action is produced at later stages. Here, the authors test the hypothesis that the effect of general anesthetics on synaptogenesis depends upon the efficacy of GABA receptor type A (GABA A)-mediated inhibition controlled by the developmental up-regulation of the potassium-chloride (K-Cl) cotransporter 2 (KCC2). Methods: In utero electroporation of KCC2 was used to prematurely increase the efficacy of (GABA A)-mediated inhibition in layer 2/3 pyramidal neurons in the immature rat somatosensory cortex. Parallel experiments with expression of the inward-rectifier potassium channel Kir2.1 were done to reduce intrinsic neuronal excitability. The effects of these genetic manipulations (n = 3 to 4 animals per experimental group) were evaluated using iontophoretic injection of Lucifer Yellow (n = 8 to 12 cells per animal). The total number of spines analyzed per group ranged between 907 and 3,371. Results: The authors found a robust effect of the developmental up-regulation of KCC2-mediated Cl - transport on the age-dependent action of propofol on dendritic spines. Premature expression of KCC2, unlike expression of a transport-inactive KCC2 variant, prevented a propofol-induced decrease in spine density. In line with a reduction in neuronal excitability, the above result was qualitatively replicated by overexpression of Kir2.1. Conclusions: The KCC2-dependent developmental increase in the efficacy of GABA A -mediated inhibition is a major determinant of the age-dependent actions of propofol on dendritic spinogenesis.

  20. K-Cl Cotransporter 2–mediated Cl− Extrusion Determines Developmental Stage–dependent Impact of Propofol Anesthesia on Dendritic Spines

    KAUST Repository

    Puskarjov, Martin

    2017-03-16

    Background: General anesthetics potentiating γ-aminobutyric acid (GABA)-mediated signaling are known to induce a persistent decrement in excitatory synapse number in the cerebral cortex when applied during early postnatal development, while an opposite action is produced at later stages. Here, the authors test the hypothesis that the effect of general anesthetics on synaptogenesis depends upon the efficacy of GABA receptor type A (GABA A)-mediated inhibition controlled by the developmental up-regulation of the potassium-chloride (K-Cl) cotransporter 2 (KCC2). Methods: In utero electroporation of KCC2 was used to prematurely increase the efficacy of (GABA A)-mediated inhibition in layer 2/3 pyramidal neurons in the immature rat somatosensory cortex. Parallel experiments with expression of the inward-rectifier potassium channel Kir2.1 were done to reduce intrinsic neuronal excitability. The effects of these genetic manipulations (n = 3 to 4 animals per experimental group) were evaluated using iontophoretic injection of Lucifer Yellow (n = 8 to 12 cells per animal). The total number of spines analyzed per group ranged between 907 and 3,371. Results: The authors found a robust effect of the developmental up-regulation of KCC2-mediated Cl - transport on the age-dependent action of propofol on dendritic spines. Premature expression of KCC2, unlike expression of a transport-inactive KCC2 variant, prevented a propofol-induced decrease in spine density. In line with a reduction in neuronal excitability, the above result was qualitatively replicated by overexpression of Kir2.1. Conclusions: The KCC2-dependent developmental increase in the efficacy of GABA A -mediated inhibition is a major determinant of the age-dependent actions of propofol on dendritic spinogenesis.

  1. p18(Hamlet) mediates different p53-dependent responses to DNA-damage inducing agents.

    Science.gov (United States)

    Lafarga, Vanesa; Cuadrado, Ana; Nebreda, Angel R

    2007-10-01

    Cells organize appropriate responses to environmental cues by activating specific signaling networks. Two proteins that play key roles in coordinating stress responses are the kinase p38alpha (MAPK14) and the transcription factor p53 (TP53). Depending on the nature and the extent of the stress-induced damage, cells may respond by arresting the cell cycle or by undergoing cell death, and these responses are usually associated with the phosphorylation of particular substrates by p38alpha as well as the activation of specific target genes by p53. We recently characterized a new p38alpha substrate, named p18(Hamlet) (ZNHIT1), which mediates p53-dependent responses to different genotoxic stresses. Thus, cisplatin or UV light induce stabilization of the p18(Hamlet) protein, which then enhances the ability of p53 to bind to and activate the promoters of pro-apoptotic genes such as NOXA and PUMA leading to apoptosis induction. In a similar way, we report here that p18(Hamlet) can also mediate the cell cycle arrest induced in response to gamma-irradiation, by participating in the p53-dependent upregulation of the cell cycle inhibitor p21(Cip1) (CDKN1A).

  2. The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a positive effect of caffeine and potential role of microtubules.

    Science.gov (United States)

    Tariqul Islam, A F M; Yue, Haicen; Scavello, Margarethakay; Haldeman, Pearce; Rappel, Wouter-Jan; Charest, Pascale G

    2018-08-01

    To study the dynamics and mechanisms controlling activation of the heterotrimeric G protein Gα2βγ in Dictyostelium in response to stimulation by the chemoattractant cyclic AMP (cAMP), we monitored the G protein subunit interaction in live cells using bioluminescence resonance energy transfer (BRET). We found that cAMP induces the cAR1-mediated dissociation of the G protein subunits to a similar extent in both undifferentiated and differentiated cells, suggesting that only a small number of cAR1 (as expressed in undifferentiated cells) is necessary to induce the full activation of Gα2βγ. In addition, we found that treating cells with caffeine increases the potency of cAMP-induced Gα2βγ activation; and that disrupting the microtubule network but not F-actin inhibits the cAMP-induced dissociation of Gα2βγ. Thus, microtubules are necessary for efficient cAR1-mediated activation of the heterotrimeric G protein. Finally, kinetics analyses of Gα2βγ subunit dissociation induced by different cAMP concentrations indicate that there are two distinct rates at which the heterotrimeric G protein subunits dissociate when cells are stimulated with cAMP concentrations above 500 nM versus only one rate at lower cAMP concentrations. Quantitative modeling suggests that the kinetics profile of Gα2βγ subunit dissociation results from the presence of both uncoupled and G protein pre-coupled cAR1 that have differential affinities for cAMP and, consequently, induce G protein subunit dissociation through different rates. We suggest that these different signaling kinetic profiles may play an important role in initial chemoattractant gradient sensing. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. 21-O-Angeloyltheasapogenol E3, a Novel Triterpenoid Saponin from the Seeds of Tea Plants, Inhibits Macrophage-Mediated Inflammatory Responses in a NF-κB-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Woo Seok Yang

    2014-01-01

    Full Text Available 21-O-Angeloyltheasapogenol E3 (ATS-E3 is a triterpenoid saponin recently isolated from the seeds of the tea tree Camellia sinensis (L. O. Kuntze. ATS-E3 has several beneficial properties including anti-inflammatory, antidiabetic, antiatherosclerotic, and anticancer effects. Unlike other phenolic compounds isolated from tea plants, there are no studies reporting the pharmacological action of ATS-E3. In this study, we therefore aimed to explore the cellular and molecular inhibitory activities of ATS-E3 in macrophage-mediated inflammatory responses. ATS-E3 remarkably diminished cellular responses of macrophages such as FITC-dextran-induced phagocytic uptake, sodium nitroprusside- (SNP- induced radical generation, and LPS-induced nitric oxide (NO production. Analysis of its molecular activity showed that this compound significantly suppressed the expression of inducible NO synthase (iNOS, nuclear translocation of nuclear factor- (NF- κB subunits (p50 and p65, phosphorylation of inhibitor of κB kinase (IKK, and the enzyme activity of AKT1. Taken together, the novel triterpenoid saponin compound ATS-E3 contributes to the beneficial effects of tea plants by exerting anti-inflammatory and antioxidative activities in an AKT/IKK/NF-κB-dependent manner.

  4. Myristoylated α subunits of guanine nucleotide-binding regulatory proteins

    International Nuclear Information System (INIS)

    Buss, J.E.; Mumby, S.M.; Casey, P.J.; Gilman, A.G.; Sefton, B.M.

    1987-01-01

    Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the α subunits of G/sub s/ (stimulatory) (α 45 and α 52 ), a 41-kDa subunit of G/sub i/ (inhibitory) (α 41 ), a 40-kDa protein (α 40 ), and the 36-kDa β subunit. No protein that comigrated with the α subunit of G 0 (unknown function) (α 39 ) was detected. In cells grown in the presence of [ 3 H]myristic acid, α 41 and α 40 contained 3 H label, while the β subunit did not. Chemical analysis of lipids attached covalently to purified α 41 and α 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein β and γ subunits and of G/sub t/ (Transducin) subunits (α, β, and γ) failed to reveal fatty acids. The fatty acid associated with α 41 , α 40 , and α 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins

  5. The Subunit Principle in Scar Face Revision.

    Science.gov (United States)

    Elshahat, Ahmed; Lashin, Riham

    2017-06-01

    Facial scaring is considered one of the most difficult cosmetic problems for any plastic surgeon to solve. The condition is more difficult if the direction of the scar is not parallel to relaxed skin tension lines. Attempts to manage this difficult situation included revisions using geometric designs, Z plasties or W plasties to camouflage the straight line visible scaring. The use of long-lasting resorbable sutures was tried too. Recently, the use of botulinum toxin during revision improved the results. Fractional CO2 lasers, microfat grafts, and platelet-rich plasma were added to the armamentarium. The scar is least visible if placed in the junction between the facial subunits. The aim of this study is to investigate the use of the subunit principle to improve the results of scar revision. Four patients were included in this study. Tissue expansion of the intact part of the subunit allowed shifting the scar to the junction between the affected subunit and the adjacent one. Tissue expansion, delivery of the expanders, and advancement of the flaps were successful in all patients. The fact that this is a 2-stage procedure and sacrifices some of the intact skin from the affected facial subunit, makes this technique reserved to patients with ugly facial scars who are ambitious to improve their appearance.

  6. Effects of visual deprivation during brain development on expression of AMPA receptor subunits in rat’s hippocampus

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    Sayyed Alireza Talaei

    2015-06-01

    Conclusion: Dark rearing of rats during critical period of brain development changes the relative expression and also arrangement of both AMPA receptor subunits, GluR1 and GluR2 in the hippocampus, age dependently.

  7. Cell adhesion-mediated radioresistance (CAM-RR). Extracellular matrix-dependent improvement of cell survival in human tumor and normal cells in vitro

    International Nuclear Information System (INIS)

    Cordes, N.; Meineke, V.

    2003-01-01

    Background: Cell-extracellular matrix (ECM) contact is thought to have great impact on cellular mechanisms resulting in increased cell survival upon exposure to ionizing radiation. Several human tumor cell lines and normal human fibroblastic cell strains of different origin, all of them expressing the wide-spread and important integrin subunit β1, were irradiated, and clonogenic cell survival, β1-integrin cell surface expression, and adhesive functionality were investigated. Material and Methods: Human tumor cell lines A172 (glioblastoma), PATU8902 (pancreas carcinoma), SKMES1 (lung carcinoma), A549 (lung carcinoma), and IPC298 (melanoma) as well as normal human skin (HSF1) and lung fibroblasts (CCD32) and human keratinocytes (HaCaT) were irradiated with 0-8 Gy. Besides colony formation assays, β1-integrin cell surface expression by flow cytometry and adhesive functionality by adhesion assays were analyzed. Results: All cell lines showed improved clonogenic survival after irradiation in the presence of fibronectin as compared to plastic. Irradiated cells exhibited a significant, dose-dependent increase in β1-integrin cell surface expression following irradiation. As a parameter of the adhesive functionality of the β1-integrin, a radiation-dependent elevation of cell adhesion to fibronectin in comparison with adhesion to plastic was demonstrated. Conclusion: The in vitro cellular radiosensitivity is highly influenced by fibronectin according to the phenomenon of cell adhesion-mediated radioresistance. Additionally, our emerging data question the results of former and current in vitro cytotoxicity studies performed in the absence of an ECM. These findings might also be important for the understanding of malignant transformation, anchorage-independent cell growth, optimization of radiotherapeutic regimes and the prevention of normal tissue side effects on the basis of experimental radiobiological data. (orig.)

  8. In cellulo examination of a beta-alpha hybrid construct of beta-hexosaminidase A subunits, reported to interact with the GM2 activator protein and hydrolyze GM2 ganglioside.

    Directory of Open Access Journals (Sweden)

    Incilay Sinici

    Full Text Available The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively, and the GM2-activator protein (GM2AP, encoded by the GM2A gene. Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750. Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1 and a new more extensive hybrid (H2, with our documented in cellulo (live cell- based assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.

  9. The Mediator co-activator complex regulates Ty1 retromobility by controlling the balance between Ty1i and Ty1 promoters.

    Science.gov (United States)

    Salinero, Alicia C; Knoll, Elisabeth R; Zhu, Z Iris; Landsman, David; Curcio, M Joan; Morse, Randall H

    2018-02-01

    The Ty1 retrotransposons present in the genome of Saccharomyces cerevisiae belong to the large class of mobile genetic elements that replicate via an RNA intermediary and constitute a significant portion of most eukaryotic genomes. The retromobility of Ty1 is regulated by numerous host factors, including several subunits of the Mediator transcriptional co-activator complex. In spite of its known function in the nucleus, previous studies have implicated Mediator in the regulation of post-translational steps in Ty1 retromobility. To resolve this paradox, we systematically examined the effects of deleting non-essential Mediator subunits on the frequency of Ty1 retromobility and levels of retromobility intermediates. Our findings reveal that loss of distinct Mediator subunits alters Ty1 retromobility positively or negatively over a >10,000-fold range by regulating the ratio of an internal transcript, Ty1i, to the genomic Ty1 transcript. Ty1i RNA encodes a dominant negative inhibitor of Ty1 retromobility that blocks virus-like particle maturation and cDNA synthesis. These results resolve the conundrum of Mediator exerting sweeping control of Ty1 retromobility with only minor effects on the levels of Ty1 genomic RNA and the capsid protein, Gag. Since the majority of characterized intrinsic and extrinsic regulators of Ty1 retromobility do not appear to effect genomic Ty1 RNA levels, Mediator could play a central role in integrating signals that influence Ty1i expression to modulate retromobility.

  10. Activation of glutathione peroxidase via Nrf1 mediates genistein's protection against oxidative endothelial cell injury

    International Nuclear Information System (INIS)

    Hernandez-Montes, Eva; Pollard, Susan E.; Vauzour, David; Jofre-Montseny, Laia; Rota, Cristina; Rimbach, Gerald; Weinberg, Peter D.; Spencer, Jeremy P.E.

    2006-01-01

    Cellular actions of isoflavones may mediate the beneficial health effects associated with high soy consumption. We have investigated protection by genistein and daidzein against oxidative stress-induced endothelial injury. Genistein but not daidzein protected endothelial cells from damage induced by oxidative stress. This protection was accompanied by decreases in intracellular glutathione levels that could be explained by the generation of glutathionyl conjugates of the oxidised genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone. Both isoflavones evoked increased protein expression of γ-glutamylcysteine synthetase-heavy subunit (γ-GCS-HS) and increased cytosolic accumulation and nuclear translocation of Nrf2. However, only genistein led to increases in the cytosolic accumulation and nuclear translocation of Nrf1 and the increased expression of and activity of glutathione peroxidase. These results suggest that genistein-induced protective effects depend primarily on the activation of glutathione peroxidase mediated by Nrf1 activation, and not on Nrf2 activation or increases in glutathione synthesis

  11. Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal subunit in Escherichia coli cells

    International Nuclear Information System (INIS)

    Shigeno, Yuta; Uchiumi, Toshio; Nomura, Takaomi

    2016-01-01

    Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly, cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation. - Highlights: • We constructed an in vivo functional assay system for Escherichia coli ribosomal protein L6. • Growth of an E. coli ΔL6 mutant was biphasic when L6 levels were depleted. • The ΔL6 mutant accumulated 50S ribosomal subunit precursors that sedimented at 45S. • L6 is a key player in the late stage of E. coli 50S subunit assembly.

  12. Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal subunit in Escherichia coli cells

    Energy Technology Data Exchange (ETDEWEB)

    Shigeno, Yuta [Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567 (Japan); Uchiumi, Toshio [Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181 (Japan); Nomura, Takaomi, E-mail: nomurat@shinshu-u.ac.jp [Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567 (Japan)

    2016-04-22

    Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly, cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation. - Highlights: • We constructed an in vivo functional assay system for Escherichia coli ribosomal protein L6. • Growth of an E. coli ΔL6 mutant was biphasic when L6 levels were depleted. • The ΔL6 mutant accumulated 50S ribosomal subunit precursors that sedimented at 45S. • L6 is a key player in the late stage of E. coli 50S subunit assembly.

  13. Suppression of 19S proteasome subunits marks emergence of an altered cell state in diverse cancers.

    Science.gov (United States)

    Tsvetkov, Peter; Sokol, Ethan; Jin, Dexter; Brune, Zarina; Thiru, Prathapan; Ghandi, Mahmoud; Garraway, Levi A; Gupta, Piyush B; Santagata, Sandro; Whitesell, Luke; Lindquist, Susan

    2017-01-10

    The use of proteasome inhibitors to target cancer's dependence on altered protein homeostasis has been greatly limited by intrinsic and acquired resistance. Analyzing data from thousands of cancer lines and tumors, we find that those with suppressed expression of one or more 19S proteasome subunits show intrinsic proteasome inhibitor resistance. Moreover, such proteasome subunit suppression is associated with poor outcome in myeloma patients, where proteasome inhibitors are a mainstay of treatment. Beyond conferring resistance to proteasome inhibitors, proteasome subunit suppression also serves as a sentinel of a more global remodeling of the transcriptome. This remodeling produces a distinct gene signature and new vulnerabilities to the proapoptotic drug, ABT-263. This frequent, naturally arising imbalance in 19S regulatory complex composition is achieved through a variety of mechanisms, including DNA methylation, and marks the emergence of a heritably altered and therapeutically relevant state in diverse cancers.

  14. A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis

    DEFF Research Database (Denmark)

    Siegel, Gabriele; Obernosterer, Gregor; Fiore, Roberto

    2009-01-01

    of acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including the alpha(13) subunits of G proteins (Galpha(13)). RNA-interference-mediated knockdown of APT1 and the expression of membrane-localized Galpha(13) both...... suppress spine enlargement caused by inhibition of miR-138, suggesting that APT1-regulated depalmitoylation of Galpha(13) might be an important downstream event of miR-138 function. Our results uncover a previously unknown miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized...

  15. Efficient expression of functional (α6β22β3 AChRs in Xenopus oocytes from free subunits using slightly modified α6 subunits.

    Directory of Open Access Journals (Sweden)

    Carson Kai-Kwong Ley

    Full Text Available Human (α6β2(α4β2β3 nicotinic acetylcholine receptors (AChRs are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β22β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β22β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells.

  16. Inhibition of the 26S proteasome blocks progesterone receptor-dependent transcription through failed recruitment of RNA polymerase II.

    Science.gov (United States)

    Dennis, Andrew P; Lonard, David M; Nawaz, Zafar; O'Malley, Bert W

    2005-03-01

    In the present study, we investigated the involvement of protein degradation via the 26S proteasome during progesterone receptor (PR)-mediated transcription in T-47D cells containing a stably integrated MMTV-CAT reporter construct (CAT0 cells). Progesterone induced CAT and HSD11beta2 transcription while co-treatment with the proteasome inhibitor, MG132, blocked PR-induced transcription in a time-dependent fashion. MG132 treatment also inhibited transcription of beta-actin and cyclophilin, but not two proteasome subunit genes, PSMA1 and PSMC1, indicating that proteasome inhibition affects a subset of RNA polymerase II (RNAP(II))-regulated genes. Progesterone-mediated recruitment of RNAP(II) was blocked by MG132 treatment at time points later than 1 h that was not dependent on the continued presence of PR, associated cofactors, and components of the general transcription machinery, supporting the concept that proteasome-mediated degradation is needed for continued transcription. Surprisingly, progesterone-mediated acetylation of histone H4 was inhibited by MG132 with the concomitant recruitment of HDAC3, NCoR, and SMRT. We demonstrate that the steady-state protein levels of SMRT and NCoR are higher in the presence of MG132 in CAT0 cells, consistent with other reports that SMRT and NCoR are targets of the 26S proteasome. However, inhibition of histone deacetylation by trichostatin A (TSA) treatment or SMRT/NCoR knockdown by siRNA did not restore MG132-inhibited progesterone-dependent transcription. Therefore, events other than histone deacetylation and stability of SMRT and NCoR must also play a role in inhibition of PR-mediated transcription.

  17. Interleukin-10 overexpression promotes Fas-ligand-dependent chronic macrophage-mediated demyelinating polyneuropathy.

    Directory of Open Access Journals (Sweden)

    Dru S Dace

    Full Text Available BACKGROUND: Demyelinating polyneuropathy is a debilitating, poorly understood disease that can exist in acute (Guillain-Barré syndrome or chronic forms. Interleukin-10 (IL-10, although traditionally considered an anti-inflammatory cytokine, has also been implicated in promoting abnormal angiogenesis in the eye and in the pathobiology of autoimmune diseases such as lupus and encephalomyelitis. PRINCIPAL FINDINGS: Overexpression of IL-10 in a transgenic mouse model leads to macrophage-mediated demyelinating polyneuropathy. IL-10 upregulates ICAM-1 within neural tissues, promoting massive macrophage influx, inflammation-induced demyelination, and subsequent loss of neural tissue resulting in muscle weakness and paralysis. The primary insult is to perineural myelin followed by secondary axonal loss. Infiltrating macrophages within the peripheral nerves demonstrate a highly pro-inflammatory signature. Macrophages are central players in the pathophysiology, as in vivo depletion of macrophages using clodronate liposomes reverses the phenotype, including progressive nerve loss and paralysis. Macrophage-mediate demyelination is dependent on Fas-ligand (FasL-mediated Schwann cell death. SIGNIFICANCE: These findings mimic the human disease chronic idiopathic demyelinating polyneuropathy (CIDP and may also promote further understanding of the pathobiology of related conditions such as acute idiopathic demyelinating polyneuropathy (AIDP or Guillain-Barré syndrome.

  18. Alternative Splicing of AMPA subunits in Prefrontal Cortical Fields of Cynomolgus Monkeys following Chronic Ethanol Self-Administration

    Directory of Open Access Journals (Sweden)

    Glen eAcosta

    2012-01-01

    Full Text Available Functional impairment of the orbital and medial prefrontal cortex underlies deficits in executive control that characterize addictive disorders, including alcohol addiction. Previous studies indicate that alcohol alters glutamate neurotransmission and one substrate of these effects may be through the reconfiguration of the subunits constituting ionotropic glutamate receptor (iGluR complexes. Glutamatergic transmission is integral to cortico-cortical and cortico-subcortical communication and alcohol-induced changes in the abundance of the receptor subunits and/or their splice variants may result in critical functional impairments of prefrontal cortex in alcohol dependence. To this end, the effects of chronic ethanol self-administration on glutamate receptor ionotropic AMPA (GRIA subunit variant and kainate (GRIK subunit mRNA expression were studied in the orbitofrontal cortex (OFC, dorsolateral prefrontal cortex (DLPFC and anterior cingulate cortex (ACC of male cynomolgus monkeys. In DLPFC, total AMPA splice variant expression and total kainate receptor subunit expression were significantly decreased in alcohol drinking monkeys. Expression levels of GRIA3 flip and flop and GRIA4 flop mRNAs in this region were positively correlated with daily ethanol intake and blood ethanol concentrations averaged over the six months prior to necropsy. In OFC, AMPA subunit splice variant expression was reduced in the alcohol treated group. GRIA2 flop mRNA levels in this region were positively correlated with daily ethanol intake and blood ethanol concentrations averaged over the six months prior to necropsy. Results from these studies provide further evidence of transcriptional regulation of iGluR subunits in the primate brain following chronic alcohol self-administration. Additional studies examining the cellular localization of such effects in the framework of primate prefrontal cortical circuitry are warranted.

  19. Heterotrimeric G proteins-mediated resistance to necrotrophic pathogens includes mechanisms independent of salicylic acid-, jasmonic acid/ethylene- and abscisic acid-mediated defense signaling.

    Science.gov (United States)

    Trusov, Yuri; Sewelam, Nasser; Rookes, James Edward; Kunkel, Matt; Nowak, Ekaterina; Schenk, Peer Martin; Botella, José Ramón

    2009-04-01

    Heterotrimeric G proteins are involved in the defense response against necrotrophic fungi in Arabidopsis. In order to elucidate the resistance mechanisms involving heterotrimeric G proteins, we analyzed the effects of the Gβ (subunit deficiency in the mutant agb1-2 on pathogenesis-related gene expression, as well as the genetic interaction between agb1-2 and a number of mutants of established defense pathways. Gβ-mediated signaling suppresses the induction of salicylic acid (SA)-, jasmonic acid (JA)-, ethylene (ET)- and abscisic acid (ABA)-dependent genes during the initial phase of the infection with Fusarium oxysporum (up to 48 h after inoculation). However, at a later phase it enhances JA/ET-dependent genes such as PDF1.2 and PR4. Quantification of the Fusarium wilt symptoms revealed that Gβ- and SA-deficient mutants were more susceptible than wild-type plants, whereas JA- and ET-insensitive and ABA-deficient mutants demonstrated various levels of resistance. Analysis of the double mutants showed that the Gβ-mediated resistance to F. oxysporum and Alternaria brassicicola was mostly independent of all of the previously mentioned pathways. However, the progressive decay of agb1-2 mutants was compensated by coi1-21 and jin1-9 mutations, suggesting that at this stage of F. oxysporum infection Gβ acts upstream of COI1 and ATMYC2 in JA signaling. © 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.

  20. Mode of ATM-dependent suppression of chromosome translocation

    Energy Technology Data Exchange (ETDEWEB)

    Yamauchi, Motohiro, E-mail: motoyama@nagasaki-u.ac.jp [Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Suzuki, Keiji; Oka, Yasuyoshi; Suzuki, Masatoshi; Kondo, Hisayoshi; Yamashita, Shunichi [Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan)

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer We addressed how ATM suppresses frequency of chromosome translocation. Black-Right-Pointing-Pointer We found ATM/p53-dependent G1 checkpoint suppresses translocation frequency. Black-Right-Pointing-Pointer We found ATM and DNA-PKcs function in a common pathway to suppress translocation. -- Abstract: It is well documented that deficiency in ataxia telangiectasia mutated (ATM) protein leads to elevated frequency of chromosome translocation, however, it remains poorly understood how ATM suppresses translocation frequency. In the present study, we addressed the mechanism of ATM-dependent suppression of translocation frequency. To know frequency of translocation events in a whole genome at once, we performed centromere/telomere FISH and scored dicentric chromosomes, because dicentric and translocation occur with equal frequency and by identical mechanism. By centromere/telomere FISH analysis, we confirmed that chemical inhibition or RNAi-mediated knockdown of ATM causes 2 to 2.5-fold increase in dicentric frequency at first mitosis after 2 Gy of gamma-irradiation in G0/G1. The FISH analysis revealed that ATM/p53-dependent G1 checkpoint suppresses dicentric frequency, since RNAi-mediated knockdown of p53 elevated dicentric frequency by 1.5-fold. We found ATM also suppresses dicentric occurrence independently of its checkpoint role, as ATM inhibitor showed additional effect on dicentric frequency in the context of p53 depletion and Chk1/2 inactivation. Epistasis analysis using chemical inhibitors revealed that ATM kinase functions in the same pathway that requires kinase activity of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to suppress dicentric frequency. From the results in the present study, we conclude that ATM minimizes translocation frequency through its commitment to G1 checkpoint and DNA double-strand break repair pathway that requires kinase activity of DNA-PKcs.

  1. Coping mediates the effects of cognitive-behavioral therapy for alcohol use disorder among out-patient clients in Project MATCH when dependence severity is high.

    Science.gov (United States)

    Roos, Corey R; Maisto, Stephen A; Witkiewitz, Katie

    2017-09-01

    There is inconsistent evidence that alcohol-specific coping is a mechanism of change in cognitive-behavioral therapy (CBT) for alcohol use disorder (AUD). Our primary aim was to test whether baseline dependence severity moderates the mediational effect of CBT on drinking outcomes via coping. Secondary data analysis of Project MATCH , a multi-site alcohol treatment trial in which participants, recruited in out-patient and aftercare arms, were randomized to three treatments: CBT, motivational enhancement therapy (MET) and Twelve-Step facilitation (TSF). Nine research sites in the United States. A total of 1063 adults with AUD. The primary outcomes were percentage days abstinent and percentage heavy drinking days at the 1-year follow-up. Coping was assessed with the Processes of Change Questionnaire . Dependence severity was measured with the Alcohol Dependence Scale . Among the full available sample (across treatment arms), there were no significant moderated mediation effects. Double moderated mediation analyses indicated that several moderated mediation effects were moderated by treatment arm (all P cognitive-behavioral therapy for alcohol use disorder was conditional on dependence severity. End-of-treatment coping mediated the positive treatment effects of cognitive-behavioral therapy on 1-year drinking outcomes among out-patient clients when dependence severity was high, but not when dependence severity was low or moderate. © 2017 Society for the Study of Addiction.

  2. The thermal structural transition of alpha-crystallin modulates subunit interactions and increases protein solubility.

    Directory of Open Access Journals (Sweden)

    Giuseppe Maulucci

    Full Text Available BACKGROUND: Alpha crystallin is an oligomer composed of two types of subunits, alpha-A and alpha-B crystallin, and is the major constituent of human lens. The temperature induced condensation of alpha-crystallin, the main cause for eye lens opacification (cataract, is a two step-process, a nucleation followed by an aggregation phase, and a protective effect towards the aggregation is exhibited over the alpha crystallin phase transition temperature (Tc = 318.16 K. METHODS/RESULTS: To investigate if a modulation of the subunit interactions over Tc could trigger the protective mechanism towards the aggregation, we followed, by using simultaneously static and dynamic light scattering, the temperature induced condensation of alpha-crystallin. By developing a mathematical model able to uncouple the nucleation and aggregation processes, we find a previously unobserved transition in the nucleation rate constant. Its temperature dependence allows to determine fundamental structural parameters, the chemical potential (Δμ and the interfacial tension (γ of the aggregating phase, that characterize subunit interactions. CONCLUSIONS/GENERAL SIGNIFICANCE: The decrease of both Δμ and γ at Tc, and a relative increase in solubility, reveal a significative decrease in the strenght of alpha-crystallin subunits interactions, which protects from supramolecolar condensation in hypertermic conditions. On the whole, we suggest a general approach able to understand the structural and kinetic mechanisms involved in aggregation-related diseases and in drugs development and testing.

  3. PRKACA: the catalytic subunit of protein kinase A and adrenocortical tumors

    Directory of Open Access Journals (Sweden)

    Annabel Sophie Berthon

    2015-05-01

    Full Text Available Cyclic-AMP (cAMP-dependent protein kinase (PKA is the main effector of cAMP signaling in all tissues. Inactivating mutations of the PRKAR1A gene, coding for the type 1A regulatory subunit of PKA, are responsible for Carney complex and primary pigmented nodular adrenocortical disease (PPNAD. PRKAR1A inactivation and PKA dysregulation have been implicated in various types of adrenocortical pathologies associated with ACTH-independent Cushing syndrome (AICS from PPNAD to adrenocortical adenomas and cancer, and other forms of bilateral adrenocortical hyperplasias (BAH. More recently, mutations of PRKACA, the gene coding for the catalytic subunit C alpha (Cα, were also identified in the pathogenesis of adrenocortical tumors. PRKACA copy number gain was found in the germline of several patients with cortisol-producing BAH, whereas the somatic Leu206Arg (c.617A>C recurrent PRKACA mutation was found in as many as half of all adrenocortical adenomas associated with AICS. In vitro analysis demonstrated that this mutation led to constitutive Cα activity, unregulated by its main partners, the PKA regulatory subunits. In this review, we summarize the current understanding of the involvement of PRKACA in adrenocortical tumorigenesis, and our understanding of PKA’s role in adrenocortical lesions. We also discuss potential therapeutic advances that can be made through targeting of PRKACA and the PKA pathway.

  4. Genetic analysis of the cytoplasmic dynein subunit families.

    Science.gov (United States)

    Pfister, K Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M C

    2006-01-01

    Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

  5. Genetic analysis of the cytoplasmic dynein subunit families.

    Directory of Open Access Journals (Sweden)

    K Kevin Pfister

    2006-01-01

    Full Text Available Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

  6. Effects of the β1 auxiliary subunit on modification of Rat Na{sub v}1.6 sodium channels expressed in HEK293 cells by the pyrethroid insecticides tefluthrin and deltamethrin

    Energy Technology Data Exchange (ETDEWEB)

    He, Bingjun [College of Life Sciences, Nankai University, Tianjin 300071 (China); Soderlund, David M., E-mail: dms6@cornell.edu [Department of Entomology, Cornell University, Geneva, NY 14456 (United States)

    2016-01-15

    We expressed rat Na{sub v}1.6 sodium channels with or without the rat β1 subunit in human embryonic kidney (HEK293) cells and evaluated the effects of the pyrethroid insecticides tefluthrin and deltamethrin on whole-cell sodium currents. In assays with the Na{sub v}1.6 α subunit alone, both pyrethroids prolonged channel inactivation and deactivation and shifted the voltage dependence of channel activation and steady-state inactivation toward hyperpolarization. Maximal shifts in activation were ~ 18 mV for tefluthrin and ~ 24 mV for deltamethrin. These compounds also caused hyperpolarizing shifts of ~ 10–14 mV in the voltage dependence of steady-state inactivation and increased in the fraction of sodium current that was resistant to inactivation. The effects of pyrethroids on the voltage-dependent gating greatly increased the size of sodium window currents compared to unmodified channels; modified channels exhibited increased probability of spontaneous opening at membrane potentials more negative than the normal threshold for channel activation and incomplete channel inactivation. Coexpression of Na{sub v}1.6 with the β1 subunit had no effect on the kinetic behavior of pyrethroid-modified channels but had divergent effects on the voltage-dependent gating of tefluthrin- or deltamethrin-modified channels, increasing the size of tefluthrin-induced window currents but decreasing the size of corresponding deltamethrin-induced currents. Unexpectedly, the β1 subunit did not confer sensitivity to use-dependent channel modification by either tefluthrin or deltamethrin. We conclude from these results that functional reconstitution of channels in vitro requires careful attention to the subunit composition of channel complexes to ensure that channels in vitro are faithful functional and pharmacological models of channels in neurons. - Highlights: • We expressed Na{sub v}1.6 sodium channels with or without β1 subunits in HEK293 cells. • Tefluthrin and deltamethrin

  7. Iron Loading Selectively Increases Hippocampal Levels of Ubiquitinated Proteins and Impairs Hippocampus-Dependent Memory.

    Science.gov (United States)

    Figueiredo, Luciana Silva; de Freitas, Betânia Souza; Garcia, Vanessa Athaíde; Dargél, Vinícius Ayub; Köbe, Luiza Machado; Kist, Luiza Wilges; Bogo, Maurício Reis; Schröder, Nadja

    2016-11-01

    Alterations of brain iron levels have been observed in a number of neurodegenerative disorders. We have previously demonstrated that iron overload in the neonatal period results in severe and persistent memory deficits in the adulthood. Protein degradation mediated by the ubiquitin-proteasome system (UPS) plays a central regulatory role in several cellular processes. Impairment of the UPS has been implicated in the pathogenesis of neurodegenerative disorders. Here, we examined the effects of iron exposure in the neonatal period (12th-14th day of postnatal life) on the expression of proteasome β-1, β-2, and β-5 subunits, and ubiquitinated proteins in brains of 15-day-old rats, to evaluate the immediate effect of the treatment, and in adulthood to assess long-lasting effects. Two different memory types, emotionally motivated conditioning and object recognition were assessed in adult animals. We found that iron administered in the neonatal period impairs both emotionally motivated and recognition memory. Polyubiquitinated protein levels were increased in the hippocampus, but not in the cortex, of adult animals treated with iron. Gene expression of subunits β1 and β5 was affected by age, being higher in the early stages of development in the hippocampus, accompanied by an age-related increase in polyubiquitinated protein levels in adults. In the cortex, gene expression of the three proteasome subunits was significantly higher in adulthood than in the neonatal period. These findings suggest that expression of proteasome subunits and activity are age-dependently regulated. Iron exposure in the neonatal period produces long-lasting harmful effects on the UPS functioning, which may be related with iron-induced memory impairment.

  8. Exploring the Mediational Role of Coping Motives for Marijuana Use in Terms of the Relation between Anxiety Sensitivity and Marijuana Dependence

    OpenAIRE

    Johnson, Kirsten; Mullin, Jennifer L.; Marshall, Erin C.; Bonn-Miller, Marcel O.; Zvolensky, Michael

    2010-01-01

    The current study evaluated the prediction that coping motives for marijuana use would mediate the relation between anxiety sensitivity and a marijuana dependence diagnosis after controlling for other co-occurring marijuana use motives. Participants were 136 current marijuana users (47.1% women; Mage = 21.9, SD = 7.2). Results were consistent with a mediational effect, with the relation between anxiety sensitivity and marijuana dependence being explained by the addition of coping motives into...

  9. Structural insights into RipC, a putative citrate lyase β subunit from a Yersinia pestis virulence operon

    International Nuclear Information System (INIS)

    Torres, Rodrigo; Chim, Nicholas; Sankaran, Banumathi; Pujol, Céline; Bliska, James B.; Goulding, Celia W.

    2011-01-01

    Comparison of the 2.45 Å resolution crystal structure of homotrimeric RipC, a putative citrate lyase β subunit from Y. pestis, with structural homologs reveals conserved RipC residues that are implicated in CoA binding. Yersinia pestis remains a threat, with outbreaks of plague occurring in rural areas and its emergence as a weapon of bioterrorism; thus, an improved understanding of its various pathogenicity pathways is warranted. The rip (required for intracellular proliferation) virulence operon is required for Y. pestis survival in interferon-γ-treated macrophages and has been implicated in lowering macrophage-produced nitric oxide levels. RipC, one of three gene products from the rip operon, is annotated as a citrate lyase β subunit. Furthermore, the Y. pestis genome lacks genes that encode citrate lyase α and γ subunits, suggesting a unique functional role of RipC in the Y. pestisrip-mediated survival pathway. Here, the 2.45 Å resolution crystal structure of RipC revealed a homotrimer in which each monomer consists of a (β/α) 8 TIM-barrel fold. Furthermore, the trimeric state was confirmed in solution by size-exclusion chromatography. Through sequence and structure comparisons with homologous proteins, it is proposed that RipC is a putative CoA- or CoA-derivative binding protein

  10. Anxiogenic properties of an inverse agonist selective for α3 subunit-containing GABAA receptors

    OpenAIRE

    Atack, John R; Hutson, Peter H; Collinson, Neil; Marshall, George; Bentley, Graham; Moyes, Christopher; Cook, Susan M; Collins, Ian; Wafford, Keith; McKernan, Ruth M; Dawson, Gerard R

    2005-01-01

    α3IA (6-(4-pyridyl)-5-(4-methoxyphenyl)-3-carbomethoxy-1-methyl-1H-pyridin-2-one) is a pyridone with higher binding and functional affinity and greater inverse agonist efficacy for GABAA receptors containing an α3 rather than an α1, α2 or α5 subunit. If doses are selected that minimise the occupancy at these latter subtypes, then the in vivo effects of α3IA are most probably mediated by the α3 subtype.α3IA has good CNS penetration in rats and mice as measured using a [3H]Ro 15-1788 in vivo bi...

  11. Three α-Subunits of Heterotrimeric G Proteins and an Adenylyl Cyclase Have Distinct Roles in Fruiting Body Development in the Homothallic Fungus Sordaria macrospora

    Science.gov (United States)

    Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich

    2008-01-01

    Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different α-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Δgsa1, Δgsa2, and Δgsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Gα-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Δgsa1Δgsa2 and Δgsa1Δgsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Gα-subunits, two recently generated Δpre strains were crossed with all Δgsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three ΔgsaΔsac1 double mutants and one Δgsa2Δgsa3Δsac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1–GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Gα-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora. PMID:18723884

  12. Roles of the β subunit hinge domain in ATP synthase F1 sector: Hydrophobic network formed by introduced βPhe174 inhibits subunit rotation

    International Nuclear Information System (INIS)

    Nakanishi-Matsui, Mayumi; Kashiwagi, Sachiko; Kojima, Masaki; Nonaka, Takamasa; Futai, Masamitsu

    2010-01-01

    The ATP synthase β subunit hinge domain (βPhe148 ∼ βGly186, P-loop/α-helixB/loop/β-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F 1 with the βSer174 to Phe mutation in the domain lowered the γ subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F 1 sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698-20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the βMet159, βIle163, and βAla167 residues of the β subunit are involved together with the mutant βPhe174. The network is expected to stabilize the conformation of β DP (nucleotide-bound form of the β subunit), resulting in increased activation energy for transition to β E (empty β subunit). The modeling further predicts that replacement of βMet159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of βS174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the β subunit hinge domain is pertinent for the rotational catalysis.

  13. The autophosphorylation and p34cdc2 phosphorylation sites of casein kinase-2 beta-subunit are not essential for reconstituting the fully-active heterotetrameric holoenzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Issinger, O G

    1993-01-01

    Two mutants of human casein kinase-2 beta-subunit with short deletions at either their amino (delta 1-4) or carboxy (delta 209-215) terminal side have been created that have lost the capability to undergo autophosphorylation and p34cdc2 mediated phosphorylation, respectively. Both mutants give rise...

  14. Studies on the subunits of human glycoprotein hormones in relation to reproduction

    International Nuclear Information System (INIS)

    Hagen, C.

    1977-01-01

    In this review summarising present knowledge of the biological and immunological activity of the subunits of human glycoprotein hormones, the specificity of the α-subunit and β-subunit radioimmunoassays are discussed. The crossreaction studies performed with the α-subunit radioimmunoassays are aummarised in one table while those with the β-subunit radioimmunoassays are presented in a second table. (JIW)

  15. Arabidopsis cryptochrome 1 is a soluble protein mediating blue light-dependent regulation of plant growth and development

    International Nuclear Information System (INIS)

    Lin ChenTao; Ahmad, M.; Cashmore, A.R.

    1996-01-01

    Cryptochrome 1 (CRY1) is a flavin-type blue type receptor of Arabidopsis thaliana which mediates inhibition of hypocotyl elongation. In the work described in this report it is demonstrated that CRY1 is a soluble protein expressed in both young seedlings grown either in the dark or under light, and in different organs of adult plants. The functional role of CRY1 was further investigated using transgenic Arabidopsis plants overexpressing CRY1. It is demonstrated that overexpression of CRY1 resulted in hypersensitivity to blue, UV-A, and green light for the inhibition of hypocotyl elongation response. Transgenic plants overexpressing CRY1 also exhibited a dwarf phenotype with reduced size in almost every organ. This was in keeping with the previous observation of reciprocal alterations found in hy4 mutant plants and is consistent with a hypothesis that CRY1 mediates a light-dependent process resulting in a general inhibitory effect on plant growth. In addition, transgenic plants overexpressing CRY1 showed increased anthocyanin accumulation in response to blue, UV-A, and green light in a fluence rate-dependent manner. This increase in anthocyanin accumulation in transgenic plants was shown to be concomitant with increased blue light-induction of CHS gene expression. It is concluded that CRY1 is a photoreceptor mediating blue light-dependent regulation of gene expression in addition to its affect on plant growth. (author)

  16. Bisphenol-A rapidly enhanced passive avoidance memory and phosphorylation of NMDA receptor subunits in hippocampus of young rats

    International Nuclear Information System (INIS)

    Xu Xiaohong; Li Tao; Luo Qingqing; Hong Xing; Xie Lingdan; Tian Dong

    2011-01-01

    Bisphenol-A (BPA), an endocrine disruptor, is found to influence development of brain and behaviors in rodents. The previous study indicated that perinatal exposure to BPA impaired learning-memory and inhibited N-methyl-D-aspartate receptor (NMDAR) subunits expressions in hippocampus during the postnatal development in rats; and in cultured hippocampal neurons, BPA rapidly promotes dynamic changes in dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDAR subunit NR2B. In the present study, we examined the rapid effect of BPA on passive avoidance memory and NMDAR in the developing hippocampus of Sprague-Dawley rats at the age of postnatal day 18. The results showed that BPA or estradiol benzoate (EB) rapidly extended the latency to step down from the platform 1 h after footshock and increased the phosphorylation levels of NR1, NR2B, and mitogen-activated extracellular signal-regulated kinase (ERK) in hippocampus within 1 h. While 24 h after BPA or EB treatment, the improved memory and the increased phosphorylation levels of NR1, NR2B, ERK disappeared. Furthermore, pre-treatment with an estrogen receptors (ERs) antagonist, ICI182,780, or an ERK-activating kinase inhibitor, U0126, significantly attenuated EB- or BPA-induced phosphorylations of NR1, NR2B, and ERK within 1 h. These data suggest that BPA rapidly enhanced short-term passive avoidance memory in the developing rats. A non-genomic effect via ERs may mediate the modulation of the phosphorylation of NMDAR subunits NR1 and NR2B through ERK signaling pathway. - Highlights: → BPA rapidly extended the latency to step down from platform 1 h after footshock. → BPA rapidly increased pNR1, pNR2B, and pERK in hippocampus within 1 h. → ERs antagonist or MEK inhibitor attenuated BPA-induced pNR1, pNR2B, and pERK.

  17. Role of desensitization and subunit expression for kainate receptor-mediated neurotoxicity in murine neocortical cultures

    DEFF Research Database (Denmark)

    Jensen, J B; Schousboe, A; Pickering, D S

    1999-01-01

    ) toxicity mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and (3) toxicity that can be mediated by kainate receptors when desensitization of the receptors is blocked. The indirect action at NMDA receptors was discovered because (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H...... nedioxy-5H-2,3-benzodiazepine (GYKI 53655), a selective AMPA receptor antagonist, abolished the remaining toxicity. These results indicated that kainate- and domoate-mediated toxicity involves both the NMDA and the AMPA receptors. Pretreatment of the cultures with concanavalin A to prevent desensitization...

  18. Multiple thyrotropin β-subunit and thyrotropin receptor-related genes arose during vertebrate evolution.

    Directory of Open Access Journals (Sweden)

    Gersende Maugars

    Full Text Available Thyroid-stimulating hormone (TSH is composed of a specific β subunit and an α subunit that is shared with the two pituitary gonadotropins. The three β subunits derive from a common ancestral gene through two genome duplications (1R and 2R that took place before the radiation of vertebrates. Analysis of genomic data from phylogenetically relevant species allowed us to identify an additional Tshβ subunit-related gene that was generated through 2R. This gene, named Tshβ2, present in cartilaginous fish, little skate and elephant shark, and in early lobe-finned fish, coelacanth and lungfish, was lost in ray-finned fish and tetrapods. The absence of a second type of TSH receptor (Tshr gene in these species suggests that both TSHs act through the same receptor. A novel Tshβ sister gene, named Tshβ3, was generated through the third genomic duplication (3R that occurred early in the teleost lineage. Tshβ3 is present in most teleost groups but was lostin tedraodontiforms. The 3R also generated a second Tshr, named Tshrb. Interestingly, the new Tshrb was translocated from its original chromosomic position after the emergence of eels and was then maintained in its new position. Tshrb was lost in tetraodontiforms and in ostariophysians including zebrafish although the latter species have two TSHs, suggesting that TSHRb may be dispensable. The tissue distribution of duplicated Tshβs and Tshrs was studied in the European eel. The endocrine thyrotropic function in the eel would be essentially mediated by the classical Tshβ and Tshra, which are mainly expressed in the pituitary and thyroid, respectively. Tshβ3 and Tshrb showed a similar distribution pattern in the brain, pituitary, ovary and adipose tissue, suggesting a possible paracrine/autocrine mode of action in these non-thyroidal tissues. Further studies will be needed to determine the binding specificity of the two receptors and how these two TSH systems are interrelated.

  19. Apoptotic role of TGF-β mediated by Smad4 mitochondria translocation and cytochrome c oxidase subunit II interaction.

    Science.gov (United States)

    Pang, Lijuan; Qiu, Tao; Cao, Xu; Wan, Mei

    2011-07-01

    Smad4, originally isolated from the human chromosome 18q21, is a key factor in transducing the signals of the TGF-β superfamily of growth hormones and plays a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-β, but the mechanisms by which Smad4 induces apoptosis are elusive. Here we report that Smad4 directly translocates to the mitochondria of apoptotic cells. Smad4 gene silencing by siRNA inhibits TGF-β-induced apoptosis in Hep3B cells and UV-induced apoptosis in PANC-1 cells. Cell fractionation assays demonstrated that a fraction of Smad4 translocates to mitochondria after long time TGF-β treatment or UV exposure, during which the cells were under apoptosis. Smad4 mitochondria translocation during apoptosis was also confirmed by fluorescence observation of Smad4 colocalization with MitoTracker Red. We searched for mitochondria proteins that have physical interactions with Smad4 using yeast two-hybrid screening approach. DNA sequence analysis identified 34 positive clones, five of which encoded subunits in mitochondria complex IV, i.e., one clone encoded cytochrome c oxidase COXII, three clones encoded COXIII and one clone encoded COXVb. Strong interaction between Smad4 with COXII, an important apoptosis regulator, was verified in yeast by β-gal activity assays and in mammalian cells by immunoprecipitation assays. Further, mitochondrial portion of cells was isolated and the interaction between COXII and Smad4 in mitochondria upon TGF-β treatment or UV exposure was confirmed. Importantly, targeting Smad4 to mitochondria using import leader fusions enhanced TGF-β-induced apoptosis. Collectively, the results suggest that Smad4 promote apoptosis of the cells through its mitochondrial translocation and association with mitochondria protein COXII. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. The mediating role of self-criticism and dependency in the association between perceptions of maternal caring and depressive symptoms.

    Science.gov (United States)

    Campos, Rui C; Besser, Avi; Blatt, Sidney J

    2010-12-01

    This study examined a theoretically based mediation model including participants' perceptions of early relationships with their mother, self-criticism, dependency, and current depressive symptoms. We expect that (a) early relationships characterized by low levels of care and high levels of overprotection will be positively associated with both current depressive state and self-criticism and dependency; (b) high levels of self-criticism and dependency will be positively associated with depressive symptoms; and (c) self-criticism and dependency will play a mediating role in the association between participants' perceptions of early relationships characterized by low levels of care and high levels of overprotection and their current depressive symptoms. A nonclinical community sample of 200 Portuguese adults participated in the study. Perceptions of early relationships were measured using the mother scales of the Parental Bonding Instrument (Parker et al. [1979: Br J Med Psychol 52:1-10]), levels of self-criticism and dependency were measured using the Depressive Experiences Questionnaire (Blatt et al. [1976: J Abn Psy 6:383-389]), and depressive symptoms were measured using the Center for the Epidemiological Studies of Depression Scale (Radloff [1977: Appl Psychol Meas 1:385-401]. Structural equation modeling showed that the link between participants' perceptions of early caretaking relationships with their mothers and their current depressive symptoms is mediated by high levels of self-criticism--a personality trait associated with vulnerability to depression--but not Dependency. However, an ancillary analysis indicated that the link between participants' perceptions of early maternal overprotective relationships and their current depressive symptoms is mediated by high levels of Neediness. Findings underscore the role of perceived early relationships in psychological vulnerability to depression among highly self-critical and among highly needy individuals and

  1. Mechanisms of the gabapentinoids and α 2 δ-1 calcium channel subunit in neuropathic pain.

    Science.gov (United States)

    Patel, Ryan; Dickenson, Anthony H

    2016-04-01

    The gabapentinoid drugs gabapentin and pregabalin are key front-line therapies for various neuropathies of peripheral and central origin. Originally designed as analogs of GABA, the gabapentinoids bind to the α 2 δ-1 and α 2 δ-2 auxiliary subunits of calcium channels, though only the former has been implicated in the development of neuropathy in animal models. Transgenic approaches also identify α 2 δ-1 as key in mediating the analgesic effects of gabapentinoids, however the precise molecular mechanisms remain unclear. Here we review the current understanding of the pathophysiological role of the α 2 δ-1 subunit, the mechanisms of analgesic action of gabapentinoid drugs and implications for efficacy in the clinic. Despite widespread use, the number needed to treat for gabapentin and pregabalin averages from 3 to 8 across neuropathies. The failure to treat large numbers of patients adequately necessitates a novel approach to treatment selection. Stratifying patients by sensory profiles may imply common underlying mechanisms, and a greater understanding of these mechanisms could lead to more direct targeting of gabapentinoids.

  2. Crystallization and preliminary X-ray diffraction analyses of several forms of the CfaB major subunit of enterotoxigenic Escherichia coli CFA/I fimbriae

    International Nuclear Information System (INIS)

    Li, Yong-Fu; Poole, Steven; Rasulova, Fatima; McVeigh, Annette L.; Savarino, Stephen J.; Xia, Di

    2009-01-01

    Three fusion proteins were generated in order to resolve the atomic structure of the CFA/I fimbriae of enterotoxigenic E. coli. CfaEB is a fusion of the minor and major CFA/I subunits, while CfaBB and CfaBBB are tandem fusions of two and three repeats, respectively, of the major subunit. Each protein was crystallized and the crystal structures of each of these fusions were determined successively by the molecular-replacement method using the CfaE crystal structure as an initial phasing model. Enterotoxigenic Escherichia coli (ETEC), a major global cause of diarrhea, initiates the pathogenic process via fimbriae-mediated attachment to the small intestinal epithelium. A common prototypic ETEC fimbria, colonization factor antigen I (CFA/I), consists of a tip-localized minor adhesive subunit CfaE and the stalk-forming major subunit CfaB, both of which are necessary for fimbrial assembly. To elucidate the structure of CFA/I at atomic resolution, three recombinant proteins were generated consisting of fusions of the minor and major subunits (CfaEB) and of two (CfaBB) and three (CfaBBB) repeats of the major subunit. Crystals of CfaEB diffracted X-rays to 2.1 Å resolution and displayed the symmetry of space group P2 1 . CfaBB exhibited a crystal diffraction limit of 2.3 Å resolution and had the symmetry of space group P2 1 2 1 2. CfaBBB crystallized in the monoclinic space group C2 and diffracted X-rays to 2.3 Å resolution. These structures were determined using the molecular-replacement method

  3. Development of a Subunit Vaccine for Contagious Bovine ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Their work has set the stage for commercial development of a sub-unit vaccine. ... The sub-unit vaccine will be cost-effective, easy to produce, and safe. How it will make a ... IDRC invites applications for the IDRC Doctoral Research Awards.

  4. Core Mediator structure at 3.4 Å extends model of transcription initiation complex.

    Science.gov (United States)

    Nozawa, Kayo; Schneider, Thomas R; Cramer, Patrick

    2017-05-11

    Mediator is a multiprotein co-activator that binds the transcription pre-initiation complex (PIC) and regulates RNA polymerase (Pol) II. The Mediator head and middle modules form the essential core Mediator (cMed), whereas the tail and kinase modules play regulatory roles. The architecture of Mediator and its position on the PIC are known, but atomic details are limited to Mediator subcomplexes. Here we report the crystal structure of the 15-subunit cMed from Schizosaccharomyces pombe at 3.4 Å resolution. The structure shows an unaltered head module, and reveals the intricate middle module, which we show is globally required for transcription. Sites of known Mediator mutations cluster at the interface between the head and middle modules, and in terminal regions of the head subunits Med6 (ref. 16) and Med17 (ref. 17) that tether the middle module. The structure led to a model for Saccharomyces cerevisiae cMed that could be combined with the 3.6 Å cryo-electron microscopy structure of the core PIC (cPIC). The resulting atomic model of the cPIC-cMed complex informs on interactions of the submodules forming the middle module, called beam, knob, plank, connector, and hook. The hook is flexibly linked to Mediator by a conserved hinge and contacts the transcription initiation factor IIH (TFIIH) kinase that phosphorylates the carboxy (C)-terminal domain (CTD) of Pol II and was recently positioned on the PIC. The hook also contains residues that crosslink to the CTD and reside in a previously described cradle. These results provide a framework for understanding Mediator function, including its role in stimulating CTD phosphorylation by TFIIH.

  5. Calcium-mediated signaling and calmodulin-dependent kinase regulate hepatocyte-inducible nitric oxide synthase expression.

    Science.gov (United States)

    Zhang, Baochun; Crankshaw, Will; Nesemeier, Ryan; Patel, Jay; Nweze, Ikenna; Lakshmanan, Jaganathan; Harbrecht, Brian G

    2015-02-01

    Induced nitric oxide synthase (iNOS) is induced in hepatocytes by shock and inflammatory stimuli. Excessive NO from iNOS mediates shock-induced hepatic injury and death, so understanding the regulation of iNOS will help elucidate the pathophysiology of septic shock. In vitro, cytokines induce iNOS expression through activation of signaling pathways including mitogen-activated protein kinases and nuclear factor κB. Cytokines also induce calcium (Ca(2+)) mobilization and activate calcium-mediated intracellular signaling pathways, typically through activation of calmodulin-dependent kinases (CaMK). Calcium regulates NO production in macrophages but the role of calcium and calcium-mediated signaling in hepatocyte iNOS expression has not been defined. Primary rat hepatocytes were isolated, cultured, and induced to produce NO with proinflammatory cytokines. Calcium mobilization and Ca(2+)-mediated signaling were altered with ionophore, Ca(2+) channel blockers, and inhibitors of CaMK. The Ca(2+) ionophore A23187 suppressed cytokine-stimulated NO production, whereas Ethylene glycol tetraacetic acid and nifedipine increased NO production, iNOS messenger RNA, and iNOS protein expression. Inhibition of CaMK with KN93 and CBD increased NO production but the calcineurin inhibitor FK 506 decreased iNOS expression. These data demonstrate that calcium-mediated signaling regulates hepatocyte iNOS expression and does so through a mechanism independent of calcineurin. Changes in intracellular calcium levels may regulate iNOS expression during hepatic inflammation induced by proinflammatory cytokines. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. DNA-dependent protein kinase inhibits AID-induced antibody gene conversion.

    Directory of Open Access Journals (Sweden)

    Adam J L Cook

    2007-04-01

    Full Text Available Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID-dependent hypermutation of Ig V(DJ rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(DJ regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(DJ hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs. Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.

  7. The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis.

    Science.gov (United States)

    Aoubala, M; Holt, J; Clegg, R A; Rowlands, D J; Harris, M

    2001-07-01

    Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system--under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.

  8. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    DEFF Research Database (Denmark)

    Schmidt, Signe Tandrup; Foged, Camilla; Korsholm, Karen Smith

    2016-01-01

    be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode......The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens...... of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the specific PRR expression profile of the target APCs. Here, we review state-of-the-art formulation approaches employed for the inclusion of immunostimulators and subunit...

  9. TLR-mediated inflammatory responses to Streptococcus pneumoniae are highly dependent on surface expression of bacterial lipoproteins.

    Science.gov (United States)

    Tomlinson, Gillian; Chimalapati, Suneeta; Pollard, Tracey; Lapp, Thabo; Cohen, Jonathan; Camberlein, Emilie; Stafford, Sian; Periselneris, Jimstan; Aldridge, Christine; Vollmer, Waldemar; Picard, Capucine; Casanova, Jean-Laurent; Noursadeghi, Mahdad; Brown, Jeremy

    2014-10-01

    Streptococcus pneumoniae infections induce inflammatory responses that contribute toward both disease pathogenesis and immunity, but the host-pathogen interactions that mediate these effects are poorly defined. We used the surface lipoprotein-deficient ∆lgt pneumococcal mutant strain to test the hypothesis that lipoproteins are key determinants of TLR-mediated immune responses to S. pneumoniae. We show using reporter assays that TLR2 signaling is dependent on pneumococcal lipoproteins, and that macrophage NF-κB activation and TNF-α release were reduced in response to the ∆lgt strain. Differences in TNF-α responses between Δlgt and wild-type bacteria were abrogated for macrophages from TLR2- but not TLR4-deficient mice. Transcriptional profiling of human macrophages revealed attenuated TLR2-associated responses to ∆lgt S. pneumoniae, comprising many NF-κB-regulated proinflammatory cytokine and chemokine genes. Importantly, non-TLR2-associated responses were preserved. Experiments using leukocytes from IL-1R-associated kinase-4-deficient patients and a mouse pneumonia model confirmed that proinflammatory responses were lipoprotein dependent. Our data suggest that leukocyte responses to bacterial lipoproteins are required for TLR2- and IL-1R-associated kinase-4-mediated inflammatory responses to S. pneumoniae. Copyright © 2014 The Authors.

  10. Novel time-dependent vascular actions of Δ9-tetrahydrocannabinol mediated by peroxisome proliferator-activated receptor gamma

    International Nuclear Information System (INIS)

    O'Sullivan, Saoirse E.; Tarling, Elizabeth J.; Bennett, Andrew J.; Kendall, David A.; Randall, Michael D.

    2005-01-01

    Cannabinoids have widespread effects on the cardiovascular system, only some of which are mediated via G-protein-coupled cell surface receptors. The active ingredient of cannabis, Δ 9 -tetrahydrocannabinol (THC), causes acute vasorelaxation in various arteries. Here we show for the first time that THC also causes slowly developing vasorelaxation through activation of peroxisome proliferator-activated receptors gamma (PPARγ). In vitro, THC (10 μM) caused time-dependent vasorelaxation of rat isolated arteries. Time-dependent vasorelaxation to THC was similar to that produced by the PPARγ agonist rosiglitazone and was inhibited by the PPARγ antagonist GW9662 (1 μM), but not the cannabinoid CB 1 receptor antagonist AM251 (1 μM). Time-dependent vasorelaxation to THC requires an intact endothelium, nitric oxide, production of hydrogen peroxide, and de novo protein synthesis. In transactivation assays in cultured HEK293 cells, THC-activated PPARγ, transiently expressed in combination with retinoid X receptor α and a luciferase reporter gene, in a concentration-dependent manner (100 nM-10 μM). In vitro incubation with THC (1 or 10 μM, 8 days) stimulated adipocyte differentiation in cultured 3T3L1 cells, a well-accepted property of PPARγ ligands. The present results provide strong evidence that THC is a PPARγ ligand, stimulation of which causes time-dependent vasorelaxation, implying some of the pleiotropic effects of cannabis may be mediated by nuclear receptors

  11. Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development.

    Science.gov (United States)

    Beier, Anna; Teichert, Ines; Krisp, Christoph; Wolters, Dirk A; Kück, Ulrich

    2016-06-21

    The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. The striatin-interacting phosphatase and kinase (STRIPAK) complex is highly conserved from yeasts to humans and is an important regulator of numerous eukaryotic developmental processes, such as cellular signaling and cell development. Although functional insights into the STRIPAK complex are accumulating, the detailed molecular mechanisms of single subunits are only partially understood

  12. Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates

    Directory of Open Access Journals (Sweden)

    Zhong Tao P

    2007-07-01

    Full Text Available Abstract Background Action potential generation in excitable cells such as myocytes and neurons critically depends on voltage-gated sodium channels. In mammals, sodium channels exist as macromolecular complexes that include a pore-forming alpha subunit and 1 or more modulatory beta subunits. Although alpha subunit genes have been cloned from diverse metazoans including flies, jellyfish, and humans, beta subunits have not previously been identified in any non-mammalian species. To gain further insight into the evolution of electrical signaling in vertebrates, we investigated beta subunit genes in the teleost Danio rerio (zebrafish. Results We identified and cloned single zebrafish gene homologs for beta1-beta3 (zbeta1-zbeta3 and duplicate genes for beta4 (zbeta4.1, zbeta4.2. Sodium channel beta subunit loci are similarly organized in fish and mammalian genomes. Unlike their mammalian counterparts, zbeta1 and zbeta2 subunit genes display extensive alternative splicing. Zebrafish beta subunit genes and their splice variants are differentially-expressed in excitable tissues, indicating tissue-specific regulation of zbeta1-4 expression and splicing. Co-expression of the genes encoding zbeta1 and the zebrafish sodium channel alpha subunit Nav1.5 in Chinese Hamster Ovary cells increased sodium current and altered channel gating, demonstrating functional interactions between zebrafish alpha and beta subunits. Analysis of the synteny and phylogeny of mammalian, teleost, amphibian, and avian beta subunit and related genes indicated that all extant vertebrate beta subunits are orthologous, that beta2/beta4 and beta1/beta3 share common ancestry, and that beta subunits are closely related to other proteins sharing the V-type immunoglobulin domain structure. Vertebrate sodium channel beta subunit genes were not identified in the genomes of invertebrate chordates and are unrelated to known subunits of the para sodium channel in Drosophila. Conclusion The

  13. The effect of NMDA-NR2B receptor subunit over-expression on olfactory memory task performance in the mouse.

    Science.gov (United States)

    White, Theresa L; Youngentob, Steven L

    2004-09-17

    The N-methyl-D-aspartate (NMDA) receptor in the forebrain is thought to modulate some forms of memory formation, with the NR2B subunit being particularly relevant to this process. Relative to wild-type mice, transgenic animals in which the NR2B subunit was over-expressed demonstrate superior memory in a number of behavioral tasks, including object recognition [Nature 401 (1999) 63]. The purpose of the present study was to explore the generality of such phenomena, interpreted as the effect of increasing NR2B expression on the retention of other types of sensory-related information. To accomplish this, we focused our evaluation on the highly salient sensory modality of olfaction. In the first experiment, mice performed both a novel-object-recognition task identical to that performed by Tang et al. [Nature 401 (1999) 63] and a novel-odor-recognition task analogously constructed. Although the results of the object recognition task were consistent with the previous literature, there was no evidence of an effect of NR2B over-expression on the retention of odor recognition memory in the specific task performed. As it was possible that, unlike object recognition memory, novel odor recognition is not NMDA-receptor-dependent, a second task was designed using the social transmission of food preference paradigm. In contrast to the foregoing olfactory task, there is evidence that the latter procedure is, indeed, NMDA-dependent. The results of the second study demonstrated that transgenic mice with NR2B over-expression had a clear memory advantage in this alternative odor memory paradigm. Taken together, these results suggest the NR2B subunit is an important component in some but not all forms of olfactory memory organization. Moreover, for those functions that are NMDA-receptor-dependent, these data support the growing literature demonstrating the importance of the NR2B subunit.

  14. Chlorpromazine-induced hepatotoxicity during inflammation is mediated by TIRAP-dependent signaling pathway in mice

    International Nuclear Information System (INIS)

    Gandhi, Adarsh; Guo, Tao; Shah, Pranav; Moorthy, Bhagavatula; Ghose, Romi

    2013-01-01

    Inflammation is a major component of idiosyncratic adverse drug reactions (IADRs). To understand the molecular mechanism of inflammation-mediated IADRs, we determined the role of the Toll-like receptor (TLR) signaling pathway in idiosyncratic hepatotoxicity of the anti-psychotic drug, chlorpromazine (CPZ). Activation of TLRs recruits the first adaptor protein, Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) to the TIR domain of TLRs leading to the activation of the downstream kinase, c-Jun-N-terminal kinase (JNK). Prolonged activation of JNK leads to cell-death. We hypothesized that activation of TLR2 by lipoteichoic acid (LTA) or TLR4 by lipopolysaccharide (LPS) will augment the hepatotoxicity of CPZ by TIRAP-dependent mechanism involving prolonged activation of JNK. Adult male C57BL/6, TIRAP +/+ and TIRAP −/− mice were pretreated with saline, LPS (2 mg/kg) or LTA (6 mg/kg) for 30 min or 16 h followed by CPZ (5 mg/kg) or saline (vehicle) up to 24 h. We found that treatment of mice with CPZ in presence of LPS or LTA leads to ∼ 3–4 fold increase in serum ALT levels, a marked reduction in hepatic glycogen content, significant induction of serum tumor necrosis factor (TNF) α and prolonged JNK activation, compared to LPS or LTA alone. Similar results were observed in TIRAP +/+ mice, whereas the effects of LPS or LTA on CPZ-induced hepatotoxicity were attenuated in TIRAP −/− mice. For the first time, we show that inflammation-mediated hepatotoxicity of CPZ is dependent on TIRAP, and involves prolonged JNK activation in vivo. Thus, TIRAP-dependent pathways may be targeted to predict and prevent inflammation-mediated IADRs. -- Highlights: ► Inflammation augments the toxicity of an idiosyncratic hepatotoxin chlorpromazine. ► Activation of Toll-like receptors by LPS or LTA induces chlorpromazine toxicity. ► Sustained stress kinase (JNK) activation is associated with chlorpromazine toxicity. ► These studies provide novel mechanistic

  15. Chlorpromazine-induced hepatotoxicity during inflammation is mediated by TIRAP-dependent signaling pathway in mice

    Energy Technology Data Exchange (ETDEWEB)

    Gandhi, Adarsh, E-mail: adarsh.gandhi@nih.gov [University of Houston, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, 1441 Moursund Street, Room 517, Houston, TX 77030 (United States); Guo, Tao, E-mail: tguo4@jhu.edu [University of Houston, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, 1441 Moursund Street, Room 517, Houston, TX 77030 (United States); Shah, Pranav [University of Houston, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, 1441 Moursund Street, Room 517, Houston, TX 77030 (United States); Moorthy, Bhagavatula [Baylor College of Medicine, Department of Pediatrics, 1102 Bates Avenue, Suite 530, Houston, TX 77030 (United States); Ghose, Romi, E-mail: rghose@uh.edu [University of Houston, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, 1441 Moursund Street, Room 517, Houston, TX 77030 (United States)

    2013-02-01

    Inflammation is a major component of idiosyncratic adverse drug reactions (IADRs). To understand the molecular mechanism of inflammation-mediated IADRs, we determined the role of the Toll-like receptor (TLR) signaling pathway in idiosyncratic hepatotoxicity of the anti-psychotic drug, chlorpromazine (CPZ). Activation of TLRs recruits the first adaptor protein, Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) to the TIR domain of TLRs leading to the activation of the downstream kinase, c-Jun-N-terminal kinase (JNK). Prolonged activation of JNK leads to cell-death. We hypothesized that activation of TLR2 by lipoteichoic acid (LTA) or TLR4 by lipopolysaccharide (LPS) will augment the hepatotoxicity of CPZ by TIRAP-dependent mechanism involving prolonged activation of JNK. Adult male C57BL/6, TIRAP{sup +/+} and TIRAP{sup −/−} mice were pretreated with saline, LPS (2 mg/kg) or LTA (6 mg/kg) for 30 min or 16 h followed by CPZ (5 mg/kg) or saline (vehicle) up to 24 h. We found that treatment of mice with CPZ in presence of LPS or LTA leads to ∼ 3–4 fold increase in serum ALT levels, a marked reduction in hepatic glycogen content, significant induction of serum tumor necrosis factor (TNF) α and prolonged JNK activation, compared to LPS or LTA alone. Similar results were observed in TIRAP{sup +/+} mice, whereas the effects of LPS or LTA on CPZ-induced hepatotoxicity were attenuated in TIRAP{sup −/−} mice. For the first time, we show that inflammation-mediated hepatotoxicity of CPZ is dependent on TIRAP, and involves prolonged JNK activation in vivo. Thus, TIRAP-dependent pathways may be targeted to predict and prevent inflammation-mediated IADRs. -- Highlights: ► Inflammation augments the toxicity of an idiosyncratic hepatotoxin chlorpromazine. ► Activation of Toll-like receptors by LPS or LTA induces chlorpromazine toxicity. ► Sustained stress kinase (JNK) activation is associated with chlorpromazine toxicity. ► These studies

  16. INTRINSIC REGULATION OF HEMOGLOBIN EXPRESSION BY VARIABLE SUBUNIT INTERFACE STRENGTHS

    Science.gov (United States)

    Manning, James M.; Popowicz, Anthony M.; Padovan, Julio C.; Chait, Brian T.; Manning, Lois R.

    2012-01-01

    SUMMARY The expression of the six types of human hemoglobin subunits over time is currently considered to be regulated mainly by transcription factors that bind to upstream control regions of the gene (the “extrinsic” component of regulation). Here we describe how subunit pairing and further assembly to tetramers in the liganded state is influenced by the affinity of subunits for one another (the “intrinsic” component of regulation). The adult hemoglobin dimers have the strongest subunit interfaces and the embryonic hemoglobins are the weakest with fetal hemoglobins of intermediate strength, corresponding to the temporal order of their expression. These variable subunit binding strengths and the attenuating effects of acetylation contribute to the differences with which these hemoglobin types form functional O2-binding tetramers consistent with gene switching. PMID:22129306

  17. The rice eukaryotic translation initiation factor 3 subunit f (OseIF3f is involved in microgametogenesis

    Directory of Open Access Journals (Sweden)

    Qi eLi

    2016-04-01

    Full Text Available Microgametogenesis is the postmeiotic pollen developmental phase when unicellular microspores develop into mature tricellular pollen. In rice, microgametogenesis can influence grain yields to a great degree because pollen abortion occurs more easily during microgametogenesis than during other stages of pollen development. However, our knowledge of the genes involved in microgametogenesis in rice remains limited. Due to the dependence of pollen development on the regulatory mechanisms of protein expression, we identified the encoding gene of the eukaryotic translation initiation factor 3, subunit f in Oryza sativa (OseIF3f. Immunoprecipitation combined with mass spectrometry confirmed that OseIF3f was a subunit of rice eIF3, which consisted of at least 12 subunits including eIF3a, eIF3b, eIF3c, eIF3d, eIF3e, eIF3f, eIF3g, eIF3h, eIF3i, eIF3k, eIF3l and eIF3m. OseIF3f showed high mRNA levels in immature florets and is highly abundant in developing anthers. Subcellular localization analysis showed that OseIF3f was localized to the cytosol and the endoplasmic reticulum in rice root cells. We further analyzed the biological function of OseIF3f using the double-stranded RNA-mediated interference (RNAi approach. The OseIF3f-RNAi lines grew normally at the vegetative stage but displayed a large reduction in seed production and pollen viability, which is associated with the down-regulation of OseIF3f. Further cytological observations of pollen development revealed that the OseIF3f-RNAi lines showed no obvious abnormalities at the male meiotic stage and the unicellular microspore stage. However, compared to the wild type, OseIF3f-RNAi lines contained a higher percentage of arrested unicellular pollen at the bicellular stage and a higher percentage of arrested unicellular and bicellular pollen, and aborted pollen at the tricellular stage. These results indicate that OseIF3f plays a role in microgametogenesis.

  18. Mediated and Moderated Effects of Neurocognitive Impairment on Outcomes of Treatment for Substance Dependence and Major Depression

    Science.gov (United States)

    Worley, Matthew J.; Tate, Susan R.; Granholm, Eric; Brown, Sandra A.

    2015-01-01

    Objective Neurocognitive impairment has not consistently predicted substance use treatment outcomes but has been linked to proximal mediators of outcome. These indirect effects have not been examined in adults with substance dependence and co-occurring psychiatric disorders. We examined mediators and moderators of the effects of neurocognitive impairment on substance use among adults in treatment for alcohol or drug dependence and major depression (MDD). Method Participants were veterans (N =197, mean age = 49.3 years, 90% male, 75% Caucasian) in a trial of two group interventions for alcohol/drug dependence and MDD. Measures examined here included intake neurocognitive assessments and percent days drinking (PDD), percent days using drugs (PDDRG), self-efficacy, 12-step affiliation, and depressive symptoms measured every 3 months from intake to the 18-month follow-up. Results Greater intake neurocognitive impairment predicted lower self-efficacy, lower 12-step affiliation, and greater depression severity, and these time-varying variables mediated the effects of impairment on future PDD and PDDRG. The prospective effects of 12-step affiliation on future PDD were greater for those with greater neurocognitive impairment. Impairment also interacted with depression to moderate the effects of 12-step affiliation and self-efficacy on PDD. Adults with greater impairment and currently severe depression had the strongest associations between 12-step affiliation/self-efficacy and future drinking. Conclusions Greater neurocognitive impairment may lead to poorer outcomes from group therapy for alcohol/drug dependence and MDD due to compromised change in therapeutic processes. Distal factors such as neurocognitive impairment can interact with dynamic risk factors to modulate the association between therapeutic processes and future drinking outcomes. PMID:24588403

  19. The Mediator complex: a master coordinator of transcription and cell lineage development.

    Science.gov (United States)

    Yin, Jing-wen; Wang, Gang

    2014-03-01

    Mediator is a multiprotein complex that is required for gene transcription by RNA polymerase II. Multiple subunits of the complex show specificity in relaying information from signals and transcription factors to the RNA polymerase II machinery, thus enabling control of the expression of specific genes. Recent studies have also provided novel mechanistic insights into the roles of Mediator in epigenetic regulation, transcriptional elongation, termination, mRNA processing, noncoding RNA activation and super enhancer formation. Based on these specific roles in gene regulation, Mediator has emerged as a master coordinator of development and cell lineage determination. Here, we describe the most recent advances in understanding the mechanisms of Mediator function, with an emphasis on its role during development and disease.

  20. Marburg Virus Glycoprotein GP2: pH-Dependent Stability of the Ectodomain α-Helical Bundle†

    Science.gov (United States)

    Harrison, Joseph S.; Koellhoffer, Jayne F.; Chandran, Kartik; Lai, Jonathan R.

    2012-01-01

    Marburg virus (MARV) and Ebola virus (EBOV) constitute the family Filoviridae of enveloped viruses (filoviruses) that cause severe hemorrhagic fever. Infection by MARV is required for fusion between the host cell and viral membranes, a process that is mediated by the two subunits of the envelope glycoprotein GP1 (surface subunit) and GP2 (transmembrane subunit). Upon viral attachment and uptake, it is believed that the MARV viral fusion machinery is triggered by host factors and environmental conditions found in the endosome. Next, conformational rearrangements in the GP2 ectodomain result in the formation of a highly stable six-helix bundle; this refolding event provides the energetic driving force for membrane fusion. Both GP1 and GP2 from EBOV have been extensively studied, but there is little information available for the MARV glycoproteins. Here we have expressed two variants of the MARV GP2 ectodomain in Escherichia coli and analyzed their biophysical properties. Circular dichroism indicates that the MARV GP2 ectodomain adopts an α-helical conformation, and one variant sediments as a trimer by equilibrium analytical ultracentrifugation. Denaturation studies indicate the α-helical structure is highly stable at pH 5.3 (unfolding energy, ΔGunf H2O, of 33.4 ± 2.5 kcal/mol and melting temperature, Tm, of 75.3 ± 2.1 °C for one variant). Furthermore, we found the α-helical stability to be strongly dependent on pH with higher stability under lower pH conditions (Tm values ranging from ~92 °C at pH 4.0 to ~38 °C at pH 8.0). Mutational analysis suggests two glutamic acid residues (E579 and E580) are partially responsible for this pH-dependent behavior. Based on these results, we hypothesize that pH-dependent folding stability of the MARV GP2 ectodomain provides a mechanism to control conformational preferences such that the six-helix bundle ‘post-fusion’ state is preferred under conditions of appropriately matured endosomes. PMID:22369502

  1. DNA requirements for interaction of the C-terminal region of Ku80 with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs).

    Science.gov (United States)

    Radhakrishnan, Sarvan Kumar; Lees-Miller, Susan P

    2017-09-01

    Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation induced DNA double strand breaks (DSBs) in human cells. Critical to NHEJ is the DNA-dependent interaction of the Ku70/80 heterodimer with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the DNA-PK holoenzyme. However, precisely how Ku recruits DNA-PKcs to DSBs ends to enhance its kinase activity has remained enigmatic, with contradictory findings reported in the literature. Here we address the role of the Ku80 C-terminal region (CTR) in the DNA-dependent interaction of Ku70/80 with DNA-PKcs using purified components and defined DNA structures. Our results show that the Ku80 CTR is required for interaction with DNA-PKcs on short segments of blunt ended 25bp dsDNA or 25bp dsDNA with a 15-base poly dA single stranded (ss) DNA extension, but this requirement is less stringent on longer dsDNA molecules (35bp blunt ended dsDNA) or 25bp duplex DNA with either a 15-base poly dT or poly dC ssDNA extension. Moreover, the DNA-PKcs-Ku complex preferentially forms on 25 bp DNA with a poly-pyrimidine ssDNA extension.Our work clarifies the role of the Ku80 CTR and dsDNA ends on the interaction of DNA-PKcs with Ku and provides key information to guide assembly and biology of NHEJ complexes. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. A conserved phenylalanine as a relay between the α5 helix and the GDP binding region of heterotrimeric Gi protein α subunit.

    Science.gov (United States)

    Kaya, Ali I; Lokits, Alyssa D; Gilbert, James A; Iverson, Tina M; Meiler, Jens; Hamm, Heidi E

    2014-08-29

    G protein activation by G protein-coupled receptors is one of the critical steps for many cellular signal transduction pathways. Previously, we and other groups reported that the α5 helix in the G protein α subunit plays a major role during this activation process. However, the precise signaling pathway between the α5 helix and the guanosine diphosphate (GDP) binding pocket remains elusive. Here, using structural, biochemical, and computational techniques, we probed different residues around the α5 helix for their role in signaling. Our data showed that perturbing the Phe-336 residue disturbs hydrophobic interactions with the β2-β3 strands and α1 helix, leading to high basal nucleotide exchange. However, mutations in β strands β5 and β6 do not perturb G protein activation. We have highlighted critical residues that leverage Phe-336 as a relay. Conformational changes are transmitted starting from Phe-336 via β2-β3/α1 to Switch I and the phosphate binding loop, decreasing the stability of the GDP binding pocket and triggering nucleotide release. When the α1 and α5 helices were cross-linked, inhibiting the receptor-mediated displacement of the C-terminal α5 helix, mutation of Phe-336 still leads to high basal exchange rates. This suggests that unlike receptor-mediated activation, helix 5 rotation and translocation are not necessary for GDP release from the α subunit. Rather, destabilization of the backdoor region of the Gα subunit is sufficient for triggering the activation process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Structural characterization of recombinant crustacyanin subunits from the lobster Homarus americanus

    International Nuclear Information System (INIS)

    Ferrari, Michele; Folli, Claudia; Pincolini, Elisa; McClintock, Timothy S.; Rössle, Manfred; Berni, Rodolfo; Cianci, Michele

    2012-01-01

    The two recombinant apo subunits H1 and H2 from H. americanus have been structurally characterized. Reconstitution studies with astaxanthin reproduced the bathochromic shift of 85–95 nm typical of the natural crustacyanin subunits. Crustacean crustacyanin proteins are linked to the production and modification of carapace colour, with direct implications for fitness and survival. Here, the structural and functional properties of the two recombinant crustacyanin subunits H 1 and H 2 from the American lobster Homarus americanus are reported. The two subunits are structurally highly similar to the corresponding natural apo crustacyanin CRTC and CRTA subunits from the European lobster H. gammarus. Reconstitution studies of the recombinant crustacyanin proteins H 1 and H 2 with astaxanthin reproduced the bathochromic shift of 85–95 nm typical of the natural crustacyanin subunits from H. gammarus in complex with astaxanthin. Moreover, correlations between the presence of crustacyanin genes in crustacean species and the resulting carapace colours with the spectral properties of the subunits in complex with astaxanthin confirmed this genotype–phenotype linkage

  4. Identification of the subunit of cAMP receptor protein (CRP) that functionally interacts with CytR in CRP-CytR-mediated transcriptional repression

    DEFF Research Database (Denmark)

    Meibom, K L; Kallipolitis, B H; Ebright, R H

    2000-01-01

    At promoters of the Escherichia coli CytR regulon, the cAMP receptor protein (CRP) interacts with the repressor CytR to form transcriptionally inactive CRP-CytR-promoter or (CRP)(2)-CytR-promoter complexes. Here, using "oriented heterodimer" analysis, we show that only one subunit of the CRP dimer......, the subunit proximal to CytR, functionally interacts with CytR in CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes. Our results provide information about the architecture of CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes and rule out the proposal that masking of activating region 2 of CRP...

  5. The S100A10 subunit of the annexin A2 heterotetramer facilitates L2-mediated human papillomavirus infection.

    Science.gov (United States)

    Woodham, Andrew W; Da Silva, Diane M; Skeate, Joseph G; Raff, Adam B; Ambroso, Mark R; Brand, Heike E; Isas, J Mario; Langen, Ralf; Kast, W Martin

    2012-01-01

    Mucosotropic, high-risk human papillomaviruses (HPV) are sexually transmitted viruses that are causally associated with the development of cervical cancer. The most common high-risk genotype, HPV16, is an obligatory intracellular virus that must gain entry into host epithelial cells and deliver its double stranded DNA to the nucleus. HPV capsid proteins play a vital role in these steps. Despite the critical nature of these capsid protein-host cell interactions, the precise cellular components necessary for HPV16 infection of epithelial cells remains unknown. Several neutralizing epitopes have been identified for the HPV16 L2 minor capsid protein that can inhibit infection after initial attachment of the virus to the cell surface, which suggests an L2-specific secondary receptor or cofactor is required for infection, but so far no specific L2-receptor has been identified. Here, we demonstrate that the annexin A2 heterotetramer (A2t) contributes to HPV16 infection and co-immunoprecipitates with HPV16 particles on the surface of epithelial cells in an L2-dependent manner. Inhibiting A2t with an endogenous annexin A2 ligand, secretory leukocyte protease inhibitor (SLPI), or with an annexin A2 antibody significantly reduces HPV16 infection. With electron paramagnetic resonance, we demonstrate that a previously identified neutralizing epitope of L2 (aa 108-120) specifically interacts with the S100A10 subunit of A2t. Additionally, mutation of this L2 region significantly reduces binding to A2t and HPV16 pseudovirus infection. Furthermore, downregulation of A2t with shRNA significantly decreases capsid internalization and infection by HPV16. Taken together, these findings indicate that A2t contributes to HPV16 internalization and infection of epithelial cells and this interaction is dependent on the presence of the L2 minor capsid protein.

  6. The S100A10 subunit of the annexin A2 heterotetramer facilitates L2-mediated human papillomavirus infection.

    Directory of Open Access Journals (Sweden)

    Andrew W Woodham

    Full Text Available Mucosotropic, high-risk human papillomaviruses (HPV are sexually transmitted viruses that are causally associated with the development of cervical cancer. The most common high-risk genotype, HPV16, is an obligatory intracellular virus that must gain entry into host epithelial cells and deliver its double stranded DNA to the nucleus. HPV capsid proteins play a vital role in these steps. Despite the critical nature of these capsid protein-host cell interactions, the precise cellular components necessary for HPV16 infection of epithelial cells remains unknown. Several neutralizing epitopes have been identified for the HPV16 L2 minor capsid protein that can inhibit infection after initial attachment of the virus to the cell surface, which suggests an L2-specific secondary receptor or cofactor is required for infection, but so far no specific L2-receptor has been identified. Here, we demonstrate that the annexin A2 heterotetramer (A2t contributes to HPV16 infection and co-immunoprecipitates with HPV16 particles on the surface of epithelial cells in an L2-dependent manner. Inhibiting A2t with an endogenous annexin A2 ligand, secretory leukocyte protease inhibitor (SLPI, or with an annexin A2 antibody significantly reduces HPV16 infection. With electron paramagnetic resonance, we demonstrate that a previously identified neutralizing epitope of L2 (aa 108-120 specifically interacts with the S100A10 subunit of A2t. Additionally, mutation of this L2 region significantly reduces binding to A2t and HPV16 pseudovirus infection. Furthermore, downregulation of A2t with shRNA significantly decreases capsid internalization and infection by HPV16. Taken together, these findings indicate that A2t contributes to HPV16 internalization and infection of epithelial cells and this interaction is dependent on the presence of the L2 minor capsid protein.

  7. Characterization of Mediator Complex and its Associated Proteins from Rice.

    Science.gov (United States)

    Samanta, Subhasis; Thakur, Jitendra Kumar

    2017-01-01

    The Mediator complex is a multi-protein complex that acts as a molecular bridge conveying transcriptional messages from the cis element-bound transcription factor to the RNA Polymerase II machinery. It is found in all eukaryotes including members of the plant kingdom. Increasing number of reports from plants regarding different Mediator subunits involved in a multitude of processes spanning from plant development to environmental interactions have firmly established it as a central hub of plant regulatory networks. Routine isolation of Mediator complex in a particular species is a necessity because of many reasons. First, composition of the Mediator complex varies from species to species. Second, the composition of the Mediator complex in a particular species is not static under all developmental and environmental conditions. Besides this, at times, Mediator complex is used in in vitro transcription systems. Rice, a staple food crop of the world, is used as a model monocot crop. Realizing the need of a reliable protocol for the isolation of Mediator complex from plants, we describe here the isolation of Mediator complex from rice.

  8. Crystal structure of the P pilus rod subunit PapA.

    Directory of Open Access Journals (Sweden)

    Denis Verger

    2007-05-01

    Full Text Available P pili are important adhesive fibres involved in kidney infection by uropathogenic Escherichia coli strains. P pili are assembled by the conserved chaperone-usher pathway, which involves the PapD chaperone and the PapC usher. During pilus assembly, subunits are incorporated into the growing fiber via the donor-strand exchange (DSE mechanism, whereby the chaperone's G1 beta-strand that complements the incomplete immunoglobulin-fold of each subunit is displaced by the N-terminal extension (Nte of an incoming subunit. P pili comprise a helical rod, a tip fibrillum, and an adhesin at the distal end. PapA is the rod subunit and is assembled into a superhelical right-handed structure. Here, we have solved the structure of a ternary complex of PapD bound to PapA through donor-strand complementation, itself bound to another PapA subunit through DSE. This structure provides insight into the structural basis of the DSE reaction involving this important pilus subunit. Using gel filtration chromatography and electron microscopy on a number of PapA Nte mutants, we establish that PapA differs in its mode of assembly compared with other Pap subunits, involving a much larger Nte that encompasses not only the DSE region of the Nte but also the region N-terminal to it.

  9. Discovery and characterization of a novel lachrymatory factor synthase in Petiveria alliacea and its influence on alliinase-mediated formation of biologically active organosulfur compounds.

    Science.gov (United States)

    Musah, Rabi A; He, Quan; Kubec, Roman

    2009-11-01

    A novel lachrymatory factor synthase (LFS) was isolated and purified from the roots of the Amazonian medicinal plant Petiveria alliacea. The enzyme is a heterotetrameric glycoprotein comprised of two alpha-subunits (68.8 kD each), one gamma-subunit (22.5 kD), and one delta-subunit (11.9 kD). The two alpha-subunits are glycosylated and connected by a disulfide bridge. The LFS has an isoelectric point of 5.2. It catalyzes the formation of a sulfine lachrymator, (Z)-phenylmethanethial S-oxide, only in the presence of P. alliacea alliinase and its natural substrate, S-benzyl-l-cysteine sulfoxide (petiveriin). Depending on its concentration relative to that of P. alliacea alliinase, the LFS sequesters, to varying degrees, the sulfenic acid intermediate formed by alliinase-mediated breakdown of petiveriin. At LFS:alliinase of 5:1, LFS sequesters all of the sulfenic acid formed by alliinase action on petiveriin, and converts it entirely to (Z)-phenylmethanethial S-oxide. However, starting at LFS:alliinase of 5:2, the LFS is unable to sequester all of the sulfenic acid produced by the alliinase, with the result that sulfenic acid that escapes the action of the LFS condenses with loss of water to form S-benzyl phenylmethanethiosulfinate (petivericin). The results show that the LFS and alliinase function in tandem, with the alliinase furnishing the sulfenic acid substrate on which the LFS acts. The results also show that the LFS modulates the formation of biologically active thiosulfinates that are downstream of the alliinase in a manner dependent upon the relative concentrations of the LFS and the alliinase. These observations suggest that manipulation of LFS-to-alliinase ratios in plants displaying this system may provide a means by which to rationally modify organosulfur small molecule profiles to obtain desired flavor and/or odor signatures, or increase the presence of desirable biologically active small molecules.

  10. Purification, subunit characterization and ultrastructure of three soluble bovine lectins: conglutinin, mannose-binding protein and the pentraxin serum amyloid P-component

    DEFF Research Database (Denmark)

    Andersen, Ove; Friis, P; Holm Nielsen, E

    1992-01-01

    affinity chromatography and selective elution was developed. The purification was monitored by SDS-PAGE, Western blotting and electron microscopy. Binding of the lectins to Sephadex-iC3b, their collagenase sensitivity, and the size and antibody reactivity of their subunits was investigated....... The demonstration, by SDS-PAGE, of 25-kDa subunits, which were unaffected by collagenase treatment but bound to Sephadex-iC3b and antibodies to human SAP, indicated the existence of bovine SAP. Bovine conglutinin (BK) also showed calcium-dependent binding to Sephadex-iC3b, whereas bovine MBP did not. The binding...... of BK was inhibitable with GlcNAc. A 3000-fold increase in BK activity (ELISA) was obtained in eluates from Sephadex-iC3b. SDS-PAGE analyses of BK and MBP revealed subunits with an Mr of 43 kDa and 30 kDa, respectively. These subunits were sensitive to collagenase treatment which reduced the Mr to 20 k...

  11. Discoidin Domain Receptor 1 Mediates Myosin-Dependent Collagen Contraction

    Directory of Open Access Journals (Sweden)

    Nuno M. Coelho

    2017-02-01

    Full Text Available Discoidin domain receptor 1 (DDR1 is a tyrosine kinase collagen adhesion receptor that mediates cell migration through association with non-muscle myosin IIA (NMIIA. Because DDR1 is implicated in cancer fibrosis, we hypothesized that DDR1 interacts with NMIIA to enable collagen compaction by traction forces. Mechanical splinting of rat dermal wounds increased DDR1 expression and collagen alignment. In periodontal ligament of DDR1 knockout mice, collagen mechanical reorganization was reduced >30%. Similarly, cultured cells with DDR1 knockdown or expressing kinase-deficient DDR1d showed 50% reduction of aligned collagen. Tractional remodeling of collagen was dependent on DDR1 clustering, activation, and interaction of the DDR1 C-terminal kinase domain with NMIIA filaments. Collagen remodeling by traction forces, DDR1 tyrosine phosphorylation, and myosin light chain phosphorylation were increased on stiff versus soft substrates. Thus, DDR1 clustering, activation, and interaction with NMIIA filaments enhance the collagen tractional remodeling that is important for collagen compaction in fibrosis.

  12. The electrically silent Kv6.4 subunit confers hyperpolarized gating charge movement in Kv2.1/Kv6.4 heterotetrameric channels.

    Directory of Open Access Journals (Sweden)

    Elke Bocksteins

    Full Text Available The voltage-gated K(+ (Kv channel subunit Kv6.4 does not form functional homotetrameric channels but co-assembles with Kv2.1 to form functional Kv2.1/Kv6.4 heterotetrameric channels. Compared to Kv2.1 homotetramers, Kv6.4 exerts a ~40 mV hyperpolarizing shift in the voltage-dependence of Kv2.1/Kv6.4 channel inactivation, without a significant effect on activation gating. However, the underlying mechanism of this Kv6.4-induced modulation of Kv2.1 channel inactivation, and whether the Kv6.4 subunit participates in the voltage-dependent gating of heterotetrameric channels is not well understood. Here we report distinct gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels, compared to Kv2.1 homotetramers, as revealed by gating current recordings from mammalian cells expressing these channels. The gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels displayed an extra component around the physiological K(+ equilibrium potential, characterized by a second sigmoidal relationship of the voltage-dependence of gating charge movement. This distinct gating charge displacement reflects movement of the Kv6.4 voltage-sensing domain and has a voltage-dependency that matches the hyperpolarizing shift in Kv2.1/Kv6.4 channel inactivation. These results provide a mechanistic basis for the modulation of Kv2.1 channel inactivation gating kinetics by silent Kv6.4 subunits.

  13. Chemotherapy inhibits skeletal muscle ubiquitin-proteasome-dependent proteolysis.

    Science.gov (United States)

    Tilignac, Thomas; Temparis, Sandrine; Combaret, Lydie; Taillandier, Daniel; Pouch, Marie-Noëlle; Cervek, Matjaz; Cardenas, Diana M; Le Bricon, Thierry; Debiton, Eric; Samuels, Susan E; Madelmont, Jean-Claude; Attaix, Didier

    2002-05-15

    Chemotherapy has cachectic effects, but it is unknown whether cytostatic agents alter skeletal muscle proteolysis. We hypothesized that chemotherapy-induced alterations in protein synthesis should result in the increased incidence of abnormal proteins, which in turn should stimulate ubiquitin-proteasome-dependent proteolysis. The effects of the nitrosourea cystemustine were investigated in skeletal muscles from both healthy and colon 26 adenocarcinoma-bearing mice, an appropriate model for testing the impact of cytostatic agents. Muscle wasting was seen in both groups of mice 4 days after a single cystemustine injection, and the drug further increased the loss of muscle proteins already apparent in tumor-bearing animals. Cystemustine cured the tumor-bearing mice with 100% efficacy. Surprisingly, within 11 days of treatment, rates of muscle proteolysis progressively decreased below basal levels observed in healthy control mice and contributed to the cessation of muscle wasting. Proteasome-dependent proteolysis was inhibited by mechanisms that include reduced mRNA levels for 20S and 26S proteasome subunits, decreased protein levels of 20S proteasome subunits and the S14 non-ATPase subunit of the 26S proteasome, and impaired chymotrypsin- and trypsin-like activities of the enzyme. A combination of cisplatin and ifosfamide, two drugs that are widely used in the treatment of cancer patients, also depressed the expression of proteasomal subunits in muscles from rats bearing the MatB adenocarcinoma below basal levels. Thus, a down-regulation of ubiquitin-proteasome-dependent proteolysis is observed with various cytostatic agents and contributes to reverse the chemotherapy-induced muscle wasting.

  14. Casein kinase 2 down-regulation and activation by polybasic peptides are mediated by acidic residues in the 55-64 region of the beta-subunit. A study with calmodulin as phosphorylatable substrate

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Issinger, O G

    1994-01-01

    to substitute for wild-type beta-subunit as a suppressor of activity toward calmodulin. The only mutations that reduced the ability of the beta-subunit to suppress calmodulin phosphorylation activity, though being compatible with normal reconstitution of CK2 holoenzyme, were those affecting Asp55, Glu57...... are conversely ineffective. The latent "calmodulin kinase" activity of CK2 can also be specifically unmasked by a peptide (alpha[66-86]) reproducing a basic insert of the catalytic subunit. This effect is reversed by equimolar addition of a peptide (beta[55-71]) including the 55-64 acidic stretch of the beta......-subunit. Comparable polylysine stimulation was observed with the holoenzymes reconstituted with either beta wt or the beta mutants capable of assembling with the alpha-subunit, with the notable exception of those bearing Ala substitutions for acidic residues at positions 55, 57, and 59-61. These were nearly...

  15. The GABAB1a isoform mediates heterosynaptic depression at hippocampal mossy fiber synapses

    DEFF Research Database (Denmark)

    Guetg, Nicole; Seddik, Riad; Vigot, Réjan

    2009-01-01

    )) receptors mediate heterosynaptic depression of MF transmission, a physiological phenomenon involving transsynaptic inhibition of glutamate release via presynaptic GABA(B) receptors. Our data demonstrate that the difference in GABA(B1a) and GABA(B1b) protein levels at MF terminals is sufficient to produce...... a strictly GABA(B1a)-specific effect under physiological conditions. This consolidates that the differential subcellular localization of the GABA(B1a) and GABA(B1b) proteins is of regulatory relevance.......GABA(B) receptor subtypes are based on the subunit isoforms GABA(B1a) and GABA(B1b), which associate with GABA(B2) subunits to form pharmacologically indistinguishable GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Studies with mice selectively expressing GABA(B1a) or GABA(B1b) subunits revealed...

  16. Enhanced killing of chordoma cells by antibody-dependent cell-mediated cytotoxicity employing the novel anti-PD-L1 antibody avelumab

    OpenAIRE

    Fujii, Rika; Friedman, Eitan R.; Richards, Jacob; Tsang, Kwong Y.; Heery, Christopher R.; Schlom, Jeffrey; Hodge, James W.

    2016-01-01

    Chordoma, a rare bone tumor derived from the notochord, has been shown to be resistant to conventional therapies. Checkpoint inhibition has shown great promise in immune-mediated therapy of diverse cancers. The anti-PD-L1 mAb avelumab is unique among checkpoint inhibitors in that it is a fully human IgG1 capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) of PD-L1-expressing tumor cells. Here, we investigated avelumab as a potential therapy for chordoma. We examined 4 ch...

  17. 25-Hydroxycholesterol promotes fibroblast-mediated tissue remodeling through NF-κB dependent pathway

    International Nuclear Information System (INIS)

    Ichikawa, Tomohiro; Sugiura, Hisatoshi; Koarai, Akira; Kikuchi, Takashi; Hiramatsu, Masataka; Kawabata, Hiroki; Akamatsu, Keiichiro; Hirano, Tsunahiko; Nakanishi, Masanori; Matsunaga, Kazuto; Minakata, Yoshiaki; Ichinose, Masakazu

    2013-01-01

    Abnormal structural alterations termed remodeling, including fibrosis and alveolar wall destruction, are important features of the pathophysiology of chronic airway diseases such as chronic obstructive pulmonary disease (COPD) and asthma. 25-hydroxycholesterol (25-HC) is enzymatically produced by cholesterol 25-hydorxylase (CH25H) in macrophages and is reported to be involved in the formation of arteriosclerosis. We previously demonstrated that the expression of CH25H and production of 25HC were increased in the lungs of COPD. However, the role of 25-HC in lung tissue remodeling is unknown. In this study, we investigated the effect of 25-HC on fibroblast-mediated tissue remodeling using human fetal lung fibroblasts (HFL-1) in vitro. 25-HC significantly augmented α-smooth muscle actin (SMA) (P 1 production (P 1 release. These results suggest that 25-HC could contribute to fibroblast-mediated lung tissue remodeling by promoting myofibroblast differentiation and the excessive release of extracellular matrix protein and MMPs via an NF-κB-TGF-β dependent pathway

  18. The V-ATPase a2-subunit as a putative endosomal pH-sensor.

    Science.gov (United States)

    Marshansky, V

    2007-11-01

    V-ATPase (vesicular H(+)-ATPase)-driven intravesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor-ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which luminal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding, coat formation and fusion are unknown. Here, we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway. According to our model, V-ATPase is responsible for: (i) the generation of a pH gradient between vesicular membranes; (ii) sensing of intravesicular pH; and (iii) transmitting this information to the cytosolic side of the membrane. We also propose the hypothetical molecular mechanism involved in function of the V-ATPase a2-subunit as a putative pH-sensor. Based on extensive experimental evidence on the crucial role of histidine residues in the function of PSPs (pH-sensing proteins) in eukaryotic cells, we hypothesize that pH-sensitive histidine residues within the intra-endosomal loops and/or C-terminal luminal tail of the a2-subunit could also be involved in the pH-sensing function of V-ATPase. However, in order to identify putative pH-sensitive histidine residues and to test this hypothesis, it is absolutely essential that we increase our understanding of the folding and transmembrane topology of the a-subunit isoforms of V-ATPase. Thus the crucial role of intra-endosomal histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with cytosolic small GTPases and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be determined.

  19. Muscular subunits transplantation for facial reanimation

    Directory of Open Access Journals (Sweden)

    Hazan André Salo Buslik

    2006-01-01

    Full Text Available PURPOSE: To present an alternative technique for reconstruction of musculocutaneous damages in the face transferring innervated subsegments(subunits of the latissimus dorsi flap for replacement of various facial mimetic muscles. METHODS: One clinical case of trauma with skin and mimetic muscles damage is described as an example of the technique. The treatment was performed with microsurgical transfer of latissimus dorsi muscle subunits. Each subunit present shape and dimensions of the respective mimetic muscles replaced. The origin, insertions and force vectors for the mimicmuscle lost were considered. Each subsegment has its own arterial and venous supply with a motor nerve component for the muscular unit. RESULTS: Pre and one year postoperative photos registration of static and dynamic mimic aspects, as well as digital electromyography digital data of the patients were compared. The transplanted muscular units presented myoeletric activity, fulfilling both the functional and cosmetic aspect. CONCLUSION: This technique seems to be a promising way to deal with the complex musculocutaneous losses of the face as well as facial palsy.

  20. Association of C-Type Lectin Mincle with FcεRIβγ Subunits Leads to Functional Activation of RBL-2H3 Cells through Syk.

    Science.gov (United States)

    Honjoh, Chisato; Chihara, Kazuyasu; Yoshiki, Hatsumi; Yamauchi, Shota; Takeuchi, Kenji; Kato, Yuji; Hida, Yukio; Ishizuka, Tamotsu; Sada, Kiyonao

    2017-04-10

    Macrophage-inducible C-type lectin (Mincle) interacts with the γ-subunit of high-affinity IgE receptor (FcεRIγ) and activates Syk by recognizing its specific ligand, trehalose-6,6'-dimycolate, a glycolipid produced by Mycobacterium tuberculosis. It has been suggested that mast cells participate in the immune defense against pathogenic microbes including M. tuberculosis, although the functions are still uncertain. In this study, we examined the Mincle-mediated signaling pathway and cellular responses using RBL-2H3 cells. Mincle formed a protein complex with not only FcεRIγ but also FcεRIβ in a stable cell line expressing myc-tagged Mincle. In addition, engagement of Mincle increased the levels of protein tyrosine phosphorylation and ERK phosphorylation. A pull-down assay demonstrated that cross-linking of Mincle induced binding of FcεRIβγ subunits to the Src homology 2 domain of Syk. Pharmacological and genetic studies indicated that activation of Syk was critical for Mincle-mediated activation of phospholipase Cγ2, leading to the activation of ERK and nuclear factor of activated T cells. Moreover, engagement of Mincle efficiently induced up-regulation of characteristic mast cell genes in addition to degranulation. Taken together, our present results suggest that mast cells contribute to Mincle-mediated immunity through Syk activation triggered by association with the FcεRIβγ complex.

  1. HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove.

    Science.gov (United States)

    Filbin, Megan E; Kieft, Jeffrey S

    2011-07-01

    Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem-loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit's decoding groove. This configuration depends on the sequence and structure of a different stem-loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis.

  2. The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation.

    Science.gov (United States)

    Malik, Sohail; Roeder, Robert G

    2010-11-01

    The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action of numerous other co-activators and co-repressors, including those acting at the level of chromatin. These interactions ultimately allow the Mediator to deliver outputs that range from maximal activation of genes to modulation of basal transcription to long-term epigenetic silencing.

  3. Inhibition of K+ transport through Na+, K+-ATPase by capsazepine: role of membrane span 10 of the α-subunit in the modulation of ion gating.

    Science.gov (United States)

    Mahmmoud, Yasser A; Shattock, Michael; Cornelius, Flemming; Pavlovic, Davor

    2014-01-01

    Capsazepine (CPZ) inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity. In this study we have investigated the functional effects of CPZ on Na+,K+-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na+,K+-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na+,K+-ATPase current in the presence of extracellular K+. In contrast, CPZ stimulated pump current in the absence of extracellular K+. Similar conclusions were attained using HEK293 cells loaded with the Na+ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na+,K+-ATPase indicated that CPZ stabilizes ion interaction with the K+ sites. The distal part of membrane span 10 (M10) of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na+ congener at the Na+ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na+,K+-ATPase. CPZ increased oxonol VI fluorescence in the absence of K+, reflecting increased Na+ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K+, likely indicating opening of an intracellular pathway selective for K+. As revealed by the recent crystal structure of the E1.AlF4-.ADP.3Na+ form of the pig kidney Na+,K+-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K+ sites in Na+,K+-ATPase.

  4. ASIC2 Subunits Target Acid-Sensing Ion Channels to the Synapse via an Association with PSD-95

    OpenAIRE

    Zha, Xiang-ming; Costa, Vivian; Harding, Anne Marie S.; Reznikov, Leah; Benson, Christopher J.; Welsh, Michael J.

    2009-01-01

    Acid-sensing ion channel-1a (ASIC1a) mediates H+-gated current to influence normal brain physiology and impact several models of disease. Although ASIC2 subunits are widely expressed in brain and modulate ASIC1a current, their function remains poorly understood. We identified ASIC2a in dendrites, dendritic spines, and brain synaptosomes. This localization largely relied on ASIC2a binding to PSD-95 and matched that of ASIC1a, which does not co-immunoprecipitate with PSD-95. We found that ASIC2...

  5. Pituitary glycoprotein hormone a-subunit secretion by cirrhotic patients

    Directory of Open Access Journals (Sweden)

    Oliveira M.C.

    1999-01-01

    Full Text Available Secretion of the a-subunit of pituitary glycoprotein hormones usually follows the secretion of intact gonadotropins and is increased in gonadal failure and decreased in isolated gonadotropin deficiency. The aim of the present study was to determine the levels of the a-subunit in the serum of patients with cirrhosis of the liver and to compare the results obtained for eugonadal cirrhotic patients with those obtained for cirrhotic patients with hypogonadotropic hypogonadism. Forty-seven of 63 patients with cirrhosis (74.6% presented hypogonadism (which was central in 45 cases and primary in 2, 7 were eugonadal, and 9 women were in normal menopause. The serum a-subunit was measured by the fluorimetric method using monoclonal antibodies. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively, with an intra-assay coefficient of variation (CV of less than 5% and an interassay CV of 5%, and sensitivity limit of 4 ng/l. The serum a-subunit concentration ranged from 36 to 6253 ng/l, with a median of 273 ng/l. The median was 251 ng/l for patients with central hypogonadism and 198 ng/l for eugonadal patients. The correlation between the a-subunit and basal LH levels was significant both in the total sample (r = 0.48, P<0.01 and in the cirrhotic patients with central hypogonadism (r = 0.33, P = 0.02. Among men with central hypogonadism there was a negative correlation between a-subunit levels and total testosterone levels (r = 0.54, P<0.01 as well as free testosterone levels (r = -0.53, P<0.01. In conclusion, although the a-subunit levels are correlated with LH levels, at present they cannot be used as markers for hypogonadism in patients with cirrhosis of the liver.

  6. Antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC) of breast cancer cells mediated by bispecific antibody, MDX-210.

    Science.gov (United States)

    Watanabe, M; Wallace, P K; Keler, T; Deo, Y M; Akewanlop, C; Hayes, D F

    1999-02-01

    MDX-210 is a bispecific antibody (BsAb) with specificity for both the proto-oncogene product of HER-2/neu (c-erbB-2) and FcgammaRI (CD64). HER-2/neu is overexpressed in malignant tissue of approximately 30% of patients with breast cancer, and FcgammaRI is expressed on human monocytes, macrophages, and IFN-gamma activated granulocytes. We investigated phagocytosis and cytolysis of cultured human breast cancer cells by human monocyte-derived macrophages (MDM) mediated by BsAb MDX-210, its partially humanized derivative (MDX-H210), and its parent MoAb 520C9 (anti-HER-2/neu) under various conditions. Purified monocytes were cultured with GM-CSF, M-CSF, or no cytokine for five or six days. Antibody dependent cellular phagocytosis (ADCP) and cytolysis (ADCC) assays were performed with the MDM and HER-2/neu positive target cells (SK-BR-3). ADCP was measured by two-color fluorescence flow cytometry using PKH2 (green fluorescent dye) and phycoerythrin-conjugated (red) monoclonal antibodies (MoAb) against human CD14 and CD11b. ADCC was measured with a non-radioactive LDH detection kit. Both BsAb MDX-210 (via FcgammaRI) and MoAb 520C9 (mouse IgG1, via FcgammaRII) mediated similar levels of ADCP and ADCC. ADCP mediated by BsAb MDX-H210 was identical to that mediated by BsAb MDX-210. Confocal microscopy demonstrated that dual-labeled cells represented true phagocytosis. Both ADCP and ADCC were higher when MDM were pre-incubated with GM-CSF than when incubated with M-CSF. BsAb MDX-210 is as active in vitro as the parent MoAb 520C9 in inducing both phagocytosis and cytolysis of MDM. MDX-210 and its partially humanized derivative, MDX-H210, mediated similar levels of ADCP. GM-CSF appears to superior to M-CSF in inducing MDM-mediated ADCC and ADCP. These studies support the ongoing clinical investigations of BsAb MDX-210 and its partially humanized derivative.

  7. Immunochemical analysis of Micrococcus lysodeikticus (luteus) F1-ATPase and its subunits.

    Science.gov (United States)

    Urban, C; Salton, M R

    1983-08-31

    The F1-ATPase from Micrococcus lysodeikticus has been purified to 95% protein homogeneity in this laboratory and as all other bacterial F1S, possesses five distinct subunits with molecular weights ranging from 60 000 to 10 000 (Huberman, M. and Salton, M.R.J. (1979) Biochim. Biophys. Acta 547, 230-240). In this communication, we demonstrate the immunochemical reactivities of antibodies to native and SDS-dissociated subunits with the native and dissociated F1-ATPase and show that: (1) the antibodies generated to the native or SDS-dissociated subunits react with the native molecule; (2) all of the subunits comprising the F1 are antigenically unique as determined by crossed immunoelectrophoresis and the Ouchterlony double-diffusion techniques; (3) antibodies to the SDS-denatured individual delta- and epsilon-subunits can be used to destabilize the interaction of these specific subunits with the rest of the native F1; and (4) all subunit antibodies as well as anti-native F1 were found to inhibit ATPase activity to varying degrees, the strongest inhibition being seen with antibodies to the total F1 and anti-alpha- and anti-beta-subunit antibodies. The interaction of specific subunit antibodies may provide a new and novel way to study further and characterize the catalytic portions of F1-ATPases and in general may offer an additional method for the examination of multimeric proteins.

  8. HTLV-1 Tax Oncoprotein Subverts the Cellular DNA Damage Response via Binding to DNA-dependent Protein Kinase*S⃞

    Science.gov (United States)

    Durkin, Sarah S.; Guo, Xin; Fryrear, Kimberly A.; Mihaylova, Valia T.; Gupta, Saurabh K.; Belgnaoui, S. Mehdi; Haoudi, Abdelali; Kupfer, Gary M.; Semmes, O. John

    2008-01-01

    Human T-cell leukemia virus type-1 is the causative agent for adult T-cell leukemia. Previous research has established that the viral oncoprotein Tax mediates the transformation process by impairing cell cycle control and cellular response to DNA damage. We showed previously that Tax sequesters huChk2 within chromatin and impairs the response to ionizing radiation. Here we demonstrate that DNA-dependent protein kinase (DNA-PK) is a member of the Tax·Chk2 nuclear complex. The catalytic subunit, DNA-PKcs, and the regulatory subunit, Ku70, were present. Tax-containing nuclear extracts showed increased DNA-PK activity, and specific inhibition of DNA-PK prevented Tax-induced activation of Chk2 kinase activity. Expression of Tax induced foci formation and phosphorylation of H2AX. However, Tax-induced constitutive signaling of the DNA-PK pathway impaired cellular response to new damage, as reflected in suppression of ionizing radiation-induced DNA-PK phosphorylation and γH2AX stabilization. Tax co-localized with phospho-DNA-PK into nuclear speckles and a nuclear excluded Tax mutant sequestered endogenous phospho-DNA-PK into the cytoplasm, suggesting that Tax interaction with DNA-PK is an initiating event. We also describe a novel interaction between DNA-PK and Chk2 that requires Tax. We propose that Tax binds to and stabilizes a protein complex with DNA-PK and Chk2, resulting in a saturation of DNA-PK-mediated damage repair response. PMID:18957425

  9. Novel time-dependent vascular actions of {delta}{sup 9}-tetrahydrocannabinol mediated by peroxisome proliferator-activated receptor gamma

    Energy Technology Data Exchange (ETDEWEB)

    O' Sullivan, Saoirse E [School of Biomedical Sciences, E Floor, Queen' s Medical Centre, University of Nottingham, Nottingham NG7 2UH (United Kingdom); Tarling, Elizabeth J [School of Biomedical Sciences, E Floor, Queen' s Medical Centre, University of Nottingham, Nottingham NG7 2UH (United Kingdom); Bennett, Andrew J [School of Biomedical Sciences, E Floor, Queen' s Medical Centre, University of Nottingham, Nottingham NG7 2UH (United Kingdom); Kendall, David A [School of Biomedical Sciences, E Floor, Queen' s Medical Centre, University of Nottingham, Nottingham NG7 2UH (United Kingdom); Randall, Michael D [School of Biomedical Sciences, E Floor, Queen' s Medical Centre, University of Nottingham, Nottingham NG7 2UH (United Kingdom)

    2005-11-25

    Cannabinoids have widespread effects on the cardiovascular system, only some of which are mediated via G-protein-coupled cell surface receptors. The active ingredient of cannabis, {delta}{sup 9}-tetrahydrocannabinol (THC), causes acute vasorelaxation in various arteries. Here we show for the first time that THC also causes slowly developing vasorelaxation through activation of peroxisome proliferator-activated receptors gamma (PPAR{gamma}). In vitro, THC (10 {mu}M) caused time-dependent vasorelaxation of rat isolated arteries. Time-dependent vasorelaxation to THC was similar to that produced by the PPAR{gamma} agonist rosiglitazone and was inhibited by the PPAR{gamma} antagonist GW9662 (1 {mu}M), but not the cannabinoid CB{sub 1} receptor antagonist AM251 (1 {mu}M). Time-dependent vasorelaxation to THC requires an intact endothelium, nitric oxide, production of hydrogen peroxide, and de novo protein synthesis. In transactivation assays in cultured HEK293 cells, THC-activated PPAR{gamma}, transiently expressed in combination with retinoid X receptor {alpha} and a luciferase reporter gene, in a concentration-dependent manner (100 nM-10 {mu}M). In vitro incubation with THC (1 or 10 {mu}M, 8 days) stimulated adipocyte differentiation in cultured 3T3L1 cells, a well-accepted property of PPAR{gamma} ligands. The present results provide strong evidence that THC is a PPAR{gamma} ligand, stimulation of which causes time-dependent vasorelaxation, implying some of the pleiotropic effects of cannabis may be mediated by nuclear receptors.

  10. Differential regulation of thyrotropin subunit apoprotein and carbohydrate biosynthesis by thyroid hormone

    International Nuclear Information System (INIS)

    Taylor, T.; Weintraub, B.D.

    1985-01-01

    The regulation of TSH apoprotein and carbohydrate biosynthesis by thyroid hormone was studied by incubating pituitaries from normal and hypothyroid (3 weeks post-thyroidectomy) rats in medium containing [ 14 C]alanine and [ 3 H] glucosamine. After 6 h, samples were sequentially treated with anti-TSH beta to precipitate TSH and free TSH beta, anti-LH beta to clear the sample of LH and free LH beta, then anti-LH alpha to precipitate free alpha-subunit. Total proteins were acid precipitated. All precipitates were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, which were then sliced and assayed by scintillation spectrometry. In hypothyroid pituitaries plus medium, [ 14 C]alanine incorporation in combined and free beta-subunits was 26 times normal and considerably greater than the 3.4-fold increase seen in total protein; combined and free alpha-subunits showed no specific increase in apoprotein synthesis. [ 3 H]Glucosamine incorporation in combined alpha- and beta-subunits in hypothyroid samples was 13 and 21 times normal, respectively, and was greater than the 1.9-fold increase in total protein; free alpha-subunit showed no specific increase in carbohydrate synthesis. The glucosamine to alanine ratio, reflecting relative glycosylation of newly synthesized molecules, was increased in hypothyroidism for combined alpha-subunits, but not for combined beta-subunits, free alpha-subunits, or total proteins. In summary, short term hypothyroidism selectively stimulated TSH beta apoprotein synthesis and carbohydrate synthesis of combined alpha- and beta-subunits. Hypothyroidism also increased the relative glycosylation of combined alpha-subunit. Thus, thyroid hormone deficiency appears to alter the rate-limiting step in TSH assembly (i.e. beta-subunit synthesis) as well as the carbohydrate structure of TSH, which may play important roles in its biological function

  11. Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development

    Directory of Open Access Journals (Sweden)

    Anna Beier

    2016-06-01

    Full Text Available The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora. Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general.

  12. Intranuclear Delivery of a Novel Antibody-Derived Radiosensitizer Targeting the DNA-Dependent Protein Kinase Catalytic Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Xiong Hairong [Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA (Georgia); State Key Laboratory of Virology, Institute of Medical Virology, Wuhan University School of Medicine, Wuhan (China); Lee, Robert J. [Division of Pharmaceutics, College of Pharmacy, Ohio State University, Columbus, OH (United States); Haura, Eric B. [Thoracic Oncology and Experimental Therapeutics Programs, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL (United States); Edwards, John G. [Apeliotus Technologies, Inc., Atlanta, GA (United States); Dynan, William S. [Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA (Georgia); Li Shuyi, E-mail: sli@georgiahealth.edu [Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA (Georgia); Apeliotus Technologies, Inc., Atlanta, GA (United States)

    2012-07-01

    Purpose: To inhibit DNA double-strand break repair in tumor cells by delivery of a single-chain antibody variable region fragment (ScFv 18-2) to the cell nucleus. ScFv 18-2 binds to a regulatory region of the DNA-dependent protein kinase (DNA-PK), an essential enzyme in the nonhomologous end-joining pathway, and inhibits DNA end-joining in a cell-free system and when microinjected into single cells. Development as a radiosensitizer has been limited by the lack of a method for intranuclear delivery to target cells. We investigated a delivery method based on folate receptor-mediated endocytosis. Methods and Materials: A recombinant ScFv 18-2 derivative was conjugated to folate via a scissile disulfide linker. Folate-ScFv 18-2 was characterized for its ability to be internalized by tumor cells and to influence the behavior of ionizing radiation-induced repair foci. Radiosensitization was measured in a clonogenic survival assay. Survival curves were fitted to a linear-quadratic model, and between-group differences were evaluated by an F test. Sensitization ratios were determined based on mean inhibitory dose. Results: Human KB and NCI-H292 lung cancer cells treated with folate-conjugated ScFv 18-2 showed significant radiosensitization (p < 0.001). Sensitization enhancement ratios were 1.92 {+-} 0.42 for KB cells and 1.63 {+-} 0.13 for NCI-H292 cells. Studies suggest that treatment inhibits repair of radiation-induced DSBs, as evidenced by the persistence of {gamma}-H2AX-stained foci and by inhibition of staining with anti-DNA-PKcs phosphoserine 2056. Conclusions: Folate-mediated endocytosis is an effective method for intranuclear delivery of an antibody-derived DNA repair inhibitor.

  13. Propofol effectively inhibits lithium-pilocarpine- induced status epilepticus in rats via downregulation of N-methyl-D-aspartate receptor 2B subunit expression

    Science.gov (United States)

    Wang, Henglin; Wang, Zhuoqiang; Mi, Weidong; Zhao, Cong; Liu, Yanqin; Wang, Yongan; Sun, Haipeng

    2012-01-01

    Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine. The inhibitory effects of propofol on status epilepticus in rats were judged based on observation of behavior, electroencephalography and 24-hour survival rate. Propofol (12.5–100 mg/kg) improved status epilepticus in a dose-dependent manner, and significantly reduced the number of deaths within 24 hours of lithium-pilocarpine injection. Western blot results showed that, 24 hours after induction of status epilepticus, the levels of N-methyl-D-aspartate receptor 2A and 2B subunits were significantly increased in rat cerebral cortex and hippocampus. Propofol at 50 mg/kg significantly suppressed the increase in N-methyl-D-aspartate receptor 2B subunit levels, but not the increase in N-methyl-D-aspartate receptor 2A subunit levels. The results suggest that propofol can effectively inhibit status epilepticus induced by lithium-pilocarpine. This effect may be associated with downregulation of N-methyl-D-aspartate receptor 2B subunit expression after seizures. PMID:25737709

  14. The Mediator subunit SFR6/MED16 controls defence gene expression mediated by salicylic acid and jasmonate responsive pathways.

    Science.gov (United States)

    Wathugala, Deepthi L; Hemsley, Piers A; Moffat, Caroline S; Cremelie, Pieter; Knight, Marc R; Knight, Heather

    2012-07-01

    • Arabidopsis SENSITIVE TO FREEZING6 (SFR6) controls cold- and drought-inducible gene expression and freezing- and osmotic-stress tolerance. Its identification as a component of the MEDIATOR transcriptional co-activator complex led us to address its involvement in other transcriptional responses. • Gene expression responses to Pseudomonas syringae, ultraviolet-C (UV-C) irradiation, salicylic acid (SA) and jasmonic acid (JA) were investigated in three sfr6 mutant alleles by quantitative real-time PCR and susceptibility to UV-C irradiation and Pseudomonas infection were assessed. • sfr6 mutants were more susceptible to both Pseudomonas syringae infection and UV-C irradiation. They exhibited correspondingly weaker PR (pathogenesis-related) gene expression than wild-type Arabidopsis following these treatments or after direct application of SA, involved in response to both UV-C and Pseudomonas infection. Other genes, however, were induced normally in the mutants by these treatments. sfr6 mutants were severely defective in expression of plant defensin genes in response to JA; ectopic expression of defensin genes was provoked in wild-type but not sfr6 by overexpression of ERF5. • SFR6/MED16 controls both SA- and JA-mediated defence gene expression and is necessary for tolerance of Pseudomonas syringae infection and UV-C irradiation. It is not, however, a universal regulator of stress gene transcription and is likely to mediate transcriptional activation of specific regulons only. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  15. Electrophysiology and Beyond: Multiple roles of Na+ channel β subunits in development and disease

    Science.gov (United States)

    Patino, Gustavo A.; Isom, Lori L.

    2010-01-01

    Voltage-gated Na+ channel (VGSC) β subunits are not “auxiliary.” These multifunctional molecules not only modulate Na+ current (INa), but also function as cell adhesion molecules (CAMs) – playing roles in aggregation, migration, invasion, neurite outgrowth, and axonal fasciculation. β subunits are integral members of VGSC signaling complexes at nodes of Ranvier, axon initial segments, and cardiac intercalated disks, regulating action potential propagation through critical intermolecular and cell-cell communication events. At least in vitro, many β subunit cell adhesive functions occur both in the presence and absence of pore-forming VGSC α subunits, and in vivo β subunits are expressed in excitable as well as non-excitable cells, thus β subunits may play important functional roles on their own, in the absence of α subunits. VGSC β1 subunits are essential for life and appear to be especially important during brain development. Mutations in β subunit genes result in a variety of human neurological and cardiovascular diseases. Moreover, some cancer cells exhibit alterations in β subunit expression during metastasis. In short, these proteins, originally thought of as merely accessory to α subunits, are critical players in their own right in human health and disease. Here we discuss the role of VGSC β subunits in the nervous system. PMID:20600605

  16. Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes

    DEFF Research Database (Denmark)

    Petersen, Rasmus Koefoed; Madsen, Lise; Pedersen, Lone Møller

    2008-01-01

    AMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho......-kinase impaired proadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates a new mechanism of c......AMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response....

  17. Voltage Dependence of a Neuromodulator-Activated Ionic Current123

    Science.gov (United States)

    2016-01-01

    Abstract The neuromodulatory inward current (IMI) generated by crab Cancer borealis stomatogastric ganglion neurons is an inward current whose voltage dependence has been shown to be crucial in the activation of oscillatory activity of the pyloric network of this system. It has been previously shown that IMI loses its voltage dependence in conditions of low extracellular calcium, but that this effect appears to be regulated by intracellular calmodulin. Voltage dependence is only rarely regulated by intracellular signaling mechanisms. Here we address the hypothesis that the voltage dependence of IMI is mediated by intracellular signaling pathways activated by extracellular calcium. We demonstrate that calmodulin inhibitors and a ryanodine antagonist can reduce IMI voltage dependence in normal Ca2+, but that, in conditions of low Ca2+, calmodulin activators do not restore